Total synthesis of LewisX using a late-stage crystalline intermediate

Article abstract of DOI:10.1016/j.carres.2015.05.017

Abstract Herein, we report on a highly efficient synthesis of a crystalline protected Lewis^X trisaccharide that was converted to Lewis^X following global deprotection. The trisaccharide was prepared in a highly convergent synthesis (seven steps, longest linear sequence) and in a 38% overall yield using a strategy that involved the regioselective glycosylation of a GlcNAc acceptor with a galactose thioglycoside donor, followed by fucosylation of the remaining free GlcNAc hydroxyl as key steps. The core trisaccharide also has the potential to be converted to other members of the Type-2 Lewis family of antigens due to the orthogonal nature of the protecting groups employed.

Full text of DOI:10.1016/j.carres.2015.05.017

Accepted Manuscript
X
Total synthesis of Lewis using a late-stage crystalline intermediate
Stefan Munneke, Gavin F. Painter, Graeme J. Gainsford, Bridget L. Stocker, Mattie
S.M. Timmer
PII:
S0008-6215(15)00185-8
10.1016/j.carres.2015.05.017
CAR 7022
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 5 April 2015
Accepted Date: 23 May 2015
Please cite this article as: Munneke S, Painter GF, Gainsford GJ, Stocker BL, Timmer MSM, Total
X
synthesis of Lewis using a late-stage crystalline intermediate, Carbohydrate Research (2015), doi:
10.1016/j.carres.2015.05.017.
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ACCEPTED MANUSCRIPT
Total synthesis of LewisX using a late-stage crystalline intermediate
Stefan Munneke,a Gavin F. Painter,b Graeme J. Gainsford,c Bridget L. Stocker,a,* and
Mattie S. M. Timmera,*
a School of Chemical and Physical Sciences, Victoria University of Wellington, PO
Box 600, Wellington, New Zealand
b Ferrier Institute, Victoria University of Wellington, PO Box 600, Wellington, New
Zealand
c Callaghan Innovation, PO Box 31310, Lower Hutt, New Zealand
Email: mattie.timmer@vuw.ac.nz; phone: +64 4 463 6529; fax: +64 4 4 463 5241
Abstract:
Herein, we report on a highly efficient synthesis of a crystalline protected LewisX
trisaccharide that was converted to LewisX following global deprotection. The
trisaccharide was prepared in a highly convergent synthesis (7 steps, longest linear
sequence) and in a 38% overall yield using a strategy that involved the regioselective
glycosylation of a GlcNAc acceptor with a galactose thioglycoside donor, followed
by fucosylation of the remaining free GlcNAc hydroxyl as key steps. The core
trisaccharide also has the potential to be converted to other members of the Type-2
Lewis family of antigens due to the orthogonal nature of the protecting groups
employed.
Keywords
LewisX, Carbohydrate, Synthesis, Glycosylation.
Graphical Abstract
1

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1. Introduction
The Lewis antigens (Le) are a related set of glycans characterised by the presence of
α1→4 or α1→3 fucosylated GlcNAc residues, termed the Type-1 and Type-2 Lewis
antigens, respectively.1 The Type-1 antigens, also known as the Lewis blood group
antigens (e.g. LeA and LeB, Figure 1), are found on erythrocytes, in blood plasma, and
in various tissues, while Type-2 Lewis antigens, [e.g. LeX and LeY, and their
sialylated forms] are serological characters widely distributed in human and animal
organisms.2 Lewis glycans have important roles in biology, and indeed, the Lewis
blood group antigens owe their name to the Lewis family who suffered from a red
blood cell incompatibility.3 Members of both subsets of Lewis antigens have been
implicated in favourable and unfavourable selectin-dependent leukocyte and cell
adhesion processes2 with, for example, Sialyl LeA and Sialyl LeX (SLeX) being
upregulated during cancer metastasis,4,5,6,7 while SLeX also has an essential role in
human fertilisation.8 Targeting the dendritic cell (DC) C-type lectin, DC-SIGN, which
binds Lewis antigens (i.e. LeX, LeY, LeB and LeA), has been shown to increase antigen
cross-presentation and enhance vaccine efficacy for cancer and intracellular
pathogens.9,10,11,12,13 Accordingly, there has been much interest in targeting this lectin
using antigen-conjugated antibodies or antigen-conjugated Lewis glycans (e.g. LeX),
whereby the latter approach is thought to be advantageous due to reduced side-effects
and the ease of synthesis.14
Figure 1. Representative Lewis antigens
Although various synthetic strategies can be envisaged for the synthesis of Type-2
Lewis glycans, the incorporation of an orthogonally protected trisaccharide LewisX
intermediate has particular merit, as this would allow access to a variety of Type-2
Lewis antigens, including sialylated and sulfated derivatives. There are three general
retrosynthetic approaches that can be used for the construction of LewisX derivatives
2

