Modular synthesis of core fucosylated N-glycans

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FULL PAPER

DOI: 10.1002/ejoc.201200468

Modular Synthesis of Core Fucosylated N-Glycans

Dimitri Ott,^[a]Joachim Seifert,^[a]Ingo Prahl,^[a]Mathäus Niemietz,^[a]Joanna Hoffman,^[a]

Janna Guder,^[a]Manuel Mönnich,^[a]and Carlo Unverzagt*^[a]

Dedicated to Professor Frieder Lichtenthaler on the occasion of his 80th birthday

Keywords: Carbohydrates / Glycosylation / Oligosaccharides / Glycoproteins / Stereoselectivity

A modular synthesis of complex-type N-glycans containing

the core fucosyl motif was optimized. The core trisaccharide

building block was protected by a methoxyphenyl group for

convenient core fucosylation. The trisaccharide was obtained

on a large scale from the glycosylation of the corresponding

chitobiosyl azide with a glucosyl donor followed by intramo-

lecular inversion. Improved methods were established for the

synthesis of the monosaccharide building blocks and for their

couplings. The inversion to the β-mannoside was ac-

companied by previously unnoticed side-reactions resulting

in the hydrolytic ring-opening of the iminocarbonate inter-

mediate. The benzylidene-protected core trisaccharide was

elongated into a biantennary N-glycan heptasaccharide by

two regio- and stereoselective couplings. The final fucosyl-

ation also gave some of the β anomer, which could be re-

moved by HPLC to give an α1,6-fucosylated N-glycan octa-

saccharide.

these features with another core modification, the bisecting

GlcNAc moiety,^[14]allows the generation of virtually all the

basic structures of complex-type N-glycans by a common

approach. This has been shown for a highly substituted

penta-antennary, bisected and core-fucosylated undecasac-

charide.^[15]In order to be able to introduce the core fucosyl

residue at an appropriate stage in the synthesis, we have

developed a modified core trisaccharide bearing a p-meth-

oxyphenyl group,^[16]which can be cleaved selectively.^[13]

This approach facilitates the introduction of a labile fucosyl

moiety in one of the last glycosylations,^[14b,17]whereas other

strategies rely on an earlier fucosylation^[18]or a different

disconnection mode.^[19]In an alternative approach, α1,6-

fucosyltransferases from several organisms have been

cloned, and these may be used for enzymatic core fucosyl-

ation of complex-type non-galactosylated N-glycans.^[20]

Introduction

The glycosylation of asparagine residues in proteins (N-

glycosylation) is known to modify the biochemical and bio-

physical properties of the proteins.^[1]Recent studies on N-

glycoprotein variants have revealed stabilizing effects

caused by N-glycan–protein interactions.^[2]The diversity of

N-glycans in glycoproteins (microheterogeneity) is also

gaining attention. In particular cases, the biological activity

of single glycoforms can be related to individual oligosac-

charide structures.^[3]A frequently occurring N-glycan

modification contributing to microheterogeneity is core fu-

cosylation,^[4]which has been found to be essential for nor-

mal growth in animals.^[5]Core fucosylation is known to af-

fect the efficiency of serum antibodies,^[6]the binding to lec-

tins,^[7]and the activity of receptors,^[8]and it also serves as

a tumor marker.^[9]Due to the difficulties in the isolation of

sufficient amounts of homogeneous N-glycans of a given

structure,^[10]the chemical and enzymatic synthesis of N-gly-

cans has become a straightforward alternative.^[11]By means

of a modular approach, multi-branched N-glycans^[12]have

become accessible, and the introduction of a core fucosyl

moiety is possible concomitantly.^[13]The combination of

Results and Discussion

In order to obtain N-glycans and glycopeptides with a

core fucosyl moiety (i.e., A; Scheme 1), a route to key build-

ing block B was developed.^[13]Core trisaccharide B is func-

tionalized for regio- and stereoselective elongations of the

central β-mannoside in a modular fashion, using donors

similar to building block C. The selective removal of the p-

methoxyphenyl group permits core fucosylation using do-

nor D.

[a] Bioorganische Chemie, Universität Bayreuth,

Gebäude NW1, 95440 Bayreuth, Germany

Fax: +49-921-55-5365

E-mail: carlo.unverzagt@uni-bayreuth.de

Homepage: www.boc.uni-bayreuth.de/de/index.html

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201200468.

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Scheme 1. The chemical synthesis of complex-type N-glycans with a core fucosyl moiety can be accomplished by using modular building

blocks B–D. Core trisaccharide B is set up for regio- and stereoselective elongations of the β-mannoside using donor C and for core

fucosylation (donor D) after removal of the p-methoxyphenyl group; MPM = p-methoxybenzyl.

As the core trisaccharide building block B could also be

The p-methoxyphenyl group of the core trisaccharide B

used for the synthesis of core-fucosylated N-glycans bearing is selectively cleavable by oxidation, and this allows conve-

an additional bisecting GlcNAc moiety,^[14b,15]a more ef- nient core fucosylation even at a late stage of the N-glycan

ficient route to B was needed. While investigating a large- synthesis. The presence of an azido group at the reducing

scale synthesis of B, we successfully shortened several key end simplifies the attachment of the N-glycan to amino ac-

steps of the initial approach,^[13]providing improved access ids or spacers by an amide bond.^[21]The highly function-

to B and its derivatives.

alized GlcNAc building block 7 was envisioned for the re-

Scheme 2. (a) NaOMe, MeOH, 98%; (b) benzaldehyde dimethyl acetal, pTsOH/H₂O, CH₃CN, 78%; (c) BnBr, NaH, DMF, 67%;

(d) pTsOH/H₂O, CH₂Cl₂, MeOH, H₂O, 85%; (e) Tf₂O, 2,6-lutidine, CH₂Cl₂, –80 °C; (f) p-methoxyphenol, 2 n NaOH, aliquat 336; (e)–

(f) 89%; (g) BF₃·OEt₂, CH₂Cl₂; (h) NIS, TfOH, CH₂Cl₂; (i) K₂CO₃, CH₂Cl₂, MeOH; (g) + (i) 87%; (h) + (i) 70%; CA = chloroacetyl.

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Modular Synthesis of Core Fucosylated N-Glycans

ducing end (Scheme 2), but initially, it was only accessible of the sterically hindered base 2,6-dimethylpyridine.^[25]On

by a tedious multistep procedure.^[13]In this first-generation warming up, triflate 6 reacted smoothly with aqueous so-

approach, a 3-O-benzylated glycosyl fluoride intermediate dium p-methoxyphenolate under phase-transfer conditions

was synthesized, and the p-methoxyphenyl group (PMP) to give the desired PMP ether 7 in 89% yield in a one-pot

was introduced by a Mitsunobu reaction^[22]prior to the reaction. This fast and easily scaleable approach consider-

conversion of the fluoride to the desired azide (i.e., 7). Car- ably improved the availability of acceptor 7.

rying out the required conversions in a different order led

Subsequently, the synthesis of chitobioside 10 was inves-

to a shorter sequence but also to undesired by-products. In tigated. Use of fluoride 8^[26]and boron trifluoride–diethyl

particular, the partial halogenation of the p-methoxyphenyl ether as an activator gave a high conversion to disaccharide

group during the transformation of the thioglycoside into 10, but purification at this stage was not practical, since

a glycosyl fluoride by using the N-bromosuccinimide-HF/ acceptor 7 could not be separated from the product by flash

pyridine method was troublesome.^[23]It was found that a chromatography. After subjecting the crude glycosylation

halogenated p-methoxyphenyl group could still be cleaved mixture to a mild dechloroacetylation, the separation im-

by oxidation, but the additional heterogeneity complicated proved and yielded disaccharide 11 in 87% over two steps.

NMR spectroscopy and MS analysis throughout the entire A shorter route to 11 was tried by glycosylation of 7 with

synthesis.

thioglycoside 9.^[26]Compound 9 serves as a direct precursor

We were thus seeking an improved synthesis of acceptor for fluoride 8, which is difficult to purify on larger scales

7 in which the different protecting groups could be installed due to a strong tendency of 8 to crystallize during flash

in a more direct manner. A short approach was found by chromatography. Despite the presence of the readily haloge-

starting from readily available azide 1,^[24]requiring only a nated PMP group in 7, the mild activation of thioglycoside

few protecting-group manipulations. In order to selectively 9 provided disaccharide 11 in 70% yield overall without by-

introduce the 3-O-benzyl group, the acetates of 1 were re- products. This was accomplished by using only a small ex-

moved, followed by 4,6-O-benzylidenation. The purified cess of NIS in combination with a quenching workup at

benzylidene acetal (i.e., 3) was benzylated to give 4, which low temperatures.

could be isolated conveniently on a larger scale by crystalli-

The installation of β-mannosides remains a difficult

zation. After acidic hydrolysis of the benzylidene acetal, the task,^[27]and we carried it out by glycosylation with glucosyl

diol (i.e., 5) was first tosylated at O-6, but nucleophilic sub- donor 12,^[26]followed by an intramolecular inversion ac-

stitution of the tosylate with p-methoxyphenolate was un- cording to the methodology developed by Kunz.^[28]When

successful. Thus, the more reactive 6-triflate was generated promoting the coupling of donor 12 and chitobiosyl ac-

at low temperature by using triflic anhydride in the presence ceptor 11 with TMSOTf, trisaccharide 13 appeared to form

Scheme 3. (a) TMSOTf (0.6 equiv.), CH₂Cl₂, 0 °C, 30 min; 23 °C, 4 h; (b) K₂CO₃, CH₂Cl₂, MeOH; (a)–b) 50%; (c) (1) hydrazine acetate,

DMF, 0 °C, 71%, (2) CF₃(C=NPh)Cl, DBU, CH₂Cl₂, 0 °C, 92%; (d) TMSOTf (0.05 equiv.), CH₂Cl₂, 0 °C, 75%; (e) (1) TMSOTf

(0.05 equiv.), CH₂Cl₂, 0 °C, 40 min, (2) TfOH, CH₂Cl₂, 0 °C, 40 min, 89%; (f) K₂CO₃, CH₂Cl₂, MeOH; (e)–(f) 89%; PMP = p-meth-

oxyphenyl.

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readily (Scheme 3). The isolation of the trisaccharide re-

quired a dechloroacetylation step to give 14, which im-

proved the separation from unreacted acceptor 11. How-

ever, the final yields of 14 were variable and rarely exceeded

50%. This led us to the assumption that the reaction might

proceed through a stable orthoester intermediate, since it

appeared to be essential to stir the glycosylation reaction

mixture at room temperature for some time if reasonable

yields were to be achieved. The formation of orthoesters by

using 3-O-phenylcarbamoylated glucosyl donors was de-

scribed by Kunz^[28a]and has similarly been observed for a

2,3-di-O-chloroacetylated thioglucoside donor,^[29]which

also served as a precursor for a β-mannoside. As an alterna-

tive glucosyl donor, we synthesized N-phenylimidate 16^[30]

from precursor 15.^[26]Donor 16 showed a rapid reaction

with 11 to give an orthoester intermediate, which slowly

reacted to form the desired trisaccharide (i.e., 13). In this

case, the conversion could be monitored by TLC and

HPLC-MS. When the reaction temperature was kept below

0 °C and only a minimal amount of TMSOTf (0.05 equiv.)

was used, the reaction yielded mainly orthoester 13a.

Orthoester 13a could be purified by flash chromatog-

raphy, and data from its ¹H and ¹³C NMR spectra, i.e., J_1,2

= 5.5, J_C-1,H-1= 192 Hz and a downfield ¹³C shift of the

glucose C-2 to δ = 78.2 ppm, were consistent with the glu-

cose orthoester structure. After confirming the presence of

an orthoester in 13a, we attempted to improve its conver-

sion to β-glucosyl trisaccharide 13 by directly isomerizing

the orthoester in situ. This required several rounds of opti-

mization, which were followed by HPLC-MS. Acceptor 11

was quickly converted into orthoester 13a when using

0.05 equiv. of TMSOTf at 0 °C. The most effective in situ

rearrangement conditions for orthoester 13a were found to

be the subsequent addition of triflic acid (0.4 equiv.) to the

reaction mixture. The overall glycosylation sequence was

prone to side-reactions, which only occurred when leaving

the temperature window of 0–5 °C, when adding too much

TfOH for the rearrangement, or when using TfOH for both

the activation and the isomerization steps. When the glyc-

osylation was promoted by TMSOTf alone, a significant

amount of silylated acceptor formed, which reduced the to-

tal yield. By using the optimized conditions for the β-gluco-

sylation of chitobiosyl acceptor 11, the isolated yield of tri-

saccharide 14 reproducibly increased from 50 to 89%.

We next investigated the conversion of β-glucoside 14 to

β-mannoside B. The multistep inversion procedure gave

variable yields in our hands. As depicted (Scheme 4a), the

intramolecular inversion from the gluco to the manno con-

figuration is initiated by formation of a secondary triflate

using triflic anhydride/pyridine.

Scheme 4. (a) Four-step inversion sequence of gluco-configured tri-

saccharide 14 to β-mannoside B. (b) Traces of water can lead to

undesired ring-opening of cyclic iminocarbonate 14b in DMF/pyr-

idine.

