there are three common 
 types of protein glycosylation that modify 
 large numbers of protein substrates in mammalian cells .
proteins localized 
 to the secretory pathway and the cell surface or secreted into the 
 extracellular space can be modified by oligosaccharide structures , 
 such as n - linked glycosylation ( linked through asparagine ) 
 or mucin o - linked glycosylation ( linked through serine 
 and threonine ) .
additionally , cytoplasmic , nuclear , and mitochondrial 
 proteins can be substrates for the addition of the single monosaccharide n - acetyl - glucosamine , termed o - glcnac modification 
 ( o - glcnacylation , linked through serine and threonine ) .
it is added to protein substrates by one of three isoforms 
 of o - glcnac transferase ( ogt ) and removed by two 
 isoforms of o - glcnacase ( oga ) .
the expression of these enzymes is also required for embryonic 
 development in mice and drosophila.(68)o - glcnac modification displays significant crosstalk 
 with other posttranslational modifications ( ptms ) , most significantly 
 phosphorylation and ubiquitination , setting up o - glcnacylation 
 as a key regulator of cellular pathways . a wide variety of proteins
have been shown to be o - glcnac modified , including regulators of transcription and translation , 
 cytoskeletal proteins , signaling proteins , and metabolic enzymes . 

the specific consequences of most of these modifications are unknown ; 
 however , limited biochemical analyses demonstrate that o - glcnac modification can change protein localization , stability , 
 molecular interactions , and activity .
critically , o - glcnacylation is also misregulated in alzheimer s disease 
 and cancer .
for example , in neurodegenerative disorders such as alzheimer s 
 disease , o - glcnacylation levels are diminished directly 
 leading to protein aggregation and cell death , and we have demonstrated that it likely plays a similar 
 role in parkinson s disease
. finally , 
 higher levels of o - glcnac modification are a common 
 feature of many cancers and are necessary for tumorigenesis and proliferation . to identify and characterize o - glcnac modifications , 
 complementary chemical methods have been developed .
in general , these technologies take advantage of bioorthogonal chemistries , 
 such as the copper(i)-catalyzed azide  alkyne cycloaddition 
 ( cuaac or 
this reaction relies upon small , abiotic chemical reporters ( azides 
 and alkynes ) that can be selectively reacted with alkyne- and azido - probes , 
 respectively , for the installation of visualization and affinity tags . 

one of these methods , initiated by the bertozzi laboratory , takes 
 advantage of monosaccharide analogues that directly incorporate azides 
 or alkynes into their structures .
these 
 analogues , termed metabolic chemical reporters ( mcrs ) , are taken up by cells through carbohydrate salvage 
 pathways and subsequently feed into the biosynthesis of nucleotide 
 sugar - donors for use by glycosyltransferases .
for example , the first o - glcnac - targeted mcr , n - azidoacetyl - glucosamine 
 ( glcnaz , figure 1b ) , has been used for the 
 visualization and proteomic identification of labeled proteins . unlike other methods ( e.g. , western blotting ) , mcrs do not necessarily 
 read - out on endogenous levels of o - glcnacylation , 
 as they must compete with glcnac in the cell . however , because they 
 must be metabolically transformed before their incorporation onto 
 proteins , they not only report on o - glcnac modification 
 but also on the integration of upstream metabolic pathways . additionally , 
 they can be used much like radioactivity to isolate new modification 
 events and subsequent rates of removal in pulse and pulse - chase labeling 
 experiments . despite the clear utility of this technology , the previous 
 iterations have limitations .
until recently , glcnaz and other mcrs 
 were presumed to label only one type of glycosylation ( i.e. , glcnaz 
 treatment results in o - glcnacylation labeling ) , but 
 several enzymatic pathways exist that can interconvert different monosaccharides , 
 raising the possibility that mcrs are converted in the same manner . upon careful characterization , it was demonstrated 
 that glcnaz can be readily transformed to n - azidoacetyl - galactosamine 
 ( galnaz , figure 1b ) and vice versa , resulting 
 in the labeling of both o - glcnacylated and mucin o - linked glycosylated proteins .
furthermore , we showed that glcnaz 
 treatment leads to labeling of n - linked glycosylation .
this can be overcome using cellular fractionation ; however , since we have complete chemical control 
 over the mcr , we predicted that structural alterations can limit this 
  off - target  labeling and produce an o - glcnacylation - specific reporter .
indeed , we previously demonstrated 
 that an alternative mcr , n - pentynyl - glucosamine ( glcnalk ) , 
 which contains a larger functional group at the n - acetyl position , is not converted to the galactosamine derivative 
 and therefore could not label mucin o - linked glycoproteins .
unfortunately , glcnalk was still incorporated 
 into n - linked glycans , preventing its use as a completely 
 selective o - glcnacylation reporter .
we report here the development 
 and application of 6-azido-6-deoxy - n - acetyl - glucosamine 
 ( 6azglcnac , figure 1b and scheme s1 and figure s7 in supporting information 
 ( si ) ) as a mcr in living cells .
cellular analysis of this mcr 
 using cuaac and fluorescent probes demonstrated that , unlike previous 
 reporters , it is highly selective for o - glcnacylated 
 proteins , allowing for the robust visualization of o - glcnac modifications using in - gel fluorescence scanning .
furthermore , 
 comparative proteomics using 6azglcnac , glcnaz , and galnaz confirmed 
 the specificity of 6azglcnac toward o - glcnac modifications . 

6azglcnac - labeling resulted in the enrichment of zero proteins , out 
 of 367 , which are annotated to have exclusively extracellular or lumenal 
 localization .
in contrast , glcnaz and galnaz identified 9 and 72 such 
 proteins , respectively .
finally , we also demonstrate that 6azglcnac 
 can bypass an assumed biosynthetic roadblock by being phosphorylated 
 by the enzyme phosphoacetylglucosamine mutase .
our previous data using mcrs demonstrated that even 
 small alterations in chemical structure can have dramatic effects 
 on the distribution of chemical reporters into different types of 
 glycosylation .
therefore , to find a specific 
 mcr of o - glcnacylation , we synthesized a small panel 
 of o - acetylated n - acetyl - glucosamine 
 analogues bearing azides at different positions ; the acetate protecting - groups 
 allow diffusion across the cell membrane and are subsequently removed 
 by endogenous lipases / hydrolases .
nih3t3 cells were treated with these 
 compounds at 200 m concentrations for 16 h , followed by lysis , 
 cuaac with an alkyne - containing rhodamine dye ( alk - rho ) , and analysis 
 by in - gel fluorescent scanning .
one of these compounds , ac36azglcnac ( figure 1b ) , gave a protein - labeling 
 pattern that was subjectively similar in both intensity and pattern 
 to ac4glcnaz ( figure 2a ) . to further characterize this mcr ,
nih3t3 cells were treated with 
 various concentrations of ac36azglcnac or ac4glcnaz for 16 h before reaction with alk - rho . in - gel
fluorescence 
 scanning revealed labeling of a wide - range of proteins in concentrations 
 as low as 50 m and maximal labeling achieved at approximately 
 200 m ( figure 2b ) , consistent with our 
 other mcrs of glycosylation . to examine the toxicity of ac36azglcnac , the viability 
 of nih3t3 cells was measured after treatment with 200 m ac36azglcnac for 16 or 72 h using a mts cell - proliferation assay . 

only minimal loss of cell growth / survival was seen even after 72 h 
 of treatment ( figure 2c ) . to determine if 6azglcnac 
 could report on o - glcnac modifications , we treated 
 nih3t3 cells with ac36azglcnac ( 200 m ) for 16 h. 
 the cells were then lysed and reacted with an alkyne - containing cleavable 
 affinity tag ( alk - azo - biotin , scheme s2 and figures 
 s8s12 ( si ) ) using cuaac .
enriched 
 proteins were then subjected to western blotting using antibodies 
 against the known o - glcnac modified proteins nedd4 , pyruvate kinase , and nucleoporin 62 
 ( nup62 ) .
all three proteins were selectively 
 enriched using 6azglcnac ( figure 2d and figure s1 ( si ) ) , showing that the mcr does label 
 known o - glcnacylated proteins .
( a ) nih3t3 
 cells were treated with ac4glcnaz ( 200 m ) , ac36azglcnac ( 200 m ) , or dmso vehicle for 16 h , followed 
 by cuaac and analysis by in - gel fluorescence scanning .
( b ) nih3t3 
 cells were treated with varying concentrations of ac4glcnaz 
 or ac36azglcnac for 16 h , followed by cuaac and analysis 
 by in - gel fluorescence scanning .
( c ) nih3t3 cells were treated with 
 ac36azglcnac ( 200 m ) , or dmso vehicle for the times 
 indicated and were tested for toxicity using an mts assay .
( d ) proteins 
 modified by 6azglcnac were enriched from nih3t3 cells treated with 
 ac36azglcnac ( 200 m ) or dmso vehicle using cuaac 
 with alkyne - azo - biotin and analyzed by western blotting .
mcrs are enzymatically transformed 
 into their nucleotide sugar - donors 
 by monosaccharide salvage pathways .
previous o - glcnac 
 mcrs are thought to largely utilize the glcnac salvage pathway ( figure s2a ( si ) ) .
the first step of this pathway is the phosphorylation of mcrs at 
 the 6-position of the carbohydrate ring by n - acetylglucosamine 
 kinase ( gnk ) .
this is followed by enzymatic mutation of the phosphate 
 to the 1-position and conversion to the uridine - diphosphate ( udp ) 
 sugar donor by n - acetylglucosamine - phosphate mutase 
 ( agm1 ) and uridine - diphosphate - n - acetylglucosamine 
 pyrophosphorylase ( agx1/2 ) , respectively .
although udp-6azglcnac is 
 known to be accepted by ogt , 6azglcnac 
 can not be phosphorylated at the 6-position , as we have replaced the 
 6-hydroxyl functionality with an azide .
therefore , we first took a 
 candidate - based approach to identify a kinase that could directly 
 phosphorylated 6azglcnac at the 1-position and chose n - acetyl - galactosamine kinase ( galk2 ) , which performs this reaction 
 on n - acetylgalactosamine ( galnac ) and poorly on glcnac . to test this possibility ,
nih3t3 cells were 
 stably transformed with five different short - hairpin rna vectors targeting 
 galk2 using retroviral infection and then treated with ac36azglcnac ( 200 m ) for 16 h. subsequent cuaac with alk - rho 
 and in - gel fluorescent scanning showed no loss of fluorescent signal , 
 despite a clear reduction of galk2 mrna as measured by semiquantitative 
 rt - pcr ( figure s3 ( si ) ) , suggesting that 
 galk2 is not the enzyme responsible for 6azglcnac metabolism . to confirm 
 this result , we subjected 6azglcnac ( scheme s3a 
 and figures s13s15 ( si ) for details of synthesis and 
 characterization ) to in vitro phosphorylation by recombinant galk2 .
specifically , galk2 was incubated with 40 mm 
 concentrations of galnac , glcnac , or 6azglcnac and [ p]atp 
 ( 5 mm ) . at these elevated substrate - concentrations ,
galk2 readily 
 phosphorylated both galnac and glcnac but gave no detectable modification 
 of 6azglcnac ( figure 3a ) .
next , we tested whether 
 phosphoacetylglucosamine mutase ( agm1 ) could directly generate 6azglcnac-1-phosphate . 

