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Azides in

Hemling s,c. Beckmllll1l and Valemin WiUmanll

Facll~rdch Cltemie. Un il'usiliit K(JII$/(IIIt. U,,;vusiIiJustr. la, D-78457 KOIIS/(/II Zo GenllulI)'

16.1 Introduction

The fi rst az.ide-contain ing sugar, a glycosyl azide. was reponed in 1930 by Bertho. j Since that lime various methods have been developed for the introduction of azides at different positions of sugars. A survey of available methods is given in Section 16.2. Unlillhc lale I 970s, azidcs contai ned in carbohydrate deri vatives were simply used as accessible syn­ thons [or am ities because of their easily performed red uction to amines. Due 10 their stability agai nst a variety of reaction conditions, al.ides often can serve as masked amincs during the course of . The development of the diazo tmnsfer reac­ tion faci litated the use of azides also as temporary protecting group for amines. Thi s was extensively applied during the preparation of nrninoglycoside deri vati ves (Section 16.3). Ove r the lasl th ree decades, azides became un im portant tool especially for the synthesis of and -proteins. In 1978 Paulsen el 0/. developed the 'a1.idc method' for the preparation of I ,2-cis-glyeosides of glycosamine deri vatives using 2-nzido-2-deoxy­ donors (Secti on 16.4). This reaction is widely used for the synthesis of O-li nked glycosyl building blocks. In N-, the glycan chains are attached to the protein via a {j-glycosyl amide. Staudinger-type reactions offer a conven ient access to such structures and are applied since the 19905 for the synthesis of a- and {j-glycosyl amides directly from glycosyl azides (Section 16.5). An enormous impact on the field of duri ng the last decade hurl the devel ­ opment of two bioorlhogonal reactions based on uzidcs: thc coppcr-catalyzed azide­ alkync 13+2J cycloudcli tion unci the Staudinger ligution . Togcther with the possibility of ill vivo incorpor:ltion of azidc and alkync tags in to glycuns and protci ns, these reactions offcr ncw options for selective labelin8 and man ipul ation of biomoleculcs even within 470 Organic Azides: Synfheses and Applica/

living cells. Especially the azide- cycloaddition has been extensively applied for the of neoglycoconjugatcs such as and mimics or multivalent glycoclustcrs (Section 16.6). Metabolic engi neer­ ing uses the biosynthetic pathways for the introduction of azide- (and alkyne-)tagged sugar moieties into the glycan.s of cells thut can subsequently be lubeled by a detectable probe. This upproach is discussed in Section 16.7.

16.2 Synthesis of Azide-Containing Carbohydrutes

A common way for the introduction of azides into is the nucleoph ilic replacement of leaving groups by the azide . These reactions can be divided into three groups: substitutions at the anomeric center leading to glycosy l azides, substitutions at pri mary, and substitu tions at secondary carbon . A widely used method for the preparation of glycosyl azides~ is the conversion of acetylated haJogenoses, such as I, by treat ment with sodium a1.ide based on Bertho's initial work (Scheme 16.1A).' While homogeneous onc-phase reactions in DMF often requ ire elevated temperatures,' phase-transfer enables milder conditions.6 One limitation of th is methodology is the instability of glyeosyl halides. Thus, sequential one­ pot procedures have been developed that avoid the isolation of glycosyl halides.' An alternative. which circumvents the preparation of glyeosyl halides completely. is the direct conversion of glycosyl acetates into the corresponding glycosyl azides using trimethylsi­ Iyl azide under Lewis acid catalysis (Scheme 16. IB ).· Glycosy l azides with 1,2-1rarls­ configurat ion arc easily obtained by the described methods using acyl protccting groups due to their neighboring group part icipation. Glycosyl azidcs with 1.2-cis-configuration can be prepared from 1.2-trans-glycosyl halidcs in an SN2-type reaction or from ether­ protected glycosyl acetates by treatment with tri methylsi lyl azide .~

A NaN3, DMF, 100 ·C. 2 h

75% ;~ / ""'Bc NaN3. Bll.tNHS04, 1 CH2CI2"sat. NaHC~. rt. 1-2 h .. %

B 01>£ ~~OAc TMSN3• snCl4 0,", C~CI2. rt. 24 h quanl. 3 4

Scheme 16. 1 Preparalion of glycosyl azidcs from (A) pe!acetylated glycosyl halides under classical homogeneous conditionsJ and undc! mild phasc·transfc! c.l1alysis6 and (8) from peracetylalcd sugars' Azides in Carbohydrale Chemisfry 471

