precise molecular control of protein glycosylation would advance the development of biopharmaceuticals as well as fundamental studies of glycosylation - dependent biological processes .
biological methods on their own have failed to achieve this long sought - after goal , as cellular biosynthetic pathways produce glycoproteins as a heterogeneous mixture of glycoforms .
glycoprotein structures with optimal bioactivity are difficult to identify from such complex mixtures and impossible to generate exclusively using conventional cell hosts .
the tools of synthetic biology have been harnessed to construct heterologous glycan biosynthetic pathways in simple host cells .
while these systems have produced glycoproteins that are globally modified with a single glycan type , no recombinant expression system can yet produce proteins with discrete glycans at individual sites and with complete homogeneity .
chemical synthesis is currently the only plausible approach to achieve homogeneously glycosylated proteins bearing tailored glycans at specific sites .
several groups have successfully combined frontier protein ligation techniques with chemical or enzymatic glycan and glycopeptide synthesis methods to construct glycoproteins that duplicate natural structures .
the valor of these efforts should not be underestimated since the difficulties inherent to glycan and protein syntheses amplify each other when both biopolymers coexist in the same synthetic target .
a more practical , though less biologically authentic , approach involves conjugating glycans to recombinant proteins via unnatural but synthetically tractable linkages .
site - specificity in the modification can be ensured by outfitting the protein with a noncanonical amino acid side chain capable of chemoselective ligation with an appropriately functionalized glycan .
we and others have explored the oxime - forming reaction of aminooxy glycans with peptide - associated aldehydes or ketones as a means of site - specific glycosylation .
this chemistry proceeds in mildly acidic aqueous buffers that are compatible with most proteins and poses little risk of side reactivity with endogenous protein or glycan functionalities . despite promising initial work
, however , the generality of the approach remains limited by two challenges : ( 1 ) the need for a practical means of introducing aldehydes or ketones into recombinant proteins using standard expression systems , and ( 2 ) a reliable route to synthesize higher - order aminooxy glycans .
the recently developed aldehyde tag methodology answers the first challenge , as it enables site - specific introduction of aldehyde - bearing formylglycine ( fgly ) residues into any recombinant protein of interest using conventional escherichia coli(32 ) or mammalian(33 ) expression systems .
the aldehyde tag is a five - residue consensus sequence ( cxpxr , where x is variable ) that is recognized by the formylglycine generating enzyme ( fge ) .
cotranslationally , fge oxidizes the genetically encoded cysteine residue to fgly , thereby providing a uniquely reactive site for chemical modification .
although fge is native to most cell types , coexpression of additional fge alongside a recombinant protein substrate ensures high cys - to - fgly conversion yields .
use of the aldehyde tag method simply requires cloning the consensus sequence into the protein s gene at the desired site of modification , followed by protein production in fge - expressing cells .
in addition to its practicality , the aldehyde tag method benefits from the resemblance of the fgly side chain to naturally glycosylated amino acid side chains .
most secreted and cell - surface vertebrate glycoproteins are modified at asn residues with n - glycans or at ser / thr residues with o - glycans ( figure 1a ) .
we envisioned oxime - linked glycoproteins deriving from conjugation of the fgly aldehyde with synthetic aminooxy glycans as illustrated in figure 1b .
protein linkages comprising fgly - derived oximes are close structural mimics of n - glycan linkages and just one atom longer than o - glycan linkages .
natural glycan linkages resemble the linkage produced by protein glycoengineering via the reaction of aminooxy sugars with the aldehyde tag .
( a ) linkages naturally found in the most abundant mammalian n- and o - linked glycan structures .
( b ) the linkage formed upon the reaction of an aminooxy glycan with the aldehyde of an fgly - containing protein .
the resulting oxime side chain bears high structural similarity to the natural glycoprotein attachments . to capitalize on the aldehyde tag as a tool for site - specific protein glycosylation
, we sought to answer the second challenge  a practical and general route to synthesize complex aminooxy glycans . here
we report on the use of glycosyl n - pentenoyl hydroxamates as intermediates in the synthesis of higher - order aminooxy glycans .
these analogues function well in both chemical and enzymatic glycosylation reactions , enabling the efficient and high - yielding production of both simple and elaborated aminooxy glycans as substrates for protein conjugation .
the new synthetic approach , together with the aldehyde tag technology , enabled the generation of recombinant proteins modified site - specifically with homogeneous glycans .
in previous work , aminooxy glycosides were synthesized by displacement of a glycosyl halide with n - hydroxysuccinimide ( nhs ) or n - hydroxyphthalimide ( nhpht ) .
reaction yields were acceptable using monosaccharide substrates but , in our hands , fell precipitously with increasing glycan complexity . additionally , glycosylations of nhs and nhpht with other donors , such as glycosyl acetimidates and thioglycosides , were low yielding , even with monosaccharide substrates .
in addition to falling short as glycosyl acceptors , nhs and nhpht were inadequate aminooxy protecting groups . they were labile to basic and reductive conditions , which undermined their use during complex glycan synthesis .
moreover , we envisioned using one - pot multienzyme methods(39 ) to convert simple aminooxy glycans into more complex structures
. however , such reactions would require protection of the aminooxy group to avoid oxime formation with the latent aldehydes or ketones associated with free sugars .
the lability of the nhs and nhpht groups undermined our efforts to deprotect late - stage glycan intermediates without loss of the aminooxy protecting group .
