Synthesis of non-hydrolysable mimics of glycosylphosphatidylinositol (GPI) anchors

Article abstract of DOI:10.1039/c3ob42116c

Synthesis of first generation non-hydrolysable C-phosphonate GPI analogs, viz., 6-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol-1-O-(sn-3,4- bis(palmitoyloxy)butyl-1-phosphonate) 23a and 6-O-(2-amino-2-deoxy-α-d- glucopyranosyl)-d-myo-inositol-1-O-(sn-2,3-bis(palmitoyloxy)propyl-1- phosphonate) 23b, is reported. The target compounds were synthesized by the coupling of α-pseudodisaccharide 21 with phosphonic acids 18a and 18b respectively in quantitative yield followed by de-protection. These synthetic C-phosphonate GPI-probes were resistant to phosphatidylinositol specific phospholipase C (PI-PLC) and also showed moderate inhibition of the enzyme activity. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob42116c

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of non-hydrolysable mimics of
glycosylphosphatidylinositol (GPI) anchors†
Mahipal Yadav,a Riya Raghupathy,b,c Varma Saikam,a,d Saidulu Dara,a,d
Parvinder Pal Singh,*a Sanghapal D. Sawant,a Satyajit Mayorb and
Ram A. Vishwakarma*a,d
Cite this: Org. Biomol. Chem., 2014,
12, 1163
Synthesis of first generation non-hydrolysable C-phosphonate GPI analogs, viz., 6-O-(2-amino-2-deoxy-
α-D-glucopyranosyl)-D-myo-inositol-1-O-(sn-3,4-bis(palmitoyloxy)butyl-1-phosphonate) 23a and 6-O-
(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol-1-O-(sn-2,3-bis(palmitoyloxy)propyl-1-phos-
phonate) 23b, is reported. The target compounds were synthesized by the coupling of α-pseudo-
disaccharide 21 with phosphonic acids 18a and 18b respectively in quantitative yield followed by de-
protection. These synthetic C-phosphonate GPI-probes were resistant to phosphatidylinositol specific
phospholipase C (PI-PLC) and also showed moderate inhibition of the enzyme activity.
Received 24th October 2013,
Accepted 3rd December 2013
DOI: 10.1039/c3ob42116c
www.rsc.org/obc
Glycosylphosphatidylinositols (GPIs) are a unique class of phosphodiester linkage with the assistance of the nucleophilic
natural glycophospholipids which provide an alternative mech- 2-OH of D-myo-inositol. In some instances this cleavage
anism for anchoring of a number of specialized proteins to the releases important second messengers such as diacylglycerol
outer leaflet of the eukaryotic plasma membrane.1–3 The GPI- (DAG) and inositol 1,4,5-triphosphate (IP3) which activate
anchored proteins (GPI-APs) along with cholesterol and sphingo- numerous important cellular processes such as DNA synthesis,
lipids create functional nanodomains (lipid rafts) which are cell proliferation and neuronal activity.9 In this direction, the
in turn enriched in specific regions of the cell by a mechanism synthesis of non-hydrolysable PLC-resistant synthetic GPI
that involves dynamic actin activity.4 These nano-domain probes will greatly help in deciphering the role of the GPI-head
enriched regions are likely to act as signaling platforms at the group in the formation of signaling platforms or functional
cell surface.5 The understanding of nano-domain enriched domains.10 A variety of non-hydrolysable PLC-resistant PI sub-
regions has provided a new functional dynamic view of the strates have been synthesized following several approaches,
plasma membrane generating significant interest in cell and viz., modification of nucleophilic the 2-OH of inositol,11 repla-
membrane biology as to how the GPI anchored proteins cement of the scissile P–O bond with a C–P bond12 and confor-
(GPI-APs) interact with cholesterol leading to the formation of mationally constrained analogs.13 Only one report towards the
ordered domains in biological membranes.6,7 The molecular synthesis of a non-hydrolysable PLC resistant GPI analog was
understanding of this mechanism of the localized clustering reported by Morris et al., in which GPI analogs, viz. glucos-
of GPI-anchored proteins4 and cholesterol in such domains aminyl(α1-6)-D-myo-inositol-1-acyl-phosphonates, were used to
requires synthetic and metabolically stable GPI probes.
study the role of glycan for recognition by GPI-PLC;8a however,
Due to a very short half-life in biological systems, GPI the synthesis of this analog was never published. Our contin-
anchors are cleaved by specific phospholipases (GPI-specific ued interest in the chemistry and biology of GPI molecules4,14
phospholipase C and PI-specific phospholipase C)8 at the motivated us to initiate efforts towards synthesis of non-hydro-
lysable PLC resistant GPI analogs where normal scissile P–O
bond of the phosphodiester linkage was replaced with a C–P
bond.
aMedicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal
Road, Jammu 180 001, India. E-mail: ram@iiim.res.in, ppsingh@iiim.ac.in;
We now report a new synthetic strategy for preparation of
Fax: +91-191-2569333; Tel: +91-191-2569111
bNational Centre for Biological Sciences, Tata Institute of Fundamental Research,
first generation non-hydrolysable PLC resistant GPI analogs
23a and 23b, and demonstrate that the C–P containing GPI
anchors were not hydrolyzed by PI-PLC.
Our approach to the synthesis of non-hydrolysable GPI
anchors 23a and 23b is outlined in Scheme 1, and comprises
three main stages: (i) synthesis of suitably protected glucos-
aminyl(α1-6)-D-myo-inositol 21; (ii) synthesis of optically pure
Bangalore 560 065, India
cShanmugha Arts, Science, Technology and Research Academy, Thanjavur 613 401,
India
dAcademy of Scientific and Innovative Research, Jammu 180001, India
†Electronic supplementary information (ESI) available: 1H and 13C NMR and
HRMS spectra of all compounds and the details of inhibition experiments. See
DOI: 10.1039/c3ob42116c
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Scheme 1 Retro-synthetic overview.
Scheme 3 Reagents and conditions: (a) Tf2O, TEA, CH2Cl2, −20 °C, 1 h,
98% for 8a; PPh3, CBr4, CH2Cl2, 0 °C then rt, 3 h, 99% for 8b; PPh3, l2,
imidazole, toluene, 90 °C, 2 h, 95% for 8c; p-TsCl, pyridine, rt, 24 h, 70%
for 8d; (b) CH3P(OBn)2, base, THF, −78 °C, 2 h.
desired product 9. Similarly, bromo 8b, iodo 8c, tosylate 8d
were also prepared from 2,3-isopropylidene glycerol 7 and
tried coupling with dibenzyl methylphosphonate. However in
this case the desired nucleophilic substitution reactions were
not successful in our case, as previously reported in the
literature.15
Scheme 2 Reagents and conditions: (a) i. TfN3, H2O, K2CO3, CH2Cl2,
MeOH, CuSO4, −10 °C to rt, 24 h; ii. Ac2O, pyridine, DMAP, 24 h, 80%
over 2 steps; (b) hydrazine acetate, DMF, 0 °C to rt, 1 h, 85%; (c) Cl3CCN,
CH2Cl2, K2CO3, rt, 2 h, 84%.
phosphonic acid donors 18a and 18b; and (iii) coupling of
Later, a different approach for the synthesis of 3,4-di-O-pal-
pseudo-disaccharide 21 with phosphonic acid donors 18a and mitoyloxybutylphosphonate 18a was tested out starting from
18b respectively.