According to the published procedure,^[28a]the triflate 14a

was heated in DMF/pyridine leading to an intramolecular

attack by the carbonyl oxygen atom of the carbamate. The

resulting cyclic iminocarbonate (i.e., 14b) was usually ac-

companied by some hydrolysis product 14c. Mild acidic hy- weakly basic conditions to give the desired β-mannosyl tri-

drolysis of iminocarbonate 14b completed the conversion to saccharide (i.e., B) in 53% yield. The β-manno configura-

carbonate 14c without affecting the benzylidene acetal. The tion of core trisaccharide B was confirmed by a C-1,H-1

cyclic carbonate was removed by methanolysis under coupling constant of 163 Hz, which is typical for this link-

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Modular Synthesis of Core Fucosylated N-Glycans

age.^[31]The reported yield for the corresponding inversion curred only at pH Ͻ 6, whereas more basic conditions gave

sequence on a disaccharide was much higher.^[28a]Since the increasing amounts of carbamates. The reaction conditions

four reactions can be performed in a one-pot manner, a during the inversion of 14a in DMF/pyridine can be consid-

significant loss of product could be due to side-reactions. ered to be weakly basic, which mainly resulted in hydrolysis

We noticed the occurrence of by-products with an R_fvalue to 14c, but also led to a pH-dependent fraction of carb-

close to that of the starting material (i.e., 14). After meth- amates 14e,f.

anolysis, these by-products could be isolated by flash

We then searched for an inversion protocol that would

1

chromatography; their H NMR spectrum revealed a com- minimize these side-reactions by screening alternative sol-

plex mixture with no starting material 14 present. This indi- vents (dichloromethane, acetonitrile, THF). Evaluation by

cated that hydrolysis of the triflate in 14a to give 14 had HPLC-MS suggested dichloromethane to be a promising

not taken place under the reaction conditions, and that the solvent; however, the reaction rate of the inversion was low,

by-products should thus derive from triflate 14a or the en- even under refluxing conditions. When utilizing a pressure-

suing intermediates.

stable glass vial, the sample could be heated to 65 °C, and

According to HPLC-MS, the two major by-products had the reaction proceeded to completion within 21 h. Accord-

the same mass as trisaccharide 14. After purification of ing to HPLC-MS, the inversion of crude 14a in dichloro-

these by-products by HPLC, they were structurally charac- methane gave almost exclusively the desired iminocarbonate

terized by a series of 2D-NMR experiments.^[32]The com- (i.e., 14b), which hydrolyzed to the desired carbonate (i.e.,

plete NMR assignments indicated that two isomeric β- 14c) under TLC or LC-MS conditions. The formation of

manno-configured carbamates 14e and 14f had formed in a very small quantities of by-products can be attributed to

1:1 ratio (Scheme 4b). These by-products may arise through the anhydrous conditions of the inversion step and the suffi-

base-catalyzed ring opening of the cyclic iminocarbonate in ciently acidic conditions during controlled hydrolysis at

14b.^[33]In the presence of trace amounts of water, labile room temperature. We also tried to avoid the pressure-stable

tetrahedral intermediate 14d can form, and this can lead vial by using the higher boiling solvents chloroform or 1,2-

either to hydrolysis of the imine (forming 14c) or to ring- dichloroethane for triflate formation, followed by heating

opening. The latter process can generate the two regioiso- to 65 °C using a reflux condenser plugged by a balloon for

meric phenylcarbamates (i.e., 14e,f) of the manno-con- pressure equilibration. Disappointingly, by-products 14e

figured core trisaccharide.

and 14f were formed in amounts similar to those of the

The pH-dependent ring-opening of cyclic N-phenyl- DMF/pyridine procedure. This outcome may be attributed

iminocarbonate model compounds has been studied in de- to traces of water, which were not rigorously excluded in

tail.^[33]It was found that hydrolysis of the imine bond oc- this unsealed approach.

Scheme 5. (a) Optimized conditions for the inversion of 14. (b) HPLC comparison of crude B obtained after standard inversion in DMF/

pyridine or optimized inversion conditions in CH₂Cl₂.

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The one-pot protocol was thus simplified by generating trace amounts of by-products, which is clear when compar-

the triflate 14a in dichloromethane, followed by an in situ ing the HPLC profiles of the different inversion procedures

inversion in the same solvent at elevated temperature (Scheme 5b). The decrease in the amount of by-products

(Scheme 5a). An increased amount of sodium methoxide raised the final yield of B from 53 to 79%.

during removal of the cyclic carbonate prevented the forma-

With sufficient quantities of core trisaccharide B pre-

tion of methyl carbonate intermediates. Under these condi- pared, the attachment of the two antennae was investigated.

tions, the optimized four-step inversion protocol gave only Elongation with trichloroacetimidate donor C^[34]proceeded

Scheme 6. (a) BF₃·OEt₂, CH₂Cl₂, 79%; (b) Ac₂O, pyridine; (c) pTsOH/H₂O, CH₃CN; (b)–(c) 80%; (d) C, BF₃·OEt₂, CH₂Cl₂, 73%;

(e) Ac₂O, pyridine; (f) CAN, toluene, CH₃CN, water; (e)–(f) 90%; (g) D, CuBr₂, Bu₄NBr, DMF, CH₂Cl₂, 85% after flash chromatography,

77% after HPLC.

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Modular Synthesis of Core Fucosylated N-Glycans

NMR experiments.^[32]For mass spectra, a Varian CH5 instrument

was used in the fast atom bombardment mode (FAB) with an m-

nitrobenzyl alcohol matrix (NBA). ESI-TOF mass spectra were re-

corded with a Micromass LCT instrument coupled to an Agilent

1100 HPLC apparatus. Flash chromatography was performed on

silica gel 60, (230–400 mesh, Merck Darmstadt). The reactions

were monitored by thin layer chromatography on coated aluminum

plates (silica gel 60 GF₂₅₄, Merck Darmstadt). Spots were detected

by UV light or by charring with a 1:1 mixture of 2 n H₂SO₄/0.2%

resorcinol monomethyl ether in ethanol.

in a regio- and steroselective manner to give the desired

α1,3-linked pentasaccharide 17 with a J_C-1,H-1value of

173.8 Hz for the newly established α-mannosidic linkage

(Scheme 6). Activation of trichloroacetimidate^[35]C by

using boron trifluoride–diethyl ether is fully compatible

with the presence of the PMP group in the acceptor and

did not lead to glycosylation at multiple sites. After acetyl-

ation of OH-2³, intermediate pentasaccharide 18 was de-

benzylidenated directly. The resulting diol (i.e., 19) was

treated with donor C in a second regio- and stereoselective

reaction to give biantennary heptasaccharide 20, as indi-

cated by a J_C-1,H-1value of 173.5 Hz for the 1,6-linked α-

mannoside. The remaining 4-hydroxy group of the β-man-

noside was acetylated in order to prevent fucosylation at

this position. Oxidative removal of the PMP group^[36]was

2-Deoxy-2-phthalimido-β-D-glucopyranosyl Azide (2): A solution of

sodium (0.72 g, 31.3 mmol) dissolved in absolute methanol (68 mL)

was added to a suspension of azide 1 (78.2 g, 169.9 mmol) in abso-

lute methanol (670 mL). The mixture was stirred at room tempera-

ture under argon for 25 min. After complete reaction (TLC: cyclo-

hexane/acetone, 1:1), the pH was adjusted to 6–7 by adding Am-

carried out by using cerium(IV) ammonium nitrate (CAN) berlyst^®15. The mixture was filtered, the solvent was removed in

under biphasic conditions.^[37]Heptasaccharide acceptor 22

vacuo, and the residue was dried under high vacuum to give crude

2 (55.9 g, 98.4%). R_f= 0.29 (cyclohexane/acetone, 1:1). [α]²_D³

=

was then fucosylated with p-methoxybenzyl-protected thio-

fucoside D^[38]by using CuBr₂in combination with tetra-

butylammonium bromide in DMF/CH₂Cl₂.^[39]Careful

HPLC-MS analysis showed that the desired α-fucosylated

octasaccharide was accompanied by some β-anomer (α/β

ratio 7:1). The formation of β-fucosides has also been noted

by others when primary alcohols were used as ac-

ceptors.^[17b,18]Despite intensive attempts to separate the an-

–31.5 (c = 1.0, CH₃OH). ¹H NMR (270 MHz, [D₆]DMSO): δ =

7.94–7.87 (m, 4 H, Pht), 5.54 (d, J_OH,3= 4.9 Hz, 1 H, 3-OH), 5.33

(d, J_1,2= 9.5 Hz, 1 H, 1-H), 5.30 (d, J_OH,4= 7.3 Hz, 1 H, 4-OH),

4.73 (dd, J_OH,6= 5.7 Hz, 1 H, 6-OH), 4.08 (m, 1 H, 3-H), 3.79–

3.68 (m, 2 H, 6a-H, 2-H), 3.54 (m, 1 H, 6b-H), 3.43 (m, 1 H, 5-H),

3.27 (m, 1 H, 4-H) ppm. ¹³C NMR (68 MHz, [D₆]DMSO): δ =

168.4 (C=O), 168.1 (C=O), 135.4 (Pht), 135.3 (Pht), 131.8 (Pht-

C_q), 131.5 (Pht-C_q), 124.0 (Pht), 123.8 (Pht), 85.8 (C-1), 80.2 (C-

omers by flash chromatography, an HPLC purification was 5), 70.9 (C-3), 70.6 (C-4), 61.1 (C-6), 57.2 (C-2) ppm. ESI-MS: m/z

= 357.23 [M + Na]⁺.

required at this stage by using either a C8 reverse-phase

column and an acetonitrile/water gradient or a diol column

4,6-O-Benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranosyl Azide

with ethyl acetate/hexane as the eluent. The final yields of

pure α-anomer 23 were 68 and 77%, respectively, by these

two purification methods. After deprotection, the core-

(3): Benzaldehyde dimethyl acetal (83.5 mL, 559.6 mmol) and p-

toluenesulfonic acid monohydrate (0.72 g, 3.79 mmol) were added

to a suspension of crude triol 2 (53.6 g, 160.3 mmol) in anhydrous

fucosylated octasaccharide was coupled to a spacer and acetonitrile (560 mL) under argon. Undissolved starting material

elongated enzymatically.^[21]These conjugates were incorpo-

was solubilized by sonicating the reaction mixture in an ultrasonic

bath. After stirring at room temperature for 15 min, p-toluenesul-

fonic acid monohydrate (3.20 g, 16.8 mmol) was added, and the

reaction mixture was stirred for 90 min. After complete reaction

(TLC: cyclohexane/acetone, 2:1), the pH was adjusted to a value

rated into neoglycoproteins, which were used for biological

studies focusing on the role of core fucosylation.^[7]

of 9 by adding triethylamine (4.0 mL, 28.9 mmol). After removing

the solvent in vacuo, the residue was diluted with CH₂Cl₂(1.5 L).

Conclusions

The organic phase was washed with water (1 L), dried with MgSO₄

and concentrated. The residue was purified by flash chromatog-

We have optimized the synthesis of a biantennary core-

fucosylated N-glycan of the complex type. The key building

block is a selectively functionalized core trisaccharide,

which allows high-yielding glycosylations of the β-manno-

side in a modular way, and permits fucosylation at any de-

sired stage of the synthesis. The optimizations were guided

by mechanistic analysis, and resulted in robust protocols

suitable for large-scale synthesis of this versatile core trisac-

charide.

raphy (cyclohexane/acetone, 9:1Ǟ5:1Ǟ2:1) to give

3 (52.8 g,

78.0%). R_f= 0.36 (cyclohexane/acetone, 2:1). [α]²_D⁴= –43.9 (c = 1.0,

CHCl₃). ¹H NMR (270 MHz, [D₆]DMSO): δ = 7.97–7.88 (m, 4 H,

Pht), 7.48–7.37 (m, 5 H, Ar), 5.84 (d, J_3,OH= 5.2 Hz, 1 H, 3-OH),

5.68 (s, 1 H, PhCH=), 5.54 (d, J_1,2= 9.6 Hz, 1 H, 1-H), 4.42–4.28

(m, 2 H, 3-H, 6a-H) 3.91–3.63 (m, 4 H, 2-H, 6b-H, 5-H, 4-H) ppm.

¹³C NMR (68 MHz, [D₆]DMSO): δ = 167.7, 167.3 (C=O), 137.4

(C_q), 134.9 (Pht), 131.2, 130.8 (C_q), 129.0, 128.1, 126.3 (Ar), 123.5,

123.3 (Pht), 100.8 (PhCH=), 85.7 (C-1), 80.6 (C-4), 68.4 (C-5), 67.4

(C-6), 67.1 (C-3), 56.9 (C-2) ppm. ESI-MS: m/z = 439.37 [M +

NH₄]⁺, 444.32 [M + Na]⁺.

Experimental Section

General Methods: Solvents were dried according to standard meth-

ods. Molecular sieves were activated prior to use by heating under

high vacuum. Optical rotations were measured with a Perkin–El-

mer 241 polarimeter at 589 nm. NMR spectra were recorded with

Bruker AC 250, Avance 360, AMX 500 and DMX 500 instruments

or a Jeol JNM-EX-270 spectrometer. Coupling constants are re-

ported in Hz. The compounds were clearly characterized by full

4,6-O-Benzylidene-3-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyr-

anosyl Azide (4): Benzyl bromide (30.1 mL, 248.3 mmol) was added

to a solution of benzylidene acetal 3 (50.0 g, 118.4 mmol) in anhy-

drous N,N-dimethylformamide (106 mL) under argon. The reaction

mixture was cooled to 0 °C, and sodium hydride (60% dispersion

in mineral oil, 8.05 g, 201.3 mmol) was added in portions. The mix-

ture was stirred at 0 °C for 1.5 h, followed by stirring at room tem-

perature for 2 h. After complete reaction (TLC: cyclohexane/ace-

1

assignment of the H and ¹³C resonances from a set of 1D and 2D

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C. Unverzagt et al.

