Patent Application: US-201113109562-A

Abstract:
one aspect of the present invention relates to solution - phase approaches to gpi synthesis . another aspect of the present invention relates to key building blocks , and syntheses thereof , useful for gpi assembly . yet another aspect of the invention relates to an automated method for the synthesis of gpis and fragments thereof .

Description:
the invention will now be described more fully with reference to the accompanying examples , in which certain preferred embodiments of the invention are shown . this invention may , however , be embodied in many different forms and should not be construed as limited to the embodiments set forth herein ; rather , these embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . solution phase synthesis of a gpi malaria toxin for use as a vaccine the malaria parasite is a formidable opponent for the human immune system . it proceeds through four distinct life cycles during the course of an infection , displaying different antigens to the immune system at each stage . over 5000 proteins are displayed as the parasite changes from sporozoite to gametocyte . therefore , a vaccine based on a protein found on the surface of sporozoites is ineffective once the parasite differentiates into a gametocyte . this stands in contrast to bacteria and viruses , where a consistent coating of antigens is displayed to the cells of the immune system . repeated exposure to the parasite over time ( as the result of repeated infection ), or infection with weakened sporozoites can both lead to antibody - based protection against infection . the former is called naturally acquired immunity ( nai ), while the latter is referred to as the attenuated sporozoite model . 13 it is known that adults in malaria endemic areas can have parasites in their bloodstream yet be asymptomatic ( i . e . they are naturally immune ). if purified antibodies from the blood of these individuals are transferred to children who have high parasite loads , the parasite load is diminished , and the child is protected from subsequent infection . the attenuated sporozoite model is based on the observation that infection with sporozoites from mosquitoes irradiated with uv light does not lead to infection . rather , individuals vaccinated with these attenuated sporozoites are protected against subsequent infections for up to 9 months . these two models are the main tenets behind modem vaccine design , and have led to four main schools of thought regarding vaccines : anti - infection , anti - transmission , anti - growth rate , and anti - toxin . in the case of malaria , these treatments all target the parasite at a different stage of its life cycle — the first three have defined protein targets derived from the cell surface of the parasite , while the last targets a toxin released following parasite replication . the limitation of the first three strategies are their specificity and inherent ineffectiveness once the parasite differentiates past the targeted life cycle stage . the parasite is versatile , and has shown the ability to vary its surface coating over time , rendering a previously effective vaccine useless . anti - infection , - transmission , and - growth vaccine approaches are greatly aided by the bounty of information known about proteins on the cell surface of the parasite . the anti - toxin strategy has the advantage that the target appears to be invariable . 14 neutralization of the toxin would work regardless of the protein coating on the surface of the parasite . anti - toxin vaccines target the toxin that is released following erythrocyte rupture , and represent the second form of protection against blood - stage parasites . release of this toxin is thought to initiate an inflammatory cascade in the host , resulting in the release of harmful cytokines such as tumor necrosis factor ( tnfα ). an effective anti - toxin vaccine would prevent the inflammatory cascade in the host through antibody sequestration and neutralization of the parasite toxin ; as the cascades caused by the toxin are necessary for parasite success , evolved resistance should be blunted . a gpi of parasite origin ( 1 ) was recently isolated that induced several of the pathological effects associated with severe malaria and was thus a candidate toxin ( fig6 ). 15 purified gpi induced tnfα expression and no output in macrophages , both of which occur during real infections and lead to clinical manifestations of malaria . in addition , a recent study found that adults with resistance to malaria had high levels of persistent anti - gpi antibodies , while susceptible children had low levels or lacked these protective anti - gpi antibodies . 16 the absence of anti - gpi antibody response correlated with malaria specific anemia and fever , suggesting that anti - gpi antibodies played a major protective role against malaria . what remained to be established was if an anti - toxin vaccine based on the structure of the isolated gpi would reduce pathogenesis or fatalities in any disease condition . anti - toxin vaccines have been discussed for some time , 17 but never reduced to practice . we sought to determine whether immunization with gpi oligosaccharide fragment 3 , prepared by chemical synthesis , could prevent pathology and fatalities in a rodent model of severe malaria ( fig7 ). one facet of the vaccine design deserves mention : our proposed synthetic fragment 2 differs from the authentic malarial gpi 1 in the phosphorylation of the inositol ring . selective installation of a phosphate on the 1 - position of an inositol ring in the course of a gpi synthesis requires preparation of an inositol building block that is orthogonally protected on the 1 - position ( i . e . leading to 1 ). this is not a trivial operation , and the search for new methods to solve this problem has been the subject of intense research . 18 we chose to install a cyclic phosphate on the 1 , 2 - position on potential vaccine precursor 2 rather than focusing on developing new methodology . we developed a synthetic route for 2 that employed five differentially - protected glycosyl donors , one inositol and one phosphoramidite building block ( fig8 ). while the initial synthesis of 2 was carried out in solution , our ultimate goal was automated solid - phase synthesis , 19 which helped guide our selection of protecting groups and glycosyl donors . building blocks 4 , 20 7 , 7a 9 , 7a and 10 7b were prepared following known procedures , while thiodonor 5 and imidates 6 and 8 required novel syntheses . the route to myo - inositol acceptor 4 ( fig9 ) followed literature precedent . 20a selective mono tritylation 21 of the methyl - α - d - glucopyranoside 11 followed by benzylation and removal of the temporary trityl ether gave 12 . swern oxidation , 22 formation of a mixture of isomeric enol acetates ( ac 2 o , k 2 co 3 ), and ferrier reaction 23 under the catalysis of mercuric acetate provided hydroxy - ketone 13 in 53 % for 3 steps . internal - delivery reduction with sodium triacetoxyborohydride gave the anti diol in 64 % yield . cleavage of the acetate with naome / meoh provided a known triol , 24 and protection of the cis alcohols as their isopropylidene under thermodynamic control gave acceptor 4 . alternatively , protection of the hydroxyls of diol 14 as ethoxyethyl ethers , replacement of acetyl by allyl , and removal of the acetals allowed production of differentiated acceptor 15 . this molecule should allow use of non - cyclic phosphodiesters . for preparation of glucosamine building block 5 , we started from known phthalimide - protected triol 25 ( fig1 ). protection of the 4 , 6 - diol as a benzylidene ring proceeded under the agency of tetraflubroboric acid 26 in 65 % yield , followed by benzylation of the remaining alcohol ( 84 % yield ). regioselective opening of the benzylidene using triethylsilane and trifluoroacetic acid 27 afforded 3 , 6 di - benzyl thioglycoside 17 in 71 % yield . the next transformation en route to the desired donor was an amine protecting group switch , from phthalamide to azide . phthlamide groups are not compatible with the conditions used for cleavage of acetate esters ( vide infra ), and would also favor an undesirable β - linkage in the initial coupling event . cleavage of the phthlamide with hydrazine monohydrate was followed by treatment of the crude amine with freshly prepared triflic azide . 28 treatment of the crude product with acetic anhydride and dmap followed by chromatography provided thiodonor 5 in 83 % yield for the three steps . the synthesis of imidate 6 began with per - benzylated methyl mannopyranoside 18 29 ( fig1 ). conversion of both the anomeric methoxy group and the 6 - o - benzyl group to acetates was accomplished using concentrated sulfuric acid with acetic anhydride as solvent . 30 the anomeric acetate was cleaved using ammonia , and the resulting lactol was converted into trichloroacetimidate 31 6 using trichloroacetonitrile and catalytic dbu . the additional degree of orthogonality required of building block 8 necessitated the use of orthoester 19 . 32 regioselective protection of the 6 - position as a silyl ether was followed by dibenzylation in 90 % yield over two steps . aqueous acetic acid was employed for the opening of the orthoester ( 82 %), resulting in formation of a 2 - o — ac anomeric lactol . preparation of imidate 8 proceeded in high yield using trichloroacetonitrile and dbu . the first glycosidic linkage to be formed was the difficult union between thiodonor 5 and inositol derived acceptor 4 ( fig1 a and fig1 b ). activation with n - iodosuccinimide / silver triflate provided pseudo - disaccharide product 20 in 70 % yield as a separable 1 . 2 : 1 mixture in favor of the desired α - isomer . deprotection with sodium methoxide removed the acetyl group to give acceptor 21 , which was then coupled to trichloroacetimidate 6 in 75 % yield . cleavage of the acetate of the resultant pseudo - trisaccharide 22 provided acceptor 23 , which was positioned for the next coupling with building block 7 . this coupling proceeded in high yield ( 92 %) and furnished exclusively α - linked pseudo - tetrasaccharide acceptor 25 following deprotection with sodium methoxide in methanol . coupling of acceptor 25 and donor 8 proceeded in excellent yield under slightly milder conditions ( tbsotf ), and was followed by deprotection to give pseudo - pentasaccharide acceptor 27 . the addition of a terminal mannose subunit proceeded in high yield ( 84 %) with donor 9 , but afforded a mixture of anomers that were inseparable by silica gel chromatography . deprotection of the isopropylidene acetal 7b on the inositol ring proved to be a difficult transformation , providing mixtures of starting material , desired diol 29 , and triol arising from concomitant tips cleavage . after multiple recycling steps and tedious column chromatography 29 was isolated in a modest yield of 67 %, albeit still as a mixture of isomers from the 5 + 1 coupling event . the difficulties encountered in the final coupling event and problematic hydrolysis of the isopropylidene group prompted us to explore an alternate route to a protected diol such as 29 . replacing per - benzylated donor 9 with 2 - acetate donor 7 would be a straightforward solution to the first problem . following saponification and benzylation the protected pseudo - hexasaccharide should be isolable as a single isomer . for a solution to the sluggish hydrolysis reaction , we turned to an earlier gpi synthesis by frick et al . 10a instead of trying to prevent concomitant silyl ether cleavage in the course of acetal cleavage , the authors had hydrolysed all acid labile groups ( fig1 ). after workup , the silyl ether had been regioselectively re - installed on the primary alcohol to give 31 . a model trisaccharide bearing the appropriate functionality was constructed to examine the hydrolysis reaction ( fig1 ). disaccharide acceptor 32 was reacted with donor 33 to give a 67 % yield of desired pseudo - trisaccharide 34 . saponification and benzylation proceeded smoothly in a combined 62 % yield for the two steps , and set the stage for the model hydrolysis . hydrolysis of the tips ether and isopropylidene groups was accomplished using 0 . 