Chemoenzymatic Synthesis of a Library of Human Milk Oligosaccharides

Article abstract of DOI:10.1021/acs.joc.6b00478

Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification, and biofunctional studies of HMOs remain a great challenge due to their diversity and complexity. The accessibility of a homogeneous HMO library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely core synthesis/enzymatic extension (CSEE), for rapid production of diverse HMOs was reported. On the basis of 3 versatile building blocks, 3 core structures were chemically synthesized via consistent use of oligosaccharyl thioether and oligosaccharyl bromide as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to up to 11 HMOs by 4 robust glycosyltransferases. A library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications.

Full text of DOI:10.1021/acs.joc.6b00478

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Article
Chemoenzymatic synthesis of a library of human milk oligosaccharides
Zhongying Xiao, Yuxi Guo, Yunpeng Liu, Lei Li, Qing Zhang, Liuqing Wen, Xuan Wang, Shukkoor
Muhammed Kondengaden, Zhigang Wu, Jun Zhou, Xuefeng Cao, Xu Li, Cheng Ma, and Peng George Wang
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00478 • Publication Date (Web): 15 Jun 2016
Downloaded from http://pubs.acs.org on June 16, 2016
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Chemoenzymatic synthesis of a library of human milk
oligosaccharides
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Zhongying Xiao‡, Yuxi Guo‡, Yunpeng Liu, Lei Li, Qing Zhang, Liuqing Wen, Xuan Wang,
Shukkoor Muhammed Kondengaden, Zhigang Wu, Jun Zhou, Xuefeng Cao, Xu Li, Cheng Ma
and Peng George Wang*
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Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University,
Atlanta, GA 30303.
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Abstract
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Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist
in human milk as one of the major components. Characterization, quantification and
biofunctional studies of HMOs remain a big challenge due to their diversity and complexity. The
accessibility of homogenous HMOs library is essential to solve these issues which have beset
academia for several decades. In this study, an efficient chemoenzymatic strategy, namely Core
Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse HMOs was reported.
Based on 3 versatile building blocks, 3 core structures were chemically synthesized via
consistent use of oligosaccharyl thioether and oligosaccharyl bromide as glycosylation donors in
a convergent fragment coupling strategy. Each of these core structures was then extended to
up to 11 HMOs by 4 robust glycosyltransferases. A library of 31 HMOs were
chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a
practical approach to harvest structurally defined HMOs for various applications.
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β4
β6
β3
β6
β3
β6
β3
β4
β4
β4
β4
β4
β4
β4
α3
α2
α2
α6 β4
α6 β4
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Introduction
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Human milk, as the sole dietary source, contains all the necessary nutrients for the infant to
thrive in the first few months. More importantly, human milk can also provide ingredients with
health benefits which traditional food could not do. Human milk oligosaccharides (HMOs) are
the third major component in human milk, only after lactose and lipid.1 Concentrations and
components of HMOs vary depending on the stages of lactation.2 Particularly, one liter mature
human milk contains about 12ꢀ14 grams HMOs.3 The structures of about 130 discovered HMOs
have been elucidated.4 The major building blocks of HMOs are 5 monosaccharides, including Dꢀ
glucose (Glc), Dꢀgalactose (Gal), NꢀacetylꢀDꢀglucosamine (GlcNAc), LꢀFucose (Fuc), and Nꢀ
acetyl neuraminic acid (Neu5Ac). Even though HMOs were first discovered and confirmed in
1950s, a comprehensive understanding of their functions is still out of reach, due to their inherit
diversity and complexity. Increasing evidences show that HMOs can provide significant
beneficial effects to the health of breastꢀfed infants through several mechanisms. For instance,
HMOs could serve as prebiotics to promote the growth of desired bacteria in infant’s intestine.1c,
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2a,
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Besides, HMOs are antiadhesive antimicrobials by serving as a receptor to prevent
pathogen attachment to infant mucosal surfaces.6 In addition, evidence has demonstrated that
HMOs can modulate epithelial and immune cell responses, and reduce excessive mucosal
leukocyte infiltration and activation, which in turn decreases the risk of necrotizing enterocolitis
(NEC), one of the most common fatal disorders in preterm infants.7 Furthermore, sialylated
HMOs may also provide necessary nutrients for the development of brain and cognition of
infants.8
Even though the general functions of HMOs has been explored and discovered, the
functional roles of individual HMOs are far less clear because of very limited accesses towards
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sufficient amount of structurally defined HMOs. To date, only a handful of shortꢀchain HMOs can
be produced in large scale and the supply of more complicated and branched HMOs is highly
demanded.
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Until now, only a few approaches has been developed for the synthesis of a small number of
wellꢀdefined HMOs.2a, 9 For example, Schmidt developed strategies to synthesize some highly
branched HMOs by solutionꢀphase and solidꢀphase synthesis.9b, 9d, 9e Enzymatic methods have
also been employed to achieve relatively simple structures.9a One of the biggest roadblocks in
previous synthesis remains to be small quantity and limited variety of HMOs needed for
biofunctional studies. Recently, we have developed an efficient Core Synthesis/Enzymatic
Extension (CSEE) strategy for rapid preparation of Nꢀglycan libraries.10 In this study, similar
strategy was successfully applied for HMOs synthesis. Briefly, 3 core oligosaccharides with one
or two GlcNAc residue(s) at the nonꢀreducing end were first synthesized by convergent
assembly of 3 simple building blocks followed by extension of the cores by 4 robust
glycosyltransferases to produce a library of 31 HMOs (Figure. 1).
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Figure 1. HMOs synthesized by the Core Synthesis/Enzymatic Extension (CSEE) strategy starting with 3
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chemically prepared core structures (boxed).
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Results and Discussion
1 Convergent Core Synthesis
Previous studies highlighted the complexity and challenges associated with synthesizing
HMOs via block coupling strategy. Schmidt developed sequential synthesis of lactoseꢀ
containing oligosaccharides, including HMO lactoꢀNꢀtetraoside based on SolidꢀPhase Synthesis
concept.9d Madsen used oneꢀpot glycosylations to achieve several human milk
oligosaccharides.9c Both of the methods can synthesize linear and simple oligosaccharides with
obvious limitations in achieving more complex HMOs, especially highly branched ones. In this
study, we developed an efficient and versatile methodology that utilized oligosaccharyl
thioethers and oligosaccharyl bromides as convergent donors for glycosylation, enabling
branching assembly in one or two steps of glycosylations with excellent stereoselectivity and
yields.
We envisaged that protected lactose 1 (Figure 2) would be a versatile precursor for the
synthesis of core structures, including symmetric and asymmetric ones, as C4, C6ꢀhydroxy
groups (OH) on Gal are protected by benzylidene and the C3ꢀOH is unprotected for chemical
glycosylation. In order to achieve the selective protection of building block 1, C3ꢀOH group was
selectively protected by 4ꢀmethoxybenzyl ether (PMB) using standard condition, followed by C4,
C6ꢀOH protection with benzylidene group. In order to furnish the target cores, two
oligosaccharyl thioethers and oligosaccharyl bromide were prepared (Figure 2).
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Figure 2. The three versatile building blocks for the assembly of the three core structures.
2 Synthesis of Building Blocks
The synthesis of precursor 1 began with lactose peracetate, which was converted to the βꢀ
lactoside 4 by reaction with benzyl alcohol in the presence of BF3ꢁEt2O, followed by
deacetylation with NaOMe/MeOH condition to furnish compound 5. Then 5 was treated with
dibutyltin oxide, followed by the reaction with 4ꢀmethoxybenzyl chloride to provide selective 3’ꢀ
OꢀPMB protected lactoside with fair yield, which has been extensively studied.11 The following
benzylidene protection on 4’,6’ꢀOH was conducted with benzaldehyde dimethyl acetal,
catalyzed by camphorsulfonic acid (CSA), to get compound 6. The perbenzylation of the
remaining hydroxyls of 6 was performed by using sodium hydride and benzyl bromide in
anhydrous DMF to give compound 7. After removing PMB protecting group of 7 treated with 2,
3ꢀdichloroꢀ5, 6ꢀdicyanoꢀ1, 4ꢀbenzoquinone (DDQ), synthesis of building block 1 was achieved
(Scheme 1).
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Scheme 1. Synthesis of Building Block 1. Reagents and conditions: (a) (i) BF3ꢁEt2O, BnOH, DCM, 4 Å MS;
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(ii) NaOMe, MeOH, 50% over two steps; (b) (i) nꢀBu2SnO, MeOH, reflux; (ii) PMBCl, TBAI, Toluene, 4 Å
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MS reflux, 40%; (c) PhCH(OMe)2, DMF, CSA, 75%; (d) BnBr, NaH, DMF, 95%; (e) DDQ, DCM/PBS
Buffer=9:1, 90%.
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Lactosamine building block 3 was initially envisioned to be synthesized using a
straightforward fashion by coupling monosaccharide 9 and 10 (Scheme 2A). Unfortunately, the
desired product was isolated in minor amount by silica gel chromatography, and substantial
amount of byproduct 11 was generated through a thioether migration reaction.12 Therefore, a
Koenigs–Knorr reaction coupling approach was carried out by installing C4 hydroxy group of
glucosamine with high yield (Scheme 2B). Building block 2 would be conveniently used in the
synthesis of two asymmetric core oligosaccharides.
