Enzymatic and Chemoenzymatic Syntheses of Disialyl Glycans and Their Necrotizing Enterocolitis Preventing Effects

Article abstract of DOI:10.1021/acs.joc.7b02167

Necrotizing enterocolitis (NEC) is one of the most common and devastating intestinal disorders in preterm infants. Therapies to meet the clinical needs for this special and highly vulnerable population are extremely limited. A specific human milk oligosaccharide (HMO), disialyllacto-N-tetraose (DSLNT), was shown to contribute to the beneficial effects of breastfeeding as it prevented NEC in a neonatal rat model and was associated with lower NEC risk in a human clinical cohort study. Herein, gram-scale synthesis of two DSLNT analogs previously shown to have NEC preventing effect is described. In addition, four novel disialyl glycans have been designed and synthesized by enzymatic or chemoenzymatic methods. Noticeably, two disialyl tetraoses have been produced by enzymatic sialylation of chemically synthesized thioethyl β-disaccharides followed by removal of the thioethyl aglycon. Dose-dependent and single-dose comparison studies showed varying NEC-preventing effects of the disialyl glycans in neonatal rats. This study helps to refine the structure requirement of the NEC-preventing effect of disialyl glycans and provides important dose-dependent information for using DSLNT analogs as potential therapeutics for NEC prevention in preterm infants.

Full text of DOI:10.1021/acs.joc.7b02167

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Article
Enzymatic and Chemoenzymatic Syntheses of Disialyl
Glycans and Their Necrotizing Enterocolitis Preventing Effects
Hai Yu, Xuebin Yan, Chloe A Autran, Yanhong Li, Sabrina Etzold, Joanna
Latasiewicz, Bianca M. Robertson, Jiaming Li, Lars Bode, and Xi Chen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02167 • Publication Date (Web): 10 Nov 2017
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Enzymatic and Chemoenzymatic Syntheses of Disialyl Gly-
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Hai Yu,a,b,†* Xuebin Yan,b,c† Chloe A Autran,d Yanhong Li,a,b Sabrina Etzold,d Joanna Latasiewicz,d
Bianca M. Robertson,d Jiaming Li,b,e‡ Lars Bode,d Xi Chenb*
aGlycohub, Inc., 4070 Truxel Road, Sacramento, CA 95834, USA
bDepartment of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
cCollege of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan, 450001, China
dDivision of Neonatology and Division of Gastroenterology and Nutrition, Department of Pediatrics, and Larsson-Rosenquist Foundation
Mother-Milk-Infant Center of Research Excellence (LRF MoMI CoRE), University of California-San Diego, La Jolla, CA 92093, USA
eDepartment of Chemistry, Zhejiang University, Hangzhou, Zhejiang, 310027, China
KEYWORDS: enzymatic synthesis, chemoenzymatic synthesis, oligosaccharide, necrotizing enterocolitis, disialyl glycan, sialic acid
ABSTRACT: Necrotizing enterocolitis (NEC) is one of the most common and devastating intestinal disorders in preterm infants.
Therapies to meet the clinical needs for this special and highly vulnerable population are extremely limited. A specific human milk
oligosaccharide (HMO), disialyllacto-N-tetraose (DSLNT), was shown to contribute to the beneficial effects of breastfeeding as it
prevented NEC in a neonatal rat model and was associated with lower NEC risk in a human clinical cohort study. Herein, gram-scale
synthesis of two DSLNT analogs previously shown to have NEC preventing effect is described. In addition, four novel disialyl
glycans have been designed and synthesized by enzymatic or chemoenzymatic methods. Noticeably, two disialyl tetraoses have been
produced by enzymatic sialylation of chemically synthesized thioethyl -disaccharides followed by removal of the thioethyl aglycon.
Dose-dependent and single-dose comparison studies showed varying NEC-preventing effects of the disialyl glycans in neonatal rats.
This study helps to refine the structure requirement of the NEC-preventing effect of disialyl glycans and provides important dose-
dependent information for using DSLNT analogs as potential therapeutics for NEC prevention in preterm infants.
