Solid-phase synthesis of 2-aminoethyl glucosamine sulfoforms

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Solid-Phase Synthesis of 2-Aminoethyl

Glucosamine Sulfoforms

Runhui Liu ^a& Alexander Wei ^a

^aDepartment of Chemistry , Purdue University , West Lafayette ,

IN , 47907-2084 , USA

Published online: 02 Jul 2012.

To cite this article: Runhui Liu & Alexander Wei (2012) Solid-Phase Synthesis of 2-Aminoethyl

Glucosamine Sulfoforms, Journal of Carbohydrate Chemistry, 31:4-6, 384-419, DOI:

10.1080/07328303.2012.658274

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Journal of Carbohydrate Chemistry, 31:384–419, 2012

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ISSN: 0732-8303 print / 1532-2327 online

DOI: 10.1080/07328303.2012.658274

Solid-Phase Synthesis of

2-Aminoethyl Glucosamine

Sulfoforms

Runhui Liu and Alexander Wei

Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA

Mono- and disaccharides of sulfonated glucosamines (GlcN sulfoforms) conjugated to

2-aminoethyl linkers were generated by solid-phase synthesis. Orthogonally protected

intermediates were tethered onto tritylated polystyrene resin beads, subjected to a mod-

ular sequence of deprotection and sulfonation steps, then cleaved from solid support

without degradation of N- or O-sulfate esters using solvolytic conditions, and ﬁnally

puriﬁed by reverse-phase HPLC to afford the title compounds.

Keywords Sulfated carbohydrates; Solid-phase synthesis; Protecting groups; Gly-

cosaminoglycans; Heparan sulfate

INTRODUCTION

Heparan sulfate proteoglycans (HSPGs) are well known for their many impor-

tant roles in cellular function.^[1,2]HSPGs are recognized by a diverse range

of heparin-binding proteins in key biological processes, such as angiogene-

sis (ﬁbroblast growth factors [FGFs]),^[3]pathogenic infection (herpes simplex

virus glycoprotein D),^[4]and inﬂammation (L-selectin).^[5]However, so far only

a handful of HSPG sequences have been elucidated, due to the heterogene-

ity of structures that exist within heparin. Many studies have shown that

the speciﬁcity of HS binding is deﬁned in large part by sulfonation patterns,

or sulfoforms. For example, FGF1 binding requires glucosamine-2,6-disulfate

(GlcNS,6S), whereas FGF2 binding requires GlcNS and iduronate-2-sulfate

(IdoA-2S).^[6–8]

The relevance of sulfoforms within HS and particularly glucosamine

(GlcN) to protein binding suggests that even relatively simple sulfoforms are

likely to exhibit sufﬁcient binding avidity to serve as potential antagonists

Received December 18, 2011; accepted January 12, 2012.

Address correspondence to Alexander Wei, Department of Chemistry, Purdue Univer-

sity 560 Oval Drive, West Lafayette, IN 47907-2084. E-mail: alexwei@purdue.edu

384

2-Aminoethyl Glucosamine Sulfoforms

385

OR

O

OBn

O

3

O

BnO

RO

BnO

RO

O

1

2

O

BnO

NH₂

RNH

OBn

O

NH₂

OR

O

BnO

RO

OR

O

OBn

4

RNH

BnO

RO

O

NH₂

O

BnO

RNH

NH₂

OBn

Figure 1: GlcN sulfoforms with 2^ꢁ-aminoethyl linkers (1–4), prepared by solid-phase synthesis.

R = H, Ac, or SO₃⁻; for speciﬁc sulfoforms, see Table 2.

in HS-mediated signaling. Recent examples in the scientiﬁc literature sup-

port this notion: a disaccharide sulfoform containing GlcN has been found

to have signiﬁcant binding to FGF2,^[6]and a clinical study showed that den-

drimers presenting multiple GlcNAc-6S units can prevent scar tissue forma-

tion.^[9]However, structure–afﬁnity relationships between HS-like compounds

and heparin-binding proteins remain rare and are limited by challenges in

identifying or producing structurally well-characterized ligands in sufﬁcient

quantities for study.

To address this bottleneck, we have introduced a method of generating

GlcN sulfoforms using solid-phase synthesis.^[10]Performing deprotections and

sulfonations on resin-immobilized intermediates greatly simpliﬁes the puriﬁ-

cation process and reduces the attrition in yield caused by multiple extraction

and chromatography steps. Our previous study established proof of concept,

but the glycol linker associated with these sulfoforms was not amenable to fur-

ther conjugation. Here we further optimize the solid-phase synthesis approach

in order to generate a number of mono- and disaccharide GlcN sulfoforms bear-

ing a 2^ꢁ-aminoethyl linker (Fig. 1) and to facilitate the preparation of glycocon-

jugates for subsequent protein-binding studies.

RESULTS AND DISCUSSION

Synthesis of Orthogonally Protected GlcN Monosaccharide

and Disaccharide

The preparation of orthogonally protected α-GlcN monosaccharides with

ethanolamine linkers is described in Scheme 1. Phthalimide-protected GlcN

derivative 5 was converted in 85% yield over three steps to 2-azido-2-

deoxyglucoside,^[10]followed by reductive ring opening of the 4,6-benzylidene

acetal using BH₃/Bu₂BOTf^[11]and formation of 6-O-TBDPS ether to afford 6

R. Liu and A. Wei

386

OTBDPS

O

OTBDPS

O

STol

N₃

O

a, b

c

NHR

Ph

O

NPhth

BnO

N₃

α:β = 4.2:1

OH

OAc

5

6

7a,b: R=Fmoc

8a,b: R=H

d

Scheme 1: Synthesis of orthogonally protected GlcN with 2^ꢁ-aminoethyl linker. Reaction

conditions: (a) (i) NH₂(CH₂)₂NH₂, BuOH, 100^◦C; (ii) TfN₃, K₂CO₃, CuSO₄, CH₂Cl₂/MeOH/H₂O,

0^◦C to rt; (iii) Ac₂O/Py, 0^◦C to rt (85% over three steps). (b) (i) BH₃, Bu₂BOTf, THF, 0^◦C; (ii)

TDBPS-Cl, imidazole, DMF, rt (62% over two steps). (c) NIS/TfOH, AE-Fmoc, Cl(CH₂)₂Cl/Et₂O,

−20^◦C (80%). (d) 20% piperidine in DMF, rt (99%). Abbreviations: AE-Fmoc =

2^ꢁ-aminoethyl-2-(9H-ﬂuorenyl)methoxycarbamate; Phth = phthalimidyl; TBDPS =

tert-butyldiphenylsilyl; Tol = p-tolyl.

in 62% yield over two steps. Glycoside 6 was subjected to glycosylation with 9-

ﬂuorenylmethoxycarbonyl (Fmoc)-protected ethanolamine to yield 7 as a mix-

ture of anomers (4.2:1 α:β ratio) in 99% yield. The major and minor isomers

were carefully separated by silica gel chromatography, then treated with 20%

piperidine in DMF to provide amine-terminated GlcN derivatives 8a and 8b,

respectively, each in 99% isolated yield. The selective cleavage of Fmoc in the

presence of other protecting groups was essential for conducting our orthogo-

nal deprotection/sulfonation strategy on a solid-phase support.

In order to prepare β-aminoethyl-linked GlcN(β1→4)Glc derivative 12, the

trichloroethoxycarbonyl (Troc) group was installed at the C2 position of GlcN to

provide anchimeric assistance during glycosyl coupling (Sch. 2). Troc-protected

GlcN derivative 9 was prepared from 6 in 94% overall yield, then subjected to

NIS/TfOH-promoted glycosylation with 15 to yield β-linked disaccharide 10

in 90% yield, followed by selective deprotection with indium powder in aque-

ous NH₄Cl^[12]and Cu²⁺-mediated diazo transfer to yield 11 in 91% yield over

three steps. Selective Fmoc deprotection with 20% piperidine in DMF gave 2^ꢁ-

aminoethyl GlcN derivative 12 in 99% isolated yield.

OBn

O

NHFmoc

NHR

OTBDPS

O

OTBDPS

O

OTBDPS

O

STol

O

OBn

O

OBn

c

a

b

OBn

O

OBn

6

BnO

N₃

BnO

N

H

O

CCl₃

N

H

CCl₃

OAc

d

OAc

11: R=Fmoc

12: R=H

9

10

Scheme 2: Synthesis of GlcN(β1→4)Glc disaccharide with β-linked 2^ꢁ-aminoethyl group.

Reaction conditions: (a) (i) NaOMe, MeOH, rt; (ii) Bu₃P, CH₂Cl₂, rt; (iii) H₂O:CH₂Cl₂1:1, rt; (iv)

Troc-Cl, pyridine, rt; (v) Ac₂O/Py, rt (94% over 5 steps). (b) 15 (see Sch. 3), NIS/TfOH, CH₂Cl₂,

−30^◦C (90%). (c) (i) In powder, NH₄Cl, EtOH/H₂O, 98^◦C; (ii) TfN₃, CuSO₄, K₂CO₃,

CH₂Cl₂/MeOH, 0^◦C to rt (91% over two steps). (d) Piperidine, DMF, rt (99%). Abbreviations:

Troc = trichloroethoxycarbonyl.

2-Aminoethyl Glucosamine Sulfoforms

387

The β-aminoethyl-linked GlcN(α1→4)Glcβ derivative 17 was synthesized

as described in Scheme 3. 2^ꢁ-Azidoethyl β-glucoside tetraacetate 13 was

prepared in 46% yield from glucose pentaaceate by glycosylation with 2-

bromoethanol and azide substitution.^[20]Saponiﬁcation, 4,6-O-benzylidene

acetal formation, benzylation of the C2 and C3 hydroxyls, and regioselective

reductive ring opening with Et₃SiH and TfOH yielded glycosyl acceptor 14 in

56% yield over four steps.^[13]The azide of 14 was converted into Fmoc deriva-

tive 15 in 54% yield over three steps, and NIS/TfOH-promoted glycosyl cou-

pling of 15 and thioglucoside 6 yielded α-1,4-linked disaccharide 16 in 60%

yield. The Fmoc group was efﬁciently cleaved to produce 2^ꢁ-aminoethyl deriva-

tive 17 in 99% yield.

OBn

OAc

O

OBn

NHR

O

OTBDPS

O

a, b

d

N₃

R

O

OBn

AcO

OAc

OBn

HO

OBn

OAc

OBn

BnO

N₃

14: R=N₃

15: R=NHFmoc

OAc

e

13

c

16: R=Fmoc

17: R=H

Scheme 3: Synthesis of GlcN(α1→4)Glc disaccharide with β-linked 2^ꢁ-aminoethyl group.

