Kilogram scale chemical synthesis of 2′-fucosyllactose

Article abstract of DOI:10.1016/j.carres.2019.03.006

A scalable synthetic procedure to high quality 2′-fucosyllactose, the most abundant oligosaccharide in human breast milk, has been designed and validated in kilogram scale. The synthetic route has been developed to suit industrial environment and contains

Full text of DOI:10.1016/j.carres.2019.03.006

Accepted Manuscript
Kilogram scale chemical synthesis of 2′-fucosyllactose
Karoly Agoston, Markus Jondelius Hederos, Istvan Bajza, Gyula Dekany
PII:
S0008-6215(19)30068-0
https://doi.org/10.1016/j.carres.2019.03.006
CAR 7688
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 29 January 2019
Revised Date: 12 March 2019
Accepted Date: 14 March 2019
Please cite this article as: K. Agoston, M.J. Hederos, I. Bajza, G. Dekany, Kilogram scale chemical
synthesis of 2′-fucosyllactose, Carbohydrate Research (2019), doi: https://doi.org/10.1016/
j.carres.2019.03.006.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Kilogram scale chemical synthesis of 2’-
fucosyllactose
Karoly Agostona*, Markus Jondelius Hederos*, Istvan Bajzab and Gyula Dekanyc
Glycom A/S, Kogle Alle 4, DK-2970 Hørsholm, Denmark
KEYWORDS: oligosaccharide synthesis, carbohydrates, Human milk oligosaccharides,
fucosylation, fucosyllactose
ABSTRACT: A scalable synthetic procedure to high quality 2`-fucosyllactose, the most
abundant oligosaccharide in human breast milk, has been designed and validated in kilogram
scale. The synthetic route has been developed to suit industrial environment and contains only a
single chromatographic purification step.
1.
INTRODUCTION
A correlation between infant health and breastfeeding has been undoubtful since the very first
use of human milk substitutes developed by Justus von Liebig [1]. The health benefits of natural
human milk over the synthetic substitutes are claimed to be the reduced number of bacterial and
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viral infections in the breastfed infants. The specific protecting agents in human milk responsible
for these effects are considered to be multiple such as secreted antibodies [2], enzymes such as
lactoferrin or lysozyme [3] and/or oligosaccharides, so-called human milk oligosaccharides
(HMOs) [4]. The mode of action of these oligosaccharides are both their prebiotic activity[5]
and ability to bind to the host-cell receptors of the pathogens inhibiting the adhesion onto the
host cells [6]. To date more than 140 different HMOs have been identified in human milk and
this number is still increasing [7]. One of the most important HMOs is 2’-fucosyllactose (2’-FL),
generally accepted as the HMO in highest concentration in mothers milk [8]. Several biological
effects of 2’-FL have been suggested including but not limited to its prebiotic-, antibacterial- and
antiviral effects [6] - making it an attractive target for nutritional applications. Since the
publication [9] of the first chemical synthesis of 2’-FL several other chemical-, [10] chemo-
enzymatic- [11] and enzymatic [12] approaches have been published, all together suffering from
the limitation to offer a feasible route for large-scale synthesis of 2’-FL. In principle, all
chemical and chemo-enzymatic synthesis contains multiple chromatographic purifications,
which makes them unsuitable beyond laboratory scale. Sole enzymatic syntheses require the
UDP sugars as glycosyl donors or genetically modified organisms as “catalyst” for the syntheses.
Neither of these possibilities provides an unambiguous solution yet although, certain
biotechnological methods have recently proven to be commercially attractive solutions [13].
However, chemical synthesis can provide suitable amount target compound within short time
period if scale-up difficulties can be solved. Furthermore, chemical synthesis provides higher
purity product for biological studies including well-defined impurity profile. In the chemical
synthesis of 2’-FL the two most critical problems are purification of intermediates by column
chromatography and the final isolation of the end-product.
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Typically, in the literature wherein articles are claiming “large scale” synthesis of an
oligosaccharide, the quantities of the target compound in the ends only milligrams (enzymatic
methods [14]) or in max. ~10 g scale (chemical methods [15]). Within our earlier program and
efforts to access high purity and large quantities (> 1 kg) of 2’-FL to drive pre-clinical / clinical
programs and regulatory processes - we have extensively investigated the possibilities of the
chemical synthesis of 2’-FL in kilogram scale. Similar to the results described herein has - to our
best knowledge - never been published providing the targeted oligosaccharide in such a scale and
purity.
2.
RESULTS AND DISCUSSION
As starting point, our synthetic plan for the preparation of the glycosyl acceptor relied on an
earlier published route [10b] where a lactose acceptor can be generated in two steps from
commercially available lactose. In contrast to the 1-O-nitrophenyl fucose donor used for the
synthesis [10b] we have selected 1-S-phenyl donor which can be readily prepared from L-fucose
in four steps (Figure 1). One key focus area was to identify a 6’-O protecting group on the
lactose acetonide acceptor with the ambition to obtain a crystalline acceptor with the option of
purification of the crude glycosyl acceptor before the glycosylation step in large scale. Numerous
different acyl- and alkyl groups (including but not limited to Ac, Bz, Piv, Lev, ClAc, Bn, Tr)
were tried, but none of them provided a crystalline lactose acceptor. Finally and as a
compromise, the choice fall on the 6’-O-pivaloate ester since it could be introduced with very
high selectively (6’ >> 2’) and opened up the possibility of the use of the crude acceptor (3, R=
Piv) directly in the glycosylation, even though purification by crystallization was not performed.
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D-lactose
2 steps
O
O
OR'
3 steps
O
SR
OBn
O
O
L-fucose
4 steps
O
OMe
OMe
O
BnO
OBn
O
+
OH
O
8 R = Ph
3 R' = Piv
OH
OH
OH
OH
HO
HO
O
O
O
O
O
O
OH
HO
HO
OH
HO
HO
O
OH
O
OH
O
12
11
O
OBn
OH
BnO
HO
OH
OBn
Crystalline, key derivative for purification
Figure 1. The synthetic route to 2’-FL
On the donor side several crystalline fucosyl donors have been published but in our hands - the
thiophenyl derivative (8, R= Ph) was providing the best yields. For the activation of the glycosyl
donor the traditional Lemieux`s method was selected because of the requirement of mild
glycosylation reaction conditions due to the acid labile protecting groups. Direct activation of the
donor using thiophyl promoters were considered, but not implemented at this stage. After the
glycosylation three different type of protecting groups needed to be removed, i.e. acetals, esters
and benzylic groups. From literature [9] it is known that one of the possible final intermediates,
compound (11), is a crystalline compound. The glycosylation product compound (9) and
partially deprotected compound (10) (See them below in Scheme 3) turned out not to be
crystalline – even after purification and isolation by flash chromatography these two compounds
failed to be crystallized. We anticipated that performing three consequently steps in a row (i.e.
glycosylation, basic ester- and acidic acetal cleavages) and purifying afterwards would be very
challenging, but not impossible due to the big difference in the polarity of the compounds in the
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crude mixture. For example, all carbohydrate impurities derived from the fucosyl donor should
still be very apolar due to the three benzyl groups, while the carbohydrate contaminants from the
acceptor molecule would be very polar upon losing all the protecting groups. In that manner we
assumed the polarity of the trisaccharide derivative (11) to be significantly different from above
mentioned impurities which were targeted to be removed by means of extractive work-up.
Another impurity to be eliminated is the β-2’-anomer of 2’-FL that can be formed during
glycosylation and would render the β-2’ stereoisomer of (11) after deprotection. The
glycosylation method chosen is known to give about 10% beta isomer [16], which possibly could
be removed at the stage of crystallization of final intermediate (11). Final product (12) has been
described as a solid when isolated from natural sources [17] and there is no reason to believe that
the synthetic product would be different.
2.1. Synthesis of lactose acceptor
For the preparation of the glycosyl acceptor anhydrous lactose was used as a starting material in
laboratory scale. In one step all hydroxyl functions but two were protected as acetonide ketal, or
as dimethyl-acetal (1→2, Scheme 1). Compound 2 was isolated from the crude reaction mixture
by crystallization with the yield of 65%. Upon execution in industrial scale a few parameters
were revised; lactose monohydrate was used as starting material for the reaction due to the
