The chemical synthesis of human milk oligosaccharides: Lacto-N-tetraose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc)

Article abstract of DOI:10.1016/j.carres.2019.107824

The total chemical synthesis of lacto-N-tetraose (LNT) has been completed using both convergent and linear strategies. Similarly to that of our previous HMO syntheses, the donor-acceptor protecting-leaving group combinations were found to be of paramount

Full text of DOI:10.1016/j.carres.2019.107824

Journal Pre-proof
The chemical synthesis of human milk oligosaccharides: Lacto-N-tetraose
(Galβ1→3GlcNAcβ1→3Galβ1→4Glc)
Mithila D. Bandara, Keith J. Stine, Alexei V. Demchenko
PII:
S0008-6215(19)30426-4
DOI:
https://doi.org/10.1016/j.carres.2019.107824
CAR 107824
Reference:
To appear in: Carbohydrate Research
Received Date: 17 July 2019
Revised Date: 21 September 2019
Accepted Date: 22 September 2019
Please cite this article as: M.D. Bandara, K.J. Stine, A.V. Demchenko, The chemical synthesis of human
milk oligosaccharides: Lacto-N-tetraose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc), Carbohydrate Research
(2019), doi: https://doi.org/10.1016/j.carres.2019.107824.
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The chemical synthesis of human milk oligosaccharides: lacto-N-
tetraose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc)
Mithila D. Bandara, Keith J. Stine, and Alexei V. Demchenko*
Department of Chemistry and Biochemistry, University of Missouri – St. Louis, One University Boulevard, St. Louis, Mis-
souri 63121, USA
Supporting Information Placeholder
S-benzoxazolyl (SBox) galactosyl donor 429,30 and glucosamine
ABSTRACT: The total chemical synthesis of lacto-N-tetraose
acceptor 5. SBox galactosyl donor 4 was very instrumental in
avoiding the unwanted aglycone transfer side reaction12,26 that
was taking place in other previously investigated building
blocks.25 As in our previous HMO synthesis, we chose benzyl
ethers as semi-permanent protecting groups.
(LNT) has been completed using both convergent and linear strat-
egies. Similarly to that of our previous HMO syntheses, the do-
nor-acceptor protecting-leaving group combinations were found
to be of paramount significance to achieving successful glycosyla-
tions and minimizing side reactions.
Scheme 1. Retrosynthesis analysis of LNT 1.
Carbohydrates are essential biomolecules that become our first
food.1 Oligosaccharides present in human milk (HMO) can supply
building blocks for the development of the infants’ cognition,2 act
as prebiotics3 and antimicrobials.4,5 Thanks to advances in glyco-
sciences, chemical structures of 162 HMO have been elucidated
to date,6,7 but our understanding of how HMO function is incom-
plete.8,9 All HMO are composed of five monosaccharides: includ-
ing glucose (Glc), galactose (Gal), N-acetylglucosamine (Glc-
NAc), fucose, and sialic acid.10 Many efforts to prepare HMO
enzymatically or chemically have been reported,11-13 and im-
portance of including HMO in infant formulas has been acknowl-
edged.14
The total amount and composition of HMO varies between
women and are dependent on maternal genetics, environment, and
geographic location.15 Lacto-N-tetraose (LNT) 1 represents one of
the most common and abundant core structures and is classified as
The synthesis of glucosamine thioglycoside acceptor 5 was
achieved from known building block 6 as depicted in Scheme 2.27
First, precursor 6 was reacted with tert-butyldimethylsilyl chloride
(TBDMSCl) in the presence of imidazole in DMF at 90 oC to
obtain compound 7 in 97% yield. The latter was then converted to
intermediate 8 via the reductive regioselective opening of the
benzylidene acetal by reaction with 1 M BH3 in THF in the pres-
ence of catalytic TMSOTf in 82% yield. Subsequently, 6-OH in
compound 8 was benzylated with BnBr in the presence of NaH in
DMF to afford compound 9 in 77% yield. In order to minimize
the formation of side products, temperature control is highly im-
portant in this reaction (see the experimental part for further de-
tails). Finally, the silyl group of compound 9 was removed with
BF3-Et2O in CH3CN at 0 oC to afford the desired glucosamine
thioglycoside acceptor 5 in 87% yield.
a
type
I
HMO.
It
comprises
a
Galβ1→3GlcNAcβ1→3Galβ1→4Glc sequence shown in Scheme
1. More specifically, LNT is a linear tetrasaccharide wherein the
reducing end lactose disaccharide (Galβ1→4Glc) is elongated
with lacto-N-biose disaccharide residue (Galβ1→3GlcNAc).
Chemical16-18 and enzymatic syntheses19 of LNT have been re-
ported, and several of its derivatives have been synthesized using
chemical synthesis in solution and on solid phase.20-24 Despite
being one of the most abundant HMO core structures in human
milk, LNT is not yet available in large quantities and at reasona-
ble prices for research and application.
Previously, we reported the total synthesis of lacto-N-
neotetraose that has been completed using both linear and conver-
gent approaches.25 Along the way, we developed the synthesis of
key building blocks, accessed scalability, and refined coupling
procedures to obtain different glycosidic linkages and sequences.
Notably, the donor and acceptor protecting/leaving group combi-
nations were found to be key parameters. In further synthetic stud-
ies of HMO in our lab reported herein is the synthesis of LNT 1.
First, we decided to investigate a convergent (2+2) synthetic strat-
egy, according to which we chose to converge the protected lacto-
N-biose donor 2 and lactose acceptor 3.25 Based on our previous
synthetic endeavors and preliminary refinement of reaction condi-
tions,25 for the synthesis of disaccharide 2 we chose superarmed
We next turned our attention to the assembly of LNB disaccha-
ride 2. Selective activation of the SBox leaving group in glycosyl
donor 4 over thioethyl anomeric moiety of acceptor 5 was
achieved in the presence of silver trifluoromethanesulfonate
(AgOTf) to afford β-linked disaccharide 2 in excellent yield of
99%. Direct coupling of disaccharide 2 with acceptor 3 was prov-
en inefficient, perhaps due to a fairly unreactive donor and accep-
tor combination. We consistently saw incomplete reactions lead-
ing to very low yields of the tetrasaccharide product. To bypass

this, we chose to employ the corresponding phosphate donor 10
that was obtained from thioglycoside 2 in 90% yield via an effi-
cient one-step protocol developed by Seeberger and co-workers.28
The coupling of phosphate donor 10 with lactose acceptor 3 in the
presence of TMSOTf was more successful, and tetrasaccharide 11
was obtained in a moderate yield of 51% with complete β-
stereoselectivity.
