Synthesis of Glycosyltransferase Inhibitors · REVIEW 3179 Synthesis of Glycosyltransferase...

REVIEW 3179

Synthesis of Glycosyltransferase InhibitorsGlycosyltransferase InhibitorsTetsuya Kajimoto,*a Manabu Nodeb

a Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, JapanFax +81(72)6901084; E-mail: [email protected]; E-mail: [email protected]

b Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan Received 12 May 2009; revised 20 June 2009

SYNTHESIS 2009, No. 19, pp 3179–3210xx.xx.2009Advanced online publication: 03.09.2009DOI: 10.1055/s-0029-1216976; Art ID: E24009SS© Georg Thieme Verlag Stuttgart · New York

Abstract: Glycosyltransferases and glycosidases work together toconstruct the oligosaccharide moieties of biologically active glyco-conjugates. Although many excellent glycosidase inhibitors havebeen developed, some of which are in clinical use, there are relative-ly few promising candidates of glycosyltransferase inhibitors. Inthis review, we summarize the current state of the development ofglycosyltransferase inhibitors.

1 Introduction2 Basic Strategy for Designing Glycosyltransferase Inhibitors3 Sugar Nucleotide Analogues4 Analogues of Acceptor Oligosaccharides (Acceptor

Analogues)5 Bisubstrate Inhibitors6 High-Throughput Screening: Discovering Structurally

Simple Inhibitors7 Antisense Inhibitors8 Questions and Future Directions

Key words: glycosyltransferases, inhibitors, transition-state ana-logues, sugar nucleotides, oligosaccharides

1 Introduction

Developments in carbohydrate chemistry and glycobiolo-gy in the last two decades reveal the essential roles thatoligosaccharides play in biologically important events. Inparticular, functions of glycolipids and glycoproteins ex-pressed on the surface of mammalian cells have beenstudied with much interest relating to fertilization, im-mune responses, and metastasis in tumor cells.1 The oli-gosaccharide moieties of the glycoconjugates areconstructed by precisely controlled actions of glycosid-ases and glycosyltransferases. The former degrade imma-ture oligosaccharides in the endoplasmic reticulum andGolgi apparatus. The latter transfer monosaccharidesfrom sugar nucleotides to the non-reducing end of appro-priate oligosaccharides in the Golgi.2 Moreover, bacterialoligosaccharides of cell walls, being essential for prolifer-ation and protection against osmotic pressure, have alsoattracted chemists because they include, as a component,unique monosaccharides that are not found in animalcells. The monosaccharides are incorporated into the cellwalls by action of distinctive glycosyltransferases in bac-teria.

Whilst many excellent glycosidase inhibitors have beendeveloped and some are now in clinical use,3 only a fewpromising glycosyltransferase inhibitors have been re-ported to date. Why the development of the glycosyltrans-ferase inhibitors has been slow can be attributed to severalfactors, including: (1) the lack of an available three-dimensional structure of glycosyltransferases until that ofb(1,4)-galactosyltransferase was disclosed using X-raycrystallography in 1999;4 (2) the complexity of the transi-tion state that involves a sugar nucleotide, an acceptorsubstrate, and a metal ion; (3) the weakness of the bindingaffinity between substrates and glycosyltransferases; and(4) the absence of facile assay methods due to no changesin UV absorption or fluorescence intensity during glyco-syltransferase-catalyzed reactions.5

Herein, we review recent progress in the synthesis of gly-cosyltransferase inhibitors, in terms of efforts made to en-hance their activity and selectivity.6

2 Basic Strategy for Designing Glycosyltrans-ferase Inhibitors

Glycosyltransferases are enzymes which transfer amonosaccharide from a sugar nucleotide to the non-reduc-ing end of an oligosaccharide. As glycosyl donors in gly-cosyltransferase-catalyzed reaction, sugar nucleotidesshould be composed of a proper combination of a nucleo-tide and a monosaccharide. Namely, galactose and N-acetylglucosamine can be glycosyl donors only whenlinked with UDP to form UDP-galactose (UDP-Gal) andUDP-N-acetylglucosamine (UDP-GlcNAc). Similarly,mannose and fucose can be glycosyltransferase substrateswhen bound with GDP to form GDP-mannose (GDP-Man) and GDP-fucose (GDP-Fuc), respectively. N-Acetylneuraminic acid (NeuAc) as well as N-glycosyl-neuraminic acid and 2-keto-3-deoxynonic acid (KDN),often referred to generally as sialic acid derivatives, canbe donor substrates, termed CMP-sialic acid or CMP-NeuAc.

The substrate specificity of acceptor oligosaccharides var-ies, depending on the source of each glycosyltransferase.This specificity is used to divide a family of glycosyl-transferases, which employ the same sugar nucleotide asthe donor substrate, into several classes.

This review divides the inhibitors into four categories: (1)analogues of sugar nucleotides; (2) analogues of acceptorsubstrates; (3) bisubstrate inhibitors in which the sugar

3180 T. Kajimoto, M. Node REVIEW

Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York

nucleotide and acceptor substrate are linked via covalentbonds; and (4) other inhibitors developed recently.

3 Sugar Nucleotide Analogues

Glycosyltransferases seem to bind more tightly with thenucleotide moiety than with the sugar moiety, as they arepotently inhibited by nucleotides produced as a side prod-uct in the corresponding enzymatic glycosylation.7 There-fore, the sugar or sugar phosphate moieties have beenmore intensively modified than the nucleotide moieties inthe design of sugar nucleotide analogues.

3.1 Fluorine-Containing Analogues

The introduction of fluorine at the C-2 position hindersthe hydrolysis of glycosidic linkages.8 The strong elec-tronegativity of fluorine destabilizes the oxocarbeniumion generated in the transition state. Furthermore, a sec-ond isotope effect was observed in a b(1,4)-galactosyl-transferase-catalyzed reaction using UDP-[1-2H]-Gal asthe donor substrate and an a(1,3)-fucosyltransferase-cata-lyzed reaction using GDP-[1-2H]-Fuc as the substrate.9

This suggested that oxocarbenium intermediates are gen-erated during the enzymatic glycosylation, and thereforethat UDP-Gal and GDP-Fuc fluorinated at C-2 could beinhibitors of the corresponding glycosyltransferases. Onthis basis, UDP-[2F]-Gal (1; Scheme 1) and GDP-[2F]-Fuc (5a and 5b; Scheme 2) could be galactosyltransferaseand fucosyltransferase inhibitors, respectively.

Wong and colleagues synthesized 2-deoxy-2-fluorogalac-tose (3) from 3,4,6-tri-O-acetyl-D-galactal (2a) by fluori-nation with xenon difluoride10 in the presence of borontrifluoride diethyl ether complex and acid hydrolysis(Scheme 1). Meanwhile, 3 was also prepared in one stepby direct fluorination of D-galactal (2b) in an aqueous so-lution.11

Scheme 1 Chemoenzymatic synthesis of UDP-[2F]-Gal (1).Reagents and conditions: (a) XeF2, BF3·OEt2, Et2O–benzene, r.t., 2.5h; (b) 2 M HCl, 90 °C, 2 h; (c) XeF2 (1.8 equiv), H2O, r.t., 1.5 h; (d)galactokinase; (e) pyruvate kinase; (f) galactose-1-phosphate uridyl-transferase; (g) UDP-glucose phosphorylase; (h) pyrophosphatase.

The 2-fluorinated galactose (3) was converted into UDP-[2F]-Gal (1) via a series of enzymatic reactions, namelyphosphorylation by galactokinase to produce galactose-1-phosphate (4), and successive reactions catalyzed by ga-lactose-1-phosphate uridyltransferase to replace the 1-

Tetsuya Kajimoto wasborn in 1960 and receivedhis B.S. in 1982 fromTokushima University. Hethen entered the GraduateSchool of Kyoto Universityand received his M.S. in1984 and Ph.D. in 1989. Hewas then appointed as As-sistant Professor at Kuma-moto University. After twoyears of postdoctoral study

(1990–1991) with ProfessorChi-Huey Wong at TheScripps Research Institutein San Diego (USA), hemoved to the Frontier Re-search Program at the Insti-tute of Physical andChemical Research (RIK-EN) (1991–1996). He wasAssociate Professor atShowa University (1996–1999), Tokyo University of

Agriculture and Technology(1999–2003), and KyotoPharmaceutical University(2004–2007). Then he wasappointed as Professor at theSuzuka University of Medi-cal Science (2008). He isnow a visiting researcher atthe Osaka University ofPharmaceutical Sciences.

Manabu Node was born in1945 and received his B.S.in 1967 from TokushimaUniversity. He then movedto Kyoto University and re-ceived his M.S. in 1970 andPh.D. in 1973. He was ap-pointed as Assistant Profes-sor at the Institute ofChemical Research at

Kyoto University. After ayear of postdoctoral studywith Professor Jung at theUniversity of Californa LosAngeles (USA), he was pro-moted to AssociateProfessor in 1983. He thenmoved to Kyoto Pharma-ceutical University in 1990,where he is currently Pro-

fessor of PharmaceuticalManufacturing Chemistry.He was a recipient of theYoung Scientist Awardfrom the PharmaceuticalSociety of Japan (1985) andthe Miyata FoundationAward (2000). His researchinterest is in the area ofasymmetric synthesis.

Biographical Sketches

cOHO

FHO

OH

OHO

AcO

FAcO

OAc

F

O

AcO

AcO OAc

ATPH2 ADPH2

PEPPyr

OHO

FOH

OH

OPO3= UDP-Glc Glc-1-P

UTPH2PPi

OHO

FHO

OH

OP

OP

O

O OO– O–

O

HO OH

N

NH

O

O

a b

2a 3

d f

g

3

1

4

Ki = 149 μM against β(1,4)-GalTase (Mn2+ 1 mM)

30%78% 83%

e

h

Pi

58% 67%

2b

O

HO

HO OH

REVIEW Glycosyltransferase Inhibitors 3181


phosphate moiety of UDP-Glc with 4.12 By employing arecycling system of coenzymes,13 only a small amount ofthe expensive UDP-glucose (UDP-Glc) was required tostoichiometrically convert 3 into 1.14 The Ki value (149mM) of 1 against b(1,4)-galactosyltransferase was thesame as the Km value (127 mM) of UDP-Gal. As the Km

value depends on the concentration of Mn2+,15,16 the Ki

value of the inhibitors could be affected by metal ions. Infact, the inhibitory activity of 1 was enhanced as the con-centration of Mn2+ was increased and the Ki value reached7 mM in the presence of 10 mM of Mn2+,7,15b while onlyweak inhibition was observed against a(1,3)-galactosyl-transferase under the same conditions.17

In the synthesis of GDP-[2F]-Fuc (5a and 5b), 6b – a bis-triflate substitute of 1-chloromethyl-4-fluoro-1,4-diazobi-cyclo[2.2.2]octane bistetrafluoroborate (F-TEDA-BF4,Selectfluor®; 6a) – was chosen as the fluorinating agent.18

In this sequence, fucoglycal 7a was treated with 6b and(BnO)2PO2H, and deprotection afforded 2-deoxy-2fluoro-a-L-fucosylphosphate (8), which was further treated withGMP-morpholidate (9) in the presence of 1H-tetrazole togive GDP-[2F]-Fuc (5a).19,20 The anomer 5b was alsosynthesized by a protocol initiated with fluorination offucoglycal 7b (Scheme 2).9b,20

Next, GDP-[6F]-Fuc (10) was synthesized from L-galac-tose (11) using (diethylamino)sulfur trifluoride (DAST)to fluorinate the C-6 hydroxy group. The inhibitors 5a, 5b,and 10 all had potent inhibitory effects on fucosyltrans-ferases. Moreover, 5a, in which the configuration of theanomeric carbon was reversed relative to the naturalGDP-Fuc, was found to be a potent inhibitor of fucosyl-transferase V and VI. Interestingly, 5a was a substrate aswell as inhibitor of fucosyltransferase III.20,21

The same strategy was applied to the design and synthesisof sialyltransferase inhibitors. The methyl ester 12b of2,3-dehydro-N-acetylneuraminic acid tetraacetate (12a)was converted into a mixture of 13a and 13b (1:3) in 80%yield by fluorination with 6.22 The application of themethod reported by Chappell and Halcomb23 using phos-phoroamidite 14 afforded CMP-[3F]-NeuAc (15)(Scheme 3).22 Although the Ki value of 15 against a(2,6)-sialyltransferase was small (5.7 mM), the inhibition wasnot potent as the Km value of CMP-NeuAc was also rela-tively small (15 mM).24

Along the same line, ADP-[2F]-L-glycero-b-D-gluco-hep-topyranose (16) was recently synthesized as an inhibitorof heptosyltransferase, a key enzyme in the constructionof the lipopolysaccharide of Gram-negative bacterial cellwalls, targeting a candidate of novel antibiotics. L-Hepto-syl-glycal 17, prepared from D-glucose in 15 steps, wassubjected to fluorophosphorylation using Selectfluor (6a)and dibenzyl phosphate. The resulting fluorophosphate 18was deprotected to give 19, which was further convertedinto 16 by way of a tetrazole-catalyzed morpholidate cou-pling procedure (Scheme 4). Although the natural hepto-syltransferase substrate requires its sugar nucleotide 20 tobe derived from manno-heptoside and ATP,25 16 dis-played competitive inhibition against the heptosyltrans-ferase from pathogenic E. coli with an IC50 of 30 mM.26

In addition to the 2-fluoro and 6-fluoro substituents ofsugar nucleotides, 5-fluoro substituents would destabilizethe putative oxocarbenium ion generated in the transitionstate of a glycosyltransferase-catalyzed reaction. In spiteof the need for a flexible method of producing 5-fluoroglycosides for the preparation of a variety of substratesand inhibitors – not only for glycosyltransferases but alsofor glycosidases, dehydrogenases, dehydratases, and epi-merases, which catalyze the transformation of monosac-charides at the C-1, C-4, C-5, and C-6 positions – only afew methods have been developed to date. Withers and

Scheme 2 Synthesis of GDP-[2F]-Fuc (5a and 5b) and GDP-[6F]-Fuc (10). Reagents and conditions: (a) 6b then (BnO)2PO2H; (b) H2,Pd/C; (c) cyclohexylamine; (d) 1H-tetrazole, 9; (e) XeF2, BF3·OEt2;(f) 2 M HCl, 90 °C; (g) Ac2O, pyr; (h) HBr, AcOH; (i) (BnO)2PO2H,Ag2CO3; (j) H2, Pd/C; (k) cyclohexylamine, MeOH; (l) AG 50W-X2(Et3N); (m) CuSO4, H2SO4 (cat.), acetone; (n) DAST, collidine; (o)AcOH, H2O; (p) BzCl, pyridine; (q) HBr, AcOH, Ac2O; (r) AG 50W-X2 (Et3N), 9, 1H-tetrazole, pyridine.

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

O

O–O

OH

N

N

HOF

O

ORRO

a

P

O

OOBn

OOBz

BzOF

OBnb, c

7a: R = Bz7b: R = Ac

P

O

OO–

O

OHHO

F

O–

NH3+

2

d

8

O

AcOOAc

F

F

O

AcOOAc

F PO

OOBn

OBnO

AcOOAc

F

OAc

e

f, g

j–l, dO

N

NH

O

NH2

OHHO

OPO

O

O–

P

O

O

O–

OOH

N

N

HOF

5a

5b

54% 92%

h, i

Ki = 36 μM against FucTase VKi = 2 μM against FucTase VI

N N FClCH2 2X++

6a:

O

OHHO

OPN

O

O–

GO

9

OHOOH

HOOH

m, n

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

O

O–

OOH

N

N

HO

10

70 %

HO

11

OO

OO

O

F

O

OBzBzO

OBz

F

O P(OBn)2

O

36 %

o–q, i

b, c, r

F

Ki = 4–38 μM against FucTase III, V–VII

56 %

Ki = 1–22 μM against FucTase III, V–VII

X = BF4–

OH

6b: TfO–,



colleagues employed radical bromination at C-5 to intro-duce fluorine; however, substrates applicable to this reac-tion are limited to those having electron-withdrawinggroups at the C-1 position, and yields are usually onlymodest.8c Moreover, commonly used protective groupsfor carbohydrates, such as benzyl groups, are incompati-ble because of improper radical stabilization.

More recently, Hartman and Coward reported a morewidely applicable method of incorporating a fluoro groupat the C-5 position of the sugar in the synthesis of a fluor-inated sugar nucleotide, UDP-5-fluoro-N-acetylglu-cosamine (UDP-[5F]-GlcNAc; 22).27 Their strategy tookadvantage of selenoxide elimination, which provides 5,6-anhydro-N-acetylglucosamine derivatives from a 6-sele-nyl glycoside.