FULL PAPER

1

tone, 1.5:1), the pH was adjusted to 7–8 by adding acetic anhydride = 0.49 (hexane/acetone, 1.5:1). [α]²_D¹= +14.0 (c = 0.2, CH₂Cl₂). H

(46.8 mL, 499.7 mmol), and the mixture was then stirred at room

temperature for 1 h. The solvents were removed in vacuo, and the

residue was dissolved in CH₂Cl₂(1 L), washed with water (1 L),

dried with MgSO₄, and concentrated in vacuo. Recrystallization

from diethyl ether gave crystalline 4 (34.07 g). The mother liquor

was concentrated and purified by flash chromatography (CH₂Cl₂/

acetone, 100:1) to give additional 4 (6.66 g). The total yield of 4

was 40.73 g (67.1%). R_f= 0.56 (cyclohexane/acetone, 1.5:1); R_f=

0.62 (CH₂Cl₂/acetone, 100:1). M.p. 133–136 °C. [α]²_D⁴= +39.7 (c =

NMR (270 MHz, [D₆]DMSO): δ = 7.90–7.70 (m, 4 H, Pht), 7.00–

6.85 (m, 9 H, PMP, Ph), 5.90 (d, J_4,OH= 7.1 Hz, 1 H, 4-OH), 5.48

(d, J_1,2= 9.4 Hz, 1 H, 1-H), 4.80 (d, J_gem= 12.0 Hz, 1 H, OCH₂),

4.44 (d, J_gem= 12.0 Hz, 1 H, OCH₂), 4.30–4.10 (m, 3 H, 6a-H, 3-

H, 6b-H), 3.92–3.82 (m, 2 H, 2-H, 5-H), 3.75–3.62 (m, 4 H, OCH₃,

4-H) ppm. ¹³C NMR (68 MHz, [D₆]DMSO): δ = 167.40 (C=O),

153.55, 152.54 (C-i PMP, C-4 PMP), 138.15 (C-i Ph), 134.79 (C-4/

5 Pht), 130.66 (C-1/2 Pht), 127.79, 127.36 (C-2/6 Ph, C-3/5 Ph),

127.18 (C-4 Ph), 123.42 (C-3/6 Pht), 115.63, 114.69 (C-2/6 PMP,

C-3/5 PMP), 85.10 (C-1), 78.25 (C-3), 76.94 (C-5), 73.67 (OCH₂),

1

1.0, CHCl₃). H NMR (270 MHz, [D₆]DMSO): δ = 7.89–7.80 (m,

4 H, Pht), 7.51–7.38 (m, 5 H, Ar), 7.00–6.86 (m, 5 H, Ar), 5.79 (s, 70.98 (C-4), 67.56 (C-6), 55.30 (OCH₃), 54.60 (C-2) ppm. ESI-MS:

1 H, =CHPh), 5.57 (d, J_1,2= 9.4 Hz, 1 H, 1-H), 4.71 (d, J_gem

=

m/z = 548.64 [M + NH₄]⁺, 553.63 [M + Na]⁺.

12.2 Hz, 1 H, CH₂O), 4.41 (d, J_gem= 12.2 Hz, 1 H, CH₂O), 4.36–

4.32 (m, 2 H, 3-H, 6a-H), 4.01–3.86 (m, 3 H, 2-H, 4-H, 6b-H), 3.77

(m, 1 H, 5-H) ppm. ¹³C NMR (68 MHz, [D₆]DMSO): δ = 167.1

(C=O), 137.6 (C_q), 137.3 (C_q), 134.9 (Pht), 130.6, (C_q), 128.9,

128.2, 127.9, 127.5, 127.4, 126.0 (Ar), 123.5 (Pht), 100.2 (=CHPh),

85.6 (C-1), 81.3 (C-4), 74.3 (C-3), 73.3 (CH₂O), 67.8 (C-5), 67.4 (C-

6), 54.6 (C-2) ppm. ESI-MS: m/z = 535.27 [M + Na]⁺, 551.25 [M

+ K]⁺, 1047.55 [2 M + Na]⁺.

3,6-Di-O-benzyl-4-(chloroacetyl)-2-deoxy-2-phthalimido-β-D-gluco-

pyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-methoxyphenyl)-2-

phthalimido-β- -glucopyranosyl Azide (10) and 3,6-Di-O-benzyl-2-

deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-de-

D

oxy-6-O-(p-methoxyphenyl)-2-phthalimido-β-D-glucopyranosyl Az-

ide (11)

Method A (via Fluoride 8): A suspension of azide 7 (3.23 g,

6.1 mmol), fluoride 8 (4.5 g, 7.9 mmol), and freshly activated

ground molecular sieves (4 Å) (5.4 g) in absolute CH₂Cl₂(80 mL)

was stirred for 60 min. Subsequently, boron trifluoride–diethyl

ether (0.37 mL, 2.9 mmol) was added. After 90 min (TLC: hexane/

acetone, 2:1), the suspension was diluted with CH₂Cl₂, filtered

through Celite and washed with dilute KHCO₃solution. The or-

ganic phase was dried with MgSO₄and concentrated. The crude

mixture was subjected to dechloroacetylation since the acceptor

could not be removed efficiently at this stage. R_f(10) = 0.24 (hexane/

acetone, 2:1).

3-O-Benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl Azide (5): p-

Toluenesulfonic acid monohydrate (38.0 g, 200 mmol) was added

to a solution of 4 (22.33 g, 43.6 mmol) in a mixture of absolute

CH₂Cl₂and absolute methanol (1:1, 700 mL). After stirring at

room temperature for 15 min, water (2 mL) and p-toluenesulfonic

acid monohydrate (23.9 g, 126 mmol) were added. The mixture was

stirred for 1.5 h, and then more water (9 mL) was added. After

1.5 h (TLC: cyclohexane/ethyl acetate, 1.5:1), the pH was adjusted

to 7 by adding triethylamine (17.8 mL). The solvent was removed

in vacuo, and the residue was dried under high vacuum. The crude

product was purified by flash chromatography (cyclohexane/ethyl

acetate, 3:1Ǟ1:1Ǟ1:3Ǟ100 % ethyl acetate) to give 5 (15.80 g,

85.4%). R_f= 0.34 (cyclohexane/ethyl acetate, 1:1.5). [α]²_D⁴= +22.3

(c = 1.0, CHCl₃). ¹H NMR (270 MHz, [D₆]DMSO): δ = 7.87–7.76

(m, 4 H, Pht), 6.96–6.84 (m, 5 H, Ar), 5.64 (d, J_OH,4= 6.2 Hz, 1

The crude mixture containing 10 was dissolved in absolute CH₂Cl₂

(160 mL) and absolute methanol (80 mL). Ground K₂CO₃(1.6 g)

was added, and the suspension was stirred for 10 min (TLC:

CH₂Cl₂/methanol, 80:1). The solids were filtered off, and the fil-

trate was adjusted to a pH of 5–6 with acetic acid (0.6 mL) and

then concentrated in vacuo. The residue was purified by flash

chromatography (hexane/acetone, 2.5:1) to give 11 (5.32 g, 87.3%

over two steps). R_f= 0.20 (hexane/acetone, 2:1). [α]²_D²= +28.6 (c =

1, CH₂Cl₂).

H, 4-OH), 5.38 (d, J_1,2= 9.4 Hz, 1 H, 1-H), 4.79 (dd, J_OH,6a,b

=

5.6 Hz, 1 H, 6-OH), 4.76 (d, J_gem= 12.2 Hz, 1 H, CH₂O), 4.42 (d,

J_gem= 12.2 Hz, 1 H, CH₂O), 4.11 (dd, J_2,3= 10.4, J_3,4= 7.6 Hz, 1

H, 3-H), 3.84–3.75 (m, 2 H, 2-H, 6a-H), 3.64–3.46 (m, 3 H, 6b-H,

4-H, 5-H) ppm. ¹³C NMR (68 MHz, [D₆]DMSO): δ = 167.4

(C=O), 167.1 (C=O), 138.2 (C_q), 134.8 (Pht), 130.6, (C_q), 127.8,

127.3, 127.1 (Ar), 123.4 (Pht), 85.1 (C-1), 79.4 (C-5), 78.3 (C-3),

73.5 (CH₂O), 70.9 (C-4), 60.2 (C-6), 54.7 (C-2) ppm. ESI-MS: m/z

= 442.28 [M + NH₄]⁺, 447.21 [M + Na]⁺, 866.40 [2 M + NH₄]⁺,

871.36 [2 M + Na]⁺.

Method B (via Thioglycoside 9): A suspension of azide 7 (1.83 g,

3.45 mmol), thioglycoside 9 (2.74 g, 4.5 mmol), and freshly acti-

vated ground molecular sieves (4 Å) (4.76 g) in absolute CH₂Cl₂

(59 mL) was cooled to –30 °C. N-Iodosuccinimide (0.97 g,

4.3 mmol) was added, and the mixture was stirred for 30 min. Tri-

fluoromethanesulfonic acid (55 μL, 0.6 mmol) was added, and stir-

ring was continued for 30 min at –35 to –30 °C (TLC: toluene/ace-

tone, 8:1). The reaction was stopped by filtering the suspension

through Celite into a stirred solution of sodium thiosulfate (10%,

cooled to 0 °C). The aqueous phase was extracted with CH₂Cl₂,

and the combined organic extracts were washed with dilute

KHCO₃solution and dried with MgSO₄. The solvent was removed,

and the residue was dried under high vacuum to give 4.43 g of

residue, which was used in the following reaction without further

purification. R_f(10) = 0.52, (toluene/acetone, 8:1). ESI-MS: m/z =

1095.57 [M + NH₄]⁺, 1100.53 [M + Na]⁺, 1116.56 [M + K]⁺.

3-O-Benzyl-2-deoxy-6-O-(p-methoxyphenyl)-2-phthalimido-β-D-gluco-

pyranosyl Azide (7): A solution of diol 5 (6.3 g, 14.8 mmol) and

2,6-lutidine (14.0 mL, 118.2 mmol) in absolute CH₂Cl₂(350 mL)

was cooled to –80 °C under argon. Trifluoromethanesulfonic anhy-

dride (4.13 mL, 24.7 mmol) was added dropwise, and after 20 min,

the starting material had been completely converted into the triflate

6 (TLC: hexane/acetone, 1.2:1). Subsequently, dilute NaOH (2 n,

105 mL), p-methoxyphenol (12.8 g, 103.0 mmol) and phase-trans-

fer catalyst aliquat 336 (3.5 mL) were added separately, and the

reaction mixture was allowed to gradually warm up to room tem-

perature whilst being stirred. After 5 h of vigorous stirring, more

p-methoxyphenol (1.0 g, 8.1 mmol) was added, and after a further

30 min, complete consumption of 6 was observed (TLC: hexane/

The residue (4.43 g) was dissolved in absolute CH₂Cl₂(93 mL) and

absolute methanol (48 mL) under argon. Ground K₂CO₃(0.95 g),

which had been activated prior to use by heating under high vac-

acetone, 1.5:1). The reaction mixture was washed with dilute HCl uum, was added. The mixture was stirred for 30 min (TLC: toluene/

and dilute KHCO₃solution. The organic phase was dried with acetone, 8:1), and then the K₂CO₃was removed by filtration

MgSO₄, concentrated, and purified by flash chromatography (hex-

ane/ethyl acetate, 4:1Ǟ3:1Ǟ1:1Ǟ1:2) to give 7 (7.0 g, 88.9%). R_f

through Celite. The pH of the filtrate was adjusted to 6–7 by adding

acetic acid (0.12 mL). After concentration in vacuo, the residue was

8

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Modular Synthesis of Core Fucosylated N-Glycans

purified by flash chromatography (cyclohexane/acetone, 5:1Ǟ3:1)

to give 11 (2.42 g, 70.1% over two steps). R_f= 0.40 (toluene/ace-

tone, 8:1), R_f= 0.50 (hexane/acetone, 1.5:1). H NMR (500 MHz,

and concentrated to give crude 13 (5.67 g). R_f(11) = 0.27, R_f(13a)

= 0.33, R_f(13) = 0.40 (toluene/acetone, 8:1).

1

The crude trisaccharide 13 (5.67 g) was dissolved in absolute

CH₂Cl₂(60 mL) and absolute methanol (30 mL) under argon.

Ground K₂CO₃(1.38 g), which had been activated prior to use by

heating under high vacuum, was added. The mixture was stirred

for 1 h (TLC: toluene/acetone, 8:1), and then the K₂CO₃was re-

moved by filtration through Celite. The pH of the filtrate was ad-

justed to 5–6 by adding acetic acid (0.4 mL). After concentration

in vacuo, the residue was purified by flash chromatography (cyclo-

hexane/acetone, 3.5:1) to give 14 (3.64 g, 88.8% over two steps). R_f

[D₆]DMSO): δ = 7.85–7.70 (m, 8 H, Pht), 7.33–7.24 (m, 7 H, Ar),

6.99–6.71 (m, 12 H, Ar), 5.63 (d, J_4,OH= 7.2 Hz, 1 H, 4²-OH), 5.34

(d, J_1,2= 9.5 Hz, 1 H, 1¹-H), 5.22 (d, J_1,2= 8.2 Hz, 1 H, 1²-H),

4.84 (d, J_gem= 12.3 Hz, 1 H, OCH₂), 4.74 (d, J_gem= 12.1 Hz, 1 H,

OCH₂), 4.63 (d, J_gem= 12.3 Hz, 1 H, OCH₂), 4.52 (d, 1 H, OCH₂),

4.38 (d, 1 H, OCH₂), 4.36 (d, 1 H, OCH₂), 4.22–4.14 (m, 2 H, 4¹-

H, 3¹-H), 4.03 (dd, J_2,3= 9.5, J_3,4= 8.2 Hz, 1 H, 3²-H), 3.98–3.95

(m, 2 H, 2²-H, 6a¹-H), 3.88–3.78 (m, 4 H, 6a²-H, 2¹-H, 6b¹-H, 5¹-

H), 3.72 (s, 3 H, OCH₃), 3.58 (dd, J_5,6b= 5.5, J_gem= 11.0 Hz, 1 H,

6b²-H), 3.49–3.41 (m, 2 H, 4²-H, 5²-H) ppm. ¹³C NMR (125 MHz,

[D₆]DMSO): δ = 167.9, 167.5, 167.1 (C=O), 153.7, 151.8 (C-i PMP,

C-4 PMP), 138.2, 138.0, 137.3 (C-i Ph), 134.9 134.7 (C-4/5 Pht),

130.7, 130.6 (C-1/2 Pht), 128.2, 127.9, 127.8, 127.4, 127.2 (C-2/6

Ph, C-3/5 Ph, C-4 Ph), 123.5, 123.3 (C-3/6 Pht), 115.5, 114.6 (C-2/

6 PMP, C-3/5 PMP), 96.3 (C-1²), 84.8 (C-1¹), 78.5 (C-3²), 76.2 (C-

3¹, C-5²), 75.0 (C-5¹), 74.6 (C-4¹), 73.5 (OCH₂), 72.3 (OCH₂), 71.6

(C-4²), 69.0 (C-6²), 66.3 (C-6¹), 55.9 (C-2²), 55.3 (OCH₃), 54.5 (C-

2¹) ppm. ESI-MS: m/z = 1024.43 [M + Na]⁺. C₅₆H₅₁N₅O₁₃

(1002.0): calcd. C 67.12, H 5.13, N 6.99; found C 67.34, H 5.24, N

6.59.