5 m hcl in methanol over 12 hours , followed by alkaline workup . following reaction with tbs - cl and imidazole ( imid . ), a 73 % yield of desired diol 37 was isolated . the good yield of the model reaction coupled with its operational simplicity prompted us to apply these hydrolysis conditions . starting from pseudo - pentasaccharide acceptor 27 , 2 - ac donor 7 was installed to give a quantitative yield of the desired pseudo - hexasaccharide as a single isomer ( fig1 a and fig1 b ). the structure of 38 was confirmed using several 2d - nmr experiments ( cosy , hsqc , hmbc , tocsy ). 33 deprotection gave a 72 % yield of the pseudo - hexasaccharide 39 before benzylation afforded compound 40 in 96 % yield . following the conditions used for our model trisaccharide , hydrolysis and re - installation of the silyl ether proceeded smoothly to afford 67 % of 41 . using a mixture of methyldichlorophosphate in pyridine , 34 pseudo - hexasaccharide cyclic phosphate could be isolated after acidic workup . the crude material was subjected to silyl ether cleavage using tbaf to give a 70 % yield of 42 . reaction with phosphoramidite 10 7b and oxidation provided bis - phosphate 43 as a mixture of diastereomers . dbu was used to cleave the β - cyanoethoxy blocking group , and removal of the benzyl ethers , benzyloxy ( cbz ) carbamate , and azide was accomplished in a single step with na / nh 3 to afford desired gpi 2 . see fig1 . the final product was characterized by 1 h , and 31 p nmr , as well as by maldi - tof mass spectrometry . to prepare an immunogen , the synthetic gpi glycan 3 was treated with 2 - iminothiolane ( fig1 ) to introduce a sulfhydryl at the primary amine within the ethanolamine phosphate , desalted , and conjugated to maleimide - activated ovalbumin ( ova , in molar ratio 3 . 2 : 1 ) or keyhole limpet haemocyanin ( klh , in molar ratio 191 : 1 ), and used to immunize mice . the synthetic malarial gpi glycan was immunogenic in rodents . antibodies from klh - glycan 3 immunized animals gave positive igg titres against ova - glycan but not sham - conjugated ova - cysteine 45 containing identical carrier and sulfhydryl bridging groups ( fig1 ). no reactivity to gpi glycan was detected in pre - immune sera or in animals receiving sham - conjugated klh . more significantly , antibodies raised against synthetic p . falciparum gpi glycan bound to native gpi as judged by several methods . the murine p . berghei anka severe malaria model has salient features in common with the human severe and cerebral malaria syndromes and is the best available small animal model of clinically severe malaria . to determine whether anti - gpi immunization prevents systemic and cerebral pathogenesis in this pre - clinical model , c57b16 mice primed and twice boosted with 6 . 5 μg klh - glycan ( 0 . 18 μg glycan ) or klh - cysteine in freund &# 39 ; s adjuvant were challenged with p . berghei anka , and the course of disease monitored ( fig1 a ). 100 % of both sham - immunized and naïve control mice died within 5 - 8 days . there were no differences between naïve and sham - immunized mice indicating exposure to carrier protein alone in freund &# 39 ; s adjuvant does not influence disease rates . in contrast , mice immunized with synthetic p . falciparum gpi glycan coupled to klh were substantially protected against cerebral malaria , with significantly reduced death rates ( 75 % survival , fig1 a ). in four separate additional experiments , results over the range of 58 . 3 - 75 % survival over this time - period in vaccine recipients ( n = 50 total ) vs . 0 - 8 . 7 % survival in sham - immunized controls ( n = 85 ) were obtained . parasitaemias were not significantly different between test and control groups in these experiments , demonstrating that prevention of fatality by anti - gpi vaccination does not operate through effects on parasite growth rates ( fig1 b ). severe malaria in both humans 35 and rodents 36 may be associated with additional organ - specific and systemic symptoms , including pulmonary edema and serum acidosis . our collaborators sought to determine whether anti - gpi vaccination protects against these additional non - cerebral disease syndromes in mice . both sham - immunized and naive individuals developed pronounced pulmonary edema by day 6 post - infection , as measured by lung dry : wet weight ratios , and this symptom was markedly reduced in vaccine recipients ( fig1 c ). similarly , whereas sham - immunized and unimmunized mice developed significant acidosis as shown by reduced blood ph at day 6 post - infection , in vaccinated mice blood ph was maintained at physiological levels ( fig1 d ). immunizing against gpi clearly prevented the development of pulmonary edema and acidosis as well as cerebral malaria in p . berghei infection . the findings of the mouse study demonstrated that gpi is the dominant endotoxin of p . falciparum and p . berghei origin . a synthetic gpi oligosaccharide coupled to carrier protein was immunogenic and provided significant protection against malarial pathogenesis and fatalities in a preclinical rodent model . it is therefore possible that gpi contributes to life - threatening disease in human malaria . the data suggest that an anti - toxin vaccine against malaria might be feasible and that synthetic fragments of the p . falciparum gpi may be developed further toward that goal . the mouse trials using anti - toxin vaccine 3 showed that synthetic gpi fragments are highly immunogenic when applied to malaria infection . the minimal structure required for an effective anti - toxin vaccine based on the malarial gpi was not defined . the rapid synthesis of gpi fragments for subsequent biological evaluation would be highly desirable , and could lead to a more potent anti - toxin vaccine . application of our automated carbohydrate synthesizer 19 to this problem seemed natural . using our solution phase synthesis as a guide , we contemplated the automated synthesis of 2 . while it would be ideal to prepare the entire skeleton on solid - phase , the α - linkage between the inositol and glucosamine residues presented a serious impediment to a fully automated synthesis . relatively few methods are available for the preparation of 1 , 2 - cis glycosidic linkages . 37 previous gpi syntheses addressed this problem by either separating mixtures of isomers , or utilizing completely α - selective coupling methods followed by protecting group manipulations . 38 neither of these solutions was amenable to solid - phase , which led us to dissect gpi 2 into two fragments : a known disaccharide 21 not readily accessible on solid phase , and a tetra - mannosyl fragment ( 46 ) readily prepared using our automated solid - phase methodology ( fig2 a and fig2 b ). the two fragments could be joined using n - pentenyl glycoside ( npg ) coupling methodology , 39 or via hydrolysis and conversion into tetrasaccharide trichloroacetimidate 47 . tetrasaccharide 46 was accessed on solid - phase using four readily available trichloroacetimidate building blocks 6 - 9 ( fig2 ). the automated synthesis was carried out on a modified abi 433a peptide synthesizer using octenediol - functionlized merrifield resin 48 . each coupling cycle ( fig2 ) relied on double glycosylations to ensure high coupling efficiencies and a single deprotection event . coupling of 6 to resin 48 using catalytic tmsotf was followed by removal of the acetate ester upon exposure to naome . based on a solution - phase model , we did not anticipate a selective coupling between donor 6 and the functionalized resin ; the stereochemistry of this first coupling was inconsequential since the npg resulting from the automated synthesis was to be coupled at the reducing end . elongation of the oligosaccharide chain was achieved using monosaccharide 7 7a , followed by deprotection of the 2 - o - acetate using naome . coupling of building block 8 employing catalytic tmsotf and deprotection with naome proceeded smoothly to create a resin - bound trisaccharide , before the final coupling with donor 9 . 1a cleavage of the octenediol linker using grubbs &# 39 ; catalyst 40 under an atmosphere of ethylene provided crude n - pentenyl tetrasaccharide 46 . hplc analysis of the crude reaction products revealed two major peaks ( fig2 ): the desired tetrasaccharide 46 ( 44 % relative area ) and deletion sequences ( 15 % relative area ). the crude material was purified by hplc to provide 46 as a mixture of α - and β - anomers . prior to attempting the crucial 4 + 2 coupling , a model coupling between npg - monosaccharide 49 and disaccharide 21 was carried out but failed to produce the desired product ( fig2 ). the failure of 49 as an effective glycosyl donor led us to examine glycosyl trichloroacetimidate 47 as a coupling partner . conversion of 46 into glycosyl donor 47 proceeded smoothly over two steps . reaction of trichloroacetimidate 47 with disaccharide 21 afforded the desired hexasaccharide 40 in modest yield . the material produced via the automated route was identical in all regards to material made previously in solution , and constitutes a formal synthesis of anti - toxin malaria vaccine 2 . to allow other phosphorylation patterns , use of the inositol acceptors with different protection patterns was necessary . coupling acceptor 15 with thiodonor 5 under the same conditions as with acceptor 4 gave , following deacetylation , a separable anomeric mixture of disaccharide acceptors 50 . ( fig2 ). tetramannose component 46 can also be prepared in solution , if large quantities are required ( fig2 ). sequential coupling / deprotection of donors 7 , 8 , and 7 to acceptor 51 , followed by benzylation of the free hydroxyl , gave 46 as expected . the modest 4 + 2 coupling yields seen above are partially due to the instability of the highly - activated tribenzyl donor being used . furthermore , mammalian gpi structures have a phosphoethanolamine on the 2 - oh of the first mannose residue . both of these issues were addressed simultaneously by the use of an ester on the 2 - position of the reducing - end mannose in the tetrasaccharide donor ; the presence of ester attenuates the activity of the donor , resulting in less decomposition , and it is removed to enable installation of phosphoethanolamine . our route is shown in fig2 : from known tetrabenzylmannose bromide , closure to the allyl orthoester takes place in the presence of the alcohol and 2 , 6 - lutidine ; debenzoylation and selective silylation of the primary hydroxyl gave 56 in good yield . benzylation was followed by opening of the orthoester ( in the presence of excess allyl alcohol ) and acid - catalyzed desilation gave acceptor 57 . this was coupled to 7 under standard conditions ; selective removal of the acetate ester using magnesium methoxide gave 59 in 90 % yield , based on the recovery of a small amount of starting material . polymer extension proceeded using the same techniques , and allyl removal used standard conditions , providing the new tetrasaccharide donor 61 . the synthesis of a pseudo - hexasaccharide glycosylphosphatidylinositol has been reduced to practice , both in solution and using a combination of solution and automated solid - phase methodologies . the material made in solution was covalently attached to a protein carrier and used to vaccinate mice . innoculated mice were substantially protected against a subsequent challenge with plasmodium parasites . this discovery further implicates gpi as the dominant toxin in malaria infections , and lays the groundwork for future trials in human volunteers . combinations of solution and automated solid - phase synthetic methodologies will see continued usage in this context , and are expected to lead to the rapid generation of more potent vaccines for malaria and other maladies . one aspect of the invention relates to compounds represented by formula i : r represents independently for each occurrence h , alkyl , aryl , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , or — si ( alkyl ) 3 ; r 1 and r 2 are independently h , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , — si ( alkyl ) 3i or r 1 and r 2 taken together are c ( ch 3 ) 2 , p ( o ) oh , or p ( o ) or 5 ; r 4 represents independently for each occurrence h , alkyl , aryl , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , — si ( alkyl ) 3 , or — p ( o )( or 5 ) 2 ; r 5 represents independently for each occurrence h , li + , li + , na + , k + , rb + , cs + , aryl , or an optionally substituted alkyl group ; and in certain embodiments , the present invention relates