Design of Glycosyltransferase Inhibitors
FULL PAPER
tracted with EtOAc (4ꢃ60 mL). The combined organic layers were
washed with a saturated aqueous solution of Na2CO3 (60 mL), H2O
(60 mL), and brine (60 mL), dried (Na2SO4), and concentrated. The resi-
due was purified by silica gel column chromatography (petroleum ether
to EtOAc) to afford the desired amide.
moieties, but also frequently to the pyrophosphate moiety.
Nevertheless, neutral inhibitors are now being investigated
to provide cell-permeable substrates for potential cellular
studies or in vivo applications as fundamental tools for biol-
ogy or as potential drugs. The design and synthesis of ten
GT inhibitors was performed from a desymmetrized pyri-
dine motif by using a combination of conjugations through
O-glycoside, amide bond, or triazole functionalities. Their
inhibition towards five enzymes provided useful structure–
activity relationships for such GTs. The inhibitions observed
were always weaker than for UDP and this could represent
a limitation for applications in cellular assays. More interest-
ingly, co-crystals of three inhibitors in the active site of
AAGlyB could be obtained, which showed the chelation of
the manganese ion with the pyridine portion of the designed
neutral GT inhibitors. The carbohydrate moieties of these
three co-crystallized inhibitors did not occupy the expected
position as in the natural UDP-Gal substrate. Rather the
carbohydrate occupied the “hexose pocket” and pointed
into the solvent. Limited contacts with the enzyme also ex-
plain the poor selectivity observed with respect to the
hexose (galactose or glucose) present in the inhibitor.
General protocol for the Meldal CuAAC conjugation: iPr2NEt
(0.036 mmol, 0.25 equiv) was added into a flask containing the azide
(0.14 mmol, 1 equiv), the alkyne (0.14 mmol, 1 equiv), and CuI
(0.01 mmol, 0.1 equiv) in DMF (2 mL). The reaction was stirred at RT
overnight. After 24 h, the solution was diluted with EtOAc (50 mL),
washed with a saturated aqueous solution of Na2CO3 (2ꢃ25 mL) and
H2O (30 mL). The combined aqueous layers were extracted with EtOAc
(3ꢃ30 mL). The combined organic layers were dried (Na2SO4) and con-
centrated. The residue was purified by silica gel column chromatography
to afford the desired triazole.
General protocol for the Sharpless CuAAC conjugation: CuSO4 (0.
11 mmol, 0.6 equiv) and sodium ascorbate (0.228 mmol, 1.2 equiv) were
added to a solution of the alkyne (0.19 mmol, 1 equiv) and the azide
(0.19 mmol, 1 equiv) in tBuOH/H2O (1:1, 5.6 mL:280 mL). The reaction
mixture was stirred at 358C for 24 h, then diluted with water (20 mL),
and extracted with CH2Cl2 (3ꢃ30 mL). The combined organic layers
were dried (Na2SO4) and concentrated. The residue was purified by silica
gel column chromatography to afford the desired triazole.
General protocol for the Sonogashira reaction: The bromo-arene
(1.0 mmol, 1 equiv), [PdACHTNUTRGNEUNG(PPh3)4] (0.1 mmol, 0.1 equiv), and CuI
(0.1 mmol, 0.1 equiv) were dissolved in toluene (25 mL) and the solution
was degassed with argon. Then, trimethylsilylacetylene (3.0 mmol,
3 equiv) and diisopropylamine (2.2 mmol, 2.2 equiv) were added. The re-
action was stirred for 48 h at RT protected from light and then poured
into a saturated aqueous solution of NH4Cl (100 mL). The aqueous layer
was extracted with CH2Cl2 (3ꢃ100 mL). The combined organic layers
were washed with H2O (100 mL) and brine (100 mL), dried (Na2SO4),
and concentrated. The residue was then purified by silica gel column
chromatography to afford the desired product.
Experimental Section
Materials and general methods: All reagents were obtained from com-
mercial sources and used without further purification. Dichloromethane
and acetonitrile were distilled over CaH2. Methanol was distilled over
Mg/I2. All reactions were performed under an argon atmosphere unless
otherwise stated. Thin-layer chromatography (TLC) was carried out on
aluminum sheets coated with silica gel 60 F254 (Merck). TLC plates were
inspected by UV light (l=254 nm) and developed by treatment with a
mixture of 10% H2SO4 in EtOH/H2O (1:1 v/v) followed by heating.
Silica gel column chromatography was performed with silica gel Si 60
(40–63 mm). NMR spectra were recorded at 293 K, unless otherwise
stated, by using Bruker 300, 400, or 500 MHz spectrometers. Chemical
shifts are referenced relative to deuterated solvent residual peaks. The
following abbreviations are used to explain the observed multiplicities:
s=singlet, d=doublet, t=triplet, q=quadruplet, m=multiplet, and br=
broad singlet. Complete signal assignments were based on 1D and 2D
NMR (COSY, HSQC, and HMBC correlations). High-resolution (HR-
ESI-QToF) mass spectra were recorded by using a Bruker MicroToF-Q
II XL spectrometer. Optical rotations were measured by using a Perkin–
Elmer polarimeter and values are given in 10ꢀ18cm2 gꢀ1. AAGlyB was
cloned and expressed in Escherichia coli (E. coli) by using standard muta-
genesis techniques.[71,72] All other GalTs were expressed and purified as
previously described,[35,73,74] apart from b-1,4-GalT, which was obtained
commercially from Sigma–Aldrich.
Radiochemical inhibition assay: Radiochemical enzyme assays were per-
formed in a final volume of 15 mL, containing the corresponding enzyme,
radioactive-labeled UDP-[3H]Gal, the requisite acceptor a-l-Fucp-(1!
2)-b-d-Galp-OC8H17, b-d-GlcNAcp-O-(CH2)8-CO2Me, or b-d-Lacp-O-
(CH2)8-CO2Me, and the inhibitor (0–3 mm). The reaction mixture was in-
cubated in 3-morpholinopropanesulfonic acid (MOPS) buffer (50 mm),
MnCl2 (20 mm), pH 7.0, and bovine serum albumin (1 mgmLꢀ1) for a cer-
tain time at 378C (a time for which linear rates are obtained by using
these assay conditions), and the reaction was quenched with cold water
(400 mL). The enzymatic product was isolated by purification with Sep-
Pak RC C-18 cartridges (Waters). Radioactivity was measured by using a
Beckman Coulter LS 6500 multi-purpose scintillation counter.
Crystallization and structure refinement: Metal-free protein AAGlyB
was crystallized at 208C by the sitting drop method with drops containing
1.6 mL of the protein stock and 1.6 mL of the reservoir solution containing
13 or 15% polyethylene glycol (PEG) 3350, 50 or 150 mm ammonium
sulfate, and 50 mm MOPS, pH 7. Seeding was performed with a horse
hair from previous similar drops, and the drops were allowed to equili-
brate over 500 mL of the reservoir solution. Crystals of the apoform of
the protein grew to a final size of approximately 200 mm in one week.
Drop wells containing the best crystals were opened, 2.4 mL of 50 mm
MOPS, pH 7, 50 mm MnCl2, and 100 mm inhibitor were added followed
by 0.8 mL of a 200 mm a-l-Fucp-(1!2)-b-d-Galp-OC8H17 solution. The
drops were mixed, resealed, allowed to equilibrate for 45 min, then
opened again, and 3 mL of the reservoir solution mixed 1:1 with glycerol
were added as cryoprotectant. After one minute, the crystals were
mounted in Mitegen loops and flash frozen in liquid nitrogen. Diffraction
data for crystals with the inhibitors 8-Gal, 17, and 8-Glc were collected in
Beamline ID23-2 at ESRF at a wavelength of l=0.873 ꢀ with a crystal-
to-detector distance of 167.5 mm with 18 oscillations, 180 images per crys-
tals, and exposure times of 0.1 to 0.5 seconds per image. Diffraction data
was integrated and scaled with XDS.[75] The structures were refined by
using PDB ID:3IOI[35] as the starting model followed by rigid body re-
finement in lieu of molecular replacement. Geometry descriptions for the
General protocol for the Staudinger–Vilarrasa conjugation: The acid
(0.466 mmol, 1 equiv) and HOBt (0.839 mmol, 1.8 equiv) were co-evapo-
rated with toluene (3ꢃ5 mL) and THF (3ꢃ5 mL). The mixture was dried
under vacuum for 1 h. The mixture was dissolved in dry THF (4 mL)
under argon and cooled to 08C. DIC (0.839 mmol, 1.8 equiv) was added
dropwise at 08C. After addition, the ice bath was removed and the reac-
tion was stirred at RT for 30 min. Meanwhile, the azide (0.699 mmol,
1.5 equiv) was dissolved in dry THF (4 mL) under argon and cooled to
08C. PMe3 (0.932 mmol, 2 equiv) was added and the reaction was stirred
at 08C. After 30 min, the solution was transferred into the flask contain-
ing the acid/HOBt solution at 08C. The flask was washed with THF
(4 mL) and the solution was transferred. The resulting reaction mixture
was stirred at 08C for 1 h then allowed to reach RT and stirred for addi-
tional 16 h. The reaction mixture was diluted with water (60 mL) and ex-
Chem. Eur. J. 2013, 19, 15346 – 15357
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
15355