Toward the Development of the Next Generation of a Rapid Diagnostic Test: Synthesis of Glycophosphatidylinositol (GPI) Analogues of Plasmodium falciparum and Immunological Characterization

Article abstract of DOI:10.1021/acs.bioconjchem.6b00542

A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols (GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium falciparum GPIs have been considered as potential vaccine candidates be

Full text of DOI:10.1021/acs.bioconjchem.6b00542

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Article
Towards the Development of next generation of Rapid Diagnostic
Test: Synthesis of Glycophosphatidylinositol (GPI) analogues
of Plasmodium falciparum and immunological characterization
Bharat P Gurale, Yun He, Xikai Cui, Hieu Dinh, Abasaheb N. Dhawane,
Naomi W Lucchi, Udhayakumar Venkatachalam, and Suri S. Iyer
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00542 • Publication Date (Web): 28 Oct 2016
Downloaded from http://pubs.acs.org on October 30, 2016
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Towards the Development of next generation of
Rapid Diagnostic Test: Synthesis of
Glycophosphatidylinositol (GPI) analogues of
Plasmodium falciparum and immunological
characterization
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Bharat P. Gurale1, Yun He2, Xikai Cui1, Hieu Dinh1, Abasaheb N.
Dhawane1, Naomi W. Lucchi3, Udhayakumar Venkatachalam3 and Suri S.
Iyer1 *
1 788 Petit Science Center, Department of Chemistry, Center for Diagnostics and
Therapeutics, Georgia State University, Atlanta, GA 30302. Email: siyer@gsu.edu.
Fax: 404ꢀ413ꢀ5505
2 Atlanta Research and Education Foundation, Atlanta, GA 30329
3 Malaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health,
Centers of Disease Control and Prevention, Atlanta, GA 30329
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Abstract:
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A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols
(GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium
falciparum GPIs have been considered as potential vaccine candidates as these molecules are
involved in inducing inflammatory responses in human hosts and natural antiꢀGPI antibody
responses have been shown to be associated with protection against severe disease. GPIs can
also be considered as targets for rapid diagnostic tests. As isolation of native GPIs in large
quantities is challenging, development of synthetic GPI molecules can facilitate further
exploration of GPI molecules for diagnostics. Here, we report synthesis and immunological
characterization of a panel of malaria specific GPI analogues. Three GPI analogues were
chemically synthesized and conjugated to a carrier protein to immunize and generate antibodies
in rabbits. The rabbit immune sera showed reactivity with synthetic GPIs and native GPIs
extracted from P. falciparum parasite as determined by Luminex and ELISA methods.
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Introduction:
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Malaria parasites, particularly Plasmodium falciparum, contribute to over 200 million clinical
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infections and about 500,000 deaths per annum.1,
The lack of effective vaccines and
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emergence of drug and insecticide resistance pose major challenges for malaria control. Rapid
and accurate diagnosis of malaria is critical for the success of any malaria control program.
Indeed, the World Health Organization (WHO) recommends that malaria treatment should be
based on diagnostic confirmation of infection.3 Although microscopic diagnosis of malaria has
been a standard practice for a long time, Rapid Diagnostic Tests (RDTs) have more recently
become very useful tools for malaria diagnosis especially in Africa where most malaria cases
occur. Commercial RDTs detect three different antigens, namely histidine rich protein II
(HRP2), lactose dehydrogenase (LDH) and aldolase.4 LDH and aldolase tests perform poorly at
low antigen concentrations (<1000 parasites/ ꢁL) and therefore the HRP2 test, which is specific
to P. falciparum and more sensitive (~200 parasites/ ꢁL) is preferred. Unfortunately, significant
parasite populations with HRP2 deletions have emerged in several endemic regions e.g.
Amazon basin of South America, leading to high false negatives and undesirable clinical
outcomes. Furthermore, residual HRP2 levels persisting after the parasite has been eliminated
from the bloodstream as determined by other methods leads to false positives, which makes it
difficult to determine if patients have completely cleared parasites after treatment in resource
poor settings.5, 6 To overcome limitations related to current RDTs, we have attempted to identify
new targets and in this report, we focus on an unique target, which is malaria GPI
(Glycophosphatidylinositol) anchors.
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GPI anchors are ubiquitous in eukaryotic cells and are particularly abundant in parasites in
the form of free lipids or attached to proteins. It is well known that GPIs are one of the parasite
products involved in modulating the host immune system to produce inflammatory mediators.
Development of natural antibodies to GPI is associated with clinical immunity to malaria.7 We
reasoned that GPIs could be targets for RDTs for the following reasons. 1) Compared to the
functionally unknown HRP2, GPIs are unlikely to be absent throughout the parasite life cycle
due to their functional importance. Specifically, GPIs function as anchors for a variety of
important surface proteins such as MSPꢀ1, MSPꢀ2 and MSPꢀ4. GPI anchors account for over
95% of postꢀtranslational carbohydrate modifications in malaria parasite.8 2) GPIs are abundant
and present on the surface of infected red blood cells (RBCs) or the parasite, which makes it
easy for the antibody to capture the parasite. 3) GPIs from P. falciparum are structurally distinct
from human GPIs. Figure 1 shows the difference between P. falciparum and mammalian GPIs.
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Therefore, antibodies raised against malaria are not expected to recognize human or other
parasite GPIs.
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Figure 1. i) Structure of natural Malarial GPI and Human GPIs and ii) unnatural PseudoꢀGPI
analogs and corresponding KLH protein conjugates
GPI anchors isolated from different species share a unique core structure; a
phosphatidylinositol (PI) moiety attached to the reducing terminal of a glucosamine residue
through an α1ꢀ6 glycoside linkage, and a distal nonꢀreducing mannose residue linked to an
ethanolamine phosphate bridge at the Cꢀ6 hydroxyl group of mannose, through which proteins
are attached. (Figure 1) Several groups have analyzed the structure of P. falciparum GPIs.
Gerold et al. first identified the glycan moiety structure of two mature GPIs: ethanolamineꢀ
phophateꢀ6 (Man1ꢀ2) Manα1ꢀ2Manα1ꢀ6Man1ꢀ4GlcNꢀPI and ethanolamineꢀphophateꢀ6Manα1ꢀ
2Manα1ꢀ6Man1ꢀ4GlcNꢀPI, which were confirmed later by a mass spectrometric analysis.9, 10
The latter study also revealed heterogeneity in fatty acyl substituents on the inositol ring, with
palmitic (major) and myristic (minor) acids at the Cꢀ2 position of myo-inositol. A synthetic GPI
NH2ꢀCH2ꢀCH2ꢀOPO3ꢀ(Manα1ꢀ2)6Manα1ꢀ2Manα1ꢀ6Manα1ꢀ4GlcNH2α1ꢀ6myoꢀinositolꢀ1,2ꢀ
cyclicꢀphosphate was evaluated as candidate antiꢀtoxic vaccine and the antiꢀGPI antibodies
generated were found to effectively neutralize proꢀinflammatory activity in a rodent malarial
model.11
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Encouraged by these studies, we envisioned the development of antibodies to P. falciparum
GPIs for integration in RDTs to accurately detect the parasite in blood samples. Unfortunately,
isolating GPIs from the parasite in sufficient amounts with consistency and high purity is not a
trivial endeavor. Fortunately, advances in glycan synthesis can generate extremely pure
chemically defined complex glycoconjugates (human and parasite GPI anchors) in appreciable
quantities.11-25 Here, we present the design, synthesis of natural and pseudo P. falciparum GPI
analogues, generation of antibodies using GPIꢀprotein conjugates and initial immunological
characterization.
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Results and Discussion:
The structures of the target compounds are depicted in Figure 1. We focused our studies on
the synthesis of the naturally occurring P. falciparum GPI hexasaccharide 3 and two unnatural
analogs, the hexasaccharide, 1 and the pentasaccharide, 2. The latter were synthesized since
the diols leading to the inositol analogs were relatively easy to synthesize in large amounts.
Studies using these analogs are important in understanding the role of inositol in antibody
recognition. We used a short alkyl chain instead of longer lipids present in naturally occurring
GPI anchors primarily because the lipids are embedded in the lipid membrane, leaving the
glycan region exposed. Antibodies raised against these compounds are expected to recognize
the exposed glycan portion of the GPI anchor. If our working hypothesis that longer lipid chains
are not significant for antibody recognition is proven to be incorrect, we can readily produce
GPIs with longer lipids because the modular nature of our synthetic strategy allowed us to
incorporate lipids at a later stage in the synthesis. The primary amine of the
phosphoethanolamine in all three compounds was conjugated with a carrier protein to generate
antibodies. We synthesized GPI anchor analogs 1, 2, 3 and their corresponding Keyhole Limpet
Hemocyanin (KLH) protein conjugates GPI1, GPI2, and GPI3.
We envisioned the synthesis of compounds 1ꢀ3 using a common trisaccharide fragment 6a.
The allyl group in 6a could be removed under mild conditions and the resulting hemiacetal could
be converted to the imidate derivative, 6b. 6b would be ready for further elaboration to the
pentasaccharide 2. 6a could also lead to the tetrasaccharide 8b, which can be used to generate
compounds 1 and 3 through a series of chemical modifications.
To generate 6a, we synthesized suitably protected Dꢀmannose monosaccharide derivatives,
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4a,b,c as described previously.
(Scheme 1) We choose the allyl group as a protecting
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group in several of the fragments because it can be readily removed to provide the hemiacetal
which can be further elaborated or it can be hydrogenated to provide a three carbon spacer
present in the final compounds. Additionally, the allyl group is compatible with several of the
protection/deprotection strategies employed in our syntheses. Coupling of 4c with 4a resulted in
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the desired exclusive α product in high yield. Removal of the acetate group provided 5b.
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0
Glycosylation of the disaccharide acceptor 5b26 and mannose imidate donor 4b at 0 C using
TMSOTf as the promoter yielded trisaccharide 6a as the exclusive αꢀisomer in 90% yield. Next,
the allyl group in the triꢀsaccharide 6a was deꢀprotected by using PdCl2/AcOH and the resulting
alcohol was converted to the corresponding imidate donor 6b in 60% yield over two steps.
