One-pot glycosylations in the synthesis of human milk oligosaccharides

Article abstract of DOI:10.1002/ejoc.201400164

Human milk oligosaccharides contain a well-defined core structure that makes them interesting synthetic targets for one-pot glycosylation strategies. In this investigation, a one-pot procedure was studied in which a galactose thioglycoside is coupled chem

Full text of DOI:10.1002/ejoc.201400164

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
FULL PAPER
DOI: 10.1002/ejoc.201400164
One-Pot Glycosylations in the Synthesis of Human Milk Oligosaccharides
Camilla Arboe Jennum,[a] Thomas Hauch Fenger,[a] Linda M. Bruun,[a] and
Robert Madsen*[a]
Keywords: Carbohydrates / Oligosaccharides / Glycosylation / Thioglycosides
Human milk oligosaccharides contain a well-defined core
structure that makes them interesting synthetic targets for
one-pot glycosylation strategies. In this investigation, a one-
pot procedure was studied in which a galactose thioglycoside
is coupled chemoselectively to a phthalimide-protected gluc-
osamine thioglycoside, and the resulting disaccharide is
then coupled to the 3Ј-position in lactose. Very high yields of
the tetrasaccharide product can be obtained when a pro-
tected lactosamine thioglycoside is formed in the first cou-
pling. In contrast, it was not possible to extend the one-pot
process to the synthesis of the corresponding 3ЈЈ-linked struc-
tures, due to an inefficient synthesis of the lacto-N-biose thi-
oglycoside in the first glycosylation. This is explained by the
reactivity difference between the 3- and 4-positions in
phthalimide-protected glucosamine. The one-pot procedure
has been applied in an efficient synthesis of the penta-
saccharide lacto-N-neofucopentaose I, which is composed of
N-acetyllactosamine, lactose, and fucose. On the other hand,
a stepwise approach was found to be the preferred synthetic
pathway for preparation of the isomeric lacto-N-fucopenta-
ose I, which contains a lacto-N-biose moiety.
Introduction
Human milk oligosaccharides are a class of structurally
distinct glycans that are present in human milk in concen-
trations ranging from 5 to 25 g/L, depending on the
lactational stage.[1] These oligosaccharides are unique to
human milk, and they participate in the development and
protection of newborn infants.[2] They are not digested by
intestinal enzymes, and they reach the large intestine intact,
where they stimulate the growth of healthy gut bacteria.[3]
Furthermore, human milk oligosaccharides are known to
prevent pathogens from binding to their receptors by acting
as soluble ligands,[4] and they are believed to serve as nutri-
ents for early brain development in infants.[1] So far, more
than 100 different human milk oligosaccharides have been
identified, and they share some common structural fea-
tures.[1] With a few exceptions, they all contain a backbone
in which lactose is found at the reducing end (Figure 1).
Consecutive elongations with N-acetyllactosamine (Galβ1–
4GlcNAc) and/or lacto-N-biose (Galβ1–3GlcNAc) through
β(1Ǟ3) and/or β(1Ǟ6) linkages then make up the back-
bone. This is then decorated with α(1Ǟ2/3/4) fucose and/or
α(2Ǟ3/6) sialic acid units to give oligosaccharides contain-
ing between 3 and 32 monosaccharides.[2]
Figure 1. Generic structure of human milk oligosaccharides.
Due to their health benefits for infants, human milk
oligosaccharides have attracted significant attention over
the past two decades, especially for use as potential addi-
tives in infant formulas.[3] Unfortunately, large quantities of
these oligosaccharides are not easily available. Microbial
and enzymatic approaches have been used to prepare
human milk oligosaccharides, but the scale, purity, and cost
continue to be a challenge.[5]
Chemical synthesis has also been used, and so far this
has led to the preparation of more than 15 different struc-
tures ranging from tetrasaccharides to octasaccharides.[6–8]
The syntheses can be divided into three categories de-
pending on the structure of the target molecule. The back-
bone has been assembled by various approaches including
solid-phase methods.[6] Fucosylated[7] and sialylated[8] oligo-
saccharides make up the two other groups. Interestingly, a
similar retrosynthetic strategy, in which a lactosamine or a
lacto-N-biose donor is prepared separately and then cou-
pled to a partially protected lactose acceptor, has been used
in the vast majority of the syntheses. A reactivity-based syn-
thesis was first developed recently in which galactose and
glucosamine tolyl thioglycosides 1 and 2 were coupled selec-
tively, followed by reaction with lactose acceptor 3 in the
[a] Department of Chemistry, Technical University of Denmark,
2800 Kgs. Lyngby, Denmark
E-mail: rm@kemi.dtu.dk
http://www.kemi.dtu.dk
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201400164.
same pot to give tetrasaccharide
4
in 40% yield
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
C. Arboe Jennum, T. Hauch Fenger, L. M. Bruun, R. Madsen
FULL PAPER
(Scheme 1).[7a] The TBS (tert-butyldimethylsilyl) protecting thioglycosides as substrates, since they are often crystalline
group in donor 1 played an important role in enhancing and more easily available on a large scale.[10] Thus for the
its reactivity, since the corresponding 3-O-benzyl-protected initial studies, benzylidene-protected glucosamine 5 was se-
donor gave the tetrasaccharide in only 30% yield in the lected as the coupling partner, together with partially pro-
same sequence.[7a] One-pot glycosylation approaches to tected lactose acceptor 6 (Figure 3). The latter was prepared
oligosaccharides are a valuable way of reducing the number in six straightforward steps from lactose, with a regioselec-
of steps required to assemble a target molecule.[9] Due to tive stannylene-mediated allylation of the 3Ј-position as the
the conserved structure of the human milk oligosacchar- key step.[11] To assemble the backbone tetrasaccharide by a
ides, one-pot approaches to this class of glycans may also one-pot process, a galactose donor must be identified that
render the syntheses more flexible, since the separate prepa- couples selectively to 5. The resulting disaccharide can then
ration of lactosamine and lacto-N-biose building blocks can react further with 6 in the same mixture. Several easily
be avoided. We decided to further investigate the one-pot available galactose thioglycosides 7–9 were selected, and
approach for assembling the backbone structure, and to ex- their coupling to 5 was investigated (Figure 3). However,
plore the scope and limitations of this strategy. In this regardless of the promoter, these couplings only led to low
paper, we report a full account on our synthetic studies yields of the desired disaccharide, probably due to an insuf-
towards human milk oligosaccharides, exemplified by the ficient reactivity difference between the donors and the ac-
syntheses of lacto-N-fucopentaose I and lacto-N-neofuco- ceptor.
pentaose I (Figure 2).
Figure 3. Glycosyl donors and acceptors 5–9.
To circumvent the chemoselectivity problem, imidate do-
nor 10 was studied next, and with this donor, glycosylation
with 5 could be achieved in good yield (Scheme 2). The re-
sulting disaccharide (i.e., 11) was subsequently coupled with
lactose acceptor 6 to give tetrasaccharide 12 in 69% yield.
Unfortunately, when the two steps were combined in a one-
Scheme 1. A reactivity-based one-pot synthesis of the tetrasac-
charide backbone.[7a] NIS = N-iodosuccinimide; TESOTf = trieth-
pot sequence, the overall yield of 12 dropped to a mere 8%.
This is presumably due to inhibition of the second coupling
by the trichloroacetamide formed in the first step. As a re-
sult, imidate donors were abandoned, and attention turned
back to thioglycoside donors.
ylsilyl trifluoromethanesulfonate; Tol = 4-methylphenyl.
Figure 2. Structures of lacto-N-fucopentaose I and lacto-N-neo-
fucopentaose I.
Scheme 2. Synthesis of tetrasaccharide 12.
Thus, a more reactive thioglycoside donor was needed,
and the recently presented donors with 2-O-benzoyl-3,4,6-
tri-O-benzyl protection,[12] such as 13, were therefore con-
Results and Discussion
We have previously used one-pot syntheses for the prepa- sidered (Figure 4). Thioglycoside 13 was prepared in eight
ration of several galactans. In that work, we used phenyl steps from galactose, with the formation and rearrangement
2
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
Synthesis of Human Milk Oligosaccharides
of a 1,2-orthoester as the key steps.[13] However, to our dis-
appointment, the coupling between 13 and 5 in the presence
of NIS and AgOTf led to only a 28% yield of the desired
disaccharide.
Figure 4. Glycosyl donors and acceptors 13–15.
Scheme 4. Sequential and one-pot synthesis of tetrasaccharide 19.
As a result, it was decided to change the benzylidene
acetal in 5 to two benzyl ethers, which would also make it
possible to address the two different glycosidic linkages in
the backbone. Accordingly, thioglycosides 14 and 15 (Fig-
ure 4) were prepared from 5, and the chemoselective cou-
plings with 13 were investigated. Gratifyingly, the glycosyl-
ation of 14 with 13 gave a remarkable 95% yield of disac-
charide 16 (Scheme 3), which was very promising for the
one-pot synthesis. Further coupling with lactose acceptor 6
was uneventful, and gave tetrasaccharide 17 in 64% yield.
Indeed, when the two steps were combined in a one-pot
process, the overall yield of 17 was a satisfying 72%, a
higher yield than that obtained over the two-step sequence.
The same experiments were carried out with thioglycoside
15, which first reacted with donor 13 to give disaccharide
18 in 44% yield (Scheme 4). Although this was an improve-
ment over the coupling between 13 and 5, it was not pos-
sible to increase this yield further by modifying the condi-
tions. These results illustrate the known observation that
the 3-position in phthalimide-protected β-glucosamines is
the least reactive.[14] Further reaction with lactose acceptor
6 gave tetrasaccharide 19 in 80% yield, but when a one-pot
sequence was run in this case, the overall yield of 19 was
only 7%. Thus, the one-pot strategy may be used for the
synthesis of the backbone with the β(1Ǟ4) linkage, but the
β(1Ǟ3) linkage is more difficult, due to a moderate yield in
the first coupling.