Reaction conditions: (a) (i) NaOMe, MeOH, rt; (ii) PhCH(OMe)₂, TsOH, THF, reﬂux; (iii) NaH,

BnBr, DMF, 0^◦C to rt (75% over three steps). (b) Et₃SiH, TfOH, 4A mol sieves, CH₂Cl₂, −78^◦C

(75%). (c) (i) Bu₃P, CH₂Cl₂, rt; (ii) H₂O:CH₂Cl₂1:1, rt; (iii) Fmoc-Cl, NaHCO₃, CH₂Cl₂, 0^◦C to rt

(54% over three steps). (d) Donor 6, NIS, TfOH, Cl(CH₂)₂Cl/Et₂O, −20^◦C (60%). (e) Piperidine,

DMF, rt (99%).

Loading of Orthogonally Protected GlcN Derivatives onto Resins

The loading of 2^ꢁ-aminoethyl GlcN derivatives onto trityl-PS resins was

initially accompanied by multiple side reactions, presumably because of the

nucleophilicity of the terminal amine. Moreover, the recovery and puriﬁcation

of excess aminoethyl-linked GlcNs from the reaction mixture was problematic.

Using a lower concentration of 2^ꢁ-aminoethyl GlcN derivatives in dry pyridine

at 60^◦C produced much cleaner results, but the resin loadings were commen-

surately lower.

Aminoethyl-linked GlcN derivatives 8a (GlcN-α), 8b (GlcN-β), 12

(GlcN(β1→4)Glc-β), and 17 (GlcN(α1→4)Glc-β) were each loaded onto trityl-

PS resin in the presence of 0.4 equivalents of DMAP (Table 1). Introducing

the amine-terminated derivatives 8a, 8b, and 12 at 50% loading capacity

(0.75 mmol/g) and 17 at 33% loading capacity (0.5 mmol/g) produced resin-

immobilized GlcN 18a, 18b, 19, and 20, with loading ratios of 0.38, 0.27, 0.18,

and 0.18 mmol/g, respectively.

R. Liu and A. Wei

388

Table 1: Loading of 2^ꢁ-aminoethyl-linked derivatives onto trityl-PS resin

GlcN derivative

Product (loading ratio)

18a (0.38 mmol/g)

18b (0.27 mmol/g)

19 (0.18 mmol/g)

20 (0.18 mmol/g)

Reaction conditions: Cl-Trityl–PS resin, DMAP, pyridine, 60^◦C, 48 h.

Generation of GlcN Sulfoforms

We ﬁrst investigated conditions for cleaving 2^ꢁ-aminoethyl-linked glyco-

sides from the trityl–PS resins. No detectable cleavage was found when

using 0.3 M BF₃·Et₂O in CH₂Cl₂at −20^◦C, the condition used to cleave

glycol-linked GlcNs, and higher temperatures (above 0^◦C) would result in the

signiﬁcant decomposition of N-sulfate esters.^[10]Fortunately, 30% hexaﬂuo-

roisopropanol (HFIP) in CH₂Cl₂at room temperature provided an ideal alter-

native: the cleavage was remarkably efﬁcient and selective, yet mild enough to

leave both O and N-sulfate esters unaffected.^[14]This condition was also found

to be effective for cleaving glycol-linked sulfoforms (trityl ethers).

The solid-phase deprotection and sulfonation conditions developed in our

earlier protocol^[10]were applied toward resin-immobilized monosaccharide

18a, to produce fully deprotected α-linked GlcN 1a in 87% yield after six opera-

tions, and sulfoforms 1b–1h in overall yields ranging from 76% (1b) to 44% (1f)

after eight operations (Table 2). This method was also used to generate GlcN

sulfoforms 2(b,f), 3(b,f), and 4(b,f) in comparable yields from 18b, 19, and 20,

respectively. The relatively low yields obtained for GlcNS,6S sulfoforms 1f–4f

2-Aminoethyl Glucosamine Sulfoforms

389

Table 2: Sulfoforms generated by orthogonal deprotection and sulfonation on

trityl–PS resins

Sulfate pattern^a

Product,^byield, no. ops^c

1a, 87%, 6 ops (a, b, c, d, g, k)

1b, 76%, 8 ops (a, b, c, f, g, h, k, l)

2b, 72%, 8 ops (a, b, c, f, g, h, k, l)

3b, 67%, 8 ops (a, b, c, f, g, h, k, l)

4b, 69%, 8 ops (a, b, c, f, g, h, k, l)

1c, 64%, 8 ops (a, b, c, g, h, d, k, l)

1d, 69%, 7 ops (a, b, i, d, g, k, l)

1e, 68%, 8 ops (a, b, c, d, g, j, k, l)

1f, 44%, 8 ops (d, a, b, h, i, g, k, l)

2f, 42%, 8 ops (d, a, b, h, i, g, k, l)

3f, 41%, 8 ops (d, a, b, h, i, g, k, l)

4f, 42%, 8 ops (d, a, b, h, i, g, k, l)

1g, 68%, 7 ops (g, a, b, j, e, k, l)

(Continued on next page)

R. Liu and A. Wei

390

Table 2: Sulfoforms generated by orthogonal deprotection and sulfonation on

trityl–PS resins (Continued)

Sulfate pattern^a

Product,^byield, no. ops^c

1h, 64%, 7 ops (d, g, a, b, j, k, l)

^a1: GlcN-α; 2: GlcN-β; 3: GlcN(β1→4)Glc-β; 4: GlcN(α1→4)Glc-β (see Fig. 1).

^bAll products were isolated as sodium salts.

^cEach step was conducted at rt for 24 h, unless otherwise noted. Reagents and conditions:

(a) Bu₃P, CH₂Cl₂, 5 h; (b) 95% aq THF; (c) Ac₂O, pyridine; (d) TBAF (pH ≥ 10), THF; (e) TBAF

(pH ≥ 10), THF/DMF; (f) TBAF (pH 7), THF; (g) NaOMe, MeOH/CH₂Cl₂; (h) SO₃·Py, DMF, 10 h; (i)

SO₃·Py, Py/Et₃N; (j) SO₃·Py, Py/Et₃N, 55^◦C, 40 h; (k) 30% HFIP, CH₂Cl₂, 1 h; (l) Na ion-exchange

chromatography.

may be due to the premature removal of the 3-O-acetate or acetyl transfer from

O3 to N2, with subsequent sulfonation of the C3 alcohol. This problem may be

circumvented by protecting the C3 alcohol as an SEM ether, which previously

demonstrated excellent orthogonality with several other protecting groups in

solution-phase synthesis.^[15]

It is worth mentioning that in our earlier studies, we performed ion ex-

change with tetrabutylammonium bromide before releasing sulfoforms from

the resin, based on the belief that the hydrophobic Bu₄N counterion (1) im-

proved the stability of sulfate esters during cleavage and (2) aided in the

swelling of resins in organic solvents.^[16]However, we have subsequently deter-

mined that sulfonation and cleavage does not require Bu₄N counterions to en-

hance process chemistry. This was conﬁrmed by subjecting resin-immobilized

GlcNS-3S,6S 1h (containing a mixture of PyH⁺and Et₃NH⁺counterions) to

HFIP cleavage without prior treatment with Bu₄N-Br, followed by passage

through a Na ion-exchange column. Good yields of the Na salts were achieved

after reverse-phase HPLC puriﬁcation, and no degradation byproducts were

observed. We thus do not need to perform Bu₄N ion exchange in our optimized

solid-phase synthesis protocol.

Our optimized solid-phase synthesis generated sulfoforms with minimal

byproduct formation in nearly all cases. To take one example, the resin-

immobilized GlcN 18a was subjected to seven operations involving depro-

tection, sulfonation, and HFIP cleavage to generate GlcNAc-3S,6S (1e) with

2-Aminoethyl Glucosamine Sulfoforms

391

Figure 2: ¹H NMR spectrum (400 MHz, CD₃OD) of sulfoform 1e (3,6-di-O-sulfate,

bis-triethylammonium salt) after cleavage from solid-phase support, prior to Na ion

exchange and HPLC puriﬁcation (color ﬁgure available online).

1

Et₃NH⁺counterions. The clean H NMR of the as-isolated product supports

the high quality of each conversion (Fig. 2), validating this method of generat-

ing sulfoforms from a single, orthogonally protected precursor.

The modular deprotection/sulfonation steps can efﬁciently generate sulfo-

forms on trityl–PS resins with up to three sulfate esters per molecule, but gen-

erating derivatives with a larger number of sulfate esters is more challenging.

The high charge density of such compounds creates a highly heterogeneous

environment within the resin and can raise the barrier to ionic exchange. To

test the limits of the solid-phase approach with trityl–PS resins, we attempted

to generate a hexasulfate ester from a resin-immobilized GlcN disaccharide

derivative (GlcNS,3S,6S(α1→4)GlcNS,3S,6S) but were unable to achieve com-

plete sulfonation (unpublished results). This limitation might be addressed by

using less-hydrophobic substrates such as PEG-grafted PS resins, which have

been used to assemble protected HS oligosaccharides.^[17]To our knowledge, on-

resin deprotection and sulfonation steps for such derivatives have not yet been

reported, although we note a case in which a sulfate ester was installed on a

synthetic tetrasaccharide immobilized on a polymeric PEG support.^[18]

In closing, we have demonstrated the generation of GlcN sulfoforms with

2^ꢁ-aminoethyl linkers using solid-phase synthesis methods and orthogonally

protected scaffolds. Each operation can be performed in high yields without

the need for ion exchange, and the amine-terminated sulfoforms can be cleaved

from trityl–PS resins under mild conditions.

R. Liu and A. Wei

392

EXPERIMENTAL DETAILS

All starting materials and reagents were obtained from commercial sources

and used as received unless otherwise noted. All solvents were freshly distilled

prior to use. All glucosamine sulfoforms were puriﬁed by HPLC using a Hydro-

RP C18 reverse-phase column, with UV detection at 214 nm. IR spectra were

acquired from NaCl plate, using a Thermo-Nicolet Nexus 670 FT-IR spectrom-

eter. ¹H NMR spectra were recorded at 300, 400, or 500 MHz; ¹³C NMR spectra

were recorded at 75, 100, or 125 MHz. Chemical shifts were referenced to the

solvent used (δ 7.27 and 77.00 for CDCl₃, δ 3.31 and 49.15 for CD₃OD, and

δ 4.80 in D₂O). Mass spectra were acquired using either a Hewlett-Packard

5989B or a Finnigan 40000 mass spectrometer. Optical rotations were mea-

sured by polarimetry at rt. Silica gel chromatography was performed with ICN

SiliTech 32-63D.

Thiotolyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-

glucopyranoside (5)

Glucosamine hydrochloride (50 g, 232 mmol) was mechanically agitated

with 1 M NaOMe in MeOH (232 mL) for 2 h at rt, followed by addition of

ﬁnely powdered phthalic anhydride (19 g, 128 mmol) and Et₃N (35.5 mL, 255

mmol). The mixture was stirred for 45 min, then treated with a second portion

of phthalic anhydride (19 g, 128 mmol) and stirred for 24 h at rt. The mixture

was cooled to −20^◦C for 4 h, ﬁltered, washed with cold MeOH, and dried un-

der reduced pressure to yield a lighter yellow solid. The solid was redispersed

in pyridine (500 mL) followed by slow addition of Ac₂O (330 mL) at 0^◦C and

continuous agitation at rt for 48 h. The crude was concentrated under reduced

pressure and puriﬁed by recrystallization in 95% EtOH to afford 1,3,4,6-tetra-

O-acetyl-2-phthalimido-2-deoxy-β-D-glucopyranoside as an amorphous white

solid (76 g, 69% over 3 steps).^{[19] 1}H NMR (300 MHz, CDCl₃): δ 7.84 (dd, 2H, J =

3, 6 Hz), 7.73 (dd, 2H, J = 3, 5.7 Hz), 6.49 (d, 1H, J = 8.7 Hz), 5.86 (dd, 1H,

J = 9.3, 10.5 Hz), 5.19 (t, 1H, J = 9.3 Hz), 4.44 (dd, 1H, J = 8.7, 10.5 Hz), 4.34

(dd, 1H, J = 3.9, 12.3 Hz), 4.11 (dd, 1H, J = 2.4, 12.3 Hz), 4.00 (ddd, 1H, J =

2.4, 4.8, 10.8 Hz), 2.08 (s, 3H), 1.97 (s, 3H), 1.84 (s, 3H); ¹³C NMR (75 MHz,

CDCl₃): δ 170.70, 170.08, 169.55, 168.71, 167.44, 134.57, 131.26, 123.87, 89.82,

72.67, 70.56, 68.32, 61.58, 53.55, 20.82, 20.68, 20.48.

A solution of the above tetraacetate (20 g, 41.9 mmol) and p-thiocresol

(6.8 g, 54.5 mmol) in CH₂Cl₂(40 mL) was cooled to 0^◦C and treated with

BF₃·Et₂O (12 mL). The mixture was stirred at rt for 24 h, quenched with satu-

rated NaHCO₃solution (100 mL), extracted with CH₂Cl₂(3 × 200 mL), washed

with brine (50 mL), then dried over Na₂SO₄and concentrated. The crude prod-

uct was puriﬁed by recrystallization in 95% EtOH to afford thiotolyl 3,4,6-

O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside as an amorphous white

2-Aminoethyl Glucosamine Sulfoforms

393

solid (19.7 g, 87%).^{19 1}H NMR (300 MHz, CDCl₃): δ 7.86 (dd, 2H, J = 2.7,

5.1 Hz), 7.75 (dd, 2H, J = 3, 5.4 Hz), 7.29 (d, 2H, J = 7.2 Hz), 7.06 (d, 2H,

J = 7.5 Hz), 5.77 (t, 1H, J = 9.9 Hz), 5.64 (d, 1H, J = 10.5 Hz), 5.11 (t, 1H,

J = 9.6 Hz), 4.24–4.30 (m, 2H), 4.19 (dd, 1H, J = 2.4, 12.3 Hz), 3.87 (ddd,

1H, J = 3, 5.4, 10.8 Hz), 2.31 (s, 3H), 2.09 (s, 3H), 2.01 (s, 3H), 1.82 (s, 3H);

¹³C NMR (75 MHz, CDCl₃): δ 170.76, 170.22, 169.58, 167.95, 167.08, 138.89,

134.57, 134.43, 134.04, 131.71, 131.29, 129.77, 127.05, 123.82, 83.22, 75.95,

71.77, 68.79, 62.31, 53.69, 21.29, 20.90, 20.73, 20.54; IR (NaCl): 1749, 1719,

1384, 1227, 1037 cm⁻¹; [α]²⁰= +40.3 (c 1.01, CH₂Cl₂); HRESI-MS: m/z calcd

D

for C₂₇H₂₇NNaO₉S [M+Na]⁺: 564.1304; found: 564.1301.

The triacetate above (15.8 g, 29.1 mmol) was dissolved in 2:3

CH₂Cl₂:MeOH (80 mL) and cooled to 0^◦C, then treated with 1 M NaOMe so-

lution in MeOH (10.5 mL, 10.5 mmol). The mixture was stirred at 0^◦C for

3 h, neutralized with activated Dowex 50X-W-H⁺ion-exchange resin, ﬁltered,

concentrated, and dried under reduced pressure. A solution of the crude triol

in THF (97 mL) was treated with benzaldehyde dimethyl acetal (13 mL, 87.2

mmol) and TsOH·H₂O (554 mg, 2.91 mmol). The mixture was heated to re-

ﬂux for 10 h, quenched with saturated NaHCO₃solution (30 mL), extracted

with CH₂Cl₂(3 × 150 mL), dried over Na₂SO₄, and concentrated under re-

duced pressure. Puriﬁcation by recrystallization in 95% EtOH yielded 4,6-O-

benzylidene acetal 5 as an amorphous white solid (12.5 g, 85% over two steps).

¹H NMR (300 MHz, CDCl₃): δ 7.82 (dd, 2H, J = 5.4, 9.9 Hz), 7.64–7.72 (m,

2H), 7.41–7.51 (m, 2H), 7.22–7.35 (m, 5H), 7.06 (d, 2H, J = 8.4 Hz), 5.58 (d,

1H, J = 10.5 Hz), 5.52 (s, 1H), 4.55 (dt, 1H, J = 3.6, 9.9 Hz), 4.33 (dd, 1H,

J = 4.2, 10.2 Hz), 4.27 (t, 1H, J = 10.5 Hz), 3.77 (t, 1H, J = 9.9 Hz), 3.58 (dt,

1H, J = 4.5, 8.7 Hz), 3.51 (t, 1H, J = 9.3 Hz), 3.30 (d, 1H, J = 3 Hz), 2.30

(s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.31, 167.61, 138.39, 136.96, 134.23,

133.22, 131.54, 129.74, 129.35, 128.37, 127.95, 126.37, 123.85, 123.37, 101.87,

84.49, 81.79, 70.28, 69.61, 68.54, 55.71, 21.15; IR (NaCl): 3474, 1776, 1713,

1386, 1092 cm⁻¹; [α]²⁰= +31.2 (c 1.10, CH₂Cl₂); HRESI-MS: m/z calcd for

D

C₂₈H₂₅NNaO₆S [M+Na]⁺: 526.1300; found: 526.1292.

Thiotolyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-

(tert-butyldiphenylsilyl)-2-deoxy-β-D-glucopyranoside (6)

Preparation of TfN₃: A solution of NaN₃(73 g, 1125 mmol) in water (30 mL)

and CH₂Cl₂(30 mL) at 0^◦C was treated with Tf₂O (38 mL, 225 mmol). The

mixture was stirred for 2 h at 0^◦C, extracted with CH₂Cl₂(2 × 35 mL), washed

with saturated aqueous Na₂CO₃solution (75 mL) and then brine (30 mL), and

used immediately.

A solution of 5 (12.5 g, 24.9 mmol) and ethylene diamine (33 mL, 500 mmol)

in n-butanol was stirred at 100^◦C for 10 h in a pressure tube. The mixture

was concentrated, azeotroped with toluene, and dried under reduced pressure.

R. Liu and A. Wei

394

This was redispersed in 66% aqueous MeOH (231 mL) and treated at 0^◦C with

K₂CO₃(5.2 g, 37.5 mmol), CuSO₄·5 H₂O (312 mg, 1.25 mmol), and freshly

prepared TfN₃(70 mL, 3.2 M solution in CH₂Cl₂). An additional 30 mL of

MeOH was added to homogenize the mixture, which was slowly warmed up

to rt over 3 h and continually stirred for another 10 h at rt, concentrated and

azeotroped with toluene, then dried under reduced pressure. The crude azide

was dissolved in pyridine (150 mL) followed by addition of Ac₂O (75 mL) at 0^◦C.

The mixture was warmed up to rt over 2 h and continually stirred for another

10 h, concentrated, and dried under reduced pressure. Puriﬁcation by silica gel

chromatography (6% EtOAc in hexanes) yielded thiotolyl 3-O-acetyl-2-azido-

4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside as an amorphous white solid

1

(9.3 g, 85% over three steps). H NMR (300 MHz, CDCl₃): δ 7.48 (d, 1H, J =

7.5 Hz), 7.37–7.45 (m, 2H), 7.33–7.36 (m, 2H), 7.19 (d, 2H, J = 7.5 Hz), 5.47

(s, 1H), 5.24 (t, 1H, J = 10.2 Hz), 4.55 (d, 1H, J = 10.2 Hz), 3.38 (dd, 1H,

J = 3.9, 10.5 Hz), 3.77 (t, 1H, J = 10.5 Hz), 3.48–3.58 (m, 2H), 3.39 (t, 1H,

J = 9.9 Hz), 2.39 (s, 3H), 2.12 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 169.72,

139.37, 136.82, 134.49, 130.11, 129.26, 128.37, 126.71, 126.23, 101.56, 87.10,

78.37, 73.12, 70.76, 68.51, 63.57, 21.38, 20.93; IR (NaCl): 2110, 1754, 1368,

1216, 1099 cm⁻¹; [α]²⁰= −121.0 (c 2.00, CH₂Cl₂); HRESI-MS: m/z calcd for

D

C₂₂H₃₃N₃NaO₅S [M+Na]⁺: 464.1256; found: 464.1246.

