Here we report the synthesis of a 150-member library of Lewis x 'glycofluoroforms' with site-specific fluorination on each monosaccharide component, by employing a diversity-orientated enzymatic assembly process (Figure ), featuring a wide substrate scope. Our design strategy incorporates azido-propyl handles to enable facile conversion of the synthetic Lewis x library into lipid-linked glycan probes (neoglycolipids, NGLs) or into glyconanoparticles (Figure ) for probing protein binding both in solid-phase high-throughput glycan microarray screening analyses and in solution-phase nanoparticle-based interaction studies. We investigate a 24-member subset of this library, for unique 'fluorine-codes' that may be discerned by Lewis x binding proteins. These include antibodies and closely related glycan-binding proteins and bacterial toxins. The position of each fluorine substitution is indicated by the bond angle, and anomeric configuration is shown as solid (beta) or dashed (alpha) lines, both following the Oxford Nomenclature System. The glycan library was constructed using a panel of chemically synthesised monosaccharide derivatives based on glucosamine 2a, galactose 3a and fucose 4a. N-Acetyl/N-trifluoroacetyl glucosamine derivatives included mono-and di-deoxyfluorination at C-6 (Figure ), and galactose and fucose derivatives were monodeoxyfluorinated at positions 3, 4 or 6. D-Fucose 3e was also included as 6-deoxy-D-galactose, and Darabinose 4e as a de-methylated L-fucose analogue. The fluorinated/deoxy UDP-Gal analogues were then synthesised using a one-pot multienzyme system (Figure ), with each galactose analogue (3a-3e) first converted to the corresponding sugar-1-phosphate using Streptococcus pneumoniae galactokinase (SpGalK), before transfer of UMP from UDP-Glc by E. coli galactose-1-phosphate uridylyltransferase (EcGalPU) and in-situ regeneration of UDP by Bifidobacterium longum UDP-sugar pyrophosphorylase (BlUSP) (Figure ; SI). Crude preparations of each UDP derivative were used in subsequent glycosylation reactions. To aid assembly of the LacNAc disaccharide core, an electrospray-mass spectrometry (ESMS) screening protocol was established to investigate the substrate scope for enzymatic galactosylation of GlcNAc library 2a-2f with UDP-Gal library UDP-3a-3e using Homo sapiens b(1,4)-galactosyltransferase (Hsb4GalT1), which showed high tolerance to fluorination at the GlcNAc 6-position and trifluorination of the N-acetyl group, and could be easily expressed in E. coli as a fusion protein with maltose-binding protein. Conversion of the azidopropyl GlcNAc aglycon (Figure ; R = azidopropyl) to a positively charged imidazolium-based tag (ITag, Figure ) 42 (see SI) BfFKP Bacteroides fragilis l-fucokinase/lfucose-1-P guanylyltransferase. enabled semi-quantitative analysis of the MS data by ensuring that both the starting material and product were fully ionised and thus detectable by ESMS with comparable efficiency. Formation of all thirty LacNAc analogues (Figure ) was thus confirmed. All six GlcNAc derivatives were accepted as substrates by the Hsb4GalT1 enzyme, with high conversion when using the natural UDP-Gal donor, and all other UDP-Gal analogues also accepted but with significant differences in conversion based on the position of fluorination/deoxygenation. Specifically, UDP-Gal, UDP-4FGal and UDP-6dGal were all substrates for HsB4GalT1 with non-optimised conversions generally over 60%. Whilst UDP-3FGal and UDP-6FGal were poorer substrates with most conversions below 40%, which can be partially rationalised by crystal structures of b(1,4)-galactosyltransferase bound to UDP-Gal (SI Fig SXX ). The library of thirty LacNAc acceptors was then glycosylated with fucoses 4a-4e, to give a total of 150 possible Lewis x glycofluoroforms (Figure ). A one-pot, two-step glycosylation reaction in which the crude product mixture from galactosylation of 2a-2f was directly fucosylated using 1 molar equivalent of an α1-3 fucosyltransferase from H. pylori and in-situ generated GDP-fucose donors. Fucosylation proved to be much more efficient than galactosylation, with the percentage conversion for fucosylation >80% for 148 out of 150 reactions. Increased fluorination generally led to lower conversion although LacNAc analogues containing 6,6-diFGlcNTFA showed 100% conversion in several cases. While Hp-α1-3FucT has previously shown promiscuity, it was satisfying to find that it accepted our broad range of modified LacNAc acceptors. The crystal structure of Hp-α1-3FucT bound to GDP-Fuc shows that C-6 is surface exposed and therefore modifications at this position would be less likely to affect interactions between enzyme and substrate (SI Fig SXX ). In contrast, both the O-3/O-4 positions of GDP-Fuc form potential H-bonds to the enzyme so interruption of their ability to act as hydrogen bond donors would be expected to disrupt binding to the enzyme however both 3FFuc-GDP and 4FFuc-GDP were successfully turned over. The two notable exceptions to this observation were addition of 3FFuc and 4FFuc to 6dGalβ-1,4-GlcNAcOR. Of the 150 Lewis x glycofluoroforms identified by ESMS, 140 are novel compounds including, to the best of our knowledge, the first examples of fluorination to more than one monosaccharide in a Lewis x analogue.