to the aforementioned compound , wherein n is 1 , 2 , or 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein n is 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein r is h . in certain embodiments , the present invention relates to the aforementioned compound , wherein r 1 and r 2 taken together are p ( o ) or 5 . in certain embodiments , the present invention relates to the aforementioned compound , wherein r 3 is n 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein r 3 is — nh 3 x . in certain embodiments , the present invention relates to the aforementioned compound , wherein r 4 represents independently for each occurrence h , — ch 2 ph , or — si ( alkyl ) 3 ; in certain embodiments , the present invention relates to the aforementioned compound , wherein r 4 represents independently for each occurrence h , — ch 2 ph , or — p ( o ) or 5 ; and r 5 is an optionally substituted alkyl group . in certain embodiments , the present invention relates to the aforementioned compound , wherein said compound of formula i is selected from the group consisting of : another aspect of the invention relates to compounds represented by formula ii : r represents independently for each occurrence h , alkyl , aryl , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , or — si ( alkyl ) 3 ; r 1 is —( ch 2 ) m ch ═ ch 2 or trichloroacetimidate ; and in certain embodiments , the present invention relates to the aforementioned compound , wherein n is 2 or 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein n is 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein m is 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein r represents independently for each occurrence — ch 2 - aryl or — si ( alkyl ) 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein r represents independently for each occurrence benzyl or — si ( ipr ) 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein r 1 is trichloroacetimidate and r represents independently for each occurrence benzyl or — si ( ipr ) 3 . in certain embodiments , the present invention relates to the aforementioned compound , wherein said compound of formula ii is selected from the group consisting of : one aspect of the present invention relates to a method of preparing glycosylphosphatidylinositol glycans as depicted in scheme 5 : r represents independently for each occurrence h , alkyl , aryl , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , or — si ( alkyl ) 3 ; r 1 and r 2 are independently h , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , — si ( alkyl ) 3 ; or r 1 and r 2 taken together are c ( ch 3 ) 2 , p ( o ) oh , or p ( o ) or 5 ; r 5 represents independently for each occurrence h , li + , li + , na + , k + , rb + , cs + , aryl , or an optionally substituted alkyl group ; r 7 is alkyl , aryl , — ch 2 - aryl , — c ( o )- alkyl , — c ( o )- aryl , or — si ( alkyl ) 3 ; and in certain embodiments , the present invention relates to the aforementioned method , wherein r is — ch 2 - aryl . in certain embodiments , the present invention relates to the aforementioned method , wherein r 1 and r 2 taken together are c ( ch 3 ) 2 . in certain embodiments , the present invention relates to the aforementioned method , wherein r 3 is — n 3 . in certain embodiments , the present invention relates to the aforementioned method , wherein r 6 is alkyl . in certain embodiments , the present invention relates to the aforementioned method , wherein r 7 is — c ( o )- alkyl . in certain embodiments , the present invention relates to the aforementioned method , wherein r is benzyl , r 1 and r 2 taken together are c ( ch 3 ) 2 , and r 3 is — n 3 . in certain embodiments , the present invention relates to the aforementioned method , wherein r is benzyl , r 1 and r 2 taken together are c ( ch 3 ) 2 , r 3 is — n 3 , and r 6 is ethyl . method of preparing glycosylphosphatidylinositol glycans using automatic synthesis on solid support one aspect of the present invention relates to a method of preparing glycosylphosphatidylinositol glycans , comprising the steps of : binding a mannopyranoside to a solid support to provide a first substrate , reacting said first substrate with a mannopyranose trichloroacetimidate to give a disaccharide bound to said solid support , reacting said disaccharide with a mannopyranose trichloroacetimidate to give a trisaccharide bound to said solid support , reacting said trisaccharide with a mannopyranose trichloroacetimidate to give a tetrasaccharide bound to said solid support , and cleaving said tetrasaccharide from said solid support . in certain embodiments , the present invention relates to the aforementioned method , wherein said mannopyranoside is bound to said solid support through a glycosidic linkage . in certain embodiments , the present invention relates to the aforementioned method , wherein said tetrasaccharide is cleaved from said solid support using grubbs &# 39 ; catalyst . for convenience , certain terms employed in the specification , examples , and appended claims are collected here . the articles “ a ” and “ an ” are used herein to refer to one or to more than one ( i . e . to at least one ) of the grammatical object of the article . by way of example , “ an element ” means one element or more than one element . for purposes of this invention , the chemical elements are identified in accordance with the periodic table of the elements , cas version , handbook of chemistry and physics , 67th ed ., 1986 - 87 , inside cover . the term “ alkyl ” refers to the radical of saturated aliphatic groups , including straight - chain alkyl groups , branched - chain alkyl groups , cycloalkyl ( alicyclic ) groups , alkyl substituted cycloalkyl groups , and cycloalkyl substituted alkyl groups . in preferred embodiments , a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone ( e . g ., c 1 - c 30 for straight chain , c 3 - c 30 for branched chain ), and more preferably 20 of fewer . likewise , preferred cycloalkyls have from 4 - 10 carbon atoms in their ring structure , and more preferably have 5 , 6 or 7 carbons in the ring structure . unless the number of carbons is otherwise specified , “ lower alkyl ” as used herein means an alkyl group , as defined above , but having from one to ten carbons , more preferably from one to six carbon atoms in its backbone structure . likewise , “ lower alkenyl ” and “ lower alkynyl ” have similar chain lengths . the terms “ alkenyl ” and “ alkynyl ” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above , but which contain at least one double or triple carbon - carbon bond , respectively . as used herein , the term “ amino ” means — nh 2 ; the term “ nitro ” means — no 2 ; the term “ halogen ” designates — f , — cl , — br or — i ; the term “ thiol ” means — sh ; the term “ hydroxyl ” means — oh ; the term “ sulfonyl ” means — so 2 —; and the term “ organometallic ” refers to a metallic atom ( such as mercury , zinc , lead , magnesium or lithium ) or a metalloid ( such as silicon , arsenic or selenium ) which is bonded directly to a carbon atom , such as a diphenylmethylsilyl group . the terms “ amine ” and “ amino ” are art - recognized and refer to both unsubstituted and substituted amines , e . g ., a moiety that can be represented by the general formula : wherein r 9 , r 10 and r ′ 10 each independently represent a group permitted by the rules of valence . the term “ acylamino ” is art - recognized and refers to a moiety that can be represented by the general formula : wherein r 9 is as defined above , and r ′ 11 represents a hydrogen , an alkyl , an alkenyl or —( ch 2 ) m — r 8 , where m and r 8 are as defined above . the term “ amido ” is art - recognized as an amino - substituted carbonyl and includes a moiety that can be represented by the general formula : wherein r 9 , r 10 are as defined above . preferred embodiments of the amide will not include imides which may be unstable . the term “ alkylthio ” refers to an alkyl group , as defined above , having a sulfur radical attached thereto . in preferred embodiments , the “ alkylthio ” moiety is represented by one of — s - alkyl , — s - alkenyl , — s - alkynyl , and — s —( ch 2 ) m — r 8 , wherein m and r 8 are defined above . representative alkylthio groups include methylthio , ethyl thio , and the like . the term “ carbonyl ” is art - recognized and includes such moieties as can be represented by the general formula : wherein x is a bond or represents an oxygen or a sulfur , and r 11 represents a hydrogen , an alkyl , an alkenyl , —( ch 2 ) m — r 8 or a pharmaceutically acceptable salt , r ′ 11 represents a hydrogen , an alkyl , an alkenyl or —( ch 2 ) m — r 8 , where m and r 8 are as defined above . where x is an oxygen and r 11 or r ′ 11 is not hydrogen , the formula represents an “ ester ”. where x is an oxygen , and r 11 is as defined above , the moiety is referred to herein as a carboxyl group , and particularly when r 11 is a hydrogen , the formula represents a “ carboxylic acid ”. where x is an oxygen , and r ′ 11 is hydrogen , the formula represents a “ formate ”. in general , where the oxygen atom of the above formula is replaced by sulfur , the formula represents a “ thiolcarbonyl ” group . where x is a sulfur and r 11 or r ′ 11 is not hydrogen , the formula represents a “ thiolester .” where x is a sulfur and r 11 is hydrogen , the formula represents a “ thiolcarboxylic acid .” where x is a sulfur and r 11 ′ is hydrogen , the formula represents a “ thiolformate .” on the other hand , where x is a bond , and r 11 is not hydrogen , the above formula represents a “ ketone ” group . where x is a bond , and r 11 is hydrogen , the above formula represents an “ aldehyde ” group . the terms “ alkoxyl ” or “ alkoxy ” as used herein refers to an alkyl group , as defined above , having an oxygen radical attached thereto . representative alkoxyl groups include methoxy , ethoxy , propyloxy , tert - butoxy and the like . an “ ether ” is two hydrocarbons covalently linked by an oxygen . accordingly , the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl , such as can be represented by one of — o - alkyl , — o - alkenyl , — o - alkynyl , — o —( ch 2 ) m — r 8 , where m and r 8 are described above . the term “ sulfonate ” is art - recognized and includes a moiety that can be represented by the general formula : in which r 41 is an electron pair , hydrogen , alkyl , cycloalkyl , or aryl . the term “ sulfonylamino ” is art - recognized and includes a moiety that can be represented by the general formula : the term “ sulfamoyl ” is art - recognized and includes a moiety that can be represented by the general formula : the term “ sulfonyl ”, as used herein , refers to a moiety that can be represented by the general formula : in which r 44 is selected from the group consisting of hydrogen , alkyl , alkenyl , alkynyl , cycloalkyl , heterocyclyl , aryl , or heteroaryl . the term “ sulfoxido ” as used herein , refers to a moiety that can be represented by the general formula : in which r 44 is selected from the group consisting of hydrogen , alkyl , alkenyl , alkynyl , cycloalkyl , heterocyclyl , aralkyl , or aryl . the term “ sulfate ”, as used herein , means a sulfonyl group , as defined above , attached to two hydroxy or alkoxy groups . thus , in a preferred embodiment , a sulfate has the structure : in which r 40 and r 41 are independently absent , a hydrogen , an alkyl , or an aryl . furthermore , r 40 and r 41 , taken together with the sulfonyl group and the oxygen atoms to which they are attached , may form a ring structure having from 5 to 10 members . the term “ trichloroacetimidate ” refers a moiety that can be represented by the general structure : analogous substitutions can be made to alkenyl and alkynyl groups to produce , for example , alkenylamines , alkynylamines , alkenylamides , alkynylamides , alkenylimines , alkynylimines , thioalkenyls , thioalkynyls , carbonyl - substituted alkenyls or alkynyls , alkenoxyls , alkynoxyls , metalloalkenyls and metalloalkynyls . the term “ aryl ” as used herein includes 4 -, 5 -, 6 - and 7 - membered single - ring aromatic groups which may include from zero to four heteroatoms , for example , benzene , naphthalene , anthracene , pyrene , pyrrole , furan , thiophene , imidazole , oxazole , thiazole , triazole , pyrazole , pyridine , pyrazine , pyridazine and pyrimidine , and the like . those aryl groups having heteroatoms in the ring