Scheme 2. Synthesis of Building Block 3. (A) Failed reaction; (B) Successful reaction. Reagents and
conditions: (a) Et3SiH, TfOH, 4Å MS, ꢀ78°C, 85%; (b) TMSOTf, DCM, 4Å MS,ꢀ20°C; (c) AgOTf,
DCM/toluene,70%.
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3 Synthesis of Core Oligosaccharides
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With all building blocks in hand, we began to assemble the three core oligosaccharides using
our convergent strategy. The synthesis of HMO1 was depicted in Scheme 3. Disaccharide 13
was obtained in a good yield (85%) by selective opening of the benzylidene ring at C6 of
building block 1 using Et3SiH/PhBCl2. Oligosaccharyl bromide 2 was freshly prepared using
HBr/AcOH condition. Silver triflate (AgOTf) promoted glycosylation of 13 with donor 2 in
dichloromethane resulted in the formation of tetrasaccharide 14 in an excellent yield of 85%.
Deprotection of 14 with ethylene diamine, followed by treatment with acetic anhydride, furnished
peracetylated tetrasaccharide 15 in 75% yield. Oꢀdeacetylation of compound 15 was performed
under Zemplén conditions, followed by the global deprotection of benzyl group (Bn) by catalytic
hydrogenolysis with Pd(OH)2/H2 in MeOH/H2O (10 : 1). The core structure HMO1 was produced
in a total yield of 80% over the two steps.
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Scheme 3. Synthesis of HMO1. Reagents and conditions: (a) Et3SiH, PhBCl2, 4 Å MS, DCM, ꢀ78°C, 85%;
(b) 2, AgOTf, 2,4,6ꢀcollidine, ꢀ20 °C, 85%; (c) (i) ethanediamine, nꢀBuOH; (ii) Ac2O, pyridine, DMAP; (d) (i)
NaOMe/MeOH 75% over two steps; (ii) H2, Pd(OH)2/C, 80% over two steps.
Glycosylation of 3’ꢀOꢀunprotected acceptor 1 with donor 3 proceeded at ꢀ20°C under
AgOTf/NIS conditions to furnish the desired tetrasaccharide 16 in 85% yield. Then selective
opening of the benzylidene ring at C6 of 16 using Et3SiH/PhBCl2 condition provided 6’ꢀOꢀ
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unprotected acceptor 17 in 80% yield. The fully protected pentasaccharide was initially
attempted to be synthesized by a convergent glycosylation of acceptor 17 and thiol donor 18.
Unfortunately, the desired product was not detected by TLC and ESI mass spectrometry
analysis (Scheme 4A). Several other donors including oligosaccharyl trichloroacetimidate and
thioethers donors were tried to install the pentasaccharide, but no product was detected. The
big challenge should be caused by the bulky benzyl group on 4’ꢀOH position, which has very
large steric hindrance and stops the glycosylation on 6’ꢀOH position, even though primary
alcohol is very nucleophilic. Therefore, 4’, 6’ꢀOꢀunprotected lactoꢀNꢀtetraose 19 was proposed
as acceptor for the glycosylation. Removal of the 4’, 6’ꢀOꢀbenzylidene of 16 by treatment with
ethanethiol in the presence of TsOH afforded acceptor 19. Glycosylation of acceptor 19 with
glycosyl bromide 2 to achieve the protected target pentasaccharide 20 proceeded smoothly and
regioselectively by use of AgOTf as Lewis acid at ꢀ20 °C in an excellent yield (85%). The two
phthalimides of 20 were then converted into acetamides 21, followed by the global deprotection
of Ac and Bn groups. The core oligosaccharide HMO2 was produced in a total yield of 53% over
the four steps (Scheme 4B).
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A)
Ph
Ph
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O
OAc
O
O
OAc
OBn
AcO
OBn
O
O
BnO
O
AcO
BnO
O
O
O
a
O
O
b
O
O
SEt
AcO
O
BnO
O
+
HO
O
O
OBn
AcO
OBn
BnO
OAc
BnO
BnO
OBn
OAc
OBn
NPhth
NPhth
OBn
OBn
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NPhth
BnO
BnO
O
OBn
OAc
O
O
OBn
O
OAc
AcO
AcO
BnO
OBn
O
OBn
OH
O
AcO
AcO
BnO
BnO
BnO
O
O
O
O
BnO
O
BnO
O
O
O
O
BnO
OBn
c
SEt
O
O
BnO
OBn
+
OBn
BnO
BnO
OAc
OBn
OAc
NPhth
17
NPhth
OBn
NPhth
OBn
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B)
Ph
O
OAc
OAc
OH
HO
OBn
O
AcO
OBn
O
AcO
AcO
BnO
O
BnO
O
O
O
O
O
O
O
d
O
BnO
O
AcO
e
O
O
O
BnO
OBn
OBn
BnO
BnO
OAc
OAc
OBn
OBn
NPhth
NPhth
OBn
OBn
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19
NHAc
R1
HO
HO
AcO
AcO
O
OH
O
OAc
O
O
OH
OAc
O
OH
O
OBn
O
HO
HO
HO
AcO
AcO
R2
HO
BnO
g
O
O
O
O
O
O
O
O
O
O
HO
O
OH
OBn
HO
BnO
BnO
OH
OH
OAc
OBn
NHAc
R1
OH
OBn
20 R1=NPhth, R2=OH
f
HMO2
R1=NHAc, R2=OAc
21
Scheme 4. Synthesis of HMO2. (A) Failed reaction; (B) Successful reaction. Reagents and conditions: (a)
NIS, TfOH, DCM, 4 Å MS,ꢀ20°C, 85%; (b) Et3SiH, PhBCl2, DCM, ꢀ78 °C. 80%; (c) NIS, AgOTf, DCM, 4 Å
MS,ꢀ20°C; (d) TsOH, EtSH, DCM, 90%; (e) 2, AgOTf, 2,4,6ꢀcollidine, DCM, 4 Å MS, ꢀ20°C 85%; (f) (i)
ethanediamine, nꢀbuOH; (ii) Ac2O, pyridine, DMAP 62% over two steps; (g) (i) NaOMe/MeOH; (ii) H2,
Pd(OH)2/C, 80% over two steps.
AgOTf promoted glycosylation of 3’ꢀOꢀunprotected acceptor 1 with donor 2 proceeded at ꢀ20°C
to furnish the trisaccharide 22 in a good yield of 85%. Then deprotection of the 4’, 6’ꢀOꢀ
benzylidene of 22 by treatment of EtSH/TsOH provided the dialcohol 23. Glycosylation of
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acceptor 23 with thiol donor 3 by treatment of AgOTf/NIS condition at ꢀ20°C to achieve the
protected target pentasaccharide 24 in 70% yield. The two phthalimides of 24 were then
converted into acetamides 25. Complete deprotection of 25 was achieved by hydrogenolytic
debenzylation (Pd(OH)2/C, H2) and complete deꢀOꢀacetylation using sodium methoxide in
methanol, resulting in core oligosaccharide HMO3 in a total yield of 67% over the four steps
(Scheme 5).
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Scheme 5. Synthesis of HMO3. Reagents and conditions: (a) AgOTf, 2, 4, 6ꢀcollidine, DCM, 4Å MS, 85%;
(b) TsOH, EtSH, DCM, 80%; (c) 3, NIS, AgOTf, DCM, 4Å MS, ꢀ20°C,70%; (d) (i) ethanediamine, nꢀBuOH;
(ii) Ac2O, pyridine, DMAP; 70% over two steps; (e) (i) NaOMe, MeOH; (ii) Pd(OH)2, H2. 95% over two
steps.
4 Enzymatic Extension of Core Structures
A total of 31 HMOs were enzymatically synthesized starting from the 3 core structures
(HMO1, HMO2, HMO3) via an enzymatic extension approach using 4 robust GTs: β1,4
galactosylꢀtransferase from Neisseria meningitidis (LgtB),13 α2,6ꢀsialyltransferase from
Photobacterium damselae (Pd2,6ST),14 Cꢀterminal 66 amino acids truncated α1,3ꢀ
fucosyltransferase from Helicobacter pylori (Hpα1,3FT),15 and α1,2ꢀfucosyltransferase from
Helicobacter mustelae (Hmα1,2FT).16 All GTs were from bacteria and had high expression
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levels in Escherichia coli, high activity, and relatively relaxed substrate tolerance. As shown in
Figure 3A, glycans HMO11ꢀHMO16 were prepared starting with the chemically prepared core
HMO1. Briefly, in a 2 mL reaction system, 30 mg of HMO1 (20 mM) was incubated with Gal (20
mM), MgCl2 (20 mM), ATP (20 mM), UTP (20 mM) and variant amount of BiGalK, BiUSP and
LgtB.17 (Figure 4) After overnight reaction, the mixture was terminated by boiling for 10min and
analyzed by MALDIꢀMS which shows a single peak at m/z 1095.748, corresponding to HMO11
[M+Na]+. Meanwhile, on the HPLCꢀELSD (Evaporative Light Scattering Detector) profile, a new
peak (TR = 11.946 min) was observed. The reaction mixture was purified by HPLC using a
water/acetonitrile gradient elution, yielding 40mg of HMO11 (93% yield). The purified HMO11
(99% pure) was then utilized for the syntheses of HMO12ꢀHMO16 (Figure 3A) catalyzed by
Pd2,6ST, and α1,2FT, α1,2FT respectively (see ESI for details). Interestingly α1,3FT can
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specifically distinguish the GlcNAc from terminal Galβ1,4GlcNAc and α1,2FT preferentially
attach to terminal Gal. We basically use this feature to biosynthesize Lewis X (Lex) and Lewis Y
(Ley). In addition, difucosylated LacNAc motif [Fucα1, 2ꢀGalꢀβ1, 4ꢀ(Fucα1, 3ꢀ)GlcNAc] was also
generated while using Hmα1,2FT. The synthesis of asymmetric biꢀantennary HMO2x and
HMO3x (Figure 3B&C) were carried out by enzymatic extension of core HMO2 and HMO3.
