Regioselective phosphorylation of: Myo -inositol with BINOL-derived phosphoramidites and its application for protozoan lysophosphatidylinositol

Article abstract of DOI:10.1039/c6ob01062h

A regioselective phosphorylation method for myo-inositol was developed by utilizing readily preparable BINOL-derived phosphoramidites. The method also facilitated the complete separation of the diastereomeric products by simple chromatography. Based on this phosphorylation and Ni-catalyzed alkyl-alkyl cross-coupling reaction for long fatty acids, we achieved the first synthesis of a lysophosphatidylinositol, EhPIa having long fatty acid C30:1, as a partial structure of glycosylphosphatidylinositol (GPI) anchor from the cell membrane of a protozoa, Entamoeba histolytica.

Full text of DOI:10.1039/c6ob01062h

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Regioselective phosphorylation of myo-inositol
with BINOL-derived phosphoramidites
and its application for protozoan
lysophosphatidylinositol†
Cite this: DOI: 10.1039/c6ob01062h
Received 16th May 2016,
Accepted 7th June 2016
Toshihiko Aiba,a,b Masaki Sato,a Daichi Umegaki,a Takanori Iwasaki,c
DOI: 10.1039/c6ob01062h
www.rsc.org/obc
Nobuaki Kambe,c Koichi Fukase*a and Yukari Fujimoto*a,b
A regioselective phosphorylation method for myo-inositol was have been shown to stimulate natural killer T (NKT) cells and
developed by utilizing readily preparable BINOL-derived phos- to induce selective production of IFN-γ but not IL-4 in a CD1d-
phoramidites. The method also facilitated the complete separation restricted manner in murine systems in the case of EhPIb.1 It
of the diastereomeric products by simple chromatography. Based has also been shown that EhLPPG has interesting biological
on this phosphorylation and Ni-catalyzed alkyl–alkyl cross-coup- activities, which induces characteristic selective IFN-γ pro-
ling reaction for long fatty acids, we achieved the first synthesis of duction influenced by the host’s testosterone.2 We are particu-
a lysophosphatidylinositol, EhPIa having long fatty acid C30:1, as a larly interested in the structure of its inositol phospholipid
partial structure of glycosylphosphatidylinositol (GPI) anchor from moieties, which have a characteristic lysophosphatidylinositol
the cell membrane of a protozoa, Entamoeba histolytica.
structure acylated by a long-chain fatty acid, although the
2′ position configuration of the glycerol moiety was not clearly
determined by spectroscopic data and was inferred based on
the previously reported configurations of similar types of pro-
tozoan glycerol moiety, as the sn-1 position of the glycerol is
connected to a long fatty acid chain, as observed in 1a.1 We
therefore synthesized both EhPIa stereoisomers to elucidate
the further detailed biological activities, including the struc-
ture–activity relationships at the configuration of the glycerol
moiety. Although numerous syntheses of phosphatidylinositol
and phosphoinositides have been reported,3 only a few
examples of lysophosphatidylinositol synthesis have been
reported.4,5 The characteristic lysophosphatidylinositol struc-
ture of EhPIa has characteristic long fatty acids, such as C28:0
and C30:1, and it is also necessary to introduce a monophos-
phate to a certain position of the inositol moiety. Controlling
selective phosphorylation is one of the fundamental issues in
biomolecules, including carbohydrates, peptides/proteins and
lipids, because of the importance of the site-specific phos-
phorylation for many physiological events.6 There have thus
been some investigations reported of the various roles of phos-
phates in natural7–9 and designed molecules.10,11
Entamoeba histolytica is a protozoan parasite that causes the
disease amebiasis and amebic liver abscess (ALA) in humans.
Its cell surface contains a complex glycoconjugate, lipopeptido-
phosphoglycan (EhLPPG), which is composed of a glycosylphos-
phatidylinositol (GPI) anchor and the inositol phospholipid
moieties, EhPIa and EhPIb (Fig. 1). The inositol phospholipids
Fig. 1 Inositol phospholipids, EhPIa and EhPIb, from Entamoeba
histolytica.