availability and price figure of anhydrous lactose. Furthermore, the concentration was increased
by two-fold and the solvent used for the crystallization was changed from diethyl ether into
MTBE as safety precaution. With these process adaptions the overall yield of the reaction
dropped by 13 %, but still acceptable (52%).
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Scheme 1. Preparation of the glycosyl acceptor.
a) 2,2-Dimethoxypropane, pTsOH, reflux; b) Pivaloyl chloride, pyridine, DCM, -5 °C.
As discussed earlier, the pivaloyl ester was used to protect the 6’ hydroxyl function of the lactose
acetonide. The reaction was carried out in DCM using pyridine as base and pivaloyl chloride as
acylating agent. Compound 3 was purified by column chromatography both in laboratory- and
pilot scale (1 kg). In order to investigate the industrial feasibility of the whole process, a parallel
experiment was done in pilot scale (~500 g) whereupon compound 3 was not purified by column
chromatography using the crude reaction mixture directly in the glycosylation. This test batch
was processed further to the final intermediate stage 11 to see the impact on eliminating the only
chromatographic purification in the whole production, e.g. recovery yield, final intermediate
product purity and process parameters such as extraction work-up performance.
2.2. Synthesis of fucosyl donor
The preparation of the glycosyl donor was done in four steps from commercially available L-
fucose (Scheme 2). Both in laboratory and pilot scale the first three steps were done in “one pot”.
Conventional acetylation (4→5) followed by the introduction of the thiophenyl leaving group
(5→6) and the catalytic removal of the acetate esters (6→7) were done in the same way both in
laboratory and pilot scale. Only the solvent used for the crystallization of compound 7 was
changed but had no significant influence on the product recovery yield.
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Scheme 2. Preparation of the glycosyl donor.
a) Ac2O, pyridine, 0 °C; b) HSPh, BF3/Et2O, DCM, 0 °C; c) NaOMe, MeOH; d) BnBr, NaH,
DMF; or BnBr, KOtBu, THF, 10 °C.
The benzylation of compound 7 was performed in DMF using NaH as base and benzyl bromide
as alkylating agent in laboratory scale. The glycosyl donor (8) was isolated by crystallization
from the mixture of Et2O and hexane. In pilot scale the base has been changed to KOtBu and the
solvent to THF resulted in safer reaction conditions. The crystallization of the product was
achieved from a mixture of EtOAc, MeOH and water allowing further simplification on the
work-up procedure. The isolated yield of compound 8 in pilot scale was not significantly lower
than in laboratory scale with the advantage of avoiding the use of sodium hydride [72% vs 67%].
2.3. Glycosylation and deprotection to final intermediate
For all the glycosylations conducted in pilot scale only crystalline donor compound 8 was used.
In terms of the preparation of acceptor, two different qualities of acceptor were used. In all
glycosylations conducted but one, the flash chromatographed and purified acceptor 3 was used.
In one case and in parallel, the crude and non-purified (only extractive work-up) was used in the
glycosylation with donor 8. Typically, in a glycosylation sequence, compounds 9 and 10 – as
being syrups – were only isolated in small scale for analytical purposes. At the stage of
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compound 11 the preparative purification was conducted and the overall yields were determined.
It was also at this stage the yields of using different quality of acceptors were compared. As a
result of the low yield of using crude acceptor for the glycosylation only the experiment using
the purified acceptor was scaled up to kg level.
In Scheme 3 is depicted the conditions for the glycosylation and deprotection to final end-
product 2’-FL. The overall yield of the three steps procedure from glycosylation to compound 11
was 27.3% in the scale of 600 g of compound 3 or 19.8% in the scale of 5.0 kg of compound 3 In
both cases the acceptor was purified by column chromatography before the glycosylation step.
This equals to the yield of ~70% per step, which was acceptable considering the difficult
glycosylation step. In case of using non-purified acceptor (3c) the isolated yield of compound 11
dropped to 8.5% over 4 steps (including the pivaloylation step). This means that the average
yield is down to 54% per step, which was below our expectation. Clearly it would be a huge
advantage to eliminate all chromatographic purifications, but the price paid for that is too high in
this process. At this stage of the scale-up we still wanted to keep the last remaining
chromatographic purification as a part of the overall synthetic procedure.
Scheme 3. Synthesis of 2`-FL.
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a) i: Br2, DCM, 5 °C, ii: TBABr, DMF, rt.; b) NaOMe, MeOH; c) AcOH, H2O, 65 °C; d) Pd/C,
H2, IPA, MeOH, AcOH, water.
The reaction conditions used for all experiments (glycosylations and deprotections) were
identical. First, compound 8 was dissolved in DCM and converted into bromosugar by adding
bromine at 5 °C in DCM. The excess of Br2 was neutralized by the addition of cyclohexene to
the mixture. The solution of acceptor (3) and tetrabutylammonium bromide in DMF were added
to the crude solution of the bromosugar. The mixture was stirred for 2 days and after work-up
procedure the pivaloate ester was removed in MeOH using NaOMe, followed by the cleavage of
the acetonide groups and the dimethyl-acetal with 60% acetic acid in water at elevated
temperature. All the impurities derived from the acceptor were removed in this stage by
extraction. Compound 11 was isolated by crystallization from EtOAc and purified further by re-
crystallization from MeOH. The final intermediate was isolated with the yield of 67-73% per
step by using the chromatographed acceptor whilst the use of “crude” acceptor resulted in 54%
yield per step. No beta anomer (2’-β-fucosyl-lactose) was identified in either of the final
intermediates by NMR or HPLC methods.
2.4. Final deprotection and isolation of targeted compound
The final deprotection step has been done in the mixture of isopropanol/methanol/water/acetic
acid to provide solubility for both the starting compound and for the product. The benzyl groups
were removed by catalytic hydrogenation using Pd/C and H2. During the scale-up the
concentration of the reaction mixture was increased, but that resulted in significantly slower
reaction. Most probably the large amount of toluene being formed in the reaction afforded the
aggregation of the catalyst and addition of fresh catalyst was necessary to drive the reaction to
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completion. The final compound was isolated by precipitation using TBME in methanol. In Pilot
scale a trituration in isopropanol was added to the procedure at the final stage to remove the
solvent residues. 2`-Fucosyllactose was isolated in kilogram scale as a white amorphous solid
and fully characterized by NMR and analyzed by HPLC and LCMS.
3.
CONCLUSION
A kilogram scale preparation of 2`-fucosyllactose has been performed by synthesis of a fucosyl
donor in ~10 kg scale and a lactose acceptor in ~1 kg scale. The developed route involves only
one purification step using column chromatography contrary to the previously published routes
which involve 4 to 8 chromatographic purification for the same target. At intermediate scale the
synthesis has been conducted omitting chromatographic purification, but the overall yield turned
out to be too low. For the glycosylation Lemieux`s method has been selected to provide high
selectivity under mild reaction condition. The purification by crystallization of the final
intermediate was the key for the success of synthesis. After the final hydrogenation step the
product was isolated as an amorphous solid with high purity. The developed and presented
procedure could provide the baseline for the synthesis of 2’-fucosyllactose in industrial scale.
4.
EXPERIMENTAL
4.1. General Methods: Commercially available starting materials were used without further
purification. Solvents were dried according to standard methods. Pilot scale synthesis for
compounds 2, 3 and 9-12 were carried out in ERCOM Kft, Hungary and for compounds 5-8
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were carried out in NITRO Kft, Hungary. Both places the reactions were performed in 20-50 L
double jacketed glass reactors except compound 2 which was prepared in 3000 L enameled
reactor. NMR spectra were recorded on a Bruker AMX-400 (100.62 MHz for 13C) or DRX-500
(125.83 MHz for 13C) spectrometer using CDCl3, DMSO-d6, and D2O as solvents. All chemical
shifts are quoted in ppm downfield using solvent peaks as references (CDCl3: 1H: 7.26 ppm, 13C:
77.1 ppm, DMSO-d6: 1H: 2.50 ppm, 13C: 39.4 ppm, D2O: 1H: 4.79 ppm). Kieselgel 60 (E. Merck,
Darmstadt, Germany) was used for column chromatography. Optical rotations were measured on
-1
25
a Perkin Elmer 241 polarimeter and the concentrations are given in units of g 100 mL . [α]
D
