Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines Andrei V. Nikolaev, a Nawaf Al-Maharik a and Olga V. Sizova b DOI: 10.1039/9781849730891-00101 Dedicated to the memory of Professor Vladimir N. Shibaev 1. Introduction Leishmania are a genus of sandfly-transmitted protozoan parasites that cause a spectrum of debilitating and often fatal diseases in humans throughout the tropics and subtropics. The disease (termed leishmaniasis) torments over 12 million people worldwide, making 2.4 million people disabled and causing about 60000 deaths annually. The WHO estimations show that 350 million people are thought to be at risk from the disease. 1–3 Although leishmaniasis is most common in tropical regions, it has also been diagnosed in overseas travellers and U.S. Gulf War veterans 4 and has emerged as an opportunistic infection of HIV patients. 5 The geographical distribution and pathology of leishmaniasis varies according to the species of the parasite. For instance, Leishmania donovani causes ‘visceral leishmaniasis’ (also known as kala azar), characterised by an enlarged liver and splin, that is often fatal, whereas L. major and L. tropica (in the Old World) and L. mexicana species complex, consisting of L. mexicana, L. amazonensis and L. venezuelensis mostly (in the New World), cause ‘cutaneous leishmaniasis’ (known as oriental sore and characterised by self-limiting skin lesions). 1 This form is the most common leishmanial disease, and is often fairly mild in comparison to the others. Widespread, chronic, non-ulcerative skin lesions resembling leprosy are in- dicative of ‘diffuse cutaneous leishmaniasis’ 6,7 that is commonly an effect of L. aethiopica (Old World) and L. mexicana species complex (New World) infection. ‘Mucocutaneous leishmaniasis’ (or espundia) is due to L. (V.) braziliensis (Viannia subgenus) infection and most prevalent in Brazil, Bo- livia and Peru. It causes lesion in the mucous membranes, leading to dev- astation of tissues and extreme facial disfigurement. 1,8 Secondary bacterial infections are common. L. (L.) mexicana causes ‘‘Chiclero’s ulcer’’, which can involve almost total destruction of the external ear. The mechanism of infection 9 poses a serious challenge for therapeutic approaches. The vector for Leishmania is a sandfly of Phlebotomus (Old World) or Lutzomyia (New World) female species that inject the parasite when taking a blood meal. To initiate and propagate the infection, parasites then actively invade and reside within macrophages, the very part of the immune system that is designed to destroy them. The majority of the current anti-leishmanial drugs, such as Sb(V) based medications Pentostam and Glucantime or non-antimonial Amphotericin B, Paromomicin and a College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee DD1 5EH, UK, E-mail: [email protected]b N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Carbohydr. Chem. , 2010, 36, 101–126 | 101 c The Royal Society of Chemistry 2010 Downloaded by Stanford University on 08/04/2013 05:31:34. Published on 01 August 2010 on http://pubs.rsc.org | doi:10.1039/9781849730891-00101
Transcript
Synthetic glycoconjugates based onLeishmania lipophosphoglycan structures aspotential anti-leishmaniasis vaccines
Andrei V. Nikolaev,a Nawaf Al-Maharika and Olga V. Sizovab
DOI: 10.1039/9781849730891-00101
Dedicated to the memory of Professor Vladimir N. Shibaev
1. Introduction
Leishmania are a genus of sandfly-transmitted protozoan parasites that causea spectrum of debilitating and often fatal diseases in humans throughout thetropics and subtropics. The disease (termed leishmaniasis) torments over 12million people worldwide, making 2.4 million people disabled and causingabout 60 000 deaths annually. The WHO estimations show that 350 millionpeople are thought to be at risk from the disease.1–3 Although leishmaniasisis most common in tropical regions, it has also been diagnosed in overseastravellers and U.S. Gulf War veterans4 and has emerged as an opportunisticinfection of HIV patients.5 The geographical distribution and pathology ofleishmaniasis varies according to the species of the parasite. For instance,Leishmania donovani causes ‘visceral leishmaniasis’ (also known as kalaazar), characterised by an enlarged liver and splin, that is often fatal, whereasL. major and L. tropica (in the Old World) and L. mexicana species complex,consisting of L. mexicana, L. amazonensis and L. venezuelensismostly (in theNew World), cause ‘cutaneous leishmaniasis’ (known as oriental sore andcharacterised by self-limiting skin lesions).1 This form is the most commonleishmanial disease, and is often fairly mild in comparison to the others.Widespread, chronic, non-ulcerative skin lesions resembling leprosy are in-dicative of ‘diffuse cutaneous leishmaniasis’6,7 that is commonly an effect ofL. aethiopica (Old World) and L. mexicana species complex (New World)infection. ‘Mucocutaneous leishmaniasis’ (or espundia) is due to L. (V.)braziliensis (Viannia subgenus) infection and most prevalent in Brazil, Bo-livia and Peru. It causes lesion in the mucous membranes, leading to dev-astation of tissues and extreme facial disfigurement.1,8 Secondary bacterialinfections are common. L. (L.) mexicana causes ‘‘Chiclero’s ulcer’’, whichcan involve almost total destruction of the external ear.
