Synthesis of 3′-, 4′ and 6′-deoxy and other analogues of D-glucosaminylphosphatidylnositol

Article abstract of DOI:10.1016/S0040-4039(00)01901-8

Deoxy and other analogues of D-glucosaminylphosphatidylinositol I have been synthesised and tested as substrates or inhibitors of a de-N-acetylase and mannosyltransferase (MT-1) involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) membra

Full text of DOI:10.1016/S0040-4039(00)01901-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 121–123
Synthesis of 3%-, 4%- and 6%-deoxy and other analogues of
-glucosaminylphosphatidylinositol
D
Charles N. Borissow,a Terry K. Smith,b Michael A. J. Fergusonb and John S. Brimacombea,*
aDepartment of Chemistry, University of Dundee, Dundee DD1 4HN, UK
bDepartment of Biochemistry, University of Dundee, Dundee DD1 4HN, UK
Received 25 August 2000; accepted 26 October 2000
Abstract—Deoxy and other analogues of D-glucosaminylphosphatidylinositol 1 have been synthesised and tested as substrates or
inhibitors of a de-N-acetylase and mannosyltransferase (MT-1) involved in the biosynthesis of the glycosylphosphatidylinositol
(GPI) membrane anchor of the parasite Trypanosoma brucei. © 2000 Elsevier Science Ltd. All rights reserved.
The ability of the parasite Trypanosoma brucei to evade
a mammalian host’s immune response is due to a
uniformly dense protective coat1 made up of ꢀ107
copies of a 55 kDa variant surface glycoprotein (VSG)
on the cell surface.2 This surface coating of VSGs not
only protects the parasite from lysis by the alternative-
and lectin-mediated complement pathways of the host,
but also, by the process of antigenic variation, from the
humoral immune response. The VSGs of T. brucei form
homodimers attached to the cell surface by means of
glycosylphosphatidylinositol (GPI) membrane anchors.3
The absolute dependence of the trypanosome on its
VSG coat, and the dependence of the latter on its GPI
anchors, makes the parasite GPI biosynthetic pathway
an attractive therapeutic target. However, in the view of
the presence and importance of GPI-anchored glyco-
proteins in mammals,4 parasite-specific inhibitors of
GPI biosynthetic enzymes are desirable. With this in
mind, we have sought to define the substrate specific-
ities of two enzymes, viz. a de-N-acetylase and a man-
nosyltransferase (MT-1), that exert their catalytic
activities early on in the GPI biosynthetic pathway.3,5
In systematically extending our earlier studies,6 it be-
came necessary to test the 3%-, 4%- and 6%-deoxy ana-
logues of 1 as potential substrate/inhibitors of these
enzymes. Having outlined the synthesis of the requisite
glycosyl donors in the preceding Letter, we now pro-
ceed to the synthesis of the monodeoxygenated GPI
analogues 2–4 and a related 4-O-methyl analogue 5.
Coupling of the 2-azido-2,3-dideoxyglycosyl fluoride 67
with the
-myo-inositol derivative 78 in dry toluene:1,4-
D
dioxane (3:1) in the presence of zirconocene dichloride
and silver perchlorate afforded the a-(1“6)-linked
pseudodisaccharide 8 (J1%,2%=3.0 Hz) in 35% yield after
Keywords: glycosylphosphatidylinositol (GPI) membrane anchors;
D-glucosaminylphosphatidylinositol analogues; GPI biosynthesis.
* Corresponding author. Fax: (44) 1382-345 517; e-mail: j.s.brimacombe@dundee.ac.uk
0040-4039/01/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01901-8