agm1 typically converts glcnac-6-phosphate to glcnac-1-phosphate during 
 the biosynthesis of udp - glcnac . as part of its enzymatic cycle ,
agm1 
 removes the 6-phosphate from substrate sugars , resulting in a phosphoenzyme 
 intermediate ( figure 3b ) . therefore ,
once loaded , phosphorylated agm1 might be capable 
 of phosphorylating 6azglcnac . to test this possibility ,
the enzyme was then incubated 
 with 6azglcnac ( 2.25 mm ) with or without different  cofactors  
 that could generate phosphorylated agm1 , specifically glucose-6-phosphate 
 or glucose-1,6-bisphosphate or glcnac-6-phosphate ( all at 1 mm ) .
to 
 isolate any 6azglcnac-1-phosphate that had been produced , the enzymatic 
 reactions were first subjected to copper - free click chemistry with 
 a fluorescein - conjugated cyclooctyne tag .
fluorescein - labeled compounds 
 ( i.e. , 6azglcnac and 6azglcnac-1-phosphate ) were then separated from 
 the phosphorylated - cofactors by paper chromatography . finally , any 
 fluorescent - spots were eluted and analyzed by mass spectrometry ( lc  ms , figure s4 ( si ) ) .
incubation of agm1 with 6azglcnac 
 alone , or with glucose-6-phosphate or glucose-1,6-bisphosphate , resulted 
 in no detectable formation of 6azglcnac-1-phosphate .
however , in the 
 presence of glcnac-6-phosphate as a cofactor , the formation of 6azglcnac-1-phosphate 
 was unambiguously detected .
this demonstrates that direct phosphorylation 
 of 6azglcnac by agm1 represents one pathway that circumvents the gnk 
 biosynthetic - roadblock .
however , because the conversion is very low 
 ( < 1% conversion to product based on ion - intensities in esi - ms ) , 
 agm1 may not be the only enzyme that can produce 6azglcnac-1-phosphate 
 in living cells .
we next analyzed the final enzyme in the biosynthetic 
 pathway , udp - n - acetylhexosamine pyrophosphorylase 
 ( agx1 ) , by first synthesizing 6azglcnac-1-phosphate ( scheme s3b and figures s16s30 ( si ) for synthetic details 
 and characterization ) .
recombinant agx1 was then incubated with different 
 concentrations of glcnac-1-phosphate or 6azglcnac-1-phosphate and 
 [ h]utp .
subsequent michaelis  menten kinetic analysis 
 demonstrated that 6azglcnac-1-phosphate is a substrate of agx1 , although 
 at a significantly lower efficiency than glcnac-1-phosphate ( figure 3c ) .
taken together , these data suggest that in living 
 cells 6azglcnac can be directly phosphorylated by agm1 and enter the 
 remainder of the glcnac salvage pathway to generate udp-6azglcnac 
 ( figure s2b ( si ) ) .
( a ) the indicated monosaccharides 
 ( 40 mm concentration ) were tested as substrates for purified galk2 
 in vitro .
( b ) proposed mechanism by which agm1 directly phosphorylates 
 6azglcnac in the presence of glcnac-6-phosphate .
( c ) kinetic constants 
 for the enzymatic production of udp sugar donors from glcnac-1-phosphate 
 and 6azglcnac-1-phosphate by the enzyme udp - n - acetylhexosamine 
 pyrophosphorylase ( agx1 ) .
next , to explore the generality of 6azglcnac 
 as a mcr , we labeled a panel of different cell lines .
specifically , 
 cos-7 , h1299 , hek293 , hela , mcf7 , mouse embryonic fibroblasts ( mefs ) , 
 and nih3t3 cells were treated with ac36azglcnac ( 200 m ) 
 for 16 h. in - gel fluorescence scanning after cuaac with alk - rho showed 
 labeling in all the cell lines examined and a diversity of the pattern 
 and intensity of modified proteins ( figure 4 ) . to qualitatively compare 6azglcnac to previous mcrs of o - glcnacylation
, the same panel of cell lines was treated 
 with 200 m ac4glcnaz or ac4galnaz for 
 16 h ( figure s5 ( si ) ) .
in - gel fluorescence 
 scanning showed incorporation of previously characterized mcrs in 
 each cell line with varying intensities and patterns , which were more 
 pronouncedly different for galnaz , when compared to 6azglcnac and 
 glcnaz . indicated cell lines
were treated with 200 m ac36azglcnac for 16 h before modified proteins were subjected to cuaac 
 with alk - rho and analysis by in - gel fluorescence scanning .
as stated above , mcrs only report on modifications 
 that occur during 
 the labeling time , raising the possibility that they can be used to 
 isolate o - glcnacylation events in a short time frame 
 via a pulse - labeling experiment . to determine the kinetics of protein 
 labeling by 6azglcnac ,
nih3t3 cells were treated with ac36azglcnac ( 200 m ) for different lengths of time .
the cells 
 were then lysed , reacted with alk - rho using cuaac , and analyzed by 
 in - gel fluorescence scanning ( figure 5a ) .
modified 
 proteins can be clearly visualized over background in 2 to 4 h , slightly 
 slower than the kinetics of protein labeling by ac4glcnaz 
 at 200 m ( figure 5a ) .
mcrs also have 
 the ability to read - out on the turnover of protein modifications using 
 a pulse - chase format .
accordingly , we treated nih3t3 cells with either 
 ac36azglcnac or ac4glcnaz at concentrations 
 of 200 m .
after 16 h , the cells were washed and fresh media 
 containing ac4glcnac ( 200 m ) was added .
cells were 
 collected after different lengths of time , lysed , and subjected to 
 cuaac with alk - rho . in - gel
fluorescence scanning showed a steady loss 
 of protein labeling over the course of 48 h ( figure 5b ) .
o - glcnacase ( oga ) is responsible for 
 the dynamic removal of o - glcnac from substrate proteins . 
 to demonstrate that 6azglcnac is incorporated into o - glcnacylation and a substrate for oga
, cells were first treated 
 with ac36azglcnac ( 200 m ) or dmso for 5 h. media 
 was then exchanged for fresh media containing 200 m ac4glcnac with or without thiamet - g ( 10 m ) , a potent and 
 highly selective oga inhibitor .
after 
 12 h , cells were harvested and subjected to cuaac with alk - rho . in - gel 

fluorescence scanning showed that cells that were treated with thiamet - g 
 maintained higher levels of 6azglcnac labeling compared to those without 
 ( figure 5c ) , demonstrating that 6azglcnac is 
 incorporated into o - glcnac modifications that can 
 be subsequently removed by oga .
( a ) nih3t3 cells were 
 treated with 200 m ac36azglcnac or ac4glcnaz for the indicated times , followed by cuaac and analysis by 
 in - gel fluorescence scanning .
( b ) nih3t3 cells were treated with 200 
 m ac36azglcnac or ac4glcnaz for 16 h , 
 at which time media was exchanged for fresh media containing 200 m 
 ac4glcnac .
cells were harvested after the indicated lengths 
 of time , subjected to cuaac , and analyzed by in - gel fluorescence scanning . 

( c ) hela cells were treated with 200 m ac36azglcnac 
 or ac4glcnaz for 5 h , at which time media was exchanged 
 for fresh media containing 200 m ac4glcnac and 10 
 m of the oga inhibitor thiamet - g or dmso .
cells were harvested 
 at the times indicated and subjected to cuaac before being analyzed 
 by in - gel fluorescence scanning . as noted above , previous 
 mcrs of o - glcnacylation are not selective for o - glcnac modifications because they are also incorporated 
 into either n - linked or mucin o - linked 
 glycans or both . to determine if 6azglcnac specifically 
 modifies o - glcnacylated proteins , we first took advantage 
 of the chimeric , secreted protein glycam - igg that contains both an n - linked and multiple mucin o - linked glycosylation 
 sites .
nih3t3 cells that stably express glycam - igg via retroviral 
 transformation were treated with ac36azglcnac , ac4glcnaz , or ac4glcnac at 200 m concentrations for 
 48 h. at this time , glycam - igg was immunoprecipitated from the media 
 using protein - a - conjugated beads .
in - gel fluorescence scanning , following 
 cuaac with alk - rho , showed that while glcnaz robustly labels glycam - igg , 
 as expected based on our previous results , 6azglcnac does not ( figure 6a and figure s6a ( si ) for the full - gel image ) .
this 
 demonstrates that while glcnaz does label the major types of cell - surface 
 glycosylation , 6azglcnac does not .
next , to confirm that 6azglcnac 
 labels o - glcnacylated proteins , we treated nih3t3 
 cells that were stably transfected with the flag - tagged transcription 
 factor foxo1 with ac36azglcnac , ac4glcnaz , or 
 ac4glcnac at 200 m concentrations for 24 h. in contrast 
 to glycam - igg , in - gel fluorescence showed that both mcrs robustly 
 labeled foxo1 ( figure 6b and figure s6b ( si ) for the expanded - gel images of replicate experiments ) , 
 demonstrating that 6azglcnac is a highly selective mcr of o - glcnac modifications . to rule out the possibility 
 that 6azglcnac was excluded from glycam - igg 
 but labeled other cell - surface glycoproteins , nih3t3 cells were treated 
 with ac36azglcnac , ac4glcnaz , or ac4galnaz at 200 m for 16 h before being harvested and submitted 
 to copper - free click chemistry using commercially available dbco - biotin . 

after subsequent incubation with fitc - conjugated avidin , cell - surface 
 glycoprotein labeling by each chemical reporter was analyzed using 
 flow cytometry .
no labeling over background was observed with ac36azglcnac , while labeling was observed with ac4glcnaz and ac4galnaz ( figure 6c ) . 

