Utilizing staudinger llgation for the synthesis of glycoamino acid building blocks and other glycomimetics

Article abstract of DOI:10.1002/ejoc.200900437

The synthesis of amide-linked glycomimetics is of interest in the glycosciences as they represent a typical linkage in biologically important glycoconjugates. We have addressed the synthesis of three types of N-glycosyl amide architectures, which were tar

Full text of DOI:10.1002/ejoc.200900437

FULL PAPER
DOI: 10.1002/ejoc.200900437
Utilizing Staudinger Ligation for the Synthesis of Glycoamino Acid Building
Blocks and Other Glycomimetics
Alexander Schierholt,[a] Harun A. Shaikh,[a] Jörn Schmidt-Lassen,[a] and
Thisbe K. Lindhorst*[a]
Keywords: Ligation / Amino acids / Glycomimetics / Glycoconjugates / Azides
The synthesis of amide-linked glycomimetics is of interest in
the glycosciences as they represent a typical linkage in biolo-
gically important glycoconjugates. We have addressed the
synthesis of three types of N-glycosyl amide architectures,
which were targeted using Staudinger ligation. This facile
method has allowed us to synthesize a collection of N-glycos-
yloctanamides, N-glycosyloxyethyl amino acids and multi-
valent glycoclusters. The last two classes represent mole-
cules that can be used as building blocks in solid-phase pep-
tide synthesis for the preparation of multivalent glycoconju-
gates.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
The glycocalyx of eukaryotic cells is of high molecular
complexity[1] and of central biological importance in cell
communication processes.[2] Among other glycoconjugates,
O- and N-linked glycoproteins are constituents of this com-
plex glyco environment and key to many of the unsolved
problems relating to glycocalyx function.[3]
The isolation of N-glycoproteins and -peptides from
physiological sources is problematic[4] and their chemical
synthesis difficult.[5] Therefore, the modification of natural,
highly branched glycoconjugates into various glycomimet-
ics, such as glycodendrimers and -clusters, has become an
important strategy in glycoscience.[6] In N-linked glycopep-
tides the saccharide portion is attached through a glycosyl
amide bond to an asparagine side-chain of the peptide
backbone. This type of linkage has been utilized for the
synthesis of a variety of branched glycomimetics[7] and re-
cently we found that Staudinger ligation is a versatile
method for accessing amide-linked glycomimetics such as
the wedged glycosyl amide glycocluster shown in
Scheme 1.[8]
Scheme 1. Three-component Staudinger ligation allows the conver-
sion of dendron-functionalized glycosyl azides into wedged gly-
coamides.[8]
which involves the formation of an amide bond from the
reaction between an azide and a carboxylic acid in the pres-
ence of trialkyl- or triarylphosphanes.[10] A simple and ef-
ficient method for utilizing this ligation method for the syn-
thesis of N-linked glycoamino acids was developed by Inazu
and Kobayashi in 1993.[11] Since then a number of modifi-
cations to this approach have been established in which acti-
vated carboxylic acids were employed for the synthesis of
glycosyl amides,[8,12] including Bertozzi and co-workers’
“traceless” Staudinger ligation in which the aza-ylide inter-
mediate is trapped by a methoxycarbonyl group.[13]
Based on our good experiences with the Staudinger lig-
ation method,[8] which proceeds under mild reaction condi-
tions and tolerates both protected as well as unprotected
substrates, it became our goal to utilize three-component
Staudinger ligation to access amide-linked glycomimetics of
three types: (i) N-glycosyloctanamides, (ii) N-mannosyloxy-
ethyl amino acids and (iii) trivalent glycocluster amino ac-
ids (Figure 1). These glycomimetics are needed for func-
tional studies with multivalent carbohydrate assemblies.
Although the Staudinger reaction was developed by
Staudinger and Meyer in 1919 for the conversion of azides
into the corresponding amines,[9] it was not until 65 years
later that Garcia et al. first reported on Staudinger ligation,
[a] Otto Diels Institute of Organic Chemistry, Christiana Albertina
University of Kiel,
Otto-Hahn-Platz 4, 24098 Kiel, Germany
Fax: +49-431-8807410
E-mail: tklind@oc.uni-kiel.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900437.
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A. Schierholt, H. A. Shaikh, J. Schmidt-Lassen, T. K. Lindhorst
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the amphiphilic target glycosyl amides. We employed oc-
tanoic acid in combination with tri-n-butylphosphane, DIC
(diisopropylcarbodiimide) and HOBt (1-hydroxybenzotri-
azole), thus saving one step and allowing milder reaction
conditions. It is important to conduct the reaction at a low
temperature because when the reaction was carried out at
room temp., for example, no ligation product was ob-
served.[8]
Accordingly, the well-known β-glycosyl azides 1 and 2
and the α-mannosyl azide 3[16] led to the corresponding
amides 4, 6 and 8, which could be deprotected according to
Zemplén and Pacsu[17] to furnish the corresponding amphi-
philes 5, 7 and 9 in high yields. Yields were in the same
range regardless of the sugar series employed. However, in
the case of the α-mannosyl azide 3, anomerization occurred
at the anomeric centre, leading to an α,β mixture of prod-
ucts in the ratio of 3:1. Anomerization is a frequently re-
ported problem in this reaction and proceeds via the glyco-
syl iminophosphorane intermediate (Scheme 3).[18] In the
case of the β-glycosyl amides 7 and 8 the stereochemistry
at the anomeric position was retained.
Figure 1. Three types of target molecules approached by the Staud-
inger ligation: (i) N-glycosyloctanamides, (ii) N-mannosyloxyethyl
amino acids and (iii) glycocluster amides.
Anomeric mixtures could be separated by HPLC, how-
ever, to circumvent the problem of the anomerization of
glycosyl azides we aimed to use azidoalkyl glycosides as al-
ternative substrates in the Staudinger ligation.
