Synthesis of 1-d-6-O-[2-(N-hydroxyaminocarbonyl)amino-2-deoxy-α-d-glucopyranosyl]-myo-inositol 1-(n-octadecyl phosphate): a potential metalloenzyme inhibitor of glycosylphosphatidylinositol biosynthesis

Article abstract of DOI:10.1016/j.carres.2008.03.028

1-d-6-O-[2-(N-Hydroxyaminocarbonyl)amino-2-deoxy-α-d-glucopyranosyl]-myo-inositol 1-(n-octadecyl phosphate) was prepared to probe the reaction mechanism of the putative zinc-dependent metalloenzyme 2-acetamido-2-deoxy-α-d-glucopyranosyl-(1→6)-phosphatidyl

Full text of DOI:10.1016/j.carres.2008.03.028

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1478–1481
Note
Synthesis of 1-D-6-O-[2-(N-hydroxyaminocarbonyl)amino-2-deoxy-
a-D-glucopyranosyl]-myo-inositol 1-(n-octadecyl phosphate): a
potential metalloenzyme inhibitor of glycosylphosphatidylinositol
biosynthesis
Arthur T. Crossman, Michael D. Urbaniak and Michael A. J. Ferguson*
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, The University of Dundee,
DD1 5EH Dundee, Scotland, United Kingdom
Received 25 January 2008; received in revised form 3 March 2008; accepted 24 March 2008
Available online 30 March 2008
Abstract—1-D-6-O-[2-(N-Hydroxyaminocarbonyl)amino-2-deoxy-a-D-glucopyranosyl]-myo-inositol 1-(n-octadecyl phosphate) was
prepared to probe the reaction mechanism of the putative zinc-dependent metalloenzyme 2-acetamido-2-deoxy-a-D-glucopyran-
osyl-(1?6)-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol biosynthesis.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Glycosylphosphatidylinositol (GPI) membrane anchors; N-Hydroxyurea; Zinc metalloenzyme inhibitor; N-Acetyl-D-glucosaminylphos-
phatidylinositol de-N-acetylase
Glycoconjugates on the cell surface of parasitic proto-
OH
zoa of the trypanosomatidae (including African trypan-
osomes, American trypanosomes and Leishmania spp.
that are human and animal pathogens) have a crucial
role in determining parasite survival and infectivity.1–3
Many glycoconjugates are attached to the plasma mem-
brane by means of glycosylphosphatidylinositol (GPI)
anchors,1,4 whose principal function is to provide stable
association of protein or oligosaccharide with the lipid
bilayer. Although this type of anchor is not confined
to the protozoa, it does appear to be used with a much
greater frequency in these organisms than in higher
eukaryotes.1 A key, early step in the biosynthesis of
the GPI anchors involves the de-N-acetylation of 2-acet-
amido-2-deoxy-a-D-glucopyranosyl-(1?6)-phosphatidy-
linositol,5 [a-D-GlcpNAc-PI (1, Fig. 1)], to form a-D-
GlcpNH2-PI (2, Fig. 1).
O
HO
HO
HO
OH
NH
OH
OH
R1
O
O
O
OR2
P
O
R2
R1
OCOC19H31
OCOC17H35
1
Ac
H
H2C
H2C
OCOC19H31
OCOC17H35
2
3
CONHOH
C18H37
De-N-acetylation is a prerequisite for subsequent pro-
cessing of 2 that leads to mature GPI anchor precur-
Figure 1.
sors.6 In Trypanosoma brucei, the causative agent of
African sleeping sickness, de-N-acetylation, is followed
by mannosylation and subsequent inositol-acylation of
*
Corresponding author. Tel.: +44 1382 384 219; fax: +44 1382 348
896; e-mail: m.a.j.ferguson@dundee.ac.uk
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.03.028

A. T. Crossman et al. / Carbohydrate Research 343 (2008) 1478–1481
1479
2,7 whereas in mammalian cells the order of these reac-
tions is reversed.8 Disruption of GPI biosynthesis is fatal
to the bloodstream form parasite in culture,9–11 and a
conditional null mutant of the gene encoding the a-D-
GlcpNAc-PI de-N-acetylase in T. brucei (TbGPI12)
has genetically validated this enzyme as a drug target.10
Recently, we have shown12 that mammalian and try-
panosomal a-D-GlcpNAc-PI de-N-acetylases are zinc
metalloenzymes, proposed a mechanism of action simi-
lar to that of zinc peptidases and postulated that known
zinc binding moieties13 such as N-hydroxyurea could act
as inhibitors. As previous studies have demonstrated
that the diacylglycerol portion of 1 is not specifically
recognised by the enzyme, and may be efficiently
replaced by an octadecyl chain,14 we synthesised, there-
fore, the N-hydroxyurea analogue 1-D-6-O-[2-(N-hydro-
xyaminocarbonyl)amino-2-deoxy-a-D-glucopyranosyl]-
myo-inositol 1-(n-octadecyl phosphate) (3, Fig. 1).
