the major insect vectors are aphids ( aphididae ; hemiptera ) , 
 who transmit nearly 50% of insect - borne plant viruses . for aphids to persistently transmit luteovirids , such as 
 pea enation mosaic virus ( pemv ) , two events are necessary .
first , 
 virions ingested with phloem sap bind a receptor for transcytosis 
 across the mid or hindgut epithelium for release into the hemocoel 
 where the virions circulate in a nonpropagative manner .
a second receptor - mediated transcytosis event 
 occurs at the accessory salivary glands ( asg ) from which virus particles 
 are secreted with saliva to inoculate the plant phloem during subsequent 
 feeding and thereby infect a new plant .
these interactions between aphids and luteovirids needed for viral 
 transmission are mediated by the viral capsid proteins , which are 
 made of one major coat protein ( cp , 22 kda ) and one minor coat protein 
 ( read - through domain , cp - rtd , 3555 kda ) .
the 
 rtd is not required for virus particle assembly or for uptake of virus 
 from the gut into the aphid hemocoel , but both cp and rtd are essential 
 for aphid transmission and are the sole determinants of vector specificity .
pea enation mosaic virus ( pemv ) provides 
 an ideal model virus for the study of luteovirid  aphid interactions 
 as it is the only luteovirid that is not phloem - limited and is thus 
 mechanically transmissible to plants .
although there are several examples of animal viruses using 
 glycan 
 or proteoglycan receptors for binding to host cells , relatively little is known about the role of glycans in plant virus 
. however , evidence for the involvement of glycans in 
 the uptake of pathogens into insect vectors is increasing .
the malaria parasite plasmodium has been shown to require midgut proteoglycans for cell invasion 
 in the mosquito vector and antibodies 
 targeting d - mannose residues on the mosquito midgut can block plasmodium development .
protein 
 glycosylation in insects consists primarily of high mannose or paucimannose 
 structures , although more complex glycans have been identified.d - mannose (  ) residues are common in several blood - feeding 
 insects that vector human and livestock disease .
mannose residues constitute the most abundant glycans in 
 the aphid gut , and mannose - binding lectins 
 targeting glycoprotein receptors on the surface of the insect gut 
 exhibit insecticidal activity against a variety of hemipteran pests .
the requirement for luteovirid interaction with receptor proteins 
 on the aphid gut epithelium and the abundance of glycoproteins in 
 the aphid gut lead to the hypothesis that glycans are involved in 
 luteovirid  aphid interactions .
in contrast to animal - infecting 
 viruses , plant viruses are mostly 
 nonenveloped , and the vast majority of nonenveloped viruses do not 
 contain glycoproteins . there is evidence for the presence of glycosylated 
 residues on structural proteins of potato virus x ( alphaflexiviridae ) and plum pox virus ( potyviridae ) .
the pemv coat protein sequence contains five 
 putative n - glycosylation sites predicted using scanprosite 
 ( http://www.expasy.ch/tools/scanprosite/ ) .
a previous study 
 on turnip yellows virus ( tuyv , luteoviridae ) showed that deglycosylation 
 of virions diminished aphid transmission of the virus , suggesting 
 that glycosylated structural proteins could be involved in virus recognition 
 and movement in the aphid .
however , a 
 later study refuted this idea demonstrating that the structural proteins 
 of tuyv and cucurbit aphid - borne yellow virus ( cabyv , luteoviridae ) 
 were not glycosylated and argued against the role of glycosylated 
 viral proteins in aphid transmission .
the glycosylation of pemv structural proteins themselves and the 
 ability of these viral surface proteins to bind to aphid - associated 
 carbohydrates has not yet been investigated . to explore the 
 possible role of glycans in virus  aphid interactions , 
 we needed information on the ability of insect protein - associated 
 glycans to bind to pemv .
a gut receptor for pemv is aminopeptidase n , which is heavily glycosylated 
 and bound by the plant lectin galanthus nivalis agglutinin ( gna).n - linked glycans are found on glycoproteins in all eukaryotes , including 
 insects .
the most common insect n - glycan structures share a terminal trimannoside structure 
 containing a -mannoside with two -mannosides attached 
 to the o-3 and o-6 positions .
we have previously 
 demonstrated the synthesis of the challenging -mannopyranoside 
 linkage by combining a -directing c-5 carboxylate methodology with fluorous tag ( f - tag)-assisted automated solution - phase synthesis .
herein , we report the development of the first 
 solution - based automated strategy for synthesis of a -mannoside - containing 
 insect n - glycan terminal branched trimannoside and 
 assays to test the affinity of this branched trisaccharide to pemv 
 using both fluorous microarray and isothermal 
 titration calorimetry ( itc ) .
to synthesize 
 the insect n - glycan terminal trimannoside 
 by automated solution - phase synthesis , we needed two building blocks : 
 a building block to construct the two 
 -linked termini and a branch point building block capable of 
 primarily making the more challenging -linkage . to obtain the 
 branch point building block
, we designed mannuronate building block 6 ( scheme 1 ) with a 3-o - p - methoxybenzyl ( pmb ) 
 group as a temporary capping group for the attachment of the -mannose 
 at the o-3 position , a c-5 carboxylate as the -directing group , and a temporary blocking group for the 6-position 
 branch point . a hydride reduction of the methyl ester should unmask 
 a free 6-oh for the attachment of the other -mannose .
the synthesis 
 of the building block started from the known 4,6-o - benzylidene acetal - protected thiol - linked mannoside 1 , which could be synthesized from d - mannose in seven steps .
after a selective cleavage of the 
 benzylidene acetal by borane tetrahydrofuran complex ( bh3thf ) and dibutylboron triflate ( bu2botf ) , compound 2 with a free hydroxyl 
 group at the 6-position was formed .
the combination of 2,2,6,6-tetramethyl-1-piperidinyloxy 
 free radical ( tempo ) and ( diacetoxyiodo)benzene ( baib ) oxidized primary 
 alcohol 2 to mannuronic acid 3 .
after esterification of the carboxylic acid , 
 the anomeric thiophenyl group of the methyl mannuronate 4 was removed by n - bromosuccinimide ( nbs ) to give 5 with a free anomeric hydroxyl group .
the subsequent formation of the trichloroacetimidate at 
 the anomeric hydroxyl provided desired donor 6 ready 
 for the automated solution - phase synthesis .
we have previously shown that our automated solution phase 
 oligosaccharide 
 synthesis strategy could successfully construct linear oligomers of 
 -mannuronate and -mannan up to hexasaccharide .
compared to automated solid - phase oligosaccharide 
 synthesis , the automated solution - phase 
 methodology had multiple significant advantages due to its homogeneous 
 reaction conditions : ( 1 ) lower loadings of the precious glycosyl donor 
 for each glycosylation cycle , ( 2 ) real - time monitoring of the reaction 
 by tlc or other means , and ( 3 ) intermediate purification any time 
 during the synthesis sequence with the option for reintroduction of 
 the purified compound for additional automated synthesis cycles .
the automated solution - phase synthesis of the insect glycan ( scheme 2 ) started from the 
 glycosylation of the allyl c8f17 fluorous tag 
 ( f - tag ) 13(31a ) and donor 6 ( 3.0 equiv ) catalyzed by trimethylsilyl trifluoromethanesulfonate 
 ( tmsotf , 0.1 equiv ) at 20 c .
after 45 min , the reaction 
 was quenched by triethylamine ( tea ) , and the solvent was removed by 
 an evaporation cycle .
the pmb group was removed by ceric ammonium 
 nitrate ( can ) , and the reaction mixture was purified with fluorous 
 solid - phase extraction ( fspe ) to remove nonfluorinated impurities 
 and retain the f - tag - modified product ; then , the product was eluted 
 from the fspe cartridge and transferred out of the synthesis platform 
 for further benchtop purification to isolate the -mannuronate 
 product from any minor mixtures of anomers .
obtaining pure -mannuronate 
 at this stage simplified the purification of the final oligosaccharide . 