The introduction of azides at the primary carbon of carbohydrates is conveniently carried out by an S/'f2 reaction. The generation of a good leaving group, such as a sul fo­ na te, is often possible in a selecti ve way without need for protection of the secondary hydroxy groups as was shown for GJcNAc derivative 5 (Scheme 16.2).10 Subsequent substi tut ion with sod ium azide usua ll y proceeds li t elevllted temperat ures with good yiclds. In cont mst, S/'f2 reactions at secondary carbons of the sugll r ring system are more complex. The SllCCCSS of such reactions is strongly dcpendcnt on the type of sugllr (stcrcochcmistry), thc position at which the SN2 reaction is cnrri cd out , anomcric confi gu­ ration, and used protecting groups. Nevertheless, this approach is widcly npplied for the introd uction of azido groups at the ring systcm. For instance, thc IIlcsy late of gluco­ side 7 WllS substit uted yielding 4-azido galactoside 8 under inversion of confi guration (Scheme 16.3)." Epoxides are :!Iso useful prccursors for the incorporati on of nzido groups by nucleo­ philic ntlnck. According to the Fi.i rst-Platlner rule,ll ring opening of sugar eflOxides by azide ions preferentially leads to the diaxial product. For ins tnnce. 2-azido cOlllflOund to is obtnincd regioselectively by opening of Cemy epoxide 9 with sodium azide (Scheme 16.4).u to was further converted into the suitably protected 11 , which was applied in the synthesis of a hepar-Ill sulfate synthon by 1.2-cis-glyeosylation (er. Section 16.4). Azides can also be introduced by radical addition to glycals. The classic:!1 azidonitra­ tion, developed by Lem ieux et at. in 1979, is a powerful method for the prcparntion of

OH 1)1 .2eqTsCI.pyr HO-S":O 2) NaN3, DMF, 80 ·C. 4 h _\~~ HO~CN H~O~CN NHAc 86% NHAc 5 6

Scheme 16.2 Regioselcclive introduction of an azido group aI /he primary carbon of 5 via nucleophilic replacement of a sulfonate intermeeliate 10 pyr '" pyridine

OPMB MsO 0 NaN3, DMF. 130 ·C. 32 h ~~MB BoO BnO 86% BnO~ ~OMe BnOOMe 7 8

Scheme 76.3 Replacement of a mesylate by an azido group (melL" inversion of configuration al .1 secondary center of Ihe sugar ring11

NaN3. OMF/H~. OAc 120 · C,12h PM80~\~O p;J ,,% BnO~O ...... CCI3 OPMB N, o" 9 11

Schem e 16.4 Regio- and slercose/ective opening of Cerny epoxide 9 le.1ds 10 2-azido com­ pound 10, which can be fur/her converted into glycosyl donor rr 1J 472 Organic Azides: Syntheses and Applications

AcO OAc OAo MO OAo CAN, NaN3, NE\,jCI, MaCN,-1S ' C.8h MO~ MeC N, 5 h AcO ~ ONC/:2 . AcO 0 ONC/:2 ACO~ N, ,<% 12 13 (75%) 14 (8%) " Scheme 16.5 Azidonitration of ga/actal 72 leads to an epimeric mixture of the 2-azido-l­ nitro-pyranoses 13 and 14 from which glycosyl donor 15 can be prep.1red directfy.l ~ CAN = cerium(lV) ammonium ni/rate

1) TfN3. 1 mol% CU S04, K2C03. OH OA' CH2CI2. MeOH, H20, 15 mln HO~\':'~ "'0 0 HO~OH 2) A~O. DMAP, pyr AeO OAe NH, ~N, 'HCI 16 82% 17

Scheme 16.6 Typical procedure for the Cu(ll)-catafyzed diazo /ransfer. 26 DMAP = 4- ( dimethyfami/Jo)pyridine

2-azido sugars that is still frequently used (Scheme 16.5). 14 It is especiall y useful for the synthesis of those 2-azido derivati ves, whose corresponding glycosamines lack accessibil­ ity from natural sources as in the case of galactosamine. However, while the reaction is highly regioselective, in most cases epimeric mixtures of the 2-azido compounds are formed. The ratio of the epimers strongly depends on the employed glyeal s ub s tr ate. ' ~ The obtained l-nitro-pyranoses can easily be converted into glycosy l donors, such as gJycosy l halides,1 4 trich lorOllcetimidates, 16 n-pentenyl ,17 or thioglycosides,18-20 which are valuable building blocks for the preparation of I ,2-cis glycosides of N-acetyl­ glycosamines (cf. Secti on 16.4). Similar methods for the synthesis of2-azido sugars using 21 22 13 radical addition to glycals are the lI zidochlorination and the azidophenylselenation. . Another possibi lity for the synthesis of organic azidcs is thc diazo transfcr usi ng trin yl azide.24 In conlrast 10 the methods descri bed above, not the entire azido group is incor­ porated into a but an N2 moiety is transfcrred onto an ex isting amine under retention of configuration. The first diazo Iransfer onto ami no sugars was reportcd in 1991 by Vasella el aes They treated different unprotected glycosamines with freshl y prepared tril1 yl azide under basic conditions. Aft er subsequent acetylation. the 2-azido sugars were isolated in good yields. This methodology was further improved by the addition of catalytic amounts of copper sulfate which leads to a much faster and more rel iable reaction (Scheme 16.6).26.27 Using the diazo transfer, it is possible to employ azides not onl y as amine synthons but also as temporary protecting groups for am ines . This has been applied for example to the synthesis of aminoglycosides (Section 16.3), heparan 29 lO sulfate fragmenls,l8 heparin fragments. . hyaluronan neoglycopolymers,lland N-acely l­ neuraminic acid dcri vali vcs. 12

