Synthesis of Orthogonally Protected Bacterial, Rare-Sugar and D

© 2013 Nature America, Inc. All rights reserved. Providencia alcalifaciens Providencia of polysaccharides wall cell the of capsules the ref. gonorrhoeae N. example, For positions. O6 or O4 O3, at sugars other various to connected are and sites acetyl or glycoproteinshave unusualmonosaccharides attachedto acetyl development vaccine carbohydrate-based host cell and and drug discovery offers avenues for target-specific the and pathogen the between discrimination allows difference structural peculiar This surface. cell human the on present not deoxy comprise glycoconjugates atypical amino sugars, which are bacterial The structures. glycan their in differ two However,the is also common in prokaryotes that protein glycosylation lished estab well is it now and refuted, been has notion this years, 20 eukaryotes to restricted be to believed function, once was and structure protein modulate to proteins carbohydrates of by modification post-translational the Glycosylation, I disaccharide fragment of the zwitterionic polysaccharide (Z T from after a single chromatographic purification. respectively, as orthogonally protected thioglycoside building blocks on a gram scale in 1–2 d, in 54–85% overall yields, T one-pot, and double-serial double-parallel bydisplacements azide, phthalimide, acetate and nitrite ions as nucleophiles. O b galactose ( (2,4-diacetamido-2,4,6-trideoxyhexose (D products. Here we describe a detailed protocol for the synthesis of orthogonally protected bacterial deoxy amino hexopyranoside sufficient purity in acceptable amounts, and therefore chemical synthesis is a crucial step toward the development of these good targets for drug discovery and carbohydrate-based vaccine development. Bacterial glycoconjugates comprise atypical deoxy amino sugars that are not present on the human cell surface, making them Published online 5 September 2013; Chemistry,of Department Indian Institute Technologyof Bombay, Mumbai, India. Correspondence should be addressed to S.S.K. ( Madhu Emmadi & Suvarn S Kulkarni rare-sugar and Synthesis of orthogonally protected bacterial, 1870 cereus AAT is present in the lipopolysaccharide of of lipopolysaccharide the in present AATis example, For residues. GalNAc or GlcNAc branched with along AATcontain ( bacteria as of a key component surfaces (FucNAc) ( of ellin Similarly, pentasaccharide. ( GalNAc core bacillosamine a prise NTRO hese hese procedures have been applied to the synthesis of hus, protocol 3 3 acylation. - In contrast to their eukaryotic counterparts that contain contain that counterparts eukaryotic their to contrast In d l -thiophenylmannoside -thiophenylmannoside was first converted into the corresponding 2,4-diols via deoxygenation or silylation at 6 troie ( -threonine

), whereas whereas ), | b d 1 VOL.8 NO.10VOL.8 d d D 5 -rhamnosyl- -rhamnosyl- and - -glucosamine (GlcNAc) -glucosamine residues are placed at the interior . Vaccine candidates against these bacteria are very much are very bacteria these . against Vaccine candidates d -galactosamine (GalNAc) at the reducing end, bacterial bacterial end, reducing the at (GalNAc) -galactosamine UCT Pseudomonas aeruginosa Pseudomonas -thiophenylmannoside -thiophenylmannoside in four steps via AAT Fig. 1 I ON T )), )), he he 2,4-diols were converted into 2,4-bis-trifluoromethanesulfonates, which underwent highly regioselective, and and N Streptococcus pneumoniae Streptococcus c

Fig. Fig. ; ref. -linked glycans of of glycans -linked | 2013 | d -glucosamine -glucosamine and N. meningitidis N. 1 8 ), whereas the branched GalNAc or or GalNAc branched the whereas ), ). In addition, several ZPSs located on the d | O 1

-mannosyl 2,4-diols can be efficiently transformed into various rare sugars and natureprotocols 4 -linked glycans located on the pilin of of pilin the on located glycans -linked , and in the repeating unit of of unit repeating the in and , doi:10.1038/nprot i. 1 Fig. S. mitis S. contain contain consist of DATDH ( of consist b Campylobacter jejuni Campylobacter d ) attached to a branched branched a to attached ) O -galactosamine building -galactosamine blocks starting from d lne gyas n flag on glycans -linked 1 -glycosamine building blocks 2 3– , 1 AT 0 N S. pneumoniae S. 5 .2013.11 and and . T Shigella sonnei Shigella -acetyl fucosamine fucosamine -acetyl DH), DH), his his would otherwise take 1–2 weeks. 1 C B. fragilis B. . Over the past past the Over . 2 2 displacement of triflates by azide in 2 d and in 66–70% overall yields. d 3 -bacillosamine, Fig. Fig. 1d l l Bacillus Bacillus Fig. 1 Fig. -serine-linked trisaccharide -serine-linked oftrisaccharide 7 -serine 1 com 1 3 1 9 PS and and , on on , , on on , – N N f ) ) a ), 2 - - - - - ; A .

1 1 (Z d d for strategy protection orthogonal one-pot elegant, regioselective an established Through co-workers and Hung approach, sugars. pioneering their of protection regioselective for devis in strategies ing engaged are groups Various groups. benzyl or acetyl acceptors containing glycosyl of a of variety synthesis allow rapid Et using by ars sug benzylated fully of mono-debenzylation regioselective a on protocol colleagues’ and Iadonisi hand, other the On strategies. chemoenzymatic or enzymatic via disaccharides and monosaccharides peracetylated of deacetylation regioselective a co-workers and Palomo sugars. protected fully of tection monodepro were for approaches regioselective reported notable oselective deprotection or protection of sugars as glycosyl used donors or acceptors be in complex glycan assembly,can via regi regioselectively that blocks access building to monosaccharide protected protocols efficient of development vaccines. glycoconjugate of development the toward step crucial the is synthesis chemical orthogo nally of protected building monosaccharide blocks through efficient Availability isolation. by amounts and purity able in However,demand. in accept are not accessible sugars the rare and the bacterial rare sugars is still missing. This is mainly mainly is This missing. still is sugars rare bacterial the and of blocks building protected orthogonally of routes diverse using by purpose this for procedures one-pot catalytic and tive tion glycosyla one-pot for acceptors and donors glycosyl protected selectively both access to catalyst, single a as (TMSOTf) fonate trifluoromethanesul trimethylsilyl using by hexopyranosides, -fucosamine, and 2-acetamido-4-amino-2,4,6-trideoxy- PS -glucose Over past few years, intense efforts have been devoted for the the for devoted been have efforts intense years, few past Over

2 A 6 1) 1) of . Subsequently, several other groups reported regioselec reported groups other several Subsequently, . 21,2 U Bacteroides Bacteroides fragilis nfortunately, they cannot be isolated with 2 27–3 , d d 3 b -Glucosamine -Glucosamine building blocks can be prepared -galactose SiH and I and SiH 2 - . However, a general protocol for the synthesis . synthesis the for However, protocol a general d -thiophenylmannoside. Neisseria Neisseria meningitidis 2 2 3 reagent combination. These routes routes These combination. reagent , d . -glucosamine [email protected] d -galactosamine, and a rare R 2 eadily eadily available 4 16–1 C and 6, 6, followed by d

-galactosamine -galactosamine 8 . Recently, two d ). -mannose

is based based is d -

1 9

used used

2 ------5

© 2013 Nature America, Inc. All rights reserved. cheaply available natural monosaccharide such as as such and monosaccharide natural abundant available an cheaply from derived intermediate, common envisioned a We that purifications. column-chromatographic three d the for especially over improvement, needed still it improvement ones, existing the marked a offered procedure the Although and to access procedure relay inversion C2,C4 double stepwise, efficient, highly a developed first we goal, Towardthis and rare-sugar the all accessing diastereomers. of separation intermittent and steps of number a entail also routes and AAT from deoxy sugars amino related and other expensive A materials. and/or starting purifications chromatographic and workup tedious manipulations, protecting-group multistep extensive with lengthy routes involve mostly approaches those strategies However, blocks carbohydrate building traditional by synthesized monosaccharide be can these of Many sugars. rare d the because rare are sugars not by available and isolation, because Figure 1 could be efficiently transformed into various rare-sugar and and rare-sugar various into transformed efficiently be could -galactosamine is very expensive. very is -galactosamine -galactosamine building blocks, which entailed eight steps and steps eight entailed which blocks, building -galactosamine We were interested in developing a short and general route for route general and a short Wein developing interested were bacterial the of synthesis the for reported are routes few A of N l d O -linked glycan HO

-Garner aldehyde -Garner C. jejuni OH -galactosamine building blocks from from blocks building -galactosamine HO HO H HO HO

HO | N. meningitidis O-linked glycanof HO Structures of various bacterial glycans containing deoxy amino sugars. AcHN H N O OH HO O NH HO O OH OH OH O AcHN O OH HO O OH O O AcHN O CO O AcHN P. aeruginosa O-linked glycanof 2 pilin OH H HO O OH de novo de HO O HO GalNAc O O 4 O 2 AcHN AcHN , respectively. These conceptually novel novel conceptually These ,respectively. OH HO HO O O O strategy was recently developed for for developed recently was strategy O O HN AcHN AcHN OH HO O DATDH d CO O -glycosamine building blocks. blocks. building -glycosamine O AcHN 2 FucNAc AcHN O O Ba HO O O HN NH c NH 2 d O HN CO O P. alcalifacien O-Polysaccharide repeatingunitof -thiomannoside OH O P 2 l O CO d -threonine O HO -glucosamine -glucosamine AcHN 2 d OH repeating unitof Zwitterionic polysaccharideA1 H -mannose, -mannose, HO O HO O 3 HO N HO s AcHN OH O2 HO B. cereu Polysaccharide repeatingunitof O O OH 33–3 2 O HO O O OH O 40,4 O O O AcHN OH O 4 O GlcNAc 3 9 s 2 1 OH H . . C AAT strainsATCCand14579

O 2 O

N B. fragilis O O O regioselective and one-pot nucleophilic displacements. Figure 2 3- the accessible accessible than C4-OTf for attack by a nucleophile. Thus, selective in C2-OTf axial the that ipated displacements using azide or nitrite anions, respectively. We antic shown that the presence of the could be achieved by adjusting the reaction conditions. It has been NO and derivative 2,4-bis-triflate double-parallel and double-serial inversions of the corresponding to rapidly access all the unusual sugars ( eoinversions and/or a double inversion at C2 and/or C4 positions at O3. We envisioned that 2,4-diol selective deoxygenation at C6, followed by tin-mediated acylation HO O HO HO O NHAc NH AA HO HO 2 O AcHN 1 OH D T O 4 -mannose Acylation Deoxygenation O 6 O 3 O OH

O -acyl group -acyl 5 | 2 1 OH A strategy to synthesize rare deoxy amino sugars via 2 O anions as nucleophiles, if the desired regioselectivity regioselectivity desired the if nucleophiles, as anions NHAc AA O 1 T O SPh R selective Regio =AcorBz reactions 43,48,4 natureprotocols We selected the readily available available readily the blocks. selected We building protected rare-sugar bacterial orthogonally of Synthesis Protocol development C6. at deoxygenation regioselective available cheaply from obtained easily be turn, in can, derivative The nucleophiles. as ions nitrite and acetate phthalimide, azide, by (OTf, trilfate) trifluoromethanesulfonates d the of displacement double-parallel and double-serial one-pot a involves strategy rare-sugar d bacterial orthogonally various protected, of for protocol synthesis the expedient an present we Here purifications. chromatographic and workup the obviate and steps the of number down cut would approach an Such reactions. one-pot and regioselective out galactosamine building blocks, by carrying obtained from from obtained uisite req The bacillosamine. with comparison in alone C2 at stereochemistry opposite DATDH, of AAT that and FucNAc, it has whereas as C4 and opposite C2 at and stereochemistry C5, and C3 at chemistry Compound sugars. its rare to the with relationship owing stereochemical material starting suitable thiomannoside -galactosamine -mannosyl or or -mannosyl 9 is essential for successful nucleophilic nucleophilic successful for essential is 3 PO 4 by using azide, phthalimide, acetate acetate phthalimide, azide, using by RO 2 3 D

-rhamnose Inversion d 4 P P = =Tf β 6 -rhamnose derivative derivative -rhamnose 3 -linkage -linkage in β H 5 2 OP - 2 O d could be manipulated by ster 1 -rhamnose -rhamnose 2 3 SPh 1 4 Inversion 1

by carrying out a regio a out carrying by | 4 ) ) via sequential, one-pot, (ref. (ref. VOL.8 NO.10VOL.8 5 d building blocks. Our Our blocks. building reactions One-pot 1 ransl 2,4-bis- -rhamnosyl d has the same stereo same the has -mannoside 1 X 4 d = 2 6 protocol -mannose via a via -mannose would be more more be would N ; ; 3 RO Fig. Fig. , PhthN,OH,AcO

d 4 Rare sugars | 2013 | X -rhamnose -rhamnose 2 3 could be be could 6 2 4 5 2 43,4 ) as the the as ) 4 O N 4 | 1 3 and and