The mechanism of infection9 poses a serious challenge for therapeuticapproaches. The vector for Leishmania is a sandfly of Phlebotomus (OldWorld) or Lutzomyia (New World) female species that inject the parasitewhen taking a blood meal. To initiate and propagate the infection, parasitesthen actively invade and reside within macrophages, the very part of theimmune system that is designed to destroy them. The majority of the currentanti-leishmanial drugs, such as Sb(V) based medications Pentostam andGlucantime or non-antimonial Amphotericin B, Paromomicin and
aCollege of Life Sciences, Division of Biological Chemistry and Drug Discovery, University ofDundee, Dundee DD1 5EH, UK, E-mail: [email protected]. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
Carbohydr. Chem., 2010, 36, 101–126 | 101
�c The Royal Society of Chemistry 2010
Dow
nloa
ded
by S
tanf
ord
Uni
vers
ity o
n 08
/04/
2013
05:
31:3
4.
Publ
ishe
d on
01
Aug
ust 2
010
on h
ttp://
pubs
.rsc
.org
| do
i:10.
1039
/978
1849
7308
91-0
0101
Miltefosine, seem to be inadequate due to factors such as cost, resistanceand toxic side effects.10–14 Thus, a vaccine that would protect the humanhost by annihilating the parasites upon transfer from the insect vectorwould be welcome. Several different designs have been recently explored,15
but no generally applicable vaccine for leishmaniasis has yet come to light.In principle, any unique feature Leishmania expose on its cell surface
could be investigated and exploited for the development of an anti-leish-maniasis vaccine.16 Leishmania are digenetic organisms that alternate be-tween the insect vector and a mammalian host. The most abundantmacromolecule on the surface of the insect infectious stage of all Leishmaniaspecies is a complex glycoconjugate called lipophosphoglycan (LPG, seeSection 3). Although killed or attenuated parasites did not result a validvaccine, the LPG itself has been suggested to be a perspective vaccinecandidate as the protective effect of the purified LPG fraction on micechallenged with L. major was demonstrated.17–19 Here we discuss chemicalsynthesis of Leishmania phosphoglycans and the preparation of syntheticglycoconjugates (neoglycoproteins and neoglycolipids) based on variousLPG structures as potential anti-leishmaniasis vaccines as well as the im-munological evaluation for some of them.
2. Glycoconjugate synthetic vaccines
The carbohydrate vaccine technology is an area developing rapidly becauseof modern bioconjugation techniques,20–22 which allow an effective for-mation of carbohydrate-protein and carbohydrate-lipid conjugates. Tradi-tional polysaccharide vaccines, i.e., vaccination with highly purifiedimmunogenic bacterial polysaccharides [usually capsular polysaccharides(CPS)],23,24 have been associated with the short duration of the inducedimmunity and proved to be poorly immunogenic in infants and youngchildren.25,26 That happens because the polysaccharide antigens are T-cellindependent: they induce an immune response without the involvement ofT-cells. The response lacks several important features that characterise theT-cell dependent immune response, such as immunological memory, anantibodies class switch from IgM to IgG, and affinity maturation.23 Con-jugation of the bacterial polysaccharide to an immunogenic protein carrier(detoxified versions of strongly immunogenic proteins like diphtheria andtetanus toxins are often used) converts it to a T-cell dependent antigen withenhanced immunogenicity. Thus, the new generation of glycoconjugatevaccines against bacterial infections are immunogenic in infants and inducelong lasting immunological memory resulting in a boostable response.27 In1990s, four anti-meningitis glycoconjugate vaccines against Haemophilusinfluenzae b (Hib) were introduced with great result: the Hib-inducedmeningitis has virtually been eradicated in countries with high immunisa-tion coverage.28 Similarly, a tetravalent meningococcal glucoconjugatevaccine against Neisseria meningitidis serogroups A, C, Y and W135 hasrecently been developed by Sanofi-Pasteur and licensed in the USA.29
The glycan parts of modern glycoconjugate vaccines are, normally, nativeor functionalised bacterial CPS. These naturally derived carbohydratepolymers are heterogeneous mixtures that may include impurities and
contaminants. The use of synthetic carbohydrate structures, which can bechemically produced as single compounds in a controlled manner withoutbatch-to-batch variability, can eliminate these problems. Recently, a fullysynthetic hexasaccharide conjugated to keyhole limpet hemocyanin (KLH)was shown to be an effective vaccine against prostate cancer.30 The meth-odology has been used by Verez-Bencomo et al.31 for the preparation of thefirst commercial synthetic anti-meningitis glycoconjugate vaccine againstHib. The results of clinical testing in the target population demonstrated thatthe glycovaccine containing fully synthetic phosphoglycans (which areCPS fragments) is as effective as its natural counterpart. The vaccine wasregistered in Cuba in 2003 and is now part of the nation immunizationprogramme. Similarly, a synthetic vaccine against Shigella dysenteriae type 1based on a synthetic oligosaccharide conjugated to a protein was found to bemore immunogenic than a glycoconjugate prepared from the native poly-saccharide.32 Synthetic preparation of the glycan part of the glycoconjugatevaccines also allows for chemical modifications of the structure that may notbe possible to perform on the native material.
3. Cell-surface and secreted phosphoglycans of Leishmania
During the parasite life cycle, which involves a promastigote stage(s) in themidgut of the insect vector and an amastigote stage in the phagolysosomesof the mammalian macrophage, Leishmania survive and proliferate inhighly hostile environments. Their survival strategies involve the formationof an elaborate and dense cell-surface glycocalyx composed of diverse stage-specific glycoconjugates that form a protective barrier.33,34 Some of thesemacromolecules (including various phosphoglycans) were shown to be es-sential for virulence of the parasite.35–37
Lipophosphoglycan (LPG) is a predominant cell-surface glycoconjugateof Leishmania promastigotes. Structurally, this is a poly(glycosyl phos-phate) consisting of the Gal-b-(1-4)-Man-a-1-OPO3 repeating units(Fig. 1), where the nature of the X and Y substituents varies according tothe species. L. donovani34,38 and L. infantum synthesise a linear phos-phoglycan, whereas those of L. mexicana,39,40 L. major41–43 and L. aethio-pica44 have branched structures bearing non-stoichiometric mono- and/ordi-saccharide components (X) linked at O-3 of the b-D-Galp residue. InL. major, the composition of the side chains varies and depends on thedevelopmental stage of the parasite. In the LPG produced by L. aethiopicathere are also some additional a-D-Manp residues (Y) linked to D-mannoseresidues in the main chain. All LPG molecules are capped at thenon-reducing terminus with a unique tetrasaccharide phosphate unit(GalMan3-phosphate, where terminal b-D-Galp and a-D-Manp residues arenon-stoichiometric) and contain a glycan core (not shown) linked to aninositolphospholipid anchor33 at the reducing end of the chain.