notably , this corroborates live - cell flow cytometry data from the 
 bertozzi lab where they used the staudinger ligation to observe some 
 cell - surface labeling with glcnaz and no labeling with 6azglcnac . to identify the proteins 
 labeled by 6azglcnac and compare them to those enriched by the previous 
 mcrs glcnaz and galnaz ,
nih3t3 cells were treated in triplicate with 
 either ac36azglcnac , ac4glcnaz , ac4galnaz , or ac4glcnac as a control ( all at 200 m ) 
 for 16 h. at this time cells were lysed using denaturing conditions 
 ( 4% sds ) and subjected to cuaac conditions with an alkyne - bearing 
 biotin tag .
equivalent amounts of the differentially labeled proteomes 
 were then reduced , alkylated , and subjected to biotin - enrichment using 
 streptavidin - conjugated beads .
after extensive washing to remove unlabeled 
 proteins , on - bead trypsinolysis afforded peptides that were analyzed 
 using lc 
ms / ms , identified using proteome discoverer and mascot , 
 and quantified by spectral counting .
labeled proteins were identified 
 as those that met the following threshold criteria : first , proteins 
 must have been identified by at least 1 unique peptide in each of 
 the three data sets and a total of 3 spectral counts in the sum of 
 three replicate data sets .
second , the sum of spectral counts of the 
 mcr - treated samples must be 3-times greater than those in the glcnac 
 labeled samples .
finally , the number of spectral counts in the mcr 
 treated sample compared to the control must be statistically significant 
 ( p < 0.05 , t test ) . using these 
 criteria ,
366 proteins were identified as being labeled by 6azglcnac 
 ( table s1 ( si ) ) , including many known o - glcnacylated proteins , such as the three annotated in 
 black in figure 6a ( map4 , nedd4 , and hcf1 ) . 

glcnaz and galnaz labeling identified 359 proteins ( table s2 ( si ) ) and 348 proteins ( table 
 s3 ( si ) ) , respectively .
in contrast to 6azglcnac , these lists 
 included both known o - glcnacylated proteins and proteins 
 that are exclusively localized to the extracellular space or the lumen 
 of the secretory pathway and lysosome , such as the three annotated 
 in red in figure 7a ( fibronectin , calumenin , 
 and -glucosidase ) .
comparison of the three proteomics lists 
 showed that 6azglcnac has greater overlap with glcnaz than galnaz 
 ( figure 7b ) , consistent with with previous 
 studies that show more efficient incorporation of galnaz versus glcnaz 
 into cell - surface glycoproteins .
importantly , 
 many of the proteins that were identified by 6azglcnac have been previously 
 identified in other o - glcnac proteomic studies ( see tables s1 , s2 , and s3 ( si ) for references ) .
nih3t3 cells stably expressing 
 either glycam - igg ( a ) or flag - tagged foxo1 ( b ) were treated with the 
 indicated mcrs or ac4glcnac , followed by immunoprecipitation , 
 cuaac , and analysis by in - gel fluorescence scanning .
( c ) nih3t3 cells were treated with 
 ac36azglcnac , ac4glcnaz , ac4galnaz , 
 or ac4glcnac ( all at 200 m ) for 16 h , at which time 
 cells were harvested and subjected to copper - free click chemistry 
 with dbco - biotin .
after incubation with fitc - avidin , live - cell surface 
 labeling was analyzed by flow cytometry .
we next annotated the proteins in our lists based on their 
 characterized 
 localizations ( figure 7c ) .
consistent with specific 
 labeling of o - glcnacylated proteins , 6azglcnac treatment 
 enriched 350 exclusively intracellular proteins ( i.e. , nuclear , cytosolic , 
 and mitochondrial ) and 9 proteins that can be localized to both the 
 cytosol and extracellular space or lumenal compartments ( e.g. , transmembrane 
 proteins ) .
in contrast , 9 and 72 exclusively extracellular or lumenal 
 proteins were found using glcnaz and galnaz treatment , respectively 
 ( figure 7c ) , reenforcing the data demonstrating 
 the nonspecific labeling of multiple types of glycosylation by glcnaz 
 and galnaz .
the use of mcrs for the 
 visualization and identification of protein 
 glycosylation has expanded the ability to investigate these key posttranslational 
 modifications .
however , recent evidence from our lab and others has 
 demonstrated that many mcrs of protein glycosylation lack specificity , 
 as they are incorporated into multiple types of glycans .
we previously showed that small 
 changes to the chemical structure of mcrs can have a large impact 
 on their distribution into different glycans . following this chemical - optimization theme further , we identified 
 a mcr ( 6azglcnac ) that robustly labeled a variety of proteins in living 
 mammalian cells . using a fluorescent alkyne tag
, we compared 6azglcnac 
 to the previous mcr , glcnaz , and demonstrated that 6azglcnac is efficiently 
 incorporated onto proteins allowing visualization in as little as 
 2 to 4 h after treatment ( figure 5a ) .
furthermore , 
 6azglcnac removal from proteins is dependent on the activity of oga , 
 demonstrating that it is dynamically incorporated into o - glcnacylated proteins ( figure 5c ) . using 
 two reporter proteins , we next showed that while glcnaz labels both 
 secreted glycoproteins and o - glcnacylated proteins , 
 6azglcnac is specific for o - glcnac modifications 
 ( figure 6a and b ) .
this is consistent with 
 our flow cytometry data ( figure 6c ) and previous 
 reports that both showed essentially no cell - surface labeling by 6azglcnac 
 and that chemically synthesized udp-6azglcnac is a substrate for recombinant o - glcnac transferase . unlike glcnaz , 
 6azglcnac can not be metabolized to the corresponding 
 udp - sugar donor by the canonical glcnac salvage - pathway ( figure s2 ( si ) ) , as the first step involves phosphorylation 
 at the 6-hydroxyl of the monosaccharide .
therefore , an alternative 
 enzyme must directly phosphorylate 6azglcnac at the 1-hydroxyl to 
 bypass this roadblock . taking a candidate - based approach
, we tested 
 galk2 and agm1 in vitro to determine if they could generate 6azglcnac-1-phosphate . 

we did not observe any product formation using galk2 , and knockdown 
 of galk2 in living cells using shrna did not result in reduced protein - labeling 
 by 6azglcnac ( figure s3 ( si ) ) . notably , 
 however , we found that agm1 is capable of directly generating 6azglcnac-1-phosphate 
 when its normal substrate , glcnac-6-phosphate , is added to the reaction 
 mixture ( figures 3b and s4 ( si ) ) .
( a ) nih3t3 cells were treated with ac36azglcnac , 
 ac4glcnaz , ac4galnaz , or ac4glcnac 
 ( all at 200 m ) for 16 h. at this time , the corresponding cell - lysates 
 were subjected to cuaac with alkyne - biotin , enrichment with streptavidin - coated 
 beads , and on - bead trypsinolysis .
ms / ms 
 are graphically presented as total number of positive minus total 
 number of control spectral counts .
three known o - glcnacylated 
 proteins are annotated in black , and three known extracellular / lumenal 
 proteins are annotated in red . ( b ) overlap between proteins identified 
 using 6azglcnac , glcnaz and , galnaz .
( c ) graphical representation 
 of enriched proteins based on whether their localization is exclusively 
 intracellular ( i.e. , cytoplasmic , nuclear , or mitochondrial ) , exclusively 
 extracellular or lumenal ( i.e. , er , golgi , lysosome ) , or have domains 
 in both ( e.g. , transmembrane protein ) . on the basis of the enzymatic mechanism , we conclude that 
 agm1 
 removes the phosphate from glcnac-6-phosphate to generate the known 
 phosphoenzyme intermediate , followed 
 by binding of 6azglcnac and phosphorylation of the 1-hydroxyl .
this 
 is consistent with the reversible nature of agm1 s activity , 
 where the monosaccharide substrates can bind the active site with 
 either the 1- or 6-hydroxyl groups oriented toward the catalytic serine . 

however , given the low levels of 6azglcnac turnover by agm1 , it is 
 reasonable to assume that additional enzymes may also phosphorylate 
 6azglcnac in living cells . additionally , we also showed that once 
 6azglcnac-1-phosphate is formed it can be enzymatically transformed 
 to udp-6azglcnac by agx1 ( figure 3c ) .
we do 
 not know if udp-6azglcnac can be epimerized to udp-6azgalnac in cells ; 
 however , even if this metabolite is formed , previous studies by bertozzi 
 and co - workers demonstrated that udp-6azgalnac is not a substrate 
 for the polypeptide - n - acetyl - galactosamine transferases .
together , these results suggest an unappreciated 
 metabolic flexibility in mammalian cells . agm1 and potentially other , 
 yet unidentified , small - molecule phosphotransferases may contribute 
 to the salvaging of natural monosaccharides from the environment . 

