Synthesis of a Fluorinated Sialophorin Hexasaccharide–Threonine Conjugate for Fmoc Solid-Phase Glycopeptide Synthesis

Article abstract of DOI:10.1002/ejoc.201600523

The decoding of the mechanisms underlying glycan-mediated recognition in disease and health requires access to structurally well-defined oligosaccharides as molecular probes. Owing to their often favourable properties, deoxyfluorosugars have emerged as a promising class of selectively modified carbohydrates for biological and immunological studies. In particular the enhanced metabolic stability and intrinsic immunogenicity of fluorinated carbohydrates has spurred research on their use for vaccine design. Herein, a first total synthesis of an orthogonally protected and fluorinated hexasaccharide–threonine conjugate of the natural sialophorin antigen has been accomplished. Starting from readily available monosaccharide building blocks, the targeted glycosyl amino acid 1 was assembled by a [3+3′]-block glycosylation strategy. Together with its (1→4)-linked regioisomer the glycan mimic can be applied to solid-phase glycopeptide synthesis to access novel sialophorin-derived molecular tools for functional and biomedical studies.

Full text of DOI:10.1002/ejoc.201600523

DOI: 10.1002/ejoc.201600523
Full Paper
Fluorinated Hexasaccharides
Synthesis of a Fluorinated Sialophorin Hexasaccharide–
Threonine Conjugate for Fmoc Solid-Phase Glycopeptide
Synthesis
Markus Daum,[a] Frederik Broszeit,[a] and Anja Hoffmann-Röder*[a]
Dedicated to Professor Horst Kunz on the occasion of his 75th birthday
Abstract: The decoding of the mechanisms underlying glycan- orthogonally protected and fluorinated hexasaccharide–
mediated recognition in disease and health requires access to threonine conjugate of the natural sialophorin antigen has
structurally well-defined oligosaccharides as molecular probes. been accomplished. Starting from readily available monosac-
Owing to their often favourable properties, deoxyfluorosugars charide building blocks, the targeted glycosyl amino acid 1 was
have emerged as a promising class of selectively modified assembled by a [3+3′]-block glycosylation strategy. Together
carbohydrates for biological and immunological studies. In par- with its (1→4)-linked regioisomer the glycan mimic can be ap-
ticular the enhanced metabolic stability and intrinsic immuno- plied to solid-phase glycopeptide synthesis to access novel
genicity of fluorinated carbohydrates has spurred research on sialophorin-derived molecular tools for functional and biomedi-
their use for vaccine design. Herein, a first total synthesis of an cal studies.
Introduction
profound expertise in chemical oligosaccharide synthesis. This
holds particularly true when analogues are used to mimic the
Regarding the eminent role of carbohydrates in many physio- bioactive function of carbohydrates.[1] In recent years, selec-
logical and pathophysiological processes, a detailed under- tively fluorinated carbohydrates have emerged as valuable tools
standing of the molecular recognition processes between for structural, functional and mechanistic studies, e.g. to map
glycans and their specific binding partners, such as lectins, anti- antibody–antigen interactions,[2] to probe lectin binding[3] and
bodies, and enzymes is indispensable. Functional studies rely to elucidate enzyme mechanisms.[4] Moreover, fluorinated gly-
on structurally well-defined glycans, which typically requires coconjugates are increasingly investigated as promising
Figure 1. Disialylated hexasaccharide building block for naturally occurring sialophorin and its novel fluorinated analogue.
[a] Center for Integrated Protein Science Munich (CIPSM) at the Department of
Chemistry, Ludwig-Maximilians-Universität,
Butenandtstr. 5-13, 81377 Munich, Germany
E-mail: Anja.Hoffmann-Roeder@cup.lmu.de
http://www.cup.lmu.de/oc/hoffmann-roeder/
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/ejoc.201600523.
building blocks for the development of synthetic vaccines with
improved metabolic stabilities and immunogenicities.[5] This,
however, requires further expansion of synthetic methodology
to allow the preparation of diverse fluorinated glycans and
fluoroglycopeptides.
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The highly glycosylated mucin-like glycoprotein sialophorin block glycosylation, i.e. by coupling of glycosyl donor 2 with
(leukosialin, CD 43)[6] is presented on the surface of hematopoi- fluorinated glycosyl acceptor 3. The final trisaccharide coupling
etic cells and participates in many important events during im- partners in turn were accessed from the following monosac-
charides: galactose-derived glycosyl donors 4[10] and 5,[11] the
TN antigen derivative 6,[12] sialic acid xanthate 7,[13] as well as
glucosamine derivative 8 (Scheme 1). Thereby, and in contrast
to previous work by Singh et al.,[9] a modified synthetic route
was followed abstaining from the use of participating groups
at C3 of the sialic acid donors, which were also introduced at a
later stage of the synthesis.
mune response. For instance, sialophorin is involved in activa-
tion, differentiation, adhesion and migration of T-cells.[7] More-
over, overexpression of sialophorin has been associated with
certain pathologies including rheumatoid arthritis, leukemia,
the Wiskott-Aldrich syndrome, and acquired immune deficiency
syndrome.[8] The low abundance and heterogeneity of naturally
occurring sialophorin glycoforms, however, has hampered in-
vestigations on the exact role of its glycans. Although chemical
synthesis of the sialophorin hexasaccharide was accomplished
by Singh et al. in 1999,[9] functional studies using synthetic
glycopeptide antigens have not yet been reported. With regard
to the general advantages of carbohydrate analogues in bio-
medical studies and in order to provide access to novel sialo-
phorin-based mimetics and antigen analogues, the first fluorin-
ated hexasaccharide–threonine conjugate is now devised bear-
ing a fluorine substituent at C6′ of the lower branch galactose
residue (Figure 1). Previous work on structurally related anti-
gens[5e,5h,10] has indicated this position to be readily addressa-
ble and suitable for enhancement of both immunogenicity and
hydrolytic stability.
Key building block 2 was assembled in a stepwise manner
by starting from protected glucosamine precursor 9[14] as the
central building block (Scheme 2). Introduction of a levulinoyl
ester to orthogonally block the 3-OH group was followed by
regioselective ring-opening of the benzylidene acetal under
well-established conditions[15] to afford the desired glucos-
aminyl acceptor 8 in 48 % yield (2 steps) after column chroma-
tography. Subsequent ꢀ-galactosylation with trichloroacetimi-
date 5[11] activated by trimethylsilyl trifluoromethanesulfonate
(TMSOTf) proceeded smoothly to furnish disaccharide 10 in
79 % yield. Notably, formation of the corresponding orthoester
of 10[16] was not observed. Cleavage of the acetate protecting
groups under mild Zemplén conditions[17] furnished glycosyl
acceptor 11, which was regio- and stereoselectively sialylated
at the more reactive 3-OH group under kinetically controlled
reaction conditions. Thus, activation of sialic acid xanthate 7
(2 equiv.) with methylsulfenyl triflate[12b,18] (MeSOTf), generated
Results and Discussion
Retrosynthetic analysis indicated a strategy towards fully pro-
tected fluorinated antigen analogue 1 for solid-phase glyco- in situ from methylsulfenyl bromide (MeSBr) and silver(I) triflate
peptide synthesis (Fmoc-SPPS) based on a convergent [3+3′]- (AgOTf) at –45 °C in a mixture of acetonitrile (MeCN) and di-
Scheme 1. Retrosynthetic analysis of the sialophorin–threonine building block 1.
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Scheme 2. Reagents and conditions: (a) (i) levulinic acid, DCC, 4-DMAP, CH2Cl2, room temp., 2.5 h; (ii) Et3SiH, TfOH, CH2Cl2, Et2O, –78 °C, 4 h, 48 % (2 steps);
(b) 5,[11] TMSOTf, CH2Cl2, molecular sieves (MS) (4 Å), –78 °C, 2.5 h, 79 %; (c) NaOMe, MeOH, pH 8.5–9.0, room temp., 18 h, 92 %; (d) 7,[13] AgOTf, MeSBr,
MeCN/CH2Cl2 (2:1), MS (4 Å), –45 °C, 24 h; (e) (i) hydrazine acetate, toluene/EtOH (2:1), room temp., 4 h; (ii) pyridine/Ac2O (2:1), 4-DMAP, room temp., 20 h,
55 % (3 steps); (f) (i) TBAF, AcOH, THF, 0 °C to room temp., 48 h; (ii) Cl3CCN, DBU, MS (4 Å), CH2Cl2, 0 °C to room temp., 6 h, 51 % (2 steps).
chloromethane (CH2Cl2),[19] provided α-sialoside 12. In this reac- as before. Thus, MeSOTf-promoted α-sialylation with xanthate
tion, attachment of the sialic acid moiety occurred exclusively 7 in MeCN/CH2Cl2 under kinetic control led to the exclusive
at 3-OH, which was unambiguously proven by heteronuclear formation of the trisaccharide 16. Again, 2D NMR spectroscopy
2D shift correlations on the basis of a cross-peak between C2– confirmed the presence of the desired C3′-linked α-sialoside,
Sia (δ = 96.9 ppm) and H3–Gal (δ = 4.63 ppm) in an HMBC which was obtained in 26 % yield after column flash chroma-
experiment (see Supporting Information). Similarly, the strong tography. Unfortunately, all attempts to improve the sialylation
coupling of H3ax–Sia (δ = 1.69 ppm) and C1–Sia (δ = 167.2 ppm) reaction by using a recently published activated N-acetylox-
confirmed the required α-stereochemistry of the newly formed azolidinone-functionalized phosphate donor of Wang et al.[23]
glycosidic bond. However, owing to difficulties in the purifica- failed, disclosing the intrinsic low reactivity of disaccharide ac-
tion of 12, the latter was directly subjected to hydrazinolysis to ceptor 11 to be mainly responsible for the insufficient yield of
liberate the 3-OH group.[20] Subsequent global acetylation with this step. Standard protecting-group manipulations (acetylation
Ac2O in pyridine then allowed isolation of trisaccharide 13 by and benzylidene acetal cleavage) then converted 16 into the
flash chromatography in high purity and with an excellent yield targeted acceptor building block 3 in 73 % yield over two steps.
of 55 % over three steps. Cleavage of the anomeric silyl ether
with tetrabutylammonium fluoride (TBAF) in the presence of
acetic acid in tetrahydrofuran furnished the reducing trisac-
charide derivative, which was readily converted into key tri-
chloroacetimidate donor 2 (51 % yield over two steps) by em-
ploying 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and trichloro-
acetonitrile.[21]
The final glycosylation reaction of trichloroacetimidate donor
2 and the fluorinated trisaccharide acceptor 3 was performed
with promotion of TMSOTf at –50 °C → –40 °C in CH2Cl2 to
yield the desired hexasaccharide backbone (Scheme 4). With a
view to previous findings,[18,24] this reaction was again expected
to occur regioselectively at the more reactive primary hydroxy
group of C6. Moreover, the neighboring-group participation of
The synthesis of the second trisaccharide building block 3 the N-(2,2,2-trichloroethoxycarbonyl) group (Troc) at C2 of the
commenced with the stereoselective coupling of known TN an- glucosamine moiety should lead to stereoselective formation
tigen derivative 6 with 6F-galactosyl bromide 4 (Scheme 3).[10] of the desired ꢀ-configured product. It should be noted that
Subsequent Zemplén transesterification of compound 14 at pH deviating from standard procedure, the precious glycosyl ac-
8.5[22] provided disaccharide–threonine conjugate 15 in 74 % ceptor 3 was used in slight excess (1.2 equiv.) in this reaction
yield, which was regio- and stereoselectively sialylated at 3-OH, providing two regioisomeric products 17 and 18 with the same
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Scheme 3. Reagents and conditions: (a) 4,[10] Hg(CN)2, CH2Cl2/MeNO2 (3:2), MS (4 Å), 100 W, 80 °C, 2 h, 85 %; (b) (i) NaOMe, MeOH, pH 8.5, room temp., 18 h;
(ii) FmocOSu, DIPEA, CH2Cl2, room temp., 15 h, 74 % (2 steps); (c) 7,[13] AgOTf, MeSBr, MeCN/CH2Cl2 (2:1), MS (4 Å), –45 °C, 24 h, 26 %; (d) (i) pyridine/Ac2O
(2:1), room temp., 16 h; (ii) AcOH, 80 °C, 1 h, 73 % (2 steps).
molecular mass. Isolation of both products succeeded effi- lap, cross-peaks between C1 of the glucosamine moiety and
ciently by column chromatography on silica gel with the major either H4 or H6a/b of the galactosamine were not detectable
product 18 (55 %) eluting first, followed by the minor com- in the HMBC spectrum of 18. Similarly, no cross-peaks between
pound 17 (45 %) and remaining acceptor 3. Whereas ꢀ-stereo- glucosamine H1 and either C4 or C6 of the galactosamine moi-
selectivity was again unambiguously proven by the large cou- ety were observed. However, high-field shift of the signal of the
pling constant between H1 and H2 of the glucosamine residue methylene group at C6 of the latter (δ = 70.5 ppm → 56.2 ppm,
after acetylation (7.7 Hz, see Supporting Information), the regio- Figure 2) suggested the presence of a (1→4)-linked product,
isomeric assignments were less clear-cut. Owing to signal over- which was later confirmed again through acetylation (19, see
Scheme 4. Reagents and conditions: (a) TMSOTf, CH2Cl2, MS (4 Å), –50 °C to –40 °C, 4 h, 45 % (for 17), 55 % (for 18); (b) pyridine/Ac2O (2:1), room temp., 16 h,
89 %.