The intermediate above (5 g, 11.3 mmol) was dissolved in a 1 M solution

of BH₃in THF (113 mL), then treated with a 1 M solution of Bu₂BOTf in

THF (11.9 mL) at 0^◦C in the presence of 2,6-lutidine (2.63 mL, 22.6 mmol).^[11]

The mixture was stirred for 10 h at 0^◦C, then cooled to −78^◦C and quenched

with Et₃N (3.14 mL, 22.6 mmol), followed by the slow addition of cold MeOH

(60 mL). The mixture was slowly warmed to rt, concentrated, then puriﬁed by

silica gel chromatography (20% EtOAc in hexanes) to yield the intermediate C6

alcohol (4.04 g, 10.68 mmol). This was dissolved in DMF (14 mL) and treated

with imidazole (2.2 g, 32.04 mmol) and TBDPS-Cl (5.56 mL, 21.36 mmol). The

mixture was stirred for 12 h at rt, quenched with saturated NaHCO₃solution

(40 mL), extracted with CH₂Cl₂(2 × 100 mL), washed with brine (40 mL),

dried over Na₂SO₄, then concentrated under reduced pressure. Puriﬁcation

by silica gel chromatography (6.25% EtOAc in hexanes) yielded 6-O-TBDPS

1

ether 6 as an amorphous white solid (5.04 g, 62% over two steps). H NMR

(300 MHz, CDCl₃): δ 7.82 (dd, 2H, J = 1.8, 7.5 Hz), 7.73 (dd, 2H, J = 1.2,

7.5 Hz), 7.54 (d, 2H, J = 8.4 Hz), 7.25–7.45 (m, 9H), 7.15–7.18 (m, 2H), 7.07

(d, 2H, J = 8.1 Hz), 5.18 (t, 1H, J = 9.3 Hz), 4.61 (dd, 2H, J = 11.1, 19.8 Hz),

4.47 (d, 1H, J = 10.2 Hz), 4.02 (dd, 1H, J = 1.2, 11.7 Hz), 3.94 (dd, 1H, J =

3, 11.7 Hz), 3.79 (t, 1H, J = 9.3 Hz), 3.42 (d, 1H, J = 9.3 Hz), 3.35 (t, 1H, J =

9.9 Hz), 2.34 (s, 3H), 2.02 (s, 3H), 1.14 (s, 9H); ¹³C NMR (75 MHz, CDCl₃): δ

169.94, 138.81, 137.77, 135.97, 135.72, 134.26, 133.36, 132.83, 129.97, 128.62,

127.97, 127.89, 127.36, 86.14, 80.08, 76.32, 75.48, 74.91, 63.21, 62.37, 27.05,

21.35, 21.01, 19.547; IR (NaCl): 2030, 2857, 2109, 1753, 1218, 1112, 1045 cm⁻¹

;

2-Aminoethyl Glucosamine Sulfoforms

395

[α]²⁰= −60.1 (c 1.21, CH₂Cl₂); HRESI-MS: m/z calcd for C₃₈H₄₃N₃NaO₅SSi

[M+^DNa]⁺: 704.2590; found: 704.2595.

2^ꢀ-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl

3-O-acetyl-2-azido-4-O-benzyl-6-O-

(tert-butyldiphenylsilyl)-2-deoxy-α-D-glucopyranoside (7a)

A solution of ethanolamine (130 µL, 2.13 mmol) in CH₂Cl₂(6 mL) was

treated at 0^◦C with Fmoc-Cl (500 mg, 1.93 mmol) in the presence of NaHCO₃

(974 mg, 11.6 mmol). The mixture was warmed up to rt over 2 h and contin-

uously stirred for another 8 h, extracted with CH₂Cl₂(3 × 100 mL), washed

with water (30 mL) and brine (30 mL), and dried over Na₂SO₄, then concen-

trated to afford Fmoc-protected ethanolamine in 96% yield as an amorphous

white solid

A mixture of thioglycoside 6 (500 mg, 0.73 mmol) and 2^ꢁ-hydroxyethyl 2-

(9H-ﬂuoren-9-yl)methylcarbamate (270 mg, 0.95 mmol) was azeotroped with

toluene (3 × 4 mL), dried under reduced pressure for 1 h, then dissolved in

1:2 (CH₂Cl)₂:Et₂O (6 mL) and stirred with activated 4A molecular sieves for

1 h at rt under Ar. The mixture was cooled to −20^◦C and treated with N-

iodosuccinimide (180 mg, 0.8 mmol) and TfOH (10 µL, 0.11 mmol). The mixture

was stirred continuously for another 5 h at −20^◦C, quenched with Et₃N (35 µL,

0.22 mmol), warmed to rt, ﬁltered through Celite, extracted with CH₂Cl₂(3 ×

70 mL), washed with brine (40 mL), dried over Na₂SO₄, then concentrated un-

der reduced pressure to yield the corresponding Fmoc-protected 2^ꢁ-aminoethyl

glycoside as a 4.2:1 mixture of anomers. Puriﬁcation by silica gel chromatogra-

phy (11% EtOAc in hexanes) yielded α-GlcN derivative 7a (486 mg, 80%) and

β-GlcN derivative 7b (116 mg, 19%) as colorless oils.

1

Major isomer 7a: H NMR (400 MHz, CDCl₃): δ 7.77 (d, 2H, J = 7.5 Hz),

7.72 (t, 4H, J = 6.7 Hz), 7.61 (dd, 2H, J = 4.6, 7.1 Hz), 7.37–7.49 (m, 8H),

7.27–7.35 (m, 5H), 7.20 (dd, 2H, J = 2.5, 7.6 Hz), 5.63 (m, 1H), 5.39 (t, 1H,

J = 5.4 Hz), 5.01 (d, 1H, J = 3.3 Hz), 4.67 (s, 2H), 4.37 (d, 2H, J = 7.3 Hz),

4.22 (t, 1H, J = 7.1 Hz), 3.90–3.98 (m, 2H), 3.77–3.86 (m, 3H), 3.58 (t, 2H,

J = 8.4 Hz), 3.35 (m, 1H), 3.17 (dd, 1H, J = 3.4, 10.6 Hz), 2.09 (s, 3H), 1.12

(s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.00, 156.35, 143.95, 143.82, 141.22,

137.63, 135.74, 135.53, 133.27, 132.85, 129.75, 128.44, 127.84, 127.71, 127.62,

127.00, 125.16, 119.86, 98.39, 74.89, 72.10, 71.83, 67.65, 66.87, 62.37, 61.50,

47.10, 40.60, 26.82, 20.90, 19.29; IR (NaCl): 2929, 2108, 1726, 1517, 1226,

1114, 1041 cm⁻¹; [α]²⁰= +80.1 (c 0.89, CH₂Cl₂); HRESI-MS: m/z calcd for

D

C₄₈H₅₂N₄NaO₈Si [M+Na]⁺: 863.3452; found: 863.3462.

1

Minor isomer 7b: H NMR (400 MHz, CDCl₃): δ 7.65–7.80 (m, 6H), 7.57

(dd, 2H, J = 7.5, 11.5 Hz), 7.26–7.46 (m, 13H), 7.20 (dd, 2H, J = 2.1, 7.5 Hz),

5.27 (t, 1H, J = 5.7 Hz), 5.07 (t, 1H, J = 9.9 Hz), 4.60–4.70 (m, 2H), 4.40 (dd,

2H, J = 4.1, 7.0 Hz), 4.32 (d, 2H, J = 8.0 Hz), 4.18 (t, 1H, J = 6.8 Hz), 3.89

R. Liu and A. Wei

396

(m, 2H), 3.81 (t, 1H, J = 9.5 Hz), 3.68 (m, 1H), 3.40–3.55 (m, 3H), 3.32 (d, 1H,

J = 9.5 Hz), 2.05 (s, 3H), 1.09 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.01,

156.60, 144.03, 141.42, 137.79, 135.98, 135.65, 133.49, 132.89, 129.96, 128.66,

128.10, 127.95, 127.93, 127.85, 127.78, 127.17, 125.25, 125.17, 120.15, 102.30,

75.88, 75.04, 74.00, 69.67, 66.71, 64.44, 62.32, 47.39, 41.17, 26.99, 21.09, 19.47;

found: 863.3461; IR (NaCl): 2932, 2110, 1752, 1726, 1225, 1050 cm⁻¹; [α]²⁰

=

D

−5.3 (c 1.57, CH₂Cl₂); HRESI-MS: m/z calcd for C₄₈H₅₂N₄NaO₈Si [M+Na]⁺:

863.3452.

2^ꢀ-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-

(tert-butyldiphenylsilyl)-2-deoxy-α-D-glucopyranoside (8a)

Fmoc-protected 7a (840 mg, 1 mmol) was dissolved in 1:4 piperidine:DMF

(5 mL) and stirred at rt for 30 min. The mixture was concentrated and pu-

riﬁed by silica gel chromatography (EtOAc) to afford 2^ꢁ-aminoethyl α-GlcN

1

derivative 8a as a colorless oil (612 mg, 99%). H NMR (400 MHz, CDCl₃):

δ 7.72 (dt, 4H, J = 1.1, 8.3 Hz), 7.36–7.46 (m, 6H), 7.25–7.36 (m, 3H), 7.20

(dd, 2H, J = 2.1, 7.7 Hz), 5.61 (m, 1H), 5.03 (d, 1H, J = 3.6 Hz), 4.66 (s, 2H),

3.90–4.03 (m, 2H), 3.83 (d, 2H, J = 5.6 Hz), 3.77 (m, 1H), 3.50 (m, 1H), 3.15 (dd,

1H, J = 3.5, 10.8 Hz), 2.80–2.95 (m, 2H), 2.06 (s, 3H), 1.11 (s, 9H); ¹³C NMR

(100 MHz, CDCl₃): δ 167.93, 137.71, 135.76, 135.53, 133.35, 132.92, 129.71,

128.42, 127.79, 127.70, 127.63, 127.59, 98.26, 76.21, 74.80, 72.16, 71.58, 70.65,

62.40, 61.55, 41.56, 26.82, 20.89, 19.30; IR (NaCl): 2929, 2856, 2106, 1748,

1225, 1111, 1042 cm⁻¹; [α]²⁰= +96.5 (c 1.33, CH₂Cl₂); HRESI-MS: m/z calcd

D

for C₃₃H₄₃N₄O₆Si [M+H]⁺: 619.2952; found: 619.2955.

2^ꢀ-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-

(tert-butyldiphenylsilyl)-2-deoxy-β-D-glucopyranoside (8b)

Fmoc-protected 7b (266 mg, 0.32 mmol) was dissolved in 1:4 piperi-

dine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated

and puriﬁed by silica gel chromatography (EtOAc) to afford 2^ꢁ-aminoethyl β-

GlcN derivative 8b as a colorless oil (194 mg, 99%). ¹H NMR (400 MHz, CDCl₃):

δ 7.75 (dd, 2H, J = 1.6, 8.0 Hz), 7.70 (dd, 2H, J = 1.0, 7.5 Hz), 7.44 (t, 2H,

J = 7.2 Hz), 7.38 (t, 4H, J = 7.1 Hz), 7.25–7.35 (m, 3H), 7.20 (dd, 2H, J = 2.1,

7.6 Hz), 5.08 (t, 1H, J = 9.8 Hz), 4.66 (q, 2H, J = 11.1 Hz), 4.42 (d, 1H, J =

8.0 Hz), 3.90–4.01 (m, 3H), 3.83 (t, 1H, J = 9.5 Hz), 3.62 (m, 1H), 3.46 (dd, 1H,

J = 8.0, 10.3 Hz), 3.39 (td, 1H, J = 2.5, 9.6 Hz), 2.90–3.06 (m, 2H), 2.04 (s, 3H),

1.43 (br, 2H), 1.11 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.83, 137.60, 135.81,

135.46, 133.36, 132.79, 129.71, 128.44, 127.85, 127.72, 127.70, 127.54, 101.99,

75.75, 75.66, 74.79, 73.94, 72.46, 64.36, 62.24, 41.85, 26.78, 20.88, 19.29; IR

(KBr): 2930, 2857, 2110, 1752, 1226, 1112, 1047 cm⁻¹; [α]²⁰= −13.8 (c 0.72,

D

2-Aminoethyl Glucosamine Sulfoforms

397

CH₂Cl₂); HRESI-MS: m/z calcd for C₃₃H₄₃N₄O₆Si [M+H]⁺: 619.2952; found:

619.2949.

