Azides in Carbohydrate Chemistry

Azides in Carbohydrate Chemistry

Hemling

s,c.

Beckmllll1l and Valemin WiUmanll

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

16.1 Introduction

The first az.ide-containing 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 contained in carbohydrate derivatives were simply used as accessible syn- thons [or amities because of their easily performed reduction to amines. Due 10 their stability against a variety of reaction conditions, al.ides often can serve as masked amincs during the course of carbohydrate synthesis. The development of the diazo tmnsfer reac- tion facilitated the use of azides also as temporary protecting group for amines. This was extensively applied during the preparation of nrninoglycoside derivatives (Section 16.3).

Over the lasl three decades, azides became un important tool especially for the synthesis of glycopeptides and -proteins. In 1978 Paulsen el 0/. developed the 'a1.idc method' for the preparation of I ,2-cis-glyeosides of glycosamine derivatives using 2-nzido-2-deoxy- donors (Section 16.4). This reaction is widely used for the synthesis of O-linked glycosyl amino acid building blocks. In N-glycoproteins, the glycan chains are attached to the protein via a {j-glycosyl amide. Staudinger-type reactions offer a convenient 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 glycobiology during the last decade hurl the devel- opment of two bioorlhogonal reactions based on uzidcs: thc coppcr-catalyzed azide- alkync 13+2J cycloudclition unci the Staudinger ligution. Togcther with the possibility of ill vivo incorpor:ltion of azidc and alkync tags into glycuns and protcins, these reactions offcr ncw options for selective labelin8 and manipulation of biomoleculcs even within

Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-110645

URL: http://kops.ub.uni-konstanz.de/volltexte/2010/11064 First publ. in: Organic azides : syntheses and applications / eds. Stefan

Bräse .... - Chichester : Wiley, 2010, pp. 469-490 'The definitive version is available at www3.interscience.wiley.com'

470 Organic Azides: Synfheses and Applica/ions

living cells. Especially the azide-alkyne cycloaddition has been extensively applied for the chemical synthesis of neoglycoconjugatcs such as glycopeptide and glycoprotein mimics or multivalent glycoclustcrs (Section 16.6). Metabolic oligosaccharide engineer- 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 carbohydrates is the nucleophilic replacement of leaving groups by the azide ion. These reactions can be divided into three groups: substitutions at the anomeric center leading to glycosyl azides, substitutions at primary, and substitutions at secondary carbon atoms.

A widely used method for the preparation of glycosyl azides~ is the conversion of acetylated haJogenoses, such as I, by treatment with sodium a1.ide based on Bertho's initial work (Scheme 16.1A).' While homogeneous onc-phase reactions in DMF often require elevated temperatures,' phase-transfer catalysis enables milder conditions.⁶ One limitation of this 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).· Glycosyl azides with 1,2-1rarls- configuration arc easily obtained by the described methods using acyl protccting groups due to their neighboring group participation. 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 trimethylsilyl azide.~

A

;~

""'Bc

1

B

/

01>£

~~OAc

0,", 3

NaN3, DMF, 100 ·C. 2 h 75%

NaN3. Bll.tNHS04 ,

CH2CI2"sat. NaHC~. rt. 1-2 h

.. %

TMSN3• snCl4

C~CI2. rt. 24 h quanl.

4

Scheme 16.1 Preparalion of glycosyl azidcs from (A) pe!acetylated glycosyl halides under classical homogeneous conditions^J 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 sulfo- nate, is often possible in a selective way without need for protection of the secondary hydroxy groups as was shown for GJcNAc derivative 5 (Scheme 16.2).10 Subsequent substitution with sodium azide usually proceeds lit elevllted temperatures with good yiclds.

In contmst, S/'f2 reactions at secondary carbons of the sugllr 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 cnrricd out, anomcric configu- ration, and used protecting groups. Nevertheless, this approach is widcly npplied for the introduction of azido groups at the ring systcm. For instance, thc IIlcsylate of gluco- side 7 WllS substituted yielding 4-azido galactoside 8 under inversion of configuration (Scheme 16.3)."

Epoxides are :!Iso useful prccursors for the incorporation of nzido groups by nucleo- philic ntlnck. According to the Fi.irst-Platlner rule,ll ring opening of sugar eflOxides by azide ions preferentially leads to the diaxial product. For instnnce. 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 glycosyl donor 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 Lemieux et

at.

in 1979, is a powerful method for the prcparntion of

OH

HO-S":O HO~CN

NHAc 5

1)1.2eqTsCI.pyr

2) NaN3, DMF, 80 ·C. 4 h 86%

_\~~

H~O~CN

NHAc 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 BoO

BnOOMe ^86%

7

~~MB

BnO~

BnOOMe 8

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

p;J

OPMB 9

NaN3. OMF/H~.