1. INTRODUCTION
disialyl hexasaccharide analogs of DSLNT, disialyllacto-N-ne-
otetraose (DSLNnT, 2) and DS'LNT (3), have shown promising
effects in preventing NEC in a preclinical neonatal rat model.12
Necrotizing enterocolitis (NEC) is one of the most common
and devastating intestinal disorders in preterm infants.1,2 It af-
fects nearly 10% of all very low-birth-weight infants and leads
to a severe and often fatal destruction in the infant’s intestine.3
Therapies to meet the clinical needs for this special and highly
vulnerable population are extremely limited. Current treatment
of NEC includes cessation of enteral feeding, antibiotic therapy,
and surgical removal of the necrotic intestine, but all of these
treatments are accompanied by devastating long-term compli-
cations.4-6
Herein, gram-scale synthesis of the two promising com-
pounds DSLNnT (2) and DS’LNT (3) using highly efficient
one-pot multienzyme (OPME) systems is described. Both con-
tain two 2–6-linked N-acetylneuraminic acid (Neu5Ac), the
most common sialic acid form. These compounds have been
used for dose-dependent studies in the established NEC neona-
tal rat model.12
To search for additional compounds with potentially en-
hanced NEC-preventing function, four additional novel disialyl
glycans 4–7 (Fig. 1) have been designed and synthesized by
chemoenzymatic or enzymatic methods. Their NEC-preventing
effects have been compared to HMOs, DSLNT (1), DSLNnT
(2), and DS'LNT (3). These include Neu5Gc-DS'LNT (4), a dis-
ialyl hexaose designed to replace the Neu5Ac residues in
DS'LNT (3) by a non-human sialic acid form, N-glycolylneu-
raminic acid (Neu5Gc), which is presented in most animals and
animal products including cow milk. The compound is designed
to probe whether the sialic acid form affects the NEC-
preventing effect of disialyl glycan. DS'LNnT (5) is also in-
cluded. It is a disialyl glycan mimic of DSLNnT (2) in which
the 2–6-sialyl linkage at the non-reducing end is replaced by
an 2–3-sialyl linkage and the resulting terminal disaccharide
structure resembles DSLNT (1) in human milk better. In addi-
It has been known that formula-fed infants have up to 10-
fold higher risk of developing NEC than breast-fed infants.7,8
A
major difference between infant formula and human milk is the
lack of human milk oligosaccharides (HMOs) in common infant
formula. This led to the hypothesis that HMOs contribute to the
protective effects of breast-feeding against NEC.9 Indeed, re-
cent studies have shown that a specific human milk oligosac-
charide (HMO) called disialyllacto-N-tetraose (DSLNT, 1, in
Fig. 1) contributes to the beneficial effects of breastfeeding in
preventing NEC in preclinical neonatal rat model studies9 and
in a human clinical cohort study.10 DSLNT has also been shown
to be a potential marker for NEC-diagnosis.11 Due to challenges
in obtaining DSLNT in large amounts for potential clinical ther-
apeutic applications, five synthetic disialyl glycans have been
produced by convenient enzymatic methods. Among these, two
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tion, disialyl T-antigen tetraose (DSTa, 6) and disialyl galacto- Gram-scale synthesis of disialyl LNT (DS'LNT) hexaose
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biose (DSGalB, 7) are designed to mimic the tetrasaccharide
structure at the non-reducing end of DSLNT (1).
(1.79 g) containing two Neu5Ac residues, Neu5Ac2–6Gal1–
3GlcNAc1–3(Neu5Ac2–6)Gal1–4Glc (3), was completed
similarly in an excellent yield (95%) using the OP2E sialylation
system containing NmCSS and Pd2,6ST with a ratio of 3 to 1
for Neu5Ac to LNT (Scheme 1B). The yield for the gram-scale
synthesis was also comparable to the previous 98% yield for
268 mg-scale synthesis.12
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Scheme 1. One-Pot Two-Enzyme (OP2E) Synthesis of A)
DSLNnT (2) and B) DS'LNT (3).
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A)
B)
Reagents and conditions: (a) Neu5Ac, CTP, MgCl2 (20 mM), Tris-HCl
buffer (pH 8.5), NmCSS, Pd2,6ST, 37 °C. Enzymes used: NmCSS, Neis-
seria meningitidis CMP-sialic acid synthetase; Pd2,6ST, Photobacterium
damselae 2–6-sialyltransferase.
Enzymatic Synthesis of Neu5Gc-Containing DS'LNT
(4). In addition to the most abundant and the most common si-
alic acid form N-acetylneuraminic acid (Neu5Ac), non-human
sialic acid N-glycolylneuraminic acid (Neu5Gc) is also broadly
presented in tissues of animals other than human.15 Neu5Gc has
also been shown to be expressed on glycoproteins and gangli-
osides in human melanoma, colon, retinoblastoma and breast
cancers.16 As rats present both Neu5Ac and Neu5Gc on cell sur-
faces, it is interesting to test whether Neu5Ac and Neu5Gc-con-
taining DS'LNT have noticeable differences in protecting neo-
natal rats from NEC.