structure may also be referred to as “ aryl heterocycle ”. the aromatic ring can be substituted at one or more ring positions with such substituents as described above , as for example , halogens , alkyls , alkenyls , alkynyls , hydroxyl , amino , nitro , thiol amines , imines , amides , phosphonates , phosphines , carbonyls , carboxyls , silyls , ethers , thioethers , sulfonyls , selenoethers , ketones , aldehydes , esters , or —( ch 2 ) m — r 7 , — cf 3 , — cn , or the like . the terms “ heterocycle ” or “ heterocyclic group ” refer to 4 to 10 - membered ring structures , more preferably 5 to 7 membered rings , which ring structures include one to four heteroatoms . heterocyclic groups include pyrrolidine , oxolane , thiolane , imidazole , oxazole , piperidine , piperazine , morpholine . the heterocyclic ring can be substituted at one or more positions with such substituents as described above , as for example , halogens , alkyls , alkenyls , alkynyls , hydroxyl , amino , nitro , thiol , amines , imines , amides , phosphonates , phosphines , carbonyls , carboxyls , silyls , ethers , thioethers , sulfonyls , selenoethers , ketones , aldehydes , esters , or —( ch 2 ) m — r 7 , — cf 3 , — cn , or the like . the term “ heteroatom ” as used herein means an atom of any element other than carbon or hydrogen . preferred heteroatoms are nitrogen , oxygen , sulfur , phosphorus and selenium . the terms ortho , meta and para apply to 1 , 2 -, 1 , 3 - and 1 , 4 - disubstituted benzenes , respectively . for example , the names 1 , 2 - dimethylbenzene and ortho - dimethylbenzene are synonymous . as used herein , the term “ substituted ” is contemplated to include all permissible substituents of organic compounds . in a broad aspect , the permissible substituents include acyclic and cyclic , branched and unbranched , carbocyclic and heterocyclic , aromatic and nonaromatic substituents of organic compounds . illustrative substituents include , for example , those described hereinabove . the permissible substituents can be one or more and the same or different for appropriate organic compounds . for purposes of this invention , the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms . this invention is not intended to be limited in any manner by the permissible substituents of organic compounds . the phrase “ protecting group ” as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations . examples of such protecting groups include esters of carboxylic acids , silyl ethers of alcohols , and acetals and ketals of aldehydes and ketones , respectively . the field of protecting group chemistry has been reviewed ( greene , t . w . ; wuts , p . g . m . protective groups in organic synthesis , 2 nd ed . ; wiley : new york , 1991 ). the invention now being generally described , it will be more readily understood by reference to the following examples , which are included merely for purposes of illustration of certain aspects and embodiments of the present invention , and are not intended to limit the invention . all reactions were performed in oven - dried glassware under an atmosphere of nitrogen unless noted otherwise . reagent grade chemicals were used as supplied except where noted . phosphate buffered saline ( pbs ) was purchased from boehringer mannheim and diluted to the desired concentration . pd - 10 columns were purchased from pharmacia . trimethylsilyl trifluoromethanesulfonate ( tmsotf ) was purchased from acros chemicals . n , n - dimethylformamide ( dmf ) was obtained from aldrich chemical co . ( sure - seal grade ) and used without further purification . merrifield &# 39 ; s resin ( 1 % crosslinked ) was obtained from novabiochem . dichloromethane ( ch 2 cl 2 ) and tetrahydrofuran ( thf ) were purchased from j . t . baker ( cycletainer ™) and passed through neutral alumina columns prior to use . toluene was purchased from j . t . baker ( cycletainer ™) and passed through a neutral alumina column and a copper ( ii ) oxide column prior to use . pyridine , triethylamine and acetonitrile were refluxed over calcium hydride and distilled prior to use . analytical thin - layer chromatography was performed on e . merck silica column 60 f 254 plates ( 0 . 25 mm ). compounds were visualized by dipping the plates in a cerium sulfate - ammonium molybdate solution followed by heating . liquid column chromatography was performed using forced flow of the indicated solvent on silicycle 230 - 400 mesh ( 60 å pore diameter ) silica gel . ir spectra were obtained on a perkin - elmer 1600 series ftir spectrometer . optical rotations were recorded on a perkin - elmer 241 polarimeter using a sodium lamp ( 589 nm ). 1 h nmr spectra were obtained on a varian vxr - 300 ( 300 mhz ), a varian vxr - 500 ( 500 mhz ) or a bruker ( 400 mhz ) spectrometer and are reported in parts per million ( δ ) relative to chcl 3 ( 7 . 27 ppm ). coupling constants ( j ) are reported in hertz . 13 c nmr spectra were obtained on a varian vxr - 300 ( 75 mhz ), a varian vxr - 500 ( 125 mhz ) or a bruker ( 100 mhz ) spectrometer and are reported in δ relative to cdcl 3 ( 77 . 23 ppm ) as an internal reference . 31 p nmr spectra were obtained on a varian vxr - 300 ( 120 mhz ) and are reported in δ relative to h 3 po 4 ( 0 . 0 ppm ) as an external reference . ethyl - 4 - o - acetyl - 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - thio - β - d - glucopyranoside 5 . [ α ] 24 d : − 43 . 5 ° ( c 1 . 07 , ch 2 cl 2 ); ir ( thin film ) 2916 , 2108 , 1743 , 1222 , 1047 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 29 ( m , 10h ), 5 . 06 - 5 . 02 ( m , 1h ), 4 . 86 ( d , j = 11 . 3 hz , 1h ), 4 . 68 ( d , j = 11 . 3 hz , 1h ), 4 . 52 ( s , 2h ), 4 . 34 ( d , j = 9 . 5 hz , 1h ), 3 . 59 - 3 . 48 ( m , 6h ), 2 . 83 - 2 . 73 ( m , 2h ), 1 . 87 ( s , 3h ), 1 . 35 ( t , j = 7 . 3 hz , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 169 . 9 , 138 . 0 , 137 . 8 , 128 . 8 , 128 . 6 , 128 . 3 , 128 . 1 , 128 . 0 , 84 . 5 , 82 . 8 , 77 . 9 , 75 . 9 , 73 . 8 , 71 . 1 , 69 . 9 , 66 . 0 , 25 . 0 , 21 . 0 , 15 . 4 ; fab ms m / z ( m + na ) + calcd 494 . 1726 , found 494 . 1716 . 6 - o - acetyl - 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranose trichloroacetimidate 6 . [ α ] 24 d : + 34 . 4 ° ( c 2 . 18 , ch 2 cl 2 ); ir ( thin film ) 2938 , 2880 , 1707 , 1683 , 1220 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 8 . 53 ( s , 1h ), 7 . 38 - 7 . 21 ( m , 14h ), 6 . 29 ( d , j = 2 . 1 hz , 1h ), 4 . 91 ( d , j = 10 . 7 hz , 1h ), 4 . 73 ( s , 2h ), 4 . 61 - 4 . 55 ( m , 3h ), 4 . 33 ( dd , j = 2 . 1 , 12 . 2 hz , 1h ), 4 . 24 ( dd , j = 4 . 3 , 11 . 9 hz , 1h ), 4 . 00 - 3 . 90 ( m , 4h ), 3 . 84 - 3 . 83 ( m , 1h ), 2 . 00 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 8 , 160 . 3 , 137 . 8 , 137 . 8 , 137 . 7 , 128 . 5 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 0 , 127 . 8 , 127 . 8 , 95 . 6 , 78 . 8 , 75 . 3 , 73 . 6 , 73 . 2 , 72 . 7 , 72 . 5 , 72 . 2 , 63 . 0 , 20 . 8 ; fab ms m / z ( m + na ) + calcd 658 . 1137 , found 658 . 1123 . 2 - o - acetyl - 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranose trichloroacetimidate 8 . [ α ] 24 d : + 43 . 4 ° ( c 2 . 20 , ch 2 cl 2 ); ir ( thin film ) 2940 , 2865 , 1752 , 1674 , 1229 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 8 . 63 ( s , 1h ), 7 . 38 - 7 . 26 ( m , 10h ), 6 . 26 ( d , j = 1 . 8 hz , 1h ), 5 . 46 ( dd , j = 2 . 1 , 3 . 1 hz , 1h ), 4 . 93 ( d , j = 10 . 4 hz , 1h ), 4 . 75 ( d , j = 11 . 3 hz , 1h ), 4 . 71 ( d , j = 10 . 7 hz , 1h ), 4 . 62 - 4 . 57 ( m , 1h ), 4 . 17 ( t , j = 9 . 8 hz , 1h ), 4 . 09 - 4 . 05 ( m , 2h ), 3 . 95 ( d , j = 11 . 3 hz , 1h ), 3 . 85 ( dd , j = 1 . 5 , 9 . 8 hz , 1h ), 2 . 16 ( s , 3h ), 1 . 15 - 1 . 05 ( m , 22h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 8 , 160 . 7 , 139 . 1 , 138 . 3 , 129 . 1 , 129 . 1 , 129 . 0 , 128 . 8 , 128 . 6 , 128 . 5 , 96 . 2 , 91 . 6 , 77 . 9 , 76 . 3 , 74 . 0 , 72 . 8 , 68 . 1 , 62 . 8 , 21 . 6 , 18 . 7 , 18 . 6 , 12 . 8 ; fab ms m / z ( m + na ) + calcd 724 . 2007 , found 724 . 2006 . o -( 4 - o - acetyl - 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 20 . a mixture of thiodonor 5 ( 3 . 21 g , 6 . 82 mmol ) and inositol 4 ( 2 . 78 g , 5 . 68 mmol ) were azeotroped with toluene ( 3 × 100 ml ) and dried under vacuum for 16 h . the oil was dissolved in et 2 o ( 110 ml )/ ch 2 cl 2 ( 30 ml ) and freshly dried 4 å molecular sieves ( 4 g ) were added . after stirring for 30 min at room temperature , the cloudy mixture was cooled to − 40 ° c ., and nis ( 2 . 04 g , 9 . 09 mmol ) was added . a 0 . 5 m solution of agotf in toluene ( 4 . 54 ml , 2 . 27 mmol ) was added via cannula , and the flask was covered with aluminum foil and allowed to warm to room temperature . after 15 h , the sieves were filtered off and the orange solution was diluted with ch 2 cl 2 ( 100 ml ), washed with sat . aqueous nahco 3 ( 2 × 100 ml ) and brine ( 1 × 100 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 20 - 930 % etoac / hexanes ) to afford 20 ( 3 . 56 g , 70 %) as a 1 . 2 : 1 α / β mixture . α - isomer : [ α ] 24 d : + 58 . 4 ° ( c 1 . 92 , ch 2 cl 2 ); ir ( thin film ) 2106 , 1742 , 1454 , 1229 , 1042 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 22 ( m , 20h ), 5 . 62 ( d , j = 3 . 4 hz , 1h ), 5 . 14 ( app t , 1h ), 4 . 88 - 4 . 73 ( m , 5h ), 4 . 68 ( d , j = 11 . 3 hz , 1h ), 4 . 57 ( d , j = 11 . 0 hz , 1h ), 4 . 46 ( d , j = 11 . 9 hz , 1h ), 4 . 33 ( d , j = 11 . 9 hz , 1h ), 4 . 25 ( app t , 1h ), 4 . 18 ( app t , 1h ), 4 . 13 - 4 . 09 ( m , 2h ), 3 . 94 ( app t , 1h ), 3 . 88 ( t , j = 9 . 8 hz , 1h ), 3 . 72 ( dd , j = 3 . 7 , 8 . 5 hz , 1h ), 3 . 46 - 3 . 40 ( m , 2h ), 3 . 27 ( dd , j = 2 . 4 , 11 . 0 hz , 1h ), 3 . 12 ( dd , j = 4 . 0 , 11 . 0 hz , 1h ), 1 . 74 ( s , 3h ), 1 . 55 ( s , 3h ), 1 . 36 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 0 , 139 . 0 , 138 . 9 , 138 . 7 , 138 . 5 , 138 . 3 , 129 . 2 , 129 . 2 , 129 . 1 , 128 . 9 , 128 . 9 , 128 . 8 , 128 . 7 , 128 . 7 , 128 . 6 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 1 , 111 . 0 , 96 . 0 , 81 . 7 , 81 . 2 , 79 . 8 , 78 . 3 , 77 . 9 , 77 . 4 , 75 . 9 , 75 . 4 , 75 . 0 , 74 . 0 , 73 . 9 , 71 . 0 , 69 . 2 , 68 . 5 , 63 . 4 , 28 . 4 , 26 . 5 , 21 . 5 ; fab ms m / z ( m + na ) + calcd 922 . 3891 , found 922 . 3864 . o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 21 . α - disaccharide 20 ( 1 . 85 g , 2 . 0 mmol ) was dissolved in ch 2 cl 2 ( 8 ml ) and a 0 . 5 m solution of sodium methoxide in methanol ( 8 ml , 4 . 0 mmol ) was added . after 21 h , the clear solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 10 → 40 % etoac / hexanes ) to afford 21 ( 1 . 24 g , 70 %). [ α ] 24 d : + 47 . 2 ° ( c 2 . 76 , ch 2 cl 2 ); ir ( thin film ) 2105 , 1496 , 1454 , 1044 , 726 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 09 ( m , 25h ), 5 . 57 ( d , j = 3 . 7 hz , 1h ), 4 . 88 - 4 . 74 ( m , 8h ), 4 . 67 ( d , j = 10 . 7 hz , 1h ), 4 . 48 ( d , j = 12 . 2 hz , 1h ), 4 . 40 ( d , j = 12 . 2 hz , 1h ), 4 . 25 ( dd , j = 4 . 0 , 5 . 5 hz , 1h ), 4 . 19 - 4 . 16 ( m , 1h ), 4 . 07 ( dd , j = 7 . 0 , 10 . 1 hz , 1h ), 4 . 00 - 3 . 97 ( m , 1h ), 3 . 95 ( t , j = 8 . 6 hz , 1h ), 3 . 78 - 3 . 69 ( m , 3h ), 3 . 48 - 3 . 41 ( m , 3h ), 3 . 31 ( dd , j = 3 . 7 , 9 . 8 hz , 1h ), 1 . 55 ( s , 3h ), 1 . 35 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 139 . 1 , 138 . 8 , 138 . 8 , 138 . 7 , 138 . 6 , 129 . 3 , 129 . 2 , 129 . 1 , 129 . 1 , 128 . 9 , 128 . 8 , 128 . 8 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 4 , 128 . 4 , 110 . 9 , 96 . 2 , 81 . 6 , 81 . 3 , 80 . 0 , 79 . 9 , 78 . 0 , 77 . 8 , 77 . 5 , 76 . 1 , 75 . 8 , 75 . 6 , 75 . 4 , 74 . 0 , 73 . 2 , 70 . 3 , 70 . 1 , 63 . 4 , 28 . 4 , 26 . 5 ; fab ms m / z ( m + na ) + calcd 880 . 3785 , found 880 . 3769 . o -( 6 - o - acetyl - 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 22 . a mixture of disaccharide acceptor 21 ( 1 . 24 g , 1 . 17 mmol ) and donor 6 ( 890 mg , 1 . 4 mmol ) were azeotroped with toluene ( 3 × 40 ml ) and dried under vacuum for 16 h . the mixture was dissolved in ch 2 cl 2 ( 11 ml ) and tmsotf ( 11 μl , 0 . 058 mmol ) was added . after 30 min , the orange solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 10 → 30 % etoac / hexanes ) to afford 22 ( 592 mg , 47 %) and 75 ( 797 mg , 75 % based on recovered 74 ). [ α ] 24 d : + 55 . 2 ° ( c 1 . 01 , ch 2 cl 2 ); ir ( thin film ) 2923 , 2104 , 1740 , 1454 , 697 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 39 - 7 . 