values are given in units of 10-1 deg cm2 g-1
4.2. Compound 2
4.2.1 Laboratory scale: p-Toluenesulphonic acid monohydrate (3.0 g, 16 mmol) was added to a
suspension of anhydrous lactose (100 g, 0.29 mol) in 2,2-dimethoxypropane (700 mL). The
mixture was heated up to reflux and stirred at reflux temperature for 1½ h. The mixture was
cooled to RT and evaporated quickly at 40°C. The residue was dissolved in DCM (1 L) and
washed thoroughly with water (4 × 500 mL) and with NaHCO3 solution (saturated, 500 mL),
dried (MgSO4), filtered and evaporated in vacuo to give a yellow oil. The residue was dissolved
in 230 ml Et2O and a seeding crystal was added, left at 5°C overnight to crystallize. The crystals
were filtered and washed with cold Et2O (2 × 50 mL) to give 96.5 g white solid (yield: 65 %).
4.2.2 Industrial scale: Lactose monohydrate (328 kg, 0.91 kmol) was added to a solution of
pTsOH (3.2 kg, 16.8 mol) in 2,2 dimethoxy-propane (975 kg). The suspension was warmed up to
reflux temperature (85 oC mantle, 63.2 oC inner) and after this temperature was reached the
mixture was stirred for 2 h. at reflux. Then the mixture was cooled down to 20-25 oC (inner
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temperature) and at that temperature the volatile components were distilled off in vacuo. (614 kg
solvents were collected). DCM (2160 kg) was added to the residue and extracted with water (3 ×
1000 kg) then with a solution of NaHCO3 (70 kg) in water (1000 kg) and finally with water
(1000 kg). The organic phase was concentrated in vacuo. (1595 kg solvents were collected, at 40
°C mantle temperature). To the residue MTBE (370 kg) was added and the mixture was
homogenized at 25 °C than cooled down to 0 °C (inner temperature) and stirred in this
temperature for 8 h. The formed crystals were separated by centrifugation (each centrifuge batch
was washed with 14 kg MTBE) affording 243 kg (yield: 52%) dry crystals. mp 131-133 °C, lit
[18] mp 133-134 °C; [α] D +38.8 (c 1, CHCl3), lit [18] [α] +39.1 (CHCl3); 1H NMR. (CDCl3,
25
25
D
400 MHz) δ: 4.56 (dd, 1 H, J1,2 6.7 Hz, J2,3 8.0 Hz, H-2), 4.39 (d, 1 H, J1´,2´ 8.3 Hz, H-1´), 4.33
(d, 1 H, H-1), 4.28 (m, 1 H, H-5´), 4.16 and 3.98 (each m, 2 H, H-6), 4.02 (m, 1 H, H-5), 3.94
(m, 1 H, H-4´), 3.92 and 3.65 (each m, 2 H, H-6`), 3.86 (m, 1 H, H-3), 3.74 (m, 1 H, H-4), 3.48
(m, 1 H, H-3´), 3.47 (m, 1 H, H-2´), 3.45 and 3.45 (2 s, each 3 H, 2 × OMe), 1.46, 1.46, 1.35,
1.35, 1.28 and 1.27 (6 s, each 3 H, 6 × -CH3). 13C NMR (CDCl3, 100 MHz) δ: 110.4, 109.8 and
108.2 (3 × -C(CH3)2), 107.0 (C-1), 103.3 (C-1´), 79.3 (C-5), 78.1 (C-3), 77.4 (C-5´), 75.7 (C-4´),
75.2 (C-2), 74.5, 74.0 and 73.4 (C-4, C-2´and C-3´), 64.4 and 62.3 (C-6 and C-6´), 57.4 and 54.3
(2 × OMe), 28.0, 27.0, 26.1, 26.1, 25.5 and 23.8 (6 × -CH3). Anal. Calcd for C23H40O12: C,
54.32; H, 7.93. Found: C, 54.37; H, 7.91.
4.3. Compound 3
4.3.1. Laboratory scale: Pyridine (16.4 mL, 202 mmol) was added to a solution of compound 2
(15 g, 29.5 mmol) in DCM (105 mL). The mixture was cooled down to -5 °C and pivaloyl
chloride (5.7 mL, 46.3 mmol) in DCM (5 mL) was added and stirred for 15 h at -5 °C. MeOH (6
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mL) was added to the mixture and stirred for 30 min. then extracted with water (75 mL), 1 N
HCl solution (2 × 75 mL), water (75 mL) and with NaHCO3 solution (75 mL). The organic phase
was dried, filtered and concentrated. The crude mixture was purified by column chromatography
(PE:EtOAc, 3:2) giving 13.8 g white foam (3a, 79%). [α] +35.4 (c 0.1, CHCl3); 1H NMR
25
D
(CDCl3, 400 MHz): δ: 4.44 (d, 1 H, J1`,2` 8.2 Hz, H-1`), 4.41 (dd, 1 H, J2,3 7.3 Hz, H-2), 4.36 (d,
1 H, J1,2 6.0 Hz, H-1), 4.31 (dd, 1H, J5,6 6.8 Hz, Jgem 11.2 Hz, H-6`), 4.28 (m, 1 H), 4.24 (dd, 1
H, J5`,6` 6.3 Hz, Jgem 11.1 Hz, H-6`), 4.17 - 4.03 (m, 4 H), 3.99 (dd, 1 H, H-6), 3.95 (ddd, 1 H),
3.91 (dd, 1 H), 3.55 (dd, 1 H), 3.43, 3.42 (2 s, each 3 H, 2 × OMe), 1.50, 1.48, 1.37, 1.37, 1.32,
1.31 (6 s, each 3 H, 6 × -C(CH3)2), 1.20 (s, 9 H, OPiv). 13C NMR (CDCl3, 100 MHz): δ = 178.0
(C=O), 110.1, 110.1 and 108.2 (3 × -C(CH3)2), 104.9 and 103.4 (C1 and C-1’), 78.7, 77.8, 77.7,
77.6, 75.9, 74.9, 74.1 and 72.9 (C-2, 3, 4, 5, 2’, 3’, 4’ and 5’), 64.5 and 62.7 (C-6 and 6’) 56.2
and 53.1 (2 × OMe), 38.6, 28.0, 27.1, 27.0, 26.3, 26.1, 25.6, 24.4 and 24.3 (9 × -CH3). Anal.
Calcd for C28H48O13: C, 56.74; H, 8.16. Found: C, 56.81; H, 8.19.
4.3.2. Pilot scale with purification: Pyridine (1.64 L, 20.2 mol) was added to a solution of
compound 2 (1.5 kg, 2.95 mol) in DCM (10 L). The mixture was cooled down to -5 °C and
pivaloyl chloride (570 mL, 4.63 mol) in DCM (500 mL) was added and stirred for 15 h between
0 and -5 °C. MeOH (600 mL) was added to the mixture and stirred for 30 min then extracted
with water (10 L), 3 N HCl solution (10 L), water (10 L) and with NaHCO3 solution (10 L). The
organic phase was dried, filtered and concentrated. The crude mixture was purified by column
chromatography (PE:EtOAc, 7:3, on 10 kg silicagel) giving 1.1 kg white foam (3b, 63%)
4.3.3. Pilot scale without purification: Compound 2 (515g, 1.01 mol) was dissolved in dry
DCM (2.5 L) and cooled to -5oC. Then pyridine (556 mL, 6.85 mol) was added at -5oC. To this
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solution pivaloyl chloride (200 mL, 1.62 mol) was added over a period of 2.5 h at -5oC
(exotermic). During the addition the clear solution turned to an off white suspension. The
mixture was stirred at -5oC for 6 h, then warmed up to 20 oC, and stirred for 12 h. Then the
mixture was cooled to 0 oC, and methanol (200 mL) was added slowly while the suspension
turned to clear solution. Then the mixture was allowed to warm up to 20 °C and stirred for 40
min then washed with water (2 × 1.3 L), 1 N HCl (3 × 1.3 L), water (2 × 1.2 L), sat. NaHCO3
(1.5 L) and water (1.5 L). Then the mixture was concentrated to dryness and dry DMF (1.2 L),
TBABr (23 g) and toluene (0.5 L) were added and again concentrated to remove the toluene.
This mixture (3c) was used without any further purification for glycosylation later.
4.4. Preparation of Compound 8 from 4.
4.4.1. Laboratory Scale: L-Fucose (4) (4.23 g, 25.7 mmol) was suspended in pyridine (20 mL).
The mixture was cooled down to 0 °C and Ac2O (20 mL) was added over a period of 30 min.
then allowed to warm up to r.t. and stirred for 10 h. The mixture was concentrated and the
residue was dissolved in DCM (150 mL) and washed with 1 N HCl (2 × 100 mL), water (75 mL)
and with NaHCO3 solution (75 mL). The organic phase was dried, filtered and concentrated. The
crude residue (5, 8.45 g, colourless oil) was used for the next reaction.
Thiophenol (3.2 g, 29 mmol) was added to the solution of crude compound 5 (8.45 g, 25.4
mmol) in DCM (20 mL). The mixture was cooled down to 0 °C and BF3/Et2O (9.45 mL, 76.6
mmol) was added over a period of 10 min. The mixture was stirred for 2 h at 0 °C and 10 h at 5
°C. Then the mixture was diluted with DCM (200 mL) and washed with water (2 × 100 mL) and
NaHCO3 solution (100 mL). The organic phase was dried, filtered and concentrated affording 9.8
g crude product (6) as a pale yellow oil.
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To the solution of the crude compound 6 (9.8 g) in MeOH (100 mL) NaOMe (150 mg) was
added and the mixture was stirred for 6 h at 40 °C. Amberlite IR 120 (H+) resin was added,
stirred and the resin was filtered off. The methanol was removed in vacuo affording the crude
product (6.25 g, yellow foam). The foam was dissolved in MeCN (30 mL) and washed with
petrolether (3 × 60 mL) then the MeCN phase was concentrated and re-dissolved in warm EtOH
(50 mL) followed by slow addition of petrolether (200 mL). The suspension was cooled to 0 °C
and the formed crystals (7) were separated by filtration. (4.03 g, 61%, over 3 steps).
NaH (5.26 g, 109 mmol, 50% in mineral oil) was washed with hexane and suspended in dry
DMF (10 mL) then the mixture was cooled down to 0 °C keeping the system all times under Ar.
Slowly, over a period of 10 min, a solution of compound 7 (6.2 g, 24.5 mmol) in DMF (20 mL)
was added followed by the addition of BnBr (13 mL, 109 mmol). The ice bath was removed and
the mixture was stirred under Ar for 10 h. The mixture was cooled to 0 °C and MeOH (8 mL)
was added over a period of 10 min. The mixture was stirred for 30 min, then concentrated. The
residue was dissolved in DCM (150 mL) and washed with water (3 × 75 mL). The organic phase
was dried, filtered and concentrated. The product was isolated by crystallization from the
mixture of Et2O and hexane (18 mL, each) affording a white solid (8, 9.1 g, 72%).
4.4.2. Pilot Scale: L-Fucose (4) (5 kg, 30.5 mol) was suspended in pyridine (12 L) in a 35 L
glass reactor. The suspension was cooled down to 0 − 5 °C and Ac2O (12 L) was added over a
period of 3 h. The cooling was stopped and the mixture was allowed to warm up. (The
temperature raised to 30 °C over a period of 2 h, then slowly cooled down to 21 °C. (ambient
temperature)) The mixture was concentrated on a 20 L rota. The reaction was repeated with a
same batch-size once more and the two residues after concentration were combined and
15