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Scheme 5. Reagents and conditions: (a) (i) Zn, AcOH, room temp., 20 h; (ii) pyridine/Ac2O (2:1), room temp., 2 d, 89 % (2 steps).
Supporting Information). Although unexpected at first, a com- the targeted sialophorin analogue 1 in 89 % over two steps
parable lack of regioselectivity has been reported for slow glyc- after column chromatography (Scheme 5). In the corresponding
osylation reactions of complex coupling partners before.[14,25]
HMBC spectrum of this compound a distinct cross-peak be-
tween H4–GalNAc (δ = 5.25 ppm) and 4-OAc–GalNAc (δ =
169.7 ppm) suggests that position C6 of the galactosamine resi-
due is glycosylated. This was further confirmed by the NOESY
spectrum displaying cross-peaks from H1–GalNAc (δ
=
4.76 ppm) and H6a/b–GalNAc (δ = 3.67 ppm and 3.61 ppm),
respectively.
Conclusions
The synthesis of fluorinated analogue 1 of the sialophorin
hexasaccharide–threonine epitope, as well as of its (1→4)-
linked regioisomer 18, both compatible to Fmoc-SPPS, was ac-
complished for the first time and by means of a convergent
[3+3′]-glycosylation strategy. The requisite trisaccharide build-
ing blocks are accessible from readily available monosacchar-
ides including sialyl xanthate 7 and TN threonine conjugate 6.
In principle, the synthetic route disclosed herein is well suited
for the preparation of novel fluorinated sialophorin-derived gly-
copeptides for biological and medical studies, including the de-
velopment of improved glycoconjugate vaccines for cancer im-
munotherapy.
Experimental Section
General Remarks: Solvents for moisture-sensitive reactions (tolu-
ene, acetonitrile, dichloromethane, nitromethane and 1,2-dichloro-
ethane) were distilled and dried according to standard procedures.
Glycosylations were carried out under argon in flame-dried glass-
ware. Commercially available reagents and solvents were used with-
out further purification. Reactions were monitored by TLC with pre-
coated silica gel 60 F254 aluminium plates (Merck KGaA, Darmstadt)
using UV light as the visualizing agent and by dipping the plate
Figure 2. (A) Extract of the 1H,13C HSQC NMR spectrum of 17 in CDCl3 for
assignment of C6-GalNAc. (B) Extract of the 1H,13C HSQC NMR spectrum of
18 in CDCl3 for assignment of C6-GalNAc.
into a 1:1 mixture of 1
M H2SO4 in EtOH and 3 % 4-methoxyphenol
solution in EtOH followed by heating. The crude products were puri-
fied by standard column chromatography using silica gel (35–
70 μm) from Acros Organics. Analytical RP-HPLC was carried out
with a JASCO system using a Phenomenex Luna C18 column (5 μm,
250 × 4.6 mm) and Phenomenex Jupiter C18 column (5 μm,
250 × 4.6 mm). Semipreparative RP-HPLC was carried out with a
The desired (1→6)-linkage of target compound 17 was fi-
nally confirmed by NMR analysis after protecting-group manip-
ulations. Thus, removal of the Troc group under mild reductive
conditions by treatment with activated zinc dust in acetic
acid[26] and subsequent acetylation (Ac2O/pyridine) furnished JASCO system using a Phenomenex Luna C18 column (10 μm,
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250 × 30 mm) and a Phenomenex Jupiter C18 column (10 μm,
250 × 30 mm). In all cases mixtures of water and acetonitrile were
used as solvents; if required 0.1 % trifluoroacetic acid was added.
Low-resolution (ESI) and high-resolution (HR-ESI) mass spectra were
recorded with a Thermo Finnigan LTQ FT instrument. 1H, 13C, 19F
and 2D NMR spectra were recorded with a Varian 400 MHz and
ethyl acetate, 2:1). HPLC (Luna, MeCN/H2O, 90:10 → 100:0, 20 min,
flow 1 mL/min): tR = 7.05 min, λ = 214 nm. [α]2D1 = –5.9 (c = 1.00,
CHCl3). 1H NMR (600 MHz, CDCl3, COSY, HSQC): δ = 7.71 (d, JCH,CH
=
7.3 Hz, 2 H, HAr-Ph, -Bn), 7.64 (d, JCH,CH = 7.4 Hz, 2 H, HAr-Ph, -Bn),
7.44–7.36 (m, 1 H, HAr-Ph), 7.35–7.22 (m, 10 H, HAr-Ph, -Bn), 4.86 (d,
JNH,H2 = 9.9 Hz, 1 H, NH-GlcN), 4.79 (t, JH4,H3 ≈ JH4,H5 = 9.5 Hz, 1 H,
600 MHz spectrometer or with a Bruker Avance III 800 MHz spec- H4-GlcN), 4.75 (d, JCH,CH = 12.1 Hz, 1 H, CH2a-Troc), 4.62 (d, JCH,CH
=
trometer. The chemical shifts are reported relative to the signal of 11.9 Hz, 1 H, CH2b-Troc), 4.52–4.43 (m, 3 H, CH2-Bn {4.48, 4.44, 2 ×
the deuterated solvent. Multiplicities are given as: s (singlet), br. s
(broad singlet), d (doublet), t (triplet), and m (multiplet). Assign-
ments of proton and carbon signals were achieved by additional
COSY, TOCSY, NOESY, HSQC, and HMBC experiments when noted.
The signals of molecule-fragments were denoted as follows: N-
d, JCH,CH = 12.1 Hz, JCH,CH = 11.8 Hz}, H1-GlcN {4.46, d, JH1,H2 =
8.1 Hz}), 3.85 (dd, JH2,H1 = 9.8 Hz, JH2,H3 = 9.5 Hz, 1 H, H2-GlcN), 3.75
(t, JH3,H2 ≈ JH3,H4 = 9.5 Hz, 1 H, H3-GlcN), 3.64–3.57 (m, 2 H, H6a-
GlcN {dd, JH6a,H6b = 10.6 Hz, JH6a,H5 = 5.0 Hz}, H6b-GlcN {dd,
JH6b,H6a = 10.5 Hz, JH6b,H5 = 4.1 Hz}), 3.18 (mc, 1 H, H5-GlcN), 2.75 (t,
acetyl-
(Gal),
raminic acid (Sia), N-acetyl-
and glycosylated threonine (Thr). Optical rotations were measured
at 598 nm with a Perkin–Elmer polarimeter 241.
D
-galactosamine (GalN),
-galactose linked to GalN (Gal′) link to GalN, N-acetyl-
-neuraminic acid linked to Gal′ (Sia′)
D
-glucosamine (GlcN),
D
-galactose
JCH,CH = 6.4 Hz, 2 H, CH2-Lev), 2.53 (d, JCH,CH = 6.3 Hz, 2 H, CH2-Lev),
D
D
-neu-
2.15 (s, 3 H, CH3-Lev), 1.07 (s, 9 H, CH3-tBu-Si) ppm. 13C NMR
(150 MHz, CDCl3, HSQC, HMBC): δ = 207.4 (C=O-Lev), 173.4 (C=O-
Lev), 154.4 (C=O-Troc), 138.0 (Cq-Bn), 136.1, 136.0 (CAr-Ph, -Bn),
133.1, 132.7 (Cq-Ph), 130.2, 130.0, 128.5, 127.8, 127.6 (CAr-Ph, -Bn),
96.5 (C1-GlcN), 95.6 (CCl3), 75.9 (C4-GlcN), 74.8 (CH2-Troc), 74.4 (C5-
GlcN), 73.8 (CH2-Bn), 70.4 (C3-GlcN), 69.8 (C6-GlcN), 57.7 (C2-GlcN),
38.4 (CH2-Lev), 29.9 (CH3-Lev), 28.3 (CH2-Lev), 26.9 (CH3-tBu-Si), 19.3
(Cq-tBu-Si) ppm. MS (ESI+): m/z calcd. for C37H48Cl3N2O9Si [M +
NH4]+ 797.21, found 797.21. HR-MS (ESI+): m/z calcd. for
C37H44Cl3NO9SiK [M + K]+ 818.1482, found 818.1493.
D
Typical Procedure for the Synthesis of Sialylated Compounds
(TP1): A solution of sialic acid xanthate 7[13] (2.5 equiv.) in a mixture
of dry acetonitrile/dichloromethane (2:1, 20 mL) was stirred with
activated, powdered molecular sieves (4 Å) under argon at room
temperature for 24 h. Then glycosyl acceptor (1.0 equiv.), dissolved
in dry acetonitrile/dichloromethane (2:1, 20 mL) was added, and
the reaction mixture was stirred at room temperature for 1 h. The
suspension was cooled to –45 °C, and silver trifluoromethanesulf-
onate (AgOTf) (2.5 equiv.) in dry acetonitrile/dichloromethane (2:1,
6 mL) was added. After stirring for 30 min, a precooled solution of
methylsulfenyl bromide (2.5 equiv., TP2) was added. The reaction
mixture was stirred under argon with protection from light at –45 °C
for 24 h, neutralized with DIPEA, stirred at –45 °C for 1 h, warmed
to room temperature, and filtered through a pad of Celite. The sol-
vents were evaporated, and the crude product was purified by flash
chromatography (SiO2).
tert-Butyldiphenylsilyl 4-O-(2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-
galactopyranosyl)-6-O-benzyl-2-deoxy-3-O-levulinoyl-2-(N-
2,2,2-trichloroethoxycarbonylamino)-ꢀ- -glucopyranoside (10):
D-
D
A mixture of compound 8 (2.00 g, 2.56 mmol), compound 5 (2.08 g,
3.84 mmol, 1.5 equiv.) and 5.0 g of powdered molecular sieves (4 Å)
in 100 mL of dry dichloromethane was stirred under argon at room
temperature for 1 h. The mixture was cooled to –78 °C and treated
with 69 μL (0.38 mmol, 0.1 equiv. based on 5) of trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf) in 1.0 mL of dry dichlorometh-
ane. The solution was warmed up to room temperature within 2.5 h,
diluted with 50 mL of dichloromethane, and filtered through a pad
of Celite. The filtrate was washed twice with 50 mL of NaHCO3 and
brine, dried (MgSO4) and concentrated in vacuo. The residue was
purified by column chromatography on silica (cyclohexane/ethyl
acetate, 2:1) to provide 10 as a colorless, amorphous solid (2.33 g,
2.10 mmol, 79 %). Rf = 0.33 (cyclohexane/ethyl acetate, 2:1). HPLC
Typical Procedure for the Preparation of Methylsulfenyl Brom-
ide Solution (1.6
M) (TP2): A dry and argon-flushed 25 mL flask
equipped with a magnetic stirrer and a septum was charged under
argon with a solution of dimethyl disulfide (709 μL, 4.99 mmol,
1.0 equiv.) in dry 1,2-dichloroethane (10 mL). Then bromine (410 μL,
4.99 mmol, 1.0 equiv.) was added at room temperature, and the
reaction mixture was stirred with protection from light for 20 h.