a significant advantage of this automated solution - phase approach 
 over more standard solid - phase approaches to biopolymer synthesis 
 is the ability to carry out such off - platform purification protocols 
 prior to the end of the total synthesis without removal of the growing 
 biopolymer chain from the support used for automated purification . 
 by this approach ,
this fluorous - tagged carbohydrate 
 was then reinjected into the synthesis platform , and the methyl ester 
 was reduced by lithium triethylborohydride ( litebh ) at 0 c . 

after fluorous solid phase extraction ( fspe ) , the diol was glycosylated 
 with known 2-o - acetyl - mannosyl - trichloroacetimidate 
 donor 8 ( 6.0 equiv ) activated 
 by tmsotf ( 0.1 equiv ) to form two -mannosidic linkages via 
 neighboring group participation .
the crude trisaccharide 9 was transferred out of the synthesis platform ; however , the desired 
 trisaccharide was more readily isolated from the mixture after deacetylation 
 under zempln conditions .
ultimately , trimannoside 10 was obtained in 50% yield from compound 7 over two 
 automation steps and one benchtop deprotection step .
the final deprotection 
 of the benzyl groups on 10 was 
 facilitated by hydrogenolysis catalyzed by 10% pd / c under 1000 psi 
 hydrogen at 20 c for 48 h to provide , in 51% yield , the fully 
 deprotected trimannoside 11 containing a fluorous tag 
 required for adherence of the compound to a fluorous microarray surface ( scheme 3 ) .
to avoid any possible interactions of the hydrophobic fluorous 
 tag with the viral surface in the measuring of their binding via calorimetry , 
 the additional insect glycan 12 with a short linker to 
 maintain the beta - linkage was made .
the synthesis of trimannoside 12 started from the cleavage of the f - tag of 10 by olefin cross - metathesis catalyzed by grubbs catalyst second generation 
 under ethylene atmosphere to provide the allyl trimannoside .
hydrogenolysis 
 catalyzed by 10% pd / c and pd black under a 1000 psi hydrogen atmosphere 
 converted the allyl trimannoside into the fully deprotected n - propyl trimannoside 12 in 91% over two steps 
 for itc experiments with unlabeled pemv . with the required insect - associated 
 glycans in hand ,
the insect n - glycan 
 trimannoside 11 was immobilized using its fluorous tag 
 onto a commercial c8f17-coated glass slide for 
 probing with fitc - labeled pemv . as a positive control , known f - tag - modified 
 -mannoside and -galactoside
were probed with fitc - labeled mannose binding protein concanavalin 
 a ( fitc - cona ) .
each of the f - tagged sugars were spotted on the fluorous 
 glass slide by a microarray spotter in a humidified chamber .
the resulting 
 slide was then incubated with solutions of fitc - pemv or fitc - cona 
 for 1 h. the following rinsing step removed the unbound fitc - pemv 
 or fitc - cona ; then , the slide was air - dried for fluorescence scanning . 
 from the results of the microarray control experiment of f - tagged 
 -mannoside , -galactoside , and 11 incubated 
 with fitc - cona , the -mannoside and 11 showed strong 
 binding and the -galactoside showed no binding as predicted . 

interestingly , the fitc - pemv incubated slide also showed no binding 
 with any of the saccharides , even with elevated fitc - pemv concentrations 
 ( figure 1 , figure s4 ) .
fluorous microarray of f - tag - attached 
 saccharides incubated with 
 ( a ) 0.2 m fitc - cona or ( b ) 1.6 m fitc - pemv .
to confirm this lack of binding interaction found 
 in the fluorous 
 microarray experiment , we commenced with itc experiments .
n - propyl trimannoside 12 was used for the itc 
 experiment with the commercially available methyl -mannoside 
 as the control group .
the itc experiments involved titration of 
 the ligand ( saccharide ) solution into the mixing cell with the protein 
 ( cona or pemv ) solution . by comparing the temperature of the mixing 
 cell and the reference cell , the machine inputs an energy pulse to 
 equalize the temperature in the two cells after each injection of 
 the ligand solution . at the end of each titration experiment , kd ,
h , s , and n could be extracted from the resulting 
 data . in the itc experiments with the two saccharides and cona
, there 
 was obvious specific binding ( kd = 4.65 
 m for methyl -mannoside , and kd = 3.27 m for 12 , figures s5 , s6 ) as expected based on prior work .
however , with pemv , both methyl -mannoside 
 and 12 did not show any noticeable overall positive binding 
 ( figures s7 , s8 ) .
this result combined 
 with the fluorous microarray data provides strong evidence that there 
 is no observable specific binding with the insect n - glycan terminal trimannoside and the pea enation mosaic virus .
the 
 glycosylation state of the pemv structural protein was also tested 
 by itc using cona . by titrating pemv into the cona solution ,
the result 
 showed weak binding between the pemv and cona , which suggests that 
 the pemv does not contain accessible terminal -mannoside or 
 -glucoside moieties ( figure s9 ) .
the first automated solution - phase syntheses of branched n - glycan - related structures using a -directing mannuronate 
 strategy was successful at producing the insect - associated n - glycan trimannosides 11 and 12 . interestingly ,
neither fluorous microarray nor itc experiments 
 showed any evidence for specific binding between the trimannoside 
 motif and the pemv in a multivalent or monovalent presentation format . 