16.3 Azides as Protecting Groups dUl'ing Aminoglycoside Synthesis

Protecting groups commonly employed for masking amino groups include alkyl carba­ mates such as benzyl-, left-butyl-, and 9-tluorenylmeth yl carbamalcs. If used for the Azidcs in 473

protecti on of containing multiple ami no groups, however, carbamate protecting groups can seriously complicate the interpretati on of NM R spectra. This is due to the occurre nce of ElZ rotamers that are in slow interconversion leading to multiple sets of signals. The use ofazides as protecting groups circum ve nts th is problem. Azides are easily reduced to amines. for example by catalytic hydrogenation or by reaction with thiols or complex hy dri des.·.J)~ A widely appli ed method in carbohydrate chemistry is the Staudinger reduct ion using triaryl- or trialkylphosphines.)' This mild procedure enables the selec ti ve reduction of azides in the presence of esters and benzy l ethers which arc frequentl y used as OH-protecting groups. Furthermore. azides can be di rectly converted into carbamate-protected amines using a variant of the Staudinger reaction (cr. Section 16.5 ). 17.36.37 Aminoglycosides are highly potent. broil.d-spectru m antibiotics, containing several amino groups presented on an oligosaccharide-like core ..l...... a Due to the appearance of bacterial strains resistanlto these drugs and due to their relatively high tox icity. lhe syn­ thesis of ami noglycoside derivatives wi th improved properties is of great intercsl.41 Several syntheses of aminoglycoside deri vatives using azides as amine protecting groups were reported,·Hl for instance the preparation of analogs of neomyci n B as shown in Scheme 16.7.21."t'I' Start ing fro m commercially available neomycin B (18), all six amino groups were converted into azides by diazo tra nsfer. After chemical derivllti1..ation of the structure, amines were regenerated by Staudinger reduction. In the course of these studies, it was observed that the regioselec ti ve reduction of a si ngle azide of multiple azide-containing molecules is feasible if only onc equi valent of phosphine is used.l7 Reduction of neamine derivative 23. for example, gave mono-amine 24 in a yield of 46 % (Scheme 16.8). Strong evidence was presented that the selectivity is primarily dctermim..'d by electronic factors wi th electron-deficient mddes being reduced more rapidl y and efficientl y than electron-rich azides. In compound 23 this is the case for

TfNJ• CuSO• . ElaN..,. ISh

I )~.THF.HiO.N~ 2) NB, ~ TI-F . EIOH ''''

Schcmc 16.7 SyntheSiS of aminoglycoside derivative 11 using azides as protecting groups for amines. First the amino groups of 18 were converted into azides by diazo /r.lnsfer. l1 After chemical remodeling of the aminoglycoside (one amino group was replaced by a hydroxy grotlp), the amines were regenerated by Stiludinger reduction" 474 Organic Azides: Sylllheses and Applicalions

PM~ (1.3 equiv), THF, H~, NaOH

46%

23 24

PM e3 (1. 1 equiv), toluene, BnO~O Boc-ON, - 78 ·C 10 10 ·C B'O~Nb BnO~ N BnO N N, ' 45% °9N'O~N', 0 0 °90~o NH0 BOC :1 ~ :1 ~ Cl Y Cl Y 25 Cl 26 Cl

Sd,cme 16.8 Regioselective reduction or tetra·azidt·s 2311 and 2S. ~<1 Boc·ON 2- (lcrl -butoxycarOOt'yJoxyimino)-2-phenylacetonitrile

the 2'-azide adjacent to the anomeric center. It was shown that the regioselectiv ity can be predicted on the basis of 13N and. to some extent. 'H NMR chemical shifts. Conse­ quentl y, by introducti on of electron withdrnwing 4-chlorobenzoyl protecting groups in the 5- and 6-position. the selecti vit y can be tuned in favor for reducti on of the I-azide (25 ~ 26).46."

16.4 Azidcs as Non-Participating Ncighboring Groups in GIycosyIatiolls

Although I ,2-cis glycosides of 2-am ino-2-dcoxys ugurs are less frequently found in natural products compared to their 1,2-trall.f isomers. they arc a common motive in important structures. In mucin-type O-glycoproteins. e.g. the glycan chains are attached to protein via an a -glycosidic linkage of N-acetyl-D-galactosamine to the fJ-hydroxy group of either serine or threonine, and a-glycosides of 2-acetamido-2-deoxY-I)- are found in the glycosaminoglycan heparan sul phate.4I-31 For the pre paration of these 1.2-cis glycosides, the commonl y employed N-acyl protecting groups are not suited because they lead to I ,2-tralls products 29 via neighboring group participati on (Scheme 16.9A).'UUJ In 1978 Pau lsen et al. showed that 2-azido-2-deoxy-glycosy l halides are suitable donors in 1,2-cis glycosylnlions. This approach preferentially leads to a -glycosides 32 either directly from J3.g lycosyl halides 30 (Scheme 16.9B)'"'.!IS or by in situ anomeriz3tion-'6 of a -glycosyl hnlides 33 (Scheme 16.9C).s7 Since then, the al.ide method has been widely used 'B llS-6.l and expanded by use of other glycosyl donors, sueh as trich loroacelirnidntes,'6 n-penlenyl glycosides,11 and th ioglycosides "-20 j ust 10 name n few. The req uired 2-azido-2-deoxy sugars are usuall y prepared by azidonitration of glyculs or by diazo transfer reacti on of Ihe corresponding glycosam ines as descri bed above. After , the azide can Azidcs in Carbohydrate Chemistry 475