N-acetylation can be carried out with amino alcohols, these
results made us reconsider the use of the 2-benzoate in the
galactose donor. A 2-acetate may be a better choice, since
it can usually be removed in the presence of a phthalimide,
and may also give a better coupling yield due to a lower
steric demand.
Consequently, the corresponding donor (i.e., 20) was
prepared in six steps from galactose.[13b] Chemoselective
glycosylation with acceptor 14 gave disaccharide 21 in 58%
yield, together with 26% of the corresponding ethyl thio-
glycoside (Scheme 5). Aglycon transfer of thiols is a known
side-reaction in some glycosylations with thioglycosides,
and is hard to predict,[15] but it came as a surprise that the
transfer was much more pronounced with acetate donor 20
than with benzoate donor 13. However, it was not a major
concern, since both thioglycosides would undergo a subse-
quent glycosylation reaction. Therefore, the mixture was
treated with lactose acceptor 6, and tetrasaccharide 22 was
isolated in 73% yield. When the two glycosylations were
carried out in a one-pot sequence, the overall yield in-
creased to a gratifying 94%.
Scheme 5. Sequential and one-pot synthesis of tetrasaccharide 22.
The same series of reactions was carried out with thio-
glycoside 15, where the first glycosylation produced disac-
charide 23 in 62% yield, together with 14% of the corre-
Scheme 3. Sequential and one-pot synthesis of tetrasaccharide 17.
Attempts to remove the benzoate selectively from 17 and sponding ethyl thioglycoside (Scheme 6). Again, the reac-
19 under Zemplén conditions were unsuccessful, since the tion was accompanied by significant aglycon transfer, but
deprotections were accompanied by ring-opening and cleav- the coupling yield with acetate donor 20 was noticably bet-
age of the phthalimide. Complete removal of both the ter than that obtained with benzoate 13. Further glycosyl-
benzoate and the phthalimido group could be achieved with ation between disaccharides 23 and 6 gave tetrasaccharide
hydrazine in ethanol at reflux (for 17), or under Zemplén 24 in 58% yield. Unfortunately, when the two glycosylations
conditions at room temperature (for 19). Although selective were carried out in a one-pot sequence, tetrasaccharide 24
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
C. Arboe Jennum, T. Hauch Fenger, L. M. Bruun, R. Madsen
FULL PAPER
was only obtained in trace amounts, and could not be suffi- moved under the basic conditions, similarly to what was
ciently purified. This result together with the coupling in observed with benzoate 19. The reaction was very slow, and
Scheme 5 clearly illustrate that the backbone with the we therefore decided to use ethylene diamine instead, fol-
β(1Ǟ3) linkage cannot be assembled by the present one- lowed by selective N-acetylation to give alcohol 29
pot approach, but must be prepared in a stepwise manner.
(Scheme 8). Glycosylation with fucose donor 26 then gave
pentasaccharide 30, which was deprotected to give lacto-N-
fucopentaose I.
Scheme 6. Sequential and one-pot synthesis of tetrasaccharide 24.
Tetrasaccharides 22 and 24 were both converted into the
target human milk oligosaccharides by four similar trans-
formations. Selective removal of the acetate in 22 could now
be carried out in a good yield under Zemplén conditions
without affecting the phthalimide (Scheme 7). Subsequent
glycosylation with fucose thioglycoside 26 gave penta-
saccharide 27 in high yield, although the product was con-
taminated by small amounts of an impurity, which could
not be removed before the next step. Cleavage of the phthal-
imido group followed by N-acetylation and hydrogenolysis
of the benzyl ethers then gave lacto-N-neofucopentaose I.
Only two previous syntheses of lacto-N-neofucopentaose I
are known, and in those syntheses, the final deprotections
were not carried out.[7d,7i] Compared to these two earlier
syntheses, the present synthesis offers a significant re-
duction in the total number of steps.
Scheme 8. Final steps in the synthesis of lacto-N-fucopentaose I.
Since the 3-position in glucosamine is the least reactive,
it may be possible to perform a selective glycosylation at
the 4-position in a 3,4-unprotected substrate. Indeed, when
glucosamine thioglycoside 31 was submitted to the one-pot
process, tetrasaccharide 32 was obtained in 57% overall
yield (Scheme 9). The position of the new interglycosidic
linkage was verified by NMR spectroscopy, where the
HMBC spectrum showed a correlation between C-1ЈЈЈ and
H-4ЈЈ. Unfortunately, it was not possible to glycosylate 32
with fucose donor 26; this resulted only in decomposition
of the donor and recovery of the acceptor. Hence, the lower
reactivity of the 3-position favors the one-pot reaction with
31 at position 4, but limits the synthetic value of the re-
sulting product (i.e., 32). Similar results were obtained with
acetate donor 20 and glucosamine acceptor 31, and this ap-
proach is therefore not useful for synthesis of another
human milk oligosaccharide.
Scheme 9. One-pot synthesis of tetrasaccharide 32.
Scheme 7. Final steps in the synthesis of lacto-N-neofucopentaose I.
Conclusions
In the same way, isomer 24 was also subjected to depro-
tection with sodium methoxide in methanol. In this case,
In summary, we have described a one-pot process for as-
however, both the acetate and the phthalimide were re- sembling the lactosamine–lactose backbone in human milk
4
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
Synthesis of Human Milk Oligosaccharides
(dd, J = 9.7, 4.4 Hz, 1 H), 3.45 (dd, J = 7.9, 6.2 Hz, 1 H), 2.06 (s,
3 H), 1.90 (s, 3 H), 1.83 (s, 3 H), 1.50 (s, 3 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 170.3, 170.0, 170.0, 168.9, 163.7, 136.8,
132.7, 131.2, 129.3, 128.9, 128.3, 128.2, 126.0, 101.4, 100.3, 84.1,
80.7, 76.5, 70.9, 70.4, 70.2, 69.1, 68.5, 66.5, 60.7, 54.2, 20.6, 20.5,
20.4, 20.0 ppm. NMR spectroscopic data are consistent with litera-
ture values.[20]
oligosaccharides, in which galactose and glucosamine thio-
glycosides are first coupled chemoselectively followed by
glycosylation with lactose. The transformation gives a
higher yield of the tetrasaccharide than the stepwise ap-
proach, and it has been applied in the synthesis of lacto-N-
neofucopentaose I. The one-pot process could not be ex-
tended to the corresponding lacto-N-biose–lactose back-
bone, due to a more difficult coupling in the first step. In
this case, the stepwise approach is still the favored pathway,
as shown with the synthesis of lacto-N-fucopentaose I.
Benzyl 2,3,4,6-Tetra-O-acetyl-β-
benzylidene-2-deoxy-2-phthalimido-β-
2,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-
β- -glucopyranoside (12): Compound 11 (921 mg, 1.12 mmol) and
D
-galactopyranosyl-(1Ǟ3)-4,6-O-
D
-glucopyranosyl-(1Ǟ3)-
D
D
lactose derivative 6 (939 mg, 0.96 mmol) were dissolved in CH2Cl2
(15 mL), the mixture was cooled to –20 °C, and then NIS (280 mg,
1.25 mmol) and TfOH (8.5 μL, 0.10 mmol) were added. After 1 h,
Et3N was added. The mixture was stirred for 15 min, and then it
was filtered through Celite and concentrated. The residue was puri-
fied by flash chromatography (EtOAc/heptane, 1:2) to give com-
Experimental Section
General Remarks: All reactions were carried out under an argon
atmosphere. Molecular sieves (MS) were flame dried before use.
Dichloromethane was dried with 4 Å MS. Phenyl 4,6-O-benzyl-
1
pound 12 (1.13 g, 0.67 mmol, 69%) as a colorless solid. H NMR
idene-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside
(5),[16]
(300 MHz, CDCl3): δ = 7.76 (br. s, 1 H), 7.59 (br. s, 1 H), 7.58–
7.50 (m, 2 H), 7.46–7.19 (m, 35 H), 7.17–7.06 (m, 3 H), 6.91 (dd,
J = 6.6, 2.5 Hz, 2 H), 5.63 (s, 1 H), 5.45 (d, J = 8.5 Hz, 1 H), 5.21
(d, J = 3.2 Hz, 1 H), 5.06–4.95 (m, 2 H), 4.93–4.81 (m, 4 H), 4.78–
4.67 (m, 2 H), 4.61–4.20 (m, 13 H), 4.19–4.01 (m, 2 H), 3.99–3.79
(m, 6 H), 3.73 (dd, J = 9.7, 4.7 Hz, 1 H), 3.64–3.57 (m, 1 H), 3.56–
3.46 (m, 4 H), 3.43–3.27 (m, 5 H), 2.93 (d, J = 10.0 Hz, 1 H), 2.08
(s, 3 H), 1.94 (s, 3 H), 1.86–1.83 (m, 3 H), 1.49 (s, 3 H) ppm. 13C
NMR (75 MHz, CDCl3): δ = 170.2, 170.0, 170.0, 168.7, 139.1,
138.9, 138.5, 138.4, 138.3, 138.1, 137.4, 136.9, 128.4, 128.3, 128.3,
128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 127.7,
127.7, 127.6, 127.6, 127.4, 127.3, 127.0, 126.8, 126.0, 126.0, 102.4,
102.3, 101.4, 100.3, 99.8, 82.9, 81.5, 81.1, 80.9, 78.8, 76.3, 75.8,
75.4, 75.2, 75.0, 75.0, 74.9, 74.7, 73.7, 73.3, 72.9, 72.9, 70.9, 70.7,
70.2, 68.9, 68.0, 67.5, 67.0, 66.5, 66.0, 60.7, 55.7, 20.6, 20.5, 20.4,
20.0 ppm. HRMS: calcd. for C96H99NO26 [M + H]+ 1682.6528;
found 1682.6554.