Thiotolyl 3-O-acetyl-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-

2-deoxy-2-(trichloro- ethylcarbonylamino)-β-

D-glucopyranoside (9)

3-O-acetate 6 (600 mg, 0.88 mmol) was dissolved in anhydrous MeOH

(20 mL) and treated at rt with 1 M NaOMe solution in MeOH (264 µL, 0.26

mmol). The mixture was stirred at rt for 3 h, neutralized with activated Dowex

50X-W-H⁺ion-exchange resin, ﬁltered, concentrated, and dried under reduced

pressure. A solution of the crude alcohol in CH₂Cl₂(10 mL) was treated at rt

with Bu₃P (241 µL, 0.97 mmol), followed by addition of H₂O (10 mL) after 3 h.

The mixture was stirred at rt for 1 day, concentrated, azeotroped with toluene

(3 × 10 mL), and dried under reduced pressure. The crude amine was redis-

persed in pyridine (10 mL) and treated with Troc-Cl (127 µL, 0.92 mmol) at

rt. The mixture was stirred at rt for 10 h and concentrated. The crude product

was redispersed in pyridine (20 mL) and treated with Ac₂O (10 mL) at rt. The

mixture was stirred at rt for 10 h, concentrated, and puriﬁed by silica gel chro-

matography (20% EtOAc in hexanes) to afford Troc-protected derivative 9 as

1

an amorphous white solid (581 mg, 94%). H NMR (400 MHz, CDCl₃): δ 7.83

(dd, 2H, J = 1.2, 7.9 Hz), 7.75 (dd, 2H, J = 1.0, 7.9 Hz), 7.51–7.37 (m, 8H),

7.16–7.23 (m, 2H), 7.07 (d, 4H, J = 8.1 Hz), 5.70 (d, 1H, J = 9.9 Hz), 5.28 (t,

1H, J = 9.8 Hz), 4.89 (d, 1H, J = 12.1 Hz), 4.78 (d, 1H, J = 12.0 Hz), 4.64 (d,

1H, J = 12.7 Hz), 4.62 (s, 2H), 3.77–3.95 (m, 4H), 3.40 (d, 1H, J = 9.6 Hz), 3.34

(s, 3H), 2.01 (s, 3H), 1.15 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 171.06, 154.33,

137.99, 137.59, 135.93, 135.56, 133.43, 133.22, 132.68, 129.70, 129.63, 128.94,

128.44, 127.88, 127.77, 127.68, 95.65, 87.14, 79.56, 76.39, 75.50, 74.88, 74.45,

62.17, 55.15, 26.88, 21.16, 20.94, 19.27; IR (NaCl): 2930, 2858, 1749, 1728,

1242, 1112, 1085, 1035 cm⁻¹; [α]²⁰= −24.8 (c 0.81, CH₂Cl₂); HRESI-MS: m/z

D

calcd for C₄₁H₄₆Cl₃NNaO₇SSi [M+Na]⁺: 852.1728; found: 852.1723.

2^ꢀ-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl 4-O-[3-O-

acetyl-4-O-benzyl-2-deoxy -6-O-(tert-butyldiphenylsilyl)-2-

(trichloroethylcarbonylamino)-β-D-glucopyranosyl]-2,3,6-tri-

O-benzyl-β-D-glucopyranoside (10)

A mixture of glycosyl donor 9 (560 mg, 0.68 mmol) and glycosyl acceptor

15 (580 mg, 0.81 mmol) was azeotroped with toluene (3 × 10 mL), dried under

reduced pressure for 1 h, then redissolved in 1:2 (CH₂Cl)₂:Et₂O (3 mL) with

freshly activated 4A molecular sieves and stirred at rt for 1 h. NIS (198 mg,

0.88 mmol) was added and the mixture was cooled to −30^◦C, followed by ad-

dition of TfOH (9 µL, 0.1 mmol). The mixture was stirred at −30^◦C for 6 h,

R. Liu and A. Wei

398

quenched with Et₃N (32 µL, 0.2 mmol), ﬁltered through Celite, treated with

aqueous Na₂S₂O₃(30 mL), extracted with CH₂Cl₂(3 × 100 mL), washed with

brine (20 mL), then dried over Na₂SO₄and concentrated. The crude product

was puriﬁed by silica gel chromatography (20% EtOAc in hexanes) to afford

1

1,4-β-linked disaccharide 10 as an amorphous white solid (860 mg, 90%). H

NMR (400 MHz, CDCl₃): δ 7.78–7.90 (m, 4H), 7.73 (d, 2H, J = 6.0 Hz), 7.64

(d, 2H, J = 7.4 Hz), 7.30–7.47 (m, 24H), 7.15–7.27 (m, 6H), 5.57 (t, 1H, J =

6.3 Hz), 4.97 (t, 1H, J = 10.4 Hz), 4.68–4.90 (m, 7H), 4.68 (s, 2H), 4.40–4.57

(m, 5H), 4.35 (d, 1H, J = 8.0 Hz), 4.24 (t, 1H, J = 5.6 Hz), 4.00 (t, 1H, J =

8.9 Hz), 3.58–3.95 (m, 9H), 3.33–3.53 (m, 4H), 3.18 (d, 1H, J = 9.7 Hz), 2.00 (s,

3H), 1.14 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.55, 156.53, 154.26, 144.00,

143.92, 141.30, 139.00, 138.28, 137.79, 137.44, 135.90, 135.65, 133.20, 132.90,

129.75, 128.72, 128.45, 128.30, 128.03, 128.00, 127.80, 127.64, 127.38, 127.04,

125.08, 125.03, 120.00, 103.83, 100.31, 95.64, 81.86, 77.30, 76.38, 75.84, 75.78,

74.82, 74.87, 74.82, 74.58, 74.33, 74.10, 73.60, 69.87, 68.20, 66.49, 62.21, 57.15,

47.27, 41.51, 26.93, 20.85, 19.22; IR (NaCl): 2941, 2858, 1748, 1729, 1520, 1452,

1236, 1109, 1052 cm⁻¹; [α]²⁰= −15.5 (c 0.84, CH₂Cl₂); ESI-MS: m/z calcd for

D

C₇₈H₈₃Cl₃N₂NaO₁₅Si [M+Na]⁺: 1443.45; found: 1443.44.

2^ꢀ-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl

4-O-[3-O-acetyl-2-azido-4-O-benzyl-

2-deoxy-6-O-(tert-butyldiphenylsilyl)-β-D-glucopyranosyl]-

2,3,6-tri-O-benzyl-β-D-glucopyranoside (11)

Troc-protected 10 (282 mg, 0.2 mmol) was dissolved in 3:2 EtOH:H₂O

(2 mL), treated with indium metal powder (46 mg, 0.4 mmol) and NH₄Cl

(32 mg, 0.6 mmol), then stirred for 6 h at 98^◦C in a high-pressure reaction ves-

sel. The mixture was concentrated, redispersed in 2:1 MeOH:H₂O (15 mL) with

K₂CO₃(41 mg, 0.3 mmol) and CuSO₄·5H₂O (5 mg, 0.02 mmol), then treated at

0^◦C with fresh prepared 0.03 M TfN₃solution in CH₂Cl₂(60 mL). The mixture

was warmed to rt slowly over 2 h and stirred at rt for another 8 h. The mix-

ture was quenched with solution of NaHCO₃(50 mL), ﬁltered through Celite,

extracted with CH₂Cl₂(3 × 50 mL), washed with brine (20 mL), then dried

over Na₂SO₄and concentrated. The crude product was puriﬁed by silica gel

chromatography (20% EtOAc in hexanes) to afford azide 11 as an amorphous

white solid (230 mg, 91%). ¹H NMR (400 MHz, CDCl₃): δ 7.80 (dd, 4H, J = 2.7,

7.4 Hz), 7.72 (dd, 2H, J = 1.1, 7.9 Hz), 7.62 (d, 2H, J = 7.5 Hz), 7.46 (dd, 4H,

J = 7.7, 9.0 Hz), 7.40 (t, 4H, J = 8.1 Hz), 7.27–7.38 (m, 15H), 7.25–7.25 (m,

7H), 5.58 (t, 1H, J = 5.4 Hz), 5.03 (t, 1H, J = 9.3 Hz), 4.89 (d, 2H, J = 11.1 Hz),

4.84 (d, 1H, J = 12.5 Hz), 4.76 (d, 1H, J = 11.1 Hz), 4.57–4.64 (m, 3H), 4.49 (d,

2H, J = 8.0 Hz), 4.46 (d, 1H, J = 4.5 Hz), 4.41 (dd, 2H, J = 6.7, 11.2 Hz), 4.23

(t, 1H, J = 6.7 Hz), 4.08 (t, 1H, J = 9.3 Hz), 3.60–3.92 (m, 8H), 3.38–3.55 (m,

2-Aminoethyl Glucosamine Sulfoforms

399

4H), 3.32 (dd, 1H, J = 8.3, 10.4 Hz), 3.05 (d, 1H, J = 9.6 Hz), 2.08 (s, 3H), 1.12

(s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.84, 156.51, 144.00, 143.90, 141.28,

138.38, 138.24, 137.71, 137.56, 135.87, 135.60, 133.13, 132.75, 129.75, 128.44,

128.32, 128.03, 127.98, 127.87, 127.81, 127.65, 127.63, 127.10, 127.01, 125.09,

125.01, 119.94, 103.82, 100.39, 81.86, 81.74, 77.26, 75.75, 74.85, 74.49, 74.30,

74.17, 73.43, 69.98, 68.53, 66.45, 65.27, 61.94, 47.26, 41.52, 26.92, 20.90, 19.20;

IR (NaCl): 2930, 2858, 2109, 1752, 1722, 1357, 1222, 1111, 1053 cm⁻¹; [α]²⁰

=

D

−4.9 (c 0.71, CH₂Cl₂); MALDI-MS: m/z calcd for C₇₅H₈₀N₄NaO₁₃Si [M+Na]⁺:

1295.5389; found: 1295.4942.