Specifically, 1-O-b-tert-butyldimethylsilyl N-phthaloyl-4,6-benzylidene glucosamine (23), prepared from N-phth-aloyl-1,3,4,6-tetra-O-acetyl-D-glucosamine (24) in foursteps, was subjected to bromination with N-bromosuccin-imide in the presence of barium carbonate to afford the 6-bromo derivative 25. The hydroxy group at C-3 of 25 wasprotected with a benzyloxymethyl (BOM) group to give26, in which the bromide group at C-6 was replaced witha phenylselenyl group and successive deprotection of ph-thaloyl and benzoyl groups with hydrazine gave 27. Tri-fluoroacetylation of the amino group of 27 withtrifluoroacetic anhydride followed by acetylation of theC-4 hydroxy group afforded 28. It is worth noting that tri-fluoroacetylation of the C-2 amino group was employedto stabilize a triester of phosphate at the C-1 position,which would be introduced in a further step.28 Desilyla-tion of 28 with HF.pyridine, followed by the formation oflithium alkoxide and further treatment with tetraben-zylpyrophosphate (TBPP),29 gave 29 in high yield. Oxida-tion of 29 with sodium periodate followed by thermalselenoxide elimination gave the alkene 30. Treatment of30 with dimethyldioxorane (DMDO) afforded a diaste-reomeric mixture of epoxides (3:2), the fluoridolysis ofwhich with HF·pyridine, followed by acetylation, gave a

separable mixture of 5-fluorinated epimers, 31a and 31b.Next, the D-gluco isomer 31a was subjected to deprotec-tion of the benzyloxymethyl and benzyl groups by hydro-genation to yield the glycosyl phosphate monoester 32,the treatment of which with methanolic ammonia fol-lowed by acetamide formation afforded 33. Finally, treat-ment of 33 with UMP-morpholidate (34) in the presenceof 1H-tetrazole20 afforded the desired UDP-[5F]-GlcNAc(22) (Scheme 5).27

Hartman and colleagues recently analyzed the inhibitoryactivity of 22 against chitobiosylpyrophosphoryl lipid

Scheme 3 Synthesis of CMP-[3F]-NeuAc (15). Reagents and conditions: (a) 6b, DMF–H2O (3:1), 60 °C; (b) 14, 1H-tetrazole, MeCN; (c)TBHP, Et3N; (d) Pd(PPh3)4, i-Pr2NH; (e) NaOMe; (f) NaOH.

ON

N

O

OHHO

OP

O

OO–

NH2

O CO2H

HO OHOH

AcHN

HO

OCO2R

AcO OAcOAc

AcHN

AcO

12a: R = H

OH

O CO2Me

AcO OAcOAc

AcHN

AcOF

ON

N

O

OAcAcO

OPAllO

i-Pr2N

NHAc

14

ON

N

O

OAcAcO

OPAllO

NHAc

O

O CO2Me

AcO OAcOAc

AcHN

AcOF

a b

F

13b

15

80% 60%

c–f58%

Ki = 5.7 μM against α(2,6)-STase

OH

O CO2H

AcO OAcOAc

AcHN

AcO F

13a

12b: R = Me

Scheme 4 Synthesis of ADP-[2F]-heptulose (16). Reagents andconditions: (a) TBSCl, imidazole, DMF; (b) BnBr, NaH, DMF; (c)TBAF, THF; (d) (COCl)2, DMSO, Et3N, CH2Cl2, –78 to 0 °C; (e)(Ph)3(Me)P+Br–, n-BuLi; (f) OsO4, NMO, acetone, H2O; (g) TBSCl,DMAP, pyridine, CH2Cl2; (h) Tf2O, pyridine, CH2Cl2; (i) CsOAc, 18-crown-6, toluene, ultrasonication; (j) H2SO4, Ac2O, CHCl3; (k) HBr,AcOH, Ac2O, CH2Cl2; (l) Zn, CuSO4, NaOAc, AcOH, H2O; (m)NaOMe, MeOH; (n) PivCl, DMAP, pyridine; (o) 6a, (BnO)2P(O)OH;(p) H2, Pd/C; (q) Bu4N

+HO–, H2O; (r) 21, 1H-tetrazole, pyridine.

O

N

OHHO

OPO

O

O–

P

O

O

O–

OHO

HOOR

fa–e

IC50 = 30 μM against heptosylTase

OH

HO 58%

g–j

OP OR2

OR2

O

o

58%

92% 48%

R = H

R = Me

OBnO

BnOOMeBnO

OBnO

BnOOMeBnO

OHHO

OAcO

AcOOAc

AcO

OAcAcO

k, l

91%

OR1O

R1O

OR1

R1O

R1 = Ac

17: R1 = Piv (95%)m, n

OR1O

R1OF

OR1

R1O

18: R1 = Piv, R2 = Bnp, q

19: R1 = R2 = H

r

56%

OHO

HOR4

OHHO

NN

N

NH2

O

A

OHHO

OPN

O

O–

O

21

16: R3 = H, R4 = F

R3

20: R3 = OH, R4 = H



synthetase (CLS), which transfers GlcNAc from UDP-GlcNAc to GlcNAc-P-P-Dol.30 UDP-[5F]-GlcNAc (22)behaved as a competitive or mixed inhibitor of CLS, withits IC50 estimated at approximately 1.25 mM at a constantconcentration of UDP-GlcNAc, 300 mM (~Km).

Although no inhibitor with a modified nucleic base moi-ety has been reported so far, we synthesized CMP-sialicacid analogue 35 having 5-fluorouracil (5-FU; 36a), anantitumor agent, as the nucleic base as sialyltransferaseinhibitor. The lactam structure of 36a could be tautomer-ized to the lactim form 36b due to the strong electronega-tivity of fluorine, and 36b could hydrogen-bond withguanine as cytosine does.

In advance of the study of 35, we had already succeededin the synthesis of another CMP-NeuNAc analogue 37 us-ing an L-threonine aldolase catalyzed reaction, which fac-ilely afforded b-hydroxy-a-L-amino acid 38 from a-cytocylacetaldehyde (39) and glycine. However, 37showed only weak inhibitory activity,31 which was attrib-uted to the threo-configuration of its b-hydroxy-a-L-ami-no acid moiety.

Fortunately, the enzymatic aldol reaction of glycine andthe aldehyde 40 derived from 36a gave the b-hydroxy-a-L-amino acid 41 with erythro-configuration as a majorproduct. Thus, the protocol used for 37 was applied to thesynthesis of 35 (Scheme 6). The inhibitory activity of 35(IC50 = 0.15 mM) was two-fold stronger than that of 37.32

In addition, 42 is another sialyltransferase inhibitor thathas 5-FU (36) as a substitute for cytosine.33

3.2 Carbasugar Analogues

In terms of tolerance toward cleavage of the glycosylbond, the replacement of the sugar nucleotide’s sugarmoiety with a carbasugar would be more effective, sincean alkyl phosphate would be more stable than an acetalphosphate and would remain intact under acidic or enzy-matic conditions.

The chemoenzymatic method for the synthesis of carba-sugar-containing sugar nucleotide analogues was first es-tablished in the synthesis of 43 (Scheme 7) by modifyingthe synthetic route of UDP-[2F]-Gal (1) shown inScheme 1.34 Namely, carba-a-D-galactose-1-phosphate(44) prepared from enatiomerically pure 45 via tetra-O-benzyl-carba-Gal [(1S,2R,3S,4S,5R)-2,3,4-(trisbenzyl-oxy)-5-(benzyloxymethyl)cyclohexan-1-ol] (46)35 wasincubated with UMP, ATP, magnesium chloride, acetylphosphate, and glucose-1-phosphate in the presence ofacetate kinase, UMP kinase, glucose-1-phosphate uridyl-transferase, and galactose-1-phosphate uridyltransferasein Tris buffer.

UDP-carba-GlcNAc (47) was synthesized from 48, whichwas in turn derived from 49,35 in the presence of GlcNAc-1-phosphate uridyltransferase and UTP (Scheme 8). In asimilar way, GDP-carba-Man (50) was synthesized from51. Specifically, carbasugar diol 52 was converted into 53via orthoacetate, bromoacetoxy,36 and b-1,2-epoxide in-termediates. Benzyl protection of 53 followed by removalof the allyl group with palladium(II) chloride and sodiumacetate in aqueous acetic acid afforded the 1-hydroxy in-termediate, which was phosphorylated and subsequentlysubjected to removal of the protecting groups. The result-ing carba-a-D-manno-1-phosphate (51) was transformedinto the desired 50 with GTP in the presence of mannose-1-phosphate guanylyltransferase (Scheme 8).

Inhibitory activities of 43, 47, and 50 were not reported bythe research group; however, Hindsgaul and colleagues,who synthesized 43 by chemical methods (Scheme 9), es-timated its Ki value to be 58 mM against b(1,4)-galactosyl-transferase from bovine milk.37

GDP-carba-Fuc (55) was synthesized by Toyokuni andcolleagues, starting with L-fucolactone 56. The enone 57derived from 56 via intramolecular Horner–Emmons re-action was reduced to carba-fucose 58 in two steps. Sub-sequent tetrazole-catalyzed coupling of 58 with 9 afforded55, which showed inhibition against a(1,3/4)-fucosyl-transferases (from human colon cancer cells, Colo 205)with an IC50 of 0.3 mM (Scheme 10).38

Scheme 5 Synthesis of UDP-[5F]-GlcNAc (22). Reagents and con-ditions: (a) (H2NCH2)2, HOAc, THF; (b) TBSCl, imidazole, DMF; (c)NaOMe, MeOH; (d) PhCH(OMe)2, TsOH, MeCN; (e) NBS, BaCO3,CCl4; (f) BOMCl, DIPEA, THF; (g) PhSeH, Et3N, THF; (h) H2NNH2,EtOH, 100 °C; (i)TFAA, pyridine; (j) Ac2O, pyridine; (k) HF·pyridi-ne, THF; (l) LDA; (m) TBPP, THF; (n) NaIO4, MeOH–H2O; (o) re-flux, DHP; (p) DMDO, CH2Cl2; (q) H2, Pd(OH)2/C, CH2Cl2, MeOH;(r) NH3; (s) Ac2O, Et3N, MeOH; (t) 34, 1H-tetrazole, pyridine.

ON

NHO

OHHO

OPOO

O–

PO

O

O–

OAcO

AcOOAc

g–h

22

a–d

IC50 = 1.25 mM against CPS, a kind of GnTase

OAc

NPhth

OOHO

OTBSO

NPhth

Ph e

24 23

OBzO

ROOTBS

Br

NPhth

25: R = H

OHO

BOMOOTBS

SePh

NH2

26: R = BOMf

OAcOBOMO

O

SePh

NH

CF3

O P OBnOBn

O

71%

i–j OAcOBOMO

OTBS

SePh

NHCOCF3

k–m

OAcOBOMO

ONH

CF3

O P OBnOBn

O

n–o

27 28

29 30

OAcOBOMO

O

OAc

NH

CF3

O P OBnOBn

O

31a: D-gluco

p, k, j

F

OR1O

HO

O

OR1

NH

R2 P O–O–

O

32: R1 = Ac, R2 = CF3CO

q

F

t

O

OHO

HO

OH

NHFO

82% 45%

95%

78%

88% 76%

89% 52%

33: R = H, R2 = MeCO (82%)

34

OU

OHHO

OPN

O

O–

O

(78 %)

31b: L-ido

31a

r, s



Scheme 6 Synthesis of the CMP-NeuAc analogues 35 and 37 and structure of 42. Reagents and conditions: (a) BrCH2CH2(OEt)2; (b) HCl(aq); (c) L-threonine aldolase, glycine.

N

NH

O

F

O

N

NH

OF

O

H2N

OH

HO2C

*

CHOO

HO

AcHN

OH

HO

O

HO

CO2H

NH

N

N

OHF

OO O2C

OH

41

N

N

O

H2N

OH

HO2C

*

38

NH2

O

HO

AcHN

OH

HO

O

HO

CO2H

NH

N

N

NH2

OO O2C

OH

37

35

erythro

threo

a, b

c

N

N

O

O

HF

N

N

HN

O

H

R R

N

N

O

O

F

R

H

lactim form of 5-FU (36b) cytosine

hydrogen acceptor

hydrogen donor hydrogen donor

hydrogen acceptor

5-FU (36a)

hydrogen acceptor

hydrogen donor

hydrogen acceptor

40

N

N

NH2

O

CHO

c

39

O

HO

AcHN

OH

HO

HO

CO2H

42

ON

NH

O

OO

O

OF

Scheme 7 Chemoenzymatic synthesis of UDP-carba-Gal (43). Reagents and conditions: (a) NaHCO3, KI, I2, H2O; (b) DBU, THF; (c)NaHCO3, MeOH, reflux; (d) Novozym 435, vinyl acetate; (e) BnOP(Ni-Pr)2, 1H-imidazole; (f) m-CPBA; (g) H2, Pd/C; (h) acetate kinase; (i)uridine monophosphate kinase; (j) glucose-1-phosphate uridyltransferase; (k) galactose-1-phosphate uridyltransferase.

ATPADP

HO

HOHO

OH

OPO3=

UDP-Glc Glc-1-P

UTPPPi

HO

HOHO

OH

OP

OP

O

O OO– O–

O

HO OH

N

NH

O

O

i

h

j

44

43

BnO

BnOBnO

OBn

OH

46

UDP

AcPiacetate

UMP

AcPi acetate

h

k

HO

O

MeO

OOH

++

d

MeO

OOH

MeO

OOAc

+

e–g

45

+

O

I

O

ab, c



3.3 Analogues with a Planar Sugar Moiety in the Transition State

In this type of analogue, the chair form of the sugar moi-eties would be distorted to a planar conformation and theanomer carbons of the nucleotides would exhibit sp2-char-acter in the transition state. Toyokuni and co-workers de-signed and synthesized 59, an analogue of GDP-Fuc, withthe fucose moiety replaced by carbasugar 60 that has adouble bond in the ring (Scheme 11). With this in hand,they focused on the planarity of the sugar moiety in thetransition state.38

Comparison of the inhibitory activities of 55 and 59against the a(1,3/4)-fucosyltransferases revealed that 59,with a planar structure in the sugar moiety, was more po-tent (IC50 = 0.1 mM) than 55 (IC50 = 0.3 mM), wherein

the fucose moiety was mimicked with a saturated carba-sugar (for reference, the IC50 of GDP is 0.6 mM). The re-sult also supported the suggestion that thefucosyltransferase-catalyzed reaction forces the fucosemoiety of GDP-Fuc to maintain a planar conformation inthe transition state.9b

Scheme 8 Chemoenzymatic synthesis of UDP-carba-GlcNAc (47) and GDP-carba-Man (50). Reagents and conditions: (a) MsCl, pyridine,CH2Cl2; (b) CsOAc; (c) H2S and then Ac2O; (d) NaOMe; (e) BnOP(Ni-Pr)2, 1H-tetrazole; (f) m-CPBA; (g) H2, Pd/C; (h) acetate kinase; (i)uridyl monophosphate kinase; (j) GlcNAc-1-phosphate uridyltransferase; (k) MeC(OMe)3, PPTS; (l) AcBr; (m) K2CO3, MeOH; (n) BF3·OEt2,CH2=CHCH2OH; (o) PdCl2, NaOAc, AcOH; (p) BnOP(Ni-Pr)2, 1H-tetrazole; (q) m-CPBA; (r) H2, Pd/C; (s) guanidyl monophosphate kinase;(t) mannose-1-phosphate guanylyltransferase.

ATPADP

HO OH

HO

OH

OPO3=

GTP PPi

HO OH

HO

OH

OP

OP

O

O OO– O–

O

HO OH

N

HN

O

s

h

t

51 50

BnO

OH

BnO

OBn

OR

52: R = H

GDPAcPi

acetate

GMP

AcPi acetate

h

N

N

H2N

ATP ADP

HO

AcHNHO

OH

OPO3= UTP PPi

HO

AcHNHO

OH

OP

OP

O

O OO– O–

O

HO OH

N

N

O

O

i

h

j

48

47

BnON3

BnO

OBn

OH

49

UDPAcPi

acetate

UMP

AcPiacetate

a–g

k–n

n–q

45

53: R = All

Scheme 9 Chemical synthesis of UDP-carba-Gal (43). Reagents andconditions: (a) MsCl, pyridine; (b) NaH, DMF; (c) Hg(OAc)2, AcOH,NaCl, acetone, H2O; (d) MEMCl, DIPEA, MeCN; (e) Tebbe reagent;(f) BH3·THF; (g) NaOH, H2O2; (h) H2, Pd/C ; (i) Ac2O, pyridine; (j)Me2BBr; (k) 54, 1H-tetrazole; (l) m-CPBA; (m) CDI; (n) 34, DMF;(o) Et3N–MeOH–H2O (7:3:1).

OBnO

BnOOMe

f–ja, b

Ki = 58 μM against β(1,4)GalTase(Km for UDP-Gal: 25 mM)

OH

OBn

c, d

R = O

AcO

AcO

OH

OAc

AcO

R = CH2

e

77% 42%55%

(78%)

OBnO

BnOOMe

OBn

BnOR

BnOOMEMBnO

k, l

53%

AcO

AcOO

OAc

AcOh, m–o

85%P

OO

O

PO

OEt2N

43

54

Scheme 10 Synthesis of GDP-carba-Fuc (55). Reagents and condi-tions: (a) LiCH2P(O)(OMe)2; (b) NaBH4; (c) DMSO, TFAA, Et3N;(d) NaH; (e) (Ph3PCuH)6; (f) (BnO)2P(Ni-Pr)2, 1H-tetrazole; (g) m-CPBA; (h) Li, NH3 (liq); (i) Dowex 50X8-400 (Et3NH+); (j) 9, pyri-dine.

