Synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of O-antigen repeating units

DOI: 10.1016/j.carres.2009.12.022

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Carbohydrate Research p. 586 - 597 (2010)

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Riley, John G.

Xu, Changchang

Brockhausen, Inka

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Article abstract of DOI:10.1016/j.carres.2009.12.022

O-antigens of Gram negative bacteria are polysaccharides covalently attached to lipopolysaccharides (LPS) that have roles as virulence factors. Due to the lack of defined substrates for in vitro assays only a few of the enzymes involved in the biosynthesis of O-antigens have been studied. Many O-antigens have GlcNAc at the reducing end of the oligosaccharide chain linked to pyrophosphate-lipid. We therefore designed and synthesized a series of GlcNAc-pyrophosphate-lipid analogs of the natural GlcNAc-pyrophosphate-undecaprenol acceptor substrate for studies of the acceptor specificities of O-antigen biosynthetic enzymes. We synthesized analogs with modifications of the pyrophosphate bond as well as the lipid chain. These compounds will be useful for the specificity studies of many bacterial glycosyltransferases. Knowledge of the substrate specificities is the basis for the development of specific glycosyltransferase inhibitors that could block O-antigen biosynthesis.

Full text of DOI:10.1016/j.carres.2009.12.022

Carbohydrate Research 345 (2010) 586–597

Contents lists available at ScienceDirect

Carbohydrate Research

journal homepage: w ww.elsevier.com/locate/carres

Synthesis of acceptor substrate analogs for the study of glycosyltransferases

involved in the second step of the biosynthesis of O-antigen repeating units

John G. Riley, Changchang Xu, Inka Brockhausen

Department of Medicine, Division of Rheumatology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6

a r t i c l e i n f o

a b s t r a c t

Article history:

O-antigens of Gram negative bacteria are polysaccharides covalently attached to lipopolysaccharides

(LPS) that have roles as virulence factors. Due to the lack of deﬁned substrates for in vitro assays only

a few of the enzymes involved in the biosynthesis of O-antigens have been studied. Many O-antigens

have GlcNAc at the reducing end of the oligosaccharide chain linked to pyrophosphate-lipid. We there-

fore designed and synthesized a series of GlcNAc-pyrophosphate-lipid analogs of the natural GlcNAc-

pyrophosphate-undecaprenol acceptor substrate for studies of the acceptor speciﬁcities of O-antigen bio-

synthetic enzymes. We synthesized analogs with modiﬁcations of the pyrophosphate bond as well as the

lipid chain. These compounds will be useful for the speciﬁcity studies of many bacterial glycosyltransfer-

ases. Knowledge of the substrate speciﬁcities is the basis for the development of speciﬁc glycosyltrans-

ferase inhibitors that could block O-antigen biosynthesis.

Received 25 September 2009

Received in revised form 21 December 2009

Accepted 22 December 2009

Available online 29 December 2009

Keywords:

Chemical synthesis

Undecaprenol-pyrophosphate-sugar

Glycosyltransferases

Substrate speciﬁcity

Acceptor substrate analogs

O-antigen repeating unit

1. Introduction

transferases that complete the synthesis of the repeating unit. In

the biosynthetic pathway of heteropolymeric O-antigens, depen-

Lipopolysaccharides (LPS) of Gram negative bacteria are

important components of the outer bacterial membrane where

they function to support the structural integrity of the organism

and protect the cell from external chemical attack.^1,2LPS are anti-

genic, act as pro-inﬂammatory agents, and are considered to be

important virulence factors. The outermost portion of the LPS is

the O-antigenic polysaccharide (O-antigen) which is of crucial

importance, since it determines the pathogenicity by controlling

host–bacterial interactions.³The O-antigen is thought to be assem-

bled as repeating units of shorter oligosaccharides linked to pyro-

phosphate-lipids.^1,2The natural acceptor substrate for the ﬁrst step

of repeating unit biosynthesis is undecaprenol-phosphate which is

enzymatically converted to sugar-pyrophosphate-undecaprenol by

the reversible transfer of sugar-phosphate from nucleotide sugar.

The biosynthetic enzymes required for the transfer of sugars to

form the repeating unit are thought to reside on the cytosolic side

of the inner bacterial membrane. Many of the O-antigens contain

GlcNAc as the ﬁrst sugar at the reducing end of the repeating unit

dent on polymerase Wzy, the repeating unit is ﬂipped to the

periplasm, polymerized, and then transferred from undecaprenol-

phosphate to the outer core oligosaccharide of lipid A to form

LPS. The completed LPS is then translocated to the outer

membrane.

A number of proteins are involved in the regulation of this pro-

cess, and several different mechanisms of assembly have been pro-

posed.¹Many of the genes encoding biosynthetic enzymes have

been identiﬁed in O-antigen gene clusters. The location of the

genes and the structures of O-antigens suggest the pathways that

are involved in the assembly of O-antigen repeating units. Genes

that potentially encode glycosyltransferases have been assigned

mainly by comparison of the sequences and overall folds of the

gene products to those of other glycosyltransferases. However,

very few of the glycosyltransferases have been assayed and bio-

chemically characterized.^4–9

We have recently synthesized GlcNAca-PO₃-PO₃-(CH₂)₁₁-

O-phenyl (GlcNAc-PP-PhU)⁴as a natural substrate analog and have

shown that the GlcNAc derivative was an excellent acceptor sub-

strate for enzymes from Escherichia coli, Salmonella, and Shigella

that catalyze the second reaction of the repeating unit assembly

by transferring a sugar residue to GlcNAc. This synthetic substrate

allowed the characterization of these enzymes. In another syn-

which has been transferred as GlcNAca-phosphate from UDP-Glc-

NAc. Subsequently, glycosyltransferases add a second sugar resi-

due to the nonreducing end of GlcNAc, followed by other

thetic method, UDP-GlcNAc which has GlcNAc in the

tion can be conveniently used as the starting material for the

synthesis of GlcNAc

-pyrophosphate substrate analogs.¹⁰

a-conﬁgura-

* Corresponding author. Tel.: +1 613 533 2927; fax: +1 613 549 2529.

E-mail address: brockhau@queensu.ca (I. Brockhausen).

doi:10.1016/j.carres.2009.12.022

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

587

Table 1

O-antigen structures of different serotypes of Gram negative bacteria, terminating in GlcNAc at the reducing end of the O-antigen repeating unit (ECODAB), and the putative

enzymes expected to add a second sugar to GlcNAc-PP-R in the synthesis of the repeating unit

Serotype

Linkage

Putative enzyme

Short name

Starting NTP

Donor

Escherichia coli

b3-Gal-T

b4-Gal-T

WbbD

WbgM

UDP-Glc

UDP-Gal

-Galb1-3

-Galb1-4

O55

O64; O114; O153

O83; O136

O1; O1B; O3; O10; O18A1; O18ac;

O18B; O18B1; O18A, O18A1;

O69; O113; O126

a3-Gal-T

-Gal

a1-3

O111

3-Gal-T

WbbP

UDP-Glc

UDP-Gal

-Gal

1-3

O116

O65

3-D-GalA-T

UDP-GlcA

UDP-GalA

D-GalA

a1-3

b3-

D-GalA-T

D-GalAb1-3

O23A; O121

O138

3-GalNAc-T

UDP-GlcNAc

UDP-GalNAc

-GalNAc 1-3

a3-GalNAcA-T

UDP-GalNAcA

D-GalNAcA

1-3

O56

b3-Glc-T

WfaP

WfgD

UDP-Glc

-Glcb1-3

O152

O173

O148

a3-Glc-T

WbbG

-Glc

a1-3

O126

O98

3-GlcNAc-T

UDP-GlcNAc

-GlcNAc

1-3

a3-

-QuiNAc-T

WbwW

UDP-

D-GlcNAc

UDP-L-QuiNAc

-QuiNAc

O6; O44; O66; O77

O78; O88

O58

b3-Man-T

GDP-Man

-Manb1-3

-Man 1-3

-Man2Acb1-3

-Man6Ac 1-3

-Fuc 1-3

-FucNAc

3-Man-T

GDP-Man

3-Man2Ac-T

3-Man6Ac-T

3-Fuc-T

UDP-GlcNAc

GDP-Fuc

UDP-Man2Ac

UDP-Man6Ac

O141

O159

GDP-

-Fuc

O4; O25; O26

3-FucNAc-T

UDP-GlcNAc

UDP-

L-FucNAc

a1-3

O105

b3-

b4-

-Rha-T

dTDP-

-Rha

dTDP-

-Rha

-Rhab1-3

-Rhab1-4

O1A; O2; O149

O16; O31; O75;

O119; O139

O16

a3-

-Rha-T

-Rhaa1-3

a3-

-Rha2Ac-T

dTDP-

-Rha

dTDP-

-Rha

-Rha2Aca1-3

Salmonella

O35 (23)

b4-

-Gal-T

-GalNAc-T

-FucNAm-T

-Gal-T

UDP-Glc

UDP-Gal

-Galb1-4

-GalNAc

O43

a3-

WfbG

UDP-GlcNAc

UDP-Glc

UDP-GalNAc

1-3

O145 (105)

O111 (88)

UDP-L-FucNAc

-FucNAm

1-3

UDP-Gal

D-Gala1-3

Shigella

O7 (83); O114 (85); O40

b3-

b4-

b3-

-Gal-T

-Galf-T

UDP-Glc

UDP-Gal

-Galb1-3

-Galb1-4

-Galfb1-3

-GalNAcA

-Glcb1-3

WbdH

WfeD

UDP-Glc

UDP-Gal

O136 (B14)

O167 (32)

O121 (78)

O152 (D12)

O148

UDP-Glc

UDP-Gal

UDP-Glc

UDP-Galf

UDP-GalNAcA

UDP-Glc

b3-

-GalNAcA-T

-Glc-T

-GlcA-T

-Man-T

-Man2Ac-T

-Fuc-T

-FucNAc-T

UDP-GlcNAc

UDP-Glc

1-3

WbbG

UDP-Glc

-Glc

-GlcAb1-3

-GlcA 1-3

-Manb1-3

-Man2Ac

-Fuc 1-3

-FucNAcb1-3

-FucNAc 1-3

a1-3

O143 (43)

B5; B9

UDP-Glc

UDP-GlcA

UDP-Man

UDP-Man2Ac

UDP-GlcA

UDP-Man

UDP-GlcNAc

GDP-Fuc

B16

O58 (84)