furthermore , they have potentially important implications for the 
 metabolism of bacterial or abiotic carbohydrates that would otherwise 
 be assumed to not enter mammalian biosynthetic pathways .
finally , 
 our results challenge a dogma in mcr design , which relies on well - established 
 metabolic pathways and directly resulted in the previous dismissal 
 of 6azglcnac as a viable mcr in living cells .
to further confirm the specificity of 6azglcnac and demonstrate 
 any advantages over other mcrs previously used to study o - glcnacylated proteins , we performed a proteomics experiment using 
 6azglcnac , glcnaz , and galnaz in combination with alkyne - biotin and 
 on - bead trypsinolysis ( figure 7 ) .
we found 
 that enrichment with 6azglcnac resulted in the identification of essentially 
 only intracellular proteins that can not contain glycans ( e.g. , n - linked or mucin o - linked ) that are added 
 in the secretory pathway .
this confirms the high degree of specificity 
 of 6azglcnac for o - glcnacylated proteins .
consistent 
 with our fluorescence data , glcnaz was less selective , resulting in 
 the enrichment of 28 transmembrane proteins and 9 exclusively extracellular 
 or lumenal proteins . finally , galnaz was the least selective , since 
 it enriched only 226 exclusively intracellular proteins and 72 proteins 
 that are only extracellular or lumenal .
we believe that this lack 
 of selectivity is one reason why a recent study using galnaz required 
 subcellular fractionation and two - dimensional electrophoresis to identify 
 the potential o - glcnacylation of the voltage - dependent 
 anion - selective channel protein 2 ( vdac2 ) , while the same protein 
 was readily identified by 6azglcnac labeling without any biochemical 
 manipulations ( table s1 ( si ) ) .
this specificity 
 is an improvement over other mcrs that require biochemical manipulations 
 ( e.g. , cell fractionation ) to exclude cell - surface glycoproteins .
previous direct comparisons of the selectivity of different 
 glycoprotein 
 mcrs are somewhat limited .
the bertozzi lab reported that 
 galnaz has superior o - glcnac labeling efficiency 
 compared to glcnaz due to more efficient metabolic conversion of galnaz 
 to udp - galnaz and subsequent epimerization to udp - glcnaz .
our in - gel fluorescence data do not support 
 these data , as glcnaz and galnaz resulted in qualitatively similar 
 levels of protein labeling in a variety of cell lines ( figure s5 ( si ) ) .
interestingly , only a minority 
 of cell lines show similar global - patterns of labeling between glcnaz 
 and galnaz ( e.g. , mcf7 ) , while most are significantly different .
this 
 also is true of 6azglcnac , which often shows different labeling patterns 
 and intensities from both glcnaz and galnaz ( figure 4 and figure s5 ( si ) ) .
this supports 
 our results that each of the mcrs is incorporated into different types 
 of glycoproteins and utilizes independent metabolic enzymes for the 
 generation of the corresponding udp donor sugars .
notably , while 6azglcnac 
 is the most selective reporter of o - glcnacylation , 
 it requires longer labeling - times to achieve the same signal - to - noise 
 as glcnaz ( figure 5a ) , highlighting a potential 
 trade - off between labeling efficiency and specificity .
however , based 
 on our results , we predict that any bottlenecks in the metabolism 
 of 6azglcnac will not dramatically hamper the visualization and identification 
 of o - glcnacylated proteins .
furthermore , the extent 
 of o - glcnacylation and identity of modified proteins 
 has been shown to be dependent on the cellular concentration of udp - glcnac .
therefore , it could be advantageous to have limited metabolic conversion 
 of an mcr to minimize the chances of altering the endogenous repertoire 
 of o - glcnacylated proteins , as long as the labeling 
 is above the detection limit .
we are currently exploring if different 
 concentrations of mcr treatment change the overall levels of o - glcnacylation . despite the increased labeling efficiency 
 of glcnaz and galnaz compared to 6azglcnac , approximately the same 
 total number of spectral counts were found in our comparative proteomics 
 experiment .
we believe that this could be due to an excess of input 
 that exceeded the capacity of the streptavidin beads , resulting in 
 equal total levels of protein enrichment prior to trypsinolysis and 
 identification .
coupled with the ever - growing toolkit of commercially 
 available 
 and custom azide - reactive tags , including terminal alkynes , cyclooctynes , 
 and phosphines , we predict that 6azglcnac will become the most powerful 
 and readily used mcr for the study of o - glcnacylation . 

in particular , metabolic labeling strategies have the unique ability 
 to isolate time - resolved protein modifications that only occur during 
 cell labeling .
furthermore , mcrs can be used in pulse - chase experiments 
 to measure the dynamic removal of o - glcnac modifications 
 in living cells .
finally , the successful application of synthetic 
 chemistry to identify a selective mcr of o - glcnacylation 
 suggests that the same chemical strategy could be used to create reporters 
 that are specific for other types of protein glycosylation .
coupled 
 with new bioorthogonal reactions ( e.g. , tetrazene clycoadditions ) , 
 which enable more diverse functional groups to be incorporated into 
 mcrs , we predict that a library of mcrs can be created 
 to enable the specific visualization and identification of the several 
 types of glycosylation in mammals and other organisms .
all reagents 
 used for chemical 
 synthesis were purchased from sigma - aldrich , alfa aesar or emd millipore 
 unless otherwise specified and used without further purification . 

analytical thin - layer chromatography 
 ( tlc ) was conducted on emd silica gel 60 f254 plates with 
 detection by ceric ammonium molybdate ( cam ) , anisaldehyde or uv . for 
 flash chromatography ,
h spectra were obtained at 400 , 500 , or 600 mhz on a varian 
 spectrometers mercury 400 , vnmrs-500 , or -600 .
c spectra were 
 obtained at 100 , 125 , or 150 mhz on the same instruments .
cos-7 , hek293 , hela and mcf7 cells were 
 cultured in dmem media ( corning ) enriched with 10% fetal bovine serum 
 ( atlanta biologicals ) .
amphopack-293 retroviral packaging cells ( clontech ) 
 were cultured in dmem media ( corning ) enriched with 10% fetal bovine 
 serum ( hyclone , thermoscientific ) . nih3t3 and
mef cells were cultured 
 in high - glucose dmem media ( corning ) enriched with 10% fetal calf 
 serum ( hyclone , thermoscientific ) .
h1299 cells were cultured in rpmi 
 media enriched with 10% fetal bovine serum ( hyclone , thermoscientific ) . 

all cell lines were maintained in a humidified incubator at 37 c 
 and 5.0% co2 . to cells at 8085% 
 confluency , 
 media containing ac4glcnac , ac4glcnaz , ac4galnaz , ac36azglcnac ( 1000 stock in dmso ) , 
 or dmso vehicle was added as indicated . for chase experiments ,
existing 
 media was replaced with media supplemented with 200 m ac4glcnac ( sigma ) or 200 m ac4glcnac ( sigma ) 
 plus 10 m thiamet - g ( 1000 x stock in dmso ) as indicated .
the cells were 
 collected by trypsinization and pelleted by centrifugation at for 
 4 min at 2000 g , followed by washing 2 with 
 pbs ( 1 ml ) .
cell pellets were then resuspended in 100 l of 
 1% np-40 lysis buffer [ 1% np-40 , 150 mm nacl , 50 mm triethanolamine 
 ( tea ) ph 7.4 ] with complete , mini , edta - free protease inhibitor cocktail 
 tablets ( roche ) for 20 min and then centrifuged for 10 min at 10  000 g at 4 c . the supernatant ( soluble cell lysate ) was 
 collected and the protein concentration was determined by bca assay 
 ( pierce , thermoscientific ) .
cell lysate ( 200 g ) was diluted 
 with cold 1% np-40 lysis 
 buffer to obtain a desired concentration of 1 g l .
newly made click chemistry cocktail ( 12 l ) 
 was added to each sample [ alkynyl - rhodamine tag ( 100 m , 10 
 mm stock solution in dmso ) ; tris(2-carboxyethyl)phosphine hydrochloride 
 ( tcep ) ( 1 mm , 50 mm freshly prepared stock solution in water ) ; tris[(1-benzyl-1-h-1,2,3-triazol-4-yl)methyl]amine ( tbta ) ( 100 m , 
 10 mm stock solution in dmso ) ; cuso45h2o ( 1 mm , 50 mm freshly prepared stock solution in water ) for a total 
 reaction volume of 200 l .
the reaction was gently vortexed 
 and allowed to sit at room temperature for 1 h. upon completion , 1 
 ml of ice cold methanol was added to the reaction , and it was placed 
 at 20 c for 2 h to precipitate proteins .
the reactions 
 were then centrifuged at 10  000 g for 10 min 
 at 4 c .
the supernatant was removed , the pellet was allowed 
 to air - dry for 15 min , and then 50 l 4% sds buffer ( 4% sds , 
 150 mm nacl , 50 mm tea ph 7.4 ) was added to each sample .
the mixture 
 was sonicated in a bath sonicator to ensure complete dissolution , 
 and 50 l of 2 sds - free loading buffer ( 20% glycerol , 
 0.2% bromophenol blue , 1.4% -mercaptoethanol , ph 6.8 ) was then 
 added .
the samples were boiled for 5 min at 97 c , and 40 g 
 of protein was then loaded per lane for sds - page separation ( any kd , criterion gel , bio - rad ) .
following sds - page separation , 
 gels were scanned on a typhoon 9400 variable mode imager ( ge healthcare ) 
 using a 532 nm for excitation and 30 nm bandpass filter centered at 
 610 nm for detection .
pcr was conducted in an eppendorf 
 mastercycler thermocycler . to a 0.2 ml thermo - walled pcr tube
was 
 added 2 reaction mix ( superscript one - step rt - pcr with platinum taq , invitrogen ) , template rna from nih3t3 cells ( 1000 ng ) , 
 sense and antisense primers for galk2 ( 10 m ) , sense and antisense 
 primers for gapdh ( 10 m ) , water , and taq enzyme ( superscript 
 one - step rt - pcr with platinum taq , invitrogen ) .
the 
 provided pcr cycle was used according to superscript one - step rt - pcr 
 with platinum taq ( invitrogen ) with an extension 
 time of 32 s ( 1 min / kbp ) .
products were diluted with 6 sample 
 loading dye ( bio - rad ) and analyzed by electrophoresis on a 5% agarose 
 gel ( 500 mg of agarose in 1 tae buffer , tris / acetic acid / edta , 
 bio - rad ) .
the gel was subsequently visualized using a chemidoc xrs+ 
 molecular imager ( bio - rad ) .
recombinant galk2 ( 8 g ml ) was incubated 
 in triplicate with galnac , glcnac or 6azglcnac ( 40 mm ) in 25 l 
 reaction buffer ( 10 mm mgcl2 , 50 mm tris hcl ph 8.0 ) containing 
 1 mg / ml bsa and 5 mm [ p]atp ( 1000 cpm / nmol ) for 
 60 min at 37 c . after this time
, reactions were terminated by 
 the addition of water ( 0.75 ml ) and applied to a dowex 1  8 
 ( cl- ) column ( 0.7 cm  3.0 cm ) .
unreacted starting material 
 was eluted by washing with 2 ml of 25 mm nh4hco3 before eluting the sugar-1-phosphates with 100 mm nh4hco3 . the fractions ( 2 ml ) were counted in a liquid scintillation 
 counter ( beta , perkinelmer ) .
column profiles were compared to detect 
 the presence of overlapping radioactive peaks corresponding to degradation 
 products . if present , these peaks were subtracted from the assay chromatogram .
homo sapiens phosphoacetylglucosamine mutase 3 ( agm1/pgm3 ) 
 cdna was obtained from biovalley ( marne - la - valle , france ) , 
 amplified by pcr , sequenced and cloned in ptrchis a ( invitrogen ) . 