Asymmetric Core HMO2 and HMO3 can more efficiently take advantage of different substrate
specificities of GTs over symmetric HMO1 via coupling a Gal to the terminal GlcNAc of one
antennary. For example, to obtain HMO311, the Gal from β6GlcNAc branch was sequentially
extended by Hmα1,2FT, Hpα1,3FT and LgtB (Figure 3C). In contrast, HMO310 were
sequentially synthesized by Hmα1,2FT, LgtB and Hpα1,3FT (Figure 3C). Such synthetic
routes were designed according to the substrate specificities of GTs to avoid undesirable
glycosylation. Each glycosylation step was further described in detail in Figure 4.
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A)
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b
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HMO12
HMO13
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HMO1
HMO11
HMO14
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d
e
HMO15
HMO16
B)
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a
HMO25
HMO21
HMO22
HMO23
a
HMO26
HMO27
a
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HMO2
d
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HMO28
HMO29
HMO210
e
HMO24
a
HMO211
C)
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HMO31
HMO32
HMO35
HMO36
a
a
d
HMO33
HMO37
HMO38
HMO3
a
d
HMO310
e
HMO34
d
a
HMO39
HMO311
Figure 3. Enzymatic extension of human milk oligosaccharides.
To avoid tedious purification steps of producing much more complex HMO structures, oneꢀpot
multi enzyme systems(OPME) were used for LgtB and Pd2,6ST catalyzed glycosylation step
(Figure 4a&b&
c).18 LgtB, which can specifically recognize terminal GlcNAc, was combined
with enzymes (BiGalK and AtUSP) involved in the biosynthesis of their corresponding sugar
nucleotide (UDPꢀGal) to produce the target glycan with desired β1,4 linkage (Figure 4a).
Similarly, Pd2,6ST was combined with NmCSS, a CMPꢀsialic acid synthetase, to form α2,6
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configuration (Figure 4b
&c). Both Hmα1,2FT and Hpα1,3FT were simply using Pure GDPꢀ
5
6
Fuc to form α1,2 and α1,3 linkage respectively (Figure 4d&e).
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a
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d
e
Figure 4. (a) LgtB, BiGalK, AtUSP, Gal, ATP, UTP, Mg2+; (b) Pd2,6ST, NmCSS, Neu5Ac, CTP, Mg2+; (c)
Pd2,6ST, NmCSS, Neu5Gc, CTP, Mg2+; (d) Hpα1,3FT, GDPꢀFuc, Mn2+; (e) Hmα1,2FT, GDPꢀFuc, Mn2+.
LgtB, Neisseria meningitidis β1,4 galactosyltransferase; BiGalk, Bifidobacterium infantis galactokinase;
AtUSP, Arabidopsis thaliana pyrophosphorylase; Pd2,6ST, Photobacterium damselae α2,6ꢀsialyltransꢀ
ferase; NmCSS, Neisseria meningitidis CMPꢀsialic acid synthetase; Hpα1,3FT, Cꢀterminal 66 amino acids
truncated Helicobacter pylori α1,3ꢀfucosyltransferase; Hmα1,2FT, Helicobacter mustelae α1,2ꢀ
fucosyltransferase.
Conclusion
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In summary, we have utilized our wellꢀdeveloped CSEE strategy for efficient synthesis of a
library of structureꢀdefined HMOs, which was assisted with rapid HPLC purification. The
combination of CSEE and HPLC purification allows us to deliver diverse and high purity of 31
homogenous HMOs. These HMOs are valuable materials for bioactivity evaluation as well as
glycan analysis. In this work, oligosaccharyl thioethers and oligosaccharyl bromide were
consistently utilized as chemical glycosylation donors for the convergent installation of branched
lactoseꢀterminated antennae. This general and efficient method furnished 3 core
oligosaccharides with high stereoselectivity and excellent yields. This work further confirmed
that any GlcNAcꢀterminated glycans could be extended to 5 or more glycans, including LeX and
SLeX, which are very important epitopes in glycobiology. The CSEE demonstrated a practical
way to harvest diverse and complex HMOs with defined structures for various applications. The
“mass” production of more homogenous HMOs and bioactivity evaluation are underway.
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Experimental Sections
General Methods: All chemicals were purchased as reagent grade and used without further
purification. Anhydrous dichloromethane (CH2Cl2), acetonitrile (CH3CN), tetrahydrofuran (THF),
N, Nꢀdimethyl formamide (DMF), toluene, and methanol (MeOH) were purchased from a
commercial source without further distillation. Pulverized molecular sieves MSꢀ4 Å (Aldrich) for
glycosylation was activated by heating at 350 °C for 3 h. All reactions were performed with dry
solvents and under nitrogen unless otherwise stated. Thinꢀlayer chromatography (TLC) with 60
F254 silica gel plastic plates was detected visualized under UV (254 nm) and/or by staining with a
solution of 10 ml anisaldehyde and 10 ml 95% H2SO4 in 400 mL Ethanol, followed by heating on
a hot plate. Column chromatography was carried out on silica gel (EMD 230ꢀ400 mesh ASTM)
and P2 gel. Optical rotation values were measured using a polarimeter at the ambient
1
temperature in specified solvents. H NMR spectra were recorded on a 400 MHz or 500 MHz
NMR spectrometer at 25 °C. All 1H Chemical shifts (in ppm) were assigned according to CDCl3
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(δ= 7.24 ppm) or D2O (δ = 4.79 ppm). 13C NMR spectra were obtained with 400 MHz NMR
spectrometer and calibrated with CDCl3 (δ = 77.00 ppm). Coupling constants (J) are reported in
hertz (Hz). Splitting patterns are described using the following abbreviations: s, singlet; brs,
broad singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. 1H NMR
spectra are reported in the following order: chemical shift, multiplicity, coupling constant(s), and
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number(s) of protons. All NMR signals were assigned on the basis of H NMR and 13C NMR
experiments. High resolution MALDI mass spectra were recorded on ESIꢀorbitrap or MALDIꢀ
TOF spectrometer.
Neu5Ac and Neu5Gc were purchased from Carbosynth Limited. ATP and CTP were
purchased from Sigma. Thermosensitive Alkaline Phosphatase from shrimp (FastAP) was
purchased from Thermo Scientific. Other enzymes including Neisseria meniningitidis β1,4ꢀ
galactosyltransferase (NmLgtB), α2,6ꢀsialyltransferase from Photobacterium damslae
(Pd2,6ST) , Cꢀterminal 66 amino acids truncated Helicobacter pylori α1,3ꢀfucosyltransferase
(Hpα1,3FT) , Helicobacter mustelae α1,2ꢀfucosyltransferase (Hmα1,2FT), CMPꢀsialic acid
synthetase from N. meningitides (NmCSS) were expressed and purified as previously described.
Enzymes were then desalted against 50 mM TrisꢀHCl, 100 mM NaCl, and 50% glycerol, and
stored at ꢀ20 °C for long term use. Sugar nucleotide guanoside 5’ꢀdiphosphoꢀLꢀfucose (GDPꢀ
Fuc) were prepared as described in our previously paper.19
General Procedures
A) Transformation of NꢀPhth to NHAc procedure: A mixture of NꢀPhth protected
oligosaccharide was dissolved in nꢀBuOH at room temperature, followed by addition of
ethylenediamine (nꢀBuOH: ethylenediamine = 2:1). After being stirred at 90 °C for 12 h, the
mixture was evaporated in vacuo to give a residue for the next step without further purification.
To a solution of the residue in pyridine was added Ac2O. After being stirred at room temperature
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for 12 h, the solution was diluted with EtOAc and washed with aqueous 1 M HCl, saturated
aqueous NaHCO3, and brine solution. The organic layer was dried over Na2SO4, filtered, and
evaporated in vacuo to give a residue, which was purified by silica gel column chromatography
to give NHAc compound.
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B) Deacetylation procedure: Acꢀprotected oligosaccharide was dissolved in MeOH, and
NaOMe in MeOH was added until pH was about 10. After stirring at room temperature for 12 h,
the solution was neutralized with ionꢀexchange resin (H+), and then filtered. The residue was
concentrated under vacuo to afford the desired deacetylated product.
C) Deprotection of benzyl group: Pd(OH)2 on carbon was added to a solution of protected
oligosaccharide in MeOH/H2O (10/1). The mixture was stirred under 1 atmosphere of hydrogen.