O159 (15); O168(D4)

O29 (D11)

O172 (4)

B11; O150(D13)

O149 (B1); B4

D10

a3-

1-3

a3-

GDP-

UDP-

-Fuc

b3-

UDP-GlcNAc

-FucNAc

a3-

-FucNAc-T

b3-

b4-

L-Rha-T

dTDP-

-Rha

dTDP-

-Rha

-Rhab1-3

-Rhab1-4

L-Rha-T

a4-

-Rha-T

-Rha

a1-4

B13

a3-

-QuiNAc-T

UDP-GlcNAc

UDP-

L-QuiNAc

-QuiNAc

a1-3

Yersinia

O98 (86)

L-QuiNAc

a1-3

a3-

-QuiNAc-T

UDP-D-GlcNAc

UDP-

L-QuiNAc

(continued on next page)

588

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

Table 1 (continued)

Serotype

Linkage

Putative enzyme

Short name

WbwW

Starting NTP

UDP- -GlcNAc

dTDP- -Rha

Donor

UDP- -QuiNAc

dTDP- -Rha

O11, O23, O24

O139 (46)

a3-

-QuiNAc-T

-Rha-T

-QuiNAc

a1-3

a3-

-Rha 1-3

Vibrio cholerae

O136 (14)

b4-

D-Gal-T

UDP-Gal

D-Gala1-3

Linkage between ﬁrst sugar (GlcNAc-alpha or beta) and second sugar; starting NTP, sugar donor substrate precursor; –T, -transferase; donor, expected donor substrate for the

transferase; -FucNAm, 2-acetamidoylamino-2,6-dideoxy- -galactose; -FucNAc, 2-acetamidino-2,6-dideoxy- -galactose; QuiNAc, 4-acetylamido-4,6-dideoxy-glucose; GalA,

galacturonic acid; GalNAcA, N-acetylgalacturonic acid.

Table 1 shows examples of O-antigens containing a GlcNAc res-

idue as the ﬁrst sugar residue at the reducing end of the repeating

unit but having different linkages between the second sugar and

GlcNAc. Data banks of glycosyltransferases (CAZy) suggest many

putative glycosyltransferases that are expected to transfer a sugar

residue to GlcNAc, with examples from E. coli, Salmonella, Yersinia,

Vibrio cholerae, and Shigella, as shown in Table 1. A few of the genes

have been assigned to speciﬁc glycosyltransferases^4–6but most of

the enzyme activities have not been demonstrated or character-

ized. With the synthesis of substrate analogs reported here, it is

Scheme 1a. Synthesis of pyridinium 6-phenoxy-1-hexyloxydiphospho(1)-2-acetamido-2-deoxy-a-D-glucopyranose (7). Reagents and conditions: (a) PhOH, K₂CO₃/DMF,

60 °C, 95%; (b) HOP(O)(OBn)₂, PPh₃, DIAD/CH₂Cl₂, 53%; (c) H₂, Pd/C, MeOH, 95%; (d) (i) DIPA, 1,1⁰-carbonyldiimidazole, THF; (ii) MeOH; (iii) DIPA, 5, THF, 62%; (e) (i) NaOMe/

MeOH; (ii) pyridinium resin, 95%.

Scheme 1b. Synthesis of pyridinium 16-phenoxy-1-hexadecyloxydiphospho(1)-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-a-D-glucopyranose (15). Reagents and conditions:

(a) (i) Cl₂(CO)₂, DMF/CH₂Cl₂; (ii) MeOH, 100%; (b) PhOH, K₂CO₃/DMF, 60 °C, 81%; (c) LAH/ether, 95%; (d) HOP(O)(OBn) ₂, PPh₃, DIAD/CH₂Cl₂, 53%; (e) H₂, Pd/C, MeOH, 95%; (f)

(i) DIPA, 1,1⁰-carbonyldiimidazole, THF; (ii) MeOH; (iii) DIPA, 5, THF, 62%; (g) (i) NaOMe/MeOH; (ii) pyridinium resin, 95%.

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

589

now possible to develop speciﬁc assays for a multitude of biosyn-

thetic enzymes, allowing enzyme puriﬁcation and studies of the

enzyme mechanisms, speciﬁcities, properties, and regulation. The

synthetic methods help to design additional substrate analogs for

speciﬁcity studies, and allow the design of acceptor analog inhibi-

tors that block the second step in the synthesis of the repeating

unit and thus prevent the synthesis of a speciﬁc O-antigen.

phosphate is then debenzylated by hydrogenolysis and the result-

ing dihydrogen phosphate (4 and 13, respectively) is activated by

forming the phosphorimidazolidate which, by condensation with

the acetylated sugar moiety 5, gives the corresponding O-acety-

lated glycosides 6 and 14, respectively.^17,18At the end, the acetyl

groups are removed using NaOCH₃in MeOH; ion exchange gave

the ﬁnal product as a pyridinium salt (7 and 15, respectively).¹⁹

Scheme 2 shows the synthesis of the acetylamino gluco-

pyranoside derivatives 20

a and b. The condensation of the trichlo-

2. Results and discussion

roacetimidate 16

and b, with the corresponding tail fragment⁴

11-phenoxyundecanol 17, was based on the methodology de-

scribed.²⁰The subsequent deprotection of the sugar moiety with

NaOCH₃in MeOH gave the target compounds. Further puriﬁcation

of compounds can be done using a C18 Sep-Pak cartridge.

2.1. Synthesis of substrate analogs

Methods for the synthesis of GlcNAc-PP-lipid analogs were

based on the synthesis of GlcNAc-PP-PhU⁴by linking GlcNAc-phos-

phate- or GlcNAc-derivative to lipid or lipid-phosphate. Phos-

phates were named according to IUPAC-IUB rules.¹¹

The synthesis of the malonate derivative 24a is shown in

Scheme 3.

The tail fragment 21 was obtained by reacting Meldrum’s acid

(2,2-dimethyl-1,3-dioxane-4,6-dione) with the corresponding

alcohol 17 in reﬂuxing toluene.²¹The condensation with the sugar

The synthesis of phospholipid glycosides 7 and 15 is shown in

Scheme 1a and b, respectively.

The preparation of the tail fragment requires initial treatment of

bromo alcohol 1 or bromo ester derivative 9^12–15giving the corre-

sponding phenoxy alcohols 2 and 10, respectively. The ethers are

subsequently treated with dibenzyl phosphate and triphenylphos-

phine, followed by the addition of diisopropylazodicarboxylate

(DIAD), giving the dibenzyl phosphate derivatives 3 and 12.¹⁶The

moiety 22a (3,4,6-tri-O-benzyl-2-acetamido-2-deoxy-a/b-D-gluco-

pyranoside²²) was based on a Mitsunobu-type reaction¹⁶in the

presence of dehydrating agent N,N⁰-dicyclohexylcarbodiimide

(DCC) in tetrahydrofuran (THF). Debenzylation by hydrogenolysis

gave the desired compound 24a.

Scheme 2. Synthesis of 11-phenoxyundecyl 2-acetylamino-2-deoxy-

a,b-

D-glucopyranoside (20a and 20b). Reagents and conditions: (a) 17, TMSOTf/CH₂Cl₂, 53%; (b) (i) H₂,

Pd/C, MeOH; (ii) Ac₂O/pyridine, = 32%, b = 41%; (c) NaOMe/MeOH,

a = 94%, b = 100%.

Scheme 3. Synthesis of 2-acetamido-2-deoxy-

a-D-glucopyranosyl 11-phenoxyundecyl malonate (24a). Reagents and conditions: (a) Meldrum’s acid/toluene, reﬂux, 98%; (b)

22a, DCC, DMAP/THF/DMF, = 62%, b = 9%; (c) H₂, Pd/C, MeOH, 92%.

590

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

Scheme 4. Synthesis of [hydroxy (11-phenoxyundecyloxy)phosphinyl]acetyl 2-acetamido-2-deoxy-

a-D-glucopyranoside (29). Reagents and conditions: (a) 17, PPh₃, DIAD/

CH₂Cl₂, 83%; (b) NaOH/H₂O/EtOH, 100%; (c) 22a, DCC, DMAP/THF/DMF, 39%; (d) H₂, Pd/C, MeOH, 85%.

Scheme 5. Synthesis of 2-acetamido-2-deoxy-1-O-(11-phenoxyundecyloxy)phosphoryl-

a-D-glucopyranose (32). Reagents and conditions: (a) (2-cyanoethoxy)bis(diisopro-

pylamino)phosphine ([iPr₂N]₂-POCH₂CH₂CN), tetrazole/DIPA (iPr₂NH), CH₂Cl₂; (b) 22b tetrazole, CH₂Cl₂at 0 °C?rt, then mCPBA, CH₂Cl₂?0 °C; (c) NH₄OH, MeOH.

The synthesis of 29 is shown in Scheme 4. The coupling of the

organophosphorus tail 27 and the sugar head group 22a was done

based on a Mitsunobu-type condensation reaction followed by

deprotection by hydrogenolysis.

were active using GlcNAc-PP-lipids with the aliphatic chain having

6–16 carbons, in addition to an O-phenyl group (compounds 7, 15,

33). Compound 32, having only one phosphate group linked to Glc-

NAc, was generally poor as a substrate, while compound 29, having

a phosphate group distant from GlcNAc, was not active. All other

compounds, including GlcNAc-phenylundecyl 20, compound 24,

and a series of GlcNAc-benzyl, -naphthyl and -quinolinyl deriva-

tives²³that lack phosphate were inactive as substrates for these

bacterial glycosyltransferases (Table 2). Therefore, as a general

rule, the enzymes (listed in Table 1) that add the second sugar of

the repeating unit require GlcNAc linked to pyrophosphate for full

activity. In contrast, the lipid moiety of the acceptors appears to

The synthesis of the monophosphate GlcNAca-P-PhU (com-

pound 32) is shown in Scheme 5. A diisopropylaminophosphine

30 was synthesized ﬁrst. The coupling with the sugar head group

22b yielded 31 which was deprotected to yield the sugar-mono-

phosphate-lipid 32.