the 6his - tagged pgm3 protein was expressed in escherichia 
 coli dh5 ( invitrogen ) cultured for 24 h at 18 c 
 in 2ty medium supplemented with 1 mm iptg and 2 mm mgcl2 .
bacteria were lysed in y - per ( thermoscientific ) and the lysate 
 diluted with 5 volumes of 50 mm phosphate buffer ph 8.0 , 300 mm nacl , 
 10 mm imidazole , 0.1 mm pmsf and 0.1 mm tcep .
after application of 
 the lysate on a histrap ff 5 ml column ( ge healthcare ) , agm1 was eluted 
 with 250 mm imidazole in 25 mm phosphate buffer ph 8.0 , 150 mm nacl , 
 0.1 mm tcep .
agm1 activity was checked in a coupled assay by 2 h incubation 
 at 37 c with glcnac-6-phosphate ( 2 mm ) , in 75 mm tris hcl ph 
 8.8 , 5 mm mgcl2 , containing 0.1 mg bsa and 2 l of 
 the pgm3 enzyme solution , and coupling with agx1 ( 0.66.3 g 
 ml , 2.222 mu ml ) and 
 yeast inorganic pyrophosphatase ( 1.6 g ml , 3 u ml ) in the presence of [ h]utp 
 ( perkinelmer , 2 mm , 260 cpm nmol ) .
after agm1/agx1/yeast 
 inorganic phosphatase heat denaturation , calf intestinal alkaline 
 phosphatase ( neb , 80 u ml ) was added in order 
 to hydrolyze all the utp and udp present , and the products were monitored 
 as described below for agx1 enzymatic tests ( separation on paper chromatography ) . 

finally , the production of udp - glcnac was estimated by scintillation 
 counting . under these conditions ,
the recombinant agm1 activity was 
 estimated to 20 nmol h g of the agm1 enzyme solution .
6azglcnac 
 ( 2.25 mm final concentration ) was incubated in 100 l of 75 
 mm tris hcl ph 8.8 , 5 mm mgcl2 , containing 0.1 mg bsa and 
 10 g of the pgm3 enzyme solution .
incubations were run at 37 
 c for 1 h 30 min either without cofactor or in the presence 
 of glc-6p , glc1,6-dip or glcnac-6p ( 1.00 mm final concentration ) . 

after thawing , 
 the reaction mixtures were incubated for 1 h at 37 c with bicyclo[6.1.0]nonyne-(poe)3-nh - dye495 
 conjugate ( synaffix , oss , netherlands ) .
the reaction mixtures which 
 contain the azido sugars coupled through their azido moiety to the 
 bcn fluorescent dye495 , were then laid onto a 46  57 cm sheet 
 of whatman 3 mm paper ( ge healthcare ) and run for descending chromatography 
 in ethyl acetate / formic acid / water ( 70:20:10 ) for 5 h. after drying , 
 fluorescent spots ( rf  0.8 ) were cut out and the products eluted from the paper 
 in 50% methanol .
( 0.66.3 g ml , 2.222 mu ml ) was incubated with glcnac-1p 
 or 6azglcnac-1p in 25 l reaction buffer ( 1 mm mgcl2 , 75 mm tris hcl ph 8.8 ) containing 1 mg ml bsa 
 and 2 mm [ h]utp ( 260 cpm nmol ) for 
 10 min at 37 c .
yeast inorganic pyrophosphatase ( 1.6 g 
 ml , 3 mu ml ) was also added 
 to inhibit the reverse reaction .
to hydrolyze excess utp and ump , calf intestinal 
 alkaline phosphatase ( new england biolabs , 80 u / ml ) was added to the 
 reaction mixture and incubated for 2 h at 37 c .
the reaction 
 was spotted onto whatman 3 mm chromatography paper and submitted to 
 descending chromatography in ethyl acetate / formic acid / water ( 70:20:10 ) 
 to remove [ h]uridine ( rf = 0.25 ) .
spots with [ h]udp - sugars ( rf = 0.01 ) were cut out of the chromatography 
 paper and counted in a liquid scintillation counter .
control samples 
 without sugar-1p received the same treatment in order to deduct background 
 radioactivity .
km and kcat were calculated using the enzyme kinetic 
 module 1.3 of sigmaplot 10 , from plots obtained with different concentrations 
 of sugar-1p and different amounts of agx1 .
nih3t3 
 cells were pretreated with 200 m 
 ac36azglcnac or dmso for 72 h prior to plating .
nih-3t3 
 cells ( 1  10 cells ) were plated per well in a 96-well , 
 white bottom dish 24 h before treatment with 200 m ac4glcnac or ac36azglcnac for 16 h in triplicate .
celltiter 
 96 aqueous non - radioactive cell proliferation assay ( promega , madison , 
 wi ) was used according to the provided protocol .
absorbance at 490 
 nm was read using a biotek synergy h4multi - mode microplate reader .
nih3t3 cells grown in 6-well plates at 8085% confluency were 
 treated with 200 m ac4glcnac , ac4glcnaz , 
 ac4galnaz or ac36azglcnac in triplicate for 
 16 h at which time media was removed and cells were gently washed 
 with pbs before being detached from the plate with 1 mm edta in pbs . 

cells were collected by centrifugation ( 5 min , 300 g at 4 c ) and were washed three times with pbs ( 5 min , 300 g at 4 c ) .
cells were then resuspended in 200 l 
 pbs containing dbco - biotin ( click chemistry tools , 60 m ) for 
 1 h , after which time they were washed three times with pbs ( 5 min , 
 300 g at 4 c ) before being resuspended in ice - cold 
 pbs containing fluorescein isothiocynate ( fitc ) conjugated avidin 
 ( sigma , 5 g / ml , 30 min at 4 c ) .
cells were then washed 
 three times in pbs ( 5 min , 300 g at 4 c ) before 
 being resuspended in 400 l pbs for flow - cytometry analysis . 

a total of 10  000 cells [ dead cells were excluded by treatment 
 with propidium iodide ( 2.5 g ml in water , 
 30 min ) ] were analyzed on a bd sorp lsrii flow cytometer using the 
 488 nm argon laser .
nih3t3 cells stably 
 expressing flag - tagged 
 foxo1 were treated with 200 m ac4glcnaz , ac36azglcnac ( 1000 x stock in dmso ) or dmso and allowed to incubate 
 overnight .
cell pellets were resuspended in 100 l of 1% np-40 
 lysis buffer [ 1% np-40 , 150 mm nacl , 50 mm triethanolamine ( tea ) ph 
 7.4 ] with complete mini , edta - free protease inhibitor cocktail tablets 
 ( thermo scientific ) for 20 min and then centrifuged at 4 c for 
 10 min at 10  000 g .
the supernatant was collected 
 and the protein concentration was determined by bca assay ( pierce , 
 thermoscientific ) .
total cell lysate ( 1.5 mg ) was diluted as necessary 
 to a final volume of 1 ml with 1% np-40 buffer with complete mini , 
 edta - free protease inhibitor cocktail tablets ( thermo scientific ) . 

ezview red anti - flag m2 affinity beads ( 30 l , sigma ) , prewashed 
 with cold np-40 buffer 2 followed by cold pbs 2 , were 
 added to each sample .
beads were collected by centrifugation at 2000 g for 2 min at 4 c , and the supernatant was carefully 
 removed .
beads were then washed with cold pbs by rotating for 5 min 
 before centrifuging 2 min at 2000 g .
the final pbs 
 wash was carefully removed , and the beads were suspended in 40 l 
 of 4% sds buffer and boiled for 5 min at 97 c . the appropriate 
 amount of click chemistry cocktail was added , and the reaction was 
 allowed to proceed for 1 h after which time 30 l of 2 
 loading buffer was added .
protein samples ( 40 g ) were then loaded per lane for sds - page 
 separation ( any kd criterion gel , bio - rad ) 
 and imaged by in - gel fluorescence scanning .
nih3t3 cells stably expressing 
 glycam - igg in 6-well dishes at 8085% confluency were treated 
 in dmem with 10% fcs and 200 m ac4glcnaz , ac36azglcnac ( 1000 stock in dmso ) or dmso for 24 h. the 
 media from each sample was collected by centrifugation at 3000 g for 10 min at 4 c to remove cell debris .
the supernatant 
 ( 1 ml ) was incubated with 50 l of recombinant protein g sepharose 
 beads ( invitrogen ) in 100 mm tea ph 8 overnight .
beads were collected 
 by centrifugation at 2000 g for 2 min at 4 c . 

glycam - ig was 
 eluted by addition of 50 l of 4% sds buffer ( 4% sds , 150 mm 
 nacl , 50 mm tea ph 7.4 ) and boiling for 5 min at 97 c .
final 
 sds concentration was diluted to 0.5% by addition of 50 mm tea ph 
 7.4 .
the appropriate amount of click chemistry cocktail was added 
 and the reaction was allowed to proceed for 1 h after which time 4 
 loading buffer ( 200 mm tris hcl , 4% sds , 40% glycerol , 0.4% bromophenol 
 blue , 1.4% -mercaptoethanol , ph 6.8 ) was added .
samples were 
 boiled for 5 min at 97 c , and 50 g were loaded for sds - page 
 separation ( any kd criterion gel , bio - rad ) .
proteins were separated by sds - page 
 before being transferred to pvdf membrane ( bio - rad ) using standard 
 western blotting procedures .
all western blots were blocked in tbst 
 ( 0.1% tween-20 , 150 mm nacl , 10 mm tris ph 8.0 ) containing 5% nonfat 
 milk for 1 h at rt .
the blots were then incubated with the appropriate 
 primary antibody in blocking buffer for 1 h at rt .
the anti - flag antibody 
 ( thermo ) and anti - mab414 antibody ( covance ) were used at a 1:5000 
 dilution and 1:1000 for detection of foxo1 and p62 , respectively . 

