Inhibitors Incorporating Zinc-Binding Groups Target the GlcNAc-PI de-N-acetylase in Trypanosoma brucei, the Causative Agent of African Sleeping Sickness

Article abstract of DOI:10.1111/j.1747-0285.2011.01300.x

Disruption of glycosylphosphatidylinositol biosynthesis is genetically and chemically validated as a drug target against the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. The N-acetylglucosamine-phosphatidylinositol de-N-acetylase (deNAc) is a zinc metalloenzyme responsible for the second step of glycosylphosphatidylinositol biosynthesis. We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups. Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme. Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC₅₀ values 0.1-1.5mm, and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the α1-4-mannose transferase with an apparent IC₅₀=19±0.5μm.

Full text of DOI:10.1111/j.1747-0285.2011.01300.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01300.x
Chem Biol Drug Des 2012; 79: 270–278
Research Article
Inhibitors Incorporating Zinc-Binding Groups
Target the GlcNAc-PI de-N-acetylase in
Trypanosoma brucei, the Causative Agent of
African Sleeping Sickness
Glycosylphosphatidylinositol (GPI)-anchored proteins are abundant in
the protozoan parasite Trypanosoma brucei, the causative agent of
African sleeping sickness in humans and the related disease
Nagana in cattle (1), and disruption of GPI biosynthesis has been
genetically (2–4) and chemically (5) validated as a drug target. The
clinically relevant bloodstream form of T. brucei expresses approxi-
mately 5 · 106 GPI-anchored variant surface glycoprotein homodi-
mers that form a dense surface coat, protecting the parasite from
the complement pathway of the host and undergoing antigenic vari-
ation to evade specific immune responses (6,7). African sleeping
sickness is invariably fatal if untreated and kills about 50 000 peo-
ple each year (8). Current drugs are toxic, expensive, and difficult
to administer, leaving an urgent need for new therapeutic agents.
Nuha Z. Abdelwahab, Arthur T. Crossman,
Lauren Sullivan, Michael A. J. Ferguson
and Michael D. Urbaniak*
Division of Biological Chemistry and Drug Discovery, College of Life
Sciences, University of Dundee, Dundee DD1 5EH, UK
*Corresponding author: Michael D. Urbaniak,
m.d.urbaniak@dundee.ac.uk
Disruption of glycosylphosphatidylinositol biosyn-
thesis is genetically and chemically validated as a
drug target against the protozoan parasite Trypan-
osoma brucei, the causative agent of African
sleeping sickness. The N-acetylglucosamine-phos-
phatidylinositol de-N-acetylase (deNAc) is a zinc
metalloenzyme responsible for the second step of
The structure, biosynthesis, and function of GPIs and related mole-
cules have been extensively reviewed (1,9–11). The basic conserved
GPI core of NH2CH2CH2PO4H-6Mana1-2Mana1-6Mana1-4GlcNa1-6-
D-myo-inositol-1-HPO4-lipid, where the lipid can be diacylglycerol, al-
kylacylglycerol, or ceramide, is often further decorated with addi-
tional ethanolamine phosphate and ⁄ or carbohydrate groups in a
species- and tissue-specific manner. Biosynthesis of GPI, which
occurs in the endoplasmic reticulum, is initiated by the transfer of
N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylino-
sitol (PI) to generate N-acetylglucosamine-phosphatidylinositol (Glc-
NAc-PI 1, Figure 1), which is de-N-acetylated by the enzyme
GlcNAc-PI de-N-acetylase (EC3.5.1.89) to give GlcN-PI 2 (12). This
de-N-acetylation is a prerequisite for the subsequent mannosylation
of GlcN-PI that leads to mature GPI anchor precursors (13). From
GlcN-PI onwards, there are significant differences in the GPI biosyn-
thetic pathways of T. brucei and mammalian cells (14–17).
glycosylphosphatidylinositol
biosynthesis.
We
recently reported the synthesis of eight deoxy-
2-C-branched monosaccharides containing carbox-
ylic acid, hydroxamic acid, or N-hydroxyurea
substituents at the C2 position that may act as
zinc-binding groups. Here, we describe the synthe-
sis of a glucocyclitol-phospholipid incorporating a
hydroxamic acid moiety and report the biochemi-
cal evaluation of the monosaccharides and the
glucocyclitol-phospholipid as inhibitors of the try-
panosome deNAc in the cell-free system and
against recombinant enzyme. Monosaccharides
with carboxylic acid or hydroxamic acid substitu-
ents were found to be the inhibitors of the try-
panosome deNAc with IC50 values 0.1–1.5 mM, and
the glucocyclitol-phospholipid was found to be a
dual inhibitor of the deNAc and the a1-4-mannose
transferase with an apparent IC50 = 19 0.5 lM.
No high-resolution structural data exist for any of the enzymes of
the GPI biosynthetic pathway, and given that the enzymes contain
between one and 13 predicted transmembrane domains and ⁄ or are
components of multi-protein complexes, such structural data may
prove difficult to obtain. Instead, the substrate specificity of the
enzymes of the T. brucei and HeLa GPI biosynthetic pathways has
been examined in vitro using a substrate analog approach (5,13–
16,18–21). The T. brucei enzymes have less stringent substrate rec-
ognition than those of the mammalian pathway, enabling substrate-
based species-specific inhibitors to be designed (16,18).
Key words: carbohydrates, glycosylphosphatidylinositol, lipid,
mechanism-based drug design, metalloenzymes, Trypanosoma
brucei
Abbreviations: deNAc, N-acetylglucosamine-phosphatidylinositol
de-N-acetylase; GPI, glycosylphosphatidylinositol; GlcNAc-PI, N-acetyl-
glucosamine-phosphatidylinositol; GlcN-PI, glucosamine phosphatidylino-
sitol; hptlc, high-performance thin-layer chromatography; ZBG, zinc-
binding group.
Received 22 September 2011, revised 30 November 2011 and accepted
for publication 5 December 2011.
