Synthesis of a Glycosylphosphatidylinositol Anchor Derived from Leishmania donovani That Can Be Functionalized by Cu-Catalyzed Azide-Alkyne Cycloadditions

Article abstract of DOI:10.1021/acs.orglett.7b01703

A flexible assembly strategy has been developed for the synthesis of Leishmania donovani GPI anchors that bear a clickable alkyne tag. This strategy is based on the use of the 2-naphthylmethyl (Nap) ethers and levulinoyl (Lev) ester for permanent protection of hydroxyls. Removal of seven Nap ethers by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone made it possible to prepare GPIs having an alkyne functionality that could be modified by Cu(I)-catalyzed [3 + 2] cycloadditions to install tags for imaging studies.

Full text of DOI:10.1021/acs.orglett.7b01703

Letter
pubs.acs.org/OrgLett
Synthesis of a Glycosylphosphatidylinositol Anchor Derived from
Leishmania donovani That Can Be Functionalized by Cu-Catalyzed
Azide−Alkyne Cycloadditions
Ning Ding,†,‡ Xiuru Li,‡ Zoeisha S. Chinoy,‡,§ and Geert-Jan Boons*,‡,§,∥
†Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
§
‡Complex Carbohydrate Research Center and Department of Chemistry, The University of Georgia, 315 Riverbend Road, Athens,
Georgia 30602, United States
∥Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, and Bijvoet Center for
Biomolecular Research, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
S
* Supporting Information
ABSTRACT: A flexible assembly strategy has been developed for the synthesis of Leishmania donovani GPI anchors that bear a
clickable alkyne tag. This strategy is based on the use of the 2-naphthylmethyl (Nap) ethers and levulinoyl (Lev) ester for
permanent protection of hydroxyls. Removal of seven Nap ethers by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone made it possible
to prepare GPIs having an alkyne functionality that could be modified by Cu(I)-catalyzed [3 + 2] cycloadditions to install tags for
imaging studies.
he protozoan parasite Leishmania donovani causes fatal
visceral leishmaniasis, which afflicts millions of people
anchor motif, which is highly conserved among all the Leishmania
LPGs.
T
worldwide.1 All parasites of the Trypanosomatid family,
including Leishmania, express a highly complex lipophosphogly-
can (LPG),2 which plays critical pleiotropic roles in parasite
survival and infectivity in both the sandfly vector and the
mammalian host. LPG circumvents the lysis of the parasite by the
host complement system.3 It facilitates intracellular parasite
survival by interfering with the pro-inflammatory host cell
responses via binding of Toll-like receptor (TLR) 2 and 4 of
macrophages and NK cells.4 It also blocks the assembly of
NADPH oxidase and prevents recruitment of proton ATPases at
the phagosomal membrane.5 This function has been attributed to
the localization of LPG at the membrane of the Leishmania-
containing phagosome.
Here, we report a facile strategy for the chemical synthesis of a
GPI anchor derived from L. donovani (e.g., compound 1a)
bearing an alkyne tag, which facilitated the attachment of various
tags such as fluorophores (e.g., compound 1b).6 The GPI anchor
domain of 1 has an unusual structure and consists of an alkyl
phosphatidylinositol having a single saturated aliphatic chain. It is
expected that the synthetic compounds will make it possible to
establish correlations between molecular structure and the
intracellular localization of LPG in host cells. Another emerging
area of research is “GPIomics”, which seeks to develop proteomic
tools for identifying proteins carrying a GPI anchor as a
posttranslational modification. Such research also requires
tagged GPI derivatives.7 Well-defined GPI fragments will also
make it possible to establish which domains are responsible for
modulation of TLR responsiveness.8
A priority for understanding the disease process of L. donovani
is to elucidate the structure−activity relationship of LPG. This
membrane-bound glycoconjugate is composed of four distinct
domains including a phosphatidylinositol lipid anchor, a
phosphosaccharide core, a repeating phosphorylated saccharide
region, and a terminating oligosaccharide cap.1 The first two
domains are also termed the glycosylphosphatidylinositol (GPI)-
Received: June 6, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01703
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Organic Letters
Letter
The preparation of compounds such as 1a (Scheme 1) is
challenging because benzyl ethers, which are commonly
We report here a strategy for the preparation of GPI anchor
derived pseudo-oligosaccharides, such as 1a, employing Nap
ethers and Lev esters for permanent protection. Surprisingly, as
many as seven Nap ethers could be cleanly removed by oxidation
with DDQ using an appropriate solvent mixture. Lev esters were
used for permanent protection of mannosyl donors and have as
attractive features that when present at C-2 can perform
neighboring group participation to selectively introduce 1,2-
trans glycosides, while they can be removed under neutral
conditions using hydrazine acetate14 without affecting fatty acids
that are commonly part of GPI structures. Furthermore, PMB,
allyl (All), and tert-butyldiphenylsilyl (TBDPS) ethers were used
as a set of temporary protecting groups that could be selectively
removed at a later stage of the synthesis to allow further
functionalization with phosphate diesters. On the basis of these
considerations, target compound 1a was assembled from
monosaccharide building blocks 7−11 (Scheme 1).