122
C. N. Borissow et al. / Tetrahedron Letters 42 (2001) 121–123
radial-band chromatography. Demethoxybenzylation
with 90% trifluoroacetic acid then gave the alcohol 9.
Activation of the H-phosphonate 109 with 3 equivalents
of pivaloyl chloride in pyridine and coupling with 9
furnished the phosphodiester 11 (isolated as the triethyl-
ammonium salt) after oxidation with iodine. Finally,
removal of the benzyl groups and simultaneous conver-
sion of the azido group into an amino group was
achieved by hydrogenolysis with 10% palladium hy-
droxide on carbon and gave 3-dGlcpN-PI 2a.10
tives 2b–5b on treatment with acetic anhydride (triti-
ated for studies with the de-N-acetylase) in 2:1
THF–MeOH containing 2% triethylamine.
Details of the enzymic studies11 with the GlcpN(Ac)-PI
analogues 2–5 will be published elsewhere in due
course, so that only a re´sume´ of the results is provided
here. Neither of the 3%-deoxy analogues 2b or 2a is a
substrate for either the T. brucei de-N-acetylase or
MT-1, respectively, unlike the 6%-deoxy analogues 4
which are good substrates for their respective enzymes.
Because the 4%-position is unavailable, neither 3a nor 5a
can function as a substrate for MT-1, which transfers a
An identical approach was used to incorporate the
2-azido-2,6-dideoxyglycosyl fluoride 127 into 6dGlcpN-
PI 4a10 (J1%,2%=4.1 Hz), following coupling with the
D
-mannose residue to this position from dolichol phos-
phate -mannose, but both of them are potential in-
D
D
-myo-inositol derivative 7.8
hibitors of MT-1. Of the two, 4dGlcpN-PI 3a proved to
be a considerably better inhibitor than GlcpN4Me-PI
5a, which showed only weak inhibition.
Coupling of the 2-azido-2,4-dideoxyglycosyl fluoride
137 with 78 provided the pseudodisaccharide derivative
14 (J1%,2%=3.6 Hz) in 35.5% yield, which, following
de-O-acetylation and benzylation at the 6%-position, was
converted into 4dGlcpN-PI 3a10 in the usual way. An
analogous procedure, commencing with the glycosyl
fluoride 15,7 provided GlcpN4Me-PI 5a10 (J1%,2%=3.7
Hz). This and the dGlcpN-PI analogues were readily
transformed into the corresponding N-acetyl deriva-
These results are highly significant in the design of the
next generation of inhibitors of the de-N-acetylase and
MT-1 of the bloodstream form of T. brucei. For in-
stance, they show that neither enzyme tolerates the
removal of 3%-OH from GlcpN(Ac)-PI 1, whereas the
removal of 6%-OH, as with 6dGlcpN(Ac)-PI 4, leaves an
entirely acceptable substrate and augers well for the

C. N. Borissow et al. / Tetrahedron Letters 42 (2001) 121–123
123
introduction of fluorogenic substituents at the 6%-position
as biological studies progress. Also encouraging is the
strong inhibition of MT-1 shown by 4dGlcpN-PI 3a.
7. The synthesis of this compound is outlined in: Dix, A. P.;
Borissow, C. N.; Ferguson, M. A. J.; Brimacombe, J. S.
Tetrahedron Lett. 2001, 42, 117.
8. Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J. J. Chem.
Soc., Perkin Trans. 1 1993, 2945.
9. Lindh, I.; Stawinski, J. J. Org. Chem. 1989, 54, 1338;
Crossman, A.; Brimacombe, J. S.; Ferguson, M. A. J. J.
Chem. Soc., Perkin Trans. 1 1997, 2769.
Acknowledgements
We thank the Wellcome Trust for support and the
BBSRC for a studentship (to C.N.B.).
10. ES-MS(−) data: 2a m/z 954.3000 [100%, M−Et3N−H]−
(requires m/z 954.5919); 3a m/z 954.4623 [100%, M−
Et3N−H]− (requires m/z 954.5919); 4a m/z 954.1000
[100%, M−Et3N−H]− (requires m/z 954.5919); 5a m/z
984.2988 [100%, M−Et3N−H]− (requires m/z 984.6025).
These compounds also exhibited 1H and 31P NMR spectra
compatible with the assigned structures.
References
1. Vikerman, K.; Luckins, A. G. Nature 1969, 224, 1125.
2. Cross, G. A. M. Parasitology 1975, 71, 393.
3. McConville, M. J.; Ferguson, M. A. J. Biochem. J. 1993,
294, 305.
4. Tarutani, M.; Itami, S.; Okabe, M.; Ikawa, M.; Tezuka,
T.; Yoshikawa, K.; Kinoshita, T.; Takeda, J. Proc. Natl.
Acad. Sci. USA 1997, 94, 7400.
5. Ferguson, M. A. J.; Brimacombe, J. S.; Brown, J. R.;
Crossman, A.; Dix, A.; Field, R. A.; Gu¨ther, M. L. S.;
Milne, K. G.; Sharma, D. K.; Smith, T. K. Biochim.
Biophys. Acta 1999, 1455, 327.
11. These studies were conducted with a trypanosomal cell-free
system to which the various dGlcpN(Ac)-PI analogues
were added as exogenous substrates. The action of the
de-N-acetylase was followed by the release of acetic-3H3
acid from analogues with tritium labelling of the N-acetyl
group, whilst that of MT-1 was assessed from the extent
of transfer of the tritium label from dolichol phosphate
D
-mannose-2-3H to the various analogues. The analogues
were also tested for their ability to inhibit the processing
of exogenous synthetic GlcpN(Ac)-PI 1 by the trypanoso-
mal cell-free system.
6. See Ref. 2 in: Dix, A. P.; Borissow, C. N.; Ferguson, M.
A. J.; Brimacombe, J. S. Tetrahedron Lett. 2001, 42, 117.
.