benzyl 2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-
benzyl-β-d-glucopyranoside (6),[11] 2,3,4,6-tetra-O-acetyl-α-d-
galactopyranosyl trichloroacetimidate (10),[7j] ethyl 2-O-benzoyl-
3,4,6-tri-O-benzyl-1-thio-β-d-galactopyranoside (13),[13a] phenyl
3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyrano-
side (14),[17] ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-β-d-galacto-
pyranoside (20),[13b] phenyl 2,3,4-tri-O-benzyl-1-thio-β-l-fucopyr-
anoside (26),[16] and phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-
thio-β-d-glucopyranoside (31)[18] were synthesized according to lit-
erature procedures. TLC was carried out on aluminum plates
coated with silica gel 60. The plates were visualized with UV light
or by dipping into a solution of cerium(IV)sulfate (2.50 g) and am-
monium molybdate (6.25 g) in sulfuric acid (10 %; 250 mL) fol-
lowed by heating. Purification was carried out with HPLC grade
solvents by flash chromatography on silica gel (Merck 40–63 mi-
cron) or by dry column chromatography[19] on silica gel (Merck
15–40 micron). Reverse-phase chromatography was carried out on
silica gel (YMC–C18, 120 Å, 5–10–20 μm). NMR spectra were re-
corded with a Varian Mercury 300, a Bruker Ascend 400, or a
Varian Unity Inova 500 instrument. Chemical shifts were calibrated
General Procedure for One-pot Glycosylation: A super-armed ga-
lactose donor and a glucosamine acceptor were dissolved in
CH2Cl2 (1 mL/100 mg reactants), and the solution was stirred un-
der argon with 4 Å MS for 1 h. The mixture was cooled to –30 °C,
and NIS (1.15 equiv.) and AgOTf (cat.) were added. The mixture
was stirred for 15 min, after which time TLC revealed full conver-
sion of the acceptor (toluene/acetone, 9:1). Then a solution of lact-
ose acceptor 6 in CH2Cl2 (0.5 mL/0.1 mmol) was added together
with NIS (1.05 equiv.) and TESOTf (0.1 equiv.). The mixture was
stirred for 40 min, after which time TLC showed full conversion of
the acceptor. The reaction was quenched with Et3N. The mixture
was stirred for 15 min, and then filtered through Celite and concen-
trated. The residue was purified by chromatography.
using the residual solvent signal in CDCl3 (δH = 7.26 ppm, δC
=
77.16 ppm) or tetramethylsilane. HRMS analyses were carried out
with an Agilent 1100 LC system equipped with a diode array detec-
tor and a Luna C18 column (3 μm, 50 mmϫ2 mm). The LC was
coupled to a Micromass LCT orthogonal time-of-flight mass spec-
trometer equipped with lock mass probe and operating in positive
electrospray mode.
Phenyl 2,3,4,6-Tetra-O-acetyl-β-
benzylidene-2-deoxy-2-phthalimido-1-thio-β-
D
-galactopyranosyl-(1Ǟ3)-4,6-O-
-glucopyranoside (11):
D
Imidate 10 (423 mg, 0.86 mmol) and thioglycoside 5 (300 mg,
0.61 mmol) were coevaporated with toluene (2ϫ 5 mL), and then
dissolved in CH2Cl2 (18 mL). The mixture was cooled to –40 °C,
and TESOTf (27.7 μL, 0.12 mmol) was added. The reaction was
stirred overnight, during which time it reached room temperature.
Then Et3N (171 μL, 1.23 mmol) was added. The stirring was con-
tinued for an additional 15 min, then the mixture was filtered
through Celite and concentrated. The residue was purified by flash
chromatography (EtOAc/heptane, 1:1) to give compound 11
General Procedure for NIS/AgOTf Glycosylation: A super-armed
galactose donor and a glucosamine acceptor were dissolved in
CH2Cl2 (1 mL/100 mg reactants), and the solution was stirred un-
der argon with 4 Å MS for 1 h. The mixture was cooled to the
temperature indicated, and then NIS (1.15 equiv.) and AgOTf (cat.)
were added. The mixture was allowed to stir for 40 min, after which
time TLC showed full conversion of the acceptor (toluene/acetone,
9:1). The reaction was quenched with Et3N. The mixture was
stirred for 15 min, and then filtered through Celite and concen-
trated. The residue was purified by chromatography.
1
(380 mg, 76%) as a colorless solid. H NMR (300 MHz, CDCl3):
δ = 7.88–7.75 (m, 4 H), 7.50–7.43 (m, 2 H), 7.40–7.31 (m, 5 H),
7.28–7.21 (m, 3 H), 5.57 (s, 1 H), 5.56 (d, J = 10.8 Hz, 1 H, 1-H),
5.18 (d, J = 2.9 Hz, 1 H, 4-H), 4.96 (dd, J = 10.4, 8.1 Hz, 1 H),
4.79–4.68 (m, 2 H), 4.54 (d, J = 7.9 Hz, 1 H, 1Ј-H), 4.44–4.32 (m,
2 H), 4.01 (dd, J = 10.8, 8.2 Hz, 1 H), 3.88–3.76 (m, 3 H), 3.72
General Procedure for NIS/TESOTf Glycosylation: A donor and
lactose acceptor 6 were dissolved in CH2Cl2 (1 mL /100 mg reac-
tants), and the solution was stirred under argon with 4 Å MS for
1 h. The mixture was cooled to –30 °C, and then NIS (1.15 equiv.)
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
C. Arboe Jennum, T. Hauch Fenger, L. M. Bruun, R. Madsen
FULL PAPER
and TESOTf (0.1 equiv.) were added. The mixture was allowed to
stir for 40 min, after which time TLC showed full conversion of the
acceptor (toluene/acetone, 9:1). The reaction was quenched with
Et3N. The mixture was stirred for 15 min, and then filtered through
Celite and concentrated. The residue was purified by chromato-
graphy.
(m, 28 H), 6.91 (d, J = 7.3 Hz, 2 H), 5.50–5.39 (m, 1 H), 5.31 (d,
J = 10.5 Hz, 1 H), 5.02 (d, J = 10.7 Hz, 1 H), 4.84 (d, J = 11.4 Hz,
1 H), 4.78 (dd, J = 10.1, 7.8 Hz, 1 H), 4.53–4.37 (m, 6 H), 4.28–
4.20 (m, 3 H), 4.15 (d, J = 11.7 Hz, 1 H), 3.85 (d, J = 2.3 Hz, 1
H), 3.74 (d, J = 10.7 Hz, 1 H), 3.69 (dd, J = 10.7, 4.3 Hz, 1 H),
3.63–3.56 (m, 2 H), 3.39 (t, J = 8.1 Hz, 1 H), 3.34–3.25 (m, 3 H)
ppm. 13C NMR (75 MHz, CDCl3): δ = 165.4, 138.7, 138.68, 138.4,
138.1, 137.5, 134.1, 133.0, 132.8, 131.8, 131.5, 130.4, 130.1, 128.9,
128.5, 128.4, 128.3, 128.3, 128.1, 128.1, 128.1, 128.0, 127.9, 127.8,
127.6, 127.5, 127.3, 100.8, 84.0, 80.3, 79.4, 77.9, 77.3, 74.9, 74.8,
73.6, 73.5, 73.4, 72.9, 72.5, 71.7, 69.3, 67.9, 55.1 ppm. HRMS:
calcd. for C68H63NO12S [M + Na]+ 1140.3963; found 1140.3955.
Phenyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-β-
(1Ǟ4)-3,6-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
D
-galactopyranosyl-
-glucopyrano-
D
side (16): Synthesized by the general NIS/AgOTf procedure using
13 (117 mg, 0.19 mmol) and 14 (103 mg, 0.18 mmol). Purified by
flash chromatography (toluene/acetone, 15:1) to give 16 (188 mg,
1
95%) as a colorless solid. H NMR (300 MHz, CDCl3): δ = 8.07–
Benzyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-β-
(1Ǟ3)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-
(1Ǟ3)-2,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-
benzyl-β- -glucopyranoside (19): Synthesized by the general NIS/
D
-galactopyranosyl-
7.94 (m, 2 H), 7.82 (d, J = 5.3 Hz, 1 H), 7.73–7.55 (m, 4 H), 7.53–
7.41 (m, 2 H), 7.39–7.08 (m, 26 H), 7.03–6.94 (m, 2 H), 6.91–6.82
(m, 2 H), 5.67 (dd, J = 10.0, 7.9 Hz, 1 H), 5.44 (d, J = 10.3 Hz, 1
H), 5.06–4.82 (m, 2 H), 4.73–4.54 (m, 4 H), 4.53–4.44 (m, 2 H),
4.41–4.20 (m, 5 H), 4.08–3.99 (m, 2 H), 3.75–3.62 (m, 1 H), 3.61–
3.40 (m, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 168.1, 167.6,
165.3, 139.0, 138.6, 138.2, 138.0, 134.1, 134.0, 133.4, 132.6, 132.5,
131.9, 131.8, 130.1, 129.3, 129.0, 128.9, 128.7, 128.2, 128.1, 128.0,
127.9, 127.8, 127.6, 127.0, 125.6, 123.7, 123.6, 101.1, 83.7, 80.0,
79.2, 78.4, 77.8, 75.1, 74.7, 73.7, 73.6, 72.8, 72.7, 71.6, 68.4,
68.2 ppm.