After being stirred for 24 h, the mixture was filtered through a PTFE syringe filter and
concentrated in vacuo. The residue was purified by BioꢀGel Pꢀ2 (BIOꢀRAD) column
chromatography using water as eluent. The product was then lyophilized to get target
compound as white powder.
D) Production of oligosaccharyl bromide: Add peracetylated oligosaccharide portionwise to a
stirred solution of HBr (33%) in glacial acetic acid (20.0 ml) at 0 °C. After all the sugar has been
added, the reaction mixture was stirred at room temperature for 45 min. TLC analysis (Hexanes:
Ethyl acetate=1:1) indicates formation of product and consumption of starting material. Then the
reaction was quenched by ice water (200 ml) and then extract the product with DCM (2 × 200
ml). Wash the combined organic extracts with a solution of NaHCO3 (aq., sat., 2 × 200 ml), dry
with Na2SO4, filter and then concentrate in vacuo. The crude product was used without further
purification.
E) General methods for enzyme treatment and HPLC purification: In general, 31 HMOs were
enzymatically synthesized by 4 glycosyltransferases (NmLgtB, Pd2,6ST, Hpα 1,3FT, Hmα
1,2FT) in the nearly same reaction condition. Reactions contain 50 mM TrisꢀHCl (pH 8.0), 10
mM of acceptor HMOs, 12 mM of sugar nucleotide (or its corresponding synthetase), 10 mM of
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MnCl2, and varying amounts of glycotransferases. FastAP (1 U/200 ꢀL) was also added to
digest the reaction byproduct UDP to drive reaction forward. Reactions incubated at 37 °C for
overnight, and monitored by HILICꢀELSD (Waters XBridge BEH amide column, 130 Å, 4.6 mm
× 250 mm under a gradient running condition (solvent A: 100 mM ammonium formate, pH 3.4;
solvent B: acetonitrile; flow rate: 1 mL/min; B%: 65ꢀ50% within 25 min)). The desired products
were detected by a highꢀefficient ELSD (evaporative light scattering detector) which increases
the sample concentration to minimize the noise and deliver higher sensitivity. After over 90%
acceptor converted, the reaction was quenched by boiling for 10 min, followed concentration by
rotary evaporator. HPLCꢀA210nm was then used to purify target HMOs using a semiꢀ
preparative column (Waters XBridge BEH amide column, 130 Å, 5 ꢀm, 10 mm × 250 mm) under
a gradient running condition which are solvent A: 100 mM ammonium formate, pH 3.4; solvent B:
acetonitrile; flow rate: 4 mL/min; B%: 65ꢀ50% within 25 min.10 MS data for purified HMOs were
obtained by ESIꢀMS and MALDIꢀMS.
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Benzyl Oꢀ(4, 6ꢀOꢀbenzylideneꢀ3ꢀOꢀ(4ꢀmethoxybenzyl)ꢀ βꢀDꢀgalactopyranosyl)ꢀ(1 4)ꢀ βꢀ
→
Dꢀglucopyranoside (6)
A suspension of benzyllactose 5 (12.0 g, 27.78 mmol) and Bu2SnO (7.6 g, 30.54 mmol) in
anhydrous MeOH (100 mL) was heated to reflux and stirred for 8 h. The solvent was removed in
vacuo. Then the residue was dissolved in dry toluene (100 mL). pꢀmethoxybenzyl chloride (3.76
mL, 20.37 mmol), tetrabutylammonium iodide (2.05 g, 11.10 mmol), and 4 Å molecular sieves (5
g) were added. The resulting mixture was heated to reflux for another 8 h and then cooled to
room temperature. The suspension was filtered through a Celite pad and the filtrate was
concentrated and chromatographied (dichloromethaneꢀmethanol=6:1) to afford 9.2 g of crude
product (60% yield).
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Benzaldehyde dimethyl acetal (2.75 mL, 18.33 mmol) was added to a solution of the above
crude product (7.8 g, 14.10 mmol) in anhydrous dimethylformamide (100 ml), then
camphorsulfonic acid was added to adjust the PH to about 2.0ꢀ3.0. The reaction mixture was
stirred overnight and then quenched with triethylamine. The mixture was concentrated in
vacuum. The residue was purified by flash column chromatography (dichloromethaneꢀ
methanol=10:1) to give 6 as a white solid (8.47 g, 87.0%). [α]D20 = +6.7 (c 1.0, CH2Cl2). 1H NMR
(CDCl3, 400 MHz): δ 7.48ꢀ7.51 (dd, 2H), 7.28ꢀ7.38 (m, 10 H), 6.85ꢀ6.87 (m, 2 H), 5.34 (s, 1 H),
4.87 (d, J = 11.95 Hz, 1 H), 4.59ꢀ4.63 (m, 4 H), 4.45 (d, J = 7.8 Hz, 1 H), 4.36 (d, J = 8.1 Hz, 1
H), 4.21 (d, J = 14.0 Hz, 1 H), 4.10 (s, 1 H), 3.92ꢀ4.00 (m, 3 H), 3.79ꢀ3.89 (m, 2 H), 3.78 (s, 3 H),
3.60ꢀ3.69 (m, 3 H), 3.40ꢀ3.50 (m, 2 H), 3.27ꢀ3.40 (m, 4 H); 13C NMR (CDCl3, 100 MHz): δ
137.6,137.2, 130.0,129.5, 128.5, 128.3, 128.2, 127.9, 126.3, 113.9, 103.6, 101.8, 101.1, 78.8,
74.9,74.7, 73.5, 72.7, 71.3, 71.2, 69.1, 69.0, 66.9, 61.9, 55.3. HRMS (ESIꢀorbitrap): [M + Na]+
C34H40NaO12 calcd for 663.2417, found 663.2420.
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Benzyl Oꢀ(2ꢀOꢀbenzylꢀ4, 6ꢀOꢀbenzylideneꢀ3ꢀOꢀ(4ꢀmethoxybenzyl)ꢀ βꢀDꢀgalactopyranosꢀ
yl)ꢀ(1 4)ꢀ2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀglucopyranoside (7)
→
60% NaH (2.25 g, 56.25 mmol) and BnBr (6.66 ml, 56.25 mmol) were added to a stirred
solution of 6 (6.0g, 9.38 mmol) in DMF (60 mL) cooled at 0°C. The color of the solution changed
to light yellow. The reaction mixture was maintained at room temperature for 4 h. Then the
solution was quenched with MeOH. The mixture was diluted with EtOAc and washed with water.
The organic layer was dried with Na2SO4 and concentrated. The residue was purified on a silica
gel column (Hexanes: EtOAc = 6:1) to afford the product 7 (1.85 g, 92.5%) as white powder.
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[α]D = +7.4 (c 1.0, CH2Cl2). H NMR (CDCl3, 400 MHz): δ 7.57ꢀ7.59 (m, 2 H), 7.51ꢀ7.54 (m, 2
H), 7.42ꢀ7.45 (m, 2 H), 7.31ꢀ7.38 (m,23 H), 7.23ꢀ7.24 (m, 2 H), 6.90ꢀ6.92 (m, 2 H), 5.51 (s, 1H),
5.25 (d, J = 11.0 Hz, 1 H), 4.98ꢀ5.03 (m, 2 H), 4.90 (d, J = 11.1 Hz, 1 H), 4.79ꢀ4.85 (m, 3
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H),4.71ꢀ4.74 (m, 3 H), 4.63 (d, J = 12.0 Hz, 1 H), 4.52ꢀ4.55 (m, 2 H), 4.43 (d, J = 12.0 Hz, 1 H),
4.26 (dd, J = 1.4 Hz, 12.4 Hz, 1 H), 4.04ꢀ4.08 (m, 2 H), 3.96 (dd, J = 4.2, 11.3 Hz, 1 H), 3.90 (dd,
J = 1.8, 12.5 Hz, 1 H), 3.85 (s, 3 H), 3.79ꢀ3.83 (m, 2 H), 3.69 (t, J = 8.8 Hz, 1 H), 3.56ꢀ3.60 (m, 1
H), 3.40ꢀ3.46 (m, 2 H); 13C NMR (CDCl3, 100 MHz): δ 159.3, 139.0, 139.0, 138.7, 138.6, 138.2,
137.6, 130.5, 129.4, 128.9, 128.7, 128.4, 128.3, 128.1, 128.0, 127.8, 127.8, 127.6, 127.6, 127.5,
127.4, 127.3, 126.6, 113.8, 103.0, 102.6, 101.4, 83.1, 81.9, 79.4, 78.9, 77.7, 75.9, 75.3, 75.2,
75.1, 73.8, 73.0, 71.4, 71.1, 69.0, 68.3, 66.4, 55.3. HRMS (ESIꢀorbitrap): [M + Na]+ C62H64NaO12
calcd for 1023.4295, found 1023.4285.