In addition to cell-surface LPG, Leishmania promastigotes produce anumber of secreted glycoproteins, where a peptide core is modified (glyco-sylated) with LPG-type phosphoglycans.46,47 L. donovani secretes an acidphosphatase (sAP) in which the peptide chains are heavily glycosylated onC-terminal serine/threonine domains. The glycans (which are, in fact,
phosphoglycans) consist of the same disaccharide phosphate repeating unitsfound in the corresponding LPG (Fig. 1) and are phosphodiester-linked toselect serine residues.34 All species of Leishmania promastigotes synthesisesecreted filamentous proteophosphoglycan (fPPG), which forms a highlyviscous gel surrounding the parasite cells. Compositionally fPPG consistsof 95% phosphoglycans in a species-specific manner (Fig. 1). The poly(glycosyl phosphate) chains are attached to serine residues of the peptidecomponent via phosphodiester groups.
Hydrophilic phosphoglycans have been found in culture supernatantsof Leishmania promastigotes and have been structurally characterized forL. major,48 L. mexicana49 and L. donovani.50 They are essentially the cor-responding LPG molecules without the glycan core-inositolphospholipidregion.
L. mexicana amastigotes synthesise large amounts of a macromolecularamastigote-specific proteophosphoglycan (aPPG) and secrete it (in mg/mLconcentrations!) into the phagolysosome of the mammalian macrophage.45
Some phosphoglycan chains are similar to those found in L. mexicana LPG,but the majority represent novel stage-specific highly-branched structures(Fig. 1), including (1-3)-linked glucobiose and glucotriose moieties andlong phosphorylated side chains. The phosphoglycans are attached to serineresidues of the protein backbone, most likely through the phosphate groups.The aPPG is believed to activate the complement system via the mannose-binding pathway. It may also contribute to the binding of Leishmania tohost cells and play a role in modulation of the biology of the infectedmacrophage.34
4. Chemical synthesis of parasitic phosphoglycans of Leishmania
The most distinctive part of Leishmania lipophosphoglycans (LPG) is theirvariable phosphoglycan domain, made of phosphodisaccharide repeats ofthe Gal-b-(1-4)-Man-a-1-OPO3 structure linked to each other through aphosphodiester group between the anomeric OH of the D-mannose of onerepeat and the 6-OH of the D-galactose of the adjoining repeat (see Section3). The dynamic structure of the phosphoglycans and their role in host-parasite interaction led to significant biological interest. This upholds theneed to develop routes for the chemical preparation of these biopolymers.The development of synthetic approaches to phosphoglycans is challenging,because the phosphoglycans are hydrolytically labile due to anomericphosphodiester linkage present between the repeating units. Synthesis ofanomeric phosphodiesters is complicated, as both the correct stereo-chemistry at C-1 and the lability of anomeric phosphodiester linkages mustbe taken into consideration. For these reasons, only few syntheses ofanomerically-linked phosphoglycans have been reported51 so far.
4.1 Syntheses of phoshoglycans using stepwise chain elongation
The first successful syntheses of Leishmania phosphoglycans were achievedby the Dundee University group (Nikolaev et al.52,53) by exploiting theH-phosphonate chemistry. The glycosyl H-phosphonate route was provento be the method of choice for the efficient and reliable assembly of various
phosphodiester linkages in natural phosphoglycans.51 The L. donovaniphosphoglycans 1–3 [linear chains, built up from disaccharide phosphaterepeating units in (1-6)-phosphodiester linkage and varying in length andby the presence of the Man2-phosphate cap in 3; see Scheme 1] were con-structed in a stepwise chain elongation manner from the disaccharideH-phosphonate derivatives 4 and 5 (for the consecutive introduction of thedisaccharide phosphate repeats) and the disaccharide monohydroxyl block6. The 6-O-dimethoxytrityl (DMT) was chosen as a temporary protectinggroup in the D-galactose moiety since it could be removed in very mild acidicconditions (1% TFA in DCM, 0 1C, 1–2min, followed by aqueous work up)without interfering with the labile glycosyl phosphate linkages. Benzoatesand acetates served for permanent O-protection. The disaccharides 4–6were, in turn, prepared (Scheme 2) from simple monosaccharides followedby protecting group remodelling. Thus, glycosylation of the decenyl man-noside acceptor 10 with acetobromogalactose 9 in the presence of AgOTfgave the corresponding (b1-4)-linked disaccharide (67%; some of the
a-linked isomer, 15%, was also isolated), which was then de-O-acetylated(with HCl in MeOH)54 and converted to the monohydroxyl block 6 (71%)via successive 60-O-dimethoxytritylation, benzoylation and detritylation.