D
-glucopyranosyl-
D
D
TESOTf procedure using 18 (126 mg, 0.11 mmol) and 6 (104 mg,
0.11 mmol). Purified by dry column chromatography (acetone/tolu-
ene, 0 to 75% in steps of 5%) to give 19 (170 mg, 80%) as a color-
less oil. In addition, synthesized by the general one-pot glycosyl-
ation procedure using 13 (124 mg, 0.21 mmol), 15 (110 mg,
0.19 mmol), and 6 (184 mg, 0.19 mmol). Purified by flash column
chromatography (acetone/toluene, 1:19) to give 19 (27 mg, 7%) as
a colorless oil. 1H NMR (400 MHz, CDCl3): δ = 7.68 (dd, J = 8.2,
1.2 Hz, 2 H), 7.59–7.53 (m, 1 H), 7.41–7.02 (m, 62 H), 7.00–6.94
(m, 2 H), 6.88–6.81 (m, 2 H), 5.51 (dd, J = 9.9, 7.9 Hz, 1 H), 5.22
(d, J = 8.4 Hz, 1 H), 5.12 (d, J = 10.6 Hz, 1 H), 4.99–4.81 (m, 6
H), 4.69 (d, J = 10.9 Hz, 1 H), 4.58–4.40 (m, 10 H), 4.36–4.11 (m,
10 H), 3.97 (d, J = 2.9 Hz, 1 H), 3.94 (d, J = 2.6 Hz, 1 H), 3.86–
3.65 (m, 6 H), 3.53–3.28 (m, 12 H), 3.25 (d, J = 9.4 Hz, 1 H), 2.92–
2.84 (m, 1 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 165.5, 139.5,
139.1, 138.8, 138.7, 138.7, 138.7, 138.5, 138.3, 138.2, 138.1, 137.7,
137.6, 133.6, 132.7, 131.1, 130.3, 130.0, 128.5, 128.5, 128.4, 128.4,
128.3, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.1, 128.1,
128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.7, 127.6, 127.6,
127.6, 127.5, 127.3, 127.2, 126.8, 126.4, 102.5, 102.5, 100.7, 99.6,
83.0, 81.7, 81.5, 80.3, 78.7, 77.5, 76.7, 76.3, 76.0, 75.5, 75.1, 75.1,
75.0, 75.0, 74.8, 74.8, 73.8, 73.7, 73.5, 73.4, 73.3, 73.3, 73.1, 72.9,
72.9, 71.7, 70.9, 69.5, 68.6, 67.9, 67.6, 56.4 ppm. HRMS: calcd. for
C123H121NO23 [M + Na]+ 2002.8222; found 2002.8276.
Benzyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-β-
(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-
(1Ǟ3)-2,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-
benzyl-β- -glucopyranoside (17): Synthesized by the general NIS/
D
-galactopyranosyl-
D
-glucopyranosyl-
D
D
TESOTf procedure using 16 (116 mg, 0.10 mmol) and 6 (111 mg,
0.11 mmol). Purified by dry column chromatography (acetone/tolu-
ene, 0 to 75% in steps of 5%) to give 17 (132 mg, 64%) as a color-
less oil. In addition, synthesized by the general one-pot glycosyl-
ation procedure using 13 (124 mg, 0.21 mmol), 14 (110 mg,
0.19 mmol), and 6 (184 mg, 0.19 mmol). Purified by flash column
chromatography (acetone/toluene, 1:19) to give 17 (270 mg, 72%)
as a colorless oil. 1H NMR (500 MHz, CDCl3): δ = 7.99 (d, J =
7.4 Hz, 2 H), 7.62 (t, J = 7.4 Hz, 2 H), 7.48 (t, J = 7.7 Hz, 3 H),
7.39–7.10 (m, 53 H), 7.07 (t, J = 7.3 Hz, 2 H), 6.94 (d, J = 6.8 Hz,
2 H), 6.90–6.76 (m, 5 H), 5.67 (dd, J = 9.8, 8.1 Hz, 1 H), 5.30 (d,
J = 8.3 Hz, 1 H), 5.04 (d, J = 11.4 Hz, 1 H), 4.99 (d, J = 11.6 Hz,
1 H), 4.94 (d, J = 12.1 Hz, 1 H), 4.91–4.81 (m, 3 H), 4.73–4.62 (m,
3 H), 4.60–4.52 (m, 4 H), 4.52–4.19 (m, 14 H), 4.16 (d, J = 11.9 Hz,
1 H), 4.06 (t, J = 9.2 Hz, 1 H), 4.04–3.97 (m, 2 H), 3.95 (d, J =
1.8 Hz, 1 H), 3.83 (t, J = 9.0 Hz, 1 H), 3.70 (dd, J = 10.9, 3.3 Hz,
1 H), 3.55 (dd, J = 10.1, 2.6 Hz, 1 H), 3.53–3.24 (m, 14 H), 2.92
(d, J = 7.6 Hz, 1 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 167.9,
167.8, 165.2, 139.5, 139.1, 139.0, 138.8, 138.7, 138.6, 138.4, 138.4,
138.3, 138.0, 137.8, 137.7, 133.5, 133.2, 131.3, 130.0, 128.5, 128.5,
128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8,
127.7, 127.6, 127.5, 127.5, 127.2, 127.1, 126.8, 126.7, 126.5, 123.1,
102.5, 102.5, 100.9, 99.7, 83.0, 82.1, 81.7, 79.9, 78.8 77.8, 77.4, 76.9,
76.1, 75.5, 75.1, 75.1, 74.8, 74.8, 74.6, 74.5, 74.1, 73.6, 73.6, 73.4,
73.3, 73.0, 73.0, 72.6, 72.6, 71.4, 70.9, 68.3, 68.1, 68.1, 67.7,
56.4 ppm. HRMS: calcd. for C123H121NO23 [M + Na]+ 2002.8222;
found 2002.8191.
Phenyl 2-O-Acetyl-3,4,6-tri-O-benzyl-β-
3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
(21a) and Ethyl 2-O-Acetyl-3,4,6-tri-O-benzyl-β-
pyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
D
-galactopyranosyl-(1Ǟ4)-
-glucopyranoside
-galacto-
D
D
D-
glucopyranoside (21b): Synthesized by the general NIS/AgOTf pro-
cedure using 20 (113 mg, 0.21 mmol) and 14 (110 mg, 0.19 mmol).
Purified by dry column chromatography (acetone/toluene, 0 to 60%
in steps of 3%) to give 21a (87 mg, 44%), 21b (10 mg), and a mix-
ture of the two (7:11, 72 mg).
Data for 21a: 1H NMR (500 MHz, CDCl3): δ = 7.81 (d, J = 6.7 Hz,
1 H), 7.78–7.60 (m, 3 H), 7.43–7.12 (m, 25 H), 7.03–6.90 (m, 2 H),
6.90–6.75 (m, 3 H), 5.51 (d, J = 10.0 Hz, 1 H), 5.36 (dd, J = 10.1,
7.9 Hz, 1 H), 4.91 (d, J = 11.6 Hz, 1 H), 4.84 (d, J = 12.2 Hz, 1
H), 4.73–4.62 (m, 2 H), 4.55–4.45 (m, 4 H), 4.42 (d, J = 12.2 Hz,
1 H), 4.36–4.21 (m, 4 H), 4.02–3.95 (m, 1 H), 3.93 (d, J = 2.5 Hz,
Phenyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-β-
(1Ǟ3)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
anoside (18): Synthesized by the general NIS/AgOTf procedure
D-galactopyranosyl- 1 H), 3.84–3.74 (m, 2 H), 3.60 (d, J = 10.0 Hz, 1 H), 3.49–3.33 (m,
D
-glucopyr-
4 H), 2.00 (s, 3 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 169.4,
168.1, 167.5, 138.9, 138.8, 138.3, 138.2, 138.1, 134.0, 133.9, 132.6,
using 13 (118 mg, 0.20 mmol) and 15 (110 mg, 0.19 mmol). Purified 132.4, 131.8, 131.8, 128.9, 128.6, 128.5, 128.5, 128.2, 128.0, 128.0,
by dry column chromatography (acetone/toluene, 0 to 50% in steps 127.9, 127.9, 127.8, 127.8, 127.8, 127.8, 127.5, 127.4, 126.9, 123.6,
of 2.5 %) to give 18 (94 mg, 44 %) as a colorless oil. 1H NMR 123.5, 100.9, 83.6, 80.5, 79.5, 78.1, 77.8, 77.4, 77.2, 76.9, 74.9, 74.6,
(500 MHz, CDCl3): δ = 7.67 (d, J = 7.4 Hz, 2 H), 7.62–7.56 (m, 3 73.6, 73.4, 72.8, 72.1, 71.8, 68.2, 54.9, 21.2 ppm. HRMS: calcd. for
H), 7.53 (t, J = 7.4 Hz, 1 H), 7.33 (t, J = 7.7 Hz, 3 H), 7.29–6.94 C63H61NO12S [M + Na]+ 1078.3807; found 1078.3806.
6
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
Synthesis of Human Milk Oligosaccharides
Data for 21b: 1H NMR (300 MHz, CDCl3): δ = 7.84–7.64 (m, 4 procedure using 23 (270 mg, 0.26 mmol) and 6 (207 mg,
H), 7.40–7.15 (m, 20 H), 7.02–6.76 (m, 5 H), 5.36 (dd, J = 10.0, 0.21 mmol). Purified by dry column chromatography (EtOAc/tolu-
8.0 Hz, 1 H), 5.22 (d, J = 10.0 Hz, 1 H), 4.97–4.80 (m, 2 H), 4.77–
ene, 0 to 33% in steps of 1.65%) to give 24 (240 mg, 58%) as a
4.62 (m, 2 H), 4.54–4.19 (m, 9 H), 4.08–3.97 (m, 1 H), 3.93 (d, J colorless oil. 1H NMR (500 MHz, CDCl3): δ = 7.72–7.52 (m, 2 H),
= 2.6 Hz, 1 H), 3.85–3.71 (m, 2 H), 3.59 (t, J = 9.4 Hz, 1 H), 3.49– 7.47–7.01 (m, 60 H), 6.96–6.80 (m, 2 H), 5.31–5.21 (m, 2 H), 5.08–
3.32 (m, 4 H), 2.75–2.52 (m, 2 H), 2.01 (s, 3 H) 1.17 (t, J = 7.4 Hz, 4.99 (m, 2 H), 4.94–4.79 (m, 5 H), 4.69 (d, J = 10.9 Hz, 1 H), 4.63–
3 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 169.5, 168.0, 167.7,
138.9, 138.9, 138.3, 138.2, 138.1, 133.9, 132.6, 131.8, 128.9, 128.6,
128.5, 128.5, 128.2, 128.0, 127.9, 127.9, 127.8, 127.4, 127.4, 126.9,
123.5, 123.4, 100.8, 81.2, 80.4, 79.5, 78.0, 77.8, 74.8, 74.5, 73.6,
4.39 (m, 9 H), 4.38–4.14 (m, 10 H), 4.08 (d, J = 7.9 Hz, 1 H), 4.01
(d, J = 2.8 Hz, 1 H), 3.93–3.75 (m, 5 H), 3.70 (ddd, J = 9.9, 4.6,
1.8 Hz, 1 H), 3.66–3.60 (m, 1 H), 3.53–3.43 (m, 4 H), 3.43–3.24 (m,
8 H), 3.04 (dd, J = 10.1, 2.7 Hz, 1 H), 2.90 (ddd, J = 9.9, 3.4,
73.6, 73.4, 72.7, 72.1, 71.7, 68.2, 54.9, 24.0, 21.2, 15.1 ppm. HRMS: 1.7 Hz, 1 H), 1.92 (s, 3 H) ppm. 13C NMR (125 MHz, CDCl3): δ
calcd. for C63H61NO12S [M + Na]+ 1030.3807; found 1030.3809.