A

activator PGO~X PGO~OR ROH HN 'f0 HN'f0 27 R 29 R

• activator .--0 .--0 _ .--0 PGO-~X PGO-"'1:;':1 - PGO-;"::'Jj ROH N3 AcHN N, 0R OR 30 31 32 c ,-...-0 Rl,N+X- PGO- --r.-l - 31 32 N,X activator, ROH [PGC~xl 33 30

SchOOl c 16.9 Preparation of D-g/yeasides of 2-amino-2-deoxysugars. (A) Use of N-acyl­ prolcclCd donors 27 results in ' ,2-trans glyeasylation due to neighOOring group participafion. (8) 1,2-cis glycosyla/ion produc/s 32 from fJ-glycosy/ halides 30~'ss or (C) by in situ anom­ crizalionst. of a-glyeasyl halidcs 33~ 1

be transformed to the natural acetamido function either in two steps by reduction of the azide and subsequent acetylation or in one step by reductive acetylati on using thioacetic acid.61.64 A successful approach for the synthesis of O-glycopeptides is the assembly of pre­ formed, more or less complex glycosyl amino acid building blocks by solid phase peptide synthesis (S PPS).(i().(i2.M-6J Based on initial work of FelTari68 and Pauisen,69 the azide method is extensively used for the preparation of such glycosyl amino acid building blocks. Especially the synthesis of complex glycosy l amino acids is chnllenging. Usually, glycosylation is pcrfonlled wi th followed by attac hment of fun her sugm' residues beeause glycosy lation reactions with oligosaccharide donors and serine or th reo­ nine acceptors often proceed with unpredictable . Nevertheless, oligosac­ charides have been successfully used in many glyeosylations as illustrated by the synthesis of glycosyl threonine building block 38 reponed by Danishefsky and coworkers (Scheme 16. 10).601

16.5 Glycosyl Azidcs as Precursors for Glycosyl Am ides

Beside the O-linked glycoproteins, the more prevalent form of glycosylation of proteins is N-linked glycosylation:'8.7o.11 N-G lycoprotei ns are characteri 7..cd by a /J-N-glycosidic linkage of the terminal N-acetylglucosamine of the pcntasaccha ri de core stmcture to the amide nitrogen of asparagine. The conventional synthetic strategy for the preparation of sllch glycosyl mn ides starts from glycosyl amincs which arc reactcd wi th activated and 476 Organic Azides: SYnlheses and Applications

"Y FmocHN C~Bn glycopeplide ""COO,,..

Scheme 16.70 SYnlhesis of glycosyl/hrconine building block 38 using the azide me/hod.I>< The 2-azido group is inlroduced by azic/onilration of 34 followed by preparation of donor 36. Glycosylation using threonine as acceptor leads 10 1,2-cis 37. After conversion of the azide group 10 an N-acetyf group by reciucfive acetylation, 38 was used as building block in glycopeptide synthesis

suitably protected aspartic acid derivatives 10 fonn the amide linkage.6O-6lM-61 Glycosyl amines are commonl y pre pared either by reduction of glycosyl azides (cf. Section 16.3) or by aminalion of unprotected reducing sugars with saturated nqueous ammonium bicar­ bonnte.ll Recently, improved variants of the laller procedure employing microwave irra­ 7J 14 di ntion • and ammonium carbamate,,,·76 respectively, have been published. Drawbllcks of this method li re the instabi lity of glycosyl ll mines lmd thei r propensit y for dimeri zation and unolllerization . Also, the preparation of a-glycosyl amides is a synthetic challenge. While the classical Staudinger reaction)J leads to iminophosphoranes which can be hyd rolyzed to amines under nqueous conditions (Staudinger reduction, cf. Section 16.3), the addition of acyl dOllors under dry conditions resul ts in amide forlll ntion.n .78 This proced ure was repeatedly applied for the synthesis of glycosyl umides, thus circunlventing the preparation of glycosyl amines. Initia ll y, Ihrcc-component reactions employing gly­ cosyl nzide, acti vated carboxyl derivat ive and phosphine were reported (Scheme 16, 11). The reaction starts fro m the ~ (39) or a-glycoside 45 with the formation of an imino­ phosphorane (40 and 42, respectively), which is then trapped by an acylati ng agent in the second step. The resulting acylami nophosphoni um salt (43/46) yields the corresponding glycosyl amide (44/47) upon . The intermediate iminophosphorane can undergo anomerizmion via open-chain fo nn 41 preferring ~con(iguratio n . TIle degree of isomeri­ zation is dependent on the efficiency of iminophosphorane trapping by the acylating agent. Differently activated carboxylic acids, such as carboxylic halides,79.1IO anhydrides,IIO.81 and carbodii mide-act ivated acids,82.8l have been employed as acylali ng agents. While ~ glycosyl mnides 44 can be obtained easily from ~g l ycosyl azides 39, the stereospecific Azides in Carbohydrate Chemistry 477