2^ꢀ-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-

(tert-butyldiphenylsilyl)-β-D-glucopyranosyl]

-2,3,6-O-benzyl-β-D-glucopyranoside (12)

Fmoc-protected 11 (184 mg, 0.14 mmol) was dissolved in 1:4 piperi-

dine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated

and puriﬁed by silica gel chromatography (5% MeOH in EtOAc) to afford 2^ꢁ-

aminoethyl β-glycoside 12 as a colorless oil (150 mg, 99%). ¹H NMR (400 MHz,

CDCl₃): δ 7.80 (d, 2H, J = 4.9 Hz), 7.70 (d, 2H, J = 7.2 Hz), 7.20–7.52 (m, 17H),

7.17–7.25 (m, 5H), 5.02 (t, 1H, J = 9.8), 4.94 (d, 1H, J = 11.0 Hz), 4.85–4.90

(m, 2H), 4.79 (d, 1H, J = 10.9 Hz), 4.71 (d, 1H, J = 12.2 Hz), 4.64 (dd, 2H, J =

11.3, 22.9 Hz), 4.57 (d, 1H, J = 12.3 Hz), 4.50 (dd, 2H, J = 8.3, 17.9 Hz), 4.17 (t,

1H, J = 8.7 Hz), 3.98 (m, 1H), 3.85 (s, 2H), 3.60–3.85 (m, 5H), 3.55 (t, 2H, J =

8.7 Hz), 3.31 (t, 1H, J = 9.4 Hz), 3.07 (d, 1H, J = 8.7 Hz), 2.95 (br, 1H), 2.08 (s,

3H), 1.13 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.84, 138.89, 138.33, 137.74,

135.87, 135.61, 133.13, 132.75, 129.75, 128.44, 128.29, 128.01, 127.95, 127.83,

127.62, 127.06, 103.64, 100.26, 81.89, 81.66, 77.33, 75.78, 75.74, 75.53, 74.86,

74.47, 74.17, 73.44, 72.41, 68.54, 65.17, 61.99, 26.92, 20.91, 19.19; IR (NaCl):

2930, 2856, 2109, 1752, 1223, 1109, 1055 cm⁻¹; [α]²⁰= −5.1 (c 0.55, CH₂Cl₂);

D

ESI-MS: m/z calcd for C₆₀H₇₁N₄O₁₁Si [M+H]⁺: 1051.49; found: 1051.36.

2^ꢀ-Azidoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (13)²⁰

To a solution of β-D-glucose pentaacetate (10 g, 25.6 mmol), 2-bromoethanol

(5.5 mL, 76.9 mmol), and N, N-diisopropylethylamine (223 µL, 1.3 mmol) in

CH₂Cl₂(60 mL) at 0^◦C was added a 1 M BF₃·Et₂O solution in CH₂Cl₂(4.9 mL,

4.9 mmol) slowly. The mixture was slowly warmed to rt over 2 h, stirred con-

tinually at rt for another 10 h, quenched with saturated NaHCO₃solution

(100 mL), extracted with CH₂Cl₂(3 × 200 mL), washed with brine (50 mL),

then dried over Na₂SO₄and concentrated. The crude product was puriﬁed by

silica gel chromatography (25% EtOAc in hexanes) to afford an amorphous

white solid (5.6 g). This was redispersed with NaN₃(3.2 g, 49.5 mmol) in

CH₂Cl₂and heated to reﬂux for 12 h, extracted with CH₂Cl₂(3 × 200 mL),

R. Liu and A. Wei

400

washed with brine (50 mL), then dried over Na₂SO₄and concentrated to af-

ford 2^ꢁ-azidoethyl β-glucoside 13 as an amorphous white solid (4.9 g, 46%). ¹H

NMR (400 MHz, CDCl₃): δ 5.19 (t, 1H, J = 9.5 Hz), 5.08 (t, 1H, J = 7.2 Hz),

5.01 (dd, 1H, J = 8.0, 9.6 Hz), 4.58 (d, 1H, J = 7.8 Hz), 4.23 (dd, 1H, J =

4.6, 12.3 Hz), 4.13 (dd, 1H, J = 2.4, 12.3 Hz), 4.01 (ddd, 1H, J = 1.0, 4.7,

10.9 Hz), 3.65–3.72 (m, 2H), 3.47 (ddd, 1H, J = 3.6, 8.4, 12.8 Hz), 3.26 (ddd,

1H, J = 3.6, 4.8, 12.8 Hz), 2.07 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H);

¹³C NMR (100 MHz, CDCl₃): δ 170.55, 170.16, 169.29, 100.54, 72.65, 71.79,

70.92, 68.48, 68.16, 61.69, 50.39, 20.63, 20.58, 20.49; IR (NaCl): 2945, 2106,

1756, 1369, 1226, 1039 cm⁻¹; [α]²⁰= −31.2 (c 1.14, CH₂Cl₂); ESI-MS: m/z

D

calcd for C₁₆H₂₃N₃NaO₁₀[M+Na]⁺: 440.1281; found: 440.1290.

2^ꢀ-Azidoethyl 2,3,6-tri-O-benzyl-β-D-glucopyranoside (14)

A solution of tetraacetate 13 (4.9 g, 11.8 mmol) was dissolved in anhydrous

MeOH (40 mL) and treated at rt with 1 M NaOMe solution in MeOH (3.5 mL).

The mixture was stirred at rt for 3 h, neutralized with activated Dowex 50X-

W-H⁺ion-exchange resin, ﬁltered, concentrated, and dried under reduced pres-

sure. A solution of the crude in THF (80 mL) was treated with benzaldehyde

dimethyl acetal (5.3 mL, 35.4 mmol) and TsOH·H₂O (225 mg, 1.18 mmol). The

mixture was heated to reﬂux for 10 h, quenched with saturated NaHCO₃so-

lution (30 mL), extracted with CH₂Cl₂(3 × 150 mL), dried over Na₂SO₄, and

concentrated under reduced pressure. The crude product was puriﬁed by sil-

ica gel chromatography (50% EtOAc in hexanes) to afford an amorphous white

solid (3.7 g). This was redispersed with BnBr (5.1 mL, 42.3 mmol) in DMF and

treated at 0^◦C with NaH (1.3 g, 32.5 mmol). The mixture was slowly warmed to

rt over 2 h and continuously stirred at rt for another 10 h, quenched with sat-

urated NH₄Cl solution (100 mL), extracted with CH₂Cl₂(3 × 200 mL), washed

with brine (50 mL), then dried over Na₂SO₄and concentrated. The crude prod-

uct was puriﬁed by silica gel chromatography (14% EtOAc in hexanes) to afford

the desired 2,3-di-O-benzyl glucoside as an amorphous white solid (4.5 g, 75%).

¹H NMR (400 MHz, CDCl₃): δ 7.52 (dd, 2H, J = 2.2, 7.9 Hz), 7.28–7.48 (m, 13H),

5.60 (s, 1H), 4.49 (dd, 2H, J = 1.7, 11.9 Hz), 4.82 (dd, 2H, J = 4.4, 11.4 Hz), 4.56

(d, 1H, J = 7.8 Hz), 4.38 (dd, 1H, J = 4.8, 10.4 Hz), 4.06 (ddd, 1H, J = 4, 6.4,

10.4 Hz), 3.62–3.86 (m, 2H), 3.38–3.55 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ

138.37, 138.27, 137.19, 128.92, 128.31, 128.26, 128.20, 128.04, 127.97, 127.69,

127.60, 125.94, 103.99, 101.07, 82.03, 81.31, 80.75, 75.39, 75.07, 68.64, 68.49,

66.01, 50.91; IR (NaCl): 2883, 2104, 1094, 1072 cm⁻¹; [α]²⁰= −33.5 (c 0.79,

CH₂Cl₂); ESI-MS: m/z calcd for C₂₉H₃₀N₃NaO₆[M+Na]⁺^D: 540.2111; found:

540.2102.

A solution of the protected intermediate above (4.4 g, 8.6 mmol) and Et₃SiH

(5.5 mL, 34.2 mmol) in CH₂Cl₂(86 mL) was cooled to −78^◦C and treated with

TfOH (3 mL, 34.2 mmol) in the presence of 4A molecular sieves. The mixture

2-Aminoethyl Glucosamine Sulfoforms

401

was stirred at −78^◦C for 6 h, quenched with Et₃N (5.4 mL, 38.5 mmol) and

MeOH (5 mL), warmed up to rt slowly over 1 h, ﬁltered through Celite, ex-

tracted with CH₂Cl₂(3 × 100 mL), washed with brine (20 mL), then dried over

Na₂SO₄and concentrated. The crude product was puriﬁed by silica gel chro-

matography (20% EtOAc in hexanes) to afford 2,3,6-tri-O-benzyl glucoside 14

1

as an amorphous white solid (3.3 g, 75%). H NMR (400 MHz, CDCl₃): δ 7.34

(m, 15H), 4.96 (t, 2H, J = 10.9 Hz), 4.74 (dd, 2H, J = 3.8, 11.0 Hz), 4.59 (dd, 2H,

J = 11.6, 17.2 Hz), 4.46 (dd, 1H, J = 2.4, 5.6 Hz), 4.06 (ddd, 1H, J = 3.6, 5.6,

10.4 Hz), 3.78 (dd, 1H, J = 3.5, 10.4 Hz), 3.67–3.79 (m, 2H), 3.59 (d, 1H, J =

8.8 Hz), 3.40–3.55 (m, 5H), 2.51 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 138.47,

138.30, 137.80, 128.50, 128.37, 128.33, 128.06, 127.90, 127.81, 127.69, 127.65,

103.64, 83.85, 81.64, 75.21, 74.76, 74.10, 73.59, 71.21, 70.04, 68.12, 50.96; IR

(NaCl): 3442, 2872, 2103, 1452, 1064 cm⁻¹; [α]²⁰= −15.3 (c 0.90, CH₂Cl₂);

D

ESI-MS: m/z calcd for C₂₉H₃₃N₃NaO₆[M+Na]⁺: 542.2267; found: 542.2272.

