Synthesis of new fluorescently labeled glycosylphosphatidylinositol (GPI) anchors

Article abstract of DOI:10.1016/j.tetlet.2011.06.005

The borondipyrromethene (BODIPY) labeled new glycosylphosphatidylinositol (GPI) molecules were synthesized as cellular probes to study the chemical basis of microdomain organization of GPI-anchored proteins and cholesterol in plasma membrane. The synthesi

Full text of DOI:10.1016/j.tetlet.2011.06.005

Tetrahedron Letters 52 (2011) 4277–4279
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of new fluorescently labeled glycosylphosphatidylinositol
(GPI) anchors
Varma Saikam a, Riya Raghupathy b, Mahipal Yadav a, Veeranjaneyulu Gannedi a, Parvinder Pal Singh a,
Naveed A. Qazi a, Sanghapal D. Sawant a, Ram A. Vishwakarma a,
⇑
a Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Council of Scientific and Industrial Research, Jammu 180 001, India
b National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560 065, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The borondipyrromethene (BODIPY) labeled new glycosylphosphatidylinositol (GPI) molecules were syn-
thesized as cellular probes to study the chemical basis of microdomain organization of GPI-anchored pro-
teins and cholesterol in plasma membrane. The synthesis enabled by a new stereo-selective glycosylation
Received 16 January 2011
Revised 31 May 2011
Accepted 2 June 2011
Available online 13 June 2011
of myo-D-inositol acceptor led to the preparation of optically pure glucosaminyl-(1-6)-a-phosphatidyl-
myo- -inositol and its unnatural stereoisomer.
D
Ó 2011 Published by Elsevier Ltd.
Keywords:
Glycosylphosphatidylinositol anchors
BODIPY fluorescent probe
Membrane micro-domains
D
and
L-myo-inositol
Unnatural GPI analogs
Glycosylphosphatidylinositols (GPIs) are a unique class of natu-
ral glycophospholipids which provide an alternative mechanism
for anchoring of a number of specialized proteins to the outer leaf-
let of the eukaryotic plasma membrane.1–3 The GPI-anchored pro-
teins along with cholesterol and sphingolipids create functional
microdomains (lipid-rafts) as signaling platforms at the cell sur-
face.4 The lipid-raft model has provided a new view of the plasma
membrane generating significant interest in cell and membrane
biology as to how the GPIs interact with cholesterol leading to
the formation of ordered domains in biological membranes.5,6
The molecular understanding of the mechanism of the localized
clustering of GPI-anchored proteins7 and cholesterol in lipid rafts
requires synthetic probes based on GPI structures. In view of the
great interest in the biochemistry and cell biology of GPI anchors,
a number of GPI analogs have been synthesized.8–11 In our research
on chemistry and biology of GPI molecules, we have designed new
approaches for the synthesis of the full-length GPI anchors and
their structural and functional mimics and used them to address
specific questions of GPI biosynthesis, inhibitor design, and mem-
brane lipid-raft organization.7,12–17 First synthesis of a head-group
labeled GPI intermediate was reported by Schmidt et al.18 followed
by our synthesis of lipid-tail labeled GPIs.13 In continuation to our
collaborative work7 showing that nanoclusters of GPI-anchored
proteins are formed by cortical-actin driven activity and to eluci-
date the stereo-chemical role of GPIs in clustering of cholesterol-
rich ordered domains, we required a BODIPY labeled fluorescent
GPI probe 15a and its unnatural stereoisomer 15b. The rationale
for designing the GlcN-PI structure was based on: (a) the glucosa-
minylation of phosphatidylinositol by UDP-GlcNAc is the first com-
mitted step of GPI bio-synthesis and (b) this step imparts
asymmetric transbilayer distribution of GPI biosynthetic interme-
diates across the plasma-membrane.13 The unique feature of GPI
anchor, compared to other glyco-conjugates in the living cells, is
the presence of a ‘free amine’ containing glucosamine residue in-
stead of the N-acetylglucosamine. Interestingly, during the biosyn-
thesis of GPIs, the N-acetylglycosamine is first transferred to a
phosphatidylinositol (PI) precursor from UDP-GlcNAc to generate
GlcNAc-PI intermediate. The N-acetyl group is then removed by a
unique enzyme N-de-acetylase to produce GlcNH2-PI (with free
amine). This N-de-acetylation is a critical and necessary require-
ment for further mannosylation steps, toward full length GPI
anchor.3 From our previous work,7,13 we have built a hypothesis
that the positive charge on glucosamine and the stereochemical
orientation of the
volved in cholesterol recognition. Therefore, it would be of great
interest to have both isomers of the probe, GlcN-(1-6)- -myo-
inositol and GlcN-(1-6)- -myo-inositol.