Results and Discussion
N-Glycosyloctanamides
To study the supramolecular behaviour of amphiphilic
glycoconjugates with varied sugar configurations we aimed
to synthesize the three different N-glycosyl amides 4, 6 and
8 (Scheme 2). These types of molecules are typically synthe-
sized by the reaction of glycosylamines with activated car-
N-Mannosyloxyethyl Amino Acids
For our studies on mannose-specific bacterial ad-
boxylic acid derivatives.[14] However, glycosylamines are rel- hesion,[19] we require a variety of glycoamino acid mimetics.
atively unstable and thus this method has often been shown We employed 2-azidoethyl mannoside 10 (Scheme 4) as the
to be rather unsatisfactory. Staudinger ligation provides a starting material in the Staudinger-type ligation reactions
facile alternative in this case. Boullanger et al. utilized oc- to obtain mannose-modified amino acids and to exclude
tanoyl chloride under Staudinger conditions[12a,15] to access the problem of anomerization at the anomeric centre.
Scheme 2. Synthesis of N-glycosyloctanamides using the Staudinger ligation strategy. Reagents and conditions: a) Octanoic acid, DIC,
HOBt, (nBu)3P, THF, 0 °CǞr.t., overnight; 81% for 4, 71% for 6, 67% for 8 (α/β = 3:1); b) NaOMe, dry MeOH, 24 h, r.t.; 99% for 5,
84% for 7, 95% for 9.
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Glycoamino Acid Building Blocks and Other Glycomimetics
Scheme 3. Anomerization during Staudinger ligation can occur via the intermediate glycosyl iminophosphorane.
Scheme 4. Synthesis of N-mannosyloxyethyl amino acids. Reagents and conditions: General procedure: amino acid derivative, 10, DIC,
HOBt, (nBu)3P, THF, 0 °CǞr.t. a) Fmoc-Asp-OH, 32%; b) Fmoc-Asp(OH)-OtBu, 72%; c) Ph3P instead of (nBu)3P, Fmoc-Glu-(OH)-
OtBu, 64%, d) Fmoc-Cys(Trt)-OH, 61%.
Thus, mannoside 10, which was easily obtained from free acid Fmoc-Asp-OH, ligation of both carboxy groups
mannose in three steps,[20] was treated with the corre- was intended. Indeed, Staudinger ligation led to the di-
sponding Fmoc-protected amino acid building blocks de- meric amide 11 as the main coupling product (32%) to-
rived from aspartic acid, glutamic acid or cysteine, respec- gether with the glycoamino acid 12 (28%). Overall, this
tively, and treated with DIC and HOBt to activate the acid result is satisfactory as both ligation products could be
and tributylphosphane, activating the azido-modified easily separated from one another and 12 could be con-
mannoside (Scheme 4). Two different aspartic acid deriva- verted into 11 after an additional ligation step. Even
tives were employed, Fmoc-protected aspartic acid (Fmoc- though 12 is an undesired product of this reaction, it is an
Asp-OH) and the corresponding tert-butyl ester Fmoc- appealing building block en route to mixed glycoclusters
Asp(OH)-OtBu. In the latter case the monoligated product or for capping the N-terminus of various glycopeptide de-
13 was targeted and obtained in high yield. With the rivatives.
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FULL PAPER
In analogy to the Staudinger ligation of 10 with Fmoc- peptide-coupling due to the risk of OǞN acetyl group mi-
Asp(OH)-OtBu, reaction with the glutamic acid derivative gration. Staudinger ligation allows the protected azidoethyl
Fmoc-Glu(OH)-OtBu proceeded equally well, delivering mannoside 10 to be utilized in peptide-coupling with the
the glycoamino acid derivative 14. The protected cysteine triacid 16 (Scheme 5). To activate the acid, again a combi-
building block Fmoc-Cys(Trt)-OH led to the ligation prod- nation of DIC and HOBt was used, but trimethylphos-
uct 15 under the same conditions. The mannosidic cysteine phane was employed instead of the more hindered tri-n-
15 has the potential to dimerize as it can form the corre- butylphosphane. At 0 °C no product formation was ob-
sponding disulfide after deprotection of the thio function.
served, whereas at –70 °C the amide-linked glycocluster 17
The glycoamino acid derivatives thus obtained in was obtained in over 50% yield. In addition to the desired
Scheme 4 will be employed in the solid-phase synthesis of product, the formation of a defect derivative carrying only
glycopeptides, for which it is advantageous to use O-pro- two mannoside moieties was observed. When the Fmoc-
tected building blocks because they are more stable than protected cluster 17 was submitted to a deprotection/acety-
the OH-unprotected analogues, are more easily cleaved lation/de-O-acetylation sequence, the OH-free cluster glyco-
from the resin and purification of the cleaved products is side 18 was obtained, which is of interest as an inhibitor of
facilitated in comparison with unprotected glycopeptides. type 1 fimbriae-mediated bacterial adhesion.[23]
Staudinger ligation has proved its worth in achieving the
Reaction of the triacid 16 with 2,3,4,6-tetra-O-benzoyl-
required O-acylated glycoamino acid building blocks for α--mannosyl azide[24] under analogous conditions led to
SPPS. Otherwise, synthesis of the products in Scheme 4 by the trivalent glycocluster 19 in somewhat moderate yield af-
standard peptide-coupling utilizing the acetyl-protected ter size-exclusion chromatography. Surprisingly, no anomer-
mannoside requires additional protection and deprotection ization occurs in this case, according to careful NMR
steps to avoid undesired OǞN acetyl group migration.
analysis.
Conclusions
Glycocluster Amides
Protected glycoamino acids are convenient building
blocks in carbohydrate chemistry and for SPPS (solid-phase
peptide synthesis). However, their synthesis by the standard
peptide-coupling of an amine and an activated carboxylic
acid utilizing O-acetyl-protected glycoside intermediates is
afflicted by undesired OǞN acetyl group migration. This
problem can be solved by Staudinger ligation, which allows
the formation of amide-linked glycomimetics from carbo-
hydrate azides and carboxylic acid derivatives, such as
amino acids, mediated by a combination of DIC, HOBt and
a suitable phosphane. This allowed us to prepare a collec-
tion of new mannosidic amino acids 11–15 by employing
the acetylated azidoethyl mannoside 10. A slightly modified
ligation protocol also allowed us to prepare glycocluster
amides 17–19, which are quite sophisticated building blocks
for the construction of multivalent glycoconjugates. Indeed
it can be concluded that Staudinger ligation allows a direct
and fast access to versatile amide-linked glycoconjugates in
a one-pot process, including amphiphilic glycosyl amides 5,
7 and 9.