optically pure S-(−)-malic acid 10 as given in Scheme 4. The
First, the synthesis of optically pure 1-O-p-methoxybenzyl- S-(−)-malic acid was converted into S-(−)-dimethyl malate 11
2,3,4,5-tetra-O-benzyl-D-myo-inositol 2 (Scheme 2, detail given which was then reduced with LiBH4 and gave S-(−)-2-deoxy-
in ESI†) was successfully achieved as per our recently pub- erythritol 12. The S-(−)-erythritol 12 on treatment with acetone
lished method.14i The 2-azido glycosyl donor 6 was synthesized in the presence of p-TSA gave a regioselective five-membered
from D-glucosamine 3 (Scheme 2) which on treatment with S-(−)-1,2-O-isopropylidene product 13.16 Compound 13 was con-
triflic azide followed by per acetylation gave 2-azido-2-deoxy- verted into an iodo compound 14a followed by treatment with
1,3,4,6-tetra-O-acetyl-D-glucosamine (4). The anomeric acetyl triethylphosphite which gave S-(−)-diethyl phosphonate 15a.
group of peracetylated compound 4 was selectively deprotected Since S-(−)-diethyl phosphonate (15a) had the same polarity as
by hydrazine acetate at 0 °C, which on treatment with triethyl phosphite or ethyl diethylphosphonate, the chromato-
trichloroacetonitrile in the presence of potassium carbonate graphic separation became very cumbersome. Therefore, the
gave 2-azido-glycosyl trichloroacetimide donor 6 (α : β).
isopropylidene protection was removed and the product
The synthesis of 2-deoxyerythrityl based C-phosphonic acid S-(−)-diethyl 3,4-dihydroxybutylphosphonate (16a) could be
i.e., 3,4-di-O-palmitoyloxybutylphosphonate 18a was initially easily purified by column chromatography. Further, diethyl
visualized as per the strategy shown in Scheme 3. The 2,3-iso- 3,4-dihydroxybutylphosphonate (16a) when treated with palmi-
propylidene glycerol 7 was first converted into triflate 8a by tic acid in the presence of DCC gave S-(−)-diethyl 3,4-di-O-
treating with triflic anhydride. The coupling of triflate 8a with palmitoyloxybutyl-1-phosphonate (17a). The compound 17a on
dibenzyl methylphosphonate under various basic conditions hydrolysis in the presence of TMSBr gave 2,3-di-O-palmitoyl-
(n-BuLi, LDA, LiHMDS at −78 °C and −40 °C) did not give the oxybutyl-1-phosphonic acid (18a). Similarly, glyceryl based
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Scheme 4 Reagents and conditions: (a) MeOH, PTSA, rt, 12 h, 86%; (b) LiBH4, THF, rt then reflux, 2 h, 95%; (c) acetone, PTSA, reflux, 2 h, 87%; (d)
PPh3, l2, imidazole, toluene, 90 °C, 2 h 98%; (e) P(OEt)3, reflux, 6 h; (f) MeOH, PTSA, rt, 12 h, 82% over two steps; (g) palmitic acid, DCC, DMAP,
CH2Cl2, rt, 12 h, 88%; (h) TMSBr, CH2Cl2, rt, 6 h then THF–H2O, reflux, 1 h, 98%.
Scheme 5 Reagents and conditions: (a) TMSOTf, CH2Cl2, MS 4 Å, −78 °C, 2 h, 90%; (b) NaOMe, MeOH–CH2Cl2, rt, 8 h; (c) BnBr, DMF, NaH, rt, 3 h,
80% over two steps; (d) DDQ, H2O, 0 °C to rt, 3 h, 55%; (e) 18a or 18b, Cl3CCN, pyridine, 60 °C, 48 h, 77% for 22a and 50% for 22b; (f) 10% Pd(OH)2/
C, H2, CH2Cl2–MeOH–H2O, rt, 24 h, 80%.
C-phosphonic acid i.e., 2,3-di-O-palmitoyloxypropyl-1-phospho- (23a). An identical approach was utilized to synthesize 6-O-(2-
nic acid (18b) was prepared from isopropylidene protected gly- amino-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol-1-O-(sn-2,3-
cerol 7 following the same sequence of steps (Scheme 4) as bis(palmitoyloxy)propyl-1-phosphonate) (23b) from its respect-
discussed above.
ive intermediates 21 and 18b.
Further, the inositol acceptor i.e., 1-O-4-methoxybenzyl-
To determine the stability of synthetic C-phosphonates con-
2,3,4,5-tetra-O-benzyl-D-myo-inositol (2) was coupled with taining GPI anchors 23a and 23b, these analogs were subjected
2-azido glycosyl donor 6 in the presence of catalytic TMSOTf at to PI-PLC (phosphatidylinositol-specific phospholipase C)
−78 °C resulting in formation of the α-pseudodisaccharide 19 enzymatic reaction17 on a small scale. The enzymes PI-PLC
in excellent yield (Scheme 5).14i The acetyl group of the glycosyl and GPI-PLC are known to cleave the PI (phosphatidylinositol)
moiety of α-pseudodisaccharide 19 was replaced with a benzyl as well as GPI substrates at the sn-3 phosphodiester bond.8b,18
protecting group (19–20). The PMB group was then removed We chose fluorophore tagged PI (24, sn-2-NBD labeled PI) as a
(20→21) and the resulting free 1-OH was successfully coupled control substrate for the PI-PLC reaction; the substrate NBD
with S-(−)-3,4-di-O-palmitoyloxybutylphosphonic acid (18a) to labeled PI (24) was synthesized by a method reported by us.14c
produce fully protected C-phosphonates containing the GPI Since the sn-2 position of PI 24 was tagged to the NBD fluoro-
intermediate 22a.16 The compound 22a was finally subjected phore, the enzymatic reaction could be easily monitored by
to global benzyl deprotection and 2-azido reduction by Pearl- TLC and MS. The NBD-PI 24 as well as C-phosphonate GPI
man’s hydrogenolysis method [Pd(OH)2, CH2Cl2–MeOH–H2O, probes 23a and 23b were treated with PI-PLC19 for 20 minutes
H2], which provided 6-O-(2-amino-2-deoxy-α-D-glucopyranosyl)- at 37 °C and checked for the completion of the reaction by
D-myo-inositol-1-O-((sn-3,4-bis(palmitoyloxy)butyl-1-phosphonate) TLC and MALDI-MS analysis. As evident from the TLC and
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Fig. 1 PI-PLC enzymatic reaction of C-phosphonate GPI analogs 23a and 23b along with the standard PI-NBD 24.
Fig. 2 (A) Represents the structure of NBD-GPI probes 26. (B and C) Represent the IC50 of 23a and 23b for the PI-PLC inhibition activity.
mass analysis, NBD-PI 24 underwent complete hydrolysis substrate (without inhibitors) to calculate the percentage
(peak at 770.1328 observed corresponding to the hydrolyzed of inhibition. The IC50 values of 0.39 mM and 1.6 mM
fragment 25, Fig. 1) whereas the C-phosphonate GPI analogues were obtained for 23a and 23b respectively (Fig. 2),
did not get hydrolyzed (peaks at 970.6192 and 956.6042 corres- confirming that these GPI probes moderately inhibited the
ponding to 23a and 23b respectively, Fig. 1).
PI-PLC activity (details of the experiments are given in Fig. 3
Since both the non-hydrolysable C-phosphonate GPI probes and 4 of ESI†).
23a/23b were substrate mimics of PI-PLC reaction, it was
In conclusion, we synthesized the first generation C-phos-
important to see if they also inhibited the enzyme. For this, we phonate containing GPI probes 23a and 23b and demonstrated
carried out inhibition experiments with the GPI probes 23a that these synthetic probes are resistant to hydrolysis by
and 23b which required a synthetic fluorescent labeled PI-PLC and inhibited the enzyme moderately at high concen-
enzyme substrate. We decided to use a NBD-labeled GPI tration, the attributes making them suitable for cell biology
analog 26 (Fig. 2A) as the substrate and synthesized it by a syn- experiments. Efforts towards synthesizing advanced mem-
thetic method designed14b by us earlier to address the mech- brane-stable GPI biosynthetic intermediates and their fluo-
anism of flip-flop of GPIs across the endoplasmic reticulum rescence labeled counterparts are currently underway. This
(ER). For inhibition experiments, various concentrations of strategy provides the method of synthesis of non-hydrolysable
23a and 23b (7.5 mM to 0.1 mM) were added to the enzyme GPI probes to study the chemical basis of the nanodomain
mix and the corresponding percentage cleavages of NBD- organization of GPI-anchored proteins and cholesterol in
labeled GPI substrate 26 were calculated (TLC) which were plasma membrane to decipher the chemical basis of the GPI
then subtracted from the control reaction of the NBD-GPI lipid raft organization.