= 169.9, 139.6, 139.2, 138.8, 138.8, 138.5, 138.4, 138.3, 138.1, 137.9,
137.8, 133.9, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.4, 128.4,
128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 128.0,
127.9, 127.8, 127.8, 127.8, 127.7, 127.6, 127.6, 127.6, 127.5, 127.3,
127.2, 126.9, 126.4, 102.6, 102.5, 100.8, 99.5, 83.2, 81.7, 81.7, 80.4,
78.8, 77.1, 76.8, 76.3, 76.1, 75.6, 75.3, 75.2, 75.1, 75.0, 74.9, 74.8,
73.9, 73.8, 73.6, 73.4, 73.4, 73.3, 73.2, 72.7, 71.6, 71.4, 70.9, 69.5,
68.5, 67.8, 67.7, 56.6, 21.0 ppm. HRMS: calcd. for C118H119NO23
[M + Na]+ 1940.8065; found 1940.8030.
Benzyl 2-O-Acetyl-3,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-
D
3,6-di-O-benzyl-2-deoxy-2-phthalimido-β- -glucopyranosyl-(1Ǟ3)-
2,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-
β- -glucopyranoside (22): Synthesized by the general NIS/TESOTf
D
D
D
procedure using a mixture of 21a/21b (300 mg, ca. 0.284 mmol) and
6 (215 mg, 0.221 mmol). Purified by flash column chromatography
(EtOAc/heptane, 1:2) to give 22 (310 mg, 73%) as a colorless oil.
In addition, synthesized by the general one-pot glycosylation pro-
cedure using 20 (686 mg, 1.3 mmol), 14 (620 mg, 1.1 mmol), and 6
(1.00 g, 1.0 mmol). Purified by flash column chromatography
(EtOAc/heptane, 1:2) to give 22 (1.86 g, 94%) as a colorless oil. 1H
NMR (500 MHz, CDCl3): δ = 7.42–7.04 (m, 58 H), 6.94–6.76 (m,
6 H), 5.37 (m, 2 H), 5.07 (d, J = 11.4 Hz, 1 H), 4.95–4.82 (m, 5
H), 4.72–4.62 (m, 3 H), 4.57–4.22 (m, 18 H), 4.17 (d, J = 11.9 Hz,
1 H), 4.06–3.98 (m, 3 H), 3.93 (d, J = 2.7 Hz, 1 H), 3.88–3.83 (m,
1 H), 3.82–3.74 (m, 2 H), 3.63–3.57 (m, 1 H), 3.54 (dd, J = 9.8,
3.0 Hz, 1 H), 3.53–3.31 (m, 12 H), 2.98–2.88 (m, 1 H), 2.01 (s, 3
H) ppm. 13C NMR (125 MHz, CDCl3): δ = 169.4, 139.6, 139.2,
139.0, 138.9, 138.8, 138.6, 138.5, 138.5, 138.2, 138.2, 138.1, 137.7,
133.5, 128.6, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2,
128.2, 128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7,
127.6, 127.6, 127.5, 127.4, 127.3, 127.2, 126.9, 126.8, 126.6, 102.6,
101.0, 99.9, 83.1, 82.1, 81.8, 80.5, 78.9, 78.0, 76.8, 76.8, 76.1, 75.6,
75.2, 75.2, 74.9, 74.9, 74.8, 74.6, 74.2, 73.8, 73.6, 73.4, 73.4, 73.2,
73.1, 72.8, 72.1, 71.8, 71.0, 68.4, 68.4, 68.2, 67.8, 56.5, 21.2 ppm.
MS: m/z = 1940.8 [M + Na]+. NMR spectroscopic data are consis-
tent with literature values.[7i]
Benzyl 3,4,6-Tri-O-benzyl-β-
benzyl-2-deoxy-2-phthalimido-β-
O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-β-
D
-galactopyranosyl-(1Ǟ4)-3,6-di-O-
-glucopyranosyl-(1Ǟ3)-2,4,6-tri-
-gluco-
D
D
D
pyranoside (25): Compound 22 (1.55 g, 0.81 mmol) was dissolved
in CH2Cl2 (10 mL), and a solution of NaOMe [from Na (40 mg,
1.74 mmol) and MeOH (100 mL)] was added. The mixture was
stirred at room temperature for 48 h, then it was quenched with
Amberlite IR 120 H+. It was stirred for an additional 2 h, then it
was filtered and concentrated in vacuo. The residue was purified
by flash column chromatography (EtOAc/heptane, 1:3) to give 25
1
(1.23 g, 81%) as a colorless oil. H NMR (500 MHz, CDCl3): δ =
7.49–6.77 (m, 64 H), 5.37 (d, J = 8.4 Hz, 1 H), 5.06 (d, J = 11.4 Hz,
1 H), 4.92–4.81 (m, 5 H), 4.73–4.67 (m, 2 H), 4.65–4.60 (m, 2 H),
4.58–4.44 (m, 8 H), 4.38 (d, J = 12.3 Hz, 1 H), 4.35–4.11 (m, 10
H), 4.08 (dd, J = 11.3, 3.5 Hz, 1 H), 4.02–3.81 (m, 6 H), 3.72 (ddd,
J = 9.9, 3.2, 1.9 Hz, 1 H), 3.59–3.26 (m, 13 H), 2.92 (ddd, J = 9.9,
3.6, 1.7 Hz, 1 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 139.5,
139.2, 139.0, 138.9, 138.8, 138.7, 138.5, 138.5, 138.3, 138.0, 138.0,
137.7, 133.5, 128.6, 128.5, 128.5, 128.4, 128.4, 128.4, 128.3, 128.3,
128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.8, 127.8, 127.7, 127.7,
127.6, 127.5, 127.4, 127.3, 127.2, 126.9, 126.8, 126.5, 123.2, 103.6,
102.6, 102.5, 100.0, 83.1, 82.1, 82.0, 81.8, 79.0, 78.4, 78.1, 76.8,
76.1, 75.6, 75.2, 75.2, 74.9, 74.8, 74.7, 74.6, 74.1, 73.8, 73.6, 73.6,
73.5, 73.2, 73.1, 72.9, 72.4, 72.3, 70.9, 68.8, 68.4, 68.4, 67.8,
56.6 ppm. HRMS: calcd. for C116H117NO22 [M + Na]+ 1898.7959;
found 1898.7934.
Phenyl 2-O-Acetyl-3,4,6-tri-O-benzyl-β-
4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
D
-galactopyranosyl-(1Ǟ3)-
-glucopyranoside
D
(23): Synthesized by the general NIS/AgOTf procedure using 15
(150 mg, 0.26 mmol) and 20 (167 mg, 0.31 mmol). Purified by dry
column chromatography (acetone/toluene, 0 to 50 % in steps of
2.5 %) to give 23 (171 mg, 62 %) as a colorless oil. 1H NMR
(300 MHz, CDCl3): δ = 7.94–7.72 (m, 4 H), 7.48–7.01 (m, 30 H),
5.44 (d, J = 10.5 Hz, 1 H), 5.31 (dd, J = 10.1, 7.9 Hz, 1 H), 5.04
(d, J = 10.3 Hz, 1 H), 4.92 (d, J = 11.3 Hz, 1 H), 4.85–4.73 (m, 1
H), 4.63 (d, J = 12.0 Hz, 1 H), 4.60–4.18 (m, 8 H), 4.14 (d, J =
7.9 Hz, 1 H), 3.88 (d, J = 2.7 Hz, 1 H), 3.85–3.77 (m, 2 H), 3.71–
3.60 (m, 2 H), 3.48 (t, J = 10.0 Hz, 1 H), 3.40–3.30 (m, 2 H), 3.10
(dd, J = 10.1, 2.7 Hz, 1 H), 2.02 (s, 3 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 169.9, 168.6, 167.2, 138.7, 138.4, 138.3, 138.0, 137.8,
134.4, 132.6, 132.0, 129.1, 128.9, 128.5, 128.4, 128.4, 128.3, 128.2,
128.0, 128.0, 128.0, 127.8, 127.7, 127.6, 127.5, 127.3, 127.2, 125.4,
123.6, 100.8, 83.7, 80.2, 79.4, 77.6, 76.7, 75.2, 74.7, 73.5, 73.5, 73.4,
72.6, 71.5, 71.3, 69.1, 67.7, 55.1, 21.0 ppm. HRMS: calcd. for
C63H61NO12S [M + Na]+ 1078.3807; found 1078.3820.
