NEW PEGYLATION TECHNOLOGY
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Name |
Chemical Structure |
Application |
| Maleimide Derivative of PEG |
PEG-methyl maleimide |
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Thiol-specific pegylation of antibodies, viruses, peptides and proteins |
| Aldehyde Derivatives of PEG |
PEG-butyraldehyde |
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N-terminal specific pegylation of proteins |
| PEG-pentaldehyde |
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| PEG-amido-propionaldehyde |
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| PEG-urethano-propioaldehyde |
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| Multi-arm PEG |
6-arm PEG |
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More reactive component in hydrogel formulation |
PEG-METHYL MALEIMIDE
FOR THIOL-SPECIFIC PEGYLATION OF ANTIBODIES AND PROTEINS
- MUCH MORE STABLE THAN PEG-MALEIMIDE -
PEG modification requires activation of the PEG polymer that is accomplished by the introduction of an electrophilic center. The PEG reagent is now susceptible to nucleophilic attack, predominantly by the nucleophilic epsilon-amino group of a lysyl residue. Because of the number of surface lysines present in most proteins, the PEGylation process can result in random attachments leading to mixtures which are difficult to purify and which may not be desirable for pharmaceutical use.
There are a large variety of PEG reagents that have been developed for the modification of proteins. This involves the covalent attachment of a PEG molecule via the formation of a linking group between the PEG polymer and the protein. Some of these reagents are, to various degrees, unstable in the aqueous medium in which the PEGylation reaction occurs. In addition, the conjugation process often results in the loss of in vitro biological activity that is due to several factors foremost of which being a steric interaction with the protein's active sites. The pegylation of a free cysteine moiety with a PEG-maleimide reagent is the primary method by which the site-specific pegylation of a protein may be realized. A PEG-sulfhydryl reactive derivative may react with a cysteine via a Michael addition to form a stable 3-thiosuccidimidyl ether linkage. The selective derivatization of a cysteine moiety is made possible by the fact that a maleimide specific sulfhydryl reagent can form a covalent bond with a cysteine residue about 1000-fold faster than with a corresponding amine.
Maleimides such as described by compound I, can add water across the double bond and can be slowly hydrolyzed to a non-reactive cis-maleamic acid derivative. The reaction of I with a sulfhydryl derivative gives the Michael addition product II. The resultant thioether is very stable and cannot be reversed under physiological conditions. However, the slow hydrolytic cleavage of the succinimide ring in compound II can occur resulting in the formation of the succinamide acid derivatives IIIa and IIIb.
A desired property of a new PEG-maleimide reagent would be one that has excellent stability in an aqueous medium and can form with alacrity a cysteine addition product that has enhanced resistance to hydrolytic cleavage. Based on preliminary data, an N-PEG-3-methylmaleimide derivative IV may be considered such a reagent. Compound IV functions as a thiol receptor to produce an addition product which is considerably more stable than the corresponding standard maleimide derivative II.
Given that the hydrolysis of the succinimide ring in compound V involves a transition state in which water is added to an electron-deficient carbonyl to produce a tetrahedral adduct intermediate, it would be expected that the effect of the methyl group both sterically and electronically, would be to decrease significantly the rate of hydrolysis of the succinimide ring in compound V as compared to the unsubstituted derivative II.
NOVEL ALDEHYDE DERIVATIVES OF PEG
A PEG aldehyde of structure 1 may react selectively with a protein amine (P-NH2), at a single site which is located at the N-terminus of the protein. This can be done by carrying out the reaction of the compound of formula 1 with P-NH2 at a pH of from 5.5 to 7.5 to form an intermediate Schiff base of formula 2. In carrying out this reaction, various buffers which maintain the reaction media at a pH of from 5.5 to 7.5 can be used. If one desires the amination to proceed on more than one amino site on the protein, then one carries out the reaction at a pH of 8.0 and above, preferably at a pH of from 8 to 10. This imine linkage of the compound of formula 2 is then reduced to an amine through the use of reducing agents such as cyanoborohydride to give the saturated conjugated protein of formula 3. In this manner, lysine amino groups, as well as the N-terminal amino group on the protein may be reductively aminated with a corresponding PEG aldehyde [Eq.(1)].
In the case of the PEG-acetaldehyde 4, the electronic nature of the -oxy substituent has a pronounced effect on the overall efficacy of the reductive amination process. The alpha- oxyaldehyde of structure 4 can readily participate as either a nucleophilic or electrophilic reagent. Because of the electron withdrawing nature of the -oxy moiety, the compound has a reactive enolizable proton as well as an aldehyde function that is susceptible to an addition reaction. The acidity and nucleophilicity of the -oxyaldehyde 4 are the requirements for a facile aldol condensation and the formation of compound 5 [Eq.(2)].
Although compound 4 has been successfully used for the reductive amination reaction as shown in equation (1), it is generally used in large excess of the protein since the aldol reaction of equation (2) is believed to be competitive with the formation of the Schiff base 2. By the insertion of an additional methylene group between the aldehyde and the ether oxygen, the effect of the -oxy group is less pronounced, resulting in a propionaldehyde derivative 6 which is less prone for an aldol condensation and which exhibits a decreased rate of reaction with a protein amine.
We have developed a novel synthesis which allows us to prepare not only the propionaldehyde 6, but a series of PEG aldehydes as described by structure 7.
These aldehydes are very stable in an aqueous medium but as expected, are somewhat less reactive for Schiff base formation. The design of these compounds makes possible the choice of a stable PEG aldehyde whose reactivity may be best suited for reaction with a particular protein amine. That is, the overall affect is expected to enable a greater selectivity in the reductive amination reaction. The less the electrophilic nature of the aldehyde a more nucleophilic amine is required for reaction.
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