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
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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–,
3182 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
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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
3184 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
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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
3186 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
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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
3188 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
+
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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)
3190 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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%)
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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
3192 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
Ki = 780 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
REVIEW Glycosyltransferase Inhibitors 3193
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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%
3194 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
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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
3196 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
Ki = 84 μM against α(2,6)STase
a–c
53%
OH
79%
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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
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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
REVIEW Glycosyltransferase Inhibitors 3199
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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
3200 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
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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
3202 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
REVIEW Glycosyltransferase Inhibitors 3203
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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.
3204 T. Kajimoto, M. Node REVIEW
Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
REVIEW Glycosyltransferase Inhibitors 3205
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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
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Synthesis 2009, No. 19, 3179–3210 © Thieme Stuttgart · New York
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
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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
S
CO2HO
S
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N
CO2H
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3208 T. Kajimoto, M. Node REVIEW
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