The synthesis of the N-hydroxyurea analogue 3 began
by reducing the azide to the amine of the known pseudo-
disaccharide15 4 by a Staudinger reaction,16 followed by
immediate treatment with 1-(4-nitrophenol)-N-(O-
benzylhydroxy) carbamate13 in the presence of triethyl-
amine to give the crude O-benzyl protected N-hydroxy-
urea 5 (Scheme 1).
ded the a-coupled compound 6 in 82% yield over three
steps.
The phospholipid moiety was introduced by the reac-
tion of 1-OH of the benzylated pseudodisaccharide 6
with triethylammonium n-octadecyl hydrogenphospho-
nate17 in the presence of pivaloyl chloride in pyridine
to give a mixture of diastereoisomeric phosphonic dies-
ters that was converted into the phosphoric diester 7 on
oxidation in situ with iodine in wet pyridine.18 The final
transformation (7?3) was accomplished by hydrogenol-
ysis over 20% Pd(OH)2 on carbon. Details of the results
of enzymic studies with the N-hydroxyurea analogue 3
will be reported elsewhere.
1. Experimental
1.1. General methods
Chemicals were purchased from commercial sources and
were used without further purification. 1H and 13C
NMR plus COSY, HSQC and DEPT45 spectra were re-
corded on a Bruker Avance 500 MHz spectrometer
using tetramethylsilane as the internal standard. 31P
NMR spectra used 85% phosphoric acid in D2O as the
external standard. Optical rotations were measured
using a Perkin Elmer 343 polarimeter. High resolution
Trifluoroacetic acid removal of the 4-methoxybenzyl
group followed by radial-band chromatography affor-
OBn
O
OBn
O
BnO
OBn
BnO
OBn
BnO
BnO
BnO
BnO
OBn
OBn
OBn
OBn
a, b
O
O
N3
NH
O
MBnO
MBnO
4
BnOHN
5
OBn
c
O
BnO
BnO
OBn
BnO
OBn
NH
O
OBn
OBn
BnOHN
BnO
BnO
O
P
O
O
d, e
O
BnO
OBn
NH
O
OC18H37
TEA
OBn
OBn
BnOHN
O
O
HO
6
7
f
OH
O
HO
HO
HO
OH
NH
OH
OH
HOHN
O
O
O
O
OC18H37
TEA
P
O
3
Scheme 1. Reagents and conditions: (a) Ph3P, THF/H2O {10:1}, 60 °C, 3 h; (b) 1-(4-nitrophenol)-N-(O-benzylhydroxy) carbamate,13 Et3N, rt,
overnight; (c) TFA, CH2Cl2, rt, 2 h, 82% over 3 steps; (d) triethylammonium n-octadecyl hydrogenphosphonate,17 PivCl, pyridine, rt, 2 h; (e) I2,
pyridine/H2O {19:1}, rt, 45 min, 58%; (f) H2, 20% Pd(OH)2/C, n-propanol/THF {1:1}, 3 atm, rt, 3 h, 94%.

1480
A. T. Crossman et al. / Carbohydrate Research 343 (2008) 1478–1481
electrospray ionisation mass spectra (HRESIMS) were
recorded with a Bruker microTOF spectrometer. HPLC
was performed using a Dionex P680 HPLC pump, an
Alltech ELSD 800 detector and a Vydac protein C4 col-
umn (250 mm ꢀ 46 mm) employing a gradient of 10%
n-propanol + 0.05% TFA in water ? 95% n-propa-
nol + 0.05% TFA in water. TLC was performed on
Kieselgel 60 F254 (Merck) with detection under UV light
or by charring with sulfuric acid–water–ethanol
(15:85:5). Radial-band chromatography (RBC) was
performed using a Chromatotron (model 7924T, TC
Research, UK) with TLC standard grade silica gel
(2–25 lm) (Aldrich) as the adsorbent.