7 β 1871 and SPh - d - - - -

© 2013 Nature America, Inc. All rights reserved. it was necessary to carry out a brief aque brief a out carry to necessary was it transformation, this In yield. overall 57% block AAT building the afford to ide phthalim potassium excess by displaced The exclusively. remaining C4-OTf was concomitantly (OTf), group triflyloxy C-2 the of displacement a regioselective highly (CH lammonium azide (TBAN with a stoichiometric amount of tetrabuty treatment upon which ing 2,4-bis-triflate, correspond the into converted first was manner. one-pot tially flates by sodium azide (NaN S double-parallel and (82%) (DATDH) derivatives 2,4-diazido the 2a manner. one-pot essentially an in (azide, acetate) or C4-OTf phthalimide of displacement philic allows reagents to of addition nucleo achieve the sequential subsequent which C2-OTf, of displacement first the in regioisomer single a generate to conditions reaction the of tuning careful the bacillosamine the block building access to ions azide by C4-OTf the of ment the of 4-OH fucosamine (v) triflation derivative fucosamine 3-OH obtain to group C3-OAc by by azide, followed by an S internal ative of the C4-OTf by nitrite ions to generate 4-OH fucosamine deriv S by followed azide, by C2-OTf of displacement block building AAT generate to ions phthalimide by C4-OTf the of placement dis concomitant and ions azide by C2-OTf the of displacement 5a/5b by to ions excess DATDHazide form blocks bis-triflates building (i) of triflation in of displacements nucleophilic oselective blocks building rare-sugar various to construct as intermediates common served (ref. acylation O3 ( sugar C6-deoxy corresponding the at C6 to obtain deoxygenated tively on ion nitrite by C4-OTf the of displacement nucleophilic by lowed sequential fol conditions, controlled under C2-OTf, of displacement azide Figure 3 1872 to as so triflation, the after workup ous protocol For the double-serial inversion, diol diol inversion, double-serial the For Accordingly, as in shown To test this hypothesis, compound and Figure Figure 3

HO 7 | CN) at CN) HO ; (ii) triflation of of ; triflation (ii) VOL.8 NO.10VOL.8 HO ; (iv) triflation of of ; (iv) triflation 2b

d | Preparation of -rhamnose were individually converted into 4 OH 1 and and 5b O

−30 °C smoothly underwent underwent °C smoothly −30 β (85%), respectively, via a via respectively, (85%), 2a/2b 6 4 - 9 SPh 4 ; (iii) triflation of of ; triflation (iii) d . The key to the success of this approach lies in in lies approach this of success the to key The . . The and conditions reaction the results of regi

Table N | 2013 | -rhamnose skeleton, seemed plausible. seemed skeleton, -rhamnose 5 2 displacement of tri of displacement 2 7 2. LiAlH 1. TsCl(1.1equiv),Py,RT,84% 3. RCl(1.1equiv),cat.Me 50–5 followed by S ) afforded 2,4-diols 2,4-diols afforded ) d 89% DIPEA (2.0equiv),THF,RT -rhamnosyl 2,4-diols 2b 1 2a 6 |

), which upon a highly regioselective regioselective highly a upon which ), . Our protocol involves the following: following: the involves protocol Our . natureprotocols ( followed by a highly regioselective S regioselective by a highly followed 4 3 R , nucleophilic displacement of C2-OTf C2-OTf of ,displacement nucleophilic (3.0equiv),THF,72% ) ) in acetonitrile =Ac)or92% Figure Figure 3 ) ) in an essen N 2b N 4 2 displacement of 2 the of C4-OTf displacement 2 2 displacement of the formed , diols 1 , regioselective nucleophilic nucleophilic , regioselective ( ( R 7 6 2 Fig. =Bz) followed by S SnCl in in 2b 5a 2a/2b ------2a 2a/2b

2 3 , and and ) ) was first regioselec . HO Figure 4 RO HO AcO are summarized summarized are 2b 2a N 2b 2 displacement displacement 2 R R =Ac =Bz . These diols diols These . OH 8 N

O | 2 2 displace O N Efficient synthesis of bacterial deoxy amino sugar thioglycoside building blocks. 3 SPh SPh 8 ; and and ; N 2 62% over3steps 3. CH 2. TBAN 1. Tf ------

H 2 2 O, 65 O, Py,DCM 3 CN, –30 by using nitrite anions. Accordingly, triflation of compound compound of triflation Accordingly, anions. nitrite using by reaction NaN of addition the before done was (DCM) dichloromethane of evaporation only inversions parallel double the all for of whereas product, crude weight the accurate the out find to product crude the of drying vacuum and workup aqueous brief a out carried we double inversions, serial the all for general, In temperature. low a at ment displace the exact conducting an and reagent using of amount by stoichiometric attained be could regioselectivity of tuning 2a/2b to the attributed of C-2-OTf the that be anticipated It is factors. can steric case this in observed regioselectivity The TBAN of equivalent triflate exact an add accordingly and triflate crude the of weight exact the obtain the the C3-acetate access derivative the generate and 4-OTf the displace to pot (TBANO nitrite reaction, tetrabutylammonium the of completion the After first. conducted was lier, TBAN with treatment concomitant its and prepared in 1–2 d, from a common intermediate intermediate common a from d, 1–2 in prepared the rare bacterial sugar building blocks building bacillosamine block the afforded C4-OTf of displacement (ref. C4 at inversion with group C4-triflyloxy the displace to ring sugar the of face top the along 3- the transformation, 3-hydroxy fucosamine generate to refluxed the was into reaction the water and of pot same amount small a added we Then, derivative. ner to (quinovosamine) obtain the 2-azido-4-OTf corresponding 3 , (1.0equiv), To access the Tothe access Finally, of triflation sequential

° C is more accessible as compared with the C-4-OTf. Further Further C-4-OTf. the with compared as accessible more is 9 ° d C in an essentially one-pot manner (81%). In this way, all this In (81%). manner one-pot essentially an in 58–6 -fucosamine derivative with a 3-OH free. Starting with with Starting free. 3-OH a with derivative -fucosamine 5b 5a 7 0

(60%) exclusively. We used a different strategy to to strategy different a used We exclusively. (60%) 8 R R , which involves nucleophilic displacement of triflate (62%) as a single product. In this water-mediated water-mediated this In product. single a as (62%) =Ac,82%over2steps HO =Bz,85%over2steps BzO RO 2a RO HO d N ,we ,we repeated the first two steps in a similar man 2b 2a -fucosamine derivative, we used a Lattrell-Dax Lattrell-Dax a used we derivative, -fucosamine 3

R R 7 3 =Ac OH =Bz . 2. NaN 1. Tf 60%over3step 3. TBANO CH 2. TBAN 1. Tf O O O N N 3 O 3 2 2 -acetyl group migrated from C3 to C4 C4 to C3 from migrated group -acetyl SPh SPh SPh O, Py,DCM O, Py,DCM 3 CN, –30 3 , DM 3 (1.0 equiv) 2 , –30 F 57%over3steps 3. PhthNK,DMF,RT CH 2. TBAN 1. Tf 2. NaN 1. Tf 6 ° 7 81% (2steps) C ° 2 1 followed by concomitant azide C toRT O, Py,DCM 3 s 2 ). CN, –30 O, Py,DCM 3 , 3 3 , DM (1.0 equiv) to obtain a single product. product. single a obtain to 5 2 – ) was added in the same same the in added was ) F 9 ° C could be conveniently d 3 , -fucosamine 4-OH 4-OH -fucosamine , as described ear described as ,

BzO N PhthN BzO 3 2a or or 9 β 6 -isomers -isomers O N O N 3 3 2b SPh SPh via via 2b - - -

© 2013 Nature America, Inc. All rights reserved. a T described earlier for for earlier protocol described migration acetate the Finally, ( respectively yields, overall 56% from obtained derivatives galactose 2-hydroxy 4-azido, the °C)), 25–30 (RT; and allowing it to reach room temperature TBAN of equivalents 3.0 adding TBANO of equivalents 3.0 adding first (i.e., reagents the of addition the of order the swapping By respectively. and protected orthogonally generated equivalents) (3.0 ion– nitrite TBANO using C4 at inversion one-pot mediated sequential a and group at C2-triflyloxy the of displacement philic 10b of double–serial triflation protocol, the inversion of use the manner. Similarly, one-pot essentially an in tion, purifica chromatographic single a after respectively, yields, overall 84% and 85% derivatives galactosamine 4-azido the afforded (DMF) formamide NaN using 2,4-bis-triflates corresponding the of flation are summarized in the results of regioselective nucleophilic displacements of tion at O3 and in conditions The excellent yields. overall reaction benzoyla or acetylation tin-mediated catalytic, highly by lowed from prepared ner d diols nosyl blocks. ing protected orthogonally of Synthesis purifications. column-chromatographic three involves available cheaply ( yields overall 57–85% in purification chromatographic single a after reactions, one-pot regioselective It involvesonlyC4OTf displacement bysodiumazide. 7 2a 2b 2b 2b 2a S a -galactosamine thioglycoside building blocks in a similar man in a similar blocks building thioglycoside -galactosamine ubstrate a b 4 le le , followed by a regioselective nucleo regioselective a by followed , 5 12b . For this purpose, first compounds 1 1 | , in 60% and 61% overall yields, yields, overall 61% and 60% in ,

−30 −30 °C by stoichiometric TBAN

10a Synthesis of bacterial rare-sugar building blocks. d -galactosamine derivatives -galactosamine y sn ti pooo, e rnfre te man the transformed we protocol, this using By N / ucleophile 1 10a/10b 10b 1 10a TBAN TBAN TBAN ( NaN NaN NaN d followed by a double-parallel displacement of of displacement by a double-parallel followed Fig. Fig. Figure -mannose, the entire process takes 4–5 d and and d 4–5 takes process entire the -mannose, and and 3 3 3 3 3 3 2a 13a into various orthogonally protected protected orthogonally various into 5 ) via regioselective silylation at O6, fol O6, at silylation regioselective via ) worked well on on well worked 10b 6 and and 11a and 2 at 0 °C, then then °C, 0 at N in 54% and and 54% in ucleophile 2 and and Table TBANO PhthNK 13b NaN NaN 3 10a OAc at 0 °C °C 0 at — Box Box 11b were were 3 3 and and 2 2 12a . As shown in 1 in in D 10a ). 3 Table - 2 glcoaie build -galactosamine ,

and P roduct Figure 6 3 5b 5a HO 1 in in 9 8 7 6 AcO ). Starting from from Starting ). 10b N,N Figure were readily OTBDPS

| Yield (%) 14 O N Efficient synthesis of -dimethyl -dimethyl 3 10a 81 62 60 57 85 82 SPh 6 / , tri 10b 3. 2. TBAN 1. Tf ------

H CH 56% over3steps 2 2 from O, 65 O, Py,CH 3 CN, –30 benzylidenation and (iii) C2 epimerization via nucleophilic nucleophilic via epimerization C2 (iii) and benzylidenation of acylation O3 regioselective highly a (i) omannoside protected orthogonally protected oglycosides. orthogonally of Synthesis 13 new compounds, which are thoroughly characterized by using All blocks in building reported the this monosaccharide study are from acetal in acetal other orthogonal Compound derivatives. cation. This route is useful for particularly obtaining glucosamine purifi respectively, chromatographic yields, a after overall single compounds to obtain purification could be out carried on a 2-g scale in 1 d anywithout intermittent sequence four-step the Alternatively, respectively. yields, overall gives step each at chromatography This column group. involving procedure C2-triflyloxy stepwise corresponding the of displacement well as well manner. one-pot a in 10a Figure 5 3

(1.0equiv), 10a C and 2D NMR spectral analysis. spectral NMR 2D and C

Thus, a complete set of orthogonally protected rare sugars, as as sugars, rare protected orthogonally of set complete a Thus, ° C to deliver smoothly the 3-hydroxy GalN only d

2 -galactosamine -galactosamine thioglycoside building blocks. ° d Cl C d -mannose in a highly efficient manner ( manner efficient highly a in -mannose

2 | HO HO -galactosamine building blocks, was rapidly synthesized synthesized rapidly was blocks, building -galactosamine Preparation of 17a HO d -GlcN / A simple protocol for the preparation of of preparation the for protocol simple A 1 HO 17b RO RO is outlined in in outlined is OH RO 13b 13a 1 O 11b 11a 10b 10a N N (Over 2steps) can be manipulated by manipulated be can d

3 3 3 R R

building blocks blocks building -glycosamine building blocks. The benzylidene SPh OTBDPS R =Bz,56% R R R = Ac,54% OTBDPS OTBDPS OH =Ac85% =Bz84 =Ac =Bz O OH O O N d 2. NaN 1. Tf natureprotocols 3. TBAN 2. TBANO 1. Tf -mannosyl 2,4-diols 3 DIPEA 2. RCl,Me 1. TBDPSCl,Py d CH 17a/17b 2 SPh SPh SPh 2 -glucosamine building blocks from thi blocks building -glucosamine O, Py,CH O, Py,CH % 3

CN, 0 over 3steps over 3steps , DMF,RT 3 (3.0equiv),0 Figure Figure 2 (3.0equiv), 2 3. TBANO 2. TBAN 1. Tf ° SnCl C 2 2 (3.0 equiv) can be elaborated into various various into elaborated be can CH Cl Cl 2 2 2 2 O, Py,CH 17a 3 17a , CN, –30 7 10b 10a 3 ( (1.0equiv), TBDPSO 2 ° and and

and and Box Box ,

R 10a/10b R | =Ac84%over2steps

VOL.8 NO.10VOL.8 =Bz87%over2steps regioselective reductive reductive regioselective HO 2 ° RO Cl C D 1 2 3 17b 17b 2 derivative derivative guoaie thi -glucosamine , (ii) a rapid 4,6- rapid a (ii) , ; ; ref. . in 45% and 44% 44% and 45% in in 66% and 70% in and 66% protocol OH Tables 1 1 Tables O RO 4 12b 12a HO 3