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Benzyl Oꢀ(2ꢀOꢀbenzylꢀ4, 6ꢀOꢀbenzylideneꢀ βꢀDꢀgalactopyranosyl)ꢀ(1
→
4)ꢀ2, 3, 6ꢀtriꢀOꢀ
benzylꢀ βꢀDꢀglucopyranoside (1)
2, 3ꢀDichloroꢀ5, 6ꢀdicyanoꢀ1, 4ꢀbenzoquinone (2.75 g, 12.10 mmol) was added to a solution
of 7 (6.0 g, 6.05 mmol) in 9:1 CH2Cl2– phosphateꢀbuffered saline (200 mL) was added. The
solution was stirred for 1.5h at room temperature and diluted with CH2Cl2. The solution was
washed with aqueous NaHCO3, brine, dried over Na2SO4, and concentrated. The residue was
was purified on a silica gel column (Hexanes: EtOAc = 5:1) to afford the product 1 (5.02 g, 95%)
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as white powder. [α]D = +15.4 (c 1.0, CH2Cl2). H NMR (CDCl3, 400 MHz): δ 7.52ꢀ7.55 (m, 4
H), 7.20ꢀ7.45 (m, 26 H), 5.56 (s, 1 H), 5.21 (d, J = 8.0 Hz 1 H), 4.97ꢀ5.02 (m, 2 H), 4.78ꢀ4.83 (m,
4 H), 4.65ꢀ4.73 (m, 2 H), 4.51ꢀ4.54 (m, 2 H), 4.46 (d, J = 12.3 Hz, 1 H), 4.30 (d, J = 12.3 Hz, 1
H), 4.06ꢀ4.13 (m, 2 H), 3.93ꢀ3.97 (m, 2 H), 3.79 (dd, J = 1.3, 11.0 Hz, 1 H), 3.68 (m, 1 H), 3.55ꢀ
3.60 (m, 3 H), 3.38ꢀ3.41 (m, 1 H), 3.14 (s, 1 H) ; 13C NMR (CDCl3, 100 MHz): δ 138.9, 138.7,
138.6, 138.5, 137.9, 137.5, 129.2, 128.8, 128.4, 128.2, 128.0, 127.8, 127.6, 127.4, 126.5, 102.8,
102.6, 101.5, 83.1, 81.9, 80.2, 77.6, 75.9, 75.7, 75.2, 75.1, 73.1, 72.9, 71.0, 68.9, 68.2, 66.5.
HRMS (ESIꢀorbitrap): [M + Na]+ C54H56NaO11 calcd for 903.3720, found 903.3725.
Ethyl 3, 6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (9)
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A mixture of compound 8 (2 g, 4.09 mmol) and 4 Å molecular sieves (2 g) in dry CH2Cl2 was
stirred at room temperature under nitrogen for 2 h. Triethylsilane (2.1 mL, 13.1 mmol) and TfOH
(1.05 mL, 11.9 mmol) were sequentially added at ꢀ 78 °C. The reaction mixture was stirred at ꢀ
78 °C for 2 h and then quenched with MeOH (2 mL) and Et3N (2 mL). The resulting mixture was
filtered. The filtrate was diluted with CH2Cl2 and washed with aqueous NaHCO3, brine, dried
over Na2SO4, and concentrated. The residue was purified on a silica gel column (Hexanes:
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EtOAc = 5:1) to afford the product 9 (1.85 g, 92.5%) as white powder. [α]D = +91.0 (c 1.0,
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 7.82 (d, J = 5.9 Hz, 1 H), 7.66ꢀ7.72 (m, 3 H), 7.28ꢀ7.42 (m,
5 H), 7.03ꢀ7.10 (m, 2 H), 6.92ꢀ7.00 (m, 3 H), 5.32 (d, J = 9.9 Hz, 1 H), 4.80 (d, J = 12.0 Hz, 1 H),
4.54ꢀ4.70 (m, 3 H), 4.25ꢀ4.34 (m, 2 H), 3.80ꢀ3.89 (m, 3 H), 3.70ꢀ3.73 (m, 1 H), 2.59ꢀ2.72 (m, 2
13
H), 1.19 (t, J = 7.3 Hz, 3 H); C NMR (CDCl3, 100 MHz): δ 168.1, 167.6, 138.2, 137.7, 134.0,
133.9, 131.6, 128.5, 127.9, 127.9, 127.8, 127.5, 123.5, 123.3, 81.2, 79.7, 78.0, 74.5, 74.2, 73.8,
70.7, 54.5, 24.0, 15.0. HRMS (ESIꢀorbitrap): [M + Na]+ C30H31NNaO6S calcd for 556.1770, found
556.1760.
Ethyl 2, 3, 4, 6ꢀtetraꢀOꢀacetylꢀ βꢀDꢀglucopyranosylꢀ(1 4)ꢀ3, 6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀ2ꢀ
→
phthalimidoꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (3)
2, 3, 4, 6ꢀTetraꢀOꢀacetylꢀβꢀDꢀgalactosyl bromide (3.35 g, 8.17 mmol) was prepared by
following general procedure (D). Then the bromide donor (3.35 g, 8.17 mmol) and 3, 6ꢀdiꢀOꢀ
benzylꢀ2ꢀdeoxyꢀ 2ꢀphthalimidoꢀ1ꢀthioꢀβꢀDꢀglucopyranoside 9 (2.64 g. 5.43 mmol) were dissolved
in a mixture of dry toluene and CH2Cl2 (1:1, 30 mL). Powdered molecular sieves (4 Å) were
added and the mixture was stirred under nitrogen for 1 h. The flask was wrapped in aluminum
foil and cooled to −45°C. AgOTf (2.79 g, 10.86 mmol) dissolved in dry toluene (20 mL) was
added during 1 h under the exclusion of light. After additional stirring for 30 min at −45°C, the
reaction mixture was quenched by aqueous Na2S2O3. The mixture transferred to a separatory
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funnel via a Celiteꢀpacked glass filter funnel. The organic phase was separated, dried with
Na2SO4, filtered, and concentrated. Purification of the residue by silica gel column
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chromatography (Hexanes:EtOAc=4:1) gave compound 3 (5.81 g, 80%). [α]D = +31.0 (c 1.0,
9
CHCl3). 1H NMR (CDCl3, 400 MHz): δ 7.77 (d, J = 6.3 Hz, 1 H), 7.62ꢀ7.66 (m, 3 H), 7.29ꢀ743(m,
5 H), 7.01(d, J = 7.0 H), 6.82ꢀ6.92 (m, 3 H), 5.13ꢀ5.28 (m, 3 H), 4.78ꢀ4.87 (m, 3 H), 4.62 (d, J =
7.9 Hz, 1 H), 4.43ꢀ4.51 (m, 2 H), 4.20ꢀ4.28 (m, 2 H), 4.09 (t, J = 9.5 Hz, 1 H), 3.89ꢀ4.02 (m, 2 H),
3.79 (s, 2 H), 3.64 (t, J = 6.8 Hz, 1 H), 3.56 (d, J = 10.0 Hz, 1 H), 2.55ꢀ2.75 (m, 2 H), 2.06 (s, 3
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H), 2.02 (s, 6 H), 1.97 (s, 3 H); 13C NMR (CDCl3, 100 MHz): δ 170.3, 170.2, 170.0, 169.2, 167.9,
167.4, 138.5, 137.9, 133.9, 133.7, 131.6, 128.6, 128.0, 127.9, 127.9, 127.1, 123.4, 123.3, 100.3,
81.1, 79.1, 77.8, 77.6, 74.5, 73.6, 70.4, 69.5, 67.7, 66.9, 60.7, 54.7, 23.9, 20.8, 20.7, 20.6, 20.6,
14.9. HRMS (ESIꢀorbitrap): [M + Na]+ C44H49NNaO15S calcd for 886.2721, found 886.2729.
Benzyl Oꢀ(2, 4ꢀdiꢀOꢀbenzylꢀβꢀDꢀgalactopyranosyl)ꢀ(1
→
4)ꢀ2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀ
glucopyranoside (13)
A mixture of compound 1 (2 g, 2.27 mmol) and 4 Å molecular sieves (2 g) in dry CH2Cl2 was
stirred at room temperature under nitrogen for 2 h. Triethylsilane (0.69 mL, 4.34 mmol) and
PhBCl2 (0.56 mL, 4.34 mmol) were sequentially added at ꢀ 78 °C. The reaction mixture was
stirred at ꢀ78 °C for 2 h and then quenched by the addition of MeOH (2 mL) and Et3N (2 mL).