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dried over MgSO4, filtered and the solvent was removed
in vacuo to yield brown oil. Flash chromatography of hexanes–
EtOAc (7 : 3) afforded a colorless oil (mixture of α/β) (27.5 g,
Experimental section
General methods
Solvents were purified according to the standard procedures, 0.074 mol, 80%). Rf = 0.25 (hexane–EtOAc, 7 : 3); 1H NMR
and the reagents used were of the highest purity available. All (400 MHz, CDCl3): δ 5.54 (d, J = 8.6 Hz, 1H), 5.10–5.00 (m, 2H),
reactions were performed in flame-dried glass apparatus under 4.29 (dd, J = 12.5, 4.5 Hz, 1H), 4.07 (dd, J = 12.5, 2.1 Hz, 1H),
an argon/nitrogen atmosphere unless mentioned otherwise. 3.81–3.77 (m, 1H), 3.65 (t, J = 9.5 Hz, 1H), 2.18 (s, 3H), 2.08 (s,
Anhydrous solvents like CH2Cl2, Et2O, THF, CH3OH, CH3CN, 3H), 2.06 (s, 3H), 2.01 (s, 3H); HRMS (MALDI): calcd for
DMF, pyridine, Et3N were freshly dried using standard C14H19N3O9 (M + Na)+ 396.1014, found 396.1020.
methods. NMR measurements (1H, 13C and 31P) were recorded
3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α/β-D-glucopyranoside (5).20
on a 400 or 500 MHz NMR-spectrometer fitted with a pulse- To an ice cold solution of 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-
field gradient probe, and tetramethylsilane (TMS) or residual α/β-D-glucopyranoside 4 (10 g, 0.027 mol) in anhydrous DMF
resonance of deuterated solvent was used as the internal refer- (100 mL) was added hydrazine acetate (4 g, 0.054 mol). After
ence. Chemical shifts are expressed in (δ) parts per million addition, the reaction mixture was warmed to room temp-
and coupling constants J in hertz. Mass spectra were recorded erature and stirred for 30 min. Then it was diluted with EtOAc
either with an LCMS-QTOF instrument or with a MALDI-TOF/ (200 mL) and washed with NaHCO3 (2 × 100 mL) and brine
TOF mass spectrophotometer using 2,5-dihydroxy benzoic solution (2 × 100 mL) respectively. The organic phase was
acid/α-cyano-4-hydroxy cinnamic acid as the matrix in aceto- dried over Na2SO4 and concentrated in vacuo. The crude was
nitrile–water containing 0.01% TFA. Optical rotations were purified by flash chromatography eluting with hexanes–EtOAc
measured on a digital polarimeter. Analytical thin layer chrom- (7 : 3) to give 7.6 g (85%) of 5 as a colorless oil. Rf = 0.20
atography (TLC) was performed on precoated UV-active alumi- (hexane–EtOAc, 7 : 3); HRMS (MALDI): calcd for C12H17N3O8
num backed silica plates, visualized with a UV254 lamp and (M + Na)+ 354.0908, found 354.0926.
stained with 20% phosphomolybdic acid in ethanol or 20%
3,4,6-Tri-O-acetyl-2-azido-2-deoxy-1-trichloroacetimido-α/β-D-
H2SO4 in methanol. Silica column chromatography was glucopyranoside (6).14b To a solution of 3,4,6-tri-O-acetyl-2-
carried out with silica gel (100–200 mesh) or flash silica gel azido-2-deoxy-α/β-D-glucopyranoside 5 (2 g, 6.04 mmol) in
(230–400 mesh). The enzyme PI-PLC was purified as reported anhydrous CH2Cl2 (20 ml), trichloroacetonitrile (0.72 ml,
earlier17 in the National Centre for Biological Sciences (Tata 7.2 mmol) and previously dried K2CO3 (1.67 g, 12.08 mmol)
Institute of Fundamental Research), Bangalore.
were added and stirred for 1 h at room temperature. The reac-
tion mixture was filtered through a celite pad, concentrated
in vacuo and subjected to flash chromatography with hexanes–
A. Procedures and spectroscopic analytical data
1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-α/β-D-glucopyranoside EtOAc (7 : 3) to provide compound 6 {(α : β: 0.25 : 0.75), 2.41 g,
(4).20 Preparation of TfN3: CH2Cl2 (250 mL) was added to a 84% yield} as a colorless solid. Rf = 0.25 (hexane–EtOAc, 7 : 3);
solution of NaN3 (59.5 g, 0.92 mol) in water (150 mL) at 0 °C. 1H NMR (500 MHz, CDCl3, peaks labelled for β isomer only):
The mixture was stirred vigorously and treated with trifluoro- δ 8.82 (s, 1H), 5.72 (d, J = 8.5 Hz, 1H), 5.14–5.11 (m, 2H), 4.32
methanesulfonic anhydride (31.0 mL, 0.19 mol) over a period (dd, J = 12.3, 4.3 Hz, 1H), 4.13 (dd, J = 12.8, 1.9 Hz, 1H),
of 3 h at 0 °C. After the complete addition of trifluoromethane- 3.84–3.81 (m, 2H), 2.11 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H);
sulfonic anhydride, the reaction mixture was stirred at 0 °C for HRMS (MALDI): calcd for C14H17Cl3N4O8 (M + Na)+ 497.0004,
2.5 h. The aqueous phase was extracted with CH2Cl2 (2 × found 497.0026.
100 mL) and the combined organic layers were washed with
saturated Na2CO3 and saved for use in the next step. [Caution: acid 10 (7 g, 0.05 mol) in MeOH (100 mL) was added a catalytic
TfN3 is explosive when not in solution!]. amount of p-TsOH·H2O (0.5 g) and the reaction mixture was
(S)-(−)-Dimethyl malate (11).16 To a solution of (S)-(−)-malic
CuSO4 (140 mg, 0.88 mmol) and K2CO3 (19.2 g, 0.14 mol) stirred overnight at room temperature. After completion, the
were added to a solution of glucosamine hydrochloride 3 reaction mixture was neutralized with triethyl amine and con-
(20.0 g, 0.092 mol) in water (300 mL). Methanol (600 mL) was centrated in vacuo. The crude product was purified by flash
added to the reaction mixture followed by the addition of the chromatography eluting with CH3COCH3–CH2Cl2 (1 : 19) to
TfN3 solution. Methanol was added until the solution was give 7.28 g (86%) of 11 as a colorless oil. Rf = 0.3 (hexane–
1
homogeneous (∼300 mL). The clear blue solution was allowed EtOAc, 7 : 3); H NMR (400 MHz, CDCl3): δ 4.54–4.50 (m, 1H),
to stir for 24 h at room temperature. Glycine (70 g) was added 3.82 (s, 3H), 3.72 (s, 3H), 3.31 (d, J = 4.3 Hz, 1H), 2.84 (qd, J =
and the reaction mixture was again allowed to stir for 24 h. 16.4, 5.3 Hz, 2H); HRMS (ESI): calcd for C6H10O5 (M + Na)+
The glycine was filtered off and the solvent was removed 185.0420, found 185.0422.