(Luna, MeCN/H2O, 90:10 → 100:0, 20 min, flow 1 mL/min): tR
=
10.5 min, λ = 214 nm. [α]2D0 = –7.5 (c = 1.00, CHCl3). 1H NMR
tert-Butyldiphenylsilyl 6-O-Benzyl-2-deoxy-3-O-levulinoyl-2-(N- (600 MHz, CDCl3, COSY, HSQC): δ = 7.74–7.70 (m, 2 H, HAr-Ph, -Bn),
2,2,2-trichloroethoxycarbonylamino)-ꢀ- -glucopyranoside (8): 7.65–7.60 (m, 2 H, HAr-Ph, -Bn), 7.43–7.39 (m, 2 H, HAr-Ph, -Bn), 7.37–
According to ref.[27] a mixture of 14.0 g (18.0 mmol, 1.0 equiv.) of 7.24 (m, 14 H, HAr-Ph, -Bn), 5.38 (dd, JH4,H3 = 3.5 Hz, JH4,H5 = 1.2 Hz,
compound 20[28] and 15.0 g of powdered molecular sieves (4 Å) in
1 H, H4–Gal), 4.93 (dd, JH2,H3 = 10.4 Hz, JH2,H1 = 7.9 Hz, 1 H, H2-Gal),
250 mL of dry dichloromethane were stirred under argon at room 4.85 (dd, JH3,H2 = 10.2 Hz, JH3,H4 = 3.5 Hz, 1 H, H3–Gal), 4.82 (dd,
temperature for 1 h. The mixture was cooled to –78 °C; sequentially JH3,H2 = 10.2 Hz, JH3,H4 = 9.5 Hz, 1 H, H3-GlcN), 4.60 (d, JCH,CH
were added 4.31 mL (27.0 mmol, 1.5 equiv.) of triethylsilane (Et3SiH) 12.2 Hz, 1 H, CH2a-Troc), 4.53 (d, JCH,CH = 12.0 Hz, 2 H, CH2-Bn), 4.44
D
=
and within 15 min a cooled solution of 2.37 mL (27.0 mmol,
1.5 equiv.) of trifluoromethanesulfonic acid (TfOH) in diethyl ether.
It was stirred at –78 °C for 4 h; after 2 h, additional 1.5 equiv. of
TfOH in diethyl ether were added. The reaction was quenched by
addition of 15 mL of methanol, the mixture neutralized with DIPEA
in dichloromethane at –78 °C, and filtered through a pad of Celite.
The filtrate was then washed three times with 100 mL of dichloro-
methane, two times with 50 mL of aqueous sodium hydrogen carb-
onate (NaHCO3) and brine. The aqueous layer was extracted with
dichloromethane (2 × 100 mL), and the combined organic layers
(d, JH1,H2 = 7.9 Hz, 1 H, H1–Gal), 4.41 (d, JH1,H2 = 10.1 Hz, 1 H, H1-
GlcN), 4.41 (d, JCH,CH = 11.9 Hz, 2 H, CH2-Bn), 4.37 (d, JCH,CH
=
12.2 Hz, 1 H, CH2b-Troc), 3.94 (t, JH4,H3 = JH4,H5 = 9.4 Hz, 1 H, H4-
GlcN), 3.87 (dd, JH2,H3 = 10.2 Hz, JH2,H1 = 10.1 Hz, 1 H, H2-GlcN), 3.68
(ddd, JH5,H6a = 7.5 Hz, JH5,H6b = 5.7 Hz, JH5,H4 = 1.3 Hz, 1 H, H5-Gal),
3.56 (dd, JH6a,H6b = 11.1 Hz, JH6a,H5 = 3.3 Hz, 1 H, H6a-GlcN), 3.51
(dd, JH6a,H6b = 9.3 Hz, JH6a,H5 = 5.6 Hz, 1 H, H6a-Gal), 3.41–3.36 (m,
2 H, H6b-Gal, H6b-GlcN), 3.04 (ddd, JH5,H4 = 9.8 Hz, JH5,H6a = 3.3 Hz,
JH5,H6b = 1.8 Hz, 1 H, H5-GlcN), 2.65–2.57 (m, 2 H, CH2-Lev), 2.51–
2.46 (m, 2 H, CH2-Lev), 2.11 (s, 3 H, CH3-Lev), 2.01, 1.94, 1.88 (3 × s,
were dried with MgSO4, filtered, and concentrated in vacuo. The 9 H, CH3-OAc), 1.07 (s, 9 H, CH3-tBu-Si) ppm. 13C NMR (150 MHz,
crude product was purified by column chromatography on silica
CDCl3, HSQC, HMBC): δ = 206.0 (C=O-Lev), 172.5, 170.2, 170.1 (C=
(cyclohexane/ethyl acetate, 2:1) to yield compound 8 as a colorless,
O-Ac), 169.1 (C=O-Lev), 154.3 (C=O-Troc), 138.0, 137.6 (Cq-Bn, -Ph),
amorphous solid (8.29 g, 10.6 mmol, 59 %). Rf = 0.30 (cyclohexane/ 136.1, 136.0 (CAr-Bn, -Ph), 133.1, 132.6, 130.2, 130.0, 128.6, 128.0,
Eur. J. Org. Chem. 0000, 0–0
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128.0, 127.8, 127.6 (CAr-Bn, -Ph), 100.5 (C1–Gal), 96.5 (C1-GlcN), 95.6
(CCl3), 74.9 (C5-GlcN), 74.8 (C4-GlcN), 73.9 (CH2-Troc), 73.5, 73.4
(CH2-Bn), 73.0 (C3-GlcN), 71.8 (C5-Gal), 71.5 (C3-Gal), 69.6 (C2–Gal),
170.0 (5 × C=O-Ac, -NHAc), 167.6 (C1–Sia), 154.4 (C=O-Troc), 138.6,
138.1 (3 × Cq-Bn), 136.1, 136.0 (Cq-Ph), 134.4, 133.2, 132.7, 130.1,
129.9, 129.1, 128.9, 128.8, 128.6, 128.5, 128.3, 127.9, 127.7, 127.5,
67.4 (C4-Gal), 67.2 (C6-Gal), 67.2 (C6-GlcN), 58.1 (C2-GlcN), 38.0 (CH2- 127.4 (CAr-Bn, -Ph), 102.3 (C1–Gal), 97.9 (C2–Sia), 96.6 (C1-GlcN), 95.6
Lev), 29.8 (CH3-Lev), 28.1 (CH2-Lev), 26.7 (CH3-tBu-Si), 20.7, 20.7, 20.6 (CCl3), 77.4 (C3-Gal), 75.1 (C5-GlcN), 74.8 (CH2-Troc), 74.0 (C4-GlcN),
(CH3-OAc), 19.3 (Cq-tBu-Si) ppm. HR-MS (ESI+): m/z calcd. for
73.2 (C3-GlcN), 73.1, 73.1 (CH2-Bn), 72.8 (C5-Gal), 72.8 (C6-Sia), 69.7
(C2–Gal), 69.3 (C6-Gal), 68.6 (C4-Sia), 68.5 (C8-Sia), 68.4 (CH2-Bn),
68.4 (C4-Gal), 67.7 (C6-GlcN), 66.9 (C7-Sia), 62.2 (C9-Sia), 57.9 (C2-
GlcN), 49.6 (C5-Sia), 37.9 (C3-Sia), 37.8 (CH2-Lev), 29.9 (CH3-Lev), 28.1
(CH2-Lev), 26.9 (CH3-tBu-Si), 23.3 (CH3-NHAc), 21.3, 20.9, 20.8, 20.7
(CH3-OAc), 19.2 (Cq-tBu-Si) ppm. HR-MS (ESI+): m/z calcd. for
C76H95Cl3N3O26Si [M + NH4]+ 1598.5033, found 1598.5030.
C56H70Cl3N2O17Si [M + NH4]+ 1175.3504, found 1175.3493.
tert-Butyldiphenylsilyl 4-O-(6-O-Benzyl-ꢀ-
6-O-benzyl-2-deoxy-3-O-levulinoyl-2-(N-2,2,2-trichloroethoxy-
carbonylamino)-ꢀ- -glucopyranoside (11): A solution of 2.33 g
D-galactopyranosyl)-
D
(2.01 mmol) of compound 10 in 25 mL of methanol was treated
with a freshly prepared solution of sodium methoxide (NaOMe) in
methanol, and the reaction mixture was adjusted to pH = 8.5. The
solution was readjusted at regular intervals and stirred at room tem-
perature for 4 h. It was neutralized with Amberlyst® 15, filtered, and
washed with methanol (50 mL). The solution was concentrated in
vacuo, the residue was co-evaporated twice with 10 mL of dichloro-
methane and purified by column chromatography on silica (cyclo-
hexane/ethyl acetate, 1:4) to give compound 11 as a colorless,
amorphous solid (1.91 g, 1.85 mmol, 92 %). Rf = 0.49 (cyclohexane/
ethyl acetate, 1:4). HPLC (Luna, MeCN/H2O, 90:10 → 100:0, 20 min,
flow 1 mL/min): tR = 8.63 min, λ = 214 nm. [α]2D2 = –1.1 (c = 1.00,
CHCl3). MS (ESI+): m/z calcd. for C50H64Cl3N2O14Si [M + NH4]+
1049. 32, fou nd 10 49 . 3 2. HR -M S ( ES I+ ): m/ z c al cd. for
C50H60Cl3NO14SiNa [M + Na]+ 1054.2746, found 1054.2728.
tert-Butyldiphenylsilyl 4-O-(2,4-Di-O-acetyl-6-O-benzyl-ꢀ-
actopyranosyl)-3-O-[benzyl (5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-α- -glycero- -galacto-non-2-ulopyranosyl)-
onate]-3-O-acetyl-6-O-benzyl-2-deoxy-2-(N-2,2,2-trichloroeth-
oxycarbonylamino)-ꢀ- -glucopyranoside (13): Cleavage of levu-
D-gal-
D
D
D
linic acid: A solution of 3.73 g (max. 1.85 mmol) of slightly contami-
nated compound 12 in 60 mL of a mixture of toluene/ethanol (2:1)
was treated with 204 mg (2.22 mmol, 1.2 equiv.) of hydrazine acet-
ate and stirred at room temperature for 4 h; 10 mL of acetone and
50 mL of water were added, and the solution was extracted with
ethyl acetate (3 × 80 mL). The organic phase was dried (MgSO4),
filtered, and the solvent was removed under reduced pressure. The
residue was co-evaporated with dichloromethane (30 mL) and used
without further purification in the next step. HPLC (Luna, MeCN/
H2O, 90:10 → 100:0, 20 min, flow 1 mL/min): tR = 8.66 min, λ =
tert-Butyldiphenylsilyl 4-O-(6-O-Benzyl-ꢀ-
3-O-[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
-glycero-
deoxy-3-O-levulinoyl-2-(N-2,2,2-trichloroethoxycarbonyl-
amino)-ꢀ- -glucopyranoside (12): Sialylated compound 12 was
D-galactopyranosyl)-
D
D
-galacto-non-2-ulopyranosyl)onate]-6-O-benzyl-2- 214 nm. HR-MS (ESI+): m/z calcd. for C71H89Cl3N3O24Si [M + NH4]+
1500.4665, found 1500.4659. The residue was dissolved in 40 mL of
pyridine and 20 mL of acetic anhydride, and 226 mg (1.85 mmol,
1.0 equiv.) of 4-DMAP was added. The solution was stirred at room
temperature for 20 h, before it was concentrated in vacuo. The resi-
due was co-evaporated with toluene (3 × 50 mL) and dichloro-
methane (3 × 50 mL). The crude product was purified by column
chromatography on silica (dichloromethane/methanol, 25:1) to pro-
vide 13 as a colorless, amorphous solid (1.63 g, 1.01 mmol, 55 %,
D
prepared according to TP1. Sialic acid xanthate 7 (3.11 g,
4.63 mmol), molecular sieves (6.0 g), glycosyl acceptor 11 (1.91 g,
1.85 mmol), AgOTf (1.19 g, 4.63 mmol), and methylsulfenyl bromide
(2.89 mL, 4.63 mmol, TP2) were allowed to react. The product was
purified twice by column chromatography on silica (dichloro-
methane/methanol, 25:1 → ethyl acetate) to provide 12 as a color-
less, amorphous solid (3.73 g, slightly contaminated). For further based on 11). Rf = 0.39 (dichloromethane/methanol, 25:1). HPLC
decontamination, compound 12 was purified by preparative re-
versed-phase HPLC [Luna, MeCN/H2O (90:10) → (100:0) in 20 min].