The resulting mixture was filtered. The filtrate was diluted with CH2Cl2 and washed with aqueous
NaHCO3, brine, dried over Na2SO4, and concentrated. The residue was purified on a silica gel
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column (Hexanes: EtOAc = 5:1) to afford the product 13 (1.70 g, 85%) as white powder. [α]D
=
1
+8.9 (c 1.0, CH2Cl2). H NMR (CDCl3, 400 MHz): δ 7.35ꢀ7.52 (m, 27 H), 7.26ꢀ7.34 (m, 3 H),
5.03ꢀ5.16 (m, 3 H), 4.78ꢀ4.97 (m, 6 H), 4.70ꢀ4.75 (m, 2 H), 4.59ꢀ4.65 (m, 2 H), 4.52 (d, J = 7.0
Hz, 1 H), 4.08 (t, J = 8.7 Hz, 1 H), 3.92 (d, J = 2.6 Hz, 2 H); 13C NMR (CDCl3, 100 MHz): δ 138.9,
138.7 (2 C), 138.5, 137.6, 128.6, 128.6, 128.5, 128.5, 128.4, 128.1, 127.9, 127.8, 127.7, 127.6,
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102.8, 102.6, 82.8, 81.9, 80.5, 77.0, 75.8, 75.5, 75.3, 75.2 (2 C), 75.1, 74.4, 73.4, 71.1, 68.4,
61.7. HRMS (ESIꢀorbitrap): [M + Na]+ C54H58NaO11 calcd for 905.3877, found 905.3867.
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Benzyl Oꢀ(3, 4, 6ꢀtriꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucopyranosyl)ꢀ(1 3)ꢀ[ 3, 4, 6ꢀ
→
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triꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucopyranosyl]ꢀ(1
→
6)ꢀ(2, 4ꢀdiꢀOꢀbenzylꢀβꢀDꢀ
galacꢀtopyranosyl)ꢀ(1 4)ꢀ2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀglucopyranoside (14)
→
3, 4, 6ꢀtriꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucosyl bromide was prepared by following
the general procedure (D). Powdered molecular sieves (4 Å) (3.0 g) was added to a solution of
above bromide donor (4.80 g, 5.44 mmol) and 13 (800 mg, 0.907 mmol) in anhydrous
dichloromethane (20 mL). The suspension was stirred under nitrogen for 1.5 h at room
temperature and then cooled to ꢀ30°C. Then 2, 4, 6ꢀcollidine (0.72 mL, 5.44 mmol), and freshly
dried AgOTf (1.40 g, 5.44 mmol) was sequentially added to the reaction mixture. After stirring
for 2 h at ꢀ30°C, the mixture was allowed to warm up to rt overnight, diluted with CH2Cl2, filtered
through Celite. The filtrate was diluted with CH2Cl2 and washed with aqueous NaHCO3, brine,
dried over Na2SO4, and concentrated. The residue was purified on a silica gel column (Hexanes:
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EtOAc = 5:1) to afford the product 14 (1.33 g, 85%) as white powder. [α]D = +6.4 (c 1.0,
CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.85 (t, J = 4.01 Hz, 1 H), 7.72ꢀ7.74 (m, 2 H), 7.43ꢀ7.56
(m, 2 H), 7.18ꢀ7.40 (m, 27 H), 7.08ꢀ7.16 (m, 3 H), 6.97 (m, 2 H), 5.76ꢀ5.87 (m, 2 H), 5.62 (d, J =
8.24 Hz, 1 H), 5.39(d, J = 8.43, 1H), 5.16ꢀ5.24 (m, 2 H), 4.85ꢀ4.93 (m, 4 H), 4.75 (d, J = 10.6 Hz,
1 H), 4.55ꢀ4.66 (m, 3 H), 4.35ꢀ4.44 (m, 5 H), 4.19ꢀ4.33 (m, 5 H), 4.12 (d, J = 12.7 Hz, 1 H), 3.91
(t, J = 10.1 Hz, 1 H), 3.71ꢀ3.86 (m, 5 H), 3.47ꢀ3.59 (m, 5 H), 3.33ꢀ3.47 (m, 2 H), 3.21 (t, J = 5.9
Hz, 1 H), 3.05(dd, J = 3.2, 9.7 Hz, 1 H), 2.09 (s, 3 H), 2.09 (s, 3 H), 2.02 (s, 3 H), 1.98 (s, 3 H),
1.87(d, 6 H); 13C NMR (CDCl3, 100 MHz): δ 170.7, 170.6, 170.1, 170.0, 169.5, 169.5, 139.1,
139.0, 138.6, 138.5, 138.4, 137.7, 134.4, 134.0, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.8,
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127.6, 127.5, 127.4, 127.4, 127.1, 127.0, 126.9, 126.5, 102.4, 102.3, 99.0, 97.5, 83.1, 81.7, 81.7,
78.6, 76.2, 76.1, 75.6, 75.2, 75.0, 74.9, 74.1, 73.1, 72.8 (2 C), 71.7, 71.6, 70.8, 70.8 (2 C), 70.5,
68.9, 68.8, 68.0, 66.8, 61.7, 61.6, 55.1, 54.7, 20.8, 20.7, 20.7, 20.6, 20.5, 20.4. HRMS (ESIꢀ
orbitrap): [M + Na]+ C94H96N2NaO29 calcd for 1739.5996, found 1739.5980.
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Benzyl Oꢀ(3, 4, 6ꢀtriꢀOꢀacetylꢀ2ꢀdeoxyꢀacetamidoꢀβꢀDꢀglucopyranosyl)ꢀ(1 3)ꢀ[ 3, 4, 6ꢀtriꢀ
→
Oꢀacetylꢀ2ꢀdeoxyꢀacetamidoꢀβꢀDꢀglucopyranosyl]ꢀ(1
→
6)ꢀ(2,4ꢀdiꢀOꢀbenzylꢀβꢀDꢀgalactopyrꢀ
anosyl)ꢀ(1 4)ꢀ2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀglucopyranoside (15)
→
Following the general procedure (A) compound 14 (1.16 g, 0.66 mmol) yielded the compound
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15 (762mg, 75% over two steps). [α]D = ꢀ1.9 (c 0.4, CH2Cl2). H NMR (CDCl3, 400 MHz): δ
7.25ꢀ7.48 (m, 27 H), 7.10ꢀ7.18 (m, 3 H), 5.78 (d, J = 9.5 Hz, 1 H), 5.01ꢀ5.16 (m, 6 H), 4.90ꢀ5.00
(m, 3 H), 4.75ꢀ4.91 (m, 4 H), 4.60ꢀ4.71 (m, 3 H), 4.41ꢀ4.55 (m, 4 H), 4.29 (d, J = 3.5 Hz, 1 H),
3.96ꢀ4.15 (m, 5 H), 3.64ꢀ3.86 (m, 8 H), 3.52ꢀ3.64 (m, 4 H), 3.45ꢀ3.51 (m, 1 H), 3.23 (d, J = 9.7
Hz, 1 H), 2.07 (s, 3 H), 2.06 (s, 3 H), 2.05 (s, 3 H), 2.00 (s, 3 H), 1.99 (s, 3 H), 1.98 (s, 3 H), 1.51
13
(s, 3 H); C NMR (CDCl3, 100 MHz): δ 170.9, 170.8, 170.6, 170.5, 170.3, 169.8, 169.3, 169.2,
139.4, 138.9, 138.8, 138.6, 137.9, 137.4, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.9, 127.8, 127.7, 127.6, 127.5, 126.2, 102.6, 102.5, 102.4, 101.4, 84.3, 82.4, 82.1, 79.5, 76.7,
76.4, 76.2, 75.2, 74.9, 74.7, 74.5, 74.4, 73.3, 73.2, 72.7, 72.0, 70.9, 70.6, 68.8, 68.5, 67.9, 67.5,
62.0, 61.6, 54.4, 53.7, 23.6, 22.8, 20.8, 20.8, 20.7, 20.6 (3 C). HRMS: [M + Na]+ C82H96N2NaO27
calcd for 1563.6098, found 1563.6126.
2ꢀdeoxyꢀacetamidoꢀβꢀDꢀglucopyranosylꢀ(1→3)ꢀ[ 2ꢀdeoxyꢀacetamidoꢀβꢀDꢀglucopyranosꢀ
yl]ꢀ(1 6)ꢀ βꢀDꢀgalactopyranosylꢀ(1 4)ꢀα,βꢀDꢀglucopyranose (HMO1)
→
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Following the general procedure (B) and (C), compound 15 (400 mg, 0.26 mmol) yielded the
compound HMO1 (152 mg, 80% over two steps). 1H NMR (D2O, 400 MHz): δ 5.15 (d, J = 3.6 Hz,
0.55 H, Glcꢀ1 Hꢀ1 of α form), 4.53ꢀ4.62 (m, overlap with D2O, 2.45 H, GlcNAcꢀ1 Hꢀ1, GlcNAcꢀ2
Hꢀ1 and Glcꢀ1 Hꢀ1 of β form), 4.36 (d, J = 7.7 Hz, 1 H, Galꢀ1 Hꢀ1), 4.07(d, J = 2.4 Hz, 1 H), 3.58ꢀ
3.95 (m, 13 H), 3.45ꢀ3.58 (m, 5 H), 3.32ꢀ3.45 (m, 4 H), 3.16ꢀ3.26 (m, 1 H), 1.99 (s, 3 H), 1.96 (s,
3 H); 13C NMR (D2O, 100 MHz): δ 174.9, 174.6, 102.9, 102.8 (GlcNAcꢀ1, GlcNAcꢀ2, Cꢀ1),101.1
(Galꢀ1, Cꢀ1), 95.7 (Glcꢀ1, Cꢀ1 of β form), 91.8 (Glcꢀ1, Cꢀ1 of α form), 81.7, 78.8, 75.8 (2 C), 75.6,
73.8, 74.7, 74.3, 73.8, 73.5, 73.4, 69.9, 69.8, 69.6, 68.7, 68.4, 60.7, 60.5, 55.6, 55.5, 22.4, 22.2.
HRMS (ESIꢀorbitrap): [M + Na]+ C28H48N2NaO21 calcd for 771.2647, found 771.2665.