07 ( m , 33h ), 5 . 63 ( d , j = 3 . 7 hz , 1h ), 5 . 20 ( d , j = 1 . 5 hz , 1h ), 4 . 94 - 4 . 90 ( m , 2h ), 4 . 82 - 4 . 74 ( m , 4h ), 4 . 68 ( d , j = 10 . 7 hz , 1h ), 4 . 65 - 4 . 50 ( m , 3h ), 4 . 43 ( d , j = 8 . 8 hz , 1h ), 4 . 41 ( d , j = 8 . 8 hz , 1h ), 4 . 30 ( d , j = 11 . 9 hz , 1h ), 4 . 27 - 4 . 25 ( m , 1h ), 4 . 22 - 4 . 06 ( m , 6h ), 3 . 93 - 3 . 88 ( m , 2h ), 3 . 83 - 3 . 78 ( m , 3h ), 3 . 74 - 3 . 67 ( m , 3h ), 3 . 57 ( d , j = 2 . 4 hz , 1h ), 3 . 45 - 3 . 41 ( m , 1h ), 3 . 37 - 3 . 34 ( m , 1h ), 1 . 96 ( s , 3h ), 1 . 55 ( s , 3h ), 1 . 35 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 5 , 138 . 2 , 138 . 1 , 138 . 0 , 137 . 9 , 137 . 8 , 137 . 8 , 137 . 4 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 8 , 127 . 7 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 4 , 127 . 3 , 127 . 2 , 127 . 0 , 126 . 8 , 126 . 5 , 110 . 0 , 100 . 6 , 95 . 1 , 80 . 6 , 80 . 4 , 79 . 5 , 79 . 2 , 78 . 8 , 78 . 0 , 78 . 0 , 77 . 1 , 76 . 3 , 75 . 7 , 75 . 0 , 74 . 8 , 74 . 7 , 74 . 4 , 74 . 0 , 73 . 8 , 73 . 0 , 72 . 7 , 71 . 8 , 71 . 7 , 70 . 7 , 69 . 6 , 68 . 2 , 63 . 2 , 62 . 9 , 29 . 4 , 27 . 3 , 25 . 5 , 20 . 6 ; fab ms m / z ( m + na ) + calcd 1354 . 5827 , found 1354 . 5859 . o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 23 . trisaccharide 22 ( 1 . 42 g , 1 . 06 mmol ) was dissolved in ch 2 cl 2 ( 11 ml ) and a 0 . 5 m solution of sodium methoxide in methanol ( 2 . 12 ml , 1 . 06 mmol ) was added . after 2 h , the clear solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 10 → 40 % etoac / hexanes ) to afford 23 ( 973 mg , 71 %). [ α ] 24 d : + 42 . 0 ° ( c 1 . 76 , ch 2 cl 2 ); ir ( thin film ) 2291 , 2104 , 1454 , 1360 , 735 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 10 ( m , 42h ), 5 . 61 ( d , j = 3 . 7 hz , 1h ), 5 . 24 ( d , j = 2 . 1 hz , 1h ), 4 . 90 - 4 . 88 ( m , 2h ), 4 . 83 - 4 . 67 ( m , 6h ), 4 . 63 - 4 . 54 ( m , 4h ), 4 . 51 ( s , 2h ), 4 . 44 ( d , j = 11 . 9 hz , 1h ), 4 . 34 ( d , j = 11 . 9 hz , 1h ), 4 . 25 ( dd , j = 3 . 7 , 5 . 5 hz , 1h ), 4 . 20 - 4 . 17 ( m , 1h ), 4 . 15 - 4 . 03 ( m , 3h ), 3 . 94 - 3 . 79 ( m , 5h ), 3 . 77 ( dd , j = 2 . 4 , 8 . 85 hz , 1h ), 3 . 74 - 3 . 72 ( m , 1h ), 3 . 66 - 3 . 62 ( m , 3h ), 3 . 59 - 3 . 56 ( m , 2h ), 3 . 49 - 3 . 47 ( m , 1h ), 3 . 42 ( dd , j = 8 . 2 , 9 . 8 hz , 1h ), 3 . 36 ( dd , j = 3 . 7 , 10 . 1 hz , 1h ), 2 . 16 ( bs , 1h ), 1 . 56 ( s , 3h ), 1 . 36 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 139 . 3 , 139 . 2 , 139 . 1 , 139 . 0 , 138 . 9 , 138 . 7 , 138 . 5 , 129 . 4 , 129 . 3 , 129 . 3 , 129 . 2 , 129 . 2 , 129 . 1 , 129 . 0 , 129 . 0 , 129 . 0 , 128 . 9 , 128 . 8 , 128 . 8 , 128 . 8 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 2 , 128 . 0 , 127 . 9 , 127 . 7 , 111 . 0 , 101 . 5 , 96 . 1 , 81 . 6 , 81 . 3 , 80 . 8 , 79 . 9 , 79 . 9 , 78 . 3 , 78 . 0 , 77 . 3 , 77 . 3 , 76 . 8 , 76 . 6 , 75 . 9 , 75 . 8 , 75 . 6 , 75 . 5 , 75 . 4 , 74 . 9 , 74 . 8 , 74 . 8 , 74 . 6 , 74 . 1 , 74 . 0 , 73 . 4 , 73 . 2 , 73 . 0 , 72 . 7 , 71 . 0 , 68 . 7 , 64 . 0 , 63 . 1 , 61 . 7 , 30 . 4 , 28 . 4 , 26 . 5 ; fab ms m / z ( m + na ) + calcd 1312 . 5722 , found 1312 . 5680 . o -( 2 - o - acetyl - 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 24 . a mixture of trisaccharide acceptor 23 ( 970 mg , 0 . 75 mmol ) and donor 7 ( 1 . 00 g , 1 . 50 mmol ) were azeotroped with toluene ( 3 × 30 ml ) and dried under vacuum for 1 h . the mixture was dissolved in ch 2 cl 2 ( 7 ml ) and a 0 . 5 m solution of tmsotf in ch 2 cl 2 ( 76 μl , 0 . 038 mmol ) was added . after 15 min , the orange solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 10 → 40 % etoac / hexanes ) to afford 24 ( 1 . 22 g , 92 %). [ α ] 24 d : + 58 . 9 ° ( c 3 . 30 , ch 2 cl 2 ); ir ( thin film ) 3029 , 2105 , 1745 , 1454 , 1237 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 10 ( m , 54h ), 5 . 62 ( d , j = 3 . 4 hz , 1h ), 5 . 49 - 5 . 48 ( m , 1h ), 5 . 26 ( s , 1h ), 4 . 94 - 4 . 88 ( m , 4h ), 4 . 85 - 4 . 75 ( m , 7h ), 4 . 69 ( d , j = 10 . 9 hz , 1h ), 4 . 62 ( d , j = 12 . 2 hz , 1h ), 4 . 58 - 4 . 55 ( m , 2h ), 4 . 54 - 4 . 46 ( m , 4h ), 4 . 44 - 4 . 36 ( m , 4h ), 4 . 33 - 4 . 30 ( m , 1h ), 4 . 28 - 4 . 23 ( m , 3h ), 4 . 20 - 4 . 09 ( m , 3h ), 3 . 98 - 3 . 79 ( m , 10h ), 3 . 74 - 3 . 66 ( m , 5h ), 3 . 61 ( dd , j = 3 . 7 , 10 . 7 hz , 1h ), 3 . 56 ( s , 2h ), 3 . 52 - 3 . 42 ( m , 4h ), 3 . 35 ( dd , j = 3 . 7 , 9 . 2 hz , 1h ), 2 . 12 ( s , 3h ), 1 . 54 ( s , 3h ), 1 . 35 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 1 , 138 . 7 , 138 . 6 , 138 . 5 , 138 . 4 , 138 . 3 , 138 . 2 , 138 . 1 , 138 . 0 , 137 . 8 , 137 . 7 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 128 . 0 , 127 . 9 , 127 . 9 , 127 . 8 , 127 . 7 , 127 . 7 , 127 . 7 , 127 . 6 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 4 , 127 . 3 , 127 . 3 , 127 . 2 , 127 . 1 , 127 . 0 , 110 . 2 , 100 . 3 , 98 . 3 , 95 . 2 , 80 . 9 , 80 . 6 , 79 . 8 , 79 . 1 , 77 . 7 , 77 . 2 , 76 . 6 , 75 . 9 , 75 . 3 , 75 . 1 , 75 . 0 , 74 . 7 , 74 . 6 , 74 . 2 , 74 . 0 , 73 . 8 , 73 . 3 , 72 . 9 , 72 . 2 , 72 . 1 , 71 . 9 , 71 . 3 , 71 . 2 , 69 . 8 , 68 . 7 , 68 . 6 , 68 . 2 , 66 . 4 , 62 . 9 , 29 . 7 , 27 . 5 , 25 . 7 , 21 . 1 ; fab ms m / z ( m + na ) + calcd 1786 . 7764 , found 1786 . 7710 . o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 25 . tetrasaccharide 24 ( 1 . 22 g , 0 . 70 mmol ) was dissolved in ch 2 cl 2 ( 7 ml ) and a 0 . 5 m solution of sodium methoxide in methanol ( 1 . 4 ml , 0 . 70 mmol ) was added . after 2 h , the clear solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 20 → 40 % etoac / hexanes ) to afford 25 ( 820 mg , 69 %). [ α ] 24 d : + 54 . 7 ° ( c 2 . 32 , ch 2 cl 2 ); ir ( thin film ) 2923 , 2105 , 1454 , 1046 , 735 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 09 ( m , 51h ), 5 . 64 ( d , j = 3 . 7 hz , 1h ), 5 . 24 ( d , j = 1 . 5 hz , 1h ), 5 . 00 ( s , 1h ), 4 . 93 - 4 . 89 ( m , 2h ), 4 . 84 - 4 . 75 ( m , 6h ), 4 . 68 ( d , j = 10 . 7 hz , 1h ), 4 . 64 - 4 . 40 ( m , 12h ), 4 . 30 - 4 . 25 ( m , 2h ), 4 . 20 - 4 . 09 ( m , 4h ), 4 . 04 ( s , 1h ), 3 . 96 ( t , j = 9 . 5 hz , 1h ), 3 . 92 - 3 . 89 ( m , 1h ), 3 . 86 - 3 . 77 ( m , 6h ), 3 . 74 - 3 . 60 ( m , 6h ), 3 . 58 - 3 . 54 ( m , 4h ), 3 . 46 - 3 . 42 ( m , 1h ), 3 . 36 ( dd , j = 4 . 0 , 9 . 2 hz , 1h ), 2 . 20 ( bs , 1h ), 1 . 55 ( s , 3h ), 1 . 36 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 139 . 5 , 139 . 2 , 139 . 1 , 139 . 0 , 139 . 0 , 138 . 9 , 138 . 8 , 138 . 7 , 138 . 5 , 138 . 4 , 129 . 3 , 129 . 2 , 129 . 1 , 129 . 1 , 129 . 0 , 129 . 0 , 128 . 9 , 128 . 8 , 128 . 6 , 128 . 5 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 6 , 111 . 0 , 101 . 2 , 100 . 6 , 96 . 0 , 81 . 6 , 81 . 3 , 80 . 6 , 80 . 3 , 80 . 2 , 79 . 8 , 78 . 1 , 77 . 3 , 76 . 9 , 76 . 1 , 75 . 8 , 75 . 7 , 75 . 6 , 75 . 4 , 75 . 0 , 74 . 8 , 74 . 6 , 74 . 1 , 74 . 0 , 73 . 6 , 73 . 4 , 73 . 0 , 72 . 8 , 71 . 9 , 71 . 8 , 70 . 5 , 69 . 5 , 69 . 4 , 68 . 4 , 66 . 9 , 63 . 7 , 30 . 4 , 28 . 3 , 26 . 5 ; maldi - tof [ m + na ] + 1746 . o -( 2 - o - acetyl - 3 , 4 - di - o - benzyl - 6 - o - triisopropyllsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 26 . a mixture of tetrasaccharide acceptor 25 ( 615 mg , 0 . 36 mmol ) and donor 8 ( 502 mg , 0 . 71 mmol ) were azeotroped with toluene ( 3 × 10 ml ) and dried under vacuum for 16 h . the mixture was dissolved in ch 2 cl 2 ( 4 ml ) and a 0 . 5 m solution of tbsotf in ch 2 cl 2 ( 36 μl , 0 . 018 mmol ) was added . after 10 min , the orange solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 20 → 40 % etoac / hexanes ) to afford 26 ( 789 mg , 98 %). [ α ] 24 d : + 38 . 7 ° ( c 2 . 57 , ch 2 cl 2 ); ir ( thin film ) 2924 , 2105 , 1743 , 1454 , 735 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 40 - 7 . 09 ( m , 55h ), 5 . 61 ( d , j = 3 . 7 hz , 1h ), 5 . 48 - 5 . 47 ( m , 1h ), 5 . 28 ( d , j = 1 . 5 hz , 1h ), 5 . 08 ( d , j = 1 . 5 hz , 1h ), 4 . 95 - 4 . 74 ( m , 10h ), 4 . 69 - 4 . 66 ( m , 3h ), 4 . 62 ( d , j = 11 . 3 hz , 1h ), 4 . 58 - 4 . 34 ( m , 12h ), 4 . 28 - 4 . 25 ( m , 2h ), 4 . 20 - 4 . 04 ( m , 7h ), 4 . 00 ( dd , j = 3 . 0 , 9 . 8 hz , 1h ), 3 . 95 - 3 . 66 ( m , 15h ), 3 . 60 - 3 . 38 ( m , 6h ), 3 . 35 ( dd , j = 3 . 7 , 10 . 1 hz , 1h ), 2 . 07 ( s , 3h ), 1 . 53 ( s , 3h ), 1 . 34 ( s , 3h ), 1 . 09 - 1 . 07 ( m , 20h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 0 , 138 . 9 , 138 . 8 , 138 . 6 , 138 . 4 , 138 . 4 , 138 . 3 , 138 . 3 , 138 . 2 , 138 . 2 , 138 . 1 , 138 . 1 , 138 . 0 , 137 . 7 , 128 . 5 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 1 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 7 , 127 . 6 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 4 , 127 . 4 , 127 . 4 , 127 . 3 , 127 . 3 , 127 . 2 , 127 . 1 , 127 . 1 , 127 . 0 , 110 . 2 , 100 . 3 , 98 . 9 , 98 . 8 , 95 . 1 , 80 . 8 , 80 . 6 , 80 . 0 , 79 . 8 , 79 . 1 , 78 . 0 , 77 . 0 , 76 . 6 , 76 . 0 , 75 . 4 , 75 . 1 , 75 . 1 , 75 . 0 , 74 . 6 , 74 . 5 , 74 . 3 , 73 . 8 , 73 . 7 , 73 . 3 , 73 . 1 , 72 . 8 , 72 . 2 , 72 . 0 , 71 . 8 , 71 . 8 , 71 . 7 , 71 . 4 , 69 . 6 , 68 . 9 , 68 . 8 , 66 . 3 , 63 . 0 , 62 . 4 , 27 . 5 , 25 . 7 , 21 . 0 , 18 . 0 , 17 . 9 , 12 . 0 ; maldi - tof [ m + na ] + 2285 . o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 27 . pentasaccharide 26 ( 746 mg , 0 . 33 mmol ) was dissolved in ch 2 cl 2 ( 4 ml ) and a 0 . 5 m solution of sodium methoxide in methanol ( 660 μl , 0 . 33 mmol ) was added . after 2 . 5 h , the light yellow solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 10 → 30 % etoac / hexanes ) to afford 27 ( 604 mg , 83 %). [ α ] 24 d : + 30 . 6 ° ( c 1 . 21 , ch 2 cl 2 ); ir ( thin film ) 2358 , 2104 , 1454 , 1045 , 696 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 33 - 7 . 03 ( m , 59h ), 5 . 56 ( d , j = 3 . 7 hz , 1h ), 5 . 24 ( d , j = 1 . 8 hz , 1h ), 5 . 09 ( s , j = 1 . 2 hz , 1h ), 4 . 87 - 4 . 68 ( m , 11h ), 4 . 64 - 4 . 61 ( m , 3h ), 4 . 55 - 4 . 28 ( m , 16h ), 4 . 20 - 4 . 18 ( m , 2h ), 4 . 12 - 4 . 01 ( m , 7h ), 3 . 95 - 3 . 60 ( m , 20h ), 3 . 54 - 3 . 47 ( m , 3h ), 3 . 41 - 3 . 37 ( m , 4h ), 3 . 28 ( dd , j = 3 . 7 , 9 . 8 hz , 1h ), 2 . 16 ( d , j = 3 . 7 hz , 1h ), 1 . 47 ( s , 3h ), 1 . 29 ( s , 3h ), 1 . 05 - 0 . 97 ( m , 23h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 138 . 7 , 138 . 7 , 138 . 6 , 138 . 5 , 138 . 3 , 138 . 3 , 138 . 3 , 138 . 1 , 138 . 1 , 138 . 0 , 138 . 0 , 138 . 0 , 137 . 6 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 128 . 0 , 127 . 8 , 127 . 8 , 127 . 7 , 127 . 7 , 127 . 6 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 4 , 127 . 3 , 127 . 3 , 127 . 3 , 127 . 2 , 127 . 2 , 127 . 2 , 127 . 1 , 127 . 1 , 126 . 9 , 110 . 1 , 100 . 5 , 100 . 1 , 98 . 9 , 95 . 0 , 80 . 8 , 80 . 6 , 80 . 0 , 79 . 8 , 79 . 7 , 79 . 7 , 79 . 0 , 76 . 9 , 76 . 8 , 76 . 5 , 75 . 9 , 75 . 3 , 75 . 0 , 74 . 9 , 74 . 9 , 74 . 6 , 74 . 4 , 74 . 3 , 73 . 8 , 73 . 7 , 73 . 3 , 73 . 2 , 73 . 1 , 73 . 0 , 72 . 7 , 72 . 2 , 72 . 0 , 71 . 9 , 71 . 9 , 71 . 8 , 71 . 5 , 69 . 6 , 68 . 9 , 68 . 8 , 68 . 5 , 66 . 3 , 62 . 9 , 62 . 6 , 27 . 4 , 25 . 7 , 18 . 0 , 18 . 0 , 11 . 9 ; maldi - tof [ m + na ] + 2243 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 28 . a mixture of pentasaccharide acceptor 27 ( 767 mg , 0 . 35 mmol ) and donor 9 ( 473 mg , 0 . 69 mmol ) were azeotroped with toluene ( 3 × 25 ml ) and dried under vacuum for 1 . 5 h . the mixture was dissolved in ch 2 cl 2 ( 4 ml ) and a 0 . 5 m solution of tmsotf in ch 2 cl 2 ( 34 μl , 0 . 017 mmol ) was added . after 30 min , the orange solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 10 → 40 % etoac / hexanes ) to afford 28 ( 793 mg , 84 %) as a 3 . 1 : 1 α : β mixture . ir ( thin film ) 2863 , 2104 , 1496 , 1454 , 735 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 39 - 7 . 08 ( m , 75h ), 5 . 59 ( d , j = 3 . 4 hz , 1h ), 5 . 30 - 5 . 21 ( m , 2h ), 5 . 06 ( d , j = 12 . 2 hz , 0 . 6h ), 4 . 94 - 4 . 65 ( m , 14h ), 4 . 60 - 4 . 31 ( m , 19h ), 4 . 28 - 4 . 18 ( m , 4h ), 4 . 16 - 3 . 37 ( m , 36h ), 3 . 33 - 3 . 30 ( m , 1h ), 3 . 24 - 3 . 20 ( m , 0 . 5h ), 1 . 52 ( s , 2h ), 1 . 48 ( s , 0 . 64h ), 1 . 34 ( s , 2h ), 1 . 32 ( s , 0 . 88h ), 1 . 05 - 1 . 00 ( m , 20h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 140 . 0 , 139 . 8 , 139 . 8 , 139 . 7 , 139 . 6 , 139 . 6 , 139 . 5 , 139 . 4 , 139 . 3 , 139 . 3 , 139 . 3 , 139 . 2 , 139 . 1 , 139 . 0 , 139 . 0 , 138 . 9 , 138 . 