the anti - nedd4 antibody ( millipore ) was used at a 1:10  000 
 dilution to detect nedd4 and the anti - pyruvate kinase antibody ( abcam ) 
 was used at 1:1000 .
the blots were then washed three times in tbst 
 for 10 min and incubated with the horseradish peroxidase ( hrp)-conjugated 
 secondary antibody for 1 h in blocking buffer at rt .
hrp - conjugated 
 antimouse , antirabbit , antigoat and antihuman antibodies ( jackson 
 immunoresearch ) were used at 1:10  000 dilutions .
after being 
 washed three more times with tbst for 10 min , the blots were developed 
 using ecl reagents ( bio - rad ) and the chemidoc xrs+ molecular imager 
 ( bio - rad ) .
nih3t3 
 cell - pellets labeled with ac36azglcnac , ac3glcnaz , 
 ac3galnaz or ac4glcnac for 16 h were resuspended 
 in 200 l h2o , 60 l pmsf in h2o 
 ( 250 mm ) , and 500 l of 0.05% sds buffer ( 0.05% sds , 10 mm tea 
 ph 7.4 , 150 mm nacl ) with complete mini protease inhibitor cocktail 
 ( roche biosciences ) . to this was added 8 l benzonase ( sigma ) , 
 and the cells were incubated on ice for 30 min . then
, 4% sds buffer 
 ( 2000 l ) was added , and the cells were briefly sonicated in 
 a bath sonicator followed by centrifugation ( 20  000 g for 10 min at 15 c ) .
soluble protein concentration 
 was normalized by bca assay ( pierce , thermoscientific ) to 1 mg ml , and 10 mg of total protein was subjected to the 
 appropriate amount of click chemistry cocktail containing alkyne - peg3-biotin 
 ( 5 mm , click chemistry tools ) for 1 h , after which time 10 volumes 
 of ice - cold meoh were added .
precipitated proteins were centrifuged at 5200 g for 30 min at 0 c and washed 3 times with 40 ml of ice - cold 
 meoh , with resuspension of the pellet each time .
the pellet was then 
 air - dried for 1 h. to capture the biotinylated proteins by streptavidin 
 beads , the air - dried protein pellet was resuspended in 2 ml of resuspension 
 buffer ( 6 m urea , 2 m thiourea , 10 mm hepes ph 8.0 ) by bath sonication . 
 to cap cysteine residues , 100 l of freshly made tcep ( 200 mm 
 stock solution , thermo )
was then added and the mixture incubated for 
 30 min , followed by 40 l of freshly prepared iodoacetamide 
 ( 1 m stock solution , sigma ) and incubation for a further 30 min in 
 the dark .
steptavadin beads ( 250 l of a 50% slurry per sample , 
 thermo ) were washed 2 with 1 ml pbs and 1 with 1 ml of 
 resuspension buffer and resuspended in resuspension buffer ( 200 l ) . 

each sample was combined with streptavadin beads and incubated on 
 a rotator for 2 h. these mixtures were then transferred to mini bio - spin 
 columns ( bio - rad ) and placed on a vacuum manifold .
captured proteins 
 were then washed with agitation 5 with resuspension buffer ( 10 
 ml ) , 5 pbs ( 10 ml ) , 5 with 1% sds in pbs ( 10 ml ) , 30 
 with pbs ( 1 ml per wash , vacuum applied between each wash ) , and 5 
 2 m urea in pbs ( 1 ml per wash , vacuum applied between each wash )
. 
 beads were then resuspended in 2 m urea in pbs ( 1 ml ) , transferred 
 to screw - top tubes , and pelleted by centrifugation ( 2000 g for 2 min ) . at this time ,
800 l of the supernatant was removed , 
 leaving a volume of 200 l . to this bead - mixture
was added 2 
 l of cacl2 ( 200 mm stock , 1 mm final concentration ) 
 and 2 l of 1 mg ml sequence grade trypsin 
 ( promega ) and incubated at 37 c for 18 h. the resulting mixtures 
 of tryptic peptides and beads were transferred to mini bio - spin columns 
 ( bio - rad ) and the eluent was collected by centrifugation ( 1000 g for 2 min ) .
any remaining peptides were eluted by addition 
 of 100 l of 2 m urea in pbs followed by centrifugation as immediately 
 above .
the tryptic peptides were then applied to c18 spin columns 
 ( pierce ) according to manufacturer s instructions , eluted with 
 70% acetonitrile in h2o , and concentrated to dryness on 
 a speedvac .
peptides were desalted 
 on a trap column following separation on a 12 cm/75um reversed phase 
 c18 column ( nikkyo technos co. , ltd .
japan ) . a 3 h gradient increasing 
 from 10% b to% 45% b in 3 h ( a : 0.1% formic acid , b : acetonitrile/0.1% 
 formic acid )
was delivered at 150 nl min . the 
 liquid chromatography setup ( dionex , boston , ma , usa )
was connected 
 to an orbitrap xl ( thermo , san jose , ca , usa ) operated in top-5-mode . 

acquired tandem ms spectra ( cid ) were extracted using proteomediscoverer 
 v. 1.3 ( thermo , bremen , germany ) and queried against the human uniprot 
 protein database using mascot 2.3.02 ( matrixscience , london , uk ) . 

all lc  ms / ms analysis were carried 
 out at the proteomics resource center at the rockefeller university , 
 new york , ny , usa . excel files containing identified proteins will 
 be made available upon request .
the fluorescent 
 detection tag alk - rho and the oga inhibitor 
 thiamet - g were also synthesized according 
 to published procedures .
commercially 
 available n - acetyl - glucosamine ( 1.00 g , 4.52 mmol ) 
 was dissolved in anhydrous pyridine under nitrogen and cooled to 20 
 c .
p - toluenesulfonyl chloride ( 1.04 g , 5.43 
 mmol ) was dissolved in anhydrous pyridine ( 3 ml ) , and the solution 
 was added dropwise over 20 min to the above reaction mixture . upon 
 completion of the addition
, the reaction was warmed to rt and stirred 
 for 18 h. the mixture was concentrated by vacuum and used without 
 further purification .
compound 1 was 
 resuspended in n , n - dimethylformamide 
 ( 20 ml ) under a nitrogen atmosphere .
sodium azide ( 1.47 g , 22.6 mmol ) 
 was added and the reaction was stirred for 3 d at 50 c .
the 
 reaction mixture was then concentrated by vacuum and resuspended in 
 pyridine ( 30 ml ) . acetic anhydride ( 10.0 ml , 66.0 mmol )
was added 
 and the mixture was stirred for 16 h at rt . upon completion ,
solvent 
 was removed under reduced pressure and the resulting mixture was redissolved 
 in ch2cl2 ( 200 ml ) and washed with 1 m hcl ( 2 
  100 ml ) , saturated aqueous sodium bicarbonate ( 2  100 
 ml ) and water ( 2  100 ml ) .
the resulting crude mixture was purified by column chromatography 
 ( 65% ethyl acetate in hexanes ) to afford 1.01 g of the product in 
 60% yield over three steps as an alpha - beta mixture : h 
 nmr ( 400 mhz , cd3od ) of pure alpha - anomer  ( ppm ) 
 6.18 ( d , j = 3.7 hz , 1h ) , 5.61 ( d , j = 8.9 hz , 1h ) , 5.255.10 ( m , 2h ) , 4.46 ( ddd , j = 10.9 , 9.0 , 3.7 hz , 1h ) , 3.943.92 ( m , 1h ) , 3.383.32 
 ( m , 1h ) , 3.29 ( dd , j = 13.5 , 5.5 hz , 1h ) , 2.19 ( s , 
 3h ) , 2.04 ( d , j = 1.3 hz , 6h ) , 1.92 ( s , 3h ) .
hydroquinone 
 ( 11.0 g , 0.910 mmol ) and propargyl 
 chloride ( 7.50 g , 0.101 mmol ) were dissolved in ethanol ( 20 ml ) under 
 an argon atmosphere in a three - neck flask equipped with an addition 
 funnel .
koh ( 0.10 m in water ) was added dropwise through the addition funnel . 

the mixture was then stirred for 20 h upon which time the reaction 
 was cooled and solvent was removed by vacuum .
the resulting crude 
 mixture was dissolved in ch2cl2 and extracted 
 with dilute , aqueous koh .
the aqueous layer was then brought to a 
 neutral ph by the addition of 1 m hcl and subsequently extracted with 
 ch2cl2 .
the organic layer was washed with water 
 and dried over sodium sulfate , filtered and concentrated .
the crude 
 mixture was purified by column chromatography ( 10% ethyl acetate : hexanes ) 
 to afford the pure product ( 4.07 g , 28% ) : h nmr ( 400 mhz , 
 cdcl3 )  ( ppm ) 6.85 ( dd , j = 9.1 , 
 1.1 hz , 2h ) , 6.78 ( d , j = 9.0 hz , 2h ) , 4.61 ( dd , j = 2.4 , 1.0 hz , 2h ) , 2.50 ( t , j = 2.4 
 hz , 1h ) . to a suspension 
 of methyl-4-amino - benzoate in 6 m hcl was added sodium nitrite at 
 0 c .
compound 3 ( 1.30 g , 18.9 mmol ) 
 was dissolved in water : thf ( 2:1 ) and cooled to 0 c .
potassium 
 carbonate ( 52.0 g , 376 mmol ) was added and reaction let stir for 30 
 min upon which time 4-(methoxycarbonyl)benzenediazonium chloride was 
 added dropwise .
the reaction was allowed to warm to rt and was stirred 
 for 18 h. the reaction was poured over water and extracted with ethyl 
 acetate ( 3  200 ml ) . the organic layer was dried over sodium 
 sulfate , filtered , and concentrated .
the resulting crude mixture was 
 purified by column chromatography by first starting at 10% ethyl acetate : hexanes 
 and increasing to 20% ethyl acetate : hexanes to elute the product . 