To synthesize the tertasaccharide, the acetate group in 6a was selectively deprotected by
Mg(OMe)2 to give trisaccharide acceptor 7 in 65% yield. Glycosylation of trisaccharide acceptor
7 with mannose imidate donor 4c yielded tetrasaccharide 8a using reported conditions.26
However, the yield were poor, approximately 20ꢀ30%. Analysis of the major byproducts
suggested that the silyl group was unstable under the reaction conditions. Attempts to improve
the yield by using a variety of conditions failed; despite our best efforts, we couldn’t produce 8a
in sufficient amounts for further elaboration. Therefore, we decided to synthesize the
tetrasaccharide 8b using a [2+2] approach. First, glycosylation of
Dꢀmannose monosaccharide
acceptor 4c and donor 4d yielded 9a with exclusive α selectivity in decent yields. The allyl group
in 9a was deꢀprotected by using PdCl2/AcOH and the resulting alcohol was converted to the
corresponding imidate donor 9b in 60% yields over two steps. 2+2 glycosylation of the Dꢀ
mannose disaccharide acceptor 5b26 and donor 9b in the presence of catalytic amount of
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TMSOTf in dichloromethane at ꢀ40 C produced the tetrasaccharide 8a in 80% yield. The allyl
group in tetrasaccharide 8a was selectively deprotected by PdCl2 and the alcohol was converted
to the corresponding imidate donor 8b. The trisaccharide and tetrasaccharide fragments (6b
and 8b) were ready for further elaboration to the target compounds, 1ꢀ3. (Scheme 1)
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Scheme 1. Synthesis of triꢀ and tetraꢀsaccharide imidate donor (6b and 8b). Reagents and
conditions: (a) DCM, TMSOTf, ꢀ10 °C to rt, (94% for 5a; 90% for 6a; <30% for 8a from 7+4c;
80% for 8a from 5b+9b; all coupled products were α isomers. (b) AcCl, MeOH, 90%; (c) i)
AcOH, PdCl2, NaOAc, H2O, rt, 6 h; ii) DCM, DBU Cl3CCN, rt, 2 h, 58ꢀ65% (for two steps); (d)
Mg(OMe)2, MeOH, THF, 65%.
Next, we synthesized suitably protected analog glucosamine pseudoꢀdisaccharide, 16a.
First, we developed a synthetic route to 12. (Scheme 2) The commercially available 1R, 2Rꢀ
transꢀcyclohexane diol 10 was selectively protected using TBDMS ether in 60% yield. Allyl
protection followed by removal of the TBDMS group yielded the monoprotected diol 12 in 53%
yield over two steps. Glycosylation of the 1ꢀO-allylꢀcyclohexaneꢀ1,2ꢀdiol 12 as acceptor and
3,4,6 triacetateꢀ2ꢀazidoꢀαꢀDꢀglucosamine trichloroacetimidate donor 1328 at ꢀ10 0C using
TMSOTf as the promoter yielded 14 as a mixture of anomers in a 1:2 (α:β) ratio. The α anomer
resonates at 5.12 ppm with a coupling constant of 2.0 Hz, whereas the β anomer resonates at
4.84 ppm with a coupling constant of 8.4 Hz. With further optimization using a mixture of
solvents DCM: diethyl ether (2:1), we obtained a higher selectivity of 1:1 (α:β ratio; as
determined by 1H NMR spectroscopy). Purification of the disaccharide by column
chromatography proved problematic because of inseparable impurities, which we ascertained
as byproducts of the donor. Fortunately, these impurities were separated later in the synthesis.
Deprotection of the acetate groups using Zemplén conditions, followed by protection of the 4,6ꢀ
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hydroxyl and 3ꢀhydroxyl group by benzylidene acetal and benzyl ether, respectively, yielded the
completely protected disaccharides, 15a (α) and 15b (β). The α, β isomers were purified by
column chromatography at this stage and characterized by 1D and 2D NMR spectroscopy. The
anomeric proton in the α isomer resonates at δ 5.04 ppm and resonates at δ 4.84 ppm in the βꢀ
anomer. (Supporting information). Next, the benzylidene acetal in 15a was selectively opened
by sodium cyanoborohydride to yield the desired disaccharide acceptor 16a in 74% yield.
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Scheme 2. Synthesis of pseudoꢀdisaccharide 16a. Reagents and conditions: (a) THF,
imidazole, TBDMSCl, rt, 12 h, 61%; (b) i) DMF, NaH, AllBr, rt, 3 h; ii) THF, HCl, rt, 3 h (53% for
two steps); (c) 13, DCM, TMSOTf, ꢀ40 °C to rt, 2 h; (d) i) MeOH, NaOMe, rt, 1 h; ii) CH3CN,
PhCH(OMe)2, CSA, rt, 12 h; iii) DMF, NaH, BnBr (15a 36%; 15b 38% for four steps); (e) THF,
NaCNBH3, HCl, rt, 3 h, 74%.
With the two fragments in hand, we reacted the tetrasaccharide donor 8b and disaccharide
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acceptor 16a at ꢀ10 C by using TMSOTf (0.1 equivalents) as promoter to afford the desired
hexasaccharide 17. The alpha isomer was the only isomer isolated, however the yields were
low and variable.15 Careful examination of the byproducts indicated that the TIPS group in the
donor was being deprotected under the reaction conditions, presumably by the acidic promoter.
To avoid this side reaction, we lowered the concentration of TMSOTf and decreased the
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reaction temperature to ꢀ40 C to obtain a higher yield of 71%. (Scheme 3) Next, the acetate
and benzoate esters in 17 were hydrolyzed using Zemplén conditions and the hydroxyl groups
in 18 were protected as the benzyl ether to provide 19. The silyl group in 19 was deprotected
using AcCl in MeOH to yield the free hydroxyl 20 in quantitative yield. The next step, which was
the introduction of the phosphoethanolamine was problematic. We followed literature
procedures, which suggested mixing the two fragments (20 and 21) in a suitable solvent,
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followed by the addition of pivaloyl chloride to introduce the phosphoethanolamine unit to 20 via
the free hydroxyl group.15 Unfortunately, the only product isolated was the pivaloate ester of the
alcohol 20. Fortunately, we found a solution to this problem by modifying the addition sequence.
We premixed the Hꢀphosphonate ester 21 with pivaloyl chloride in anhydrous pyridine, followed
by addition of 20 to yield the desired product 21 in quantitative yield. Global deprotection of all
the benzyl ethers and azide reduction in 21 using Pd(OH)2/H2 under atmospheric conditions,
followed by purification using a Biogel Pꢀ2 column yielded the fully deprotected GPI analog 1 in
quantitative yield.
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Scheme 3. Synthesis of pseudoꢀhexasaccharide 1: Reagents and conditions: a) DCM,
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TMSOTf, ꢀ10 °C to rt, 71%,α isomer only. b) NaOMe, DCM:MeOH (1:1), 50 C, 20 h, 80%; c)
NaH, BnBr, DMF, rt, 12 h, 85%; d) AcCl, DCM:MeOH (3:1), rt, 12 h, 91%; e) PivCl, py., rt, 12 h
then Iodine, py:H2O (19:1), rt, 12 h, 92%; f) Pd(OH)2/C, H2 (balloon), MeOH, 12 h, 94%.
For the synthesis of 2, glycosylation of the trisaccharide donor 6b and pseudoꢀdisaccharide
acceptor 16a was performed using the optimized conditions as described for 17 to afford
pseudoꢀpentasaccharide 23 in 74% yield with exclusive α selectivity. (Scheme 4) Removal of
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the acetate and benzoate groups followed by protection of the corresponding hydroxyls to the
benzyl ether yielded 24 in appreciable yield. Silyl deprotection in 24 by AcCl/MeOH afforded
hydroxyl 25 and the phosphoethanolamine unit was introduced using a procedure as described
for 22 to generate 26 in high yield. Global deprotection of all the benzyl ethers and azide
reduction in 26 using Pd(OH)2/H2, followed by purification using a Biogel Pꢀ2 column yielded
GPI analog 2 in quantitative yield.
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Scheme 4. Synthesis of pseudoꢀpentasaccharide 2: Reagents and conditions: a) DCM,
TMSOTf, ꢀ10 °C to rt, 74%, α isomer only; b) i) NaOMe, DCM:MeOH (1:1), 50 oC, 20 h; ii) NaH,
BnBr, DMF, rt, 12 h, 76% (2 steps); c) AcCl, DCM:MeOH (3:1), rt, 12 h, 89%; d) PivCl, py., rt, 12
h then Iodine, py:H2O (19:1), rt, 12 h, 90%; e) Pd(OH)2/C, H2 (balloon), MeOH, 12 h, 90%.
For the synthesis of GPI analog 3, the desired inositolꢀglucosamine pseudoꢀdisaccharide 27
was synthesized by the procedure reported in the literature.18 The subsequent steps: 1)
Glycosylation of disaccharide acceptor 27 with tetramannoside donor 8b; 2) Deprotection
acetate and benzoate ester; 3) Benzyl protection; 4) TIPS deprotection; 5) Introduction of
phosphoethanol unit and 6) global deprotection was carried out in a similar manner to the
synthesis of GPI analogs 1 and 2 to afford GPI analog 3. (Scheme 5) All intermediates and
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final products were characterized by HNMR, C13NMR, HSQC NMR and HRMS spectroscopy.
(Supporting information)
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Scheme 5. Synthesis of hexasaccharide 3: Reagents and conditions: a) DCM, TMSOTf, ꢀ
o
20 °C to rt, 70%, α isomer only; b) i) NaOMe, DCM:MeOH (1:1), 50 C, 20 h; ii) NaH, BnBr,
DMF, rt, 12 h, 72% (2 steps); c) AcCl, DCM:MeOH (3:1), rt, 12 h, 87%; d) PivCl, py., rt, 12 h
then Iodine, py:H2O (19:1), rt, 12 h, 92%; e) Pd(OH)2/C, H2 (balloon), MeOH, 12 h, 94%.
The three synthesized GPI moieties were coupled to keyhole limpet haemocyanin (KLH), a
commonly utilized protein molecule as a carrier protein. The use of a larger carrier protein is
necessary in immunization protocols since a small molecule such as a short peptide or, in our
case, GPI moiety may not be immunogenic enough to elicit a strong immune response.
Coupling glycans to a larger carrier molecule such as the keyhole limpet haemocyanin (KLH)
has been shown to lead to successful immunization. KLH is the most commonly used carrier
protein because it has a significantly higher immunogenicity as compared to other proteins. To
this end, compounds 1, 2 and 3 were conjugated with preꢀactivated ImmꢀLink KLH (Novobios) to
provide GPI1, GPI2 and GPI3, respectively, according to manufacturer’s protocol. (Scheme 6).