1.3. Triethylammonium 1-D-6-O-[2-(N-benzyloxyamino-
carbonyl)amino-2-deoxy-3,4,6-tri-O-benzyl-a-D-gluco-
pyranosyl]-2,3,4,5-tetra-O-benzyl-myo-inositol 1-(n-
octadecyl phosphate) (7)
Each of compounds 6 (60 mg, 0.05 mmol) and triethyl-
ammonium
n-octadecyl
hydrogenphosphonate17
(47 mg, 0.11 mmol) was dried overnight over P2O5 in a
vacuum desiccator, whereafter anhyd pyridine (5 mL)
was evaporated therefrom. They were then dissolved in
dry pyridine (5 mL), pivaloyl chloride (42 lL,
0.34 mmol) was added and the resulting solution was
stirred under argon at rt for 2 h. A freshly prepared
solution of iodine (53 mg. 0.21 mmol) in 19:1 pyridine–
water (10 mL) was then added and stirring of the reac-
tion was continued for 45 min. After the addition of
CH2Cl2 (25 mL), the organic solution was washed suc-
cessively with 5% NaHSO3 (25 mL), water (25 mL)
and 1 M TEAB buffer solution (3 ꢀ 15 mL), dried
(MgSO4) and concentrated under reduced pressure. Col-
umn chromatography (19:1 CHCl3–MeOH) of the resi-
due afforded the TEA phosphate derivative 7 (45 mg,
1.2. 1-D-6-O-[2-(N-Benzyloxyaminocarbonyl)amino-2-
deoxy-3,4,6-tri-O-benzyl-a-D-glucopyranosyl]-2,3,4,5-
tetra-O-benzyl-myo-inositol (6)
To a stirred a,b mixture of 415 (50 mg, 0.04 mmol) in
10:1 THF–water (3 mL) at 60 °C was added Ph3P
(31 mg, 0.12 mmol). After 3 h, the reaction mixture
was cooled to rt and then Et3N (78 lL, 0.56 mmol)
and 1-(4-nitrophenol)-N-(O-benzylhydroxy) carba-
mate13 (29 mg, 0.10 mmol) were added. Stirring of the
mixture was continued overnight, whereafter it was
25
1
58%); ½aꢁD +40 (c 1.0, CHCl3); H NMR (500 MHz,
CDCl3): d 11.60 (s, 1H, NH(CH2CH3)3), 9.65 (s, 1H,
0
NHOBn), 7.40–6.95 (40H, 8 ꢀ Ph), 6.35 (d, 1H, J2 ;NH
evaporated
and
co-evaporated
with
toluene
8.8 Hz, NHC(O)NHOBn), 5.16 (s, 1H, H-10), 5.03–
4.34 (14H, 7 ꢀ CH2Ph), 4.30 (s, 1H, H-2), 4.23 (d, 1H,
J1,6 11.0 Hz, H-1), 4.12–3.99 (m, 5H, H-20, 4, 6, CH2Ph),
(3 ꢀ 10 mL) under reduced pressure. A solution of the
residue in EtOAc was percolated through a short col-
umn of silica gel (further elution with EtOAc) and the
eluent was concentrated under reduced pressure to give
the crude derivative 5. A solution of this methoxybenzyl
compound 5 in CH2Cl2 (2 mL) containing TFA (27 lL,
0.36 mmol) was set aside at rt for 2 h, whereafter it was
neutralised with Et3N, washed successively with water
and brine, dried (MgSO4) and concentrated under
reduced pressure. RBC of the residue (elution first with
hexane and then with 1:1 hexane–EtOAc) afforded the