1

= 0.24 (toluene/acetone, 8:1). H NMR (500 MHz, [D₆]DMSO): δ

= 9.66 (s, 1 H, NH), 7.83–7.60 (m, 8 H, Pht), 7.43–7.24 (m, 15 H,

Ph), 6.99–6.70 (m, 14 H, PMP, Ph), 5.88 (d, J_2,OH= 5.4 Hz, 1 H,

2³-OH), 5.53 (s, 1 H, PhCH=), 5.31 (d, J_1,2= 9.5 Hz, 1 H, 1¹-H),

5.20 (d, J_1,2= 8.4 Hz, 1 H, 1²-H), 4.97 (dd, J_2,3= J_3,4= 9.5 Hz, 1

H, 3³-H), 4.83 (d, J_gem= 12.5 Hz, 1 H, OCH₂), 4.74 (d, J_gem

=

11.8 Hz, 1 H, OCH₂), 4.63 (d, J_gem= 12.0 Hz, 1 H, OCH₂), 4.62

(d, J_1,2= 7.5 Hz, 1 H, 1³-H), 4.55 (d, 1 H, OCH₂), 4.36 (d, 1 H,

OCH₂), 4.26 (d, 1 H, OCH₂), 4.18 (dd, J_3,4= 9.9, J_4,5= 8.8 Hz, 1

H, 4¹-H), 4.16–4.11 (m, 2 H, 3²-H, 3¹-H), 4.07 (m, 1 H, 6a³-H),

4.00–3.88 (m, 5 H, 6a²-H, 2²-H, 4²-H, 6a¹-H, 6b²-H), 3.81 (dd, J_2,3

= 10.2 Hz, 1 H, 2¹-H), 3.79–3.76 (m, 2 H, 6b¹-H, 5¹-H), 3.71 (s, 3

H, OCH₃), 3.61 (dd, J_3,4= J_4,5= 9.5 Hz, 1 H, 4³-H), 3.58 (m, 1 H,

6b³-H), 3.55 (m, 1 H, 5²-H), 3.41 (m, 1 H, 2³-H), 3.32 (m, 1 H, 5³-

H) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ = 168.0, 167.3, 167.1

(C=O), 153.6, 151.8 (C-i, C-4 PMP), 152.9 (C=O Phcm), 139.1,

138.6, 138.1, 137.9, 137.3 (C-i Ph, C-4 PhCH=), 135.3, 134.1 (C-4/

5 Pht), 130.5 (C-1/2 Pht), 129.3, 128.9, 128.7, 128.4, 128.0, 127.8,

127.6, 127.4, 127.2, 126.7, 125.4 (C-2/6 Ph, C-3/5 Ph, C-4 Ph, C-3/

5 Phcm), 124.1 (C-3/6 Pht), 122.8 (C-4 Phcm), 118.8, 117.5 (C-2/6

Phcm), 115.1, 114.8 (C-2/6 Mp), 103.1 (C-1³), 100.3 (PhCH=), 96.4

(C-1²), 84.7 (C-1¹), 78.1 (C-4³), 78.0 (C-4²), 76.4 (C-3²), 76.3 (C-

3¹), 74.9 (C-4¹, C-5¹), 74.8 (C-5²), 74.0 (C-3³), 73.7 (OCH₂), 72.5

(C-2³), 72.1 (OCH₂), 67.7 (C-6²), 67.6 (C-6³), 66.3 (C-6¹), 65.6 (C-

5³), 55.9 (C-2²), 55.3 (OCH₃), 54.9 (C-2¹) ppm. ESI-MS: m/z =

1393.42 [M + Na]⁺.

4,6-O-Benzylidene-2-O-(chloroacetyl)-3-(phenylcarbamoyl)-β-

D-gluco-

pyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-

D-gluco-

pyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-methoxyphenyl)-2-

phthalimido-β- -glucopyranosyl Azide (13) and 4,6-O-Benzylidene-

3-(phenylcarbamoyl)-β- -glucopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-

deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-de-

D

oxy-6-O-(p-methoxyphenyl)-2-phthalimido-β-D-glucopyranosyl Az-

ide (14)

Method A (via Donor 12): A suspension of disaccharide azide 11

(4.36 g, 4.4 mmol), imidate 12 (5.76 g, 9.5 mmol), and freshly acti-

vated ground molecular sieves (4 Å) (5.0 g) in absolute CH₂Cl₂

(180 mL) was stirred at 0 °C for 30 min. Subsequently, TMSOTf

(470 μL, 2.6 mmol) was added. The reaction mixture was allowed

to warm to ambient temperature, and then stirred for an additional

4 h (TLC: cyclohexane/acetone, 1.5:1), diluted with CH₂Cl₂, and

filtered through Celite. After washing with dilute KHCO₃solution,

the organic phase was dried with MgSO₄and concentrated in

vacuo. The crude mixture was directly subjected to dechloroacet-

ylation since the product could not be purified efficiently at this

stage. R_f(13) = 0.45 (cyclohexane/acetone, 1.5:1).

4,6-O-Benzylidene-2-O-chloroacetyl-3-O-(N-phenylcarbamoyl)-α,β-

D

-glucopyranosyl-(N-phenyl)trifluoroacetimidate (16): Compound

15 (17.05 g, 31.6 mmol) was dissolved in DMF (25.6 mL) at room

temperature under an argon atmosphere. The solution was stirred

in an ice bath for 30 min, and then hydrazine acetate (1.45 g,

15.7 mmol) was added. After 5 min, more hydrazine acetate (1.45 g,

15.7 mmol) was added. After 1 h (TLC: hexane/acetone, 2:1), ace-

tone (34 mL) was added, and the reaction mixture was stirred at

0 °C for 15 min. The DMF was removed by coevaporation with

toluene under high vacuum. The residue was dissolved in CH₂Cl₂,

washed with water (2ϫ), brine (2ϫ), and dilute KHCO₃solution.

The organic phase was dried with MgSO₄and concentrated in

vacuo. The residue was purified by flash chromatography (cyclo-

hexane/acetone, 4:1) to give the intermediate hemiacetal (10.4 g,

70.9%). R_f= 0.25 (hexane/acetone, 2:1).

The crude mixture containing 13 was dissolved in absolute CH₂Cl₂

(90 mL) and absolute methanol (40 mL). Ground K₂CO₃(2.0 g)

was added, and the suspension was stirred for 30 min (TLC: cyclo-

hexane /acetone, 2:1). The solids were then filtered off, and the fil-

trate was adjusted to a pH of 5–6 with acetic acid (0.15 mL) and

then concentrated in vacuo. The residue was purified by flash

chromatography (cyclohexane/acetone, 4:1) to give 14 (2.99 g,

50.1% over two steps). R_f= 0.10 (cyclohexane/acetone, 2:1). [α]²_D²

= +11.3 (c = 1, CH₂Cl₂).

Method B (via Donor 16): Disaccharide 11 (3.0 g, 3 mmol), donor

16 (2.85 g, 4.5 mmol), and ground molecular sieves (4 Å) (6.0 g) in

The hemiacetal (13.0 g, 28.0 mmol) was dissolved in CH₂Cl₂

(700 mL) under argon. N-Phenyltrifluoroacetimidoyl chloride

absolute CH₂Cl₂(126 mL) were stirred at room temperature under (9.1 mL, 56.1 mmol) and DBU (4.17 mL, 28.0 mmol) were sequen-

argon for 30 min. The mixture was cooled in an ice bath for 10 min,

and then dilute TMSOTf (150 μmol, 540 μL of TMSOTf/CH₂Cl₂,

1:19) was added over 5 min. After 40 min (TLC: toluene/acetone,

8:1), TfOH (109 μL, 1.23 mmol) was added, and stirring was con-

tinued for a further 40 min. The reaction was stopped by filtration

through Celite into a stirred dilute KHCO₃solution. The organic

phase was washed with dilute KHCO₃solution, dried with MgSO₄,

tially added at 0 °C, and the reaction mixture was stirred for 40 min

(TLC: hexane/acetone, 2:1). The solvent was removed in vacuo, and

the residue was purified by flash chromatography (cyclohexane/

acetone, 5:1Ǟ4:1Ǟ2:1) to give 16 (anomeric mixture α/β = 1:3,

16.34 g, 91.8%) R_f= 0.41 (hexane/acetone, 2:1). [α]²_D⁵of α-anomer

= +37.5 (c = 0.15, CH₂Cl₂); [α]²_D²of β-anomer = +38.6 (c = 0.5,

CH₂Cl₂).

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C. Unverzagt et al.

FULL PAPER

1

α-Anomer: H NMR (360 MHz, CDCl₃, 50 °C): δ = 7.49–7.30 (m,

11 H, Ar), 7.15–7.07 (m, 2 H, Ar), 6.83–6.81 (m, 2 H, Ar), 6.57 (s,

1 H, NH), 6.55 (br. s, 1 H, 1-H), 5.64 (dd, J_2,3= J_3,4= 9.8 Hz, 1

H, 3-H), 5.56 (s, 1 H, PhCH=), 5.27 (dd, J_1,2= 3.7, J_2,3= 9.8 Hz,

1 H, 2-H), 4.37 (dd, J_5,6= 4.9, J_gem= 10.4 Hz, 1 H, 6a-H), 4.14

(m, 1 H, 5-H), 4.06 (s, 2 H, CH₂Cl), 3.85–3.77 (m, 2 H, 4-H, 6b-

H) ppm. ¹³C NMR (90 MHz, CDCl₃, 50 °C): δ = 167.0 (C=O),

152.2 (C=O), 143.2, 137.6, 136.9 (C-i Ar), 129.4, 129.3, 129.1,

128.5, 126.5, 125.1, 124.2, 119.6, 119.4 (C-Ar), 102.2 (PhCH=),

92.9 (C-1), 78.9 (C-4), 72.1 (C-2), 70.5 (C-3), 68.7 (C-6), 65.5 (C-

5), 40.4 (CH₂Cl) ppm.

4,6-O-Benzylidene-β-

D

-mannopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-

deoxy-2-phthalimido-β-D-glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-de-

oxy-6-O-(p-methoxyphenyl)-2-phthalimido-β-D-glucopyranosyl Az-

ide (B)

Method A (by Inversion in DMF): A solution of trisaccharide 14

(12.73 g, 9.3 mmol) in absolute CH₂Cl₂(450 mL) and absolute pyr-

idine (6 mL) was cooled to –40 °C. Subsequently, trifluoromethane-

sulfonic anhydride (3.22 mL, 19.6 mmol) was added dropwise. The

mixture was allowed to warm up to 0 °C over 10 min, and it was

stirred for 2 h until the starting material disappeared (TLC: hexane/

acetone, 1.2:1). The mixture was concentrated in a rotary evapora-

tor by maintaining a bath temperature of 5 °C, and then dried un-

der high vacuum for 20 min.

1

β-Anomer: H NMR (360 MHz, CDCl₃, 50 °C): δ = 7.45–7.06 (m,

14 H, Ar), 6.86 (m, 1 H, Ar), 6.61 (s, 1 H, NH), 5.93 (br. s, 1 H,

1-H), 5.53 (s, 1 H, PhCH=), 5.37–5.29 (m, 2 H, 3-H, 2-H), 4.39

(dd, J_5,6= 4.9, J_gem= 10.5 Hz, 1 H, 6a-H), 4.05 (s, 2 H, CH₂Cl),

3.86–3.78 (m, 2 H, 4-H, 6b-H), 3.58 (m, 1 H, 5-H) ppm. ¹³C NMR

(90 MHz, CDCl₃, 50 °C): δ = 166.4 (C=O), 152.3 (C=O), 143.1,

137.5, 136.8 (C-i Ar), 129.4, 129.3, 129.1, 128.5, 126.4, 125.1, 124.3,

119.5 (C-Ar), 102.1 (PhCH=), 95.0 (C-1), 78.3 (C-4), 73.4 (C-3),

73.0 (C-2), 68.6 (C-6), 67.4 (C-5), 40.4 (CH₂Cl) ppm. ESI-MS: m/z

= 657.96 [M + Na]⁺, 673.98 [M + K]⁺.

The residue, which contained triflate 14a, was dissolved in dry

DMF (60 mL) and dry pyridine (6 mL) and stirred at 65 °C for 3 h

(TLC: CH₂Cl₂/methanol, 50:1). The solvents were evaporated un-

der high vacuum at 45 °C. The residue was dried under high vac-

uum, dissolved in CH₂Cl₂(500 mL), washed with KHCO₃solution,

dried with MgSO₄, and concentrated.

The residue, which contained the imino carbonate 14b, was dis-

solved in dioxane (60 mL), and a mixture of acetic acid (18 mL)

and water (12 mL) was added. The mixture was kept at 4 °C for

10 h, followed by room temperature for 3 h (TLC: hexane/acetone,

2:1). After concentration, the remaining volatiles were coevapo-

rated with toluene (2 ϫ 20 mL). The residue was dissolved in

CH₂Cl₂(600 mL). The solution was washed with dilute HCl (2ϫ)

and dilute KHCO₃solution, dried with MgSO₄, and concentrated

in vacuo. The residue, which contained the cyclic carbonate 14c,

was dried under high vacuum and subsequently dissolved in abso-

lute CH₂Cl₂(400 mL). A solution of methanolic sodium methoxide

(20 mL, 0.1 %) was added. The mixture was stirred for 50 min

(TLC: hexane/acetone, 1.5:1) and then neutralized with acetic acid.

After evaporation of the solvents, the residue was coevaporated

with toluene (20 mL) and then dissolved in CH₂Cl₂(600 mL). The

solution was washed with dilute KHCO₃solution, dried with

MgSO₄, and concentrated in vacuo. The final product was purified

by flash chromatography (cyclohexane/acetone, 3:1 Ǟ 2:1) to give

B (6.12 g, 52.6% over four steps). R_f(14a) = 0.42 (hexane/acetone,

1.2:1); R_f(14b) = 0.58 (CH₂Cl₂/methanol, 50:1); R_f(14c) = 0.33

(hexane/acetone, 1.5:1); R_f(14e,f) = 0.52 (cyclohexane/ethyl acetate,

1:1); R_f(B) = 0.15 (cyclohexane/ethyl acetate, 1:1); R_f(B) = 0.24

(hexane/acetone, 2:1). [α]²_D²= +24.5 (c = 1.0, CH₂Cl₂).