2.2. Use of synthetic derivatives as enzyme substrates

The compounds shown in Table 2 were synthesized and sepa-

rated well by reverse-phase HPLC. After the enzymatic addition

of a second sugar, the enzyme product was eluted earlier than

the substrate and could be well separated. The synthesized com-

pounds were tested as acceptor substrates with bacterial b3-Gal-

transferase WbbD from E. coli serotype O7,^4,7,8,10b3-Glc-transfer-

ases WfaP from E. coli O56, WfgD from E. coli O152⁵, and b4-Gal-

transferase WfeD from Shigella B14.⁶Thus far, all these enzymes

that catalyze the second reaction of the repeating unit assembly

play a minor role. Similar results were reported for an a1,3-Gal-

NAc-transferase from E. coli O86 that adds GalNAc as the second

sugar to GalNAc-PP-PhU.⁹

Based on these ﬁndings, we propose that assays are now

available for the characterization of a large number of glycosyl-

transferases including those listed in Table 1 which are expected

to act on GlcNAc-PP-lipid as acceptor substrates. The assays uti-

lizing GlcNAc-PP-lipid-based substrates can serve to synthesize

partial O-antigen repeating units which could be substrates for

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

591

Table 2

List of GlcNAc analogs synthesized and tested as a substrate for the second step of O-antigen synthesis

Compound

Structure

Activity (%)

WbbD

WfaP

WfgD

183

WfeD

7 GlcNAc

-PP-(CH₂)₆-O-Ph

O^-

15 GlcNAc

a-PP-(CH₂)₁₆-O-Ph

156

174

O^-

GlcNAc

-(CH₂)₁₁-O-Ph

20b GlcNAcb-(CH₂)₁₁-O-Ph

GlcNAc

-malonate-(CH₂)₁₁-O-Ph

29 GlcNAc

32 GlcNAc

-hydroxy-phosphinyl-(CH₂)₁₁-O-Ph

O^-

-P-(CH₂)₁₁-O-Ph

33 GlcNAc

a-PP-(CH₂)₁₁-O-Ph

100

O^-

(continued on next page)

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J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

Table 2 (continued)

Compound

Structure

Activity (%)

WbbD

WfaP

WfgD

150

WfeD

34 GlcNAc

-PP-(CH₂)₉-CH3

O^-

( )₈

O^-

35 GlcNAcb-Bn

36 GlcNAca-Bn

37 GlcNAcb-O-2-naphthyl

38 GlcNBub-O-2-naphthyl

39 GlcNAcb-3-isoquinolinyl

40 GlcNBub-S-2-naphthyl

The enzymes tested were WbbD from E. coli O7, WfaP from E. coli O56, WfgD from E. coli O152, and WfeD from Shigella B14.^5–8,10The activity is shown relative to that using

the active substrate GlcNAc-PP-(CH₂)₁₁-O-Ph, compound 33, at 0.2 mM (or *0.5 mM) concentration in the assay. nd, not determined.

the third and subsequent steps of the biosynthetic pathways of

O-antigen repeating unit synthesis. However, the subsequently

acting glycosyltransferases (beyond the second step) may not re-

quire the pyrophosphate bond for activity and may instead act

on simpler oligosaccharide substrates.

3. Experimental

3.1. Materials

The syntheses of GlcNAca-pyrophosphate bound to a pheno-

A future goal is to synthesize pyrophosphate-sugar analogs con-

taining a short lipid chain to produce water-soluble compounds.

Although this may not necessarily lead to high enzyme activity,

it can facilitate enzyme assays, as well as further enzyme–

substrate-binding complex study by crystallization. The series of

compounds described here could also be synthesized based on

GalNAc-PP-PhU for studies of multiple enzymes that are expected

to utilize GalNAc-pyrophosphate-lipids as acceptors.⁹

xyhexyl moiety (7) and a phenoxyhexadecyl moiety (15), the phen-

oxyundecyl glycosides 20 and 20b, the phenoxyundecyl malonate

glycoside 24 , GlcNAc -hydroxyphosphinyl bound to phenoxyun-

decyloxy moiety (29), and phenoxyundecylphosphoryl glycoside

32 are described below. Some of the compounds used in the syn-

thesis were obtained as described in the corresponding references:

compound 5, 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-

a-D-gluco-

pyranose dihydrogen phosphate;⁴compounds 16

,b, 3,4,6-tri-

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

593

O-acetyl-2-azido-2-deoxy-

date,²⁴compound 17, 11-phenoxyundecanol;⁴compound 22a,

3,4,6-tri-O-benzyl-2-acetamido-2-deoxy-

-glucopyranoside,²²

22b, 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-

-glucopyranoside,⁴

compound 25, (benzyloxyhydroxyphosphoryl)acetic acid benzyl

ester.²⁵Compound 33, phenoxyundecylpyrophosphate-

-GlcNAc,

,b-

-glucopyranosyl trichloroacetimi-

3.4. Synthesis of substrate analogs

3.4.1. Dibenzyl 6-phenoxyhexyl phosphate (3)

a-D

Compound 2 (6-phenoxy-1-hexanol) was synthesized from 6-

bromo-1-hexanol 1 with phenol in dimethylformamide (DMF)

and potassium carbonate according to Suzuki et al.^12,13and Chang

et al.¹⁴A solution of compound 2 (385 mg, 2.0 mmol), dibenzyl

phosphate (828 mg, 3.0 mmol), and triphenylphosphine (PPh₃)

(935 mg, 3.6 mmol) in CH₂Cl₂(5 mL) was stirred under N₂atmo-

sphere for 30 min at rt. The solution was then cooled to 0 °C and

treated with DIAD (0.690 mL, 3.6 mmol), stirred for 30 min, and

then warmed up to rt. After 2 h the mixture was quenched with

1 mL MeOH, stirred for 5 min, and concentrated. The resulting res-

idue was puriﬁed by reverse-phase preparative chromatography

(C18 stationary phase) using acetonitrile/water (70:30) as the mo-

bile phase to give the title compound as a colorless transparent so-

lid (480 mg, 53%); ¹H NMR (CDCl₃) d 1.38–1.47 (m, 4H), 1.66 (m,

2H), 1.76 (m, 2H), 3.94 (t, J = 6.4 Hz, 2H, PhOCH₂), 4.03 (q,

J = 6.6 Hz, 2H, POCH₂), 5.04 (m, 4H, 2 ꢀ OCH₂Ph), 6.94 (m, 3H),

7.30–7.45 (m, 12H); ¹³C NMR (CDCl₃) d 25.2, 25.6, 29.2, 30.1,

was synthesized as described.⁴The benzyl-, naphthyl-, and quin-

olinyl derivatives were synthesized as described.²³All other re-

agents were purchased from Sigma Chemical Co., St. Louis, MO,

unless otherwise indicated. In most cases, the GlcNAc sugar head

group and the fragment tail were assembled separately and then

coupled using well-established methodologies. For use in enzyme

assays, compounds were subjected to puriﬁcation by C18 Sep-Pak

columns. After puriﬁcation, compounds were analyzed by HPLC for

purity and to determine the best conditions for enzyme product

separation.

3.2. General methods

The ¹H, ¹³C, and ³¹P NMR spectra were recorded on Bruker

Avance 100-, 200-, 300-, 400-, and 500-MHz spectrometers. Chem-

ical shifts (d) are reported in parts per million (ppm), and signals

are described as s (singlet), d (doublet), dd (doublet of doublets),

t (triplet), q (quartet), quin (quintet), and m (multiplet). Proton

chemical shifts are given relative to those of internal standards

CHCl₃, CH₃OD, and dimethylsulfoxide (DMSO): d = 7.26, 3.30 or

2.25 ppm, respectively. Carbon chemical shifts are given relative

to those of CDCl₃, CD₃OD, or DMSO: d = 77, 49 or 39.5 ppm, respec-

tively. Phosphorus chemical shifts are given relative to that of 85%

phosphoric acid: d = 0 ppm. High-resolution MALDI and electro-

spray mass spectra were recorded on an Applied Biosystems/

MDS Sciex QSTAR XL spectrometer with an Agilent HP1100 Cap-

LC system. Samples were run in 50% aqueous MeOH at a ﬂow rate

67.2 (d, J_CP= 5.2 Hz), 67.6 (PhOCH₂), 67.8 (d, J_CP= 6.1 Hz), 69.2

(d, J_CP= 5.5 Hz), 114.5, 120.6, 127.1, 127.5, 127.9, 127.9, 128.0,

128.4, 128.5, 128.6, 129.4, 129.8, 134.5, 135.2, 159.1 (Ar-C); ³¹P

NMR d ꢁ0.60; electrospray ionization mass spectrometry (ESMS)

m/z 455 [M+H]⁺; high resolution MS (HRMS) [M+H]⁺, Found:

455.1985. C₂₆H₃₂O₅P requires m/z, 455.1987.

3.4.2. 6-Phenoxyhexyl dihydrogen phosphate (4)

A suspension of compound 3 (154 mg, 0.3 mmol) and 10% Pd/C

(0.154 g) in MeOH (2.5 mL) was stirred under H₂atmosphere for

16 h. The Pd/C was removed by ﬁltration through a Celite plug,

washed with MeOH, and the ﬁltrate was concentrated under re-

duced pressure to give the title compound as a colorless solid

(88 mg, 95%); ¹H NMR (CD₃OD) d 1.52 (m, 4H), 1.72 (m, 2H), 1.79

(m, 2H), 3.97 (m, 4H, 2 ꢀ OCH₂), 6.90 (m, 3H), 7.25 (m, 2H); ¹³C

of 6 lL/min. Melting points were determined on a Mel-Temp II

melting point apparatus and are uncorrected. Optical rotations

were measured using an Autopol^ÒII automatic polarimeter for

solutions in a 1-dm cell at room temperature (rt). Thin-layer chro-

matography was performed using glass- or aluminum-backed Sili-

cycle Silica Gel 60 F₂₅₄plates.

Compounds dissolved in MeOH were separated by reverse-phase

HPLC using an analytical C18 column and acetonitrile/water mix-

tures as the mobile phase at 1 mL/min ﬂow rate. The elution of com-

pounds was monitored by measuring the absorbance at 195 nm.

NMR d 25.1, 25.4, 29.0, 30.1, 66.3 (d, J_CP= 6.0 Hz), 67.3, 114.1,

120.1, 129.0, 159.2; ³¹P NMR d 0.46; ESMS m/z 273 [MꢁH]^ꢁ; HRMS

[MꢁH]^ꢁ, Found: 273.0890. C₁₂H₁₈O₅P requires m/z, 273.0892.