~ ::;..."'" H,O PGO--S-~ -= [ PGO-..-!LN.~::;...~ 00.] PGO&NHCOR' 40 oiI~ -R,PO .. I " >I' ,£:0OPG0 PGO-~N acyIami~ium salt 41 I (/)'''"' ::;..."';' .co, ",0 ~~ PGO-4 '"0,,--\ if; >1'] -R3PO .. ['GO~R'OC.N.~ NHCOR' _00" ."" .. " Scheme 76. ' 1 Mechanism of the S/audinger reaction wilh glycosyl azides

01>£ AcO---\~;- R 11 CHf 72% (~only) AcO~ NHCOR OAc R '" CF3: 76% (~only) • 48

R "CH3: 72% (~only) 48 2) (RCO),o R = CFf 61% (alP'" 85:15)

Scheme 16. 12 Three-componcmSt<1udingcr-type reaction wilh {J-g/yeasy' azide 4 stereos!!­ Icclively leads 10 the fJ-glycosyf amides 48. a-Clycosyl amides call only be obtained from a-glycosyl azide 49 wilh strong aerlaling agents to prevent complcle aflomerization of the intermediate iminophosphoranc fJO synthesis of a -glycosyl amides 47 starting fro m a-glycosy l azides 45 is only possible with strong acylaling agents which trap the intermediate iminophosphorane 42 before anorncri wtion can take place.so Rcpresentat ive examples for the three-componcnt Staudinger reaction are shown in Scheme 16.12. Rarely. the Staudinger reaction with reactivc alkylphosphines and free carboxy lic acids has been reported.l-I·c In this case. amide-bond fonnation is assumed to proceed in a concerted reaction without generation of an irninophosphorane intermediate. Recentl y, the synthesis of glycosyl amidcs has also been achievcd cmployi ng the trace­ 9 less two-component Staudinger ligation .16,17 developed in thc laboratories of Bertozzi" 19 90 and Raines · (Scheme 16.1 3). Starting from glycosyl azidcs 50 and 55, respectively, the initiall y formed iminophosphorane 52157 reacts with an intramolecular (thio)ester group 10 foml the llcylaminophosphoni um salt 53/58 from which the phosphine moiety is rcmoved by hydro lysis with watcr. Using benzyl protected a-glycosyl azides such as 50 478 Organic Azides: Syntheses ,1nd Applications

_\o~ B %?O~NH OMF,rt H20 BnO .J.-... - o. ~ "" BocHN CO:!Bn S9 (55 %. Ponly) ""6BocHN ~BIl " Scheme 16. 13 Two.-componelll Iraceless Slaudinger ligalions using phosphinc·derivatized eSler 5 1 (A)~ or lhiocsler 56 (Br

and stable phosphine 51 or similar esters in polar aprotic solve nts such as DMF, the reac­ tion proceeded stereo conservatively to yield predominantly a -glycosyl amides (Scheme 16.1 3A).9 The use of acetyl protected a -glycosy l azides, however, resulted only in ~ glycosyl amides due to isomerization of the less reacti ve iminophosphorane. All me thods described above have been used for the preparation of the ~g l ycosy l amide linkage between N-acety lglucosamine and the side chain of as paragine in both S4 tU 82 8l three-component reacti ons using free · or activated • carboxylic acids and two­ 9 87 component reactions as shown in Scheme 16. 13. . The obtained protected glycosyl amino acids can be used as building blocks in SPPS of N-linked glycopeptides.91 .92 It was also shown that deprotected sugars can be attached to amino acids and whole peptides using the thrcc-component reaction.9l Beside Staudinger-type reactions, another route towards the synthesis of glycosyl amides is the reaction of glycosyl azides with thiocar­ boxylic acids. 9J

16.6 Synthesis of Glycoconjugates via Azide-Alkync [3+2] Cycloaddition

Although the azide-alkyne l3+2J cycloaddition9-1 (cr. Chapter 9) is known in carbohydrate chemi stry for more than 50 years,9S its application for the preparation of glyeoconjugntes became particularly attractive with the development of the copper(l)-catalyzed variant by 96 97 Melda 1 and Sharpless. The copper(I)-catal yzed azide-alkyne cycloadd ition (CuAA q9\l,9') enables the regioselecti ve formation of 1,4-di substituted 1,2,3-tria zoles under very mild conditions even in a biological contex t. However, the cellulm· toxicity of the copper cata­ lyst precl udes ap plications wherei n cell s must remai n vi able. Therefore, as an al tern ative Azidcs in Carbohydraw Chemistry 479