Rf = 0.46 (ethyl acetate), 0.23 (dichloromethane/methanol, 25:1).
(Luna, MeCN/H2O, 90:10 → 100:0, 20 min, flow 1 mL/min): tR =
11.7 min, λ = 214 nm. [α]2D2 = +5.2 (c = 1.00, CHCl3). 1H NMR
(800 MHz, CDCl3, COSY, HSQC): δ = 7.71–7.67 (m, 2 H, HAr-Ph, -Bn),
[α]2D2 = –1.3 (c = 1.00, CHCl3). 1H NMR (600 MHz, CDCl3, COSY, HSQC): 7.63–7.59 (m, 2 H, HAr-Ph, -Bn), 7.43–7.23 (m, 21 H, HAr-Ph, -Bn), 5.47
δ = 7.73–7.67 (m, 2 H, HAr-Ph, -Bn), 7.65–7.59 (m, 2 H, HAr-Ph, -Bn), (dd, JH8,H7 = 7.5 Hz, JH8,H9 = 2.9 Hz, 1 H, H8-Sia), 5.43–5.34 (m, 2 H,
7.40–7.21 (m, 21 H, HAr-Ph, -Bn), 5.40–5.35 (m, 2 H, H8-Sia {5.36},
NH-GlcN), 5.30 (dd, JH7,H8 = 8.6 Hz, JH7,H6 = 1.6 Hz, 1 H, H7-Sia),
NH-GlcN {5.42}, H7-Sia {5.36}), 5.19–5.13 (m, 1 H, NH-Sia {5.14}), 5.12
(d, JH4,H3 = 3.3 Hz, 1 H, H4–Gal), 4.92–4.82 (m, 2 H, H2-Gal {4.87},
H4–Sia {4.82}), 4.81 (d, JCH,CH = 12.0 Hz, 1 H, CH2a-Troc), 4.74 (d,
5.19–5.15 (m, 1 H, NH-Sia), 4.91 (td, JH4,H5/H3ax = 10.2 Hz, JH4,H3eq
=
4.3 Hz, 1 H, H4–Sia), 4.86 (dd, JH3,H2 = 10.2 Hz, JH3,H4 = 9.6 Hz, 1 H,
JH1,H2 = 8.2 Hz, 1 H, H1–Gal), 4.54 (dd, JH3,H2 = 12.3 Hz, JH3,H4
=
H3-GlcN), 4.74 (d, JCH,CH = 12.0 Hz, 1 H, CH2a-Troc), 4.65 (d, JCH,CH
12.0 Hz, 1 H, CH2b-Troc), 4.54–4.50 (m, 2 H, CH2-Bn {d, JCH,CH
11.9 Hz}), 4.49 (d, JCH,CH = 12.3 Hz, 2 H, CH2-Bn), 4.45 (d, JCH,CH
=
=
=
3.3 Hz, 1 H, H3–Gal), 4.53 (d, JCH,CH = 12.0 Hz, 1 H, CH2b-Troc), 4.52
(d, JCH,CH = 11.9 Hz, 2 H, CH2-Bn), 4.46–4.41 (m, 4 H, CH2-Bn {4.44,
4.41}), 4.38–4.35 (m, 1 H, H1-GlcN {4.36}), 4.29 (dd, JH9a,H9b = 12.5 Hz,
12.4 Hz, 2 H, CH2-Bn), 4.42 (d, JH1,H2 = 7.7 Hz, 1 H, H1–Gal), 4.39 (d,
JH1,H2 = 8.1 Hz, 1 H, H1-GlcN), 4.22 (dd, JH9a,H9b = 12.4 Hz, JH9a,H8
2.5 Hz, 1 H, H9a-Sia), 4.07 (dd, JH6,H5 = 10.8 Hz, JH6,H7 = 1.8 Hz, 1 H,
J
H9a,H8 = 2.9 Hz, 1 H, H9a-Sia), 4.12–4.06 (m, 3 H, H6-Sia {4.09}, H9b-
Sia {4.08}, H5-Sia {4.07}), 4.04–3.85 (m, 3 H, H3-GlcN {4.03}, H4-GlcN
{3.96}, H2-GlcN {3.87}), 3.77 (mc, 1 H, H5-Gal), 3.58 (dd, JH6a,H6b
11.2 Hz, JH6a,H5 = 4.3 Hz, 1 H, H6a-GlcN), 3.49–3.42 (m, 2 H, H6b-
=
=
H6-Sia), 4.04–4.00 (m, 2 H, H9b-Sia {4.00}, H4-GlcN {4.00}), 3.99–3.95
(m, 1 H, H5-Sia), 3.92–3.86 (m, 2 H, H3–Gal {3.89}, H2-GlcN {3.87}), GlcN {3.46}, H6a-Gal {3.54}), 3.33 (dd, JH6b,H6a = 9.7 Hz, JH6b,H5
=
3.75 (dd, JH6a,H6b = 11.4 Hz, JH6a,H5 = 3.7 Hz, 1 H, H6a-GlcN), 3.57–
3.52 (m, 3 H, H6b-GlcN {3.53}, H6a-Gal {3.53}, H6b-Gal {3.52}), 3.51–
3.47 (m, 1 H, H2-Gal), 3.44 (d, JH4,H3 = 3.3 Hz, 1 H, H4–Gal), 3.42 (t,
JH5,H6a/b = 6.1 Hz, 1 H, H5-Gal), 3.20–3.15 (m, 1 H, H5-GlcN), 2.75
(dd, JH3eq,H3ax = 13.0 Hz, JH3eq,H4 = 4.6 Hz, 1 H, H3eq-Sia), 2.63–2.36
7.6 Hz, 1 H, H6b-Gal), 3.14–3.11 (m, 1 H, H5-GlcN), 2.62 (dd,
H3eq,H3ax = 12.7 Hz, JH3eq,H4 = 4.6 Hz, 1 H, H3eq-Sia), 2.12, 2.11, 2.10,
2.06, 2.02, 1.97, 1.89 (7 × s, 21 H, CH3-OAc), 1.95–1.91 (m, 1 H,
H3ax–Sia), 1.86 (s, 3 H, CH3-NHAc), 1.05 (s, 9 H, CH3-tBu-Si) ppm. 13
NMR (200 MHz, CDCl3, HSQC, HMBC): δ = 170.8, 170.6, 170.3, 170.2,
J
C
(m, 4 H, CH2-Lev), 2.15 (s, 3 H, CH3-Lev), 2.09, 2.04, 2.00, 1.93 (4 × s, 170.2, 170.1 (8 × C=O-NHAc, -OAc), 168.1 (C1–Sia), 154.2 (C=O-Troc),
12 H, CH3-OAc), 2.03–1.99 (m, 1 H, H3ax–Sia), 1.87 (s, 3 H, CH3-NHAc),
1.05 (s, 9 H, CH3-tBu-Si) ppm. 13C NMR (150 MHz, CDCl3, HSQC,
HMBC): δ = 207.0 (C=O-Lev), 172.9 (C=O-Lev), 170.9, 170.6, 170.5,
138.6, 137.9 (Cq-Bn, -Ph), 136.1, 136.0, 135.2, 135.1, 134.9, 129.0,
128.8, 128.8, 128.7, 128.7, 128.7, 128.6, 128.4, 128.3, 127.9, 127.8,
127.6, 127.4 (CAr-Bn, -Ph), 100.7 (C1–Gal), 98.9 (C2–Sia), 97.2 (C1-
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GlcN), 95.5 (CCl3), 75.0 (C4-GlcN), 74.9 (C5-GlcN), 74.6 (CH2-Troc), 9.6 Hz, 1 H, H4-GlcN), 4.14 (ddd, JH2,H3 = 11.0 Hz, JH2,NH = 9.2 Hz,
73.9 (C2–Gal), 73.7 (C3-GlcN), 73.5, 73.4 (CH2-Bn), 72.5 (C6-Sia), 71.8
(C3-Gal), 71.6 (C5-Gal), 69.2 (C4-Sia), 68.4 (CH2-Bn), 67.9 (C6-GlcN),
JH2,H1 = 3.7 Hz, 1 H, H2-GlcN), 4.04–3.99 (m, 2 H, H5-Sia {4.03}, H5-
GlcN {4.02}), 3.90 (dd, JH9b,H9a = 12.4 Hz, JH9b,H8 = 6.0 Hz, 1 H, H9b-
67.7 (C4-Gal), 67.7 (C8-Sia), 67.6 (C6-Gal), 67.4 (C7-Sia), 62.4 (C9-Sia), Sia), 3.84 (ddd, JH5,H6a = 6.9 Hz, JH5,H6b = 5.5 Hz, JH5,H4 = 1.1 Hz, 1
58.1 (C2-GlcN), 49.4 (C5-Sia), 38.3 (C3-Sia), 26.8 (CH3-tBu-Si), 23.3 H, H5-Gal), 3.81 (dd, JH6a,H6b = 11.5 Hz, JH6a,H5 = 3.8 Hz, 1 H, H6a-
(CH3-NHAc), 21.2, 21.0, 21.0, 20.9, 20.9, 20.8, 20.7 (CH3-OAc), 19.2 GlcN), 3.72 (dd, JH6b,H6a = 11.6 Hz, JH6b,H5 = 1.8 Hz, 1 H, H6b-GlcN),
(Cq-tBu-Si) ppm. HR-MS (ESI+): m/z calcd. for C77H95Cl3N3O27Si [M +
3.50 (dd, JH6a,H6b = 9.6 Hz, JH6a,H5 = 5.4 Hz, 1 H, H6a-Gal), 3.46 (dd,
JH6,H5 = 10.8 Hz, JH6,H7 = 2.7 Hz, 1 H, H6-Sia), 3.39 (dd, JH6b,H6a
9.6 Hz, JH6b,H5 = 7.3 Hz, 1 H, H6b-Gal), 2.61 (dd, JH3eq,H3ax = 12.6 Hz,
H3eq,H4 = 4.7 Hz, 1 H, H3eq-Sia), 2.11, 2.11, 2.03, 2.02, 1.99, 1.98, 1.97
(7 × s, 21 H, CH3-OAc), 1.84 (s, 3 H, CH3-NHAc), 1.67 (t, JH3ax,H3eq
NH4]+ 1626.4982, found 1626.4985.
=
4-O-(2,4-Di-O-acetyl-6-O-benzyl-ꢀ-
[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
glycero- -galacto-non-2-ulopyranosyl)onate]-3-O-acetyl-6-O-
benzyl-2-deoxy-2-(N-2,2,2-trichloroethoxycarbonylamino)-α/ꢀ-
-glucopyranosyl Trichloroacetimidate (2): A mixture of 1.63 g
D-galactopyranosyl)-3-O-
J
D
-
=
D
JH3ax,H4 = 12.4 Hz, 1 H, H3ax–Sia) ppm. 13C NMR (200 MHz, CDCl3,
HSQC, HMBC): δ = 171.1, 170.7, 170.5, 170.4, 170.2, 169.8, 169.7
(8 × C=O-NHAc, -OAc), 167.4 (C1–Sia), 161.0 (C=NH-TCI), 154.4 (C=
O-Troc), 138.4, 137.9 (3 × Cq-Bn), 135.0, 129.1, 128.8, 128.5, 128.4,
127.9, 127.9, 127.6, 127.6 (CAr-Bn), 100.5 (C1–Gal), 97.1 (C2–Sia), 95.5
(CCl3), 94.8 (C1-GlcN), 91.0 (CCl3-TCI), 74.5 (CH2-Troc), 74.2 (C4-GlcN),
73.5, 73.4 (CH2-Bn), 73.2 (C5-GlcN), 72.2 (C6-Sia), 71.9 (C3-Gal), 71.7
(C5-Gal), 71.3 (C3-GlcN), 70.7 (C2–Gal), 69.2 (C4-Sia), 68.5 (CH2-Bn),
67.9 (C8-Sia), 67.8 (C4-Gal), 67.7 (C6-Gal), 67.6 (C6-GlcN), 67.1 (C7-
Sia), 62.2 (C9-Sia), 54.3 (C2-GlcN), 48.9 (C5-Sia), 37.5 (C3-Sia), 23.2
(CH3-NHAc), 21.6, 21.3, 20.9, 20.8, 20.8, 20.7, 20.7 (CH3-OAc) ppm.