3.4.3. Bis(diisopropylammonium) 6-phenoxy-1-hexyloxydi-

phospho(1)-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-a-D-

glucopyranose (6)

Both 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-a-D-glucopyranose

dihydrogen phosphate 5 (101 mg, 0.2 mmol)⁴and compound 4

(71 mg, 0.3 mmol) were converted into the corresponding diiso-

propylamine (DIPA) salt by co-evaporation with toluene/DIPA

(1 mL toluene/ﬁve drops DIPA, ꢀ2). Compound 4 was then dis-

solved in dry THF (5 mL) and 1,1⁰-carbonyldiimidazole (186 mg,

1.1 mmol) was added to the solution. The reaction mixture was

3.3. Glycosyltransferase assays

For glycosyltransferase assays, bacteria were cultured and the

homogenates prepared in 50 mM sucrose as described.^7,8Diluted

homogenates were used as the enzyme source. Standard assays

were carried out in 10 min incubations utilizing radioactive su-

gar–nucleotides as donor substrates.^4–8For Gal-transferases WbbD

and WfeD, UDP-[³H]Gal was used as the donor substrate and for

Glc-transferases WfaP and WfgD, UDP-[¹⁴C]Glc was used as the do-

nor substrate. Under these conditions, initial rates were measured.

Radioactive enzyme products having hydrophobic groups were

separated using C18 Sep-Pak. Assay mixtures were loaded on small

Sep-Pak cartridges in water, followed by washing with 4 mL water,

eluting nucleotide sugars and free sugars. Enzyme products and

substrates were then eluted with 3 mL MeOH as described.⁴En-

zyme products from assays utilizing neutral substrates were also

separated by anion exchange chromatography on AG1ꢀ8.²³HPLC

was used to separate substrates and products and to conﬁrm that

enzyme product was formed. Eluates were analyzed by measuring

the absorbance at 195 nm and the radioactivity of collected frac-

tions, in comparison to standard compounds.⁷

stirred at rt for 90 min, quenched with dry MeOH (50 lL), and stir-

red for an additional 30 min. The solvent was removed to give a

colorless oil that was dried under vacuum for 30 min. Compound

5 was then dissolved in dry THF (15 mL) and the solution was

added to the imidazolidate. The reaction was left stirring for 48 h

and the solvent was removed under reduced pressure. The result-

ing residue was puriﬁed by column chromatography on silica gel

(CHCl₃/MeOH, 2:1 v/v) to give the title compound as a colorless

oil (131 mg, 62%); ¹H NMR (CD₃OD) d 1.34, 1.35 (2s, 24H,

8 ꢀ CH₃DIPA), 1.44 (m, 2H, CH₂), 1.51 (m, 2H, CH₂), 1.69 (m, 2H,

CH₂), 1.79 (m, 2H, CH₂), 2.00, 2.03, 2.06, 2.07 (4s, 12H, 4 ꢀ CH₃CO),

3.46 (m, 4H, 4 ꢀ CH DIPA), 3.97 (t, 2H, OCH₂, J = 6.5 Hz), 4.03 (br s,

2H, OCH₂), 4.19 (m, 1H), 4.33–4.45 (m, 3H), 5.12 (t, J = 9.4 Hz, 1H),

5.35 (t, J = 9.8 Hz, 1H), 5.58 (m, 1H, H-1), 6.89 (m, 3H), 7.25 (t,

J = 7.9 Hz, 2H); ¹³C NMR d 19.0, 19.2, 19.3, 19.3 (4 ꢀ CH₃CO),

25.3, 25.5, 28.4, 29.0 (4 ꢀ CH₂), 47.0 (CH, DIPA), 51.8, 61.4, 65.8,

67.4, 67.7, 68.4, 71.7, 94.4 (C-1), 114.1, 120.0, 129.0, 159.1, 169.9,

594

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

170.4, 171.1, 172.7; ³¹P NMR d ꢁ13.4, ꢁ10.4; ESMS m/z 682 [MꢁH]

^ꢁ; HRMS [MꢁH]^ꢁ, Found: 682.1699. C₂₆H₃₈NO₁₆P₂requires m/z,

682.1665.

30 min at rt. The solution was then cooled to 0 °C and treated with

DIAD (0.523 mL, 2.7 mmol), stirred for 30 min, and then warmed up

to rt. After 2 h, the mixture was treated with MeOH (3 mL), stirred

for 5 min, and concentrated. The resulting residue was puriﬁed by

column chromatography on silica gel (hexanes/ethyl acetate, 2:1

v/v) to give the title compound as a colorless oil (955 mg, 89%);

¹H NMR (CDCl₃) d 1.23–1.39 (m, 22H), 1.47 (m, 2H), 1.62 (m, 2H),

1.81 (m, 2H), 4.00 (m, 4H, 2 ꢀ OCH₂CH₂), 5.06 (m, 4H, OCH₂Ph),

6.93 (m, 3H), 7.29 (m, 2H), 7.38 (m, 10H); ¹³C NMR (CDCl₃) d 25.4,

26.1, 29.1, 29.3, 29.4, 29.5, 29.6, 29.6, 29.6, 29.7, 29.7, 30.2, 30.2,

3.4.4. Pyridinium 6-phenoxy-1-hexyloxydiphospho(1)-2-

acetamido-2-deoxy-

-glucopyranose (7)

mol) was dissolved in a solution of so-

Compound 6 (31 mg, 45

dium methoxide (NaOMe)/MeOH (2 mL, 0.05 M) and stirred for

25 min at rt. The reaction mixture was then quenched with an ex-

cess of IR-120 ion exchange resin (pyridinium form) and stirred for

an additional 20 min. The suspension was then ﬁltered and con-

centrated under reduced pressure to give the title compound

(21 mg, 95%) as a colorless oil; ¹H NMR (CD₃OD) d 1.41 (m, 2H),

1.48 (m, 2H), 1.68 (m, 2H), 1.76 (m, 2H), 2.05 (s, 3H), 3.45 (m,

2H), 3.73 (m, 1H), 3.82 (m, 1H), 3.91–4.05 (m, 6H), 5.64 (br s, 1H,

H-1), 6.89 (m, 3H), 7.25 (t, J = 8.1 Hz, 2H), 8.03 (br s, 3H), 8.55 (br

s, 1H), 8.93 (br s, 2H); ¹³C NMR d 21.5, 25.1, 25.2, 28.3, 29.0,

53.7, 61.2, 67.3, 67.7, 70.5, 71.5, 73.8, 95.3 (C-1), 114.1, 120.0,

126.8, 129.0, 142.7, 145.1, 156.0, 159.2, 173.0. ³¹P NMR d ꢁ12.7,

ꢁ10.6. ESMS m/z 556 [MꢁH]^ꢁ; HRMS [MꢁH]^ꢁ, Found: 556.1339.

C₂₀H₃₂NO₁₃P₂requires m/z, 556.1348.

67.9 (PhOCH₂), 68.1 (d, J_CP= 6.1 Hz), 69.1 (d, J_CP= 5.5 Hz), 114.5,

120.4, 127.9, 128.5, 128.6, 129.4, 136.0, 136.0, 159.1 (Ar-C); ³¹P

NMR d ꢁ0.63; ESMS m/z 617 [M+Na]⁺; HRMS [M+Na]⁺, Found:

617.3393. C₃₆H₅₁O₅NaP requires m/z, 617.3372.

3.4.8. 16-Phenoxyhexadecyl dihydrogen phosphate (13)

A suspension of compound 12 (153 mg, 0.3 mmol) and 10% Pd/C

(200 mg) in MeOH (4 mL) was stirred under H₂atmosphere for 1 h.

The Pd/C was removed by ﬁltration through a Celite plug, washed

with MeOH (3 ꢀ 10 mL), and the ﬁltrate was concentrated under

reduced pressure to give the title compound as a colorless solid

(90 mg, 84%); ¹H NMR (DMSO-d₆) d 1.23–1.37 (m, 22H), 1.40 (m,

2H), 1.53 (m, 2H), 1.69 (m, 2H), 3.78 (m, 2H, POCH₂), 3.93 (t,

J = 6.4 Hz, 2H, PhOCH₂), 6.90 (m, 3H), 7.27 (t, J = 7.8 Hz, 2H); ¹³C

NMR d 25.4, 25.6, 29.1, 29.2, 29.2, 29.5, 30.4, 65.7 (POCH₂), 67.7

(PhOCH₂), 114.8, 120.8, 129.9, 159.1; ³¹P NMR d ꢁ0.99; ESMS m/z

413 [MꢁH]^ꢁ; HRMS [MꢁH]^ꢁ, Found: 413.2459. C₂₂H₃₈O₅P requires

m/z, 413.2457.

3.4.5. Methyl 16-phenoxyhexadecanoate (10)

Methyl 16-bromohexadecanoate (9) was prepared from 16-

bromohexadecanoic acid 8 as described.¹⁵To a solution of com-

pound 9 (900 mg, 2.6 mmol) and phenol (364 mg, 3.9 mmol) in

DMF (6 mL), potassium carbonate (1.07 g, 7.7 mmol) was added

under stirring, at 60 °C. The suspension was stirred for 5 h, cooled

to rt, and diluted with ethyl acetate (30 mL) and water (300 mL).

The organic layer was separated and washed successively with

water (2 ꢀ 30 mL), a solution of NaOH 1.0 M (2 ꢀ 30 mL), water

(30 mL), and brine (30 mL). The organic layer was then dried

(Na₂SO₄), ﬁltered, and concentrated. The resulting residue was

puriﬁed by column chromatography on silica gel (hexanes/ethyl

acetate, 8:1 v/v) to give the title compound as a colorless solid

(756 mg, 81%); ¹H NMR (CDCl₃) d 1.23–1.39 (m, 22H), 1.48 (m,

2H), 1.65 (m, 2H), 1.81 (m, 2H), 2.33 (t, J = 7.6 Hz, CH₂CO), 3.69

(s, 3H, OCH₃), 3.97 (t, J = 6.6 Hz, 2H, PhOCH₂), 6.93 (m, 3H), 7.29

(m, 2H); ¹³C NMR (CDCl₃) d 25.0, 26.1, 29.2, 29.3, 29.3, 29.5, 29.5,

29.6, 29.7, 34.1, 51.4 (CH₂CO), 67.9 (PhOCH₂), 114.5, 120.4, 129.4,

159.2 (Ar-C), 174.3 (CO); ESMS m/z 385 [M+Na]⁺; HRMS [M+Na]⁺,

Found: 385.2714. C₂₃H₃₈O₃Na requires m/z, 385.2719.

