Thus, the two C2 carboxylate groups found in the nonreducing terminal sialic acids of the decasaccharide lactol422cwere converted into amides with 4(4,6dimethoxy1,3,5triazin2yl)4methylmorpholinium chloride (DMTMM).30Through the use of an elevated temperature (3750C) and an excess of the ammonium salt and coupling agent we were able to drive amidation to completion to give5ae. %) erhalten, wenn nichtspezifische, nichtenzymatische Reaktionen (Glykierung) zurckgedrngt wurden. Glykosylierung mit nichtnatrlichen Zuckern ermglichte ferner das regioselektive Anbringen einer Fracht. Protein glycosylation is the most common and varied posttranslational modification, critically influencing protein function, 1and yet is currently difficult to maslinic acid control.2Endoglycosidase (ENGase or Endo)catalyzed glycosylation is an attractive strategy to access homogeneous glycoproteins (Figure1a);3the initially limited scope of glycoproteins was expanded to antibodies (Abs) by the use of EndoS,4a family 18 glycoside hydrolase (GH) fromStreptococcus pyogenescapable of glycosylating immunoglobulins (Igs).5Monoclonal Abs (mAbs) and antibodydrug conjugates (ADCs) are a rapidly growing class of therapeutics.6Glycans in Abs7modulate stability, the rate of clearance, and the pharmacokinetic profile;8aggregation, folding, and immunogenicity;9complement activation;10binding to Fc receptors maslinic acid and Abdependent cellmediated cytotoxicity;11and Abmediated inflammation.12They are therefore vital functional switches that cannot yet be controlled cleanly (see the Supporting Information for an extended discussion). == Figure 1. == a) Endoglycosidasecatalyzed glycosylation with activated sugar donors may lead to competing chemical glycation. b) Current mAbs are formed as mixtures of glycoforms; G0F, G1F, and G2F predominate. c) EndoSWT cleaves the core of mixed Nglycans and can subsequently catalyze glycosylation with oxazoline donors,5ato give, in principle, purer glycoform distributions. d) Payload molecules may also, in principle, be introduced directly through glycosylation or indirectly by the incorporation of reactive handles in sugars and a subsequent selective reaction. Antibodies are all Nglycosylated in the Fc region of each of two heavy chains. All therapeutic Abs are currently produced from cells as mixtures (Figure1b); more than 20 different glycoforms are typically identified.13By contrast, the chemoenzymatic ENGase method could potentially be used to access pure Abs. However, until now it has been Mouse monoclonal to Plasma kallikrein3 assumed that this method will necessarily give rise to homogeneous glycoforms by virtue of the direct reversal of selective enzymatic hydrolytic activity (Figure1). maslinic acid Herein we demonstrate that this assumption is incorrect: not only do nonspecific background chemical modifications compete, but we now reveal optimized methods that allow access to essentially homogenous (90 % pure) glycoforms of a key therapeutic mAb. Our preliminary studies5ahad indicated that wildtype (WT) EndoS could be successfully used to trim glycans from mixtures of glycoforms of human IgG to reveal single GlcNAc moieties (Figure1c, left). Subsequent treatment of the resulting IgGGlcNAc with WT EndoS and an appropriately activated sugar oxazoline donor led to the formation of a new glycosidic linkage (Figure1c, right).5aHowever, the inherent hydrolytic activity of EndoS prevented fully efficient reactions. To overcome this limitation, we explored the use of mutated variants of EndoS to access enzymes with enhanced transglycosylation:hydrolysis (T:H) activity ratios. Similar strategies14,15have proven successful in other ENGase systems, by partial analogy with synthases described by Withers and coworkers.16,17Sequence alignment (see the Supporting Information) with other family 18 and 85 GHs18suggested residues D233, E235, Q303, and Y305, which enhance the role of the C2 amide in reactions involving oxazolinium intermediates (D233), act as a general acid/base (E235), or assist substrate binding (Q303, Y305).19We generated EndoS mutants and assessed combined T:H activities (100:1 [Ab]:[EndoS]; T:H=35:30 (D233A), 65:25 (D233A/Q303E), nd:100 (Y305F), 75:55 (D233E), 80:20 (D233Q), 10:100 (WT); nd=not determined; see the Supporting Information). Although, in our hands, none displayed completely abolished hydrolytic activity, it was substantially decreased in EndoSD233Q as compared to EndoSWT, thus giving rise to a T:H activity of 80:20. We therefore selected EndoSD233Q. During the course of this study, Wang and coworkers also suggested that EndoSD233Q and EndoSD233A mutants possess useful synthase activity. 5bThe mutant EndoSD233Q is sufficiently stable to be produced on scale. We chose the therapeutic mAb Herceptin as a highly representative substrate (see the Supporting Information). Our analysis of.