reactions corresponding to entries 17 were conducted in ch2cl2 ( unless specified ) at 0 c , and those corresponding to entries 813 were performed at 20 c .
therefore , we sought to identify an alternative aminooxy protecting group that possessed better chemical stability overall but was readily removable under mild conditions that are compatible with free sugars .
ideally , the nucleophilicity of the masked aminooxy group would be superior to that of the n - hydroxyimides as well , thus enhancing reactivity as a glycosyl acceptor .
after screening a variety of options , we found the n - pentenoyl protecting group to be an excellent candidate .
originally introduced by fraser - reid and co - workers , the pentenoyl amide is stable to many reaction conditions and is removed by gentle treatment with aqueous iodine . despite its potential as a synthetic substrate , n - pentenoyl hydroxamic acid ( 1 , table 1 , also termed n - hydroxypentenamide , which we abbreviate nhpent ) has seldom been utilized in synthesis(43 ) and , to our knowledge , never as a masked aminooxy group . to determine the scope of compound 1 's reactivity as a glycosyl acceptor , we subjected it to glycosylation reactions using donor substrates that included glycosyl bromides and fluorides , thioglycosides , and glycosyl acetimidates ( table 1 ) .
the highest yields were obtained using glycosyl n - phenyl trifluoroacetimidates , as previously observed in the syntheses of glycosyl amides(44 ) and hydroxamates . by contrast , the less stable glycosyl trichloroacetimidates gave low yields in glycosylation reactions with 1 under similar conditions ( data not shown ) .
we anticipated that the mild conditions reported for cleavage of n - pentenoyl amides would be compatible with unprotected glycans . to confirm that such conditions can cleave n - alkoxypentenamides without harm to free sugars , we performed model reactions on the test substrate n - pentenoyl aminooxy lactose ( 15 , prepared from the known disaccharide aminooxy lactose(28 ) as shown in scheme s1 ) .
treatment of n - pentenoyl aminooxy lactose with 3 equiv of i2 in 1:1 tetrahydrofuran ( thf)/h2o as reported(41 ) resulted in iodocyclization of the nhpent group , but hydrolysis of the iodoimidate intermediate to afford the free aminooxy disaccharide was sluggish .
fortunately , the use of nonaqueous solvent mixtures and acid catalysis , as shown previously , alleviated this problem .
we identified the optimal deprotection conditions to be 3 equiv of i2 in wet mecn / meoh with 0.1% formic acid ( fa ) at room temperature , which afforded quantitative deprotection of n - pentenoyl aminooxy lactose in under 1 h. the utility of nhpent glycosides as substrates for complex aminooxy glycan synthesis was demonstrated with the construction of the trisaccharide lewis x ( gal1,4(fuc1,3)glcnac ) , a leukocyte and stem cell marker ( scheme 1 ) .
we began the synthesis with the coupling of known trichloroacetimidate 16 to thioglycoside 17 to generate the lactosamine precursor 18 following related , published routes .
as seen with similarly protected glucosamine derivatives , only glycosylation at the more reactive 4-hydroxyl of 17 was observed .
the fucose residue was introduced in the form of 19 , which was outfitted with a p - methoxybenzyl ( pmb ) ether at the 2-position to enhance cis - glycosylation .
similar to a previous report,(55 ) tfoh - catalyzed coupling of 19 with the free c-3 hydroxyl of 18 proceeded smoothly to afford 20 .
the pmb group was removed with 10% trifluoroacetic acid , thereby avoiding reductive or oxidative conditions that are incompatible with the pentenoyl and thioglycoside groups , and the 2-hydroxyl group of the fucose residue was acetylated to afford 21 .
motivated by the results in table 1 , we converted thioglycoside 21 to the corresponding glycosyl n - phenyl trifluoroacetimidate , 22 , for coupling with compound 1.(46 ) the glycosylation reaction was conducted with 1 equiv of tmsotf in dichloromethane at 20 c to afford n - hydroxypentenoyl trisaccharide 23 .
the troc protecting group was removed with zn in acidic mecn , and the amino group was acetylated .
notably , we observed no reduction of the n  o bond during the zn reduction step , in sharp contrast to our previous experience with nhpth glycosides .
removal of the tbdps and acetyl groups of 24 proceeded in high yield and without damage to the n - hydroxypentenamide as well .
finally , aminooxy lewis x was obtained by treatment of 25 with i2 in acidic mecn / meoh .
enzymatic elaboration of synthetic core structures is an increasingly popular strategy for complex glycan assembly .
enzymes provide tight stereo- and regiochemical control and obviate the need for intermediate protecting group manipulations and harsh chemical conditions .
the impact of enzymatic glycosylation methods is most evident in cases where the chemical counterpart is notoriously difficult , such as in the addition of terminal sialic acid residues to a complex glycan.(57 ) given the biological importance of sialosides,(58 ) we sought to construct sialylated aminooxy glycans as substrates for glycoprotein engineering .
trisaccharides 29 and 30 ( scheme 3 ) , termed aminooxy 2,6- and 2,3-sialyllactose , respectively , were chosen as initial targets with which to evaluate the performance of nhpent glycosides as substrates for enzymatic sialylation .
we used the one - pot , three - enzyme system introduced by chen and co - workers to append sialic acid to n - pentenoyl aminooxy lactose ( 15 , scheme 2 ) .