in vacuo to afford brown oil. The oil was taken up in pyridine
(S)-1,2,4-Butanetriol (12).16 (S)-(−)-Dimethyl malate 11 (7 g,
(95 mL), cooled to 0 °C and DMAP (∼30 mg) and acetic anhy- 0.04 mol) dissolved in 80 ml of dry tetrahydrofuran was added
dride (86 mL, 0.91 mol) were added. The solution was stirred dropwise to a solution of lithium borohydride (23 mL of 2 M
for 12 h at room temperature. The reaction was quenched with solution, 0.04 mol) in 20 mL of dry tetrahydrofuran with the
saturated NaHCO3 and the aqueous phase was extracted with help of a cannula under a nitrogen atmosphere (place an ice
CH2Cl2 (3 × 1000 mL). The organic phases were combined, bath if more heat is generated towards the end of the
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addition). After complete addition, the reaction mixture was (dd, J = 8.6, 5.4 Hz, 1H), 3.25 (dd, J = 9.8, 4.6 Hz, 1H), 3.15
refluxed for 2 h. Addition of water to the reaction mixture gave (dd, J = 9.8, 8.5 Hz, 1H), 1.46 (s, 3H), 1.35 (s, 3H); HRMS (ESI):
a white precipitate, which was filtered and washed with four calcd for C6H11IO2 (M + H)+ 242.9876, found 242.9887.
30 ml portions of dry ethanol. The combined solution was
(S)-Diethyl-[O-3,4-isopropylidene-3,4-dihydroxybutyl]phos-
evaporated to near dryness in vacuo. The inorganic material phonate (15a).16 A mixture of 14a (1 g, 4.78 mmol) and triethyl
contained in the residual oil was removed by short column phosphite (4.1 mL, 23.91 mmol) was stirred at 135–150 °C for
chromatography (flash silica). [The column was first packed in 8 h, at which time the excess triethyl phosphite was removed
chloroform followed by elution with pure acetone, and then in vacuo. The residual oil was a mixture of 15a and diethyl
the compound was loaded by dissolving it in MeOH– ethylphosphonate. The S-(−)-diethyl phosphonate 15a has the
CH3COCH3 (1 : 9) followed by elution with a gradual increase same polarity as diethyl ethylphosphate and made chromato-
in percentage of MeOH from MeOH–CH3COCH3 (1 : 9) to graphic separation very cumbersome; therefore the oil was
MeOH–CH3COCH3 (3 : 7)]. Removal of the solvent gave 4.35 g subjected to the next step without purification.
(95%) of 12 as a slightly yellow oil, indicated to be practically
pure by NMR. Rf
0.7 (EtOAc–MeOH–CH3COCH3–H2O, 15b23 from 14b.
7 : 1 : 1 : 1); 1H NMR (400 MHz, CD3OD): δ 3.80–3.71 (m, 3H), (S)-Diethyl-(3,4-dihydroxybutyl)phosphonate (16a).16 A solu-
An identical procedure was followed for the preparation of
=
3.54–3.45 (m, 2H), 1.79–1.71 (m, 1H), 1.66–1.57 (m, 1H); tion of the above oil containing 15a (2.14 g) and p-TsOH·H2O
HRMS (ESI): calcd for C4H10O3 (M + Na)+ 129.0522, found (120 mg) in CH3OH (30 mL) was stirred overnight at room
129.0526.
temperature. NaHCO3 was added to neutralize the solution,
(S)-1,2-O-Isopropylidenebutane-1,2,4-triol (13).16 (S)-1,2,4- and the stirring was continued for another 10 min. The
Butanetriol 12 (4 g, 0.037 mol) was dissolved in acetone mixture was filtered, and the filtrate was concentrated in vacuo.
(100 ml) and a catalytic amount of p-toluenesulfonic acid The residue was dissolved in CHCl3 (10 mL) and filtered
(0.5 g) was added to it. Then the reaction mixture was refluxed through celite. Concentration of the filtrate left a colorless oil,
for 2.5 h. After completion, the reaction mixture was neutral- which was purified by flash chromatography eluting with
ized with triethyl amine and acetone was evaporated to CHCl3–CH3OH (5 : l) to give 1 g (82% over two steps) of 16a as
dryness. The residue was taken up in ethyl acetate and washed a colorless oil. Rf = 0.3 (CHCl3–CH3OH, 9 : 1); 1H NMR
with aqueous solutions of sodium bicarbonate and sodium (400 MHz, CD3OD): δ 4.15–4.06 (m, 4H), 3.63–3.57 (m, 1H),
chloride respectively, and the organic layer was dried over 3.46 (dd, J = 5.6, 1.9 Hz, 2H), 2.08–1.75 (m, 4H), 1.33 (t, J =
MgSO4. After removal of the solvent, flash chromatography 7.1 Hz, 6H); 31P NMR (161 MHz, CD3OD): δ 33.79; HRMS (ESI):
CH3COCH3–CH2Cl2 (2 : 8) of the residue gave 4.79 g (87%) of calcd for C8H19O5P (M + H)+ 227.1043, found 227.1046.
13 as a colorless oil. Rf = 0.3 (hexane–EtOAc, 7 : 3); 1H NMR
An identical procedure followed for the preparation of
(400 MHz, CDCl3): δ 4.31–4.24 (m, 1H), 4.10 (dd, J = 8.0, 16b23 from 15b (yield, 82%). Rf = 0.3 (CHCl3–CH3OH, 9 : 1);
6.0 Hz, 1H), 3.80 (t, J = 5.5 Hz, 2H), 3.60 (t, J = 7.6 Hz, 1H), 1H NMR (400 MHz, CDCl3): δ 4.15–4.10 (m, 5H), 3.67 (dd, J =
2.34 (brs, 1H), 1.85–1.80 (m, 2H), 1.43 (s, 3H), 1.37 (s, 3H); 11.2, 3.2 Hz, 1H), 3.53 (dd, J = 11.3, 5.8 Hz, 1H), 2.10–1.92
HRMS (ESI): calcd for C7H14O3 (M + Na)+ 169.0841, found (m, 2H), 1.33 (t, J = 7.1 Hz, 6H); 31P NMR (161 MHz, CDCl3):
169.0846.
δ 30.05; HRMS (ESI): calcd for C7H17O5P (M + H)+ 213.0886,
found 213.0873.
(S)-1-Iodo-O-isopropylidenebutane-3,4-diol (14a).21 To
a
solution of (S)-1,2-O-isopropylidenebutane-1,2,4-triol 13
(S)-Diethyl 3,4-bis(palmitoyloxy)butylphosphonate (17a).24
(1.46 g, 10 mmol) in toluene (20 mL) were added PPh3 (recrys- To a solution of 16a (0.5 g, 2 mmol), palmitic acid (1.12 g,
tallized from acetone, 3.14 g, 12 mmol), imidazole (2.04 g, 4.38 mmol), and DMAP (56 mg, 0.46 mmol) in anhydrous
30 mmol) and iodine (3.29 g, 13 mmol). The reaction CH2Cl2 (5 mL) was added DCC (0.94 g, 4.56 mmol), and the
mixture was stirred at 90 °C for 2 h. After completion of the resulting mixture was stirred overnight at room temperature.