this specific case of carbohydrate interaction discussed in this paper 
 is therefore likely not involved in the transmission of pemv in aphid 
 vectors and , consequently , the development of compounds targeting 
 this interaction for an antiadhesive strategy for viral control would be ineffective .
in addition , a lectin - binding 
 study with pemv does not show evidence for accessible terminal -mannosides 
 or -glucosides on the viral surface that could be used for 
 interaction with an aphid - associated carbohydrate binding protein . 

related studies have now been initiated to test whether the nonglycosylated 
 receptor proteins have any specific binding interaction with pemv 
 or whether both the peptide and glycan of the receptor are responsible 
 for this interaction .
tetrahydrofuran ( thf ) was collected from a solvent 
 purification system prior to the reactions .
all other commercial solvents 
 and reagents were reagent grade and used as received without further 
 purification .
the reactions were monitored by thin layer chromatography 
 ( tlc ) with 250 m silica gel tlc plates .
the hydrogenation reaction 
 under 1000 psi hydrogen was operated in a high pressure reactor .
the 
 developed tlc plates were visualized by stain with p - anisaldehyde solution followed by heating on a hot plate .
flash 
 column chromatography was performed with silica gel ( 4063 
 m particle size ) .
the automated solution phase synthesis was 
 performed in the automated synthesis platform with a hood , 16 reactor 
 vials ( 13 ml capacity each ) , and a heating / cooling unit ( 200 c 
 to 20 c ) machined to hold the spe cartridges at the 
 iowa state university machine shop .
h and c nmr spectra were obtained at 400 and 100 mhz and also at 600 and 
 150 mhz .
chemical shifts (  ) were reported in 
 parts per million ( ppm ) relative to cdcl3 and cd3od as internal references .
mass spectra were obtained on a triple 
 quadrupole mass spectrometer fitted with an esi interface .
hplc traces 
 were obtained from an hplc system using a 3.9  150 mm silica 
 column ( 4 m particle size ) . mannuronate donor building block 
 ( 0.20 g , 0.30 mmol ) was dissolved in anhydrous ch2cl2 ( 1.6 ml ) in the 13 ml vial and placed in the inert reagent 
 rack under an argon atmosphere . a 0.055 m tmsotf solution ( 5.0 ml ) 
 in anhydrous ch2cl2 was prepared in an 8 ml 
 vial and placed as indicated in figure s1 on the inert reagent rack under an argon atmosphere .
acetonitrile 
 ( mecn , 100 ml ) was placed in a stock solution bottle at the stock 
 solution station .
toluene ( 1.0 l ) in a stock solution bottle was placed 
 at the reservoir bottle rack with tubing as a reservoir solution for 
 rinsing .
anhydrous ch2cl2 ( 20 ml ) in the 50 
 ml vial was placed on the inert reagent rack under an argon atmosphere . 

anhydrous thf ( 10 ml ) in the 50 ml vial was placed on the inert reagent 
 rack under an argon atmosphere .
an 80% methanol / water ( 100 ml ) stock 
 was prepared in the 100 ml vial and placed on the inert reagent rack . 

the fluorous - tagged acceptor molecule ( 50 mol ) was dissolved 
 with anhydrous ch2cl2 ( 0.8 ml ) in a wheaton 
 8 ml e - z extraction vial ( conically bottomed ) and flushed with argon , 
 capped with a septa , and placed on the reagent rack .
methanol ( meoh , 
 8.0 ml ) was transferred to an 8 ml - vial capped with septa and placed 
 at the reagent rack .
tea ( 5.0 ml ) was added to an 8 ml vial capped 
 with a septa and placed on the reagent rack .
dmso ( 8.0 ml ) was transferred 
 to an 8 ml vial capped with a septa and placed on the reagent rack . 

a 0.45 m can solution in acetonitrile / water ( 9:1 ) ( 5.0 ml ) was prepared 
 in an 8 ml vial capped with a septa and placed on the reagent rack . 