The T. brucei GlcNAc-PI de-N-acetylase (deNAc) has been geneti-
cally validated as a drug target through the generation of a condi-
270

Trypanosoma brucei GlcNAc-PI de-N-acetylase Inhibitors
lane as the internal standard, unless otherwise indicated. All
coupling constants (J) are given in Hz. High-resolution electrospray
ionization mass spectra (HRESIMS) and liquid chromatography mass
spectra were recorded with a Bruker microTof spectrometer. Melting
points were determined on a Reichert hot-plate apparatus and are
uncorrected. Optical rotations were measured with a Perkin-Elmer
343 polarimeter. Thin-layer chromatography (TLC) was performed on
Kieselgel 60 F254 (Merck, Nottingham, UK) with various solvent sys-
tems as developers, followed by detection under UV light or by
charring using either sulfuric acid ⁄ water ⁄ ethanol (15:85:5), phospho-
molybdic acid, orcinol, or ninhydrin spray reagents. Flash column
chromatography (FCC) was performed on Kieselgel 60
(0.040)0.063 mm) (Merck, Notttingham, UK). Radial-band chroma-
tography (RBC) was performed using a Chromatotron (model 7924T;
TC Research, Norwich, UK) with silica gel F254 TLC standard grade
as the adsorbent. All reactions were carried out in commercially
available dry solvents, unless otherwise stated.
Figure 1: The GlcNAc-PI de-N-acetylase substrates and products
used in this study.
tional null mutant (3) and has been shown to be a zinc metalloen-
zyme (22). Owing to their role in the progression of various human
diseases, zinc metalloenzymes have gained much interest as poten-
tial drug targets (23,24). Synthetic inhibitors of zinc metalloenzymes
typically consist of a backbone and a zinc-binding group (ZBG). The
backbone is typically a drug-like structure that interacts with the
protein through non-covalent interactions and contributes to both
the affinity and selectivity of the inhibitor for its target. The ZBG
coordinates to the zinc divalent cation and primarily contributes to
the binding affinity of the inhibitor–metalloenzyme complex.
Substrates and deoxy-2-C-branched
monosaccharides
The synthesis of D-GlcN-a(1–6)-D-myo-inositol-1-octadecyl phosphate
(GlcN-IPC18, 3) (27) and the monosaccharides 12–19 has been
described previously (26). The corresponding N-acetyl derivate GlcNAc-
IPC18 4 was prepared by treatment with acetic anhydride (14). The con-
centration of stock solutions of 3 and 4 was determined by measure-
ment of the inositol content by selected ion-monitoring GC-MS (28).
We have postulated that ZBGs could act as inhibitors of the T. bru-
cei GlcNAc-PI de-N-acetylase (22). As part of our efforts to test this
hypothesis, we have previously reported the synthesis of a GlcNAc-
PI analog incorporating an N-hydroxyurea zinc-binding moiety (1-D-6-
O-[2-(N-hydroxyaminocarbonyl)amino-2-deoxy-a-D-glucopyranosyl]-
myo-inositol 1-(n-octadecyl phosphate) (25). Unfortunately, this com-
pound proved to be unstable under the conditions employed in the
activity assay and was therefore judged to be unsuitable for further
study. We recently reported the synthesis of eight deoxy-2-C-
branched monosaccharides incorporating ZBGs, Figure 2 (26). Here,
we describe the synthesis of a glucocyclitol-phospholipid incorporat-
ing a ZBG and report the biological evaluation of the monosaccha-
rides and the glucocyclitol-phospholipid as inhibitors of the
trypanosome GlcNAc-PI de-N-acetylase.
Phenyl 3,4,6-tri-O-acetyl-2-C-(carboxymethyl
N-benzyloxyamide)-2-deoxy-1-thio-D-
glucopyranoside (6)
EDAC (58.7 mg, 0.31 mmol) and TEA (40 lL, 0.30 mmol) were
added successively to a solution of the carboxylic acid 5 (26)
(90 mg, 0.21 mmol) and O-benzylhydroxylamine hydrochloride
(39 mg, 0.25 mmol) in CH2Cl2 (3 mL) under argon at room tempera-
ture. After 2 h, TLC showed the complete disappearance of the
starting carboxylic acid whereby CH2Cl2 (10 mL) was added, and the
resulting solution was washed with water (5 mL), brine (5 mL),
dried with Na2SO4, filtered, and concentrated under reduced pres-
sure. The crude product was purified by RBC (5:1 fi 1:1 hex-
ane)EtOAc) to give the benzyloxyamide 6 (89 mg, 80%): Rf 0.27
(1:1 hexane)EtOAc); 1H NMR (CDCl3, 500 MHz): d 9.60, 9.40
(2 · s, a & b NH), 7.50)7.20 (m, 4 · Ph), 5.70 (d, J1,2 3.8 Hz,
Materials and Methods
General methods
1H, 13C, 31P NMR spectra were recorded on a Bruker AVANCE spec-
trometer using deuteriochloroform as a solvent and tetramethylsi-
Figure 2: Structures of the deoxy-2-C-branched monosaccharides.
Chem Biol Drug Des 2012; 79: 270–278
271

Abdelwahab et al.
H-1a), 5.26 (t, J2,3 = J3,4 9.9 Hz, H-3b), 5.15 (t, J2,3 = J3,4 9.9 Hz,
H-3a), 5.02 (t, J4,5 10.1 Hz, H-4a), 4.95)4.77 (m, H-1b, H-4b,
CH2Ph), 4.57)4.52 (m, H-5a), 4.26–4.17 (m, H-6aa, H-6a,bb), 4.10–
4.00 (m, H-6ba, CH2Ph), 3.70)3.65 (m, H-5b), 3.03)2.97 (m, H-2a),
2.40)2. 32 (m, H-2b, H-7a,bb), 2.29)2.20 (m, H-7a,ba), 2.13, 2.03,
1.99, 1.98,1.97, 1.96 (6 · s, 6 · CH3CO); 13C NMR (CDCl3,
125 MHz): d 174.2, 173.7, 170.8, 170.7, 170.5, 169.8, 169.8, 168.0
(8 · C=O), 135.5)127.6 (C-Ph), 87.5 (C-1a), 86.4 (C-1b), 79.3, 79.1
(CH2Ph), 75.2 (C-5b), 74.1 (C-3b), 72.2 (C-3a), 69.9 (C-4a), 69.7 (C-
4b), 68.7 (C-5a), 62.6 (C-6b), 62.4 (C-6a), 41.8 (C-2b), 41.5 (C-2a),
32.6 (C-7a), 31.5 (C-7b), 21.0, 20.9, 20.8, 20.7, 20.7, 20.6 (6 · s,
6 · CH3CO). HRESIMS: Calcd for [C27H31NO9S + H]+: 546.1792.
Found m ⁄ z: 546.1805.
a further 5 min at )78 ꢀC, a solution of 1R,2R-trans-cyclohexanediol
(32 mg, 0.28 mmol) in THF)CH2Cl2 (1:1 0.5 mL) was added dropwise.