HR-MS (ESI+): m/z calcd. for C63H77Cl6N4O27 [M + NH4]+ 1531.2885,
found 1531.2901.
D
(1.01 mmol) of compound 13 in 100 mL of tetrahydrofurane (THF)
was cooled to 0 °C, followed by simultaneous addition of 15.2 mL
(397 mg, 1.52 mmol, 1.5 equiv.) of a solution of tetrabutylammo-
nium fluoride (TBAF) in THF (0.1
M) and 15.0 mL (3.03 mmol,
3.0 equiv.) of a solution of 0.17 mL of glacial acetic acid and 14.8 mL
of THF within 15 min. The reaction mixture was stirred at 0 °C for
30 min, warmed up to room temperature, and stirred for 48 h. The
solution was diluted with dichloromethane (100 mL) and washed
with NaHCO3 and brine (3 × 50 mL). The aqueous phase was ex-
tracted with dichloromethane (3 × 50 mL), the combined organic
layers were dried (MgSO4) and concentrated in vacuo. Purification
of the crude product by column chromatography on silica (dichloro-
methane/methanol, 25:1) provided the desired compound as a col-
orless, amorphous solid (1.21 g, 0.88 mmol, 87 %). Rf = 0.22/0.29
(dichloromethane/methanol, 25:1). HPLC (Luna, MeCN/H2O, 90:10
→ 100:0, 20 min, flow 1 mL/min): tR = 3.83 min/4.12 min, λ =
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido)-2-deoxy-
4,6-O-benzylidene-3-O-[6-deoxy-6-fluoro-ꢀ-
D-galactopyran-
osyl]-α- -galactopyranosyl- -threonine tert-Buyl Ester (15):
D
L
2.54 g (2.59 mmol) of compound 14 in 50 mL of methanol was
treated with a freshly prepared solution of NaOMe in MeOH. The
214 nm. HR-MS (ESI+): m/z calcd. for C61H77Cl3N3O27 [M + NH4]+ reaction mixture was adjusted to a pH value of 8.5, and it was
1388.3805, found 1388.3795. HR-MS (ESI–): m/z calcd. for
62H74Cl3N2O29 [M + HCO2]– 1415.3448, found 1415.3438. A solu-
readjusted at regular intervals. The solution was stirred at room
temperature for 18 h before it was neutralized with AcOH. The sol-
C
tion of 700 mg (0.51 mmol) of crude product in 60 mL of dry di- vent was removed under reduced pressure, and the residue was
chloromethane was added to 1.0 g of powdered molecular sieves
(4 Å) under argon and stirred at room temperature for 30 min.
The reaction mixture was cooled to 0 °C and treated with 511 μL
(5.10 mmol, 10.0 equiv.) of trichloroacetonitrile and four drops of
DBU. The mixture was warmed up to room temperature while being
stirred for 6 h, during which after 3 h one drop of DBU was added.
It was diluted with dichloromethane (20 mL), filtered through a pad
of Celite, and concentrated in vacuo. The crude product was puri-
fied by column chromatography on silica (cyclohexane/ethyl acet-
ate, 1:9) to obtain 2 as a colorless, amorphous solid (460 mg,
0.30 mmol, 51 % over 2 steps) in an anomeric mixture (α/ꢀ, 1:1.8,
determined by analytic reversed-phase HPLC). Rf = 0.41/0.52 (cyclo-
co-evaporated with toluene (3 × 50 mL) and dichloromethane (2 ×
50 mL). The crude product was dissolved in 100 mL of dry dichloro-
methane and 847 mg (2.59 mmol, 1.0 equiv.) of Fmoc-OSu was
added. The pH value was adjusted to 9.5 with DIPEA, and the reac-
tion mixture was stirred under argon at room temperature for 15 h.
The solution was neutralized with Amberlite IR120 and filtered. The
solvent was removed under reduced pressure, and the crude prod-
uct was purified by column chromatography on silica (cyclohexane/
ethyl acetate → ethyl acetate → ethyl acetate/ethanol, 20:1) to give
compound 15 as a colorless, amorphous solid (1.63 g, 1.92 mmol,
74 %). Rf = 0.26 (ethyl acetate/ethanol, 20:1). HPLC (Luna, MeCN/
H2O, 30:70 → 77:23 in 30 min → 100:0 in 60 min, flow 1 mL/min):
1
hexane/ethyl acetate, 1:9). [α]2D2 = +34.4 (c = 0.50, CHCl3, α-anomer). tR = 21.6 min, λ = 214 nm. [α]D22 = +77.3 (c = 1.00, CHCl3). H NMR
1H NMR (800 MHz, CDCl3, COSY, HSQC): α-anomer: δ = 8.70 (s, 1 H,
NH-TCI), 7.44–7.41 (m, 2 H, HAr-Bn), 7.40–7.35 (m, 3 H, HAr-Bn), 7.35–
7.30 (m, 10 H, HAr-Bn), 6.41 (d, JH1,H2 = 3.7 Hz, 1 H, H1-GlcN), 5.49
(ddd, JH8,H7 = 8.8 Hz, JH8,H9b = 6.0 Hz, JH8,H9a = 2.8 Hz, 1 H, H8-Sia),
5.43 (d, JCH,CH = 12.0 Hz, 1 H, CH2-Bn), 5.37 (dd, JH3,H2 = 11.0 Hz,
(800 MHz, CDCl3, COSY, HSQC): δ = 7.81–7.73 (m, 2 H, H4-, H5-
Fmoc), 7.64 (d, JH1/H8,H2/H7 = 7.3 Hz, 2 H, H1-, H8-Fmoc), 7.52 (d,
JCH,CH = 6.9 Hz, 2 H, HAr-Bzn), 7.45–7.27 (m, 7 H, H2-, H3-, H6-, H7-
Fmoc, HAr-Bzn), 6.33 (d, JNH,H2 = 8.9 Hz, 1 H, NH-GalN), 5.55 (d,
JNH,Tα = 8.5 Hz, 1 H, NH-urethane), 5.51 (s, 1 H, CH-Bzn), 4.96 (d,
JH3,H4 = 9.2 Hz, 1 H, H3-GlcN), 5.30 (dd, JH7,H8 = 8.7 Hz, JH7,H6
2.8 Hz, 1 H, H7-Sia), 5.26 (d, JNH,H2 = 9.2 Hz, 1 H, NH-GlcN), 5.15 (dd,
H4,H3 = 3.4 Hz, JH4,H5 = 1.1 Hz, 1 H, H4–Gal), 5.09 (d, JCH,CH = 12.0 Hz,
1 H, CH2-Bn), 4.96 (dd, JH2,H3 = 9.8 Hz, JH2,H1 = 7.9 Hz, 1 H, H2-Gal),
4.94 (d, JNH,H5 = 9.5 Hz, 1 H, NH-Sia), 4.87 (d, JH1,H2 = 7.9 Hz, 1 H,
=
JH1,H2 = 4.9 Hz, 1 H, H1-GalN), 4.67–4.54 (m, 2 H, H2-GalN, H6a-Gal′),
4.53–4.45 (m, 4 H, H2-Gal′, H6b-Gal′, CH2-Fmoc), 4.43–4.36 (m, 1 H,
H4-GalN), 4.30–4.22 (m, 3 H, Tα, H1-Gal′, H9-Fmoc), 4.22–4.15 (m, 2
H, H6a-GalN, Tꢀ), 4.08–4.02 (m, 1 H, H6b-GalN), 3.77–3.73 (m, 1 H,
H3-GalN), 3.72–3.68 (m, 1 H, H5-GalN), 3.62–3.58 (m, 1 H, H4-Gal′),
3.57–3.52 (m, 1 H, H5-Gal′), 3.31–3.24 (m, 1 H, H3-Gal′), 2.04 (s, 3 H,
CH3-NHAc), 1.46 (s, 9 H, CH3-tBu), 1.28 (d, JTγ,Tꢀ = 6.2 Hz, 3 H, Tγ)
ppm. 13C NMR (200 MHz, CDCl3, HSQC, HMBC): δ = 172.3, 170.4 (2 ×
C=O-NHAc, -ester), 156.5 (C=O-urethane), 143.8, 143.7 (C1a-, C8a-
Fmoc), 141.4, 141.4 (C4a-, C5a-Fmoc), 136.7 (Cq-Bzn), 128.9, 128.8
(C2-, C7-Fmoc), 128.3, 128.3, 127.9 (CAr-Bzn), 127.2 (C3-, C6-Fmoc),
126.4, 126.2 (CAr-Bzn), 125.1, 125.0 (C1-, C8-Fmoc), 120.2, 120.2
J
H1–Gal), 4.84 (ddd, JH4,H3ax = 12.1 Hz, JH4,H5 = 10.3 Hz, JH4,H3eq
=
4.6 Hz, 1 H, H4–Sia), 4.71 (d, JCH,CH = 12.0 Hz, 1 H, CH2a-Troc), 4.69
(d, JCH,CH = 12.0 Hz, 1 H, CH2b-Troc), 4.64 (d, JCH,CH = 11.8 Hz, 1 H,
CH2-Bn), 4.61 (dd, JH3,H2 = 10.0 Hz, JH3,H4 = 3.4 Hz, 1 H, H3–Gal),
4.56 (d, JCH,CH = 11.8 Hz, 1 H, CH2-Bn), 4.53 (d, JCH,CH = 11.8 Hz, 1
H, CH2-Bn), 4.44 (d, JCH,CH = 11.8 Hz, 1 H, CH2-Bn), 4.29 (dd, JH9a,H9b
12.4 Hz, JH9a,H8 = 2.8 Hz, 1 H, H9a-Sia), 4.18 (t, JH4,H3 = JH4,H5
=
=
Eur. J. Org. Chem. 0000, 0–0
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8
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
(C4-, C5-Fmoc), 105.3 (C1-Gal′), 101.4 (CH-Bzn), 100.6 (C1-GalN), 83.4
(Cq-tBu), 83.4 (d, JC6,F = 168.1 Hz, C6-Gal′), 77.8 (C3-GalN), 76.9 (Tꢀ),
75.8 (C4-GalN), 73.4 (d, JC5,F = 20.2 Hz, C5-Gal′), 73.3 (C3-Gal′), 69.1
(C6-GalN), 68.4 (d, JC4,F = 7.3 Hz, C4-Gal′), 67.2, 67.2 (C2-Gal′, CH2-
Fmoc), 63.7 (C5-GalN), 59.1 (Tα), 48.2 (C2-GalN), 47.3 (C9-Fmoc), 28.1
(CH3-tBu), 21.4 (CH3-NHAc), 19.0 (Tγ) ppm. 19F NMR (376 MHz,
CDCl3): δ = –228.8 (dd, JF,H6 = 47.1 Hz, JF,H5 = 15.0 Hz, F6-Gal′) ppm.
glycero-
anosyl-
D
-galacto-non-2-ulopyranosyl)onate]}-α-
D-galactopyr-
L
-threonine tert-Buyl Ester (3): 541 mg (0.38 mmol) of
compound 16 was dissolved in 20 mL of pyridine, and 10 mL of
acetic anhydride was added dropwise. The solution was stirred at
room temperature for 16 h before it was concentrated in vacuo.
The residue was co-evaporated with toluene (3 × 20 mL) and di-
chloromethane (2 × 20 mL); 20 mL of aqueous acetic acid (80 %)
HR-MS (ESI+): m/z calcd. for C44H57FN3O14 [M + NH4]+ 870.3819, was added, and the reaction mixture was stirred at 80 °C for 1 h.
found 870.3819.