In the present study, we developed two key reactions for the
synthesis of these inositol phospholipids; Ni-catalyzed alkyl–
alkyl cross-coupling reaction and regioselective phosphoryl-
ation of myo-inositol using binaphthyl phosphoryl/phosphora-
midite reagents. In many phosphatidylinositol syntheses,
including the abovementioned lysophosphatidylinositol syn-
thesis, one of main methods for introducing a phosphate
group is that myo-inositol was first desymmetrized through
aDepartment of Chemistry, Graduate School of Science, Osaka University,
1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
E-mail: fujimotoy@chem.keio.ac.jp
bDepartment of Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
cDepartment of Applied Chemistry, Graduate School of Engineering, Osaka
University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ob01062h
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formation of diastereomeric derivatives.12–14 A phosphoryl group
was subsequently transferred to the liberated hydroxy group.15
Miller and co-workers reported the regioselective phosphoryl-
ation using chiral peptide catalysis.16,17 Utilizing molecular
catalysts designed for those particular inositol moieties, the
reaction displayed high selectivity. It has also been reported
that chiral inositol derivative syntheses were enabled with
using a commercially available chiral inositol building block,
2,3:5,6-bis-O-(1-methylethylidene)-D-myo-inositol 1,4-dibutano-
ate, as the starting material,18,19 although the compound is
quite costly and the protecting groups are not always con-
venient for the subsequent synthesis. Jessen and co-workers
reported examples of desymmetrization of meso-inositol
derivatives using a chiral phosphoramidite reagent containing
mandelic acid derivative as the chiral auxiliary. However, no
selectivity was obtained in the earlier reports20,21 and then
higher selectivity (1.0 : 2.5, in 66% yield) was reported in their
latest research using pentafluorophenol (PFP)–phosphite tri-
mester for higher reactivity.22
Scheme 1 Synthesis of acyl glycerol 7a and 7b via Ni catalyzed alkyl–
alkyl cross-coupling reaction.
In the present study, we used BINOL as the chiral auxiliary
for the phosphoramidite reagent for desymmetrization of
meso-inositol. The reagent was readily prepared with the usual
phosphoramidite synthesis and can be selectively cleaved with
using simple cleavage conditions for the aryl phosphate
linkage, while maintaining the alkyl phosphate linkage. It was
also advantageous that BINOL recycling was relatively easy,
which would be important in the case of a larger synthesis
scales. In this study, concerning EhPIa synthesis, highly enan-
tiomerically pure compounds are necessary for the biological
studies. We thus developed the method for the regioselective
phosphorylation using the chiral phosphorylation reagents
with BINOL as the auxiliary, which facilitated the selective
phosphorylation and also the complete separation of the dia-
stereomeric products by simple chromatography.
Table 1 Phosphorylation of myo-inositol with liberated 1,3,5-hydroxy
groups using BINOL-containing phosphorylation reagents
To prepare the characteristic long fatty acids, a Ni-catalyzed
alkyl–alkyl cross-coupling reaction was also developed,23 and
applied to the total synthesis of EhPIa. To prepare the acyl-
glycerol 7a and 7b (Scheme 1), the long fatty acid derivative 4
was synthesized with a Ni-catalyzed alkyl–alkyl cross-coupling
reaction using bromide 2 and Grignard reagent 3 to obtain 4
with a 64% yield. Due to the very low solubility of the Grignard
reagent 3 at lower temperature, the coupling reaction was per-
formed through a reverse-addition procedure described in the
previous report.23 After cleaving the tBu ester, condensation
with the alcohol 6a and 6b was carried out. The TBDPS group
was subsequently cleaved with TBAF to generate the acyl gly-
cerols 7a and 7b.
Product, yield,
Entry Reagent (eq.)
Time dr(1-P : 3-P)a
1
2
3
4
5
11 (5.0), Et3N (2.0), DMAP (0.5) 1 d
12 (5.0), tetrazole (5.0)
12 (3.0), HOBt (6.0)
12 (3.0), CF3-HOBt (6.0)
13 (5.0), tetrazole (5.0)
9a, 40%, dr (50 : 50)
1 d
12 h
1 h
9a, 74%, dr (79 : 21)b
9a, 79%, dr (67 : 33)
9a, 53%, dr (75 : 25)
2.5 h 10a, 49%,c dr (90 : 10)
a Determined by HPLC analysis. b Determined by NMR analysis.