ACCEPTED MANUSCRIPT
dissolved in DCM (50 L) and washed with 1 N HCl (2 × 20 L), water (20 L) and with NaHCO3
solution (20 L). The organic phase was dried (MgSO4) and filtered resulted in a 60 L paled
yellow solution which was used for the next reaction without any further treatment.
Thiophenol (7.8 L, 75.9 mol) was added to the solution of crude compound 5 (60 L pale yellow
solution in DCM, estimated 61 mol) in a 400 L enamelled reactor. The mixture was cooled down
to 0 °C and BF3/Et2O (23 L, 186 mol) was added over a period of 3 h. The mixture was stirred
for 8 h keeping the temperature between 0 − 5 °C. Then the mixture was washed with water (2 ×
40 L), 1 N NaOH solution (2 × 40 L) and NaHCO3 solution (1 × 20 L). The organic phase was
dried, filtered and concentrated affording 25 kg crude product (6, yellow oil).
To the solution of the crude compound 6 (25 kg) in MeOH (40 L) NaOMe (300 mL, 30 %
solution in MeOH) was added and the mixture was stirred for 6.5 h at 21 °C. Amberlite IR 120
(H+) resin (5 kg) was added, stirred and the resin was filtered off. The methanol was removed in
vacuo affording the crude product (14.3 kg, yellow foam). The foam was dissolved in warm
EtOH (60 L) followed by slow addition of petrolether (70 L). The suspension was cooled to 0 °C
and the formed crystals were separated by filtration (8.2 kg). The mother liquor was concentrated
and crystallized from EtOH (18 L) and petrolether (14 L) affording 2.3 kg product (7). Total
yield: 10.5 kg, 67.2%, over 3 steps.
KOtBu (16 kg, 142.6 mol) was added to a solution of compound 7 (8.2 kg, 32 mol) in THF (60
L) over a period of 3 h in a 400 L enamelled reactor at 0 − 5 °C. After the addition was finished
the mixture was stirred for 1 h then BnBr (23.04 kg, 134.8 mol) was added over a period of 4 h
keeping the temperature below 5 °C. After the addition was finished the temperature was raised
to 10 °C and stirred for 16 h at 10 °C. The mixture was cooled to 0 °C and MeOH (10 L) was
16