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Benzyl Oꢀ(2, 3, 4, 6ꢀtetraꢀOꢀacetylꢀβꢀDꢀgalactopyranosyl)ꢀ(1
→
4)ꢀ 3, 6ꢀdiꢀOꢀbenzylꢀ2ꢀ
deoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucopyranosylꢀ(1
→
3)ꢀ(2ꢀOꢀbenzylꢀ4, 6ꢀOꢀbenzylideneꢀβꢀDꢀ
galacꢀtopyranosyl)ꢀ(1 4)ꢀ2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀglucopyranoside (16)
→
Powdered molecular sieves (4 Å) (3.0 g) was added to a solution of Compound 3 (871 mg,
0.96 mmol) and 1 (652 mg, 0.74 mmol) in anhydrous dichloromethane (20 mL). The
suspension was stirred under nitrogen for 1.5 h at room temperature and then cooled to ꢀ30°C.
Then NIS (260mg, 1.15mmol), and TMSOTf (35 ꢂl, 0.19 mmol) was sequentially added to the
reaction mixture. After stirring for 2 h at ꢀ30°C, the mixture was allowed to warm up to rt.
overnight, diluted with CH2Cl2, filtered through Celite. The filtrate was diluted with CH2Cl2 and
washed with aqueous NaHCO3, brine, dried over Na2SO4, and concentrated. The residue was
purified on a silica gel column (Hexanes: EtOAc = 5:1) to afford the product 16 (1.04 g, 86%) as
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white powder. [α]D = +12.9 (c 1.0, CH2Cl2). H NMR (CDCl3, 400 MHz): δ 7.53ꢀ7.55 (m, 2 H),
7.45ꢀ7.50 (m, 2 H), 7.27ꢀ7.45 (m, 27 H), 7.19 (t, J = 3.6 Hz, 2 H), 7.09ꢀ7.16 (m, 3 H), 7.02ꢀ7.06
(m, 2 H), 6.84ꢀ6.94 (m, 5 H), 5.49 (s, 1 H), 5.47 (d, J = 7.4 Hz, 1 H), 5.35 (d, J = 3.2 Hz, 1 H),
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5.25 (dd, J = 7.9, 10.4 Hz, 1 H), 5.09 (d, J = 10.7 Hz, 1 H), 4.88ꢀ4.99 (m, 3 H), 4.85 (d, J = 12.0
Hz, 1 H), 4.65ꢀ4.78 (m, 4 H), 4.61 (d, J = 12.0 Hz, 1 H), 4.44ꢀ4.55 (m, 3 H), 4.30ꢀ4.39 (m, 4 H),
4.18ꢀ4.28 (m, 5 H), 4.09 (t, J = 9.3 Hz, 1 H), 3.98ꢀ4.04 (m, 2 H), 3.89ꢀ3.96 (m, 1 H), 3.80ꢀ3.89
(m 2 H), 3.66ꢀ3.79 (m, 3 H), 3.53ꢀ3.64 (m, 2 H), 3.44ꢀ3.51 (m, 3 H), 3.38 (d, J = 10.1 Hz, 1 H),
2.93ꢀ3.02 (m, 2 H), 2.12 (s, 3 H), 2.10 (s, 3 H), 2.05 (s, 3 H), 2.03 (s, 3 H); 13C NMR (CDCl3, 100
MHz): δ 170.3, 170.2, 170.1, 169.2, 139.0, 138.6 (2 C), 138.5, 138.3, 137.8, 137.6, 133.5, 131.3,
128.7, 128.6 (2 C), 128.4, 128.3 (2 C), 128.2, 128.1 (2 C), 128.0, 127.9, 127.8, 127.7, 127.6,
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127.5, 127.2, 127.1, 126.7, 126.5, 126.3, 123.1, 102.4 (2 C), 100.7, 100.6, 99.6, 83.1, 81.8, 80.9,
78.4, 77.7, 76.0, 75.7, 75.0, 74.8, 74.7, 74.5, 74.3, 73.8 (2 C), 73.0 (2 C), 71.1, 70.9 (2 C), 70.6,
69.7, 68.8, 68.7, 67.9, 67.0, 66.4, 60.8, 55.8, 20.9, 20.7 (2 C), 20.6. HRMS (ESIꢀorbitrap): [M +
Na]+ C96H99NNaO26 calcd for 1704.6353, found 1704.6383.
Benzyl Oꢀ(2, 3, 4, 6ꢀtetraꢀOꢀacetylꢀβꢀDꢀgalactopyranosyl)ꢀ(1
→
4)ꢀ 3, 6ꢀdiꢀOꢀbenzylꢀ2ꢀ
deoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucopyranosylꢀ(1
→
3)ꢀ(2ꢀOꢀbenzylꢀβꢀDꢀgalactopyranosyl)ꢀ(1 4)ꢀ
→
2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀglucopyranoside (19)
To a solution of compound 16 (800 mg, 0.49 mmol) in anhydrous MeOH (10 mL) was added
TsOH (8.4 mg) and EtSH (0.21 ml, 2.93 mmol). The reaction mixture was stirred at rt. for 6 h
and then quenched with triethylamine and evaporated under reduced pressure. The mixture
was purified with silica column (Hexanes: Acetone = 5:1) to get white power compound 19 (702
20
1
mg, 90%). [α]D = +18.4 (c 0.5, CH2Cl2). H NMR (CDCl3, 400 MHz): δ 7.38ꢀ7.52 (m, 11 H),
7.25ꢀ7.36 (m, 16 H), 7.22ꢀ7.26 (m, 2 H), 7.03ꢀ7.14 (m, 3 H), 6.99ꢀ7.03 (m, 2 H), 6.85ꢀ6.92 (m, 2
H), 6.78 (d, J = 7.1 Hz, 2 H), 5.29ꢀ5.31 (m, 2 H), 5.19ꢀ5.25 (m, 1 H), 4.86ꢀ4.98 (m, 4 H), 4.83 (d,
J = 12.2 Hz, 1 H), 4.68ꢀ4.77 (m, 3 H), 4.65 (d, J = 8.3 Hz, 1 H), 4.57ꢀ4.62 (m, 2 H), 4.46 (t, J =
12.4 Hz, 2 H), 4.34ꢀ4.40 (m, 1 H), 4.26ꢀ4.34 (m, 3 H), 4.17ꢀ4.26 (m, 3 H), 3.95ꢀ4.08 (m, 3 H),
3.81ꢀ3.88 (m, 1 H), 3.72ꢀ3.79 (m, 3 H), 3.64ꢀ3.72 (m, 2 H), 3.47ꢀ3.55 (m, 2 H), 3.34ꢀ3.46 (m, 5
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H), 3.17ꢀ3.23 (m, 1 H), 3.00ꢀ3.06 (m, 1 H), 2.87 (s, 1 H), 2.11 (s, 3 H), 2.08 (s, 3 H), 2.05 (s, 3
H), 2.02 (s, 3 H); 13C NMR (CDCl3, 100 MHz): δ 170.3, 170.2, 170.1, 169.2, 138.9, 138.4, 138.3
(2 C), 137.6, 137.5, 133.6, 131.1, 128.8, 128.4, 128.3 (3 C), 128.2 (2 C), 128.1, 127.9, 127.8,
127.7, 127.6, 127.5, 127.4, 127.2, 126.7, 126.4, 123.2, 102.4, 102.1, 100.6, 99.0, 83.8, 82.8,
81.5, 78.3, 77.9, 76.6, 76.4, 75.6, 74.9, 74.6, 74.3, 73.8, 73.7, 73.1, 71.0, 70.9, 70.7, 69.6, 68.3,
67.9, 67.7, 66.9, 62.2, 60.8, 55.6, 20.8, 20.68, 20.6, 20.6. HRMS (ESIꢀorbitrap): [M + Na]+
C89H95NNaO26 calcd for 1616.6040, found 1616.6065.
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Benzyl Oꢀ(2, 3, 4, 6ꢀtetraꢀOꢀacetylꢀβꢀDꢀgalactopyranosyl)ꢀ(1 → 4)ꢀ 3, 6ꢀdiꢀOꢀbenzylꢀ2ꢀ
deoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucopyranosylꢀ(1 → 3) )ꢀ[ 3, 4, 6ꢀtriꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀ
phthalimidoꢀβꢀDꢀglucopyranosyl]ꢀ(1→6)ꢀ(2ꢀOꢀbenzylꢀβꢀDꢀgalactopyranosyl)ꢀ(1→4)ꢀ2, 3, 6ꢀ
triꢀOꢀbenzylꢀ βꢀDꢀglucopyranoside (20)
3, 4, 6ꢀTriꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucosyl bromide was prepared by following
the general procedure (D). Powdered molecular sieves (4 Å) (3.0 g) was added to a solution of
19 (460 mg, 0.29 mmol), 2, 4, 6ꢀcollidine (76 ꢂL, 0.58 mmol), and freshly dried AgOTf (150 mg,
0.58 mmol) in anhydrous dichloromethane (20 mL). The suspension was stirred under nitrogen
for 1.5 h at room temperature and then cooled to ꢀ30°C. Then a solution of above bromide
donor (460 mg, 0.29 mmol) in dichloromethane (5.0 mL) was added dropwise during 30 min to
the reaction mixture. After stirring for 2 h at ꢀ30°C, the mixture was allowed to warm up to rt.