After cooling to room temperature, the solution was concentrated
in vacuo, and the residue was co-evaporated with toluene (3 ×
20 mL) and dichloromethane (2 × 20 mL). The crude product was
purified by column chromatography on silica (dichloromethane/
ethanol, 20:1 → dichloromethane/ethanol, 10:1) to provide com-
pound 3 as a colorless, amorphous solid (395 mg, 0.28 mmol, 73 %).
Rf = 0.18 (dichloromethane/ethanol, 20:1). HPLC (Luna, MeCN/H2O,
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-{2-acetamido-4,6-O-
benzylidene-2-deoxy-3-O-(6-deoxy-6-fluoro-ꢀ-
anosyl)-3-O-[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-α- -glycero- -galacto-non-2-ulopyranosyl)onate]}-α-
galactopyranosyl- -threonine tert-Buyl Ester (16): Sialylated
D-galactopyr-
D
D
D-
L
compound 16 was prepared according to TP1. Sialic acid xanthate
7 (1.93 g, 2.87 mmol), molecular sieves (3.0 g), glycosyl acceptor 15
(1.22 g, 1.44 mmol), AgOTf (740 mg, 2.87 mmol), and methylsulfenyl
bromide (1.80 mL, 2.88 mmol, TP2) were allowed to react. The prod-
uct was purified by column chromatography on silica (1 × dichloro-
methane/ethanol, 20:1, 1 × cyclohexane/ethyl acetate, 1:4) to pro-
vide 16 as a colorless, amorphous solid (534 mg, 0.38 mmol, 26 %).
Rf = 0.16 (dichloromethane/ethanol, 20:1). HPLC (Luna, MeCN/H2O,
30:70 → 77:23 in 30 min → 100:0 in 60 min, flow 1 mL/min): tR
=
23.6 min, λ = 214 nm. [α]2D2 = +39.5 (c = 1.00, CHCl3). 1H NMR
(800 MHz, CDCl3, COSY, HSQC): δ = 7.76 (d, JH4/H5,H3/H6 = 7.5 Hz, 2
H, H4-, H5-Fmoc), 7.62–7.56 (m, 2 H, H1-, H8-Fmoc), 7.45–7.34 (m,
7 H, HAr-Bn, H3-, H6-Fmoc), 7.32–7.27 (m, 2 H, H2-, H7-Fmoc), 6.35
(d, JNH,H2 = 9.6 Hz, 1 H, NH-GalN), 5.75 (d, JNH,Tα = 9.7 Hz, 1 H, NH-
urethane), 5.59 (dt, JH8,H7 = JH8,H9a = 8.3 Hz, JH8,H9b = 2.6 Hz, 1 H,
H8-Sia′), 5.44 (d, JCH,CH = 11.9 Hz, 1 H, CH2-Bn), 5.21 (dd, JH7,H8
=
30:70 → 77:23 in 30 min → 100:0 in 60 min, flow 1 mL/min): tR
=
8.6 Hz, JH7,H6 = 2.8 Hz, 1 H, H7-Sia′), 5.11–5.03 (m, 2 H, H2-Gal′, CH2-
Bn), 5.01–4.99 (m, 1 H, H4-Gal′), 4.97 (d, JNH,H5 = 10.2 Hz, 1 H, NH-
22.4 min, λ = 214 nm. [α]2D2 = +46.0 (c = 1.00, CHCl3). 1H NMR
(800 MHz, CDCl3, COSY, HSQC): δ = 7.81–7.78 (m, 2 H, H4-, H5-
Fmoc), 7.64, 7.61 (2 × d, JH1/H8,H2/H7 = 7.5 Hz, 2 H, H1-, H8-Fmoc),
Sia′), 4.98 (d, JH1,H2 = 3.8 Hz, 1 H, H1-GalN), 4.83 (dt, JH4,H3ax
JH4,H5 = 10.3 Hz, JH4,H3eq = 4.5 Hz, 1 H, H4-Sia′), 4.65 (d, JH1,H2
8.0 Hz, 1 H, H1-Gal′), 4.59 (dt, JH2,NH = JH2,H3 = 10.5 Hz, JH2,H1
=
=
=
7.58–7.53 (m, 4 H, HAr-Bn, -Bzn), 7.43 (t, JH3/H6,H2/H7 = JH3/H6,H4/H5
7.5 Hz, 2 H, H3-, H6-Fmoc), 7.41–7.30 (m, 8 H, H2-, H7-Fmoc,
HAr-Bn, -Bzn), 6.52 (d, JNH,H2 = 8.9 Hz, 1 H, NH-GalN), 6.04 (d, JNH,Tα
9.9 Hz, 1 H, NH-urethane), 5.56 (s, 1 H, CH-Bzn), 5.46–5.43 (m, 1 H,
H7-Sia′), 5.27–5.19 (m, 4 H, H8-Sia′, NH-Sia′, CH2-Bn), 5.03 (d, JH1,H2
3.9 Hz, 1 H, H1-GalN), 4.93–4.88 (m, 1 H, H4-Sia′), 4.75 (dt, JH2,NH
JH2,H3 = 9.6 Hz, JH2,H1 = 3.8 Hz, 1 H, H2-GalN), 4.59–4.48 (m, 2 H,
H6a-Gal′, H9a-Sia′), 4.48–4.40 (m, 2 H, CH2-Fmoc), 4.38–4.29 (m, 4 H,
Tα, Tꢀ, H4-GalN, H6b-Gal′), 4.26 (t, JH9,CH2 = 7.3 Hz, 1 H, H9-Fmoc),
4.25–4.21 (m, 1 H, H6a-GalN), 4.19 (d, JH1,H2 = 7.7 Hz, 1 H, H1-Gal′),
4.16–4.11 (m, 1 H, H5-Sia′), 4.10–4.06 (m, 1 H, H6b-GalN), 4.02–3.95
(m, 3 H, H6-Sia′, H9b-Sia′, H3-Gal′), 3.76–3.68 (m, 2 H, H5-GalN, H3-
GalN), 3.66–3.59 (m, 1 H, H2-Gal′), 3.53–3.49 (m, 1 H, H5-Gal′), 3.23
=
3.2 Hz, 1 H, H2-GalN), 4.53 (dd, JH3,H2 = 10.3 Hz, JH3,H4 = 3.3 Hz, 1
H, H3-Gal′), 4.44–4.35 (m, 4 H, CH2-Fmoc, H6a-Gal′, H9a-Sia′), 4.34–
4.19 (m, 4 H, H6b-Gal′, Tα, Tꢀ, H9-Fmoc), 4.18–4.13 (m, 1 H, H4-GalN),
4.04 (q, JH5,H4 = JH5,H6 = JH5,NH = 10.4 Hz, 1 H, H5-Sia′), 3.92–3.85
(m, 3 H, H5-Gal′, H5-GalN, H6a-GalN), 3.84–3.77 (m, 2 H, H9b-Sia′,
H6b-GalN), 3.76–3.72 (m, 1 H, H3-GalN), 3.54–3.50 (m, 1 H, H6-Sia′),
2.61 (dd, JH3eq,H3ax = 12.6 Hz, JH3eq,H4 = 4.6 Hz, 1 H, H3eq-Sia′), 2.28,
2.13, 2.08, 2.06, 2.04, 2.00, 1.98, 1.82 (8 × s, 24 H, CH3-NHAc, -OAc),
1.65 (t, JH3ax,H3eq = JH3ax,H4 = 12.4 Hz, 1 H, H3ax-Sia′), 1.48 (s, 9 H,
CH3-tBu), 1.31 (d, JTγ,Tꢀ = 6.3 Hz, 3 H, Tγ) ppm. 13C NMR (200 MHz,
CDCl3, HSQC, HMBC): δ = 171.7, 171.2, 170.7, 170.4, 170.0, 170.0,
169.9, 169.9, 168.8 (9 × C=O-NHAc, -OAc, -ester), 167.5 (C1-Sia′),
156.8 (C=O-urethane), 143.9, 143.7 (C1a-, C8a-Fmoc), 141.5, 141.4
(C4a-, C5a-Fmoc), 134.7 (Cq-Bn), 129.1, 128.9, 128.8, 127.9 (C2-, C7-
Fmoc, CAr-Bn), 127.1 (C3-, C6-Fmoc), 125.2, 125.1 (C1-, C8-Fmoc),
120.2 (C4-, C5-Fmoc), 102.3 (C1-Gal′), 99.9 (C1-GalN), 96.9 (C2-Sia′),
83.3 (Cq-tBu), 81.5 (d, JC6,F = 172.1 Hz, C6-Gal′), 78.5 (C3-GalN), 76.0
(Tꢀ), 72.5 (C6-Sia′), 72.1 (d, JC5,F = 21.8 Hz, C5-Gal′), 71.2 (C3-Gal′),
69.9 (C5-GalN), 69.8 (C4-GalN), 69.2 (C7-Sia′), 69.0 (C2-Gal′), 68.7
(CH2-Bn), 67.9 (C8-Sia′), 67.7 (C4-Sia′, C4-Gal′), 67.4 (CH2-Fmoc), 63.4
(C9-Sia′), 63.1 (C6-GalN), 59.3 (Tα), 48.9 (C5-Sia′), 47.4 (C2-GalN), 47.3
(C9-Fmoc), 37.3 (C3-Sia′), 28.3 (CH3-tBu), 23.5, 23.4 (CH3-NHAc), 21.6,
21.3, 21.1, 20.9, 20.9, 20.8 (6 × CH3-OAc), 19.4 (Tγ) ppm. 19F NMR
(376 MHz, CDCl3): δ = –230.3 (dt, JF,H6 = 46.9 Hz, JF,H5 = 13.6 Hz, F6-
Gal′) ppm. HR-MS (ESI+): m/z calcd. for C67H84FN3O28Na [M + Na]+
1420.5118, found 1420.5111.
=
=
=
(br. s, 1 H, H4-Gal′), 2.86 (br. s, 1 H, 2-OH-Gal′), 2.72 (dd, JH3eq,H3ax
=
12.8 Hz, JH3eq,H4 = 4.7 Hz, 1 H, H3eq-Sia′), 2.13 (s, 3 H, CH3-OAc),
2.07–2.05 (m, 4 H, CH3-NHAc, H3ax-Sia′ {2.05}), 2.04, 1.99, 1.87 (4 ×
s, CH3-NHAc, -OAc), 1.48 (s, 9 H, CH3-tBu), 1.31 (d, JTγ,Tꢀ = 6.4 Hz, 3
H, Tγ) ppm. 13C NMR (200 MHz, CDCl3, HSQC, HMBC): δ = 170.8,
170.4, 169.9, 169.8, 169.2, 169.2, 168.8 (7 × C=O-NHAc, -OAc, -ester),
166.7 (C1-Sia′), 155.8 (C=O-urethane), 142.7, 142.7 (C1a-, C8a-Fmoc),
140.3 (C4a-, C5a-Fmoc), 140.2, 136.6 (Cq-Bn, -Bzn), 127.9, 127.1
(C2-, C7-Fmoc, CAr-Bn, -Bzn), 126.1 (C3-, C6-Fmoc), 125.5 (CAr-Bn,
-Bzn), 124.2, 124.0 (C1-, C8-Fmoc), 119.0 (C4-, C5-Fmoc), 105.3 (C1-
Gal′), 100.0 (CH-Bzn), 99.5 (C1-GalN), 96.6 (C2-Sia′), 82.2 (Cq-tBu),
81.9 (d, JC6,F = 168.6 Hz, C6-Gal′), 77.2 (C3-GalN), 75.0 (Tꢀ), 74.8 (C4-
GalN), 74.2 (C3-Gal′), 72.5 (C6-Sia′), 72.0 (d, JC5,F = 21.2 Hz, C5-Gal′),
68.7 (C7-Sia′), 68.1 (C6-GalN), 67.7 (C4-Sia′), 67.2, 67.2 (C8-Sia′, CH2-
Bn, C2-Gal′), 66.7 (d, JC4,F = 7.5 Hz, C4-Gal′), 66.3 (CH2-Fmoc), 62.7
(C9-Sia′), 62.6 (C5-GalN), 58.2 (Tα), 48.1 (C5-Sia′), 46.8 (C2-GalN), 46.1
(C9-Fmoc), 36.3 (C3-Sia′), 28.1 (CH3-tBu), 23.2, 21.4 (CH3-NHAc), 20.2,
20.1, 19.8, 19.7 (CH3-OAc), 18.4 (Tγ) ppm. 19F NMR (376 MHz, CDCl3):
δ = –229.0 (dt, JF,H6 = 47.1 Hz, JF,H5 = 15.0 Hz, F6-Gal′) ppm. HR-MS
(ESI+): m/z calcd. for C70H88FN4O26 [M + NH4]+ 1419.5665, found
1419.5657.