). It involves ).involves It SPh | 2013 |

R R (Over 2steps) =Ac60% =Bz61% OTBDPS 14 O N and 3 (56%) |

1873 SPh 1

O 2 H, ). - - - -

© 2013 Nature America, Inc. All rights reserved. derivatives derivatives Compounds protection. O6 selective and hydrolysis via or opening O6 or O4 T 1874 10a 10b 10a 10b 10a 10b 10a S a protocol Box 1 76.2, 68.0,62.4,60.8,27.0,19.3. 3.89-3.77 (m,3H,H-5,H-6a&H-6b), 1.06(s, 9H, 5.40 (dd, 13b 60.3, 26.9,20.9,19.3. 1H, H-5),2.19(s, 3H,CH 1H, H-3),4.50(d, 13a ANAL Total time for steps1–10:15.5h;allshould beperformed inasingle day. Steps 6–10:5h Steps 3–5:4h Steps 1–2:6.5h S ● 10. Characterize the product  9. Drythe residue under vacuumtoobtain compound 8. Collectthe fractions and evaporate the solvent byusing arotary evaporator. 254 nmorbyspraying withaCAMstainand subsequently heating onahot plate. acetate ataratio of 9:1(vol/vol)for 7. Identify the fractions containing compound 6. Work upasdescribed inPROCEDUREStep7A(v–x). mixture for stirring atRTfor 3h. 5. Afterthe completeconsumption of the starting material (asmonitored byTLC),add TBAN 4. Add 18.0mmol of TBANO 3. Add acetonitrile(12ml)tothe compound obtained from step2and coolthe flaskto0°C. 2. Perform PROCEDUREStep7B(i–v). 1. Perform PROCEDURESteps2–6(starting with6.0mmol of 2,4-bis triflatesbyusing TBANO The 4-azido galactosederivatives ubstrate b

ynthesis of 4-azidogalactose derivative 13aand13b

le le

13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( T PAUSE

H NMR(400MHz,CDCl H NMR(400MHz,CDCl | IMI

C NMR(100MHz,CDCl C NMR(100MHz,CDCl VOL.8 NO.10VOL.8 2 2 TLC petroleumether:ethylacetate9:1(vol/vol) TLC petroleumether:ethylacetate9:1(vol/vol) Y T | N I

Synthesis of galactosamine building blocks. CAL G

J 12a PO | N

Synthesis of 4-azido galactose derivatives 13a and 13b = 17a ucleophile 1 I D

9.6,3.6Hz1H,H-3),4.59(d, NT and TBANO TBANO m/z m/z ATA TBAN TBAN TBAN NaN NaN and and

Compounds | 2013 | ): [M ): [M 12b J 3 3

3 3 3

2 2 = 17b

9.6Hz,1H,H-1),4.19(d, via benzylidene hydrolysis, benzylidene via |

+ + natureprotocols 3 were also converted to galactosamine galactosamine to converted also were

3 3 Na] Na] 3 3 ), 1.06(s, 9H, ) ) N ) ) 2 δ δ (3.0equivalents) and keep the reaction mixture atthe same temperature for 30min. ucleophile 2 δ δ 13a 13a 8.14-8.12(m,2H,ArH),7.68-7.58 (m,5H,ArH),7.52-7.3610H,7.30-7.273H, 7.67-7.65(m,4H,ArH),7.50-7.389H,7.29-7.27 2H,ArH),5.07(dd,

166.2,135.7,133.8,133.0,132.8, 131.8,130.3,130.1,129.2,129.1,128.7,128.3,128.0,89.5, 77.4, 170.6,135.7,133.0,132.9,132.8,131.6,130.1,129.2,128.4, 128.0,89.3,77.28,76.0,67.5,62.4, + + TBANO TBANO

TBAN TBAN calculatedfor C calculatedfor C 2 NaN NaN OAc followed byTBAN or and 13a 3 3 13b 3 3 2 2 13a and 13b from step9byNMRspectroscopy using deuterated chloroform asthe solvent. (CH (R canbestored atRTfor several months without marked decomposition. 13b P f 3 J 0.4)and roduct 13b 12b 11b ) 13a 12a 11a

3 canbesynthesized inaone-pot manner bydouble–serial displacement of 13a = 30 35 CSi).

9.6Hz,1H,H-1),4.28(d, H H J or 35 3 37

R (CH regioselective regioselective

. = N N f

f 3.4Hz,1H,H-4),3.85-3.76(m,3H,H-2,H-6a&H-6b),3.70(t, 0.4 13a 0.4 3 3 13b Yield (%) 3 13b O O ) 5 5 3 NaSSi, 600.1964;found 600.1965. NaSSi, 662.2121; found 662.2158. (yield 54%)or CSi). 56 56 54 61 60 84 85 bysilica gel TLCdeveloped withamixture of petroleum ether and ethyl (R 10a f 0.4).The products are visualized bymeasuring UVabsorbance at or 10b charide charide moiety of disaccharide key the obtain to promoter a as (AgOTf) acceptor coside glycosyl thiogly the with glycosylated corresponding orthogonally was which bromide, the generate to bromine with treated blocks building in found A1 ZPS the of moiety disaccharide the of synthesis we a show herein short our methodology, of tion applica an As oligosaccharides. and glycoproteins bacterial the assemble to stereoselectively as acceptors or donors used glycosyl be stable could blocks building thioglycoside rare-sugar The the Applications approach of for followed be earlier purpose. this described procedure one-pot recommend the we and that lengthy, is sequence this However, condi tions. Lattrell-Dax under epimerization C4 and silylation O6 ). B. fragilis B. 13b J

(yield 56%)asapaleyellowish liquid.

= 18

3.6Hz,1H,H-4),3.97(t, is suitable for elaboration into the tetrasaccharide tetrasaccharide the into elaboration for suitable is

18 (81%, (81%, 6 12a and and by using silver trifluoromethanesulfonate trifluoromethanesulfonate silver using by 3 (3.0equivalents) and keep the reaction 12a α -only). Orthogonally protected disac protected Orthogonally -only). ( Fig. Fig. 8 ● ; ref. ref. ; J

T =

9.6Hz,1H,H-5), IMI 4 5 ). Thioglycoside Thioglycoside ). J

= B. fragilis B.

9.6,3.4Hz, G J ~15.5 h

= β

- 6.4Hz, d -mannosyl by using using by

was was - - - -

© 2013 Nature America, Inc. All rights reserved. protocol. Conceptually, this is the first example in which such a such which in example first the is this Conceptually, protocol. 6 blocks building the both that noted be should It unit. repeating Figure 7 and and HO Box 2  Next, quench the reaction mixture withtriethylamine. 17. Add benzaldehyde dimethylacetal (1.2equivalents) dropwise under N  Check the pHof the solution; itshould bebetween1 and 2. 16. Add 7mlof acetonitrileand keep the magnetic stirrer on.To thissolution, add 0.25equivalents of 10-camphor sulfonic acid. the flaskwithanitrogen balloon. 15. Drythe flaskunder vacuum for 1h,fillitwithnitrogen, remove it from vacuum manifold and capit with arubberseptum.Equip 14. Weigh 1.8mmol of S 13. Characterize the products 12. Drythe residue under vacuumtoobtain 11. Collectthe fractions and evaporate the solvent byusing arotary evaporator. subsequently heating onahot plate. ratio of 3:2(vol/vol)(R 10. Identify the fractions containing 9. Elutethe product withamixture of petroleum ether and ethyl acetateataratio of 3:7(vol/vol)and collectfractions of 20–30ml. rotary evaporator toobtainafree-flowing solid; loaditontop of the silica gel bed. 8. Dissolvethe crude product obtained from step6with10mlof ethyl acetateand add 1.5gof silica gel. Evaporate the solvent ona a ratio of 3:7(vol/vol). 7. Pack achromatography column(4cmi.d. ×25cmlength) withsilica gel byusing amixture of petroleum ether and ethyl acetate at 6. Extract withethyl acetateasdescribed inPROCEDUREStep7A(v–vii). (vol/vol) HCltoquench the reaction. 5. Keep the reaction mixture atRTfor 2.5h.Aftercompleteconsumption of starting material (asindicated byTLC),add 25mlof 3% for 4. To thissolution, add DIPEA(15.0mmol), Me 3. Add THF:H Equip the flaskwithanitrogen balloon. 2. Drythe flaskunder vacuum for 1h,fillitwithnitrogen, remove it from vacuum manifold and capitwitharubberseptum. 1. Weigh 7.5mmol of S PROCE protective gloves/protective clothing/eye protection/face protection. Camphor-10-sulfonic acid (Sigma-Aldrich, cat.no. 147923) respiratory and skinirritation. Donot breathe the vapor. Benzaldehyde dimethyl acetal(Sigma-Aldrich, cat.no. 226076) A regioselective O3acylation, 4,6- d

HO Ph HO -glucosamine building blockscanbeconveniently prepared from ynthesis of 3- ynthesis of benzylideneprotected mannosederivatives 16aand16b DDI

CR CR 15b 16b 16a 12a RO O T I I

T T | O I D

OH 1 R R Efficient synthesis of ONAL I I . O URE =Ac(90% CAL CAL =Bz(90% are prepared from from prepared are | OH SPh Synthesisoforthogonallyprotected

O STEP STEP M 2 O (48ml,19:1)and startthe magnetic stirrer. ATER O SPh ) ) RT, 2.5 THF: DIPEA (2equiv) BzCl orAcCl(1.1equiv Me -acyl-mannose derivatives 15aand15b The reaction mixture should not bekept for along time. The pHof the reaction mixture should beacidic. 2 SnCl I H ALS 2 1 h O 19:1 2 15a (1.0equivalents) into a50-mlround-bottom flaskand add a Teflon-coated magnetic stirbar. RT, 1 2. NaN DCM, 0°C,10min 1. Tf f (0.05equiv) 0.5).The products are visualized bymeasuring UVabsorbance at254nmorbyspraying withaCAMstainand d 2 or -glucosamine thioglycoside building blocks. O, pyridine h 3 , DMF, 15a d 15b -mannose by using the one-pot one-pot the using by -mannose O ) -benzylidenation and C2inversion via azide displacement of the C2-triflate. and (1.0equivalents) into a25-mlround-bottom flaskand add a Teflon-coated magnetic stirbar. 15a HO HO RO 15b 15a 15b 17b 17a and

R Ph R from step12byNMRspectroscopy, using deuterated chloroform asthe solvent. =Ac(90%) =Bz(92%)

R R 15a OH =Ac(82%over2steps) =Bz(85%over2steps) O 15b RO O 2 O (yield 90%,foam) and SnCl SPh bysilica gel TLCdeveloped withamixture of petroleum ether and ethyl acetateata 2 (0.37mmol) and 1.1equivalents of acetylchloride for O N 10–15 mi CH cat. CS PhCH(OMe) 3 3 CN, RT SPh

● ! A

n CAUT

T 2 IMI !

CAUT β I N - ON d G -thiomannoside ref. the of as such sugar abundant rally natu and a common from synthesized is disaccharide complex ing ing block date exclusive offered is spared. donors Of the various glycosyl donors tried, the trichloroacetimi individual of synthesis separate that so modification anomeric by selective trichloroacetimidates), halides, sulfoxides, glycosyl (e.g., donors of types various between switch to ibility flex the offer they Advantageously, months. RT for at stored be can which thioglycosides, stable all are blocks building the that acid amino the of OH primary the with monosaccharide the of coupling in ~10h Thiscompound causessevere skinburns and eyedamage; wear I 15b ON By using our protocol, we also accomplished the first synthesis synthesis first the we accomplished protocol, our By also using 2

4 atmosphere and keep for stirring atRTfor 10min. ● Thiscompound isharmfulifswallowed;itcauseseye, 4 d (yield 92%,whitesolid).

). Here a major challenge was achieving ). was achieving Here a challenge major α T -glucosamine buildingblocks IMI - l 5a -serine-linked trisaccharide of of trisaccharide -serine-linked N l was into transformed G -serine. One of the salient features of our method is method our of features salient the of One -serine. ~6h 1 via afour-step sequence involving natureprotocols α -selectivity. First, the thioglycoside build First, the -selectivity. thioglycoside d -mannose.

trichloroacetimidate donor trichloroacetimidate 15a

| VOL.8 NO.10VOL.8 orbenzoylchloride N. N. meningitidis α -stereoselectivity -stereoselectivity protocol

(continued) | 2013 |

(

Fig. Fig. |

1875 19, 9 - - - - ;

© 2013 Nature America, Inc. All rights reserved. 1876 protocol Total time for steps26–41:12h;allshould beperformed in asingle day. Steps 36–41:5h Steps 30–35:5h Steps 26–29:2h S Total time for steps14–25:6h;allshould beperformed inasingle day. Steps 23–25:1h Steps 18–22:3h Steps 14–17:2h S Total time for steps1–13:10h;allshould beperformed inasingle day. Steps 8–13:5h Steps 6and 7:1h Steps 1–5:4h S ● products  41. Drythe residue under vacuumtoobtain 40. Collectthe fractions and evaporate the solvent byusing arotary evaporator. stain and subsequently heating them onahot plate. a ratio of 9:1(vol/vol)(R 39. Identify the fractions containing 38. Purifybycolumnchromatography asdescribed inPROCEDUREStep7A(viii–x). 37. Extract withethyl acetateasdescribed inPROCEDUREStep7A(v–vii). mixture withethyl acetate(15ml). by measuring UVabsorbance at254nmorbyspraying withaCAMstainand subsequently heating onahot plate),dilutethe reaction 36. Aftercompleteconsumption of starting material (asmonitored byTLC,developing withhexane:ethyl acetate(8:2)and visualizing 35. To thissolution, add NaN 34. Add DMF(3.5ml)tothe compound obtained from step33and turnthe magnetic stirrer on. 33. Keep the residue under vacuumfor 2h,and then fillthe flaskwithnitrogen and capitwitharubberseptum. 32. Remove the sodiumsulfate and the DCMasdescribed inStep7A(vi,vii). 31. Perform asolvent extraction asdescribed inPROCEDUREStep7B(i,ii).  the product byUVabsorbance at254nmorbyspraying withaCAMstainand subsequently heating onahot plate. 30. Afterthe completeconsumption of starting material (asmonitored byTLC,developing withhexane:ethyl acetate(8:2)),visualize 29. Add 3.12mmol of pyridine and keep the reaction mixture atthe same temperature for 10min. cool the flaskto 28. Add dry DCM(9ml)byusing asyringe and turnthe magnetic stirrer on.Add trifluoromethanesulfonic anhydride (3.12mmol) and Equip the flaskwithanitrogen balloon. 27. Drythe flaskunder vacuum for 1h,fillitwithnitrogen, remove it from the vacuum manifold and capitwitharubberseptum. 26. Weigh 1.6mmol of S 25. Characterize the products 24. Drythe residue under vacuumtoobtain 23. Collectthe fractions and evaporate the solvent byusing arotary evaporator. heating onahot plate. (vol/vol) (R 22. Identify the fractions containing 21. Elutethe product withamixture of petroleum ether and ethyl acetateataratio of 7:3(vol/vol)and collectfractions of 20–30ml. rotary evaporator toobtainafree-flowing solid; loaditontop of the silica gel bed. 20. Dissolvethe crude product obtained from step18with5mlof ethyl acetateand add 0.5gof silica gel. Evaporate the solvent ona at aratio of 7:3(vol/vol). 19. Pack achromatography column(2cmi.d. ×15cmlength) withsilica gel byusing amixture of petroleum ether and ethyl acetate 18. Evaporate the solvents onarotary evaporator under reduced pressure. Box 2

ynthesis of glucosaminederivatives 17aand17b ynthesis of glucosaminederivatives 17aand17b ynthesis of benzylideneprotected mannosederivatives 16aand16b ynthesis of 3-