a-linked O-benzyl protected N-hydroxyurea pseudodi-
3.91 (d, 1H, J4 ;5 9.3 Hz, H-50), 3.77–3.60 (m, 4H, H-30,
40, OCH2), 3.42 (d, 1H, J3,4 9.8 Hz, H-3), 3.30 (t, 1H,
0
0
0
0
J4,5 = J5,6 8.2 Hz, H-5), 2.95 (dd, 2H, J6 a,6 b 11.0 Hz,
H-60a,b), 2.77 (m, 6H, 3 ꢀ CH2CH3), 1.50 (m, 2H,
OCH2CH2), ꢂ1.15 (30H, [CH2]15), 1.00 (t, 9H, J
7.2 Hz, 3 ꢀ CH2CH3), 0.81 (t, 3H, J 7.0 Hz, CH2CH3);
13C NMR (125 MHz, CDCl3): d 160.1 (C@O), 138.4–
126.2 (C–Ph), 98.9 (C-10), 81.2, 81.1, 80.0 (C-5), 79.8
(C-3), 77.3, 76.6, 76.3, 76.0, 75.8, 75.5 (C-2), 74.8,
74.2, 74.1, 73.5, 72.1, 71.4, 69.8 (C-50), 66.8 (C-60), 64.6
(OCH2), 52.3, 44.1 (3 ꢀ CH2CH3), 30.9, 30.2, 30.1,
29.3, 28.7, 28.5, 28.4, 25.5, 25.0, 21.7, 13.1 (CH2CH3),
7.3 (3 ꢀ CH2CH3); 31P NMR (202 MHz, CDCl3): dP
25
saccharide 6 (37 mg, 82% over 3 steps); ½aꢁD +63 (c
1
1.1, CHCl3); H NMR (500 MHz, CDCl3): d 7.40–6.90
0
(40H, 8 ꢀ Ph), 6.25 (d, 1H, J2 ;NH 9.3 Hz, NHC(O)N-
1
HOBn), 5.39 (d, 1H, J1 ;2 3.4 Hz, H-10), 5.05–4.16
ꢃ1.68 (with H heteronuclear decoupling); HRESIMS:
0
0
(16H, 8 ꢀ CH2Ph), 4.12 (m, 1H, H-20), 4.03 (t, 1H,
calcd for [C93H124N3O15PꢃNEt3ꢃH]ꢃ: 1451.7493.
0
0
J3,4 = J4,5 9.8 Hz, H-4), 3.94 (br d, 1H, J4 ;5 9.9 Hz,
Found m/z: 1451.7459.
H-50), 3.85 (t, 1H, J1,6 = J5,6 9.4 Hz, H-6), 3.81 (t, 1H,
J1,2 = J2,3 2.2 Hz, H-2), 3.76 (t, 1H, J3 ;4 9.7 Hz, H-40),
1.4. Triethylammonium 1-D-6-O-[2-(N-hydroxyamino-
carbonyl)amino-2-deoxy-a-D-glucopyranosyl]-myo-inosi-
tol 1-(n-octadecyl phosphate) (3)
0
0
3.69 (t, 1H, J2 ;3 9.2 Hz, H-30), 3.31 (dd, 1H, H-3),
0
0
3.25 (dd, 1H, J5 ;6 a 2.8, J6 a;6 b 11.3 Hz, H-60a), 3.19 (m,
2H, H-5, 60b), 3.09 (dt, 1H, H-1), 2.33 (d, 1H, J1,OH
9.6 Hz, OH); 13C NMR (125 MHz, CDCl3): d 158.6
(C@O), 138.0–126.0 (C–Ph), 97.4 (C-10), 80.6 (C-4),
80.1 (C-5), 80.0 (C-3, 30), 77.7 (C-6), 77.4, 76.8 (C-40),
76.6, (C-2), 76.3, 76.0, 75.8, 74.8, 74.4, 73.9, 73.8, 73.6,
72.2, 72.0 (C-1), 71.9, 69.9 (C-50), 66.9 (C-60), 52.4
(C-20); HRESIMS: calcd for [C69H72N2O12+H]+:
1121.5158. Found m/z: 1121.5158.
0
0
0
0
A solution of the TEA salt 7 (30 mg, 0.02 mmol) in 1:1
n-propanol–THF (10 mL) containing 20% Pd(OH)2 on
carbon (20 mg) was stirred under 3 atm of hydrogen
for 3 h before it was percolated through a short column
of Chelex 100 on a bed of Celite (further elution with 1:1
n-propanol–THF). The eluent was concentrated under
reduced pressure and then purified via HPLC to give

A. T. Crossman et al. / Carbohydrate Research 343 (2008) 1478–1481
1481
25
Milne, K. G.; Sharma, D. K.; Smith, T. K. Biochim.
Biophys. Acta 1999, 1455, 327–340.