O

OBnBnOOBn

O a, bOH

OBnBnOOBn

CH2P(OMe)2

OH O

O

OBnBnOOBn

CH2P(OMe)2

OO

c

d

OBnBnOOBn

O

OBnBnOOBn

Oe

b

OBnBnOOBn

OH

58

ON

NHO

NH2

OHHO

OPO

O

O–P

O

OO–

N

N

55

OHHOOH

94% 93%

99% 46%

f–j

85% 82%

IC50 = 0.3 mM against α(1,3/4)-FucTase

L-fucose

56

57

Scheme 11 Synthesis of the GDP-Fuc analogue 59. Reagents andconditions: (a) CeCl3, NaBH4; (b) (BnO)2P(Ni-Pr)2, 1H-tetrazole; (c)m-CPBA; (d) Li, NH3 (liq); (e) Dowex 50X8-400 (Et3NH+); (f) 9, py-ridine.

ON

NH

O

NH2

OHHO

OPO

O

O–P

O

O

O–

N

N

OBnBnOOBn

OH OHHOOH

59

b–f57

a

91% 47%

IC50 = 0.1 mM against α(1,3/4)-FucTase60



Schmidt and colleagues expected the sialic acid moiety ofCMP-NeuAc to form a planar structure in the transitionstate in sialyltransferase-catalyzed reactions, and synthe-sized a CMP-sialic acid analogue in which the anomercarbon of sialic acid was composed from an sp2-carbon.Interestingly, the analogues 61a and 61b, in which thesialic acid moiety was replaced with an aromatic ring – ei-ther benzene or furan – were synthesized.39 The synthesisstarted with the reaction of dibenzylphosphonic acid andan aromatic aldehyde, such as benzaldehyde or furfural, toafford the a-hydroxyphosphonic acids 62a and 62b.These were derived to the target molecules 61a and 61bby the Kajiwara–Hashimoto method40 using cyanoeth-ylphosphoramidite 63 as a nucleotide precursor. Schmidtand colleagues synthesized phosphoramidate derivativeslike 61c starting from chiral a-aminophosphonic acid 62c.In addition, the other derivatives 64a and 64b, in whichthe phosphonate moiety of 61a and 61b was replaced withcarboxylic acid, were synthesized in a similar way startingfrom optically pure mandelic acid. The inhibitory activi-ties of these compounds toward a(2,6)-sialyltransferasewere not affected by the configuration of the carbon on thephosphonic acid, but were reduced somewhat by the re-

placement of phosphonate with carboxylate or phosphor-amidate (Scheme 12).

CMP-NeuAc analogues 65–69, with sialic acid installed,were synthesized by the same research group at almost thesame time. The route was rather simple: 2,3-Dehydro-NeuAc tetraacetate 12a was converted into thioester 12cwith p-thiocresol and 1,1¢-carbonyldiimidazole (CDI),which was reduced to neuraminol 70. Subsequent cou-pling of 70 with CMP using Kajiwara and Hashimoto’smethod40 followed by deacetylation afforded 65. Mean-while, oxidation of 70 with Dess–Martin periodinane ledto the aldehyde 71. Next, 71 and dibenzyl phosphonatewere condensed in the presence of triethylamine to affordhydroxyphosphonic acid 72a, which was bound to CMPusing the Kajiwara–Hashimoto method40 (Scheme 13).The resulting phosphodiester 73a was subjected to hydro-genolysis followed by hydrolysis with sodium methoxidein methanol, resulting in 67. Here, the diene 68 was isolat-ed as a minor product, which originated from a base-pro-moted deacetoxy-phosphorylation. The inhibitory activityof 68 was not relatively potent and the Ki value (6 mM)was only a few times smaller than the Km value of CMP-NeuAc.41

Scheme 12 Synthesis of the CMP-NeuAc analogues 61a,b and 64–69. Reagents and conditions: (a) 1H-tetrazole; (b) TBHP; (c) Et3N; (d) Pd/C, H2, or Pd(PPh3)4, dimedone; (e) NH3.

ON

N

O

OHHO

OP

O

O

O–

NH2

Ar P(OBn)2

XH

O

62a: X =O, Ar = Ph62b: X = O, Ar = furyl62c: X = NH, Ar = Ph

ON

N

O

OAcAcO

OP

O

i-Pr2N

NHAc

NC

+P

Ar

O OH

O–

61a: X = O, Ar = Ph: Ki = 0.2 μM, 1.0 μM61b: X = O, Ar = furyl: Ki = 0.28 μM, 1.0 μM61c: X = NH, Ar = Ph: Ki = 68 μM, 140 μM

ON

N

O

OHHO

O

P

O

OO–

NH2

O RAcHN

HO

HO

OH

68: R = H

a–e

Ki = 40 nM against α(2,6)STase

63

Ki = 6 μM

ON

N

O

OHHO

O

P

O

OO–

NH2

67: R = PO3H2

O

RH

HOAcHN

HO OH

OH

Ki = 0.35 μM

65: R = H Ki = <2000 μM

69: R = PO3H2

ON

N

O

OHHO

OP

O

O

O–

NH2

CO2H

Ar

64a: Ar = Ph: Ki (R) = 10 μM Ki (S) = 7 μM64b: Ar = furyl: Ki (R) = 15 μM Ki (S) = 23 μM

66 Ki = 400 μM

ON

N

O

OHHO

OP

O

O–

NH2

O

OHHOAcHN

HO OHOH



Replacement of the hydrogen atom with phosphonate ledto a dramatic enhancement of the inhibitory activity,41 asobserved in a structural comparison of 65 with 67. Thus,the novel compound 69, with a phosphonate group inplace of the exo-olefinic proton of the diene 68, was de-signed as a more promising inhibitor. The synthesis of thetarget molecule 69 began with the reaction of 71 with di-allyl phosphonate to give the a-hydroxyphosphonate 72b,which was coupled with CMP as described above. The di-astereomer mixture containing 73b was treated with DBUto eliminate acetate followed by deallylation withPd(PPh3)4 in the presence of dimedone.42 Subsequent hy-drolysis with a mild base yielded 69, wherein the two de-cisive groups had the desired geometry (Scheme 13). Thephosphonate 69 was the most potent inhibitor of sialyl-transferases (Ki = 40 nM) reported so far.

Later, Schwörer and Schmidt made a further modificationto 67 and 68 by replacing the hydrophilic C-7–C-9 sidechain with a hydrophobic alkoxy or aryloxy group in or-der to develop a more potent inhibitor.43 The target mole-cules (74a,b and 75a,b) shown here bear an N-acetylglucosamine derivative as a sugar moiety, linked tothe phosphate group of CMP through a rotation of theC-4–O axis by 180° (Scheme 14).

Thus, in the beginning of the synthesis of 74a,b and75a,b, phenyl N-acetyl-b-D-glucosaminide (76a)44,45 and2-benzoyloxyethyl N-acetyl-b-D-glucosaminide (76b), it-

self prepared by the glycosylation of 2-benzoyloxyetha-nol with the N-triethoxycarbonyl (Teoc)-protected donor7746 in the presence of trimethylsilyl triflate followed byprotection and deprotection procedures, were convertedinto the phenyl and 2-benzoyloxyethyl 3,4-benzoyloxy-N-acetylglucosaminides 78a and 78b, respectively. Theprimary alcohols of glucosanimides 78 were oxidized un-der Pfitzner–Moffatt conditions to the aldehydes 79,which were treated with dially phosphonate in the pres-ence of triethylamine to give the alcohols 80. Applicationof the Kajiwara–Hashimoto method40 to 80 afforded theprecursors 81 of the target molecules. Treatment of com-pounds 81 with aqueous ammonia followed by deallyla-tion with Pd(PPh3)4 in the presence of dimedone42 yielded74a,b, while the deallylation and subsequent deacetyla-tion with aqueous ammonia furnished 75a,b.

Two stereoisomers 74a,b and two geoisomers 75a,b wereseparated and inhibitory activities against a(2,6)-sialyl-transferases (rat liver) were measured. As expected,74a,b, which have both a phosphate group and a phospho-nate group, were more potent inhibitors than 75a,b, whichhave only a phosphate group (Scheme 14).

Schmidt and colleagues prepared the UDP-Gal analogue82, with an sp2-anomeric carbon, using a similar method(Scheme 15). The Ki value for 82 was 62 mM againstb(1,4)-galactosyltransferases from bovine milk.47

Scheme 13 Synthesis of the CMP-NeuAc analogues 65–69. Reagents and conditions: (a) CDI, 4-MeC6H4SH; (b) NaBH4; (c) 63, 1H-tetra-zole; (d) TBHP; (e) Et3N; (f) NaOMe, MeOH; (g) Dess–Martin periodinane; (h) HP(O)(OBn)2, Et3N; (i) H2, Pd/C; (j) Ac2O, pyridine;(k) HP(O)(OAll), Et3N; (l) DBU; (m) Pd(PPh3)4, dimedone; (n) NH3, MeOH; (o) IR-120 (Na+); (p) O2, O3, N4-triacetylcytosine, DCC, DMAP.

O

OAcAcHN

AcO OAcOAc

C

12a: R = OH

12c: R = SC6H4Me (78%)

O

OAcAcHN

AcO OAcOAc

70

OH

O

OAc

AcHN

AcO OAcOAc

ON

N

O

OAcAcO

OP

O

OO–

NHAc

O

OAc

AcHN

AcO OAcOAc

CHO

O

OAcAcHN

AcO OAcOAc P(OR)2

7172a: R = Bn (99% from 71)

72b: R = All

OH

O

O

OAc

AcHN

AcO OAcOAc

ON

N

O

OAcAcO

OP

O

O O–

NHAc

73a: R = Bn

73b: R = All

H

P(OR)2

O

O

OAcAcHN

AcO OAcOAc

P(OBn)2

OAc

O

65

O

OAcAcHN

AcO OAcOAc

ON

N

O

OAcAcO

OP

O

O–

NHAc

72a R-isomer

OAc

66

6867

69

+

a

b

73%

c–e

90% f

67%

g

82%

h c–e

73%

i, f

(88%) (5%)

k c–e l, j, m–o

56%

i, pf

70 %

j

quant. 88%

O

R

70



3.4 Analogues with an Elongated Sugar–Phosphate Bond in the Transition State

The structure of the sugar nucleotides changes not only atthe anomeric center but also at the linkage of sugar andphosphate in the transition state of a glycosyltransferase-catalyzed reaction – specifically, the bond would lengthenas the reaction proceeds. This structural change in thetransition state was not of interest, however, until the syn-thesis of the potent sialyltransferase inhibitor 69.39 Horen-stein and colleagues synthesized unique sialyltransferaseinhibitors 83a and 83b, focusing on the extended bondlength between the sugar and the nucleotide moieties. TheKi values of 83a and 83b for a(2,6)- and a(2,3)-sialyl-transferases were 10–20 mM (Scheme 16).48 In addition, a

UDP-GlcNAc analogue having an azabicyclo[3.1.0]hex-ane skeleton was also synthesized.49

Scheme 14 Synthesis of the CMP-NeuAc analogues 74a,b and 75a,b. Reagents and conditions: (a) HO(CH2)2OBz, TMSOTf, CH2Cl2; (b)Zn, Ac2O, AcOH–THF; (c) NaOMe, MeOH; (d) TBDMSCl, Et3N, DMAP; (e) BzCl, pyridine; (f) HCl, MeOH; (g) DMSO, DCC, Et3N; (h)HP(O)(OAll)2, Et3N; (i) 63, 1H-tetrazole; (j) TBHP; (k) Et3N; (l) NH3, H2O; (m) Pd(PPh3)4, dimedone; (n) RP-18 HPLC; (o) IR-120 (Na+).

O

OAcAcHN

ROP(OAll)2

80a: R = Ph (quant.)

80b: R = (CH2)2OBz (87%)

OH

O

O

OAcAcHN

ROO

N

NO

OAcAcO

OP

O

O O–

NHAc

81a: R = Ph (82%)

81b: R = (CH2)2OBz (55%)

P(OAll)2

O

i–k O

OHAcHN

ROO

N

NO

OHHO

OP

O

O O–

NH2

74a: R = Ph

74b: R = (CH2)2OBz

P

O

O–

O–

OAcHNRO O

N

NO

OHHO

OP

O

O O–

NH2

75a: R = Ph

75b: R = (CH2)2OH

H

OOAc

AcOAcO TeocHN

O

NH

CCl3

OOH

HOHO NHAc

O(CH2)2OBz

OOH

HOHO NHAc

OPh

OOH

BzOBzO NHAc

OR

77

76a

76b

78a: R = Ph (69%)

78b: R = (CH2)2OBz (71%)

OOHC

NHAc

ORBzO

79a: R = Ph (85%)

79b: R = (CH2)2OBz (48%)

a–c

44% d–f g h

9–36%

l–o

m, l, n, o 6–35%

two separable stereoisomers

Ki = 29 nM 690 nM

Ki = 38 nM 59 nM

Ki = 158 μM (E), 25 μM (Z)

Ki = 2.4 μM (E), 3.5 μM (Z) against α(2,6)STase

Scheme 15 Synthesis of the UDP-Gal analogue 82. Reagents andconditions: (a) Raney Ni, NaH2PO4, AcOH–pyridine–H2O; (b)NaBH4; (c) MsCl, Et3N; (d) NaBr; (e) P(OTMS)3; (f) NaOH; (g) IR-120 (Et3NH+); (h) 34.

ON

NHO

OHHO

OPO

O–

O

a b–fOCN

AcO OAc

AcO OAc

OCHO

AcO OAc

AcO

OHO OH

HO PO

O–

O–

2 Na+

g, h

OHO OH

HO P

O

OO–

82

40% 66%

Ki = 62 μM against β(1,4)GalTase

Scheme 16 Synthesis of the sialyltransferase inhibitors 83.Reagents and conditions: (a) TBAF; (b) 63, 1H-tetrazole; (c) TBHP;(d) Et3N; (e) NaOMe, MeOH (aq).

CO2H

HO

N

N

O

OAcAcO

OPO

OO–

NHAc

HOTBS

CO2Me

Et3NH+

a–c

51%

d, e

30 %

O

HO OHOH

AcHNHO

CO2–

OP

O

–O O cytidine

CO2H

HO

N

N

O

OHHO

OPO

OO–

NH2

83a

Ki = 10–20 μM against α(2,3)- and α(2,6)-STases

extended

CO2H

HO

N

N

O

OHHO

OPO

OO–

NH2

83b

transition state

+



Schäfer and Thiem designed and synthesized the UDP-Gal analogue 84a, in which one methylene was insertedbetween galactose and diphosphate moieties, by the cou-pling of uridine phosphomorpholidate (34) with a-C-ga-lactoside 85,50 which was prepared from the methylgalactoside 86 and propargyl-TMS (Scheme 17).51 Ananalogue of UDP-GlcNAc, 87, was also synthesized.51

Later, Schmidt and co-workers reported that neither 84anor its anomeric isomer 84b showed inhibitory activityagainst a(1,3)-galactosyltransferase from a pig at a con-centration of 50 mM.52

Elongation of the bond between the sugar and phosphatemoieties of GDP-Fuc in the transition state was focusedon earlier by a research group at Smith-Kline Pharmaceu-tical Co. Ltd.,53 who synthesized the novel GDP-Fuc ana-logues 88 and 89a,b as fucosyltransferase inhibitors(Scheme 18). In this approach, a C-fucoside with a meth-ylene chain on the anomer carbon was linked with GDP.Specifically, a-L-fucopyranoside peracetate (90) was sub-jected to siloxymethylation54 catalyzed by cobalt ion to af-ford 91, which was further converted into 88.

Alternative L-fucose derivatives bearing an ethylenegroup on the anomeric carbon were synthesized by theozonolysis of C-allyl-L-fucosides 92a and 92b, whichwere prepared by allylation using allylsilane in the pres-

ence of Lewis acid. Treatment of 90 with allylsilane in thepresence of trimethylsilyl triflate afforded 92a as a majorproduct (92a:92b = 14:1) when conducted in ni-tromethane with trimethylsilyl triflate,55 while a 1:1 mix-ture of 92a and 92b (a- and b-anomers) was obtainedusing zinc(II) bromide as a Lewis acid.56 Ozonolysis fol-lowed by reduction with sodium borohydride afforded theprimary alcohols 93a and 93b, which were further derivedto phosphate and linked with 9 to give 89a and 89b, re-spectively (Scheme 18). Unfortunately, the inhibitory ac-tivities of 88 and 89a,b were not reported.53

Later, Wong and co-workers speculated that the analogue94, which has both features of a half-chair conformationof the fucose moiety and an extended sugar–phosphatelinkage, would be more potent fucosyltransferase inhibi-tors than 88, 89a,b, and 59.57

The D-mannose derivative was chosen as a starting mate-rial for the L-fucose moiety in the synthesis of 94 sincethree contiguous secondary alcohols of these two naturalsugars share the same configuration. The p-methoxyphe-nyl group of the fully protected mannose 95, which wasderived from p-methoxyphenyl a-D-mannoside (96)58 intwo steps, was cleaved with ceric ammonium nitrate to re-veal the anomeric hydroxy group. Subsequent Albright–Goldman oxidation of 95 afforded the lactone 97 andtreatment with lithium dimethyl methylphosphonate thenyielded the tertiary alcohol 98. Reductive ring opening of98 with sodium borohydride, subsequent Swern oxida-tion, and an intramolecular Horner–Emmons reactiongave the carbacyclic enone 99. A pseudo-equatorial hy-droxy group generated by the subsequent Luche reductionof 99 was methylated to give 100, the tert-butyldiphenyl-

Scheme 17 Synthesis of the UDP-Gal analogues 84a,b and UDP-GlcNAc analogue 87. Reagents and conditions: (a) propargyl-TMS,BF3·OEt2, MeCN; (b) O3; (c) NaBH4; (d) PO(OPh)2Cl, pyridine,DMAP; (e) H2, Pd/C; (f) H2, PtO2, MeOH; (g) 34, 1H-tetrazole, pyri-dine; (h) 10% Et3N, CH2Cl2, i-PrOH; (i) SOCl2; (j) BuLi, THF; (k) Li-Naph; (l) CO2; (m) MeI, DMF.