T PAUSE CR

| IMI VOL.8 NO.10VOL.8 I T N I CAL G 17a

PO | f (continued)

0.5).Products are visualized bymeasuring UVabsorbance at254nmorbyspraying withaCAMstainand subsequently I STEP and NT O

Compounds | 2013 |

− -acyl-mannose derivatives 15aand15b Ensure thatthe starting material iscompletely consumed; ifnot, keep the reaction mixture for anadditional 10min. 17b

0 °C. from step41byNMRspectroscopy, using deuterated chloroform asthe solvent. 16a | natureprotocols f 0.4).The products are visualized bymeasuring UVabsorbance at254nmorbyspraying them withaCAM or 13a 3 16a (3.12mmol) and keep the reaction mixture for stirring atRTfor 1h. 16b and and (1.0equivalents) into a25-mLround-bottom flaskand add a Teflon-coated magnetic stirbar. 16a 17a 13b 16b and and canbestored atRTfor several months without marked decomposition. Characterize the from step24byNMRspectroscopy, using deuterated acetone asthe solvent. 16a 17a 17b 16b (yield 82%,Whitesolid) and (yield 90%,solid) and bysilica gel TLC,developed withamixture of petroleum ether and ethyl acetateat bysilica gel TLCdeveloped withamixture of petroleum ether and ethyl acetate3:2

●

T IMI N G ~12h 16b (yield 90%,whitesolid). 17b (yield 85%,whitesolid). (continued) © 2013 Nature America, Inc. All rights reserved. tant glycoconjugates, including mucin type type mucin including glycoconjugates, tant The glycans. and GalNAc bacterial various synthesizing for used be can blocks building rare-sugar way,other similar a In yield. overall upon global deprotection delivered the target molecule (ref. of glycosylation acceptor acid amino glycosyl afforded methanol) and (triethylamine group acetate O3 of removal tive single a generate cleanly to the with coupled concomitantly was which Synthesis of these glycosamine-containing glycoconjugates can can glycoconjugates glycosamine-containing these of Synthesis N 5.35 (s, 1H,H-1),5.07(dd, 16a H-2), 4.21 (t, (t, 15b 4.30 (d, 15a ANAL (m, 6H,ArH),5.72(s, 1H,benzylidene), 5.46(s, 1H,H-1),5.40(dd, 16b 69.0, 20.9. 1H, H-5),2.03(s, 3H,CH 4.27 (dd, 87.3, 78.5,73.6,70.9,68.5,64.1. H-6ax), 3.71(t, 1H, H-3),5.47(s, 1H,benzylidene), 4.69(d, 17b 63.7, 20.9. H-5), 3.41(t, H-3), 4.61(d, 17a 88.67, 76.5,75.0,72.0,71.6,69.1. H-5). (t, - Box 2

lcpoen, lb- ad agisd cne antigens cancer ganglioside and globo-H glycoproteins, 1 HRMS calculatedfor C 13 1 HRMS calculatedfor C 13 1 1 HRMS calculatedfor C 13 HRMS calculatedfor C 13 1 HRMS calculatedfor C 13 1 HRMS calculatedfor C 13 4 J J H NMR(400MHz,(CD H NMR(400MHz,(CD H NMR(400MHz,CDCl H NMR(400MHz,(CD H NMR(400MHz,CDCl H NMR(400MHz,CDCl

C NMR(100MHz,CDCl C NMR(100MHz,(CD C NMR(100MHz,(CD C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,(CD

= TLC petroleumether:ethylacetate 9:1(vol/vol) TLC petroleumether:ethylacetate7:3(vol/vol) TLC petroleumether:ethylacetate3:7(vol/vol) TLC petroleumether:ethylacetate 9:1(vol/vol) TLC petroleumether:ethylacetate 7:3(vol/vol) TLC petroleumether:ethylacetate3:7(vol/vol) = Y ) generated exclusively exclusively ) generated

7.5 Hz, 2H, ArH), 7.23 (t, T 10.0Hz,1H,H-4),4.31(dd, I J CAL

| =

d =

(continued) 3.0Hz,1H,H-2),4.25(t, D

-GlcNAc motifs are also present in several impor several in present also are motifs -GlcNAc 10.0,5.0Hz,1H,H-6eq),4.19(t, J J J ATA

J =

9.8 Hz, 1H, H-4), 3.92 (dd,

10.0Hz,1H,H-2),2.11(s, 3H,CH

with the di-galactosyl di-galactosyl the with 10.0Hz,1H,H-1),4.39(dd,

9.6Hz,1H,H-4),3.63(dt, 26 21 26 21 19 14 3 3 3 3 3 3 3 3 3 3 ) ) ) H H H H H H 3 3 3 ). ) ) ) ) ) ) 2 2 2 ) ) ) 2 2 2 22 20 18 23 21 24 α CO) CO) CO) CO) CO) CO) δ δ δ J δ δ δ -isomer (92%), which upon selec upon which (92%), -isomer O O O O O O

8.08(d, 7.59-7.55(m,2H,ArH),7.42-7.338H,5.48(s, 1H,benzylidene), 5.24(t, 7.42-7.39(m,2H,ArH),7.23-7.193H,4.94(s, 1H,H-1),4.83(dd, α 165.4,136.7,133.8,133.5,130.8,130.0,129.4,129.3,129.1, 128.9,128.5,128.3,126.2,101.5, 169.7,136.8,134.0,130.7,129.39,129.33,129.03,128.4,126.2,101.6,87.1,78.4,73.1,70.8,68.5, 171.4,134.2,131.0,129.2,127.6,87.3,80.3,76.7,71.0,63.6,61.2,21.2.

= 6 6 6 5 6 5 -linked product product -linked S [M S [M S [M SN S [M SN

δ δ δ 10.0,3.3Hz1H,H-3),5.00(dd, δ δ δ J 8.08(d, 7.49-7.44(m,4H,ArH),7.37-7.305H,7.26-7.221H,5.65(s, 1H,benzylidene), 8.08(d, 166.3,138.9,136.8,134.0,131.0(2C),130.5,129.9,129.6,129.3,128.8,127.5,127.1,102.3, 170.8,138.9,136.7,130.4,129.8,129.6,128.8,127.5,127.2,102.4,88.46,76.3,74.3,71.9,71.4, 166.7,137.0,133.8,131.3,130.5,130.3,129.8,129.2,127.3,87.7,82.0,79.0,71.4,65.3,62.5.

3 3

= [M [M

7.5 Hz, 1H, ArH), 5.27 (s, 1H, H-1), 5.10 (dd,

J + + + +

Na] Na] Na] Na]

= J

+ + J

10.0,5.0Hz,1H,H-6eq),3.90(t,

Na] Na] =

21 = α

J J 7.2Hz,2H,ArH),7.62-7.56(m,3H,7.40-7.2710H,5.50(t,

+ + + + 10.0Hz,1H,H-4),3.93(bs, 2H,H-6),3.37-3.35(m,1H,H-5),2.14(s, 3H,CH

-glycosyl chloride chloride -glycosyl (80%). Subsequent Subsequent (80%). 487.1191;found 487.1171. 425.1035;found 425.1026. 399.0878;found 399.0880. 337.0722;found 337.0711. l

= =

J + + -serine acceptor acceptor -serine J

7.2Hz,2H,ArH),7.62(t, 7.0Hz,2H,ArH),7.62(t,

450.1100;found 450.1079. 512.1256;found 512.1264.

= O J

23 J 12.0, 2.5 Hz, 1H, H-6a), 3.81 (dd, J

9.6Hz,1H,H-1),4.39(dd,

-glycoproteins, -glycoproteins,

= (80%), which R R R R R R

11.2,4.4Hz,1H,H-6eq),3.78(t,

9.6,4.4Hz1H,H-5),3.58(t,

10.0Hz,1H,H-4),3.84(t, f f f 3 f f f 0.5 0.5 0.4 0.5 0.5 0.4 ). 24 in 51% 20 22 6 d 2 - - - . ,

5.7,0.8Hz,OH;exchangeable byD J bio-orthogonal probes for live-cell imaging live-cell for probes bio-orthogonal using further them tag to order in glycans surface bacterial into selectively incorporated metabolically be can that intermediates sugar azido varied structurally provide also would It assembly. synthesis oligosaccharide automated with in blocks conjunction building rare-sugar tected pro orthogonally such of availability Ready protocol. an expedient by of facilitated greatly be Synthesis would candidates candidates. vaccine antigenic vaccine glycoconjugate potential as T tected block building used Toward protocol. this end, we have this through be achieved also

= Bacterial glycoproteins and oligosaccharides are emerging emerging are oligosaccharides and glycoproteins Bacterial

10.0,3.3Hz1H,H-3),4.58(d, J J

= = J

7.5Hz,1H,ArH),7.52-7.48(m,4H,7.31 7.5Hz,1H,ArH),7.52-7.41(m,6H,7.35-7.23

= J N

10.0Hz,1H,H-6ax),3.79(dt,

cancer antigen cancer = J

9.6,5.0Hz1H,H-6eq),3.80(t,

= J J

= 10.0Hz,1H,H-6ax),3.69(dt,

= =

9.8, 3.3 Hz, 1H, H-3), 4.43 (d,

12.0, 5.1 Hz, 1H, H-6b), 3.60-3.56 (m, 1H, H-5). 9.6Hz,1H,H-2). J

10.0Hz,1H,H-4),3.55-4.52(m,2H,H-6a, 14 natureprotocols for the synthesis of orthogonally pro orthogonally of synthesis the for 4 6 5 3 . would expedite the bacterial glycan glycan bacterial the expedite would 2 J O), 4.43-4.38(m,1H,H-2),

= J

3.3Hz,1H,H-2),4.39

J |

10.0,3.0Hz1H,H-3), VOL.8 NO.10VOL.8

10.0,5.0Hz,1H, J J 64–6

J = =

= 9.6Hz,1H, 10.0Hz,1H,

= 6

protocol 10.0,5.0Hz,

3.3 Hz, 1H, . J

= 3 ).

9.6Hz, | 2013 |

1877 - - © 2013 Nature America, Inc. All rights reserved. • • • • andunderglassware nitrogen atmosphere. out inafumehood,reactions carried shouldbe usingclean anddefect-free ! REAGENTS M a glycan. of bacterial particular synthesis a planning when considered be should aptitude migratory This implications. useful with pair form complementary a groups OAc and OBz the Together, group. acetate extent the same as the to migrate not does group 3-OBz the because of place diol contrast, In conditions. these under fucosamine in successfully place of compound that is method our of limitations the of One form. to starts compound diazido corresponding the than higher not be raised step should this of protocol. The temperature serial double- the during step displacement first the in regioselectivity desired the obtain to as so conditions the temperature the reactions, observe critically to one-pot necessary is it that regioselective remember should the experimenter of success the For design Experimental B. fragilis Figure 8 1878

protocol allergic skinreaction. allergic swallowed; itcausessevereif andeye skinburns damage andmay causean Benzoyl chloride (Spectrochem, cat. no. 0102214) severe and eye skinburns damage; wear protective gloves/eye protection. flammable water; liquid, itisahighly with reacts violently and itcauses Acetyl chloride (Spectrochem, cat. no. 0101168) gloves/protective clothing/eye protection/face protection. fire;gas, it under handle inert protect itfrom moisture and wear protective water, contactpossible with reaction flash andpossible violent of because compound causessevere andeye skinburns damage; keep itaway from any Lithium aluminum hydride (Spectrochem, cat. no. 011208) protection. ! p CAUT ATER

-Toluenesulfonyl chloride (TsCl; Spectrochem, cat. no. 0120142) CAUT

| I VOL.8 NO.10VOL.8 ON I I

| . ON ALS A short synthesis of the disaccharide moiety of the ZPS A1 from 2a Always wear eye protection, alabapron andhandgloves. the All PhthN There is a risk of serious damage to the eyes; wear eye/face eye/face wear eyes; the to damage serious of risk a is There BzO for synthesizing the 3-OH fucosamine derivative derivative fucosamine 3-OH the synthesizing for 7

− , as the 3-OAc group in in group 3-OAc the as ,

30 °C. The regioselectivity is lost at at lost is regioselectivity The °C. 30 6

AgOTf, 4ÅMS,sym-collidine | 2013 | O N 3 SPh –30 2b | °

C, 2h,81% natureprotocols for the of preparation AAT CH repeating unitof Disaccharide moietyoftheZPSA1 Br 2 PhthN Cl 2 BzO 2 PhthN BzO AcO N O 3 , O B. fragilis 2a 18 AcO OTBDPS N ! is prone to migration migration to prone is O N 3 O

HO 2b CAUT ! Br 3

CAUT cannot be used in in used be cannot 12a SPh 2a OTBDPS I O N ON cannot be used used be cannot I 3 ON Acetyl chloride ! SPh