MeOH. Subsequent N-acetylation with acetic anhydride in MeOH
furnished tetrasaccharide intermediate 16 in 92% yield. Subse-
quently, benzyl ethers were hydrogenated the presence of 10%
Pd/C in wet ethanol to afford the target trisaccharide 1 in 81%
yield.
Scheme 3. The linear synthesis of tetrasaccharide 11 and
its deprotection to obtain LNT 1.
Scheme 2. Convergent synthesis of protected LNT 11.
OBn
FmocCl
O
5
BnO
LG
NPhth
LG = SEt
Py, CH2Cl2
2 h, rt
FmocO
12
13
LG = OPO(OBu)2
3
HOPO(OBu)2, NIS/TfOH
CH2Cl2, molec. sieves 3Å
0 oC, only 94%
TMSOTf, CH2Cl2
molec. sieves 3Å
-30 oC -> rt, 15 min,
only, 86%
BnO
O
OPico
OBn
OBn
O
O
BnO
OBn
BnO
RO
O
O
OBn
BzO
PhthN
14
R = Fmoc
R = H
4
15
30% Et3N/CH2Cl2
rt, 2 h, 89%
AgOTf, CH2Cl2
molec. sieves 3Å
-30 oC -> rt, 15 min
89%
11
i) NH2.NH2.H2O
MeOH, 80 oC, 48 h
ii) Ac2O, MeOH, rt, 16 h
92%
BnO
O
BnO
OH
O
OBn
OBn
O
OBn
O
OBn
BnO
O
BnO
O
O
BnO
HO
OBn
AcNH
HO
16
Pd/C, H2, EtOH
rt, 16 h
81%
HO
O
HO
OH
O
OH
OH
OH
O
OH
O
HO
O
HO
O
OH
O
HO
Despite numerous attempts involving modifying the reaction
condition, and replacing the glycosyl donor with O- and S-
imidoyl leaving groups, we failed to improve the outcome of this
reaction. We were also unable to elucidate structures of by-
products forming alongside the desired tetrasaccharide 11. Not
being satisfied with the outcome of the convergent synthesis, we
were curious to investigate whether the linear approach would be
more successful in achieving a better outcome. Only a minimal
strategic adjustment was required, and this involved conversion of
building block 5 into its 4-O-Fmoc derivative 12 that was subse-
quently transformed into phosphate donor 13. Glycosylation be-
tween donor 13 and lactose acceptor 3 in the presence of TMSOTf
afforded trisaccharide 14 in 86% yield. The Fmoc protecting
group was removed with 30% Et3N in CH2Cl2 and glycosylation
of the resulting trisaccharide acceptor 15 with SBox donor 4 in the
presence of AgOTf afforded the desired β-linked tetrasaccharide
11 in 89% yield. Overall, the three-step linear assembly of 11
involving glycosylation of acceptor 3 with glycosyl donor 13,
interim Fmoc deprotection, followed by glycosylation with donor
4 proceeded with 68% overall yield for the synthesis of tetrasac-
charide 11. In contrast, the convergent approach was much less
efficient, 45% over three steps, primarily due to the very low-
yielding last coupling step between disaccharides 3 and 10.
HO
AcHN
HO
1
Lacto-N-tetraose
In summary, the total synthesis of lacto-N-tetraose has been
completed using both linear and convergent synthesis approaches.
The linear approach was significantly more effective in this appli-
cation. Along the way, we have developed new synthetic proto-
cols for different glycosidic linkages. Notably, the donor and ac-
ceptor protecting group and the leaving group combinations were
found to be of paramount significance to successful glycosyla-
tions. The protecting groups in precursors used for the synthesis
of the key building block 3 were chosen to provide access to vari-
able glycosylation sites. In this application, 3’-OH acceptor 3 was
achieved via the Fmoc group removal,25 but the same precursors
could also be used to achieve 6’-OH via the OPico group removal,
or provide access to 3’,6’-diol for the synthesis of branched
HMO. Further synthetic studies of HMO are underway in our
laboratory. We expect that new methods for obtaining individual
HMO will boost practical applications of these important biomol-
ecules.
Experimental
General methods. The reactions were performed using com-
mercial reagents and the ACS grade solvents were purified and
dried according to standard procedures. Column chromatography
was performed on silica gel 60 (70-230 mesh) and Sephadex G-25
size exclusion resin, reactions were monitored by TLC on Kie-
selgel 60 F254. The compounds were detected by examination
With the key tetrasaccharide intermediate 11 we endeavored to
carry out its deprotection steps to obtain the target LNT tetrasac-
charide 1. Deprotection of the phthalimido and the ester groups
was performed in the presence of NH2NH2-H2O in refluxing

2
under UV light and by charring with 10% sulfuric acid in metha-
nol. Solvents were removed under reduced pressure at <40 oC.
CH2Cl2 was distilled from CaH2 directly prior to application. Mo-
lecular sieves (3Å), used for reactions, were crushed and activated
in vacuo at 390 °C during 8 h in the first instance and then for 2-3
h at 390 °C directly prior to application. AgOTf was co-
evaporated with toluene (3 x 10 mL) and dried in vacuo for 2-3 h
directly prior to application. Optical rotations were measured
4.80 (dd, 2H, J = 11.7 Hz, CH2Ph), 5.40 (d, 1H, J1,2 = 10.6 Hz,
H-1), 7.27-7.95 (m, 9H, aromatic) ppm; 13C NMR (75 MHz,
CDCl3): δ, -4.6, -4.0, 15.0, 17.7, 24.3, 25.7 (×3), 56.8, 62.0, 73.3,
74.7, 79.7 (×2), 81.2, 123.3, 123.7, 127.3 (×2), 127.6, 128.4 (×2),
131.7, 132.1, 134.3 (×2), 138.1, 167.6, 168.8 ppm; ESI TOF
LCMS [M+Na]+ calcd for C29H39NNaO6SSi 580.2165, found
580.2163.