Coupling of the same donor 9 with the mannose 4-OH derivative 7,followed by similar reaction sequence with the exception of the detritylationstep, provided the disaccharide derivative 8, which was transformed into theH-phosphonate 4 on successive anomeric debenzoylation55,56 with Me2NH(77%), 1-O-phosphitylation55,56 with PCl3-imidazole-Et3N system and mildhydrolysis (92%). Compound 8 was also resynthesised recently57 byTMSOTf assisted coupling the 6-O-TBS protected trichloroacetimidate 11
and the acceptor 7 (82%) and subsequent 60-O-reprotection. Finally, themannobiosyl H-phosphonate 5 was made up from synthons 12 and 13 bythe glycosylation reaction, followed by anomeric deacetylation andH-phosphonylation (as it was done for 8).
With all the building blocks in hand, the assembly of the phosphoglycanswas only one step away (Scheme 1). Adamantane-1-carbonyl chloride as-sisted condensation of the disaccharide H-phosphonate 4 with the alcohol 6,followed by in situ oxidation with iodine in aqueous pyridine and subsequentmild acidolysis of the DMT group afforded the phosphotetrasaccharide 14
Scheme 2 Disaccharide building blocks for the phosphoglycan synthesis.
(81%). Coupling of 14 with the same H-phosphonate 4, employing theprescribed route of the oxidation and de-O-tritylation, provided the phos-phohexasaccharide 15 (75%), which on coupling with the mannobiosyl H-phosphonate 5 followed by oxidation (89%) and global deprotection usingMeONa in MeOH furnished the octasaccharide triphosphate phosphoglycan3 (99.5%). Compounds 14 and 15 were also debenzoylated to provide theshorter phosphoglycans 1 and 2, respectively.
All the synthetic phosphoglycans prepared by the Dundee group containa dec-9-enyl aglycone moiety as they were purposely designed for (1)studying Leishmania biosynthetic enzymes and (2) the preparation of neo-glycoconjugates, which could be tested as potential anti-leishmaniasis vac-cines.53 The hydrophobicity of the dec-9-enyl chain has been exploited forassaying, first, the mannosylphosphate transferases58,59 and then thegalactosyltransferases60,61 involved in the LPG biosynthesis. As for thebioconjugation, the high lability of the anomeric phosphodiester bridges inphosphoglycans was given the top priority, while selecting techniques forthe neoglycoconjugate preparation. It has been decided to avoid the use ofany condensing reagents (which could easily interfere with the phosphategroups and facilitate the cleavage of the glycosyl phosphate bonds) andprofit from the ozonolysis of the double bond in the dec-9-enyl aglyconelinker instead, thus transforming it into an aldehyde functionality, whichcould be coupled to a protein or a lipid carrier by reductive amination (seeSection 5).
The branched heptaglycosyl triphosphate phosphoglycan 17 (Scheme 3)from L. mexicana (containing b-D-glucose moiety in the side chain) wasassembled via stepwise chain elongation from the H-phosphonate deriva-tives 4 and 23.62 The trisaccharide 23 was prepared starting from the glu-cosyl bromide 18 and the disaccharide acceptor 19 via their coupling andacid hydrolysis (-20), followed by the orthogonal 60-O-DMT protectionand anomeric debenzoylation (-22). The hemiacetal 22 was converted tothe H-phosphonate 23 by phosphitylation with PCl3-imidazole-Et3N systemand subsequent hydrolysis. Condensation of the disaccharide H-phospho-nate 4 and dec-9-en-1-ol in the presence of a condensing reagent (ada-mantane-1-carbonyl chloride) followed by oxidation (I2) and mild acidicdetritylation gave the phosphodiester 60-OH block 21 (90%). Coupling ofthe disaccharide phosphate 21 with the trisaccharide H-phosphonate 23,followed by oxidation and de-O-tritylation furnished 24 (71%), which onfurther elongation with the H-phosphonate 4 (79%) afforded, after globaldeprotection, the desired branched phosphoglycan 17. The disaccharidephosphodiester 21 is the key synthon that was used for further preparationof branched phosphoglycans from L. major (see below). It was alsodebenzoylated63 to furnish the shortest Leishmania synthetic phosphoglycan16, containing only one phosphosaccharide repeat.
A matching strategy, in which adamantane-1-carbonyl chloride was re-placed with trimethylacetyl chloride as a condensing reagent, was employedin the assembly of the branched phosphoglycans 27–2964,65 and 30–3266–68
from L. major (Scheme 4). The phosphoglycan molecules were synthesisedfrom the disaccharide 4, trisaccharide 25 and tetrasaccharide 26 H-phos-phonate building blocks and the 60-OH phosphodiester 21.
The synthesis of the decaglycosyl triphosphate 32, which is the largestmolecule of the set, is shown in Scheme 5. The disaccharide phosphate 21
was first elongated with the tetrasaccharide H-phosphonate 26 to providethe monohydroxyl phosphoglycan derivative 39 (89%). One more chainextension capitalizing on the H-phosphonate 26 (83%), followed by globaldeprotection gave the targeted phosphoglycan 32. The tetrasaccharidesynthon 26 was prepared in advance by making use of the thiogalactoside33, the galactosyl trichloroacetimidate 35 and the arabinosyl chloride 37 asglycosyl donors in a 16-step linear synthesis starting from the mannoseacceptor 7.66 Its coupling with the galactose donor 33 in the presence ofMeOTf (80%) and subsequent de-O-chloroacetylation gave the disacchar-ide 34, which, in turn, was glycosylated with the trichloroacetimidate 35
(69%), followed by protecting group remodelling to form the trisaccharide
acceptor 36. This was coupled with the arabinosyl donor 37 (76%) profitingby the Lemieux’s halide inversion protocol,69 followed by partial depro-tection and introduction of the 60-TBS ether (-38). Tetrasaccharide 38
was benzoylated, followed by replacement of the TBS protection with with
60-O-DMT group and standard introduction of the H-phosphonate moietyat C-1 to afford the required 26.