120·C,12h

,,%

PM80~\~O

OAc

BnO~O ... CCI3

N,

o

"

11

Scheme 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 rr1J

472 Organic Azides: Syntheses and Applications

MO OAo CAN, NaN3, AcO OAc OAo NE\,jCI,

ACO~

^{MaCN,-1S'C.8h AcO}

^~

_N, ^ONC/:2

.

AcO

^MO~

0 ONC/:2 ^{MeCN, 5 h} ,<%

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

HO~\':'~

OH HO~OH

NH, 'HCI 16

1) TfN3. 1 mol% CUS04, K2C03.

CH2CI2. MeOH, H20, 15 mln 2) A~O. DMAP, pyr

82%

~

^OA'

"'0 0

AeO OAe

N, 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 especially useful for the synthesis of those 2-azido derivatives, 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 substrate.'~ The obtained l-nitro-pyranoses can easily be converted into glycosyl donors, such as gJycosyl halides,14 trichlorOllcetimidates, 16 n-pentenyl glycosides,17 or thioglycosides,18-20 which are valuable building blocks for the preparation of I ,2-cis glycosides of N-acetyl- glycosamines (cf. Section 16.4). Similar methods for the synthesis of2-azido sugars using radical addition to glycals are the lIzidochlorination21 and the azidophenylselenation.²².13

Another possibility for the synthesis of organic azidcs is thc diazo transfcr using trinyl azide.²⁴ In conlrast 10 the methods described above, not the entire azido group is incor- porated into a molecule but an N2 moiety is transfcrred onto an existing amine under retention of configuration. The first diazo Iransfer onto amino sugars was reportcd in 1991 by Vasella el

ae

^s They treated different unprotected glycosamines with freshly prepared tril1yl azide under basic conditions. After 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 reliable reaction (Scheme 16.6).26.27 Using the diazo transfer, it is possible to employ azides not only as amine synthons but also as temporary protecting groups for amines. This has been applied for example to the synthesis of aminoglycosides (Section 16.3), heparan sulfate fragmenls,l8 heparin fragments.²⁹.lO hyaluronan neoglycopolymers,lland N-acelyl- neuraminic acid dcrivalivcs.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-tluorenylmethyl carbamalcs. If used for the

Azidcs in Carbohydrate Chemistry 473

protection of molecules containing multiple amino groups, however, carbamate protecting groups can seriously complicate the interpretation of NMR spectra. This is due to the occurrence of ElZ rotamers that are in slow interconversion leading to multiple sets of signals. The use ofazides as protecting groups circumvents this problem. Azides are easily reduced to amines. for example by catalytic hydrogenation or by reaction with thiols or complex hydrides.·.J)~ A widely applied method in carbohydrate chemistry is the Staudinger reduction using triaryl-or trialkylphosphines.)' This mild procedure enables the selective reduction of azides in the presence of esters and benzyl ethers which arc frequently used as OH-protecting groups. Furthermore. azides can be directly 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-spectrum 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 toxicity. lhe syn- thesis of aminoglycoside derivatives with improved properties is of great intercsl.⁴¹ Several syntheses of aminoglycoside derivatives using azides as amine protecting groups were reported,·Hl for instance the preparation of analogs of neomycin B as shown in Scheme 16.7.21."t'I' Starting from commercially available neomycin B (18), all six amino groups were converted into azides by diazo transfer. After chemical derivllti1..ation of the structure, amines were regenerated by Staudinger reduction.

In the course of these studies, it was observed that the regioselective reduction of a single azide of multiple azide-containing molecules is feasible if only onc equivalent 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 with electron-deficient mddes being reduced more rapidly and efficiently 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

23

BnO~O

BnO~ N

°9N'O~N'

^{, 0 0}

:1 ~

Cl

Y

25 Cl

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

46%

PMe3 (1.1 equiv), toluene, Boc-ON, -78 ·C 10 10 ·C

45%

24

B'O~ Nb

BnO N

N, '

°90~NHBOC ^o

⁰

:1 ~

Cl

Y

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 regioselectivity can be predicted on the basis of 13N and. to some extent. 'H NMR chemical shifts. Conse- quently, by introduction of electron withdrnwing 4-chlorobenzoyl protecting groups in the 5-and 6-position. the selectivity can be tuned in favor for reduction of the I-azide (25 ~ 26).46."

16.4 Azidcs as Non-Participating Ncighboring Groups in GIycosyIatiolls

Although I ,2-cis glycosides of2-amino-2-dcoxysugurs 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)-glucose are found in the glycosaminoglycan heparan sulphate.4I-31 For the preparation of these 1.2-cis glycosides, the commonly employed N-acyl protecting groups are not suited because they lead to I ,2-tralls products 29 via neighboring group participation (Scheme 16.9A).'UUJ In 1978 Paulsen et al. showed that 2-azido-2-deoxy-glycosyl halides are suitable donors in 1,2-cis glycosylnlions. This approach preferentially leads to a-glycosides 32 either directly from J3.glycosyl 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^'BllS-6.l

and expanded by use of other glycosyl donors, sueh as trichloroacelirnidntes,'6 n-penlenyl glycosides,11 and thioglycosides"-²⁰ just 10 name n few. The required 2-azido-2-deoxy sugars are usually prepared by azidonitration of glyculs or by diazo transfer reaction of Ihe corresponding glycosamines as described above. After glycosylation, the azide can