litebh solution in thf ( 1.0 m , 3 ml ) was transferred to an 8 ml vial 
 capped with a septa and placed on the reagent rack .
water ( 8.0 ml ) 
 was added to an 8 ml vial capped with a septa and placed on the reagent 
 rack . a fluorous solid - phase extraction ( fspe ) cartridge ( 2.0 g , 10 
 cc ) was preconditioned with 80% methanol / water and placed on the machined 
 fspe block .
an empty wheaton 8 ml e - z extraction vial was placed underneath 
 the fspe cartridge ( see figure s1 for the 
 rack positions ) . before introduction 
 of reagents ,
the 
 reactor vials were cleaned , dried , and flushed with argon by running 
 the cleaning cycle . during the cleaning cycle ,
each of the 16 reactor 
 vials ( 13 ml capacity each ) was rinsed with toluene ( 8.0 ml ) and methanol 
 ( 8.0 ml ) 3 times .
after the solvent was removed , the reactor vials 
 were dried under vacuum and purged with argon for 45 min .
reagent 
 solutions were prepared by azeotropic removal of water from each building 
 block donor with toluene ; the resulting building block donors were 
 then dried under high vacuum .
after the cleaning cycles were done , 
 the reagents were transferred into their respective reagent vials , 
 which were then placed on the inert condition reagent rack and general 
 atmosphere reagent rack .
the needle transferred 
 the acceptor molecule 
 ( f - tag ) solution ( 0.80 ml ) to the reaction vial , followed by the transfer 
 of the donor molecule solution ( 0.80 ml ) .
the mixture was vortexed 
 under ambient temperature at 800 rpm for 20 min . then
, the reactor 
 vials were cooled to 20 c during the 60 min wait time 
 by the heat transfer oil with an 800 rpm vortex rate .
the tmsotf solution 
 ( 0.10 ml ) was transferred into the reactor vial at a 200 rpm vortex 
 rate .
after each individual transfer , the needle ( inside and outside ) 
 was rinsed with toluene ( 2.0 ml ) before operating the next task .
the 
 reaction mixture was vortexed at 800 rpm for 45 min at 0 c under 
 an argon atmosphere . after the reaction time
, the needle withdrew 
 20 l of the solution from the reaction mixture and placed it 
 into the first well of the microtiter plate for thin layer chromatography 
 monitoring .
tea ( 0.050 ml ) was added to the solution for quenching , 
 and the solvent was evaporated under reduced pressure . to the dried residue after the glycosylation 
 was added the can solution ( 1.0 ml ) in the reactor vial .
the reaction 
 mixture was vortexed at 800 rpm for 1 h at ambient temperature .
after 
 the reaction time , the needle withdrew 20 l of the solution 
 from the reaction mixture and placed it into the second well of the 
 microtiter plate for thin layer chromatography monitoring .
the reaction 
 mixture ( 1.2 ml ) was carried to the fspe cartridge at the fspe block 
 and dispensed at a speed of 1.0 ml / s via the 10 ml syringe .
then , 
 80% methanol ( 2.0 ml ) was used to rinse the empty reactor vial .
the 
 80% methanol solution was removed from the reactor vial and delivered 
 to the fspe cartridge .
an additional 80% methanol solution ( 4.0 ml , repeated 
 2 times ) was used to rinse the fspe cartridge . during the 80% methanol 
 rinse
acetonitrile ( mecn ) ( 2.0 ml , repeated 
 3 times ) was used to wash the fspe cartridge for eluting the desired 
 compound . during the task
spe 
 collect  to be placed right above the 8 ml vial for collection 
 of the sample .
after the task , the position of the spe rack was changed 
 to  spe direct  for the needle to withdraw the collected 
 sample from the conically bottomed vial and to deliver it to the clean 
 reactor vial for the next reaction .
anhydrous thf ( 1.0 ml ) was added to 
 the sample and vortexed at 800 rpm at 0 c under an argon atmosphere 
 for 30 min .
the litebh ( 0.30 ml , 1.0 m in thf ) was added to the reaction 
 solution , and the mixture was vortexed for 30 min at 800 rpm at 0 
 c under an argon atmosphere . after the reaction
, the needle 
 withdrew 20 l of the solution from the reaction mixture and 
 placed it into the fourth well of the microtiter plate for thin layer 
 chromatography monitoring .
meoh ( 0.50 ml ) was added to quench the 
 reaction , and the solvent was removed under reduced pressure .
crude product ( < 0.30 g ) was dissolved in 1.0 ml 
 dimethyl sulfoxide ( dmso ) and loaded onto the 80% meoh preconditioned 
 2 g fspe cartridge .
the cartridge was washed with 80% meoh ( 4.0 ml 
  3 times ) .
mechanical 
 infection of pea plants ( pisum sativum ) with pemv 
 was performed as described in liu et al .
the method used to purify pemv from plants was modified from liu 
 et al . infected pea plants ( 150 g )
were 
 frozen in liquid nitrogen and homogenized in a blender with 0.2 m 
 sodium acetate buffer ( ph 6.0 ) ( 1 ml/1 g tissue ) and an equal volume 
 of chloroform .
the supernatant was transferred 
 to clean tubes and centrifuged at 3000 g for 5 min . 

the supernatant was again transferred to clean tubes and then centrifuged 
 at 17,200 g for 2 h. pellets from this step were saved , 
 and the supernatant was centrifuged at 140,500 g for 
 2.5 h. virus pellets were soaked in 0.2 m sodium acetate buffer ( ph 
 7.0 ) overnight at 4 c and resuspended the following day .
after 
 resuspension , the soluble fraction was centrifuged at 145,000 g through a 30% sucrose cushion made in the same buffer . 

the final pellet was washed three times with buffer to remove excess 
 sucrose and resuspended in 0.2 m sodium acetate ( ph 7.0 ) .
the final 
 virus solution was frozen in liquid nitrogen and stored at 80 
 c .
the protein concentration of pemv was determined by densitometric 
 analysis of the coomassie stained bands with reference to known bsa 
 concentrations resolved by sds 
purified pemv was labeled with 
 fluorescein isothiocyanate ( fitc ) using a fitc antibody labeling kit 
 following the manufacturer s protocol .
the labeling reaction 
 was completed in 0.2 m sodium acetate buffer ( ph 7.0 ) in place of 
 the recommended 50 mm sodium borate at ph 8.5 .
to confirm fitc labeling 
 of pemv , we separated the virions by sds - page , and the gel was scanned 
 using a gel imager in the green - excited mode ( 532 nm ) .
microarrays 
 were printed by a microarray printer equipped with a 
 robotic pin ( 0.35 mm ) .
the f - tag attached sugar solution with a concentration 
 of 222 m in meoh / dmso / h2o ( v : v : v = 1:3.5:1.15 ) was 
 printed onto the fluorous glass slides .
the printed slide was dried 
 in a humid chamber for 20 h and ready for incubation with protein 
 solution .
the protein solution was prepared in various concentrations 
 in 1 pbs buffer with 1% bovine serum albumin ( bsa ) . for fitc - cona , 