The New Delhi group (Vishwakarma and coworkers) reported similarsyntheses of L. donovani70,71 (Scheme 6) and L. major72 (not discussed here)phosphoglycans using the glycosyl H-phosphonate method, but startingfrom D-lactal 4073 for preparing the Gal-(b1-4)-Man disaccharide. Noneof the prepared phosphoglycans contained an aglycone spacer (which isstrongly preferable, but not strictly indispensable for bioconjugation) andno preparation of a neoglycoconjugate therefrom was reported. Dibutyltinoxide mediated selective 6-O-silylation of the galactose moiety in compound40, followed by m-CPBA assisted D-glycal-D-mannose transformation andconventional acetylation led to the desired disaccharide 41, which served asa starting material for the disaccharide H-phosphonate 42 as well as for thealcohol 43 preparation, for iterative assembly of the phosphoglycan repeats.Selective de-O-acetylation at the anomeric position with Me2NH, followed
Scheme 5 Stepwise synthesis of a branched phosphoglycan of L. major.
by phosphitylation with PCl3-imidazole-Et3N system provided the H-phosphonate 42, which in pivaloyl chloride assisted coupling with the al-cohol 43 and subsequent oxidation with iodine led to the fully protectedphosphotetrasaccharide 44 (75%). The latter compound has become asubject to further chain elongation either upstream (at the non-reducing6w-end) or downstream (at the reducing 1-OH end). After removal of the6w-O-TBS group, the resulting tetrasaccharide alcohol was coupled withthe H-phosphonate 42 to provide the phosphohexasaccharide 45 (63%). Onthe other hand, selective deacetylation at the reducing-end anomeric pos-ition of 44, followed by direct conversion to the corresponding glycosylH-phosphonate (86%) and its coupling with the alcohol 43 provided thesame protected hexasaccharide diphosphate 45 (61%), which on successive
desilylation with aq. HF in MeCN and deacetylation with Et3N in MeOH-water provided the targeted L. donovani phosphoglycan 46.
4.2 Blockwise chain elongation
In addition to the stepwise chain elongation using protected glycosylH-phosphonates for the successive introduction of the sugar phosphateresidues, the Dundee group has also developed a blockwise approach63 thatinvolves condensation of two phosphodiester blocks (Scheme 7). Thisattractive approach was successfully adapted in the synthesis of theL. donovani phosphoglycan 50 composed of two repeats and the Man2-phosphate cap. Adamantane-1-carbonyl chloride mediated condensation ofthe tetrasaccharide H-phosphonate phosphodiester 49 and the mono-hydroxyl phosphodiester block 21, followed by oxidation with iodine andglobal deacylation afforded the hexaglycosyl triphosphate 50 in 59% overallyield. Compound 49 was prepared by coupling of the H-phosphonate 5 anddisaccharide 47, followed by anomeric deprotection (-48, 78%) and con-version of 1-OH to the anomeric H-phosphonate.
4.3 Polymer-supported syntheses
Synthesis of the L. donovani phosphohexasaccharide 55 (Scheme 8) wassuccessfully developed by the Dundee group building on a polymer-sup-ported methodology74 that employed the glycosyl H-phosphonates 4 and 54
for consecutive chain elongations, DMT ether as the orthogonal 6- and 60-protecting group and monomethyl polyethylene glycol (MPEG, with massof 5000Da) as a polymer support. The advantage of MPEG is that it allowsfor both solid-phase and solution-phase techniques: the MPEG-boundproducts are soluble in most organic solvents and water, however, they canbe precipitated (quantitatively) from ether or cold ethanol greatly simpli-fying their purification. The succinic ester linker was used for binding thegalactoside 51 to the polymer in the presence of 1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MS-NT) in 91% yield. Successive acetylation of anyresidual OH-groups and acidic detritylation afforded the MPEG-boundhydroxyl acceptor 52. Standard chain-elongation cycle (i.e., condensation,oxidation and detritylation) engaging the disaccharide H-phosphonate de-rivative 4 was applied twice to provide the polymer 53. The mannosylH-phosphonate 54 was then used to terminate the chain extension. Aftertotal deprotection with methanolic NaOMe, which also cleaved the productfrom MPEG, the hexaglycosyl triphosphate 55 (57% from 51, corres-ponding to an average 95% per step) was isolated by anion-exchangechromarography.
Alternatively, a solid-phase synthesis of the L. donovani phosphohex-asaccharide 58 (Scheme 9) was reported by the New Delhi group,71 whichused modified Merrifield resin and the disaccharide H-phosphonate 42.
Their methodology was based on the application of a cis-but-2-en-1,4-diyl-1-O-phosphoryl linker that enabled the selective cleavage of the firstanomeric phosphodiester linkage from the resin with Wilkinson’s catalystwithout affecting the glycosyl phosphate bond. The pivaloyl chloride me-diated coupling efficiency of each of the three iterative cycles (-56; -57)was more than 90%. Cleavage of the phosphoglycan from the resin, fol-lowed by desilylation (aq. HF in CH3CN) and deacetylation, provided thetargeted hexasaccharide triphosphate 58 in 70% yield.