8 , 138 . 8 , 138 . 7 , 138 . 5 , 129 . 5 , 129 . 4 , 1294 , 129 . 3 , 129 . 3 , 129 . 2 , 129 . 2 , 129 . 1 , 129 . 0 , 129 . 0 , 129 . 0 , 128 . 9 , 128 . 9 , 128 . 9 , 128 . 8 , 128 . 8 , 128 . 7 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 6 , 128 . 5 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 8 , 111 . 0 , 101 . 1 , 100 . 9 , 100 . 4 , 99 . 8 , 95 . 9 , 81 . 7 , 81 . 5 , 81 . 1 , 80 . 8 , 80 . 7 , 79 . 9 , 78 . 7 , 78 . 1 , 78 . 1 , 77 . 9 , 77 . 8 , 77 . 7 , 77 . 6 , 77 . 4 , 76 . 8 , 76 . 4 , 76 . 2 , 76 . 2 , 75 . 9 , 75 . 8 , 75 . 8 , 75 . 7 , 75 . 5 , 75 . 5 , 75 . 2 , 75 . 1 , 75 . 0 , 75 . 0 , 74 . 8 , 74 . 6 , 74 . 4 , 74 . 3 , 74 . 2 , 74 . 1 , 74 . 0 , 74 . 0 , 73 . 6 , 73 . 5 , 73 . 3 , 73 . 2 , 73 . 1 , 73 . 1 , 73 . 0 , 72 . 9 , 72 . 9 , 72 . 8 , 72 . 8 , 72 . 6 , 72 . 3 , 71 . 5 , 70 . 5 , 69 . 7 , 69 . 5 , 69 . 5 , 67 . 0 , 63 . 8 , 63 . 4 , 28 . 3 , 28 . 3 , 26 . 6 , 21 . 7 , 19 . 0 , 18 . 9 , 18 . 9 , 18 . 8 , 12 . 9 , 12 . 8 , 12 . 7 ; maldi - tof [ m + na ] + 2767 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - d - myo - inositol 29 . to hexasaccharide 28 ( 546 mg , 0 . 20 mmol ) in ch 3 cn ( 15 ml )/ ch 2 cl 2 ( 15 ml ) was added ethylene glycol ( 1 . 55 ml , 0 . 14 mmol ), followed by csa ( 276 mg , 1 . 19 mmol ). after 6 h , the clear solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ). the aqueous phase was back extracted with ch 2 cl 2 ( 2 × 50 ml ), and the combined organic phases were washed with brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 20450 % etoac / hexanes ) to afford 28 ( 131 mg ) and 29 ( 330 mg , 81 % based on recovered 28 ) as a 3 . 3 : 1 α : β mixture . ir ( thin film ) 3029 , 2864 , 2106 , 1458 , 1362 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 36 - 7 . 07 ( m , 75h ), 5 . 56 ( m , 0 . 3h ), 5 . 46 ( m , 0 . 2h ), 5 . 44 ( m , 0 . 2h ), 5 . 41 ( d , j = 3 . 4 hz , 1h ), 5 . 30 - 5 . 28 ( m , 2h ), 5 . 21 ( s , 1h ), 5 . 08 - 5 . 03 ( m , 0 . 6h ), 4 . 93 - 4 . 60 ( m , 16h ), 4 . 58 - 4 . 41 ( m , 14h ), 4 . 40 - 4 . 14 ( m , 11h ), 4 . 10 - 4 . 06 ( m , 1h ), 4 . 02 - 3 . 72 ( m , 17h ), 3 . 70 - 3 . 55 ( m , 8h ), 3 . 49 - 3 . 32 ( m , 10h ), 3 . 22 - 3 . 19 ( m , 0 . 42h ), 2 . 95 - 2 . 91 ( m , 0 . 43h ), 2 . 52 ( s , 1h ), 1 . 02 - 1 . 00 ( m , 20h ); 13 c - nmr ( 125 mhz , cdcl 3 ) 139 . 8 , 139 . 7 , 139 . 6 , 139 . 6 , 139 . 4 , 139 . 3 , 139 . 3 , 139 . 2 , 139 . 2 , 139 . 2 , 139 . 1 , 139 . 1 , 138 . 8 , 138 . 7 , 138 . 5 , 138 . 3 , 138 . 3 , 129 . 3 , 129 . 3 , 129 . 2 , 129 . 2 , 129 . 1 , 129 . 1 , 129 . 1 , 129 . 0 , 129 . 0 , 129 . 0 , 128 . 9 , 128 . 9 , 128 . 9 , 128 . 8 , 128 . 8 , 128 . 7 , 128 . 7 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 6 , 128 . 6 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 1 , 128 . 0 , 128 . 0 , 128 . 0 , 127 . 9 , 127 . 8 , 127 . 7 , 127 . 7 , 100 . 8 , 100 . 3 , 99 . 9 , 98 . 8 , 82 . 3 , 81 . 7 , 81 . 4 , 81 . 1 , 80 . 7 , 80 . 6 , 80 . 5 , 76 . 6 , 76 . 0 , 75 . 8 , 75 . 6 , 75 . 5 , 75 . 2 , 75 . 1 , 74 . 9 , 74 . 2 , 74 . 1 , 74 . 0 , 74 . 0 , 73 . 9 , 73 . 5 , 73 . 5 , 73 . 3 , 73 . 1 , 72 . 9 , 72 . 8 , 72 . 7 , 72 . 7 , 72 . 3 , 71 . 5 , 70 . 4 , 69 . 7 , 69 . 5 , 65 . 0 , 18 . 9 , 18 . 8 , 18 . 8 , 12 . 8 , 12 . 8 , 12 . 7 ; maldi - tof [ m + na ] + 2726 . o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - β - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 32 . [ α ] 24 d : − 38 . 2 ° ( c 3 . 88 , ch 2 cl 2 ); ir ( thin film ) 2871 , 2361 , 2339 , 2109 , 1071 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 32 - 7 . 16 ( m , 22h ), 4 . 87 ( d , j = 10 . 7 hz , 1h ), 4 . 82 ( d , j = 10 . 1 hz , 1h ), 4 . 79 ( d , j = 11 . 6 hz , 1h ), 4 . 75 - 4 . 65 ( m , 6h ), 4 . 45 ( d , j = 11 . 9 hz , 1h ), 4 . 41 ( d , j = 11 . 9 hz , 1h ), 4 . 16 ( at , 3h ), 3 . 98 - 3 . 92 ( m , 2h ), 3 . 85 ( at , 3h ), 3 . 65 - 3 . 57 ( m , 4h ), 3 . 41 ( at , 1h ), 3 . 37 ( dd , j = 8 . 3 , 10 . 1 hz , 1h ), 3 . 28 - 3 . 24 ( m , 1h ), 3 . 08 - 3 . 04 ( m , 1h ), 2 . 69 ( d , j = 1 . 8 hz , 1h ), 1 . 41 ( s , 3h ), 1 . 25 ( s , 3h ); 13 c - nmr ( 100 mhz , cdcl 3 ) δ 139 . 2 , 138 . 8 , 138 . 7 , 138 . 1 , 129 . 3 , 129 . 2 , 129 . 2 , 129 . 2 , 129 . 2 , 128 . 9 , 128 . 9 , 128 . 7 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 5 , 128 . 4 , 110 . 6 , 101 . 9 , 83 . 1 , 83 . 1 , 81 . 9 , 79 . 7 , 78 . 1 , 77 . 7 , 76 . 1 , 75 . 9 , 75 . 7 , 74 . 9 , 74 . 5 , 74 . 1 , 74 . 0 , 73 . 4 , 71 . 4 , 66 . 3 , 28 . 5 , 26 . 4 ; esi ms m / z ( m + na ) + calcd 880 . 3780 , found 880 . 3786 . o -( 2 - o - acetyl - 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 4 )- 0 -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - β - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 34 a mixture of pseudo - disaccharide acceptor 32 ( 554 mg , 0 . 65 mmol ) and donor 33 ( 681 mg , 0 . 97 mmol ) were azeotroped with toluene ( 3 × 10 ml ) and dried under vacuum for 16 h . the mixture was dissolved in ch 2 cl 2 ( 9 ml ) and a 0 . 5 m solution of tmsotf in ch 2 cl 2 ( 64 μl , 0 . 032 mmol ) was added . after 30 min , the orange solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 3 → 5 % etoac / toluene ) to afford 34 ( 604 mg , 67 %) as a colorless foam . [ α ] 24 d : − 8 . 10 ° ( c 0 . 68 , ch 2 cl 2 ); ir ( thin film ) 2865 , 2361 , 2339 , 2110 , 1741 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 39 - 7 . 19 ( m , 32h ), 5 . 37 ( dd , j = 1 . 8 , 3 . 0 hz , 1h ), 5 . 27 ( d , j = 1 . 2 hz , 1h ), 4 . 93 ( d , j = 11 . 0 hz , 1h ), 4 . 90 ( d , j = 10 . 4 hz , 1h ), 4 . 88 - 4 . 83 ( m , 2h ), 4 . 80 - 4 . 69 ( m , 6h ), 4 . 65 - 4 . 62 ( m , 2h ), 4 . 48 - 4 . 42 ( m , 3h ), 4 . 25 ( at , 1h ), 4 . 12 - 4 . 04 ( m , 2h ), 4 . 01 ( at , 1h ), 3 . 94 ( at , 1h ), 3 . 87 - 3 . 82 ( m , 3h ), 3 . 74 - 3 . 71 ( m , 1h ), 3 . 68 - 3 . 61 ( m , 3h ), 3 . 55 - 3 . 46 ( m , 3h ), 3 . 29 - 3 . 22 ( m , 2h ), 1 . 95 ( s , 3h ), 1 . 49 ( s , 3h ), 1 . 31 ( s , 3h ), 1 . 04 ( s , 21h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 170 . 0 , 138 . 8 , 138 . 4 , 138 . 1 , 138 . 0 , 138 . 0 , 137 . 7 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 1 , 128 . 0 , 128 . 0 , 128 . 0 , 128 . 0 , 128 . 0 , 127 . 9 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 6 , 127 . 6 , 127 . 5 , 127 . 4 , 127 . 4 , 127 . 4 , 109 . 8 , 101 . 1 , 98 . 5 , 83 . 4 , 82 . 4 , 81 . 2 , 78 . 8 , 77 . 9 , 76 . 8 , 75 . 2 , 75 . 2 , 75 . 1 , 74 . 4 , 74 . 4 , 74 . 1 , 73 . 7 , 73 . 5 , 73 . 4 , 73 . 4 , 73 . 2 , 71 . 7 , 69 . 2 , 68 . 8 , 66 . 2 , 61 . 9 , 27 . 6 , 25 . 6 , 20 . 7 , 18 . 0 , 17 . 9 , 12 . 0 ; esi ms m / z ( m + na ) + calcd 1420 . 6687 , found 1420 . 6693 . o -( 2 , 3 , 4 - tri - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - β - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 36 . trisaccharide 34 ( 600 mg , 0 . 43 mmol ) was dissolved in ch 2 cl 2 ( 5 ml ) and a 0 . 75 m solution of sodium methoxide in methanol ( 571 μl , 0 . 43 mmol ) was added . after 1 . 5 h , the clear solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous aq . nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering , and concentration the crude product was dissolved in dmf ( 5 ml ) and benzyl bromide ( 78 μl , 0 . 65 mmol ) was added . the clear solution was cooled to 0 ° c ., and nah ( 60 % dispersion in mineral oil , 26 mg , 0 . 65 mmol ) was added in one portion . after 14 h , meoh ( 5 ml ) was added , and the mixture was poured into h 2 o ( 50 ml ) and washed with et 2 o ( 3 × 50 ml ). the combined organic phases were washed with brine ( 1 × 50 ml ), dried ( na 2 so 4 ), filtered and concentrated . purification by flash silica column chromatography ( 0 → 8 % etoac / toluene ) afforded 36 ( 386 mg , 62 %). [ α ] 24 d : − 11 . 0 ° ( c 1 . 07 , chcl 3 ); ir ( thin film ) 2865 , 2361 , 2338 , 2110 , 1067 cm − ; 1 h nmr ( cdcl 3 ) δ 7 . 40 - 7 . 15 ( m , 45h ), 5 . 22 ( d , j = 1 . 0 hz , 1h ), 4 . 93 ( d , j = 10 . 7 hz , 1h ), 4 . 91 - 4 . 87 ( m , 2h ), 4 . 84 ( d , j = 9 . 5 hz , 1h ), 4 . 80 ( d , j = 3 . 4 hz , 1h ), 4 . 78 - 4 . 73 ( m , 4h ), 4 . 65 ( d , j = 10 . 7 hz , 1h ), 4 . 58 ( d , j = 11 . 9 hz , 1h ), 4 . 55 - 4 . 49 ( m , 3h ), 4 . 41 ( d , j = 11 . 6 hz , 1h ), 4 . 39 ( d , j = 12 . 2 hz , 1h ), 4 . 28 - 4 . 25 ( m , 2h ), 4 . 12 - 4 . 05 ( m , 3h ), 3 . 95 ( at , 1h ), 3 . 86 ( dd , j = 3 . 7 , 11 . 0 hz , 1h ), 3 . 82 - 3 . 79 ( m , 1h ), 3 . 76 - 3 . 67 ( m , 6h ), 3 . 57 ( dd , j = 2 . 1 , 9 . 5 hz , 1h ), 3 . 52 - 3 . 46 ( m , 2h ), 3 . 30 - 3 . 27 ( m , 1h ), 3 . 14 ( at , 1h ), 1 . 51 ( s , 3h ), 1 . 33 ( s , 3h ), 1 . 10 - 0 . 99 ( m , 26h ); 13 c - nmr ( 100 mhz , cdcl 3 ) δ 138 . 9 , 138 . 8 , 138 . 6 , 138 . 6 , 138 . 3 , 138 . 2 , 138 . 1 , 128 . 5 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 7 , 127 . 6 , 127 . 3 , 127 . 1 , 110 . 0 , 101 . 2 , 99 . 9 , 83 . 2 , 82 . 5 , 81 . 3 , 79 . 7 , 78 . 9 , 77 . 0 , 76 . 9 , 76 . 0 , 75 . 3 , 75 . 2 , 75 . 1 , 74 . 9 , 74 . 3 , 74 . 3 , 74 . 3 , 74 . 2 , 73 . 4 , 73 . 3 , 72 . 2 , 72 . 1 , 69 . 5 , 66 . 1 , 62 . 6 , 27 . 7 , 25 . 8 , 18 . 1 , 18 . 1 , 12 . 1 ; esi ms m / z ( m + na ) + calcd 1468 . 7051 , found 1468 . 7059 . o -( 2 , 3 , 4 - tri - o - benzyl - 6 - o - tert - butyldimethylsilyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - β - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - d - myo - inositol 37 . trisaccharide 36 ( 375 mg , 0 . 26 mmol ) was dissolved in ch 2 cl 2 ( 4 ml ) and a 0 . 5 m solution of hcl in meoh ( 9 . 2 ml , 4 . 61 mmol ) was added . after 8 h , the yellow solution was concentrated several times with ch 2 cl 2 , then diluted with ch 2 cl 2 ( 50 ml ) and washed with h 2 o ( 2 × 50 ml ), sat . aqueous nahco 3 ( 2 × 50 ml ), and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering , and concentration the crude product was dissolved in ch 2 cl 2 ( 3 ml ). imidazole ( 35 mg , 0 . 52 mmol ) and tert - butyldimethylchlorosilane ( 59 mg , 0 . 39 mmol ) were added , and the cloudy suspension was stirred at room temperature for 1 . 5 h . meoh ( 5 ml ) was added , and the solution was diluted with ch 2 cl 2 ( 50 ml ), and washed with sat . aqueous aq . nahco 3 ( 2 × 50 ml ), and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering , and concentration the crude product was purified by flash silica column chromatography ( 5 → 15 % etoac / toluene ) to afford 37 ( 258 mg , 73 %) as a colorless oil . [ α ] 24 d : − 2 . 47 ° ( c 1 . 74 , ch 2 cl 2 ); ir ( thin film ) 2926 , 2111 , 1454 , 1361 , 1111 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 33 - 7 . 11 ( m , 33h ), 5 . 19 ( d , j = 2 . 1 hz , 1h ), 4 . 95 ( d , j = 11 . 0 hz , 1h ), 4 . 92 ( d , j = 10 . 4 hz , 1h ), 4 . 83 - 4 . 80 ( m , 3h ), 4 . 75 ( d , j = 10 . 7 hz , 1h ), 4 . 72 ( d , j = 12 . 2 hz , 1h ), 4 . 67 ( d , j = 11 . 9 hz , 1h ), 4 . 59 - 4 . 46 ( m , 7h ), 4 . 41 ( d , j = 11 . 6 hz , 1h ), 4 . 37 ( d , j = 12 . 2 hz , 1h ), 4 . 22 ( d , j = 12 . 2 hz , 1h ), 4 . 18 ( at , 1h ), 4 . 02 ( d , j = 9 . 5 , 1h ), 4 . 00 - 3 . 91 ( m , 3h ), 3 . 78 - 3 . 63 ( m , 8h ), 3 . 53 - 3 . 49 ( m , 2h ), 3 . 45 - 3 . 38 ( m , 4h ), 3 . 23 ( app t , 1h ), 2 . 45 ( s , 1h ), 0 . 82 ( s , 9h ), − 0 . 01 ( s , 3h ), − 0 . 02 ( s , 3h ); 13 c - nmr ( 100 mhz , cdcl 3 ) δ 138 . 7 , 138 . 6 , 138 . 5 , 138 . 0 , 137 . 9 , 137 . 7 , 129 . 1 , 128 . 5 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 1 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 8 , 127 . 7 , 127 . 6 , 127 . 2 , 127 . 1 , 101 . 9 , 100 . 0 , 82 . 9 , 82 . 6 , 82 . 1 , 81 . 5 , 79 . 5 , 79 . 4 , 76 . 1 , 75 . 9 , 75 . 8 , 75 . 5 , 75 . 0 , 74 . 6 , 74 . 4 , 74 . 2 , 73 . 5 , 72 . 4 , 72 . 3 , 72 . 2 , 70 . 6 , 69 . 0 , 68 . 9 , 66 . 3 , 62 . 4 , 26 . 0 , 18 . 4 , − 5 . 0 , − 5 . 2 ; esi ms m / z ( m + na ) + calcd 1386 . 6268 , found 1386 . 6255 . o -( 2 - o - acetyl - 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 38 . a mixture of pentasaccharide acceptor 27 ( 454 mg , 0 . 20 mmol ) and donor 7 ( 260 mg , 0 . 41 mmol ) were azeotroped with toluene ( 3 × 7 ml ) and dried under vacuum for 16 h . the mixture was dissolved in ch 2 cl 2 ( 3 ml ) and a 0 . 5 m solution of tmsotf in ch 2 cl 2 ( 20 μl , 0 . 010 mmol ) was added . after 30 min , the orange solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering and concentration the crude product was purified by flash silica column chromatography ( 10 → 40 % etoac / hexanes ) to afford 38 ( 560 mg , quant .) as a colorless oil . [ α ] 24 d : + 52 . 1 ° ( c 0 . 