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(I, Scheme 1). First, the target trisaccharide can be constructed through
galactosylation of an α-L-Fuc-(1→3)-D-GlcNAc acceptor, which in turn is accessible
via reductive ring opening of benzylidene protected dimer II (Route A).15 Dimer II is
conveniently constructed by 3-O-fucosylation of a benzylidene protected GlcNAc
acceptor. A number of elegant syntheses of LewisX have been achieved in this
manner, including the synthesis by Stahl et al. using an N-acetylglucosamine building
block,16 and the one-pot synthesis by Boons and co-workers using more advanced
building blocks.17 The lability of the fucose residue during the 2+1 coupling,
however, can be troublesome,18,19,20 and this has instigated the need for more complex
fucose building blocks that contain electron-withdrawing protecting
groups.15,17,18,21,22,23 Alternatively, LeX (I) can be constructed from the 2+1 coupling
of a fucose donor with a β-D-Gal-(1→4)-D-GlcNAc acceptor III, which is derived
from a protected GlcNAc precursor (Route B).24 Again, some remarkable syntheses of
the Type-2 Lewis glycans have been achieved using this approach, including the
synthesis of SLeX by Seeberger and co-workers.25 The most efficient route to LeX,
however, involves a regioselective coupling at the 4-position of a glucosamine
precursor (Route C), as elegantly shown by Roy and co-workers,26 and later adapted
Scheme 1. Retrosynthetic approaches that have been used for the synthesis of LeX.
3

ACCEPTED MANUSCRIPT
by others.27,28,29,30 Accordingly, we envisioned using this third regioselective
glycosylation approach to develop a highly efficient synthesis suitable for the
preparation of LeX, as well as other Type-2 Lewis glycans. To this end, we sought to
avoid the use of a glucosamine N-protecting group, such as Troc or Phth, as this
would require a late stage N-deprotection and acetylation step. For the fucose donor,
we opted for a perbenzylated thioglycoside, as the synthesis of a donor with electron-
withdrawing groups would require a more complex building block synthesis and
additional late-stage deprotection steps.
2. Results and discussion
To begin the synthesis of LeX, we selected three building blocks that could not only
be readily synthesised, but moreover, would provide an orthogonally protected LeX
core that could be used for the construction of the full family of Type-2 Lewis
antigens. The three building blocks, all of which can be synthesised in 2-5 steps, are
depicted in Scheme 2. The simplest of these to prepare was the GlcNAc acceptor,
which was synthesised on a multi gram scale in two-steps from GlcNAc (1). Here,
Fisher glycosylation of lactol 1 with benzyl alcohol gave the crystalline benzyl
glucoside 2,31 which was then selectively protected at the 6-position using tert-
butyldiphenylsilyl (TBDPS) chloride to yield protected GlcNAc acceptor 3 in 70%
over the two steps.32 We observed the TBDPS-protection was sluggish at room
temperature and that warming the reaction to 40 °C increased the solubility of the
Scheme 2. Synthesis of key building blocks
4

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sugar and reaction yields significantly. Next, the galactose donor was prepared via a
5-step synthesis that commenced with the peracetylation of D-galactose (4), followed
by installation of the anomeric thiophenol and deacetylation to give crystalline
thiogalactoside 5 in 80% yield over the three steps.33 Installation of the 4,6-
benzylidine acetal protecting group,33 followed by benzoylation of the remaining free
hydroxyls then gave the required crystalline donor 6,34 equipped with a 2-O-acyl
protecting group to allow for neighbouring group participation and β-selective
glycosylation. Finally, the fucose acceptor was also prepared in five-steps. L-Fucose
(7) was per-acetylated, converted to the anomeric bromide with subsequent
conversion to the thioglycoside, and deacetylated to give triol 8. Perbenzylation then
afforded the crystalline target donor 9 in 56% overall yield.35
With the target building blocks in hand, the assembly of the LeX trisaccharide began
with the regioselective glycosylation of GlcNAc acceptor 3 and the galactose donor 6
(Scheme 2). It was observed that the slow addition of the donor (1.2 equivalents) to a
mixture of the acceptor and activator at -50 °C resulted in a high yielding (74%)
regioselective glycosylation, however, when the reaction was performed at higher
temperatures (or allowed to warm to 0 °C) small amounts of the over-glycosylated
trimer were formed. Given that the acceptor is obtained in fewer steps compared to
the donor, the reaction was repeated using a slight excess of acceptor (1.1 equivalents
of GlcNAc) at -50 °C for 1 hour, which, following purification by silica gel flash
column chromatography, gave disaccharide 10 in 78%. NMR analysis was used to
confirm that the resulting glycoside was the β-pyranoside, whereby 1H NMR analysis
Scheme 3. Assembly of the LeX core 11
5

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revealed a J1´´,2´´ = 8.2 Hz, indicative of the β-anomer, while the regioselectivity of the
glycosylation was confirmed using 2D NMR analysis in which HMBC correlations
between both H-1´´/C-4´, and H-4´/C-1´´ were observed.
With the disaccharide acceptor in hand, the glycosylation between disaccharide 10
and fucose donor 6 was explored. Activation of the thioethyl donor 6 using Ogawa’s
conditions36 resulted in the smooth formation of the desired α-fucosylated
trisaccharide 11 in excellent yield (92%) and excellent α-selectivity (1JC,H = 170
Hz37). Much to our delight, trisaccharide 11 could be purified via silica gel
chromatography and/or readily crystallised from methanol (Figure 2),38 which
allowed for rapid purification. The obtained crystal structure also confirmed the regio-
and stereo-selectivity of both glycosylation reactions. While one other crystalline LeX
intermediate has been reported (though no crystal structure was obtained),39 the
synthetic route employed was more complex and did not readily allow for the further
derivatisation of the LeX scaffold into other Type-II Lewis glycans. Moreover, our
yield over the two glycosylation reactions (72%) is comparable to other reported
strategies. While there are several notable syntheses of protected LeX
trisaccharides,25,29,30,40 with for example an 80% yield over the two coupling steps,30
these strategies employ N-protected glucosamine acceptors, thereby requiring more
complex building block syntheses and late stage functional group interconversions.
Comparing our route to the synthesis by Roy and co-workers who pioneered the
regioselective glycosylation of an N-acetylglucosamine acceptor,26 we obtained
similar glycosylation yields, however, our crystalline derivative contains orthogonally
protected 6´- and 6´´-positions, thereby allowing for the synthesis of 6-sulfo- or 6´-
sulfo-LeX derivatives.41 In addition, deacetylation of 11 gives the 2´´,3´´-diol which in
turn can be used en route towards dimeric LeX,42 3´-sulfo-LeX,43 as well as SLeX
derivatives.44 Taken as a whole, our short synthetic sequence (longest linear sequence
of 7 steps) and high overall yield (38%) for the synthesis of orthogonally protected
LeX 11 provides a highly efficient and versatile route for the synthesis of Type-2
Lewis glycans.
6