Orthoester 13a: A suspension of disaccharide azide 11 (200 mg,

0.2 mmol), imidate 16 (192 mg, 0.3 mmol), and freshly activated

ground molecular sieves (4 Å) (0.4 g) in absolute CH₂Cl₂(8.4 mL)

was stirred at ambient temperature under argon for 30 min. The

mixture was cooled in an ice bath for 10 min, and dilute TMSOTf

(10 μmol, 36 μL of TMSOTf/CH₂Cl₂, 1:19) was added over 5 min.

After 30 min (TLC: toluene/acetone, = 8:1), the reaction was

stopped by filtration through Celite into a stirred dilute KHCO₃

solution. The mixture was extracted, and the organic phase was

dried with MgSO₄. After removal of the solvent in vacuo, the resi-

due was dried under high vacuum to give crude orthoester 13a.

The crude product was dissolved in absolute CH₂Cl₂(4.2 mL) and

absolute methanol (2.1 mL). Ground K₂CO₃(92 mg), which had

been activated prior to use by heating under high vacuum, was

added. The reaction mixture was stirred for 1 h (TLC: toluene/ace-

tone, 8:1), and then the solids were removed by filtration through

Celite. The pH of the filtrate was adjusted to 5–6 by adding acetic

acid (16 μL). After concentration in vacuo, the residue was purified

by flash chromatography (cyclohexane/acetone, 4:1) to give 13a

(212 mg, 75.0%). R_f= 0.33 (toluene/acetone, 8:1). [α]²_D⁵= +27.4 (c

= 0.5, CH₂Cl₂). ¹H NMR (360 MHz, [D₆]DMSO): δ = 9.90 (s, 1

H, NH), 7.84–7.50 (m, 8 H, Pht), 7.40–7.16 (m, 14 H, Ar), 6.98–

6.60 (m, 15 H, Ar), 6.20 (d, J_1,2= 5.5 Hz, 1 H, 1³-H), 5.70 (s, 1 H,

PhCH=), 5.32 (d, J_1,2= 9.6 Hz, 1 H, 1¹-H), 5.22 (dd, J_2,3= 3.4,

J_3,4= 9.1 Hz, 1 H, 3³-H), 5.17 (d, J_1,2= 7.5 Hz, 1 H, 1²-H), 4.90

(d, J_gem= 12.0 Hz, 1 H, OCH₂), 4.87–4.83 (m, 2 H, OCH₂, 2³-H),

4.65 (d, J_gem= 12.3 Hz, 1 H, OCH₂), 4.48 (d, J_gem= 12.3 Hz, 1 H,

OCH₂), 4.41–4.34 (m, 2 H, OCH₂, 6a³-H), 4.17–3.72 (m, 19 H, 3²-

H, 3¹-H, 4¹-H, OCH₂, 2²-H, CH₂Cl, 5³-H, 6a²-H, 6a¹-H, 4³-H, 4²-

H, 2¹-H, 6b¹-H, 6b³-H, 5¹-H, OCH₃), 3.57 (m, 1 H, 6b²-H), 3.45

(m, 1 H, 5²-H) ppm. ¹³C NMR (90 MHz, [D₆]DMSO): δ = 168.0,

167.3, 167.1 (C=O), 153.6, 152.3, 151.7, 138.7, 138.6, 138.0, 137.1

(C-q Ar, C=O Phcm), 134.8, 134.5, 134.4 (C-4/5 Pht), 130.7, 130.6,

130.4 (C-q Ar), 129.0, 128.7, 128.1, 127.7, 127.6, 127.5, 127.2,

127.1, 126.8, 126.1, 123.4, 123.3, 122.7 (Ar), 119.1 (C-q orthoester),

118.4, 115.4, 114.5 (Ar), 100.4 (PhCH=), 98.9 (C-1³), 96.4 (C-1²),

84.8 (C-1¹), 78.5 (C-3²), 78.2 (C-2³), 76.1 (C-3¹, C-4³), 75.2 (OCH₂),

75.1 (C-4¹), 74.9 (C-5¹), 74.8 (C-5²), 73.7 (OCH₂), 73.4 (C-3³), 73.1

Method B (by Inversion in CH₂Cl₂): A solution of trisaccharide 14

(1 g, 729 μmol) in absolute CH₂Cl₂(18 mL) and absolute pyridine

(470 μL, 5.8 mmol) was stirred in a pressure-stable glass vial at 0 °C

for 15 min. Trifluoromethanesulfonic anhydride (250 μL,

1.5 mmol) was added dropwise, and the mixture was stirred at 0 °C

for 45 min until the starting material disappeared (TLC: cyclohex-

ane/acetone, 2:1). The solution was heated in the pressure-stable

glass vial (CAUTION!) for 22 h with a bath temperature of 65 °C

until the reaction was complete (TLC: CH₂Cl₂/methanol, 40:1).

Subsequently, the mixture was acidified with acetic acid (1.25 mL,

21.8 μmol). Water (836 μL) was added, and the biphasic mixture

was vigorously stirred at ambient temperature for 6 h. After com-

plete reaction (TLC: CH₂Cl₂/methanol, 40:1), the solvents were

evaporated. The crude carbonate was dissolved in CH₂Cl₂, washed

with 1 m HCl and 2 m KHCO₃solution, dried with MgSO₄, and

concentrated.

(C-4²), 72.3 (OCH₂), 68.5 (C-6²), 67.5 (C-6³), 66.3 (C-6¹), 62.6 (C- Subsequently, the residue was dissolved in absolute CH₂Cl₂

5³), 55.8 (C-2²), 55.3 (OCH₃), 54.5 (C-2¹), 46.8 (CH₂Cl) ppm. ESI- (18 mL), and a solution of sodium methoxide in absolute methanol

MS: m/z = 1464.04 [M + NH₄]⁺, 1468.92 [M + Na]⁺, 1484.90 [M

(3.6 mL, 109 mm) was added. The solution was stirred at ambient

temperature for 3 h. Further sodium methoxide in absolute meth-

+ K]⁺.

10

www.eurjoc.org

Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20468

/KAP1

Date: 24-07-12 10:53:25

Pages: 16

Modular Synthesis of Core Fucosylated N-Glycans

anol (3.6 mL, 109 mm) was added, and stirring was continued for

128.9, 128.6, 128.2, 128.0, 127.9, 127.7, 127.6, 127.3, 127.2, 127.1,

126.9, 126.3 (C Ar), 123.4, 123.3 (C-3/6 Pht), 122.2, 118.2 (C Ar),

a further 2 h until the reaction was complete (TLC: cyclohexane/

ethyl acetate, 1:1). The solution was neutralized with acetic acid 115.4, 114.4 (C-2/6, C-3/5 PMP), 101.1(=CH-Ph), 99.4 (C-1³), 96.4

(1 mL). After removal of the solvents, the residue was purified by

flash chromatography (cyclohexane/ethyl acetate, 1.5:1 Ǟ 1:1 Ǟ

1:2) to give B (722 mg, 79.1%). R_f(14a) = 0.33 (cyclohexane/ace-

tone, 2:1); R_f(14b) = 0.38 (CH₂Cl₂/methanol, 40:1); R_f(14c) = 0.43

(CH₂Cl₂/methanol, 40:1); R_f(B) = 0.11 (cyclohexane/ethyl acetate,

1:1). ¹H NMR (500 MHz, [D₆]DMSO): δ = 7.85–7.61 (m, 8 H,

Pht), 7.41–7.25 (m, 10 H, Ar), 6.94–6.73 (m, 14 H, Ar), 5.51 (s, 1

(C-1²), 84.7 (C-1¹), 78.5 (C-3³), 78.5 (C-4³), 78.3 (C-4²), 76.5 (C-

3¹), 76.2 (C-3²), 75.3 (C-4¹), 75.0 (C-5¹), 74.1 (C-5²), 73.6 (OCH₂),

73.6 (OCH₂), 72.5 (C-2³), 72.1 (OCH₂), 67.8 (C-6²), 67.8 (C-6³),

66.6 (C-5³), 66.6 (C-6¹), 55.9 (C-2²), 55.3 (OCH₃), 54.4 (C-2¹) ppm.

ESI-MS: m/z = 1393.39 [M + Na]⁺.

Data for 14f: R_f= 0.35 (CH₂Cl₂/methanol, 30:1). [α]²_D²= +6.4 (c =

1

0.7, CH₂Cl₂). H NMR (360 MHz, [D₆]DMSO): δ = 9.85 (s, 1 H,

H, PhCH=), 5.34 (d, J_1,2= 9.5 Hz, 1 H, 1²-H), 5.21 (d, J_1,2

=

NH), 7.90–7.73 (m, 7 H, Pht), 7.64–7.57 (m, 1 H, Pht), 7.51–7.21

(m, 14 H, Ar), 6.97 (dd, J = 7.5, J = 7.4 Hz, 1 H, Ar), 6.94–6.70

(m, 14 H, Ar), 5.56 (s, 1 H, =CH-Ph), 5.41 (d, 1 H, J_OH,2= 5.1 Hz,

2³-OH), 5.35 (d, J_1,2= 9.5 Hz, 1 H, 1¹-H), 5.20 (d, J_1,2= 8.4 Hz,

1 H, 1²-H), 4.89–4.77 (m, 4 H, 3³-H, OCH₂, OCH₂, 1³-H), 4.65 (d,

J_gem= 12.2 Hz, 1 H, OCH₂), 4.59 (d, J_gem= 12.2 Hz, 1 H, OCH₂),

4.38 (d, J_gem= 12.5 Hz, 1 H, OCH₂), 4.33 (d, J_gem= 12.2 Hz, 1 H,

OCH₂), 4.24–4.10 (m, 4 H, 3¹-H, 4¹-H, 2³-H, 3²-H), 4.09–3.91 (m,

5 H, 6a³-H, 4³-H, 2²-H, 4²-H, 6a¹-H), 3.90–3.77 (m, 4 H, 6a²-H,

2¹-H, 6b¹-H, 5¹-H), 3.76–3.68 (m, 4 H, 6b²-H, OCH₃), 3.67–3.58

(m, 1 H, 6b³-H), 3.50–3.43 (m, 1 H, 5²-H), 3.37–3.27 (m, 1 H, 5³-

H) ppm. ¹³C NMR (90 MHz, [D₆]DMSO): δ = 167.1 (C=O Pht),

153.6, 152.8, 151.7 (C-4 PMP, C-i PMP, C=O Phcm), 139.0, 138.2,

137.9, 137.5 (C-i Ar), 134.8, 134.7 (C-4/5 Pht), 130.6 (C-1/2 Pht),

128.9, 128.7, 128.3, 128.0, 127.8, 127.7, 127.6, 127.4, 127.1, 127.0,

126.1 (C Ar), 123.4 (C-3/6 Pht), 122.4, 118.1 (C Ar), 115.4, 114.5

(C-2/6, C-3/5 PMP), 100.8 (=CH-Ph), 100.2 (C-1³), 96.5 (C-1²),

84.8 (C-1¹), 78.1 (C-4²), 76.3 (C-4¹), 76.2 (C-3²), 75.1 (C-4³), 74.9

(C-3¹), 74.8 (C-5¹), 74.6 (C-5²), 73.7 (OCH₂), 73.7 (OCH₂), 72.7

(C-3³), 72.3 (OCH₂), 68.6 (C-2³), 68.3 (C-6²), 67.7 (C-6³), 66.5 (C-

5³), 66.4 (C-6¹), 55.9 (C-2²), 55.3 (OCH₃), 54.5 (C-2¹) ppm. ESI-

MS: m/z = 1388.43 [M + NH₄]⁺.

8.4 Hz, 1 H, 1¹-H), 4.92 (d, J_2,OH= 4.4 Hz, 1 H, 2³-OH), 4.90 (d,

J_3,OH= 6.9 Hz, 1 H, 3³-OH), 4.83 (d, 1 H, OCH₂), 4.81 (d, 1 H,

OCH₂), 4.62 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1³-H), 4.62–4.55 (2 d, J_gem

=

12.1 Hz, 2 H, OCH₂), 4.36 (d, J_gem= 12.5 Hz, 1 H, OCH₂), 4.33

(d, J_gem= 11.9 Hz, 1 H, OCH₂), 4.20 (dd, J_3,4= J_4,5= 8.9 Hz, 1

H, 4²-H), 4.18–4.10 (m, 2 H, 3²-H, 3¹-H), 4.02 (dd, J_gem= 10.1,

J_5,6a= 4.7 Hz, 1 H, 6a³-H), 3.98–3.94 (m, 3 H, 2¹-H, 4¹-H, 6a²-H),

3.86–3.78 (m, 5 H, 6a¹-H, 6b²-H, 2²-H, 5²-H, 2³-H), 3.72 (s, 3 H,

OCH₃), 3.72–3.68 (m, 2 H, 6b¹-H, 4³-H), 3.56 (dd, J_gemϽ 1 Hz, 1

H, 6b³-H), 3.52 (m, 1 H, 3³-H), 3.44 (m, 1 H, 5-H), 3.09 (m, 1 H,

5³-H) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ = 167.9, 167.1

(C=O), 153.6, 151.7 (C-i PMP, C-4 PMP), 138.3, 138.2, 137.9,

137.8 (C-i Ar), 134.8, 134.6 (C-4/5 Pht, C-4 PhCH=), 130.5 (C-1/2

Pht), 128.7, 128.2, 127.9, 127.7, 127.6, 127.3, 127.1, 127.0, 126.3

(C-2/6 Ph, C-3/5 Ph), 123.4 (C-3/6 Pht), 115.4, 114.5 (C-2/6 PMP,

C-3/5 PMP), 100.9 (PhCH=), 100.4 (J_C-1,H-1= 162.8 Hz from a

coupled HMQC-spectrum, C-1³β), 96.5 (C-1²), 84.8 (C-1¹), 78.3

(C-4³), 77.4 (C-4¹), 76.3 (C-3²), 76.1 (C-3¹), 74.9 (C-4², C-5²), 74.6

(C-5¹), 73.6 (OCH₂), 72.1 (OCH₂), 70.8 (C-2²), 69.9 (C-3³), 68.0

(C-6¹), 67.8 (C-6³), 66.8 (C-5³), 66.4 (C-6²), 55.9 (C-2²), 55.3

(OCH₃), 54.5 (C-2¹) ppm. ESI-MS: m/z = 1274.0 [M + Na]⁺.

