ABSTRACT. Cell-surface oligosaccharides appended to proteins and

lipids are key binding epitopes in many critical biological processes, including bacterial infection, cell development and the immune response. Understanding these processes at the molecular level requires access to oligosaccharides of known structure to support investigations of enzyme substrate specificity, to screen for carbohydrate binding proteins, and to develop assays for enzymic activity. These oligosaccharides are currently unavailable in the commercial sector at affordable prices and high purities, thus impeding progress in this field. To address these deficiencies, large-scale syntheses of oligosaccharides derived from the parent high-mannose N-glycan precursor (14-mer) were developed. Forty-one (41) oligosaccharides ranging in size from tri- to nonasaccharides and containing both Man and GlcNAc residues were regioselectively synthesized with minimally protected mannosyl acceptors. These oligosaccharides were prepared in 10-100 mg quantities in >98% chemical purity, as determined by NMR, HRMS, HPLC, and capillary electrophoresis (CE). Samples will be used by the NIH to construct glycan arrays for the screening of carbohydrate binding proteins, and for other biochemical or biomedical applications. [This work was supported by NIH/NCI SBIR HHSN261201300038C and HHSN261201500020C].

INTRODUCTION. Deciphering the relationships between saccharide

structure and function lies at the heart of modern glycobiology. It is

widely recognized that saccharides covalently attached to membrane-

associated proteins and lipids play major roles in human biochemical

processes (Hermansson et al., 2011). These saccharides are

commonly attached to proteins (or lipids) as N- or O-glycosides, with

the asparagine (Asp) side chain involved in the former, and serine (Ser)

and threonine (Thr) side chains involved in the latter (Varki, 1993).

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N- and O-linked glycoproteins are synthesized in vivo in different ways.

N-Linked oligosaccharide is installed cotranslationally, and involves a

consensus sequence on the protein (Asn-X-Ser/Thr) that is recognized

by oligosaccharyl transferase (OST). OST catalyzes the en-bloc

transfer of the parent 14-residue oligosaccharide 1, biologically

activated by dolichol phosphate (Lehle et al., 2006), to the polypeptide.

This 14-mer precursor is then modified in the Golgi to produce high-

mannose, complex and hybrid type N-glycans (Helenius & Aebi, 2001)

as shown in 2–4.

SYNTHESIS. Chemical glycosylation can be accomplished

with either an orthogonally protected acceptor that is

regiospecific but requires many steps to synthesize, or with a

partially protected acceptor requiring fewer steps to prepare

but regioselectivity is required during glycosylation. This

SBIR project involves the preparation of a wide range of

nested fragments of the high-mannose parent N-glycan 1 and

the related structure 2. Considering the number of branched

structures targeted in this project, glycosylation with minimally

protected acceptors will likely expedite the overall synthesis.

SUMMARY AND FUTURE DIRECTIONS Forty-one (41) homo-mannose oligosaccharides, including six

trisaccharides, eight tetrasaccharides, nine pentasaccharides, nine

hexasaccharides, six heptasaccharides, three octasaccharides and one

nonasaccharide were prepared during NIH-funded SBIR Phase I and II

projects in quantities ranging from a few milligrams to hundreds of

milligrams and in high purities (typically >97%).

Glycosylation with minimally protected acceptors simplified the

oligosaccharide syntheses and the strategy is currently being applied to

the preparation of GlcNAc-containing oligosaccharides (up to 13-mers).

Capillary electrophoresis has proven to be an effective analytical

method to determine oligosaccharide purity due to its unique ability to

resolve structurally similar isomers.

Forty-one (41) homo-mannose oligosaccharides (Schemes

1 and 2) ranging from tri- to nonasaccharides were

synthesized from these three glycosyl acceptors

(compound numbers are from the original proposal). NMR,

HPLC, HRMS, and CE were the primary analytical tools

used to establish the identities and purities of protected

precursors and final oligosaccharide products.