Cui (5 equiv) i-P'2NEt (5 equlv) MoOH

>90%

61 62

Schcmc 16.14 Coupling of azide-substituwd galacloside 60 to illkYllc-modifted C,. hydro­ carbon 61 nOflcovalclltly bound 10 Ihe micrOliler well surface'f1l

to CuAAC. strain-promoted azide-alkyne P+2J cycloadditions hn ve been developed that proceed at room temperature wi thout the need for n clItalysl. 'OO.IOl These reactions are discussed in the next section dealing with mcta bolic oligosaccharide engineering. Another eX lUllple of metal-free Iriazolc formation by a tandem (3+2J cycloaddition-retro-Diels­ Alder reaction has been developed by van Berk el et al. although no carbohydrate-rellllcd application was reportcd_ 1Ol CuAAC reactions have been extensively lIppJied in carbohydrate chemistry including the synthesis of simplc glycoside and oligosacchllride mimetics. glyco-macrocycles. gly­ coconjugates. glycoclusters. and for the attachmcnt of carbohydratcs to surfaces. TIle field has been thoroughly reviewed'Jl&.lOJ-101 and, thercfore. we will focus on a fcw selected examples which arc of special interest for glycobiology_ One of thc firs t applicati ons of CuAAC in carbohydratc chem istry was - beside thc 96 one in the seminal paper of Meldal - the immobilization of azide-substituted sugars on microtiter plates (Scheme 16.1 4).'01:1 The surface-bound sugnrs such as 62 were screened wi th various Icctins and could be elongated by glycosylt ransfcrase-catalyzed fucosylation. The tcchni(IUC was later on improved by incorporation of a clcavublc disu Hide bond in the linker allowing mass spectrometnc chll racterization of the carbohydrate ,may_'OO Neoglycopeptides and -protcins'lo differ from naturally occurring structures by replace­ ment of the natuml carbohydrate-peptide li nkage wi th a non-natural one. Th is not on ly lI110ws sludying the influcnce of distinct structu ral elcments on biological ac ti vity. but has mllny practi cal applications as well. Use of chemoselecti ve ligation react ions such as 480 Organic Azides: Synlheses and Applic.1lions

OR OR Cu(OAc>2. RO - S'::O ,N:': N RO--\:O ~)" Na·ascorbate RO~N 3 • PG-HN ~OMe RO~N~X )" X I-BUCH, H20 o PG.HN OMe 63 64 o R = Ac, Bn, Bz PG '" Boc, Fmoc, Ts .5 X" OR, NHAc n"'1,2, 3

Scheme J 6, 15 Applica tion o( CuAAC (or (he prepar,llion of triazofc-linked gfyr:osyl amino acids 65'"

o Cull)

" 68

Scheme 76,76 Preparation of tyrocidine derivatives 68 by CuAAC o( propargyfgfyr:ine­ containing cyclic pep/ides 66 and aZido-funClionafized sugars 67" J

CuAAC makes glycoconjugates accessible to a broader community, Furthermore, the non-natural linkage often is more stable (both chemically and with respect to enzymatic degradation) which can lead to an increased half life of a glycoconjugate within a biologi­ cal system . Scheme \6.15 depicts the sy nthe s i.~ of triazole-li nked glycosyl amino acids 65 starting from glycosy[ azides 63 and different a[kyne-containing amino acids 64 which lll m can be used as bu ilding blocks in peptide synthesis. . Un and Walsh applied CuAAC for the attachment of2[ di fferent azido-functiona\ized monosaccharides 67 to [3 derivatives 66 of the cyclic decapeptide tyrocidine containing one to three propargyJg lyci ne residues at positions 3- 8 (Scheme 16.16). lI l Head-to-tai l cyclization of the peptides was accomplished using a thiocsterase domain from tyrocidine synthetase. Ant ibacterial and hemolyti c assays showed that the two best glycopeptide mimetics had a 6-fold better therapeutic index than the nntural tyrocidine. CuAAC has ll 6 further been used to attach carbohydrates to whole virus partic1es • . m and DNA." More challenging is the modification of bacteriall y expressed proteins by si te-speci fi c attachment of carbohydrates. Crucial step is the in troduction of a chemical tag. which can be chellloseJecliveJy modified. into the protei n. It has been shown thal alkyne- and azido-modificd amino acids, such as 2-amino-5-hcxynoic acid (homopropargylglycine, Hpg),lI7 4-azidohomoalanine (Aha),1l1.1I 9 lInd wi lh less effi ciency also l-azidoalanine, 5-azidonorvaline. and 6-azidonorleucine.1lO act as methionine surrogates that arc actio Azides in C.ubohydratc Chemistry 48 I

vated by the methionyt-tRNA synthetase of E. coli and replace meth ion ine in proteins expressed in methionine-depleted bacterial cultures. This, together with other methods for the incorpor

