Table 1.

Results of donor substrate specificity studies for the α2–6-sialyltransferase activity of NmSiaD_W using in situ generated CMP-sialic acids and analogs.

	Donor precursor	Percentage conversion (%)
		CMP-Sialic acid	Sialyltransfer
			10 μg/mL, 10 min	3.3 mg/mL, 10 h
1	Neu5Ac	aCMP-Neu5Ac (Quant.)	30±0.3	Quant.
2	Neu5Gc	aCMP-Neu5Gc (Quant.)	32±2	Quant.
3	Neu5Ac8OMe	aCMP-Neu5Ac8OMe (Quant.)	0	83±7
4	Neu4,5Ac2	a,cCMP-Neu4,5Ac2 (Quant.)	0	0
5	Neu5,9Ac2	a,cCMP-Neu5,9Ac2 (Quant.)	0	0
6	Kdn	aCMP-Kdn (Quant.)	0	98±2
7	ManNAz	bCMP-Neu5NAz (Quant.)	28±2	Quant.
8	ManNAc4N3	bCMP-Neu5Ac7N3 (Quant.)	0	78±3
9	ManNAc6N3	bCMP-Neu5Ac9N3 (Quant.)	0	90±2
10	ManNAc6NAc	bCMP-Neu5Ac9NAc (Quant.)	0	18±1
11	Man2N3	bCMP-Neu5N3 (Quant.)	0	64±1

Step 1 of the reaction was carried out with a monosaccharide (1–11, 1.2 equiv.) in the presence of NmCSS (1.5 mg/mL) and CTP (1 equiv.) for 4 h ^awithout (for 1–6) or ^bwith (for 7–11) PmAldolase (4 mg/mL) and sodium pyruvate (5 equiv.). Tris-HCl (pH 8.5) was used for all reactions except for entries 4 and 5 where ^cTris-HCl (pH 7.5) was used to minimize de-O-acetylation which would take place at pH 8.5.