Ethyl
4,6-di-O-benzyl-2-deoxy-2-phthalimido-3-O-tert-
butyldimethylsilyl-1-thio-β-D-glucopyranoside (9). NaH (0.51
g, 0.021 mmol) was added portionwise to a cooled (-20 C) solu-
tion of 8 (3.80 g, 7.06 mmol) in DMF (30 mL) and the resulting
mixture was stirred under argon at -20 C until gas evolution has
ceased. After that, BnBr (1.06 mL, 9.18 mmol) was added and the
resulting mixture was stirred for 6 h at -15 C. The reaction mix-
1
using a Jasco polarimeter. H NMR spectra were recorded at 300
MHz or 600 MHz, and 13C NMR spectra were recorded at 75
o
1
MHz or 151 MHz. The H chemical shifts are referenced to the
o
signal of the residual TMS (δH = 0.00 ppm) for solutions in CDCl3
or the signal of the residual D2O (δH = 4.79 ppm) for solutions in
D2O. The 13C chemical shifts are referenced to the central signal
of CDCl3 (δC = 77.16 ppm) for solutions in CDCl3 or the central
signal of CD3COCD3 δC = 29.84 ppm) for solutions in D2O. Accu-
rate mass spectrometry determinations were performed using
Agilent 6230 ESI TOF LCMS mass spectrometer.
o
ture was cooled to -40 oC and glacial acetic acid (~2 mL) was
added dropwise. The resulting mixture was allowed to attain rt,
then diluted with EtOAc (~500 mL) and washed with water (50
mL), sat. aq. NaHCO3 (50 mL) and water (2 x 50 mL). The or-
ganic phase was separated, dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (ethyl acetate - hexane gradient elution) to afford the
title compound as a colorless syrup in 77% yield (3.65 g, 5.42
mmol). Analytical data for 9: Rf = 0.80 (ethyl acetate/hexane,3/7,
v/v); [α]D23 +39.3 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): δ,
-0.36, 0.00 (2 s, 6H, 2 x SiCH3), 0.79 (s, 9H, SitBu), 1.26 (t, 3H, J
= 7.4 Hz, CH2CH3), 2.73 (m, 2H, CH2CH3), 3.63-3.71 (m, 2H, H-
4, 5), 3.77-3.86 (br d, 2H, H-6a, 6b), 4.35 (dd, 1H, J2,23 = 10.0 Hz,
Preparation of monosaccharide building blocks
Ethyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-
2-phthalimido-1-thio-β-D-glucopyranoside (7).
TBDMSCl
(0.37 g, 2.44 mmol) and imidazole (0.16 g, 2.44 mmol) were add-
ed to
a
solution of ethyl 4,6-O-benzylidene-2-deoxy-2-
phthalimido-1-thio-β-D-glucopyranoside27 (6, 0.54 g, 1.22 mmol)
o
in DMF (7.0 mL) and the resulting mixture was heated at 90 C
for 3 h. After that, the reaction mixture was cooled to rt, diluted
with CH2Cl2 (~250 mL) and washed with water (40 mL), sat. aq.
NaHCO3 (40 mL), and water (40 mL). The organic phase was
separated, dried over MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(ethyl acetate - hexane gradient elution) to afford the title com-
H-2), 4.58 (dd, 1H, J3,4 = 7.6 Hz, H-3), 4.64 (d, 2H, J = 9.6 Hz,
2
CH2Ph), 4.79 (dd, 2H, J = 12.0 Hz, CH2Ph), 5.38 (d, 1H, J1,2
=
10.5 Hz, H-1), 7.21-7.99 (m, 14H, aromatic) ppm; 13C NMR (75
MHz, CDCl3): δ, -4.6, -4.1, 15.1, 17.7, 23.9, 25.8 (×3), 56.8, 69.0,
73.4, 73.5, 74.6, 79.5, 80.0, 80.8, 123.2, 123.7, 127.1 (×2), 127.4,
127.6, 127.8 (×2), 128.4 (×4), 131.8, 132.2, 134.3 (×2), 138.3,
138.4, 167.7, 168.8 ppm; ESI TOF LCMS [M+Na]+ calcd for
C36H45NNaO6SSi 670.2635, found 670.2633.
pound as a white form in 97% yield (0.65 g, 1.18 mmol). Analyti-
23
cal data for 7: Rf = 0.60 (ethyl acetate/hexane, 3/7, v/v); [α]D
-
2.5 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): δ, -0.28, -0.12 (2
s, 6H, 2 x SiCH3), 0.59 (s, 9H, SitBu), 1.19 (t, 3H, J = 7.4 Hz,
CH2CH3), 2.69 (m, 2H, CH2CH3), 3.59 (t, 1H, J4,5 = 8.8 Hz, H-4),
3.71 (m, 1H, J5,6a = 10.0 Hz, J5,6b = 4.4 Hz, H-5), 3.81 (dd, 1H,
J6a,6b = 10.0 Hz, H-6a), 4.32 (dd, 1H, J2,3 = 9.6 Hz, H-2), 4.39 (dd,
Ethyl
4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-
glucopyranoside (5). BF3-Et2O (1.05 mL, 8.29 mmol) was added
to a solution of 9 (5.07 g, 7.54 mmol) in dry CH3CN (90 mL) and
the resulting mixture was stirred under argon for 20 min at 0 C.
o
1H, H-6b), 4.67 (dd, 1H, J3,4 = 8.8 Hz, H-3), 5.37 (d, 1H, J1,2
=
10.7 Hz, H-1), 5.54 (s, 1H, CHPh), 7.31-7.94 (m, 9H, aromatic)
ppm; 13C NMR (75 MHz, CDCl3): δ, -5.1, -4.0, 15.0, 17.8, 24.2,
25.5 (×3), 56.7, 68.8, 70.6, 70.7, 81.9, 82.8, 102.1, 123.3, 123.8,
126.5 (×2), 128.3 (×2), 129.2, 131.7, 131.9, 134.3, 134.4, 137.2,
167.7, 168.4 ppm; ESI TOF LCMS [M+Na]+ calcd for
C29H37NNaO6SSi 578.2009, found 578.1997.