It isharmful

CAUT − 6 and 4-OH

15 °C and and °C 15

I ON This This 8

• • • • • of Figure 9 if swallowed; wearif protective gloves/eye protection/face protection. ! Trifluoromethanesulfonic anhydride (Spectrochem, cat. no. 0120199) protection. protection/face clothing/eye gloves/protective protective wear product; this to water add Never damage. eye and burns skin severe causes (H Sulfuric acid causing cancer; wear suitable protective clothing andgloves. itisswallowed; itcausesskinirritation, if of anditissuspected harmful Chloroform (Merck, cat. no. 82226592001730) from ignition. sources of swallowed, anditcausessevere andeye skin burns damage; keep itaway ! protection. clothing/eye gloves/protective protective wear life; aquatic to toxic very is it and ! Dimethyltin dichloride (Me N

N. N. meningitidis CAUT CAUT CAUT , N -diisopropylethylamine (DIPEA; Spectrochem, cat. no. 0104261) I I I

ON ON ON | Application of the protocol. Total synthesis of the trisaccharide This compound reacts violently with water, compound This with reacts violently anditisharmful flammable liquid DIPEAvapor. and isahighly It is toxic if irritation, respiratory cause may dichloride Dimethyltin AcO 2. Et 1 . 20 AcO 3 , TMSOTf,–78 N, MeOH,80% 2 . 3. NaOMe,MeOH, 2. NaBrO thenAcCl,Py,72% 1. PPh SO AcO OA H H O 4 2 2 BnO 22 c ; Merck, cat. no. 61752790301730) O, 55 O, EtOAc,82% 3 AcO HO , AcO O H HO 3 , Na 2 ° O, Py,THF BnO C, 87% OBz 2 ° SnCl 2 AcO AcO OH C, 92% AcO O OAc HO S HO Cl 2 O O O BnO

N N N HO 4 2 3 3 , 3 CbzHN ; Sigma-Aldrich, cat. no. 288012) 24 19 5a O O 21 N 2.CCl 1. NBS,THF, N N CbzHN sym-collidine 22 O O O BnO 3 3 3 OBz O O OH HO , AgOTf,–30 23 HO O O 80% 3 SPh NH O O CN, DBU,80% N NHAc CO 20 CCl 3 CbzHN CbzHN AcHN 2 CO Me ! 3 N

O H O CAUT 3 2 O 2 O Me ° O, 88% C CO I COO ON !

2 CAUT Me Chloroform is H I ON H

© 2013 Nature America, Inc. All rights reserved. ( reactions using thisstarting material are described in 2a 1| PROCE • • • • • • • • • • • • • • • • • • • • if swallowed or if inhaled; wear swallowed protective or if if gloves. ! 2,4,6-Trimethylpyridine (sym-collidine; Sigma-Aldrich, cat. no. 27690) protective clothing andgloves. ! Silver trifluoromethanesulfonate (AgOTf; Sigma-Aldrich, cat. no. 176435) flames/hot surfaces; itcausesdamage to organs. flammable liquidvapor; and highly it keep away from heat/sparks/open Methanol (Merck, cat. no. 60600825001730) eye/face protection. from ignition. sources of Wear suitable protective clothing, gloves and swallowed andcausessevere burns; keep itinacool place, andkeep itaway flammable,highly on inhalation, harmful with skin or if on contact Triethylamine (Merck, cat. no. 8083520100) protective contact occurs. eyes/skin immediately clothing andrinse if Molecular sieves causeeye, wear suitable irritation; skinandrespiratory Molecular sieves, 3Å(Sigma-Aldrich, cat. no. 233641) wear suitableirritation; protective clothing, gloves andeye/face protection. no. 91741) Trimethylsilyl(TMSOTf; trifluoromethanesulfonate Sigma-Aldrich, cat. protective clothing, gloves andeye/face protection. ! 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU; Spectrochem, cat. no. 010438) to theenvironment. acetonitrile istoxic long-lastingeffects; to avoid aquatic life with itsrelease Trichloroacetonitrile (Spectrochem, cat. no. 0120238) flammable liquidvapor, and highly eye irritation. anditcausesserious Tetrahydrofuran Spectrochem, (THF; cat. no. 0120176) N N protection. gloves/protective clothing/eye protection/face protection andrespiratory toxic to aquatic life; avoid itsrelease to theenvironment. Wear protective severe andeye skinburns damage. itisinhaled, It andisvery isfatalif Bromine (Merck, cat. no. 60194500201730) protective clothing, gloves andeye/face protection. ! tert ! Tetrabutylammonium (TBANO nitrite eye irritation. andserious Phthalimide potassiumsaltcausesskinirritation Phthalimide potassiumsalt(Sigma-Aldrich, cat. no. 160385) inhaled. skinor if with swallowed, if flammable itisharmful liquidvapor; and highly on contact Acetonitrile (Merck, cat. no. 61848805001730) environment. ! Tetrabutylammonium azide (TBAN skin,with eye irritation. anditcausesserious ! toxic; exposure harmful. skincontact very canbe via azideSodium (NaN protective clothing andgloves. not breathe itsvapor, avoid skinandeyes andwear contact suitable with CaH Dichloromethane (DCM;Merck, cat. no. over 60604990251730)(dried swallowed. skinor if contact with with water; on inhalation, itisharmful violently flammable and reacts on (Merck,Pyridine cat. no. 82230125001730) A N,N

CAUT CAUT CAUT CAUT CAUT CAUT CAUT

-Bromosuccinimide swallowed; wear if protective isharmful gloves. -Bromosuccinimide (Spectrochem, cat. no. 010230) and ) (i) -Butylchlorodiphenyl Spectrochem, silane(TBDPSCl; cat. no. 0102208) S There are twostarting materials thatcanbeusedfor thismain protocol: option Adescribes the synthesis of compounds 2 -dimethyl formamide (DMF; Merck, cat. no. 61836005001730) ) ynthesis of 2aor2b a Teflon-coated magnetic stir bar. Weigh 35.0 mmol of phenyl-1-thio- D I I I I I I I ! ON ON ON ON ON ON ON

2b CAUT URE This compound avoid This causesskinirritation; itsrelease to the on DMFisaflammable itisharmful liquidvapor; and contact 2,4,6-Trimethylpyridine isaflammable liquid, anditisharmful AgOTf causeseye, wear suitable andskinirritation; respiratory DBU causessevere andeye skinburns damage; wear suitable wear suitable irritation; andrespiratory TBDPSCl causesburns TBANO ! , and option Bdescribes the synthesis of compounds

CAUT I ON DCM has limited evidence of acarcinogenic effect; do haslimited of evidence DCM I ON 3 2 ; Merck, cat. no. 61824401001730) causeseye, system respiratory andskinirritation. TMSOTf isflammable, TMSOTf and causes burns respiratory

● 3

; Sigma-Aldrich, cat. no. 651664) T IMI 2 ; Sigma-Aldrich, cat. no. 86884) N ! ! !

G CAUT

CAUT

CAUT CAUT ! 30.5h

CAUT I I I β ON ON I ON ON - I ! ! d ! Pyridine is highly ishighly Pyridine ON Bromine causes

This reagent This is -mannopyranoside ! CAUT CAUT

! Methanol isa CAUT

CAUT CAUT Acetonitrile isa ! I I I ON

ON I CAUT ON I ON ON

Trichloro

It isvery THFisa Box

2 - . 1 10a • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • EQUIPMENT • • (1.0equivalents) into a250-mlround-bottom flaskand add Petroleum ether (Merck, cat. no. 61782225001730) no.61782225001730) cat. (Merck, ether Petroleum flammable liquid; it keep away from flames/hot heat/sparks/open surfaces. Toluene (Merck, cat. no. 61836605001730) away from ignition. sources of or cracking, andvapors maydryness causedrowsiness anddizziness; keep it flammable;eye highly itcauses irritation; repeated exposure may causeskin Ethyl acetate (Merck, cat. no. 61757092001730) bicarbonate(Merck,Sodium cat. no. 61752090501730) chlorideSodium (Merck, cat. no. 61751990201730) sulfateSodium (Merck, cat. no. 61752190511730) andgloves HCl. handling while goggles wear safety irritation; ! Hydrochloric (HCl; acid 35%(vol/vol), Merck, cat. no. 61752690251730) protective clothing/eye protection/face protection. eye damage; andmay itisself-heating catch fire; wear protectivegloves/ methoxide water; itcausessevere with reacts violently and skinburns methoxideSodium (Spectrochem, cat. no. 011913) toxic gases, sokeep itaway from heat, andflames. sparks dithionite isself-heating, anditmay catch fire;acids liberates with contact dithioniteSodium (Fisher Scientific, cat. no. 7775146) combustible contact with materialirritation; may causefire. may intensify fire. It isan oxidizer. swallowed andcausesskin if It isharmful bromateSodium (Merck, cat. no. 8450660250) or repeated inhaled; wear exposure protective if gloves. reaction, aswelldamage to (Nervous organs system) through prolonged Triphenylphosphine swallowed; itmay skin if isharmful causeanallergic Triphenylphosphine (Spectrochem, cat. no. 0120102) Vacuum lineandnitrogen source (Scam) Conical flasks, 25-ml volume (Scam) Addition (Scam) funnel Chromatography column (Scam) Hot stirrer (Heidolph) plate magnetic Glass oven (Büchi) Dewar flask(Scam) Ultra-low temperature chiller (Julabo) Micro-volume (Hamilton) syringe (Glassvan) Glass syringe (Sigma-Aldrich) Septum Teflon-coated bars (Sigma-Aldrich) stir magnetic Mass spectrometer (Bruker-QTOF) NMR spectrometer (Bruker, AV-400) (Norell)NMR tube (Merck, cat. no. 105715) Silica gel60F254thin-layer chromatography (TLC) plates, 20cm× (Büchi) evaporator Rotary UV lamp(Superfit India) (Borosil) funnel Separatory Filter papers(Asco) Funnel (Borosil) Vacuum pump(Hindhivac) Round-bottomed flask(Borosil) cat. no. 60118201001730) Ammonium molybdate tetrahydrate ((NH sulfateCerium(IV) ((Ce(SO fertility. impaired of risk possible a also is there environment; aquatic the in effects adverse long-term cause may and organisms aquatic to toxic is It inhalation. through exposure prolonged by health to damage serious cause may and harmful is It irritation. skin causes It flammable. highly is ether

CAUT and I ON 10b HCl iscorrosive, causessevere andmay burns causerespiratory . The synthesis of natureprotocols 4 ) 2 ); Sigma-Aldrich, cat. no. 359009) 15a , 4 ! )

15b 6 CAUT Mo

| ! VOL.8 NO.10VOL.8 !

CAUT 7 CAUT and the subsequent O I ON ! 24

! CAUT ; Merck, !

Toluene isahighly I CAUT !

I ON CAUT

ON protocol CAUT Sodium bromate Sodium I Ethyl acetate is ON I I

ON ON I | 2013 |

Sodium Sodium ON Petroleum Petroleum

Sodium Sodium |

1879

1880 (xxiii) (xviii) (xxvi) (xxiv) protocol (xxii) (xvii) (xxv) (xiii) (viii) (xxi) (xiv) (xvi) (xiv) (vii) (xx) (xv) (xii) (iii) (ix) (vi) (iv) (xi) (ii) (v) (x) Remove the ice bath, immerse the reaction flask into a preheated oil bath at 80 °C and reflux the reaction mixture for 3 h.

| VOL.8 NO.10VOL.8 sulfate, shaking itmildlyfor 30sand allowing ittostand for about10min. Wash the organic phasewithbrine (20ml).Separate the organic layerand dry itbyadding about1gof sodium Dissolve the residue obtained from Step1A(iv)in80mlof chloroform and transfer ittoaseparatory funnel. prepared inStep1A(viii). round-bottom flask. This should take about30–45min. remove the solvents under reduced pressure byusing arotary evaporator atawaterbathtemperature of 45 Dissolve the compound obtained from Step1A(vii)in150mlof THFand cannulate itdropwise into the coldsolution Filter the mixture through afilterpaperonfunnel to remove the sodiumsulfate and collectthe filtrate ina100-ml Keep the reaction mixture atRTfor 6h.Afterthe completeconsumption of starting material (asindicated byTLC), cap itwitharubberseptum.Equipthe flaskwithanitrogen balloon. Dry the compound under vacuumfor 1h.Fillthe flaskwithnitrogen, remove the flask from the vacuum manifold and high vacuumfor 2h. Evaporate the solvent byusing arotary evaporator at~35°Cunder vacuum(0–10mbar).Drythe compound under white solid. petroleum ether and ethyl acetateataratio of 2:3(vol/vol). Dry the residue under vacuumtoobtainphenyl 6-deoxy-1-thio- Pack achromatography column(4-cminternal diameter (i.d.) ×30cmlength) withsilica gel byusing amixture of round-bottom flask. Teflon-coated magnetic stir bar and cap the flask with a rubber septum. Add 60 ml of dry THF and cool the flask to 0 °C. Filter the mixture through afilterpaperonfunnel to remove the sodiumsulfate and collectthe filtrate ina250-ml Weigh 100 mmol of lithium aluminum hydride under nitrogen atmosphere into a 500-ml round-bottom flask, add a copy byusing deuterated acetone asasolvent. solvent onarotary evaporator toobtainafree-flowing solid, and then loaditontop of the silica gel bed. Dissolve the crude product obtained from Step1A(xiv)with10mlof DCMand add 3gof silica gel. Evaporate the Evaporate the solvent byusing arotary evaporator at~35°Cunder vacuum(0–10mbar). Characterize the product phenyl 6-deoxy-1-thio- Collect the fractions and evaporate the solvent by using arotary evaporator. Add pyridine (80ml)withasyringe and coolthe flaskto0°C. To this, add asolution of TsCl (38.6mmol, 1.1 equivalents) inpyridine (54ml)dropwise byusing anaddition funnel. Afterthe addition, remove the ice bath. Transfer the solution into aseparatory funnel and washthe aqueouslayerwithethyl acetate(50ml×2). Combine the organic phase and dry itoveranhydrous sodiumsulfate (1.5g) for 10min. flask withanitrogen balloon. 100-ml round-bottom flask,add a Teflon-coated magnetic stirbar and cap the flaskwitharubber septum.Equip the 30–40 ml. Weigh 7.8mmol of phenyl 6-deoxy-1-thio- Elute the product withamixture of petroleum ether and ethyl acetateataratio of 2:3(vol/vol);collectfractions of add 20mlof 3%(vol/vol) HCl toquench the reaction. Keep the reaction mixture atRTfor 1h.Aftercompleteconsumption of starting material (asindicated byTLC), H heating onahot plate. The CAMstainstocksolution isprepared bymixing Ce(SO by measuring UVabsorbance at254nmorbyspraying withceric ammonium molybdate (CAM)stainand subsequently with amixture of petroleum ether and ethyl acetate ataratio of 3:2(vol/vol)(R ! of ethyl acetatefollowed byafewdrops of water. Add 40mlof THFand turnonthe magnetic stirrer. Identify the fractions containing phenyl 6-deoxy-1-thio- After 3h,coolthe reaction mixture to0°Cand quench the excess lithiumaluminum hydride withthe slowaddition benzoyl chloride for To thissolution, add DIPEA(15.6mmol), Me  (100 ml×3).Combine the organic phaseand dry it overanhydrous sodiumsulfate (2g)for 10min. Add 100mlof 2NH

2 CAUT

SO CR 4 I (50ml)indistilledwater(450withconstant stirring. T I I ON CAL

| 2013 | Thisisanexothermic reaction; add the compound slowlytolithiumaluminum hydride.