the solution was with 1.0 mg / ml of cacl2 and 1.0 mg / ml 
 of mncl2 .
the incubation was carried out in an incubation 
 chamber for 1 h , and then washed twice with 1 pbs buffer and 
 once with di water .
then , the slide was dried in air and scanned at 
 the iowa state university dna facility using a microarray scanner 
 set at 488 nm .
the 10 
 m protein solution and the ligand solution ( 300 m for 
 the saccharides and 100 m for the pemv ) were both prepared 
 in ph 7.0 naoac buffer .
for titration experiments with cona , cacl2 and mncl2 ( 1.0 mg / ml ) were added to the buffer 
 for both cona and ligand solutions .
the solutions were degassed prior 
 to the experiment , and the titration was carried out at 26 c 
 with a 310 rpm stirring rate . to the phenyl 2-o - benzyl-3-o - p - methoxybenzyl-4,6-o - benzylidene - thio--d - mannopyranoside ( 1 )
( 0.80 g , 1.4 mmol ) was added a borane tetrahydrofuran complex 1.0 
 m solution in tetrahydrofuran ( 14 ml , 14 mmol ) under an argon atmosphere 
 at 0 c , and the solution was stirred until the starting material 
 was dissolved .
then , a dibutylboryl trifluoromethanesulfonate 1.0 
 m solution in ch2cl2 ( 1.7 ml , 1.7 mmol ) was 
 added dropwise , and the reaction mixture was stirred under argon atmosphere 
 at 0 c for 3 h. triethylamine ( 0.30 ml , 2.15 mmol ) was added 
 dropwise to the reaction ; then , methanol was added slowly to quench 
 the reaction at 0 c .
the solvent was removed under reduced pressure , 
 and the crude mixture was coevaporated with methanol twice .
the crude 
 product was purified by flash column chromatography on silica gel 
 using etoac / petroleum ether ( 1:2 ) as eluent .
the product was obtained 
 as a colorless syrup ( 0.69 g , 1.20 mmol , 86% ) .
h nmr ( cdcl3 , 600 mhz ) :  7.407.26 ( m , 17h , harom ) , 6.886.87 ( m , 2h , harom ) , 5.50 ( d , 1h , j = 1.6 hz , h-1 ) , 4.96 ( d , 1h , j = 10.8 
 hz , chhph ) , 4.694.64 ( m , 2h , chhph ) , 4.57 ( s , 2h , chhph ) , 4.12 ( m , 1h , h-5 ) , 4.03 
 ( t , 1h , j = 9.2 hz , h-4 ) , 3.97 ( dd , 1h , j = 2.8 , 1.6 hz , h-2 ) , 3.89 ( dd , 1h , j = 9.2 , 3.2 
 hz , h-3 ) , 3.82 ( s , 3h , co2ch3 ) , 3.813.75 ( m , 2h , 2  h-6 ) .
c nmr ( cdcl3 , 150 mhz ) :  159.4 , 138.5 , 138.0 , 134.1 , 131.9 , 130.6 , 
 130.3 , 129.6 , 129.4 , 129.2 , 128.5 , 128.1 , 128.0 , 127.9 , 127.9 , 127.9 , 
 127.7 , 114.0 , 86.1 , 79.8 , 76.6 , 76.6 , 75.3 , 74.8 , 73.4 , 72.4 , 72.0 , 
 62.2 , 55.4 . hrms ( esi ) : [ m + na ] calcd for c34h36nao6s , 595.2125 ; found , 595.2119 . to a solution of phenyl 2,4-di - o - benzyl-3-o - p - methoxybenzyl - thio--d - mannopyranoside 2 ( 0.50 g , 0.87 mmol ) in dichloromethane / water 
 ( 5.8 ml/2.9 ml ) were added tempo ( 0.030 g , 0.19 mmol ) and ( diacetoxyiodo)benzene 
 ( 0.70 g , 2.17 mmol ) , and the solution was stirred at ambient temperature . 

after 45 min , the mixture was diluted with dichloromethane ( 10 ml ) 
 and washed with a 10% na2s2o3 solution 
 ( 10 ml ) and water ( 10 ml ) .
the solvent was removed under reduced pressure , 
 and the crude product was purified by flash column chromatography 
 on silica gel using etoac / petroleum ether ( 1:2  1:0 ) as eluent . 

the product was obtained as a light yellow syrup ( 0.34 g , 0.57 mmol , 
 66% ) .
h nmr ( cdcl3 , 600 mhz ) :  7.52 
 ( d , 2h , j = 6.6 hz , harom ) , 7.337.24 
 ( m , 13h , harom ) , 7.19 ( d , 2h , j = 8.4 
 hz , harom ) , 6.83 ( d , 2h , j = 9.0 hz , harom ) , 5.67 ( d , 1h , j = 4.8 hz , h-1 ) , 4.694.65 
 ( m , 3h , chhph ) , 4.64 ( d , 1h , j = 
 6.6 hz , h-5 ) , 4.52 ( m , 3h , chhph ) , 4.21 ( t , 1h , j = 6.6 hz , h-4 ) , 3.88 ( dd , 1h , j = 4.8 , 
 2.4 hz , h-2 ) , 3.82 ( m , 1h , h-3 ) , 3.78 ( s , 3h , co2ch3 ) .
c nmr ( cdcl3 , 150 
 mhz ) :  174.7 , 159.3 , 137.8 , 137.7 , 133.8 , 131.4 , 129.7 , 129.6 , 
 129.0 , 128.4 , 128.4 , 128.4 , 128.4 , 128.1 , 127.9 , 127.9 , 127.8 , 113.8 , 
 75.7 , 72.6 , 72.3 , 72.0 , 55.3 . hrms ( esi ) : [ m + na ] calcd 
 for c34h34nao7s , 609.1917 ; 
 found , 609.1904 . to a solution of phenyl 2,4-di - o - benzyl-3-o - p - methoxybenzyl - thio--d - mannopyranosiduronic acid 3 ( 0.50 g , 0.85 mmol ) in anhydrous 
 dmf ( 4.0 ml ) were added k2co3 ( 0.12 g , 0.85 
 mmol ) and iodomethane ( 0.30 g , 2.13 mmol ) .
the reaction mixture was 
 stirred at ambient temperature under argon atmosphere for 6 h. the 
 mixture was diluted with etoac ( 10 ml ) and washed with water ( 10 ml ) . 
 the aqueous portion was separated and extracted with etoac ( 10  
 2 ml ) .
the solvent was removed under reduced pressure , and the 
 crude product was purified by flash column chromatography on silica 
 gel using etoac / petroleum ether ( 1:3 ) as eluent .
the product was obtained 
 as a light yellow syrup ( 0.47 g , 0.78 mmol , 92% ) .
h nmr 
 ( cdcl3 , 600 mhz ) :  7.52 ( d , 2h , j = 7.2 hz , harom ) , 7.357.25 ( m , 13h , harom ) , 7.19 ( d , 2h , j = 7.8 hz , harom ) , 6.83 
 ( d , 2h , j = 8.4 hz , harom ) , 5.67 ( d , 1h , j = 4.8 hz , h-1 ) , 4.664.62 ( m , 4h , chhph , h-5 ) , 4.524.46 ( m , 3h , chhph ) , 4.21 
 ( t , 1h , j = 7.2 hz , h-4 ) , 3.88 ( dd , 1h , j = 4.8 , 3.0 hz , h-2 ) , 3.81 ( m , 1h , h-3 ) , 3.78 ( s , 3h , och3 ) .
c nmr ( cdcl3 , 150 mhz ) :  
 169.7 , 159.4 , 138.1 , 137.9 , 134.0 , 131.5 , 130.0 , 129.6 , 129.6 , 129.0 , 
 128.5 , 128.5 , 128.1 , 127.9 , 127.9 , 127.8 , 127.3 , 113.8 , 76.0 , 73.1 , 
 72.4 , 72.2 , 55.3 , 52.3 . hrms ( esi ) : [ m + na ] calcd for 
 c35h36nao7s , 623.2074 ; 
 found , 623.2071 . to a solution of methyl ( phenyl 2,4-di - o - benzyl-3-o - p - methoxylbenzyl - thio--d -mannopyranoside ) uronate
4 ( 0.50 g , 0.83 mmol ) 
 in 10% water / acetone ( 10 ml ) was added n - bromosuccinimide 
 ( 0.44 g , 2.49 mmol ) .
the reaction mixture was stirred at ambient temperature 
 for 1 h. the reaction mixture was diluted with etoac ( 30 ml ) and washed 
 with a saturated nahco3 solution ( 30 ml ) .
the solvent was removed 
 under reduced pressure , and the crude product was purified by flash 
 column chromatography on silica gel using etoac / petroleum ether ( 2:3 ) 
 as eluent .
the product was obtained as a pale yellow syrup ( 0.38 g , 
 0.75 mmol , 90% ) .
h nmr ( cdcl3 , 400 mhz ) :  
 7.367.20 ( m , 12h , harom ) , 6.85 ( d , 2h , j = 8.4 hz , harom ) , 5.43 ( t , 1h , j = 4.8 hz , h-1 ) , 4.75 ( d , 1h , j = 12.0 hz , chhph ) , 4.69 ( m , 3h , chhph ) , 4.51 ( s. 2h , 
 chhph ) , 4.47 ( d , 2h , j = 6.4 hz , 