After that, the reaction was quenched with sat. aq. NaHCO3 (5
mL), and the volatiles were removed in vacuo. The residue was
diluted with CH2Cl2 (~500 mL) and washed with brine (2 x 50
mL). The organic phase was separated, dried over MgSO4, and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (ethyl acetate - hexane gradient elution)
to afford the title compound as a white amorphous solid in 87%
yield (3.49 g, 6.54 mmol). Analytical data for 5: Rf = 0.30 (ethyl
Ethyl
4-O-benzyl-2-deoxy-2-phthalimido-3-O-tert-
butyldimethylsilyl-1-thio-β-D-glucopyranoside (8). A 1 M
solution of BH3 in THF (43 mL, 43 mmol) was added to a solu-
tion of 7 (4.80 g, 8.66 mmol) in CH2Cl2 (45 mL). The resulting
solution was cooled to 0 oC, TMSOTf (0.78 mL, 4.33 mmol) was
added, and the resulting mixture was stirred for 5 h while the reac-
tion temperature was allowed to gradually increase to rt. After
that, the reaction was quenched with Et3N (~2 mL) and MeOH
(~5 mL), and the volatiles were removed in vacuo. The residue
was diluted with CH2Cl2 (~500 mL), washed with sat. aq. Na-
HCO3 (50 mL) and water (2 x 50 mL). The organic phase was
separated, dried over MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(ethyl acetate - hexane gradient elution) to afford the title com-
pound as a clear syrup in 82% yield (3.80 g, 7.06 mmol). Analyti-
22
1
acetate/hexane, 3/7, v/v); [α]D +7.3 (c 1.0, CHCl3); H NMR
(300 MHz, CDCl3): δ, 1.20 (t, 3H, J = 7.4 Hz, CH2CH3). 2.36 (d,
1H, J = 4.5 Hz, OH), 2.67 (m, 2H, CH2CH3), 3.58-3.69 (m, 2H,
H-4, 5), 3.75-3.85 (m, 2H, H-6a, 6b), 4.24 (dd, 1H, J2,3 = 10.4 Hz,
H-2), 4.48 (m, 1H, H-3), 4.63 (dd, 2H, 2J = 12.1 Hz, CH2Ph), 4.70
2
(m, 2H, J = 11.5 Hz, CH2Ph), 5.29 (d, 1H, J1,2 = 10.4 Hz, H-1),
7.15-7.90 (m, 14H, aromatic) ppm; 13C NMR (75 MHz, CDCl3):
δ, 15.1, 24.1, 55.7, 69.0, 72.8, 73.6, 74.8, 79.3 (×2), 81.2, 123.4,
123.8, 127.8, 127.9 (×2), 128.0 (×2), 128.1, 128.5 (×2), 128.7
(×2), 131.7, 131.8, 134.2 (×2), 138.2 (×2), 168.1, 168.3 ppm; ESI
TOF LCMS [M+Na]+ calcd for C30H31NNaO6S 556.1770, found
556.1767.
Ethyl
4,6-di-O-benzyl-2-deoxy-3-O-
23
cal data for 8: Rf = 0.80 (ethyl acetate/hexane, 2/3, v/v); [α]D
fluorenylmethoxycarbonyl-2-phthalimido-1-thio-β-D-
+27.4 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): δ, -0.38, 0.00
(2 s, 6H, 2 x SiCH3), 0.76 (s, 9H, SitBu), 1.20 (t, 3H, J = 7.4 Hz,
CH2CH3), 2.21 (m, 1H, OH), 2.68 (m, 2H, CH2CH3), 3.52-3.67
(m, 2H, H-4, 5), 3.73 (m, 1H, H-6a), 3.94 (m, 1H, H-6b), 4.27
(dd, 1H, J2,3 = 10.6 Hz, H-2), 4.56 (dd, 1H, J3,4 = 8.0 Hz, H-3),
glucopyranoside (12). FmocCl (3.88 g, 15.04 mmol) was added
to a solution of 5 (2.08 g, 3.90 mmol) in CH2Cl2 (50 mL) and pyr-
idine (1.72 mL) and the resulting mixture was stirred under argon
for 2 h at rt. After that, the reaction mixture was diluted with
CH2Cl2 (~500 mL) and washed with 1 M aq. HCl (50 mL) and

water (2 x 50 mL). The organic phase was separated, dried over
MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (ethyl acetate - hexane gra-
dient elution) to afford the title compound as a white amorphous
solid in 92% yield (2.70 g, 3.58 mmol). Analytical data for 12: Rf
purified by column chromatography on silica gel (acetone - tolu-
ene gradient elution) to afford the title compound as a white foam
in 99% yield (0.23 g, 0.22 mmol). Analytical data for 2: Rf = 0.55
23
1
(acetone/toluene, 1/9 v/v); [α]D +34.1 (c 1.0, CHCl3); H NMR
(300 MHz, CDCl3): δ, 1.09 (t, 3H, J = 7.4 Hz, CH2CH3). 2.56 (m,
2H, CH2CH3), 3.30-3.51 (m, 4H, H-3ʹ, 5ʹ, 6aʹ, 6bʹ), 3.57-3.72 (m,
2H, H-4, 5), 3.77 (br d, 2H, J = 2.1 Hz, H-6a, 6b), 3.94 (br d, 1H,
J3ʹ,4ʹ = 2.5 Hz, H-4ʹ), 4.26 (dd, 2H, 2J = 11.6 Hz, CH2Ph), 4.29 (d,
1H, J = 12.0 Hz, CHPh), 4.30 (dd, 1H, J2,3 = 10.3, H-2), 4.44 (d,
1H, J1ʹ,2ʹ = 7.9 Hz, H-1ʹ), 4.454.64 (m, 5H, 5 × CHPh), 4.83 (dd,
1H, J3,4 = 8.0 Hz, H-3), 4.91 (d, 1H, J = 11.3 Hz, CHPh), 5.06 (d,
1H, J1,2 = 10.4 Hz, H-1), 5.09 (d, 1H, J = 10.5 Hz, CHPh), 5.52
(dd, 1H, J2ʹ,3ʹ = 9.9 Hz, H-2ʹ), 6.92-7.78 (m, 34H, aromatic) ppm;
13C NMR (75 MHz, CDCl3): δ, 14.9, 23.7, 54.8, 67.8, 69.2, 71.7,
72.5, 72.8, 73.3, 73.4, 73.5, 74.8, 75.0, 77.4, 77.8, 79.5, 80.3,
81.0, 100.7, 127.2, 127.5 (×3), 127.6 (×2), 127.8 (×4), 128.0 (×3),
128.1 (×6), 128.2 (×3), 128.3 (×3), 128.4 (×3), 128.5 (×3), 130.0
(×2), 130.3, 131.5, 132.8, 134.0, 137.5, 138.0, 138.3, 138.7 (×2),
165.4 ppm; ESI TOF LCMS [M+Na]+ calcd for C64H63NNaO12S
1092.3969, found 1092.3981.
23
= 0.40 (ethyl acetate/hexane,2/3, v/v); [α]D +55.2 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3): δ, 1.22 (t, 3H, J = 7.4 Hz,
CH2CH3), 2.70 (m, 2H, CH2CH3), 3.73-3.86 (m, 4H, H-5, 6a, 6b,
OCOCH2CH), 3.91-4.16 (m, 3H, H-4, OCOCH2CH), 4.47 (dd,
1H, J2,3 = 10.5 Hz, H-2), 4.62 (dd, 2H, 2J = 12.1 Hz, CH2Ph), 4.64
(dd, 2H, 2J = 11.2 Hz, CH2Ph), 5.46 (d, 1H, J1,2 = 10.5 Hz, H-1),
5.75 (dd, 1H, J3,4 = 8.9 Hz, H-3), 7.07-7.88 (m, 26H, aromatic)
ppm; 13C NMR (75 MHz, CDCl3): δ, 15.1, 24.2, 46.5, 54.1, 68.8,
70.3, 73.6, 75.0, 76.6, 78.5, 79.2, 81.0, 120.0 (×2), 123.7, 123.8,
125.1, 125.3, 127.3 (×2), 127.8 (×4), 127.9 (×4), 128.4 (×2),
128.5 (×2), 131.3, 131.8, 134.1, 134.4, 137.8, 138.2, 141.1, 141.2,
143.0, 143.3, 154.8, 167.5, 168.0 ppm; ESI TOF LCMS [M+Na]+
calcd for C45H41NNaO8S 778.2451, found 778.2451.