reaction (confirmed by TLC) toluene was evaporated under After completion of the reaction a white solid was removed by
reduced pressure, the residue was dissolved in CH2Cl2, washed filtration through Celite, and the filtrate was evaporated to
with saturated Na2S2O3 (to quench the unreacted iodine), furnish a white solid, which was purified by flash chromato-
brine and dried over Na2SO4. The solvent was then removed graphy on silica gel using hexanes–EtOAc (1 : l) to afford 1.23 g
in vacuo and the residue was purified by flash column (88%) of 17a as a white solid. Rf = 0.6 (CHCl3–CH3OH, 9 : 1);
chromatography (hexane–ethyl acetate = 49 : 1) to afford 2.71 g 1H NMR (400 MHz, CDCl3): δ 5.12–5.06 (m, 1H), 4.24 (dd, J =
(98%) of compound 14a as a colorless oil. Rf = 0.7 (hexane– 11.8, 3.7 Hz, 1H), 4.14–4.01 (m, 5H), 2.32–2.28 (m, 4H),
1
EtOAc, 9 : 1); H NMR (400 MHz, CDCl3): δ 4.16–4.08 (m, 1H), 1.98–1.68 (m, 4H), 1.65–1.55 (m, 4H), 1.34–1.26 (m, 54H), 0.88
4.02 (dd, J = 7.9, 6.1 Hz, 1H), 3.52 (dd, J = 8.0, 6.5 Hz, 1H), (t, J = 7.1 Hz, 6H); 31P NMR (161 MHz, CDCl3): δ 30.65; 13C
3.21–3.14 (m, 2H), 2.08–1.93 (m, 2H), 1.34 (s, 3H), 1.29 (s, 3H); NMR (100 MHz, CDCl3): δ 173.3, 173.1, 70.8 (d, JCP = 18.1 Hz),
HRMS (ESI): calcd for C7H13IO2 (M + H)+ 257.0033, found 64.2, 61.6 (d, JCP = 6.5 Hz), 34.2, 34.0, 33.9, 31.8, 29.6, 29.6,
257.0036.
29.5, 29.4, 29.4, 29.3, 29.2, 29.2, 29.1, 25.6, 24.9, 24.9, 24.8,
In an identical procedure 14b22 was prepared from 7 (yield, 24.1 (d, JCP = 4.3 Hz), 22.6, 21.6 (d, JCP = 143.8 Hz), 16.4 (d, JCP
98%). Rf = 0.7 (hexane–EtOAc, 9 : 1); 1H NMR (400 MHz, = 6.0 Hz), 14.03; HRMS (ESI): calcd for C40H79O7P (M + H)+
CDCl3): δ 4.31–4.25 (m, 1H), 4.15 (dd, J = 8.6, 6.1 Hz, 1H), 3.79 703.5636, found 703.5602.
1168 | Org. Biomol. Chem., 2014, 12, 1163–1172
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An identical procedure was followed for the preparation of 4.48–4.43 (m, 2H), 4.28–4.23 (m, 2H), 4.15–4.12 (m, 1H), 4.06
1
17b25 from 16b (yield, 88%). Rf = 0.6 (CHCl3–CH3OH, 9 : 1); H (s, 1H), 3.81 (s, 3H), 3.62–3.59 (m, 2H), 3.49 (dd, J = 9.5, 1.8
NMR (400 MHz, CDCl3): δ 5.39–5.30 (m, 1H), 4.37 (dd, J = 11.9, Hz, 1H), 3.45 (dd, J = 9.6, 1.8 Hz, 1H), 3.43–3.39 (m, 1H),
3.2 Hz, 1H), 4.17–4.07 (m, 5H), 2.33–2.28 (m, 4H), 2.13 (dd, J = 3.15–3.11 (m, 1H), 2.07 (s, 3H), 1.96 (s, 3H), 1.83 (s, 3H);
19.0, 6.8 Hz, 1H), 1.61 (t, J = 7.0 Hz, 4H), 1.33–1.26 (m, 54H), HRMS (MALDI): calcd for C54H59N3O14 (M + Na)+ 996.3895,
0.88 (t, J = 7.1 Hz, 6H); 31P NMR (161 MHz, CDCl3): δ 25.45; found 996.3868.
13C NMR (100 MHz, CDCl3): δ 173.2, 172.7, 66.7, 64.6 (d, JCP
=
(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1–6)-
8.8 Hz), 61.0 (t, JCP = 6.1 Hz), 34.3, 34.1, 31.9, 29.7, 29.7, 29.6, 1-O-4-methoxybenzyl-2,3,4,5-tetra-O-benzyl-D-myo-inositol
29.5, 29.4, 29.3, 29.2, 29.1, 28.0, 27.9 (d, JCP = 141.8 Hz), 24.8 (20).29 The preceding pseudo disaccharide 19 (1.36 g,
(d, JCP = 8.8 Hz), 22.7, 16.4 (d, JCP = 6.0 Hz), 14.1; HRMS (ESI): 1.4 mmol) dissolved in a solvent mixture of anhydrous CH2Cl2
calcd for C39H77O7P (M + Na)+ 711.5305, found 711.5332.
(3 mL) and MeOH (12 mL) was treated with a catalytic amount
(S)-[3,4-Bis(palmitoyloxy)butyl]phosphonic acid (18a).26 To of NaOMe. The reaction was stirred for 24 h at room tempera-
a solution of 17a (0.1 g, 0.14 mmol) in anhydrous CH2Cl2 ture. After completion, the reaction was neutralized with a
(1 mL) at room temperature was added slowly over cation-exchange resin, filtered and concentrated. The residue
45 min bromotrimethylsilane (∼150 µL). After the resulting was dissolved in anhydrous DMF (32 mL) and cooled to 0 °C.
mixture was stirred at room temperature for 6 h, the solvent was This was followed by addition of benzyl bromide (2 mL) and
evaporated under reduced pressure. The residue was dissolved NaH (0.6 g). The reaction was stirred at room temperature for
in 10% aqueous THF (1 mL), and the solution was heated at 12 h, after which the reaction was quenched by addition of
reflux for 1 h. The solvent was removed under reduced MeOH (1 mL). The reaction was diluted with ethyl acetate
pressure, and the residue was dissolved in CHC13 (5 mL). The (100 mL), and the organic layer was washed with brine and
CHC13 solution was then dried (Na2SO4), filtered, and concen- water, dried over anhydrous Na2SO4, and concentrated. The
trated under reduced pressure to afford 92 mg (98%) of the residue was purified by silica gel column chromatography
phosphonic acid 18a as a white solid that was utilized in the (hexane–ethyl acetate) to give (1.25 g, 80%) 20. Rf = 0.5
next step without further purification. Rf = 0.1 (CHCl3–CH3OH, (hexane–EtOAc, 7 : 3); [α]D = +41 (c 1.0, CHCl3); 1H NMR
1
9 : 1); H NMR (400 MHz, CDCl3): δ 5.14 (brs, 1H), 4.26 (d, J = (400 MHz, CDCl3): δ 7.44–7.07 (m, 37H), 6.90 (d, J = 8.3 Hz,
9.2 Hz, 1H), 4.04 (brs, 1H), 2.36–2.28 (m, 4H), 1.93–1.71 (m, 2H), 5.79 (d, J = 3.5 Hz, 1H), 5.06–4.99 (m, 2H), 4.92–4.81 (m,
4H), 1.61–1.59 (m, 4H), 1.34–1.26 (m, 48H), 0.88 (t, J = 7.1 Hz, 5H), 4.76–4.66 (m, 4H), 4.57–4.52 (m, 3H), 4.41 (d, J = 11.0 Hz,
6H); 31P NMR (161 MHz, CDCl3): δ 33.66; HRMS (MALDI): 1H), 4.32–4.24 (m, 2H), 4.17 (t, J = 9.6 Hz, 1H), 4.07–3.97 (m,
calcd for C36H71O7P (M + Na)+ 669.4830, found 669.4847.
3H), 3.85 (s, 3H), 3.77–3.69 (m, 1H), 3.53–3.43 (m, 3H),
An identical procedure was followed for the preparation of 3.31–3.14 (m, 3H); HRMS (MALDI): calcd for C69H71N3O11
1
18b27 from 17b (yield, 98%). Rf = 0.1 (CHCl3–CH3OH, 9 : 1); H (M + Na)+ 1140.4986, found 1140.4988.