In oligosaccharide assembly, different types of linkages can form

between the anomeric centers of the donor sugars and the multiple

hydroxyl groups of the acceptors, giving rise to branched structures

such as 2. This work will make available a wide range of N-glycans to

help investigators sort out the language encoded in complex

carbohydrate sequences. It will do so by systematically and

regioselectively preparing a comprehensive set of nested fragments of

one type of N-glycan, namely, the high-mannose type shown in

structures 1 and 2 with minimally protected glycosyl acceptors.

REPRESENTATIVE DATA ANALYSIS. NMR, HPLC, CE

and HRMS were the main analytical tools used to establish

the identities and purities of the products. 1H, 13C{1H} and 2D 1H-13C gHSQC (when necessary) data were used to confirm

the structures of the oligosaccharide products. For example,

the 2D 1H-13C gHSQC spectrum (Figure 2A) was used to

establish that a product was not a-D-mannopyranosyl-

(13)-[a-D-mannopyranosyl-(16)]-D-mannopyranose

(compound 13) as expected, but rather its regioisomer, a-D-

mannopyranosyl-(14)-[a-D-mannopyranosyl-(16)]-D-

mannopyranose. The most downfield carbon signals in the

non-anomeric region correlate with two triplet proton signals

(only H4s exhibit this pattern in a Man ring), showing this

structure contained a (14) linkage. We subsequently

completed the synthesis of compound 13. The expanded

non-anomeric region of its HSQC spectrum is shown in

Figure 2B. The most downfield carbon signals in the non-

anomeric region (near 80 ppm) show correlations with

doublet proton signals (C3-H3 correlations) indicating a

(13) linkage.

Figure 2. The non-anomeric region of the 2D 1H-13C gHSQC spectrum of (A) a-D-

mannopyranosyl-(14)-[a-D-mnnopyranosyl-(16)]-D-mannopyranose) and (B) a-D-

mannopyranosyl-(13)-[a-D-mannopyranosyl-(16)]-D-mannopyranose (13) in 2H2O at 25 oC.

HPLC and CE were used to determine the purity of the final products. CE

has the potential to resolve structurally similar isomers owing to the large

number of theoretical plates in these columns. CE trace for

octasaccharide 103 is shown in Figure 3A. Impurities with a retention time

of 9.317 min were observed (not detected in HPLC). These impurities are

probably isomers of 103 because they migrate similarly as 103. The

crude mixture of compound 103 was also analyzed by CE (Figure 3B) and

additional impurities were observed, which were removed by Biogel P4

SEC column chromatography along with partial removal of the impurities

at 9.317 min.

Three minimally protected glycosyl acceptors were designed

to achieve the synthetic goals (Figure 1). Acceptor A, 1,2-O-

ethylidene-b-D-mannopyranoside, was found to be

glycosylated at the 4 and 6 positions instead of the expected

3 and 6 positions. Acceptor B, allyl 3-O-benzyl-a-D-

mannopyranoside, was expected to be regioselectively

glycosylated at the 6 position, then at the 3 position after

removal the 3-O-benzyl group. Glycosylation proceeded as

expected until the syntheses of compounds containing an a-

Man-(12)-a-Man-(12)-Man branch at their 3-positions.

Multiple regioisomers were produced during glycosylation

with the per-O-acetylated a-Man-(12)-a-Man-(12)-Man

donor due to acyl migration from the 2 and 4 positions of the

acceptors, which made final purification difficult.

Glycosylation with acceptor C, allyl 6-O-TBDPS-a-D-

mannopyranoside, was expected to be regioselectively

glycosylated at the 3 position, then at the 6 position after

removal the TBDPS group. This strategy produced the

desired product with few impurities. Figure 3. CE data for a-D-mannopyranosyl-(12)-a-D-mannopyranosyl-(13)-[a-D-

mannopyranosyl-(16)]-a-D-mannopyranosyl-(16)-[a-D-mannopyranosyl-(12)-a-D-

mannopyranosyl-(12)-a-D-mannopyranosyl-(13)]-D-mannopyranose (103). (A) Final

product. (B) Crude product.