Encouraged by these results, we set out to employ Stau-
dinger ligation in the synthesis of glycoclusters based on
Newkome and co-workers’ branched triacid 16.[21] Peptide-
coupling of unprotected 2-aminoethyl α--mannoside with
the trivalent wedge 16 has been reported previously,[22] and
led to the OH-unprotected product, which was extremely
difficult to purify. The O-acetylated 2-aminoethyl α--man-
noside, on the other hand, cannot be employed in standard
Experimental Section
General Methods: Thin-layer chromatography was performed on
silica gel plates (GF 254, Merck). Detection was effected by UV
irradiation and subsequent charring with 10% sulfuric acid in
EtOH followed by heat treatment. Flash chromatography was per-
formed on silica gel 60 (230–400 mesh, particle size 0.040–
0.063 mm, Merck) using distilled solvents. Optical rotations were
measured with a Perkin–Elmer 241 polarimeter (sodium D-line:
Scheme 5. Synthesis of amide-linked glycoclusters. Reagents and
conditions: a) 10, DIC, HOBt, P(CH3)3, –70 °CǞr.t., overnight,
52%; b) CH2Cl2, morpholine, 4 h, room temp., then Ac2O, pyr-
idine, room temp., overnight, 68%; NaOMe, MeOH, 4 h, room
temp., quant.; c) 2,3,4,6-tetra-O-benzoyl-α--mannosyl azide,[24]
DIC, HOBt, P(CH3)3, –70 °CǞr.t., overnight, 34%.
1
589 nm, length of cell: 1 dm). H and 13C NMR spectra were re-
corded with Bruker DRX-500 and AV-600 spectrometers. Chemical
shifts are reported relative to internal tetramethylsilane (δ =
0.00 ppm) or D2O (δ = 4.76 ppm). Air- and/or moisture-sensitive
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Glycoamino Acid Building Blocks and Other Glycomimetics
reactions were carried out under nitrogen. Commercial reagents
were used without purication unless otherwise noted.
mannopyranosyloxy)ethyl]-L-asparagine Amide (11): According to a
slightly altered protocol for the Staudinger ligation, mannosyl azide
10 (1.9 g, 4.5 mmol, 2.3 equiv.), Fmoc-Asp-OH (674 mg, 1.9 mmol,
1.0 equiv.), HOBt (662 mg, 4.9 mmol, 2.6 equiv.), DIC (758 µL,
4.9 mmol, 2.6 equiv.) and tri-n-butylphosphane (1.2 mL, 4.9 mmol,
2.6 equiv.) were allowed to react in a Schlenk flask under N2. The
crude product was purified by silica gel chromatography (EtOAc/
MeOH, 9:1) to yield 11 (669 mg, 0.61 mmol, 32%) as a colourless
foam and the byproduct Nα-(fluoren-9-ylmethoxycarbonyl)-Nδ-[2-
(2Ј,3Ј,4Ј,6Ј-tetra-O-acetyl-α--mannopyranosyloxy)ethyl]--aspar-
agine (12; 393 mg, 0.54 mmol, 28%) as a white foam.
General Procedure for the Staudinger Ligation: The corresponding
azide-functionalized carbohydrate (1 equiv.) and the carboxylic
acid (1.8 equiv.) were combined with HOBt (1.8 equiv.) in a
Schlenk flask and dried for more than 1 h in vacuo. This mixture
was dissolved in dry THF under nitrogen and cooled to 0 °C. Then
DIC (1.8 equiv.) was added and the solution was stirred for 10 min,
followed by the addition of tri-n-butylphosphane (1–1.8 equiv.) and
stirring for 1 h at 0 °C. Then the reaction mixture was stirred over-
night at room temp., diluted with water (50 mL) and extracted
twice with ethyl acetate (30 mL). The combined organic phases
were washed with brine, dried with MgSO4 and the mixture was
filtered and concentrated under reduced pressure. The crude prod-
uct was purified by silica gel chromatography.
Data for 11: [α]2D0 = +30.4 (c = 1.3, MeOH). H NMR (600 MHz,
1
3
3
CD3OD, TMS): δ = 7.83 (d, J = 7.5 Hz, 2 H, Ar-H), 7.69 (d, J
3
3
= 7.3 Hz, 2 H, Ar-H), 7.43 (t, J = 7.7 Hz, 2 H, Ar-H), 7.35 (t, J
= 7.4 Hz, 2 H, Ar-H), 5.33 (dd, 3J2Ј,3Ј = 3.4, J3Ј,4Ј = 10.0 Hz, 2 H,
3
3
3
2ϫ3Ј-H), 5.31 (dd, J1Ј,2Ј = 1.7, J2Ј,3Ј = 3.4 Hz, 2 H, 2ϫ2Ј-H),
General Procedure for O-Acetyl Deprotection According to Zemplén
and Pacsu:[17] The protected N-glycosyl amide was dissolved in dry
MeOH (5 mL) and sodium methoxide (1  solution in MeOH,
100 µL) was added. The mixture was stirred for 4 h, neutralized
with Amberlite IR120 (H), filtered and the solvents evaporated to
dryness. The substrate was dissolved in water and subjected to ly-
ophilization.