STEP | natureprotocols Check the final organic phasebyTLCand ensure that no compound ispresent inthe aqueouslayer. 2 2b SO 4 . and transfer itinto aseparatory funnel. Wash the aqueouslayerwithethyl acetate β - d 2 -mannopyranoside obtained from Step1A(xx)(1.0 equivalents) into a SnCl β -

2 d (0.39mmol) and 1.1equivalents of acetylchloride for -mannopyranoside derivative from Step1A(xx)by NMRspectros β - d -mannopyranoside derivative bysilica gel TLCdeveloped β - d -mannopyranoside derivative (yield 60%)asa f 4 0.35).The products are visualized ) 2 (5g),(NH 4 ) 6 Mo

7 O 24 (12.5g)and

2a °

- © 2013 Nature America, Inc. All rights reserved. Teflon-coated magnetic stirbarinto it. 2| T (B) (xxxiii) (xxviii) (xxxiv) (xxxii) riflation riflation (xviii) (xxix) (xxxi) (xvii) (xiii) (viii) (xxx)

(xvi) (xiv) (xii) (vii) (xv) (iii) (xi) (ix) (vi) (iv) (ii) (x) S (i) (v) (v) Weigh 6.0mmol of ynthesis of 10aor10b solvent onarotary evaporator toobtainafree-flowing solid, and then loaditontop of the silica gel bed. Dissolve the crude product obtained from Step1A(xxvii)with10mlof DCM,and add 2gof silica gel. Evaporate the ethyl acetateataratio of 3:2(vol/vol). high vacuumfor 1h. Pack achromatography column(3cmi.d. ×25cmlength) withsilica gel byusing amixture of petroleum ether and Evaporate the solvent byusing arotary evaporator at~35°Cunder vacuum(0–10mbar).Drythe compound under Collect the fractions and evaporate the solvent byusing arotary evaporator. Keep the reaction mixture atRTfor 1h.Aftercomplete consumption of starting material (asindicated byTLC), add 1.0gof sodiumsulfate. add 15mlof 3%(vol/vol)HCltoquench the reaction and 50mlof ethyl acetate. Separate the organic layerand solvent onarotary evaporator toobtainafree-flowing solid, and loaditontop of the silica gel bed. flask witharubberseptum.Equipthe flaskwithanitrogen balloon. Dissolve the crude product obtained from Step1B(xi) with10mlof DCM,and add 1gof silica gel. Evaporate the Add THF(40ml)tothe compound obtained from Step 1B(vii),add aTeflon-coated magnetic stirbarand capthe Weigh 4.5mmol of  ethyl acetateataratio of 3:2(vol/vol). fractions of 20–30ml. Collect the fractions and evaporate the solvent by using arotary evaporator. Elute the product withamixture of petroleum ether and ethyl acetateataratio of 3:2(vol/vol),and collect Dry the residue under vacuumtoobtain Pack achromatography column(3cmi.d. ×25cmlength) withsilica gel byusing amixture of petroleum ether and the solvent. Characterize the products with anitrogen balloon. Dry the compound under vacuumfor 1h.Fillthe flaskwithnitrogen and capitwitharubberseptum.Equipthe flask Identify the fractions containing with aCAMstainand subsequent heating onahot plate. acetate ataratio of 3:2(vol/vol)(R Dry the residue under vacuumtoobtain fractions of 20–30ml. Elute the product withamixture of petroleum ether and ethyl acetateataratio of 3:2(vol/vol),and collect  the reaction mixture atRTfor 12h. Add pyridine (5ml)withasyringe. After5min,add TBDPSCl(9.1mmol, 1.1equivalents) withasyringe and keep the solvent. benzoyl chloride for round-bottom flask. pressure byusing arotary evaporator. Characterize the products Perform Step1B(vi,vii). To thissolution, add DIPEA(9.1mmol), Me Filter the organic layerthrough afilterpaperonfunnel to remove sodiumsulfate; collectthe filtrate ina100-ml After the completeconsumption of the starting material (asindicated byTLC),remove the solvents under reduced or byspraying withaCAMstainand subsequently heating onahot plate. ethyl acetateataratio of 3:2(vol/vol)(R Identify the fractions containing for 10 min. 10 for g) (1.0 sulfate sodium over it dry and layer organic the Separate ml). (20 brine with phase organic the Wash Dissolve the residue obtained from Step 1B(iv) in 40 ml of chloroform and transfer it into a separatory funnel. funnel. separatory a into it transfer and chloroform of ml 40 in 1B(iv) Step from obtained residue the Dissolve

● PAUSE PAUSE

T IMI

N PO PO G I I ~3.5 h NT NT 2a Compounds The reaction mixture canbeleftatthistemperature overnight. or 1 10b (1.0equivalents) into a50-mlround-bottom flask;add a Teflon-coated magnetic stirbar.

2b ●

. (1.0equivalents) (or T 2a 10a IMI and 2a N and G and 21.5h 2a 10a 2b 10b

and from Step1A(xxxiii)byNMR spectroscopy, using deuterated chloroform as f 2b 0.35).The products are visualized byUVabsorbance at254nmorbyspraying and from Step1B(xvii)byNMRspectroscopy byusing deuterated chloroform as canbestored atRTfor several months without marked decomposition. 2a 10a 2b f 10b (yield 89%,whitesolid) and 0.5).The products are visualized bymeasuring UV absorbance at254nm 2 bysilica gel TLCdeveloped withamixture of petroleum ether and ethyl (yield 84%,foam) and SnCl bysilica gel TLCdeveloped withamixture of petroleum ether and 10a 2 (0.2mmol) and 1.1equivalents of acetylchloride for or 10b ) into a100-mloven-dried round-bottom flaskand add a 10b (yield 87%,whitesolid). 2b (yield 92%,whitesolid). natureprotocols

| VOL.8 NO.10VOL.8

protocol 10a

| 2013 | or

1881

© 2013 Nature America, Inc. All rights reserved. (B) ( prepared bycarrying outStep7Bfollowed byStep8C,using compound and 8 10b steps canbeusedtoprepare the 4-azido galactosamine derivatives Option Adescribes the synthesis of 7| ? additional 20 min.  absorbance at254nmorby spraying withaCAMstainand subsequently heating onahot plate. 10b 6| (6.0 equivalents). 5| ice-salt bath. 4| septum. Equipthe flaskwith anitrogen balloon. 3| 1882 (xiii) (viii) (xiv) protocol A (xii) (vii) (from triflate of (iii) TROU (xi) (ix) (vi) (iv)

(ii) (ii) ) (x) (v) CR (i) (i) . Option Bdescribes the stepscommon tothe synthesis of compounds ) isconsumed (asmonitored byTLC,developing withhexane:ethyl acetate(8:2)),visualize the product bymeasuring UV S 10b S

There are twooptions atthisstage. Bothoptions assume thatthe triflation reaction (Steps2–6)hasbeenperformed. Allow the temperature of the ice bathtoreach 10°Cgradually over2h.Afterthe starting material Add 78.4mmol of pyridine (13.0equivalents), and after5minadd 36.9mmol of trifluoromethanesulfonic anhydride Add dry DCM(55ml)byusing asyringe, and turnthe magnetic stirrer on.Coolthe flaskto Dry the flaskunder vacuum for 1h,fillitwithnitrogen, remove it from the vacuum manifold and capitwitharubber | I ynthesis of 2-azido-4- ynthesis of D VOL.8 NO.10VOL.8  ! stirring for 8hatRT. Separate the organic layerand dry it over anhydrous sodiumsulfate (1g)for 15min. a nitrogen balloon.Add DMF(40ml)and turnthe magnetic stirrer on.  ethyl acetateataratio of 9:1(vol/vol)for T measuring UVabsorbance at254nmorbyspraying withaCAMstainand subsequently heating onahot plate. ether and ethyl acetateat aratio of 9:1(vol/vol)for solvent onthe rotary evaporator toobtainafree-flowing solid, and then loaditonthe top of the silica gel bed. round-bottom flask. off the magnetic stirrer. Wash the organic layersuccessively with20mlof 1MHCl,20mlof aqueousNaHCO Dilute the reaction mixture with40mlof DCMand transfer itinto aseparatory funnel. Characterize the product Cool the flaskto0°Cand add 60.0mmol of NaN Fill the flaskwithnitrogen and remove itfrom vacuum manifold. Capthe flaskwitharubberseptum and equipitwith Evaporate the solvents byusing arotary evaporator at~35°C.Drythe compound under vacuumfor 1h. Dry the residue under vacuum toobtaincompound Collect the fractions and evaporate the solvent by using arotary evaporator. Identify the fractions containing compound Elute the product withamixture of petroleum ether and ethyl acetateataratio of 9:1(vol/vol)for Dissolve the crude product obtained from Step7A(vii) with5mlof DCM,and add 1gof silica gel. Evaporate the Pack achromatography column(2.5cmi.d. ×25cmlength) withsilica gel byusing amixture of petroleum ether and Evaporate the solvent by using arotary evaporator at~35°Cunder vacuum(0–10mbar). Filter the mixture through afilterpaperonfunnel to remove the sodiumsulfate; collectthe filtrate ina100-ml Add ethyl acetate(50ml)and water(40ml)tothe reaction mixture and transfer the mixture toaseparatory funnel. After the completeconsumption of the starting material (asmonitored byTLC),remove the nitrogen balloonand turn and collectfractions of 20–25ml. sodium sulfate (1g)for 15min. Extract the aqueouslayerwithethyl acetate(50ml×2).Combine the organic phase and dry itoveranhydrous

I B CAUT

CAL

. Ifyouare performing the lattersynthesis, youshould continue withStep8B. GalNAc-3-OH derivative LES PAUSE PAUSE

H STEP I OOT ON

PO | 2013 | Gasmay liberate vigorously during the process. Add NaN Ensure that the starting material is completely consumed; if not, keep the reaction mixture at 10 °C for an I 2a N I I AT NT NT G ). The same stepscanbeusedfor the synthesis of the GalNAc-4-OH derivatives DH derivatives 5aand5b The reaction mixture canbeleftatthistemperature overnight. Compounds | natureprotocols

OT 5a f (quinovosamine) derivative or 5a 5a 5b and and from Step7A(xii)byNMRspectroscopy, using deuterated chloroform asthe solvent. 5b 5b canbestored atRTfor several months without marked decomposition. from the corresponding triflates derived from compounds

● 5a 5a

T and IMI or 3 (10equivalents). After10min,remove the ice bathand continue N 5b 5b 5a G bysilica gel TLCdeveloped withamixture of petroleum ~18h 2a . (yield 82%)or

(R ●

f T 0.4)and IMI 11a N 10a and G ~23h 3 portionwise and slowly. 6 asastarting material. 2b 5b (from triflate of 11b (R (yield 85%)asapaleyellowishliquid. from the triflates derived from f 0.45).The products are visualized by 3 and 20mlof brine solution. 2b

), −10 °Cbyusing an 7

(from triflate of 12a 2a 2a and 5a and or and 12b 2b 14 2b 10a (or from 5b canbe . The same

and , 2b

10a

10a ) and

© 2013 Nature America, Inc. All rights reserved. ( (B) ( (option C). 8| (viii) C A (vii) (vii) Collectthe fractions and evaporate the solvent byusing arotary evaporator. (vii) (iii) (iii) (iii) (iii) (vi) (iv) (ix) Characterize the product (vi) (iv) (vi) (iv) )

(ii) (ii) (ii) ) (v) Collectthe fractions and evaporate the solvent byusing arotary evaporator. (v) (v) (i) (i) (i) S S S At thispoint, there are three options: preparation of compound ynthesis of Fuc ynthesis of Fuc ynthesis of (1.5 g)for 10 min. aqueous layer with ethyl acetate(50ml× 2).Combine the organic phaseand dry itoveranhydrous sodium sulfate ? for anadditional 30min.  (1.5 g)for 15min. aqueous layerwithethyl acetate(50ml×2).Combine the organic phaseand dry itoveranhydrous sodiumsulfate flask withanitrogen balloon.  off the magnetic stirrer. Evaporate the solvent under reduced pressure byusing arotary evaporator. one-third of itsoriginal volume. ? an additional 30min.  (3.0 equivalents) and keep the reaction mixture atRTfor 1.5h.  ? additional 1h.  with aCAMstainand subsequently heating onahot plate. acetate ataratio of 8:2(vol/vol) (R subsequently heating onahot plate. 9:1 (vol/vol).The products are visualized bymeasuring UVabsorbance at254nmorbyspraying withaCAMstainand  Dry the residue under vacuumtoobtaincompound Identify the fractions containing compound Work upasdescribed inStep7A(v–x),byusing amixture of toluene and ethyl acetate9:1(vol/vol)asthe solvent Add 12.0mmol of phthalimide potassiumsalt(2.0equivalents) and keep the reaction mixture atRTfor 10h. Add DMF(30ml)and turnthe magnetic stirrer on. Release the flaskfrom the rotary evaporator under nitrogen atmosphere and capitwitharubberseptum.Equipthe After the completeconsumption of the starting material (asmonitored byTLC),remove the nitrogen balloonand turn Add 6.0mmol of TBAN Add 160mlof acetonitriletothe flaskand coolthe flaskto Fill the flaskwithnitrogen, remove itfrom the vacuum manifold and capitwitharubberseptum.Equipthe flaskwith Dry the residue under vacuumfor 2h. Remove the sodiumsulfate and the ethyl acetateasdescribed inStep7A(vi,vii). Add ethyl acetate(50ml)and brine (40ml)tothe reaction mixture and transfer ittoaseparatory funnel. Extract the Add 22.0equivalents of water; keep the reaction mixture for reflux at65°C for 1.5h. After the completeconsumption of the starting material (asmonitored byTLC),reduce the volume of acetonitrileto Characterize the product Dry the residue under vacuumtoobtaincompound Identify the fractions containing compound Work upasdescribed inStep 7A(vi–x). Add ethyl acetate(50ml)and brine (40ml)tothe reaction mixture and transfer ittoaseparatory funnel. Extract the After the completeconsumption of the starting material (asmonitored byTLC),add 18.0mmol of TBANO system for packing the columnand eluting the product. a nitrogen balloon.