3.4.9. Bis(diisopropylammonium) 16-phenoxy-1-hexadecyloxy-

diphospho(1)-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-a-D-gluco-

pyranose (14)

Both compound

(63 mg, 0.1 mmol)⁴and compound 13

(67 mg, 0.2 mmol) were converted into the corresponding DIPA

salt by co-evaporation with toluene/DIPA (1 mL toluene/ﬁve drops

DIPA, ꢀ2). Compound 13 was then dissolved in THF/toluene (2:1,

3 mL) and 1,1⁰-carbonyldiimidazole (105 mg, 0.6 mmol) was added

to the solution. The reaction mixture was stirred at rt for 90 min,

quenched with dry MeOH (40 lL), and stirred for an additional

30 min. The solvent was then removed to give a colorless oil that

was dried under vacuum for 30 min. Compound 5 was then dis-

solved in dry THF (3 mL) and the solution was added to the imi-

dazolidate. The reaction was left stirring for 48 h and then the

solvent was removed under reduced pressure. The resulting resi-

due was puriﬁed by column chromatography on silica gel (gradient

of CHCl₃/MeOH, 2:1 to 3:2 v/v) to give the title compound as a col-

orless solid (70 mg, 53%); ¹H NMR (CD₃OD) d 1.23–1.46 (m, 46H,

10 ꢀ OCH₂, 8 ꢀ CH₃DIPA), 1.50 (m, 2H, CH₂), 1.67 (m, 2H, CH₂),

1.78 (m, 2H, CH₂), 1.95, 2.00, 2.02, 2.08 (4s, 12H, 4 ꢀ CH₃CO),

3.48 (m, 4H, 4 ꢀ CH DIPA), 3.96 (t, 2H, OCH₂, J = 6.5 Hz), 4.03 (br

s, 2H, OCH₂), 4.16 (m, 1H), 4.31–4.42 (m, 3H), 5.12 (t, J = 10.0 Hz,

1H), 5.35 (t, J = 9.9 Hz, 1H), 5.61 (m, 1H, H-1), 6.90 (m, 3H), 7.26

(t, J = 8.1 Hz, 2H), 8.80 (d, J = 8.5 Hz, 1H); ¹³C NMR d 18.0, 18.0,

19.2, 21.4 (4 ꢀ CH₃CO), 25.6, 25.8, 29.0, 29.1, 29.2, 29.3, 29.4,

47.0 (CH, DIPA), 51.8, 61.4, 65.8, 67.4, 67.5, 68.3, 71.8, 94.4 (C-1),

114.1, 120.0, 129.0, 159.1, 170.0, 170.4, 171.1, 172.8; ³¹P NMR d

ꢁ13.6, ꢁ10.6; ESMS m/z 822 [MꢁH]^ꢁ; HRMS [MꢁH]^ꢁ, Found:

822.3262. C₃₆H₅₈NO₁₆P₂requires m/z, 822.3231.

3.4.6. 16-Phenoxy-1-hexadecanol (11)

To a suspension of lithium aluminum hydride (LAH) (24 mg,

0.6 mmol) in ether (1.5 mL), a solution of compound 10 (114 mg,

0.3 mmol) in ether (1 mL) was added at 0 °C. The reaction mixture

was then warmed up to rt, stirred for 2 h, and diluted with ethyl

acetate (10 mL) and water (10 mL). The organic layer was then sep-

arated and the aqueous layer was extracted with ethyl acetate

(3 ꢀ 10 mL). The combined organic extracts were then washed

with brine (10 mL), dried (Na₂SO₄), ﬁltered, and concentrated.

The resulting residue was puriﬁed by column chromatography on

silica gel (hexanes/ethyl acetate, 2:1 v/v) to give the title com-

pound as a colorless solid (100 mg, 95%); ¹H NMR (CDCl₃) d

1.23–1.39 (m, 22H), 1.34 (m, 2H), 1.48 (m, 2H), 1.80 (m, 2H),

3.66 (t, J = 6.6 Hz, CH₂OH), 3.97 (t, J = 6.6 Hz, 2H, PhOCH₂), 6.95

(m, 3H), 7.29 (m, 2H); ¹³C NMR (CDCl₃) d 25.8, 26.1, 29.3, 29.4,

29.5, 29.6, 29.6, 29.6, 29.7, 32.8, 63.1 (CH₂OH), 67.9 (PhOCH₂),

114.5, 120.4, 129.4, 159.1 (Ar-C); ESMS m/z 335 [M+H]⁺; HRMS

[M+H]⁺, Found: 335.2939. C₂₂H₃₉O₂requires m/z, 335.2950.

3.4.10. Pyridinium 16-phenoxy-1-hexadecyloxydiphospho(1)-

3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-a-D-glucopyranose (15)

Compound 14 (42 mg, 47 mol) was dissolved in a solution of

3.4.7. Dibenzyl 16-phenoxyhexadecanyl phosphate (12)

A solution of compound 11 (603 mg, 1.8 mmol), dibenzyl phos-

phate (552 mg, 2.0 mmol), and triphenylphosphine (709 mg,

2.7 mmol) in THF (15 mL) was stirred under N₂atmosphere for

NaOMe/MeOH (4 mL, 0.05 M) and stirred for 40 min at rt. The reac-

tion mixture was then quenched with an excess of IR-120 ion ex-

change resin (pyridinium form) and stirred for an additional

20 min. The suspension was ﬁltered and concentrated under re-

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

595

duced pressure to give the title compound (35 mg, 97%) as a color-

less solid; ¹H NMR (CD₃OD) d 1.23–1.37 (m, 22H), 1.49 (m, 2H),

1.66 (m, 2H), 1.78 (m, 2H), 2.01 (s, 3H), 3.44 (m, 2H), 3.72 (m,

2H), 3.85 (m, 1H), 3.91–4.05 (m, 7H), 5.60 (br s, 1H, H-1), 6.89

(m, 3H), 7.25 (t, J = 7.7 Hz, 2H), 8.03 (br s, 3H), 8.55 (br s, 1H),

8.91 (br s, 2H); ¹³C NMR d 18.0, 21.6, 25.5, 25.8, 29.0, 29.1, 29.1,

29.3, 29.4, 29.4, 30.3, 54.0, 61.3, 66.5, 67.5, 70.5, 71.5, 73.8, 95.3

(C-1), 114.1, 120.1, 126.8, 129.0, 142.6, 145.2, 156.0, 159.2,

172.8.; ³¹P NMR dꢁ12.7, ꢁ10.7; ESMS m/z 822 [MꢁC₅H₆N]^ꢁ; HRMS

[MꢁC₅H₆N]^ꢁ, Found: 696.2930. C₃₀H₅₂NO₁₃P₂requires m/z,

696.2914. For use in the assays, the compound was further puriﬁed

by C18 Sep-Pak columns.

3.88 (m, 1H, one OCH₂), 3.97 (t, J = 6.6, 2H PhOCH₂), 4.12 (br d,

J = 12.2 Hz, 1H), 4.28 (dd, J = 12.1, 4.5 Hz, 1H), 4.70 (d, J = 8.3 Hz,

1H, H-1), 5.08 (t, J = 9.5 Hz, 1H), 5.33 (t, J = 9.8 Hz, 1H), 5.46 (d,

J = 8.5 Hz, 1H), 6.95 (m, 3H), 7.29 (m, 2H); ¹³C NMR d 20.7, 20.7,

20.8, 23.4, 25.9, 26.1, 29.4, 29.4, 29.5, 29.5, 29.6, 29.6, 54.9, 62.0,

67.9, 68.7, 70.0, 71.8, 72.4, 100.7 (C-1b), 114.5, 120.5, 129.4, 159.1,

169.4, 170.1, 170.8, 170.9 (4 ꢀ CO); ESMS m/z 594 [M+H]⁺; HRMS

[M+H]⁺, Found: 594.3278. C₃₁H₄₈NO₁₀requires m/z, 594.3260.

3.4.13. 11-Phenoxyundecyl 2-acetylamino-2-deoxy-

glucopyranoside (20

Compound 19 (20 mg, 34 lmol) was dissolved in a solution of

a-D-

NaOMe/MeOH (1 mL, 0.05 M) and stirred for 20 min at rt. The reac-

tion mixture was then quenched with an excess of IR-120 ion ex-

change resin and stirred for an additional 20 min. The suspension

was then ﬁltered and concentrated under reduced pressure to give

3.4.11. 11-Phenoxyundecyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-

a,b-

D-glucopyranoside (18a,b)

A solution of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-

,b-

D-gluco-

pyranosyl trichloroacetimidate 16

,b (189 mg, 0.4 mmol)¹⁹and

the title compound (15 mg, 94%) as a colorless solid; [a]_D= +68.0 (c

11-phenoxyundecanol 17 (126 mg, 0.5 mmol)⁴in dichloromethane

(2 mL) was stirred for 30 min under N₂atmosphere over molecular

sieves (4 Å). The solution was then cooled to 0 °C and trimethylsi-

1.0, MeOH); ¹H NMR (CDCl₃) d 1.31–1.40 (m, 12H), 1.47 (m, 2H,

OCH₂), 1.61 (m, 2H, OCH₂), 1.78 (m, 2H, OCH₂), 2.00 (s, 3H, COCH₃),

3.39 (m, 2H), 3.60 (m, 1H), 3.65–3.72 (m, 3H), 3.82 (br d,

J = 11.7 Hz, 1H), 3.89 (dd, J = 10.6, 3.3 Hz, 1H), 3.97 (t, J = 6.4 Hz,

PhOCH₂), 4.80 (d, J = 3.4 Hz, 1H, H-1), 6.90 (m, 3H), 7.26 (t,

J = 7.6 Hz, 2H); ¹³C NMR d 21.2, 25.8, 25.9, 29.0, 29.1, 29.2, 29.2,

29.3, 29.3, 29.4, 54.2, 61.4, 67.4, 67.5, 71.0, 71.4, 72.4, 97.0 (C-

lyltriﬂuoromethanesulfonate (TMSOTf) (58

added. The reaction mixture was warmed up to rt over a period

of 15 min and was quenched with Et₃N (100 L). The solution

lL, 0.3 mmol) was

was then ﬁltered and concentrated under reduced pressure. The

resulting residue was puriﬁed by column chromatography on silica

gel (CH₂Cl₂/acetone, 95:5 v/v) to give the title compounds as a mix-

1a), 114.1, 120.1, 129.0, 159.2, 172.2 (CO); ESMS m/z 490

[M+Na]⁺; HRMS [M+Na]⁺, Found: 490.2766. C₂₅H₄₁NO₇Na requires

ture of anomers (

:b, 1:1.3 (122 mg, 53%); ¹H NMR (CDCl₃) d 1.27–

m/z, 490.2781.