4.4 Phoshoglycan syntheses by polycondensation
Both groups, Dundee and New Delhi group, described the construction oflarger phosphoglycans 61 having 10 repeats75 and 62 having 19–22 repeats71
in a one-pot synthesis by pivaloyl chloride assisted polycondensation (andsubsequent oxidation) of the monohydroxyl H-phosphonate derivatives 59and 60, respectively, which served as bifunctional monomer building blocks(Scheme 10). Polycondensation of the monomer 60, containing O-acetylprotecting groups (rather than O-benzoyl in 59) seems to favour the for-mation of longer polymer chains. After deacetylation, the polymers 61 and62 were isolated by anion-exchange chromatography in 85 and 58% yield,respectively.
Polycondensation of the H-phosphonate 59 in the presence of 0.1 equiv.of the disaccharide monohydroxyl block 6 furnished (after standard oxi-dation and debenzoylation) the phosphoglycan 63 (60%) containing 6–7repeats and the dec-9-enyl aglycone moiety (a copolycondensation product).The polycondensation product 64 (25%) was also isolated.
The H-phosphonate polycondensation methodology was proven to besuccessful for the later preparation of synthetic phosphoglycans composedof ribose-ribitol-phosphate repeats (representing the CPS of Haemophilusinfluenzae b), which were then incorporated into the first synthetic anti-Hibglycoconjugate vaccine.31
5. The preparation of neoglycoconjugates based on phosphosaccharide
repeats (using reductive amination technique) and the immunological studies
As it is mentioned in Section 4.1, all (but two, see 61 and 64) the phos-phoglycans designed and synthesised in Dundee were equipped with adec-9-enyl aglycone linker to enable their future incorporation into neo-glycoconjugates via oxidation of the terminal double bond. Ozonolysis of
the phosphoglycans in methanolic solution at � 78 1C (followed by re-duction with Me2S; Scheme 11) afforded the corresponding 8-carbonyloctylglycosides (or phosphosaccharide diesters), which were immediately usedfor coupling with a protein (or a lipid) carrier by reductive amination withNaCNBH3.
76–78 The conversion of the phosphoglycans to their 8-carbo-nyloctyl derivatives was shown to be complete and proceeds without de-struction of the labile anomeric phosphodiesters.76 Ozonolysed compounds
Protein carrier: Tetanus toxin fragment C, TetC.
Recombinant, non-toxic, M = 53434 Da, 33 NH2 groups.
PROTEIN CARRIER
(H2N)33
Sug–Sug– P –Sug–Sug– P –(CH2)8CH=CH2
Sug–Sug– P –Sug–Sug– P –(CH2)8CH=O
Sug–Sug– P –Sug–Sug– P NH
PROTEIN CARRIER
n
1) O3, MeOH, -78 oC, 5 min
2) Me2S, rt, 2 h
NaCNBH3, 0.1 M phosphate
buffer, pH 7.4, 37 oC, 72 h
Hapten/Protein 1 mole of a hapten per
mole/mole (n) number of NH2 groups protein weight
65 [Gal-Man-OPO2H-Gal-Man-O(CH2)9]n–TetC 3.82 8.6 14000 Da
were mixed with NaCNBH3 in a phosphate buffer solution (PBS, pH 7.4)and added to a protein solution to obtain a molar ratio of phospho-saccharide : protein NH2 group : NaCNBH3 of 1 : 3 (if another one is notspecified): 50. After 72 h at 37 1C, the formed neoglycoconjugates wereimmediately purified by gel filtration on a column of Superdex 200 HR inPBS. The following SDS-PAGE analysis showed that no starting proteinwas observed.77,78
To develop the technique, first,76 the L. donovani phosphoglycan 3
(Scheme 1) was ozonolysed and coupled to ribonuclease A (RNAase A;M=13 690Da, 10 amino groups) using a molar ratio of phosphosaccharide: protein amino group of 1 : 5. That gave a glycoconjugate containing 1.1phosphosaccharide (hapten) chains per RNAase A molecule (i.e., 1 mole ofa hapten per 12 440Da of protein). The ozonolysed disaccharide phosphate16 (Scheme 3) was coupled to bovine serum albumin (BSA) as a carrier(M=66 382Da, 59 amino groups) using a molar ratio of phospho-saccharide : protein amino group of 1 : 1, thus producing a glycoconjugatecontaining 8 hapten chains per molecule of BSA (i.e., 1 mole of a hapten per8300Da of protein). Although this was around 3-time lower than that re-ported for oligosaccharide–BSA conjugates prepared using similar re-ductive amination techniques,79,80 in the latter cases, a large molar excess ofoligosaccharide over BSA amino groups (about 200 : 1) was used. Since (1)the synthetic phosphoglycans are rather precious and (2) a relatively lowhapten : protein ratio (like 1 mole of a hapten per 10 kDa of protein) may bedesirable from an antigen-presentation point of view,76 the developedconditions were used for the preparation77,78 of Leishmania phosphoglycan–tetanus toxin fragment C conjugates 65–71 (Scheme 11).
The engineered tetanus toxin fragment C (TetC)81 does not retain anytoxicity, but retains immunogenicity.82 It was already tested on the vaccinemarket83 and has the advantage of inducing a Th-1 response that is ne-cessary for the killing Leishmania parasites.84,85 The synthetic phos-phoglycans were coupled to TetC using a molar ratio of phosphosaccharide: protein amino group of 1 : 3.77,78 Thus, three L. donovani–TetC glyco-conjugates 65–67 were prepared from three different linear phosphoglycans1–3, correspondingly. Ozonolysis of the branched L. mexicana phos-phoglycan 17 (Scheme 3), followed by protein coupling produced theL. mexicana–TetC glycoconjugate 68, while the L. major–TetC glyco-conjugates 69 and 70 were generated from the L. major phosphoglycans 29and 32 (Scheme 4), respectively. The prepared conjugates were carryingfrom 3.37 to 4.85 phosphosaccharide haptens per molecule with, on aver-age, 1 mole of a hapten per 13300Da of protein.