5.33 (dd ≈ t, 3J4Ј,5Ј = 9.9 Hz, 2 H, 2ϫ4Ј-H), 4.89 (d, 3J1Ј,2Ј = 1.7 Hz,
3
2 H, 2ϫ1Ј-H), 4.54 (t, JH-α,H-β = 6.3 Hz, 1 H, α-H), 4.44 (dd,
3JCHH-Fmoc,CH-Fmoc = 7.2, JCHH-Fmoc,CH-Fmoc = 10.6 Hz, 1 H
2
3
CHH-Fmoc), 4.40–4.34 (m, 1 H, CHH-Fmoc), 4.26 (dd, J5Ј,6Ј
5.2, J6Ј,6ЈЈ = 12.2 Hz, 2 H, 2ϫ6aЈ-H), 4.30–4.24 (m, 1 H, CH-
Fmoc), 4.14 (dd, J5Ј,6Ј = 7.1, J6Ј,6ЈЈ = 14.2 Hz, 2 H, 2ϫ6bЈ-H),
4.10–4.04 (m, 2 H, 2ϫ5Ј-H), 3.85–3.77 (m, 2 H, 2ϫOCHHCH2),
=
2
3
2
N-Octanoyl-β-D-glucopyranosyl Amide (5): According to the gene-
3.64–3.67 (m, 2 H, 2ϫOCHHCH2), 3.51–3.46 (m, 2 H,
ral procedure for Staudinger ligation, glucosyl azide 1 (318 mg,
2ϫOCH2CHH), 3.44–3.38 (m, 2 H, 2ϫOCH2CHH), 2.76 (dd,
2
853 µmol), octanoic acid (221 mg, 1.53 mmol, 1.8 equiv.), HOBt 3JH-α,H-β = 5.7, JH-β,H-βЈ = 14.4 Hz, 1 H, β-Ha), 2.68 (dd,
2
(233 mg, 1.75 mmol, 2.1 equiv.), DIC (255 µL, 1.64 mmol,
1.9 equiv.) and tri-n-butylphosphane (212 µL) were allowed to react
in THF (40 mL). Silica gel column chromatography (cyclohexane/
EtOAc, 1:1) gave compound 4 (326 mg, 688 µmol, 81%) as an
amorphous colourless solid. The deprotected title compound was
prepared from 4 (84.0 mg, 178 µmol) according to Zemplén and
Pacsu and obtained as an amorphous colourless solid (54.0 mg,
177 µmol, 99%). The analytical data were identical to those re-
ported in the literature.[15]
3JH-α,H-βЈ = 7.7, JH-β,H-βЈ = 14.5 Hz, 1 H, β-Hb), 2.16, 2.07, 2.05,
1.98 (each s, each 6 H, 8ϫCO2CH3) ppm. 13C NMR (150 MHz,
CD3OD, TMS): δ = 173.7 (NH-CO-CH2), 172.9, 172.4, 171.6,
171.5 (8ϫCO2CH3), 158.0 (CO2-Fmoc), 145.2 (Ar-C), 142.6 (Ar-
C), 128.8 (Ar-C), 128.2 (Ar-C), 126.2 (Ar-C), 120.9 (Ar-C), 98.9
(C-1Ј), 70.8 (C-2Ј), 70.7 (C-3Ј), 70.7 (C-5Ј), 68.1 (CH2-Fmoc), 67.8
(OCH2CH2), 67.3 (C-4Ј), 63.6 (C-6Ј), 53.4 (C-α), 48.4 (CH-Fmoc),
40.3 (OCH2CH2), 38.9 (C-β), 20.8, 20.6, 20.6, 20.5
(8ϫCO2CH3) ppm. MS (MALDI-TOF): calcd. for [C51H63N3O24
+ Na]+ 1124.36; found 1124.59; calcd. for [C51H63N3O24 + K]+
1140.34; found 1140.57.
N-Octanoyl-β-D-galactopyranosyl Amide (7): According to the ge-
neral procedure for Staudinger ligation, glycosyl azide 2 (305 mg,
820 µmol, 1 equiv.), octanoic acid (207 mg, 1.44 mmol, 1.8 equiv.),
HOBt (217 mg, 1.61 mmol, 1.9 equiv.), DIC (240 µL, 1.56 mmol,
1.9 equiv.) and tri-n-butylphosphane (200 µL) were allowed to react
in THF (40 mL). After purification by silica gel column chromatog-
raphy (cyclohexane/EtOAc, 1:1) the protected ligation product 6
was obtained as a colourless oil (274 mg, 579 µmol, 71%). Depro-
tection of 6 (278 mg, 610 µmol) was achieved according to Zemplén
and Pacsu, yielding the title compound 7 as an amorphous colour-
less solid (151 mg, 494 µmol, 84%). The analytical data were iden-
tical to those reported in the literature.[15]
Nα-(Fluoren-9-ylmethoxycarbonyl)-Nδ-[2-(2Ј,3Ј,4Ј,6Ј-tetra-O-acetyl-
α-D-mannopyranosyloxy)ethyl]-L-asparagine tert-Butyl Ester (13):
According to the general procedure for Staudinger ligation, man-
nosyl azide 10 (150 mg, 0.36 mmol, 1 equiv.), Fmoc-Asp(OH)-
OtBu (324 mg, 0.78 mmol, 1.8 equiv.), HOBt (105 mg, 0.78 mmol,
1.8 equiv.), DIC (121 µL, 0.78 mmol, 1.8 equiv.) and tri-n-butyl-
phosphane (100 µL, 0.40 mmol, 1.1 equiv.) were allowed to react in
a Schlenk flask under N2. The crude product was purified by silica
gel chromatography (cyclohexane/EtOAc, 1:1), followed by a sec-
ond chromatography (cyclohexane/EtOAc/MeOH, 5:4:1) to give 13
(207 mg, 0.26 mmol, 72%) as a colourless foam. [α]2D0 = +28.6 (c =
1.4, CH2Cl2). 1H NMR (500 MHz, CDCl3): δ = 7.76 (d, 3J =
N-Octanoyl-α,β-D-mannopyranosyl Amide (9): According to the ge-
neral procedure for Staudinger ligation, mannosyl azide 3 (303 mg,