TROU

TROU CR PAUSE CR PAUSE CR PAUSE I I I T T T I I I B B B CAL CAL CAL LES LES LES

PO PO PO AAT

H H H I I I STEP STEP STEP NT NT NT OOT OOT OOT

NA NA building block6 Compound The reaction mixture canbeleftatthistemperature overnight. The reaction mixture canbeleftatthistemperature overnight. Ensure thatthe starting material iscompletelyconsumed; ifnot, keep the reaction mixture at65°C Ensure thatthe starting material iscompletelyconsumed; ifnot, keep the reaction mixture atRTfor Ensure thatthe starting material iscompletelyconsumed; ifnot, keep the reaction mixture for an c-3- I I I c-4- N N N G G G

O O 3 (1.0equivalents) and keep the reaction mixture atthe same temperature for 20h. H derivative 8 H derivative 7

7 6 from Step8A(viii)byNMRspectroscopy byusing deuterated chloroform asthe solvent. from Step8B(vi)byNMRspectroscopy, using deuterated chloroform asasolvent. 6 canbestored atRTfor several months without marked decomposition.

f 0.3).The products are visualized byUVabsorbance at254nmorbyspraying 6 7 bysilica gel TLC,developed withamixture of petroleum ether and ethyl bysilica gel TLCdeveloped withamixture of toluene and ethyl acetate 7 6 (yield 60%)asapaleyellowishliquid. (yield 57%)asaviscousliquid.

−

30 °Cbyusing acryostat.Turn the magnetic stirrer on. 6

(option A),compound natureprotocols

7 (option B)orcompound

| VOL.8 NO.10VOL.8 protocol 2

| 2013 |

8 1883

© 2013 Nature America, Inc. All rights reserved.  22| 21| with aCAMstainand subsequently heating onahot plate. acetate ataratio of 9:1(vol/vol) (R 20| 19| aqueous layerwithethyl acetate(30ml×2).Combine the organic phaseand dry itoveranhydrous sodium sulfate (0.5g). 18| off the magnetic stirrer. 17|  16| nitrogen balloon.Add DMF(10ml)and turnthe magnetic stirrer on. 15| 14| additional 20 min.  254 nmorbyspraying withaCAMstainand subsequently heating onahot plate. (as monitored byTLC,developing withhexane:ethyl acetate(9:1)),visualize the product bymeasuring UVabsorbance at 13| (1.2 equivalents). 12| 11| septum. Equipthe flaskwithanitrogen balloon. 10| magnetic stirbar. 9|  S 1884 (viii) protocol ynthesis ynthesis of buildingbacillosamine block 9 (vii) Collectthe fractions and evaporate the solvent byusing arotary evaporator. (ix) (vi) (iv) Remove the sodiumsulfate and the ethyl acetateasdescribed inStep7A(vi,vii).

PAUSE PAUSE (v) Purifybycolumnchromatography asdescribed inStep7A(viii–x). CR CR Drythe residue under vacuum toobtaincompound Collectthe fractions and evaporate the solvent byusing arotary evaporator. Identify the fractions containing compound Perform Step7A(vi–x). Add ethyl acetate(30ml) and water(10ml)tothe reaction mixture and transfer ittoaseparatory funnel. Extract the Afterthe completeconsumption of the starting material (asmonitored byTLC),remove the nitrogen balloonand turn Coolthe flaskto0°Cand add 6.0mmol of NaN Fillthe flaskwithnitrogen, remove itfrom the vacuum manifold and capitwitharubberseptum.Equipthe flaskwitha Evaporate the solvents byusing arotary evaporator at~35°C.Drythe compound under vacuumfor 1h. Allowthe temperature of the ice bathtogradually reach 10°Cover2h.Afterstarting material Add 6.0mmol of pyridine (6.0equivalents). After5minadd 1.2mmol of trifluoromethanesulfonic anhydride Add dry DCM(8ml)byusing asyringe and turnthe magnetic stirrer on.Coolthe flaskto Drythe flaskunder vacuum for 1h,fillitwithnitrogen, remove it from the vacuum manifold and capitwitharubber

Weigh 1.0mmol (1.0equivalents) of | I I VOL.8 NO.10VOL.8  T T spraying withaCAMstainand subsequently heating onahot plate. acetate ataratio of 8:2(vol/vol)(R Characterize the product Dry the residue under vacuumtoobtaincompound Identify the fractions containing compound I I CAL

CAL PAUSE

PO PO

The starting material for thissection iscompound I I STEP NT NT

Compound The reaction mixture can be left at this temperature overnight.

PO | 2013 | Ensure that the starting material is completely consumed; if not, keep the reaction mixture at 10 °C for an I NT Compound | natureprotocols 9 can be stored at RT for several months without marked decomposition. 8 from Step8C(viii)byNMRspectroscopy, using deuterated chloroform asasolvent. 8 canbestored atRTfor several months without marked decomposition. f 0.5).Products are visualized bymeasuring UVabsorbance at254nmorbyspraying d f -fucosamine derivative 0.25).The products are visualized bymeasuring UVabsorbance at254nmorby ●

9 T bysilica gel TLC,developed withamixture of petroleum ether and ethyl IMI 8 3 bysilica gel TLCdeveloped withamixture of petroleum ether and ethyl . After10min,remove the ice bathand continue stirring for 8hatRT. N G 9 19.5 h (yield 81%). 8 (yield 62%)asapaleyellowish liquid. 7 . 7 into a50-mlround-bottom flaskand add a Teflon-coated

−

10 °C.

7 isconsumed

© 2013 Nature America, Inc. All rights reserved. T Step 7B(i–iv): 3h Step 2–6:3.5h S Total time for Steps 2–8A:22.5h;should be performed in2d Step 8A(vi–ix):3h Step 8A(v):5h Step 8A(i–iv):11h Steps 2–6:3.5h S Step 7B(v–vii):20h Step 7B(i–iv):3h S Total time for Step7A(i–xiv):14.5h;should beperformed inasingle day Step 7A(viii–xiv):4h Step 7A(iv–vii):2h Step 7A(i–iii):8.5h S Steps 2–6: 3.5 h T Total time for Step 1B: 21.5 h; should be performed in a single day Step 4 1B(xii–xviii): h Step 1B(viii–xi): 2 h Step 1B(iv–vii): 2 h Step 1B(i–iii): 13.5 h S Total time for Step 1A: 30.5 h; should be performed in 2 d Step 6 1A(xxviii–xxxiv): h Step 3 1A(xxii–xxvii): h Step 1A(xv–xxi): 6 h Step 1A(xii–xiv): 1 h Step 1A(viii–xi): 4.5 h Step 1A(iv–vii): 8.5 h Step 1A(i–iii): 1.5 h S ● Troubleshooting advice canbefound in ? 23| Step 7B(v–vii): 20h a Low Low yield of Low yield of Low yield of P riflation ynthesis of Fuc ynthesis of ynthesis of 2-azido-4- ynthesis of D ynthesis of 10aor10b ynthesis of 2aor2b

TROU

roblem b T le le IMI Characterize the product 3 3 N B |

LES G Troubleshooting table. 5a 5a 5a H AAT OOT , , , , AT 5b 5b 5b NA DH derivatives 5a,5bandsynthesis of 4-azidogalactosamine derivatives 11aand11b building block6 I , , , , , N 6, 6, 7, 8 6, 7, 8 6, 7, 8 c-4- G O OT H derivative 7andsynthesis of Gal f (quinovosamine) derivative 9 displacement Temperature fluctuation during C2 OTf upon keeping it for a long time violently with water. The reagent goes bad Trifluoromethanesulfonic anhydride reacts reaction Triflation of P from Step22byNMRspectroscopy, using deuterated chloroform as the solvent. ossible reason T able 2a and 3 . 2b is a moisture-sensitive NA

c-4-

O H derivative 12aand12b Temperature should be strictly controlled at is seen, replace the reagent The reagent is a colorless liquid; if a white precipitate Use fresh Trifluoromethanesulfonic anhydride. Moisture must be strictly avoided Reaction must be done under dry conditions. S olution natureprotocols

| VOL.8 NO.10VOL.8 protocol

| 2013 |

−

30 30 °C

1885

© 2013 Nature America, Inc. All rights reserved. (d, 4.40 (d, 5a. 128.0, 127.8,87.3,79.7,76.9, 70.9,67.2,64.9,27.0,19.4. (m, 2H,H-6),3.59-3.54 1H, H-5),2.89(d, 3.2 Hz,1H,H-3),5.00(d, 10b. 127.81, 87.2,79.7,77.0,70.7,67.0,64.9,26.8,21.3,19.3. (m, 1H,H-5),2.79(bs, 1H,OH),2.25(d, 3.2 Hz,1H,H-3),4.30-4.28(m,H-2),4.14-4.09 1H, H-4),3.97(d, 10a. 1H, H-2),3.86(t, ArH), 7.32-7.25(m,3H,5.00(dd, 2b. 2.15 (s, 3H,CH (dd, 2a. 1.29 (d, 4.20 (bs, 3H,OH),4.09(s, 1H),3.58(dd, P ANAL vaccine development and drug discovery. This expedient protocol isexpected tospeedupthe synthesis of various antigenic bacterial glycoconjugatesand facilitate ANT Total time for Steps9–23:19.5h;should beperformed inasingle day Steps 17–23:6h Steps 13–16:12h Steps 9–12:1.5h S Total time for Steps2–8C:35.5h;should beperformed in2d Step 8C(vi–ix):4h Step 8C(i–v):5h Step 7B(v–vii):20h Step 7B(i–iv):3h Steps 2–6:3.5h S Total time for Steps2–8B:34.5h;should beperformed in2d Step 8B(iv–vii):3h Step 8B(i–iii):5h 1886 protocol ynthesis of bacillosamine building block9 ynthesis of Fuc henyl 6-deoxy-1-thio- 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 H NMR (400 MHz, (CD H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R TLC(petroleum ether:ethyl acetate, 3:2vol/vol)R TLC(petroleum ether:ethyl acetate, 3:2vol/vol)R J C NMR (100 MHz, CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,(CD I

TLC(petroleum ether:ethyl acetate, 3:2vol/vol)R TLC(petroleum ether:ethyl acetate, 3:2vol/vol)R J C | Y

VOL.8 NO.10VOL.8 =

I T

PATE 6.2Hz,3H,CH I

9.6,3.2Hz1H,H-3),4.28(dd, CAL J J

= = D

D 10.0Hz,1H,H-1), 3.82(d, 6.0Hz,3H). RESULTS ATA m/z m/z m/z m/z m/z 3 ), 1.39(d,

| 2013 | NA ): [M ): [M ): [M ): [M ): [M J

c-3-

9.6Hz,1H,H-4),3.48-3.44(m,H-5),1.41(d, 3 |

natureprotocols + + + + + O b 3 3

3 ) 3 3 3 3 3 Na] Na] Na] Na] Na] H derivative 8andsynthesis of Gal 3 3 3 - ) ) 2 ) ) ) ) ) J ) ) ) D 2 J CO) δ

CO) -mannopyranoside derivative. δ δ δ δ δ

δ δ δ = 166.8, 134.0, 133.7, 131.5, 130.0, 129.4, 129.2, 128.6, 127.8, 87.1, 77.6, 76.8, 71.1, 70.7, 18.1.