5. Doering, T. L.; Masterson, W. J.; Englund, P. T.; Hart, G.
W. J. Biol. Chem. 1989, 264, 11168–11173.
the deprotected product 3 (15 mg, 94%); ½aꢁD +21 (c 1.5,
1
MeOH); H NMR (500 MHz, CD3OD): d 5.10 (d, 1H,
J1 ;2 3.2 Hz, H-10), 3.84 (m, 1H, H-40), 3.65 (m, 2H,
H-2, 60a), 3.57 (br t, 3H, J 6.5 Hz, H-6, OCH2), 3.40
(m, 2H, H-1, 60b), 3.20 (q, 6H, J 7.3 Hz, 3 ꢀ CH2CH3),
2.35 (m, 1H, H-4), 2.00–1.78 (m, 5H, H-20, 3, 30, 5, 50),
1.60 (m, 2H, OCH2CH2), ꢂ1.30 (39H, 3 ꢀ CH2CH3,
[CH2]15), 0.90 (t, 3H, J 7.5 Hz, CH2CH3); 13C NMR
(125 MHz, CD3OD): d 177.3 (C@O), 105.1 (C-10), 68.0
(C-40), 67.2, 62.8, 62.1, 47.9 (3 ꢀ CH2CH3), 33.2, 33.1,
31.8, 31.4, 30.8, 30.5, 28.9, 26.3, 24.4, 23.4, 14.5
(CH2CH3), 9.25; 31P NMR (202 MHz, CDCl3): dP 0.12
0
0
6. Guther, M. L. S.; Ferguson, M. A. J. EMBO J. 1995, 14,
¨
3080–3093.
7. Doerrler, W. T.; Ye, J.; Falck, J. R.; Lehrman, M. A. J.
Biol. Chem. 1996, 271, 27031–27038.
8. Sharma, D. K.; Smith, T. K.; Crossman, A.; Brimacombe,
J. S.; Ferguson, M. A. J. Biochem. J. 1997, 328, 171–177.
9. Nagamune, K.; Nozaki, T.; Maeda, Y.; Ohishi, K.;
Fukuma, T.; Hara, T.; Schwarz, R. T.; Sutterlin, C.;
Brun, R.; Reizman, H.; Kinoshita, T. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 10336–10341.
10. Chang, T.; Milne, K. G.; Guther, M. L. S.; Smith, T. K.;
¨
1
(with H heteronuclear decoupling); HRESIMS: calcd
Ferguson, M. A. J. J. Biol. Chem. 2002, 277, 50176–50182.
11. Lillico, S.; Field, M. C.; Blundell, P.; Coombs, G. H.;
Mottram, J. C. Mol. Biol. Cell 2003, 14, 1182–1194.
12. Urbaniak, M. D.; Crossman, A.; Chang, T.; Smith, T. K.;
van Aalten, D. M. F.; Ferguson, M. A. J. J. Biol. Chem.
2005, 280, 22831–22838.
for [C37H76N3O15PꢃNEt3ꢃH]ꢃ: 731.3737. Found m/z:
731.3749.
Acknowledgement
13. Parrish, D. A.; Zou, Z.; Allen, C. L.; Day, C. S.; King, S.
B. Tetrahedron Lett. 2005, 46, 8841–8843.
14. Smith, T. K.; Crossman, A.; Paterson, M. J.; Borissow, C.
N.; Brimacombe, J. S.; Ferguson, M. A. J. J. Biol. Chem.
2002, 277, 37147–37153.
This work was supported by a programme grant from
the Wellcome Trust (071463).
References
15. Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J.
J. Chem. Soc., Perkin Trans. 1 1993, 2945–2951.
16. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F.
Tetrahedron 1981, 37, 437–472.
17. Crossman, A., Jr.; Paterson, M. J.; Ferguson, M. A. J.;
Smith, T. K.; Brimacombe, J. S. Carbohydr. Res. 2002,
337, 2049–2059.
1. Ferguson, M. A. J. J. Cell Sci. 1999, 112, 2799–2808.
2. Guha-Niyogi, A.; Sullivan, D. R.; Turco, S. J. Glycobio-
logy 2001, 11, 45R–59R.
3. Mendonca-Previato, L.; Todeschini, A. R.; Heise, N.;
Previato, J. O. Curr. Opin. Struct. Biol. 2005, 15, 499–505.
4. Ferguson, M. A. J.; Brimacombe, J. S.; Brown, J. R.;
18. Crossman, A., Jr.; Brimacombe, J. S.; Ferguson, M. A. J.
J. Chem. Soc., Perkin Trans. 1 1997, 2769–2774.
Crossman, A.; Dix, A.; Field, R. A.; Guther, M. L. S.;
¨