The reaction mixture was continuously stirred under argon at )78 ꢀC
for 2 h and then allowed to gradually attain room temperature over a
30-min period. Afterward, the mixture was filtered through a pad of
Celite and concentrated under reduced pressure. The crude material
was purified by RBC (1:1 fi 1:3 cyclohexane)Et2O) to give first the
b-anomer 9 (36 mg, 60%): mp 111)115 ꢀC; Rf 0.22 (1:3 cyclohex-
ane)Et2O); ½aꢀD -11.0 (c 1.0, CHCl3); H NMR (CDCl3, 500 MHz): d
7.48)7.36 (m, 5H, Ph), 5.33 (dd, 1H, J2,3 11.1, J3,4 9.2 Hz, H-3), 5.03
(d, 1H, J1,2 8.6 Hz H-1), 5.00)4.86 (m, 3H, H-4 and CH2Ph), 4.24 (dd,
1H, J5,6a 5.5, J6a,6b 12.2 Hz, H-6a), 4.16 (dd, 1H, J5,6b 2.4 Hz, H-6b),
3.80 (ddd, 1H, J4,5 10.1 Hz, H-5), 3.41)3.36 (m, 1H, H-1¢), 3.34)3.29
(m, 1H, H-2¢), 3.13 (dd, 1H, J2,7a 4.8, J7a,7b 17.8 Hz, H-7a), 2.85 (dd,
1H, J2,7b 4.3 Hz, H-7b), 2.34)2.27 (m, 1H, H-2), 2.09, 2.02, 2.01
(3 · s, 9 H, 3 · CH3CO), 2.06–2.03 (m, 1H, H-6¢a), 1.89)1.84 (m, 1H,
H-3¢a), 1.66 (br, 2H, H-4¢a, H-5¢a), 1.53 (s, 9H, (CH3)3C), 1.30)1.22 (m,
2H, H-3¢b, H-4¢b), 1.20)1.16 (m, 2H, H-5¢b, H-6¢b); 13C NMR (CDCl3,
25
1
Phenyl 3,4,6-tri-O-acetyl-2-C-(carboxymethyl N-
tert-butyloxycarbonyl-N-benzyloxy-amide)-2-
deoxy-D-glucopyranoside (7)
Di-tert-butyl dicarbonate (0.30 g, 1.4 mmol) was added to a stirred
solution of the benzyloxyamide 6 (0.15 g, 0.28 mmol) and 4-(dim-
ethylamino) pyridine (DMAP) (3.4 mg, 0.03 mmol) in THF (8 mL).
Stirring was continued at room temperature for 3.5 h, and then the
reaction mixture was concentrated under reduced pressure. The
crude product was purified by RBC (6:1 fi 2:1 hexane)EtOAc) to
yield an a:b (4:1) anomeric mixture of the N-Boc derivative 7
(173 mg, 96%): Rf 0.26 (2:1 hexane)EtOAc); 1H NMR (CDCl3,
500 MHz): d 7.52)7.24 (m, 4 · Ph), 5.91 (d, J1,2 5.0 Hz, H-1a),
5.41 (dd, J2,3 10.6, J3,4 7.2 Hz, H-3b), 5.27 (d, J1,2 10.8 Hz, H-1b),
5.21 (dd, J2,3 11.4, J3,4 9.1 Hz, H-3a), 5.04 (dd, J4,5 10.2 Hz, H-4a),
4.98 (dd, J4,5 10.1 Hz, H-4b), 4.91)4.84 (m, 2 · CH2Ph), 4.59)4.55
(m, H-5a), 4.31 (dd, J5,6a 5.1, J6a,6b 12.3 Hz, H-6aa), 4.26 (dd, J5,6a
5.5, J6a,6b 12.2 Hz, H-6ab), 4.16 (dd, J5,6b 2.2 Hz, H-6bb) 4.05 (dd,
J5,6b 2.1 Hz, H-6ba), 3.80)3.76 (m, H-5b), 3.17 (dd, J2,7a 4.3, J7a,7b
17.1 Hz, H-7ab), 3.08)3.03 (m, H-7aa, H-7bb), 3.01)2.96 (m, H-2a),
2.87 (dd, J2,7b 4.1, J7a,7b 17.1 Hz, H-7ba), 2.41)2.35 (m, H-2b),
2.08, 2.05, 2.04, 2.03, 2.01, 2.00 (6 · s, 6 · CH3CO), 1.54, 1.53
(2 · s, (CH3)3C); 13C NMR (CDCl3, 125 MHz): d 170.7, 170.5, 170.4,
169.9, 169.8, 169.1, 169.0, 169.0 (8 · C=O), 151.2, 150.5
[(CH3)3COC=O], 134.4)127.6 (C-Ph), 87.5 (C-1a), 86.2 (C-1 b), 78.1,
77.8 (CH2Ph), 75.5 (C-5b), 73.6 (C-3b), 72.0 (C-3a), 69.9 (C-4a), 69.6
(C-4b), 68.6 (C-5a), 62.7 (C-6b), 62.4 (C-6a), 42.4 (C-2b), 41.5 (C-
2a), 36.6 (C-7a), 34.7 (C-7b), 28.0, 27.4 [2 · (CH3)3C], 20.8, 20.7,
20.7 (3 · s, 6 · CH3CO). HRESIMS: Calcd for [C32H39NO11S + Na]+:
668.2086. Found m ⁄ z: 668.2098.
125 MHz): d 170.8, 170.6, 169.8, 169.2 (4 · C=O), 151.2
[(CH3)3COC=O], 134.3)128.5 (C-Ph), 102.0 (C-1), 87.1 (C-2¢), 77.9
(CH2Ph), 73.2 (C-1¢), 72.5 (C-3), 71.7 (C-5), 69.6 (C-4), 62.2 (C-6), 43.6
(C-2), 32.8 (C-7), 32.2 (C-6¢), 30.9 (C-3¢), 28.0 [(CH3)3C], 24.3, 23.7 (C-4¢,
C-5¢), 20.8, 20.7, 20.7 (3 · CH3CO). HRESIMS: Calcd for
[C32H45NO13 + Na]+: 674. 2783. Found m ⁄ z: 674.2761. Then the a-
anomer 8 (7.9 mg, 13%): mp 99)103 ꢀC; Rf 0.18 (1:3 cyclohexane )
25
Et2O); ½aꢀD + 36.0 (c 1.0, CHCl3); 1H NMR (CDCl3, 500 MHz): d
7.48)7.35 (m, 5H, Ph), 5.89 (d, 1H, J1,2 5.0 Hz, H-1), 5.08)5.01 (m,
4H, H-3, H-4 and CH2Ph), 4.38 (dd, 1H, J5,6a 4.8, J6a,6b 12.4 Hz, H-6a),
4.16 (dd, 1H, J5,6b 2.5 Hz, H-6b), 4.08)4.05 (m, 1H, H-5), 3.44)3.39
(m, 1H, H-1¢), 3.20)3.15 (m, 1H, H-2¢), 2.81)2.67 (m, 3H, H-2, H-7a,b),
2.10, 2.08, 2.05 (3 · s, 9H, 3 · CH3CO), 2.09 (br, 1H, H-3¢a), 2.04–
2.00 (m, 1H, H-6¢a), 1.75)1.69 (m, 2H, H-4¢a, H-5¢a), 1.43 (s, 9H,
(CH3)3C), 1.29)1.24 (m, 2H, H-5¢b, H-6¢b), 1.24)1.11 (m, 2H, H-3¢b, H-
4¢b); 13C NMR (CDCl3, 125 MHz): d 171.8, 170.5, 170.0, 169.5
(4 · C=O), 152.5 [(CH3)3COC=O], 134.0)129.0 (C-Ph), 100.3 (C-1), 80.4
(C-2¢), 78.0 (CH2Ph), 73.4 (C-1¢), 71.5 (C-3), 70.0 (C-5), 67.3 (C-4), 61.8
(C-6), 39.9 (C-2), 34.3 (C-7), 32.2 (C-6¢), 30.3 [(CH3)3C], 29.7 (C-3¢), 24.4,
24.0 (C-4¢, C-5¢), 20.7, 20.7, 20.6 (3 · CH3CO). HRESIMS: Calcd for
[C32H45NO13 + Na]+: 674. 2783. Found m ⁄ z: 674.2761.
Triethylammonium 1R,2R-1-O-[3,4,6-tri-O-acetyl-
C-(carboxymethyl N-tert-butyloxy-carbonyl-N-
benzyloxyamide)-2-deoxy-b-D-glucopyranosyl]-
cyclohexanediol 2-(n-octa-decyl phosphate) (10)
Trimethylacetyl chloride (66 lL, 0.54 mmol) was added to a stirred
mixture of the pseudo-disaccharide 9 (56 mg, 0.086 mmol) and the
n-octadecyl hydrogenphosphonate (27) (75 mg, 0.17 mmol) in pyri-
dine (5 mL) under argon at room temperature. The reaction mixture
was stirred for 2 h, and then a solution of iodine (87 mg,
0.344 mmol) and water (0.1 mL) in pyridine (9.5 mL) was subse-
quently added. Stirring of the mixture was continued for 45 min,
after which it was diluted with dichloromethane (40 mL), washed
with 5% sodium hydrogen sulfite (20 mL), water (15 mL), 1.0 M trie-
thylammonium hydrogen carbonate (TEAB) buffer solution
(3 · 20 mL), dried (MgSO4), and concentrated under reduced pres-
sure. The residue was purified by column chromatography (5:1
CHCl3 ) MeOH) to give the TEA salt 10 (54 mg, 58%): Rf 0.28 (5:1
1R,2R-1-O-[3,4,6-Tri-O-acetyl-2-C-
(carboxymethyl N-tert-butyloxycarbonyl-N-
benzyl-oxyamide)-2-deoxy-a-D-glucopyranosyl]-
cyclohexanediol (8) and the b-anomer (9)
A freshly prepared 0.4 M benzenesulfenyl chloride solution (29)
(0.68 mL, 0.27 mmol) was added dropwise to a solution of silver
triflate (AgOTf) (83 mg, 0.32 mmol) in CH2Cl2 (1.5 mL) under argon at
)78 ꢀC. While at )78 ꢀC, the mixture was stirred for a further 5 min
and then activated powdered 4 ꢀ molecular sieves (20 mg) were
added; followed by the dropwise addition of a solution of the N-Boc
protected hydroxamate 7 (60 mg, 0.93 mmol) and 2,6-di-tert-butyl-4-
methyl pyridine (DTBMP) (67 mg, 0.33 mmol) in CH2Cl2 (0.5 mL). After
272
Chem Biol Drug Des 2012; 79: 270–278

Trypanosoma brucei GlcNAc-PI de-N-acetylase Inhibitors
CHCl3
)
MeOH); ½aꢀ2D5-8.4 (c 1.0, CHCl3); 1H NMR (CDCl3,
alents per assay) were washed twice in incorporation buffer
(50 mM NaHEPES pH 7.4, 25 mM KCl, 0.1 mM Tos-LysCH2Cl and
1 lg ⁄ mL leupeptin) and resuspended in incorporation buffer (40 lL
per assay) supplemented with 5 mM MnCl2, 5 mM MgCl2, 5 mM N-
ethylmaleimide, 0.15% n-octyl b-D-glucopyranoside, 1.25 lg ⁄ mL tu-
nicamycin and GDP-[3H]Man (0.5 lCi per assay), and briefly soni-
cated. The cell-free system was incubated with or without inhibitor
for 5 min at 30 ꢀC, transferred to tubes containing 400 pMol dry
GlcNAc-IPC18 or GlcN-C18, sonicated briefly and incubated at 30 ꢀC
for 30 min. Glycolipid products were recovered by extraction into a
chloroform ⁄ methanol ⁄ water mixture (10:10:3), evaporated to dry-
ness, partitioned between butan-1-ol and water, and analyzed by
high-performance thin-layer chromatography (hptlc).