812 µmol, 1 equiv.), octanoic acid (207 mg, 1.56 mmol, 1.8 equiv.),
HOBt (213 mg, 1.56 mmol, 1.9 equiv.), DIC (240 µL, 1.56 mmol,
1.9 equiv.) and tri-n-butylphosphane (200 µL) were allowed to react
in THF (40 mL) and the product was purified by chromatography
on silica gel (cyclohexane/EtOAc, 1:1) to yield protected 8 (259 mg,
547 µmol, 67%) as a colourless oil. The deprotected title compound
9 was prepared from 8 (203 mg, 429 µmol) according to Zemplén
and Pacsu and obtained as anomeric mixture in the form of an
amorphous colourless solid (124 mg, 406 µmol, 95%); anomeric ra-
tio according to 1H NMR, α:β = 3:1. The analytical data were
identical to those reported in the literature.[15]
3
3
7.5 Hz, 2 H, Ar-H), 7.60 (d, J = 7.2 Hz, 2 H, Ar-H), 7.39 (t, J =
3
7.4 Hz, 2 H, Ar-H), 7.30 (t, J = 7.4 Hz, 2 H, Ar-H), 6.18 (br. s, 1
3
H, CH2NHCO), 6.05 (d, JNH,H-α = 8.2 Hz, 1 H, NH-Fmoc), 5.33
(dd, J2Ј,3Ј = 3.2, J3Ј,4Ј = 9.9 Hz, 1 H, 3Ј-H), 5.29–5.24 (m, 2 H,
2Ј-H, 4Ј-H), 4.82 (d, J1Ј,2Ј = 1.6 Hz, 1 H, 1Ј-H), 4.51 (br. s, 1 H,
3
3
3
α-H), 4.41–4.37 (m, 1 H, CHH-Fmoc), 4.33–4.29 (m, 1 H, CHH-
3
2
Fmoc), 4.27 (dd, J5Ј,6Ј = 5.5, J6Ј,6ЈЈ = 12.2 Hz, 1 H, 6aЈ-H), 4.23
3
2
(m, 1 H, CH-Fmoc), 4.12 (dd, J5,6ЈЈ = 2.5, J6Ј,6ЈЈ = 12.2 Hz, 1 H,
3
3
3
6bЈ-H), 3.98 (ddd, J4Ј,5Ј = 9.9, J5Ј,6Ј = 5.5, J5Ј,6ЈЈ = 2.5 Hz, 1 H,
5Ј-H), 3.78–3.72 (m, 1 H, OCHHCH2), 3.60–3.51 (m, 2 H,
OCHHCH2, OCH2CHH), 3.46–3.37 (m, 1 H, OCH2CHH), 2.91
3
2
(dd, JH-α,H-βЈ = 5.6, JH-βa,H-βb = 15.1 Hz, 1 H, β-Ha), 2.90 (dd,
N-[2-(2Ј,3Ј,4Ј,6Ј-Tetra-O-acetyl-α-
(fluoren-9-ylmethoxycarbonyl)-Nδ-[2-(2Ј,3Ј,4Ј,6Ј-tetra-O-acetyl-α-
D
-mannopyranosyloxy)ethyl]-Nα-
3JH-α,H-βb = 5.9, 2JH-βa,H-βb = 15.1 Hz, 1 H, β-Hb), 2.14, 2.09, 2.04,
D-
1.98 (each s, each 3 H, 4ϫCO2CH3), 1.47 (s, 9 H, CCH3) ppm. 13
C
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NMR (150 MHz, CDCl3, TMS): δ = 170.6 (NHCOC-β), 170.1
(CO2-tBu), 170.0, 169.9, 169.9, 169.6 (4ϫCO2CH3), 156.2 (CO2-
Fmoc), 143.9 (Ar-C), 141.2 (Ar-C), 127.6 (Ar-C), 127.0 (Ar-C),
(CO-C-α), 169.9, 169.8, 169.7, 169.7 (4ϫCO2CH3), 155.9 (Ar-C),
144.4 (Ar-C), 143.8 (Ar-C), 143.7 (Ar-C), 143.7 (Ar-C), 129.6 (Ar-
C), 129.6 (Ar-C), 129.0 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 127.7
125.2 (Ar-C), 119.9 (Ar-C), 97.7 (C-1Ј), 82.3 (CCH3), 69.3 (C-2Ј), (Ar-C), 120.0 (Ar-C), 97.7 (C-1Ј), 69.4 (C-3Ј), 69.0 (C-2Ј), 68.7 (C-
68.9 (C-3Ј), 68.7 (C-5Ј), 67.4 (OCH2CH2), 67.1 (CH2-Fmoc), 66.2 5Ј), 67.05 (OCH2CH2), 66.2 (CH2-Fmoc), 66.2 (C-4Ј), 62.5 (C-6Ј),
(C-4Ј), 62.5 (C-6Ј), 51.4 (C-α), 47.1 (CH-Fmoc), 39.1 (OCH2CH2), 53.99 (C-α), 47.2 (CH-Fmoc), 39.1 (CH2N), 33.1 (C-β), 20.8
38.1 (C-β), 27.9 [C(CH3)3], 20.8, 20.7, 20.6, 20.6
(COCH3), 20.7 (COCH3), 20.7 (COCH3), 20.7 (COCH3) ppm.
HRMS (ESI): calcd. for [C53H54N2O13S + Na]+ 981.3244; found
981.3223.
(4ϫCO2CH3) ppm. HRMS (ESI): calcd. for [C39H48N2O15
+
Na]+ 807.2952; found 807.2964.
Nα-(Fluoren-9-ylmethoxycarbonyl)-Nδ-[2-(2Ј,3Ј,4Ј,6Ј-tetra-O-acetyl-
α- -mannopyranosyloxy)ethyl]-L-glutamine tert-Butyl Ester (14): tetra-O-acetyl-α-D-mannopyranosyloxy)-3-aza-4-oxo-hexane (17):
3-Cascade:N-fluorenylmethoxycarbonyl-aminomethane[3]:1-(2,3,4,6-
D
According to a slightly altered protocol for the Staudinger ligation,
mannosyl azide 10 (150 mg, 0.36 mmol, 1 equiv.), Fmoc-Glu(OH)-
OtBu (266 mg, 0.64 mmol, 1.8 equiv.), HOBt (105 mg, 0.78 mmol,
1.8 equiv.), DIC (121 µL, 0.78 mmol, 1.8 equiv.) and triphenylphos-
phane (170 mg, 0.64 mmol, 1.8 equiv.) were allowed to react in a
Schlenk flask under N2. The crude product was purified by silica
gel chromatography (cyclohexane Ǟ cyclohexane/EtOAc, 1:1 Ǟ
EtOAc) to give 14 (186 mg, 0.23 mmol, 64%) as a colourless foam.