ACCEPTED MANUSCRIPT
added and the mixture was stirred for 1 h. The mixture was diluted with EtOAc (60 L) and
washed with water (2 × 60 L). To the organic phase MeOH (75 L) was added followed by the
addition of water (48 L). The formed crystals were separated by filtration (11.2 kg, white solid,
25
D
25
D
67%) mp: 106-108 °C, [α] -13.8 (c 1, CHCl3) mp: lit [19] 107-109 °C, [α] lit [19] -14.0 (c
0.7, CHCl3) 1H NMR (CDCl3, 400 MHz): δ: 7.60-7.18 (m, 20 H, aromatics), 4.83, 4.76 and 4.73
(each ABq, each 2 H, 3 -CH2Ph), 4.60 (d, 1H, J1,2 7.8 Hz, H-1), 3.93 (dd, 1 H, J2,3 9.5 Hz, H-2),
3.68 (dd, 1 H, J4,5 <1 Hz, H-4), 3.59 (dd, 1 H, J3,4 2.8 Hz, H-3), 3.52 (m, 1H, H-5), 1.26 (d, 3 H,
H-6). Anal. Calcd for C33H34O4S: C, 75.83; H, 5.79. Found: C, 75.58; H, 5.76.
4.5. Compound 9, Glycosylations:
4.5.1. Experiment 1 using 600 g purified acceptor: Compound 8 (600 g, 1.14 mol) was
dissolved in dry DCM (2 L) and the solution was cooled down to 5 oC (Inner temperature, the
cooling liquid was adjusted to 0 oC). To the solution Br2 (73 mL, 1.42 mol) in dry DCM (200
mL) was added keeping the temperature between 5 and 10 oC over a period of 20 min. After the
addition was finished the mixture was stirred for 10 min then cyclohexene (130 mL) was added
to the mixture over a period of 20 min. keeping the temperature between 5 and 10 oC. The dark
red solution turned to light yellow solution. To this crude solution of the bromosugar, compound
3 (600g, 1.01 mol) and TBABr (25 g, 77.5 mmol) in DMF (1.2 L) was added over a period of 30
min, then the cooling was removed and the solution was stirred for 48 h at 20 oC. The mixture
was diluted with toluene (2 L) and washed with NaHCO3 solution (saturated, 2 × 3 L) and with
water (3 L). The organic phase was concentrated affording 2 L yellow oil. The crude mixture
(9a) used for the next reaction without any further purification.
17