OOBnBnO

BnOBnOOMe

OOBnBnO

BnOBnO

C

OOHHO

HOHO

OP(OPh)2

O

OOBn

BnOBnO

AcHN

OHO

OBn

BnOBnO

AcHNCO2Me

OOH

HOHO

AcHNOP(OPh)2

O

ON

NH

O

O

OHHO

OPOO

O–PO

OO–

OOHHO

HOHO

ON

NH

O

O

OHHO

OPOO

O–PO

OO–

OOH

HOHO

AcHN

a

86

85 84a

87

c–e

f, g

26%

i–m c, d

f, g

b OOBnBnO

BnOBnO

CHO

OOBnBnO

BnOBnO

CHOO

N

NH

O

O

OHHO

OPOO

O–PO

OO–

OOHHO

HOHO

84b

86a, b, h

Scheme 18 Synthesis of the GDP-Fuc analogues 88 and 89a,b.Reagents and conditions: (a) CO2(CO)8, CO, HSiEt2Me; (b) AcOH;(c) allylsilane, TMSOTf, MeNO2; (d) O3 and then NaBH4; (e)(PhO)2POCl, pyridine; (f) H2, PtO2; (g) K2CO3, MeOH; (h) 9, pyri-dine.

O

AcOOAcOAc

OAc

90

O

AcOOAcOAc

OSiEt2Mea

85%

c

O

AcOOAcOAc

92a (α-isomer)

91%

O

AcOOAcOAc

OH

ON

NH

O

NH2

OHHO

OPO

O

O–P

O

O–

N

NO

HOOHOH

O

88

ON

NH

O

NH2

OHHO

OPO

O

O–P

O

O–

N

N

O

HOOHOH

O

d 89%

e–h

b, e–h 78% (e–g)

91

92b (β-isomer)

93a (α-isomer)

93b (β-isomer)

89a (α-isomer)

89b (β-isomer)



silyl group of which was deprotected with tetra-n-butyl-ammonium fluoride. The primary alcohol thus generatedwas phosphorylated by a reaction with diisopropyl phos-phoramidite and subsequent oxidation with m-chloroper-oxybenzoic acid yielding 101. Synchronous deprotectionof the MOM and benzyl groups of 101 was attained bytreatment with trifluoroacetic acid in the presence ofthiophenol, and subsequent coupling of the resulting non-protected sugar phosphate with GMP by using the proto-col reported by Wittmann and Wong20 provided 94(Scheme 19).

Compound 94 showed good competitive inhibition of fu-cosyltransferases V and VI with Ki values in the mMrange. It is interesting to compare the inhibitory activitiesof 94 and 59, noting that a methylene group between thesugar and phosphate moieties enhanced the inhibitory ef-fect.

3.5 Analogues with a Positively Charged Ano-meric Carbon in the Transition State

Since the sugar moiety of sugar nucleotides would possessa positive charge in the transition state, iminosugars exist-ing as ammonium salts under physiological conditions(pH 7.4) could be excellent mimetics of the sugar moiety.The research groups of Wong and Schuster reported GDP-

Fuc analogues carrying an iminosugar instead of fucose.First, Wong and colleagues replaced the L-fucose moietyin GDP-Fuc with a six-membered homoazafucose to ob-tain analogue 102, which was designed to have an elon-gated bond between the anomeric carbon and phosphategroup in the transition state as well as a positive charge onthe fucose moiety.57 A phosphate of homoazafucose, 103,was synthesized using an FDP-aldolase-catalyzed reac-tion of dihydroxyacetone phosphate (DHAP) and alde-hyde prepared by the acid hydrolysis of 104. The reactionof 103 with GDP-morpholidate (9) gave the target mole-cule 102 (Scheme 20).57 Schuster and Blechert synthe-sized the GDP-Fuc analogue 105, in which the fucosemoiety was substituted with a five-membered iminosugar,focusing on the conformational change of the fucose inthe transition state.59 A phosphate ester of the iminosugar,106, was prepared using an FDP-aldolase-catalyzed reac-tion of chiral aldehyde 107 and DHAP followed by cata-lytic hydrogenation and subsequent reduction withsodium cyanoborohydride. Compound 106 was convertedinto 105 in the same way as 103 was converted into 102.60

Moreover, Flessner and Wong designed the novel GDP-Fuc analogue 108, which satisfied three requirements:planarity of the fucose moiety, the presence of a positivecharge around the anomeric carbon, and an elongatedbond between the fucose and phosphate groups in thetransition state.57 The benzyl protecting groups of tria-zolecarboxylate 109,61 synthesized from 2,3,4-tri-O-ben-zyl-L-fucose (110) in four steps including the [3+2]cycloaddition of azide and olefin, were removed by hy-drogenation, followed by reprotection of the generated al-cohol with a tert-butyldimethylsilyl group to give 111.Reduction of the methyl ester of 111 with lithium alumi-num hydride, followed by phosphorylation of the result-ing primary alcohol with diisopropyl phosphoramiditeand subsequent oxidation with m-chloroperoxybenzoicacid yielded the dibenzyl phosphate 112. Complete depro-tection of 112 through the treatment with tetra-n-butylam-monium fluoride and successive hydrogenation afforded asugar phosphate analogue 113, the coupling of which withGMP-morpholidate (9)20 furnished the target molecule108 (Scheme 20).

The GDP-Fuc analogues 102 and 108 showed moderateinhibitory activity against a(1,3)-fucosyltransferase andfucosyltransferases V and VI, with the Ki value for GDPbeing 29 mM and the Km value for GDP-Fuc in the rangeof 8 to 34 mM.38 In addition, the IC50 value of 105 (45–82mM) was close to that of GDP (50 mM).7 The resultseemed disappointing; however, 105 is still a more potentinhibitor than 59. Moreover, the IC50 value of 105 wasmuch smaller than that of GDP-ethanolamine (1 mM)having a positive charge at a proper distance. These re-sults support a significant contribution of imino-sugarmoieties in 102, 105, and 108 to the inhibitory effect onthe fucosyltransferase V.

Scheme 19 Synthesis of the GDP-Fuc analogue 94. Reagents andconditions: (a) TBDPSCl, DMAP, pyridine; (b) MOMCl, DIPEA,DMAP, CH2Cl2; (c) CAN, MeCN (aq); (d) DMSO, Ac2O; (e)MeP(O)(OMe)3, n-BuLi, THF; (f) NaBH4, THF; (g) DMSO, TFAA,CH2Cl2, –78 °C; (h) NaH, diglyme; (i) NaBH4, CeCl3, MeOH; (j)NaH, MeI, THF; (k) TBAF, THF; (l) (i-Pr)2NP(OBn)2, 1H-tetrazole,CH2Cl2, NH3, H2O; (m) m-CPBA; (n) TFA, 95% aq THF, thiophenol;(o) 9, 1H-tetrazole, pyridine.

ON

OHHO

OP

O

O–

OR1O

R1O O

96: R1 = R2 = Ha, b

c, d e

f–h

95%

i, j

R2OR1O

OMOMO

MOMO

TBDPSOOMOM

O

97

OMOMO

MOMO

TBDPSOOMOM

98

OHP(OMe)2

OMOMO

O

99

OTBDPS

OMOM

MOMO

MeO

100

OTBDPS

OMOM MOMO

MeO OP(OBn)2

OMOM

O

N

N

NH

O

NH2OP

O

OO–

HO

MeO

OH

OMOM

OMOM OMOM

77%

101

94

82%

70%

k–m

71%

n, o

21%

OH

OMe

Ki = 8 μM against FucTase V

Ki = 6 μM against FucTase VI

95: R1 = MOM, R2 = TBDPS (76%)



3.6 Analogues with Other Functional Groups In Place of the Phosphate Moiety

Sugar nucleotide analogues having another functionalgroup, such as a phosphonate or malonate group, in placeof the phosphate moiety have attracted interest because oftheir stability.

3.6.1 Analogues with Phosphonate in Place of the Phosphate

There have been numerous attempts to replace phosphatewith phosphonate in the synthesis of analogues of CMP-silaic acid. Some examples were given in section 3.3.Imamura and Hashimoto reported to have synthesized114a, a phosphonate derivative of CMP-NeuAc, using atrimethylsilyl triflate catalyzed rearrangement of phos-phite, where, as a key reaction, diethyl sialyl phosphite115a62 was converted into sialyl phosphonate in the pres-

ence of dimethyl trimethylsilyl phosphite.63 Later,Schmidt and co-workers scrutinized the reaction in detail,and found that treatment of 115b64 under the same condi-tions afforded not only the b-phosphonate 116a but alsothe a-phosphonate 116b.65 Thus, they synthesized both114a and 114b by following Hashimoto’s scheme for114a. Specifically, 116a and 116b were converted into themonomethyl phosphonates 117a and 117b by treatmentwith benzenethiol and triethylamine, respectively.66 Fi-nally, 117a and 117b were further treated with triacetyl-cytidine (118a) under conditions for Mitsunobu reaction,then deprotection to afford 114a and 114b (Scheme 21).At that point, it was ascertained by comparison of spectraldata that Hashimoto had not synthesized 114a, but 114binstead. The inhibitory activities of 114a and 114b towarda(2,6)-sialyltransferases from rat liver were relativelyweak, and the Ki values were 780 mM and 250 mM, respec-tively.

Scheme 20 Synthesis of the GDP-Fuc analogues 102, 105, and 108. Reagents and conditions: (a) 0.1 M HCl, 50 °C; (b) FDP-aldolase, DHAP;(c) H2, PtO2, MeOH (aq); (d) H2, Pd/C, 0.5 M HCl; (e) NaBH3CN; (f) 9, 1H-tetrazole, pyridine; (g) methyl (triphenylphosphoranylidene)acetate,toluene, 80 °C; (h) MsCl, DMAP, pyridine; (i) NaN3, DMF, 80 °C; (j) DBU, 80 °C; (k) H2, Pd/C, MeOH–AcOH; (l) TBSOTf, 2,6-lutidine,CH2Cl2; (m) LiAlH4; (n) i-Pr2NP(OBn)2, 1H-tetrazole, CH2Cl2; (o) m-CPBA; (p) TBAF, THF; (q) H2, Pd/C, EtOH; (r) 9, 1H-tetrazole, pyridine.

EtOOH

N3EtOa, b

70%

O

HO OH

N3=O3PO

HO

c

60% N OPO3=

OHOHHO

H

103

fO

N

OHHO

OP

O

O–

N

N

NH

O

NH2OP

O

O

O–

102

NOH

OHHO

H40%

O

H

N3

b

OH OOPO3

=

N3 OHd, e

HN

OPO3=

HO OH ON

OHHO

OP

O

O–

N

N

NH

O

NH2OP

O

O

O–

105

f

107

52% 51% 40% NHOH

HO

Ki = 13 μM agaist FucTase V

Ki = 11 μM agaist FucTase VI

IC50 = 45–82 μM agaist FucTase V

104

106

ON

OHHO

OP

O

O–

g, h

74%

N

N

NH

O

NH2OP

O

O

O–

108

CO2Me

OBn

OMs

BnO

BnO

110

i, j

30%N

RO OROR

CO2MeN N

109: R = Bn

111: R = TBS (79 %)k, l

30%

m–o

N

R1O OR1

OR1

N N

OP(OR2)2

O

N

HO OHOH

N N

112: R1 = TBS, R2 = Bn

113: R1 = R2 = H (88%)p, q

r

40%

Ki = 8 μM against FucTase V

Ki = 13 μM against FucTase VI

O

BnO OBnOBn

OH



Hashimoto and colleagues synthesized 119, anotherCMP-NeuAc analogue, in which the bond between thesugar and phosphate moiety is longer than that of CMP-NeuAc, in their attempt to target a more potent inhibitor.67

The phosphonate ester 120 was synthesized in eight stepsfrom b-C-allyl sialoside 121,68 and subsequent deprotec-tion, then oxidation of the resulting alcohol to the carbox-ylic acid and its methylation with diazomethane afforded122a. After conversion of the dimethyl phosphonate 122ainto the monomethyl phosphonate 122b,66 the latter wascoupled with triacetyl cytidine (118a) in the presence ofbenzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP), demethylation of the methylphosphonate and subsequent treatment with ammoniumhydroxide to give 119. The IC50 of 119 against a(2,3)- and

a(2,6)-sialyltransferases was 47 mM and 340 mM, respec-tively (Scheme 21).

Some sugar nucleotide analogues in which the diphos-phate moiety was replaced with phosphonate had beensynthesized and reported earlier than those mentionedabove. Vaghefi and colleagues reported the synthesis ofphosphonate linked with galactose at the anomeric carbonby the reaction of 2,3,4,6-tetra-O-benzyl-1-O-acetyl-D-galactose (123) with tris(trimethylsilyl)phosphite in thepresence of trimethylsilyl triflate.69 Thereafter, the ob-tained product 124 was treated with uridine 5¢-phosphonicdibutylphosphinothionic anhydride (125)70 to yield theUDP-Gal analogue 126 (Scheme 22). The inhibitory ac-tivity of 126 against galactosyltransferase (from L1210

Scheme 21 Synthesis of the phosphonic acid analogues of CMP-NeuAc 114a,b and 119. Reagents and conditions: (a) (RO)2PX, base; (b)Me3SiOP(OMe)2, TMSOTf; (c) PhSH, Et3N; (d) 118a, DIAD, Ph3P, THF; (e) NH3/H2O, then NaOH; (f) BOMCl, DIPEA, DMF; (g) LiEt3BH;(h) TBSCl, imidazole; (i) O3 then Me2S; (j) BuLi, (MeO)2POH, then ClC(S)OPh; (k) Bu3SnH, AIBN; (l) Pd/C, HCO2NH4; (m) Ac2O, pyridine;(n) HF·pyridine; (o) RuCl3, NaIO4; (p) CH2N2; (q) PhSH, Et3N; (r) 118b, BOP, DIPEA; (s) NH4OH.

O CO2Me

AcO OAc

AcOAcHN

OAc

O CO2Me

PAcO OAc

AcOAcHN

OAc

116a: R = Me117a: R = Et3NH (95%)

O OMe

ORO

N

N

O

OHHO

O

NH2

O CO2H

PHO OH

HOAcHN

OH

O

ONa

114a

OH

O CO2Me

AcO OAc

AcOAcHN

OAc

115a: R = Et

OP OR

OR

O

CO2Me

P

AcO OAc

AcOAcHN

OAc O

OMe

ORO

N

N

O

OHHO

O

NH2

O

CO2H

P

HO OH

HOAcHN

OH OONa

114b116b: R = Me117b: R = Et3NH (90%)

a

b

d, c, e

d, c, e

c

c

64%

83%

Ki = 250 μM against α(2,6)STase


115b: R = Bn

O CO2Me

HO OH

HOAcHN

OH

121

O

PO3Me2AcO OAc

AcOAcHN

OAcOTBS

120

O CO2Me

PAcO OAc

AcOAcHN

OAc

122a: R = Me122b: R = Et3NH

O OMe

OR ON

N

O

OHHO

O

NH2

O CO2H

PHO OH

OHAcHN

OH

O

OHf–m

11947%

n–p

52%

r, q, s

32%

IC50 = 47 μM against α(2,3)STaseIC50 = 340 μM against α(2,6)STase

q

OR

OAcAcO

HO118a: R =

N

N

O

NHAc

118b: R =N

NH

O

O



leukemia cells) was relatively weak (Ki = 165 mM), con-sidering the Km value (13.7 mM) of UDP-Gal.

Another UDP-Gal analogue, in which the diphosphatemoiety was substituted with methylene diphosphonate,was also synthesized by the Vaghefi research group.71

Treatment of b-D-galactose pentaacetate (127) withdiphenylphosphonic acid (128) at high temperature andsuccessive hydrogenation on platinum oxide gave diphe-nyl phosphonate 129, which was next condensed with2¢,3¢-diacetyluridine (118b) in the presence of 1-(mesity-lene-2-sulfonyl)-3-nitro-1,2,4-triazole (MNST; 130).72

Subsequent deacetylation at the final step yielded the tar-get molecule 131, whose Ki value for the galactosyltrans-ferase from L1210 leukemia cells was 96.9 mM, making italmost as potent as 126 (Scheme 22).