The modified L. donovani–TetC glycoconjugates 71a and 71bwere preparedfrom the synthetic C-glycosyl phosphono analogue of the LPG fragmentwith a structure of Gal-(b1-4)-Man-a-(1-CH2PO2H-6)-Gal-(b1-4)-Man-a-1-CH2PO2H-O(CH2)8CH=CH2.
86 Replacing the anomeric O-1 in D-mannoseresidues with CH2 groups does not, probably, change much conformation ofthe molecule, but makes the C-glycosyl phosphono analogues significantlymore stable chemically than parent phosphoglycans and resists the actionof phosphatases as well. All of those make the phosphono analoguesof the parasitic and bacteria phosphoglycans strong candidates for synthetic
glycoconjugate vaccines.87,88 Ozonolysis of the above structure, followed bycoupling to TetC with a molar ratio of phosphonosaccharide : protein aminogroup of 1 : 3 furnished the glycoconjugate 71a containing just 2 haptenchains per molecule, while the higher loaded conjugate 71b (with 9.7 haptenchains per molecule) was prepared using a higher molar ratio of the phos-phonosaccharide over TetC amino groups of 2 : 3.
Finally, the neoglycolipid 72 (Scheme 11) was synthesised76 by couplingthe ozonolysed phosphoglycan 3 and 1,2-di-O-hexadecyl-sn-glycero-3-phosphatidylethanolamine (PE) in a mixture of chloroform-methanol-water (60 1C, 16 h) using a molar ratio of phosphosaccharide : PE :NaCNBH3 of 1 : 20: 50. After purification on octyl-Sepharose columnfollowed by Kieselgel 60, the glycoconjugate was isolated in 36% yield. Forimmunological studies, the neoglycolipids could be incorporated intoliposomes that act as a carrier and as an adjuvant.89
The glycoconjugates 66 (L. donovani–TetC), 68 (L. mexicana–TetC), 69(L. major–TetC with D-Gal side chains) and 70 (L. major–TetC with D-Ara-D-Gal side chains) were used by Bates and Rogers (University of Liverpool)as glycovaccines (without any adjuvants) to immunize BALB/c mice, whichwere then challenged by the bite of L. mexicana infected Lutzomyia long-ipalpis sand flies.90 Intriguingly, only the L. mexicana–TetC glycovaccine 68showed significant protection compared with the control TetC immuniza-tion, resulting in a 50% reduction in lesion size and a 94% (!) reduction inparasite burden. The other glycovaccines 66, 69 and 70, neither theL. mexicana phosphoglycan 17 (Scheme 3) alone, were unable to provideprotection against infected fly bite challenge. Thus, that was the first (to theauthors90 knowledge) demonstration that a synthetic glycoconjugate vac-cine can have a direct antiparasite effect in leishmaniasis or any otherparasitic disease. It seems that these glycovaccines may induce species- andglycan structure-specific protection, although the ability of the L. mexicana–TetC glycovaccine 68 to protect against other species of Leishmania has notbeen tested yet.
6. The preparation of neoglycoconjugates based on the tetrasaccharide
cap structure
The Cambridge (Massachusetts) group (Seeberger and co-workers) hasdesigned and synthesised16 two potential Leishmania vaccine constructsbased on a unique structure of the tetrasaccharide phosphate cap (Gal-Man3-phosphate), while the phosphate group being omitted thus simplify-ing strongly the conjugation technique (see Section 4.1). The preparedtetrasaccharide 73 (Scheme 12) was equipped with a 6-aminohexyl aglyconelinker to facilitate futher bioconjugation. The GalMan3 tetrasaccharideitself has previously been a subject of two synthetic approaches.91,73
Epoxidation of the double bond in the hexa-O-benzyl-D-lactal 74 with 2,20-dimethyldioxirane, followed by opening of the epoxide with ethanethiol,resulted in the thioethyl lactoside derivative 75 (51%), which was thenconverted to the Gal-(b1-4)-Man disaccharide 78 via the oxidation-reduction at C-2 (47%) and subsequent pivaloylation. The glycosyl donor78 was combined with N-Cbz-6-aminohexanol in MeOTf assisted
glycosylation (54%), while subsequent cleavage of the 2-pivaloate furnishedthe disaccharide acceptor 76. Further coupling with the thioethyl manno-side donor 7792 (66%), followed by de-O-pivaloylation gave the tri-saccharide acceptor 79, which was iteratively glycosylated with the samedonor 77 (70%) prior to a two-step global deprotection to provide the re-quired tetrasaccharide 73.