Di-O-butyl
4,6-di-O-benzyl-2-deoxy-3-O-
fluorenylmethoxycarbonyl-2-phthalimido-β-D-glucopyranosyl
phosphate (13). A mixture containing thioglycoside 12 (0.50 g,
0.66 mmol), dibutyl hydrogen phosphate (0.39 mL, 1.99 mmol),
and molecular sieves (3 Å, 1.0 g) in CH2Cl2 (10 mL) was stirred
under argon for 1h. The mixture was cooled to 0 °C, NIS (0.29 g,
1.32 mmol) and TfOH (10 µL, 0.13 mmol) were added, and the
resulting mixture was stirred under argon for 20 min at 0 °C. Af-
ter that, the solids were filtered off and washed successively with
CH2Cl2. The combined filtrate (~100 mL) was washed with 10%
aq. Na2S2O3 (15 mL), sat. aq. NaHCO3 (15 mL), and water (2 x
15 mL). The organic phase was separated, dried over MgSO4, and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (acetone - toluene gradient elution) to
afford the title compound as a white form in 94% yield (0.56 g,
0.62 mmol). Analytical data for 13: Rf = 0.45 (ethyl ace-
Di-O-butyl
O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-
galactopyranosyl)-(1‰3)-4,6-di-O-benzyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl phosphate (10). A mixture of
compound 2 (0.236 g, 0.234 mmol), dibutyl hydrogen phosphate
(0.14 mL, 0.802 mmol), and freshly activated molecular sieves (3
Å, 0.5 g) in CH2Cl2 (5.0 mL) was stirred under argon for 1 h at rt.
The mixture was cooled to 0 °C, NIS (0.104 g, 0.468 mmol) and
TfOH (4.15 µL, 0.047 mmol) were added, and the resulting mix-
ture was stirred for 20 min at 0 oC. After that, the solids were
filtered off and rinsed successively with CH2Cl2. The combined
filtrate (~100 mL) was washed with 10% aq. Na2S2O3 (15 mL)
and sat. aq. NaHCO3 (15 mL), and water (2 x 15 mL). The organic
phase was separated, dried over MgSO4, and concentrated in vac-
uo. The residue was purified by column chromatography on silica
gel (acetone - toluene gradient elution) to afford the title com-
pound as an oily syrup in 94% yield (0.267 g, 0.219 mmol). Ana-
23
tate/hexane,1/4, v/v); [α]D +43.0 (c 1.0, CHCl3); 1H NMR (300
MHz, CDCl3): δ, 0.71, 0.85 (2 t, 6H, 2 x O(CH2)3CH3), 1.07 (m,
2H, O(CH2)2CH2CH3), 1.22-1.37 (m, 4H, OCH2CH2CH2CH3),
1.48-1.60 (m, 2H, OCH2CH2CH2CH3), 3.67-4.17 (m, 11H, 2 x
OCH2CH2CH2CH3, H-4, 5, 6a, 6b, OCOCH2CH, OCOCH2CH),
23
lytical data for 10: Rf = 0.35 (acetone/toluene,1/9, v/v); [α]D
+26.2 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): δ, 0.67 (t, 3H,
J = 7.2 Hz, O(CH2)3CH3), 0.81 (t, 3H, J = 7.3 Hz, O(CH2)3CH3),
0.90-1.06 (m, 2H, O(CH2)2CH2CH3), 1.11-1.31 (m, 4H,
O(CH2)2CH2CH3, OCH2CH2CH2CH3), 1.38-1.53 (m, 2H,
OCH2CH2CH2CH3), 3.31-3.96 (m, 13H, H-3ʹ, 4, 4ʹ, 5, 5ʹ, 6a, 6b,
6aʹ, 6bʹ, 2 x OCOCH2(CH)2CH3), 4.20 (d, 1H, 2J = 11.7 Hz,
CHPh), 4.24-4.32 (m, 3H, H-2, 2 x CHPh), 4.43-4.54 (m, 5H, H-
1, 4 x CHPh), 4.59 (d, 1H, 2J = 12.0 Hz, CHPh), 4.91 (m, 2H, H-
2
4.49 (dd, 1H, J2,3 = 10.6 Hz, H-2), 4.60 (dd, 2H, J = 11.9 Hz,
2
CH2Ph), 4.64 (dd, 2H, J =11.2 Hz, CH2Ph), 5.80 (dd, 1H, J3,4
=
8.9 Hz, H-3), 6.02 (d, 1H, J1,2 = 8.3, H-1), 6.97-7.86 (m, 22H,
aromatic) ppm; 13C NMR (75 MHz, CDCl3): δ, 13.5, 13.6, 18.4,
18.6, 31.9 (d, J = 7.0 Hz), 32.0 (d, J = 7.2 Hz), 46.4, 55.3 (d, J =
8.9 Hz). 67.9 (d, J 6.1 Hz), 68.1 (d, J = 6.0 Hz), 70.3, 73.6, 74.9,
75.2, 76.0, 76.8, 77.3, 93.9 (d, J = 4.6 Hz), 120.0 (×2), 123.6,
125.0, 125.2, 125.4, 127.2 (×2), 127.7 (×3), 127.8 (×3), 127.9
(×3), 128.3, 128.4 (×3), 128.5 (×3), 129.1, 134.2, 137.6, 137.9,
141.1, 141.2, 143.0, 143.2, 154.6 ppm; ESI TOF LCMS [M+Na]+
calcd for C51H54NNaO12P 926.3281, found 926.3285.
3, CHPh), 5.09 (d, 1H, 2J = 10.5 Hz, CHPh), 5.51 (dd, 1H, J2ʹ,3ʹ
=
8.8 Hz, H-2ʹ), 5.69 (dd, 1H, J1,2 = 7.4 Hz, H-1) 6.94-7.74 (m, 36H,
aromatic) ppm; 13C NMR (75 MHz, CDCl3): δ, 13.5, 13.6, 18.3,
18.6, 31.8 (d, J = 7.1 Hz). 32.0 (d, J = 7.2 Hz), 56.1, 56.2, 67.6,
67.8 (d, J = 4.5 Hz), 68.0 (d, J = 6.4 Hz), 68.6, 71.7, 72.5, 72.8,
73.3, 73.5 (×2), 74.8, 74.9, 75.6, 76.2, 80.2, 94.1 (d, J = 4.6 Hz),
100.8, 123.5, 127.3, 127.5 (×2), 127.6 (×3), 127.7, 127.8 (×3),
128.0 (×7), 128.1 (×4), 128.2 (×4), 128.3 (×2), 128.4 (×2), 128.5
(×2), 130.0, 130.2, 131.5, 132.7, 134.0, 137.5, 138.0, 138.1,
138.6, 138.7, 165.3 ppm; ESI TOF LCMS [M+Na]+ calcd for
C70H76NNaO16P 1240.4799, found 1240.4829.