To enable the selective monophosphorylation of myo-inosi-
tol with three non-protected hydroxy groups of compound 8,24
BINOL-containing chiral phosphorylation reagents were
designed. Phosphorylation using (R)-BINOL-phosphoryl chlor-
ide (11)25 with several myo-inositol derivatives under various
c Contains ca. 50% of diphosphate.
reaction conditions obtained unsatisfactory results; e.g., the 74% yield with appropriate selectivity (1-P : 3-P = 79 : 21) with
conditions shown in Table 1 (entry 1) obtained no selectivity. (R)-BINOL. When the reaction was continued for more than
We then used the phosphoramidite reagent with (R)-BINOL one day, the 1,3-bisphosphorylated compound quantity was
(12)26 using tetrazole as the additive (entry 2) to obtain 9a in gradually increased, which is shown in entry 2.
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To increase the reactivity, the additive was changed to groups of benzyl phosphate was cleaved with LiBr in acetone.30
HOBt.27 The reaction was faster than with tetrazole, but the Acyl glycerol (7a or 7b) was then introduced using the Mitsu-
selectivity was somewhat decreased (1-P : 3-P = 67 : 33) (entry nobu reaction31 to obtain compound 17a and 17b with 31%
3). HOBt-CF3 was used to further investigate the additive. and 81% yields (for the two steps), respectively. The TBS
HOBt-CF3 enhanced the reactivity but the yield was low (53%) groups were cleaved with TBAF to obtain 18a in 88% yield and
due to the additional phosphorylation (entry 4). In the case of 18b quantitatively. Final deprotection of all protecting groups
the phosphoramidite reagent having (R)-H8-BINOL (13) and with TMSBr and BSTFA and then 30% TFA in CH2Cl2 led to
using tetrazole as the additive (entry 5), the reactivity and successful production of the desired inositol phospholipid 1a
selectivity was higher (1-P : 3-P = 90 : 10) than the (R)-BINOL and 2′-epimer 1b (43% for 1a and 31% for 1b).
(12) (entry 2), but additional phosphorylation also proceeded
In conclusion, we developed a regioselective phosphoryl-
and the diphosphates were difficult to separate. Therefore, the ation method for myo-inositol by utilizing readily preparable
condition of entry 2 was used for the following EhPIa total syn- BINOL-derived phosphoramidites, which led to higher selecti-
thesis. The configuration of 9a was determined by cleavage of vity for a desymmetrization method obtained to date. The
protecting groups obtaining the enantiomeric inositol-1-phos- obtained diastereomeric mixture was separable by standard
phate for which the optical rotation is known28 (see ESI†).
chromatography and the chiral auxiliary, BINOL, could be
The total EhPIa synthesis is shown in Scheme 2. First, two easily recycled. We also developed the Ni-catalyzed alkyl–alkyl
hydroxyl groups of phosphorylated inositol 9 (9a : 9b = 79 : 21) cross-coupling reaction for preparing long-chain fatty acids.
were protected with allyloxycarbonyl (alloc) groups for easier Based on these phosphorylation and alkyl–alkyl cross-coupling
separation using SiO2 middle-pressure column chromato- reactions, we achieved the first synthesis of immunomodula-
graphy. The protecting group was subsequently cleaved with tory lysophosphatidylinositols, EhPIa and its epimer, as the
Ru complex29 to obtain compound 9a as the single isomer. partial structure of the glycosylphosphatidylinositol (GPI)
After protecting the hydroxyl groups with TBS, BINOL was anchor from the cell membrane of a protozoa, Entamoeba his-
exchanged with the benzyl group to obtain 16 with 63% yield. tolytica. The synthetic methods would be also applicable for
The cleaved BINOL could be easily recycled. One of the benzyl various types of inositol phospholipids.
We wish to acknowledge Prof. Masato Kitamura and
Dr Shinji Tanaka for providing the Ru complex. This work was
supported in part by Grants-in-Aid for Scientific Research (no.
26282211 and 26102732) from the Japan Society for the
Promotion of Science, by ERATO Murata Lipid Active Structure
Project, by a funding program for Next Generation World-
Leading Researchers (NEXT Program; LR025) from JSPS
and CSTP, by Keio Gijuku Academic Development Funds,
by Yamada Science Foundation, and by The Sumitomo
Foundation.
Notes and references
1 H. Lotter, N. Gonzalez-Roldan, B. Lindner, F. Winau,
A. Isibasi, M. Moreno-Lafont, A. J. Ulmer, O. Holst,
E. Tannich and T. Jacobs, PLoS Pathog., 2009, 5, e1000434.
2 H. Lotter, E. Helk, H. Bernin, T. Jacobs, C. Prehn,
J. Adamski, N. Gonzalez-Roldan, O. Holst and E. Tannich,
PLoS One, 2013, 8, e55694.