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Figure 2. Crystal structure of trisaccharide 11.38
To complete the synthesis of LeX, trisaccharide 11 was globally deprotected (Scheme
4). Accordingly, the benzoyl and TBDPS groups were first removed using
NaOMe/MeOH and HF.pyridine, respectively, to give triol 12. The conditions of
Zang and co-workers were then employed for the reductive removal of both the
benzyl ether and benzylidene protecting groups.45 Here, it should be noted that the
solvent mixture THF:H2O:AcOH (4:2:1, v/v/v) was essential in obtaining near
quantitative yields of the target trisaccharide without concomitant de-fucosylation.
With an overall yield of 37% (longest linear sequence of 10-steps), our synthesis of
LeX is the highest yielding to date and should facilitate in the preparation of complex
glycoconjugates via the use of a variety of ligation strategies.46
Scheme 4. Global deprotection and total synthesis of LeX
3. Conclusion
In conclusion, the synthesis of the LeX trisaccharide was completed in excellent yield
and in few linear steps. To achieve this, highly efficient syntheses of the core building
blocks were accomplished (2-5 steps, 54-70% yield), and a regioselective
glycosylation between the glucosamine acceptor and galactose donor was performed
in 78% yield. An α-selective fucosylation then gave the resulting trisaccharide as
crystalline material in excellent (92%) yield, with global deprotection affording LeX in
near quantitative yield. It is envisioned that this highly efficient route will find further
7

ACCEPTED MANUSCRIPT
application, not only for the targeting of LeX to receptors such as DC-SIGN, but also
for the synthesis of other members of the Type-2 family of Lewis antigens.
4. Experimental
General procedure. Unless otherwise stated all reactions were performed under
argon. Prior to use, THF (Pancreac) was distilled from sodium and benzophenone,
pyridine was distilled and dried over 4Å molecular sieves (4Å MS), CH2Cl2 (Fisher)
was distilled from P2O5, DMF was distilled from BaO, and H2O was distilled.
NaOMe was freshly prepared from sodium [Aldrich] and MeOH. N-Acetyl
glucosamine (Fluka), BnOH (Ajax Chemicals), conc. HCl (Panreac), TBDPS-Cl
(Aldrich), DMAP (Pierce), N-iodosuccinimide (Aldrich), TfOH (Aldrich), TPABr
(Aldrich), CuBr2 (Chempur), HF.Pyridine (Aldrich), Ca(OAc)2 (Sigma), AcOH
(Sigma-Aldrich), Pd/C (Aldrich), Et3N (Sigma), Dowex H+ (Sigma-Aldrich 50WX8),
4Å molecular sieves (ROTH), Toluene (Panreac), Et2O (Pancreac), EtOAc
(Pancreac), petroleum ether (Pure Science), MeOH (Pure Science), CHCl3 (Pancreac),
EtOH (absolute, Pure Science), NaHCO3 (Pure Science), Na2S2O3 (Merck) and NaCl
(Pancreac) were used as received. Galactose donor 633,34 and fucose donor 935 were
prepared according to literature procedures. All solvents were removed by
evaporation under reduced pressure. Reactions were monitored by TLC-analysis on
Macherey-Nagel silica gel coated plastic sheets (0.20 mm, with fluorescent indicator
UV254) with detection by UV-absorption (short wave UV – 254 nm; long wave UV –
366 nm), by dipping in 10% H2SO4 in EtOH followed by charring at ~150 °C, by
dipping in I2 in silica, or by dipping into a solution of ninhydrin in EtOH followed by
charring at ~150 °C. Column chromatography was performed on Pure Science silica
gel (40 – 63 micron). AccuBOND II ODS-C18 (Agilent) was used for reverse phase
chromatography. Infrared spectra were recorded as thin films using a Bruker
Tensor 27 FTIR spectrometer equipped with an Attenuated Total Reflectance (ATR)
sampling accessory and are reported in wave numbers (cm-1). Nuclear magnetic
resonance spectra were recorded at 20 °C in D2O, CD3OD, CDCl3, or pyridine-d5
using either a Varian INOVA operating at 500 MHz or Varian VNMRS operating at
600 MHz. Chemical shifts are given in ppm (δ) relative to solvent residues. NMR
peak assignments were made using COSY, HSQC and HMBC 2D experiments.
8