1.5 (2 m, 28H), 1.66 (m, 4H, OCH₂), 1.79 (m, 4H, OCH₂), 2.03, 2.06,

2.10 2.11 (4s, 18H, COCH₃), 3.29 (dd, J = 3.4, 7.2 Hz, 1H), 3.47–3.59

(m, 4H), 3.66 (br m, 1H), 3.72 (q, 1H), 3.95 (m, 5H), 4.06 (m, 1H),

4.12 (br d, 1H), 4.29 (br dd, 2H), 4.39 (d, J = 8.0 Hz, 1H, H-1b),

4.96–5.08 (m, 4H), 5.51 (t, J = 10.1 Hz, 1H), 6.92 (m, 6H), 7.30 (m,

4H); ¹³C NMR d 20.6, 20.7, 25.9, 26.1, 29.3, 29.3, 29.4, 29.5, 29.5,

60.8, 61.9, 62.0, 63.8, 67.5, 67.9, 68.5, 68.6, 69.1, 70.4, 70.8, 71.8,

3.4.14. 11-Phenoxyundecyl 2-acetylamino-2-deoxy-b-

glucopyranoside (20b)

D-

Compound 19b (23 mg, 39 lmol) was dissolved in a solution of

NaOMe/MeOH (1 mL, 0.05 M) and stirred for 20 min at rt. The reac-

tion mixture was quenched with an excess of IR-120 ion exchange

resin and stirred for an additional 20 min. The suspension was then

ﬁltered and concentrated under reduced pressure to give the title

72.5, 97.9 (C-1a), 102.1 (C-1b), 114.5, 120.4, 129.4, 159.1, 170.0;

ESMS m/z 600 [M+Na]⁺; HRMS [M+Na]⁺, Found: 600.2873.

C₂₉H₄₃N₃O₉Na requires m/z, 600.2897.

compound (18 mg, 100%) as a colorless solid. [

]

_D= ꢁ6.0 (c 1.0,

MeOH); ¹H NMR (CDCl₃) d 1.31–1.40 (m, 12H), 1.52 (m, 2H, OCH₂),

1.56 (m, 2H, OCH₂), 1.78 (m, 2H, OCH₂), 2.00 (s, 3H, COCH₃), 3.28–

3.35 (m, 2H), 3.48 (m, 2H), 3.62–3.72 (m, 2H), 3.90 (m, 2H), 3.96 (t,

J = 6.4 Hz, PhOCH₂), 4.40 (d, J = 8.4 Hz, 1H, H-1), 6.91 (m, 3H), 7.26

(t, J = 8.0 Hz, 2H); ¹³C NMR d 21.6, 25.8, 25.8, 29.0, 29.1, 29.3, 29.3,

29.4, 56.1, 61.4, 67.5, 69.2, 70.8, 74.7, 76.6, 101.3 (C-1b), 114.1,

120.1, 129.0, 159.2, 172.3 (CO); ESMS m/z 468 [M+H]⁺; HRMS

[M+H]⁺, Found: 468.2954. C₂₅H₄₂NO₇requires m/z, 468.2961.

3.4.12. 11-Phenoxyundecyl 3,4,6-tri-O-acetyl-2-acetylamino-2-

deoxy-a,b-

-glucopyranoside (19

a,b)

A suspension of compounds 18

,b (106 mg, 0.2 mmol) and 10%

Pd/C (100 mg) in MeOH (3 mL) was stirred under H₂atmosphere

for 2 h. The Pd/C was removed by ﬁltration through a Celite plug,

washed with MeOH, and the ﬁltrate was concentrated under re-

duced pressure to give a colorless solid which was then dissolved

in pyridine (1 mL) and Ac₂O (1 mL) and allowed to stir at rt for

16 h. The solvents were then removed under reduced pressure

and the resulting residue was further puriﬁed by column chroma-

tography on silica gel (CHCl₃/MeOH, 98:2 v/v) to give the title com-

3.4.15. 11-Phenoxyundecyl-malonic acid (21)

A stirred solution of 11-phenoxyundecanol 17 (650 mg, 2.5 mmol)

and Meldrum’s acid (354 mg, 2.5 mmol) in toluene (5 mL) was

heated to reﬂux for 1 h. The reaction mixture was then cooled to

rt, diluted with ethyl acetate (10 mL), and washed with water

(10 mL) and brine (10 mL). The organic layer was then dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure to give

the title compound as a colorless solid (840 mg, 98%); ¹H NMR

(CDCl₃) d 1.28–1.42 (m, 12H), 1.47 (m, 2H), 1.68 (m, 2H), 1.81 (m,

2H), 3.45 (s, 2H, COCH₂), 3.98 (t, J = 6.6 Hz, 2H, PhOCH₂), 4.18 (t,

J = 6.7 Hz, 2H, OCH₂), 6.94 (m, 3H), 7.20 (m, 2H), 10.72 (br s, 1H,

CO₂H); ¹³C NMR (CDCl₃) d 21.5, 25.8, 26.1, 28.4, 29.2, 29.3, 29.4,

29.5, 29.6, 41.0 (COCH₂), 66.0 (OCH₂), 67.9 (PhOCH₂), 114.5, 120.5,

129.4, 159.1 (Ar-C), 167.5, 170.8 (2 ꢀ CO); ESMS m/z 373 [M+Na]⁺;

HRMS [M+Na]⁺, Found: 373.1974. C₂₀H₃₀O₅Na requires m/z,

373.1991.

pounds, 19

(35 mg, 32%); [

and 19b as colorless solids (80 mg, 73%). Data for 19

]

_D= +48 (c 1.0, CHCl₃); ¹H NMR (CDCl₃) d 1.27–1.40

(m, 12H), 1.47 (m, 2H, OCH₂), 1.63 (m, 2H, OCH₂), 1.80 (m, 2H,

OCH₂), 1.97, 2.04, 2.05, 2.11 (4s, 12H, 4 ꢀ COCH₃), 3.44 (dt,

J = 6.7, 3.0 Hz, 1H, one OCH₂), 3.70 (dt, J = 6.8, 2.7 Hz, 1H, one

OCH₂), 3.97 (m, 3H), 4.11 (br d, J = 12.3 Hz, 1H), 4.26 (dd, J = 12.3,

4.5 Hz, 1H), 4.36 (dt, J = 9.8, 3.6 Hz, 1H), 4.84 (d, J = 3.6 Hz, 1H, H-

1), 5.14 (t, J = 9.6 Hz, 1H), 5.23 (t, J = 9.8 Hz, 1H), 5.66 (d,

J = 9.5 Hz, 1H), 6.94 (m, 3H), 7.29 (m, 2H); ¹³C NMR d 20.6, 20.7,

20.8, 23.2, 26.1, 26.2, 29.3, 29.4, 29.4, 29.5, 29.6, 51.9, 62.0, 67.7,

67.8, 68.2, 68.6, 71.5, 97.1 (C-1a), 114.5, 120.5, 129.4, 159.1,

169.3, 169.8, 170.7, 171.4 (4 ꢀ CO); ESMS m/z 594 [M+H]⁺; HRMS

[M+H]⁺, Found: 594.3278. C₃₁H₄₈NO₁₀requires m/z, 594.3278. Data

for 19b (45 mg, 41%); [

]

_D= ꢁ4.0 (c 1.0, CHCl₃); ¹H NMR (CDCl₃) d

3.4.16. 3,4,6-Tri-O-benzyl-2-acetamido-2-deoxy-

-D

1.27–1.40 (m, 12H), 1.44 (m, 2H, OCH₂), 1.58 (m, 2H, OCH₂), 1.80

(m, 2H, OCH₂), 1.96, 2.04, 2.05, 2.10 (4s, 12H, 4 ꢀ COCH₃), 3.47

(m, one OCH₂), 3.71 (br d, J = 8.4 Hz, 1H), 3.84 (q, J = 6.9 Hz, 1H),

glucopyranosyl 11-phenoxyundecyl malonate (23

,b)

Compound 21 (190 mg, 0.5 mmol) and 3,4,6-tri-O-benzyl-2-

acetamido-2-deoxy-

a-D

-glucopyranoside, 22a (222 mg, 0.5 mmol)²²

596

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

were dissolved in a mixture of THF (3 mL) and DMF (1 mL). DCC

(201 mg, 1.0 mmol) and 4-dimethylamino)pyridine (DMAP)