2^ꢀ-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl

2,3,6-tri-O-benzyl-β-D-glucopyranoside (15)

A solution of 2^ꢁ-azidoethyl glucoside 14 (2.4 g, 4.6 mmol) in CH₂Cl₂(30 mL)

was treated at rt with Bu₃P (1.3 mL, 5.1 mmol), followed by addition of H₂O

(30 mL) after 3 h. The mixture was stirred at rt for 1 day, concentrated,

azeotroped with toluene (3 × 10 mL), and dried under reduced pressure to

afford a yellowish oil. This was redispersed with NaHCO₃(2.3 g, 27.9 mmol)

in CH₂Cl₂(35 mL) and treated at 0^◦C with Fmoc-Cl (1.2 g, 4.6 mmol) dis-

solved in CH₂Cl₂(5 mL). The mixture was slowly warmed to rt over 2 h and

stirred continually for another 10 h at rt, extracted with CH₂Cl₂(3 × 100 mL),

washed with brine (20 mL), then dried over Na₂SO₄and concentrated. The

crude product was puriﬁed by silica gel chromatography (20% EtOAc in hex-

anes) to afford Fmoc-protected 15 as an amorphous white solid (1.6 g, 54%). ¹H

NMR (400 MHz, CDCl₃): δ 7.77 (d, 2H, J = 7.5 Hz), 7.61 (d, 2H, J = 7.4 Hz),

7.20–7.55 (m, 19H), 5.61 (t, 1H, J = 5.0 Hz), 4.96 (dd, 2H, J = 11.5, 20.1 Hz),

4.78 (d, 2H, J = 11.4 Hz), 4.52 (dd, 2H, J = 12.1, 26.7 Hz), 4.35–4.45 (m, 3H),

4.19 (t, 1H, J = 6.7 Hz), 3.79–3.90 (m, 2H), 3.75 (dd, 1H, J = 3.2, 10.2 Hz), 3.66

(dd, 1H, J = 5.8, 10.1 Hz), 3.59 (t, 1H, J = 8.8 Hz), 3.25–3.52 (m, 5H), 2.45 (br,

1H); ¹³C NMR (100 MHz, CDCl₃): δ 156.52, 144.01, 143.87, 141.26, 138.46,

138.21, 137.67, 128.55, 128.42, 128.36, 127.98, 127.96, 127.89, 127.77, 127.69,

127.64, 126.99, 125.03, 119.93, 103.96, 83.92, 81.65, 75.22, 74.79, 73.97, 73.53,

71.23, 70.13, 69.96, 66.43, 47.23, 41.56; IR (NaCl): 3430, 3031, 2874, 1711,

1452, 1251, 1062 cm⁻¹; [α]²⁰= −10.9 (c 1.31, CH₂Cl₂); ESI–MS: m/z calcd for

D

C₄₄H₄₅NNaO₈[M+Na]⁺: 738.3043; found: 738.3045.

R. Liu and A. Wei

402

2^ꢀ-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl

4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-

(tert-butyldiphenylsilyl)-α-D-glucopyranosyl]-2,3,6-tri-O-

benzyl-β-D-glucopyranoside (16)

A mixture of glycosyl acceptor 15 (503 mg, 0.7 mmol) and glycosyl donor

6 (400 mg, 0.59 mmol) was azeotroped with toluene (3 × 10 mL), dried under

reduced pressure for 1 h, then redispersed in 1:2 (CH₂Cl)₂:Et₂O (3 mL) with

freshly activated 4A molecular sieves and stirred at rt for 1 h. NIS (145 mg,

0.65 mmol) was added and the mixture was cooled to −20^◦C, followed by ad-

dition of TfOH (16 µL, 0.18 mmol). The mixture was stirred at −20^◦C for

10 h, quenched with Et₃N (58 µL, 0.42 mmol), and ﬁltered through Celite,

then treated with saturated Na₂S₂O₃(30 mL), extracted with CH₂Cl₂(3 ×

100 mL), washed with brine (20 mL), then dried over Na₂SO₄and concen-

trated. The crude product was puriﬁed by silica gel chromatography (20%

EtOAc in hexanes) to afford 1,4-α-linked disaccharide 16 as an amorphous

1

white solid (447 mg, 60%). H NMR (400 MHz, CDCl₃): δ 7.77 (dd, 2H, J =

3.1, 7.3 Hz), 7.62 (ddd, 4H, J = 0.9, 8.0, 12.3 Hz), 7.56 (t, 2H, J = 7.1 Hz),

7.25–7.50 (m, 24H), 7.21 (d, 2H, J = 7.4 Hz), 6.94–7.06 (m, 4H), 5.69 (d,

1H, J = 3.7 Hz), 5.65 (t, 1H, J = 10.3 Hz), 5.10 (d, 1H, J = 10.9 Hz), 4.90

(d, 1H, J = 11.0 Hz), 4.82 (d, 1H, J = 10.8 Hz), 4.69 (d, 1H, J = 11.0 Hz),

4.66 (s, 2H), 4.54 (m, 1H), 4.35 (dd, 1H, J = 6.1, 10.6 Hz), 4.25 (d, 1H, J =

7.6 Hz), 4.23 (d, 1H, J = 15.2 Hz), 4.15 (m, 1H), 3.74–3.90 (m, 4H), 3.69 (d, 2H,

J = 11.6 Hz), 3.57 (d, 2H, J = 10.5 Hz), 3.21–3.57 (m, 7H), 3.06 (dd, 1H, J =

3.7, 10.8 Hz), 2.06 (s, 3H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 170.03,

156.58, 144.07, 143.74, 141.39, 141.33, 138.47, 138.18, 138.02, 137.49, 135.82,

135.56, 133.49, 132.87, 129.68, 128.42, 128.15, 128.04, 127.89, 127.70, 127.62,

127.45, 127.31, 127.03, 126.94, 125.03, 124.79, 119.98, 104.02, 97.96, 84.62,

82.63, 75.74, 74.81, 74.64, 74.59, 73.57, 73.21, 73.08, 72.06, 70.83, 68.77, 65.79,

61.68, 61.41, 47.44, 41.76, 26.91, 20.92, 19.32; IR (NaCl): 2929, 2858, 2108,

1749, 1730, 1227, 1043 cm⁻¹; [α]²⁰= +42.6 (c 0.29, CH₂Cl₂); ESI-MS: m/z

D

calcd for C₇₅H₈₀N₄NaO₁₃Si [M+Na]⁺: 1295.54; found: 1295.29.

2^ꢀ-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-

2-deoxy-6-O-(tert-butyldiphenylsilyl)-α-D-glucopyranosyl]-

2,3,6-tri-O-benzyl-β-D-glucopyranoside (17)

Fmoc-protected 16 (427 mg, 0.24 mmol) was dissolved in 1:4 piperi-

dine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated

and puriﬁed by silica gel chromatography (EtOAc) to afford 2^ꢁ-aminoethyl β-

glycoside 17 as a colorless oil (349 mg, 99%). ¹H NMR (400 MHz, CDCl₃): δ 7.65

(dd, 4H, J = 6.9, 15.0 Hz), 7.41–7.47 (m, 2H), 7.25–7.40 (m, 18H), 7.21 (dd, 2H,

J = 1.8, 8.1 Hz), 7.13 (dd, 2H, J = 1.8, 7.5 Hz), 7.06 (dd, 2H, J = 1.1, 5.9 Hz),

2-Aminoethyl Glucosamine Sulfoforms

403

5.73 (d, 1H, J = 3.8 Hz), 5.49 (t, 1H, J = 9.5 Hz), 5.12 (d, 1H, J = 10.9 Hz), 4.97

(d, 1H, J = 11.0 Hz), 4.83 (d, 1H, J = 10.8 Hz), 4.75 (d, 1H, J = 11.0 Hz), 4.68

(d, 1H, J = 13.9 Hz), 4.68 (d, 2H, J = 2.6 Hz), 4.46 (d, 1H, J = 7.8 Hz), 4.42

(d, 1H, J = 5.7 Hz), 4.41 (d, 1H, J = 13.9 Hz), 3.77–3.95 (m, 4H), 3.57–3.75 (m,

6H), 3.40–3.55 (m, 2H), 3.07 (dd, 1H, J = 3.8, 10.8 Hz), 2.82–2.97 (m, 2H), 2.10

(br, 1H), 2.05 (s, 3H), 1.08 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 169.94, 138.46,

138.14, 137.96, 137.73, 135.79, 135.52, 133.49, 132.88, 129.62, 128.39, 128.35,

128.15, 127.86, 127.70, 127.64, 127.60, 127.54, 127.45, 127.39, 127.25, 103.52,

97.48, 84.68, 82.57, 75.75, 74.73, 74.66, 74.50, 73.99, 73.24, 72.93, 72.20, 72.02,

71.97, 68.95, 61.76, 61.39, 41.89, 26.87, 20.87, 19.29; IR (NaCl): 3030, 2930,

2857, 2107, 1751, 1360, 1225, 1090, 1039 cm⁻¹; [α]²⁰_D= +53.9 (c 1.21, CH₂Cl₂);

ESI-MS: m/z calcd for C₆₀H₇₁N₄O₁₁Si [M+H]⁺: 1051.4889; found: 1051.4905.

2^ꢀ-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-

butyldiphenylsilyl)-2-deoxy-α-D-glucopyranoside, Bound to

Trityl–PS Resin (18a)

Cl-Trityl–PS resin beads (1.29 g, 1.93 mmol) were suspended in dry pyri-

dine (3 mL) for 5 min, followed by the addition of 2^ꢁ-aminoethyl α-GlcN glyco-

side 8a (597 mg, 0.96 mmol) and DMAP (47 mg, 0.39 mmol). The mixture was

shaken for 2 days at 60^◦C, then cooled to rt. Anhydrous MeOH (0.5 mL) was

added and the mixture was shaken at rt for 1 h. The resin was ﬁltered and

washed with 5% iPr₂NEt in 8:1 CH₂Cl₂:MeOH (2 × 4 mL), then alternatively

with DMF (2 × 4 mL) and CH₂Cl₂(2 × 4 mL), then ﬁnally with additional

CH₂Cl₂(3 × 4 mL). The resin was dried under reduced pressure in the pres-

ence of KOH pellets to afford fully resin-bound α-GlcN derivative 18a, with

a loading ratio of 0.38 mmol/g. IR (KBr): 2925, 2107, 1749, 1644, 1491, 1447,

1223, 1074 cm⁻¹. To determine the loading ratio, 50 mg of 18a was treated

with 30% HFIP in CH₂Cl₂(3 mL) at rt for 1 h. The combined washings were

concentrated under reduced pressure for recovery of free α-GlcN derivative 8a.