this methodology allows for the in situ generation of costly cytidine 5-monophosphate ( cmp)-sialic acid from mannac , pyruvate , and cytidine 5-triphosphate ( ctp ) by the combined use of e. coli aldolase and a cmp - sialic acid synthase , nmcss.(61 ) in the same pot , bacterial -2,6-sialyltransferase ( pd2,6st ) or -2,3-sialyltransferase ( pmst1 ) transferred sialic acid to 15 , affording nhpent glycosides 27 and 28 , respectively , in excellent yields .
notably , free aminooxy glycans would not be viable substrates for this procedure , as the aminooxy group would undergo side reactions with free reducing sugars as well as pyruvate . finally , we employed our previously optimized deprotection conditions to obtain trisaccharides 29 and 30 ( scheme 3 ) . a higher level of complexity was embodied in the tetrasaccharide sialyl lewis x ( sia2,3gal1,4(fuc1,3)glcnac , abbreviated sle ) , a glycan known for its involvement in cancer progression and leukocyte homing .
we envisioned generating aminooxy - functionalized sle by chemical synthesis of an nhpent - modified disaccharide core , followed by enzymatic installation of the terminal sialic acid and fucose residues . toward this end ,
compound 18 was transformed into nhpent lactosamine derivative 33 by acetylation of its one free hydroxyl group , conversion of the thioglycoside to the n - phenyl trifluoroacetimidate , and glycosylation with compound 1 ( scheme 4 ) .
deprotection and n - acetylation gave n - acetyllactosamine nhpent glycoside 35 as a substrate for enzymatic glycosylation .
after purification by size exclusion chromatography , the fucose residue was installed using the one - pot , two - enzyme system described by wu and co - workers.(64 ) briefly , guanosine 5-diphosphate ( gdp)-fucose was generated in situ from adenosine triphosphate ( atp ) , guanosine 5-triphosphate ( gtp ) , and fucose by the bacteroides fragilis fucose kinase phosphorylase ( fkp ) .
finally , deprotection of 37 with i2 in acidic mecn / meoh gave aminooxy sle38 in 65% isolated yield . with a route to complex aminooxy glycans established , we sought to combine the synthetic sugars with recombinant aldehyde - tagged proteins as shown schematically in figure 2a .
as a protein substrate , we used a previously reported construct of the human growth hormone ( hgh ) that contains a c - terminal aldehyde tag as well as an n - terminal his tag .
the native protein is a well - established biotherapeutic used in the treatment of human developmental diseases among others .
site - specific chemical modification of hgh is a potential route to improving its pharmacokinetic properties .
indeed , an artificially glycosylated hgh variant was recently shown to have a prolonged serum half - life in mice.(69 ) generation of site - specifically glycosylated hgh .
( a ) aldehyde - tagged hgh was reacted with synthetic aminooxy glycans to yield the oxime - glycosylated conjugates .
( b ) western blot of aldehyde - tagged hgh ( lane 1 ) or the ca mutant ( lane 2 ) after reaction with aminooxy lactose s1 ( 5% mecn , 0.02% fa , 0.26 mg / ml aldehyde - tagged hgh , 1 mm s1 , 16 h , 37 c ) .
the blot was probed with sba - fitc ( top ) , and total protein was detected by ponceau stain ( bottom ) .
( c ) esi - ms spectrum of the crude reaction mixture showing ions in the 20 + and 21 + charge states .
ions corresponding to aldehyde - tagged hgh ( hgh ) and glycosylated hgh ( hgh+lac ) are shown with arrows .
( d ) western blot of aldehyde - tagged hgh ( lane 1 ) or the ca mutant ( lane 2 ) after reaction with aminooxy sle38 ( 50 mm nacit , ph 3.5 , 0.26 mg / ml aldehyde - tagged hgh , 1 mm 38 , 16 h , 37 c ) .
the blot was probed with an anti - slex antibody km93 followed by a horseradish peroxidase ( hrp)-conjugated secondary antibody ( top ) , and then stripped and reprobed with aal - biotin followed by a fitc - conjugated antibiotin antibody ( middle ) .
( e ) esi - ms spectrum of the sle crude reaction mixture showing ions in the 20 + and 21 + charge states .
ions corresponding to aldehyde - tagged hgh ( hgh ) and glycosylated hgh ( hgh+sle ) are shown with arrows .
we produced aldehyde - tagged hgh with > 95% cys - to - fgly conversion in e. coli with coexpression of the fge from mycobacterium tuberculosis.(32 ) the protein was purified by chromatography on ni - nta agarose and then reacted with aminooxy lactose ( s1 ) in 5% aqueous mecn with 0.02% fa ( ph 3.8 ) .
the formation of the lactose glycoconjugate was confirmed by western blot probing with the galactose - binding lectin , soybean agglutinin conjugated to fluorescein isothiocyanate ( sba - fitc ) ( figure 2b ) .
a control conjugation reaction using an hgh construct in which the cys residue required for the aldehyde installation was mutated to ala ( ca ) produced no sba - reactive product .
this result confirmed the necessity of the cys - derived fgly residue for site - specific chemical glycosylation with aminooxy lactose .
further , we analyzed the oxime conjugate by electrospray ionization mass spectrometry ( esi - ms ) ( figure 2c ) .
ions corresponding to glycosylated ( hgh+lac ) and unreacted ( hgh ) aldehyde - tagged hgh were observed , the relative intensities of which indicated a reaction yield of 81% .
the protein glycoconjugate was found to be highly stable when stored at 20 c , and < 10% hydrolysis of the conjugate was observed after 8 days at 4 c ( table s1 ) .
we performed a similar reaction using the more complex glycan aminooxy sle ( 38 ) .