(12 mg, 0.1 mmol) were added to the solution and the reaction

mixture was stirred at rt for 90 min. The reaction mixture was ﬁl-

tered and concentrated under reduced pressure. The residue was

dissolved in ethyl acetate and washed with a solution of HCl

1.0 M (10 mL), satd aq NaHCO₃(10 mL), brine (10 mL), and water

(10 mL). The organic layer was then dried (Na₂SO₄), ﬁltered, and

concentrated under reduced pressure to give a mixture of anomers

(1.5 mL) was stirred under N₂atmosphere for 20 min at rt. The

solution was then cooled to 0 °C and treated with DIAD (86 L,

0.4 mmol), stirred for 30 min, and then warmed up to rt. After

4 h the mixture was treated with MeOH (250 L), stirred for

5 min, and concentrated. The resulting residue was puriﬁed by col-

umn chromatography on silica gel (hexanes/ethyl acetate, 2:1 v/v)

to give the title compound as a colorless oil (140 mg, 83%); ¹H NMR

(CDCl₃) d 1.23–1.42 (m, 12H), 1.48 (m, 2H), 1.60 (m, 2H), 1.79 (m,

2H), 3.05 (m, 2H, J_H,P= 21.5 Hz), 3.96–4.03 (m, 4H, 2 ꢀ OCH₂), 5.11

(m, 2H, OCH₂Ph), 5.17 (s, 2H, CO₂CH₂Ph), 6.94 (m, 3H), 7.20 (m,

2H), 7.37 (s, 10H Ph-H); ¹³C NMR (CDCl₃) d 25.4, 26.1, 29.1, 29.3,

29.4, 29.5, 29.5, 29.6, 30.3, 30.4, 34.5 (d, J_C,P= 134.8 Hz, CH₂P),

66.8 (d, J_C,P= 6.8 Hz, CH₂OP), 67.3 (CO₂CH₂Ph), 67.9 (PhOCH₂),

66.8 (d, J_C,P= 6.1 Hz, CH₂OP), 114.5, 120.5, 128.0, 128.4, 128.4,

128.5, 128.6, 128.6, 129.4, 135.3, 136.0, 136.1, 159.1 (Ar-C), 165.5

:b, 7.5:1) that was puriﬁed by column chromatography on silica

gel (ethyl acetate/hexanes, 1:1 v/v) to give both the -anomer as a

colorless oil (236 mg, 63%) and the b-anomer (32 mg, 9%) as a col-

orless oil. Data for 23 ; [

_D= +37 (c 1.0, CHCl₃); ¹H NMR (CDCl₃) d

1.27–1.40 (m, 12H), 1.47 (m, 2H, OCH₂), 1.67 (m, 2H, OCH₂), 1.80

(m, 2H, OCH₂), 1.89 (s, 3H, COCH₃), 3.46 (ABq, J_A,B= 16.5 Hz, 2H,

COCH₂), 3.71 (m, 2H), 3.78 (dd, J = 9.9, 3.2 Hz, 1H), 3.82–3.92 (br

m, 2H), 3.97 (t, J = 6.5 Hz, 2H, PhOCH₂), 4.15 (br m, 2H, OCH₂)

4.48–4.59 (br m, 3H), 4.65 (d, J = 12.2 Hz, 1H, one OCH₂Ph), 4.72

(d, J = 11.4 Hz, 1H, one OCH₂Ph), 4.85 (br t, 2H, 2 ꢀ OCH₂Ph), 6.28

(d, 3.3 Hz, 1H, H-1), 6.39 (d, 9.6 Hz, 1H), 6.95 (m, 3H), 7.28 (m,

2H), 7.31–7.37 (m, 15H); ¹³C NMR d 23.1 (COCH₃), 25.8, 26.1,

28.4, 29.2, 29.3, 29.4, 29.5, 29.6, 41.4 (COCH₂), 51.8, 66.5, 67.8,

(d, J_C,P= 6.0 Hz, COCH₂P); ³¹P NMR

d 20.1; ESMS m/z 589

[M+Na]⁺; HRMS [M+Na]⁺, Found: 589.2679. C₃₃H₄₃O₆NaP requires

m/z, 589.2695.

3.4.19. [Benzyloxy(11-phenoxyundecyloxy)phosphinyl]acetic

acid (27)

To a solution of compound 26 (140 mg, 0.2 mmol) in ethanol

(2 mL), 0.5 mL of 0.4 M NaOH were added under stirring. The reac-

tion mixture was stirred for 1 h and was quenched with 0.3 mL

1.0 M HCl. The solvents were then removed and the residue was

partitioned between CH₂Cl₂(10 mL) and brine (10 mL). The organic

layer was separated and the aqueous layer was extracted with

CH₂Cl₂(10 mL). The combined organic extracts were then dried

(Na₂SO₄), ﬁltered, and concentrated under reduced pressure to give

the title compound as a colorless transparent solid (95 mg, 100%);

¹H NMR (CDCl₃) d 1.22–1.42 (m, 12H), 1.46 (m, 2H), 1.63 (m, 2H),

1.81 (m, 2H), 3.04 (m, 2H, J_H,P= 21.6 Hz), 3.97 (t, 2H, J = 6.6 Hz,

PhOCH₂), 4.08 (m, 2H, POCH₂), 5.19 (m, 2H, OCH₂Ph), 6.94 (m,

3H), 7.28 (m, 2H), 7.32–7.38 (m, 5H Ph-H); ¹³C NMR (CDCl₃) d

24.5, 25.2, 28.2, 28.4, 28.5, 28.6, 28.6, 28.6, 33.5 (d, J_C,P= 134.8 Hz,

CH₂P), 66.4 (d, J_C,P= 6.9 Hz, CH₂OP), 67.0 (PhOCH₂), 67.7 (d,

J_C,P= 6.1 Hz, CH₂OP), 113.6, 119.6, 127.2, 127.7, 128.1, 128.5,

128.6, 128.6, 129.4, 135.0, 135.0, 158.2, 166.9 (d, J_C,P= 5.2 Hz,

COCH₂P); ³¹P NMR d 21.8; ESMS m/z 477 [M+H]⁺; HRMS [M+H]⁺,

Found: 477.2413. C₂₆H₃₈O₆P requires m/z, 477.2406.

68.2, 73.6, 74.0, 74.5, 75.0, 75.3, 80.2, 93.2 (C-1a), 114.5, 120.5,

127.8, 127.8, 128.0, 128.1, 128.1, 128.4, 128.5, 129.4, 137.8,

137.9, 138.2, 159.1, 164.3, 168.2, 170.4 (3 ꢀ CO); ESMS m/z 824

[M+H]⁺; HRMS [M+H]⁺, Found: 824.4397. C₄₉H₆₂NO₁₀requires m/

z, 824.4374. Data for 23b; [a]

_D= +20 (c 1.0, CHCl₃); ¹H NMR (CDCl₃)

d 1.27–1.40 (m, 12H), 1.47 (m, 2H, OCH₂), 1.63 (m, 2H, OCH₂), 1.80

(m, 2H, OCH₂), 1.84 (s, 3H, COCH₃), 3.41 (ABq, J_A,B= 16.2 Hz, 2H,

COCH₂), 3.69 (m, 1H), 3.76–3.86 (m, 3H), 3.96–4.00 (m, 3H),

4.10–4.17 (m, 3H), 4.52 (d, J = 12.1 Hz, 1H, one OCH₂Ph), 4.59 (d,

J = 10.9 Hz, 1H, one OCH₂Ph), 4.61 (d, J = 12.1 Hz, 1H, one OCH₂Ph),

4.69 (d, J = 11.6 Hz, 1H, one OCH₂Ph), 4.79 (d, J = 11.0 Hz, 1H, one

OCH₂Ph), 4.82 (d, J = 10.9 Hz, 1H, one OCH₂Ph), 5.41 (d, J = 8.8 Hz,

1H, H-1), 5.79 (d, J = 7.9 Hz, 1H), 6.94 (m, 3H), 7.20 (m, 2H),

7.28–7.37 (m, 15H); ¹³C NMR d 21.1 (COCH₃), 23.4, 25.8, 26.1,

28.4, 29.3, 29.3, 29.4, 29.5, 29.6, 41.3 (COCH₂), 54.0, 60.4, 65.9,

67.9, 73.5, 74.3, 74.7, 75.7, 77.5, 80.3, 93.2 (C-1b), 114.5, 120.5,

127.7, 127.9, 127.9, 128.0, 128.2, 128.4, 128.5, 128.6, 129.4,

137.8, 137.9, 138.1, 159.1, 165.3, 166.3, 170.4 (3 ꢀ CO); ESMS m/z

846 [M+Na]⁺; HRMS [M+Na]⁺, Found: 846.4205. C₄₉H₆₁NO₁₀Na re-

quires m/z, 846.4193.

3.4.20. [Benzyloxy(11-phenoxyundecyloxy)phosphinyl]acetyl

3,4,6-tri-O-benzyl-2-acetamido-2-deoxy-a-D-glucopyranoside

3.4.17. 2-Acetamido-2-deoxy-

phenoxyundecyl malonate (24

A suspension of compound 23

)

-glucopyranosyl 11-

(28)

Compound 27 (60 mg, 0.2 mmol) and 3,4,6-tri-O-benzyl-2-acet-

amido-2-deoxy-

(41 mg, 50

lmol) and 10% Pd/C

a-D

-glucopyranoside 22a (92 mg, 0.2 mmol)⁴were

(40 mg) in MeOH (4 mL) was stirred under H₂atmosphere for 16 h.

The Pd/C was removed by ﬁltration through a Celite plug, washed

with MeOH and the ﬁltrate concentrated under reduced pressure

to give the title compound as a colorless solid (25 mg, 92%);

dissolved in a mixture of THF (1.5 mL) and DMF (0.75 mL). DCC

(58 mg, 0.3 mmol) and DMAP (3 mg, 0.03 mmol) were added to

the solution, and the reaction mixture was stirred at rt for 16 h.

The reaction mixture was then ﬁltered, concentrated under re-

duced pressure and puriﬁed by column chromatography on silica

gel (ethyl acetate/hexanes, 4:1 v/v) to give the title compound as

colorless oil (57 mg, 39%); ¹H NMR (CDCl₃) d 1.24–1.40 (m, 12H),

1.47 (m, 2H, OCH₂), 1.66 (m, 2H, OCH₂), 1.81 (m, 2H, OCH₂), 1.94,

1.98 (2s, 3H, COCH₃), 2.90–3.04 (m, 2H, COCH₂P), 3.68 (dd, 1H),

3.774 (m, 1H), 3.82–3.90 (br m, 3H), 3.97 (t, J = 6.85 Hz, 2H,

PhOCH₂), 4.04 (br m, 2H, OCH₂) 4.52–4.65 (br m, 4H), 4.80–4.86

(m, 3H), 5.09–5.15 (m, 2H), 6.22, 6.24 (2d, J = 3.4, 3.4 Hz, 1H,

2 ꢀ H-1), 6.94 (m, 3H), 7.18 (m, 2H), 7.28–7.42 (m, 20H), 7.48,

7.52 (2d, 1H); ¹³C NMR d 22.8, 22.9 (2 ꢀ COCH₃), 25.0, 25.4, 25.4,

25.6, 26.1, 29.1, 29.3, 29.4, 29.5, 29.6, 30.3, 30.3, 34.9 (d,

J_C,P= 110.6 Hz, COCH₂P), 52.0, 52.1, 67.2 (d, J_C,P= 7.0 Hz), 67.5 (d,

J_C,P= 7.1 Hz), 67.8, 67.8 (2 ꢀ PhOCH₂), 68.4, 68.5 (d, J_C,P= 6.4 Hz),

68.8 (d, J_C,P= 6.3 Hz), 73.5, 74.0, 74.0, 75.2, 75.3, 75.4, 77.3, 80.0,

[a]