h-5 ) , 4.22 ( t , 1h , j = 6.8 hz , h-4 ) , 3.91 ( dd , 1h , j = 7.2 , 2.8 hz , h-3 ) , 3.80 ( s , 3h , co2ch3 ) , 3.71 ( dd , 1h , j = 4.4 , 
 2.8 hz , h-2 ) , 3.65 ( s , 3h , och3 ) , 3.18 
 ( d , 1h , j = 4.8 hz , oh ) .
c nmr ( cdcl3 , 100 mhz ) :  170.3 , 159.3 , 138.4 , 138.2 , 
 130.3 , 129.5 , 129.5 , 128.5 , 128.5 , 128.5 , 128.4 , 128.0 , 127.9 , 127.7 , 
 113.8 , 93.0 ( jc1h1 = 168.8 hz , 
 c-1 ) , 75.9 , 75.5 , 74.0 , 72.9 , 72.4 , 72.2 , 55.4 , 52.4 . hrms ( esi ) : 
 [ m + na ] calcd for c29h32nao8 , 531.1989 ; found , 531.1985 . to a solution of methyl 
 ( 2,4-di - o - benzyl-3-o - p - methoxylbenzyl--d - mannopyranose)uronate
( 5 ) ( 0.50 g , 0.98 mmol ) in dichloromethane ( 35 ml ) was added trichloroacetonitrile 
 ( 0.85 g , 5.88 mmol ) at 0 c under an argon atmosphere .
1,8-diazabicyclo[5.4.0]undec-7-ene 
 ( dbu ) ( 0.030 g , 0.2 mmol ) was then added , and the reaction mixture 
 was stirred at 0 c under argon atmosphere for 3 h. the solvent 
 was removed under reduced pressure , and the crude product was purified 
 by flash column chromatography on silica gel using etoac / petroleum 
 ether / triethylamine ( 1:2:0.1 ) as eluent .
the product was obtained 
 as a colorless syrup and a mixture of anomers ( / = 4:1 , 
 0.59 g , 0.93 mmol , 95% ) .
h nmr ( cdcl3 , 400 mhz ) : 
  9.32 ( s , 1h , hn ) , 8.62 
 ( s , 1h , hn ) , 7.417.26 ( m , 
 10h , harom ) , 7.21 ( d , 2h , j = 8.8 hz , 
 harom ) , 7.16 ( d , 2h , j = 8.4 hz , harom ) , 6.83 ( d , 2 h , j = 8.4 hz , harom ) , 6.40 ( d , 1h , j = 2.8 hz , h-1 ) , 
 5.96 ( d , 1h , j = 7.6 hz , h-1 ) , 
 4.854.74 ( m , 3h , chhph ) , 4.654.48 
 ( m , 4h , chhph , h-5 ) , 4.39 ( d , 1h , j = 8.4 , h-4 ) , 4.28 ( t , 1h , j = 
 8.4 hz , h-3 ) , 4.17 ( dd , 1h , j = 
 4.4 , 2.4 hz , h-3 ) , 3.903.87 ( m , 1h , h-2 ) , 3.833.81 ( m , 1h , h-2 ) , 
 3.80 ( s , 3h , co2ch3 3.71 ( s , 
 3h , och3 ) , 3.64 ( s , 3h ) .
c nmr ( cdcl3 , 100 mhz ) :  169.4 , 169.1 , 160.4 , 159.5 , 
 159.5 , 160.0 , 138.0 , 137.9 , 137.2 , 130.0 , 129.8 , 129.4 , 128.7 , 128.6 , 
 128.6 , 128.5 , 128.3 , 128.2 , 128.1 , 128.1 , 128.0 , 128.0 , 128.0 , 113.9 , 
 113.9 , 95.8 , 95.2 , 90.9 , 77.3 , 75.6 , 75.4 , 75.2 , 75.0 , 74.3 , 74.1 , 
 74.0 , 73.7 , 73.6 , 73.6 , 73.6 , 73.0 , 72.7 , 72.6 , 72.3 , 55.4 , 52.7 , 
 52.6 . hrms ( esi ) : [ m + na ] calcd for c31h32cl3nnao8 , 674.1086 ; found , 
 674.107 .
the crude product solution was transferred out 
 of the asw1000 after the fspe step ( 10th step ) of the first cycle . 