A

•

c

PGO~X

HN'f0

27 R

.--0

PGO-~X

N, 30

,-...-0

activator

ROH

activator

ROH

Rl,N+X- PGO---r.-l

N,X activator, ROH 33

Azidcs in Carbohydrate Chemistry 475

.--0 _

PGO-"'1:;':1 - N30R 31

PGO~OR

HN'f0 29 R

.--0 PGO-;"::'Jj

AcHNOR 32

[PGC~xl

^{- 31 32}

30

SchOOlc 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 acetylation 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 (SPPS).(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 glycosyl amino acids is chnllenging. Usually, glycosylation is pcrfonlled with monosaccharides followed by attachment of fun her sugm' residues beeause glycosylation reactions with oligosaccharide donors and serine or threo- nine acceptors often proceed with unpredictable stereochemistry. 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 Amides

Beside the O-linked glycoproteins, the more prevalent form of glycosylation of proteins is N-linked glycosylation:'8.7o.11 N-Glycoproteins are characteri7..cd by a /J-N-glycosidic linkage of the terminal N-acetylglucosamine of the pcntasaccharide core stmcture to the amide nitrogen of asparagine. The conventional synthetic strategy for the preparation of sllch glycosyl mnides starts from glycosyl amincs which arc reactcd with activated and

476 Organic Azides: SYnlheses and Applications

" Y

FmocHN C~Bn

""COO,

glycopeplide

, ..

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 glycoside 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 commonly prepared either by reduction of glycosyl azides (cf. Section 16.3) or by aminalion of unprotected reducing sugars with saturated nqueous ammonium bicar- bonnte.^{l l} Recently, improved variants of the laller procedure employing microwave irra- dintion^7J•14 and ammonium carbamate,,,·76 respectively, have been published. Drawbllcks of this method lire the instability of glycosylllmines lmd their propensity for dimerization 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 hydrolyzed to amines under nqueous conditions (Staudinger reduction, cf. Section 16.3), the addition of acyl dOllors under dry conditions results in amide forlllntion.n .⁷⁸ This procedure was repeatedly applied for the synthesis of glycosyl umides, thus circunlventing the preparation of glycosyl amines. Initially, Ihrcc-component reactions employing gly- cosylnzide, activated carboxyl derivative and phosphine were reported (Scheme 16,11).

The reaction starts from the ~ (39) or a-glycoside 45 with the formation of an imino- phosphorane (40 and 42, respectively), which is then trapped by an acylating agent in the second step. The resulting acylaminophosphonium salt (43/46) yields the corresponding glycosyl amide (44/47) upon hydrolysis. The intermediate iminophosphorane can undergo anomerizmion via open-chain fonn 41 preferring ~con(iguration. 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 carbodiimide-activated acids,82.8l have been employed as acylaling agents. While ~ glycosyl mnides 44 can be obtained easily from ~glycosyl azides 39, the stereospecific

Azides in Carbohydrate Chemistry 477

::;..."'" = [ ::;...~ 00.]

^H,O PGO&NHCOR' ^~

PGO--S-~

- PGO-..-!LN.~

40

_I

oiI~ _{" >I'} ^-R,PO

..

,£:00

OPG acyIami~ium salt PGO-~N

'''"'

41

I (/)

.. ::;..."';'

_.co,

['GO~ ^>1']

^",0

^~~

PGO- 4

_{if; -R3PO}

'"0,,--\

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

48 R "CH3: 72% ^(~only) 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 from a-glycosyl azides 45 is only possible with strong acylaling agents which trap the intermediate iminophosphorane 42 before anorncriwtion can take place.so Rcpresentative examples for the three-componcnt Staudinger reaction are shown in Scheme 16.12. Rarely. the Staudinger reaction with reactivc alkylphosphines and free carboxylic 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.

Recently, the synthesis of glycosyl amidcs has also been achievcd cmploying the trace- less two-component Staudinger ligation⁹.16,17 developed in thc laboratories of Bertozzi"

and Raines¹⁹·90 (Scheme 16.13). Starting from glycosyl azidcs 50 and 55, respectively, the initially formed iminophosphorane 52157 reacts with an intramolecular (thio)ester group 10 foml the llcylaminophosphonium salt 53/58 from which the phosphine moiety is rcmoved by hydrolysis with watcr. Using benzyl protected a-glycosyl azides such as 50

478 Organic Azides: Syntheses ,1nd Applications

OMF,rt

""6

^{BocHN ~BIl}

""

"

_\o~

B%?O~NH

H20 BnO

.J.-...

- o. ~

BocHN CO:!Bn S9 (55 %. Ponly)

Scheme 16.13 Two.-componelll Iraceless Slaudinger ligalions using phosphinc·derivatized eSler 51 (A)~ or lhiocsler 56

(Br

and stable phosphine 51 or similar esters in polar aprotic solvents such as DMF, the reac- tion proceeded stereo conservatively to yield predominantly a-glycosyl amides (Scheme 16.13A).9 The use of acetyl protected a-glycosyl azides, however, resulted only in ~ glycosyl amides due to isomerization of the less reactive iminophosphorane.

All methods described above have been used for the preparation of the ~glycosyl

amide linkage between N-acetylglucosamine and the side chain of asparagine in both three-component reactions using free^S4·tU or activated^82•8l carboxylic acids and two- component reactions as shown in Scheme 16.13.^9.87 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 chemistry 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 Melda 1⁹⁶ and Sharpless.⁹⁷ The copper(I)-catal yzed azide-alkyne cycloadd ition (CuAA q9\l,9')

enables the regioselective formation of 1,4-disubstituted 1,2,3-triazoles under very mild conditions even in a biological context. However, the cellulm·toxicity of the copper cata- lyst precludes applications wherein cells must remain viable. Therefore, as an alternative

61

Azidcs in Carbohydraw Chemistry 479

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

MoOH

>90%

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 hnve been developed that proceed at room temperature without the need for n clItalysl.'OO.IOl These reactions are discussed in the next section dealing with mctabolic oligosaccharide engineering. Another eXlUllple of metal-free Iriazolc formation by a tandem (3+2J cycloaddition-retro-Diels- Alder reaction has been developed by van Berkel 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 first applications of CuAAC in carbohydratc chemistry was - beside thc one in the seminal paper of Meldal⁹⁶ - the immobilization of azide-substituted sugars on microtiter plates (Scheme 16.14).'01:1 The surface-bound sugnrs such as 62 were screened with various Icctins and could be elongated by glycosyltransfcrase-catalyzed fucosylation.

The tcchni(IUC was later on improved by incorporation of a clcavublc disuHide bond in the linker allowing mass spectrometnc chllracterization of the carbohydrate ,may_'OO

Neoglycopeptides and -protcins^'lo differ from naturally occurring structures by replace- ment of the natuml carbohydrate-peptide linkage with a non-natural one. This not only lI110ws sludying the influcnce of distinct structural elcments on biological activity. but has mllny practical applications as well. Use of chemoselective ligation reactions such as

480 Organic Azides: Synlheses and Applic.1lions

OR RO- -\ :O

RO~N3 X 63 R =Ac, Bn, Bz X" OR, NHAc

~)"

•

PG-HN ~OMe

64

o

PG'" Boc, Fmoc, Ts n"'1,2,3

Cu(OAc>2.

Na·ascorbate

I-BUCH, H20

OR

RO- S'::O ,N:':N

RO~N~

_{X )"}

PG.HN OMe

.5 ^o

Scheme J 6, 1 5 Application 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 synthesi.~ of triazole-linked glycosyl amino acids 65 starting from glycosy[ azides 63 and different a[kyne-containing amino acids 64 which can be used as building blocks in peptide synthesis.^lll.m

Un and Walsh applied CuAAC for the attachment of2[ different azido-functiona\ized monosaccharides 67 to [3 derivatives 66 of the cyclic decapeptide tyrocidine containing one to three propargyJglycine residues at positions 3-8 (Scheme 16.16).lIl Head-to-tail cyclization of the peptides was accomplished using a thiocsterase domain from tyrocidine synthetase. Antibacterial and hemolytic assays showed that the two best glycopeptide mimetics had a 6-fold better therapeutic index than the nntural tyrocidine. CuAAC has further been used to attach carbohydrates to whole virus partic1es^ll •. m and DNA."⁶

More challenging is the modification of bacterially expressed proteins by site-specific attachment of carbohydrates. Crucial step is the introduction of a chemical tag. which can be chellloseJecliveJy modified. into the protein. It has been shown thal alkyne- and azido-modificd amino acids, such as 2-amino-5-hcxynoic acid (homopropargylglycine, Hpg),lI7 4-azidohomoalanine (Aha),1l1.1I9 lInd wilh less efficiency 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 methionine in proteins expressed in methionine-depleted bacterial cultures. This, together with other methods for the incorpor<uion of non-canonical amino acids into proteins, Ill-Ill offers the pos- sibility to use az.ide-alkyne cycloaddition (and also Staudinger ligation'^M-'16) not only for protein labeling within cells or on cell surfaces l19_.120 but also for the preparation of neoglycoproteins.

Davis and coworkers expanded the diversity of chemical protein modification by a combination of this CuAAC-based labeling and disulfide bond formation via genetically engineered cysteine (eys) residues.¹²¹ Aha and Hpg. respectively, were introduced into engineered proteins by the auxotrophy-based residue-specific mcthod. Subsequent CuAAC reactions with alkyne-and azide-substituted carbohydrates. respectively. resulted in homogeneous protein glycoconjugates. As second modificlltion reaction. conjugation of Cys residues with substituted methanethiosulfonates was chosen. Applying these two orthogonal protein modification reactions. derivatives of the LacZ reporter enzyme car- rying the tetrasaccharide sialyl Lewis X and a sulfotyrosine mimic were created that allowcd detcction of mammalian brain inflammation.

Recently. Merkel et al. reportcd efficient N-terminal glycoconjugation of proteins by the N-end rule. U8 Bulky amino acids at the second and third sequence position of the bamasc inhibitor barstar efficiently prevenl excision of N-tcrminal methionine analogue Aha introduced by thc auxotrophy-based residue-specific method. lh: created azide tag at the protein's N-terminus was subsequently conjugated to propargyl glycosides of N- acetylglucosamine and N.N'-diacetylchitobiose. respectively, by CuAAC. The obtained glycoprotein mimetics show binding affinity to the lectin wheat germ agglutinin whereas the natural activity of barstar is conserved.

Lectins are carbohydrate-binding proteins other than immunoglobulins without enzy- matic activity towards the recognized sugars.^I29-I)1 Carbohydnlte-lectin interactions are involvcd in numerous intra- and intercellular events during development, inflammation.

immune response ,lIld cancer lllelastasis. I32-^U6 Multi valency appears 10 play an important role in lectin-mediated intcractions,IJ1-I40 and many lcctins arc found to recognize indi- vidual carbohydrate epitopes only with low affinity. Prepllration of carbohydrate clusters, therefore, is a common strategy to obtain high-arfinity lectin ligllnds.141-144 Because of its robustness, CuAAC is excellently suited for the simultaneous attachment of several car- bohydrate epitopes 10 11 scaffold. Initially, Santoyo-Gonzales Hnd coworkers prepared different multivalent mannose clusters starting from propargyl rnannosides and azide- containing scaffolds. '4s This strategy as well as the opposite approach based on azide- containing carbohydrates and alkyne-bearing scaffolds have been used intensively for the preparation of glycoclusters.9l.IO.l-I07 Glycosyl azides are easily produced. however. the direct attachment of a triazole to the sugar may interfere with the recognition of the car- bohydrate by the protein and, therefore. linkers of varying length havc been introduced between the sugar and the triawle moiety. It would be far beyond the scope of this chapter to mention all applications. Exemplarily, the asymmetrical. bifunctional dendrimer 69 containing 16 ITIallnose units and two coumarin chromophorcs^l-46 and poly(mcthacrylate)- bnsed glycopolymer 70141 are depicted in Scheme 16.17.

Although orgnnic azides are stable against most reaction conditions. compounds con- taining multiple az.ide residues (like multivalent scaffolds) are potentially explosive.

Thcrefore. several one-pot procedures to generate azides in .l'illl followed by CuAAC have

482 Organic Azides: Syntheses and Applications

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

been reported.^'48-LS4 While the lIzides in most of these procedurcs arc introduced by a nucleophilic substitution 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 which allows the preparation of multivalent structures starting frorn commercially 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 diamine 71 and propar- gyl glycoside 72 as shown in Scheme 16.18.

Azides can also undergo [3+2J cycloaddition reactions with nitriles giving access to 1.5-disubstituled tetrazoles. lntennolccular reactions, 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- dered aliphatic azides by neat, thennal fusion.'SI.,YI Ifllmmolecular 13+2) cycloaddition reactions of organic azides to nitriles occur more readily.I60-16-I Still, they require high reaction temperatures and yields are with few exceptions l611 not satisfactory. When precisely positioned on a rigid carbohydrate scaffold, however. azides can undergo cyclo- addition reactions with nitriles 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 Oli gosaccharide 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 involved in numerous recognition processes such as cell adhesion and attachment of bacteria or viruses. Inside cells, glycans direct protein traf- ficking and they modulate structure and activity of proteins.^Ill-IJ6 Hence. in vivo monitor- ing of glycosylation processes is of utmost interest."· While fluorescent fusion proteins and other genetically encoded tags provide a means lor labcling specific proteins in live cells, analogous techniques are not available for secondary gene products including glycans. Metabolic oligosaccharide engineering offers the possibility 10 introduce carbo- hydrates with unnatural structural 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 tagging of the glycans by a chemoselective ligation reaction. Examples for such reporter groups include kClonesl70.lll and thiols.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 reactions. Staudinger Iigation ll4 (cf. Section 16.5) and azide-alkyne 13+2] cyclo- addition (cr. Section 16.6 and Chapter 9).

Azidc derivatization of monosaccharides represents a subtle structural change that is acccpted by several metabolic pathways. Thus, azide derivatives of N-acetylmannosamine (Le. N-(azidoacetyl)mannosamine, ManNAz), N-acetylgalactosamine (i.e. N-(azidoacetyl) galactosamine, GaINAz), N-acetylglucosarnine (Lc. N-(azidoacctyl)glucosamine, GlcNAz), and L-fucose(i.e. 6-azido-L-fucose) have been cxplored. 16I.169 Initially, ManNAz was employcd to tag sialylated cell surface glycans of mammalian cells in vitro (Scheme 16.19).114.173 Cells are grown in the presence of pcracetylated ManNAz 74 which can be takcn up by the cells more easily than ManNAz. After de-O-acctylation by cellular ester- ases, resulting ManNAz is metabolized similtlfly to its IIlltive counterpart N-acetylman- nosmnine and integrated into cellular glycans. Finally, the azide-Iabeled glycans are rellcted with a detectable probe by StHudinger ligation. GalNAz can be Illetabolically introduced at the core position of mucin-type O-linked glycoproteins.^l1• Thus, a selective labeling of mucin-type glycoproteins is possible. Both,the metabolic labeling ofsialylated glycans with ManNAzm and labeling of mucin-type O-glycoprotcins with GaINAz¹⁷⁶ can be carried out ;/1 vivo. Analogously, GlcNAz has been used for the labeling of O-GlcNAc glycosylated proteins. m Recently, cells were labeled simultaneously with an azide- and a ketone-containing sugar.l1B Using orthogonal ligation reactions, glycans bearing these sugar residues can be visualized in parallel on the same cells.

In the cases mentioned so rar. nuorcscence labeling has been achieved by a two-step procedure. First. a biotin label¹²( or FLAG tag (octapeptide Asp-Tyr-Lys-Asp-Asp-Asp- Asp_Lys)l1J·m.m is covalently attached to the azide-contllining glycan by Staudinger ligation at high concentration. In a second step. a nuorcscently labelcd rcceptor (avidin and lInti-FLAG antibody. respectively) is lidded at lower concentration. To avoid the problem of high bllckground nuorcscence caused by the application or nuorcscent dyes,

1) ~~ - -0 .

78 non-nuornscen\

Azidcs in Carbohydrate Chemistry 485

N ''-r-n-i O~

W

^OH

HOOH

79

Cu(IJ

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 of fucosylated g/ycoconjuga/es^l7'1

Wong and coworkers developed a one-step labeling method based on CuAAC ligation using fluorogcnic dyes (Scheme [6.20).179 6-Azido-L-fucose was applied for wgging of fucosylatcd proteins by metabolic oligosaccharide engineering. Reaction of alkyne-sub- stituted naphthalimide 78 and azide-modificd glycoprotcin 79 results in formation of fluo- rescent triazole 80. Since 78 is not fluorescent, it can be applied at high concentrations without producing a background signal. The method was used for cell surface glycopro- tein analysis and intracellular localization of fucosylated glycoconjugates by using fluo- rescence microscopy.

Other examples for the application of CuAAC for labcling and visualization of glyco- proteins in cells have been published by the research groups of Bertozzi^l80 and WOllg.¹⁸¹ The main advantage of CuAAC ovcr Staudingcr ligation is its much faster 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 this cyclo- addition. Bascd on observations made by Willig who described the exothennic cycload- dition of cyclooctyne with phenyl azide."² Deno7.Zi and coworkers reponed the copper-free, strain-promoted cycloaddition between azides and substituted cyclooclyne 81 for covalent modification of biomolcculcs in living systems (Scheme 16.21 ^).100 The reaction rates were lower than those of CuAAC but comparable to those of Staudinger Iigation.^'ll 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 dramllticnlly improved by introduction of electron- withdrawing fluorine substituents in

a

position of the triple bond (Scheme 16.21.82-84) with the difluorinatcd cyclooctync (DIFO) dcrivatives 83 and 84 possessing comparable kinetics to those of CuAAC.^ISJ..IS5 Similar reaction ratcs were observed with dibcnzocy- clooctyne derivative 85.101 These reactions are not rcgiosclective but proceed chemose- lectively within minutes on live cells with 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

0 ° 2

^{:::,... 1}

^{~ F "}

^{:::,... 1}

^QF

^0"

,9 ^[fF '---./~

'-""'\ °

^R

°

^{R R}

81 82 0 R 83 84 85

Scheme 16.21 Cyclooc/yne derivatives for use in copper·free, slr,lin'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 wilh different fluorophores auached, it

WilS possible to pcrfonn a spmiotcmpoml .malysis of glycan cxprcssion and trafficking.

References

[I) A. Bertho. Ber. Dlsch. Chelll. Ges. 1930,63.836-43.

121 Z, Gy6rgydeak, L. Szitagyi, H. Paulsen, J. CMbollydr. Cllelll. 1993,/2,139-63.

(3J M. Hayashi. H. Kawabala, in Recem Devel. Carbohydrate Rts .• Vol. 1 (cd.; S.G. Pandalai).

Transworld Research Nelwork. Trivandrurn, 2003, pp. 195-208.

(4) Z. Gy6rgydeak, l. Thiem. Adv. Carbollydr. Chelll. Riodrem. 2006. 60. 103-82.

(5) W. pneiderer, E. BUhlcr, Chem. Ber. 1966,99.3022-39.

16J F.D. Tropper. F.O. Andersson. S. BnLUn. R. Roy. SYlrthesis 1992.618-20.

[7J R. Kurnar, P. Tiwari, P.R. Maulik, A.K. Mism, Ellr. J. Or8. Cl/em 2006. 74-9.

(8) li. Paulsen, Z. Gy6rgydcak, M. Friedmann, Clrem. Ber. 1974, /07. 1568-78.

[9J A.. Bianehi, A. Bemardi. J. OrK, Che"" 2006. 71,4565-77.

(10) F. Sicirerl, V. Willmann, AlIgew, Clrelll .. flrl, Ed. 200S, 44. 2096-9.

(11) C, Vogcl, p, Gries, J, Carbohydr. Chem. 1994, 11.37-46.

[12) A. FUrsl, P.A. PlaUner. in Abslracts of P11IJe/'s, I2lh Il1Iemllliol1l1l CO/lgress of Pllre wrd AWlied Chemislry, New York, 1951, p. 409.

(13) S. Amdt. Le. Hsich-Wilson, Orl/. Lelt. 2003. 5, 4179-82.

[14) R.U. Lcmicux, R.M. Ratcliffc, Cw!. J. Chem. 1979,57, 1244-51.

[15) A.F.G. Bongat, A.V. Dcmchcnko, ClIrOOhYllr, Res. 2007,142,374-406.

/161 G. Grundler. R.R. Schrnidl. Uebil/sAI1/1, Chem. 1984, 1826-47.

1171 SA Svurovsky. l1. Barchi, lr., Carbohydr. Res. 2003,138. 1925-35.

118J H, Paubcn, W, Rauwald, U. Weichert. UebigsAlllr. Clrem, 1988,75-86.

[19J 1. Hansson. PJ. Garegg, S. Oscarson, 1. Org. Clrelll. 2001. 66, 6234-43.

[20/ 1D.C. Cod6e. R.EJ.N. Liljens, R. den Hcclen, ell/I., Org. Lell. 2003. 5. 1519-22.

/21) N.V. Bovin. S.E. Zurabyan. A.Y. Khorlin, Carbohydr, Res. 1981,98,25-35.

122) F. Sanloyo·Gonzalez. F.G. Calvo·Aorcs. P. Garcfa·Mendo1.a. el af" J. Or8. C/rem. 1993,58.

6122-5.

123( S. Czcmccki. E. Ayadi. D. R~mdriamandimby, J. Org. C/rem. 1994,59,8256-60.

1241 C,l. Cavender. V.J. Shiner, J. Ors. C/rem. 1972,17.3567-9.

[25] A, Vasclla, e. Wilzig. l·L, Chium, M. Munin-Lomas, He/I'. Chilli. ACll/ 1991, 74, 2073-7.

126J P,B. Alper, S,-e. Hung, e.-H. Wong, TetralledrOIl Lell. 1996,17.6029-32.

(27) P.T. Nyffclcr, e..H. Liang, K.M. Koellcr. C.-H. Wong. J, Am. Clrtm, Soc. 2002. 124, 10773-8.

(28) O. Gavard, Y. Hcrsanl. 1. Alais, et lit.. Ellr. J. Org, C/rcm. 2003,3603-20.

Azides in Carbohydrate Chemistry 487

129J Miehael F. Hailer, G.-l Boons, Ellr. J. Org. Chem. 2002,2033-8.

130J H.A. Orguciru, A. Burtolozzi. P. Schell, el (11., Chem. £lIr. J. 2003, 9, 140--69.

[311 S.lyer, S. Rclc, G. Gmsa, S. Nolan, E.L. Chaikof, Chem. COIIIIIIIIII. 2003,1518-19.

[32] N. Laurcnt, D. L<lfont, p, Boullanger, l.M. Mallet, Carbohyt/r. Res. 2005, 340,1885-92.

[33] S. Brlisc, C. Gil, K. Kneppcr, V. Zimmcn1mnn, AI/gew. Chem., 1111. Ed. 2005,44,5188- 240.

I34J E.F.V. Scriven, K. Tumbull, Chem. Rev. 1988,88,297-368.

(5) H. Staudinger, 1. Meyer, Helv. Chilli. ACla 1919, 2, 635-46.

[36J X. Ariza, F. Urpi, 1. Vilarmsa, Tetrahedrol/ fLit. 1999,40,7515-17.

[371 X. Ariza, F. Urpi, C. Viladomat, 1. Vilarrasa, Telrahedron ^fLit. 1998,39,9101-2.

[38J l.G. Silva, I. Carvalho, Cllf(. Med. Chem. 2007, /4, J 101-19.

[39J D.S. Pilch, M. Kaol, C.M. Barbieri, Top. Cllf(. Chelll. 2005, 253, 179-204.

[40J Q. ViCCIlS, E. Westhof, ChemBioChem 2003, 4, 1018-23.

[4JJ M. 11;linrichson,

r.

Nudclman, T. Baasov, Org. Biomol. Clzem. 2008, 6, 227-39.

[42J Y. Ding, E.E. SwaY7.c, S.A. Hofstadlcr, R.H. Griffey, Tetrahedron /..ell. 2000, 41. 4049-52.

(43] M. Fridman, V. Bclakhov, S. Yaron, T. Baasov, Org. Letl. 2003,5, 3575-8.

[441 P.B. Alpcr, M. Hendrix, P. Scars, C.-H. Wong, J. Am. Chem. Soc. 1998,120, 1965-78.

[451 W.A. Grccnbcrg, E.S. Priestley, P.S. Scars, et aI., 1. Am. Chem. Soc. 1999, /2/,6527- 41.

[46J 1. Li, H.-N. Chen, H. Chang, J. Wang, C.-W.T. Chang, Or8. fLlI. 2005, 7, 3061-4.

147J l. Li, F.-I. Chiang, H.-N. Chen, C.-W.T. Chang, J. Org. Clzem. 2007, 72,4055--66.

[48] V. WiUmann, in Glycoscience: Chemislryalld ClzemicalBiology, 2 cd. (eds.: B. Fmscr-Rcid, K. Tatsuta, 1. Thicm), Springer-Verlag, Berlin, 2008, pp. 1735-70.

[491 H.C. Hang, C.R. Bertozzi, Bioorg. Med. Clzem. 2005, 13,5021-34.

[50] P. Van den Steen, P.M. Rodd, R.A. Dwek, G. Opdenakker, Crit. Rev. Biacllem. Mol. Bioi.

1998,33, 151-208.

f51J 1.R. Bishop, M. Schuksz, 1.0. Esko, Nail/re 2007, 446,1030-7.

(52J 1. Banoub, P. Boulhmger, O. Lufont, Chem. Rev. 1992,92,1167--95.

(53J J. Debcnham, R. Rodebaugh, B. Fraser-Reid, Liebigs AIlIl.lReCllei/1997, 791-802.

[54J H. Paulscn, W. Stcnzel, Chem. Ber. 1978, 1/1,2334-47.

[551 H. Paulsen, C. Kolar, W. Stcnzcl, Chem. 8er. 1978, 11/,2358-69.

[56] R.U. L.cmieux, K.B. Hendriks, R.V. Stick, K. lames, J. Am. Chem. Soc. 1975, 97, 4056-62.

[57) It Palllsen. O. Lockhoff, TetmhelirolZ £,ell. 1978,4027-30.

[581 B. Palllsen, Angew. Cllelll., 1nl. £t/. ElIgl. 1982,21, 155-73.

[59] R.R. Schmidt, Angew. Chem., Int. Etl. Encl. 1986,25,212-35.

[60) H. Herzncr, T. Reipcn, M. Sehullz, H. Kunz, Chem. Rev. 2000, 100,4495-538.

[61 J M.R. Prate, C.R. Bcrtozzi, Chem. Soc. Rev. 2005,34,58-68.

[621

c.

Haase, O. Seitz, Top. Cllf(. Chem. 2007, 267, 1-36.

[63] T. ROSCII, I.M. Lico, O.T.W. Cho, J. Org. Chelll. 1988,53, 1580-2.

[64] lB. Schwarz, S.D. Kuduk, X.-T. Chen, el (11., 1. Am. Cllem. Soc. 1999, 121,2662-73. [65J B.G. Davis, Chem. Rev. 2002, 102, 579-601.

[661 T. Boskas, S. Ingalc, G.-1. Boons, Glycobiology 2006,16, I J3R-J36R.

(671 GlycOIJeplides al/d GlycoproleiflS: Sylllhesis, Slfllclllre, and ApplicmiOlz (TOI)ics in CI/rrelll Chemistry, vol. 267) (cd.: V. Winmallll), Springer-Verlag, Berlin, 2007.

[68] B. Ferrari, A.A. Paviat. Cllfbollydr. Res. 1980, 79, CI-C7.

!691 H. Paulscn, l.-P. Hoick, Carbohydr. Res. 1982, 109, 89-107.

[70] L. Lchle, S. Strahl, W. Tanner, A,zgew. Clzem., Int. Ed. 2006, 45, 6802-18.

[71] P.M. Rudd, R.A. Dwek, Crit. Rev. BiOl.:hem. Mol. Bioi. 1997,32,1-100.

[72J L.M. Likhoshcrstov, O.S. Novikova, V.A. Dcrcvitskaya, N.K. Kochetkov, C(lfbohylir. Res.

1986, /46, cI--c5.

/131 M. Bejllgam, S.L. Flitsch, Org. £,ell. 2004,6,4001-4.

[74J M.A. Brun, M.O. Disney, P.H. Sccbcrger, ChemBioClzem 2006, 7, 421-4.

[75J C.P.R. Haekenbcrgcr, M.K. O'Rcilly, B. Impcriali, 1. Org. Cllt!m. 2005. 70,3574--8.