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-[2-acetamido-6-O-(4-O-
{2,4-di-O-acetyl-6-O-benzyl-ꢀ-
[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
glycero - - galacto -non-2-ulopyranosyl)onate]-ꢀ-
D-galactopyranosyl-3-O-
D
-
-
D
D
galactopyranosyl}-3-O-acetyl-6-O-benzyl-2-deoxy-2-(N-2,2,2-tri-
chloroethoxycarbonylamino)-ꢀ- -glucopyranosyl)-3-O-(2,4-di-
O-acetyl-6-deoxy-6-fluoro-ꢀ- -galactopyranosyl)-3-O-
[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
-glycero- -galacto-non-2-ulopyranosyl)onate]-ꢀ- -galacto-
D
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-{2-acetamido-2-deoxy-
3-O-(2,4-di-O-acetyl-6-deoxy-6-fluoro-ꢀ-
O-[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
D
D-galactopyranosyl)-3-
D
-
D
D
D
Eur. J. Org. Chem. 0000, 0–0
www.eurjoc.org
9
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
pyranosyl]-2-deoxy-α-
D
-galactopyranosyl]-
L
-threonine tert-Buyl
(C1-, C8-Fmoc), 120.2, 120.2 (C4-, C5-Fmoc), 102.3 (C1-Gal′), 102.0
(C1-GlcN), 100.5 (C1-Gal), 100.2 (C1-GalN), 97.1 (C2-Sia, C2-Sia′), 96.9
(CCl3), 83.2 (Cq-tBu), 81.4 (d, JF,C6 = 172.1 Hz, C6-Gal′), 78.8 (C3-GalN),
76.2 (Tꢀ), 75.5 (C4-GlcN), 74.9 (C5-GlcN), 74.6 (CH2-Troc), 74.5 (C3-
GlcN), 73.5, 73.5 (CH2-Bn), 72.4, 72.4 (C6-Sia′, C6-Sia), 71.9 (C3-Gal),
71.9 (d, JF,C5 = 21.9 Hz, C5-Gal′), 71.6 (C5-Gal), 71.3 (C3-Gal′), 70.9
(C2-Gal), 70.5 (C6-GalN), 69.4 (C4-Sia, C4-Sia′), 69.1 (C2-Gal′), 68.7
(C6-GlcN), 68.6, 68.6 (CH2-Bn), 68.8 (C5-GalN), 68.3 (C4-GalN), 68.2
(C8-Sia), 68.0 (C8-Sia′), 67.9 (C4-Gal), 67.7 (C4-Gal′), 67.7 (C6-Gal),
67.6 (C7-Sia′), 67.4 (C7-Sia), 67.4 (CH2-Fmoc), 63.3 (C9-Sia′), 62.4 (C9-
Sia), 59.4 (Tα), 56.4 (C2-GlcN), 49.0 (C5-Sia, C5-Sia′), 47.9 (C2-GalN),
47.3 (C9-Fmoc), 37.6 (C3-Sia, C3-Sia′), 28.2 (CH3-tBu), 23.4 (CH3-
NHAc-GalN), 23.2 (CH3-NHAc-Sia, -Sia′), 21.5, 21.5, 21.2, 21.2, 21.1,
20.9, 20.9, 20.9, 20.9, 20.8, 20.8, 20.7 (13 × CH3-OAc), 19.4 (Tγ) ppm.
19F NMR (376 MHz, CDCl3): δ = –230.2 (dt, JF,H6a/b = 46.9 Hz,
JF,H5 = 13.6 Hz, F6-Gal′) ppm. HR-MS (ESI+): m/z calcd. for
Ester (17): A solution of 91 mg (0.060 mmol) of compound 2 and
100 mg (0.072 mmol, 1.2 equiv.) of compound 3 in 20 mL of dry
dichloromethane was added to 250 mg of powdered molecular
sieves (4 Å), and the mixture was stirred under argon at room tem-
perature for 1 h. The mixture was cooled to –50 °C before sequential
addition of a solution of 5 μL (0.030 mmol, 0.5 equiv.) of trimethyl-
silyl trifluoromethanesulfonate (TMSOTf) in 500 μL of dry dichloro-
methane within 5 min. The solution was warmed up to –40 °C over
a period of 4 h during which additional 250 μL (0.015 mmol,
0.25 equiv.) of TMSOTf in dry dichloromethane after 2 h was added.
It was neutralized with DIPEA in dichloromethane at –40 °C before
the mixture was diluted with dichloromethane (50 mL), filtered
through a pad of Celite, and washed with dichloromethane (4 ×
80 mL). The filtrate was washed with NaHCO3 and brine (2 × 50 mL),
the aqueous layer was extracted with dichloromethane (3 × 50 mL).
The combined organic layers were dried (MgSO4), concentrated in
vacuo, and purified by column chromatography (cyclohexane/ethyl
acetate, 1:9) to provide compound 17 as a colorless, amorphous
solid (75 mg, 0.027 mmol, 45 %). Rf = 0.08 (cyclohexane/ethyl acet-
ate, 1:9). HPLC (Luna, MeCN/H2O, 50:50 → 90:10 in 20 min → 100:0
in 40 min, flow 1 mL/min): tR = 23.1 min, λ = 214 nm. [α]2D2 = +5.2
(c = 0.10, CHCl3). 1H NMR (800 MHz, CDCl3, COSY, TOCSY, HSQC):
δ = 7.78–7.74 (m, 2 H, H4-, H5-Fmoc), 7.62–7.58 (m, 2 H, H1-, H8-
Fmoc), 7.44–7.27 (m, 22 H, HAr-Bn, H3-, H6-Fmoc), 7.28–7.23 (m, 2
H, H2-, H7-Fmoc), 6.54 (d, JNH,H2 = 9.7 Hz, 1 H, NH-GlcN), 6.47 (d,
JNH,H2 = 9.5 Hz, 1 H, NH-GalN), 5.84 (d, JNH,Tα = 9.9 Hz, 1 H, NH-
Fmoc), 5.57 (td, JH8,H7 = JH8,H9b = 8.1 Hz, JH8,H9a = 2.8 Hz, 1 H, H8-
Sia′), 5.48 (ddd, JH8,H7 = 8.8 Hz, JH8,H9b = 6.3 Hz, JH8,H9a = 2.8 Hz, 1
C
128H160Cl3FN6O54 [M + H + NH4]2+ 1384.4498, found 1384.4498.
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-[2-acetamido-6-O-(2-ac-
etamido-4-O-{2,4-di-O-acetyl-6-O-benzyl-ꢀ- -galactopyranosyl-
3-O-[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
-glycero- -galacto-non-2-ulopyranosyl)onate]-ꢀ-
galactopyranosyl}-3-O-acetyl-6-O-benzyl-2-deoxy-ꢀ- -gluco-
pyranosyl)-3-O-(2,4-di-O-acetyl-6-deoxy-6-fluoro-ꢀ- -galacto-
pyranosyl)-3-O-[benzyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-α- -glycero- -galacto-non-2-ulopyranosyl)onate]-ꢀ-
galactopyranosyl])-4-O-acetyl-2-deoxy-α- -galactopyranosyl]-
D
D
D
D-
D
D
D
D
D-
D
L-
threonine tert-Buyl Ester (1): Cleavage of the Troc protection
group: 75 mg (0.027 mmol) of compound 17 was dissolved in 20 mL
of glacial acetic acid, and 1.0 g of activated zinc was added [washed
H, H8-Sia), 5.45 (d, JCH,CH = 12.0 Hz, 1 H, CH2-Bn), 5.42 (d, JCH,CH
=
with aqueous HCl (1 M) and water (2 × 10 mL), dried with methanol
12.0 Hz, 1 H, CH2-Bn), 5.29 (dd, JH7,H8 = 8.4 Hz, JH7,H6 = 2.7 Hz, 1 H,
H7-Sia), 5.21 (dd, JH7,H8 = 8.5 Hz, JH7,H6 = 2.7 Hz, 1 H, H7-Sia′), 5.14
(dd, JH4,H3 = 3.2 Hz, JH4,H5 = 1.1 Hz, 1 H, H4-Gal), 5.10–5.00 (m, 7 H,
H3-GlcN {5.08}, CH2-Bn {5.08, 5.06, d, JCH,CH = 12.2 Hz}, H2-Gal′ {5.07},
NH-Sia {5.02}, NH-Sia′ {5.01}, H4-Gal′ {5.00}), 4.91 (dd, JH2,H3
10.1 Hz, JH2,H1 = 7.9 Hz, 1 H, H2-Gal), 4.86–4.81 (m, 3 H, H4-Sia, H4-
Sia′ {4.84}, H1-GalN {4.82}), 4.77–4.69 (m, 4 H, CH2a-Troc {4.74, d,
and diethyl ether]. The reaction mixture was stirred at room temper-
ature for 20 h, filtered through a pad of Celite, and washed with
acetic acid (4 × 50 mL). The filtrate was concentrated in vacuo, and
the residue was co-evaporated with toluene (3 × 50 mL) before the
crude compound was subjected to the final acetylation step. Rf =
0.27 (ethyl acetate/ethanol, 10:1). HPLC (Luna, MeCN/H2O, 50:50 →
90:10 in 20 min → 100:0 in 40 min, flow 1 mL/min): tR = 2.93 min,
λ = 214 nm. The crude compound was dissolved in 30 mL of a
mixture of pyridine/acetic anhydride and stirred at room tempera-
ture for 2 d. The solvents were removed under reduced pressure
before the residue was dissolved in dichloromethane (50 mL) and
washed with water and brine (4 × 50 mL). The aqueous phase was
extracted with dichloromethane (50 mL), and the combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo. Puri-
fication by column chromatography on silica (ethyl acetate/ethanol,
10:1) provided the desired compound 1. Final purification was ac-
complished by preparative reversed-phase HPLC [Rt = 21.8 min, Ju-
piter, MeCN/H2O + 0.1 % TFA (50:50) → (90:10) 20 min, → (100:0)
in 40 min] to yield 1 as a colorless, amorphous solid after lyophiliza-
tion (63 mg, 0.024 mmol, 89 % over 2 steps based on 17). Rf = 0.63
(ethyl acetate/ethanol, 10:1). HPLC (Luna, MeCN/H2O, 50:50 → 90:10
in 20 min → 100:0 in 40 min, flow 1 mL/min): tR = 15.7 min, λ =
214 nm. [α]2D0 = +20.0 (c = 0.10, CHCl3). 1H NMR (800 MHz, CDCl3,
TOCSY, NOESY, HSQC): δ = 7.77 (d, JH4,H3 = JH5,H6 = 7.9 Hz, 2 H,
H4-, H5-Fmoc), 7.60 (d, JH1,H2 = JH8,H7 = 7.5 Hz, 2 H, H1-, H8-Fmoc),
=
JCH,CH = 12.1 Hz}, H1-Gal {4.74}, CH2b-Troc {4.71, d, JCH,CH = 12.1 Hz},
H1-GlcN {4.69}), 4.67–4.62 (m, 2 H, H1-Gal′ {4.65, d, JCH,CH = 8.6 Hz},