Fmoc-protected triacid 16 (110 mg, 0.234 mmol), mannosyl azide
10 (584 mg, 1.40 mmol) and HOBt (104 mg, 772 mmol) were dried
in a Schlenk flask under vacuum for 1 h. This mixture was dis-
solved in dry THF (20 mL) under an inert atmosphere, DIC
(81 µL, 772 mmol) was added and the solution stirred for 10 min.
Then trimethylphosphane (1  solution in THF, 2.41 mL,
2.1 mmol) was added at –70 °C and it was stirred for 12 h at room
temp. The reaction was quenched by the addition of water, the
aqueous phase was washed with CH2Cl2 (3ϫ15 mL) and the com-
1
[α]2D0 = +16.4 (c = 0.8, CH2Cl2). H NMR (500 MHz, CDCl3): δ =
7.75 (d, 3J = 7.4 Hz, 2 H, Ar-H), 7.61 (d, 3J = 7.4 Hz, 2 H, Ar-H), bined organic phases dried with MgSO4. The mixture was filtered,
7.38 (mc, 2 H, Ar-H), 7.30 (mc, Ar-H), 6.47 (br., 1 H, CH2NHCO), evaporated and purification by silica gel column chromatography
3
5.67 (d, JNH,H-α = 7.9 Hz, 1 H, NH-Fmoc), 5.35 (dd, 3J2Ј,3Ј = 3.5,
(cyclohexane/EtOAc, 1:1.5) led to the pure product 17 (194 mg,
3J3Ј,4Ј = 10.0 Hz, 1 H, 3Ј-H), 5.27 (dd, 3J1Ј,2Ј = 1.6, 3J2Ј,3Ј = 3.4 Hz, 0.122 mmol, 52%) in the form of a white foam. [α]2D0 = +2.9 (c =
1 H, 2Ј-H), 5.26–5.24 (m, 1 H, 4Ј-H), 4.82 (d, 3J1Ј,2Ј = 1.6 Hz, 1 H, 0.8, CH2Cl2). 1H NMR (600 MHz, CDCl3, TMS): δ = 7.75 (d, J =
1Ј-H), 4.42–4.37 (m, 2 H, CH2-Fmoc), 4.25 (dd, 3J5Ј,6Ј = 5.5, 2J6Ј,6ЈЈ
7.15 Hz, 2 H, Ar-H), 7.62–7.61 (m, 2 H, Ar-H), 7.39 (mc, 2 H, Ar-
3
3
= 12.2 Hz, 1 H, 6aЈ-H), 4.25–4.19 (m, 2 H, CH-Fmoc, α-H), 4.05– H), 7.30 (mc, 2 H, Ar-H), 5.29 (dd, J2,3 = 3.3, J3,4 = 10.0 Hz, 3
4.01 (m, 1 H, 6bЈ-H), 3.99 (mc, 1 H, 5Ј-H), 3.80–3.75 (m, 1 H, H, 3ϫ3-H), 5.26 (d, J3,4 = 9.7 Hz, 3 H, 3ϫ4-H), 5.24 (dd, J1,2
3
3
3
3
OCHHCH2), 3.58–3.50 (m, 2 H, OCHHCH2, OCH2CHH), 3.45– = 1.7, J2,3 = 3.2 Hz, 3 H, 3ϫ2-H), 4.83 (d, J1,2 = 1.5 Hz, 3 H,
3.41 (m, 1 H, OCH2CHH), 3.16–3.03 (m, 1 H, γ-Ha), 2.98–2.78 3ϫ1-H), 4.38 (br. s, 2 H, 2ϫCH2Fmoc), 4.23 (dd, 3J5,6 = 5.6, 2J6,6Ј
(m, 1 H, γ-Hb), 2.31–2.25 (m, 2 H, β-H), 2.12, 2.11, 2.07, 1.96 = 12.2 Hz, 3 H, 3ϫ6-Ha), 4.18 (t, 3J = 6.6 Hz, 1 H, CHFmoc),
(each s, each 3 H, 4ϫCO2CH3), 1.47 (s, 9 H, CCH3) ppm. 13C
NMR (150 MHz, CDCl3, TMS): δ = 171.1 (COC-γ), 170.6 (COO-
tBu), 169.9, 169.9, 169.6, 169.5 (4ϫCO2CH3), 157.1 (CO2-Fmoc),
4.13 (dd, J5,6Ј = 2.6, J6,6Ј = 12.3 Hz, 3 H, 3ϫ6-Hb), 3.98 (ddd,
3
2
3J4,5 = 9.5, J5,6 = 5.4, J5,6Ј = 2.5 Hz, 3 H, 3ϫ5-H), 3.76 (mc, 3
3
3
H, 3ϫman-OCHHCH2), 3.57–3.50 (m, 6 H, 3ϫman-OCHHCH2,
143.9 (Ar-C), 141.3 (Ar-C), 127.7 (Ar-C), 127.0 (Ar-C), 125.2 (Ar- 3ϫman-OCH2CHH), 3.41–3.36 (m, 3 H, 3ϫman-OCH2CHH),
C), 119.9 (Ar-C), 97.8 (C-1Ј), 82.4 (CCH3), 69.3 (C-2Ј), 69.0 (C-
3Ј), 68.9 (C-5Ј), 67.3 (OCH2CH2), 67.0 (CH2-Fmoc), 66.1 (C-4Ј),
62.4 (C-6Ј), 53.8 (C-α), 49.0 (CH-Fmoc), 39.0 (OCH2CH2), 32.4
(C-γ), 29.1 (C-β), 27.9 [C(CH3)3], 20.8, 20.6, 20.6, 20.5
2.21 [mc, 6 H, 3ϫC(O)CH2CH2], 2.14, 2.09, 2.03, 1.96 (each s,
each 9 H, 12ϫCO2CH3), 1.98 [mc, 6 H, 3ϫC(O)CH2CH2] ppm.