A phosphonate analogue of GDP-Fuc, 132, was also syn-thesized with the Arbusov reaction using the bromide 133(Scheme 22).53

Recently, 134, a phosphonate analogue of UDP-GlcNAc,was synthesized by the research groups of Kirk73 andFinney.74 Kirk and colleagues started the synthesis with135, which was obtained by allylation of the chloride atthe C-1 position of N-acetylglucosamine derivative 136under radical conditions.75 After migration of the doublebond of 135 with iridium complex,76 ozonolysis affordedthe aldehyde 136. Addition of the anion of diethylphos-phite to 137 afforded the a-hydroxyphosphonate 137(dr = 95:5), which was subjected to radical deoxygenationvia methyl oxalate derivative to give phosphonate 138.Complete deprotection of 138 afforded N-acetyl-a-D-glu-cosaminylmethyl phosphonate (139), which was furthertreated with morpholidate 34 and then triethylamine toyield the target molecule 134 (Scheme 23). The Finneyroute required more steps to reach the key intermediate139 because it started with 3,4,6-tri-O-benzyl-D-glucal(140), which does not have an acetamido group at the C-2position (Scheme 23). The inhibitory activity of 134 wasunfortunately faint against O-linked N-acetylglucosami-nyltransferase (IC50 = 3.5 mM) and chitin synthetase.

Scheme 22 Synthesis of the UDP-Gal analogues 126 and 131 andGDP-Fuc analogue 132. Reagents and conditions: (a) H2O; (b)NH4Cl, cyclohexene, Pd(OH)2/C; (c) NH4OH; (d) Ag2CO3, pyridine;(e) 170 °C, vacuum; (f) H2, PtO2; (g) 130, 118b; (h) NH4OH; (i)AcOH; (j) Br2, Ph2PCl, imidazole; (k) (EtO)3P, reflux; (l) Me3SiBr;(m) H2O; (n) K2CO3, MeOH; (o) 9, pyridine.

O

AcO

AcOOAc

AcOOAc

O

HO

HO OH

HO P

O

OHO

N

NH

O

OHHO

O

O

CH2 P

O

OH

OPhP

O

PhO

HO P

O

HO

CH2

O

HO

AcOOAc

AcO OHP

O

OH

O P

O

HO

CH2

OSO2

NN

NNO2

MNST (130)

O

BnO

BnOOBn

BnOO

CH3

O

OS

O

O CF3

SiMe3

O

BnO

BnOOBn

BnO

O S

OO

CF3

MeSiO P

OSiMe3

O SiMe3

O

HO

HO OH

HO

PO

OHOH

ON

NHO

OHHO

O

O

O P

O

OH

Bu2P

SO

HO

HOOH

HOPO

OHO

N

NHO

OHHO

O

O

O P

O

OH

Ki = 165 μM to GalTase

123

:

124

+

126125

a–c

70%(4 steps)

d

53%

Ki = 96.9 μM to GalTase

O

AcOOAc

OAcBr

ON

NH

O

NH2

OHHO

OPO

O

O–P

O

O–

N

NO

HOOHOH

O

HOOHOH

PO3=

133

i, j

85 %

k–n

132

93%

131

128127 129

24%

g, h

38%

e, f

o

+

91

124

Scheme 23 Synthesis of the analogue of UDP-GlcNAc 134.Reagents and conditions: (a) Bu3SnCH2CH=CH2, AIBN, toluene;(b) Ir, THF; (c) O3 and then Me2S; (d) LiHDMS, HPO(OEt)2;(e) LiHDMS, ClCOCO2Me, THF; (f) Bu3SnH, AIBN, toluene;(g) TMSBr, CH2Cl2; (h) NaOMe, MeOH; (i) 34, 1H-tetrazole, pyri-dine; (j) Et3N, MeOH, H2O; (k) Amberlite (Na+); (l) DMDO, CH2Cl2;(m) Al(CH=CH2)3, CH2Cl2; (n) TBSOTf, 2,6-lutidine, (o) O3 and thenNaBH4; (p) MsCl, DIPA, CH2Cl2; (q) TBAI, DMF, 120 °C;(r) P(OEt)3; (s) TFA; (t) Moffat oxidation; (u) NH2OH; (v) Ac2O;(w) B2H6; (x) TMSI.

ON

NHO

O

OHHO

OPOO

O–PO

O–

OOH

HOHO

AcHN

134

e, f

i–k

b, c

OOAc

AcOAcO

AcHNR

OOAc

AcOAcO

AcHNPHO

136: R = Cl

135: R = CH2CH=CH2

a

d

O

OEtOEt

137: R = CHO (87%)

OOR1

R1OR1O

AcHNPO

OR2

OR2

138: R1 = Ac, R2 = Et

139: R1 = R2 = Hg, h

53% 54%

10–37%

OOBn

BnOBnO

o-q

OOBn

BnOBnO

R2OR1

R1 = CH=CH2, R2 = Hn

140

R1 = CH=CH2, R2 = TBS (95%)

R1 = CH2I, R2 = TBS (34%)

OOBn

BnOBnO

TBSOPO

OEtOEt

OOBn

BnOBnO

NPO

OEtOEt

AcO

l, m r

53%

s–v w, v, x139

75%



3.6.2 Analogues with an Oxycarbonylaminosulfone or a b-Sulfonyl Amide in Place of the Diphos-phate

Recently, Augé and colleagues synthesized the GDP-Fucanalogue 141 which has an oxycarbonylaminosulfonylgroup as a non-charged isostere of the diphosphategroup.77 In the beginning, they designed 141 as a surro-gate of the donor substrate for fucosyltransferase-cata-lyzed reactions, on the basis that p-nitrophenyl a-sialosidewas reported by Withers and co-workers to act an alterna-tive donor for sialyltransferase in the presence of a cata-lytic amount of CMP.78

The benzyl-protected fucose 110 was treated first withchlorosulfonyl isocyanate and then with guanosine deriv-ative 142a to afford 143 as a mixture of a- and b-isomers(4:1). Removal of benzyl and benzylidene protecting

groups by hydrogenolysis, followed by the treatment withpenicillin amidase to cleave the phenylacetamide bond,gave 141. In advance of the synthesis of 141 by Augé,Chapleur and colleagues had synthesized 144, a similarGDP-Fuc analogue, starting from D-mannose. However,neither 141 nor 144 showed significant inhibitory activityagainst fucosyltransferase III (Scheme 24).77,79 In addi-tion, Augé prepared 145, an analogue of UDP-GlcNAc,according to the method reported by Camarasa and col-leagues,80 and evaluated its inhibitory activity; however,145 showed no inhibitory activity against N-acetylglu-cosaminyltransferase (EC 2.4.1.56, Lgt A).77

3.6.3 Analogues with a Monosaccharide in Place of the Diphosphate

Tunikamycin (146), a potent inhibitor of the enzyme thattransfers GlcNAc-1-phosphate to dolichol 1-phosphate,does not have any phosphate groups such as shown in thetransition state 147.81 Careful comparison of 146 and 147revealed that the heptose unit mimics the pyrophosphatecomplex formed with Mn2+ in the transition state(Scheme 25). Only one analogue in which the sugar phos-phate moiety of the sugar nucleotide was substituted withan oligosaccharide has been synthesized. Wong and col-leagues found that 5¢-O-b-lactosyluridine (148) synthe-sized in their study inhibited b(1,4)-galactosyltransferasefrom L1210.82 Field and co-workers synthesized 148 viaanother route and reported no inhibitory activity againstthe galactosyltransferase from bovine.83 The discrepancycan be attributed to the difference in the materials used.

3.6.4 Analogues with Malonate or Tartrate in Place of the Diphosphate

Analogues 149a–c and 150, in which the phosphate groupwas substituted with malonate and tartarate derivatives,have been synthesized; however, no assay data are avail-able in the literature (Figure 1).82

Figure 1 Structure of sugar nucleotide analogues 149a–c and 150,which have malonate or tartrate in place of the diphosphate moiety

Scheme 24 Synthesis of GDP-Fuc analogues 143, 144 and structureof UDP-GlcNAc analogue 145. Reagents and conditions: (a)O=C=NSO2Cl, CH2Cl2; (b) 142a, pyridine CH2Cl2; (c) H2, Pd/C; (d)penicillin amidase; (e) Me2C(OMe)2, PTSA, DMF; (f) CrO3, pyri-dine; (g) Ph3P, CCl4, THF; (h) Raney Ni, AcOEt; (i) AcOH, H2O; (j)TsCl, pyridine; (k) BzCl, pyridine; (l) NaI, butanone; (m)HSCH2CO2Me, Cs2CO3, DMF; (n) m-CPBA, EtOAc; (o) NaOMe,MeOH; (p) 142b, BOP, Et3N; (q) TsCl, Et3N, CH2Cl2; (r) TFA–H2O(8:2).

ON

NR1

NR2R3

OO

R6

O

142a: R1 = H; R2, R3 = H, COBn, R4, R5 = Ph, H, R6 = OH

110a, b

N

N

40%

ON

NH

NHR1

OR4R3O

ON

N

O

R2OOR2 OR2

143: R1 = COBn, R2 = Bn, R3, R4 = CHPh

NH

O OS

O O O

141: R1 = R2 = R3 = R4 = H (32%)

(α/β = 9:1)

D-mannose

c, d

O

Cl

ClO

OO

OO

BzO OBzOBz

I

O

HO OHOH

S CO2HO O

R4 R5

142b: R1 = MOM; R2, R3 = CHNMe2, R4, R5 = Me, H, R6 = NH2

p–rO

HO OHOH

SO O

ON

NH

NH2

OHHO

ON

NHN

O

144

OHO

HOAcHN

OH

O ON

NH

NH2

OHHO

ON

NNH

OS

O O O

145

53% 46% 92%

48%

m–of–le–g

O

OHHO

149a: R =

N

NH

O

O O

O OR

NHO

HOOH

OH

149b: R = NHOHO

OH

OH

149c: R =

NHOHO

OH

OH

O

O

OHHO

HO HO

O

OHHO

N

NH

O

O

O O

OH

OHO

150



3.6.5 Analogues with a Peptide Bond in Place of the Phosphate

Antibiotics such as nikkomycin Z (151)84 and polyoxin J(152)85 are sugar nucleotide analogues in which the phos-phate group has been replaced with peptide bonds(Figure 2). Nikkomycin Z was reported to be an inhibitorof a kind of N-acetylglucosaminyltransferase, namelychitin synthetase.84

We designed and synthesized 153a and 153b, peptide mi-metics of GDP-Fuc, using an L-threonine aldolase cata-lyzed reaction as a key step (Scheme 26).86 Specifically,the aldehyde 154 prepared from chloroguanine 155 in twosteps was treated with glycine in the presence of L-threo-nine aldolase to afford the b-hydroxy-a-L-amino acid 156.The amino group of 156 was condensed with the fucosederivatives 157a and 157b by a conventional method us-ing EDC to form a peptide bond, and deprotection fur-nished 153a and 153b, respectively. Unfortunately, theinhibitory activities of 153a and 153b against fucosyl-transferases were weak, probably due to a lack of func-tional groups capable of making a chelate with Mn2+. Thesynthesis of 158, which could form a chelate with Mn2+,is now in progress.

3.7 Non-Sugar Analogues

To date, only a few analogues of sugar nucleotides inwhich the sugar moiety is replaced with a non-sugar com-pound have been reported. Schmidt and co-workers syn-thesized 159, a unique analogue of CMP-NeuAc in whichthe sialic acid moiety was replaced with (–)-quinic acid(160).87 In the synthesis of 159 starting with 160(Scheme 27), selective protection and differentiation ofthree hydroxy groups were required to replace the hy-droxy group at the C-3 position of 160 with an acetamidogroup. For this, Schmidt employed the transformation of160 into 3-azido g-lactone (161),88 where (–)-quinic acid(160) was converted into the corresponding lactone withresin-supported acid and azeotropic removal of water, andsubsequent tosylation of the equatorial alcohol was fol-lowed by an SN2 reaction using azide as a nucleophile togive 161. Next, the lactone 161 was converted into 162 inthree steps, whereupon the tertiary alcohol was treatedwith benzyloxychlorophosphitamide89 to afford the phos-phoramidite 163.

Further treatment of 163 with 118a in the presence of 1H-tetrazole followed by oxidation with tert-butyl hydroper-oxide (TBHP) provided 164. Debenzylation by way of hy-drogenolysis, followed by deacetylation with methoxideand saponification of the methyl ester, furnished 159. TheKi value of 159 for a(2,6)-sialyltransferase from rat liverwas estimated at 84 mM (Scheme 27).87

3.8 Analogues Developed through Combinatori-al Chemistry

The research groups of Wong and Sharpless reported thesynthesis of a very potent fucosyltransferase inhibitor us-ing a combination of combinatorial and click chemis-try,90–93 even before the three-dimensional structure offucosyltransferases was revealed.

Scheme 25 Structure of tunicamycin (146) and synthesis of 5¢-O-b-lactosyluridine (148). Reagents and conditions: (a) Ac2O, pyridine; (b)HBr in AcOH; (c) Hg(CN)2; (d) NaOMe, MeOH; (e) Dowex 50 (H+).

O O

OOAcOAc

AcO

AcO AcO

AcO AcOBr

ON

NH

O

O

OO

HO

28%

c–e+ O O

OOHOH

HO

HO HO

HO HO

ON

NH

O

O

OHHO

O

a, b

lactose

148

92%

O P O PO

O O

O O

O

HO OH

N

NH

O

OMn2+

O NHAc

HO

HOOH

OP

O

OHO-

O O O

HO OH

N

NH

O

O

O NHAc

HO

HOOH

OHHO OHNH

O

918

transition state of dolichol-pyrophosphate GlcNAc synthase catalyzed reaction

tunicamycin (146)

147

Figure 2 Structure of nikkomycin Z (151) and polyoxin J (152)

H2N O NH

O

OH

OH

NH2

O CO2H

OH

HO OH

N

NH

O

O

polyoxin J (152)

N

NH

O

O

HO2C

nikkomycin Z (151)

Ki = 2 μM (chitin synthetase)

OH

HO OH

NH

OH NH2

ONHO



According to the results of mechanistic studies, fucosyl-transferases bind most tightly with the GDP moietyamong the four components of a complex transition state,namely, the donor sugar (fucose), acceptor oligosaccha-ride, divalent metal ion (Mn2+), and nucleotide(GDP).57,94–96 Moreover, on the basis of their own study,in which several N-acetyllactosamine derivatives withdifferent aglycons were prepared and assayed for inhibi-tory activity against fucosyltransferase VI, it was revealed

that a hydrophobic pocket adjacent to the binding site ofthe acceptor substrate enhances the affinity of the accep-tor. Therefore, a library of compounds which have a GDPcore, a hydrophobic group, and a linker of varying lengthwith a triazole linker in between, were prepared by usingcopper(I)-catalyzed triazole synthesis as the key reactionof click chemistry.92,93,97 Specifically, 85 azide com-pounds 165, carrying diverse hydrophobic groups andtethered with 2–6 methylene units, were synthesized andtreated with propargyl GDP (166) in the presence of acopper(I) catalyst to afford a library of crude GDP-tria-zole compounds 167. The inhibitory assay of the librarywas performed directly in microtiter plates using the pyru-vate kinase/lactate dehydrogenase coupled enzyme assay,and 168 proved to be selective and showed the most po-tent inhibitory activity against a(1,3)-FucT VI (Ki = 62nM) (Scheme 28).98 The potent inhibitory activity of 168was later rationalized by Lin’s research group, as shownin the next section.

3.9 Analogues Derived through Precise Analyses of the Structure of Glycosyltransferases and Their Catalyzed Mechanisms

Lin and colleagues precisely analyzed the mechanism ofa(1,3)-fucosyltransferase from H. pylori on the basis ofX-ray crystallography of the GDP-Fuc complex of a C-terminal 115 residue-truncated enzyme that was com-posed of 340 amino acids.99 In the analysis, 18 well-de-fined hydrogen bonds were observed between the enzymeand GDP-Fuc, mainly in the C-terminal domain where

Scheme 26 Synthesis of the GDP-Fuc analogues 153a,b. Reagents and conditions: (a) K2CO3, BrCH2CH(OEt)2; (b) 1 M HCl; (c) L-threoninealdolase, glycine; (d) Na, NH3 (liq); (e) Ac2O, pyridine; (f) RuO2, NaIO4; (g) KOH, MeOH; (h) 157a or 157b, DCC, HOBt, NMM; (i) 0.02 MNaOMe in MeOH; (j) 0.01 M NaOH (aq).

O

AcOOAc

OAc

O CO2H

O

HOOH

OH

O

OH

NH

–O2C

NH

NN

N

O

NH2O

43%

OHCO2Bn

NH2

HN

N N

N

O

H2N

O

AcOOAc

OAcO CO2H

O

HO OHOH

O

OH

NH

–O2C

NH

NN

N

O

NH2O

83%

3

3O

BnOOBn

OBn

O3

O

BzOOBz

OBzO

3

156

153a

153b

157a

157b

N

N NH

N

H2N

Cl

CHO

HN

N N

N

O

H2N

154

a, b

c

d–f

g, e, f

h–j

h–j

O

HO OHOH

O

OH

NH

–O2C

NH

NN

N

O

NH2O

158

O

H2N

Mn2+

155

Scheme 27 Synthesis of the CMP-NeuAc analogue 159. Reagentsand conditions: (a) Amberlite IR-120 H+, DMF; (b) TsCl, pyridine;(c) NaN3; (d) NaOMe, MeOH, then Amberlite IR-120 H+; (e) Ac2O,pyridine; (f) H2, Pd/C, MeOH then Ac2O; (g) Cl(i-Pr)P(OBn),DIPEA, MeCN; (h) 118a, 1H-tetrazole, MeCN then TBHP; (i) H2,Pd/C, MeOH then Et3N; (j) NaOH, MeOH, and then NaOH, MeOH(aq).

d–f

43%

i–j

HOHO

OH OH

CO2H

160

HO

N3O

O

161

AcOAcO

NHAc

OH

CO2Me

162

g

92%

AcOAcO

NHAc

O

CO2Me

163

P OBnN-i-Pr2 h

71%AcO

AcONHAc

O

CO2Me

164

POBn O

N

NO

OAcAcO

O

NHAcO

HOHO

NHAc

O

CO2H

159

PO– O

N

NO

OHHO

O

NH2

O


a–c

53%

OH

79%



electropositive Arg-195 and Lys-250 residues neutralizethe negative charge of the diphosphate moiety.