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Benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (2). To a
solution of N-acetylglucosamine (6.40 g, 28.9 mmol) in benzyl
alcohol (50 mL, 480 mmol), conc. aq. HCl (3.0 mL) was added and
the reaction mixture was stirred at 90 °C for 3 h. The crude mixture was cooled down
to rt, poured into Et2O (500 mL) and left to crystallize at -4 °C for 18 h. The
crystalline product was filtered and washed with petroleum ether. Purification by
silica gel column chromatography (CH2Cl2:MeOH, 95:5 → 88:12, v/v) gave benzyl
glycoside 2 (6.67 gram, 21.4 mmol, 74%) as a white foam. Rf = 0.25 (CH2Cl2:MeOH,
17.8
90:10, v/v); Mp 182 – 184 °C; αD = +222 (c = 0.1, MeOH); IR (film) 3298, 3092,
2938, 2901, 2844, 1648, 1552, 1497, 1455, 1375, 1309, 1230, 1156, 1093, 1047, 778,
1
732, 695 cm-1; H NMR (500 MHz, CD3OD) δ 7.40 – 7.25 (m, 5H, CHarom), 4.85 (d,
1H, J1´,2´ = 3.6 Hz, H-1´), 4.74 (d, 1H, J1a,1b = 12.0 Hz, H-1a), 4.49 (d, 1H, J1a,1b = 12.0
Hz, H-1b), 3.89 (dd, 1H, J1´,2´ = 3.6 Hz, J2´,3´ = 10.8 Hz H-2´), 3.83 (dd, 1H, J5´,6a´
=
1.6 Hz, J6a´,6b´ = 11.4 Hz, H-6a´), 3.73 – 3.64 (m, 3H, H-6b´, H-3´, H-4´), 3.36 (dd,
1H, J4´,5´ = J5´,6b´ = 9.6 Hz, H-5´), 1.95 (s, 3H, CH3 Ac); 13C NMR (125 MHz, CD3OD)
δ 173.6 (C=O Ac), 139.0 (Cqarom), 129.4 (C-oarom), 129.3 (C-marom), 128.8 (C-parom),
97.5 (C-1´), 74.1 (C-4´), 72.7 (C-3´), 72.5 (C-5´), 70.1 (C-1), 62.7 (C-6´), 55.4 (C-2´),
22.5 (CH3 Ac); HRMS(ESI) m/z calcd. for [C15H22NO6]+: 312.1442, obsd.: 312.1446.
Benzyl 2-acetamido-6-O-tert-butyldiphenylsilyl-2-deoxy-α-D-
glucopyranoside (3). To a solution of benzyl glycoside 2 (837
mg, 8.74 mmol) in pyridine (10 mL), TBDPS-Cl (2.75 mL, 33.9
mmol) and DMAP (200 mg, 1.64 mmol) were added and the reaction mixture was
stirred at 40 °C for 18 h. The crude mixture was concentrated in vacuo and purified by
silica gel column chromatography (CH2Cl2:MeOH, 100:0 → 95:5, v/v) to give diol 3
17.8
(1.39 gram, 94%) as a white solid. Rf = 0.50 (CH2Cl2:MeOH, 95:5, v/v); αD
=
+58.4 (c = 0.5, CHCl3); IR (film) 3324, 3071, 2930, 2857, 1739, 1655, 1544, 1472,
1
1456, 1428, 1376, 1217, 1112, 1048, 1025, 823, 775, 739, 701 cm-1; H NMR (500
MHz, CDCl3) δ 7.73 – 7.68 (m, 4H, CHarom), 7.46 – 7.27 (m, 11H, CHarom), 5.84 (d,
1H, J2´,NH = 8.5 Hz, NH), 4.88 (d, 1H, J1´,2´ = 3.0 Hz, H-1´), 4.72 (d, 1H, J1a,1b = 11.8
Hz, H-1a), 4.44 (d, 1H, J1a,1b = 11.8 Hz, H-1b), 4.14 – 4.06 (m, 1H, H-2´), 3.94 – 3.85
(m, 2H, H-6a´, H-6b´), 3.77 – 3.68 (m, 2H, H-3´, H-5´), 3.63 (dd, 1H, J3´,4´ = J4´,5´
=
9.1 Hz, H-4´), 3.24 (bs, 1H, OH), 2.86 (bs, 1H, OH), 2.00 (s, 3H, CH3 Ac), 1.07 (s,
9