Fig. 1. Structures of disialyllacto-N-tetraose (DSLNT, 1) found in human
milk and disialyl glycans (2–7) synthesized and tested for NEC-preventing
effects in this study.
2. RESULTS AND DISCUSSION
Gram-Scale Enzymatic Synthesis of DSLNnT (2) and
DS'LNT (3). Gram-scale syntheses of DSLNnT and DS’LNT
were carried out by sialylating lacto-N-neotetraose (LNnT, 8)12
and lacto-N-tetroase (LNT, 9), respectively, with two Neu5Ac
residues in a one-pot two-enzyme (OP2E) system containing
Neisseria meningitidis cytidine 5'-monophosphate (CMP)-
sialic acid synthetase (NmCSS)13 and Photobacterium dam-
selae 2–6-sialyltransferase (Pd2,6ST).14 As shown in Scheme
1A, Pd2,6ST-catalyzed OP2E sialylation of LNnT using N-
acetylneuraminic acid (Neu5Ac) as the donor precursor (3
equivalents) produced 1.7 grams of the desired product
Neu5Ac2–6Gal1–4GlcNAc1–3(Neu5Ac2–6)Gal1–
4Glc (DSLNnT, 2) in an excellent 96% yield, comparable to the
previous 99% yield for 236 mg-scale synthesis.12
To obtain Neu5Gc-DS'LNT (4), the synthesis was carried
out in the OP2E sialylation system containing NmCSS and
Pd2,6ST using Neu5Gc as the donor precursor and LNT (9) as
the acceptor substrate for Pd2,6ST. As shown in Scheme 2, hex-
asaccharide Neu5Gc2–6Gal1–3GlcNAc1–3(Neu5Gc2–
6)Gal1–4Glc (Neu5Gc-DS'LNT, 4) (272 mg) was obtained in
an excellent 94% yield.
Scheme 2. One-Pot Two-Enzyme (OP2E) Synthesis of
Neu5Gc-DS'LNT (4).
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Reagents and conditions: (a) Neu5Gc, CTP, MgCl2 (20 mM), Tris-HCl
buffer (pH 8.5), NmCSS, Pd2,6ST, 37 °C. Enzymes used: NmCSS, Neis-
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seria meningitidis CMP-sialic acid synthetase; Pd2,6ST, Photobacterium
damselae 2–6-sialyltransferase.
Enzymatic Synthesis of DS'LNnT (5) Using a Sequential
One-Pot Multienzyme (OPME) Glycosylation Approach.
DS’LNnT (5) is a hexasaccharide containing the LNnT (8) core
with a Neu5Ac 2–6-linked to the internal galactose (Gal) res-
idue and a Neu5Ac 2–3-linked to the terminal Gal. This hex-
asaccharide is designed to better mimic the natural DSLNT (1)
found in human milk. Enzymatic sialylation with different si-
alyl linkages in DS’LNnT (5) was achieved by using a glyco-
sylation sequence different from the one used for the synthesis
of DSLNnT (2). Instead of carrying out 2–6-sialylation to in-
troduce two 2–6-linked Neu5Ac simultaneously after the for-
mation of LNnT using a 1–4-galactosyltransferase-catalyzed
one-pot multienzyme (OPME) reaction, the formation of 2–3-
and 2–6-linked Neu5Ac in DS’LNnT (5) was accomplished
by 2–6-sialylation of the trisaccharide core GlcNAc1–
3Gal1–4Glc (Lc3, 10) followed by sequential extension of the
core with 1–4-galactosylation and 2–3-sialylation.17 As
shown in Scheme 3, trisaccharide GlcNAc1–3Gal1–4Glc
(Lc3, 10) obtained from lactose in a one-pot four-enzyme Glc-
NAc-activation and transfer reaction12 containing recombinant
enzymes Bifidobacterium longum strain ATCC55813 N-
acetylhexosamine-1-kinase (BLNahK),18 Pasteurella multo-
cida N-acetylglucosamine uridylyltransferase (PmGlmU),19
Pasteurella multocida inorganic pyrophosphatase (PmPpA),20
and Neisseria meningitidis 1–3-N-acetylglucosaminyltrans-
ferase (NmLgtA)21 was used for 2–6-sialylation by the OP2E
system containing NmCSS and Pd2,6ST. Tetrasaccharide Glc-
NAc1–3(Neu5Ac2–6)Gal1–4Glc (11) (0.3 g) was obtained
in an excellent 96% yield. Next, galactosylation of this tetrasac-
charide using a one-pot four-enzyme galactosylation system