the formation of the glycosylated hgh product was confirmed by western blot probing with a sle - specific antibody as well as with the fucose - binding lectin , aleuria aurantia lectin ( aal ) , conjugated to biotin ( figure 2d ) . using the above conjugation conditions ( 5% mecn , 0.02% fa ) , the yield of glycosylated hgh as determined by esi - ms was a modest 2025% .
however , use of a sodium citrate ( nacit ) buffer at ph 3.5 improved the conjugation efficiency to 64% . despite the acidity of the reaction buffer
, we did not detect any loss of the potentially labile sialic acid and fucose moieties or any damage to the aldehyde - tagged protein .
in summary , the facile generation of homogeneous glycoproteins was made possible by an improved route to aminooxy glycans featuring glycosyl n - pentenoyl hydroxamate intermediates , combined with the genetically encoded aldehyde tag .
the versatility of the aldehyde tag with respect to expression system , protein target , and site of aldehyde placement makes this approach broadly useful for protein glycoengineering . in principle
, multiple aldehyde tags can be introduced into a single protein , allowing for the production of multivalent glycoforms , a work currently in progress .
the method joins a growing toolkit for site - specific modification of proteins with synthetic glycans , which combined could produce myriad protein glycoforms with discrete glycans at different positions .
other chemoselective ligation reactions , such as azide  alkyne cycloadditions and disulfide exchange reactions , have proven useful for modifying a single protein with multiple moieties.(19 ) oxime formation is orthogonal to these and other chemoselective ligation reactions ( thiol  ene additions , olefin metathesis , suzuki couplings , etc . ) . exploring
the tandem and parallel use of these reactions for multiplexed protein modification is a subject of future interest .
a solution of disaccharide 18 ( 400 mg , 0.32 mmol ) in pyridine / ac2o ( 2:1 , v : v ; 4.5 ml ) was stirred for 3 h at room temperature under n2 . upon completion ,
the crude residue was purified by silica flash chromatography ( hex / etoac gradient ) to obtain fully protected disaccharide 31 as a white powder ( 400 mg , 97% ) : h nmr ( 500 mhz , cdcl3 )  8.18 ( d , j = 7.4 hz , 2h ) , 8.07 ( d , j = 7.4 hz , 2h ) , 7.937.79 ( m , 6h ) , 7.687.49 ( m , 12h ) , 7.477.34 ( m , 5h ) , 7.337.19 ( m , 5h ) , 7.09 ( t , j = 7.5 hz , 2h ) , 7.03 ( t , j = 7.3 hz , 1h ) , 6.85 ( t , j = 7.7 hz , 2h ) , 6.69 ( d , j = 9.0 hz , 1h ) , 6.02 ( d , j = 2.9 hz , 1h ) , 5.81 ( dd , j = 10.1 , 8.2 hz , 1h ) , 5.58 ( dd , j = 10.4 , 3.1 hz , 1h ) , 5.50 ( t , j = 9.8 hz , 1h ) , 5.27 ( d , j = 8.0 hz , 1h ) , 5.11 ( d , j = 10.4 hz , 1h ) , 4.90 ( q , j = 12.1 hz , 2h ) , 4.74 ( dd , j = 11.2 , 4.6 hz , 1h ) , 4.554.39 ( m , 2h ) , 4.304.23 ( m , 1h ) , 4.09 ( q , j = 10.2 hz , 1h ) , 3.95 ( d , j = 11.2 hz , 1h ) , 3.77 ( d , j = 11.0 hz , 1h ) , 3.40 ( d , j = 9.7 hz , 1h ) , 2.24 ( s , 3h ) , 1.09 ( s , 9h ) ; c nmr ( 126 mhz , cdcl3 )  171.54 , 166.00 , 165.56 , 165.32 , 164.45 , 154.59 , 136.05 , 135.67 , 135.29 , 133.70 , 133.52 , 133.33 , 133.17 , 132.91 , 131.69 , 130.86 , 130.25 , 129.84 , 129.77 , 129.74 , 129.51 , 129.13 , 128.77 , 128.69 , 128.41 , 128.29 , 128.22 , 128.16 , 127.78 , 126.74 , 100.10 , 95.78 , 85.76 , 78.61 , 77.41 , 74.52 , 74.18 , 73.61 , 72.02 , 71.43 , 69.90 , 68.22 , 61.95 , 60.96 , 54.87 , 26.76 , 21.24 , 19.35 ; hrms ( esi ) calcd for c67h65no16cl3ssi [ m+h]m / z = 1304.2853 , found 1304.2883 .
thioglycoside 31 ( 290 mg , 0.22 mmol ) was dissolved in acetone / ch2cl2/h2o ( 15:1:1 , v : v ; 6 ml ) , and n - iodosuccinimide ( nis ) ( 75 mg , 0.33 mmol ) was added while stirring at room temperature .
the reaction was monitored by thin - layer chromatography ( tlc ) ( 2:1 , hex / etoac ) , and n - bromosuccinimide was added in 2 equiv portions until the reaction was complete . upon full hydrolysis
, the resulting hemiacetal was diluted in ch2cl2 and washed sequentially with na2s2o3 solution , nahco3 solution , and brine .
the resulting residue was dissolved in ch2cl2 ( 3 ml ) and cs2co3 ( 150 mg , 0.44 mmol ) was added , followed by n - phenyl trifluoroacetimidoyl chloride ( 72 l , 0.44 mmol ) dropwise .
