Communication pubs.acs.org/JACS Rationally Designed Short Polyisoprenol-Linked PglB Substrates for Engineered Polypeptide and Protein N‑Glycosylation † § † § ‡ § † Feng Liu, , Balakumar Vijayakrishnan, , Amirreza Faridmoayer, , Thomas A. Taylor, † † ‡ † Thomas B. Parsons, Goncalo̧ J.L. Bernardes, Michael Kowarik, and Benjamin G. Davis*, † Department of Chemistry, Chemistry Research Laboratory, Oxford University, Oxford OX1 3TA United Kingdom ‡ GlycoVaxyn AG, 8952 Schlieren, Switzerland *S Supporting Information ABSTRACT: The lipid carrier specificity of the protein N-glycosylation enzyme C. jejuni PglB was tested using a logical, synthetic array of natural and unnatural C10, C20, C30, and C40 polyisoprenol sugar pyrophosphates, including those bearing repeating cis-prenyl units. Unusual, short, synthetically accessible C20 prenols (nerylnerol 1d and geranylnerol 1e) were shown to be effective lipid carriers for PglB sugar substrates. Kinetic analyses for PglB revealed clear KM-only modulation with lipid chain length, thereby implicating successful in vitro application at appropriate concentrations. This was confirmed by optimized, efficient in vitro synthesis allowing >90% of Asn-linked β-N-GlcNAc-ylated peptide and proteins. This reveals a simple, flexible biocatalytic method for glycoconjugate synthesis using PglB N-glycosylation machinery and varied chemically synthesized glycosylation donor precursors. Figure 1. Bacterial N-linked glycosylation and designed unnatural candidate polyprenols 1a−g as alternative short lipid carriers for PglB- catalyzed glycosylation. The polyprenols were synthesized from building blocks 2a−d and 3a and 3b. rotein glycosylation is a vital co- or post-translational P modification that links glycans to proteins typically through N- or O- linkages. Such modifications exist widely in eukaryotic as the fact that C. jejuni PglB can be readily overexpressed in 16 and archaeal organisms and greatly expand the diversity of the functional form in Escherichia coli, highlight PglB’s potential as − proteome.1 3 While N-linked glycosylation had been believed to a synthetic biocatalyst. They suggest it as a potentially ideal be absent in prokaryotic systems, the discovery of the protein N- model from which to generate a ready synthetic system for in glycosyltransferase PglB in Gram-negative bacterium Campylo- vitro protein glycosylation. However, the donor substrates used bacter jejuni highlighted greater diversity; PglB is responsible for normally by PglB in vivo (C55 lipid pyrophosphoryl-linked − glycosylating over 50 different proteins.4 7 Since then PglB oligosaccharides containing the rare, bacterial sugar bacillos- orthologues have been found in Campylobacter lari,8 the amine (Bac)) would restrict this system (both in substrate Helicobacter genus (H. pullorum, H. canadensis, H. winghamensis)9 accessibility and product relevance). as well as Desulfovibrio desulfuricans10 and, very recently, In an attempt to optimize the PglB protein N-glycosylation Methanococcus voltae.11 In vivo PglB uses a C55 undecaprenylpyr- platform for practical, synthetic (and hence in vitro) use, we ophosphate-linked oligosaccharide as its substrate and glyco- designed an array of chemically generated polyisoprenol variants fi sylates the primary amide nitrogen of the asparagine side chain in to nd those simpler and shorter than the natural undecaprenol a D/E-X-N-X-T/S consensus sequence (where X can be any (Figure 1) and that might serve as alternative lipid carriers that amino acid except proline, Figure 1). In contrast to its eukaryotic could be recognized by PglB. Insightful prior work has elucidated fi 17,18 counterpart Stt3p, PglB does not require other proteins/subunits some aspects of the polyisoprenol speci city of PglB. and can apparently catalyze this N-glycosylation in vivo alone.12 However, estimated conversions for these reactions were ≤ Moreover, in vivo biosynthetic studies that allowed exposure of 20%. In addition, many of these prior substrates were prepared PglB to other lipid-linked oligosaccharides have suggested a enzymatically from polyisoprenols isolated from natural sources, possibly relaxed specificity toward the nature of glycan substrate, enabling only nmol analysis of lipid pyrophosphates containing as compared to the relatively highly specific eukaryotic N- the natural Campylobacter (GalNAc-GalNAc-BacdiNAc) gly- glycosylation enzymes.13 Recent elegant approaches flexibly using PglB in vivo have been developed to prepare Received: September 11, 2013 glycoproteins.14,15 These distinct characteristics of PglB, as well Published: December 16, 2013 © 2013 American Chemical Society 566 dx.doi.org/10.1021/ja409409h | J. Am. Chem. Soc. 2014, 136, 566−569 Journal of the American Chemical Society Communication a Scheme 1. Syntheses of Polyprenol Variants Scheme 2. Syntheses of Lipid-linked Oligosaccharides and use a in Glycopeptide Synthesis a(i) BuLi, THF, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMTP), −78 °C. Yield: 4a = 92%, 4b = 93%, 4c = 83%, 4d = 63%, 4e = 67%, 4f = 74%. (ii) (1) TBAF, THF, RT, 4 h; (2) LiEt3BH, ° − (dppp)PdCl2, THF, 