42 , ch 2 cl 2 ); ir ( thin film ) 2863 , 2361 , 2105 , 1734 , 1050 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 37 - 6 . 96 ( m , 83h ), 5 . 58 ( d , j = 3 . 7 hz , 1h ), 5 . 54 ( app s , 1h ), 5 . 27 ( app s , 1h ), 5 . 26 ( app s , 1h ), 5 . 02 ( app s , 1h ), 4 . 88 - 4 . 72 ( m , 13h ), 4 . 67 ( app t , 2h ), 4 . 56 - 4 . 31 ( m , 18h ), 4 . 26 - 4 . 09 ( m , 10h ), 4 . 06 - 4 . 00 ( m , 4h ), 3 . 97 - 3 . 64 ( m , 20h ), 3 . 60 - 3 . 34 ( m , 11h ), 3 . 32 - 3 . 29 ( m , 1h ), 2 . 10 ( s , 3h ), 1 . 50 ( s , 3h ), 1 . 25 ( s , 3h ), 1 . 07 - 1 . 00 ( m , 22h ); hsqc data 13 c ( 125 mhz )/ 1 h ( 500 mhz ): 100 . 0 / 5 . 03 ( 1a ), 68 . 9 / 5 . 56 ( 2a ), 78 . 9 / 3 . 98 ( 3a ), 68 . 9 / 3 . 59 , 3 . 43 ( 6a ), 100 . 1 / 5 . 28 ( 1b ), 75 . 6 / 4 . 05 ( 2b ), 99 . 2 / 4 . 81 ( 1c ), 72 . 8 / 4 . 14 ( 2c ), 100 . 5 / 5 . 27 ( 1d ), 80 . 2 / 3 . 76 ( 3d ), 66 . 3 / 3 . 89 , 3 . 37 ( 6d ), 95 . 3 / 5 . 60 ( 1e ), 63 . 2 / 3 . 32 ( 2e ), 77 . 2 / 4 . 12 ( 4e ); hmbc cross peaks 13 c ( 125 mhz ): 75 . 6 ( a → b ), 72 . 8 ( b → c ), 66 . 3 ( c → d ), 77 . 2 ( d → e ); esi ms m / z ( m + na ) − calcd 2717 . 2497 , found 2717 . 2450 . o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 6 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 39 . hexasaccharide 38 ( 560 mg , 0 . 21 mmol ) was dissolved in ch 2 cl 2 ( 3 ml ) and a 0 . 75 m solution of sodium methoxide in methanol ( 278 μl , 0 . 21 mmol ) was added . after 3 h , the light yellow solution was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering and concentration the crude product was purified by flash silica column chromatography ( 2 . 5 → 12 . 5 % etoac / toluene ) to afford 39 ( 396 mg , 72 %). [ α ] 24 d : + 62 . 3 ° ( c 0 . 30 , ch 2 cl 2 ); ir ( thin film ) 2361 , 2338 , 2104 , 1261 , 1050 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 37 - 6 . 97 ( m , 85h ), 5 . 57 ( d , j = 3 . 7 hz , 1h ), 5 . 29 ( app s , 1h ), 5 . 24 ( d , j = 1 . 5 hz , 1h ), 5 . 13 ( app s , 1h ), 4 . 89 - 4 . 63 ( m , 18h ), 4 . 54 - 4 . 32 ( m , 19h ), 4 . 30 - 4 . 20 ( m , 5h ), 4 . 16 - 4 . 01 ( m , 11h ), 3 . 94 - 3 . 33 ( m , 36h ), 3 . 30 ( dd , j = 3 . 7 , 10 . 1 hz , 1h ), 2 . 37 ( d , j = 2 . 1 hz , 1h ), 1 . 50 ( s , 3h ), 1 . 32 ( s , 3h ), 1 . 08 - 1 . 00 ( m , 24h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 138 . 9 , 138 . 8 , 138 . 7 , 138 . 6 , 138 . 4 , 138 . 4 , 138 . 2 , 138 . 2 , 138 . 2 , 138 . 1 , 138 . 1 , 138 . 1 , 138 . 0 , 137 . 9 , 137 . 8 , 137 . 6 , 128 . 6 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 1 , 128 . 1 , 128 . 0 , 128 . 0 , 128 . 0 , 127 . 9 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 8 , 127 . 8 , 127 . 8 , 127 . 7 , 127 . 7 , 127 . 7 , 127 . 7 , 127 . 6 , 127 . 6 , 127 . 6 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 5 , 127 . 4 , 127 . 4 , 127 . 4 , 127 . 4 , 127 . 2 , 127 . 2 , 127 . 2 , 127 . 1 , 127 . 1 , 127 . 0 , 126 . 9 , 126 . 8 , 110 . 1 , 101 . 2 , 100 . 3 , 100 . 0 , 98 . 9 , 95 . 0 , 80 . 8 , 80 . 6 , 80 . 3 , 79 . 9 , 79 . 6 , 79 . 0 , 77 . 2 , 76 . 9 , 76 . 9 , 76 . 6 , 76 . 5 , 75 . 9 , 75 . 3 , 75 . 0 , 74 . 9 , 74 . 8 , 74 . 7 , 74 . 6 , 74 . 3 , 74 . 0 , 73 . 7 , 73 . 5 , 73 . 4 , 73 . 3 , 73 . 3 , 73 . 2 , 73 . 1 , 73 . 0 , 72 . 7 , 72 . 7 , 72 . 3 , 72 . 2 , 72 . 1 , 72 . 0 , 71 . 9 , 71 . 8 , 71 . 8 , 71 . 4 , 71 . 3 , 69 . 6 , 68 . 9 , 68 . 6 , 68 . 4 , 68 . 3 , 66 . 1 , 62 . 9 , 62 . 4 , 27 . 4 , 25 . 7 , 18 . 1 , 18 . 1 , 11 . 8 ; esi ms m / z ( m + na ) + calcd 2675 . 2391 , found 2675 . 2380 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 6 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 40 . pseudo - hexasaccharide 39 ( 70 mg , 0 . 026 mmol ) was dissolved in dmf ( 2 ml ) and benzyl bromide ( 5 μl , 0 . 040 mmol ) was added . the clear solution was cooled to 0 ° c ., and nah ( 60 % dispersion in mineral oil , 2 mg , 0 . 040 mmol ) was added in one portion . after 1 h , meoh ( 5 ml ) was added , and the mixture was poured into h 2 o ( 50 ml ) and washed with et 2 o ( 3 × 50 ml ). the combined organic phases were washed with brine ( 1 × 50 ml ), dried ( na 2 so 4 ), filtered and concentrated . purification by flash silica column chromatography ( 1 . 5 → 7 . 5 % etoac / toluene ) afforded 40 ( 68 mg , 96 %). [ α ] 24 d : + 46 . 0 ° ( c 0 . 10 , ch 2 cl 2 ); ir ( thin film ) 2863 , 2104 , 1496 , 1454 , 735 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 37 - 6 . 95 ( m , 90h ), 5 . 57 ( d , j = 2 . 7 hz , 1h ), 5 . 28 ( app s , 1h ), 5 . 24 ( app s , 1h ), 5 . 20 ( app s , 1h ), 4 . 89 - 4 . 64 ( m , 15h ), 4 . 58 - 4 . 30 ( m , 20h , 4 . 25 - 4 . 20 ( m , 5h ), 4 . 16 - 4 . 04 ( m , 8h ), 4 . 00 - 3 . 34 ( m , 35h ), 3 . 31 - 3 . 29 ( m , 1h ), 1 . 50 ( s , 3h ), 1 . 32 ( s , 3h ), 1 . 03 - 1 . 00 ( m , 20h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 140 . 0 , 139 . 8 , 139 . 8 , 139 . 7 , 139 . 6 , 139 . 6 , 139 . 5 , 139 . 4 , 139 . 3 , 139 . 3 , 139 . 3 , 139 . 2 , 139 . 1 , 139 . 0 , 139 . 0 , 138 . 9 , 138 . 8 , 138 . 8 , 138 . 7 , 138 . 5 , 129 . 5 , 129 . 4 , 129 . 4 , 129 . 3 , 129 . 3 , 129 . 2 , 129 . 2 , 129 . 1 , 129 . 0 , 129 . 0 , 129 . 0 , 128 . 9 , 128 . 9 , 128 . 9 , 128 . 8 , 128 . 8 , 128 . 7 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 6 , 128 . 5 , 128 . 5 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 8 , 111 . 0 , 101 . 1 , 100 . 9 , 100 . 4 , 99 . 8 , 95 . 9 , 81 . 7 , 81 . 5 , 81 . 1 , 80 . 8 , 80 . 7 , 79 . 9 , 78 . 7 , 78 . 1 , 78 . 1 , 77 . 9 , 77 . 8 , 77 . 7 , 77 . 6 , 77 . 4 , 76 . 8 , 76 . 4 , 76 . 2 , 76 . 2 , 75 . 9 , 75 . 8 , 75 . 8 , 75 . 7 , 75 . 5 , 75 . 5 , 75 . 2 , 75 . 1 , 75 . 0 , 75 . 0 , 74 . 8 , 74 . 6 , 74 . 4 , 74 . 3 , 74 . 2 , 74 . 1 , 74 . 0 , 74 . 0 , 73 . 6 , 73 . 5 , 73 . 3 , 73 . 2 , 73 . 1 , 73 . 1 , 73 . 0 , 72 . 9 , 72 . 9 , 72 . 8 , 72 . 8 , 72 . 6 , 72 . 3 , 71 . 5 , 70 . 5 , 69 . 7 , 69 . 5 , 69 . 5 , 67 . 0 , 63 . 8 , 63 . 4 , 28 . 3 , 28 . 3 , 26 . 6 , 21 . 7 , 19 . 0 , 18 . 9 , 12 . 7 ; esi ms m / z ( m + 2na ) 2 + calcd 1394 . 1377 , found 1394 . 1358 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - tert - butyldimethylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - d - myo - inositol 41 . pseudo - hexasaccharide 40 ( 240 mg , 0 . 088 mmol ) was dissolved in ch 2 cl 2 ( 1 . 5 ml ) and a 0 . 5 m solution of hcl in meoh ( 3 . 0 ml , 1 . 49 mmol ) was added . after 14 h , the yellow solution was concentrated several times with ch 2 cl 2 , then diluted with ch 2 cl 2 ( 50 ml ) and washed with h 2 o ( 2 × 50 ml ), sat . aqueous aq . nahco 3 ( 2 × 50 ml ), and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering , and concentration the crude product was dissolved in ch 2 cl 2 ( 2 ml ). imidazole ( 12 mg , 0 . 18 mmol ) and tert - butyldimethylchlorosilane ( 20 mg , 0 . 13 mmol ) were added , and the cloudy suspension was stirred at room temperature for 40 minutes . meoh ( 5 ml ) was added , and the solution was diluted with ch 2 cl 2 ( 50 ml ), and washed with sat . aqueous aq . nahco 3 ( 2 × 50 ml ), and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtering , and concentration the crude product was purified by flash silica column chromatography ( 10 → 20 % etoac / toluene ) to afford 41 ( 156 mg , 67 %) as a colorless oil . [ α ] 24 d : + 44 . 6 ° ( c 1 . 0 , ch 2 cl 2 ); ir ( thin film ) 2926 , 2361 , 2338 , 2106 , 1052 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 30 - 6 . 93 ( m , 86h ), 5 . 36 ( d , j = 3 . 7 hz , 1h ), 5 . 22 ( d , j = 1 . 8 hz , 1h ), 5 . 19 ( s , 2h ), 4 . 88 - 4 . 80 ( m , 7h ), 4 . 75 - 4 . 67 ( m , 7h ), 4 . 63 - 4 . 58 ( m , 3h ), 4 . 55 - 4 . 35 ( m , 17h ), 4 . 29 - 4 . 15 ( m , 11h ), 4 . 11 - 4 . 01 ( m , 2h ), 3 . 96 - 3 . 53 ( m , 30h ), 3 . 45 - 3 . 27 ( m , 12h ), 2 . 47 ( s , 1h ), 0 . 79 ( m , 9h ), − 0 . 03 ( s , 3h ), − 0 . 04 ( s , 3h ); 13 c - nmr ( 125 mhz , cdcl 3 ) δ 138 . 8 , 138 . 7 , 138 . 6 , 138 . 4 , 138 . 4 , 138 . 3 , 138 . 3 , 138 . 3 , 138 . 2 , 138 . 0 , 137 . 8 , 137 . 6 , 137 . 5 , 128 . 7 , 128 . 5 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 0 , 128 . 0 , 128 . 0 , 127 . 9 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 8 , 127 . 8 , 127 . 7 , 127 . 7 , 127 . 7 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 5 , 127 . 4 , 127 . 4 , 127 . 4 , 127 . 3 , 127 . 3 , 127 . 2 , 127 . 2 , 127 . 2 , 127 . 1 , 127 . 0 , 127 . 0 , 126 . 8 , 100 . 1 , 100 . 0 , 99 . 3 , 99 . 0 , 97 . 9 , 81 . 4 , 80 . 9 , 80 . 6 , 80 . 1 , 79 . 8 , 79 . 7 , 79 . 6 , 79 . 5 , 77 . 2 , 76 . 6 , 75 . 8 , 75 . 1 , 74 . 9 , 74 . 8 , 74 . 6 , 74 . 5 , 74 . 3 , 74 . 0 , 73 . 2 , 73 . 2 , 73 . 1 , 72 . 7 , 72 . 6 , 72 . 5 , 72 . 2 , 72 . 1 , 72 . 0 , 71 . 9 , 71 . 3 , 70 . 6 , 69 . 5 , 68 . 9 , 68 . 6 , 66 . 1 , 64 . 1 , 62 . 1 , 26 . 0 , 18 . 2 , − 4 . 8 , − 5 . 5 ; esi ms m / z ( m + 2na ) 2 + calcd 1353 . 0985 , found 1353 . 0998 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - cyclic phosphate - d - myo - inositol 42 . to pyridine ( 2 ml ) was added methyl dichlorophosphate ( 48 μl , 0 . 48 mmol ). after 15 min another aliquot of methyl dichlorophosphate ( 48 μl , 0 . 48 mmol ) was added , and the resulting white cloudy solution was stirred for 30 min . hexasaccharide diol 41 ( 127 mg , 0 . 048 mmol ) in pyridine ( 2 ml ) was added via cannula , and the resulting cloudy yellow solution was stirred for 50 min , then quenched with sat . aqueous aqueous nahco 3 ( 1 . 0 ml ). the solution was concentrated in vacuo , taken up in h 2 o ( 80 ml ), acidified with 2 . 0 m hcl until the solution reached ph 1 , then extracted with etoac ( 4 × 50 ml ). the combined organic phases were dried ( na 2 so 4 ), filtered and concentrated to yield a white foam . the crude cyclic phosphate was taken up in thf ( 3 ml ) and a 1 . 0 m solution of tbaf in thf ( 130 μl , 0 . 13 mmol ) was added . the light yellow solution was stirred at room temperature for 14 h , then concentrated in vacuo . purification by flash silica column chromatography ( 100 : 7 : 1 ch 2 cl 2 : meoh : 30 % nh 3 ) afforded 42 ( 80 mg , 70 %) as a colorless oil . [ α ] 24 d : + 54 . 5 ° ( c 0 . 44 , ch 2 cl 2 ); ir ( thin film ) 3028 , 2924 , 2106 , 1453 , 1361 cm − 1 ; 1 h nmr ( 500 mhz , dmso - d 6 ) δ 7 . 42 - 6 . 87 ( m , 75h ), 5 . 52 ( s , 1h ), 5 . 21 - 5 . 10 ( m , 3h ), 4 . 94 - 4 . 91 ( m , 1h ), 4 . 85 - 4 . 04 ( m , 39h ), 3 . 92 - 3 . 14 ( m , 39h ); 31 p nmr ( 120 mhz , cdcl 3 ) δ 14 . 3 ; esi ms m / z ( m + 2na ) 2 + calcd 1338 . 0241 , found 1338 . 0235 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - o -( 6 -( 2 -[ n -( benzyloxycarbonyl ) amino ] ethyl 2 ′- cyanoethyl phosphate - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- 0 -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - cyclic phosphate - d - myo - inositol 43 . a mixture of hexasaccharide cyclic phosphate acceptor 42 ( 165 mg , 0 . 063 mmol ) and phosphoramidite 10 ( 174 mg , 0 . 44 mmol ) were azeotroped with toluene ( 3 × 6 ml ) and dried under vacuum for 3 h . a 1 : 1 mixture of ch 3 cn : ch 2 cl 2 ( 8 ml ) was added , followed by sublimed 1h - tetrazole ( 31 mg , 0 . 44 mmol ). the slightly opaque solution was stirred at room temperature for 3 h , at which time tlc analysis showed complete consumption of the acceptor . a 70 % wt . solution of tert - butyl hydroperoxide ( 86 μl , 0 . 63 mmol ) was added , and the resulting clear solution was stirred at room temperature for 2 h . the mixture was diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ), filtration and concentration the crude product was purified by flash silica column chromatography ( 93 : 6 : 1 ch 2 cl 2 : meoh : 30 % nh 3 ) to afford 43 ( 156 mg , 84 %) as a mixture of diastereomers ; ir ( thin film ) 2922 , 2362 , 1718 , 1454 , 1362 cm − 1 ; 1 h nmr ( 500 mhz , dmso - d 6 ) δ 7 . 34 - 7 . 04 ( m , 80h ), 5 . 40 - 5 . 12 ( m , 3h ), 5 . 02 - 4 . 90 ( m , 5h ), 4 . 80 - 3 . 16 ( m , 87h ), 2 . 50 - 2 . 49 ( m , 2h ); 31 p nmr ( 120 mhz , cdcl 3 ) δ 13 . 6 , − 0 . 95 , − 1 . 18 ; maldi - tof [ m + na + k ] + 2981 . o -( α - d - mannopyranosyl )-( 1 → 2 )- o -( 6 -( 2 - aminoethyl hydrogen phosphate - α - d - mannopyranosyl )-( 1 → 2 )- o -( α - d - mannopyranosyl )-( 1 → 6 )- o -( α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - amino - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 1 , 2 - cyclic phosphate - d - myo - inositol 2 . bis - phosphate 43 ( 144 mg , 0 . 049 mmol ) was dissolved in ch 2 cl 2 ( 2 ml ) and dbu ( 22 μl , 0 . 