500 MHz): d 12.91 (s, 1H, NH(CH2CH3)3), 7.47)7.35 (m, 5H, Ph),
5.35 (dd, 1H, J2,3 11.1, J3,4 9.2 Hz, H-3), 5.06 (d, 1H, J1,2 8.6 Hz, H-
1), 5.02 (dd, 1H, J4,5 9.9 Hz, H-4), 4.95 and 4.86 (ABq, 2H, J 9.2 Hz,
CH2Ph), 4.31)4.28 (m, 2H, H-2¢, H-6a), 4.09 (dd, 1H, J5,6b 2.6, J6a,6b
12.1 Hz, H-6b), 3.95)3.85 (m, 2H, OCH2), 3.82)3.78 (m, 1H, H-1¢),
3.74 (ddd, 1H, J5,6a 4.0 Hz, H-5), 3.10 (dd, 1H, J2,7a 4.4, J7a,7b
18.0 Hz, H-7a), 3.07)3.02 (m, 6H, HN(CH2CH3)3), 2.89 (dd, 1H, J2,7b
4.4 Hz, H-7b), 2.31)2.25 (m, 1H, H-2), 2.09, 2.05, 2.00 (3 · s, 9H,
3 · CH3CO), 1.99)1.93 (m, 1H, H-3¢a), 1.91)1.85 (m, 1H, H-6¢a),
1.72)1.58 (m, 4H, H-3¢b, H-4¢a, OCH2CH2), 1.52 (s, 9H, (CH3)3C),
1.50)1.45 (m, 1H, H-5¢a), 1.44)1.38 (m, 1H, H-6¢b), 1.32)1.24 (m,
H-4¢b, H-5¢b, 3 · CH2CH3, (CH2)15), 0.92)0.86 (m, 3H, CH2CH3); 13C
NMR (CDCl3, 125 MHz): d 170.9, 170.6, 169.8, 169.4 (4 · C=O),
151.0 [(CH3)3COC=O], 134.4)128.5 (C-Ph), 99.7 (C-1), 77.8 (CH2Ph),
77.5 (C-1¢), 74.4 (br, C-2¢), 72.8 (C-3), 71.5 (C-5), 69.7 (C-4), 66.1 (br,
OCH2), 62.2 (C-6), 45.2 (CH2CH3), 43.6 (C-2), 32.7 (C-7), 31.7
(OCH2CH2), 30.7 (C-6¢), 30.7 (C-3¢), 29.7 (CH2CH3), 29.5, 29.4 (CH2),
28.0 [(CH3)3C], 27.0, 25.8, 25.5, 22.7, 21.6 (CH2), 20.8, 20.7 (CH3CO),
High-performance thin-layer chromatography
Glycolipid standards and samples were applied to 10 cm aluminum-
backed silica gel 60 and developed with chloroform ⁄ methanol ⁄ 13 M
ammonia ⁄ 1 M ammonium acetate ⁄ water (180 ⁄ 140 ⁄ 9 ⁄ 9 ⁄ 23, v ⁄ v).
Dried hptlc plates were analyzed by a radiometric scanner (Bioscan
AR2000, Washington, DC, USA) and ⁄ or sprayed with En3Hanceꢁ
(PerkinElmer, Boston, MA, USA) and radiolabeled components visu-
alized by fluorography at )80 ꢀC using Kodak XAR-5 film (Sigma,
St. Louis, MO, USA) with an intensifying screen.
14.2, 8.5 (CH3); 31P NMR (CDCl3, 202 MHz): dP )0.86 (with H het-
1
eronuclear decoupling). HRESIMS: Calcd for [C50H81NO16P-NEt3-H]):
982.5298. Found m ⁄ z: 982.5380.
Triethylammonium 1R,2R-1-O-[2-C-
(carboxymethyl N-hydroxyamide)-2-deoxy-b-D-
glucopyranosyl]-cyclohexanediol 2-(n-octadecyl
phosphate) (11)
Cloning of T. brucei GlcNAc-PI de-N-acetylase
The DNA-encoding residues 24–252 of T. brucei GlcNAc-PI de-N-
acetylase (Tb11.01.3900) was amplified by PCR from genomic Try-
panosoma brucei brucei DNA (variant MITat1.4) using a 5¢ primer
(5¢-ttatactggatccatggataaggttttagatgcatcttgcgtaagt-3¢) that incorpo-
rated an BamHI site (italics) and a six residue N-terminal sequence
(MDKVLD, underlined) and a 3¢- primer (5¢-tataatgcggccgctcatgc
gacccccaat-3¢) that incorporated a NotI site (italics). The PCR frag-
ment was subcloned into pGEX-6P-1 (Invitrogen) with BamHI and
NotI to give the plasmid pGEX-TbGPI12, which appended a GST tag
to the N-terminus of the ORF.
A
0.03 M methanolic sodium methoxide solution (0.36 mL,
0.011 mmol) was added to a solution of the TEA salt 10 (15 mg,
0.014 mmol) in methanol (1 mL) under argon at room temperature,
and the reaction was stirred for 6 h. Afterward, the reaction mix-
ture was neutralized with Amberlite IR-120 (H+) ion-exchange resin,
filtered, and the filtrate was concentrated under reduced pressure
and then co-evaporated with water (5 · 5 mL). The residue was
purified by FCC (5:1 fi 3:1 CHCl3 ) MeOH) to give the hydroxamic
acid TEA salt 11 (6 mg, 56%): Rf 0.21 (3:1 CHCl3 ) MeOH);
25
1
½aꢀD )2.0 (c 0.5, CHCl3); H NMR (CDCl3, 500 MHz): d 12.30 (s, 1H,
NH(CH2CH3)3), 8.00 (s, 1H, NH), 4.42 (d, 1H, J1,2 8.6 Hz, H-1), 4.22
(br, 1H, H-2¢), 3.88)3.79 (m, 4H, H-6a, H-6b and OCH2), 3.70 (br, 1H,
H-1¢), 3.47–3.41 (m, 1H, H-4), 3.37 (dd, 1H, J2,3 9.2, J3,4 8.0 Hz, H-
3), 3.27 (br, 1H, H-5), 3.11)3.02 (m, 6H, (CH2CH3)3), 2.64 (dd, 1H,
J2,7a 4.1, J7a,7b 15.2 Hz, H-7a), 2.45 (dd, 1H, J2,7b 6.5 Hz, H-7b),
2.12)2.05 (m, 1H, H-2), 1.64)1.48 (m, 3H, H-3¢a and OCH2CH2),
1.40)1.21 (m, 46H, H-3¢b, H-4¢a,b, H-5¢a,b, H-6¢a,b, (CH2CH3)3,
(CH2)15), 0.88 (t, 3H, J 6.7 Hz, CH2CH3); 13C NMR (CDCl3, 125 MHz):
d 169.5 (CONOH), 100.6 (C-1), 76.9 (br, C-1¢), 75.6 (C-5), 74.0 (C-3),
73.7 (br, C-2¢), 70.5 (C-4), 64.7 (br, OCH2), 60.6 (C-6), 44.4
[(CH2CH3)3], 43.8 (C-2), 31.4 (C-7), 30.9)20.8 (CH2), 13.1 (CH3), 7.5
[(CH2CH3)3]; 31P NMR (CDCl3, 202 MHz): dP )0.70 (with 1H
heteronuclear decoupling). HRESIMS: Calcd for [C32H61NO11P-NEt3-
H]): 666.3988. Found m ⁄ z: 666.4045.