C₆₉H₆₅N₅O₁₈(1252.31): calcd. C 66.18, H 5.23, N 5.59; found C

66.39, H 5.25, N 5.36.

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-

(1Ǟ2)-3,4,6-tri-O-acetyl-α- -mannopyranosyl-(1Ǟ3)-4,6-O-benzyl-

idene-β- -mannopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-

phthalimido-β- -glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-

methoxyphenyl)-2-phthalimido-β- -glucopyranosyl Azide (17): A

D-glucopyranosyl-

D

4,6-O-Benzylidene-2-(phenylcarbamoyl)-β-

(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-

(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-methoxyphenyl)-2-phthalimido-β-

-glucopyranosyl Azide (14e) and 4,6-O-Benzylidene-3-(phenylcarb-

amoyl)-β- -mannopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-

phthalimido-β- -glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-

methoxyphenyl)-2-phthalimido-β- -glucopyranosyl Azide (14f): A

D

-mannopyranosyl-

D

-glucopyranosyl-

D

suspension of trisaccharide B (2.62 g, 2.09 mmol), imidate C

(3.42 g, 3.94 mmol), and freshly activated ground molecular sieves

(4 Å) (3.0 g) in absolute CH₂Cl₂(60 mL) was stirred at –25 °C for

30 min. Subsequently, boron trifluoride–diethyl ether (0.13 mL,

1.04 mmol) was added, and the mixture was allowed to warm up

to –15 °C over 2 h, and then stirred at room temperature for 1 h

(TLC: cyclohexane/acetone, 1.5:1). The suspension was diluted

with CH₂Cl₂, filtered through Celite and washed with a dilute

KHCO₃solution. The organic phase was dried with MgSO₄and

concentrated. The residue was purified by flash chromatography

D

fraction containing a mixture of 14e and 14f was obtained after

flash chromatography of the product mixture of the inversion reac-

tion of 14 in DMF. This mixture was separated by HPLC: Supelco

Ascentis C18, 5 μm (10 ϫ 250 mm), 4 mL/min, 80–85 % water/

acetonitrile + 0.1%TFA.

Data for 14e: R_f= 0.26 (CH₂Cl₂/methanol, 30:1). [α]²_D²= –0.3 (c =

1

0.7, CH₂Cl₂). H NMR (360 MHz, [D₆]DMSO): δ = 9.63 (s, 1 H, (cyclohexane/acetone, 3:1 Ǟ 2:1) to give 17 (3.23 g, 79%). R_f=

NH), 7.88–7.64 (m, 8 H, Pht), 7.54–7.21 (m, 14 H, Ar), 6.98 (dd,

0.46 (hexane/acetone, 1:1). [α]²_D³= –5.3 (c = 0.4, CH₂Cl₂). ¹H NMR

(500 MHz, [D₆]DMSO): δ = 7.91–6.69 (m, 36 H, Pht, Ar), 5.61 (s,

J = 7.2, J = 7.3 Hz, 1 H, Ar), 6.89–6.62 (m, 14 H, Ar), 5.55–5.47

(m, 2 H, 3³-OH, =CH-Ph), 5.31 (d, J_1,2= 9.6 Hz, 1 H, 1¹-H), 5.28 1 H, PhCH=), 5.51 (dd, J_2,3= J_3,4= 10.0 Hz, 1 H, 3⁵-H), 5.34 (d,

(dd, J_1,2Ͻ 1, J_2,3= 3.0 Hz, 1 H, 2³-H), 5.20 (d, J_1,2= 8.0 Hz, 1 H,

J_1,2= 9.5 Hz, 1 H, 1¹-H), 5.20–5.17 (m, 2 H, J_1,2= 9.5 Hz, 1²-H,

2³-OH), 5.01 (dd, J_2,3= 2.8, J_3,4= 10.2 Hz, 1 H, 3⁴-H), 4.94 (d,

J_1,2= 8.5 Hz, 1 H, 1⁵-H), 4.89–4.74 (m, 5 H, 4⁵-H, 4⁴-H, 1⁴-H,

1²-H), 4.91 (d, J_1,2Ͻ 1 Hz, 1 H, 1³-H), 4.82 (d, J_gem= 12.5 Hz, 1

H, OCH₂), 4.72 (d, J_gem= 12.4 Hz, 1 H, OCH₂), 4.63 (d, J_gem

=

12.1 Hz, 1 H, OCH₂), 4.56 (d, J_gem= 12.1 Hz, 1 H, OCH₂), 4.33 OCH₂), 4.56 (2 d, J_gem= 12.2 Hz, 2 H, OCH₂), 4.52 (d, J_1,2Ͻ

(d, J_gem= 12.4 Hz, 1 H, OCH₂), 4.29 (d, J_gem= 12.5 Hz, 1 H, 1.0 Hz, 1 H, 1³-H), 4.38–4.31 (2 d, 2 H, OCH₂), 4.21–4.13 (m, 2

OCH₂), 4.19–4.07 (m, 2 H, 3¹-H, 4¹-H), 4.06–3.91 (m, 4 H, 6a³-H, H, 4¹-H, 3¹-H), 4.09–3.90 (m, 9 H, 3²-H, 2⁵-H, 6a³-H, 6a⁵-H, 6a¹-

3²-H, 4²-H, 2²-H), 3.90–3.70 (m, 8 H, 6a¹-H, 6a²-H, 3³-H, 2¹-H, H, 2²-H, 2⁴-H, 5⁴-H, 4²-H), 3.89–3.68 (m, 10 H, 4³-H, 6a²-H, 6b¹-

6b²-H, 6b¹-H, 5¹-H, 4³-H), 3.68 (s, 3 H, OCH₃), 3.63–3.54 (m, 1 H, 2¹-H, 5¹-H, 2³-H, 6b⁵-H, OCH₃), 3.65 (m, 1 H, 6b²-H), 3.59–

H, 6b³-H), 3.51–3.43 (m, 1 H, 5²-H), 3.26–3.17 (m, 1 H, 5³-H)

ppm. ¹³C NMR (90 MHz, [D₆]DMSO): δ = 167.1 (C=O Pht), 3.11 (m, 1 H, 5³-H), 2.66 (m, 1 H, 5⁵-H), 2.03, 1.97, 1.95, 1.90 (4

153.6, 153.2, 151.7 (C-4 PMP, C-i PMP, C=O Phcm), 139.3, 138.4,

s, 12 H, OAc), 1.80 (s, 6 H, OAc) ppm. ¹³C NMR (125 MHz, [D₆]

138.0, 137.6 (C-i Ar), 134.8, 134.5 (C-4/5 Pht), 130.5 (C-1/2 Pht), DMSO): δ = 169.88, 169.76, 169.63, 169.19, 167.13 (C=O), 153.62,

3.52 (m, 4 H, 6a⁴-H, 6b⁴-H, 6b³-H, 3³-H), 3.40 (m, 1 H, 5²-H),

Eur. J. Org. Chem. 0000, 0–0

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Date: 24-07-12 10:53:25

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C. Unverzagt et al.

FULL PAPER

151.77 (C-4 PMP, C-i PMP), 138.33, 138.19, 137.98, 137.79 (C-i the reaction was stopped by adding pyridine (259 μL, 3.21 mmol),

Ar), 134.88 (C-4/5 Pht, C-4 PhCH=), 130.60 (C-1/2 Pht), 128.34, and then the solvents were evaporated in vacuo. The residue was

128.13, 127.82, 127.71, 127.64, 127.40, 127.09, 126.56 (C-2/6 Ph, dissolved in CH₂Cl₂, and the solution was washed with dilute

C-3/5 Ph, C-4 Ph), 123.46 (C-3/6 Pht), 115.42, 114.53 (C-2/6, C-3/5 KHCO₃solution, dried with MgSO₄, and concentrated. The resi-

PMP), 101.08 (PhCH=), 99.70 (J_C-1,H-1= 162.3 Hz from a coupled due was purified by flash chromatography (hexane/acetone, 1:1) to

HMQC spectrum, C-1³β), 97.56 (J_C-1,H-1= 173.8 Hz from a cou-

pled HMQC spectrum, C-1⁴α), 96.5 (C-1²), 95.42 (C-1⁵), 84.82 (C-

give 19 (428.3 mg, 80.0%). R_f= 0.1 (hexane/acetone, 1.2:1). [α]²_D³

+6.5 (c = 1, CH₂Cl₂). ¹H NMR (500 MHz, [D₆]DMSO): δ = 7.87–

=

1¹), 77.90 (C-3³), 77.28 (C-4³), 77.04 (C-4²), 76.34 (C-3¹), 76.00 (C- 6.68 (m, 31 H, Pht, Ar), 5.63 (dd, J_2,3= J_3,4= 9.9 Hz, 1 H, 3⁵-H),

3²), 75.00 (C-4¹), 74.90 (C-5¹), 74.66 (C-5²), 73.71, 73.61 (OCH₂),

5.43 (d, J_4,OH= 4.7 Hz, 1 H, 4³-OH), 5.29 (d, J_1,2= 8.5 Hz, 1 H,

73.01 (C-5⁴), 72.34 (OCH₂), 70.85 (C-5⁵), 69.84 (C-2³), 69.54 (C- 1⁵-H), 5.27 (d, J_1,2= 9.5 Hz, 1 H, 1¹-H), 5.13 (d, J_1,2= 8.0 Hz, 1

3⁵), 69.05 (C-4⁴), 68.33 (C-4⁵), 68.02 (C-6²), 67.66 (C-6³), 67.60 (C-

2⁴), 66.43 (C-6¹), 66.18 (C-5³), 65.13 (C-3⁴), 62.03 (C-6⁴), 61.23 (C-

6⁵), 55.83 (C-2²), 55.37 (OCH₃), 54.56 (C-2¹), 53.69 (C-2⁵), 20.39,

20.13 (OAc) ppm. ESI-MS: m/z = 1979.77 [M + Na]⁺.

H, 1²-H), 5.07 (dd, J_2,3= 3.0, J_1,2Ͻ 1.0 Hz, 1 H, 2³-H), 4.97 (dd,

J_3,4= J_4,5= 9.4 Hz, 1 H, 4⁵-H), 4.92 (dd, J_3,4= J_4,5= 10.1 Hz, 1

H, 4⁴-H), 4.87 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1⁴-H), 4.81 (d, J_gem= 12.0 Hz,

1 H, OCH₂), 4.75 (d, J_gem= 12.4 Hz, 1 H, OCH₂), 4.71 (dd, J_2,3

=

3.0, J_3,4= 10.4 Hz, 1 H, 3⁴-H), 4.61 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1³-H),

4.46 (2 d, J_gem= 12.3 Hz, 2 H, OCH₂), 4.42 (dd, J_6,OH= 5.4 Hz, 1

H, 6³-OH), 4.29 (d, J_gem= 12.4 Hz, 1 H, OCH₂), 4.22–4.15 (m, 3

H, 6a⁵-H, OCH₂, 2⁴-H), 4.12–4.09 (m, 3 H, 4¹-H, 2⁵-H, 3¹-H),

4.01–3.88 (m, 6 H, 3²-H, 6b⁵-H, 4²-H, 2²-H, 6a¹-H, 5⁵-H), 3.82–

3.56 (m, 12 H, 2¹-H, 6b¹-H, 5⁴-H, 6a²-H, 5¹-H, OCH₃, 6a³-H, 6a⁴-

H, 6b⁴-H, 6b²-H), 3.52–3.42 (m, 3 H, 4³-H, 6b³-H, 3³-H), 3.32 (m,

1 H, 5²-H), 3.03 (m, 1 H, 5³-H), 1.99, 1.95, 1.94, 1.90, 1.89, 1.84,

1.75 (7 s, 21 H, OAc) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ

= 170.07, 169.86, 169.68, 169.68, 169.22, 167.07 (C=O), 153.57,

151.74 (C-4 PMP, C-i PMP), 138.30, 138.13, 137.92 (C-i Ar),

134.83 (C-4/5 Pht), 130.55 (C-1/2 Pht), 128.19, 127.73, 127.68,

127.55, 127.34, 127.04 (C-2/6 Ph, C-3/5 Ph, C-4 Ph), 123.42 (C-3/

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-

(1Ǟ2)-3,4,6-tri-O-acetyl-α- -mannopyranosyl-(1Ǟ3)-2-O-acetyl-

4,6-O-benzylidene-β- -mannopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-de-

oxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-

6-O-(p-methoxyphenyl)-2-phthalimido-β- -glucopyranosyl Azide

D-glucopyranosyl-

D

(18): Pentasaccharide 17 (550 mg, 280.9 μmol) was dissolved in pyr-

idine/acetic anhydride (5 mL, 2:1) and stirred for 1 d. The mixture

was concentrated in vacuo, coevaporated with toluene (5 mL, 3ϫ)