7.71-7.68(m,5H,ArH),7.48-7.3510H,4.91(s, 1H,H-1),4.84(dd, 8.05-8.03(m,2H,ArH),7.56-7.541H,7.48-7.467.41-7.37 7.48-7.44(m,2H,ArH),7.31-7.243H,4.88(d, 7.60-7.58(m,2H,ArH),7.34-7.33 (m,3H,ArH),4.90(dd, 8.12-8.10(m,2H,ArH),7.73-7.724H,7.71-7.24 (m,14H,ArH),5.09(dd, =

166.6,135.87,135.0,134.2, 133.7,133.0,132.9,131.5,130.1,130.09,129.6,129.3, 128.7, 171.1,135.9,135.8,135.7,134.0,132.9,132.7,131.5, 131.3, 130.1,129.2,128.0,127.86, 171.5,133.8,131.6,129.2,127.8,87.0,77.0,76.9,70.1,70.5,21.3,18.0. + + + + +

0.8Hz,1H,H-1),4.43-4.41(m,H-2),4.25(dt,

6.0Hz,3H,CH calculatedfor C calculatedfor C calculatedfor C calculatedfor C calculatedfor C δ δ 7.45-7.43 (m, 2H, ArH), 7.31-7.28 (m, 2H, ArH), 7.22-7.18 (m, 1H, ArH), 5.04 (s, 1H, H-1), 137.6,130.0,129.7,127.0,87.5,77.0,75.7,73.8,73.3,18.4. J J J

= =

9.6,3.2Hz,1H,H-3),4.93(d,

3.2,0.8Hz,1H,H-2),3.71(t, = 3.4Hz,1H,H-4), 3.70-3.65(m,2H,H-2& H-5),2.16(s, 3H,CH

6.2Hz,1H,OH),2.18(s, 3H,CH

9.0,3.2Hz,1H),3.44(t, 3 ). 19 14 12 35 30 J

H H H H H

= 38 36 20 18 16

3.2Hz,1H,OH),2.41(d, O O O O O 6 6 5 5 4 f f f 0.4 0.35 0.35 NaSSi, 637.2056;found, 637.2054. NaSSi, 575.1900;found, 575.1860. NaS, 383.0929;found, 383.0941. NaS, 321.0773;found, 321.0776. NaS, 279.0667;found, 279.0663. f f 0.5 0.5 TLC(petroleum ether:ethyl acetate, 2:3vol/vol)R NA c-3- O H derivative 14 J

= J

J J J 9.0Hz,1H),3.40-3.34(m,

= = 6.0Hz,3H,CH 3

4.8Hz,2H,H-6a,H-6b),3.50-3.45

), 1.07(s, 9H,Si( 9.6Hz,1H,H-4),3.45-3.38(m,H-5), 0.8Hz,1H,H-1),4.40(dd, =

6.4Hz,1H,OH),1.08(s, 9H,Si( J

9.8,3.2Hz,1H,H-4),4.05-3.98 J

0.8Hz,1H,H-1),4.75

10.0,3.4Hz,1H,H-3), 3 ). CH 3 ) 3 ). J J f

3 0.35

= ), 1.35

3.2,0.8Hz,

10.0, CH J

= 3

)

9.8, 3

© 2013 Nature America, Inc. All rights reserved. 128.0, 86.5,76.7,75.8,68.4, 65.2,59.2,26.8,21.2,19.2. 3.61 (bs, 1H,OH),3.51(t, 1H, H-4),4.02(dd, ArH), 7.30-7.24(m,3H, 4.77(dd, 12a. 128.0, 86.9,77.3,75.3,62.3,60.3,59.9,26.9,19.3. 3.90-3.69 (m,3H,H-6a,6b&H-5),3.74(t, (m, 3H,ArH),5.24(dd, 11b. 62.3, 59.9,59.6,27.0,20.8,19.3. 3.84-3.70 (m,2H,H-6a,6b),3.72-3.62H-2,H-5),2.19(s, 3H,CH (m, 3H,ArH),4.95(dd, 11a. 63.6, 18.8. (t, (m, 3H,ArH),5.28(t, 9. (d, (d, 8. (t, (m, 3H,ArH),4.99(dd, 7. 73.6, 73.2,62.1,51.7,17.1. H-2 ),4.72(d, 7.49-7.27 (m,5H,ArH),5.39(dd, 6. 59.8, 17.8. 3.80-3.77 (m,2H,H-2&H-5),1.39(d, (m, 3H,ArH),5.18(dd, 5b. HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 1 HR-ESI-MS ( 13 TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl H NMR(400MHz,CDCl J J TLC(toluene:ethyl acetate, 9:1vol/vol)R J J C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl3) C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl C NMR(100MHz,CDCl

TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R

= =

10.0Hz,1H,H-4),1.45(d, 10.0Hz,1H,H-2),3.77(q,

6.4Hz,3H,CH 10.0Hz,1H,H-1),3.72-3.67(m,2H,H-3&H-5),3.49(t, m/z m/z m/z m/z m/z m/z m/z m/z J

= ): [M ): [M ): [M ): [M ): [M ): [M ): [M ): [M

9.8Hz,1H,H-1),4.00(qd, J

= 3 J

). 11.2,4.0Hz,1H,H-6a),3.92 (dd,

+ + + + + + + + J J J J

3 3 3 3 3 3 3 3 Na] Na] Na] Na] Na] Na] Na] Na]

3 3 3 3 3 3 3 3 = = = = 10.0Hz,1H,H-3),4.57(d, ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) J

10.0,3.6Hz,1H,H-3),4.39(d, 10.0,2.9Hz,1H,H-3),4.53(d, 10.0,3.5Hz1H,H-3),4.49(d, 10.0,3.5Hz,1H,H-3),4.52(d, δ δ δ δ δ δ δ δ

δ δ δ δ δ δ δ δ δ

7.66-7.63(m,4H,ArH),7.58-7.512H,7.47-7.376H,7.31-7.28 8.08-8.06(m,2H,ArH),7.63-7.593H,7.49-7.457.38-7.35 7.61-7.58(m,2H,ArH),7.33-7.303H,5.15(d, 8.07-8.04(m,2H,ArH),7.64-7.573H,7.47-7.377.36-7.34 7.89-7.83(m,3H,ArH),7.79(s, 3H,ArH),7.72-7.66(m,2H,7.64-7.611H, 8.10-8.07(m,2H,ArH),7.64-7.593H,7.50-7.467.38-7.35 7.76-7.74(m,2H,ArH),7.69-7.667.64-7.61 (m,2H,ArH),7.46-7.376H, 8.10(d, =

170.1,133.5,131.2,129.2,128.6,86.5,75.5,73.4,63.0,59.3,20.7,17.8. 170.3,135.8,135.7,133.6, 132.4, 132.0,131.2,130.27,130.23,129.24,128.5,128.1, 165.5,135.6,134.0,133.3,132.9,132.8,131.2,130.2, 130.1, 129.2,128.8,128.6,128.5, 165.5,133.9,133.8,130.9,130.1,129.3,128.9,128.8,128.7,86.3,75.3,75.1,65.9, 171.7,133.1,131.9,129.0,128.3,86.5,73.3,73.1,72.1,62.4,20.9,16.9. 165.8,133.8,133.5,131.6,130.0,129.2,128.7,128.6,86.6,76.8,74.8,69.4,59.9,16.8. 168.5,165.2,134.5,133.7,133.5,132.5,129.8,129.1,128.8,128.6,128.1,123.8,88.9, 165.6,133.9,133.4,131.2,130.1,129.1,128.7,128.5,128.4,86.6,75.8,73.6,63.1, + + + + + + + + 170.0,135.7,133.4,132.94,132.89,131.2,130.1,129.2, 128.6,128.0,86.9,77.2,75.2,

calculatedfor C calculatedfor C calculatedfor C calculatedfor C calculatedfor C calculatedfor C calculatedfor C calculatedfor C 4.0Hz,1H,H-5),2.17(s, 3H,CH J J J

= =

9.8,7.0Hz,1H,H-3),5.04(dd, J

6.4Hz,1H,H-5),1.38(d, 6.2Hz,3H,CH

J =

= 6.8Hz,2H,ArH),7.66-7.60(m,5H,7.56-7.40 10H,ArH),7.38-7.31

6.3Hz,3H,CH

10.0,2.8Hz,1H,H-3),4.47 (d, J f

0.45

= 19 14 19 27 19 14 30 35 J

10.0Hz,1H,H-2),1.05(s, 9H,

H H H H H H H H

= 16 35 36 18 17 19 22 18

6.4,2.8Hz,1H,H-5),1.20(d, N N N N N N N N f f f f 0.3 0.5 0.45 0.25 4 6 6 3 6 6 3 3 3 O O O O O O O O J f f f ). 0.45 0.5 0.5

5 3 3 5 4 3 4 4

NaS, 408.0994;found, 408.0974. NaS, 537.1209;found, 537.1134. NaS, 433.1059;found, 433.1039. NaS, 371.0902;found, 371.0905. NaSSi 600.1964; found 600.1980. NaSSi 687.2186;found 687.2171. NaS, 433.1059;found, 433.1055. NaS, 346.0837;found, 346.0819. =

10.0Hz,1H,H-1),3.49-3.44(m,2H,H-2&H-5),3.29 3 J ).

J J J =

11.2,4.0Hz,1H,H-6b),3.85 (t,

= = =

10.0Hz,1H,H-1),3.85(dd,

10.0Hz,1H,H-1),4.01(d, 10.0Hz,1H,H-1),4.29(d, 10.0Hz,1H,H-1),4.16(d, 3 ), 1.07(s, 9H, J J

= =

6.4Hz,3H,CH 10.0Hz,1H,H-2),2.13(s, 3H,CH J

= 3

7.0,2.8Hz,1H,H-4),4.91(t, ), 1.05(s, 9H, J

(CH (CH 10.0Hz,1H,H-1),4.28(d, J

3 3 natureprotocols

) = ) 3 3 3 ).

6.4Hz,3H,CH CSi). CSi). J

= (CH

3.2Hz,1H,H-4),4.45 J J J J 3

)

= = =

= 3

2.9Hz,1H,H-4),3.83 3.5Hz,1H,H-4), 3.6Hz,1H,H-4), CSi). J

3.5,1.0Hz,1H,H-4),

10.0Hz,1H,H-2),

| 3 VOL.8 NO.10VOL.8 ). 3 ) 1.38 J

= protocol J

9.8Hz,1H,

2.8Hz, | 2013 |

1887

© 2013 Nature America, Inc. All rights reserved. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprints/index.h at online available is information permissions and Reprints interests. CO data and participated in discussions. and wrote the manuscript. M.E. carried out all the experiments, collected spectral to synthesis of bacterial glycans, supervised M.E. to carry out the experiments displacement of to bis-triflates prepare deoxy amino sugars and its application AUT M.E. thanks CSIR-New Delhi for a fellowship. Scientific and Industrial Research (CSIR; grant no. 01(2376)/10/EMR-II). of Science and Technology (grant no. andSR/S1/OC-40/2009) the Council of A 127.96, 86.8,77.3,73.5,69.1,62.6,61.6,26.9,20.8,19.2. (t, 1H, H-4),4.46(d, 14. 129.2, 128.6,128.5,128.12,128.09,86.8,77.5,76.6,68.2,64.9,59.7,26.9,19.2. 4.05-3.93 (m,3H,H-6a,6b&H-2),3.60(t, (m, 3H,ArH),4.98(dd, 12b. 1888 protocol cknowle

M HR-ESI-MS ( 13 1 HRMS calculatedfor C 13 1

H NMR(400MHz,CDCl H NMR(400MHz,CDCl H J PET TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R 31 Res.Carbohydr. S from polysaccharides capsular the of lipopolysaccharide. Structuralstudies theofO-specific side-chains theof (2001). 26479–26485 the of Chem. Biol. J. bacterium, gram-negative the in glycoproteins (1995). 1201–1214 2,4-diacetamido-2,4,6-trideoxyhexose.digalactosyl (2011). 5723–5777 glycoproteins. bacterial target (2006). 1575–1580 Bacteroides fragilis Bacteroides of strain one from polysaccharides capsular two ofelucidation Structural formulation. vaccine forantigens oligosaccharide (2009). 611–622 research. glycoconjugate andoligosaccharide synthetic (2009). 5561–5577 chemistry. medicinal in frontier a Carbohydrates: Baumann, H., Tzianabos, A.O., Brisson, J.-R., Kasper, D.L. & Jennings,H.J. & Kasper,D.L. J.-R., Brisson,Tzianabos, A.O., H., Baumann, studies Structural D.A. Powell, & J. Lönngren, B., Lindqvist, B., Lindberg, Kenne,Lindberg,L., B.,Petersson, Katzenellenbogen,K., Romanowska,& E. E. characterization R.W.Structural Carlson, & F.J.P.,Cassels, Castric, N.M. Young, E. Stimson, syntheticimmunology: and Carbohydrates L. Lay, & L. Poletti, L., Morelli, in challenges and Opportunities G.-J. Boons, & T.Buskas,T.J., Boltje, P.H. Seeberger, & A. Adibekian, B., Lepenies, P., Stallforth, and discover to tools Chemical B.Wang, & K. Champasa, D.H., Dube, glycoproteomics. Bacterial A. Dell, P.G.& Hitchen, C NMR(100MHz,CDCl C NMR(100MHz,CDCl OR

TLC(petroleum ether:ethyl acetate, 9:1vol/vol)R

| = , 4081–4089 (1992). 4081–4089 , VOL.8 NO.10VOL.8 I

N CONTR 10.0Hz,1H,H-2),2.01(s, 3H,CH G G FI dgm Pseudomonas aeruginosa Pseudomonas NANC IB ents et al. et et al. et UT

m/z

277 I 78 I

tm AL

This This work was financially supported by the Department ONS Meningococcal pilin: a glycoprotein substituted with substituted glycoprotein a pilin: Meningococcal | 2013 | Structure of the of Structure , 111–117 (1980). 111–117 , using high-resolution NMR spectroscopy.NMR high-resolution using Carbohydr.Res. , 42530–42539 (2002). 42530–42539 , l ): [M I . J NTERESTS

S.S.K. S.S.K. conceived the idea of sequential double

10.0Hz,1H,H-1),3.80-3.77(m,2H,H-3&H-5),3.75-3.68H-6aH-6b),3.48 |

35 natureprotocols + J

3 3 Na] H

3 3 = ) )

) ) Chem. Commun. Chem. 37 The authors declare no competing financial declare no The financial authors competing 1244 pilin glycan. pilin 1244

10.0,2.8Hz,1H,H-3),4.55(d, δ δ

δ δ 78 O 7.65-7.61(m,6H,ArH),7.57-7.377.29-7.273H,5.43(d, 8.08(d,

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