overnight, diluted with CH2Cl2, filtered through Celite. The filtrate was diluted with CH2Cl2 and
washed with aqueous NaHCO3, brine, dried over Na2SO4, and concentrated. The residue was
purified on a silica gel column (Hexanes: EtOAc = 5:1) to afford the product 20 (495 mg, 85%)
as white powder. [α]D20 = +11.7 (c 1.0, CH2Cl2). 1H NMR (CDCl3, 400 MHz): δ 7.76ꢀ7.91 (m, 4 H),
7.49ꢀ7.56 (m, 2 H), 7.41ꢀ7.49 (m, 8 H), 7.14ꢀ7.20 (m, 2 H), 7.05ꢀ7.11 (m, 1 H), 6.97ꢀ7.04 (m, 4
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H), 6.85ꢀ6.94 (m, 3 H), 6.75 (d, J = 7.4 Hz, 2 H), 5.73 (dd, J = 9.1, 10.61 Hz, 1 H), 5.43 (d, J =
8.3 Hz, 1 H), 5.33 (d, J = 3.5 Hz, 1 H), 5.12ꢀ5.25 (m, 2 H), 4.99 (d, J = 8.9 Hz, 1 H), 4.77ꢀ4.95
(m, 3 H), 4.72ꢀ4.84 (m, 3 H), 4.61ꢀ4.71 (m, 2 H), 4.48ꢀ4.58 (m, 2 H), 4.33ꢀ4.47 (m, 3 H), 4.09ꢀ
4.31 (m, 8 H), 3.80ꢀ4.06 (m, 6 H), 3.65ꢀ3.80 (m, 5 H), 3.62 (d, J = 10.0 Hz, 1 H), 3.50ꢀ3.56 (m, 2
H), 3.36ꢀ3.45 (m, 3 H), 3.20ꢀ3.27 (m, 2 H), 3.09ꢀ3.17 (m, 2 H), 2.87 (s, 1 H), 2.14 (s, 3 H), 2.11
(s, 3 H), 2.09 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.99 (s, 3 H), 1.87(s, 3 H); 13C NMR (CDCl3,
100 MHz): δ 170.7, 170.3, 170.2, 170.0 (2 C), 169.5, 169.5, 139.1, 138.7, 138.4, 138.3 (2 C),
137.7, 137.6, 131.2, 128.8, 128.4, 128.3 (3 C), 128.2, 128.1, 127.9 (2 C), 127.8, 127.7, 127.6 (2
C), 127.4, 127.2, 126.5, 123.2, 102.4, 101.9, 100.7, 98.5, 97.6, 83.8, 83.1, 81.9, 78.4, 77.8, 76.5,
76.3, 75.7, 75.1, 74.9, 74.5, 74.4, 74.4, 73.9, 72.9 (2 C), 72.0, 71.7, 71.1, 71.0, 70.8, 70.6, 69.5,
68.8, 68.1, 67.6, 66.9, 66.6, 61.7, 60.8, 55.4, 54.7, 20.9, 20.8, 20.67, 20.6 (3 C), 20.5. HRMS
(ESIꢀorbitrap): [M + Na]+ C109H114N2NaO35 calcd for 2033.7100, found 2033.7089.
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Benzyl Oꢀ(2, 3, 4, 6ꢀtetraꢀOꢀacetylꢀβꢀDꢀgalactopyranosyl)ꢀ(1 → 4)ꢀ 3, 6ꢀdiꢀOꢀbenzylꢀ2ꢀ
deoxyꢀacetamidoꢀβꢀDꢀglucopyranosylꢀ(1→ 3)ꢀ[3, 4, 6ꢀtriꢀOꢀacetylꢀ2ꢀdeoxyꢀacetamidoꢀβꢀDꢀ
glucopyꢀranosyl]ꢀ(1→6)ꢀ(4ꢀOꢀacetylꢀ2ꢀOꢀbenzylꢀβꢀDꢀgalactopyranosyl)ꢀ(1→4)ꢀ2, 3, 6ꢀtriꢀOꢀ
benzylꢀ βꢀDꢀglucopyranoside (21)
Following the general procedure (A), compound 20 (400 mg, 0.20 mmol) yielded the
compound 21 (232 mg, 62% over two steps). [α]D20 = ꢀ1.0 (c 1.2, CH2Cl2). 1H NMR (CDCl3, 400
MHz): δ 7.51 (m, 2H), 7.21ꢀ7.43 (m, 33 H), 5.89 (d, J = 9.5 Hz , 1 H), 5.43 (d, J = 8.4 Hz, 1 H),
5.33 (d, J = 2.8 Hz, 1 H), 4.33ꢀ5.24 (m, 23 H), 4.24 (d, J = 8.2 Hz, 1 H), 3.94ꢀ4.10 (m, 6 H),
3.45ꢀ3.86 (m, 15 H), 3.23 (d, J = 9.7 Hz, 1 H), 2.12 (s, 3 H), 2.11 (s, 3 H), 2.05 (s, 3 H), 2.04 (s,
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3 H), 2.01 (s, 3 H), 2.00 (s, 3 H), 1.99 (s, 3 H), 1.97 (s, 3 H), 1.94 (s, 3 H), 1.54 (s, 3 H); C
NMR (CDCl3, 100 MHz):δ 170.8, 170.7, 170.4, 170.3, 170.2, 170.0, 170.0 (2 C), 169.7, 169.2,
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139.4, 138.7, 138.6 (2 C), 138.0, 137.7, 137.4, 128.7, 128.6, 128.4, 128.4 (2 C), 128.3, 128.2,
128.1, 128.0, 127.9 (2 C), 127.8, 127.7, 127.6, 127.0, 102.6, 102.3, 101.1, 100.7, 100.0, 83.8,
82.4, 79.5, 78.3, 77.9, 76.2 (2 C), 75.6, 75.0, 74.9, 74.6, 74.4, 73.9, 73.5, 73.3, 73.2 (2 C), 73.2,
70.9, 70.8, 70.7, 70.6, 70.0, 69.4, 68.3, 68.0, 67.9, 67.9, 67.3, 66.9, 61.5, 60.8, 54.5, 53.6, 20.9,
20.8, 20.7 (3 C), 20.6 (3 C), 20.5 (2 C). HRMS: [M + Na]+ C99H116N2NaO34 calcd for 1899.7307,
found 1899.7287.
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βꢀDꢀgalactopyranosylꢀ(1
→
4)ꢀ2ꢀdeoxyꢀacetamidoꢀβꢀDꢀglucopyranosylꢀ(1
→
3)ꢀ[ 2ꢀdeoxyꢀ
acetamidoꢀβꢀDꢀglucopyranosyl]ꢀ(1
(HMO2)
→
6)ꢀ βꢀDꢀgalactopyranosylꢀ(1 4)ꢀα,βꢀDꢀglucopyranose
→
Following the general procedure (B) and (C), compound 21 (200 mg, 0.11 mmol) yielded the
compound HMO2 (87 mg, 90%). 1H NMR (D2O, 400 MHz): δ 5.11 (d, J = 3.6 Hz, 0.42 H, Glcꢀ1
Hꢀ1 of α form), 4.53ꢀ4.62 (m, 1.58 H, GlcNAcꢀ1 Hꢀ1, Glcꢀ1 Hꢀ1 of β form), 4.50 (d, J = 8.4 Hz, 1
H, GlcNHACꢀ2 Hꢀ1), 4.36 (d, J = 7.9 Hz, 1 H, Galꢀ1 Hꢀ1), 4.31 (d, J = 8.0 Hz, 1 H, Galꢀ2 Hꢀ1),
4.03 (d, J = 3.0 Hz, 1 H), 3.77ꢀ3.92 (m, 5 H), 3.40ꢀ3.77 (m, 21 H), 3.30ꢀ3.38 (m, 2 H), 3.17 (t, J =
8.9 Hz, 1 H), 1.95 (s, 3 H), 1.92 (s, 3 H); 13C NMR (D2O, 100 MHz): δ 174.9, 174.5, 103.0, 102.8,
102.7 (GlcNAcꢀ1, GlcNAcꢀ2, Galꢀ2, Cꢀ1), 101.0 (Galꢀ1, Cꢀ1), 95.7 (Glcꢀ1, Cꢀ1 of β form), 91.8
(Glcꢀ1, Cꢀ1 of α form), 81.8, 78.9, 78.8, 78.1, 75.8, 75.3, 74.7, 74.5, 74.3, 73.8, 73.4, 72.5, 72.1,
71.4, 71.2, 70.9, 69.6, 68.5, 68.4, 61.0, 60.1, 60.0, 59.8, 55.5, 55.2, 22.4, 22.2. HRMS (ESIꢀ
orbitrap): [M + Na]+ C34H58N2NaO26 calcd for 933.3175, found 933.3195.
Benzyl Oꢀ(3, 4, 6ꢀtriꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀβꢀDꢀglucopyranosyl)ꢀ(1
→
3)ꢀ(2ꢀOꢀ
benzylꢀ4, 6ꢀOꢀbenzylideneꢀβꢀDꢀgalactopyranosyl)ꢀ(1 4)ꢀ2, 3, 6ꢀtriꢀOꢀbenzylꢀ βꢀDꢀglucopyꢀ
→
ranoside (22)
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