Synthesis of oligosaccharides
Ethyl
O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-
galactopyranosyl)-(1‰3)-4,6-di-O-benzyl-2-deoxy-2-
phthalimido-1-thio-β-D-glucopyranoside (2). A mixture of
benzoxazolyl
2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-β-D-
galactopyranoside29,30 (4, 0.20 g, 0.29 mmol), acceptor 5 (0.12 g,
0.22 mmol), and freshly activated molecular sieves (3Å, 600 mg)
in CH2Cl2 (7 mL) was stirred under argon for 2 h. The reaction
mixture was cooled to -30 oC, and freshly conditioned AgOTf
(0.15 g, 0.58 mmol) was added. The resulting mixture was stirred
for 15 min while the temperature was allowed to increase gradual-
ly. The reaction mixture was then diluted with CH2Cl2, the solids
were filtered off, and rinsed successively with CH2Cl2. The com-
bined filtrate (~50 mL) was washed with sat. aq. NaHCO3 (10
mL) and water (2 x 10 mL) The organic phase was separated,
dried over MgSO4, and concentrated in vacuo. The residue was
Benzyl
O-(4,6-di-O-benzyl-2-deoxy-3-O-
fluorenylmethoxycarbonyl-2-phthalimido-β-D-
glucopyranosyl)-(1‰3)-O-(2-O-benzoyl-4-O-benzyl-6-O-
picoloyl-β-D-galactopyranosyl)-(1‰4)-2,3,6-tri-O-benzyl-β-D-
glucopyranoside (14). A mixture of donor 13 (0.14 g, 0.15
mmol), acceptor 325 (0.12 g, 0.12 mmol) (reference from LNnT
paper), and freshly activated molecular sieves (3Å, 450 mg) in
CH2Cl2 (7.0 mL) was stirred under argon for 2 h. The mixture was
o
cooled to -30 C, TMSOTf (56 µL, 0.31 mmol) was added, and
the resulting mixture was stirred for 15 min while the temperature

was allowed to increase gradually. The reaction mixture was then
diluted with CH2Cl2, the solids were filtered off and rinsed suc-
cessively with CH2Cl2. The combined filtrate (~50 mL) was
washed with sat. aq. NaHCO3 (10 mL) and water (2 x 10 mL) The
organic phase was separated, dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (acetone - toluene gradient elution) to afford the title
compound as a white foam in 86% yield (0.17 g, 0.10 mmol).
benzyl-6-O-picoloyl-β-D-galactopyranosyl)-(1‰4)-2,3,6-tri-O-
benzyl-β-D-glucopyranoside (11). Convergent method. A mix-
ture of donor 10 (80.0 mg, 0.065 mmol), acceptor 3 (49.8 mg,
0.050 mmol),25 and freshly activated molecular sieves (3Å, 400
mg) in CH2Cl2 (7.0 mL) was stirred under argon for 2 h. The mix-
ture was cooled to -60 oC, TMSOTf (24 µL, 0.131 mmol) was
added, and the resulting mixture was stirred for 30 min while the
temperature was allowed to increase gradually. The reaction mix-
ture was then diluted with CH2Cl2, the solids were filtered off and
rinsed successively with CH2Cl2. The combined filtrate (~50 mL)
was washed with sat. aq. NaHCO3 (10 mL) and water (2 x 10 mL)
The organic phase was separated, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (acetone - toluene gradient elution) to afford
the title compound as an off-white amorphous solid in 51% yield
(66.6 mg, 0.033 mmol). Linear method. A mixture of benzoxa-
23
Analytical data for 14: Rf = 0.45 (acetone/toluene, 1/4 v/v); [α]D
1
+11.3 (c 1.0, CHCl3); H NMR (600 MHz, CDCl3): δ, 2.91 (ddd,
1H, J = 1.8, 3.0, 9.9 Hz, H-5), 3.27 (dd, 1H, J = 9.5 Hz, H-6a),
3.33 (dd, 1H, J2,3 = 8.4 Hz, H-2), 3.38-3.42 (m, 2H, H-3, 6b),
3.69-3.75 (m, 3H, H-3ʹ, 5ʹ, OCOCH2CH), 3.79-3.85 (m, 4H, H-4,
5ʹʹ, 6aʹʹ, 6bʹʹ), 3.87-3.93 (m, 2H, H-4ʹʹ, OCOCH2CH), 4.02 (dd,
1H, J = 10.5, 7.2 Hz, OCOCH2CH), 4.11 (br d, 1H, J = 2.4 Hz, H-
4ʹ), 4.18-4.24 (m, 2H, H-6aʹ, CHPh), 4.27 (d, 1H, J1,2 = 7.7 Hz, H-
1), 4.34-4.40 (m, 2H, H-2ʹʹ, 6bʹ), 4.42-4.49 (m, 3H, J1ʹ,2ʹ = 8.1 Hz,
zolyl
2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-β-D-
galactopyranoside29,30 (4, 13.3 mg, 0.0194 mmol), acceptor 15 (22
mg, 0.0149 mmol), and freshly activated molecular sieves (3Å,
100 mg) in CH2Cl2 (2.0 mL) was stirred under argon for 2 h. The
reaction mixture was cooled to -30 oC, freshly conditioned AgOTf
(10.0 mg, 0.0387 mmol) was added, and the resulting mixture was
stirred for 15 min while the temperature was allowed to increase
gradually. The reaction mixture was then diluted with CH2Cl2, the
solids were filtered off and was rinsed successively with CH2Cl2.