Scheme 1. Retrosynthetic Analysis of the Target GPI 1a
The assembly of 1a commenced with the synthesis of the α-
linked pseudo-disaccharide 12 (Scheme 2), which required the
Scheme 2. Synthesis of Pseudo-Disaccharide 5
preparation of an optically pure inositol derivative and the
formation of an α-glycosidic bond between glucosamine and
inositol, which is notoriously difficult due to the formation of
anomeric mixtures.8 To address this challenge, inositol derivative
7 was prepared using an optimized method described by Fraser-
Reid and co-workers15 (see the SI for details). To achieve a
glycosylation with acceptable α-selectivity, 2-azido-2-deoxy-D-
glucosyl donor 8 was employed in view of possible remote
participation of the Lev ester at C-6.16 First, the glycosylation of
inositol derivative 7 (see the SI) with glucosaminyl imidate 8 (see
the SI) was carried out in the presence of catalytic TBSOTf in
DCM at −30 °C, and while the pseudo-disaccharide 12 could be
isolated in acceptable yield (74%), the anomeric outcome was
unfavorable (α/β = 1.0/1.3). The use of diethyl ether as the
reaction solvent did not substantially improve the stereoselective
outcome of the glycosylation (α/β = 1/1); however, by
employing toluene/1,4-dioxane (1:3 v/v),17 the pseudo-
disaccharide 12 was isolated in 84% yield mainly as the α-
anomer (α/β = 4/1). After removal of the Fmoc protecting
group using triethylamine, the anomers could readily be
separated by silica gel column chromatography to give
anomerically pure 5. The glycosylation and Fmoc removal
could be carried out as a one-pot, two-step procedure allowing
the preparation of pseudo-disaccharide 5 in multigram
quantities.
employed as permanent protecting groups for the preparation
of GPI anchors,8 are not compatible with the presence of the
alkyne moiety. Benzoyl esters have been used as a permanent
protecting group for GPI synthesis.9 However, such a protecting
group reduces glycosyl donor and acceptor reactivities, and their
removal is not compatible with the presence of fatty esters. Guo
and co-workers have pioneered the use of p-methoxybenzyl
(PMB) ethers for the permanent protection of alcohols of GPI
intermediates.6d,10 Multiple PMB ethers could be removed by
treatment with trifluoroacetic acid (TFA) in DCM, and these
conditions were compatible with the presence of unsaturated
functionalities. This strategy did, however, not use glycosyl
donors having a C-2 ester for controlling 1,2-trans anomeric
selectivities in glycosylations.11 Furthermore, PMB ethers are
rather fragile in acid-catalyzed glycosylations, which may cause
difficulties when highly complex compounds need to be
assembled. The 2-naphthylmethyl (Nap) ether is emerging as
an attractive alternative of the PMB ether12 and recently was
employed as a permanent protecting group in the synthesis of a
GPI pseudo-disaccharide.13 In this case, removal was accom-
plished under acidic conditions using TFA in toluene.
Compounds such as 1a, which contain acid-sensitive glycosidic
linkages, are not stable to treatment under moderately strong
acidic conditions, and hence, this approach does not provide a
reliable entry into compounds such as 1a.
Monosaccharides 9−11 (see the SI for details) were employed
for the assembly of trisaccharide donor 6 (Scheme 3).
Compound 10 was used as a versatile key intermediate that
would make it possible to prepare various GPIs having a
substituent at the C-6 position of Man-II. The glycosylation of 10
with anomeric acetate 9 proceeded smoothly using SnCl4 as the
promoter in DCM to give disaccharide 13 in good yield.