147 mmol ) was added dropwise . the yellowish solution was stirred at room temperature for 2 h , then diluted with ch 2 cl 2 ( 50 ml ), washed with sat . aqueous nahco 3 ( 2 × 50 ml ) and brine ( 1 × 50 ml ). following drying ( na 2 so 4 ) the crude product was flushed through a pad of silica . the silica was washed with 93 : 6 : 1 ch 2 cl 2 : meoh : 30 % nh 3 ( 100 ml ), and the combined washed concentrated to give the crude product as an oil . ammonia ( 2 ml ) was condensed in a flame - dried 3 - neck flask . following cooling to − 78 ° c ., sodium metal ( ca . 20 mg ) was added and the resultant dark blue solution was stirred for 5 min . thf ( 1 ml ) was added , followed by crude diphosphate in thf ( 2 ml ) via cannula . the blue solution was stirred at − 78 ° c . for 15 min ( blue color must not disappear ! ), then nh 4 cl was added dropwise . following disappearance of the blue color , meoh ( 20 ml ) was added . the flask was warmed to room temperature , concentrated to ca . 30 ml , and added to a silica column of 3 : 3 : 2 ch 2 cl 2 : meoh : 30 % nh 3 . the column was washed with 200 ml of 3 : 3 : 2 ch 2 cl 2 : meoh : 30 % nh 3 , then concentrated to a white solid . the crude product was further purified by flash silica column chromatography ( 2 : 2 : 2 : 1 buoh : etoh : h 2 o : 30 % nh 3 ) to yield 2 ( 18 . 0 mg , 75 %) as a white powder following concentration and lyophilization ( h 2 o ). ir ( thin film ) 3144 , 3051 , 1407 , 1118 cm − 1 ; 1 h nmr ( 500 mhz , d 2 o ) δ 7 . 38 ( s , 1h ), 5 . 54 ( d , j = 6 . 1 hz , 1h ), 5 . 28 ( s , 1h ), 5 . 15 ( s , 1h ), 5 . 07 - 5 . 03 ( m , 3h ), 4 . 67 ( t , j = 4 . 6 hz , 1h ), 4 . 41 ( s , 1h ), 4 . 34 - 4 . 27 ( m , 1h ), 4 . 20 - 4 . 17 ( m , 1h ), 4 . 10 - 3 . 58 ( m , 34h ), 3 . 57 - 3 . 53 ( m , 1h ), 3 . 38 ( at , 1h ), 3 . 27 - 3 . 25 ( m , 2h ), 2 . 07 ( s , 1h ), 1 . 20 ( bs , 1h ); 31 p nmr ( 120 mhz , d 2 o ) δ 16 . 0 , 0 . 38 ; maldi - tof [ m + 2h + na + k ] + 1238 . n - pentenyl 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl -( 1 → 2 )- 3 , 4 - di - o - benzyl - 6 - o - trisopropylsilyl - α - d - mannopyranosyl -( 1 → 2 )- 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl -( 1 → 6 )- 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranoside 46 . octenediol functionalized resin 48 ( 50 μmol , 50 mg , 1 . 00 mmol / g loading ) was loaded into a reaction vessel and inserted into a modified abi - 433a peptide synthesizer . the resin was glycosylated using donor 6 ( 5 equiv ., 0 . 25 mmol , 160 mg loaded into cartridges ) delivered in ch 2 cl 2 ( 3 ml ) and tmsotf ( 0 . 5 equiv ., 2 . 0 ml , 0 . 0125 m tmsotf in ch 2 cl 2 ) at room temp . mixing of the suspension was performed ( 10 s vortex , 50 s rest ) for 15 min . the resin was then washed with ch 2 cl 2 ( 6 × 4 ml each ), and the glycosylation was repeated ( double glycosylation ). deprotection of the acetyl ester was carried out by treating the glycosylated resin with sodium methoxide ( 8 equiv ., 0 . 5 ml , 0 . 75 m naome in meoh ) in ch 2 cl 2 ( 5 ml ) for 30 min at room temp . the resin was then washed with ch 2 cl 2 ( 1 × 4 ml ) and subjected to the deprotection conditions a second time for 30 min . the deprotected polymer - bound c6 - oh monosaccharide was then glycosylated using donor 7 ( 5 equiv ., 0 . 25 mmol , 160 mg loaded into cartridges ) delivered in ch 2 cl 2 ( 3 ml ) and tmsotf ( 0 . 5 equiv ., 2 . 0 ml , 0 . 0125 m tmsotf in ch 2 cl 2 ) at room temp . the resin was then washed with ch 2 cl 2 ( 6 × 4 ml each ), and the glycosylation was repeated ( double glycosylation ). deprotection of the acetyl ester was carried out by treating the glycosylated resin with sodium methoxide ( 8 equiv ., 0 . 5 ml , 0 . 75 m naome in meoh ) in ch 2 cl 2 ( 5 ml ) for 30 min at room temp . the resin was then washed with ch 2 cl 2 ( 1 × 4 ml ) and subjected to the deprotection conditions a second time for 30 min . the deprotected polymer - bound disaccharide was then glycosylated using donor 8 ( 5 equiv ., 0 . 25 mmol , 175 mg loaded into cartridges ) delivered in ch 2 cl 2 ( 3 ml ) and tmsotf ( 0 . 5 equiv ., 2 . 0 ml , 0 . 0125 m tmsotf in ch 2 cl 2 ) at room temp . mixing of the suspension was performed ( 10 s vortex , 50 s rest ) for 15 min . the resin was then washed with ch 2 cl 2 ( 6 × 4 ml each ), and the glycosylation was repeated ( double glycosylation ). deprotection of the acetyl ester was carried out by treating the glycosylated resin with sodium methoxide ( 8 equiv ., 0 . 5 ml , 0 . 75 m naome in meoh ) in ch 2 cl 2 ( 5 ml ) for 30 min at room temp . the resin was then washed with ch 2 cl 2 ( 1 × 4 ml ) and subjected to the deprotection conditions a second time for 30 min . the polymer - bound trisaccharide was then glycosylated using donor 9 ( 5 equiv ., 0 . 25 mmol , 171 mg loaded into cartridges ) delivered in ch 2 cl 2 ( 3 ml ) and tmsotf ( 0 . 5 equiv ., 2 . 0 ml , 0 . 0125 m tmsotf in ch 2 cl 2 ) at room temp . mixing of the suspension was performed ( 10 s vortex , 50 s rest ) for 15 min . the resin was then washed with ch 2 cl 2 ( 6 × 4 ml each ) and vessel was removed from the synthesizer . the glycosylated resin ( 50 μmol ) was dried in vacuo over phosphorous pentoxide for 12 h and transferred to a round bottom flask . the flask was purged with ethylene and grubbs &# 39 ; catalyst ( bis ( tricyclohexylphosphine ) benzylidine ruthenium ( iv ) dichloride , 12 mg , 30 mol %) was added . the reaction mixture was diluted with ch 2 cl 2 ( 3 ml ) and stirred under 1 atm ethylene for 36 h . triethylamine ( 100 μl , 160 equiv ) and tris hydroxymethylphosphine ( 50 mg , 80 equiv ) were added and the resulting solution was stirred at room temperature for 1 h . the pale yellow reaction mixture was diluted with ch 2 cl 2 ( 5 ml ) and washed with water ( 3 × 5 ml ). the aqueous phase was extracted with ch 2 cl 2 ( 3 × 5 ml ) and the combined organics were dried over na 2 so 4 , filtered and concentrated . the crude product was analyzed by hplc using a waters model 600 pump and controller coupled to a waters model 2487 dual λ absorbance detector . analytical hplc was performed on a waters nova - pak © silica column ( 3 . 9 × 150 mm ) using a gradient of 5 → 20 % etoac / hexanes ( 20 min ) and a flow rate of 1 . 0 ml / min , monitoring at 260 nm . semi - preparative hplc was performed on a waters prep nova - pak © silica column ( 7 . 8 × 300 mm ) using a gradient of 5 → 20 % etoac / hexanes ( 20 min ) and a flow rate of 2 . 5 ml / min , monitoring at 260 nm . fractions collected during semi - preparative hplc were checked by analytical hplc for purity . clean fractions were concentrated to give 46α ( 2 . 0 mg , 2 % yield ) as a clear oil . [ α ] 24 d : + 21 . 7 ° ( c 0 . 63 , ch 2 cl 2 ); ir ( thin film ) 2361 , 2338 , 1095 , 668 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 7 . 35 - 7 . 03 ( m , 60h ), 5 . 77 - 5 . 71 ( m , 1h ), 5 . 31 ( s , 1h ), 5 . 21 ( s , 1h ), 4 . 98 - 4 . 86 ( m , 7h ), 4 . 82 ( d , j = 10 . 7 hz , 1h ), 4 . 77 ( s , 1h ), 4 . 72 - 4 . 61 ( m , 5h ), 4 . 60 - 4 . 53 ( m , 8h ), 4 . 50 - 4 . 46 ( m , 6h ), 4 . 44 ( d , j = 12 . 2 hz , 1h ), 4 . 36 ( d , j = 11 . 6 hz , 1h ), 4 . 28 ( d , j = 12 . 2 hz , 1h ), 4 . 05 ( t , j = 9 . 5 hz , 1h ), 4 . 01 - 3 . 98 ( m , 1h ), 3 . 96 - 3 . 84 ( m , 1h ), 3 . 82 - 3 . 65 ( m , 7h ), 3 . 64 - 3 . 49 ( m , 8h ), 3 . 32 - 3 . 27 ( m , 1h ), 2 . 08 - 2 . 00 ( m , 4h ), 1 . 57 - 1 . 55 ( m , 2h ), 1 . 10 - 1 . 02 ( m , 24h ); hsqc data 13 c ( 125 mhz )/ 1 h ( 500 mhz ): 100 . 5 / 5 . 31 ( 1a ), 73 . 1 / 4 . 15 ( 2a ), 80 . 1 / 3 . 94 ( 3a ), 99 . 3 / 4 . 90 ( 1b ), 75 . 1 / 4 . 16 ( 2b ), 80 . 7 / 3 . 89 ( 3b ), 98 . 0 / 4 . 77 ( 1c ), 74 . 8 / 3 . 75 ( 2c ), 80 . 7 / 3 . 88 ( 3c ), 99 . 8 / 5 . 21 ( 1d ), 74 . 9 / 3 . 86 ( 2d ), 71 . 2 / 3 . 69 ( 3d ), 66 . 8 / 3 . 59 ( 6d ); hmbc cross peaks 13 c ( 125 mhz ): 75 . 1 ( a → b ), 74 . 8 ( b → c ), 66 . 8 ( c → d ); esi ms m / z ( m + na ) + calcd 1993 . 9705 , found 1993 . 9747 . 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl -( 1 → 2 )- 3 , 4 - di - o - benzyl - 6 - o - trisopropylsilyl - α - d - mannopyranosyl -( 1 → 2 )- 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl -( 1 → 6 )- 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl trichloracetimidate 47 . n - pentenyl glycoside 46 ( 200 mg , 0 . 10 mmol ) was suspended in 8 : 1 acetonitrile / water ( 8 ml ) and n - bromosuccinimide ( 63 mg , 0 . 35 mmol ) was added . the orange mixture was stirred in the dark for 1 h , then quenched by the addition of sat . aqueous aqueous na 2 s 2 o 3 ( 5 ml ). water ( 25 ml ) was added , and the mixture was washed with ch 2 cl 2 ( 4 × 50 ml ). the combined organic phases were dried ( na 2 so 4 ), filtered and concentrated . silica gel column chromatography ( 10 - 320 % etoac / hexanes ) afforded the anomeric lactols ( 128 mg , 67 %) as a colorless oil . the lactols ( 64 mg , 34 μmol ) were dissolved in ch 2 cl 2 ( 1 ml ) and dbu ( 1 μl , 7 μmol ) and cl 3 ccn ( 3 μl , 340 μmol ) were added . after 1 h at room temperature the crude mixture was filtered through a pad of silica gel , washing with 30 % etoac / hexanes . the crude material was concentrated , then purified by silica gel column chromatography ( 10 % etoac / hexanes ) to afford 47 ( 52 mg , 75 %) as a colorless oil . [ α ] 24 d : + 10 . 9 ° ( c 0 . 90 , ch 2 cl 2 ); ir ( thin film ) 2864 , 2360 , 1454 , 1096 , 697 cm − 1 ; 1 h nmr ( 500 mhz , cdcl 3 ) δ 8 . 47 ( s , 1h ), 7 . 37 - 6 . 97 ( m , 60h ), 6 . 21 ( d , j = 2 . 1 hz , 1h ), 5 . 22 ( s , 1h ), 5 . 12 ( s , 1h ), 4 . 84 - 4 . 18 ( m , 22h ), 4 . 08 - 4 . 07 ( m , 1h ), 4 . 02 - 3 . 42 ( m , 21h ), 0 . 97 - 0 . 95 ( m , 18h ); 13 c nmr ( 125 mhz , cdcl 3 ) δ 160 . 1 , 139 . 2 , 139 . 1 , 139 . 0 , 139 . 0 , 138 . 8 , 138 . 7 , 138 . 7 , 138 . 6 , 138 . 4 , 138 . 2 , 138 . 0 , 128 . 7 , 128 . 6 , 128 . 6 , 128 . 6 , 128 . 5 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 4 , 128 . 3 , 128 . 3 , 128 . 2 , 128 . 2 , 128 . 1 , 128 . 1 , 128 . 0 , 128 . 0 , 127 . 9 , 127 . 9 , 127 . 9 , 127 . 8 , 127 . 8 , 127 . 7 , 127 . 7 , 127 . 6 , 127 . 6 , 127 . 5 , 127 . 5 , 127 . 5 , 127 . 4 , 100 . 4 , 0 . 8 , 99 . 0 , 95 . 9 , 80 . 4 , 80 . 1 , 80 . 0 , 79 . 5 , 77 . 5 , 77 . 2 , 77 . 0 , 75 . 2 , 75 . 1 , 75 . 0 , 75 . 0 , 74 . 7 , 74 . 6 , 74 . 2 , 74 . 1 , 73 . 8 , 73 . 7 , 73 . 5 , 73 . 3 , 73 . 1 , 72 . 8 , 72 . 7 , 72 . 5 , 72 . 4 , 72 . 3 , 72 . 2 , 71 . 9 , 71 . 7 , 69 . 3 , 69 . 1 , 66 . 3 , 62 . 9 , 18 . 4 , 18 . 3 , 18 . 3 , 18 . 3 , 12 . 2 , 12 . 2 ; esi ms m / z ( m + na ) + calcd 1993 . 9705 , found 1993 . 9747 . n - pentenyl 2 - o - acetyl - 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranoside 49 . [ α ] 24 d : + 27 . 2 ° ( c 2 . 5 , chcl 3 ); ir ( thin film ) 2941 , 2865 , 1746 , 1369 , 1236 cm − 1 ; 1 h nmr ( 400 mhz , cdcl 3 ) δ 7 . 38 - 7 . 27 ( m , 10h ), 5 . 80 ( ddd , j = 6 . 6 , 10 . 2 , 17 . 1 hz , 1h ), 5 . 33 ( dd , j = 1 . 8 , 3 . 2 hz , 1h ), 4 . 98 ( d , j = 10 . 2 hz , 1h ), 4 . 92 ( d , j = 10 . 7 hz , 1h ), 4 . 76 ( d , j = 1 . 6 hz , 1h ), 4 . 73 ( d , j = 11 . 2 hz , 1h ), 4 . 66 ( d , j = 10 . 7 hz , 1h ), 4 . 58 ( d , j = 11 . 2 hz , 1h ), 4 . 03 - 3 . 98 ( m , 4h ), 3 . 71 - 3 . 63 ( m , 2h ), 3 . 39 ( dd , j = 6 . 5 , 9 . 6 hz , 1h ), 2 . 13 ( s , 1h ), 2 . 11 - 2 . 07 ( m , 2h ), 1 . 69 - 1 . 62 ( m , 2h ), 1 . 17 - 1 . 05 ( m , 21h ); 13 c - nmr ( 100 mhz , cdcl 3 ) δ 170 . 8 , 138 . 8 , 138 . 3 , 138 . 2 , 128 . 6 , 128 . 3 , 128 . 2 , 127 . 9 , 127 . 9 , 115 . 1 , 97 . 6 , 78 . 4 , 75 . 5 , 74 . 4 , 73 . 1 , 72 . 0 , 69 . 3 , 67 . 1 , 62 . 9 , 30 . 5 , 28 . 8 , 21 . 3 , 18 . 2 , 18 . 2 , 12 . 2 ; esi ms m / z ( m + na ) + calcd 649 . 353 , found 649 . 351 . o -( 2 , 3 , 4 , 6 - tetra - o - benzyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 - di - o - benzyl - 6 - o - triisopropylsilyl - α - d - mannopyranosyl )-( 1 → 2 )- o -( 3 , 4 , 6 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 6 )- o -( 2 , 3 , 4 - tri - o - benzyl - α - d - mannopyranosyl )-( 1 → 4 )- o -( 2 - azido - 3 , 6 - di - o - benzyl - 2 - deoxy - α - d - glucopyranosyl )-( 1 → 6 )- 3 , 4 , 5 - tri - o - benzyl - 1 , 2 - o - isopropylidene - d - myo - inositol 40 . a mixture of acceptor 21 ( 18 . 1 mg , 21 . 2 μmol ) and donor 47 ( 18 . 4 mg , 9 μmol ) was azeotroped with toluene ( 3 × 4 ml ), then dried in vacuo for 1 . 5 h . ch 2 cl 2 ( 1 ml ) and 4 å activated molecular sieves ( 20 mg ) were added , followed by a 0 . 09 m solution of tmsotf in ch 2 cl 2 ( 10 μl , 0 . 90 ; 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