The combined filtrate (~30 mL) was washed with sat. aq. Na-
HCO3 (7 mL) and water (2 x 7 mL) The organic phase was sepa-
rated, dried over MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (acetone -
toluene gradient elution) to afford the title compound as a white
foam in 89% yield (26.7 mg, 0.0132 mmol). Analytical data for
2
H-1ʹ, 2 x CHPh), 4.5-4.69 (m, 7H, 7 x CHPh), 4.80 (d, 1H, J =
12.0 Hz, CHPh), 4.81 (d, 1H, 2J = 12.0 Hz, CHPh), 4.92 (d, 1H, 2J
2
= 10.4 Hz, CHPh), 5.12 (d, 1H, J = 11.5 Hz, CHPh), 5.36 (dd,
1H, J2ʹ,3ʹ = 10.1 Hz, H-2ʹ), 5.44 (d, 1H, J1ʹʹ,2ʹʹ = 8.3 Hz, H-1ʹʹ), 5.66
(dd, 1H, J = 8.9, 10.7 Hz, H-3ʹʹ), 6.81-8.73 (m, 56H, aromatic)
ppm; 13C NMR (151 MHz, cdcl3): δ, 46.4, 55.2, 63.8, 67.6, 68.9,
70.3, 71.0, 71.9, 72.0, 73.5 (×2), 73.6 (×2), 74.4, 74.7, 75.0 (×2),
75.2, 75.6, 76.2, 76.6, 80.6, 81.7, 82.6, 99.4, 100.4, 102.6, 120.0,
125.0, 125.3, 125.5, 126.9, 127.2 (×2), 127.3, 127.6 (×2), 127.7
(×3), 127.8 (×3), 127.9 (×5), 128.0, 128.1 (×9), 128.3 (×6), 128.4
(×3), 128.5 (×3), 128.6 (×9), 128.7 (×3), 129.4, 129.8, 132.9,
137.0, 137.6 (×2), 137.9, 138.3, 138.7 (×2), 139.0, 141.1, 141.2,
142.9, 143.3, 147.8, 150.0, 154.6, 164.5, 164.5 ppm; ESI TOF
LCMS [M+Na]+ calcd for C103H94N2NaO21 1718.6280, found
1718.6222.
22
11: Rf = 0.45 (acetone/toluene, 1/4, v/v); [α]D +2.5 (c 1.0,
1
CHCl3); H NMR (600 MHz, CDCl3): δ, 2.85 (ddd, 1H, J = 9.6
Benzyl
O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-
Hz, H-5), 3.19 (dd, 1H, J = 10.2 Hz, H-6a), 3.25-3.37 (m, 6H, H-
2, 3, 3ʹʹʹ, 6aʹʹʹ, 6b, 6bʹʹʹ), 3.43 (m, 1H, H-5ʹʹʹ), 3.58-3.66 (m, 3H, H-
3ʹ, 4ʹʹ, 5ʹ), 3.70 (m, 1H, H-5ʹʹ), 3.84-3.75 (m, 3H, H-4, 6aʹʹ, 6bʹʹ),
3.88 (br d, 1 H, J = 1.9 Hz, H-4ʹʹʹ), 3.98 (s, 1H, H-4ʹ), 4.14-4.30
(m, 9H, H-1, 1ʹʹʹ, 2ʹʹ, 6aʹ, 6bʹ, 4 x CHPh), 4.33 (d, 1H, J1ʹ,2ʹ = 8.0
glucopyranosyl)-(1‰3)-O-(2-O-benzoyl-4-O-benzyl-6-O-
picoloyl-β-D-galactopyranosyl)-(1‰4)-2,3,6-tri-O-benzyl-β-D-
glucopyranoside (15). Compound 14 (135 mg, 0.0796 mmol)
was dissolved in a mixture of Et3N in CH2Cl2 (5.0 mL, 3/7, v/v)
and the resulting solution was stirred for 2 h at rt. After that, the
reaction mixture was concentrated in vacuo, and the residue was
purified by column chromatography on silica gel (acetone - tolu-
ene gradient elution) to afford the title compound as a white foam
in 89% yield (104.8 mg, 0.0711 mmol). Analytical data for 15: Rf
2
Hz, H-1ʹ), 4.41-4.60 (m, 9H, 9 x CHPh), 4.66 (d, 1H, J = 11.0
Hz, CHPh), 4.78-4.82 (m, 3H, H-3ʹʹ, 2 x CHPh), 4.89 (d, 2H, 2J =
2
11.0 Hz, 2 x CHPh), 4.95 (d, 1H, J = 11.7 Hz, CHPh), 5.00 (d,
1H, J1ʹʹ,2ʹʹ = 8.3 Hz, H-1ʹʹ), 5.06 (d, 1H, 2J = 10.4 Hz, CHPh), 5.17
22
1
(dd, 1H, J2ʹ,3ʹ = 9.8 Hz, H-2ʹ), 5.45 (dd, 1H, J
= 8.9 Hz, H-
2ʹʹ,3ʹʹ
= 0.55 (acetone/toluene, 1/4 v/v); [α]D -11.6 (c 1.0, CHCl3); H
NMR (600 MHz, CDCl3): δ, 2.45 (d, 1H, J = 4.6 Hz, OH), 2.94
(ddd, 1H, J = 8.9 Hz, H-5), 3.29 (dd, 1H, J = 10.3 Hz, H-6a), 3.34
(dd, 1H, J2,3 = 9.0 Hz, H-2), 3.39-3.44 (m, 2H, H-3, 6b), 3.61 (dd,
1H, J3”,4” = 9.1 Hz, J4”,5” = 9.1 Hz, H-4ʹʹ), 3.67-3.76 (m, 3H, H-3ʹ,
5ʹʹ, 6aʹʹ), 3.78-3.88 (m, 3H, H-4, 5ʹ, 6bʹʹ), 4.11 (br s, 1H, H-4ʹ),
4.13-4.26 (m, 3H, H-2ʹʹ, 6aʹ, CHPh), 4.29 (d, 1H, J1.2 = 7.7 Hz, H-
1), 4.36 (dd, 1H, J = 6.1, 11.0 Hz, H-6bʹ), 4.44-4.52 (m, 4H, J1ʹ,2ʹ
= 8.0 Hz, H-1ʹ, 3ʹʹ, 2 x CHPh), 4.55-4.70 (m, 6H, 6 x CHPh), 4.74
(d, 1H, 2J = 11.4 Hz, CHPh), 4.82 (d, 2H, 2J = 12.0 Hz, CHPh × 2
2ʹʹʹ), 6.75-8.69 (m, 68H, aromatic) ppm: 13C NMR (151 MHz,
CDCl3): δ, 29.8, 31.1, 56.0, 63.8, 67.4, 67.8, 69.5, 71.0, 71.8, 72.0
(× 2), 72.6, 73.2, 73.5 (× 2), 73.6, 74.4, 74.8, 74.9, 75.0, 75.1,
75.2, 75.6, 75.8 (× 2), 76.1, 79.4, 80.2, 81.6, 82.6, 99.3, 100.4,
100.5, 102.6, 122.7, 123.7, 125.4, 126.8, 127.3, 127.4 (×2), 127.5
(×4), 127.6 (×2), 127.7, 127.8 (×7), 127.9, 128.0 (×3), 128.1 (×7),
128.2 (×8), 128.3 (×4), 128.4 (×7), 128.5 (×6), 128.6 (×2), 128.8
(×2), 129.6, 129.8, 130.0, 130.2, 130.9, 131.3, 132.7, 133.0,
133.4, 134.6, 136.9, 137.5, 137.6, 138.0, 138.1, 138.2, 138.5,
138.7, 138.8, 139.0, 147.8, 150.0, 164.3, 164.4, 165.5, 166.3,
168.4 ppm; ESI TOF LCMS [M+H]+calcd for C122H117N2O25
2010.7979, found 2010.7963.