NMR (400 MHz, CDCl3): δ 5.43–5.41 (m, 1H), 4.34–4.31 (m,
(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-α-D-glucopyranosyl)-(1–6)-
1H), 4.12–4.09 (m, 1H), 2.34–2.29 (m, 4H), 2.15–2.09 (m, 2H), 2,3,4,5-tetra-O-benzyl-D-myo-inositol (21).14b Compound 20
1.58–1.57 (m, 4H), 1.34–1.26 (m, 48H), 0.85 (t, J = 7.1 Hz, 6H); (0.25 g, 0.22 mmol) was dissolved in CH2Cl2–H2O, 99 : 1
31P NMR (161 MHz, CDCl3): δ 28.91; HRMS (ESI): calcd for (40 mL), cooled to 0 °C, treated with DDQ (0.20 g, 0.88 mmol),
C35H69O7P (M − H)− 631.4708, found 631.4707.
and stirred at room temperature for 3 h. The reaction mixture
(3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranosyl)-(1–6)- was quenched with saturated NaHCO3 solution, extracted with
1-O-4-methoxybenzyl-2,3,4,5-tetra-O-benzyl-D-myo-inositol (19).28
CH2Cl2 (3 × 100 mL) and the organic layer was dried over
A
mixture of 1-O-4-methoxybenzyl-2,3,4,5-tetra-O-benzyl-D-myo- sodium sulfate. The solvent was removed under reduced
inositol acceptor 2 (1.74 g, 2.63 mmol) and 3,4,6-tri-O-acetyl-2- pressure and the residue was purified by column chromato-
azido-2-deoxy-α/β-D glucopyranosyltrichloroacetimidate donor graphy (hexane–EtOAc) to afford 21 (0.12 g, 55%). Rf = 0.4
6 (2.1 g, 4.41 mmol), and freshly activated powdered 4 Å mole- (hexane–EtOAc, 7 : 3); [α]D = +45 (c 1.0, CHCl3); 1H NMR
cular sieves were dried by azeotropic removal of residual moist- (400 MHz, CDCl3): δ 7.32–7.00 (m, 35H), 5.37 (d, J = 3.5 Hz,
ure through toluene. The mixture was dissolved in anhydrous 1H), 4.96–4.31 (m, 14H), 4.08–4.02 (m, 2H), 3.99–3.85 (m, 3H),
CH2Cl2 (14 mL), stirred under nitrogen at room temperature 3.65 (t, J = 9.6 Hz, 1H), 3.60–3.48 (m, 2H), 3.43–3.40 (m, 1H),
for 30 min, and then cooled to −78 °C. To the above was 3.30 (t, J = 9.6 Hz, 1H), 3.15 (dd, J = 11.0, 1.8 Hz, 1H), 2.98 (d,
added a solution of TMSOTf (0.6 mL, 0.2 M in CH2Cl2) drop- J = 11.0, 1H); 13C NMR (100 MHz, CDCl3): δ 138.8, 138.6, 138.6,
wise and the mixture was stirred further for 40 min at −78 °C. 138.2, 137.9, 137.9, 137.8, 128.4, 128.4, 128.4, 128.3, 128.3,
After completion, the reaction mixture was neutralized with 128.0, 127.9, 127.8, 127.7, 127.7, 127.7, 127.6, 127.6, 127.5,
triethylamine, filtered through celite and concentrated. The 127.3, 127.3, 98.4, 83.2, 81.7, 81.0, 80.6, 80.5, 78.7, 78.0, 75.7,
silica column chromatography (hexane–ethyl acetate) provided 75.4, 75.3, 74.9, 74.8, 73.4, 73.1, 71.2, 70.6, 68.5, 63.7; HRMS
product 19 (2.3 g, 90%) as a colorless solid. Rf = 0.4 (hexane– (MALDI): calcd for C61H63N3O10 (M + Na)+ 1020.4411, found
EtOAc, 7 : 3); [α]D = +74 (c 1.0, CHCl3); 1H NMR (400 MHz, 1020.4436.
CDCl3): δ 7.40–7.21 (m, 22H), 6.86 (dd, J = 8.7, 2.4 Hz, 2H),
5.79 (d, J = 3.6 Hz, 1H), 5.45–5.40 (m, 1H), 5.14–5.11 (m, 1H), glucopyranosyl)-(1–6)-2,3,4,5-tetra-O-benzyl-1-O-(sn-3,4-di-O-
4.94–4.89 (m, 3H), 4.80–4.69 (m, 3H), 4.66–4.63 (m, 2H), palmitoyl-butyl-1-phosphonate)-D-myo-inositol (22a). To
Triethylammonium (3,4,6-tri-O-benzyl-2-azido-2-deoxy-α-D-
a
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suspension of the phosphonic acid 18a (20 mg, 0.03 mmol)
Triethylammonium (2-amino-2-deoxy-α-D-glucopyranosyl)-
obtained above and glycosyl acceptor 21 (37 mg, 0.037 mmol) (1–6)-1-O-(sn-3,4-di-O-palmitoyl-butyl-1-phosphonate)-D-myo-
in dry pyridine (1 mL) at 60 °C was added trichloroacetonitrile inositol (23a). The protected C-P GPI intermediate 22a (0.02 g,
(500 µL, 5 mmol), and the reaction mixture was stirred at 0.012 mmol) and the catalyst 20% Pd(OH)2 (0.05 g) were dis-
60 °C for 60 h. After completion, the reaction mixture was con- solved in a solvent mixture of MeOH (2 mL), CH2Cl2 (2 mL)
centrated under reduced pressure to afford a pale orange and H2O (0.1 mL). The residual and the dissolved air from the
residue. Purification of the residue by chromatography on flask were removed by repeated evacuations and the reaction
silica gel eluting with CHC13–CH3OH (9 : l) gave 40 mg (77%) mixture was stirred under a hydrogen atmosphere overnight.
of 22a as a white foam. Rf = 0.6 (CH2Cl2–MeOH, 9 : 1); [α]D
=
After completion of the reaction, the mixture was filtered
1
+25.33 (c 0.01, CHCl3); H NMR (400 MHz, CDCl3): δ 7.34–7.20 through a small celite pad, and concentrated under reduced
(m, 35H), 5.67 (d, J = 3.4 Hz, 1H), 5.08–4.98 (m, 3H), 4.93–4.81 pressure. The product was purified by quick filtration through
(m, 4H), 4.78–4.73 (m, 3H), 4.65–4.57 (m, 4H), 4.54–4.51 (m, a silica column using MeOH–CH2Cl2 (1 : 1) to provide the com-
1H), 4.48–4.44 (m, 1H), 4.39–4.32 (m, 1H), 4.13–4.01 (m, 4H), pound 23a (9.5 mg, 80%). Rf = 0.4 (EtOAc–MeOH–CH3COCH3–
3.85–3.75 (m, 3H), 3.65–3.49 (m, 4H), 3.31–3.26 (m, 1H), H2O, 7 : 1 : 1 : 1); 1H NMR (400 MHz, CDCl3–CD3OD–D2O =
2.89–2.87 (m, 6H), 2.21–2.14 (m, 4H), 1.85–1.74 (m, 4H), 4 : 4 : 1): δ 5.47 (m, 1H), 5.17 (m, 2H), 4.34 (m, 2H), 4.15–4.03
1.56–1.46 (m, 4H), 1.31–1.20 (m, 48H), 1.18 (t, J = 7.2 Hz, 9H), (m, 3H), 3.87 (m, 2H), 3.73–3.67 (m, 3H), 3.39–3.36 (m, 2H),
0.88 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, CDCl3): δ 23.48; 3.19 (q, J = 7.0 Hz, 6H), 3.14 (t, J = 5.0 Hz, 1H), 2.34–2.31 (m,
13C NMR (100 MHz, CDCl3): 173.3, 173.2, 139.5, 138.6, 138.6, 4H), 2.00–1.84 (m, 4H), 1.63–1.62 (m, 4H), 1.39–1.27 (m, 57H),
138.5, 138.4, 138.2, 138.1, 128.4, 128.3, 128.3, 128.2, 128.1, 0.89 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, CDCl3–CD3OD–
128.0, 128.0, 127.9, 127.8, 127.8, 127.6, 127.5, 127.4, 127.4, D2O = 4 : 4 : 1): δ 30.39; 13C NMR (100 MHz, CDCl3–CD3OD–
127.0, 126.9, 97.2, 84.2, 82.2, 80.8, 80.8, 80.4 (d, JCP = 6.1 Hz), D2O = 4 : 4 : 1): 175.4, 175.3, 95.4, 77.8, 73.5, 72.7, 72.6, 71.5,
78.2 (d, JCP = 5.7 Hz), 77.6 (d, JCP = 6.2 Hz), 75.7, 75.2, 75.1, 70.5, 70.4, 64.8, 64.7, 62.6, 61.3, 61.2, 54.5, 46.6, 34.3, 29.6
74.9, 73.6, 73.5, 72.3, 71.8 (d, JCP = 14.8 Hz), 71.5, 70.1, 68.3, (multiple peaks), 24.9, 22.7, 22.6, 21.8, 13.5, 8.5; HRMS (ESI):
64.4, 63.7, 45.3, 34.3, 34.3, 34.1, 31.9, 29.7, 29.7, 29.6, 29.5, calcd for C48H92NO16P (M − H)− 968.6081, found 968.6093.