2^ꢀ-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-

butyldiphenylsilyl)-2-deoxy-β-D-glucopyranoside, Bound to

Trityl–PS Resin (18b)

Cl-Trityl–PS resin beads (386 mg, 0.55 mmol) were suspended in dry pyri-

dine (3 mL) for 5 min, followed by addition of 2^ꢁ-aminoethyl β-GlcN glycoside

8b (170 mg, 0.28 mmol) and DMAP (13 mg, 0.11 mmol). The mixture was

shaken for 2 days at 60^◦C, then cooled to rt. Anhydrous MeOH (0.5 mL) was

added and the mixture was shaken at rt for 1 h. The resin was ﬁltered and

washed with 5% iPr₂NEt in 8:1 CH₂Cl₂:MeOH (2 × 4 mL), then alternatively

with DMF (2 × 4 mL) and CH₂Cl₂(2 × 4 mL), then ﬁnally with additional

R. Liu and A. Wei

404

CH₂Cl₂(3 × 4 mL). The resin was dried under reduced pressure in the pres-

ence of KOH pellets to afford fully resin-bound β-GlcN derivative 18b, with a

loading ratio of 0.27 mmol/g. To determine the loading ratio, 50 mg of 18b was

treated with 30% HFIP in CH₂Cl₂(3 mL) under standard cleavage conditions.

The combined washings were then concentrated for recovery of free β-GlcN

derivative 8b.

2^ꢀ-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-

(tert-butyldiphenylsilyl)-β-D-glucopyranosyl]-2,3,6-

tri-O-benzyl-β-D-glucopyranoside, Bound to Trityl–PS Resin (19)

Cl-Trityl–PS resin beads (294 mg, 0.44 mmol) were suspended in dry pyri-

dine (3 mL) for 5 min, followed by addition of GlcN(β1→4)-Glcβ derivative

12 (232 mg, 0.22 mmol) and DMAP (10.8 mg, 0.088 mmol). The mixture was

shaken for 2 days at 60^◦C, then cooled to rt. Anhydrous MeOH (0.3 mL) was

added and the mixture was shaken at rt for 1 h. The resin was ﬁltered and

washed with 5% iPr₂NEt in 8:1 CH₂Cl₂:MeOH (2 × 4 mL), then alternatively

with DMF (2 × 4 mL) and CH₂Cl₂(2 × 4 mL), then ﬁnally with additional

CH₂Cl₂(3 × 4 mL). The resin was dried under reduced pressure in the pres-

ence of KOH pellets to afford fully resin-bound GlcN(β1→4)-Glcβ derivative

19, with a ratio of 0.17–0.18 mmol/g. To determine the loading ratio, 50 mg

of 19 was treated with 30% HFIP in CH₂Cl₂(3 mL) under standard cleavage

conditions. The combined washings were concentrated under reduced pressure

for recovery of free GlcN(β1→4)-Glcβ derivative 12.

2^ꢀ-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-

(tert-butyldiphenylsilyl)-α-D-glucopyranosyl]-2,3,6-

tri-O-benzyl-β-D-glucopyranoside, Bound to Trityl–PS Resin (20)

Cl-Trityl–PS resin beads (267 mg, 0.40 mmol) were suspended in dry pyri-

dine (3 mL) for 5 min followed by addition of GlcN(α1→4)-Glcβ derivative 17

(140 mg, 0.13 mmol) and DMAP (6.5 mg, 0.053 mmol). The mixture was shaken

for 2 days at 60^◦C, then cooled to rt. Anhydrous MeOH (0.3 mL) was added and

the mixture was shaken at rt for 1 h. The resin was ﬁltered and washed with

5% iPr₂NEt in 8:1 CH₂Cl₂:MeOH (2 × 4 mL), then alternatively with DMF

(2 × 4 mL) and CH₂Cl₂(2 × 4 mL), then ﬁnally with additional CH₂Cl₂(3 ×

4 mL). The resin was dried under reduced pressure in the presence of KOH

pellets to afford resin-bound GlcN(α1→4)-Glcβ derivative 20, with a ratio of

0.17–0.18 mmol/g. To determine the loading ratio, 50 mg of 20 was treated

with 30% HFIP in CH₂Cl₂(3 mL) under standard cleavage conditions. The

combined washings were concentrated under reduced pressure for recovery of

free GlcN(α1→4)-Glcβ derivative 17.

2-Aminoethyl Glucosamine Sulfoforms

405

2^ꢀ-Aminoethyl 2-acetamido-4-O-benzyl-2-

deoxy-α-D-glucopyranoside (1a)

Op. 1: Resin-bound α-GlcN derivative 18a (44 mg, 0.0167 mmol sugar)

was swollen in CH₂Cl₂(3 mL) for 5 min, then treated with Bu₃P (25 µL, 0.1

mmol). The mixture was shaken for 5 h at rt, ﬁltered, then subjected to stan-

dard workup conditions to give the intermediate N-ylide. Op. 2: The resin was

treated with 95:5 THF:H₂O (4 mL), shaken for 1 day at rt, washed with THF

(2 × 3 mL), then subjected to standard workup conditions to give the resin-

bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for

5 min, treated with Ac₂O (1.5 mL), and shaken for 1 day at rt. The resin was

ﬁltered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard

workup conditions to give the N-acetylated intermediate. Op. 4: The resin was

swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL),

and shaken for 1 day at rt. The resin was ﬁltered, washed with THF (2 × 3 mL),

then subjected to standard workup conditions to give the C6 alcohol. Op. 5:

The resin was swollen in CH₂Cl₂(2 mL) for 5 min, treated with 1 M NaOMe in

MeOH (50 µL), and shaken for 5 h at rt. The resin was ﬁltered, washed with

1:1 MeOH:CH₂Cl₂(2 × 3 mL), then subjected to standard workup conditions

to give the fully deprotected resin-bound α-GlcN. Op. 6: The resin was treated

with 30% HFIP in CH₂Cl₂under standard cleavage conditions. Puriﬁcation

by reverse-phase HPLC yielded 2^ꢁ-aminoethyl α-GlcNAc 1a as an amorphous

1

white solid (5.1 mg, 87% over six operations). H NMR (400 MHz, CD₃OD): δ

7.23–7.44 (m, 5H), 4.95 (d, 1H, J = 10.8 Hz), 4.75 (d, 1H, J = 3.6 Hz), 4.65 (d,

1H, J = 11.0 Hz), 3.99 (dd, 1H, J = 3.5, 10.5 Hz), 3.84 (dd, 1H, J = 8.5, 10.4 Hz),

3.77 (dd, 1H, J = 2.0, 9.8 Hz), 3.71 (dd, 1H, J = 5.0, 19.2 Hz), 3.57–3.69 (m,

2H), 3.43 (t, 1H, J = 9.2 Hz), 3.33–3.42 (m, 2H), 2.76–2.87 (m, 2H), 1.99 (s,

3H); ¹³C NMR (100 MHz, CD₃OD): δ 173.69, 139.94, 129.28, 129.07, 128.68,

98.86, 80.02, 76.05, 73.53, 73.05, 70.27, 62.32, 55.57, 42.05, 22.57; IR (NaCl):

3292, 2920, 1656, 1576, 1315, 1068, 1029 cm⁻¹; [α]²⁰= +58.1 (c 0.31, MeOH);

D

ESI-MS: m/z calcd for C₁₇H₂₆N₂O₆Na [M+Na]⁺: 377.1689; found: 377.1697.

2^ꢀ-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-6-O-

sulfonato-α-D-glucopyranoside, Sodium Salt (1b)

Op. 1: Resin-bound α-GlcN derivative 18a (44 mg, 0.0167 mmol sugar)

was swollen in CH₂Cl₂(3 mL) for 5 min, then treated with Bu₃P (25 µL, 0.1

mmol). The mixture was shaken for 5 h at rt, ﬁltered, then subjected to stan-

dard workup conditions to give the intermediate N-ylide. Op. 2: The resin was

treated with 95:5 THF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2

× 3 mL), then subjected to standard workup conditions to give the resin-bound

primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min,

treated with Ac₂O (1.5 mL), and shaken for 1 day at rt. The resin was ﬁltered,

R. Liu and A. Wei

406

washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup

conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen

in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF adjusted to pH 7

with AcOH (2.7 mL), and shaken for 1 day at rt. The resin was ﬁltered, washed

with 5:1 THF:pyridine (2 × 3 mL), then subjected to standard workup condi-

tions to give the C6 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for

5 min, then treated with SO₃·Py (27 mg, 0.17 mmol) and shaken for 10 h at rt.

The resin was ﬁltered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then sub-

jected to standard workup conditions to give the resin-bound 6-O-sulfate. Op.

6: The resin was swollen in CH₂Cl₂(2 mL) for 5 min, treated with 1 M NaOMe

in MeOH (50 µL), and shaken for 5 h at rt. The resin was ﬁltered, washed with

1:1 MeOH:CH₂Cl₂(2 × 3 mL), then subjected to standard workup conditions to

give the fully deprotected resin-bound 6-O-sulfate. Op. 7: The resin was treated

with 30% HFIP in CH₂Cl₂under standard cleavage conditions. Op.8: The crude

was passed through an ion-exchange column (Dowex Marathon MSC, Na form)

and puriﬁed by reverse-phase HPLC to yield 2^ꢁ-aminoethyl α-GlcNAc 6-O-

sulfate 1b (sodium salt) as an amorphous white solid (5.5 mg, 76% over eight

operations). ¹H NMR (400 MHz, CD₃OD): δ 7.41 (d, 2H, J = 7.1 Hz), 7.23–7.34

(m, 3H), 4.92 (d, 1H, J = 10.8 Hz), 4.78 (d, 1H, J = 3.7 Hz), 4.72 (d, 1H, J =

10.5 Hz), 4.34 (dd, 1H, J = 1.8, 11.0 Hz), 4.12 (dd, 1H, J = 6.2, 10.9 Hz), 4.03

(dd, 1H, J = 3.8, 10.8 Hz), 3.83–3.97 (m, 2H), 3.81 (dd, 1H, J = 9.0, 10.7 Hz),

3.52 (m, 1H), 3.41 (t, 1H, J = 9.4 Hz), 3.03 (t, 2H, J = 4.8 Hz), 2.01 (s, 3H);

¹³C NMR (100 MHz, CD₃OD): δ 173.63, 139.77, 129.47, 129.24, 128.67, 99.90,

79.87, 76.10, 73.59, 71.00, 68.88, 68.00, 55.40, 41.60, 22.63; IR (NaCl): 3449,

2925, 1659, 1543, 1223, 1222, 1068, 1013 cm⁻¹; [α]²⁰= +64.7 (c 0.41, MeOH);

D

ESI-MS: m/z calcd for C₁₇H₂₅N₂O₉S [M]⁻: 433.1281; found: 433.1279.

2^ꢀ-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-3-O-

sulfonato-α-D-glucopyranoside, Sodium Salt (1c)