dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1-phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI-MS, and NMR, showing about 95% purity.

dTDP-L-rhamnose, dTDP-4-keto-6-deoxy-D-glucose, dTDP-4-keto-6-deoxy-D-glucose 3, 5-epimerase, dTDP-4-keto-rhamnose reductase

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10
5,0,0	bLRhap	95.97	72.04	73.17	72.57	70.77	16.91
5,0	P
5	P
	xXnucdT	85.61	38.74	71.28	86.35	65.88	152.13	166.91	111.13	137.75	12.12


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9	H10
5,0,0	bLRhap	5.23	3.40	3.66	3.47	4.10	1.32
5,0	P
5	P
	xXnucdT	6.35	2.40	4.62	4.20	4.20	-	-	-	7.79	1.94


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9	C10/H10
5,0,0	bLRhap	95.97/5.23	72.04/3.40	73.17/3.66	72.57/3.47	70.77/4.10	16.91/1.32
5,0	P
5	P
	xXnucdT	85.61/6.35	38.74/2.40	71.28/4.62	86.35/4.20	65.88/4.20				137.75/7.79	12.12/1.94

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Cc1cn([C@H]2C[C@H](O)[C@@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@@H](C)[C@H](O)[C@@H](O)[C@H]3O)O2)c(=O)[nH]c1=O

548.331 g/mol (C₁₆H₂₆N₂O₁₅P₂)