29.5, 29.4, 29.3, 29.3, 29.1, 25.0 (d, J = 4.9 Hz), 24.8, 22.7, 14.1,
8.5; HRMS (MALDI): calcd for C97H132N3O16P (M − H)− (1–6)-1-O-(sn-2,3-di-O-palmitoyl-propyl-1-phosphonate)-D-myo-
1625.9306, found 1625.9333. inositol (23b). The protected C-P GPI intermediate 22b (0.02 g,
Triethylammonium (2-amino-2-deoxy-α-D-glucopyranosyl)-
Triethylammonium (3,4,6-tri-O-benzyl-2-azido-2-deoxy-α-D- 0.012 mmol) and the catalyst 20% Pd(OH)2 (0.05 g) were dis-
glucopyranosyl)-(1–6)-2,3,4,5-tetra-O-benzyl-1-O-(sn-2,3-di-O- solved in a solvent mixture of MeOH (2 mL), CH2Cl2 (2 mL)
palmitoyl-propyl-1-phosphonate)-D-myo-inositol (22b). To
a
and H2O (0.1 mL). The residual and the dissolved air from the
suspension of the phosphonic acid 18b (20 mg, 0.03 mmol) flask were removed by repeated evacuations and the reaction
obtained above and glycosyl acceptor 21 (37 mg, 0.037 mmol) mixture was stirred under a hydrogen atmosphere overnight.
in dry pyridine (1 mL) at 60 °C was added trichloroacetonitrile After completion of the reaction, the mixture was filtered
(500 µL, 5 mmol), and the reaction mixture was stirred at through a small celite pad and concentrated under reduced
60 °C for 60 h. After completion, the reaction mixture was con- pressure. The product was purified by quick filtration through
centrated under reduced pressure to afford a pale orange a silica column using MeOH–CH2Cl2 (1 : 1) to provide the com-
residue. Purification of the residue by chromatography on pound 23b (9.3 mg, 80%). Rf = 0.4 (EtOAc–MeOH–CH3COCH3–
silica gel eluting with CHC13–CH3OH (9 : l) gave 25 mg (50%) H2O, 7 : 1 : 1 : 1); 1H NMR (400 MHz, CDCl3–CD3OD–D2O =
of 22b as a white foam. Rf = 0.6 (CH2Cl2–MeOH, 9 : 1); [α]D
=
4 : 4 : 1): δ 5.44 (m, 2H), 4.47–4.35 (m, 2H), 4.12–3.68 (m, 9H),
1
+28.38 (c 0.01, CHCl3); H NMR (500 MHz, CDCl3): δ 7.40–7.18 3.46–3.35 (m, 2H), 3.19 (q, J = 7.0 Hz, 7H), 2.35–2.33 (m, 4H),
(m, 35H), 5.67 (d, J = 3.4 Hz, 1H), 5.06–4.97 (m, 3H), 4.92 (d, 2.19–2.14 (m, 2H), 1.63–1.62 (m, 4H), 1.38–1.28 (m, 57H), 0.89
J = 3.4 Hz, 1H), 4.85–4.75 (m, 6H), 4.65–4.56 (m, 4H), 4.48–4.44 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, CDCl3–CD3OD–D2O =
(m, 2H), 4.39–4.34 (m, 1H), 4.14–4.00 (m, 5H), 3.83–3.74 (m, 4 : 4 : 1): δ 23.53; 13C NMR (100 MHz, CDCl3–CD3OD–D2O =
3H), 3.65–3.53 (m, 3H), 3.35–3.31 (m, 1H), 2.98 (q, J = 7.0 Hz, 4 : 4 : 1): 174.4, 174.3, 95.1, 77.7, 75.6, 72.9, 72.3, 71.8, 70.4,
6H), 2.20–2.14 (m, 4H), 1.81–1.75 (m, 2H), 1.53–1.51 (m, 4H), 70.0, 66.8, 64.9, 64.8, 60.7, 60.6, 54.1, 46.2, 33.6, 31.8–29.0,
1.25–1.21 (m, 57H), 0.88 (t, J = 7.0 Hz, 6H); 31P NMR (161 MHz, 28.5, 24.3, 22.1, 13.2, 7.9; HRMS (ESI): calcd for C47H90NO16P
CDCl3): δ 16.62; 13C NMR (125 MHz, CDCl3): 173.6, 173.2, (M + H)+ 956.6075, found 956.6042.
138.8, 138.8, 138.6, 138.5, 138.3, 138.2, 138.1, 128.5, 128.4,
128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1,
B. Method for the PI-PLC reaction17–19
128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6, The sample in CHCl3 was evaporated and re-suspended in H2O
127.6, 127.5, 127.5, 127.4, 127.3, 127.2, 127.0, 98.4, 83.0, 82.0, by bath sonication (5 min) such that the final concentration of
81.5, 80.9, 80.5, 78.5, 77.5, 75.5, 75.4, 75.1, 74.9, 74.7, 73.6, the sample was 10 mM. To 5 µL of the sample (10 mM) were
73.3, 72.2, 70.8, 70.1, 68.1, 64.1, 63.32, 45.3, 34.5, 34.4, 34.2, added 5 µL of sodium deoxycholate (0.8%, w/v), 10 µL of
31.9, 29.7, 29.7, 29.6, 29.5, 29.3, 29.2, 29.2, 22.7, 14.1, 8.4; borate buffer [0.1 M (sodium borate/HCl (pH 7.5))] and 4 µL of
HRMS (MALDI): calcd for C96H130N3O16P (M − H)− 1611.9149, enzyme PI-PLC. The reaction mixture was incubated at 37 °C
found 1611.9126.
for 20 minutes and quenched with 200 µL CHCl3–MeOH–
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Conc. HCl (66 : 33 : 1). The CHCl3 layer was then checked for
monitoring the progress of the reaction by TLC and mass
spectrometry and the results are given in the Discussion part
and Fig. 1.
5 T. S. van Zanten, A. Cambi, M. Koopman, B. Joosten,
C. G. Figdor and M. F. Garcia-Parajo, Proc. Natl. Acad.
Sci. U. S. A., 2009, 106, 18557.
6 K. Simons and M. Gerl, Nat. Rev. Mol. Cell Biol., 2010, 11,
688.
7 L. Johannes and S. Mayor, Cell, 2010, 142, 507.
8 (a) J. C. Morris, L. P. Sheng, T.-Y. Shen and K. Mensa-
Willmot, J. Biol. Chem., 1995, 270, 2517; (b) P. Butikofer,
M. Boschung, U. Brodbeck and A. K. Menon, J. Biol. Chem.,
1996, 271, 15533.