Benzyl 2,3,4-Tri-O-benzyl-α-
benzyl-β- -galactopyranosyl-(1Ǟ4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-β- -glucopyranosyl-(1Ǟ3)-2,4,6-tri-O-benzyl-β-
galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-β- -glucopyranoside
L-fucopyranosyl-(1Ǟ2)-3,4,6-tri-O-
D
D
D-
D
(27): NIS (112 mg, 0.50 mmol) and TESOTf (13 mg, 0.049 mmol)
were added to a mixture of 25 (711 mg, 0.38 mmol), 26 (253 mg,
0.48 mmol), and 4 Å MS in CH2Cl2 (7 mL) at –30 °C. The mixture
was stirred for 20 min, then the reaction was quenched with Et3N.
The mixture was stirred for 15 min, then it was filtered through
Celite and concentrated in vacuo. Purification by flash column
chromatography (acetone/toluene, 1:24) gave 27 (657 mg, 89%) as
1
a colorless oil. H NMR (400 MHz, CDCl3): δ = 7.69 (br. s, 1 H),
7.52 (br. s, 1 H), 7.39–7.04 (m, 70 H), 6.93–6.80 (m, 5 H), 6.75 (t,
J = 7.3 Hz, 2 H), 5.73 (d, J = 3.8 Hz, 1 H), 5.38 (d, J = 7.9 Hz, 1
H), 5.10 (d, J = 11.4 Hz, 1 H), 4.97 (d, J = 11.5 Hz, 1 H), 4.94–
4.16 (m, 34 H), 4.15–3.99 (m, 4 H), 3.95 (d, J = 2.5 Hz, 1 H), 3.94–
3.75 (m, 5 H), 3.64 (dd, J = 9.7, 2.8 Hz, 1 H), 3.59 (dd, J = 9.8,
Benzyl 2-O-Acetyl-3,4,6-tri-O-benzyl-β-
4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-
2,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-
β- -glucopyranoside (24): Synthesized by the general NIS/TESOTf
D
-galactopyranosyl-(1Ǟ3)-
D
-glucopyranosyl-(1Ǟ3)-
D
D
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
C. Arboe Jennum, T. Hauch Fenger, L. M. Bruun, R. Madsen
FULL PAPER
2.9 Hz, 1 H), 3.57–3.29 (m, 12 H), 3.00–2.90 (m, 1 H), 1.39 (d, J
= 6.5 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 139.5,
139.1, 138.9, 138.9, 138.8, 138.7, 138.6, 138.6, 138.5, 138.4, 138.2,
138.2, 138.1, 137.7, 133.6, 131.3, 128.6, 128.6, 128.5, 128.4, 128.4,
128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1,
128.0, 129.0, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5,
127.4, 127.3, 127.3, 127.2, 127.0, 126.9, 126.5, 126.3, 123.2, 102.5,
102.5, 100.9, 100.2, 97.7, 84.1, 83.1, 82.5, 81.8, 79.4, 78.9, 78.2,
76.8, 76.8, 76.7, 76.1, 75.8, 75.8, 75.6, 75.3, 75.2, 75.0, 75.0, 74.7,
74.5, 74.3, 73.9, 73.7, 73.6, 73.5, 73.3, 73.2, 73.1, 72.7, 72.4, 72.3,
71.0, 70.9, 68.8, 68.5, 68.3, 67.8, 66.6, 56.5, 17.0 ppm. MS: m/z =
2314.8 [M + Na]+. NMR spectroscopic data are consistent with
literature values.[7i]
ppm. 13C NMR (100 MHz, CDCl3): δ = 171.2, 139.4, 139.4, 139.2,
139.0, 138.7, 138.6, 138.5, 138.3, 138.2, 138.2, 137.6, 128.7, 128.7,
128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.0,
128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6,
127.6, 127.5, 127.4, 127.3, 127.3, 126.5, 104.3, 102.6, 102.5, 101.5,
83.0, 81.8, 81.7, 81.4, 81.1, 80.8, 76.7, 76.5, 76.4, 75.6, 75.2, 75.2,
75.1, 75.1, 74.8, 74.8, 74.6, 73.9, 73.6, 73.6, 73.6, 73.6, 73.5, 73.5,
72.5, 71.8, 71.0, 69.4, 68.6, 68.4, 68.2, 56.1, 23.2 ppm. HRMS:
calcd. for C110H117NO21 [M + Na]+ 1810.8010; found 1810.7994.
Benzyl 2,3,4-Tri-O-benzyl-α-
benzyl-β-D-galactopyranosyl-(1Ǟ3)-2-acetamido-4,6-di-O-benzyl-2-
deoxy-β- -glucopyranosyl-(1Ǟ3)-2,4,6-tri-O-benzyl-β- -galacto-
pyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-β- -glucopyranoside (30): NIS
L-fucopyranosyl-(1Ǟ2)-3,4,6-tri-O-
D
D
D
Benzyl 2,3,4-Tri-O-benzyl-α-
L
-fucopyranosyl-(1Ǟ2)-3,4,6-tri-O-
-galactopyranosyl-(1Ǟ4)-2-acetamido-3,6-di-O-benzyl-2-
-glucopyranosyl-(1Ǟ3)-2,4,6-tri-O-benzyl-β- -galacto-
-glucopyranoside (28): A
(17 mg, 0.077 mmol) and TESOTf (1.9 mg, 0.0077 mmol) were
added to a mixture of 29 (100 mg, 0.056 mmol), 26 (38 mg,
0.073 mmol), and 4 Å MS in CH2Cl2 (1.5 mL) at –20 °C. The mix-
ture was left to reach 10 °C, then the reaction was quenched with
Et3N. The mixture was stirred for 15 min, then it was filtered
through Celite and concentrated in vacuo. Purification by flash col-
umn chromatography (deactivated with Et3N; acetone/toluene, 1:9)
gave 30 (84 mg, 68 %) as a colorless oil. 1H NMR (400 MHz,
CDCl3): δ = 7.43–6.96 (m, 75 H), 5.60 (d, J = 3.7 Hz, 1 H), 5.06
(d, J = 11.1 Hz, 2 H), 4.99 (d, J = 10.6 Hz, 1 H), 4.95–4.34 (m, 31
H), 4.26 (d, J = 11.8 Hz, 1 H), 4.19 (dd, J = 9.5, 7.6 Hz, 1 H), 4.05
(dd, J = 10.5, 2.5 Hz, 1 H), 4.02–3.91 (m, 5 H), 3.89 (d, J = 1.4 Hz,
1 H), 3.87–3.37 (m, 18 H), 3.33–3.22 (m, 1 H), 1.63 (s, 3 H), 1.20
(d, J = 6.5 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 169.3,
139.7, 139.6, 139.3, 139.2, 138.9, 138.7, 138.7, 138.4, 138.3, 138.3,
138.1, 137.6, 129.0, 128.8, 128.6, 128.5, 128.5, 128.4, 128.3, 128.3,
128.2, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.9,
127.8, 127.8, 127.7, 127.6, 127.6, 127.5, 127.3, 127.2, 127.2, 127.1,
126.4, 126.3, 102.7, 102.5, 102.0, 101.6, 98.2, 84.0, 83.0, 81.8, 81.5,
80.2, 79.3, 78.1, 78.0, 77.5, 77.2, 77.1, 76.8, 76.6, 76.4, 75.7, 75.6,
75.3, 75.3, 75.2, 75.1, 75.1, 75.0, 74.8, 74.7, 74.1, 73.7, 73.6, 73.6,
73.5, 73.5, 73.4, 72.8, 72.4, 72.0, 71.2, 71.0, 69.5, 68.9, 68.4, 68.3,
66.4, 55.8, 23.6, 16.8 ppm. HRMS: calcd. for C137H145NO25 [M +
H]+ 2205.0178; found 2205.0132.
benzyl-β-
D
deoxy-β-
D
D
pyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-β-
D
mixture of 27 (475 mg, 0.207 mmol) and ethylenediamine (5.7 mL,
0.085 mmol) in tBuOH (25 mL) was stirred at 100 °C for 16 h. The
volatiles were removed in vacuo, and the residue was coevaporated
with toluene (2ϫ 10 mL) and ethanol (10 mL). The residue was
dissolved in CH2Cl2 (6 mL), and acetic anhydride (2 mL,
0.021 mol), Et3N (4 mL), and DMAP (4-dimethylaminopyridine;
25 mg, 0.21 mmol) were added at 0 °C. The mixture was stirred at
room temperature for 15 h. The residue was diluted with EtOAc
(50 mL), then washed with saturated aqueous NaHCO3 (2 ϫ
30 mL) and H2O (40 mL), dried with MgSO4, and concentrated in
vacuo. The residue was purified by flash column chromatography
(deactivated with Et3N; EtOAc/toluene, 1:9) to give 28 (336 mg,
74%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ = 7.44–
7.00 (m, 75 H), 5.71 (d, J = 3.6 Hz, 1 H), 5.10 (d, J = 8.3 Hz, 1
H), 5.05–4.97 (m, 2 H), 4.96–4.87 (m, 5 H), 4.83–4.71 (m, 5 H),
4.70–4.17 (m, 24 H), 4.07–3.91 (m, 5 H), 3.86 (d, J = 10.4 Hz, 1
H), 3.81–3.33 (m, 19 H), 3.30–3.23 (m, 1 H), 1.47 (s, 3 H), 1.27 (d,
J = 6.4 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 170.1,
139.5, 139.2, 139.1, 138.9, 138.9, 138.8, 138.7, 138.6, 138.5, 138.3,
138.2, 138.0, 137.7, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1,
128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6,
127.5, 127.4, 127.4, 127.3, 127.2, 126.9, 126.3, 102.7, 102.6, 101.8,
101.1, 97.6, 84.1, 83.0, 81.9, 81.9, 80.1, 79.4, 78.5, 78.1, 77.5, 77.2,
76.8, 76.6, 76.4, 75.9, 75.8, 75.7, 75.6, 75.3, 75.2, 75.0, 75.0, 74.8,
74.7, 74.3, 73.7, 73.6, 73.6, 73.5, 73.3, 73.3, 73.2, 72.7, 72.5, 72.4,
71.2, 71.0, 68.9, 68.3, 68.3, 68.3, 66.5, 56.4, 23.3, 17.0 ppm. HRMS:
calcd. for C137H145NO25 [M + H]+ 2205.0178; found 2226.0173.