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13
9H, CH3 tBu); C NMR (125 MHz, CDCl3) δ 172.0 (C=O), 137.1, 135.81, 135.78,
133.3, 133.2, 130.0, 128.8, 128.3, 128.2, 127.9 (Carom), 96.5 (C-1´), 74.5 (C-3´), 72.9
(C-5´), 71.5 (C-4´), 69.4 (C-1), 64.3 (C-6´), 53.8 (C-2´), 27.0 (CH3 tBu), 23.4 (CH3
Ac), 19.4 (Cq tBu); HRMS(ESI) m/z calcd. for [C31H40NO6Si]+: 550.2619, obsd.:
550.2620.
Benzyl 2-acetamido-4-O-(2,3-di-O-benzoyl-4,6-O-
benzylidene-β-D-galactopyranosyl)-2-deoxy-6-O-
tert-butyldiphenylsilyl-α-D-glucopyranoside (10).
Glycosyl acceptor 3 (680 mg, 1.24 mmol) was co-
evaporated with toluene (3 × 3 mL) and dissolved in
dry CH2Cl2 (5 mL). In a separate flask, glycosyl donor 6 (798 mg, 1.40 mmol)33, 34
was co-evaporated with toluene (3 × 3 mL) and dissolved in dry CH2Cl2 (10 mL).
Activated molsieves (4Å) were added to both the donor and the acceptor solutions,
and both solutions were stirred at rt for 30 min. N-iodosuccinamide (631 mg, 2.81
mmol) was added to the mixture with acceptor 3 and the reaction mixture was cooled
to -50 °C. Freshly distilled TfOH (124 µL) was added at -50 °C and the reaction
mixture was stirred for 15 min before adding the solution of donor 6. The crude
reaction mixture was stirred for 1 h at -50 °C and when TLC analysis showed full
conversion of the glycosyl donor, the reaction mixture was quenched by the addition
of NaHCO3 (830 mg). The crude reaction mixture was diluted with CH2Cl2 (50 mL),
washed with sat. aq. Na2S2O3 (30 mL) and the water layer was extracted with CH2Cl2
(3 × 10 mL). The combined organic layers were dried with MgSO4, filtered and
concentrated in vacuo. Purification by silica gel flash column chromatography
(PE:EtOAc 75:25 → 0:100, v/v) gave disaccharide 10 (909 mg, 73%) as a white
17.8
foam. Rf = 0.56 (EtOAc); αD = +91.1 (c = 1, CHCl3); IR (film) 3422, 3382, 3069,
2931, 2889, 2857, 1722, 1668, 1602, 1531, 1452, 1428, 1369, 1315, 1273, 1219,
1177, 1112, 1070, 1040, 1027, 821, 773, 741, 708 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.97 (d, 2H, Jo-m = 7.3 Hz, CH-o Bz), 7.76 – 7.66 (m, 6H, CHarom), 7.55 – 7.30 (m,
16H, CHarom), 7.28 – 7.14 (m, 8H, CHarom), 5.88 (dd, 1H, J1´´,2´´ = 8.2 Hz, J2´´,3´´ = 10.6
Hz, H-2´´), 5.64 (d, 1H, J2´,NH = 8.8 Hz, NH) 5.52 (s, 1H, CHPh), 5.31 (dd, 1H, J3´´,4´´
= 3.0 Hz, J2´´,3´´ = 10.7 Hz, H-3´´), 4.98 (d, 1H, J1´´,2´´ = 8.2 Hz, H-1´´), 4.88 (d, 1H,
J1´,2´ = 3.2 Hz, H-1´), 4.58 (d, 1H, J3´´,4´´ = J4´´,5´´ = 3.0 Hz, H-4´´), 4.50 (d, 1H, J1a,1b
=
10

ACCEPTED MANUSCRIPT
12.0 Hz, H-1a), 4.45 (d, 1H, J6a´,6b´ = 12.0 Hz, H-6a´), 4.34 (d, 1H, J1a,1b = 12.0 Hz, H-
1b), 4.20 – 4.00 (m, 3H, H-2´, H-6b´, H-4´), 3.87 (dd, 1H, J2´,3´ = J3´,4´ = 9.8 Hz, H-3´),
3.75 (d, 1H, J6a´´,6b´´ = 11.6 Hz, H-6a´´), 3.64 (bs, 1H, H-5´´), 3.57 (d, 1H, J6a´´,6b´´
=
11.6 Hz, H-6b´´), 3.45 (d, 1H, J4´,5´ = 9.9 Hz, H-5´), 1.99 (s, 3H, CH3 Ac), 1.08 (s, 9H,
CH3 tBu); 13C NMR (125 MHz, D2O) δ 170.3 (C=O Ac), 166.2 (C=O 3-O-Bz), 165.1
(C=O 2-O-Bz), 137. 6, 137.3, 136.1, 135.7, 134.0, 133.6, 133.3, 133.0, 130.1, 130.0,
129.8, 129.2, 129.1, 128.6, 128.5, 128.3, 128.1, 128.0, 127.8, 126.4 (Carom), 100.93
(CPh), 100.91 (C-1´´), 96.8 (C-1´), 79.0 (C-4´), 73.5 (C-4´´), 72.7 (C-3´´), 70.7 (C-
5´), 70.2 (C-3´), 69. 8 (C-1), 69.3 (C-2´´), 68.6 (C-6´), 67.0 (C-5´´), 61.7 (C-6´´), 53.4
(C-2´), 27.1 (CH3 tBu), 23.6 (CH3 Ac), 19.7 (Cq tBu); HRMS(ESI) m/z calcd. for
[C58H62NO13Si]+: 1008.3985, obsd.: 1008.4009.
Benzyl 2-acetamido-4-O-(2,3-di-O-benzoyl-4,6-O-
benzylidene-β-
3-O-(2,3,4-tri-O-benzyl-α-
deoxy-6-O-tert-butyldiphenylsilyl-α-
D
-galactopyranosyl)-
L
-fucopyranoside)-2-
D-
glucopyranoside (11). A mixture of glycosyl acceptor
10 (0.90 g, 0.89 mmol) and glycosyl donor 9 (512 mg,
1.07 mmol)35 were co-evaporated with dry DMF (3 × 3 mL) and dissolved in dry
DMF (5 mL) and dry CH2Cl2 (10 mL). Activated molsieves (4Å) were added and the
reaction mixture was stirred at rt for 30 min. TPABr (570 mg, 2.14 mmol) and CuBr2
(480 mg, 2.14 mmol) were added and the reaction mixture was stirred ar rt for 15 h.
When TLC analysis showed full conversion of the glycosyl acceptor, the crude
reaction mixture was quenched by the addition of sat. aq. Na2S2O3 (30 mL). The
water layer was extracted with CH2Cl2 (3 × 10 mL), and the combined organic layers
were washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The
residue was purified by silica gel flash column chromatography (PE:EtOAc, 9:1 →
2:1, v/v) and/or crystallized from hot methanol (200 mL) to afford trisaccharide 11
24.4
(1.05 g, 0.74 mmol, 92%) as white crystals. Rf = 0.23 (PE:EtOAc, 2:1, v/v); αD
=
+110 (c = 0.5, CHCl3); IR (film) 3088, 3064, 3032, 3007, 2932, 2894, 2859, 1735,
1671, 1497, 1453, 1428, 1367, 1315, 1273, 1250, 1250, 1219, 1165, 1143, 1098,
1
1061, 1046, 1027, 1002, 772, 708, 699 cm-1; H NMR (500 MHz, CDCl3) δ 8.05 (d,
2H, Jo,m = 7.7 Hz, CH-o Bz), 7.87 – 7.81 (m, 4H, CHarom), 7.64 – 7.07 (m, 39H,
11