and dried under high vacuum. The residue was dissolved in

CH₂Cl₂, and the solution was washed with 1 m HCl and dilute

KHCO₃solution, dried with MgSO₄, and concentrated. The crude

pentasaccharide 18 (562 mg, quant.) was used directly in a subse-

quent debenzylidenation reaction. R_f= 0.26 (hexane/acetone,

1.2:1). [α]²_D³= +1.3 (c = 1, CH₂Cl₂). ¹H NMR (500 MHz, [D₆]-

DMSO): δ = 7.90–6.68 (m, 36 H, Pht, Ar), 5.72 (s, 1 H, PhCH=),

5.52 (dd, J_2,3= J_3,4= 10.0 Hz, 1 H, 3⁵-H), 5.33 (d, J_1,2= 8.5 Hz,

1 H, 1¹-H), 5.31 (m, 1 H, 2³-H), 5.21 (d, J_1,2= 8.5 Hz, 1 H, 1²-H),

4.90–4.78 (m, 8 H, 1⁵-H, 1⁴-H, 4⁴-H, 4⁵-H, 1³-H, OCH₂, 3⁴-H),

6 Pht), 115.39, 114.47 (C-2/6, C-3/5 PMP), 97.47 (J_C-1,H-1

=

177.5 Hz from a coupled HMQC spectrum, C-1⁴α), 97.12 (J_C-1,H-1

= 163.7 Hz from a coupled HMQC spectrum, C-1³β), 96.49 (C-1²),

95.99 (C-1⁵), 84.75 (C-1¹), 76.70 (C-3²), 76.60 (C-4²), 76.47 (C-5³),

76.34 (C-3¹), 75.74 (C-3³), 75.11 (C-4¹), 74.90 (C-5¹), 74.45 (C-5²),

73.83, 73.67 (OCH₂), 73.43 (C-2⁴), 72.19 (OCH₂), 71.01 (C-5⁵),

70.53 (C-2³), 69.67 (C-3⁵), 69.07 (C-3⁴), 68.62 (C-4⁵), 68.10 (C-5⁴),

68.00 (C-6²), 66.76 (C-4³), 66.42 (C-6¹), 64.47 (C-4⁴), 61.86 (C-6)⁴,

61.65 (C-6⁵), 60.36 (C-6³), 55.89 (C-2²), 55.32 (OCH₃), 54.85 (C-

2¹), 53.64 (C-2⁵), 20.51, 20.36, 20.07 (OAc) ppm. ESI-MS: m/z =

4.60–4.54 (2 d, J_gem= 12.0 Hz, 2 H, OCH₂), 4.35 (d, J_gem

=

12.4 Hz, 1 H, OCH₂), 4.19 (d, J_gem= 11.5 Hz, 1 H, OCH₂), 4.14

(m, 2 H, 3¹-H, 4¹-H), 4.08–3.94 (m, 9 H, 3²-H, 6a³-H, 2⁵-H, 3³-H,

4²-H, 2²-H, 6a⁵-H, 6a¹-H, 2⁴-H), 3.87–3.68 (m, 11 H, 6a²-H, 6b¹-

H, 2¹-H, 4³-H, 5¹-H, 5⁴-H, 6b⁵-H, OCH₃, 6b²-H), 3.64–3.52 (m, 3

H, 6b³-H, 6a⁴-H, 6b⁴-H), 3.43 (m, 1 H, 5²-H), 3.23 (m, 1 H, 5³-H),

2.51 (m, 1 H, 5⁵-H), 2.03, 1.99, 1.98, 1.95, 1.90, 1.79, 1.78 (7 s, 21

H, OAc). ¹³C NMR (125 MHz, [D₆]DMSO): δ = 169.82, 169.73,

169.16, 169.08, 167.09 (C=O), 153.59, 151.77 (C-4 PMP, C-i PMP),

138.30, 138.02, 137.93, 137.38 (C-i Ar), 134.93, 134.79 (C-4/5 Pht,

C-4 PhCH=), 130.55 (C-1/2 Pht), 128.29, 128.17, 127.84, 127.74,

127.47, 127.32, 127.21, 127.08, 126.47 (C-2/6 Ph, C-3/5 Ph, C-4

Ph), 123.41 (C-3/6 Pht), 115.41, 114.48 (C-2/6, C-3/5 PMP), 100.96

(PhCH=), 97.52 (J_C-1,H-1= 166.4 Hz from a coupled HMQC spec-

trum, C-1³β), 96.78 (J_C-1,H-1= 176.1 Hz from a coupled HMQC

spectrum, C-1⁴α), 96.47 (C-1²), 95.14 (C-1⁵), 84.76 (C-1¹), 78.39

(C-4³), 76.90 (C-4²), 76.38 (C-3¹, C-3²), 75.19 (C-4¹), 74.91 (C-5)¹,

74.23 (C-5²), 73.93, 73.68 (OCH₂), 73.46 (C-3³), 72.73 (C-2⁴), 72.14

(OCH₂), 70.81 (C-5⁵), 70.33 (C-2³), 69.51 (C-3⁵), 68.53 (C-3⁴),

68.23 (C-5⁴), 68.09 (C-4⁵), 67.88 (C-6²), 67.54 (C-6³), 66.45 (C-6¹),

65.55 (C-5³), 64.52 (C-4⁴), 61.77 (C-6⁴), 60.96 (C-6⁵), 55.89 (C-2²),

55.33 (OCH₃), 54.50 (C-2¹), 53.54 (C-2⁵), 20.44, 20.31, 20.04 (OAc)

977.4 [M + 2 Na]²⁺

.

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-

(1Ǟ2)-3,4,6-tri-O-acetyl-α- -mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-

acetyl-2-deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ2)-3,4,6-tri-

O-acetyl-α- -mannopyranosyl-(1Ǟ6)]-2-O-acetyl-β- -mannopyr-

anosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β- -glucopyr-

anosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-methoxyphenyl)-2-phthal-

imido-β- -glucopyranosyl azide (20): A suspension of pentasac-

D-glucopyranosyl-

D

charide 19 (414 mg, 217 μmol), imidate C (280 mg, 323 μmol), and

freshly activated ground molecular sieves (4 Å) (1.0 g) in absolute

CH₂Cl₂(50 mL) was stirred at –40 °C for 45 min. Subsequently,

boron trifluoride–diethyl ether (10 μL, 81 μmol) was added over

5 min. The mixture was warmed to –10 °C over 2 h, and then

stirred at room temperature for 3 h (TLC: hexane/acetone, 1.2:1).

The suspension was diluted with CH₂Cl₂, filtered through Celite

and washed with dilute KHCO₃solution. The organic phase was

dried with MgSO₄and concentrated. The residue was purified by

flash chromatography (hexane/acetone, 1:1) to give 20 (414 mg,

73.0%). R_f= 0.10 (hexane/acetone, 1.2:1). [α]²_D²= +6.9 (c = 1,

ppm. ESI-MS: m/z = 1000.6 [M + 2 H]²⁺

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-

(1Ǟ2)-3,4,6-tri-O-acetyl-α- -mannopyranosyl-(1Ǟ3)-2-O-acetyl-β-

-mannopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-

-glucopyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-methoxyphen-

-glucopyranosyl Azide (19): p-Toluenesulfonic

.

D

-glucopyranosyl-

1

D

CH₂Cl₂). H NMR (500 MHz, [D₆]DMSO): δ = 7.83–7.70 (m, 16

D

H, Pht), 7.55–7.11 (m, 5 H, Ar), 6.82–6.60 (m, 14 H, Ar), 5.65 (dd,

J_2,3= J_3,4= 9.8 Hz, 1 H, 3⁵-H), 5.52 (dd, J_2,3= J_3,4= 9.8 Hz, 1 H,

3^5Ј-H), 5.51 (d, J_4,OH= 4.7 Hz, 1 H, 4³-OH), 5.31 (d, J_1,2= 8.5 Hz,

1 H, 1⁵-H), 5.27 (d, J_1,2= 9.5 Hz, 1 H, 1¹-H), 5.17 (d, J_1,2= 8.5 Hz,

1 H, 1^5Ј-H), 5.10 (d, J_1,2= 8.2 Hz, 1 H, 1²-H), 5.08 (dd, J_1,2Ͻ 1.0,

J_2,3= 2.7 Hz, 1 H, 2³-H), 5.00 (dd, J_3,4= J_4,5= 9.8 Hz, 1 H, 4⁵-

yl)-2-phthalimido-β-

D

acid monohydrate (560 mg, 2.9 mmol) was added to a solution of

pentasaccharide 18 (560 mg, 280 μmol) in absolute acetonitrile

(5.6 mL). After stirring for 45 min (TLC: hexane/acetone, 1.2:1),

12

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Modular Synthesis of Core Fucosylated N-Glycans

H), 4.99–4.87 (m, 4 H, 4⁴-H, 4^4Ј-H, 4^5Ј-H, 3^4Ј-H), 4.83–4.75 (m, 2

J_3,4= 9.8 Hz, 1 H, 3⁵-H), 5.58 (dd, J_2,3= J_3,4= 9.8 Hz, 1 H, 3^5Ј-

H, 1³-H, OCH₂), 4.71 (dd, J_2,3= 2.7, J_3,4= 10.2 Hz, 1 H, 3⁴-H), H), 5.29 (d, J_1,2= 8.5 Hz, 1 H, 1⁵-H), 5.26 (dd, J_1,2Ͻ 1.0, J_2,3

=

4.66–4.57 (m, 2 H, 1⁴-H, OCH₂), 4.51–4.42 (2 d, J_gem= 12.2 Hz, 2

2.7 Hz, 1 H, 2³-H), 5.23 (d, J_1,2= 9.5 Hz, 1 H, 1¹-H), 5.21 (d, J_1,2

H, OCH₂), 4.35–4.26 (m, 3 H, 1^4Ј-H, OCH₂), 4.22–3.88 (m, 13 H, = 8.5 Hz, 1 H, 1^5Ј-H), 5.20 (d, J_1,2= 8.2 Hz, 1 H, 1²-H), 5.06–

6a⁵-H, 2⁴-H, 2⁵-H, 3¹-H, 2^5Ј-H, 6a^5Ј-H, 2^4Ј-H, 4¹-H, 3²-H, 2²-H, 4.84 (m, 6 H, 4⁵-H, 4⁴-H, 4^5Ј-H, 4^4Ј-H, 3^4Ј-H, 4³-H), 4.82 (d, J_gem

4²-H, 5⁵-H, 6b⁵-H), 3.83–3.54 (m, 15 H, 6a¹-H, 2¹-H, 5⁴-H, 6a²-H, = 12.2 Hz, 1 H, OCH₂), 4.76 (dd, J_6,OH= 5.4 Hz, 1 H, 6¹-OH),

6b^5Ј-H, 6b¹-H, OCH₃, 6a⁴-H, 6b⁴-H, 5¹-H, 5^4Ј-H, 6a^4Ј-H, 6b²-H), 4.75 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1³-H), 4.72 (dd, J_2,3= 3.1, J_3,4

=

3.47–3.19 (m, 8 H, 6a³-H, 6b³-H, 4³-H, 3³-H, 5^5Ј-H, 6b^4Ј-H, 5²-H, 10.5 Hz, 1 H, 3⁴-H), 4.67 (d, J_gem= 11.9 Hz, 1 H, OCH₂), 4.56–

5³-H), 2.01, 1.97, 1.96, 1.95, 1.93, 1.90, 1.87, 1.79, 1.77, 1.76 (13 s,

4.51 (m, 3 H, 1⁴-H, OCH₂), 4.35 (2 d, J_gem= 12.3 Hz, 2 H,

OCH₂), 4.30 (m, 1 H, 1^4Ј-H), 4.24 (dd, J_5,6= 4.8, J_gem= 12.8 Hz,

39 H, OAc) ppm. ¹³C NMR (125 MHz, [D₆]DMSO): δ = 170.15,

170.09, 169.91, 169.84, 169.67, 169.63, 169.55, 169.24, 169.16, 1 H, 6a⁵-H), 4.18–3.90 (m, 13 H, 2⁵-H, 3²-H, 2⁴-H, 6a^5Ј-H, 2^5Ј-H,

167.70, 167.25 (C=O), 153.53, 151.78 (C-4 PMP, C-i PMP), 138.29, 3¹-H, 2^4Ј-H, 4¹-H, 6b⁵-H, 5⁵-H, 2²-H, 4²-H, 3³-H), 3.82–3.64 (m,

138.17 (C-i Ar), 134.83, 134.71, 134.60 (C-4/5 Pht), 130.78, 130.56 7 H, 5⁴-H, 2¹-H, 6a⁴-H, 6b^5Ј-H, 6a^4Ј-H, 6b⁴-H), 3.55–3.36 (m, 8

(C-1/2 Pht), 128.29, 127.78, 127.68, 127.44, 127.38, 127.19, 126.91

(C-2/6 Ph, C-3/5 Ph, C-4 Ph), 123.37 (C-3/6 Pht), 115.33, 114.42

(C-2/6, C-3/5 PMP), 97.88 (J_C-1,H-1= 172.8 Hz from a coupled

HMQC spectrum, C-1⁴α), 97.16 (J_C-1,H-1= 173.5 Hz from a cou-

pled HMQC spectrum, C-1^4Јα), 96.89 (J_C-1,H–1= 162.9 Hz from a

H, 5^4Ј-H, 5^5Ј-H, 6b^4Ј-H, 6b⁴-H, 6a³-H, 5²-H, 6a¹-H, 5³-H), 3.31

(m, 1 H, 5¹-H), 3.18–3.16 (m, 2 H, 6b³-H, 6b¹-H), 2.26, 2.03, 2.00,

1.99, 1.98, 1.96, 1.94, 1.93, 1.90, 1.84, 1.81, 1.80, 1.79, 1.75 (14 s,

42 H, OAc). ¹³C NMR (125 MHz, [D₆]DMSO): δ = 170.13,

170.10, 169.96, 169.89, 169.80, 169.67, 169.60, 169.55, 169.27,

coupled HMQC spectrum, C-1³β), 96.64 (C-1²), 96.23 (C-1^5Ј), 169.24, 169.16, 167.70, 167.25 (C=O), 138.35, 138.12 (C-i Ar),

96.11 (C-1⁵), 84.80 (C-1¹), 76.97 (C-4²), 76.81 (C-3¹), 76.00 (C-3²), 135.06, 134.89, 134.77, 134.60 (C-4/5 Pht), 130.75, 130.60 (C-1/2

75.73 (C-3³), 75.41 (C-4¹), 75.10 (C-5¹), 74.47 (C-5²), 74.14 (C-5³), Pht), 128.21, 127.75, 127.70, 127.53, 127.35, 127.24, 127.01 (C-2/