9 M. Katan and R. L. Williams, Cell Develop. Biol., 1997, 8,
287.
10 K. G. N. Suzuki, T. K. Fujiwara, M. Edidin and A. Kusumi,
J. Cell Biol., 2007, 177, 731.
11 (a) H.-X. Zhai, P.-S. Lei, J. C. Morris, K. Mensa-Wilmot and
T. Y. Shen, Tetrahedron Lett., 1995, 36, 7403; (b) S. P. Seitz,
R. F. Kaltenbach, R. H. Vreekamp, J. C. Calabreses and
F. W. Perrella, Bioorg. Med. Chem. Lett., 1992, 2, 171;
(c) S. F. Martin and A. S. Wagman, J. Org. Chem., 1996, 61,
8016; (d) V. R. Garigapati and M. F. Roberts, Tetrahedron
Lett., 1993, 34, 5579; (e) K. A. Lewis, V. R. Garigapati,
C. Zhou and M. F. Roberts, Biochemistry, 1993, 32,
8836.
12 (a) M. S. Shashidhar, J. J. Volwerk, J. F. W. Keana and
O. H. Griffith, Biochim. Biophys. Acta (Lipids Lipid Metab.),
1990, 1042, 410; (b) M. Ryan, M. P. Smith, T. K. Vinod,
W. L. Lau, J. F. W. Keana and O. H. Griffith, J. Med. Chem.,
1996, 39, 4366.
C. Method for the PI-PLC reaction and inhibitor reactions17–19
NBD-GPI 26 is added to borate buffer pH 7.5 containing deoxy-
cholate at 2 mM (above CMC concentration to form micelles).
To this a PI-PLC (0.0013 mg mL−1) enzyme is added and
warmed to 37 degrees to initiate the reaction. The reaction is
continued for 10 min for 50% of GPI-NBD to undergo PI-PLC
cleavage. The reaction is terminated by placing the reaction
mixture on ice. The reaction mixture is immediately spotted
on TLC sheets (silica gel 60 coated on glass plates) and run in
the 7 : 1 : 1 : 1 EtOAc–MeOH–Me2CO–H2O solvent system. This
TLC is further imaged in UV to find the concentration of the
product formed in each case. GPI-NBD on PI-PLC cleavage
gives rise to 2 products namely the glycan part and the NBD-
glycerolipid of which the latter will fluoresce in UV. Hence
from the fluorescent bands of reactants (R) and the product (P)
we calculate the percentage of the reaction (P/R × 100). Both
the reactant band and the product band are normalized to the
loading control in each lane.
In the case of inhibition reactions, to the above GPI-NBD
reaction, varying concentrations of the inhibitor (i.e., 7.5 mM
−0.1 mM in case of 23a and 20 mM–0.1 mM in case of 23b)
are added and the reaction carried out at 37 degrees for
10 minutes. For each concentration of the inhibitor added, the
corresponding % of reaction is calculated and this is sub-
tracted from that of the NBD-GPI 26 reaction (control reaction,
without inhibitor) to calculate the % of inhibition.
13 C. Mihai, X. Yue, Li. Zhao, A. Kravchuk, M.-D. Tsaibe and
K. S. Bruzik, New J. Chem., 2010, 34, 925.
14 (a) A. Ali, D. C. Gowda and R. A. Vishwakarma, Chem.
Commun., 2005, 519; (b) R. A. Vishwakarma and
A.
K.
Menon,
Chem.
Commun.,
2005,
453;
(c) R. A. Vishwakarma, S. Vehring, A. Mehta, A. Sinha,
T. Pomorski, A. Herrmann and A. K. Menon, Org. Biomol.
Chem., 2005, 3, 1275; (d) A. Ali and R. A. Vishwakarma,
Tetrahedron, 2010, 66, 4357; (e) R. A. Vishwakarma and
D. Ruhela, Enzymes, 2009, 26, 181; (f) D. Ruhela and
R. A. Vishwakarma, J. Org. Chem., 2003, 68, 4446;
(g) M. Chawla and R. A. Vishwakarma, J. Lipid. Res., 2003,
44, 594; (h) S. Chandra, D. Ruhela, A. Deb and
R. A. Vishwakarma, Expert Opin. Ther. Target, 2010, 14, 739;
(i) V. Saikam, R. Raghupathy, M. Yadav, V. Gannedi,
P. P. Singh, N. A. Qazi, S. D. Sawant and
R. A. Vishwakarma, Tetrahedron Lett., 2011, 52, 4277.
Acknowledgements
The financial support by CSIR research grant # BSC 0108 is
gratefully acknowledged. MY, VS and SD thank CSIR for
Research Fellowships. The authors thank the Instrumentation
Division of CSIR-IIIM for support.
Notes and references
1 M. A. J. Ferguson, S. W. Homans, R. A. Dwek and 15 D. B. Berkowitz, M. Eggen, Q. Shen and D. G. Sloss, J. Org.
T. W. Rademacher, Science, 1988, 239, 753. Chem., 1993, 58, 6174.
2 S. W. Homans, M. A. J. Ferguson, R. A. Dwek, 16 S. F. Martin, Y.-L. Wong and A. S. Wagman, J. Org. Chem.,
T. W. Rademacher, R. Anand and A. F. Williams, Nature,
1988, 333, 269.
1994, 59, 4821.
17 H. Ikezawa and R. Taguchi, Methods Enzymol., 1981, 71,
3 For a comprehensive review on the structure and bio-
731.
synthesis of GPI anchors see: M. J. McConville and 18 O. H. Griffith, J. J. Volwerk and A. Kuppe, Methods
M. A. J. Ferguson, Biochem. J., 1993, 294, 305. Enzymol., 1991, 197, 493.
4 D. Goswami, K. Gowrishankar, S. Bilgrami, S. Ghosh, 19 M. Ryan, M. P. Smith, T. K. Vinod, W. L. Lau,
R. Raghupathy, R. Chadda, R. A. Vishwakarma, M. Rao and
J. F. Keana and O. H. Griffith, J. Med. Chem., 1996, 39,
S. Mayor, Cell, 2008, 135, 1085.
4366.
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Paper
Organic & Biomolecular Chemistry
20 H. A. Orgueira, A. Bartolozzi, P. Schell, R. E. J. N. Litjens, 24 S.-K. Chung, K.-S. Chang, B. E. Kim and S. H. Ryu, Korean
E. R. Palmacci and P. H. Seeberger, Chem.–Eur. J., 2003, 9,
J. Med. Chem., 1993, 3, 33.
140.
25 P. Mituła and C. Wawrzeńczyk, ARKIVOC, 2012, 4, 216.
21 K. Mori and H. Watanabe, Tetrahedron, 1986, 42, 26 L. Qiao, F. Nan, M. Kunkel, A. Gallegos, G. Powis and
295. A. P. Kozikowski, J. Med. Chem., 1998, 41, 3303.
22 W. Wu, R. Li, S. S. Malladi, H. J. Warshakoon, 27 I. L. Doerr, J.-C. Tang, A. F. Rosenthal, R. Engel and
M. R. Kimbrell, M. W. Amolins, R. Ukani, A. Datta and
S. A. David, J. Med. Chem., 2010, 53, 3198.
23 B. N. Thomas, C. M. Lindemann, R. C. Corcoran,
B. E. Tropp, Chem. Phys. Lipids, 1977, 19, 185.
28 K. Pekari, D. Tailler, R. Weingart and R. R. Schmidt, J. Org.
Chem., 2001, 66, 7432.
C. L. Cotant, J. E. Kirsch and P. J. Persichini, J. Am. Chem. 29 S. Cottaz, J. S. Brimacombe and M. A. J. Ferguson, Carbo-
Soc., 2002, 127, 1227.
hydr. Res., 1995, 270, 85.
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