13C NMR (150 MHz, CDCl3, TMS): δ = 173.3 (HNCOCH2CH2),
170.7, 170.1, 169.7 (4 CO2CH3), 154.8 (NHCO2), 144.1, 144.4,
127.6, 127.0, 125.1, 120.0 (Ar-C), 97.4 (C-1), 69.5 (C-2), 69.2 (C-
3), 68.8 (C-5), 67.0 (man-OCH2CH2), 66.2 (C-4), 65.8
(NHCO2CH2), 62.6 (C-6), 57.2 (CNHFmoc), 47.5 (CH-fluorene),
39.0 (man-OCH2CH2), 31.2 [C(O)CH2CH2], 30.7 [C(O)CH2CH2],
21.0, 20.8, 20.7, 20.7 (CO2CH3) ppm. HRMS (ESI): calcd. for
[C73H96N4O35 + Na]+ 1611.5753; found 1611.5917.
(4ϫCO2CH3) ppm. HRMS (ESI): calcd. for [C40H50N2O15
+
Na]+ 821.3109; found 821.3118.
N-[2-(2Ј,3Ј,4Ј,6Ј-Tetra-O-acetyl-α-
(fluoren-9-ylmethoxycarbonyl)-S-(triphenymethyl)-
D
-mannopyranosyloxy)ethyl]-Nα-
-cysteine Amide
L
(15): According to the general procedure for Staudinger ligation,
mannosyl azide 10 (2.20 g, 6.9 mmol, 1 equiv.), Fmoc-Cys(Trt)-OH
(5.67 g, 12.4 mmol, 1.8 equiv.), HOBt (1.54 g, 12.4 mmol,
1.8 equiv.), DIC (1.77 mL, 12.4 mmol, 1.8 equiv.) and tri-n-butyl-
phosphane (1.7 mL, 6.9 mmol, 1 equiv.) were allowed to react in a
Schlenk flask under N2. The crude product was purified by silica
gel chromatography (EtOAc/toluene, 1:1) to give 15 (3.93 g,
3-Cascade:N-acetyl-aminomethane[3]:1-(α-D-mannopyranosyloxy)-
3-aza-4-oxo-hexane (18): Glycocluster 17 (80 mg, 5.03 mmol) was
dissolved in dry CH2Cl2 (5 mL) and morpholine (500 µL) under an
inert atmosphere. The reaction mixture was stirred for 4 h at room
temp. and then evaporated in vacuo. The residue was dried under
4.21 mmol, 61%) as a colourless foam. 1H NMR (500 MHz, high vacuum overnight, dissolved in acetic anhydride (1 mL) and
CDCl3, TMS): δ = 7.76–7.70 (m, 2 H, Ar-H), 7.60–7.55 (m, 2 H, pyridine (3 mL) and stirred at room temp. for 16 h. The solvent
Ar-H), 7.43–7.34 (m, 8 H, Ar-H), 7.29–7.13 (m, 11 H, Ar-H), 5.33 was evaporated and the crude product was subjected to silica gel
3
3
(dd, J2,3 = 3.6, J3,4 = 10.1 Hz, 1 H, 3Ј-H), 5.27–5.22 (m, 2 H, 2Ј- column chromatography (EtOAc/MeOH, 1:1.5) to give the peracet-
H, 4Ј-H), 4.76 (br. s, 1 H, 1Ј-H), 4.45–4.33 (m, 2 H, CH2-Fmoc),
ylated product as a slightly coloured syrup (47 mg, 0.034 mmol,
68% over two steps). This completely acetylated glycocluster was
dissolved in dry methanol (5 mL) and sodium methoxide (1  solu-
4.23 (dd, 3J5Ј,6Ј = 5.6, 2J6Ј,6ЈЈ = 12.2 Hz, 1 H, 6aЈ-H), 4.19 (t, 3JCH2-
3
= 6.5 Hz, 1 H, CH-Fmoc), 4.07 (dd, J5Ј,6ЈЈ = 2.4,
Fmoc,CH-Fmoc
2J6Ј,6ЈЈ = 12.2 Hz, 1 H, 6bЈ-H), 3.97–3.92 (m, 1 H, 5Ј-H), 3.75 (mc, tion in MeOH, 100 µL) was added. The reaction mixture was
1 H, α-H), 3.72–3.68 (m, 1 H, OCHHCH2), 3.54–3.47 (m, 1 H, stirred for 4 h at room temp., neutralized by the addition of Am-
3
OCHHCH2), 3.45–3.29 (m, 2 H, CH2N), 2.74 (dd, JH-α,H-β = 7.4, berlite IR120 (H), filtered and the solvents evaporated. The purity
2
2JH-β,H-βЈ = 12.9 Hz, 1 H, β-Ha), 2.74 (dd, 3JH-α,H-βЈ = 7.6, JH-β,H-
of the compound was confirmed by RP HPLC on a LiChrosorb
= 12.9 Hz, 1 H, β-Hb), 2.12, 2.04, 1.99, 1.97 (each s, each 3 H, RP-8 column at a flow rate of 10 mL/min using a linear acetoni-
βЈ
4ϫCO2CH3) ppm. 13C NMR (125 MHz, CDCl3, TMS): δ = 170.6
trile/water gradient of 10% to 100% acetonitrile over 80 min to
3788
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 3783–3789

Glycoamino Acid Building Blocks and Other Glycomimetics
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yield the title compound 18 as a white lyophilisate (19 mg,
0.021 mmol, quant.). [α]2D0 = +1.5 (c = 1.0, MeOH). 1H NMR
3
(600 MHz, CD3OD, TMS): δ = 4.81 (d, J1,2 = 1.6 Hz, 3 H, 3ϫ1-
2
H), 3.88 (dd, 3J5,6 = 2.2, J6,6Ј = 11.7 Hz, 3 H, 3ϫ6-Ha), 3.86 (dd,
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3
3J1,2 = 1.7, J2,3 = 3.3 Hz, 3 H, 3ϫ2-H), 3.81 (m, 3 H,
3
2
3ϫOCHHCH2), 3.75 (dd, J5,6 = 6.6, J6,6Ј = 11.6 Hz, 3 H, 3ϫ6-
3
3
Hb) 3.74 (dd, J2,3 = 3.5, J3,4 = 9.7 Hz, 3 H, 3ϫ3-H), 3.64 (dd ≈
t, 3J = 9.7 Hz, 3 H, 3ϫ4-H), 3.61–3.57 (m, 6 H, 3ϫ5-H,
3ϫOCHHCH2), 3.49–3.46 (m, 3 H, 3ϫOCH2CHH), 3.43–3.40
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André, F. Sansone, H. Kaltner, A. Casnati, J. Kopitz, H.-J. Ga-
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Murphy, Eur. J. Org. Chem. 2007, 4177–4187; c) P. Nieder-
(m,
3
H, 3ϫOCH2CHH), 2.24 [mc,
6
H, 3ϫC(O)-
CH2CH2], 2.03 [mc, 6 H, 3ϫC(O)CH2CH2], 2.01 [s, 3 H, NHC(O)-
CH3] ppm. 13C NMR (150 MHz, CD3OD, TMS): δ = 176.1
[NHC(O), HNCOCH2CH2], 101.7 (C-1), 74.8 (C-5), 72.6 (C-3),
72.1 (C-2), 68.9 (C-4), 67.2 (man-OCH2CH2), 63.0 (C-6), 59.3
(CNHAc), 40.5 (man-OCH2CH2), 31.8 [C(O)CH2CH2], 31.2
[C(O)CH2CH2], 23.5 [NHC(O)CH3] ppm. HRMS (ESI): calcd. for
[C36H64N4O22 + Na]+ 927.3910. Found, 927.3995.