The GalMan3 tetrasaccharide has been also synthesised in the form of theprotected pent-4-enyl glycoside 83 by exploiting the traditional solution-phase and exploring a novel solid-phase synthetic technique (Scheme 12).The assembly of the branched glycan from differentially protected mono-saccharide glycosyl donors (not shown here) was accomplished using an
Scheme 12 Syntheses of the LPG tetrasaccharide cap structures.
identical reaction path, but starting from either pent-4-enyl alcohol 81 (forthe traditional chemistry) or octenediol-functionalized Merrifield resin 80
(for the solid-phase synthesis)93 as acceptor for the first glycosylation. Thetraditional 7-step glycan chain assembly required at least two weeks time(with purification of all the intermediates), but proceeded smoothly andresulted in the tetrasaccharide 83 in an overall 17% yield. For the solid-phase synthesis (which was significantly faster and took 4 days only), eachchain elongation cycle relied on a double glycosylation (using catalytic orequimolar TMSOTf) to secure high coupling efficiencies followed by re-moval of an orthogonal protecting group. Simple washing of the resin wasused to remove excessive reagents. Thus, after four consecutive elongations,the polymer bound product 82 was produced. Cleavage of the octenediollinker by olefin cross-metathesis using Grubbs’ catalyst in an atmosphere ofethylene,94,95 afforded pure pent-4-enyl tetrasaccharide 83 (which wasidentical to the one made earlier) after a single silica column in 18% overallyield. Although the transformation of 83 to the 6-aminohexyl tetra-saccharide 73 has not been reported,16 it seems to be just a matter of aglysosylation reaction (as the pent-4-enyl glycoside 83 can work as a gly-cosyl donor96), followed by a two-step deprotection.
1) 0.2 M aq. NH2OH, PBS, pH 7.4, 24 h2) 2-aminoethanethiol, 24 h
K L H protein
(BrCH2COHN)m
HO OO
HO
HO
O
OHHO
HOHO
O OO
HO
HO
85
HO OOH
HO
HO
HN
OS
O
O(CH2)6
OC6F5
OS
O
73 +DMF, 68%
K L H protein
NH nNH
OSGalMan3−O(CH2)6
O
87
NH
O HNGalMan3−O(CH2)6 C15H31
OS
O
C15H31
O
O C15H31
O
73 +
HATU, i-Pr2NEtDMF, CH2Cl2, 60%
HO
O HN C15H31
OS
O
C15H31
O
O C15H31
O
[
[
86
88 89
84
Scheme 13 Synthetic neoglycoconjugates based on the tetrasaccharide cap structure fromLeishmania LPG.
Two neoglycoconjugates 87 and 89 (Scheme 13) were prepared by joiningthe tetrasaccharide antigen 73 with protein carrier keyhole limpethemocyanin (KLH) or with lipid immunostimulator tripalmitoyl-S-glycer-ylcysteine 88, respectively. The KLH proteins are known to be highly het-erogeneous and have enormous molecular masses (4500–13 000 kDa) as wellas ill-defined structures, but their immunogenic capabilities can be su-perior.97 Prior to the conjugation, the protein itself was reacted with N-succinimidyl bromoacetate to form the bromoacetate-modified carrier 86,while the 6-aminohexyl tetrasaccharide 73 was coupled with penta-fluorophenyl (acetylsulfanyl)acetate 84 to give the tetrasaccharide 85 withmasked SH functionally in the linker. Further coupling with the modifiedprotein 86 in the presence of hydroxylamine was followed by capping of anyremaining bromoacetyl groups with 2-aminoethanethiol to furnish theneoglycoprotein 87, which was purified by gel filtration on a Pd-10 column(Sephadex G-25). The reported conjugation ratio of the GalMan3 haptenchains to each KLH molecule was 55:1 (which is about 1 mole of the tet-rasaccharide hapten per 82 000–236 000Da of protein).
The lipopeptide N-palmitoyl-S-[(2R,2S)-2,3-di(palmitoyloxy)propyl]-L-cysteine 88 (tripalmitoyl-S-glycerylcysteine, Pam3Cys) has been alreadyreported as a part of a fully synthetic vaccine.98 It can work both as a carrierand an adjuvant99 and is readily available by synthesis.100 Coupling with the6-aminohexyl tetrasaccharide 73 was performed in the presence of HATUand di-isopropylethylamine and afforded the neoglycolipid 89 (60%) afterpurification on a silica column. Both synthetic glycovaccines has been testedfor their immunological evaluation in BALB/c mice, but the results have notbeen reported yet.
Acknowledgments
We are indebted to Prof. M. A. J. Ferguson (Dundee) for his interest,constant support and encouragement. Financial support from the WellcomeTrust (grant WT 076254 for partial sponsorship of N. A.-M. and grant WT083481 for the infrastructure maintenance) and the Royal Society (6-monthTravelling Fellowship for O. V. S.) is gratefully acknowledged.
References
1 B. L. Herwaldt, Lancet, 1999, 354, 1191.
2 World Health Organisation: http://www.who.int; WHO Leishmaniasis Fact-
![Page 1: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/1.jpg)
![Page 2: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/2.jpg)
![Page 3: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/3.jpg)
![Page 4: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/4.jpg)
![Page 5: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/5.jpg)
![Page 6: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/6.jpg)
![Page 7: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/7.jpg)
![Page 8: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/8.jpg)
![Page 9: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/9.jpg)
![Page 10: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/10.jpg)
![Page 11: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/11.jpg)
![Page 12: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/12.jpg)
![Page 13: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/13.jpg)
![Page 14: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/14.jpg)
![Page 15: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/15.jpg)
![Page 16: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/16.jpg)
![Page 17: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/17.jpg)
![Page 18: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/18.jpg)
![Page 19: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/19.jpg)
![Page 20: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/20.jpg)
![Page 21: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/21.jpg)
![Page 22: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/22.jpg)
![Page 23: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/23.jpg)
![Page 24: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/24.jpg)
![Page 25: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/25.jpg)
![Page 26: [Carbohydrate Chemistry] Carbohydrate Chemistry Volume 36 || Synthetic glycoconjugates based on Leishmania lipophosphoglycan structures as potential anti-leishmaniasis vaccines](https://reader031.documents.pub/reader031/viewer/2022020408/575093021a28abbf6bac51ff/html5/thumbnails/26.jpg)