Benzyl 2-O-Benzoyl-3,4,6-tri-O-benzyl-β-
(1Ǟ4)-6-O-benzyl-2-deoxy-2-phthalimido-β-
(1Ǟ3)-2,4,6-tri-O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-
benzyl-β- -glucopyranoside (32): Synthesized by the general one-
D
-galactopyranosyl-
D
-glucopyranosyl-
D
D
pot glycosylation method using 13 (120 mg, 0.20 mmol), 31 (83 mg,
0.17 mmol), and 6 (164 mg, 0.17 mmol). Purified by flash column
chromatography (EtOAc/heptane, 1:2) to give 32 (183 mg, 57%) as
Benzyl 3,4,6-Tri-O-benzyl-β-
amido-4,6-di-O-benzyl-2-deoxy-β-
O-benzyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-benzyl-β-
D
-galactopyranosyl-(1Ǟ3)-2-acet-
-glucopyranosyl-(1Ǟ3)-2,4,6-tri-
-gluco-
1
a colorless oil. H NMR (400 MHz, CDCl3): δ = 8.09–7.94 (m, 2
D
H), 7.69–6.81 (m, 62 H), 5.65 (dd, J = 10.0, 8.0 Hz, 1 H), 5.35 (d,
J = 8.4 Hz, 1 H), 4.98 (d, J = 11.4 Hz, 1 H), 4.92 (d, J = 11.7 Hz,
1 H), 4.89–4.75 (m, 3 H), 4.68–4.36 (m, 11 H), 4.31–4.13 (m, 8 H),
4.12–3.99 (m, 3 H), 3.96–3.84 (m, 3 H), 3.84–3.75 (m, 1 H), 3.67–
3.25 (m, 17 H), 2.94–2.84 (m, 1 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 167.9, 165.2, 139.5, 139.1, 138.7, 138.6, 138.5, 138.4,
138.3, 138.1, 137.7, 137.4, 137.4, 134.6, 133.7, 133.5, 133.4, 131.5,
130.2, 130.1, 129.9, 129.9, 129.1, 128.6, 128.5, 128.5, 128.4, 128.4,
128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 127.9,
127.9, 127.9, 127.7, 127.7, 127.7, 127.6, 127.6, 127.5, 127.2, 127.2,
127.1, 126.9, 126.6, 123.2, 102.5, 102.4, 102.1, 99.7, 83.0, 82.3, 82.1,
81.7, 79.8, 78.9, 76.8, 76.0, 75.5, 75.1, 75.1, 74.8, 74.6, 74.2, 74.0,
73.8, 73.7, 73.3, 73.1, 73.1, 73.1, 72.2, 72.1, 71.8, 70.9, 69.4, 68.7,
68.7, 68.4, 67.7, 56.6 ppm. HRMS: calcd. for C116H115NO23 [M +
Na]+ 1912.7752; found 1912.7687.
D
D
pyranoside (29): A mixture of 22 (301 mg, 0.162 mmol) and ethyl-
enediamine (4.7 mL, 0.070 mmol) in tBuOH (20 mL) was stirred at
100 °C for 16 h. The volatiles were removed in vacuo, and the resi-
due coevaporated with toluene (2ϫ 10 mL) and ethanol (10 mL).
The residue was dissolved in ethanol (4.5 mL), and acetic anhydride
(1.5 mL, 0.016 mol) and Et3N (3 mL) were added at 0 °C. The mix-
ture was stirred at room temperature for 15 h. The residue was
diluted with EtOAc (40 mL), then washed with saturated aqueous
NaHCO3 (2ϫ 20 mL) and H2O (30 mL), dried with MgSO4, and
concentrated in vacuo. The residue was purified by flash column
chromatography (deactivated with Et3N; EtOAc/heptane, 2:3) to
give 29 (209 mg, 72 %) as a colorless oil. 1H NMR (400 MHz,
CDCl3): δ = 7.40–7.11 (m, 60 H), 5.09 (d, J = 8.6 Hz, 1 H), 5.04–
4.87 (m, 7 H), 4.80–4.32 (m, 19 H), 4.28–4.20 (m, 2 H), 4.15 (d, J
= 7.5 Hz, 1 H), 4.03–3.85 (m, 5 H), 3.85–3.58 (m, 9 H), 3.57–3.43 α-
L
-Fucopyranosyl-(1Ǟ2)-β-D-galactopyranosyl-(1Ǟ3)-2-acetamido-
(m, 6 H), 3.42–3.34 (m, 2 H), 3.34–3.27 (m, 2 H), 1.55 (s, 3 H)
2-deoxy-β- -glucopyranosyl-(1Ǟ3)-β-
D
D
-galactopyranosyl-(1Ǟ4)-β-
8
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
Synthesis of Human Milk Oligosaccharides
-glucopyranose (Lacto-N-fucopentaose I): A solution of 30 (30 mg,
[4]
[5]
R. M. Hickey, Int. Dairy J. 2012, 22, 141–146.
D
a) N. S. Han, T.-J. Kim, Y.-C. Park, J. Kim, J.-H. Seo, Biotech-
nol. Adv. 2012, 30, 1268–1278; b) H. A. Nguyen, T.-H. Nguyen,
D. Haltrich, J. Sci. Dev. 2012, 10, 693–706.
0.014 mmol) in THF (1 mL) and MeOH (1 mL) was flushed thor-
oughly with argon, and then Pd/C (5%; 16 mg, 0.0075 mmol) and
TFA (trifluoroacetic acid; 0.05 mL, 0.65 μmol) were added. The
mixture was stirred under a hydrogen atmosphere (balloon) at
room temperature for 16 h, then it was filtered through Celite, and
the filtrate was concentrated in vacuo. The residue was purified by
reverse-phase dry column chromatography (CH2Cl2/MeOH, 0 to
100% in steps of 10%) to give lacto-N-fucopentaose I (11.5 mg,
[6]
a) I. Kovacs, I. Bajza, M. Hederos, G. Dekany, S. Demko, N.
Khanzhin (Glycom A/S), WO 2013044928 A1, 2013; b) A. Ya-
mada, K. Hatano, T. Koyama, K. Matsuoka, Y. Esumi, D.
Terunuma, Carbohydr. Res. 2006, 341, 467–473; c) F. Roussel,
M. Takhi, R. R. Schmidt, J. Org. Chem. 2001, 66, 8540–8548;
d) P. Knuhr, J. Castro-Palomino, M. Grathwohl, R. R.
Schmidt, Eur. J. Org. Chem. 2001, 4239–4246; e) F. Roussel, L.
Knerr, R. R. Schmidt, Eur. J. Org. Chem. 2001, 2067–2073; f)
L. Knerr, R. R. Schmidt, Eur. J. Org. Chem. 2000, 2803–2808;
g) M. R. E. Aly, E.-S. I. Ibrahim, E.-S. H. E. El-Ashry, R. R.
Schmidt, Eur. J. Org. Chem. 2000, 319–326; h) M. R. E. Aly,
E.-S. I. Ibrahim, E.-S. H. E. El Ashry, R. R. Schmidt, Car-
bohydr. Res. 1999, 121–132; i) H. Shimizu, Y. Ito, O. Kanie, T.
Ogawa, Bioorg. Med. Chem. Lett. 1996, 6, 2841–2846; j) A.
Maranduba, A. Veyrières, Carbohydr. Res. 1986, 151, 105–119;
k) T. Takamura, T. Chiba, S. Tejima, Chem. Pharm. Bull. 1981,
29, 2270–2276; l) T. Takamura, T. Chiba, H. Ishihara, S. Te-
jima, Chem. Pharm. Bull. 1980, 28, 1804–1809.
a) Y. Hsu, X.-A. Lu, M. M. L. Zulueta, C.-M. Tsai, K. Lin,
S.-C. Hung, C.-H. Wong, J. Am. Chem. Soc. 2012, 134, 4549–
4552; b) J.-C. Lee, C.-Y. Wu, J. V. Apon, G. Siuzdak, C.-H.
Wong, Angew. Chem. Int. Ed. 2006, 45, 2753–2757; Angew.
Chem. 2006, 118, 2819–2823; c) K. R. Love, P. H. Seeberger, J.
Org. Chem. 2005, 70, 3168–3177; d) K. R. Love, R. B. Andrade,
P. H. Seeberger, J. Org. Chem. 2001, 66, 8165–8176; e) H. M.
Kim, I. J. Kim, S. J. Danishefsky, J. Am. Chem. Soc. 2001, 123,
35–48; f) A. Chernyak, S. Oscarson, D. Turek, Carbohydr. Res.
2000, 329, 309–316; g) S. Cao, Z. Gan, R. Roy, Carbohydr. Res.
1999, 318, 75–81; h) Y.-M. Zhang, J. Esnault, J.-M. Mallet, P.
Sinaÿ, J. Carbohydr. Chem. 1999, 18, 419–427; i) W. Bröder, H.
Kunz, Bioorg. Med. Chem. 1997, 5, 1–19; j) L. Manzoni, L.
Lay, R. R. Schmidt, J. Carbohydr. Chem. 1998, 17, 739–758; k)
L. Lay, L. Manzoni, R. R. Schmidt, Carbohydr. Res. 1998, 310,
157–171; l) A. Toepfer, W. Kinzy, R. R. Schmidt, Liebigs Ann.
Chem. 1994, 449–464.