3-Cascade:N-Fluorenylmethoxycarbonylaminomethane[3]:1-(2,3,4,6-
Tetra-O-benzoyl-α-
D-mannopyranosyl)-1-aza-2-oxobutane
(19):
ˇ
hafner, M. Reinisˇ, J. Sebestík, J. Jezˇek, J. Pept. Sci. 2008, 14,
Fmoc-protected triacid 16 (59 mg, 0.12 mmol), 2,3,4,6-tetra-O-ben-
zoyl-α--mannosyl azide[24] (336 mmol, 0.540 mmol) and HOBt
(49 mg, 0.36 mmol) were dried under vacuum in a Schlenk flask
for about 1 h. Then this mixture was dissolved in dry THF (20 mL)
under an inert atmosphere, DIC (55 µL, 0.36 mmol) was added and
the solution stirred for 10 min. Then trimethylphosphane solution
(1  in THF, 540 µL, 540 mmol) was added at –70 °C and the reac-
tion mixture stirred overnight. The reaction was quenched by the
addition of water, the aqueous phase was extracted with CH2Cl2
(3ϫ15 mL) and the combined organic phases dried with MgSO4.
The mixture was filtered, the solvent was removed in vacuo and the
residue purified by silica gel column chromatography (cyclohexane/
EtOAc, 1:1) followed by GPC on LH-20 (CH2Cl2/CH3OH, 1:1) to
yield pure 19 (89 mg, 0.040 mmol, 34%) as a white amorphous so-
lid. [α]2D0 = +0.6 (c = 0.9, CH2Cl2). 1H NMR (600 MHz, CDCl3,
TMS): δ = 8.04 (mc, 6 H, Ar-H), 7.96 (mc, 5 H, Ar-H), 7.90 (mc,
6 H, Ar-H), 7.74 (mc, 5 H, Ar-H), 7.60 (mc, 2 H, Ar-H), 7.53–7.47
(m, 10 H, Ar-H), 7.40–7.30 (m, 20 H, Ar-H), 7.26 (mc, 6 H, Ar-
H), 7.20 (mc, 8 H, Ar-H), 6.85 (br. s, 3 H, NHCOCH2), 6.02 (dd
556–587.
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≈ t, 3J = 10.03 Hz, 3 H, 3ϫ4-H), 5.81 (dd, J1,2 = 1.1, J2,3
=
3
3
[16] a) Z. Györgydeak, L. Szilagyi, H. Paulsen, J. Carbohydr. Chem.
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4.0 Hz, 3 H, 3ϫ2-H), 5.74 (d, 3J = 9.53 Hz, 3 H, 3ϫ1-H), 5.68
3
3
(dd, J2,3 = 3.1, J3,4 = 10.1 Hz, 3 H, 3ϫ3-H), 5.02 (mc, 1 H,
CH2CH), 4.64 (dd, J5,6 = 2.5, J6,6Ј = 12.4 Hz, 3 H, 3ϫ6-Ha),
4.35 (dd, J5,6 = 3.7, J6,6Ј = 12.2 Hz, 3 H, 3ϫ6-Hb), 4.18 (mc, 3
H, 3ϫ5-H), 3.82 (mc, 2 H, CH2CH), 1.82 [mc, 6 H, 3ϫC(O)-
CH2CH2], 1.75 [mc, 6 H, 3ϫC(O)CH2CH2] ppm. 13C NMR
3
2
3
2
(150 MHz, CDCl3, TMS):
δ
=
172.6–165.3 (3ϫNHCO,
12ϫPhCO), 157.0 (NHCO2), 143.9–119.8 (Ar-C), 76.6 (C-1), 74.1
(C-5), 72.6 (C-3), 70.9 (C-2), 66.3 (C-4), 65.7 (NHCO2CH2), 62.4
(C-6), 57.1 (CNHFmoc), 47.2 (CH-fluorene), 31.3 [C(O)CH2CH2],
30.5 [C(O)CH2CH2] ppm. HRMS (ESI): calcd. for [C127H108N4O32
+ Na]+ 2223.6844; found 2224.8521.
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Res. 2008, 343, 1665–1674.
Supporting Information (see footnote on the first page of this arti-
cle): 13C NMR spectra of 4–9, 11, 13–15, 17–19 and mass spectra
of 11, 13–15, 17–19.
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Received: April 20, 2009
Published Online: June 22, 2009
Eur. J. Org. Chem. 2009, 3783–3789
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3789