the reaction mixture was stirred for 12 h at room temperature under n2 , after which it was diluted with ch2cl2 , filtered , and concentrated under vacuum .
the crude residue was purified by silica chromatography to give the trifluoroacetimidate 32 as a yellow oil ( 267 mg , 87% ) : h nmr ( 500 mhz , cdcl3 )  8.168.03 ( m , 4h ) , 7.877.68 ( m , 7h ) , 7.687.31 ( m , 14h ) , 7.19 ( dddd , j = 28.0 , 16.4 , 10.1 , 3.9 hz , 7h ) , 6.98 ( dt , j = 30.6 , 15.6 hz , 1h ) , 6.71 ( dd , j = 23.5 , 7.7 hz , 1h ) , 6.005.92 ( m , 1h ) , 5.835.73 ( m , 1h ) , 5.605.40 ( m , 1h ) , 5.33 ( t , j = 9.9 hz , 1h ) , 5.25 ( dd , j = 22.4 , 14.4 hz , 1h ) , 5.18 ( dt , j = 19.0 , 9.4 hz , 1h ) , 4.76 ( dt , j = 28.6 , 14.3 hz , 1h ) , 4.634.51 ( m , 2h ) , 4.474.35 ( m , 1h ) , 4.294.17 ( m , 1h ) , 4.13 ( dt , j = 13.7 , 7.1 hz , 1h ) , 4.053.69 ( m , 2h ) , 3.583.26 ( m , 1h ) ,
nmr ( 126 mhz , cdcl3 )  173.12 , 166.12 , 165.51 , 165.50 , 164.72 , 154.46 , 136.03 , 135.47 , 133.88 , 133.84 , 133.62 , 133.45 , 133.35 , 130.54 , 130.02 , 129.92 , 129.88 , 129.70 , 129.58 , 129.17 , 129.03 , 128.87 , 128.73 , 128.55 , 128.44 , 128.36 , 127.96 , 100.52 , 95.48 , 74.75 , 73.37 , 73.24 , 72.20 , 71.66 , 69.90 , 68.23 , 62.14 , 60.53 , 54.23 , 26.90 , 20.97 , 19.50 .
a mixture of trifluoroacetimidate 32 ( 257 mg , 0.186 mmol ) and n - pentenoyl hydroxamic acid 1 ( 32 mg , 0.28 mmol ) was dried by coevaporation with anhydrous toluene and left under high vacuum . to the dried mixture was added 3  ms with stirring in ch2cl2 ( 4 ml ) for 1 h at room temperature under n2 .
the solution was cooled to 20 c , and tmsotf ( 34 l , 0.19 mmol ) was added dropwise .
the reaction was allowed to warm to room temperature over 1.5 h and was quenched with triethylamine , filtered , and concentrated under vacuum .
the resulting residue was purified by silica flash chromatography ( toluene / acetone gradient ) to obtain n - hydroxypentenoyl disaccharide 33 as a white solid ( 160 mg , 66% ) : h nmr ( 500 mhz , cdcl3 )  8.45 ( s , 1h ) , 8.158.02 ( m , 4h ) , 7.837.74 ( m , 4h ) , 7.747.65 ( m , 4h ) , 7.60 ( dt , j = 21.6 , 6.4 hz , 2h ) , 7.547.36 ( m , 12h ) , 7.257.20 ( m , 2h ) , 7.207.16 ( m , 2h ) , 5.95 ( d , j = 3.2 hz , 1h ) , 5.825.69 ( m , 2h ) , 5.695.61 ( m , 1h ) , 5.48 ( dd , j = 10.3 , 3.2 hz , 1h ) , 5.20 ( t , j = 9.3 hz , 1h ) , 5.12 ( d , j = 8.0 hz , 1h ) , 5.01 ( d , j = 16.5 hz , 1h ) , 4.96 ( d , j = 10.3 hz , 1h ) , 4.86 ( d , j = 12.0 hz , 1h ) , 4.68 ( t , j = 9.1 hz , 2h ) , 4.53 ( d , j = 6.4 hz , 2h ) , 4.34 ( t , j = 9.3 hz , 1h ) , 4.17 ( t , j = 6.0 hz , 1h ) , 3.943.77 ( m , 3h ) , 3.27 ( d , j = 9.4 hz , 1h ) , 2.352.15 ( m , 4h ) , 2.12 ( s , 3h ) , 1.08 ( s , 9h ) ; c nmr ( 126 mhz , cdcl3 )  171.32 , 170.85 , 166.00 , 165.51 , 165.43 , 165.21 , 164.72 , 136.58 , 136.08 , 136.06 , 136.02 , 135.95 , 135.48 , 135.44 , 133.81 , 133.51 , 133.46 , 133.43 , 133.29 , 130.54 , 130.15 , 130.09 , 130.04 , 129.98 , 129.93 , 129.85 , 129.65 , 129.49 , 128.91 , 128.83 , 128.68 , 128.64 , 128.60 , 128.45 , 128.41 , 128.32 , 128.11 , 127.85 , 127.77 , 115.81 , 100.22 , 95.41 , 75.63 , 74.87 , 74.71 , 73.14 , 71.91 , 71.50 , 69.83 , 68.08 , 62.01 , 60.64 , 54.00 , 53.53 , 28.98 , 26.86 , 20.95 , 19.42 ; hrms ( esi ) calcd for c66h67n2o18cl3si [ m+h]m / z = 1309.3296 , found 1309.3315 . to a solution of n - hydroxypentenoyl disaccharide 33 ( 140 mg , 0.11 mmol ) in 10%
fa / mecn ( 3 ml ) was added activated zn ( 500 mg ) , and the mixture was stirred for 3 h. the suspension was filtered to remove catalyst , concentrated under vacuum , and coevaporated with toluene to remove excess fa . the crude free amine was dissolved in anhydrous meoh ( 3 ml ) , followed by dropwise addition of ac2o ( 55 l , 0.54 mmol ) and n , n - diisopropylethylamine ( dipea ) ( 22 l , 0.13 mmol ) at 0 c under n2 .