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CHarom), 5.88 (dd, 1H, J2´´,3´´ = 9.0 Hz, C-2´´), 5.60 (s, 1H, CHPh), 5.49 (d, 1H, J2´,NH
= 9.9 Hz, NH), 5.28 – 5.20 (m, 3H, H-1´´, H-1´´´, H-3´´), 4.95 (q, 1H, J5´´´,6´´´ = 6.7
Hz, H-5´´´), 4.84 – 4.77 (m, 2H, H-1´, CH2-a 2´´´-O-Bn), 4.73 (d, 1H, Ja,b = 11.7 Hz,
CH2-a 3´´´-O-Bn), 4.68 (d, 1H, Ja,b = 11.7 Hz, CH2-b 3´´´-O-Bn), 4.58 – 4.48 (m, 4H,
H-4´´, CH2-b 3´´´-O-Bn, H-2´, H-6a´), 4.41 – 4.31 (m, 3H, H-1a, H-1b, H-4´), 4.20 (d,
1H, Ja,b = 11.4 Hz, CH2-a 4´´´-O-Bn), 4.12 (d, 1H, J6a´,6b´ = 12.1 Hz, H-6b´), 4.06 –
4.01 (m, 1H, H-3´´´), 3.99 (dd, 1H, J2´,3´ = J3´,4´ = 9.7 Hz, H-3´), 3.96 – 3.89 (m, 2H,
H-2´´´, H-6a´´), 3.54 – 3.47 (m, 3H, H-6b´´, H-5´´, CH2-b 4´´´-O-Bn), 3.29 (d, 1H,
J4´,5´ = 9.8 Hz, C-5´), 3.25 (bs, 1H, H-4´´´), 1.81 (s, 3H, CH3 Ac), 1.33 (d, 3H, J5´´´,6´´´
13
= 6.2 Hz C-6´´´), 1.14 (s, 9H, CH3 tBu); C NMR (125 MHz, CDCl3) δ 169.7 (C=O
Ac), 166.0 (C=O 3-OBz), 164.8 (C=O 2-OBz), 139.9, 139.8, 138.6, 137.8, 137.1,
136.2, 135.6, 134.0, 133.6, 133.2, 132.4, 130.3, 130.1, 130.0, 129.7, 129.2, 129.1,
129.0, 128.7, 128.64, 128.55, 128.52, 128.48, 128.45, 128.35, 128.3, 128.23, 128.20,
128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.4, 127.1, 126.9, 125.9 (Carom), 99.9 (C-
1´´), 99.8 (CHPh), 97.9 (C-1´´´), 96.8 (C-1´), 79.4 (C-4´´´), 78.8 (C-3´´´), 75.3 (C-
2´´´), 75.0 (CH2 4´´´-O-Bn), 73.9 (C-4´), 73.6 (C-4´´), 72.9 (C-3´´, CH2 2´´´-O-Bn),
72.7 (C-3´), 72.0 (CH2 3´´´-O-Bn), 71.3 (C-5´), 70.1 (CH2 1-O-Bn), 69.2 (C-2´´), 69.1
(C-6´), 66.6 (C-5´´, C-5´´´), 61.3 (C-6´´), 54.1 (C-2´), 27.1 (CH3 tBu), 23.7 (CH3 Ac),
19.7 (Cq tBu), 16.4 (C-6´´´); HRMS(ESI) m/z calcd. for [C85H89NO17SiNa]+:
1446.5792, obsd.: 1446.5797.
Benzyl
2-acetamido-4-O-(4,6-O-benzylidene-β-
galactopyranosyl)-3-O-(2,3,4-tri-O-benzyl-α-
fucopyranosyl)-2-deoxy-α-
D
-
L
-
D
-glucopyranoside (12). To a
solution of trisaccharide 11 (201 mg, 141 µmol) in methanol
(3.0 mL) and CH2Cl2 (3.0 mL), 1M methanolic NaOMe (2.0
mL) was added and the reaction mixture was stirred at rt for 4 h. The reaction mixture
was quenched by the addition of Dowex H+ and the reaction mixture was filtered,
washed with MeOH and concentrated in vacuo. The crude product was used in the
next step without further purification. Rf = 0.57 (PE:EtOAc, 50:50, v/v); HRMS(ESI)
m/z calcd. for [C71H81NO15SiNa]+: 1238.5268, obsd.: 1238.5276.; The crude
trisaccharide was dissolved in pyridine (5 mL), HF.pyridine (0.5 mL) was added at 0
°C and the reaction mixture was stirred ar rt for 18 h. After TLC analysis showed
12