concentration under decreased pressure affords the product as a yellow 
 oil ( 2.00 g , 90% ) : h nmr ( 400 mhz , cdcl3 )  
 ( ppm ) 8.20 ( d , j = 8.5 hz , 2h ) , 7.92 ( d , j = 8.6 hz , 2h ) , 7.56 ( d , j = 3.1 hz , 1h ) , 
 7.10 ( dd , j = 9.1 , 3.1 hz , 1h ) , 7.00 ( d , j = 9.1 hz , 1h ) , 4.75 ( d , j = 2.3 hz , 2h ) , 
 3.97 ( s , 3h ) , 2.56 ( t , j = 2.4 hz , 1h ) ; c nmr ( 125 mhz , cdcl3 )  ( ppm ) 166.25 , 153.08 , 150.83 , 
 148.27 , 137.01 , 131.92 , 130.80 , 130.17 , 128.36 , 123.68 , 122.02 , 119.17 , 
 111.60 , 78.41 , 75.86 , 56.77 , 52.38 ; maldi - ms calculated for c17h15n2o3 [ m + h ] 295.1083 , found 293.9045 .
compound 4 ( 0.124 g , 0.421 
 mmol ) was dissolved in tetrahydrofuran ( 2 ml ) .
naoh ( 0.758 mg , 1.90 
 mmol ) dissolved in water was added and reaction let stir 18 h. upon 
 completion , a color change from purple to orange is seen .
the reaction 
 was neutralized by the dropwise addition of acetic acid and subsequently 
 concentrated under reduced pressure to remove solvent .
the resulting 
 crude mixture was dissolved in ch2cl2 and washed 
 with water ( 2  50 ml ) .
the crude mixture was column 
 purified ( 8:1.5:0.5 ethyl acetate : methanol : water ) to afford the pure 
 product as a bright orange solid ( 0.825 g , 70% ) : h nmr 
 ( 600 mhz , cd3od )  ( ppm ) 8.05 ( d , j = 8.5 hz , 2h ) , 7.85 ( d , j = 8.5 hz , 2h ) , 7.46 ( d , j = 3.0 hz , 1h ) , 7.01 ( dd , j = 9.2 , 3.0 
 hz , 1h ) , 6.88 ( d , j = 8.9 hz , 1h ) , 4.68 ( d , j = 2.3 hz , 2h ) , 2.87 ( t , j = 2.4 hz , 1h ) ; c nmr ( 125 mhz , cd3od )  ( ppm ) 216.29 , 178.12 , 
 162.96 , 160.46 , 160.22 , 148.17 , 146.80 , 140.14 , 132.53 , 132.07 , 129.05 , 
 115.05 , 89.05 , 88.03 , 65.94 , 40.41 ; maldi - ms calculated for c16h12n2o3na [ m + na ] 303.0740 , found 302.9541 . to a solution of 5 ( 0.180 g , 
 0.642 mmol ) in thf under argon
was added n - hydroxysuccinimide 
 ( 0.177 g , 1.54 mmol ) and n , n-dicyclohexylcarbodiimide 
 ( 0.317 g , 1.54 mmol ) .
the reaction was let stir for 18 h at rt at 
 which time the reaction was concentrated by vacuum .
the mixture was 
 dissolved in ethyl acetate and filtered to remove solids . the flow - through 
 was concentrated and the crude product was purified by column chromatography 
 ( 1:10 ethyl acetate : ch2cl2 ) to afford the product 
 as a dark red solid that was used in the subsequent reaction without 
 further purification .
compound 6 ( 0.040 
 g , 0.101 mmol ) was dissolved in anhydrous n , n-dimethylformamide ( 1 ml ) under argon .
ez - link 
 amine peg3-biotin ( 0.460 mg , 0.111 mmol ) ( thermo scientific ) 
 was added and reaction let stir for 18 h upon which time solvent was 
 removed by vacuum .
the resulting crude mixture was purified by rp - hplc 
 over a c18 semipreparative column ( the nest group ) using a 5.544% 
 b linear gradient over 10 min before switching to a 44100% 
 b linear gradient over 40 min , tr = 18 
 min ( buffer a : 0.1% tfa in water , buffer b : 0.1% tfa , 90% acn in water ) 
 and lyophilized to afford the pure product as an orange solid ( 0.022 
 g , 31% ) : maldi - ms calculated for c34h40n6o8s ( oxidized at the biotin cysteine ) [ m + na ] 719.2839 , found 719.2656 . commercially 
 available 2-deoxy-2-n - acetyl - glucopyranose ( 2.50 
 g , 11.3 mmol ) was coevaporated from toluene and dissolved in anhydrous 
 pyridine ( 20 ml ) .
p - toluenesulfonyl chloride ( 2.59 g , 13.6 mmol ) was then 
 dissolved is anhydrous pyridine ( 5 ml ) and added dropwise to the stirring 
 mixture . upon completion of addition
, the reaction was allowed to 
 warm to room temperature and stirred for 16 h under an argon atmosphere . 
 to purify
, the reaction was concentrated under reduced pressure and 
 the crude mixture purified by column chromatography ( 7:1:0.5 ethyl 
 acetate : methanol : water ) to afford the product as a yellow oil ( 1.78 
 g ) : h nmr ( 500 mhz , cd3od ) -anomer  
 7.74 ( d , j = 8.3 hz , 2h ) , 7.26 ( d , j = 8.2 hz , 2h ) , 5.13 ( d , j = 3.6 hz , 1h ) , 3.99 ( m , 
 1h ) , 3.90 ( dd , j = 2.2 , 10.6 hz , 1h ) , 3.73 ( m , 1h ) , 
 3.55 ( dd , j = 3.2 , 13.3 hz , 1h ) , 3.44 ( dd , j = 5.4 , 12.6 hz , 1h ) , 3.38 ( m , 1h ) , 2.39 ( s , 3h ) , 2.01 
 ( s , 3h ) . the product was used in the subsequent reaction with no further 
 characterization .
compound 8 ( 1.78 g , 4.73 mmol ) was coevaporated from 
 toluene and dissolved 
 in anhydrous n , n-dimethylformamide 
 ( 20 ml ) .
sodium azide ( 1.54 g , 23.7 mmol ) was then added and the reaction 
 warmed to 50 c .
the reaction was stirred for 3 d after which 
 time the reaction was cooled and concentrated under reduced pressure . 

the crude mixture was purified by silica gel chromatography ( 9:1:0.5 
 ethyl acetate : methanol : water ) to afford the product as a white solid 
 ( 402 mg , 14% yield over 2 steps ) .
the sugar was further purified by 
 rp - hplc over a c18 semipreparative column ( the nest group ) using a 
 515% b linear gradient over 10 min , tr = 2.5 min ( buffer a : 0.1% tfa in water , buffer b : 0.1% tfa , 
 90% acn in water ) : h nmr ( 500 mhz , ( cd3)2so ) -anomer  7.69 ( d , j = 8.3 
 hz , 1h ) , 4.94 ( app s , 1h ) , 3.78 ( m , 1h ) , 3.62 ( m , 1h ) , 3.50 ( m , 2h ) , 
 3.37 ( m , 1h ) , 3.10 ( app t , j = 9.2 hz , 1h ) , 1.83 
 ( s , 3h ) ; c nmr ( 125 mhz,(cd3)2so ) 
 -anomer  169.39 , 90.77 , 71.85 , 70.52 , 70.17 , 54.21 , 
 51.60 , 22.67 .
the procedure 
 was adapted from literature . commercially 
 available 2-deoxy-2-n - acetyl - glucopyranose ( 5.00 
 g , 22.6 mmol )
was suspended in benzyl alcohol ( 50 ml ) and concentrated 
 hcl was added ( 1 ml ) .
the solution was warmed to 75 c and stirred 
 for 4 h after which time the reaction was cooled and poured into diethyl 
 ether ( 400 ml ) with vigorous stirring . a white precipitate was observed 
 and the mixture left at 4 c for 16 h. the precipitate was then 
 filtered and washed with diethyl ether ( 50 ml ) to remove remaining 
 benzyl alcohol .
the filtrated was dried and recrystallized in a minimal 
 amount of isopropanol to afford the product as white solid ( 2.48 g , 
 7.98 mmol , 35% yield ) : h nmr ( 500 mhz , ( cd3)2so )  7.82 ( d , j = 8.2 hz , 1h ) , 
 7.387.28 ( m , 5h ) , 4.71 ( d , j = 3.5 hz , 1h ) , 
 4.68 ( d , j = 12.5 hz , 1h ) , 4.31 ( d , j = 12.5 hz , 1h ) , 3.80 ( q , j = 6.1 hz , 1h ) , 3.703.64 
 ( m , 2h ) , 3.553.47 ( m , 2h ) , 3.18 ( t , j = 9.02 
 hz , 1h ) , 1.83 ( s , 3h ) . compound 10 ( 2.36 g , 7.58 mmol ) was coevaporated from toluene and dissolved 
 in anhydrous pyridine ( 20 ml ) under an argon atmosphere . the mixture 

p - toluenesulfonyl 
 chloride ( 1.74 g , 9.10 mmol ) , freshly rescrystallized from ch2cl2 , was dissolved in pyridine ( 7 ml ) and added 
 dropwise over 20 min .
the reaction was stirred at 20 c 
 for 1 h and the dry ice bath replaced with an ice bath .
the reaction 
 was allowed to warm to room temperature over 16 h. upon completion , 
 the mixture was concentrated to remove pyridine and purified over 
 silica gel ( 9:1:0.5 etoac : methanol : water ) to afford product ( 1.85 
 g , 3.97 mmol , 52% yield ) : h nmr ( 500 mhz , cd3od )  7.85 ( d , j = 10.4 hz , 2h ) , 7.46 ( d , j = 1.1 hz , 2h ) , 7.36 ( m , 5h ) , 4.76 ( d , j = 4.5 hz , 1h ) , 4.65 ( d , j = 15 hz , 1h ) , 4.45 ( d , j = 14.8 hz , 1h ) , 4.36 ( dd , j = 2.6 , 13.6 
 hz , 1h ) , 4.27 ( dd , j = 7.3 , 13.6 hz , 1h ) , 3.88 ( dd , j = 4.6 , 13.5 hz , 1h ) , 3.813.78 ( m , 1h ) , 3.713.66 
 ( m , 1h ) , 3.353.31 ( m , 1h ) , 2.46 ( s , 3h ) , 1.97 ( s , 3h ) .
compound 11 ( 1.85 g , 3.97 mmol ) was 
 resuspended in pyridine ( 20 ml ) and acetic anhydride ( 1.12 ml , 11.01 
 mmol ) .
the reaction was stirred for 3 h at room temperature after 
 which time the reaction mixture was concentrated and purified over 
 silica gel ( 75% etoac in hexanes ) to afford product in quantitative 
 yield ( 2.18 g , 3.97 mmol ) : h nmr ( 500 mhz , cdcl3 )  7.79 ( d , j = 10.3 hz , 2h ) , 7.377.29 
 ( m , 7h ) , 5.63 ( d , j = 11.9 hz , 1h ) , 5.20 ( dd j = 11.7 , 13.5 hz , 1h ) , 4.96 ( t , j = 12.1 
 hz , 1h ) , 4.82 ( d , j = 4.6 hz , 1h ) , 4.67 ( d , j = 14.7 hz , 1h ) , 4.44 ( d , j = 14.8 hz , 
 1h ) , 4.26 ( td , j = 4.6 , 12.6 hz , 1h ) , 4.07 ( d , j = 5.0 hz , 2h ) , 4.044.00 ( m , 1h ) , 2.44 ( s , 3h ) , 
 1.98 ( d , j = 2.6 hz , 6h ) , 1.86 ( s , 3h ) . procedure adapted from published literature .
pd(oh)2/c ( 10% pd ) was added 
 and a balloon of h2 was attached .
the reaction was monitored 
 by tlc ( 75% etoac in hexanes ) and stirred for 48 h to completion . 