_D= +61 (c 1.0, CHCl₃); ¹H NMR (CDCl₃) d 1.03–1.42 (m, 12H),

1.50 (m, 2H, OCH₂), 1.68 (m, 2H, OCH₂), 1.77 (m, 2H, OCH₂), 1.99

(s, 3H, COCH₃), 3.50 (t, J = 9.3 Hz, 1H), 3.56 (br t, 1H), 3.66 to 3.82

(m, 4H), 3.97 (br t, 1H), 4.04 (dd, J = 3.2, 10.7 Hz, 1H), 4.17 (t,

J = 6.6 Hz, 2H, PhOCH₂), 6.19 (d, J = 3.2 Hz, 1H, H-1), 6.89 (m, 3H),

7.25 (t, 2H); ¹³C NMR d 21.1 (COCH₃), 25.5, 25.6, 28.2, 28.9, 29.0,

29.1, 29.2, 29.3, 29.3, 53.0, 60.7, 61.6, 65.5, 67.5, 70.0, 70.9, 75.0,

91.8 (C-1a), 114.1, 120.1, 129.0, 158.2, 165.6, 167.2, 172.5

(3 ꢀ CO); ESMS m/z 554 [M+H]⁺; HRMS [M+H]⁺, Found: 554.2968.

C₂₈H₄₄NO₁₀requires m/z, 554.2965.

3.4.18. [Benzyloxy(11-phenoxyundecyloxy)phosphinyl]acetic

acid benzyl ester (26)

A solution of (benzyloxyhydroxyphosphoryl)acetic acid benzyl

ester 25 (95 mg, 0.3 mmol),¹¹11-phenoxyundecanol 17 (118 mg,

0.4 mmol)⁴and triphenylphosphine (116 mg. 0.4 mmol) in CH₂Cl₂

80.9, 93.5, 93.6 (C-1a), 114.5, 120.5, 120.5, 127.6, 127.7, 127.7,

127.8, 127.8, 127.9, 128.0, 128.2, 128.4, 128.4, 128.6, 128.8, 128.9,

J. G. Riley et al. / Carbohydrate Research 345 (2010) 586–597

597

129.0, 129.4, 137.9, 138.0, 138.1, 138.4, 159.1, 163.7 (d, J_C,P= 6.4 Hz,

3.4.24. 2-Acetamido-2-deoxy-1-O-(11-phenoxyundecyloxy)

phosphoryl- -glucopyranose (32)

COCH₂P), 170.8 (CONH); ³¹P NMR d 21.1; ESMS m/z 950 [M+H]⁺;

a-D

HRMS [M+H]⁺, Found: 950.4595. C₅₅H₆₉NO₁₁

950.4608.

requires m/z,

To a stirred solution of compound 31 (84 mg, 0.1 mmol) in

MeOH (2 mL) was added concentrated NH₄OH (1.5 mL). The reac-

tion mixture was stirred overnight at rt and the solvent was re-

moved under reduced pressure. The resulting crude material was

puriﬁed by Sephadex G15 size exclusion chromatography, using

MeOH (0.1% NH₄CO₃) as the mobile phase to give compound 32

(572 mg, 91%). ¹H NMR (CDCl₃): d 1.4–1.7 (m, 18H), 2.026 (3H,

COCH₃), 3.4 (m, 3H), 3.72 (m, 2H), 3.89 (q, 5H), 3.97 (t, 4H), 5.46

3.4.21. [Hydroxy (11-phenoxyundecyloxy)phosphinyl]acetyl 2-

acetamido-2-deoxy- -glucopyranoside (29)

A suspension of compound 23 (42 mg, 44 lmol) and 10% Pd/C

a-D

(40 mg) in MeOH (3 mL) was stirred under H₂atmosphere for 16 h.

The Pd/C was removed by ﬁltration through a Celite plug, washed

with MeOH, and the ﬁltrate concentrated under reduced pressure

to give the title compound as a colorless solid (22 mg, 85%);

(d, 1H-GlcNAca

, J = 2.4–3.2 Hz), 6.9 (3H), 7.23–7.28 (2H); ¹³C

NMR: d (COCH₃) 22, 27.1, 31.9, 47.6, 55.5, 62.6, 66.8, 68.9, 71.9,

72.8, 74.9, 95.6, 115.5, 121.5, 130.4, 160.6, 171.8; ³¹P NMR: d 3.0;

MS(-TOF): m/z 546 [MꢁH]^ꢁin MeOH; C₂₅H₄₁NO₁₀P requires m/z

547.58.

[a]

_D= +46 (c 1.0, CHCl₃); ¹H NMR (CDCl₃) d 1.24–1.40 (m, 12H),

1.47 (m, 2H, OCH₂), 1.66 (m, 2H, OCH₂), 1.81 (m, 2H, OCH₂), 1.94,

1.98 (2s, 3H, COCH₃), 2.90–3.04 (m, 2H, COCH₂P), 3.48 (m, 2H),

3.56 (t, J = 6.6 Hz, 1H), 3.73–3.79 (m, 4H), 3.96 (t, J = 6.7 Hz, 1H),

4.08 (m, 3H), 6.14 (d, J = 2.8 Hz, 1H, H-1), 6.90 (m, 3H), 7.24 (t,

J = 7.7 Hz, 2H); ¹³C NMR d 21.3 (COCH₃), 24.7, 25.3, 25.4, 25.6,

25.8, 28.9, 29.0, 29.1, 29.2, 29.3, 30.3, 32.8 (d, J_C,P= 108.8 Hz),

Acknowledgments

The authors thank Drs. Pedro Montoya-Peleaz and Carmen Lazar

for chemical synthesis and Dr. Walter A. Szarek at the Department

of Chemistry, Queen’s University, Kingston ON, Canada for supervi-

sion of the synthetic work. We thank Dr. Lei Wang, Nankai Univer-

sity, Tianjin, China, for supplying the plasmids to study bacterial

glycosyltransferases. This research was supported by grants from

NSERC and the Canadian Cystic Fibrosis Foundation.

53.1, 60.8, 61.6, 66.0, 67.4, 70.0, 71.1, 75.0, 91.9 (C-1a), 114.1,

120.1, 129.0, 159.2, 165.6, 172.6 (2 ꢀ CO); ³¹P NMR d 19.3; ESMS

m/z 590 [M+H]⁺; HRMS [M+H]⁺, Found: 590.2716. C₂₇H₄₅NO₁₁P re-

quires m/z, 590.2730.

3.4.22. (2-Cyano-ethoxy)(11-Phenoxyundecyloxy)diisopropy-

laminophosphine (30)

To a stirred solution of compound 17 (400 mg, 1.5 mmol) in

dichloromethane (20 mL) were added (2-cyanoethoxy)bis(diiso-

propylamino)phosphine (503 mg, 1.7 mmol) and diisopropylam-

moniumtetrazolide (13 mg, 0.8 mmol). The mixture was allowed

to stir at rt for 6 h. The reaction mixture was then washed with

satd aq NaHCO₃, dried over MgSO₄, ﬁltered, and concentrated to

give crude compound 30. The resulting crude material was puriﬁed

by centrifugal chromatography (ether/hexanes, 3:1 v/v) to give

compound 30 (488 mg, 70%); ¹H NMR (CDCl₃): d 1.2–1.24 (m,

16H), 1.25 (1H), 1.32 (2H), 1.37 (m, 2H), 1.48 (m, 2H), 1.63 (m,

2H), 1.8 (m, 2H), 2.65 (m, 2H), 3.5 (m, 2H), 3.6–3.7 (m, 4H), 3.84

(m, 3H), 3.97 (m, 2H), 6.9 (m, 3H), 7.3 (m, 2H),; ¹³C NMR: d 25.1,

25.9, 26.3, 29.3, 29.5, 29.2, 30.5, 31.9, 43.0, 59.3, 68.1, 69.5,

114.3, 120.7, 129.4, 159.2; ³¹P NMR: d 140.5, 148.6; C₂₆H₄₅N₂O₃P

requires m/z 464.62.

References

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3.4.23. 3,4,6-Tri-O-acetyl-2-acetamido-1-O-[(2-cyanoethoxy)

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To a stirred solution of compound 22b (215 mg, 0.6 mmol) in

dichloromethane (60 mL) at 0 °C was added compound 30

(488 mg, 1.1 mmol) and tetrazole (219 mg, 3.1 mmol). The mixture

was warmed to rt and stirred for 6 h. The solution was then cooled

to 0 °C, treated with m-chloroperoxybenzoic acid (mCPBA) (77%,

69 mg, 3.1 mmol), and allowed to stir overnight at rt The reaction

mixture was then washed with a saturated solution of Na-sulﬁte,

followed by satd aq NaHCO₃, and brine. The organic layer was dried

over MgSO₄, and concentrated to give crude compound 31. The

material was puriﬁed by centrifugal chromatography (ethyl ace-

tate/hexanes, 2:1 v/v) to give compound 31 (3.07 mg, 68%). ¹H

NMR (CDCl₃): d 1.20–1.26 (m, 5H), 1.37–1.43 (m, 1H), 1.63–1.77

(m, 2H), 1.93 (m, 3H), 1.98–2.0 (m, 12H), 2.05 (4H), 2.75 (2H), 3.9

(m, 2H), 4.04–4.14 (m, 5H), 4.2–4.3 (m, 5H), 4.38–4.42 (m, 1H),

5.12–5.25 (m, 2H), 5.66 (m, 1H), 6.34, 6.59 (2d, 1H), 6.86 (m, 3H),

7.2 (m, 2H); ¹³C NMR (COCH₃), d 20, 22.7, 25.5, 28.9, 29.2, 29.3,

51.7, 60.2, 61.2, 62.2, 67.4, 69, 77, 96.2, 114.3, 116.4, 120.3, 129.2,

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Chem. 1998, 63, 7244–7257.

158.9, 169, 170.4, 170.9; ³¹P NMR d ꢁ2.1, ꢁ2.2; C₃₄H₅₁N₂O₁₃

requires m/z 726.75.

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