the solvent was removed under reduced pressure , and the product was 
 purified by flash column chromatography on silica gel using etoac / petroleum 
 ether ( 1:3.5 ) as eluent .
the product was obtained as a colorless syrup 
 ( 72 mg , 0.078 mmol , 78% over 2 steps ) .
h nmr ( cdcl3 , 400 mhz ) :  7.357.23 ( m , 10h , harom ) , 5.74 ( m , 2h , hc = ch ) , 
 4.96 ( d , 1h , j = 12.0 hz , chhph ) , 
 4.77 ( d , 1h , j = 11.2 hz , chhph ) , 
 4.63 ( d , 1h , j = 1.6 hz , h-1 ) , 4.61 ( d , 1h , j = 12.0 hz , chhph ) , 4.60 ( d , 1h , j = 11.2 hz , chhph ) , 4.47 ( dd , 1h , j = 12.8 , 4.8 hz , o  chhc = c ) , 
 4.24 ( dd , 1h , j = 12.8 , 6.4 hz , o  chhc =
c ) , 4.04 ( m , 2h , c = cch2-o ) , 3.99 ( t , 1h , j = 7.6 , h-4 ) , 3.90 
 ( d , 1h , j = 8.0 , h-5 ) , 3.81 ( d , 1h , j = 2.4 , h-2 ) , 3.78 ( m , 1h , h-3 ) , 3.72 ( s , 3h , co2ch3 ) , 3.49 ( m , 2h , o 
ch2ch2 ) , 2.69 ( d , 1h , j = 9.6 , 
 4-oh ) , 2.23 ( m , 2h , ch2cf2 ) , 1.88 ( m , 2h , o 
c nmr ( cdcl3 , 100 mhz ) :  
 169.4 , 138.2 , 138.2 , 130.6 , 128.7 , 128.6 , 128.4 , 128.3 , 128.2 , 128.1 , 
 128.1 , 128.0 , 100.3 ( jc1h1 = 157.7 
 hz , c-1 ) , 78.0 , 76.0 , 74.5 , 74.3 , 72.3 , 69.0 , 66.7 , 65.1 , 52.5 , 28.4 
 ( t , jc  f = 22.0 hz ) , 21.0 . hrms 
 ( esi ) : [ m + na ] calcd for c36h35f17nao8 , 941.1953 ; found , 941.1959 .
the crude mixture 
 was transferred out of the synthesis platform after the third cycle 
 ( 33rd step of automated synthesis ) , diluted with ch2cl2 ( 20 ml ) , and washed with satd nh4cl(aq ) .
the organic 
 layer was dried over na2so4 , and the solvent 
 was removed under reduced pressure .
then , the crude product was purified 
 by flash chromatography on silica gel etoac / petroleum ether ( 1:3 ) . 

the disaccharide byproduct could not be separated at this stage , and 
 the crude mixture was carried to the next step without further purification .
hrms ( esi ) : [ m + na ] calcd for c93h95f17nao19 , 1861.6088 ; found , 1861.6118 .
crude 9 ( 58 mg ) from the automated 
 synthesis was dissolved in meoh ( 2.0 ml ) , and na ( 0.50 mg , 0.22 mmol ) 
 was added .
the mixture was stirred at ambient temperature for 1 h. 
 the reaction was added by dowex 50wx8 200 acidic resin until the ph 
 paper showed neutral .
the solvent was removed under reduced pressure , 
 and the crude product was purified by flash column chromatography 
 on silica gel using etoac / petroleum ether ( 4:5  1:0 ) as eluent . 

the product was obtained as a colorless syrup ( 44 mg , 0.025 mmol , 
 50% over 2 automated synthesis steps and 1 benchtop step ) .
h nmr ( cdcl3 , 600 mhz ) : 
  7.417.14 ( m , 40 h , harom ) , 5.67 ( m 2h , 
 hc = ch ) , 5.23 ( d , 1h , j = 1.2 hz , h-1 ) , 5.08 ( d , 1h , j = 1.2 hz , h-1 ) , 4.99 ( d , 1h , j = 12.0 hz , chhph ) , 4.83 ( 2  
 d , 2h , j = 10.8 hz , chhph ) , 4.72 ( d , 1h , j = 12.6 hz ) , 4.664.59 ( m , 5h , chhph ) , 4.554.46 
 ( m , 7h , chhph ) , 4.39 ( s , 1h , h-1 ) , 4.35 ( dd , 1h , j = 12.6 , 4.8 hz , o  chhc = c ) , 4.11 ( d , 1h , j = 1.8 hz , h-2 ) , 4.09 ( dd , 1h , j = 13.2 , 6.6 hz , o  chhc = c ) , 4.023.95 
 ( m , 3h , h-2 , c = cch2o ) , 
 3.93 ( d , 1h , j = 3.0 hz ) , 3.883.84 ( m , 5h ) , 
 3.83 ( dd , 1h , j = 9.0 , 3.6 hz , h-3 ) , 3.793.74 ( m , 4h ) , 3.71 ( dd , 1h , j = 
 10.8 , 4.2 hz ) , 3.653.60 ( m , 3h ) , 3.41 ( m , 2h , och2ch2 ) , 3.33 ( m , 1h ) , 2.33 ( d , 1h , j = 
 1.8 hz , 2-oh ) , 2.30 ( d , 1h , j = 2.4 hz , 2-oh ) , 2.18 
 ( m , 2h , ch2cf2 ) , 1.83 ( m , 2h , o 
c nmr ( cdcl3 , 150 
 mhz ) :  139.2 , 138.7 , 138.7 , 138.5 , 138.4 , 138.1 , 138.0 , 130.0 , 
 128.7 , 128.6 , 128.6 , 128.5 , 128.4 , 128.4 , 128.3 , 128.1 , 128.1 , 128.1 , 
 128.0 , 128.0 , 128.0 , 128.0 , 127.9 , 127.9 , 127.9 , 127.8 , 127.8 , 127.7 , 
 127.7 , 127.5 , 101.8 ( jc1h1 = 165.0 
 hz , c-1 ) , 100.8 ( jc1h1 = 151.8 
 hz , c-1 ) , 100.2 ( jc1h1 = 170.1 
 hz , c-1 ) , 81.2 , 80.2 , 79.8 , 78.1,75.5 , 75.3 , 75.3 , 75.2 , 75.0 , 74.6 , 
 74.5 , 74.4 , 73.6 , 73.5 , 72.4 , 72.1 , 71.6 , 71.4 , 69.4 , 69.1 , 68.9 , 
 68.9 , 68.1 , 66.8 , 66.4 , 64.9 , 28.4 ( t , jc 
hrms ( esi ) : [ m + na ] calcd for c89h91f17nao17 , 
 1777.5877 ; found , 1777.5875 .
cis-4-(1h,1h,2h,2h,3h,3h - perfluoroundecyloxy)-2-butenyl 2,4-di - o - benzyl-3,6-di - o-(3,4,6-tri - o - benzyl--d - mannopyranosyl)--d - mannopyranoside 10 ( 44 mg , 0.025 mmol ) was dissolved in meoh ( 3.0 ml ) , and 
 10% pd / c ( 10 mg ) was added .
after 48 h , the mixture was 
 filtered through a short pad of celite , and the solvent was removed 
 under reduced pressure .
the desired product was collected as a white 
 foam ( 13.2 mg , 0.013 mmol , 51% ) .
h nmr ( cdcl3 , 600 mhz ) :  5.08 ( d , 1h , j = 1.2 hz , h-1 ) , 4.83 ( d , 1h , j = 1.2 hz , h-1 ) , 
 4.50 ( s , 1h , h-1 ) , 4.10 ( d , 1h , j = 3.0 hz , h-2 ) , 
 3.98 ( dd , 1h , j = 3.0 , 1.2 hz , h-2 ) , 3.953.91 
 ( m , 2h ) , 3.883.84 ( m , 3h ) , 3.833.79 ( m , 3h ) , 3.77 
 ( d , 1h , j = 9.6 hz ) , 3.743.72 ( m , 2h ) , 3.713.67 
 ( m , 2h ) , 3.653.63 ( m , 2h ) , 3.603.56 ( m , 3h ) , 3.52 
 ( t , 2h , j = 18.0 hz , och2ch2ch2cf2 ) , 3.49 ( m , 
 2h , ch2ch2o ) , 3.37 
 ( m , 1h ) , 2.28 ( m , 2h , ch2cf2 ) , 1.87 ( m , 2h , och2ch2ch2cf2 ) , 1.67 ( s , 4h , och2ch2ch2ch2o ) . c nmr ( cdcl3 , 150 mhz ) :  
 104.1 , 101.8 , 101.6 , 83.0 , 77.0 , 75.1 , 74.5 , 72.8 , 72.6 , 72.2 , 72.2 , 
 72.1 , 71.9 , 70.6 , 70.2 , 69.1 , 68.8 , 67.7 , 67.4 , 63.2 , 63.1 , 29.2 ( t , jc 
hrms 
 ( esi ) : [ m + na ] calcd for c33h45f17nao17 , 1059.2278 ; found , 1059.2269 .
cis-4-(1h,1h,2h,2h,3h,3h - perfluoroundecyloxy)-2-butenyl 2,4-di - o - benzyl-3,6-di - o-(3,4,6-tri - o - benzyl--d - mannopyranosyl)--d - mannopyranoside 10 ( 55 mg , 0.031 mmol ) was dissolved in dry ch2cl2 ( 4.0 ml ) , and grubbs catalyst second generation ( 5.4 
 mg , 6.4 mol ) was added .
the solution was stirred at 20 c , 
 and ethylene gas was bubbled through for 30 min ; then , an ethylene 
 balloon was attached , and the mixture was stirred for 24 h. the solvent 
 was evaporated , and the mixture was loaded onto a short silica gel 
 column for purification ( ch2cl2  etoac / petroleum 
 ether , 1:1 ) .
the product fraction was collected ; the solvent was evaporated , 
 and the crude product was purified by fspe following the general procedure 
 for benchtop fluorous solid - phase extraction . after the acetone fraction 
 was collected , and the solvent was removed under reduced pressure , 
 the product was dissolved in meoh ( 3.0 ml ) and 10% pd / c ( 25 mg ) was 
 added .
after 24 h , another portion of pd black ( 20 mg ) 
 and acoh ( 0.30 ml ) was added , and the mixture was stirred at 20 c 
 under 1000 psi h2 atmosphere for an additional 24 h. the 
 mixture was filtered through a small pad of celite , and the celite 
 pad was washed with meoh ( 10  3 ml ) . after the solvent was removed 
 under reduced pressure , the residue was washed with ch2cl2 ( 3  1.0 ml ) , and the fully deprotected product 
 was obtained as a white foam ( 15.6 mg , 0.028 mmol , 91% over 2 steps ) . 