after 1.5 h , the reaction was allowed to warm to room temperature , diluted with meoh , and concentrated under vacuum .
the resulting residue was purified by silica flash chromatography ( toluene / acetone gradient ) to give the n - acetylated disaccharide 34 as a white solid ( 97 mg , 77% ) : h nmr ( 500 mhz , cdcl3 )  9.32 ( s , 1h ) , 8.138.03 ( m , 4h ) , 7.827.73 ( m , 4h ) , 7.727.67 ( m , 2h ) , 7.667.54 ( m , 6h ) , 7.527.45 ( m , 8h ) , 7.417.37 ( m , 2h ) , 7.23 ( t , j = 7.8 hz , 3h ) , 7.17 ( t , j = 7.8 hz , 2h ) , 6.19 ( d , j = 8.4 hz , 1h ) , 5.96 ( d , j = 3.1 hz , 1h ) , 5.825.69 ( m , 2h ) , 5.48 ( dd , j = 10.4 , 3.4 hz , 1h ) , 5.11 ( dd , j = 17.2 , 7.9 hz , 2h ) , 5.00 ( dd , j = 17.2 , 1.3 hz , 1h ) , 4.94 ( d , j = 10.2 hz , 1h ) , 4.60 ( d , j = 8.5 hz , 1h ) , 4.584.47 ( m , 2h ) , 4.36 ( t , j = 9.1 hz , 1h ) , 4.204.10 ( m , 2h ) , 3.88 ( q , j = 11.2 hz , 2h ) , 3.26 ( d , j = 9.4 hz , 1h ) , 2.382.15 ( m , 4h ) , 2.14 ( s , 3h ) , 2.02 ( s , 3h ) , 1.06 ( s , 9h ) ; c nmr ( 126 mhz , cdcl3 )  172.53 , 171.81 , 170.88 , 166.04 , 165.46 , 164.88 , 137.93 , 136.75 , 136.06 , 135.74 , 135.43 , 133.81 , 133.56 , 133.43 , 133.40 , 131.23 , 130.58 , 130.15 , 129.94 , 129.89 , 129.85 , 129.54 , 129.11 , 128.98 , 128.82 , 128.73 , 128.65 , 128.52 , 128.41 , 128.30 , 128.26 , 127.78 , 125.38 , 115.62 , 102.03 , 100.27 , 75.89 , 72.99 , 72.35 , 71.86 , 71.59 , 69.83 , 68.17 , 62.07 , 60.56 , 52.23 , 32.87 , 29.04 , 26.82 , 23.35 , 21.53 , 21.03 , 19.41 ; hrms ( esi ) calcd for c65h68n2o17si [ m+h]m / z = 1177.4360 , found 1177.4396 . a solution of n - hydroxypentenoyl disaccharide 34 ( 94 mg , 0.080 mmol ) in thf ( 4 ml ) was cooled to 0 c , and tetrabutylammonium fluoride ( 1 m in thf , 160 l ) was added dropwise under n2 .
the mixture was allowed to warm to room temperature and stirred for 18 h , after which it was diluted with etoac and concentrated under reduced pressure .
the resulting residue was dissolved in meoh ( 4 ml ) , and naome ( 25% in meoh , 40 l ) was added dropwise with stirring at room temperature . after 8 h
, the reaction was neutralized with dowex 50w - x8 ( h ) and filtered to remove resin .
the crude product was concentrated under vacuum and purified by silica column chromatography ( 1025% meoh / ch2cl2 ) .
the purified residue was filtered to remove trace silica and lyophilized to yield the deprotected n - hydroxypentenoyl lacnac 35 as a white powder ( 32 mg , 84% ) : h nmr ( 500 mhz , d2o )  5.80 ( ddt , j = 12.8 , 10.3 , 6.5 hz , 1h ) , 5.104.98 ( m , 2h ) , 4.75 ( d , j = 8.8 hz , 1h ) , 4.44 ( d , j = 7.8 hz , 1h ) , 3.983.92 ( m , 1h ) , 3.923.80 ( m , 3h ) , 3.773.66 ( m , 5h ) , 3.63 ( dd , j = 10.0 , 3.4 hz , 1h ) , 3.613.54 ( m , 1h ) , 3.50 ( dd , j = 9.8 , 7.9 hz , 1h ) , 2.32 ( dd , j = 13.1 , 6.5 hz , 2h ) , 2.282.21 ( m , 2h ) , 2.02 ( s , 3h ) ; c nmr ( 126 mhz , cdcl3 )  174.72 , 172.85 , 136.46 , 115.89 , 103.79 , 102.82 , 77.78 , 75.33 , 75.06 , 72.45 , 72.22 , 70.91 , 68.51 , 61.00 , 59.77 , 52.90 , 31.72 , 28.81 , 22.18 ; hrms ( esi ) calcd for c19h32n2o12 [ m+h]m / z = 481.2028 , found 481.2036 . to n - pentenoyl aminooxy lacnac 35 ( 21 mg , 0.044 mmol ) were added n - acetylmannosamine ( 15 mg , 0.066 mmol ) , sodium pyruvate ( 24 mg , 0.22 mmol ) , and ctpna ( 37 mg , 0.066 mmol ) , and they were dissolved in h2o ( 3 ml ) .
