Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy

Abstract

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage.

Fig. 1.

Structure of N-glycans and a bio-inspired strategy for their preparation. a, MGAT enzymes responsible for installing GlcNAc at different branching points. b, Enzyme classes involved in the biosynthesis of complex N-glycans. c, Structure of unnatural UDP-GlcNTFA (4). d, Bio-inspired strategy for the synthesis of asymmetric N-glycans. Symmetrical bi-antennary glycan 1, which can easily be obtained from a glycopeptide isolated from egg yolk, can be further branched by recombinant MGAT4 and MGAT5. The use of unnatural UDP-GlcNTFA makes it possible to prepare 2 bearing GlcNAc, GlcN₃ and GlcNH₂ branching moieties. Compound 2 is the key intermediate for preparing complex targets such as 3. e, Transformation of GlcNTFA, installed by MGAT4 and MGAT5, into GlcNH₂ or GlcN₃ “stops” further enzymatic extension of these moieties until they are converted into natural GlcNAc (go) that can then be elaborated by glycosyltransferases into complex appendages.

Fig. 2.

Two strategies for desymmetrizing N-glycans using the branch selectivity of the sialyltransferase ST6Gal1 and the galactosidase from E. coli, and subsequent preparation of asymmetric branched bi-antennary glycans such as 13. The α2,6-sialoside of 8 blocks further modification of the MGAT1 antenna allowing selective elaboration of the MGAT2 arm. The MGAT1 and MGAT2 arms of asymmetrically branched glycan 9 can selectively be extended by exploiting that many glycosyltransferases modify LacNAc but not terminal GlcNAc moieties making it was possible to first elaborate the MGAT2 arm without affecting the MGAT1 arm. The peptide sequence of SGP is NH₂-Lys-Val-Ala-Asn-Lys-Thr-COOH with the glycan connected to Asn. Each intermediate was purified by HPLC on an XBridge HILIC column. The transformation of 10 into 13 was also performed without intermediate compound purification and by only subjecting 13 to P2 size exclusion column chromatograph, an improved yield of 79% over three steps was accomplished.

Fig. 3.

Synthesis of asymmetric branched tri-antennary glycosyl asparagines using MGAT5 and UDP-GlcNTFA. MGAT5 readily accepts UDP-GlcNTFA to give a tri-antennary glycan that upon base treatment provides a compound having a GlcNH₂ at β6-arm (15). The latter residue is not a substrate for the galactosyl transferase B4GalT1 and therefore it is possible to selectively elaborate the MGAT1 and MGAT2 arm by exploiting inherent branch selectivities of glycosidases and glycosyl transferases. Once the MGAT1 and MGAT2 arms were capped with Neu5Ac preventing these positions from further elongation, the GlcNH₂ could be acetylated to give natural GlcNAc capable of being extended by a series of glycosyl transferase. Compounds 16 - 19 were purified by HPLC using a HILIC column. Compound 19 was subjected to three enzymatic transformations to yield 22, which was purified by P2 size-exclusion chromatography.

Fig. 4.

Synthesis of asymmetric branched tetra-antennary N-glycans using MGAT4 and MGAT5 in combination with UDP-GlcNTFA and subsequent conversion of the transferred GlcNTFA into GlcN₃ or GlcNH₂. The latter moieties are temporary disabled from modification by glycosyl transferases making it possible to selectively elaborate the MGAT1 and MGAT 2 arms. At an appropriate point in the synthesis, the unnatural GlcN₃ or GlcNH₂ moieties can be converted into natural GlcNAc allowing each arm to be uniquely extended. Compounds 26, 28, and 3 were purified by HPLC using a HILIC column and derivatives 23, 2, and 24 were purified by P2 size-exclusion chromatography.