Thioglycoside 13 was converted into N-phenyl trifluoracetimi-
B
DOI: 10.1021/acs.orglett.7b01703
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Scheme 3. Synthesis of the Saccharide Scaffold 2
Scheme 4. Synthesis of the Target GPI Anchor 1a
date 15 by treatment with a mixture of NIS and TFA in wet DCM
followed by reaction of the resulting lactol with 2,2,2-trifluoro-N-
ether of 2 exposed a hydroxyl group that could be esterified with
fatty acids to give entry into a range of important GPIs.2 Target
GPI derivative 1 does, however, not contain a lipid at this
position, and therefore, the PMB ether was cleaved with 5% TFA
in DCM and the resulting hydroxyl temporarily protected as an
acetyl ester by treatment with acetic anhydride in pyridine to give
18. Next, the allyl ether of 18 was removed by a two-step
procedure involving isomerization with [Ir(COD)(PMePh2)2]-
PF6 that was activated by H2, followed by cleavage of the resulting
vinyl ether by employing HgO/HgCl2 in acetone/H2O19 to give
19 in 88% yield. Installation of the phospholipid was achieved by
using H-phosphonate 320 (see the SI for details), which was
activated with pivaloyl chloride (PivCl) followed by in situ
oxidation with I2 to furnish glycolipid 20 in 89% yield.
Subsequent removal of the TBDPS group by treatment with
diluted HF·pyridine gave alcohol 21 (83%), which was modified
by an alkynyl phosphate by coupling with H-phosphonate 4 (see
the SI) in the presence of PivCl followed by in situ oxidation with
I2 to give lipidated 22 as the triethylammonium salt (72%).
The deprotection sequence commenced with the reduction of
the azide using 1,3-propanedithiol in the presence of Et3N21 to
give the corresponding amine. Oxidative removal of the seven
phenylacetimidoyl chloride in the presence of Cs2CO3.18
A
TMSOTf-mediated glycosylation of 15 with 11 in DCM at −20
°C gave trisaccharide 16 as only the α-anomer due to the
presence of the Lev ester at C-2 of the glycosyl donor.
Compound 16 was converted into N-phenyl trifluoracetimidate
6 using a standard two-step procedure.
The glycosylation of pseudo-disaccharide 5 with 6 was
challenging due to the low reactivity of the C-4 hydroxyl of the
glucosamine moiety. A glycosylation using TMSOTf (0.1 equiv)
as the activator in DCM at 0 °C did not provide the desired
product but resulted in the hydrolysis of the donor along with
partial loss of the PMB protecting group of the acceptor. A lower
reaction temperature (−50 °C) avoided the PMB loss, but the
yield of 2 was modest (47%). Fortunately, the use of TBSOTf
(0.1 equiv)6d as the promoter in toluene/1,4-dioxane (1:3, v/
v)17 at 0 °C gave the desired pseudo-pentasaccharide 2 in 84%
yield.
Next, attention was focused on the further modification of 2
with phosphodiesters at C-2 of the inositol moiety and C-6 of the
distal mannoside (Scheme 4). Selective cleavage of the PMB
C
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Nap ethers was carried out in chloroform in the presence of
excess of DDQ, and gratifyingly, the reaction was fast and
efficient and provided the expected glycolipid product. The latter
compound was treated with sodium methoxide in DCM/MeOH
(0.1 mol/L) to remove the remaining ester protecting groups to
give target compound 1a in an overall yield of 65% over three
steps. Alternatively, the Lev esters could be cleaved with
hydrazine acetate while leaving other esters intact, and such an
approach may give entry to GPI anchors having base-sensitive
fatty acids.
To demonstrate that the alkynyl moiety of GPI 1a could be
modified by probes, azide−AlexaFluor 488 was conjugated with
1a by a Cu(I)-catalyzed [3 + 2] cycloaddition under standard
conditions to provide 1b (Scheme 5), which could potentially be
used for imaging studies.
Special Project for “Significant New Drugs Development”
(2017ZX09101-004-006-008) to N.D. and the National Institute
of General Medical Sciences (P01GM107012) from the US
National Institutes of Health to G.J.B.
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clickable alkyne tag (1a) has been accomplished using Nap
ethers and Lev esters as permanent hydroxyl protecting groups.
This approach allowed the incorporation of functionalities such
as alkynes that are incompatible with the use of benzyl ethers for
permanent protection. By using the Cu(I)-catalyzed [3 + 2]
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b01703.
Experimental procedures and NMR spectra for the
synthesis of all new compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
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*E-mail: gjboons@ccrc.uga.edu, G.J.P.H.Boons@uu.nl.
ORCID
Geert-Jan Boons: 0000-0003-3111-5954
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by the China Scholarship Council
(CSC) and the National Major Scientific and Technological
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DOI: 10.1021/acs.orglett.7b01703
Org. Lett. XXXX, XXX, XXX−XXX