Scheme 16.17 (A) Asymmetrical, bifunctional dendrimer 69 containing 16 mannose units and two coumarin chromophores T46 and (13) poly(methacrylate)-based glycopolymer 70TU prep.lred by CuAAC and used for leetin binding studies with concanavalin A

been reported. '48-LS4 While the lI zides in most of these procedurcs arc introduced by a nucleophilic substit ut ion of a lellving group in allyl, bcnzyl, glycosyl, or similar posi­ tion. 148-ls2 aliphalic 1Sol and aromalic TSJ amino groups may also serve as precursors. We reported. for example a onc-pot procedure for diazo transfer und subsequent CuAAC wh ich allows the preparat ion of mu lt ivalent structures starting frorn commerciall y avail - Azide5 in Carbohydrate Chemistry 483 :J "

Schcmc 16. J 8 Sequential one-pot procedure for diazo trallsfer alld CuAAC U~ First, diaminc 7J is 1r.1llsformed 10 Ihe corresponding diazide by Cu(II)-catalyzcd diazo transfer. After completion_ Cu{/) required for subsequellt CuMC with 72 is generatcd by <1Clditioll of reduc­ iflg agent Na ascorbate. MW = microwave; roTA = tris(bcnzyltriazo/ylmellly/)amille

able amine scaffolds without need for isolation of the azide-containing intermediates. lS4 As an example, divalent glycoconjugate 73 was synthesized from diami ne 71 and pro par­ gyl glycoside 72 as shown in Scheme 16.18. Azides can also undergo [3+2J cycloaddition reactions wi th nitriles giving access to 1.5-disubstituled tetrazoles. lntennolccular reacti ons, however, require electron delicient nitrilcs and very forcing conditions to occur with sufficiently high rcilction rutcs. m-1jl High yields have been reported for the reaction of sulronyl and acyl cyanides with unhin­ ,YI dered al iphatic azides by neat, thennal fus ion.'SI. Ifllmmolecular 13+2 ) cycloaddition reactions of organic azides to nitriles occur more read ily.I 60-16-I Still, they require high reaction temperatures and yields are wi th few exceptions l611 not satisfactory. When precisely positioned on a rigid carbohydrate scaffold , however. azides can undergo cyclo­ addition reactions with nit ri les under exceptionally mild conditions. Thus, 3-azido- I,2- O-cyanoclhylidene-3-deoxy-allopyranose was shown to fonn a tetrazole embedded in a bridged tetracyclic ring system even at room tempcrature. l66

l6.7 Metabolic Oligosaccharide Engineering

Glycosylation of proteins is an important co- and posttranslationul event that has been estimated to occur on more than 50% of eukaryotic proteins. 161 The glycan chains of cell-surface glycoproteins are in volved in numerous recogni tion processes such as cell adhesion and attachment of bacteria or viruses. Inside cells, glycans direct protein traf­ Il IJ6 ficki ng and they modu late structure and acti vity of proteins. l- Hence. in vivo monitor­ ing of glycosylation processes is of utmost interest. "· While fluorescent fu sion proteins and other geneti call y encoded tags provide a means lor labcling specific proteins in li ve cells, analogous techniques are not available for secondary gene products including glycans. Metabolic oligosaccharide engi neering offers the possibility 10 introduce carbo­ hydrates with unnatural structu ral elements into the glycans without genetic manipulation making use of the cell 's biosynthetic machinery.l69 If suitable chemical reporter groups are introduced. subsequent addition of an exogenously delivered detectable probe allows for taggi ng of the glycans by a chemoselec ti ve ligation reacti on. Examples for such reporter groups incl ude kClonesl70.lll and th iols.112 However, the azido group is much more 4B4 Organic Azides: Syntheses and Applicalions

o ,-_ ~I" OM. prObe ~ PPh~

SUludlnger" ligaHon

Scheme 16. 19 Me/abolic oligosaccharide engineering: perace/yla/ed ManNAz 74 is taken up by mammalian cells and converted into an azide-containing sialic acid derivative which is incorporaled inlO sialic acid-bearing glycans 75. In Ihe /lex/ step, a delec/able probe 76 can be aI/ached IQ 7S via S/audinger Iigalion"··11J