2
2
), 4.94 (d, 1H, J = 10.4 Hz, CHPh), 5.14 (d, 1H, J = 11.5 Hz,
CHPh), 5.22 (d, 1H, J1ʹʹ,2ʹʹ = 8.3 Hz, H-1ʹʹ), 5.34-5.39 (dd, 1H, J2ʹ,3ʹ
= 9.9 Hz, H-2ʹ), 7.00-8.75 (m, 48H, aromatic) ppm; 13C NMR
(151 MHz, CDCl3): δ, 56.8, 63.9, 67.5, 69.1, 71.0 (×2), 72.0 (×2),
73.4, 73.6 (×2), 74.3, 74.9 (×3), 75.1, 75.6, 76.1, 79.2, 80.3, 81.6,
82.6, 99.7, 100.3, 102.5, 125.4, 126.9, 127.2, 127.5, 127.7 (×3),
127.8 (×3), 127.9, 128.0 (×8), 128.1 (×4), 128.2 (×3), 128.3 (×6),
128.4 (×3), 128.5 (×3), 128.6 (×6), 128.7 (×3), 129.4, 129.6,
132.9, 133.6, 137.0, 137.5, 137.9, 138.1, 138.2, 138.6, 138.8,
139.0, 147.7, 149.9, 164.4 (×2) ppm; ESI TOF LCMS [M+H]+
calcd for C88H85N2O19 1473.5747, found 1473.5757.
Deprotection of tetrasaccharide 11
Benzyl O-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1‰3)-
O-(3,6-di-O-benzyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-
(1‰3)-O-(4-O-benzyl-β-D-galactopyranosyl)-(1‰4)-2,3,6-tri-
O-benzyl-β-D-glucopyranoside (16). Compound 11 (59.0 mg,
0.029 mmol) was dissolved in MeOH (3.0 mL), NH2NH2-H2O
(130 µL, 2.64 mmol) was added, and the resulting mixture was
o
heated at 90 C for 24 h. After that, the volatiles were removed
under reduced pressure, and the residue was dried in vacuo for 3
h. The crude residue was dissolved in a mixture of Ac2O and
MeOH (2.0 mL, 1/1, v/v) and the resulting mixture was stirred for
Benzyl
O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-
galactopyranosyl)-(1‰3)-O-(4,6-di-O-benzyl-2-deoxy-2-
phthlimido-β-D-glucopyranosyl)-(1‰3)-O-(2-O-benzoyl-4-O-

12 h at rt. The volatiles were removed under reduced pressure, the
residue was diluted with CH2Cl2 (50 mL), and washed with sat.
aq. NaHCO3 (10 mL) and 1 M HCl (10 mL). The organic phase
was separated, dried over MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(acetone - toluene gradient elution) to afford the title compound as
an off-white amorphous solid in 92% yield (42.9 mg, 0.026
mmol). Analytical data for 16: Rf = 0.50 (acetone/toluene, 3/7
v/v); [α]D23 +70.8 (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3): δ,
1.81 (s, 3H, CH3CO), 2.95-4.03 (m, 24H, H-2, 2ʹ, 2ʹʹ, 2ʹʹʹ, 3, 3ʹ, 3ʹʹ,
3ʹʹʹ, 4, 4ʹ, 4ʹʹ, 4ʹʹʹ, 5, 5ʹ, 5ʹʹ, 5ʹʹʹ, 6a, 6aʹ, 6aʹʹ, 6aʹʹʹ, 6b, 6bʹ, 6bʹʹ,6bʹʹʹ),
4.23 (d, 1H, 2J = 11.8 Hz, CHPh), 4.30-4.58 (m, 10H, H-1, 1ʹ, 1ʹʹʹ,
7 x CHPh), 4.60-4.96 (m, 11H, 11 x CHPh), 5.01-5.04 (m, 2H, H-
1ʹʹ, CHPh), 6.48 (d, 1H, J = 6.3 Hz, NHCOCH3), 7.12-7.37 (m,
50H, aromatic) ppm; 13C NMR (151 MHz, CDCl3): δ, 23.7, 58.4,
61.9, 68.2, 68.8, 69.4, 71.3, 71.6, 71.9, 72.1, 72.8, 73.5, 73.7 (×3),
73.8, 74.3, 74.7, 74.8, 74.9, 75.1 (×2), 75.2, 75.4, 76.6, 77.1, 81.9,
82.1, 83.0, 83.1, 83.7, 101.8, 102.9, 103.1, 104.5, 127.4, 127.5,
127.6 (×3), 127.7 (×2), 127.8 (×3), 127.9 (×4), 128.0 (×7), 128.2
(×8), 128.3 (×6), 128.4 (×4), 128.5 (×6), 128.7 (×3), 128.8 (×2),
137.6, 138.0 (×2), 138.1, 138.2, 138.4, 138.6, 138.7, 138.9, 139.0,
172.3 ppm; ESI TOF LCMS [M+Na]+calcd for C96H105NNaO21
1631.7110, found 1631.7121.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by the National Institute of General
Medical Sciences (U01GM120673).
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1
form/methanol/water, 2/1/0.4, v/v/v); H NMR (600 MHz, D2O):
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4.14 (d, 1H, J = 3.3 Hz), 4.43 (d, 2H, J = 7.8 Hz), 4.65 (d, 1H, J =
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ASSOCIATED CONTENT
Supporting Information
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AUTHOR INFORMATION
Corresponding Author
* Department of Chemistry and Biochemistry, University of Mis-
souri – St. Louis, One University Boulevard, St. Louis, Missouri
63121, USA; demchenkoa@umsl.edu
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script.
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7

•
The total synthesis of lacto-N-tetraose has been completed using both linear and convergent
synthesis approaches;
•
•
•
The linear approach was proven to be more efficient in this application;
New synthetic protocols for different glycosidic linkages have been developed and refined;
New methods for obtaining individual HMO help to improve understanding their roles and boost
practical applications

The authors have no conflicts to declare