h nmr ( cd3od , 600 mhz ) : 
  5.1 ( d , j = 1.2 hz , 1h , h-1 ) , 4.83 ( d , 1h , j = 1.8 hz , h-1 ) , 4.50 ( s , 1h , h-1 ) , 4.10 ( d , 1h , j = 3.0 hz , h-2 ) , 3.98 ( dd , 1h , j = 3.0 , 
 1.8 hz , h-2 ) , 3.95 ( dd , j = 10.8 , 5.4 hz ) , 3.883.80 
 ( m , 6h ) , 3.773.71 ( m , 3h ) , 3.703.60 ( m , 4h ) , 3.593.56 
 ( m , 2h ) , 3.51 ( m , 1h ) , 3.37 ( m , 1h ) , 1.64 ( m , 2h , ch2ch3 ) , 0.96 ( t , 3h , j = 7.2 
 hz , ch3 ) .
c nmr ( cd3od , 150 mhz ) :  104.1 , 101.8 , 101.5 , 83.0 , 76.9 , 75.1 , 74.5 , 
 72.7 , 72.6 , 72.5 , 72.2 , 72.1 , 72.1 , 69.0 , 68 . 7 , 67.6 , 67.3 , 63.2 , 
 63.0 , 24.0 , 11.0 .
hrms ( esi ) : [ m + na ] calcd for c21h38nao16 , 569.2052 ; found , 
 569.2040 .
 pea 
 enation mosaic virus ( pemv)a plant rna virus transmitted 
 exclusively by aphids  causes disease in multiple food crops . 
 
 however , the aphid - virus interactions required for disease transmission 
 are poorly understood . for virus transmission 
 , pemv binds to a heavily 
 glycosylated receptor aminopeptidase n in the pea aphid gut and is 
 transcytosed across the gut epithelium into the aphid body cavity 
 prior to release in saliva as the aphid feeds . to investigate the 
 role of glycans in pemv  aphid interactions and explore the 
 possibility of viral control through blocking a glycan interaction , 
 we synthesized insect n - glycan terminal trimannosides 
 by automated solution - phase synthesis . 
 the route features a mannose 
 building block with c-5 ester enforcing a -linkage , which also 
 provides a site for subsequent chain extension . 
 the resulting insect n - glycan terminal trimannosides with fluorous tags were 
 used in a fluorous microarray to analyze binding with fluorescein 
 isothiocyanate - labeled pemv ; however , no specific binding between 
 the insect glycan and pemv was detected . to confirm these microarray 
 results 
 , we removed the fluorous tag from the trimannosides for isothermal 
 titration calorimetry studies with unlabeled pemv . 
 the itc studies 
 confirmed the microarray results and suggested that this particular 
 glycan  pemv interaction is not involved in virus uptake and 
 transport through the aphid . 
Introduction
Results and Discussion
Conclusions
Experimental Section