a concentrated stock of tris - hcl buffer ph 8.5 with mgcl2 was added to a final concentration of 100 mm tris , 20 mm mgcl2 .
recombinant e. coli k12 sialic acid aldolase ( 4 u ) , neusserua meningitidis cmp - sialic acid synthetase ( 4 u ) , and pasteurella multocida -2,3-sialyltransferase ( 2 u ) were added , followed by h2o , to bring the volume to 4 ml .
the reaction mixture was incubated at 37 c for 2 h , followed by shaking at room temperature for 14 h. the reaction was monitored by tlc ( 4:2:1 etoac / meoh / h2o ) , and upon completion , calf alkaline phosphatase was added to remove remaining nucleotide phosphate . after further incubation at 37 c for 2 h ,
the reaction mixture was quenched with cold meoh ( 4 ml ) and incubated on ice for 20 min .
the mixture was centrifuged , precipitates were removed , and the solution concentrated under vacuum .
the resulting residue was passed through a biogel p-2 size exclusion column and eluted with ddh2o to obtain 36 ( 28.9 mg , 86% ) as a white , fluffy powder after lyophilization : h nmr ( 600 mhz , d2o )  5.80 ( ddt , j = 16.9 , 10.3 , 6.6 hz , 1h ) , 5.094.99 ( m , 2h ) , 4.74 ( d , j = 8.8 hz , 1h ) , 4.52 ( d , j = 7.9 hz , 1h ) , 4.08 ( dd , j = 9.9 , 3.1 hz , 1h ) , 3.96 ( app d , j = 10.7 hz , 1h ) , 3.92 ( d , j = 3.0 hz , 1h ) , 3.883.79 ( m , 5h ) , 3.763.70 ( m , 3h ) , 3.693.64 ( m , 3h ) , 3.623.52 ( m , 5h ) , 2.73 ( dd , j = 12.4 , 4.6 hz , 1h ) , 2.31 ( dd , j = 13.5 , 6.7 hz , 2h ) , 2.24 ( app t , j = 7.0 hz , 2h ) , 2.02 ( s , 3h ) , 2.00 ( s , 3h ) , 1.77 ( t , j = 12.1 hz , 1h ) ; c nmr ( 151 mhz , d2o )  175.00 , 174.73 , 173.83 , 172.51 , 136.54 , 115.87 , 103.81 , 102.54 , 99.79 , 77.73 , 75.46 , 75.16 , 75.08 , 72.87 , 72.20 , 71.74 , 69.34 , 68.31 , 68.08 , 67.45 , 62.57 , 61.01 , 59.81 , 59.42 , 52.91 , 51.67 , 39.62 , 31.73 , 28.81 , 22.19 , 22.01 ; hrms ( esi ) calcd for c30h49n3o20 [ m 
z = 770.2837 , found 770.2812 . to a 5 ml solution of 100 mm tris buffer ( ph 7.5 ) containing 5 mm l - fucose ( 0.025 mmol ) , 5 mm mgso4 ( 0.025 mmol ) , 10 mm gtpna ( 0.05 mmol ) , and 10 mm atpna ( 0.05 mmol ) were added saccharomyces cerevisiae inorganic pyrophosphatase ( 40 u ) and b. fragilis fkp ( 4 u ) .
the mixture was incubated at 37 c for 2 h to preform gdp - fucose and monitored by tlc ( 2:1:1 iproh / acoh / h2o ) . to this
was added n - pentenoyl aminooxy sialoside 36 ( 5 mg , 0.07 mmol ) and h. pylori -1,3 fucosyltransferase ( 1 u ) , followed by incubation at room temperature for 16 h. the reaction was monitored by tlc ( 4:2:1 etoac / meoh / h2o ) , and upon completion , calf alkaline phosphatase was added to remove remaining nucleotide phosphate . after further incubation at 37 c for 2 h ,
the reaction mixture was quenched with cold meoh ( 5 ml ) and incubated on ice for 20 min .
the mixture was centrifuged , precipitates were removed , and the solution was concentrated under vacuum .
the resulting residue was passed through a biogel p-2 size exclusion column and eluted with ddh2o to obtain 37 ( 4.6 mg , 78% ) as a white , fluffy powder after lyophilization : h nmr ( 600 mhz , d2o )  5.885.77 ( m , 1h ) , 5.06 ( dd , j = 26.6 , 14.1 hz , 3h ) , 4.81 ( app s , 1h ) , 4.51 ( d , j = 7.7 hz , 1h ) , 4.124.01 ( m , 2h ) , 3.97 ( dd , j = 20.8 , 10.7 hz , 2h ) , 3.923.82 ( m , 7h ) , 3.75 ( d , j = 12.5 hz , 1h ) , 3.703.55 ( m , 10h ) , 3.51 ( t , j = 8.7 hz , 1h ) , 2.792.70 ( m , 1h ) , 2.372.30 ( m , 2h ) , 2.302.22 ( m , 2h ) , 2.02 ( d , j = 4.1 hz , 6h ) , 1.78 ( t , j = 12.2 hz , 1h ) , 1.15 ( d , j = 6.1 hz , 3h ) ; c nmr ( 151 mhz , d2o )  175.04 , 174.51 , 173.79 , 172.53 , 136.55 , 115.88 , 103.70 , 101.62 , 99.64 , 98.61 , 75.65 , 75.57 , 74.91 , 74.58 , 72.92 , 71.89 , 71.83 , 69.24 , 69.19 , 68.26 , 68.12 , 67.70 , 67.30 , 66.70 , 62.62 , 61.44 , 59.52 , 59.33 , 53.56 , 51.70 , 39.78 , 31.73 , 28.80 , 22.29 , 22.03 , 15.25 ; hrms ( esi ) calcd for c36h59n3o24 [ m 
a solution of n - hydroxypentenoyl sle37 ( 4.2 mg , 0.0046 mmol ) in mecn / meoh / fa ( 3:1:0.001 , v : v ; 1.6 ml ) was stirred at room temperature with dropwise addition of i2 ( 0.014 mmol , 0.5 m solution in thf ) .