suited for this approach because azides can take part in two important bioorthogonal ligation reacti ons. Sta udi nger Iigation ll4 (cf. Section 16.5) and azide-alkyne 13+2] cyclo­ addit ion (cr. Section 16.6 and Chapter 9). Azidc de rivatization of monosaccharides represents a subtl e structu ral change that is acccpted by several metabolic pathways. Thus, azide deri vatives of N-acetylmannosamine (Le. N-(azidoacetyl)man nosamine, ManNAz), N-acetylgalactosamine (i. e. N-(azidoacetyl) galactosamine, GaI NAz), N-acetylglucosarn ine (Lc. N-(azidoacctyl)glucosamine, GlcNAz), and L- fucose (i.e. 6-azido-L- fucose) have been cxplored.16I.169 Initia ll y, ManNAz was employcd to tag sialylated cell surface glycans of mammalian cells in vit ro (Scheme 16. 19).114.173 Cells are grown in the presence of pcracetylated ManNAz 74 which can be takc n up by the cell s more eas il y than ManNAz. Aft er de-O-acctylati on by cellu lar ester­ ases, res ulting Man NAz is metabolized simi ltlfly to its IIllti ve counterpart N-acetylman­ nosmnine and integrated in to cellular glycans. Finall y, the azide-Iabeled glycans are rellc ted wi th a detectable probe by StHudinger ligation. Ga lNAz can be Ill etabolicall y l1 introduced at the core position of mucin-type O-li nked glycoproteins. • Thus, a selective labeling of muci n-type glycoproteins is possible. Both, the metabolic labeli ng of sial ylated glycans with ManNAzm and labeling of muci n-type O-glycoprotci ns with GaINAz176 can be carried out ;/1 vivo. Analogously, GlcNAz has been used for the labeling of O-GlcNAc glycosylated proteins. m Recentl y, cells were labeled simultaneously with an azide- and a ketone-contai ni ng sugar.l 1B Using orthogonal ligation reacti ons, glycans bearing these sugar residues can be visualized in parall el on the same cells. In the cases mentioned so rar. nuorcscence labeling has been achieved by a two-step 12 procedure. First. a biotin label ( or FLAG tag (octapeptide As p-Tyr-Lys-Asp-Asp-Asp­ Asp_Lys)l1J·m.m is covalently attached to the azide-contlli ning glycan by Staudinger ligation at high concentration. In a second step. a nuorcscent ly labelcd rcceptor (avidin and lInti-FLAG . respectively) is lidded at lower concentration. To avoid the problem of high bllckground nuorcscence caused by the application or nuorcscent dyes, Azidcs in Carbohydrate Chemistry 485

O~ N''-r-n-i Cu(IJ ~~ OH 1)--0 . WOH HO 79 78 non-nuornscen\

Scheme 76.20 Generation of fluorescent Iriazole 80 by CuMC of fluorogenic ctllynyl­ naphtha/imide 78 and azide-Iabeled glycoproleins 79 applicable for intracellular loca/izatiOIl l of fucosyla ted g/ycoconjuga/es 7'1

Wong and coworkers developed a one-step labeling method based on CuAAC ligation using flu orogcni c dyes (Scheme [6.20).179 6-Azido-L-fu cose was applied for wgging of fucosylatcd protei ns by metabol ic oligosaccharide engi neering. Reacti on of alkyne-sub­ stitu ted naphthalim ide 78 and azide-modifi cd glycoprotcin 79 results in formation of fluo­ rescent triazole 80. Since 78 is not fl uorescent, it can be applied at high concentrations without produci ng a background signal. The method was used for cell surface glycopro­ tei n analysis and int racellular locali zati on of fucosylated glycoconjugates by us ing flu o­ rescence microscopy. Other examples for the application of CuAAC for labcling and visuali zati on of glyco­ protei ns in cells have been publ ished by the research groups of Bertozzil80 and WOll g. 181 The main advantage of CuAAC ovcr Staudingcr ligation is its much fa ster reaction kinct­ ics . Howevcr, thc use of CuAAC for applicmions ill lIillo is limited duc to the cellular toxicity of copper ions. This led to the development of copper-free variants of thi s cyclo­ addition. Bascd on observations made by Willig who described the exothennic cycload­ dition of cyclooctyne with phenyl azide."2 Deno7.Zi and cowork ers reponed the copper-free, strai n-promoted cycloaddition between azides and substituted cyclooclyne 8 1 for covalent modification of biomolcculcs in living systems (Scheme 16.2 1).100 The reaction rates were lower than those of CuAAC but comparable to those of Staudinger Ii gation.' l l TIle validity of the approach was demonstrated by functionaliz.ation of modi­ fied Jurkat cells with a biotin derivative of 81 .'110 Reaction rates of the strain-promoted azide-alkyne cycloadditioll could be dramllticnll y improved by introduction of electron­ withdrawing fluorine substituents in a position of the triple bond (Scheme 16.2 1. 82-84) with the difluorinatcd cyclooctync (DIFO) dcrivatives 83 and 84 possessing comparable I kinetics to those of CuAAC. SJ..IS5 Similar reaction ratcs were observed wi th dibcnzocy­ clooctyne derivative 85 .101 These reactions are not rcgiosclective but proceed chemose­ lectively within minutes on li ve cells wi th no apparent tOJ(ieity.' OI.I&UIS Latest application of DlFO derivative 83 is the ill lIillo imaging of membrane-associated glycans in live 486 Organic Azides: Symheses and Applications

1 1 ~F " QF [fF 0°2:::,... :::,... 0" ,9 '---./~ '-""'\ ° R R 0R R 81 ° 82 83 84 85

Scheme 16.21 Cyclooc/yne derivatives for use in copper·free, slr,l in'promoled azide·alkyne (3+2/ cycloaddilions designed by Bcr/oui (81,'00 82,'&) 83,'&· 84'85) and Boons (85'01 ). The secolld'genera/ioll difluorillaled derivalive 84 is easier 10 synthesize th,ln 83

developing .,.cbrafish.'86 Using two derivatives 83 wi lh different flu orophores auached, it WilS possible to pcrfonn a spmiotcmpoml .malysis of glycan cx prcssion and traffi cking.

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