Briefly, the preꢀactivated KLH was treated with the large excess of glycan (KLH:glycan; 1:~200)
ratio). After conjugation, unreacted glycans were removed by dialysis against distilled water.
KLH is an approximately 400 KDa multidomain protein complex29 and characterization of
conjugates11 was not practical by mass spectroscopy. However, the synthesized conjugates
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were analyzed using colorimetric methods; the glycan loading was determined to range
between 15ꢀ17% using the procedures reported by Zou et al.30-32 (Supporting information)
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Scheme 6. Synthesis of GPIꢀKLH conjugates (GPI1 to GPI3).
Biological assays:
Polyclonal antibodies using rabbits were raised against each of GPIꢀKLH conjugate, with
two rabbits per conjugate. (Figure 2) All rabbits immunized with GPI1, GPI2 or GPI3, had
positive antibody responses, with IgG titer steadily increasing over the seventy day course. Antiꢀ
sera from rabbits 1ꢀ4, immunized with GPI1 or GPI2 exhibited reactivity against GPI1 and GP2,
but did not exhibit any cross reactivity with GP13. Similarly, antiꢀGPI3 sera from rabbits 5 and 6
exhibited reactivity with GPI3 but minimal (rabbit 5) or no (rabbit 6) cross reactivity with GPI1 or
GPI2. This pattern of reactivity, indicates that the antibodies in these sera may be directed to
inositol residue as the primary difference between GPI1/GPI2 and GPI3 is in this residue.
Interestingly, immune sera raised against GPI1 showed higher level of reactivity against GPI2
than GPI1. Structurally, GPI1 and GPI2 share an identical pseudo inositol residue except for the
presence of additional distal mannose in GPI1. This cross reactivity between GP1 and GP2
indicates the epitope recognized by the elicited IgG does not lie on the nonꢀreducing mannose
end.
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Figure 2. AntiꢀGPI antibody titers in six immunized rabbits measured by Luminex assay.
Luminex assay using three synthetic GPI analogues against rabbit antiꢀsera harvested at
different days. Six rabbits were immunized with the three generated synthetic GPIs: 1, 2 and 3.
Rabbit 1 and 2 were immunized with GPI1; rabbit 3 and 4 with GPI2; rabbit 5 and 6 with GPI3.
The rabbits were bled before the immunization procedure (day 0 sample) and subsequently on
days 7, 21, 35 and 56 post immunization. The luminex assay was used to measure the antiꢀGPI
antibodies titers elicited by the rabbits. All rabbits immunized with GPI1, GPI2 or GPI3, had
positive antibody responses, with IgG titer steadily increasing over the seventy day course. Antiꢀ
sera from rabbits 1ꢀ4, immunized with GPI1 or GPI2 exhibited reactivity against GPI1 and GPI2,
but did not exhibit any cross reactivity with GP13. Similarly, antiꢀGPI3 sera from rabbits 5 and 6
exhibited reactivity with GPI3 but minimal (rabbit 5) or no (rabbit 6) cross reactivity with GPI1 or
GPI2.
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We tested the reactivity pattern of the rabbit antisera, both preꢀimmunization (preimmune
sera) and postꢀimmunization (immune sera) against native GPI isolated from P. falciparum
(Figure 3). the immune sera from rabbits 1, 3, 5 and the hyperimmune sera used showed
statistically significant increase in reactivity compared to preimmune sera (P<0.05), suggesting
that these rabbits responded strongly to the synthetic GPI immunogen. Immune sera from
rabbit number 2 (P=0.527), rabbit 4 (P=0.125) and rabbit 6 (P=0.104) were not statistically
significant. This binding affinity was comparable to reactivity obtained with natural human hyper
immune sera used as positive control (Figure 3). Overall, this data suggests that antibodies
generated against the synthetic analogues of GPI can recognize native parasite GPI antigens
and therefore, this antigen/antibody system based on synthetic GPIs that can be exploited to
develop RDTs. Studies to demonstrate these antiꢀGPI antibodies do not cross react with GPI
moieties from other infectious diseases are ongoing.
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Figure 3.Binding affinity of P. falciparum native GPI to rabbit antiꢀGPI antibodies. ELISA
assays were performed to determine if the antiꢀGPI antibodies generated in the immunized
rabbits were capable of detecting GPIs from P. falciparum parasites. Native P. falciparum GPIs
were dissolved in methanol and coated to ELISA plates at a concentration of 40 ng/ml. Both
preimmune and immune (56 days post immunization) sera from the 6 rabbits were tested in
triplicate. As a control, human hyperimmune serum (HUMAN) was also tested. The mean
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405nm absorbance was recorded for each sample. Immune sera from rabbits 1, 3 and 5 and the
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hyperimmune human sera showed significant affinity for the native P. falciparum GPI.
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Conclusions:
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We successfully synthesized three synthetic GPI analogues and developed antibodies
against these analogues. Antibodies raised against these synthetic GPIꢀprotein conjugates
exhibited binding to the synthetic GPIs. The results also indicated that the antibodies rasied
against the pseudoꢀinositol synthetic GPI are specific to the GPIs containing this pseudoꢀinositol
moiety. Antisera raised against synthetic GPI can capture native GPI indicating the potential use
of these structures as targets for diagnostic assays.
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Experimental procedures:
General:
Glycosylation reactions were performed under argon with solvents dried using a solvent
purification system Innovative Technology. All chemical reagents were of analytical grade, used
as supplied without further purification unless indicated. The acidic ion exchange resin used was
Amberlite® IR 120 (H+) resin. Analytical thin layer chromatography (TLC) was performed on
silica gel 230ꢀ400 mesh (Sicicycle). Plates were visualized under UV light, and/or by staining
with acidic CeH8Mo3N2O12 followed by heating. Column chromatography was performed on
1
silica gel (230ꢀ400 mesh). H and 13C NMR spectra were recorded on Bruker 400MHz
1
spectrometer. Chemical shifts are reported in δ (ppm) units using 13C and residual H signals
from deuterated solvents as references. Spectra were analyzed with MestreNova® (Mestrelab
Research). Electrospray ionization mass spectra were recorded on a Micromass Q \T 2
(Waters) and data were analyzed with MassLynx® 4.0 (Waters) software. Reported yields refer
to spectroscopically and chromatographically pure compounds that were dried under high
vacuum (10–2 mbar) before analytical characterization, unless otherwise specified. ImmꢀLink
KLH (Amine) Immunogen Conjugation Kit was purchased and followed given by Novus
biotechno brand.
Abbreviations: Acetic anhydride, Ac2O; Acetic acid, AcOH; Acetyl chloride, AcCl; Benzoyl
chloride, BzCl; Ethyl acetate, EtOAc; Dichloromethane, DCM; Dibenzundcane, 1,8ꢀ
Diazabicyclo[5.4.0]undecꢀ7ꢀene; DBU; Methanol, MeOH; Pyridine, Py.; Thin layer
chromatography, TLC; Trimethylsilyl trifluoromethanesulfonate, TMSOTf; TIPSCl, Triꢀisopropyl
silyl chloride; Tertꢀbutyl dimethyl silyl chloride, TBDMSCl, Triethyl amine, NEt3; Phosphoꢀethanol
amine, PEA.
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2ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosylꢀ(1→2)ꢀ1ꢀO-trichloroacetimidylꢀ6ꢀOꢀ
triisopropylsilylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranoside (9b): To a solution of disaccharide
9a27 (4.00 g, 4.00 mmol) in AcOH (180 mL) and H2O (20.0 mL) were added NaOAc (2.00 g,
24.50 mmol) and PdCl2 (1.04 g, 6.00 mmol), and the solution stirred at rt for 12 h (monitored by
TLC). The solution was then filtered through a small silica gel plug and quenched with saturated
solution of NaHCO3. The aqueous layer was extracted with EtOAc (3 × 100 mL) and the
combined organic layers were washed with brine and dried over Na2SO4. The solvent was
removed under reduced pressure to obtain crude alcohol (3.20 g) which was used for next
reaction without further purification. The crude alcohol (3.20 g) was dissolved in dry DCM (50
mL) and cooled to 0 °C. To this solution trichloroacetonitrile (1.60 mL, 16.00 mmol) and DBU
(0.12 mL, 0.98 mmol) was added and stirred for 1 h at rt. The solvent was removed under
reduced pressure and the residue was purified by flash chromatography (EtOAc/hexane (1:4) to
give 9b (2.7 g, 60% for two steps) as a colorless oil. Rf = 0.3 (EtOA/hexane, 1:4). 1H NMR (400
MHz, CDCl3) δ 8.50 (s, 1H), 7.29ꢀ7.47 (m, 23H), 7.23 (d, J = 4 Hz, 2H), 6.37 (s, 1H), 5.64 (s,
1H), 5.18 (s, 1H), 4.99 (d, J = 10.6 Hz, 1H), 4.92 (d, J = 10.8 Hz, 1H), 4.77 (dt, J = 21.4, 10.5
Hz, 5H), 4.65 – 4.49 (m, 3H), 4.17 (d, J = 9.9 Hz, 1H), 4.14 – 3.94 (m, 7H), 3.88 (t, J = 11.6 Hz,
2H), 3.77 (d, J = 10.8 Hz, 1H), 2.20 (s, 3H), 1.10 (t, J = 6.4 Hz, 21H); 13C NMR (101 MHz,
CDCl3) δ 170.3, 160.0, 138.6, 138.5, 138.3, 138.1, 128.5, 128.5, 128.4, 128.29, 128.26, 128.2,
128.17, 128.1, 128.0, 127.9, 127.8, 127.79, 127.7, 127.5, 127.5, 100.0, 97.2, 91.0, 78.9, 78.6,
75.8, 75.4, 75.1, 74.3, 73.7, 73.55, 73.47, 72.6, 72.2, 71.8, 68.8, 68.7, 62.3, 21.2, 18.1, 12.0,
11.9; HRMS (ESI): m/z: calcd for [M+Na]+: C60H74Cl3NO12Si: 1156.3944; found 1156.3972.