0 C RT. Yield: 1a= 65%, 1b = 57%, 1c = 50%, 1d = 70%, 1e = 60%, 1f = 54%. cans. This revealed tantalizing activity for two shorter (mixed cis/ trans prenol-9 and prenol-8) variants. However, despite some − individual pioneering examples19 28 synthetic access to other prenols has been rare, and homologated families of lipids have not yet been probed. Therefore, with the intention of probing fuller lipid and sugar substrate plasticity and breadth in PglB and with the intention of creating synthetically accessible substrates, we first systematically varied the lipid carriers (Figure 1). As a starting point for lipid variation, we speculated that the use of repeating cis-prenyl units at the hydroxyl terminus would confer binding affinity due to a resemblance to the alcohol- terminus that bears the glycan-pyrophosphate found in the natural substrate and likely enters a binding pocket in PglB.29 This notion is supported by a crystal structure which has a Mg2+ bound at the active site.29 Subsequent modeling suggested that the lipid carrier would likely locate first in a narrow pocket that would tightly accommodate two isoprenyl units (and thus a − (i) 5a g, TBAP, TCA, DCM, RT; 5h, POCl3,Et3N, 2h. (ii) (1) CDI, impose tighter stereochemical requirements) and then along a DMF; (2) MeOH; (3) 6, DMF, RT, 3−5 days. (iii) NaOMe, MeOH. broader hydrophobic groove with potentially more relaxed Yield over 3 steps: 8a = 17%, 8b = 23%, 8c = 9%, 8d = 17%, 8e = 9%, requirements (Figure S12). Thus, compounds 1a−g, all bearing 8f = 38%, 8g = 29%, 8h = 32%, 13 = 22%, 14 = 30%. Extension one or more terminal repeating cis-prenyl units were designed reactions (blue arrows, 5−94%); see SI for further details. and synthesized as primary candidate lipid carriers (Figure 1). In brief (see SI for further details), compounds 1a−f were tri-O-acetyl-2-deoxy-α-D-glucopyranose 1-phosphate (6) using synthesized from head (compound 3a and 3b) and tail building carbonyldiimidazole (CDI)-mediated phosphoesterification.32,33 blocks (compound 2a−d). The requisite building blocks were Deacetylation with catalytic NaOMe in MeOH (Zempleń prepared and elongated using coupling between appropriate conditions) gave the lipid pyrophosphate-linked saccharides sulfones and allyl chloride. Utilization of a convergent synthetic (LPPS) 8a−g in 9−38% yield over three steps (Scheme 2). strategy in the case of long lipids (Scheme 1) allowed useful With these first synthetic LPPS’s in hand, 8a−h were then flexibility in the generation of lipids with different stereo- tested as substrates of PglB. The glycosylation of peptides chemistry. Removal of sulfonyl groups in one step using bearing the required D/E-X-N-X-S/T consensus motif was LiEt3BH/(dppp)PdCl2 valuably reduced both the total number determined by examining the increase of molecular weight of synthetic steps and the formation of isomeric products (16%/6 M urea tricine-SDS-PAGE)34 that corresponds to the resulting from double-bond migration and basic conditions.23,25 addition of GlcNAc (Figure 2). An extract from E. coli LPPS The resulting, pure synthetic polyprenols were shown to have a containing C. jejuni heptasaccharide-linked undecaprenyl d.r. (E/Z) > 95% according to the characteristic allylic methyl pyrophosphate was used as a positive control. Excitingly, 8a−e group signals in 1H NMR (see Figure S1); no unwanted isomeric were active substrates for PglB-catalyzed N-glycosylation of the product (<2%, 1H NMR signals at δ 2.7, 5.9 ppm)30 was fluorescent peptide Tamra-DANYTK as indicated by the detected. appearance of glycopeptide product bands with higher molecular These polyprenols were readily phosphorylated using mono- weight consistent with the addition of GlcNAc. This importantly (tetrabutylammonium) phosphate (TBAP) activated with revealed that the lipid carrier for the substrate of PglB can contain trichloroacetonitrile (TCA).31 We next attached just a single as few as two cis-head repeating units. The activity of 8c and 8e as residue atypical glycan to provide a stringent test of the catalytic substrates for PglB also indicated that PglB can tolerate lipid activity of PglB. Importantly, we used the sugar that would be carriers bearing trans geometry close to the hydroxyl terminus. found as the first N-linked residue in eukaryotic glycoproteins However, activity was lost as the length of lipid became shorter (but not prokaryotic and so not that normally employed by than four prenyl units: nerol (1g)-PP-GlcNAc (8g), cis,cis- Campylobacter): GlcNAc. The resulting polyprenyl mono- farnesol (1f)-PP-GlcNAc conjugate (8f), and citronellol-PP- phosphates 5a−g were therefore coupled to 2-acetamido-3,4,6- GlcNAc conjugate (8h)32,33 did not show any detectable activity 567 dx.doi.org/10.1021/ja409409h | J. Am. Chem. Soc. 2014, 136, 566−569 Journal of the American Chemical Society Communication phate (5d) with the 6-azido-GlcNAc (9) and 2-azidoGlcNAc (10, GlcNAz). These compounds would allow subsequent flexible postexpressional modifications on proteins that contain D/E-X-N-X-S/T tag via reaction of the introduced azide with a number of compatible methods.