D-myo-inositol residue of GPI anchor are in-
a-D
a-L
Here, we report the synthesis of fluorescent labeled glucosami-
nyl-(1-6)- -phosphatidyl-myo- -inositol 15a and glucosaminyl-
(1-6)- -phosphatidyl-myo- -inositol 15b with fluorescent label at
6-OH position of glucosamine residue. For preliminary studies,
a
D
a
L
⇑
Corresponding author. Tel.: +91 191 2569111; fax: +91 191 2569333.
E-mail address: ram@iiim.res.in (R.A. Vishwakarma).
0040-4039/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2011.06.005

4278
V. Saikam et al. / Tetrahedron Letters 52 (2011) 4277–4279
N
F
O
N
F
B
N
F
HN
N
F
O
O
B
4
HN
MeO
O
NH
4
O
MeO
NH
HO
HO
+H3N
O
P
HO
HO
OH
OH
HO OH
+H3N
O
-O
OH
OH
HO
HO
O
O
O
O
O
P
-O
15b
O
O
OCO(CH2)14CH3
OCO(CH2)14CH3
OCO(CH2)14CH3
OCO(CH2)14CH3
15a
GlcN-PI with BODIPY at 6-OH position of glucosamine was de-
signed as this position offers reasonable distance from the mem-
brane surface and also gives optional site (4-OH of GlcN residue)
for further biosynthetic elongation of GPI anchor. Furthermore,
choice of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene
(BODIPY) as a fluorophore was based on two reasons: (a) the rela-
tively small size of BODIPY probe should minimize the impact on
binding affinity of the resulting GPI probe to the membrane and
(b) BODIPY probe having good photo-physical properties (high
photo-stability, quantum yield, extinction coefficient, and narrow
emission bandwidth).19
OH
BnO
HO OH
OBn
OBn
OH
OH
ref 16
O
HO
HO
HO
1
2
a
O
OBn
O
+
BnO
In our earlier approach15 for the synthesis of key glucosamine–
inositol intermediate, the racemic 1-O-p-methoxybenzyl-2,3,4,
5-tetra-O-benzyl-myo-inositol was glycosylated with a 2-azidogly-
cosyl donor to get a pseudo-disaccharide which was resolved by
converting into 4,6-cyclic acetal protected disaccharide. Now, we
have improved this method significantly by: (a) introducing a
new simpler method for the resolution of myo-inositol intermedi-
ate 2, (b) 6-O-acetyl group directed stereo-selective glycosylation20
BnO
BnO
O
OBn
OBn
O
O
O
L-Isomer
3b
D-Isomer
3a
b
b
OH
OBn
BnO OH
OBn
OBn
BnO
BnO
O
O
of myo-inositol intermediate 7a with azidoglycosyl
a/b trichloro-
OH
HO
4a
4b
acetimidate TCA (without cumbersome isolation of minor
8
c
b-TCA isomer), and (c) improved H-phosphonate coupling of the
glycero-lipid donor.
c
Accordingly, optically pure D and L inositol intermediates 7a and
BnO OH
OH
OBn
b (Scheme 1) were obtained by reaction of 221 with camphor
dimethyl acetal.22 Now the camphor protection of myo-inositol
intermediate 3a was removed and 1-O-position was selectively
protected by a PMB group by dibutyltin chemistry followed by
2-O-benzylation to give 6a. Removal of 6-O-allyl group from 6a
OBn
OBn
O
BnO
BnO
O
PMBO
OPMB
5a
5b
d
d
BnO OBn
provided the acceptor 7a. Similarly, the L-myo-inositol intermedi-
OBn
OBn
O
OBnOBn
BnO
BnO
ate 7b was prepared from 3b. The absolute configuration of the
myo-inositol intermediates 7a and b was confirmed by removing
PMB group and comparing the optical rotation with that of the re-
ported compounds.21
PMBO
O
6a
OPMB
6b
e
e
The azidoglycosyl donor 8 was prepared by a known method.20b
Initially we isolated b-isomer of trichloroacetamidate for glycosyl-
ation with inositol acceptor 7a. However, later we found that the
BnO
OBn
OBn
OBn
OBn
OBn
HO
BnO
BnO
OH
PMBO
separation of
using /b mixture of 8 proceeds smoothly at low temperature to
give -pseudodisaccharide 9a (Scheme 2). This is the first report
a/b isomer of donor is not required and glycosylation
OPMB
7a
a
a
7b
Scheme 1. Synthesis of enantio-pure
D and L myo-inositol building block. Reagents
of such stereochemically controlled synthesis of key glucosa-
mine–inositol intermediate of the GPI anchor synthesis.20 How-
ever, such a method for directed glycosylation has been reported
for heparin synthesis.20b Therefore, the reaction of inositol acceptor
7a with donor 8 in the presence of catalytic TMSOTf at À78 °C re-
sulted in formation of the desired product 9a in excellent yield
(Scheme 2). Now the azido group of pseudo-disaccharide 9a was
transformed into the amino function by reduction with propanedi-
thiol in pyridine/water using catalytic amounts of triethylamine
followed by its protection with Boc group affording the desired
and conditions: (a) camphor dimethyl ketal, dry CH2Cl2, PTSA, reflux, 2 h, 43% (3a)
and 42% (3b); (b) PTSA, MeOH, 50 °C, 3 h, 86% (4a) and 85% (4b); (c) (i) (Bu)2SnO,
MeOH, reflux, 2 h; (ii) PMBCl, (Bu)4NBr, dry toluene, 4 Å MS, reflux 6 h, 90% (5a) and
89% (5b); (d) BnBr, NaH, DMF, rt, 3 h, 93% (6a and b); (e) (i) t-BuOK, DMSO, 80 °C,
3 h; (ii) 1 M HCl:acetone (1:9), 80 °C, 30 min, 82% (7a) and 85% (7b).
