loading . . . Iterative Bump-and-hole engineering creates a bioorthogonal reporter for N-acetylglucosaminyltransferase I Asparagine-linked protein glycosylation is among the most frequent modifications of proteins trafficking through the secretory pathway. These glycans are manufactured in an assembly line process to a common precursor that is then subject to individual modifications with different levels of complexity. An important biosynthetic modulator is the incorporation of N-acetylglucosamine (GlcNAc) at distinct positions in N-linked glycan biosynthesis, commencing with the activity of the glycosyltransferase MGAT1. While mapping of N-glycans to their corresponding protein attachment sites is generally possible, not much is known about the glycoprotein substrate choice for MGAT1 and related transferases. Analogs of GlcNAc with small bioorthogonal tags can be incorporated into N-glycans. However, due to the promiscuity of some GlcNAc transferases, incorporation is of little specificity towards individual positions. Here, we report an iterative bump-and-hole approach in the design of a bioorthogonal precision tool for the activity of MGAT1 in mammalian cells. Structure-informed protein engineering abrogated the activity of MGAT1 towards the nucleotide-sugar UDP-GlcNAc while retaining activity towards bumped, azide-modified analogs. Kinetic and computational analyses using a neural network approach informed the synthesis of a tailored UDP-GlcNAc analog with preferential acceptance by the engineered enzyme. Following substrate biosynthesis, the strategy allowed selective incorporation of a chemical tag on MGAT1 substrate proteins in living mammalian cells with little background incorporation by other GlcNAc transferases. Our work expands the toolbox for glycan-based reporter compounds. ### Competing Interest Statement C.R.B. is a co-founder and scientific advisory board member of GanNA Bio, Neuravid, Firefly Bio, Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, Grace Science and InterVenn Biosciences. C.R.B. is a member of the Board of Directors of Alnylam, Xaira Therapeutics, Acepodia and OmniAb. Wellcome Trust, https://ror.org/029chgv08, CC2127, 312750/Z/24/Z Cancer Research UK, https://ror.org/054225q67, CC2127, DRCMDP-Nov22/100011 Medical Research Council, https://ror.org/03x94j517, CC2127 UK Research and Innovation, https://ror.org/001aqnf71, EP/X042383/1, EP/Y032527/1, MR/V02213X/1, UKRI2014 Howard Hughes Medical Institute, https://ror.org/006w34k90, CC31920 Children's Tumor Foundation https://www.biorxiv.org/content/10.64898/2026.01.16.699845v1