ACCEPTED MANUSCRIPT
4.5.2. Experiment 2 using 5 kg purified acceptor: Compound 8 (5.0 kg, 9.49 mol) was dissolved
in dry DCM (20 L) and the solution was cooled down to 5 oC (Inner temperature, the cooling
liquid was adjusted to 0 oC). To the solution Br2 (500 mL, 9.72 mol) in dry DCM (200 mL) was
added keeping the temperature between 5 and 10 oC over a period of 1 h. After the addition was
finished the mixture was stirred for 10 min then cyclohexene (820 mL) was added to the mixture
over a period of 1 h. keeping the temperature between 5 and 10 oC. The dark red solution turned
to light yellow solution. To the crude solution of the bromosugar, compound 3 (5.0 kg, 8.42 mol)
and TBABr (250 g, 775 mmol) in DMF (12 L) was added over a period of 3 h, then the cooling
was removed and the solution was stirred for 48 h at 22 oC. The mixture was diluted with toluene
(20 L) and washed with NaHCO3 solution (saturated, 2 × 30 L) and with water (30 L). The
organic phase was concentrated affording 17 L yellow oil (9b). The crude mixture used for the
next reaction without any further purification.
4.5.3. Experiment 3 using 495 g crude acceptor: Compound 8 (465 g, 0.88 mol) was dissolved
in dry DCM (1.85 L) and the solution was cooled down to 5 oC (Inner temperature, the cooling
liquid was adjusted to 0 oC). To the solution Br2 (46 mL, 0.89 mol) in dry DCM (185 mL) was
added keeping the temperature between 5 and 10 oC over a period of 20 min. After the addition
was finished the mixture was stirred for 10 min then cyclohexene (76 mL) was added to the
mixture over a period of 20 min. keeping the temperature between 5 and 10 oC. The dark red
solution turned to light yellow solution. To this crude solution of the bromosugar, compound 3c
(crude from experiment Pilot scale without purification starting from 515 g of compound 2) and
TBABr (20 g, 62 mmol) in DMF (1.2 L) was added over a period of 30 min, then the cooling
was removed and the solution was stirred for 48 h at 22 oC. The mixture was diluted with toluene
(2 L) and washed with NaHCO3 solution (saturated, 2 × 3 L) and with water (3 L). The organic
18

ACCEPTED MANUSCRIPT
phase was concentrated affording 2 L yellow oil. The crude mixture (9c) used for the next
reaction without any further purification.
25
D
200 mg of the sample (from experiment 2) was purified for analysis: [α] -42.0 (c 1, CHCl3),
1H NMR. (CDCl3, 400 MHz) δ: 7.45-7.25 (m, 15 H, aromatic), 5.60 (d, 1 H, J1´´,2´´ 3.2 Hz, H-
1´´), 4.98 and 4.65 (ABq, 2 H, Jgem 11.6 Hz, -CH2Ph), 4.88 and 4.74 (ABq, 2 H, Jgem 12.0 Hz, -
CH2Ph), 4.76 (s, 2 H, -CH2Ph), 4.65 (d, 1 H, J1´,2´ 8.2 Hz, H-1´), 4.48 (dd, 1 H, J1,2 6.0, J2,3 7.4
Hz, H-2), 4.38 (d, 1 H, H-1), 4.29 (m, 2 H, H-6), 4.24 (m, 1 H, ), 4.19 (m, 1 H, H-3´), 4.08 (m, 2
H, H-4´ and H-5´), 4.08 and 3.88 (2 m, each 1 H, H-6´), 4.06 (m, 2 H, H-2´´ and H-3), 3.98 and
3.91 (2 m, each 1 H ) 3.72 (dd, 1 H, H-2´), 3.66 (bs, 1 H, ), 3.43 (2 s, each 3 H, 2 × OMe), 1.48,
1.42, 1.37, 1.36, 1.31 and 1.28 (6 s, each 3 H, 6 × -CH3), 1.21 (s, 9 H, Piv), 1.10 (d, 3 H, J5´´,6´´
6.46 Hz, H-6´´). 13C NMR (CDCl3, 100 MHz) δ: 178.2 (CO), 110.4, 110.1 and 108.8 (3 × -
C(CH3)2), 105.3 (C-1), 101.6 (C-1´), 95.3 (C-1´´), 80.4 (C-3´), 79.4, 78.2, 77.8, 77.8, 76.6, 75.6,
74.2, 72.8, 72.8, 70.6 and 66.6 (C-2, C-3, C-4, C-5, C-2´, C-4´, C-5´, C-2´´, C-3´´, C-4´´ and C-
5´´), 75.0, 73.6 and 73.4 (3 CH2Ph), 65.3 and 62.6 (C-6 and C-6´), 56.3 and 53.0 (2 × OMe), 39.0
(Piv), 28.1, 27.5, 27.4, 27.4, 27.3, 27.1, 27.0, 26.6 and 24.6 (9 × -CH3), 17.1 (C-6´´).
4.6. Compound 10
4.6.1. Experiment 1, using the crude product from 4.5.1.: NaOMe (120 g, 2.22 mol) was added
to a solution of compound 9 (9a, made from 1.01 mol compound 3) in dry MeOH (1.8 L) and
stirred for 18 h at 20 oC. (The yellow solution became turbid after 2 h.) Amberlite IR 120 resin
(H+ form, 600 g) was added to the mixture and stirred for 30 min then the resin was filtered off
and the filtrate was concentrated affording 1 L dark yellow slurry (10a). The mixture was used
for the next step without any purification.
19