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Allyl(2ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀ
triisopropylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→6)ꢀ2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranoside (8a): A mixture of disaccharide
acceptor 5b26 (1.60 g, 1.72 mmol) and mannose donor 9b (2.32 g, 2.04 mmol) was coꢀ
evaporated with toluene (3 × 3 mL) and dried under vacuuo overnight. To the mixture was
added dry DCM (5.00 mL), cooled (ꢀ20 oC) and added TMSOTf (30 ꢁL, 0.17 mmol) and stirred
o
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for 1 h at ꢀ20 C and 0.5 h at ꢀ10 C (monitored by TLC). The reaction was quenched by the
addition of Et3N (0.50 mL) and the reaction mixture was concentrated. The crude was purified
by chromatography (eluent: 20% ethyl acetate in hexane) to give 8a26 (2.40 g, 80%) as gummy
liquid. Rf = 0.3 (EtOAc/hexane (1:4). NMR data for 8a is in agreement with reported in the
literature.26
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(2ꢀOꢀAcetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀ
triisopropylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→6)ꢀ2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl trichloroacetimidate (8b): To a
solution of tetrasaccharide 8a (2.40 g, 1.24 mmol) in AcOH (90 mL) and H2O (10 mL) NaOAc
(1.52 g, 18.52 mmol) and PdCl2 (1.64 g, 9.20 mmol) was added and the mixture was stirred for
12 h at rt. The solution was then filtered through a small silica gel plug and quenched with
saturated solution of NaHCO3 and extracted with EtOAc (3 × 100 mL). The combined organic
layers were washed with brine and dried over anhydrous Na2SO4. The solvents were
evaporated on reduced pressure to obtain the crude alcohol (1.80 g) which was used for next
reaction without further purification. The crude alcohol (1.80 g) was dried under vacuum,
dissolved in DCM (20 mL) and cooled to 0 °C. trichloacetonitrile (2.40 mL, 24.00 mmol) and
DBU (0.12 mL, 0.96 mmol) was added and stirred at rt for 1 h. The reaction mixture was
concentrated on reduced pressure and the resulting crude was purified by chromatography
(EtOAc;hexane (1:9)) to afford the 8b (1.48 g, 60% over two steps). The analytical data for 8b
was in agreement with the reported in the literature.26
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Synthesis of (1R,2R)ꢀ2ꢀ((tertꢀbutyldimethylsilyl)oxy)cyclohexanꢀ1ꢀol (11): To a cooled (0
oC) solution of 1R,2Rꢀcyclohexaneꢀtransꢀdiol 10 (2.00 g, 17.20 mmol) in DCM (50 mL) imidazole
(2.34 g, 34.40 mmol), TBDMSCl (2.86 g, 18.92 mmol) was added and stirred at rt for 12 h. The
solvent was removed under reduced pressure and the residue was purified by flash
chromatography (EtOAc:hexane, 1:9) to give 11 (2.42 g, 61%) as a colorless oil. Rf = 0.3
1
(EtOAc:hexane, 1:9). H NMR (400 MHz, CDCl3) δ 3.34 (q, J = 3.8 Hz, 2H), 2.48 (s, 1H), 1.98
(td, J = 6.7, 3.1 Hz, 1H), 1.85 (t, J = 6.1 Hz, 1H), 1.68 (s, 2H), 1.25 (d, J = 7.0 Hz, 4H), 0.91 (s,
13
9H), 0.10 (d, J = 5.7 Hz, 6H) ppm; C NMR (101 MHz, CDCl3) δ 77.3, 77.1, 77.0, 76.7, 75.4,
33.5, 31.5, 25.8, 24.3, 24.0, 18.0, ꢀ4.1, ꢀ4.7 ppm.
Synthesis of (1R,2R)ꢀ2ꢀO-allylꢀcyclohexanꢀ1ꢀol (12): To a cooled (0 oC) solution of alcohol 11
(2.20 g, 9.60 mmol) in THF was added NaH (60% in dispersion oil, 0.77 g, 19.20 mmol) and
stirred for 15 min. Allyl bromide (1.20 mL, 14.20 mmol) was added drop wise and the reaction
mixture was stirred for 3 h at rt. The reaction mixture was quenched with ice cold H2O and the
aqueous layer was extracted with ethyl acetate (3 x 100 mL) and the combined organic layers
were washed with brine and dried over Na2SO4. The solvents were removed under reduced
pressure and obtained crude product 2.60 g was used for next reaction without further
purification. To a solution of crude residue in THF (10.00 mL) and water (4.00 mL) 1M HCl in
diethyl ether (10 mL) was added and stirred at rt for 3 h. The reaction mixture was quenched
with Et3N, diluted with water and the aqueous layer was extracted with diethyl ether (3 x 100
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mL). The combined organic layers were further washed with brine and dried over Na2SO4. The
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solvents were carefully removed at 20 C (since the bp of the product is very low) and purified
by column chromatography in 2% ethyl acetate in hexane to remove impurities and then eluted
with diethyl ether to give 12 (0.82 g, 53% for two steps). 1H NMR (400 MHz, CDCl3) δ 5.95 (ddt,
J = 16.4, 10.8, 5.6 Hz, 1H), 5.29 (d, J = 17.3 Hz, 1H), 5.18 (d, J = 10.3 Hz, 1H), 4.17 (dd, J =
12.7, 5.5 Hz, 1H), 3.97 (dd, J = 12.8, 5.8 Hz, 1H), 3.46 (td, J = 9.8, 5.0 Hz, 1H), 3.10 (td, J = 9.7,
9.1, 4.0 Hz, 1H), 2.79 (s, 1H), 2.14 – 1.94 (m, 2H), 1.71 (t, J = 10.2 Hz, 2H), 1.36 – 1.08 (m,
4H).
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(2ꢀAzidoꢀ3,4,6ꢀtriꢀO-acetylꢀ2ꢀdeoxyꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ (1S,2S)ꢀ2ꢀOꢀallyl cyclo
hexane (14α/β):
o
To a cooled (ꢀ10 C) solution of allyl gylcoside 12 (0.32 g, 2.05 mmol) and glucosamine
trichloroacetimidate 1328 (1.36 g, 2.87 mmol) in DCM (7.00 mL), TMSOTf (50 ꢁL, 0.21 mmol)
was added, and stirred for at ꢀ40 °C to rt for 2 h (monitored by TLC). The reaction was
quenched by the addition of Et3N (0.20 mL) and the reaction mixture was concentrated. Crude
product was purified by chromatography (EtOAc:hexane, 1:4) to give pseudoꢀdisaccharide
14α/β (1:1, 1.50 g; 1HNMR and HSQC spectra) along with trichloroꢀacetamide impurity. Rf = 0.3
(EtOAc:hexane, 1:4). See supporting information for the NMR spectra of 14α/β. HRMS (ESI):
m/z: calcd for C21H31N3O9 [M+Na]+: 492.1958; found 492.1947.
(2ꢀAzidoꢀ3ꢀO-benzylꢀ4,6ꢀO-benzylideneꢀ6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀα/βꢀDꢀglucopyranosyl)ꢀ
(1→1)ꢀ (1S,2S)ꢀ2ꢀOꢀallylꢀcyclohexanꢀ1ꢀol (15α/β): The disaccharide 14 (1.50 g) was dissolved
in MeOH (0.01 L) and NaOMe (0.5 M in MeOH, 0.60 mL, 0.30 mmol) was added and stirred at rt
for 2 h (monitored by TLC). The solution was neutralized with acidic DOWEX resin to
approximately pH 6. The organic layer was collected by filtrating off the resin, and was
evaporated to give the crude triol which was purified by short column to remove the trichloroꢀ
acetamide impurity to obtain triol (0.60 g) as a gummy liquid. The crude triol was coꢀevaporated
with toluene, vacuum dried and dissolved in dry acetonitrile (10 mL) and CSA (0.04 g, 0.18
mmol) and PhCH(OMe)2 (0.40 mL, 2.70 mmol) was added. The reaction mixture was stirred
for 12 h, before being quenched with Et3N. The solvent was then evaporated under reduced
pressure. The residue was coꢀevaporated with toluene (3 x 5 mL) and dried under high vacuum
for 1 h. The residue was then dissolved in DMF (0.02 L), followed by the addition of TBAI (0.96
g, 2.60 mmol). The reaction mixture was then cooled to 0 ºC and NaH (60% in mineral oil, 0.14
g, 3.40 mmol) was added in one portion. After 20 min, BnBr (0.31 mL, 2.60 mmol) was added
and the reaction mixture was stirred at rt for 5 h. The reaction mixture was quenched with water
and the aqueous layer was extracted with ethyl acetate (3 x 50 mL) and the combined organic
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layers were further washed with brine and dried over Na2SO4. Evaporation in vacuo led to the
crude product, which was purified by column chromatography (EtOAc:hexane, 1:9) to give the
15a (0.380 g, 36%, over four steps) and 15b (0.390 g, 38%, over four steps) as gummy liquids.
Data for αꢀisomer (15a): Rf 0.25, EtOAc:hexane, 1:9; 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J =
7.1 Hz, 2H), 7.46 – 7.29 (m, 8H), 6.00 (ddd, J = 16.7, 11.0, 5.4 Hz, 1H), 5.60 (s, 1H), 5.32 (d, J
= 17.2 Hz, 1H), 5.17 (d, J = 10.4 Hz, 1H), 5.04 (d, J = 2.8 Hz, 1H, anomeric H), 4.98 (d, J = 11.0
Hz, 1H), 4.82 (d, J = 11.1 Hz, 1H), 4.38 – 4.23 (m, 2H), 4.20 – 4.03 (m, 3H), 3.80 – 3.67 (m,
2H), 3.66 – 3.53 (m, 1H), 3.33 (q, J = 10.2 Hz, 2H), 2.08 (d, J = 12.3 Hz, 2H), 1.73 (q, J = 6.1
Hz, 2H), 1.32 (dq, J = 34.1, 11.7, 11.3 Hz, 5H) ppm; 13C NMR (101 MHz, CDCl3) δ 137.9, 137.5,
135.2, 129.0, 128.4, 128.3, 128.2, 127.8, 126.1, 117.1, 101.5, 95.5, 83.0, 79.4, 77.9, 77.4, 77.0,
76.7, 75.8, 74.9, 70.9, 68.9, 62.9, 62.5, 30.0, 28.7, 23.3, 23.2 ppm. HRMS (ESI): m/z: calcd for
C29H35N3O6N [M+Na]+: 544.2424; found 524.2440.