N-Boc protected intermediate 10a. The primary hydroxyl group
of 10a was converted to azido intermediate by 6-O-deacetylation,
mesylation followed by azide substitution (10a–11a). The PMB

V. Saikam et al. / Tetrahedron Letters 52 (2011) 4277–4279
4279
AcO
BnO
BnO
AcO
BnO
phosphate]. The final fluorescent product 15a was purified by pre-
parative TLC using EtOAc/(Me)2CO/MeOH/H2O (7:1:1:1) as eluent
followed by reversed phase HPLC. The structure of final probe
was supported by the presence of peaks at m/z 1464 (M) and
1444 (MÀF) in negative mode of MALDI mass spectrum.
BnO
OBn
O
O
OBn
OBn
HO
BnO
N3
BnO OBn
PMBO
N3
8
OBn
OBn
OTCA
7a
O
PMBO
a
9a
An identical synthetic approach was utilized to prepare the
unnatural BODIPY-GlcN-PI 15b viz. 6-O-(2-amino-2-deoxy-6-BOD-
b
IPY-amino-6-deoxy-a-D-glucopyranosyl)-myo-L-inositol-1-[sn-2,
3-bis(palmitoyloxy)propyl phosphate] from its respective
intermediate 7b (Scheme 3, Supplementary data).
In conclusion, BODIPY labeled GPI probes 15a and 15b were
synthesized in an efficient manner. Our initial cell biology experi-
ments have demonstrated the utility of the GPI probe 15a in its
insertion in plasma-membrane, the full biological study will be
published in due course.
N3
AcO
O
O
O
c
BnO
BnO
BnO
BnO
BnO OBn
BnO
OBn
BocHN
BocHN
OBn
OBn
OBn
OBn
O
PMBO
10a
PMBO
11a
d
N3
O
BnO
BnO
N3
BnO
BnO
Acknowledgments
BnO OBn
O
BocHN
-O
OBn
OBn
O
16
O
P
BnO OBn
O
BocHN
OBn
OBn
We thank Satyajit Mayor (NCBS, Bangalore) for collaboration.
V.S. and V.G. thank the CSIR for Junior Research Fellowship.
O
e
HO
O
OCO(CH2)14CH3
OCO(CH2)14CH3
12a
Supplementary data
13a
f
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.06.005.
H2N
HO
HO
O
g
HO OH
BocHN
References and notes
OH
15a
O
OH
O
P
O
O
1. Ferguson, M. A. J.; Homans, S. W.; Dwek, R. A.; Rademacher, T. W. Science 1988,
239, 753.
2. Homans, S. W.; Ferguson, M. A. J.; Dwek, R. A.; Rademacher, T. W.; Anand, R.;
Williams, A. F. Nature 1988, 333, 269.
-O
OCO(CH2)14CH3
OCO(CH2)14CH3
3. For a comprehensive review on structure and biosynthesis of GPI anchors:
McConville, M. J.; Ferguson, M. A. Biochem. J. 1993, 294, 305.
4. Lingwood, D.; Simons, K. Science 2010, 327, 46. and references cited therein.
5. Simons, K.; Gerl, M. Nat. Rev. Mol. Cell Biol. 2010, 11, 688.
6. Johannes, L.; Mayor, S. Cell 2010, 142, 507.
7. Goswami, D.; Gowrishankar, K.; Bilgrami, S.; Ghosh, S.; Raghupathy, R.; Chadda,
R.; Vishwakarma, R. A.; Rao, M.; Mayor, S. Cell 2008, 135, 1085.