ACCEPTED MANUSCRIPT
4.6.2. Experiment 2, using the crude product from 4.5.2.: NaOMe (850 g, 15.73 mol) was added
to a solution of compound 9 (9b, made from 8.42 mol compound 3) in dry MeOH (5 L) and
stirred for 18 h at 21 oC. (The yellow solution became turbid after 5 h.) Amberlite IR 120 resin
(H+ form, 5 kg) was added to the mixture and stirred for 30 min then the resin was filtered off
and the filtrate was concentrated affording 10 L dark yellow slurry. The residue was taken up in
EtOAc (30 L) and washed with brine (30 L). The organic phase was concentrated affording
compound 10 (10b) and used for the next reaction.
4.6.3. Experiment 3, using the crude product from 4.5.3.: NaOMe (81 g, 1.59 mol) was added to
a solution of compound 9 (9c, made from 1.01 mol compound 2) in dry MeOH (2 L) and stirred
for 18 h at 25 oC. Amberlite IR 120 resin (H+ form, 500 g) was added to the mixture and stirred
for 30 min then the resin was filtered off and the filtrate was concentrated affording 1 L dark
yellow slurry. The residue was taken up in DCM (2 L) and washed with water (2 × 2 L). The
organic phase was concentrated affording compound 10 (10c) and used for the next reaction.
25
D
200 mg of the sample (from experiment 2) was purified for analysis: [α] -39.0 (c 1, CHCl3),
1H NMR. (CDCl3, 400 MHz) δ: 7.42-7.26 (m, 15 H, aromatic), 5.58 (d, 1 H, J1´´,2´´ 3.2 Hz, H-
1´´), 4.98 (d, 1 H, Jgem 11.6 Hz, -CH2Ph), 4.88 (d, 1 H, Jgem 12.0 Hz, -CH2Ph), 4.77-4.61 (m, 4 H,
-CH2Ph), 4.55 (d, 1 H, J1´,2´ 8.2 Hz, H-1´), 4.48 (dd, 1 H, J1,2 6.0, J2,3 7.4 Hz, H-2), 4.36 (d, 1 H,
J1,2 7.6 Hz H-1), 4.25-4.19 (m, 2 H), 4.09-3.90 (m, 8 H), 3.85-3.62 (m, 6 H), 3.43 (2 s, each 3 H,
2 × OMe), 1.48, 1.44, 1.39, 1.37, 1.31 and 1.29 (6 s, each 3 H, 6 × -CH3), 1.10 (d, 3 H, J5´´,6´´ 6.5
Hz, H-6´´). 13C NMR (CDCl3 100 MHz) δ: 110.4, 109.7 and 108.7 (3 × -C(CH3)2), 107.6 (C-1),
101.4 (C-1´), 95.2 (C-1´´), 80.7 (C-3´), 79.0, 78.0, 77.9, 77.1, 76.3, 75.6, 74.7, 74.4, 74.3, 72.6
and 66.3 (C-2, C-3, C-4, C-5, C-2´, C-4´, C-5´, C-2´´, C-3´´, C-4´´ and C-5´´), 74.8, 73.9 and
20

ACCEPTED MANUSCRIPT
73.0 (3 CH2Ph), 65.0 and 62.4 (C-6 and C-6´), 57.7 and 54.0 (2 × OMe), 27.9, 27.0, 26.9, 26.5,
25.5 and 25.1 (6 × -CH3), 16.8 (C-6´´).
4.7. Compound 11
4.7.1. Experiment 1, using the crude product from 4.6.1.: The crude mixture (10a, made from
1.01 mol compound 3) from the depivaloylation reaction was suspended in the mixture of AcOH
(1.5 L) and water (1 L). The suspension was stirred at 65 oC (inner temperature) for 3 d. The
mixture was concentrated and taken up in EtOAc (3 L) and washed with water (2 × 4 L), the
water phases were combined and washed with EtOAc (0.5 L). The organic phases were
combined and stored in the freezer for 2 d. White crystals were formed which were isolated by
filtration resulting 300 g crude crystals. The crude product was dissolved warm MeOH (750 mL)
and cooled down. The clean product (11) was isolated after filtration (210 g, 0.276 mol, 27.3%
over 3 steps, 73% per step)
4.7.2. Experiment 2 using the crude product from 4.6.2.: The crude mixture (10b, made from
8.42 mol compound 3) from the depivaloylation reaction was suspended in the mixture of AcOH
(15 L) and water (10 L). The suspension was stirred at 65 oC (inner temperature) for 1 d. The
mixture was cooled down and extracted with 10 L hexane. The aqueous phase was concentrated
and taken up in EtOAc (15 L) and washed with water (2 × 5 L), the water phases were combined
and washed with EtOAc (2 L). The organic phases were combined and stored in the freezer for 2
d. White crystals were formed which were isolated by filtration resulting 1.95 kg crude crystals.
The crude product was dissolved warm MeOH (5 L) and cooled down. The clean product (11)
was isolated after filtration (1.27 kg, 1.67 mmol, 19.8% over 3 steps, 67% per step)
21