the mixture was then filtered over a pad of celite and the flow - through 
 evaporated to yield the product ( 710 mg , 1.55 mmol ) that was used 
 in subsequent reactions with no further characterization .
compound 13 ( 629 mg , 1.37 
 mmol ) was coevaporated with toluene and resuspended in ch2cl2 ( 10 ml ) under an argon atmosphere .
5-(ethylthio)-1h - tetrazole 
 ( 1.07 g , 8.22 mmol ) was added and the reaction stirred for 15 min . 

diallyl - n , n-diisopropylphosphoramidite 
 ( 1.00 g , 4.11 mmol ) was added dropwise , and the reaction stirred for 
 2 h until completed as determined by tlc ( 5% methanol in ch2cl2 ) . at this time , the reaction was cooled to 78 

c and freshly recrystallized m - chloroperoxybenzoic 
 acid was added ( 1.18 g , 6.85 mmol ) .
the reaction was allowed to proceed 
 for 10 min after which time the dry ice bath was replaced with an 
 ice bath , and the reaction was slowly warmed to room temperature over 
 1 h. upon completion , the reaction was diluted with ch2cl2 ( 50 ml ) and washed 2 each with saturated sodium 
 thiosulfate , saturated sodium bicarbonate , water and brine .
the organic 
 layer was then concentrated and purified over silica gel ( 35%45% 
 acetone in hexanes ) to afford the product ( 717 mg,74% yield over 2 
 steps ) : h nmr ( 500 mhz , cdcl3 )  7.70 
 ( d , j = 8.4 hz , 2h ) , 7.29 ( d , j = 
 7.8 hz , 2h ) , 6.06 ( d , j = 9.4 hz , 1h ) , 5.935.82 
 ( m , 2h ) , 5.54 ( dd , j = 3.3 , 6.3 hz , 1h ) , 5.32 ( ddd , j = 17.1 , 12.3 , 1.4 hz , 2h ) , 5.23 ( ddd , j = 10.6 , 9.6 , 1.2 hz , 1h ) , 5.12 ( dd , j = 10.9 , 9.4 
 hz , 1h ) , 4.96 ( dd , j = 10.3 , 9.5 hz , 1h ) , 4.544.49 
 ( m , 3h ) , 4.254.21 ( m , 1h ) , 4.194.15 ( m , 1h ) , 4.04 
 ( dd , j = 11.1 , 2.6 hz , 1h ) , 3.98 ( dd , j = 11.1 , 5.1 hz , 1h ) , 2.39 ( s , 3h ) , 1.94 ( s , 3h ) , 1.93 ( s , 3h ) , 1.86 
 ( s , 3h ) ; c nmr ( 125 mhz , cdcl3 )  171.08 , 
 170.31 , 169.06 , 145.21 , 132.23 , 132.12 , 132.07 , 131.91 , 131.86 , 129.86 , 
 128.05 , 119.06 , 118.93 , 95.65 , 95.60 , 69.73 , 69.37 , 68.79 , 67.67 , 
 66.99 , 51.69 , 51.63 , 22.87 , 21.63 , 20.58 , 20.45 ; p nmr 
 ( 500 mhz , cdcl3 )  2.77 ; apci - hrms calculated 
 for c25h34no13psna [ m + na ] 642.1488 , found 642.1398 compound 14 ( 669 mg , 1.08 
 mmol ) was resuspended 
 is methanol ( 10 ml ) .
the reaction was monitored by tlc ( 10% methanol 
 in ch2cl2 ) and was determined complete after 
 1.5 h. upon completion , the reaction was quenched with acetic acid 
 and concentrated to afford the crude .
silica gel chromatography ( 7% 
 methanol in ch2cl2 ) yielded the product ( 282 
 mg , 57% yield ) : h nmr ( 500 mhz , cd3od ) 7.79 
 ( d , 2h , j = 8.3 hz ) , 7.44 ( d , j = 
 8.5 hz , 2h ) , 6.025.92 ( m , 2h ) , 5.59 ( dd , j = 8.5 , 8.5 hz , 1h ) , 5.425.37 ( m , 2h ) , 5.295.26 ( m , 
 2h ) , 4.604.55 ( m , 4h ) , 4.32 ( dd , j = 11.0 , 
 1.9 hz , 1h ) , 4.21 ( dd , j = 11.0 , 5.7 hz , 1h ) , 3.933.89 
 ( m , 1h ) , 3.863.82 ( m , 1h ) , 3.64 ( dd , j = 
 10.8 , 8.8 hz , 1h ) , 3.36 ( t , j = 8.7 hz , 1h ) , 2.46 
 ( s , 3h ) , 1.98 ( s , 3h ) ; c nmr ( 125 mhz , cdcl3 )  173.75 , 146.59 , 134.05 , 133.63 , 133.58 , 133.57 , 133.53 , 
 131.02 , 129.09 , 118.89 , 118.87 , 97.39 , 97.34 , 73.35 , 71.15 , 71.12 , 
 70.22 , 69.89 , 69.84 , 69.79 , 69.75 , 55.08 , 55.01 , 22.51 , 21.62 ; p nmr ( 500 mhz , cdcl3 )  2.51 ; apci - hrms 
 calculated for c21h30no11psna [ m 
 + na ] 558.1169 , found 558.1154 compound 15 ( 282 mg , 0.527 
 mmol ) was coevaporated from toluene and resuspended in n , n-dimethylformamide ( 20 ml ) under an argon 
 atmosphere .
sodium azide ( 172 mg , 2.63 mmol ) was added , and the reaction 
 reaction warmed to 60 c .
the reaction proceeded for 48 h after 
 which time the reaction was concentrated and purified over silica 
 gel ( 7:2:1 etoac : methanol : water ) to afford the product ( 151 mg , 71% 
 yield ) : h nmr ( 500 mhz , cd3od ) 6.075.73 
 ( m , 2h ) , 5.40 ( dd , j = 7.4 , 3.3 hz , 1h ) , 5.275.20 
 ( m , 2h ) , 5.065.01 ( m , 2h ) , 4.344.31 ( m , 2h ) , 4.274.24 
 ( m , 2h ) , 3.893.81 ( m , 2h ) , 3.59 ( dd , j = 
 10.6 , 8.9 hz , 1h ) , 3.49 ( dd , j = 13.2 , 2.5 hz , 1h ) , 
 3.363.31 ( m , 2h ) , 3.223.21 ( m , 1h ) , 1.91 ( s , 3h ) ; c nmr ( 125 mhz , cdcl3 )  173.81 , 135.99 , 
 135.92 , 116.18 , 116.15 , 95.59 , 95.54 , 73.63 , 72.48 , 72.37 , 67.33 , 
 67.29 , 67.17 , 67.13 , 55.33 , 55.27 , 52.63 , 22.85 ; p nmr 
 ( 500 mhz , cdcl3 )  0.66 , 1.37 .
compound 16 ( 50 mg , 0.123 mmol ) was resuspended in 4 ml of methanol : thf 
 ( 1:1 ) under an argon atmosphere .
p - toluenesulfinic 
 acid sodium salt ( 44 mg , 0.246 mmol ) and tetrakis(triphenylphosphine)-palladium(0 ) 
 ( 11 mg , 0.095 mmol ) were added .
the the reaction was monitored by 
 tlc ( 3:2:1 n - propanol : acetic acid : water ) and determined 
 complete .
the reaction was then evaporated under reduced pressure 
 and purified by silica gel chromatography ( 3:2:1 n - propanol : acetic acid : water ) .
the sugar was further purified 
 by rp - hplc over a c18 semipreparative column ( the nest group ) using 
 a 0% b isocratic flush over 10 min followed by a 050% b linear 
 gradient from 10 to 20 min and a second linear gradient 500% 
 b 2030 min , tr = 2.54 
 min ( buffer a : 0.1% tfa in water , buffer b : 0.1% tfa , 90% acn in water ) : h nmr ( 500 mhz , d2o )  5.33 ( dd , j = 7.3 , 3.4 hz , 1h ) , 3.943.83 ( m , 2h ) , 3.68 ( dd , j = 10.5 , 9.1 hz , 1h ) , 3.643.56 ( m , 2h ) , 3.533.43 
 ( m , 2h ) , 1.95 ( s , 3h ) ; c nmr ( 125 mhz , d2o ) 
  174.63 , 163.38 , 163.10 , 162.82 , 162.53 , 119.78 , 117.46 , 115.14 , 
 112.82 , 93.43 , 93.38 , 71.34 , 70.42 , 70.35 , 58.59 , 53.73 , 53.66 , 50.67 , 
 33.71 , 21.92 ; p nmr ( 500 mhz , d2o )  
 1.65 ; esi - ms calculated for c8h14n4o8p [ m  h ] 323.04 , found 
 325.00 .
 metabolic chemical reporters ( mcrs ) 
 of glycosylation are analogues 
 of monosaccharides that contain bioorthogonal functionalities and 
 enable the direct visualization and identification of glycoproteins 
 from living cells . each mcr was initially thought to report on specific 
 types of glycosylation . 
 we and others have demonstrated that several 
 mcrs are metabolically transformed and enter multiple glycosylation 
 pathways . 
 therefore , the development of selective mcrs remains a key 
 unmet goal . 
 we demonstrate here that 6-azido-6-deoxy - n - acetyl - glucosamine ( 6azglcnac ) is a specific mcr for o - glcnacylated proteins . biochemical analysis and comparative proteomics 
 with 6azglcnac , n - azidoacetyl - glucosamine ( glcnaz ) , 
 and n - azidoacetyl - galactosamine ( galnaz ) revealed 
 that 6azglcnac exclusively labels intracellular proteins , while glcnaz 
 and galnaz are incorporated into a combination of intracellular and 
 extracellular / lumenal glycoproteins . 
 notably , 6azglcnac can not be 
 biosynthetically transformed into the corresponding udp sugar - donor 
 by the canonical salvage - pathway that requires phosphorylation at 
 the 6-hydroxyl . in vitro experiments 
 showed that 6azglcnac can bypass 
 this roadblock through direct phosphorylation of its 1-hydroxyl by 
 the enzyme phosphoacetylglucosamine mutase ( agm1 ) . 
 taken together , 
 6azglcnac enables the specific analysis of o - glcnacylated 
 proteins , and these results suggest that specific mcrs for other types 
 of glycosylation can be developed . 
 additionally , our data demonstrate 
 that cells are equipped with a somewhat unappreciated metabolic flexibility 
 with important implications for the biosynthesis of natural and unnatural 
 carbohydrates . 
Introduction
Results and Discussion
Conclusion
Experimental Procedures