Use of Functionalized Poly(Ethylene Glycol)s for Modification of Polypeptides

Abstract

The unique properties of poly(ethylene glycol) (or PEG) and its general compatibility with polypeptide materials facilitated development of a variety of different applications of this polymer. 1 -ll A marked proportion of these applications involve the use of covalently linked polypeptide-PEG adducts (reviewed else-where5-11 ). For example, a number of PEG-enzymes were shown to be useful as catalysts, soluble and active in organic solvents. 7 Due to the affinity to the upper phase of PEG/Dextran and PEG/salt two-phase systems, PEG-modified proteins were proven useful both as diagnostic tools 8 and in preparative separations of biological cells. 9 Unquestionably, the properties of polymer-polypeptide conjugates in vivo were a significant reason for the substantial amount of research reported in the area during the last two decades. It has been repeatedly demonstrated that covalent attachment of multiple strands of PEG to proteins produces conjugates with dramatically reduced immunogenicity and antigenicity. IO Such preparations also show great resistance to proteolytic digestion, and remain present in the bloodstream a considerably longer time than the parent polypeptides. These beneficial properties of modified polypeptides allowed development of a number of PEG-modified therapeutic proteins. IO Slight to moderate modification of potent allergenic proteins with PEG can often be SAMUEL ZALIPSKY and CHYI LEE· Enzon, Inc., South Plainfield, New Jersey 07080. Present address for s. Z.:

Solubilization of Hydrophobic Peptides by Reversible Cysteine PEGylation

“…A wide variety of chemistries have been developed with which to denvatize PEG for attachment to proteins (Zalipsky & Lee, 1992). Most of this development has focused on different ways to synthesize PEG derivatives that can be conjugated with free amino groups to fonn amide bonds.…”

“…Most of this development has focused on different ways to synthesize PEG derivatives that can be conjugated with free amino groups to fonn amide bonds. This method is preferred for most applications due to the abundance of free amino groups in proteins in the form of Lys residues and the stability of the amide bond formed (Zalipsky & Lee, 1992).…”

“…A large excess of reagent was required due to the tendency for the chlonnes of cyanuric chloride to spontaneously hydrolyze under the conditions required for PEGy lation of proteins (Abuchowski et al, 1977). This as well as the toxicity of the derivative and its promiscuous reactivity with the side chains of Cys and Tyr (Zalipsky et al, 1992) provided the impetus for the discovery of new chemistries of PEG denvatization. (Beauchamp et al, 1983) and MPEGphenylcarbonate (Veronese et al, 1985) were less promiscuous than the cyanuric chloriâe derivative, but suffered ffom poor reactivity and reagent toxicity, respectively.…”

“…(Beauchamp et al, 1983) and MPEGphenylcarbonate (Veronese et al, 1985) were less promiscuous than the cyanuric chloriâe derivative, but suffered ffom poor reactivity and reagent toxicity, respectively. MPEG-succinirnidyl succinate (Abuchowski et al, 1984) and MPEG-succinimidyl carbonate (Zalipsky et al, 1992) exhibit high reactivity and specificity. However, the ester bond between the succinate and the MPEG in the former is subject to degradation in aqueous solution, and the synthesis of the latter requires the use of the extremely toxic reagent phosgene.…”

“…However, reaction with Lys resuits in irreversible amide and urethane bonds (Zalipsky & Lee, 1992). This is useful when stability is an asset, but is unsuitable for any application that requires transient PEGylation.…”