CH2-Bn {4.64, d, JCH,CH = 12.2 Hz}), 4.60–4.56 (m, 3 H, H3-Gal {4.58},
CH2-Bn {4.58}, H2-GalN {4.57}), 4.54–4.49 (m, 2 H, H3-Gal′ {4.52}, CH2-
Bn {4.51}), 4.48–4.39 (m, 3 H, CH2-Fmoc {4.47, 4.40}, CH2-Bn {4.42}),
4.39–4.28 (m, 4 H, H9a-Sia′ {4.37}, H6a-Gal′ {4.37}, H9a-Sia {4.33},
H6b-Gal′ {4.31}), 4.27–4.20 (m, 3 H, Tꢀ {4.25}, Tα {4.23}, H9-Fmoc
{4.23}), 4.11 (mc, 1 H, H4-GalN), 4.06–4.00 (m, 2 H, H5-Sia′ {4.03}, H5-
Sia {4.01}), 3.94–3.89 (m, 2 H, H9b-Sia {3.91}, H4-GlcN {3.90}), 3.89–
3.64 (m, 9 H, H5-Gal′ {3.87}, H5-GalN {3.85}, H6a-GlcN {3.81}, H9b-
Sia′ {3.81}, H5-Gal {3.79}, H2-GlcN {3.74}, H3-GalN {3.73}, H6b-GlcN
{3.71}, H6a-GalN {3.70}), 3.63–3.56 (m, 2 H, H6b-GalN {3.59}, H5-GlcN
{3.58}), 3.53–3.48 (m, 2 H, H6-Sia′ {3.51}, H6a-Gal {3.49}), 3.46 (dd,
JH6,H5 = 10.7 Hz, JH6,H7 = 2.7 Hz, 1 H, H6-Sia), 3.39–3.35 (m, 1 H,
H6b-Gal), 2.60 (dd, JH3eq,H3ax = 12.6 Hz, JH3eq,H4 = 4.9 Hz, 2 H, H3eq-Sia,
H3eq-Sia′), 2.12, 2.10, 2.10, 2.07, 2.05, 2.03, 2.00, 1.98, 1.97 (13 × s,
39 H, CH3-OAc), 2.05 (s, 3 H, CH3-NHAc-GalN), 1.81 (2 × s, 6 H, CH3-
NHAc-Sia, -Sia′), 1.66 (mc, 2 H, H3ax-Sia, H3ax-Sia′), 1.44 (s, 9 H, CH3-
tBu), 1.34 (d, JTγ,Tꢀ = 6.4 Hz, 3 H, Tγ) ppm. 13C NMR (200 MHz, CDCl3,
HSQC, HMBC): δ = 170.9, 170.8, 170.8, 170.6, 170.5, 170.5, 170.4,
170.4, 170.2, 170.2, 169.8, 169.7 (17 × C=O-NHAc, -OAc, -ester),
167.5, 167.4 (C1-Sia, C1-Sia′), 156.8 (C=O-urethane), 155.1 (C=O-
Troc), 143.9, 143.8 (C1a-, C8a-Fmoc), 141.4, 141.4 (C4a-, C5a-Fmoc),
138.5, 137.9, 137.9 (4 × Cq-Bn), 135.0, 134.7, 129.1, 129.1, 128.9,
7.45–7.26 (m, 24 H, HAr-Bn, H2-, H3-, H6-, H7-Fmoc), 6.68 (d, JNH,H2
=
9.1 Hz, 1 H, NH-GalN), 5.81 (d, JNH,Tα = 9.7 Hz, 1 H, NH-Fmoc), 5.61
(td, JH8,H7 = JH8,H9b = 7.5 Hz, JH8,H9a = 2.8 Hz, 1 H, H8-Sia′), 5.53 (td,
JH8,H7 = JH8,H9b = 7.6 Hz, JH8,H9a = 2.7 Hz, 1 H, H8-Sia), 5.45 (d,
JCH,CH = 12.2 Hz, 2 H, CH2-Bn), 5.31–5.23 (m, 2 H, H4-GalN {5.25},
H7-Sia {5.24}), 5.24 (dd, JH7,H8 = 8.3 Hz, JH7,H6 = 2.6 Hz, 1 H,
128.8, 128.8, 128.5, 128.5, 128.4, 127.9, 127.9, 127.9, 127.9, 127.6, H7-Sia′), 5.14 (d, JH4,H3 = 3.2 Hz, 1 H, H4-Gal), 5.09–5.04 (m, 4 H, NH-
127.6, 127.2, 127.1 (20 × CAr, C2-, C3-, C6-, C7-Fmoc), 125.2, 125.1 Sia, NH-Sia′ {5.09, 5.08, d, JNH,H5 = 9.7 Hz}, CH2-Bn {5.06, d, JCH,CH
=
Eur. J. Org. Chem. 0000, 0–0
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Full Paper
J. A. Garnett, Y. Liu, E. Leon, S. A. Allman, N. Friedrich, S. Saouros, S. Curry,
D. Soldati-Favre, B. G. Davis, T. Feizi, S. Matthews, Protein Sci. 2009, 18,
1935–1947.
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12.1 Hz}), 5.01–4.97 (m, 2 H, H4-Gal′ {5.00}, H2-Gal′ {4.99}), 4.93–4.90
(m, 2 H, H2-Gal {4.92}, H1-GalN {4.91, d, JH1,H2 = 3.5 Hz}), 4.84 (td,
JH4,H3ax = JH4,H5 = 11.1 Hz, JH4,H3eq = 4.7 Hz, 2 H, H4-Sia, H4-Sia′),
4.81–4.75 (m, 2 H, H1-Gal {4.80}, H1-GlcN {4.76, d, JH1,H2 = 7.7 Hz }),
4.65–4.60 (m, 2 H, CH2-Bn {4.64}, H1-Gal′ {4.62, d, JH1,H2 = 7.9 Hz}),
4.57–4.45 (m, 6 H, CH2-Bn {4.59}, H3-Gal {4.58}, H2-GalN {4.54}, CH2-
Bn {4.52}, H3-Gal′ {4.51}, CH2-Fmoc {4.49}), 4.44–4.38 (m, 4 H, H6a-
Gal′ {4.43}, CH2-Bn {4.43}, CH2-Fmoc {4.41}, H9a-Sia′ {4.37}), 4.38–
4.33 (m, 1 H, H6b-Gal′), 4.30–4.21 (m, 3 H, Tꢀ {4.25}, H9-Fmoc {4.24},
Tα {4.24}), 4.21–4.13 (m, 2 H, H9a-Sia {4.19}, H5-GalN {4.18}), 4.06–
3.95 (m, 4 H, H5-Sia, H5-Sia′ {4.03}, H9b-Sia {3.99}, H3-GlcN {3.97}),
3.92 (dd, JH3,H2 = 11.1 Hz, JH3,H4 = 3.2 Hz, 1 H, H3-GalN), 3.91–3.83
(m, 4 H, H4-GlcN {3.91}, H9b-Sia′ {3.86}, H2-GlcN {3.84}, H5-Gal′
{3.84}), 3.80–3.59 (m, 6 H, H6a-GlcN {3.80}, H5-Gal {3.79}, H6b-GlcN
{3.71}, H6a-GalN {3.67}, H6b-GalN {3.61}, H5-GlcN {3.60}), 3.50 (dd,
JH6,H5 = 10.6 Hz, JH6,H7 = 2.7 Hz, 2 H, H6-Sia, H6-Sia′), 3.47 (dd,
JH6a,H6b = 10.7 Hz, JH6a,H5 = 2.7 Hz, 1 H, H6a-Gal), 3.38–3.35 (m, 1 H,
[4]
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H6b-Gal), 2.60 (dd, JH3eq,H3ax = 12.6 Hz, JH3eq,H4 = 4.6 Hz, 2 H, H3eq
-
Sia, H3eq-Sia′), 2.27, 2.19, 2.13, 2.10, 2.07, 2.05, 1.98 (7 × s, 42 H, CH3-
OAc), 2.13 (s, 6 H, CH3-NHAc-GalN, -GlcN), 1.83 (s, 6 H, CH3-NHAc-
Sia, -Sia′), 1.65 (t, JH3ax,H3eq = JH3ax,H4 = 12.1 Hz, 2 H, H3ax-Sia, H3ax-
[6]
[7]
Sia′), 1.45 (s, 9 H, CH3-tBu), 1.33 (d, JTγ,Tꢀ = 6.3 Hz, 3 H, Tγ) ppm. 13
C
NMR (200 MHz, CDCl3, HSQC, HMBC): δ = 171.7, 171.4, 170.9, 170.9,
170.8, 170.7, 170.5, 170.4, 170.1, 170.0, 169.9 (19 × C=O-NHAc, -OAc,
-ester), 167.5 (C1-Sia, C1-Sia′), 156.8 (C=O-urethane), 143.9, 143.7
(C1a-, C8a-Fmoc), 141.5, 141.4 (C4a-, C5a-Fmoc), 138.2, 137.7 (4 ×
Cq-Bn), 134.7, 129.1, 128.9, 128.8, 128.0, 127.9, 127.2, 127.2 (20 ×
CAr, C2-, C3-, C6-, C7-Fmoc), 125.2, 125.1 (C1-, C8-Fmoc), 120.2, 120.2
(C4-, C5-Fmoc), 101.1 (C1-Gal′), 100.7 (C1-GlcN), 100.1 (C1-Gal), 99.8
(C1-GalN), 96.9 (C2-Sia, C2-Sia′), 83.3 (Cq-tBu), 81.3 (d, JF,C6
=
172.1 Hz, C6-Gal′), 76.5 (Tꢀ), 73.7 (C3-GlcN), 73.6 (C3-GalN), 73.6 (C4-
GlcN), 73.5, 73.4 (CH2-Bn), 72.4 (C5-GlcN), 72.3 (C6-Sia, C6-Sia′),
71.8 (d, JF,C5 = 23.1 Hz, C5-Gal′), 71.8 (C3-Gal), 71.7 (C5-Gal), 71.4
(C3-Gal′), 70.8 (C2-Gal), 70.5 (C6-GalN), 69.9 (C4-GalN), 69.2 (C4-Sia,
C4-Sia′), 69.0 (C2-Gal′), 69.0 (C4-Gal′), 68.7, 68.7 (CH2-Bn), 68.7 (C6-
GlcN), 68.1 (C5-GalN), 68.0 (C8-Sia, C8-Sia′), 67.7 (C6-Gal), 67.7 (C4-
Gal), 67.6 (C7-Sia, C7-Sia′), 67.5 (CH2-Fmoc), 63.1 (C9-Sia, C9-Sia′),
59.8 (C2-GlcN), 59.2 (Tα), 49.0 (C5-Sia, C5-Sia′), 48.9 (C2-GalN), 47.2
(C9-Fmoc), 37.5 (C3-Sia, C3-Sia′), 28.2 (CH3-tBu), 23.2 (CH3-NHAc-Sia,
-NHAc-Sia′), 23.1 (CH3-NHAc-GalN, -NHAc-GlcN), 21.6, 21.5, 21.1,
20.9, 20.8, 20.8, 20.8 (14 × CH3-OAc), 19.2 (Tγ) ppm. 19F NMR
(376 MHz, CDCl3): δ = –230.3 (dt, JF,H6a/b = 46.8 Hz, JF,H5 = 13.7 Hz,
F6-Gal′) ppm. HR-MS (ESI+): m/z calcd. for C129H158FN5Na2O54 [M +
2Na]2+ 1353.4787, found 1353.4779.
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Acknowledgments
[13]
[14]
Support for this work by the Excellence Cluster Center of Inte-
grated Protein Science Munich (CIPSM) is gratefully acknowl-
edged. We are grateful to Dr. David Stephenson and Claudia
Dubler for assistance with NMR spectroscopy.
[15]
[16]
Keywords: Carbohydrates · Antigens · Sialophorin · Sialic
acids · Biomimetic synthesis · Fluorine
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see the Supporting Information.
To avoid any loss of material by Fmoc deprotection under basic condi-
tions, the crude product was treated with FmocOSu and DIPEA in di-
chloromethane prior to purification.
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Received: April 27, 2016
Published Online: ■
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Fluorinated Hexasaccharides
A first total synthesis of a fluorinated
hexasaccharide–threonine
antigen
M. Daum, F. Broszeit,
A. Hoffmann-Röder* ....................... 1–13
mimic has been accomplished using a
[3+3′]-block glycosylation strategy to
access novel sialophorin-derived mo-
lecular tools for biomedical and immu-
nological studies.
Synthesis of a Fluorinated Sialo-
phorin Hexasaccharide–Threonine
Conjugate for Fmoc Solid-Phase
Glycopeptide Synthesis
DOI: 10.1002/ejoc.201600523
Eur. J. Org. Chem. 0000, 0–0
www.eurjoc.org
13
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim