UDP-Galf analogs could serve as useful tools to elucidate the
bifunctional activity of GlfT2.
H-2′, H-3′), 4.30–4.27 (m, 1H, H-4′), 4.24 (ddd, 1 H, J = 11.8,
4.2, 2.6 Hz, H-5′a), 4.22–4.18 (m, 2 H, H-5′b, H-3′′), 4.15 (ddd,
1 H, J = 8.3, 4.2, 2.3 Hz, H-2′′), 3.90–3.87 (m, 1 H, H-5′′), 3.80
(dd, 1 H, J = 7.0, 6.1 Hz, H-4′′), 3.60 (dd, 1 H, J = 10.8, 3.9 Hz,
H-6′′a), 3.53 (dd, 1 H, J = 10.8, 7.2 Hz, H-6′′b), 3.39 (s, 3 H,
Experimental details
OCH3); MS (ESI) m/z 579 ([M − H]−, 5.6%), 289 ([M − 2H]2−
,
Preparation of acceptor trisaccharides and UDP-Galf donor
analogs
100%); HRMS (ESI) m/z Calcd for (M
C16H24N2O17P2: 289.0281. Found 289.0281.
−
2H)2−
Details for the synthesis of acceptor trisaccharides 2 and 3 are
reported elsewhere.37 UDP-Galf (1) and UDP-Galf analogs 4–6
were prepared as previously described.34,36,43 Details for the syn-
thesis of Galf-1-phosphate analogs 13–19 are found in the ESI.†
Uridine 5′-diphospho-5′′-O-methyl-α-D-galactofuranose (9).
(0.5 mg, <5%); H NMR (700 MHz, D2O, δH) 7.96 (d, 1 H, J =
1
8.1 Hz, H-6), 6.00–5.98 (m, 1 H, H-1′), 5.98 (d, 1 H, J = 8.1 Hz,
H-5), 5.67 (dd, 1 H, J = 6.7, 3.8 Hz, H-1′′), 4.39–4.37 (m, 2 H,
H-2′, H-3′), 4.29–4.28 (m, 1 H, H-4′), 4.24 (ddd, 1 H, J = 11.8,
4.4, 2.6 Hz, H-5′a), 4.20 (ddd, 1 H, J = 11.8, 5.6, 2.8 Hz, H-5′a),
4.16–4.12 (m, 2 H, H-2′′, H-3′′), 3.88–3.84 (m, 2 H, H-4′′, H-6′′
a), 3.62 (dd, 1 H, J = 12.5, 5.8 Hz, H-6′′b), 3.55 (s, 3 H, OCH3),
3.55–3.50 (m, 1 H, H-5′′); 13C NMR (175 MHz, D2O, δC) 167.1
(C-4), 142.5 (C-5), 103.5 (C-6), 98.7 (d, J = 5.6 Hz, C-1′′), 89.2
(C-1′), 84.2 (C-5′′), 84.1 (d, J = 8.9 Hz, H-4′), 82.1 (C-4′′), 77.6
(d, J = 7.1 Hz, H-2′′), 75.1 (C-3′′), 74.6, 70.5 (C-2′, C-3′), 65.7
(d, J = 5.9 Hz, H-5′), 60.2 (C-6′′), 59.4 (OCH3); MS (ESI) m/z
579 ([M − H]−, 18%), 289 ([M − 2H]2−, 100%); HRMS (ESI)
m/z Calcd for (M − 2H)2− C16H24N2O17P2: 289.0281. Found:
289.0281.
General procedure for chemo-enzymatic synthesis of UDP-Galf
analogs
UDP-glucose pyrophosphorylase (GalU) and resin immobilized-
galactose-1-phosphate uridyltransferase (GalPUT) were prepared
as previously described.34,35,44 To a solution of the Galf-1-phos-
phate analog (10.5 mg, 22 μmol) in 50 mM HEPES buffer pH
8.0 containing 10 mM MgCl2 and 5 mM KCl, was added UTP
(12.2 mg, 20 μmol), GalU (10 U), inorganic pyrophosphatase
(IPP, 2 U), and immobilized GalPUT (0.6 mL, ∼15 U) for a final
volume of 1 mL. The reaction was initiated by the addition of
UDP-Glc (91 μg, 0.15 μmol) and incubated at ambient tempera-
ture under a N2(g) atmosphere with gentle rotation. After 1–3
days, when analysis of the reaction by HPLC36 indicated the
complete consumption of UTP, the resin bound and soluble pro-
teins were removed by transferring the reaction mixture to a BD
column cartridge, washing with Milli-Q water (3–5 mL). The
flow through was filtered using a centrifugal filter device with a
molecular weight cut off of 10 000 Da. The resulting filtrates
were purified by semi-preparative HPLC and gel filtration
chromatography as previously described36 to give the final
UDP-Galf analogs as lyophilized white powders.
Uridine 5′-diphospho-2′′-O-methyl-α-D-galactofuranose (10).
1
(0.03 mg, <5%); H NMR (700 MHz, D2O, δH) 7.95 (d, 1 H,
J = 8.1 Hz, H-6), 5.98–5.97 (m, 1 H, H-1′), 5.97 (d, 1 H, J = 8.1
Hz, H-5), 5.78 (dd, 1 H, J = 5.5, 4.2 Hz, H-1′′), 4.38–4.35 (m, 2
H, H-2′, H-3′), 4.28–4.26 (m, 2 H, H-4′, H-3′′), 4.23 (ddd, 1 H,
J = 11.8, 4.5, 2.6 Hz, H-5′a), 4.19 (ddd, 1 H, J = 11.8, 5.7, 2.9
Hz, H-5′a), 3.94 (ddd, 1 H, J = 8.5, 4.1, 2.6 Hz, H-2′′), 3.82 (dd,
1 H, J = 7.4, 5.2 Hz, H-4′′), 3.77–3.73 (m, 1 H, H-5′′), 3.69 (dd,
1 H, J = 11.8, 4.4 Hz, H-6′′a), 3.62 (dd, 1 H, J = 11.8, 7.2 Hz,
H-6′′b), 3.49 (s, 3 H, OCH3); HRMS (ESI) m/z Calcd for
(M − H)− C16H25N2O17P2: 579.0634. Found: 579.0634.
Uridine 5′-diphospho-3′′-deoxy-α-D-xylo-hexofuranose (7).
1
(3.7 mg, 31%); H NMR (700 MHz, D2O, δH) 7.96 (d, 1 H, J =
8.1 Hz, H-6), 6.00–5.98 (m, 1 H, H-1′), 5.98 (d, 1 H, J = 8.1 Hz,
H-5), 5.60 (dd, 1 H, J = 5.3, 4.3 Hz, H-1′′), 4.39–4.36 (m, 2 H,
H-2′, H-3′), 4.35–4.31 (m, 1 H, H-2′′), 4.30–4.28 (m, 1H, H-4′),
4.25 (ddd, 1 H, J = 11.8, 4.5, 2.6 Hz, H-5′a), 4.21 (ddd, 1 H, J =
11.8, 5.7, 2.8 Hz, H-5′b), 4.10 (ddd, 1 H, J = 9.9, 6.5, 6.5 Hz,
H-4′′), 3.70 (ddd, 1 H, J = 6.8, 6.5, 3.7 Hz, H-5′′), 3.66 (dd, 1 H,
J = 12.0, 3.7 Hz, H-6′′a), 3.56 (dd, 1 H, J = 12.0, 6.8 Hz, H-6′′
b), 2.31–2.29 (m, 1 H, H-3′′a), 1.83 (app. q, 1 H, J = 10.9, H-3′′
b); 13C NMR (175 MHz, D2O, δC) 167.1 (C-4), 152.7 (C-2),
142.5 (C-5), 103.5 (C-6), 98.5 (d, 1 C, J = 6.0 Hz, C-1′′), 89.2
(C-1′), 84.1 (d, 1 C, J = 9.1 Hz, C-4′), 79.7 (C-4′′), 75.4 (C-5′′),
74.6, 70.5 (C-2′, C-3′), 72.6 (d, 1 C, J = 8.2 Hz, C-2′′), 65.7 (d,
1 C, J = 5.3 Hz, C-5′), 63.2 (C-6′′), 31.5 (C-3′′); MS (ESI) m/z
549 ([M − H]−, 30%), 274 ([M − 2H]2−, 100%); HRMS (ESI)
m/z Calcd for (M − H)− C15H23N2O16P2: 549.0528. Found:
549.0529.
Chemo-enzymatic synthesis of dTDP-Galf
Resin immobilized Cps2L protein was prepared from E. coli
BL21 (DE3) cells containing recombinant pSK001 plasmid.50
Cells were grown in LB broth (1 L) supplemented with 25 μg
mL−1 kanamycin. Production of Cps2L was induced by the
addition of 375 μM IPTG (isopropyl 1-thio-β-D-galactopyrano-
side) at a OD600 of 0.6 followed by incubation at 30 °C for 4 h.
Cells were collected by centrifugation at 11 300 × gmax for
15 min and the pellets were then re-suspended in 40 mL of
resuspension buffer (20 mM Tris-HCl, pH 8.0, containing
300 mM NaCl and 10 mM imidazole). The cells were lysed
using a benchtop cell disruptor (Constant Systems Inc., NC) set
to 20 kpsi and the lysate clarified by centrifugation (105 000 × g
for 1 h at 4 °C). The lysate was applied to a 5 mL Ni–NTA
agarose column and washed with 6 column volumes of wash
buffer (20 mM Tris-HCl, pH 8.0, containing 300 mM NaCl and
25 mM imidazole), followed by 6 column volumes of reaction
buffer (20 mM Tris-HCl, pH 8.0, containing 300 mM NaCl).
To a solution of Galf-1-phosphate (5 mg, 11 μmol) in reaction
buffer containing 2.5 mM MgCl2 was added dTTP (4.8 mg,
Uridine 5′-diphospho-6′′-O-methyl-α-D-galactofuranose (8).
1
(0.5 mg, <5%); H NMR (700 MHz, D2O, δH) 7.96 (d, 1 H, J =
8.1 Hz, H-6), 6.00–5.98 (m, 1 H, H-1′), 5.98 (d, 1 H, J = 8.1 Hz,
H-5), 5.64 (dd, 1 H, J = 5.2, 4.2 Hz, H-1′′), 4.38–4.36 (m, 2 H,
4084 | Org. Biomol. Chem., 2012, 10, 4074–4087
This journal is © The Royal Society of Chemistry 2012