Expression of recombinant T. brucei GlcNAc-PI
de-N-acetylase
Escherichia coli BL21 (DE3) transformed with pETB-TbGPI12 were
grown in Luria-Bertani medium with 50 lg ⁄ mL carbenicillin at
37 ꢀC until A600 ꢁ 0.5, induced with 250 lM isopropyl b-D-thioga-
lactoside, and cultured for a further 16 h at 21 ꢀC. Cells were har-
vested by centrifugation at 4500 · g for 20 min at 4 ꢀC,
resuspended in 10 mL buffer A (50 mM Tris–HCl pH 8.0, 200 mM
NaCl, 0.06% n-octyl-b-D-glucopyranoside, 10% glycerol v ⁄ v) per
liter, and incubated with lysozyme (1 mg ⁄ mL) on ice for 20 min.
Cells were lysed at 30 000 psi (OneShot; Constant Cell Disruption
Systems, Daventry, UK), and the lysate clarified by centrifugation at
30 000 · g for 30 min at 4 ꢀC. The supernatant was filtered
through a 0.4 lM membrane, mixed with glutathione-sepharose
beads (GE Healthcare, Chafont St. Giles, UK) for 2 h at 4 ꢀC,
washed with buffer A, eluted with 20 mM glutathione in buffer A.
Pooled fractions were concentrated and washed in buffer A in a
10 000 molecular weight cut-off spin-concentration device (Satorius,
Aubagne, France) repeatedly to remove glutathione. The concentra-
Trypanosome cell-free system inhibition assays
Bloodstream form T. brucei (variant MITat1.4) were isolated and
membranes (cell-free system) prepared as described previously and
stored at )80 ꢀC (30). Trypanosome membranes (2 · 107 cell equiv-
Chem Biol Drug Des 2012; 79: 270–278
273

Abdelwahab et al.
tion of GST-TbGPI12 was determined by absorbance at 280 nm
Results and Discussion
using a calculated e = 7.464 · 104 ⁄ cm ⁄ M.
Synthesis of the glucocyclitol-phospholipid
The chemical synthesis of the glucocyclitol-phospholipid 11, Figure 3,
began from the carboxylic acid 5 (26). An anomeric mixture of 5 was
coupled to O-benzylhydroxylamine hydrochloride in the presence of
N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide hydrochloride (EDAC)
to afford, after purification, the benzyloxyamide 6 which was treated
with di-tert-butyl dicarbonate (Boc anhydride) and a catalytic amount
of DMAP to give the N-Boc-protected hydroxamate 7. The introduc-
tion of the Boc protecting group was necessary in order to circumvent
unwanted side reactions, which were apparent in previous studies,
when activating the anomeric position for coupling to the cyclohex-
anediol moiety. The aforementioned coupling was achieved by con-
verting the thioglycoside 7 to a glycosyl triflate through treatment
with phenylsulfenyl triflate (PST) (31,32). PST was generated in situ by
the addition of a freshly prepared solution of 0.4 M benzenesulfenyl
chloride (29) to a solution of silver triflate (AgOTf) at )78 ꢀC. After
the addition of activated powdered 4 ꢀ molecular sieves, a solution
of the thioglycoside 7 and the proton scavenger DTBMP were added
at )78 ꢀC, followed by 1R,2R-trans-cyclohexanediol in THF ) CH2Cl2
(1:1). Diligent RBC provided the a- and b-anomers 8 and 9, at 13%
and 60% yields, respectively.
Tryptic peptide mass fingerprinting of GST-
TbGPI12
The protein was reductively alkylated prior to SDS-PAGE and stain-
ing with Sypro Orange, the band excised and digested in 0.1% n-
octylglucoside and 20 mM NaHCO3 with 12.5 lg ⁄ mL trypsin before
analysis by MALDI-TOF MS and MS-MS. The protein was confirmed
as T. brucei GlcNAc-PI de-N-acetylase with a Mascot score of 1162,
with 87% sequence coverage.
ES-MS ⁄ MS de-N-acetylase assay
The recombinant protein GST-TbGPI12 (500 ng ⁄ lL) was incubated in
incorporation buffer (50 lL) with or without inhibitor for 5 min at RT.
The solution was transferred to tubes containing dry GlcN-IPC18
3
(0.5 nmol), briefly vortexed, sonicated for 5 seconds, and incubated
at 37 ꢀC for 30 min. The mixture was diluted with 5% propan-1-ol
(1 mL), the glycolipids were bound to C8 resin (100 mg Isolute car-
tridge), washed (5% propan-1-ol, 5 mM NH4OAc), eluted (60% pro-
pan-1-ol, 5 mM NH4OAc), and the products analyzed directly by
electrospray tandem mass spectrometry (Micromass Quattro Ultima,
Manchester, UK) in precursor ion scanning mode (precursor of m ⁄ z
223). The m ⁄ z 223 fragment ion, [inositol-1,2-cyclic phosphate –
H2O], is common to both the GlcNAc-IPC18 substrate 3 and the GlcN-
IPC18 product 4. The ratio of the integrals for the m ⁄ z 714 [GlcNAc-
IPC18 – H]- and m ⁄ z 672 [GlcN-IPC18 – H]- precursor ions were used
to calculate the percentage of substrate conversion to product in a
given sample. Inhibitor IC50 values were calculated using a four-
parameter fit of eight-point potency curves derived from three inde-
pendent experiments, and are quoted with standard deviation.
The phosphoric diester 10 was accessible from the pseudodisac-
charide 9 by means of the hydrogenphosphonate approach (33).
Thus, condensation of the known n-octadecyl hydrogenphosphonate
salt (27) with the pseudodisaccharide 9 furnished a mixture of dia-
stereoisomeric phosphonic diesters that were converted into the
phosphoric diester 10 on oxidation in situ with iodine in wet pyri-
dine (33). An identical approach using the a-anomer 8 was unsuc-
cessful owing to inseparable contaminants in the reaction mixture
after coupling and in situ oxidation.
Figure 3: Synthesis of compound 11. Experimental conditions: (a) BnONH2ÆHCl, EDAC, TEA, CH2Cl2, room temperature, 80%; (b) Boc2O, 4-
(dimethylamino) pyridine, THF, room temperature, 96%; (c) PhSCl, AgOTf, 4 ꢀ molecular sieves, 2,6-di-tert-butyl-4-methyl pyridine, CH2Cl2,
1R,2R-trans-cyclohexanediol in 1:1 THF ) CH2Cl2, )78 ꢀC to room temperature, 13% a-anomer and 60% b-anomer; (d) i. trimethylacetyl chlo-
ride, pyridine, triethylammoniun n-octadecyl hydrogenphosphonate, room temperature, ii. I2, pyridine (9.5 mL) – H2O (0.1 mL), room tempera-
ture, 58%; (e) i. 0.03 M NaOMe, MeOH, ii. Amberlite IR 120 (H+) resin, room temperature, 56%.