1
98%) as an amorphous solid. H NMR (400 MHz, D2O): δ = 5.22
(d, J = 3.7 Hz, 0.4 H, 1α-H), 5.19 (d, J = 4.0 Hz, 1 H, 1ЈЈЈЈ-H),
4.70–4.58 (m, 2.6 H, 1β-H, 1ЈЈЈ-H, 1ЈЈ-H), 4.42 (d, J = 7.8 Hz, 1
H, 1Ј-H), 4.29 (q, J = 6.6 Hz, 1 H, 5ЈЈЈЈ-H), 4.14 (d, J = 3.2 Hz, 1
H, 4Ј-H), 4.04–3.44 (m, 27 H), 3.28 (t, J = 8.5 Hz, 0.6 H, 2β-H),
2.05 (s, 3 H), 1.23 (d, J = 6.6 Hz, 3 H) ppm. 13C NMR (100 MHz,
D2O): δ = 174.2, 103.2 (C-1ЈЈ), 102.9 (C-1Ј), 100.2 (C-1ЈЈЈ), 99.5
(C-1ЈЈЈЈ), 95.7 (C-1β), 91.8 (C-1α), 81.5, 78.2, 78.1, 77.1, 76.6, 75.2,
75.0, 74.8, 74.3, 73.8, 73.5, 71.8, 71.3, 71.1, 70.2, 69.4, 69.1, 68.6,
68.4, 68.0, 66.5, 61.1, 60.9, 60.4, 60.0, 55.9, 22.1, 15.2 ppm. MS:
m/z = 860.6 [M + Li]+. 1H NMR spectroscopic data are consistent
with literature values.[21]
[7]
α-
2-deoxy-β-
-glucopyranose (Lacto-N-neofucopentaose I): A solution of 28
L
-Fucopyranosyl-(1Ǟ2)-β-
D
-galactopyranosyl-(1Ǟ4)-2-acetamido-
D
-glucopyranosyl-(1Ǟ3)-β-
D
-galactopyranosyl-(1Ǟ4)-β-
D
(140 mg, 0.064 mmol) in THF (5 mL) and MeOH (5 mL) was
flushed thoroughly with argon, and then Pd/C (5%; 78 mg,
0.037 mmol) and TFA (0.1 mL, 0.0013 mmol) were added. The
mixture was stirred under a hydrogen atmosphere (balloon) at
room temperature for 16 h, then it was filtered through Celite, and
the filtrate was concentrated in vacuo. The residue was purified by
reverse-phase dry column chromatography (CH2Cl2/MeOH, 0 to
100% in steps of 10%) to give lacto-N-neofucopentaose I (53 mg,
1
99%) as an amorphous solid. H NMR (400 MHz, D2O): δ = 5.30
(d, J = 2.6 Hz, 1 H, 1ЈЈЈЈ-H), 5.22 (d, J = 3.7 Hz, 0.4 H, 1α-H),
4.70 (d, J = 8.4 Hz, 1 H, 1ЈЈ-H), 4.66 (d, J = 8.0 Hz, 0.6 H, 1β-H), [8]
4.55 (d, J = 7.8 Hz, 1 H, 1ЈЈЈ-H), 4.44 (d, J = 7.8 Hz, 1 H, 1Ј-H),
4.21 (q, J = 6.6 Hz, 1 H, 5ЈЈЈЈ-H), 4.14 (d, J = 3.2 Hz, 1 H, 4Ј-H),
4.01–3.54 (m, 25 H), 3.50–3.42 (m, 1 H), 3.32–3.23 (m, 0.6 H, 2β-
H), 2.04 (s, 3 H), 1.22 (d, J = 6.6 Hz, 3 H, 6ЈЈЈЈ-H) ppm. 1H NMR
spectroscopic data are consistent with literature values.[22] 13C
a) D. Schmidt, J. Thiem, Beilstein J. Org. Chem. 2010, 6, no.
18; b) C.-H. Hsu, K.-C. Chu, Y.-S. Lin, J.-L. Han, Y.-S. Peng,
C.-T. Ren, C.-Y. Wu, C.-H. Wong, Chem. Eur. J. 2010, 16,
1754–1760; c) P. K. Mandal, A. K. Misra, Tetrahedron 2008,
64, 8685–8691; d) A. A. Sherman, O. N. Yudina, Y. V. Mironov,
E. V. Sukhova, A. S. Shashkov, V. M. Menshov, N. E. Nifan-
tiev, Carbohydr. Res. 2001, 336, 13–46.
a) C.-H. Hsu, S.-C. Hung, C.-Y. Wu, C.-H. Wong, Angew.
Chem. Int. Ed. 2011, 50, 11872–11923; Angew. Chem. 2011,
123, 12076–12129; b) Y. Wang, X.-S. Ye, L.-H. Zhang, Org.
Biomol. Chem. 2007, 5, 2189–2200; c) J. D. C. Codée,
R. E. J. N. Litjens, L. J. van den Bos, H. S. Overkleeft, G. A.
van der Marel, Chem. Soc. Rev. 2005, 34, 769–782; d) B. Yu,
Z. Yang, H. Cao, Curr. Org. Chem. 2005, 9, 179–194.
a) T. H. Fenger, R. Madsen, Eur. J. Org. Chem. 2013, 5923–
5933; b) T. H. Schmidt, R. Madsen, Eur. J. Org. Chem. 2007,
3935–3941.
NMR (100 MHz, D2O): δ = 174.9, 102.9 (C-1Ј), 102.7 (C-1ЈЈ),
[9]
100.2 (C-1ЈЈЈ), 99.4 (C-1ЈЈЈЈ), 95.7 (C-1β), 91.8 (C-1α), 81.9, 78.3,
78.2, 76.4, 75.8, 75.2, 75.1, 74.8, 74.8, 74.3, 73.8 (C-2β), 73.5, 72.0,
71.6, 71.4, 71.1, 70.1, 70.0, 69.6, 69.1, 68.3 (C-4Ј), 68.2, 66.9 (C-
5ЈЈЈЈ), 61.1, 60.9, 60.0, 60.0, 55.4, 22.2, 15.3 ppm. MS: m/z = 860.6
1
[M + Li]+. H NMR spectroscopic data are consistent with litera-
ture values.[22]
[10]
Supporting Information (see footnote on the first page of this arti-
1
cle): Synthesis of thioglycoside 15 from 5, as well as copies of H
and 13C NMR spectra for all products.
[11]
[12]
A. K. Sarkar, R. K. Jain, K. L. Matta, Carbohydr. Res. 1990,
203, 33–46.
a) T. Kamkhachorn, A. R. Parameswar, A. V. Demchenko,
Org. Lett. 2010, 12, 3078–3081; b) H. D. Premathilake, L. K.
Mydock, A. V. Demchenko, J. Org. Chem. 2010, 75, 1095–1100.
a) K. Plé, Carbohydr. Res. 2003, 338, 1441–1454; b) M.-J. L.
Thijssen, K. M. Halkes, J. P. Kamerling, J. F. G. Vliegenhart,
Bioorg. Med. Chem. 1994, 2, 1309–1317.
M. L. Bohn, M. I. Colombo, P. L. Pisano, C. A. Stortz, E. A.
Rúveda, Carbohydr. Res. 2007, 342, 2522–2536, and references
cited herein.
Acknowledgments
Financial support from The Danish Council for Strategic Research
is gratefully acknowledged. The authors thank Ms. Gyrithe Lanz
for performing the one-pot reaction shown in Scheme 6.
[13]
[14]
[15]
[1] L. Bode, Glycobiology 2012, 22, 1147–1162.
[2] O. Ballard, A. L. Morrow, Pediatr. Clin. North Am. 2013, 60,
49–74.
a) M. Kato, G. Hirai, M. Sodeoka, Chem. Lett. 2011, 40, 877–
879; b) Z. Li, J. C. Gildersleeve, J. Am. Chem. Soc. 2006, 128,
11612–11619.
[3] M. Chichlowski, J. B. German, C. B. Lebrilla, D. A. Mills,
Annu. Rev. Food Sci. Technol. 2011, 2, 331–351.
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
9

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
C. Arboe Jennum, T. Hauch Fenger, L. M. Bruun, R. Madsen
FULL PAPER
[16]
[21] H. Sabharwal, B. Nilsson, M. A. Chester, F. Lindh, G.
Grönberg, S. Sjöblad, A. Lundblad, Carbohydr. Res. 1988, 178,
145–154.
J. Watts, J. Jiménez-Barbero, A. Poveda, T. B. Grindley, Can.
J. Chem. 2003, 81, 364–375.
M. R. Pratt, C. R. Bertozzi, J. Am. Chem. Soc. 2003, 125,
6149–6159.
[17]
[22] a) S. Drouillard, H. Driguez, E. Samain, Angew. Chem. Int.
Ed. 2006, 45, 1778–1780; Angew. Chem. 2006, 118, 1810–1812;
b) T. Urashima, M. Arita, M. Yoshida, T. Nakamura, I. Arai,
T. Saito, J. P. Y. Arnould, K. M. Kovacs, C. Lydersen, Comp.
Biochem. Physiol. B 2001, 128, 307–323.
[18] C. M. Gampe, H. Tsukamoto, T.-S. A. Wang, S. Walker, D.
Kahne, Tetrahedron 2011, 67, 9771–9778.
[19] D. S. Pedersen, C. Rosenbohm, Synthesis 2001, 2431–2434.
[20] R. K. Jain, R. D. Locke, K. L. Matta, Carbohydr. Res. 1993,
241, 165–176.
Received: January 30, 2014
Published Online: ᭿
10
www.eurjoc.org
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O40164
/KAP1
Date: 03-04-14 17:19:32
Pages: 11
Synthesis of Human Milk Oligosaccharides
Oligosaccharide Synthesis
C. Arboe Jennum, T. Hauch Fenger,
L. M. Bruun, R. Madsen* ............... 1–11
One-Pot Glycosylations in the Synthesis of
Human Milk Oligosaccharides
Keywords: Carbohydrates
/
Oligosac-
charides / Glycosylation / Thioglycosides
A one-pot glycosylation strategy has been
developed for assembling the lactosamine–
lactose backbone in human milk oligosac-
charides, and applied in a concise synthesis
of lacto-N-neofucopentaose I.
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
11