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complete conversion, the mixture was diluted with CH2Cl2 (25 mL) and washed with
1M aq. Ca(OAc)2 (50 mL). The aqeaous layer was extracted with CH2Cl2 (2 × 25 mL)
and the combined organic extracts were washed with brine, dried with MgSO4,
filtered and concentrated in vacuo. The residue was co-evaporated with toluene (3 × 5
mL) in order to remove traces of pyridine, and purified by silica gel flash column
chromatography (EtOAc:MeOH, 100:0 → 95:5, v/v) to afford triol 12 (137 mg, 140
24.6
µmol, 99%) as a white foam. Rf = 0.45 (EtOAc:MeOH, 8:1, v/v), αD = -24.2 (c =
0.5, CHCl3); IR (film) 3428, 3334, 3063, 3031, 2974, 2929, 2906, 2872, 1658, 1543,
1497, 1454, 1397, 1363, 1338, 1246, 1213, 1165, 1136, 1093, 1047, 966, 909, 858,
734, 697 cm-1 1H NMR (500 MHz, CDCl3) δ 7.48 – 7.42 (m, 2H, CHarom), 7.31 – 7.14
;
(m, 22H, CHarom), 7.11 (t, 1H, J = 7.6 Hz, CHarom), 7.00 (t, 2H, J = 7.6 Hz, CHarom),
6.79 (d, 1H, JNH,2´ = 7.2 Hz, NH), 5.47 (s, 1H, CHPh), 5.38 (d, 1H, J1´´,2´´ = 3.2 Hz, H-
1´´), 4.98 (d, 1H, J1´´´,2´´´ = 3.4 Hz, H-1´´´), 4.80 (d, 1H, J1´,2´ = 7.6 Hz, H-1´), 4.70 –
4.62 (m, 3H, CH2a 2-O-Bn, CH2a 3-O-Bn, CH2a 4-O-Bn), 4.60 (d, 1H, Ja,b = 10.4 Hz,
CH2b 2-O-Bn), 4.55 (d, 1H, J1a,1b = 12.0 Hz, CH2a-1-O-Bn), 4.52 (d, 1H, Ja,b = 11.7
Hz, CH2b 3-O-Bn), 4.35 (d, 1H, J1a,1b = 12.0 Hz, CH2b-1-O-Bn), 4.31 – 4.20 (m, 2H,
H-6a´´, H-5´´´), 4.16 (dd, J2´,3´ = 9.1 Hz, J3´,4´ = 10.2 Hz, H-3´), 4.10 – 4.03 (m, 2H, H-
4´, H-4´´), 4.00 (bd, J6a´,6b´ = 12.2 Hz, H-6a´), 3.98 – 3.90 (m, 3H, H-2´, H-2´´, H-
6b´´), 3.84 (dd, 1H, J3´´´,4´´´ = 2.5 Hz, J2´´´,3´´´ = 10.0 Hz, H-3´´´), 3.74 – 3.64 (m, 3H,
H-2´´, H-5´, H-6b´), 3.59 – 3.51 (m, 2H, H-4´´´, H-5´´), 3.51 – 3.44 (m, 2H, H-3´´,
OH), 2.85 (bs, 1H, OH), 1.70 (bs, 2H, 2 × OH), 1.45 (s, 3H, CH3 Ac), 1.04 (d, 3H,
J5´´´,6´´´ = 6.6 Hz, H-6´´´); 13C NMR (125 MHz, CDCl3) δ 170.3 (C=O, Ac), 138.9,
137.8, 137.5, 137.5 (CHarom), 129.2, 128.8, 128.6, 128.51, 128.50, 128.33, 128.28,
128.10, 128.06, 128.0, 127.7, 127.3, 126.5 (CHarom), 102.3 (C-1´´), 101.1 (CHPh),
98.6 (C-1´´´), 96.3 (C-1´), 79.4 (C-3´´´), 77.7 (C-4´´´), 77.5 (C-2´´´), 76. 7 (C-3´),
75.6 (C-4´), 75.3 (C-4´´), 75.1 (CH2 4-OBn), 74.7 (CH2 2-OBn), 73.2 (C-2´´), 72.8
(C-5´´), 72.2 (CH2 3-OBn), 71.1 (C-5´), 69.9 (CH2-1), 69.5 (C-6´´), 67.5 (C-5´´´),
67.0 (C-3´´), 61.0 (C-6´), 54.1 (C-2´), 22.8 (CH3 Ac), 17.1 (C-6´´´); HRMS(ESI) m/z
calcd. for [C55H64NO15]+: 978.4270, obsd.: 978.4274.
2-Acetamido-2-deoxy-3-O-(α-
L
-fucopyranosyl)-4-
O-(β- -galactopyranosyl))-
D
D-glucopyranoside
(LeX). To trisaccharide 12 (137 mg, 140 µmol) in
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distilled THF (4 mL), H2O (2 mL), AcOH (1.0 mL) and Pd/C (100 mg) were added
and hydrogen gas was bubbled though the reaction mixture at rt for 3 h. The crude
mixture was diluted with water (20 mL), filtered over paper (Whatman 42) and
lyophilised. Purification by size exclusion chromatography (BioGel P-2, 1200 × 10
mm) gave LeX (72.5 mg 137 µmol, 98%) as a white foam. Rf = 0.12
(n-butanol:AcOH:H2O, 4:1:1, v/v/v); IR (film) 3313, 2971, 2938, 1640, 1555, 1427,
1
1378, 1162, 1118, 1071, 1034, 1021, 968, 811 cm-1; H NMR (300 MHz, D2O) α-
anomer: δ 5.11 – 5.08 (m, 2H, H-1´, H-1), 4.84 (q, 1H, J5´´,6´´ = 6.7 Hz, H-5´´), 4.46
(d, 1H, J1´,2´ = 7.8 Hz, H-1´ α), 4.15 (dd, 1H, J1,2 = 3.5 Hz, J2,3 = 10.4 Hz, H-2 α), 4.03
– 3.92 (m, 4H, H-3, H-4, H-5, H-6a), 3.92 – 3.81 (m, 8H, H-6b, H-3´´, H-4´), 3.79 (d,
1H, J 3´´,4´´ = J4´´,5´´ = 3.0 Hz, H-4´´), 3.77 – 3.62 (m, 4H, H-6´a, H-6´b, H-2´´, H-3´),
3.62 – 3.57 (m, 1H, H-5´), 3.53 – 3.46 (m, 1H, H-2´), 2.03 (s, 3H, CH3 Ac), 1.19 –
1.15 (m, 3H, H-6´´); β-anomer: δ 5.11 – 5.08 (m, 1H, H-1´), 4.84 (q, 1H, J5´´,6´´ = 6.7
Hz, H-5´´), 4.72 (d, 1H, J1,2 = 8.1 Hz, H-1 β), 4.45 (d, 1H, J1´,2´ = 7.8 Hz, H-1´ β), 4.03
– 3.92 (m, 2H, H-4, H-6a), 3.92 – 3.81 (m, 5H, H-6b, H-3´´, H-4´, H-2, H-3), 3.79 (d,
1H, J 3´´,4´´ = J4´´,5´´ = 3.0 Hz, H-4´´), 3.77 – 3.62 (m, 4H, H-6´a, H-6´b, H-2´´, H-3´),
3.62 – 3.57 (m, 2H, H-5, H-5´), 3.53 – 3.46 (m, 1H, H-2´), 2.03 (s, 6H, CH3 Ac), 1.19
– 1.15 (m, 6H, H-6´´); 13C NMR (125 MHz, D2O) δ 174.4 (C=O β), 174.2 (C=O β),
101.76 (C-1´ β), 101.74 (C-1´ α), 98.6 (C-1´´ β), 98.5 (C-1´´ α), 94.7 (C-1 β), 91.0
(C-1 α), 75.4 (C-5 β), 74.91 (C-3 β), 74.85 (C-5´ α + β), 73.2 (C-4 α+β), 72.8 (C-3 α),
72.4 (C-3´ α+β), 71.8 (C-4´´ α+β), 71.3 (C-5 α), 71.0 (C-2´ α+β), 69.20 (C-3´´ α),
69.15 (C-3´´ β), 68.3 (C-4´ α+β), 67.6 (C-2´´ α+β), 66.6 (C-5´´ α+β), 61.5 (C-6´ α+β),
59.7 (C-6 β), 59.6 (C-6 α), 56.9 (C-2 β), 54.0 (C-2 α), 22.2 (CH3 Ac β), 21.9 (CH3 Ac
α), 15.2 (C-6´´ α+β); HRMS(ESI) m/z calcd. for [C20H36NO15]+: 530.2079, obsd.:
530.2093.
Acknowledgements
We thank the Wellington Medical Research Foundation for financial support
(2014/253). SM would like to acknowledge the support of Victoria University of
Wellington and Industrial Research Ltd for a post-graduate scholarship.
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Highlights
A crystalline orthogonally protected LewisX trisaccharide was synthesised
Sequential regioselective glycosylation of a GlcNAc acceptor with galactose and fucose donors
provided the trisaccharide
The efficient total synthesis of LewisX was completed