Data for βꢀisomer (15b): Rf 0.3, EtOAc:hexane, 1:9; 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J =
6.9 Hz, 3H), 7.47 – 7.22 (m, 9H), 6.12 – 5.95 (m, 1H), 5.58 (s, 1H), 5.33 (d, J = 17.3 Hz, 1H),
5.19 (d, J = 10.3 Hz, 1H), 4.93 (d, J = 11.2 Hz, 1H), 4.87ꢀ4.70 (m, 2H), 4.33ꢀ4.37 (dd, J1 = 5.2
Hz, J2 = 10.8 Hz, 1H), 4.23 – 4.04 (m, 2H), 3.94 – 3.28 (m, 9H), 2.13 (d, J = 8.8, Hz, 1H), 2.01
(d, J = 8.8, Hz, 1H), 1.61 (d, J = 6.4 Hz, 1H), 1.42 (q, J = 11.5 Hz, 1H), 1.28ꢀ1.20 (m, 4H) ppm;
13C NMR (101 MHz, CDCl3) δ 138.0, 137.2, 135.4, 129.0, 128.4, 128.3, 128.3, 128.1, 127.8,
126.0, 116.6, 102.9, 101.3, 81.7, 81.6, 80.6, 79.1, 74.9, 70.5, 68.7, 66.5, 66.0, 32.1, 30.0, 23.9,
23.4 ppm. HRMS (ESI): m/z: calcd for C29H35N3O6N [M+Na]+: 544.2424; found 524.2440.
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(2ꢀAzidoꢀ3,6ꢀdiꢀO-benzylꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ(1S,2S)ꢀ2ꢀOꢀallylꢀ
cyclohexane(16a): To a solution of pseudodisaccharide acetal 15a (0.30 g, 0.57 mmol) in THF
(5 mL) was added NaCNBH3 (1.0 M in THF, 10.00 mL, 10.00 mmol) and the solution was
cooled to 0 ºC. HCl in Et2O (2.0 M, ∼5 mL) was then added drop wise until the reaction mixture
reached PH 1. The reaction mixture was then warmed up to room temperature, and stirred for 2
h at rt. The reaction was then quenched by the addition of aq. NaHCO3 solution and the
aqueous layer was extracted with Et2O (3 x 25 mL). The combined organic extracts were
washed with brine and evaporated under reduced pressure. The residue was purified by column
chromatography (EtOAc:hexane, 1:4) to give 16a (0.22 g, 74%; Rf 0.25, EtOAc:hexane, 1:4).
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Data for 16a: H NMR (400 MHz, Chloroformꢀd) δ 7.49 – 7.29 (m, 12H), 5.95 (ddt, J = 16.5,
11.2, 5.8 Hz, 1H), 5.29 (d, J = 17.1 Hz, 1H), 5.15 (d, J = 10.4 Hz, 1H), 5.07 (d, 3.2 Hz, 1H
anomeric), 4.94 (d, J = 11.3 Hz, 1H), 4.87 (d, J = 11.3 Hz, 1H), 4.71 – 4.61 (m, 1H), 4.55 (d, J =
12.0 Hz, 1H), 4.14 (d, J = 28.2 Hz, 3H), 3.89 (t, J = 9.4 Hz, 1H), 3.84 – 3.72 (m, 2H), 3.55ꢀ71
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(m, 2H), 3.30 (d, J = 10.3 Hz, 2H), 2.60 (s, 1H, OH), 2.01ꢀ2.09 (m, 2H), 1.55ꢀ1.75 (m, 2H), 1.18ꢀ
1.42 (m, 2H) ppm; 13C NMR (101 MHz, CDCl3) δ 138.2, 137.9, 135.4, 128.6, 128.5, 128.4,
128.2, 128.0, 127.7, 127.7, 116.8, 94.4, 79.6, 79.5, 77.4, 77.1, 76.8, 74.8, 73.6, 72.5, 71.1, 69.9,
69.5, 62.6, 30.3, 28.5, 23.5, 23.2 ppm. HRMS (ESI): m/z: calcd for C29H37N3O6 [M+Na]+:
546.2580; found 546.2575.
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(2ꢀOꢀAcetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀ
triisopropylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→6)ꢀ(2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ
2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane
(17):
Pseudoꢀ
disaccharide acceptor 16a (0.20 g, 0.38 mmol) and tetramannoside donor 8b (1.15 g, 0.57
mmol) were mixed, and coꢀevaporated with toluene (3 x 6 mL), and dried under high vacuum
overnight. The mixture was then dissolved in DCM (5 mL) and cooled to ꢀ40 ºC. TMSOTf (4 ꢁL,
0.019 mmol) was added and the reaction mixture was stirred for 1 h at ꢀ40 ºC to ꢀ20 ºC before
being quenched with Et3N. The solvent was evaporated in vacuo and the residue was purified
by column chromatography (EtOAC;hexane, 1:9) to give the pseudo hexasaccharide 17 (0.64 g,
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71%). Rf = 0.4 (EtOAC;hexane, 1:4). H NMR (400 MHz, CDCl3) δ 8.05 – 7.98 (m, 2H), 7.46 –
7.03 (m, 63H), 5.98 (td, J = 10.9, 5.2 Hz, 1H), 5.73 (s, 1H), 5.61 (s, 1H), 5.54 (s, 1H), 5.35 (d, J
= 8.0 Hz, 1H), 5.19 (d, J = 10.5 Hz, 1H), 5.08 (d, J = 9.6 Hz, 2H), 4.99 – 4.70 (m, 10H), 4.68 –
4.34 (m, 14H), 4.25 (dd, J = 25.2, 12.7 Hz, 3H), 4.17 – 3.41 (m, 29H), 3.39 – 3.24 (m, 2H), 2.15
(s, 3H), 2.13 – 1.99 (m, 2H), 1.71 (q, J = 18.5, 14.4 Hz, 2H), 1.22ꢀ132 (m, 4H), 1.07 (s, 21H)
ppm; 13C NMR (101 MHz, CDCl3) δ 170.1, 165.3, 138.9, 138.6, 138.5, 138.2, 138.1, 137.9,
137.8, 137.4, 135.5, 133.2, 130.0, 129.8, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 128.0,
127.9, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.5, 127.4, 127.4, 127.3, 116.6, 100.01, 99.9,
99.1, 98.9, 93.7, 80.3, 80.2, 79.8, 79.6, 78.7, 78.69, 77.4, 77.3, 77.1, 76.7, 76.7, 75.4, 75.2,
75.1, 75.1, 74.8, 74.6, 74.4, 74.1, 73.8, 73.6, 73.4, 73.2, 72.7, 72.2, 72.2, 72.0, 71.8, 71.6, 71.5,
71.0, 69.5, 69.1, 69.0, 68.9, 68.8, 68.4, 66.5, 63.1, 62.7, 30.2, 29.7, 29.4, 28.4, 23.5, 23.2, 21.2,
18.2, 18.1, 18.0, 17.9, 12.0, 11.9 ppm. HRMS (ESI): m/z: calcd for C141H163N3O28Si [M+Na]+:
2397.1085; found 2397.1082.
(3,4,6ꢀtriꢀOꢀBenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtriisopropylsilylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ
αꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ
(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane(18): Hexasaccharide 17 (160 mg, 0.067 mmol) was
dissolved subsequently in DCM (1 mL) and MeOH (5 mL). NaOMe (0.5 M in MeOH, 1 mL) was
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added at room temperature. The reaction mixture was then heated to 50 ºC and stirred at this
temperature for 20 h. After cooling down, the reaction mixture was neutralized with acidic
DOWEX resin to approximately pH 6. The resin was removed by filtration and the filtrate was
evaporated to dryness and purification by column chromatography afforded diol 18 (120 mg,
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80%) as a gummy liquid. Rf = 0.25 (30% ethyl acetate in hexane). H NMR (400 MHz, CDCl3) δ
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7.58 – 7.10 (m, 60H), 6.11 – 5.93 (m, 1H), 5.46 (s, 1H), 5.38ꢀ5.33 (m, 2H), 5.29 – 5.19 (m, 2H),
5.16 (s, 1H), 5.08ꢀ4.80 (m, 6H), 4.85ꢀ4.48 (m, 18H), 4.44 – 4.11 (m, 9H), 4.11 – 3.48 (m, 24H),
3.45ꢀ3.30 (m, 2H), 2.54 (s, 1H, OH), 2.27 (brs, 1H, OH), 2.16ꢀ2.08 (m, 2H), 1.76 (s, 2H), 1.52 –
1.26 (m, 4H), 1.16 (s, 21H) ppm; 13C NMR (101 MHz, CDCl3) δ 139.0, 138.9, 138.7, 138.6,
138.3, 138.2, 138.1, 137.8, 137.7, 135.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0,
127.9, 127.8, 127.7, 127.7, 127.7, 127.5, 127.4, 127.3, 116.6, 101.5, 101.4, 100.0, 99.2, 93.7,
80.5, 80.2, 80.1, 79.8, 77.5, 77.3, 77.2, 76.8, 76.5, 75.6, 75.1, 75.0, 74.7, 74.4, 74.2, 73.7, 73.4,
73.2, 73.1, 72.4, 72.2, 72.0, 71.7, 71.5, 71.1, 69.6, 69.1, 68.6, 66.0, 63.0, 62.6, 60.4, 30.2, 29.7,
28.4, 23.5, 23.2, 18.2, 12.0 ppm. HRMS (ESI): m/z: calcd for C132H157N3O26Si [M+Na]+:
2251.0722; found 2251.0717
(2,3,4,6ꢀtetraꢀOꢀBenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtriisopropylsilylꢀ
αꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀ
o
glucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane(19): To a cooled (0 C) solution of
hexasaccharide diol 18 (120 mg, 0.053 mmol) in DMF (5 mL) was added NaH (60% in mineral
oil, 11 mg, 0.269 mmol) in one portion. After 20 min benzyl bromide (32 ꢁL, 0.269 mmol) was
added and the reaction mixture was stirred at rt for 12 h. The reaction mixture was quenched
with water and the aqueous layer was extracted with ethyl acetate (3 x 50 mL) and the
combined organic layers were further washed with brine and dried over anhydrous sodium
sulfate. Evaporation of the solvents gave the crude product, which was purified by column
chromatography (EtOAC:hexane, 1:4) to give 19 (110 mg, 85%) as a gummy liquid. Rf = 0.3
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(EtOAC:hexane, 1:9). H NMR (400 MHz, CDCl3) δ 7.50 – 7.10 (m, 70H), 6.13 – 6.00 (m, 1H),
5.46 (s, 1H), 5.44 – 5.33 (m, 3H), 5.29 (d, J = 6.6 Hz, 1H), 5.16 (s, 1H), 5.01 (dd, J = 10.5, 7.2
Hz, 5H), 4.94 (d, J = 10.9 Hz, 1H), 4.84 (d, J = 10.8 Hz, 1H), 4.80 – 4.54 (m, 16H), 4.54 – 4.35
(m, 6H), 4.30 (s, 2H), 4.27 – 3.80 (m, 24H), 3.77 – 3.66 (m, 4H), 3.59 (d, J = 10.8 Hz, 3H), 3.40
(d, J = 9.3 Hz, 2H), 2.15 (t, J = 13.6 Hz, 2H), 1.79 (brs, 2H), 1.38 (dt, J = 11.1, 10.7 Hz, 4H),
1.18 (d, J = 3.0 Hz, 21H) ppm; 13C NMR (101 MHz, CDCl3) δ 139.2, 139.1, 139.0, 138.8, 138.7,
138.6, 138.6, 138.5, 138.4, 138.4, 138.2, 138.0, 135.6, 128.6, 128.4, 128.4, 128.3, 128.3, 128.2,
128.1, 128.0, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.4, 127.4, 127.1, 116.7, 100.3,
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100.2, 99.7, 99.3, 93.8, 80.4, 80.3, 80.1, 80.0, 79.9, 77.6, 77.4, 77.2, 76.9, 76.6, 75.9, 75.2,
75.2, 75.0, 74.9, 74.8, 74.7, 74.5, 73.8, 73.7, 73.4, 73.3, 73.2, 72.7, 72.5, 72.3, 72.2, 72.1, 72.0,
71.9, 71.1, 69.7, 69.4, 69.1, 68.9, 66.7, 62.8, 30.3, 29.8, 28.4, 23.5, 23.3, 18.3, 12.1 ppm.