274
Chem Biol Drug Des 2012; 79: 270–278

Trypanosoma brucei GlcNAc-PI de-N-acetylase Inhibitors
The generation of the final b-glucohydroxamic acid-cyclitol-phospho-
lipid analog 11 was initially planned to proceed through Zꢁmplen
de-O-acetylation, followed by the removal of the Boc protecting
group under acidic conditions and, finally, catalytic hydrogenolysis
to remove the benzyl (Bn)-protecting group. De-O-acetylation was
carried out using sodium methoxide in methanol and when TLC indi-
cated the complete disappearance of 10, then the resulting
reaction mixture was neutralized with Amberlite IR 120 (H+) ion-
exchange resin, filtered, evaporated to dryness under reduced pres-
sure, and then subjected to column chromatography. Surprisingly,
the major fraction isolated, from the purification, corresponded to
the completely deprotected analog 11: triethylammonium 1R,2R-1-
O-[2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-b-D-glucopyranosyl]-
cyclohexanediol 2-(n-octadecyl phosphate). Thus, de-O-acetylation,
Boc removal, and benzyl deprotection occurred in a single deprotec-
separated by hptlc, quantified radiometrically, and visualized by
fluorography. The compounds 11–19 were tested at an initial
concentration of 10 mM in the cell-free system (Figure 4). Com-
pounds 13, 15, 17, and 19 produced <10% inhibition of the for-
mation of radiolabelled mannosylated products compared to the
DMSO control, while 11, 12, 15, 16, and 18 all produced
>80% inhibition. The potency of the latter compounds was then
determined using eight-point potency curves in triplicate (Table 1).
Notably, the a ⁄ b anomers 17 and 18 gave >100-fold difference
in potency. These compounds contain a thiophenyl group, pre-
dicted to adopt an axial position in the a-anomer 17, and an
equatorial position in the b-anomer 18, which could potentially
lead to differences in the abilities of the two anomers to fit into
the active site of the enzyme. This result is surprising, given that
it has previously been observed that the deNAc is able to de-N-
acetylate both the natural substrate GlcNAc-a-PI 1 and the unnat-
ural GlcNAc-b-PI (17).
1
tion step. The 13C and H NMR data indicated the loss of all the
protecting groups, which was further confirmed by the mass spec-
tral data. Under the conditions used, de-O-acetylation is indisput-
able, and although it is known from the literature that acid
treatment is the common deprotection method used for the Boc
group (34), it has also been documented that this group can be
removed under basic conditions (35,36). The removal of the benzyl-
protecting group of N-OBn, on the other hand, is not so obvious.
Possibly, after the neutralization of the reaction mixture with Am-
berlite IR 120 (H+) resin followed by its evaporation to dryness, the
conditions became sufficiently acidic to lead to the removal of the
benzyl-protecting group. This statement is pure conjecture, and the
N-OBn fi N-OH reaction pathway may never be understood under
the conditions described.
The indirect cfs assay is unable to distinguish between the inhibi-
tion of the deNAc and inhibition of the first mannosyltransferase
(MT1), because either will lead to an overall reduction in mannosy-
lated GPI species. Furthermore, to account for the observation that
priming the cfs with GlcNAc-PI is significantly more efficient than
priming with GlcN-PI, it has been postulated that substrate channel-
ing occurs between the deNAc and MT1 (14,17). Given the struc-
tural similarity of the deNAc substrate GlcNAc-PI 1 and the MT1
substrate GlcN-PI 2, the analogs 11–19 may also interact with
MT1. We assessed the specificity of the two most potent inhibitors
11 and 18 by measuring their ability to inhibit the cfs primed with
GDP-[3H]Man and either the deNAc substrate GlcNAc-PI 1 or the
MT1 substrate GlcN-PI 2. Compound 18 inhibited the formation of
mannosylated products when the cfs was primed with GlcNAc-PI 1,
but not when primed with GlcN-PI 2 (Figure 5A), suggesting that it
inhibits only the deNAc. Compound 11 inhibited the formation of
mannosylated products when the cfs was primed with either Glc-
NAc-PI 1 or GlcN-PI 2 (Figure 5A), suggesting either that it inhibits
both the deNAc and MT1, or that it inhibits MT1 only. The apparent
inhibition of MT1 by the glucocyclitol-phospholipid 11 may be due
to substrate channeling between the deNAc and MT1, such that
inhibition of the deNAc is able to prevent GlcN-PI from accessing
the MT1 active site. However, the present data do not rule out
direct inhibition of MT1 only.
Evaluation of inhibitors in the trypanosome
cell-free system
The ability of the compounds 11–19 to inhibit the deNAc was
initially assessed in vitro using the T. brucei cell-free system (cfs),
i.e., washed trypanosome membranes that are competent in GPI
biosynthesis. Because de-N-acetylation of GlcNAc-PI must precede
the addition of the three mannose residues (13), the activity of
the deNAc can be indirectly monitored by measuring production of
mannosylated GPI biosynthetic intermediates. In the assay, the cfs
is primed with GDP-[3H]Man and synthetic GlcNAc-PI 3 with and
without inhibitor, and the radiolabelled mannosylated products
Figure 4: Inhibition of Trypanosoma brucei glycosylphosphatidylinositol (GPI) biosynthesis in the cell-free system. Compounds 11–19
(10 mM) were incubated with the T. brucei cell-free system for 5 min prior to priming with GlcNAc-PI and GDP-[3H]Man to stimulate the pro-
duction of radiolabelled mannosylated GPI intermediates. Glycolipid products were extracted, separated by high-performance thin-layer chro-
matography, and visualized by fluorography. DPM – dolichol-phosphate-mannose, M1 – Man1GlcN-PI, M2 – Man2GlcN-PI, M3 – Man3GlcN-PI,
aM3 – Man3GlcN-(acyl)PI, A' – EtNPMan3GlcN-PI. PI, phosphatidylinositol.
Chem Biol Drug Des 2012; 79: 270–278
275

Abdelwahab et al.