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Supporting Information
Total synthesis of LewisX using a late-stage crystalline intermediate
Stefan Munneke,a Gavin F. Painter,b Graeme J. Gainsford,c Bridget L. Stocker,a,* and Mattie S.
M. Timmera,*
a
School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600,
Wellington, New Zealand
b Ferrier Institute, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
c
Callaghan Innovation, PO Box 31310, Lower Hutt, New Zealand
Email: mattie.timmer@vuw.ac.nz, bridget.stocker@vuw.ac.nz
Table of content
Benzyl 2-acetamido-2-deoxy-α-D-glucopyranoside (2) ....................................................SI2
Benzyl 2-acetamido-6-O-tert-butyldiphenylsilyl-2-deoxy-α-D-glucopyranoside (3) .........SI4
Benzyl
2-acetamido-4-O-(2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranosyl)-2-
deoxy-6-O-tert-butyldiphenylsilyl-α-D-glucopyranoside (10) ....................................SI6
Benzyl 2-acetamido-4-O-(2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranosyl)-3-O-
(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-2-deoxy-6-O-tert-butyldiphenylsilyl-α-D-
glucopyranoside (11) ..............................................................................................SI8
Benzyl
2-acetamido-4-O-(4,6-O-benzylidene-β-D-galactopyranosyl)-3-O-(2,3,4-tri-O-
benzyl-α-L-fucopyranosyl)-2-deoxy-α-D-glucopyranoside (12) ...............................SI10
2-Acetamido-2-deoxy-3-O-(α-L-fucopyranosyl)-4-O-(β-D-galactopyranosyl)-α-D-
glucopyranoside (LeX) ...........................................................................................SI12
SI1

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`
SI2

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SI3

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SI4

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SI5

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SI6

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SI7

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SI8

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SI9

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SI10

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SI11

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SI12