Glu-95, located in the N-terminal domain, seemed to playa vital role as a general base in the active site to abstract aproton from the hydroxy group of an acceptor substrate.On considering the reaction mechanism, N-acetyllac-tosamine (LacNAc), an acceptor model, was put in thecleft formed by Glu-95 and Glu-41. Because there is stillconsiderable distance between the donor and acceptorsites, a major conformational change of the C-terminal a-helix, where Arg-195 is present, should be induced by thebinding of GDP-Fuc and would afford a conformation inwhich the electropositive N-terminal and highly elec-tronegative C-terminal regions are close. On the basis ofthis speculation and the finding that fucosyltransferase in-teract more with GDP than with fucose,57,94–96 a variety ofGDP derivatives 169 were prepared by coupling the GDP-hexylamine 170 with 80 different carboxylic acids. TheIC50 values of inhibitors 169 against a(1,3)-fucosyltrans-ferase ranged from 10 to 100 mM (Scheme 29).

Nishimura and colleagues designed 171a, an analogue ofUDP-Gal, as a galactosyltransferase inhibitor, on the basisof docking simulation. Mass spectrometric analysis re-vealed that 310Trp residue located near the active site ofhuman galactosyltransferase can be selectively modifiedwith the naphthylmethyl group of 171a. Based on that re-sult, 171b was developed as the strongest (Ki = 1.86 mM)inhibitor of galactosyltransferase to date (Figure 3).100

4 Analogues of Acceptor Oligosaccharides (Acceptor Analogues)

4.1 Methylated and Deoxygenated Analogues

Hindsgaul and colleagues synthesized two trisaccharidederivatives to act as the sugar moiety of the N-acetylglu-cosaminyltransferase V substrate. One derivative was172, in which the hydroxy group at the C-4 position of the

mannose moiety was methylated. The other derivativewas 173, in which the hydroxy group was deoxygenated.An enzymatic assay showed that 172 was a relatively po-tent inhibitor (Ki = 14 mM) while 173 behaved as an excel-lent substrate (Km = 76 mM) (Figure 4).101

The inhibitory activity of 172 was a result of repulsion be-tween the C-6 position of the mannose unit and the donorsubstrate, caused by the methoxy group at C-4. Later, sev-

Scheme 28 Strategy for discovering fucosyltransferase inhibitors using combinatorial chemistry based on click chemistry. Reagents and con-ditions: (a) Br(CH2)nCOCl (n = 1–5); (b) NaN3; (c) H3PO3, I2, Et3N; (d) GDP-morpholidate, Oct3N, 1H-tetrazole; (e) CuI catalyst.

RNH2

168

RNHN3

O

n

a, b

OH

40–100%

c

69%

OPO3H2d

41%

GDP

e

RNHN

O

n NN

GDP

ON

NH

O

NH2

OHHO

OPO

O

O–P

O

O–

N

NON

N N

NH

O

assay

165

166

167

Ki = 62 nM against α(1,3)-FucTase VI

Scheme 29 Detailed structure of the active site of a(1,3)-fuco-syltransferase and its inhibitors 169. Reagents and conditions: (a)XCO2H, amide-forming reagent.

OH3C

HOOHOH

O

δ+δ–

O–O

249Glu

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

O–

N

N

NH3

+

250Lys

HN

+

195Arg

NH2H2N

highly electronegative region

electropositive region

H2N(CH2)nCH2O ON

NH

O

NH2

OHHO

OPO

O

O–P

O

O–

N

N

n = 2 or 5

aXCONH(CH2)nCH2O O

N

NH

O

NH2

OHHO

OPO

O

O–

P

O

O–

N

N

170

169

OO

O

OHHO

HOHO

O

AcHNO

OH

O O–

95Glu

H

–O O

41Glu

H



eral acceptor analogues 174–180, in which the key hy-droxy groups of acceptor oligosaccharides weredeoxygenated, were synthesized by the same researchgroup. It was found that not all of the deoxygenated ana-logues were inhibitors of appropriate glycosyltransferases(Figure 4), thus it was considered that these hydroxygroups were indispensable for recognition by the glyco-

syltransferases.16a Meanwhile, Hashimoto and colleaguessynthesized deoxygenated and thiolated analogues (181and 182) of LacNAc, in which the hydroxy group at the C-6¢ position was modified, and measured their inhibitoryactivity. The thiolated analogue 182 showed weak inhibi-tory activity toward a(2,6)-sialyltransferase.102

4.2 Fluorinated Analogues

Selectively fluorinated carbohydrates have been used asacceptor probes for the study of glycosyltransferases.Lowary and Hindsgaul revealed that blood group A and Bglycosyltransferases, which catalyze the reaction thattranfers N-acetylgalactosamine or galactose to the C-3 po-sition of the galactose residue in the fucosyl-a(1-2)galac-tose unit, act on the C-6-fluoro-substituted galactoside184 as well as the natural substrate 183 and 6-deoxygalac-toside 185. Regarding the inhibitory activity, the C-3-fluorinated galactoside 186 inhibited the A transferase,but not so strongly the B transferase. Against the B trans-ferase, the 3-deoxygenated galactose 187 was a more po-tent inhibitor than 186 (Figure 5).103

In addition, Hartman and Coward reported that the effectof a 5-fluoro substituent on the GlcNAc b-octyl glycoside188 against b(1,4)-galactosyltransferases was a sixfold in-crease in Km and an approximate 30% decrease in kcat.

27

Matta and colleagues synthesized partially fluorinatedmucin core 2 tetrasaccharides 189–191, modified at the C-3 or C-4 position of the pertinent galactose residue, as sia-lyltransferase acceptor inhibitors.104 With their approach,the 4-fluorinated galactose donor 192 was prepared fromthe corresponding methyl galactopyranoside 193,105 whilethe 3-fluorinated donors 194a and 194b were synthesizedfrom 1,2:5,6-diisopropylidene-a-D-gulofuranose (195a)through fluorination of the C-3 hydroxy group with DASTto afford 195b.106 Moreover, the N-phthalimido-protectedthioglycosides 196a–b and 197a–b were used as glycosyldonors to N-acetyllactosamine and N-acetylglucosamine(Scheme 30). As a result, the tetrasaccharides 189 and 190were good substrates for a(2,3)-O-sialyltransferases, and191 was a good substrate for a(2,3)-N- and a(2,6)-N-sia-lyltransferases. Meanwhile, 189 was a poor substrate for

Figure 3 Structures of galactosyltransferase inhibitors 171a,b

OHO

HOHO

R

OP

OP

O

O OO– O–

O

HO OH

N

N

O

O

Ki = 1.86 μM against β(1,4)-GalTase

O

HN

OO

O Br

O

OO

OO

171a R =

171b R =

Figure 4 Acceptor analogues 172–182 as glycosyltransferase inhi-bitors

O OR

OH

HOO NHAc

OOHHO

HO H

α(1,2)-FucTase Ki = 0.80 mM

(Km for natural sub. = 0.20 mM)

α(1,3/4)-FucTase no inhibition(Km for natural sub. = 0.07 mM)

O OR

OH

HO NHAc

OOHHO

HO OH

174

175

O OR

OH

OH NHAc

OOHHO

HO OH

α(1,3)-FucTase no inhibition(Km for natural sub. = 0.24 mM)

176

O OMe

OH

HHO NHAc

β(1,4)-GalTase(Km for natural sub. = 1.3 mM)

no inhibition

177

O OR

OH

OHO NHAc

OOHHO

H OH

α(2,3)-STase no inhibition(Km for natural sub. = 0.15 mM)

180

O OR

HHO

O NHAc

OOHHO

HO HO

β(1,6)-GnTase(Km for natural sub. = 0.08 mM)

Ki = 0.56 mM

179

O OMe

OH

OHO NHAc

ORHO

HO OH

α(2,6)-STase Ki = 0.76 mM

(Km for natural sub. = 0.9 mM)

181: R = H182: R = SH

O

O

O

R

HO

HO

O

OHOH

OHAcHN

O(CH2)7Me

O

HOHO OH

172: R = OMe Ki = 14 μM for GNTase V173: R = H Km = 76 μM

GlcNAc

O

O

O

R1

H

HO

O

OHOH

OHAcHN

OR2

O

HOHO OH

GnTase V(Km for natural sub. = 0.036 mM)

Ki = 0.063 mM

178

α(1,4)-FucTase(Km for natural sub. = 0.24 mM)

Ki = 0.54 mM

Figure 5 Fluorinated acceptor analogues as substrates and glyco-syltransferase inhibitors

183: R1 = R2 = R3 = OH

184: R1 = R2 = OH, R3 = F

OR3

R2

R1 O

O(CH2)7Me

O

HO OHOH

185: R1 = R2 = OH, R3 = H

OOHHO

HO NHAc

O(CH2)7Me

F

186: R1 = F, R2 = R3 =OH

188

187: R1 = H, R2 = R3 =OH

Km (μM)Blood A transferase Blood B transferase

1.50 21.9

4.96 55.6

7.29 68.8

Ki (μM)Blood A transferase Blood B transferase

48.9 110

68.9 14

Km 6-fold increasedkcat 30% decreased



a(2,3)-N- and a(2,6)-N-sialyltransferases, probably due tothe weakened nucleophilicity of hydroxy groups causedby the strong electronegativity of fluorine. This suggestedthat the a(2,3)-N- and a(2,6)-N-sialyltransferases transfera sialic acid to the b(1-4)galactose residue of the N-acetyl-lactosamine moiety and a(2,3)-O-sialyltransferase prefersthe b(1-3)galactose residue linked to N-acetylgalac-tosamine.

Moreover, it was interesting that the 4-fluorinated tet-rasaccharide 189 did not inhibit a(2,3)-N-sialyltransferasebut did inhibit a(2,6)-N-sialyltransferase with a high de-gree of selectivity.

5 Bisubstrate Inhibitors

Glycosyltransferases accept oligosaccharides as well assugar nucleotides as substrates. Since the active sites ofglycosyltransferases would be occupied by both sub-strates at the same time in the transition state, transition-state analogues in which acceptor oligosaccharides andsugar nucleotides are linked via a covalent bond could berational glycosyltransferase inhibitors. Inhibitors de-signed on the basis of this concept are referred to as

‘bisubstrate inhibitors’, and are expected to exhibit potentinhibitory activity and be highly selective for glycosyl-transferases. The first bisubstrate inhibitor 201 was re-ported by Hindsgaul and colleagues, and inhibited a(1,2)-fucosyltransferase (Ki = 2.3–16 mM).107 However, instructure, 201 seemed to resemble the transition state in afucosyltransferase-catalyzed reaction in that it lacked afucose moiety. Therefore, 201 could be considered ananalogue of GDP-Fuc (Figure 6).

Figure 6 The first bisubstrate inhibitor, 201, synthesized byHindsgaul and colleagues

Actually, according to Hashimoto and colleagues, evenUDP-Fuc and UDP-Man, which are composed of incom-patible combinations of sugars and nucleotides as the gly-cosyltransferase donor substrate, could markedly inhibitb(1,4)-galactosyltransferase which employs UDP-Gal asthe donor substrate.108 Thus, it is difficult to clearly ruleout that the analogue 201 inhibited the fucosyltransferaseas a sugar nucleotide mimic.7

Thereafter, bisubstrate inhibitors with improved potencyand selectivity have been exploited in the development ofsynthetic study of oligosaccharides. In this section, the in-hibitors are classified by the target glycosyltransferases,which are themselves characterized by the donormonosaccharides.

5.1 Galactosyltransferase Inhibitors

The first bisubstrate inhibitor to target b(1,4)-galactosyl-transferase was composed of an acceptor saccharide, a do-nor sugar, and a nucleotide, and was synthesized byHashimoto and colleagues.

Compound 202109 was designed so that the hydroxy groupat C-2 of UDP-Gal, a donor substrate of the galactosyl-transferase, was linked with that at C-6 of N-acetylglu-cosamine, the acceptor substrate, via a methylene in orderto mimic the transition state where the anomer carbon ofgalactose and the C-4 hydroxy group of N-acetylglu-

Scheme 30 Synthesis of the fluorinated acceptor analogues 189–191. Reagents and conditions: (a) Ac2O, H2SO4; (b) NH2NH2, AcOH,DMF, 50 °C; (c) CCl3CN, DBU, CH2Cl2; (d) 197a, TMSOTf, 4 ÅMS, CH2Cl2, –65 to –70 °C; (e) Ac2O, pyridine, DMAP; (f) DAST;(g) 60% AcOH; (h) Ac2O, pyridine; (i) 197b, TMSOTf, 4 Å MS,CH2Cl2, HCl, MeOH, –65 °C; (j) HBr, AcOH; (k) 198, Hg(CN)2,MeNO2, benzene, 65 °C; (l) 199, NIS, TfOH, 4 Å MS, CH2Cl2, –65to –60 °C; (m) NH2NH2, H2O, MeOH, 90 °C; (n) NaOMe, MeOH; (o)DDQ; (p) 200, NIS, TfOH, CH2Cl2, –45 to –40 °C.

OOAc

F

AcO AcOO

NHCCl3

192

OOAc

F

AcO AcO

OMe

193

O

OO

R1

OO

OOAcAcO

F AcOO

NH

CCl3194a

OOAc

AcO

F AcO

194b

OOAcAcO

F AcO

OOHHO

O AcHNOBnO

OAcR1

R2 AcO

OOR3

O NPhthSPh

OOHHO

R3 HO

OOHO

O AcHN

OOO

AcO

AcHN OHHO R2

R1

OBn

HOHO

196a: R1 = F, R2 = OAc, R3 = Piv

196c

195a: R1 =H, R2 = OH

a-c 46 %

d, e 63%

i, e 33%

196b: R1 = OAc, R2 = F, R3 = NAP

l, e, m, e, n (196a) (34%)

189: R1 = F, R2 = R3 = OH

Br

k, g 33%

190: R1 = R3 = OH, R2 = F

p, e, m, e, n (29%)

191: R1 = R2 = OH, R3 = F

g, h, b, c g, h, j

l, e, m, e, o, n (196b) (23%)

195b: R1 =F, R2 = Hf

R2

OOR3

NPhthSPh

HOHO

197a: R3 = Piv

197b: R3 = NAP

OOAcAcO

AcO AcO

OOHHO

O AcHNOBn

199

OO

HO AcHNOBn

O

Ph

198

OOAcAcO

AcO AcO

OOAc

OAcHN 200AcO

SPh

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

O–

N

NOH3C

HOOHOH O

201

OO

OHHO

HO OHδ+

transition state of FucTase-catalyzed reaction

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

O–

N

NH2C

O OOHHO

HO OH2C

Ki = 2.3–16 μM for α(1,2)FucTase



cosamine are a short distance apart (Scheme 31). The syn-thesis required short steps and 202 showed potentinhibitory activity with Ki values of 1.35 mM (towardGlcNAc) and 3.3 mM (toward UDP-Gal).

Scheme 31 Synthesis of 202, a b(1,4)-galactosyltransferase inhibi-tor. Reagents and conditions: (a) PhCH(OMe)2, TsOH, 60 °C; (b)MeSCH2Cl, NaH, NaI, DMF; (c) MeOTf, 4 Å MS, CH2Cl2; (d) t-BuOK, DMSO, 60 °C; (e) HgCl2; (f) n-BuLi, (BnO)2POCl, –78 °C;(g) H2, Pd/C, Bu3N, MeOH; (h) UMP-Imd, DMF.

Later, Schmidt and colleagues synthesized 203a and 203bas novel a(1,3)-galactosyltransferase bisubstrate inhibi-tors.52 The aldehydes 204a and 204b obtained from thegalactonolactone 205 in eight steps were treated with theiodide 206 in the presence of tert-butyllithium to afford207a and 207b, respectively. The newly generated hy-

droxy groups of 207a,b were deoxygenated by the Bartonmethod, which was followed by the deprotection of thetert-butyldimethylsilyl groups to give the phosphates208a,b, which were converted, in another four steps, into203a,b (Scheme 32). An inhibitory assay with a(1,3)-ga-lactosyltransferases from pig liver revealed that 203a wasa potent inhibitor (IC50 = 5 mM) while 203b showed no in-hibitory activity even at 50 mM. It is worth noting that203a is a rare inhibitor of retention enzymes (vide infra).

5.2 N-Acetylglucosaminyltransferase Inhibitors

N-Acetylglucosaminyltransferases (GnTases) V110 andIX111 have attracted interest from synthetic chemists be-cause their activities are directly related to the metastaticpotential and malignancy of tumor cells. The transferasescatalyze the reaction that transfers N-acetylglucosaminefrom UDP-GlcNAc to the C-6 hydroxy group of the ter-minal a(1-6)mannose unit, which forms a part of the tri-mannnose core by linking to the bisecting b-mannnose.Therefore, inhibitors of GnTase V and IX have to be de-signed to link UDP-GlcNAc and the C-6 hydroxy groupof a(1-6)mannoside by mimicking the transition state inthe enzyme-catalyzed reaction.

Manabe, Ito, and co-workers developed a unique methodwhereby the monomethyl ether of polyethylene glycol(MPEG) was chosen as a polymer support for the synthe-sis of oligosaccharides by taking advantage of the highpolarity and small molecular weight of MPLG.112,113 Inadvance of this study, they utilized the method for the syn-

O

OHHO

OPOO

O–PO

O–

NO

202

NH

O

O

OOHHO

BnO HOOAll

a, bO

BnOMTMO

OAll

OO

Ph

c

OOH

BnOBnO AcHN

OMe

OBnO

O OR

OO

Ph

OOMe

NHAcBnO

BnOO

R = AllR = H (79%)R = PO(OBn)2 (27%)

OHO

O

OHOH

OOMe

NHAcHO

HOO

d, e

f

g, h

46% 51%

Ki = 1.3 μM (for GlcNAc)Ki = 3.3 μM (for UDP-Gal)

Scheme 32 Bisubstrate inhibitors 203a and 203b of a(1,3)-galactosyltransferase. Reagents and conditions: (a) t-BuLi, 206; (b) NaH, CS2,imidazole; (c) MeI; (d) Bu3SnH, AIBN; (e) TBAF; (f) i-Pr2NP(OBn)2, 1H-tetrazole; (g) m-CPBA; (h) H2, Pd/C; (i) Et3N; (j) 34, pyridine, 1H-tetrazole; (k) IR-120 (Na+).

OOBnBnO

BnOBnO

CHO

OTBS

O

OBnBnO

BnO BnO

CHO

OTBS

OOBnBnO

BnO

OMe

I

OOBnBnO

BnO BnO

OTBS

OBnO OBn

OBn

OMe

O

OBnBnO

BnO BnO

TBSOO

BnO OBn

OBnOMe

206

HO

OH

OOBnBnO

BnO BnO

O

OBnO OBn

OBn

OMe

P

O

OBnOBn O

OHHO

OPO

O

O–

P

O

O–

NO

NH

O

O

O

OHHO

HO HO

OHO OH

OH

OMe

O

OBnBnO

BnO BnO

O

OBnO OBn

OBn

OMe

P

O

BnO

BnO

OOHHO

HO HO OHO OH

OHOMe

O

OHHO

OPO

O

O–

P

O

O–

NO

NH

O

O

204b 207b

a

71%

208b

203b

b–g h–k

56%

IC50 = 5 μM against α(1,3)GalTase

204a

207a

a

83%

203a

b–g h–k

56%

208a

OOBnBnO

BnOBnO

O

205

205



thesis of 209a, a GnTase IX inhibitor (see Scheme 34 be-low).114

First, b-mannosylation of MPEG was conducted by theHodosi–Kováč method115 and subsequent glycosylation,using the activation of thioglycosides 210 and 211 with N-iodosuccinimide and triflic acid as the key step, affordinga derivative of the acceptor trisaccharide 212. Many prac-tical revisions were made during their study;113 however,only the route of synthesis is discussed here. Namely, thetrisaccharide moiety of 212 was released from MPEG andconverted within six steps into disulfide 213 in good yield(Scheme 33).

Next, 213 was reduced with tris(2-carboxyethyl)phos-phine (TCEP) and the resulting thiol was linked with theGlcNAc-1-phosphate derivative 215 in the presence of di-isopropylethylamine to afford the sulfide 216a. Finally,bisubstrate inhibitor 209a was obtained by a reaction ofthe phosphate group of 216a and UMP-morpholidate (34)(Scheme 34). Compound 209a showed inhibitory activitywith a Ki value of 7.2 mM against N-acetylglucosaminyl-transferase IX.

Recently, Ito and colleagues succeeded in synthesizingfour other derivatives, 209b–e, in an attempt to find morepotent inhibitors of N-acetylglucosaminyltransferase V aswell as IX, by precisely controlling the distance betweenthe trisaccharide moiety and N-acetylglucosamine(Scheme 35).116 For the synthesis of 209b, tributylphos-

Scheme 33 Synthesis of trisaccharide disulfide 213. Reagents and conditions: (a) Et3SiH, TFA, CH2Cl2; (b) 210, NIS, TfOH, CH2Cl2, 4 ÅMS; (c) hydrazine dithiocarbonate, MeCN; (d) 211, NIS, TfOH, 4 Å MS; (e) Fmoc-Cys-Wang resin, DIPEA; (f) 4-aminomethyl piperidine; (g)60% AcOH, 60 °C; (h) 1 M KOH in EtOH–THF; (i) H2NCH2CH2NH2, BuOH, 100 °C; (j) Ac2O, pyridine; (k) 0.05 M NaOMe, MeOH; (l)TMSCHN2; (m) TsCl, DMAP, CH2Cl2; (n) H2, Pd(OH)2/C, AcOH, MeOH; (o) AcSK, DMF, 70 °C; (p) 0.05 M NaOMe in MeOH.

O

OBn

TrOBnO

BnOO

O

OMPEG

O

OBnO

BnOBnO

O

O

OMPEG

OO

O Ph

OBnClCH2CO2

O

OBnO

BnOBnO

O

O

OMPEG

OO

O Ph

OBn

OClCH2CO2

BnOO

OBn

PhthN OO

BnOO

OBn

PhthN

OS

NH2O

O

O

OBnO

BnOBnO

O

O

OMPEG

OO

O Ph

OBnO

HOBnO

O

OBn

PhthN

S

HNO

OO

O

OBnO

BnOBnO

O

O

OH

OOAcOAc

OBn

OAcO

BnOO

OBn

AcHN

OOHO

HOHO

O

O

OMe

OOTsOH

OH

OHO

HOO

OH

AcHN

OOHO

HOHO

O

O

OMe

OS

OH

OH

OHO

HOO

OH

AcHN

4

4 4

4 44 4

a, b

OO2CCH2Cl

OO

Ph

BnOSPh

OBnO

ClCH2CO2BnO

SPhPhthN

c, d e, f

g–j

88% 88%

213(46% for 6 steps)

k–n o, p

45% 90%

210

211

212 2

Scheme 34 Synthesis of 209a, an N-acetylglucosaminyltransferaseIX inhibitor. Reagents and conditions: (a) H2NNH2, AcOH, THF; (b)TBSCl, imidazole; (c) DMF, H2, Pd/C; (d) (BrCH2CO)2O, pyridine,CH2Cl2; (e) 47% aq HF, MeCN; (f) (i-Pr)2NP(OAll)2, 1H-tetrazole,CH2Cl2; (g) TBHP; (h) [Pd(PPh3)4], Et3SiH, AcOH; (i) TCEP·HCl,MeOH (aq); (j) 215, DIPEA; (k) Et3N; (l) 34, 1H-tetrazole, pyridine.

OOAc

AcOAcO

NHCbz

OAc

OOAc

AcOAcO

NH

OTBS

O

Br

OOAc

AcOAcO NH

O

Br

O P

O

OHO–

OOHO

HOHO

O

O

OMe

OS

OHOH

OHO

HOO

OH

AcHN

OOH

HOHO NH

O O P

O

OHO–

4

215

216a

O

OHHO

OPOO

O–PO

O–

NO

NH

O

O

OOHO

HOHO

O

O

OMe

OS

OHOH

OHO

HOO

OH

AcNH

OOH

HOHO NH

O

4

209a

88%

a–d e–h

i–k

45%

51%

l

78%

213

Ki = 7.2 μM to GlcNAcTase IX

214



phine was used instead of TCEP to reduce the disulfide213, and subsequent treatment with pyridyl disulfide gave217. This was then coupled with 218 (prepared from 214in six steps) to yield 216b, which was converted into 209bby the Wittmann–Wong method.20

In the synthesis of 209c, the reduction of 213 with tribu-tylphosphine was followed by treatment with 219 (pre-pared from 214 in three steps) and subsequent acetylationand removal of the tert-butyldimethylsilyl group gave220. Phosphorylation of the anomeric hydroxy group ofN-acetylglucosamine was achieved by treatment of diallyldiisopropylphosphinamidite in the presence of 1H-tetra-zole and subsequent oxidation with TBHP gave 221.Deallylation and subsequent deacetylation of 221 afford-ed 216c, which was converted into 209c by the Wittman–Wong method20 (Scheme 35).

Finally, for the synthesis of 209d and 209e, the reducedproduct of 213 was treated with 222a and 222b, respec-tively, in the presence of cesium carbonate, followed bydeacetylation with triethylamine to afford 216d and 216e.The sugar phosphates of the latter were coupled withUMP-morpholidate (34) to afford 209d and 209e, respec-tively (Scheme 36).

The inhibitory activities of 209a–e against N-acetylglu-cosaminyltransferases V and IX are summarized inTable 1.116

5.3 Sialyltransferase Inhibitors

The strategy of linking the donor and acceptor substratesof glycosyltransferases via sulfide bonds was adopted inthe design and synthesis of sialyltransferase inhibitors.

Focusing on the fact that the hydroxy groups, whichwould be glycosylated with sialic acid, should orientate in

Table 1 Inhibitory Activities of 209a–e

Inhibitor Ki (mM)

GnTase V GnTase IX

209a 71.9 10.1a

209b 119.3 4.7

209c 47.1 17.6

209d 26.9 21.5

209e 18.3 15.1

a This value was shown as 7.2 mM in Scheme 34.114

Scheme 35 Synthesis of 209b and 209c, N-acetylglucosaminyltransferase bisubstrate inhibitors. Reagents and conditions: (a) n-Bu3P, THF,H2O; (b) pyridyl disulfide, 0.5 M HCl, MeOH, H2O; (c) 219, Cs2CO3, DMF; (d) Ac2O, pyridine; (e) HF·pyridine, DMF; (f) (i-Pr)2NP(OAll)2,1H-tetrazole; (g) TBHP, then Me2S; (h) Pd(PPh3)4, Et3SiH, AcOH, toluene; (i) 218, MeOH, NH4OAc; (j) 34, 1H-tetrazole, pyridine; (k) AcSH,DIPEA, MeCN; (l) Et3N, MeOH, H2O; (m) NH2NH2, AcOH, THF; (n) BrCH2Cl, DIPEA, MeCN.

213 217

ON

NHO

OHHO

O

O

O

POO–

a, b O

O

OH

HOHO AcHN

OOH

OHSSPyr

OO

HOHO

OH

O OMe4

O

a, c–e

220: R1 = H, R2 = Ac, n = 1 (31%)

O

O

OR2

R2OR2O AcHN

OOR2

OR2

S(CH2)nS

OO

R2OR2O

OR2

O OMe4

O

221: R1 = P(O)(OAll)2, R2 = Ac, n = 1 (85%)

216b: R1 = P(O)(OH)2, R2 = H, n = 0 (47% from 217)

216c: R1 = P(O)(OH)2, R2 = H, n = 1 (45%)

f, g

h

i

j

O

O

OH

HOHO AcHN

OOH

OHS(CH2)nS

OO

HOHO

OH

O OMe4

O

OOH

HOHO NH

OO P

O

O–

209b: n = 0 (78%)

209c: n = 1 (58%)

k, e, f, g

56 % OAll

OOAc

AcOAcO NH

O O PO

OAll

AcS

h, l 51%

O–

OOH

HOHO NH

O O PO

OH

HS

Et3NH+

214

218

OOAc

AcOAcO NHCOCH2SCH2Cl

219

OTBS

OOR2

R2OR2O NH

OOR1

k, m, n



different directions in comparison to the transition statesin a(2,3)- and a(2,6)-sialyltransferase-catalyzed reac-tions, Ito and colleagues planned to synthesize selectiveinhibitors of each sialyltransferase by controlling thelength of the linear alkyl chains linking the donor and ac-ceptor substrates. According to their strategy, the acceptorhydroxy group of galactoside, the phosphate group ofCMP, and the anomeric carbon of the sialic acid could beplaced in a line to mimic the transition state of sialyltrans-ferase-catalyzed reactions by controlling the chainlength.117

Thus, first, the tosylate group at the C-6¢ position of meth-yl N-acetyllactoside or the methyl lactoside derivatives223a and 223b was replaced with a mercapto group togive 224, which were converted into thioacetals 225 bychloromethylation followed by an SN2 reaction with thio-acetate. Treatment of 223a,b with four kinds of alkane-thiol, followed by conventional acetylation, gave 226–229. A coupling reaction of the thiols derived from 225–229 and the bromohydrin derivative of sialic acid 230 inthe presence of a base afforded the sulfides of a trisaccha-ride analogue, which were linked with CMP to yield 231–235 (Scheme 37). The inhibitory activities of 231–235 areshown in Table 2.

Scheme 36 Synthesis of 209d and 209e, N-acetylglucosami-nyltransferase bisubstrate inhibitors. Reagents and conditions: (a) n-Bu3P, THF, H2O; (b) 222a or 222b, Cs2CO3, DMF; (c) Et3N, MeOH,H2O; (d) 34, 1H-tetrazole; (e) HO(CH2)nSH; (f) NBS, Ph3P, CH2Cl2;(g) 47% aq HF, MeCN; (h) (i-Pr)2NP(OAll)2, 1H-tetrazole, CH2Cl2;(i) TBHP, then Me2S; (j) Pd(PPh3)4, Et3SiH, AcOH, toluene.

ON

NHO

OHHO

O

O

O

PO

O–

213

O

O

OH

HOHO AcHN

OOH

OHS(CH2)nS

OO

HOHO

OH

O OMe4

O

OOH

HOHO NH

OOPO3

=

d

O

O

OH

HOHO AcHN

OOH

OHS(CH2)nS

OO

HOHO

OH

O OMe4

O

OOH

HOHO NH

OO P

O

O–

209d: n = 2 (58%)

209e: n = 3 (61%)

a–c

216d: n = 2 (37%)216e: n = 3 (63%)

214 OH

OOAc

AcOAcO NH

O O P

O

OH

S

g–je, f

Brn

OOAc

AcOAcO NH

O

S

Brn

OTBS

n = 2 (96%)

n = 3 (95%)

222a: n = 2 (58%)

222b: n = 3 (44%)

Scheme 37 Synthesis of 231–235, sialyl transferase inhibitors.Reagents and conditions: (a) (CHO)n, HCl, CH2Cl2; (b) KSAc, 80 °C;(c) 230, TMS2NK, MeOH; (d) HS(CH2)nSH, TMS2NK, THF, HMPA;(e) Ac2O, pyridine; (f) H2NNH2, AcOH, DMF; (g) 230, TMS2NK,MeOH; (h) 63, 1H-tetrazole; (i) TBHP; (j) DBU; (k) NaOMe;(l) LiOH.

OO

OTsAcO

AcO OAcO

AcOR

OAc

OMe OO

SHAcO

AcO OAcO

AcOR

OAc

OMe

OO

SAcO

AcO OAcO

AcOR

OAc

OMe

SAc

OO

SAcO

AcO OAcO

AcOR

OAc

OMe

SAc

n

OO

SAcO

AcO OAcO

AcOR

OAc

OMe

SH

OO

SAcO

AcO OAcO

AcOR

OAc

OMe

S

O

OH

CO2Me

AcO OAcOAc

AcHNAcO

O

OHHO

OPOO

O–

N

N

O

OO

SHO

HO OHOHO

R

OH

OMe

S

O CO2H

HO OHOH

AcHNHO

NH2

223a: R = OAc 223b: R = NHAc 224: R = OAc or NHAc

225: R = OAc or NHAc

226: n = 2, 227: n = 3 228: n = 4, 229: n = 5

R = OAc or NHAc

231a,b: n = 1 232a,b: n = 2233a,b: n = 3234a,b: n = 4235a,b: n = 5

c 21–22%

64–82%d, e

89–99%f

21–68%

g

h–l

n

nn

OH

Br

O CO2Me

AcO OAcOAc

AcHNAcO

230

R = NHAc or OH

a, b 75–92%

Table 2 Inhibitory Activities of 231–235 against a(2,3)- and a(2,6)-Sialyltransferases

Inhibitor ST6N Ki (mM) ST3N Ki (mM)

donor acceptor donor acceptor

231a232a233a234a235a

R = NHAc 1027

2071111

1348

2173196

1045

2436

29

1322

2267

110

231b232b233b234b235b

R = OH 102855442

4 3

430271260114

90

195111124

4066

158324

887851

LacNAc 2380* 2630*

CMP-NeuAc 43* 74*

* Km value.



Following Ito’s report, Hashimoto and colleagues synthe-sized 236, a sialyltransferase bisubstrate inhibitor.67 Theroute started with the demethylation of dimethyl phospho-nate 120 to yield 237, which was condensed with triace-tylcytidine (118a) to afford 238. After desilylation of 238with tetrabutylammonium fluoride, the resulting primaryalcohol was treated with the galactosyl phosphoramidite239118 followed by oxidation to phosphonate to give 240.Finally, demethylation of the methyl phosphonate moietyof 240 and subsequent deacetylation with ammonium hy-droxide furnished 236 (Scheme 38).

The inhibitory activity of 236 against a(2,3)- and a(2,6)-sialyltransferases was weaker (IC50 = 1.3 mM and 1.4mM, respectively) than that of 119, an analogue of the do-nor substrate.67

5.4 Fucosyltransferase Inhibitors

Concerning fucosyltransferase inhibitors, many uniquemethods have been developed since fucosyltransferases(FucTase) catalyze the final step in the biosynthesis ofmany oligosaccharides, namely sialyl Lewis X and sialylLewis Y, and afford crucial biological functions to glyco-conjugates. Therefore, the design and synthesis of fuco-syltransferase inhibitors are a promising strategy tocontrol biological processes by suppressing the excess ex-pression of fucosylated glycoconjugates.

In fact, van Boom and colleagues synthesized 241, a fuc-osyltransferase bisubstrate inhibitor, relatively early on byreplacing the diphosphate moiety of GDP-Fuc with mal-onamide (Figure 7); however, no inhibitory activity wasreported.119

Meanwhile, Wong and co-workers found a synergistic ef-fect on the inhibitory activity against fucosyltransferase V

when a mixture of the five-membered iminofucose 242and GDP was used as the inhibitor.7,120

The effect was due to the structural similarity of the pro-tonated iminofucose 242 and the oxocarbenium cation,which would be formed in advance of nucleophilic attackof the acceptor substrate in the transition state(Scheme 39). The GDP-Fuc sugar nucleotide analogue105 (Scheme 20) was synthesized after this phenomenonhad been reported.59

Wong and colleagues tried to apply this concept to the de-velopment of bisubstrate inhibitors. Namely, a combina-tion of GDP and a trisaccharide analogue 243, composedof iminofucose 244 and an acceptor carbohydrate, wasconsidered to inhibit a fucosyltransferase synergistically,compared with either GDP or the trisaccharide alone as aninhibitor.121 To test the hypothesis, b-L-homofuconojiri-mycin (244), obtained by the treatment of 103 with acidphosphatase, was linked with the C-3 hydroxy group of N-acetyllactosamine to afford the trisaccharide analogue243, which contains an iminosugar, in several steps(Scheme 40).120 Compound 243 behaved as a potent fuc-osyltransferase inhibitor in the presence of GDP, the con-centration of which was almost the same as that underphysiological conditions (0.030–0.050 mM).121b

Figure 7 Structure of 241, a fucosyltransferase bisubstrate inhibitor

ON

OHHO

241

N

N

NH

O

NH2O

HOHOOH

O

OHO

OH

OC6H11

NHAc

O

HN

O

HN

Scheme 38 Synthesis of 236, a sialyltransferase inhibitor. Reagents and conditions: (a) PhSH, Et3N; (b) 118a, BOP, DIPEA, DMF; (c) TBAF,THF, AcOH; (d) 239, 1H-tetrazole, MeCN; (e) TBHP; (f) NH4OH, MeOH.

120 O

PAcO OAc

AcOAcHN

OAc

237

O OMe

O–

Et3NH+ ON

N

O

OAcAcO

O

NHAc

O

PAcO OAc

AcOAcHN

OAc

O

OMeOTBS

a

OTBS

ON

N

O

OAcAcO

O

NHAc

O

PAcO OAc

AcOAcHN

OAc

O

OMe

O

OAcO

AcO AcO

OMe

OP

O

O–

ON

N

O

OHHO

O

NH2

O

PHO OH

HOAcHN

OH

O

O–

O

OHO

HO HOOMe

OP

O

O– OAcO

AcOOMe

AcO

O PCN

N(i-Pr)2

86%

239

236

b

c–e

43%

a, f

53%

IC50 = 1.3 mM to α(2,3)STase

IC50 = 2.4 mM to α(2,6)STase

238

240



Recently, Hashimoto and colleagues synthesized the firstfucosyltransferase bisubstrate inhibitor.122 They speculat-ed that the three-dimensional structure of Lex,7 the productof the a(1,3)-fucosyltransferase-catalyzed reaction, wouldresemble the transition state where the C-6 position of fu-cose and the C-6 position of galactose are in close prox-imity. On this basis, L-galactose was chosen as an L-fucose analogue, the primary alcohol of which was usedto connect with that of D-galactose via an alkyl chain.Moreover, ethylene glycol was employed as a mimic ofthe N-acetyllactosamine acceptor with vicinal hydroxy

groups at the C-3 and C-4 positions of the N-acetylglu-cosamine moiety because the C-3 hydroxy group, thatneighbors the galactosylated C-4 hydroxy group, was con-sidered to be indispensable for binding to the enzyme.Herein, two bisubstrate analogues 248a and 248b withmethylene- or ethylene-tethered saccharides were synthe-sized (see Scheme 42 below).

First, as shown in Scheme 41, a protected form of the me-thylene-tethered saccharide 249a was synthesized fromphenyl 2,3,4-tri-O-benzoyl-1-thio-b-D-galactopyrano-side (250)123 and trimethylsilylethyl 2,3,4,6-tetra-O-acetyl-b-L-galactopyranoside (251).124 In the beginning,the D-galactoside 250 was treated with dimethylsulfoxide,acetic anhydride, and acetic acid125 to afford the 6-O-methylthiomethyl ether 252, while the fully protected L-galactoside 251 was converted into the partially protectedL-galactoside 253. Treatment of 252 with 253 in the pres-ence of methyl triflate gave the methylene acetal 254,which was further treated with ethylene glycol monoace-tate in the presence of N-iodosuccinimide and triflic acidto afford 249a. Next, a protected form of the ethylene-tethered disaccharide 249b was synthesized by a ratherconventional method from 255126 and 251. The SN2 cou-pling of the tosylate 256 derived from 255 and the primaryalcohol 257 prepared from 251 gave the disaccharide 258.

Scheme 39 Mechanism of the synergistic inhibition by GDP andfive-membered iminofucose 242 against fucosyltransferases

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

–ON

N

O

HOOH

Oδ+

Mn2+

O

OH

acceptor H -:B

O

HOOH

δ+

O

OH

acceptorHCO2

δ –

–O2C

ON

NH

O

NH2

OHHO

OPO

O

O–

P

O

–ON

N–O

Mn2+

Oacceptor H -:B

NH2+

OHHO OH

242

Scheme 40 Synthesis of iminofucose-containing trisaccharide 243.Reagents and conditions: (a) 1H-tetrazole, 246; (b) 247, TMSOTf; (c)O3 in CH2Cl2–MeOH, then Me2S, and then NaBH4; (d) Tf2O, DIPEA,CH2Cl2; (e) 245; (f) NaOMe, MeOH; (g) H2, Pd(OH)2/C, MeOH–AcOH; (h) NaH, BnBr; (i) H2, Pd(OH)2/C.

N

BnOOBn OBn

OBn

O

OH

AcO

AcOAcO

OAc

OAcO

AcOAcO

OAc

O PO

O

OAcO

AcOAcO

OAc

OO

OBn

NHAcO O O

AcO

AcOAcO

OAc

OO

OBn

NHAcO

TfO

O

OAcO

AcOAcO

OAc

OO

OBn

NHAcO O

PO

OEt2N

246

92%

a

OHOAllO

AcHN

OBn

O

247

38%

b

44%

c, d

e f, g

91%

N

HOOH OH

OH

OHO

HOHO

OH

OO

OH

NHAcO O

243

NH

BnOOBn

OBn

OBn

h, iN

H

HOOH OH

OH

244 245

Scheme 41 Synthesis of tethered disaccharides 249a,b. Reagentsand conditions: (a) DMSO, Ac2O, AcOH; (b) NaOMe, MeOH; (c)TrCl, pyridine, 50 °C, then BzCl; (d) TsOH, CHCl3, MeOH; (e)MeOTf, 3 Å MS, CH2Cl2, –65 to –70 °C; (f) NIS, TfOH, 4 Å MS,CH2Cl2, HOCH2CH2OAc, –30 °C; (g) Ac2O, pyridine; (h) NaH,Br(CH2)2OTHP, DMF; (i) TsOH, CHCl3–MeOH; (j) TsCl, pyridine;(k) NaH, BnBr; (l) NaH, DMSO; (m) NBS, acetone (aq); (n) H2,Pd(OH)2/C; (o) BzCl, pyridine; (p) H2NNH2 AcOH, DMF, 60 °C; (q)Cl3CCN, Cs2CO3, CH2Cl2; (r) TMSOTf, 3 Å MS, HO(CH2)2OAc,CH2Cl2, 0 °C.

OORBzO

BzO BzO

SPh

250: R = H252: R = CH2SMe (53%)

OORBnO

BnO BnO

SPh

255: R = H256: R = CH2CH2OTs (40%)

O

R2O

R1O OR1

OCH2CH2TMSOR1

251: R1 = R2 = Ac253: R1 = Bz, R2 = H (51%)

O

R2O

R1O OR1

OCH2CH2TMSOR1

251: R1 = R2 = Ac257: R1 = Bn, R2 = H (37%)

+

OOR2O

R2O OR2

R1

OOBzBzO

OCH2CH2TMSOBz

254: R1 = SPh, R2 = Bz249a: R1 = OCH2CH2OAc, R2 = Bz (66%)

O

+

OOR1O

R1O OR1

R2

OOR1R1O

OCH2CH2TMSOR1

258: R1 = Bn, R2 = SPh

259: R1 = Bn, R2 = OAc

O

249b: R1 = Bz, R2 = OCH2CH2OAc

260: R1 = Bz, R2 = OAc (62% from 258)

a b–d

e

44%

f, g

h–j b, c, k, h

l

58%

m, g

p–r

n, o



After the conversion of thioglycoside 258 into the acetylglycoside 259, the protecting benzyl groups were changedto benzoyl groups to give 260, which was further convert-ed into 249b using the Schmidt method (Scheme 41).

Finally, the tethered disaccharides 249a,b were treatedwith trifluoroacetic acid to cleave the trimethylsilylethylgroups on the L-galactosyl moieties and subsequentlyconverted into a-imidate 261, which was further trans-formed into b-phosphate 262 according to the report bySchmidt et al.127 Cleavage of the benzyl groups by hydro-genation on palladium-on-carbon and successive treat-ment with pyridine and ammonium hydroxide gave thenon-protected sugar phosphate 263. Further treatment of263 with GMP-imidazolidate (264) in the presence ofmagnesium chloride afforded the targets 248a,b.128 Com-pounds 248a,b showed inhibitory activity toward fucosyl-transferases V and VI with Ki values in the micromolarrange (Scheme 42).

6 High-Througput Screening: Discovering Structurally Simple Inhibitors

Recently, a random screening method referred to high-throughput screening (HTS), requiring reliable non-radio-metric assays, has been developed and some non-sugar-related compounds were found to have inhibitory activityagainst glycosyltransferases.

Walker and colleagues applied a protease-protection as-say to monitor the activity of O-linked N-acetylglu-cosamine transferase (OGT),129 which catalyzes N-acetylglucosamination of proteins on Ser/Thr residues.They had based this study on the fact that OGT had recent-ly been revealed to mediate a unique type of signal trans-duction through intracellular O-glycosylation.130 Theirmethod was simply based on the fact that glycosylation ofproteins generally increases the half-life by retarding ac-cess by proteases. Using this strategy, both the C- and N-terminals of a peptide are first labeled with a compatiblepair for fluorescence resonance energy transfer (FRET),and subjected to glycosylation, and then treated with aprotease. Subsequently, the FRET signal was measured todetermine the amount of glycosylated and non-glycosy-lated peptides; in other words, the amount of non-cleavedand cleaved peptides.

Here, in order to implement the assay system, it is neces-sary to find a peptide that satisfies the following criteria.First, it must be a good substrate of OGT and should havea site that is cleavable by a unique protease adjacent to theglycosylation site. Next, there must be a major differencein the rate of proteoysis of glycosylated and non-glycosy-lated peptides. In this study, the peptide STPVSRANMKwas chosen for the assay of N-acetylglucosamine-trans-ferring efficiency by OGT, where proteinase K was em-ployed to selectively cleave non-glycosylated peptidesbetween the valine and the serine residues(Figure 8).131,132

Scheme 42 Synthesis of 248a,b, fucosyltransferase bisubstrate in-hibitors. Reagents and conditions: (a) TFA, CH2Cl2; (b) Cl3CCN,Cs2CO3, CH2Cl2; (c) (BnO)2P(O)OH, CH2Cl2; (d) H2, Pd/C, Et3N,MeOH; (e) pyridine, NH4OH; (f) 264, MgCl2, DMF.

261: R1 = OC(NH)CCl3, R2 = H, R3 = Ac, R4 = Bz

263: R1 = R3 = R4 = H, R2 = OPO3=

OOR4O

R4O OR4

O(CH2)2OR3

O

OR4R4O

R2OR4

O

n

R1

249a,b

249a: (76%) 249b: (73%)

a: n = 0 (81%) , b: n = 1 (59%)

ON

OHHO

OP

O

O–

N

N

NH

O

NH2OP

O

O–O

OOHO

HO OH

O(CH2)2OH

O

OHHOOH

O

n

248a: n = 0 (47%)

c262: R1 = H, R3 = R4 =Ac, R2 = OP(O)(OBn)2

d, e

ON

OHHO

OP

O

O–

N

N

NH

O

NH2NN

264

248b: n = 1 (21%)

IC50 = 0.26 mM Ki = 41 μM against FucTase VIC50 = 0.11 mM against FucTase VI

IC50 = 0.27 mM Ki = 43 μM against FucTase VIC50 = 0.19 mM against FucTase VI

a, b

f

Figure 8 Principles of HTS for discovering OGT inhibitors. Whenthe intact peptide is activated by excitation light (485 nm), the excita-tion energy of the N-terminal diethylaminocoumarin is transferred tothe C-terminal fluoroscein, and the resulting emission from fluore-scein (535 nm) is observed. In the cleaved peptided, the excitationenergy of the N-terminal diethylaminocoumarin is negligibly trans-ferred to fluoroscein. Thus, in priciple, the ratio of non-cleaved (gly-cosylated) peptide to cleaved (non-glycosylated) peptide can beestimated by measuring the intensity of the light at 535 nm and 485nm emitted from a sample solution.

O-sugar

glycosyltransferaseprotease

OH485 nm

O-sugar

535 nm

fluoresceindiethylaminocoumarin

S T P V S R

S T P V S R

S T P V S R

OH

S T P V S R N M KA

N M KA

N M KA

N M KA



In this assay system, a 100-fold higher concentration ofthe protease was required for the cleavage of the glycosy-lated peptide than for that of the non-glycosylated peptide.Thus, using an appropriate concentration of proteinase K,the degree of the glycosylation could be evaluated underthe condition where a glycosyltransferase inhibitor candi-date is present with the substrate peptide.

Taking advantage of this assay system, 124226 com-pounds were screened in duplicate over two days. Ofthese, 84 compounds inhibited OGT activity by 30% ormore and 38 of these compounds were confirmed. Finally,nine of the confirmed hits showed IC50 values of between0.9 and 20 mM. A subset of three validated OGT inhibitors265–267 were discovered by this method (Figure 9).

7 Antisense Inhibitors

A few papers have reported the inhibition of glycosyl-transferase expression by an antisense DNA. The humangenome is comprised of three billion base pairs. AntisenseDNA with 16 bases can bind only one site in the genome,in theory, and suppress the expression of DNA with ex-tremely high specificity. In 1997, Kemmner and col-leagues prepared an antisense DNA complementary to thesequence adjacent to the initial codon of a(2,6)-sialyl-transferase in cDNA. The antisense DNA (CAU AAUGAA GAU GUG UUC; 18 bases) suppressed the expres-sion of a(2,6)-sialyltransferase in human colon cancerHT29 cells at 2 mM.133 In 1999, Yu and colleagues pre-pared a vector which incorporated an antisense sequencetoward the 5¢-terminal fragmental code of CD3 syn-thetase, and transfected to F-11 neuroblast melanomacells. The procedure suppressed the production of GD3.134

8 Questions and Future Directions

Glycosyltransferases can be divided into two groups, re-tention enzymes that catalyze the formation of glycosidicbonds while maintaining the configuration of the anomercarbon in the corresponding donor substrates, and inver-sion enzymes that form glycosidic bonds having a differ-ent configuration from that of the sugar nucleotide. Mostof the inhibitors reviewed in this article are inhibitors ofinversion enzymes, except for 186, 187,103 and 203a.52

For instance, the inhibitor 1 acting on b(1,4)-galactosyl-transferase (an inversion enzyme) (Ki = 7 mM) showed

only weak inhibitory activity toward a(1,3)-galactosyl-transferase (a retention enzyme) under the same condi-tions (10 mM Mn2+).17

Recently, an imino-rhamnose derivative, which does nothave an appropriate nucleotide (TDP) but has b-config-ured naphthyl group as an aglycon, was reported to inhibitrhamnosyltransferase from Mycobacterium tuberculo-sis.135 In addition, [(2S,3R,4R,5S)-3,4,5-trihydroxy-2-(phenylsulfanyl)tetrahydrofuran-2-yl]methyl sulfate, de-signed on the basis of density functional theory (DFT) cal-culations and a docking study, was suggested to inhibitb(1,4)-galactosyltransferase in spite of having no nucleo-tide moiety.136 These results seem to be in conflict withthe fact that glycosyltransferases employ sugar nucleo-tides as the donor substrate and bind most strongly withthe nucleotide moiety.

More details of inhibition mechanisms against glycosyl-transferases will be revealed by further studies, and morepotent, selective, and structurally simple inhibitors arelikely to be developed.

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Figure 9 Structures of the OGT inhibitors 265–267 found by HTS

NH2

O

F

O

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CO2HO

S

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N

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