HRMS (ESI): m/z: calcd for C146H169N3O26Si [M+Na]+: 2431.1680; found 2431.1656.
(2,3,4,6ꢀtetraꢀOꢀBenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀ
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mannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀ
glucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane (20): To a solution of 19 (0.110 g,
0.046 mmol) in CH2Cl2 (4 mL) and MeOH (1 mL), AcCl (0.03 mL, 0.46 mmol) was added and
stirred at rt for 2 h. Reaction mixture was diluted with CH2Cl2 (50 mL) and washed with aq.
NaHCO3 solution, brine. The organic layer was dried over anhyd. sodium sulfate, evaporated
under reduced pressure and the residue was purified by column chromatography
(EtOAc:hexane, 1:4) to give 20 (0.095 g, 91%; Rf 0.3, EtOAc:hexane, 1:4). 1H NMR (400 MHz,
CDCl3) δ 7.71 – 6.84 (m, 78H), 6.06 (ddd, J = 22.5, 10.7, 5.5 Hz, 1H), 5.43 (d, J = 3.5 Hz, 1H),
5.30 (ddd, J = 35.5, 18.3, 4.0 Hz, 5H), 5.06 – 4.84 (m, 6H), 4.78 – 4.34 (m, 24H), 4.34 – 4.07
(m, 8H), 4.06 – 3.52 (m, 30H), 3.43 (dd, J = 10.2, 6.8 Hz, 1H), 3.30 (dd, J = 10.3, 3.5 Hz, 1H),
2.23 – 2.01 (m, 5H), 1.73 (d, J = 8.7 Hz, 2H), 1.58 – 1.46 (m, 1H), 1.34 (dd, J = 11.9, 4.6 Hz,
8H). 13C NMR (101 MHz, CDCl3) δ 138.7, 138.6, 138.6, 138.5, 138.5, 138.4, 138.3, 138.3,
138.1, 138.0, 137.9, 136.1, 135.6, 134.4, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1,
128.0, 127.97, 127.9, 127.86, 127.7, 127.6, 127.5, 127.4, 127.31, 127.0, 125.0, 116.3, 110.7,
100.4, 100.3, 100.0, 99.3, 99.1, 98.7, 84.6, 81.6, 80.9, 79.9, 79.7, 79.6, 79.4, 79.2, 77.9, 75.6,
75.2, 75.1, 74.9, 74.73, 74.6, 74.5, 74.4, 74.2, 74.0, 73.8, 73.7, 73.5, 73.4, 73.3, 73.1, 72.9,
72.7, 72.6, 72.5, 72.3, 72.1, 72.0, 71.8, 71.6, 71.4, 70.6, 70.4, 70.3, 69.9, 69.5, 69.2, 69.0, 67.4,
66.3, 64.1, 63.2, 62.6, 62.1, 61.7, 60.5, 54.7, 31.9, 29.9, 28.7, 24.0, 23.4, 21.8, 21.1, 19.6, 14.3;
HRMS (ESI): m/z: calcd for C137H149N3O26Na [M+Na]+: 2275.0327; found 2275.0322.
(2,3,4,6ꢀtetraꢀOꢀBenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀ(2ꢀ(Nꢀ
benzyloxycarbonyl)aminoethylꢀphosphonatoꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀ
azidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane
(22):
Ethanolamine Hꢀphosphonate 2118 (144 mg, 0.40 mmol) and hexasaccharide 20 (90 mg, 0.040
mmol) were coꢀevaporated with anhydrous pyridine (3 x 3 mL) in two separate 10 mL RBF and
dried under high vacuum overnight. The ethanolamine Hꢀphosphonate 21 was dissolved in
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anhydrous pyridine (1 mL) followed by Pivaloyl chloride (50 mL, 0.40 mmol) was added and
stirred for 15 min at room temperature. To that mixture a solution of hexasaccharide 20 in
anhydrous pyridine (1.5 mL) was added and the resulting mixture was stirred at room
temperature for 12 h. Iodine (100 mg, 0.40 mmol) in a mixture of pyridine/water (19:1, 0.5 mL)
was added to oxidize P(III) to P(V) and stirred for additional 6 h at room temperature. Reaction
mixture was diluted with CH2Cl2 (50 mL) and organic layer washed with sodium thiosulfate
solution, followed by 100 mM TEAB (triethyl ammonium borate) buffer solution and dried over
anhyd sodium sulfate. Solvents were evaporated on reduced pressure and the crude was
purified by flash column chromatography with Et3Nꢀdeactivated silica gel to give 22 (92 mg,
92%) as a gummy liquid. Rf 0.3 (CH2Cl2/MeOH = 9:1). 1H NMR (400 MHz, CDCl3) δ 7.53 – 6.96
(m, 77H), 6.10 – 5.98 (m, 1H), 5.34 (d, J = 16.1 Hz, 3H), 5.19 (dd, J = 12.1, 8.3 Hz, 3H), 5.02 (s,
2H), 4.98 – 4.75 (m, 5H), 4.75 – 4.42 (m, 17H), 4.31 (dd, J = 36.6, 11.8 Hz, 7H), 4.24 – 4.05 (m,
7H), 4.05 – 3.03 (m, 35H), 2.09 (d, J = 17.6 Hz, 3H), 1.67 (s, 3H), 1.47 (d, J = 8.3 Hz, 2H), 1.40
– 1.19 (m, 9H), 1.18 – 1.01 (m, 5H). 13C NMR (101 MHz, CDCl3) δ 138.42, 138.34, 138.12,
137.93, 137.63, 135.59, 131.63, 128.48, 128.37, 128.34, 128.25, 128.19, 127.96, 127.83,
127.69, 127.55, 127.48, 127.44, 127.09, 121.86, 116.24, 102.35, 98.60, 86.96, 79.87, 79.39,
78.21, 77.45, 77.13, 76.81, 76.16, 75.02, 74.59, 73.34, 73.11, 72.74, 72.45, 72.21, 71.52, 70.76,
70.56, 70.49, 70.30, 70.01, 69.60, 69.40, 68.93, 67.45, 63.07, 61.16, 31.81, 29.76, 27.22, 23.89,
23.40. 31P NMR (162 MHz, CDCl3) δ 0.30; C147H159N4O31P [MꢀH]: 2508.0804; found 2508.0799.
αꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(6ꢀOꢀaminoethylꢀphosphonato)ꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ
αꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀaminoꢀ2ꢀdeoxyꢀαꢀDꢀ
glucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀcyclohexane diol (1):
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Phosphorylated hexasaccharide 22 (90 mg, 0.035 mmol) was dissolved in a mixture MeOH (2
mL) and formic acid (5 ꢀl) followed by Pd(OH)2/C (20% Pd content) (20 mg) and the mixture
was purged with nitrogen. The mixture was then stirred in an atmosphere of hydrogen
(hydrogen balloon pressure) for 12 h. The reaction mixture was filtered through a fret filter with
methanol (10 mL). Solvents were evaporated with reduced pressure and dried under high
vacuum. 1H NMR of the crude shows the complete conversion of the starting material to
product. The product was passed through the biogel P2ꢀcolumn and lyophilized to give the
pseudoꢀGPI anchor 1 as a white solid (35 mg, 94%).
1H NMR (400 MHz, D2O) δ 5.27 (s, 1H), 5.20 (s, 1H), 5.13 (s, 1H), 5.08 (s, 1H), 5.02 (s, 1H),
4.07 (d, J = 21.2 Hz, 6H), 3.90ꢀ4.06 (m, J = 7.2 Hz, 4H), 3.89 – 3.64 (m, 18H), 3.59 (t, J = 8.6
Hz, 4H), 3.48 – 3.41 (m, 1H), 3.37 (s, 1H), 3.27 (d, J = 4.7 Hz, 3H), 2.93 (d, J = 11.6 Hz, 1H),
2.09 (s, 2H), 1.55 (dd, J = 15.0, 7.5 Hz, 2H), 1.21 (d, J = 8.5 Hz, 4H), 1.14 (d, J = 6.3 Hz, 3H),
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0.87 (t, J = 7.7 Hz, 2H); P NMR (162 MHz, CDCl3) δ 0.30 ppm; HRMS (ESI): m/z: calcd for
C41H74N2O29P [MꢀH]: 1089.4115; found 1089.4109.
(2ꢀOꢀAcetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtriisopropylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀ
cyclohexane (23): Pseudoꢀpentasaccharide 23 was synthesized in a manner as described for
17 by using trisaccharide 6b18 and pseudoꢀdisaccharide 16a with 74% yield.
1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 7.2 Hz, 2H), 7.67 – 6.94 (m, 48H), 6.02 – 5.99 (m, 1H),
5.78 (s, 1H), 5.58 (d, J = 11.3 Hz, 2H), 5.38 (d, J = 17.1 Hz, 1H), 5.27 – 5.20 (m, 2H), 5.15 (s,
1H), 5.09 – 4.84 (m, 6H), 4.80 – 4.28 (m, 14H), 4.28 – 3.47 (m, 23H), 3.38 (d, J = 6.6 Hz, 2H),
2.18 – 2.14 (m, 5H), 1.79 – 1.76 (m, 2H), 1.53 – 1.21 (m, 4H), 1.15 (s, 21H) ppm; 13C NMR (101
MHz, CDCl3) δ 170.0, 165.2, 163.6, 137.8, 137.5, 135.6, 133.3, 129.5, 128.7, 127.1, 116.6,
99.0, 94.4, 93.8, 92.1, 80.3, 80.0, 79.7, 78.8, 78.0, 75.5, 75.3, 75.2, 74.9, 74.5, 74.0, 73.7, 73.3,
72.1, 71.8, 71.6, 71.1, 69.5, 69.3, 69.1, 69.0, 66.7, 63.1, 62.6, 30.3, 28.6, 23.6, 23.3, 21.1, 18.2,
18.1, 12.1 ppm; HRMS (ESI): m/z: calcd for C114H135N3O23SiNa [M+Na]+: 1964.9153; found
1964.9148.
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2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀtriisopropylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀ
Dꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀ
Oꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane (24): 24 was
1
synthesized in a manner described for 18 and 19 with 76% yield. Data for 24: H NMR (400
MHz, CDCl3) δ 7.67 – 6.97 (m, 55H), 6.02 – 5.99 (m, 1H), 5.30 – 5.27 (m, 4H), 5.12 (d, J = 3.2
Hz, 1H), 5.07 – 4.82 (m, 5H), 4.82 – 4.29 (m, 18H), 4.30 – 4.05 (m, 6H), 4.05 – 3.47 (m, 18H),
3.47 – 3.28 (m, 2H), 2.99 – 2.88 (m, 2H), 2.43 (s, 1H), 2.13 (dd, J = 19.3, 7.5 Hz, 2H), 1.70 –
1.68 (m, 2H), 1.35 – 1.32 (m, 4H), 1.15 (s, 21H) ppm; 13C NMR (101 MHz, CDCl3) δ 141.1,
139.1, 138.9, 138.6, 138.5, 138.4, 138.2, 137.9, 135.6, 128.6, 128.5, 128.41, 128.4, 128.3,
128.3, 128.2, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 127.3, 127.0,
127.0, 116.7, 100.2, 99.3, 98.6, 93.7, 80.5, 79.9, 79.8, 75.1, 75.0, 74.7, 74.6, 73.8, 73.3, 73.1,
72.4, 72.1, 72.0, 71.7, 71.1, 69.6, 69.5, 69.1, 65.3, 63.1, 62.8, 29.8, 28.4, 23.5, 23.2, 20.0, 18.1,
18.1, 12.1 ppm; HRMS (ESI): m/z: calcd for C119H141N3O21SiNa [M+Na]+: 1998.9725; found
1998.9719.
2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀ
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Dꢀglucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane (25): 25 was synthesized in a
similar manner described for 20.
1H NMR (400 MHz, CDCl3) δ 7.66 – 6.86 (m, 55H), 5.97 ꢀ 5.94 (m, 1H), 5.31 (s, 2H), 5.11 (s,
3H), 4.91 – 4.89 (m, 5H), 4.73 – 4.21 (m, 20H), 4.11 (s, 3H), 4.01 – 3.60 (m, 20H), 3.53 (d, J =
10.9 Hz, 2H), 3.33 (d, J = 9.8 Hz, 2H), 2.06 (d, J = 11.3 Hz, 2H), 1.70 (s, 2H), 1.37 – 1.35 (m,
4H) ppm; 13C NMR (101 MHz, CDCl3) δ 138.6, 138.4, 138.2, 137.8, 135.4, 128.5, 128.40,
128.36, 128.3, 128.24, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.0, 116.7,
100.25, 99.6, 99.4, 96.9, 93.7, 80.0, 79.8, 79.5, 76.0, 75.2, 75.0, 74.8, 74.6, 74.3, 74.2, 73.8,
73.3, 73.1, 72.8, 72.6, 72.4, 72.2, 72.0, 71.9, 71.1, 69.6, 69.1, 66.4, 62.7, 62.3, 29.7; HRMS
(ESI): m/z: calcd for C110H121N3O21SiNa [M+Na]+: 1842.8390; found 1842.8408.
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2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀ(2ꢀ(Nꢀbenzyloxycarbonyl)aminoethylꢀphosphonatoꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀ
glucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ2ꢀOꢀallylꢀcyclohexane (26): 26 was synthesized in a manner
described for 22. 1H NMR (400 MHz, CDCl3) δ 7.24 – 7.18 (m, 60H), 6.00 – 5.85 (m, 1H), 5.31
(d, J = 9.2 Hz, 2H), 5.21 – 5.06 (m, 2H), 4.99 (d, J = 17.9 Hz, 3H), 4.87 (d, J = 10.9 Hz, 2H),
4.82 – 4.59 (m, 5H), 4.59 – 4.19 (m, 16H), 4.19 – 3.96 (m, 5H), 3.96 – 3.57 (m, 17H), 3.53 –
31
3.22 (m, 6H), 2.85 (s, 6H), 2.03 (s, 2H), 1.67 (s, 2H), 1.40 – 1.14 (m, 4H); P NMR (162 MHz,
CDCl3) δ ꢀ0.05 ppm; HRMS (ESI): m/z: calcd for C120H134N3O26P [M+H]+: 2077.9024; found:
2077.9018.
(6ꢀOꢀAminoethylꢀphosphonato)ꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀαꢀDꢀmannopyranosyl)ꢀ
(1→6)ꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀaminoꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→1)ꢀ(1R,2R)ꢀ
cyclohexane diol (2): Hexasaccharide 2 was synthesized by a similar manner described for 1.
1H NMR (400 MHz, D2O) δ 5.25 (d, J = 3.5 Hz, 1H), 5.14 (s, 1H), 5.04 (s, 1H), 4.95 (s, 1H), 4.18
– 3.95 (m, 8H), 3.95 – 3.44 (m, 20H), 3.33 – 3.30 (m, 2H), 3.16 – 3.01 (m, 2H), 2.95 (d, J = 7.4
Hz, 1H), 2.03 (s, 2H), 1.68 (d, J = 5.5 Hz, 2H), 1.54 – 1.50 (m, J = 37.3, 6.0 Hz, 2H), 1.18 (dd, J
= 12.8, 7.3 Hz, 4H), 0.80 (t, J = 7.4 Hz, 3H) ppm; HRMS (ESI): m/z: calcd for C35H66N2O24P
[M+H]+: 929.3743; found: 929.3738.
(2ꢀOꢀAcetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀ
triisopropylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→6)ꢀ(2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ
2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→6)ꢀ1ꢀOꢀallylꢀ 2,3,4,5ꢀtetraꢀOꢀbenzylꢀDꢀmyoꢀinositol (28):
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Pseudoꢀhexaꢀsaccharide 28 was synthesized in a manner as described for 17 by using
tetrasaccharide 8b and pseudoꢀdisaccharide 27a18 with 70% yield.
1H NMR (400 MHz, Chloroformꢀd) δ 8.11 (d, J = 6.6 Hz, 2H), 7.65 – 6.92 (m, 85H), 6.16 – 5.99
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(m, 1H, O-Allyl CH), 5.94 – 5.78 (m, 2H, anomeric H), 5.78 – 5.60 (m, 2H, anomeric H), 5.55 –
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5.28 (m, 3H), 5.27 – 5.13 (m, 2H), 5.11 – 3.26 (m, 66H), 2.24 (s, 3H), 1.16 (s, 21H); C NMR
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(101 MHz, CDCl3) δ 170.2, 165.4, 139.0, 138.9, 138.7, 138.6, 138.5, 138.3, 138.0, 137.8, 137.5,
134.4, 133.2, 130.1, 129.9, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.6, 127.6, 127.4,
127.2, 127.1, 117.2, 100.1, 100.0, 99.2, 99.1, 97.7, 82.1, 81.9, 81.4, 81.0, 80.6, 78.0, 79.7, 78.8,
75.9, 75.6, 75.4, 75.2, 74.6, 74.5, 74.2, 73.6, 73.5, 73.3, 73.0, 72.7, 72.3, 72.1, 71.9, 71.7, 71.3,
70.9, 69.7, 69.1, 68.9, 68.5, 66.3, 63.7, 29.8, 21.3, 18.3, 12.1 ppm; HRMS (ESI): m/z: calcd for
C169H187N3NaO32Si [M+Na]+: 2821.2765; found: 2821.2712.
(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtriisopropylsilylꢀ
αꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀ
glucopyranosyl)ꢀ(1→6)ꢀ1ꢀOꢀallylꢀ 2,3,4,5ꢀtetraꢀOꢀbenzylꢀDꢀmyoꢀinositol (29):
Compound 29 was synthesized in a manner described for 18 and 19 with 72% yield.
1H NMR (400 MHz, CDCl3) δ 7.64 – 6.95 (m, 92H), 6.09 – 5.91 (m, 1H), 5.85 (d, J = 3.1 Hz, 1H),
5.45 – 5.20 (m, 5H), 5.16 – 3.24 (m, 73H), 1.10 (d, J = 4.9 Hz, 21H) ppm; 13C NMR (101 MHz,
CDCl3) δ 138.8, 138.6, 138.5, 138.4, 138.0, 134.3, 128.5, 128.4, 128.3, 128.2, 128.2, 128.1,
127.9, 127.8, 127.7, 127.5, 127.5, 127.4, 127.2, 127.1, 126.9, 117.2, 100.5, 100.3, 99.7, 99.2,
97.4, 82.0, 81.5, 80.9, 80.5, 80.2, 80.1, 80.0, 79.9, 76.1, 75.9, 75.1, 74.8, 73.4, 73.2, 72.2, 72.1,
71.6, 71.5, 71.3, 70.9, 69.7, 69.0, 68.9, 68.8, 68.7, 63.3, 62.6, 18.2, 12.0 ppm; HRMS (ESI):
m/z: calcd for C174H193N3O30NaSi [M+Na]+: 2832.3438; found:. 2832.3467.
(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀazidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀ
glucopyranosyl)ꢀ(1→6)ꢀ1ꢀOꢀallylꢀ 2,3,4,5ꢀtetraꢀOꢀbenzylꢀDꢀmyoꢀinositol (30):
Compound 30 was synthesized in a manner described for 20 with 87% yield.
1H NMR (400 MHz, CDCl3) δ 7.52 – 6.97 (m, 92H), 6.04 – 5.89 (m, 1H), 5.79 (d, J = 3.6 Hz, 1H),
5.37 – 5.18 (m, 4H), 5.13 (s, 1H), 5.04 (d, J = 11.2 Hz, 1H), 4.92ꢀ4.89 (m, 9H), 4.82 – 4.72 (m,
3H), 4.60 – 4.55 (m, 16H), 4.43 – 4.25 (m, 7H), 4.23 – 3.96 (m, 10H), 3.96 – 3.32 (m, 26H), 3.26
(dd, J = 16.3, 6.5 Hz, 2H) ppm; 13C NMR (101 MHz, CDCl3) δ 138.9, 138.6, 138.1, 134.3, 128.5,
128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.5, 127.2, 127.1, 126.9, 117.3, 100.7, 100.2, 99.0,
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97.4, 81.9, 81.4, 81.0, 80.1, 78.0, 79.5, 78.9, 76.3, 76.0, 75.4, 75.1, 75.0, 74.7, 74.4, 74.1, 73.8,
73.3, 73.2, 72.8, 72.7, 72.7, 72.4, 72.3, 72.0, 71.9, 71.4, 71.0, 69.7, 68.9, 68.4, 66.1, 63.3, 62.0
ppm; ; HRMS (ESI): m/z: calcd for C165H173N3O30Na [M+Na]+: 2699.2002; found: 2699.2052.
(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀ(2ꢀ(Nꢀ
benzyloxycarbonyl)aminoethylꢀphosphonatoꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀ
benzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀ
azidoꢀ3,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀDꢀglucopyranosyl)ꢀ(1→6)ꢀ1ꢀOꢀallylꢀ2,3,4,5ꢀtetraꢀOꢀbenzylꢀ
Dꢀmyoꢀinositol (31):
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Compound 31 was synthesized in a similar manner described for 22 with 92% yield.
1H NMR (400 MHz, CDCl3) δ 7.84 (s, 4H), 7.52 – 6.85 (m, 93H), 6.00 – 5.87 (m, 1H), 5.76 (s,
1H), 5.62 (s, 1H), 5.39 – 5.08 (m, 4H), 5.08 – 2.99 (m, 80H); 13C NMR (101 MHz, CDCl3) δ
144.9, 141.7, 139.0, 138.8, 138.5, 138.3, 137.9, 134.2, 128.5, 128.4, 128.3, 128.1, 128.0, 127.9,
127.7, 127.5, 127.4, 127.1, 127.0, 117.3, 100.2, 99.0, 97.4, 96.1, 81.9, 81.5, 80.9, 79.5, 79.1,
75.8, 75.5, 75.2, 75.0, 74.4, 74.1, 73.9, 73.3, 73.2, 72.8, 72.4, 72.2, 72.1, 71.8, 71.7, 71.4, 70.9,
69.7, 68.9, 66.6, 66.2, 63.2 ppm 31P NMR (162 MHz, CDCl3) δ ꢀ0.26 ppm; HRMS (ESI): m/z:
calcd for C175H184N3O35P [MꢀH]+: 2932.2479; found: 2932.2490.
αꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(6ꢀOꢀaminoethylꢀphosphonato)ꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ
αꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀαꢀDꢀmannopyranosyl)ꢀ(1→4)ꢀ(2ꢀaminoꢀ2ꢀdeoxyꢀαꢀDꢀ
glucopyranosyl)ꢀ(1→6)ꢀ Dꢀmyoꢀinositol (3):
GPI analog 3 was synthesized in a similar manner described for 1 with 94% yield.
1H NMR (400 MHz, D2O) δ 5.28 (s, 1H), 5.21 (s, 1H), 5.16 (s, 1H), 5.02 (s, 1H), 4.97 (s, 1H),
4.23 (s, 1H), 4.04 (s, 4H), 3.99 (s, 2H), 3.94 – 3.85 (m, 4H), 3.83 – 3.30 (m, 28H), 3.21 (s, 2H),
3.13 – 3.06 (m, 1H), 1.60 – 1.48 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H) ppm; 31P NMR (162 MHz,
CDCl3) δ ꢀ0.26 ppm; HRMS (ESI): m/z: calcd for C41H74N2O33P [MꢀH]+: 1153.3911; found:
1153.3982.
Conjugation of Glycans (1, 2 and 3) with activated KLH:
GPIꢀKLH conjugates (GPI1, GPI2 and GPI3) were synthesized from 1, 2 and 3 by using Immꢀ
Link KLH (Amine) Immunogen labeling Kit and Conjugation Protocol provided by Novobios
(http://www.novusbio.com/ImmꢀLinkꢀKLHꢀ(Amine)ꢀKit_450ꢀ0500.html). Briefly, 2 mg of ImmꢀLink
KLH (molecular weight ~400 KDa; 0.0050 ꢀmol, 1 equivalent) was reacted with 1 mg glycan 1
or 2 or 3 (~200 equivalent). Glycan 1 or 2 or 3 (1 mg) dissolved in 10 mM phosphate buffer (1
mL), 50 ꢀl amine modifier provided by the manufacturer was added at rt. After 10 min, the
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solution was added to the vial containing preꢀactivated KLH (2 mg) and stirred at rt for 12 h. The
solution was transferred to dialysis cassettes with a cutoff of 7000 Dalton and dialyzed against
distilled water three times to remove unreacted material and impurities. After lyophilization, the
final conjugates (GPI1 (1.90 mg), GPI2 (1.82 mg) and GPI3 (1.98 mg) were obtained as white
solids. The glycoconjugates were analyzed by phenolꢀsulfuric acid assay method. (Supporting
information)
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Rabbit immune sera preparation
Six rabbits were immunized with the three synthetic GPIs (rabbit 1 and 2 were immunized with
GPI1; rabbit 3 and 4 with GPI2; rabbit 5 and 6 with GPI3), according to a Standard 70ꢀday rabbit
immunization protocol (Thermo Scientific). The primary injection was carried out on day 1 and
0.5mg of antigen was injected; subsequently, three booster injections with 0.25mg of antigen
were given on day 14, day 28 and day 42 respectively. The titer of elicited antiꢀGPI antibodies
from rabbit sera at various bleeding time point (I: before immunization; II: 7 days after the 1st
injection; III: 7 days after the 2nd injection; IV: 7 days after the 3rd injection; V: 14 days after the
4th injection) were measured using Luminex assay, which is a colorꢀcoded beadꢀbased binding
assay. Briefly, antigenꢀcoated beads were incubated with rabbit sera and the bounded rabbit
IgG was detected by a complex of biotinylated antiꢀrabbit IgG and Phycoerythrin (PE)ꢀ
conjugated Streptavidin.
Luminex assay
GPI analogues 1, 2 and 3 were coupled to carboxylated MagPlex® Microspheres (Luminex
Corporation). The coated beads were incubated with rabbit sera at a 1:1000 dilution at room
temperature for 1h and washed three times with phosphate buffer saline containing 0.5%
Tweenꢀ20 (PBST). The beads then were incubated with biotinylated goat antiꢀrabbit IgG
(0.5ꢂg/ml, SouthernBiotech) at room temperature for 45min and washed three times with PBST.
Next, the beads were incubated with Streptavidin Phycoerythrin (2ꢂg/ml, Lifetechnologies) at
room temperature for 30min followed by three washes with PBST. The fluorescence intensity of
beads were measured and recorded by a BioꢀPlex® MAGPIX™ Multiplex Reader. The
fluorescence intensity shown in Figure 1 is the mean value of 50 beads after subtracting the
background.
ELISA
AntiꢀKLH antibodies were removed from the total IgG by solid phase immunoꢀaffinity column
chromatography over a KLHꢀ Separopore® 4B to enrich for GPIꢀspecific antibodies. The purified
antiꢀGPI antibodies were used in the ELISA assay to determine the elicited response to native
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plasmodium GPI. P. falciparum GPIs were kindly given by Dr. D. Channe Gowda, which were
extracted from natural P. falciparum parasites and purified by HPLC. GPIs were dissolved in
methanol to yield a coating solution of 40ng/ml. 50 ꢁl of coating solution was added to each well
and the plate was dried in incubator at 37oC overnight. The wells were washed once with 100 ꢁl
of PBST and blocked with PBSꢀcasein (pH7.4, 0.5 % casein) for 1 h at rt. 50ꢁl of diluted sera
samples (1:10) in PBSꢀcasein, containing 0.05 % Tween 20 was added to the wells and
incubated for 1h at rt. The wells were washed three times with PBST and 50ꢁl of polyꢀHRP
conjugated goat antiꢀrabbit antibodies (1:200 dilution in PBSꢀcasein) was added to each well.
After 1h of incubation at rt, the wells were washed three times with 100 ꢁl of PBST. 50 ꢁl of
TMB peroxidase substrate solution (KPL) was added to each well and incubate at rt for 1 h. 50
ꢁl of 1 M sulfuric acid was added to quench the reaction and absorbance at 405 nm was
measured. Each sample was assayed in triplicate. The control human hyperimmune serum was
obtained from a previous study carried out in Kenya,33 which was pooled from samples of
patients with multiple exposures to P. falciparum infection.
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Associated content
Supporting Information
The NMR spectra available free of charge via the Internet at http://pubs.acs.org.
Author information
Corresponding Author
siyer@gsu.edu
Acknowledgment
We are grateful to the Office of the Vice President of Research, Georgia State University,
Atlanta, GA for funding.
References:
1.
2.
(2013) World Health Organization Malaria Report Geneva.
White, N. J., Pukrittayakamee, S., Hien, T. T., Faiz, M. A., Mokuolu, O. A., and Dondorp,
A. M. Malaria, The Lancet 383, 723ꢀ735.
3.
Amexo, M., Tolhurst, R., Barnish, G., and Bates, I. (2004) Malaria misdiagnosis: effects
on the poor and vulnerable, The Lancet 364, 1896ꢀ1898.
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