8. Guo, Z.; Bishop, L. Eur. J. Org. Chem. 2004, 17, 3585.
9. Paulick, M. G.; Bertozzi, C. R. Biochemistry 2008, 47, 6991.
10. Hecht, M.-L.; Tsai, Y.-H.; Liu, X.; Wolfrum, C.; Seeberger, P. H. ACS Chem. Biol.
2010, 5, 1075.
14a
Scheme 2. Synthesis of Bodipy-GlcN-PI (15a). Reagents and conditions: (a)
TMSOTf, CH2Cl2, À70 °C, 1 h, 75%; (b) 1,3-propanedithiol, pyridine, TEA, H2O, 12–
18 h and then (Boc)2O, dry toluene, 4 h, 66%; (c) (i) NaOMe, MeOH, 1 h, 95%; (ii)
mesyl chloride, pyridine, 80 °C, 3 h, 97%; (iii) NaN3, DMF, 120 °C 4 h, 96%; (d) DDQ,
CH2Cl2, H2O, 2 h, 55%; (e) 2,3-di-O-palmitoyloxy-sn-glycero-H-phosphonate (16),
pivaloyl chloride, dry pyridine, dry ACN, 1 h, and then I2, pyridine, water, 2 h, 90%;
(f) Pd(OH)2, H2, DCM:MeOH:H2O, 18 h, 80%; (g) (i) 6-((4,4-difluoro-1,3-dimethyl-5-
(4-methoxyphenyl)-4-bora-3a,4a-diaza-S-indacene-2-propionyl)amino)-hexanoic
acid, succinimidyl ester, TEA, 6–8 h; (ii). 15% TFA, 1 h, 60%.
11. Paulick, M. G.; Wise, A. R.; Forstner, M. B.; Groves, J. T.; Bertozzi, C. R. J. Am.
Chem. Soc. 2007, 129, 11543.
12. Ali, A.; Gowda, D. C.; Vishwakarma, R. A. Chem. Commun. 2005, 4, 519.
13. Vishwakarma, R. A.; Menon, A. K. Chem. Commun. 2005, 4, 453.
14. Vishwakarma, R. A.; Vehring, S.; Mehta, A.; Sinha, A.; Pomorski, T.; Herrmann,
A.; Menon, A. K. Org. Biomol. Chem. 2005, 3, 1275.
15. Ali, A.; Vishwakarma, R. A. Tetrahedron 2010, 66, 4357.
16. Vishwakarma, R. A.; Ruhela, D. Enzymes 2009, 26, 181.
17. Chandra, S.; Ruhela, D.; Deb, A.; Vishwakarma, R. A. Expert Opin. Ther. Target
2010, 14, 739.
18. Mayer, T. A.; Weingart, R.; Munstermann, F.; Kawada, T.; Kurzchalia, T.;
Schmidt, R. R. Eur. J. Org. Chem. 1999, 2563.
19. Karolin, J.; Johansson, L. B. A.; Strandberg, L.; Ny, T. J. Am. Chem. Soc. 1994, 116,
7801.
20. (a) Lu, L. D.; Shie, C.; Kulakarni, S. S.; Pan, G. R.; Lu, X.; Hung, S. C. Org. Lett. 2006,
8, 5995; (b) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Litjens, R.; Palmacci, E. R.;
Seeberger, P. H. Chem. Eur. J. 2003, 9, 140.
21. Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J. J. Chem. Soc Perkin Trans. 1 1993,
2945. and references therein.
group was now removed (11a–12a) and the resulting free 1-OH
was reacted with 2,3-di-O-palmitoyloxy-sn-glycero-H-phospho-
nate 16.12 The efficiency of this H-phosphonate coupling was sig-
nificantly improved (from low yield to >90%) by the addition of
acetonitrile during H-phosphonate coupling.
The final steps involved the global deprotection and 6-O-azide-
reduction of 13a by Pearman’s hydrogenolysis method [Pd(OH)2,
CH2Cl2–MeOH–H2O, H2] followed by coupling of resulting 14a with
the required BODIPY ester, that is, 6-((4,4-difluoro-1,3-dimethyl-
5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-S-indacene-2-propi-
onyl)amino)-hexanoic acid, succinimidyl ester (BODIPY TMR-X, SE)
and deprotection of Boc group providing the target BODIPY-GlcN-
PI 15a viz. 6-O-(2-amino-2-deoxy-6-BODIPY-amino-6-deoxy-
a-D-
22. Bruzik, K. S.; Tsai, M. D. J. Am. Chem. Soc. 1992, 114, 6361.
glucopyranosyl)-myo- -inositol-1-[sn-2,3-bis(palmitoyloxy)propyl
D