the mixture was heated to 35 c and stirred for 2 h , after which the reaction was quenched with the addition of aqueous nh4hco3 ( 500 mm ) and na2s2o3 ( 50 mm ) until the disappearance of color .
the solvent was removed under reduced pressure , and the remaining residue was purified by silica flash chromatography ( 4:2:1 etoac / meoh / h2o ) .
the purified product was dissolved in ddh2o and lyophilized to give the free aminooxy sle38 ( 2.5 mg , 65% ) as a white powder : h nmr ( 600 mhz , d2o )  5.11 ( d , j = 4.0 hz , 1h ) , 4.72 ( d , j = 8.7 hz , 1h ) , 4.52 ( d , j = 7.8 hz , 1h ) , 4.09 ( dd , j = 9.9 , 3.0 hz , 1h ) , 4.04 ( dd , j = 14.8 , 4.7 hz , 1h ) , 3.98 ( dd , j = 18.7 , 9.6 hz , 2h ) , 3.943.92 ( m , 1h ) , 3.923.87 ( m , 5h ) , 3.86 ( dd , j = 13.6 , 6.3 hz , 2h ) , 3.78 ( app d , j = 2.8 hz , 1h ) , 3.703.63 ( m , 8h ) , 3.613.56 ( m , 3h ) , 3.553.51 ( m , 1h ) , 2.76 ( dd , j = 12.4 , 4.6 hz , 1h ) , 2.03 ( s , 6h ) , 1.80 ( t , j = 12.2 hz , 1h ) , 1.17 ( d , j = 6.6 hz , 3h ) ; c nmr ( 151 mhz , d2o )  175.06 , 174.51 , 173.78 , 103.04 , 101.65 , 99.95 , 99.66 , 98.61 , 75.68 , 75.37 , 74.92 , 74.71 , 73.18 , 72.95 , 71.90 , 71.84 , 69.27 , 69.20 , 68.27 , 68.15 , 67.72 , 67.32 , 66.72 , 62.65 , 61.46 , 59.69 , 53.94 , 51.72 , 43.35 , 39.79 , 22.18 , 22.05 , 15.27 ; hrms ( esi ) calcd for c31h52n3o23 [ m 
to a solution of 5% mecn , 0.02% fa , or 50 mm nacit , ph 3.5 , was added 0.26 mg / ml aldehyde - tagged protein and 1 mm aminooxy sugar .
the reaction was incubated at 37 c for 18 h , at which point the reaction was quenched with 4 protein loading dye and resolved by sds - page .
the protein was transferred to nitrocellulose and probed with anti - sialyl lewis x igm ( km93 , chemicon , 1:500 dilution ) followed by anti - mouse igm - hrp ( southern biotech , 1:10000 ) in 5% dry milk in phosphate - buffered saline/0.1% tween ( pbst ) .
the presence of galactose was probed with sba - fitc ( ey , 1:1000 dilution ) in 3% bovine serum albumin ( bsa)/pbst . for fucose analysis ,
the nitrocellulose was probed with aal - biotin ( ey labs , 1:1000 dilution ) followed by rabbit anti - biotin fitc ( abcam , 1:1000 ) in 3% bsa / pbst .
membranes were developed by chemiluminescence using the supersignal west pico kit ( thermo ) or scanned for fluorescence by a typhoon 9410 imaging system ( amersham ) . for mass spectrometry analysis ,
the protein was reacted in 5% mecn , 0.02% fa , or nacit at 1 mg / ml with 2 mm aminooxy sugar for 18 h at 37 c .
the reaction was quenched with 1 m tris buffer ph 8 , and the crude protein was purified by reversed - phase high - performance liquid chromatography ( plrp - s , polymer laboratories ) with elution in aqueous mecn . the multicharged peaks of the full length protein were obtained by esi - ms ( bruker / agilent esquire ) and identified by mass spectrometry analysis software . for stability analysis , the purified glycoconjugate was diluted to 10 m in pbs at ph 7.2 and stored at 20 or 4 c .
aliquots were taken at various time points and analyzed by matrix - assisted laser desorption / ionization mass spectrometry to determine the remaining glycoconjugate .
 homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development . 
 the use of unnatural protein  glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins . here 
 we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages . 
 we employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde - bearing formylglycine residue at a specific site . 
 complex aminooxy glycans were synthesized using a new route that features n - pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically . 
 we demonstrated the method by constructing site - specifically glycosylated variants of the human growth hormone . 
Introduction
Results and Discussion
Conclusion
Experimental Section