Table 1: Potency of inhibitors in the indirect cell-free system
assay
Instead, an alternative construct containing a GST tag (TbGPI12-
GST) afforded an improved yield of soluble protein, with protein
identity confirmed by tryptic mass fingerprinting (Mascot score
1162, 87% coverage). The activity of TbGPI12-GST was confirmed in
the electrospray tandem mass spectrometry (ES-MS ⁄ MS) assay
using the synthetic GlcNAc-PI analog GlcNAc-IPC18 3, where the
diacyl glycerol portion of PI is replaced by a C18 alkyl chain without
affecting enzyme recognition (19). The ability of 11 and 18 to inhi-
bit TbGPI12-GST was assessed in the ES-MS ⁄ MS assay, and both
were found to inhibit with an IC50 = 600 € 300 lM (Figure 5B) and
980 € 220 lM (data not shown), respectively. Both compounds show
reduced potency against the soluble truncated protein in the ES-
MS ⁄ MS assay compared to their potency against the intact protein
in the indirect cfs assay. This may be due to the loss of interactions
with the truncated portion of the protein and ⁄ or ER membrane, or
in the case of 11, the lack of the postulated substrate channeling
effect.
a
Compound
IC50 ⁄ lM
11
12
13
14
15
16
17
18
19
19 € 3.2
290 € 100
>10 000
1500 € 200
>10 000
300 € 50
>10 000
100 € 13
>10 000
aThe IC50 values were determined from three separate eight-point potency
curves and are reported to two significant figures with standard deviation.
Evaluation of inhibitors against recombinant
T. brucei GPI de-N-acetylase
We have developed a mass spectrometry-based assay to measure
the activity of a recombinant truncated rat deNAc construct, where
the first 23 residues corresponding to the transmembrane region
are replaced with 6 residues from an orthologous E. coli protein
(22). Cloning and expression of the equivalent T. brucei deNAc con-
struct failed to produce a significant yield of soluble protein.
Conclusions and Future Directions
Disruption of GPI biosynthesis has been genetically (2–4) and chem-
ically (5) validated as a drug target against T. brucei, the causative
agent of African sleeping sickness in humans and the related dis-
A
B
Figure 5: Inhibitors target the GlcNAc-PI de-N-acetylase. (A) Inhibition of the Trypanosoma brucei cell-free system by 11 and 18 (10 mM)
when primed with either GlcNAc-PI or GlcN-PI. Conditions as Figure 4. (B) Inhibition of recombinant T. brucei GlcNAc-PI de-N-acetylase by 11
measured by electrospray tandem mass spectrometry. The intensity of the reaction product GlcN-IPC18 4 (m ⁄ z 672) is normalized to the
turnover in uninhibited control. PI, phosphatidylinositol.
276
Chem Biol Drug Des 2012; 79: 270–278

Trypanosoma brucei GlcNAc-PI de-N-acetylase Inhibitors
ease Nagana in cattle. African sleeping sickness is invariably fatal
if untreated, and there is an urgent need for new therapeutic
agents that is not being met by the pharmaceutical industry. We
have previously shown that the GPI biosynthetic enzyme GlcNAc-PI
de-N-acetylase is a zinc metalloenzyme and postulated that ZBGs
could act as inhibitors (22). As part of our efforts to develop drugs
that target GPI biosynthesis in T. brucei, we have synthesized small
molecules to probe the mechanism of the GlcNAc-PI de-N-acetylase
(25,26). Here, we report that small molecules incorporating
carboxylic acid or hydroxamic acid can inhibit the T. brucei GlcNAc-
PI de-N-acetylase, confirming our hypothesis that ZBGs can be used
to target the enzyme. However, the current compounds are neither
sufficiently potent nor drug-like to be useful therapeutics. Future
synthetic efforts will be directed toward developing a more drug-
like inhibitor backbone, to increase potency, and to introduce
species selectivity.
a therapeutic target for African sleeping sickness. Biochim Bio-
phys Acta;1455:327–340.
10. Kinoshita T., Inoue N. (2000) Dissecting and manipulating the
pathway for glycosylphosphatidylinositol-anchor biosynthesis.
Curr Opin Chem Biol;4:632–638.
11. Eisenhaber B., Maurer-Stroh S., Novatchkova M., Schneider G.,
Eisenhaber F. (2003) Enzymes and auxiliary factors for GPI lipid
anchor biosynthesis and post translational transfer to proteins.
Bioessays;25:367–385.
12. Doering T.L., Masterson W.J., Englund P.T., Hart G.W. (1989) Bio-
synthesis of the glycerol phosphatidylinositol membrane anchor
of the trypanosome variant surface glycoprotein.
Chem;264:11168–11173.
J Biol
13. Sharma D.K., Smith T.K., Crossman A., Brimacombe J.S., Fergu-
son M.A.J. (1997) Substrate specificity of the N-acetylglucosami-
nyl-phosphatidylinositol
de-N-acetylase
of
glycosylphosphatidylinositol membrane anchor biosynthesis in
African trypanosomes and human cells. Biochem J;328:171–177.
14. Smith T.K., Cottaz S., Brimacombe J.S., Ferguson M.A.J. (1996)
Substrate specificity of the dolichol phosphate mannose: gluco-
samine phosphatidylinositol alpha-1-4-mannosyltransferase of
the glycosylphosphatidylinositol biosynthetic pathway of African
trypanosomes. J Biol Chem;271:6476–6482.
Acknowledgments
This work was supported by a Wellcome Trust Programme Grant
(085622). N. Z. Adbelwahab is indebted to the BBSRC for a Ph.D
studentship.
15. Sharma D.K., Smith T.K., Weller C.T., Crossman A., Brimacombe
J.S., Ferguson M.A.J. (1999) Differences between the trypanoso-
mal and human GlcNAc-PI de-N-acetylases of glycosylphosphat-
idylinositol membrane anchor biosynthesis. Glycobiology;9:415–
422.
16. Smith T.K., Sharma D.K., Crossman A., Brimacombe J.S., Fergu-
son M.A.J. (1999) Selective inhibitors of the glycosylphosphat-
idylinositol biosynthetic pathway of Trypanosoma brucei. EMBO
J;18:5922–5930.
17. Smith T.K., Crossman A., Borrissow C.N., Paterson M.J., Dix A.,
Brimacombe J.S. et al. (2001) Specificity of GlcNAc-PI de-N-
acetylase of GPI biosynthesis and synthesis of parasite specific
suicide substrate inhibitors. EMBO J;20:3322–3332.
18. Smith T.K., Patterson M.J., Crossman A., Brimacombe J.S., Fer-
guson M.A.J. (2000) Parasite-specific inhibition of the glycosyl-
phosphatidylinositol biosynthetic pathway by stereoisomeric
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