73.92 (C-2^4Ј), 73.88 (OCH₂), 73.54 (C-2⁴), 73.53, 72.36 (OCH₂),

71.07 (C-5⁵), 70.80 (C-5^5Ј), 70.46 (C-2³), 69.76 (C-3^5Ј), 69.74 (C-

3⁵), 69.68 (C-3^4Ј), 69.08 (C-3⁴), 68.63 (C-4⁵), 68.37 (C-4^5Ј), 68.15

(C-5⁴), 67.80 (C-6²), 67.57 (C-5^4Ј), 67.11 (C-6³), 66.91 (C-4³), 66.42

(C-6¹), 64.70 (C-4⁴), 64.43 (C-4^4Ј), 61.81 (C-6⁴), 61.80 (C-6⁵), 61.64

(C-6^4Ј), 61.37 (C-6^5Ј), 55.77 (C-2²), 55.27 (OCH₃), 54.54 (C-2¹),

53.87 (C-2^5Ј), 53.78 (C-2⁵), 20.63, 20.51, 20.38, 20.36, 20.25, 20.07

6 Ph, C-3/5 Ph, C-4 Ph), 123.40 (C-3/6 Pht), 97.76 (J_C-1,H-1=

173.5 Hz from a coupled HMQC spectrum, C-1⁴α), 96.95

(J_C-1,H-1= 173.5 Hz from a coupled HMQC spectrum, C-1^4Јα),

96.87 (J_C-1,H-1= 163.7 Hz from a coupled HMQC spectrum, C-

1³β), 96.80 (C-1²), 96.08 (C-1⁵), 96.05 (C-1^5Ј), 84.85 (C-1¹), 77.33

(C-4²), 77.05 (C-5¹), 76.71 (C-3¹), 75.99 (C-3²), 74.91 (C-4¹), 74.31

(C-5²), 73.72, 73.65 (OCH₂), 73.45 (C-2^4Ј), 73.41 (C-2⁴), 73.08 (C-

3³), 72.25 (OCH₂), 71.26 (C-5³), 71.05 (C-5⁵), 70.93 (C-5^5Ј), 70.13

(C-2³), 69.78 (C-3⁵), 69.68 (C-3^5Ј), 69.53 (C-3^4Ј), 69.30 (C-4³), 68.79

(C-3⁴), 68.54 (C-4⁵), 68.59 (C-5⁴), 68.48 (C-4^5Ј), 67.99 (C-6⁴), 67.74

(C-5^4Ј), 67.06 (C-6³), 64.45 (C-4⁴), 64.43 (C-4^4Ј), 61.74 (C-6^4Ј), C-

6⁵, 61.62 (C-6^5Ј), 58.81 (C-6¹), 55.74 (C-2²), 54.61 (C-2¹), 53.85 (C-

2⁵), 53.80 (C-2^5Ј), 20.93, 20.52, 20.48, 20.36, 20.25, 20.07 (OAc)

(OAc) ppm. ESI-MS: m/z = 1331.0 [M + 2 Na]²⁺

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-

(1Ǟ2)-3,4,6-tri-O-acetyl-α-

acetyl-2-deoxy-2-phthalimido-β-

O-acetyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-di-O-acetyl-β-

pyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-

pyranosyl-(1Ǟ4)-3-O-benzyl-2-deoxy-6-O-(p-methoxyphenyl)-2-

phthalimido-β- -glucopyranosyl Azide (21) and 3,4,6-Tri-O-acetyl-

2-deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ2)-3,4,6-tri-O-

acetyl-α- -mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-acetyl-2-deoxy-2-

phthalimido-β- -glucopyranosyl-(1Ǟ2)-3,4,6-tri-O-acetyl-α- -manno-

pyranosyl-(1Ǟ6)]-2,4-di-O-acetyl-β- -mannopyranosyl-(1Ǟ4)-3,6-

di-O-benzyl-2-deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ4)-3-O-

benzyl-2-deoxy-2-phthalimido-β- -glucopyranosyl Azide (22)

.

D

-glucopyranosyl-

D

-mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-

-glucopyranosyl-(1Ǟ2)-3,4,6-tri-

-manno-

-gluco-

D

ppm. ESI-MS: m/z = 1299.0 [M + 2 Na]²⁺

.

D

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-

(1Ǟ2)-3,4,6-tri-O-acetyl-α- -mannopyranosyl-(1Ǟ3)-[3,4,6-tri-O-

acetyl-2-deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ2)-3,4,6-tri-

O-acetyl-α- -mannopyranosyl-(1Ǟ6)]-2,4-di-O-acetyl-β- -manno-

pyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β- -gluco-

pyranosyl-(1Ǟ4)-[2,3,4-tri-O-(p-methoxybenzyl)-α- -fucopyranosyl-

(1Ǟ6)]-3-O-benzyl-2-deoxy-2-phthalimido-β- -glucopyranosyl Azide

D-glucopyranosyl-

D

L

D

(23): A suspension of heptasaccharide 22 (116 mg, 45.6 μmol), ethyl

thiofucoside D (49 mg, 86.1 μmol), and freshly activated ground

molecular sieves (4 Å) (30 mg) in absolute CH₂Cl₂(410 μL) and

DMF (330 μL) was stirred at ambient temperature for 10 min. Af-

ter addition of tetrabutylammonium bromide (21 mg, 64.5 μmol)

and copper(II) bromide (15 mg, 65 μmol), the suspension was

stirred for 2 d. The suspension was then diluted with CH₂Cl₂, and

triethylamine (13.8 μL, 100 μmol) and ethanethiol (7.6 μL,

100 μmol) were added. The mixture was then filtered through Ce-

lite. The filtrate was concentrated, and the residue was purified by

flash chromatography (hexane/acetone, 1:1) to give 23 as a mixture

of anomers (119 mg, 85.3%). Further purification was achieved by

Step 1 (Acetylation): Heptasaccharide 20 (390 mg, 149 μmol) was

dissolved in pyridine/acetic anhydride (5 mL, 2:1) and stirred for

1 d. The mixture was concentrated in vacuo, coevaporated with tol-

uene (5 mL, 3ϫ) and dried under high vacuum. The residue was

dissolved in CH₂Cl₂, and the solution was washed with 1 m HCl

and dilute KHCO₃solution, dried with MgSO₄, and concentrated.

The crude product 21 (396 mg, quant.) was not purified, but was

used directly in the following step. R_f= 0.26 (hexane/acetone,

1.2:1).

Step 2 (Removal of the PMP Group): Water (2.45 mL) and CAN

(165 mg, 301 μmol) were added to a solution of crude hepta-

saccharide 21 (80 mg, 30 μmol) in toluene (2.45 mL) and acetoni- HPLC using the following conditions: Eclipse XDB-C8, 5 μm,

trile (2.82 mL), and the solution was stirred for 15 h. The mixture 4.6ϫ150 mm, 75–95% (water/acetonitrile + 0.1% HCOOH) or

was diluted with ethyl acetate (10 mL) and washed with saturated Diol-60 5 μm, 6.0ϫ300 mm, 60–70% (hexane/ethyl acetate). R_f=

1

KHCO₃solution and brine. After drying with MgSO₄, the organic 0.16 (hexane/acetone, 1.2:1). [α]²_D²= –13.9 (c = 0.43, CH₂Cl₂). H

phase was concentrated. The residue was purified by flash NMR (500 MHz, [D₆]DMSO): δ = 7.94–7.70 (m, 16 H, Pht), 7.38–

chromatography (hexane/acetone, 1:1) to give 22 (69 mg, 90%). R_f6.74 (m, 27 H, Ar), 5.64 (dd, J_2,3= J_3,4= 10.2 Hz, 1 H, 3⁵-H), 5.57

1

= 0.16 (hexane/acetone, 1.2:1). [α]²_D²= –12.0 (c = 1, CH₂Cl₂). H

(dd, J_2,3= J_3,4= 10.2 Hz, 1 H, 3^5Ј-H), 5.42 (d, J_1,2= 8.5 Hz, 1 H,

1²-H), 5.28 (d, J_1,2= 8.5 Hz, 1 H, 1⁵-H), 5.25 (dd, J_1,2Ͻ 1.0, J_2,3

= 2.4 Hz, 1 H, 2³-H), 5.18 (d, J_1,2= 9.8 Hz, 1 H, 1¹-H), 5.16 (d,

NMR (500 MHz, [D₆]DMSO): δ = 7.93–7.63 (m, 16 H, Pht),

7.34–7.12 (m, 5 H, Ar), 6.93–6.74 (m, 10 H, Ar), 5.63 (dd, J_2,3

=

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/KAP1

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Pages: 16

C. Unverzagt et al.

FULL PAPER

J_1,2= 8.5 Hz, 1 H, 1^5Ј-H), 5.03 (dd, J_3,4= J_4,5= 9.3 Hz, 1 H, 4⁵-

H), 4.98 (dd, J_3,4= J_4,5= 10.2 Hz, 1 H, 4⁴-H), 4.92 (dd, J_3,4= J_4,5

= 9.8 Hz, 1 H, 4^5Ј-H), 4.89–4.86 (m, 4 H, 4^4Ј-H, 3^4Ј-H, 4³-H,

CH₂O), 4.79 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1³-H), 4.72–4.63 (m, 6 H, 3⁴-H,

CH₂O), 4.58 (d, J_1,2= 3.0 Hz, 1 H, 1⁸α-H), 4.56 (d, J_1,2Ͻ 1.0 Hz, 1

H, 1⁴-H), 4.54–4.46 (m, 3 H, CH₂O), 4.38–4.32 (m, 3 H, CH₂O),

4.28 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1^4Ј-H), 4.26–4.21 (m, 2 H, 3²-H, 6a⁵-

H), 4.17–4.04 (m, 6 H, 2⁵-H, 4¹-H, 6a^5Ј-H, 2^5Ј-H, 2⁴-H, 3¹-H), 4.02–

3.91 (m, 6 H, 2^4Ј-H, 2²-H, 6b⁵-H, 4²-H, 3³-H, 5⁵-H), 3.86–3.78 (m,

6 H, 2¹-H, 5²-H, 5⁸-H, 5⁴-H, 6a²-H, 6b^5Ј-H), 3.74, 3.73 (2 s, 6 H,

CH₃O), 3.71 (m, 2 H, 2⁸-H, 6a⁴-H), 3.69 (s, 3 H, CH₃O), 3.65–3.56

(m, 4 H, 3⁸-H, 6b⁴-H, 6b²-H, 4⁸-H), 3.54–3.37 (m, 8 H, 5¹-H, 6a^4Ј-

H, 6a¹-H, 5^4Ј-H, 5^5Ј-H, 6b^4Ј-H, 5³-H, 6a³-H), 3.29 (m, 1 H, 6b¹-

H), 3.17 (m, 1 H, 6b³-H), 2.25, 2.07, 2.03, 1.99, 1.98, 1.97, 1.94,

1.93, 1.92, 1.83, 1.80, 1.79, 1.78, 1.75 (14 s, 42 H, OAc), 0.92 (d,

J_5,6= 6.4 Hz, 3 H, H-6⁸) ppm. ¹³C NMR (125 MHz, [D₆]DMSO):

δ = 170.02–169.10, 167.99–167.06 (C=O), 158.62, 158.60, 158.46

(C-4 MPM), 138.30, 138.09, 137.93 (C-i Ar), 134.93, 134.82, 134.54

(C-4/5 Pht, C-1 MPM), 130.89, 130.74, 130.61, 130.51 (C-1/2 Pht),

129.35–127.06 (C-Ar), 123.37 (C-3/6 Pht), 113.54, 113.44, 113.35

(C-3/5 MPM), 97.67 (J_C-1,H-1= 174.3 Hz from a coupled HMQC

spectrum, C-1⁴α), 97.30 (J_C-1,H-1= 165.1 Hz from a coupled

HMQC spectrum, C-1³β), 96.96 (J_C-1,H-1= 173.6 Hz from a cou-

pled HMQC spectrum, C-1^4Јα), 96.54 (C-1⁸), 96.26 (C-1²), 96.03

(C-1^5Ј), 96.01 (C-1⁵), 84.26 (C-1¹), 78.16 (C-2⁸), 78.15 (C-4²), 77.04

(C-4⁸), 76.23 (C-3²), 75.85 (C-3¹), 75.24 (C-5¹), 74.60 (C-4¹), 74.02

(C-5²), 73.80, 73.67, 73.63 (OCH₂), 73.39 (C-3⁸), 73.32 (C-2^4Ј),

73.26 (C-2⁴), 73.19 (C-3³), 72.25, 71.48 (OCH₂), 71.03 (C-5³), 70.98

(C-5⁵, OCH₂), 70.83 (C-5^5Ј), 69.85 (C-2³), 69.65 (C-3⁵), 69.53 (C-

3^5Ј), 69.32 (C-3^4Ј, C-4²), 69.28 (C-4³), 68.64 (C-3⁴), 68.47 (C-5⁴),

68.45 (C-5⁵), 68.38 (C-4^5Ј), 67.63 (C-5^4Ј), 67.50 (C-6²), 67.09 (C-

6³), 65.58 (C-5⁸), 64.32 (C-4⁴), 64.31 (C-4^4Ј), 63.55 (C-6¹), 61.60 (C-

6⁵), 61.59 (C-6^4Ј), 61.50 (C-6⁴), 61.46 (C-6^5Ј), 55.56 (C-2²), 54.96,

54.88, 54.81 (OCH₃), 54.65 (C-2¹), 53.69 (C-2⁵), 53.63 (C-2^5Ј),

20.85, 20.37, 20.29, 20.11, 20.02, 19.90, 19.75 (OAc), 16.06 (C-6⁸)

ppm. FAB-MS: m/z = 3081 [M + Na]⁺.

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This work was supported by the Deutsche Forschungsgemein-

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Received: April 12, 2012

Published Online: ᭿

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Pages: 16

C. Unverzagt et al.

FULL PAPER

Oligosaccharide Synthesis

D. Ott, J. Seifert, I. Prahl, M. Niemietz,

J. Hoffman, J. Guder, M. Mönnich,

C. Unverzagt* ................................. 1–16

Modular Synthesis of Core Fucosylated N-

Glycans

Keywords: Carbohydrates / Glycosylation /

Oligosaccharides / Glycoproteins / Stereo-

selectivity

A modular synthesis of core-fucosylated

N-glycans was optimized, leading to the bi-

antennary octasaccharide N-glycan A. Sev-

eral obstacles to the synthesis of func-

tionalized core trisaccharide building block

B were overcome, leading to an improved

and efficient protocol for β-mannosylation

by intramolecular inversion.

16

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Article Doi

Modular synthesis of core fucosylated N-glycans

DOI: 10.1002/ejoc.201200468

Source and publish data:

Authors:

Article abstract of DOI:10.1002/ejoc.201200468

Full text of DOI:10.1002/ejoc.201200468

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