3 M. D. Best, H. Zhang and G. D. Prestwich, Nat. Prod. Rep.,
2010, 27, 1403–1430.
4 C. Murakata and T. Ogawa, Tetrahedron Lett., 1991, 32, 101–
104.
5 E. Filthuth and H. Eibl, Chem. Phys. Lipids, 1992, 60, 253–261.
6 F. H. Westheimer, Science, 1987, 235, 1173–1178.
7 S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223–
2311.
8 P. D’Arrigo and S. Servi, Molecules, 2010, 15, 1354–1377.
9 P. Siman and A. Brik, Org. Biomol. Chem., 2012, 10, 5684–5697.
Scheme 2 Synthetic scheme for the inositol phospholipid, EhPIa. Com-
10 K. Panigrahi, M. Eggen, J. H. Maeng, Q. R. Shen and
pound 9 was a mixture of 1- and 3-phosphates in the ratio of 79 : 21,
and the diastereomer was removed while purifying compound 14.
D. B. Berkowitz, Chem. Biol., 2009, 16, 928–936.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
11 K. Panigrahi, D. L. Nelson and D. B. Berkowitz, Chem. Biol., 21 S. Capolicchio, H. C. Wang, D. T. Thakor, S. B. Shears and
2012, 19, 666–667. H. J. Jessen, Angew. Chem., Int. Ed., 2014, 53, 9508–9511.
12 P. J. Garegg, T. Iversen, R. Johansson and B. Lindberg, Car- 22 M. Duss, S. Capolicchio, A. Linden, N. Ahmed and
bohydr. Res., 1984, 130, 322–326. H. J. Jessen, Bioorg. Med. Chem., 2015, 23, 2854–2861.
13 K. S. Bruzik and G. M. Salamonczyk, Carbohydr. Res., 1989, 23 T. Iwasaki, K. Higashikawa, V. P. Reddy, W. W. S. Ho,
195, 67–73.
Y. Fujimoto, K. Fukase, J. Terao, H. Kuniyasu and
N. Kambe, Chem. – Eur. J., 2013, 19, 2956–2960.
24 A. M. Riley and B. V. L. Potter, J. Org. Chem., 1995, 60,
4970–4971.
14 P. J. Ga-regg, B. Lindberg, I. Kvarnström and
S. C. T. Svensson, Carbohydr. Res., 1988, 173, 205–216.
15 G. M. Salamonczyk and K. M. Pietrusiewicz, Tetrahedron
Lett., 1991, 32, 4031–4032.
25 N. Kato, J. Am. Chem. Soc., 1990, 112, 254–257.
16 B. R. Sculimbrene and S. J. Miller, J. Am. Chem. Soc., 2001, 26 A. H. M. deVries, A. Meetsma and B. L. Feringa, Angew.
123, 10125–10126. Chem., Int. Ed. Engl., 1996, 35, 2374–2376.
17 P. A. Jordan, K. J. Kayser-Bricker and S. J. Miller, Proc. Natl. 27 A. Ohkubo, Y. Kuwayama, T. Kudo, H. Tsunoda, K. Seio
Acad. Sci. U. S. A., 2010, 107, 20620–20624. and M. Sekine, Org. Lett., 2008, 10, 2793–2796.
18 M. Mentel, V. Laketa, D. Subramanian, H. Gillandt and 28 T. Akiyama, N. Takechi, S. Ozaki and K. Shiota, Bull. Chem.
C. Schultz, Angew. Chem., Int. Ed., 2011, 50, 3811–
3814.
19 M. X. Wu, L. S. Chong, S. Capolicchio, H. J. Jessen,
Soc. Jpn., 1992, 65, 366–372.
29 M. Kitamura, S. Tanaka and M. Yoshimura, J. Org. Chem.,
2002, 67, 4975–4977.
A. C. Resnick and D. Fiedler, Angew. Chem., Int. Ed., 2014, 30 N. O. Mahmoodi, Phosphorus, Sulfur Silicon Relat. Elem.,
53, 7192–7197. 2002, 177, 2887–2893.
20 S. Capolicchio, D. T. Thakor, A. Linden and H. J. Jessen, 31 Y. Xu, B. R. Sculimbrene and S. J. Miller, J. Org. Chem.,
Angew. Chem., Int. Ed., 2013, 52, 6912–6916.
2006, 71, 4919–4928.
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