ACCEPTED MANUSCRIPT
4.7.3. Experiment 3, using the crude product from 4.6.3.: The crude mixture (10c, made from
1.01 mol compound 2) from the depivaloylation reaction was suspended in the mixture of AcOH
(1.5 L) and water (1.0 L). The suspension was stirred at 65 oC (inner temperature) for 3 d. The
mixture was cooled down and extracted with 1 L hexane. The aqueous phase was concentrated
and taken up in EtOAc (1.5 L) and washed with water (2 × 0.5 L), the water phases were
combined and washed with EtOAc (200 mL). The organic phases were combined and stored in
the freezer for 2 d. White crystals were formed which were isolated by filtration resulting 70 g
crude crystals. The crude product was dissolved warm MeOH (200 mL) and cooled down. The
clean product (11) was isolated after filtration (65 g, 85.5 mmol, 8.5% over 4 steps, 54% per
step)1H NMR. (DMSO d6, 400 MHz) δ: 7.40-7.20 (m, 15 H, aromatic), 6.66 (d, 1 H, J 6.7 Hz 1-
OH), 5.54 (br s, 1 H, H-1), 5.00-4.42 (m, 11 H, 6 × OBn and 5 × OH), 4.40-4.15 (m, 4 H, H-1’,
H-1’’ and 2 × OH), 3.90-3.10 (m, 15 H), 2.98 (m, 1 H), 1.05 (d, 3 H, J5´´,6´´ 6.5 Hz, H-6´´). 13C
NMR (DMSO d6, 100 MHz) δ: 139.6, 139.5 and 139.4 (aromatic quat), 101.4 (C-1´), 97.0 (C-1),
96.5 (C-1´´), 79.1, 78.6, 78.3, 75.8, 75.7, 75.3, 75.1, 75.0, 74.0, 69.1 and 65.9 (C-2, C-3, C-4, C-
5, C-2´, C-3´, C-4´, C-5´, C-2´´, C-3´´, C-4´´ and C-5´´), 74.8, 72.4 and 70.7 (3 × CH2Ph), 60.7
and 60.6 (C-6 and C-6´), 16.7 (C-6´´).
4.8. Compound 12
4.8.1. Laboratory scale: Compound 11 (10 g, 13.2 mmol) was suspended in the mixture of
MeOH (100 mL), IPA (50 mL), and AcOH (5 mL). Then Pd/C (1 g, 10 %) was added in water
(10 mL). The mixture was stirred under 6 bar H2 pressure for 12 h. The H2 atmosphere was
changed into Ar and the catalyst was filtered off and the mixture was concentrated. The residue
22

ACCEPTED MANUSCRIPT
was dissolved in MeOH (5 mL) and the product was precipitated by adding MTBE (25 mL)
resulting a white powder (6.6 g, 13.1 mmol, quant.).
4.8.2. Pilot scale: Compound 11 (1.70 kg, 2.24 mol) was suspended in the mixture of MeOH (9
L), IPA (4.5 L), and AcOH (180 mL). Then Pd/C (180 g, 10 % in charcoal) was added in water
(1 L). The mixture was stirred under 4.5 bar H2 pressure for 24 h. TLC indicated that the reaction
was still incomplete. Pd/C (90 g, 10 % in charcoal) was added in MeOH (1 mL) and the mixture
was stirred under 4.5 bar H2 pressure for 5 h, then again Pd/C (40 g, 10 % in charcoal) was added
in MeOH (0.5 mL). The mixture was stirred for 12 h under H2 pressure. The H2 atmosphere was
changed into Ar and the catalyst was filtered off and the mixture was concentrated. The residue
was dissolved in MeOH (5 L) and the product was precipitated by adding MTBE (20 L) resulting
a pale yellow powder (1.3 kg). The crude product was stirred in IPA (10 L) for 2 h, then filtered
off, washed with IPA (0.5 L) and dried in vacuo affording a white solid (1.1 kg, 2.24 mmol,
25
D
25
D
quant.). [α] -47.2 (initial) → -58.0 (72 h, c 2, H2O), [α] lit. [16] -53.5 (initial) → -57.5 (72
h, c 2, H2O). MS: ESI+ 511.2 [M+Na]+, 572.2 [M+K]+; ESI- 487.4 [M-H]-, 523.2 [M+Cl]-.
NMR data: See in Table 1.
Table 1, 1H and 13C NMR data of compound 12.
Ring
1H
H-1
H-2
δ (ppm)
5.22
multiplicity
J (Hz)
4.0
13C
C-1
δ (ppm)
94.5
d
3.59
dd
9.2, 4.0
C-2
74.0
23

ACCEPTED MANUSCRIPT
H-3*
H-4
3.80
3.71
3.91
3.90
3.80
4.63
3.29
3.58
3.72
3.47
3.94
3.76
4.52
3.66
3.88
3.90
3.81
3.81
3.74
5.30
3.80
dd
dd
m
9.6, 9.2
9.6, 9.0
C-3*
C-4
72.3
77.9
73.1
α-D glucose
H-5
C-5
H-6a
H-6b
H-1
m
C-6
62.7
m
d
8.1
C-1
C-2
C-3
C-4
C-5
98.6
76.6
77.0
78.5
78.0
H-2
dd
dd
dd
ddd
dd
dd
d
9.3, 8.1
9.6, 9.3
9.8, 9.6
9.8, 5.2, 1.8
11.8, 1.8
11.8, 5.2
7.8
H-3
β-D glucose
H-4
H-5
H-6a
H-6b
H-1
C-6
62.9
C-1
C-2
C-3
C-4
C-5
102.9
79.0
76.3
71.9
74.0
H-2
dd
m
9.0, 7.8
H-3
β-D
galactose
H-4
m
H-5
m
H-6a
H-6b
H-1
m
C-6
63.8
m
d
2.0
C-1
102.0
72.4
H-2*
m
C-2*
α-L-fucose
H-3*
H-4
3.80
m
d
C-3*
C-4
70.9
74.4
69.6
18.0
3.82
1.0
H-5
4.22, 4.25
1.22
qd
d
6.0 + 1.0
6.0
C-5
CH
3
CH
3
* Interchangeable assignments due to non-first order, overlapping spin systems
24

ACCEPTED MANUSCRIPT
ASSOCIATED CONTENT
Supporting Information. The following file is available free of charge; Analytical data (NMR,
HPLC, and LCMS) of selected compounds (3, 8, 11 and 12).
AUTHOR INFORMATION
Corresponding Authors
Karoly Agostona*, Markus Jondelius Hederos*.
Present Addresses
a: Present address: Carbosynth LTD, 8&9 Old station Business Park, RG20 6NE Compton, Berkshire, UK;
b: Present address: GlycoOptim, Kemia E328, 4032 Debrecen, Hungary.
c: Present address: CarboCode Gmbh, Byk-Gulden-strasse 2, 78467, Konstanz, Germany.
ACKNOWLEDGMENT
The presented work has been founded by Glycom A/S. Authors thank to Dr. Agnes Agoston for
the HPLC analysis, Dr Gabor Tarkanyi for the NMR analysis of the final compound and to Dr.
Laszlo Drahos for the LCMS analysis of the final compound.
REFERENCES
25

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