containing Escherichia coli galactokinase (EcGalK),22
Bifidobacterium longum UDP-sugar phosphorylase (BLUSP),23
Pasteurella multocida inorganic pyrophosphatase (PmPpA),20
and Neisseria meningitidis 1–4-galactosyltransferase
(NmLgtB)20 produced pentasaccharide Gal1–4GlcNAc1–
3(Neu5Ac2–6)Gal1–4Glc (12) (0.167 g) in 94% yield. This
showed that oligosaccharides such as tetrasaccharide 11 is a bet-
ter acceptor substrate than monosaccharide GlcNAc20 for
NmLgtB. Finally, further sialylation of the pentasaccharide (12)
using an OP2E 2–3-sialylation system containing NmCSS and
Reagents and conditions: (a) Neu5Ac, CTP, MgCl2 (20 mM), Tris-HCl
buffer (pH 8.5), NmCSS, Pd2,6ST, 37 °C; (b) Gal, ATP, UTP, 20 mM
MgCl2, Tris-HCl buffer (pH 8.0), EcGalK, BLUSP, NmLgtB, PmPpA, 30
°C; (c) Neu5Ac, CTP, MgCl2 (20 mM), Tris-HCl buffer (pH 8.5), NmCSS,
PmST1_M144D, 37 °C. Enzymes used: NmCSS, Neisseria meningitidis
CMP-sialic acid synthetase; Pd2,6ST, Photobacterium damselae 2–6-si-
alyltransferase; EcGalK, Escherichia coli galactokinase; BLUSP,
Bifidobacterium longum UDP-sugar phosphorylase; PmPpA, Pasteurella
multocida inorganic pyrophosphatase; NmLgtB, Neisseria meningitidis
1–4-galactosyltransferase; PmST1_M144D, Pasteurella multocida 2–3-
sialyltransferase 1 M144D mutant.
Chemoenzymatic synthesis of tetrasaccharide DSTa (6).
The next disialyl glycan target is a tetrasaccharide containing a
Gal1–3GalNAc (T antigen or Ta) core. Ta is an essential com-
ponent of glycans presented on glycoproteins (e.g. O-GalNAc
glycan core-2 structure) and glycolipids, and also an important
carbohydrate epitope involved in cell adhesion, signaling, ferti-
lization, differentiation, development, and cancer metastasis.25
The disialyl Ta tetrasaccharide (DSTa, 6) has two 2–6-linked
Neu5Ac residues on neighboring monosaccharide units in the
disaccharide core, mimicking the situation of the terminal tetra-
saccharide component in human milk DSLNT (1). It will be in-
teresting to test whether it has NEC-preventing effects.
Gal1–3GalNAc was synthesized in gram-scale from N-
acetylgalactosamine (GalNAc), galactose (Gal), and adenosine
5'-triphosphate (ATP) using a one-pot two-enzyme (OP2E) ga-
lactosylation system containing EcGalK and Bifidobacterium
infantis D-galactosyl-1–3-N-acetyl-D-hexosamine phosphor-
ylase (BiGalHexNAcP).26 Silica gel filtration and crystalliza-
tion were used to obtain the pure product. Attempts for adding
two Neu5Ac residues simultaneously by sialylating disaccha-
ride Gal1–3GalNAc using an excess amount (three equiva-
lents) of Neu5Ac in the OP2E 2–6-sialylation system contain-
ing NmCSS and Pd2,6ST led to the formation of mono-
sialylated glycan as the major product and the disialyl glycan
was formed in a low yield. This was most likely due to the pre-
dominant presence of the -anomer at the free reducing end of
the Gal1–3GalNAc (Ta) in the crystallized disaccharide. As
Gal1–3GalNAcOR with a beta-linkage at the reducing end
of the disaccharide is a suitable glycan for direct disialylation,27
disaccharide Gal1–3GalNAcSEt with a thioethyl group at the
reducing end which can be selectively removed after the sialyla-
tion reaction was designed as the acceptor substrate for dis-
ialylation reactions. As shown in Scheme 4, GalNAcSEt (14)
was readily synthesized from galactosamine (GalNH2) using a
Pasteurella multocida 2–3-sialyltransferase
1 (PmST1)
M144D (PmST1_M144D) mutant24 produced the desired
DS’LNnT (5) (121 mg) in 93% yield.
Scheme 3. Sequential One-Pot Multienzyme (OPME) Syn-
thesis of DS'LNnT (5).
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reported method.28 The disaccharide Gal1−3GalNAcSEt
(15) was then enzymatically synthesized from GalNAcSEt via
the OP2E galactosylation reaction containing EcGalK and BiG-
alHexNAcP.26 OP2E 2–6-sialylation of Gal1−3GalNAcSEt
(15) using three equivalents of Neu5Ac formed the desired dis-
ialyl tetrasaccharide Neu5Ac2–6Gal1−3(Neu5Ac2–6)Gal-
NAcSEt (16) in an excellent 93% yield. Removal of the thi-
oethyl group in compound 16 using N-bromosuccinimide
(NBS) in acetone-water led to the formation of DSTa (6) in 94%
yield.
Disialylation of Gal1–3GalSEt (18) using the OP2E 2–
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6-sialylation system containing NmCSS and Pd2,6ST formed
the desired tetrasaccharide Neu5Ac2–6Gal1−3(Neu5Ac2–
6)GalSEt (19) in 84% yield. Removal of the thioethyl group
using N-bromosuccinimide in acetone-water followed by puri-
fication produced DSGalB (7) in 87% yield.
Scheme 5. Chemoenzymatic Synthesis of Disialyl Galactobi-
ose (DSGalB) (7).
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Scheme 4. Chemoenzymatic Synthesis of Disialyl T-Antigen
DSTa (6).
Reagents and conditions: (a) Gal, ATP, UTP, MgCl2 (20 mM), Tris-HCl
buffer (pH 8.0), EcGalK, BLUSP, PmPpA, CjCgtB, 37 C; (b) Neu5Ac,
CTP, MgCl2 (20 mM), Tris-HCl buffer (pH 8.5), NmCSS, Pd2,6ST, 37 °C;
(c) NBS, acetone, H2O. Enzymes used: EcGalK, Escherichia coli galacto-
kinase; BLUSP, Bifidobacterium longum UDP-sugar phosphorylase;
PmPpA, Pasteurella multocida inorganic pyrophosphatase; CjCgtB, Cam-
pylobacter jejuni 1–3-galactosyltransferase.
In vivo Efficacy of Disialyl Glycans in Preventing Ne-
crotizing Enterocolitis (NEC) in a Preclinical Neonatal Rat
Model. The in vivo efficacy in preventing NEC in the neonatal
rat model of the four new disialyl glycans Neu5Gc-DS'LNT (4),
DS'LNnT (5), DSTa (6), and DSGalB (7) was tested and com-
pared to two synthetic disialyl hexasaccharides DSLNnT (2),
and DS'LNT (3), as well as HMOs. A single concentration of
300 M was used for this purpose. The choice of this concen-
tration was based on the concentration of DSLNT (1) deter-
mined by HPLC in the pooled HMOs supplemented to formula
(10 mg/mL, similar to the level of HMO concentrations in ma-
ture milk) for in vivo neonatal rat feeding studies in a previous
study.9 As shown in Fig. 2, similar to previously reported find-
ings,12 DSLNnT (2) and DS'LNT (3) reduced the pathology
scores significantly, although less effectively compared to
HMOs. Replacing the Neu5Ac in DS'LNT (3) by Neu5Gc in
Neu5Gc-DS'LNT (4) decreased the efficiency slightly. Replac-
ing the terminal 2–6-linked Neu5Ac in DSLNnT (2) by the
terminal 2–3-linked Neu5Ac in DS'LNnT (5) led to moder-
ately decreased efficacy. On the other hand, disialyl tetraoses
including DSTa (6) and DSGalB (7) did not have obvious NEC-
preventing effect. These results indicate that Neu5Ac is a better
sialic acid form than Neu5Gc for NEC-preventing disialyl com-
pounds. In addition, disialyl hexasaccharides are more effective
NEC-preventing compounds than disialyl tetrasaccharides. Fur-
thermore, the distance between two sialic acid residues on the
disialyl hexasaccharide is also important.
Reagents and conditions: (a) i) Phthalic anhydride, NaOH (1 M); ii) Ac2O,
.
.
pyridine; iii) EtSH, BF3 Et2O, CH2Cl2, 44% in three steps; (b) i) N2H4 H2O,
MeOH; ii) Ac2O, MeOH, 93% in two steps; (c) Gal, ATP, MgCl2 (20 mM),
Tris-HCl buffer (pH 6.8), EcGalK, BiGalHexNAcP, 37 °C; (d) Neu5Ac,
CTP, MgCl2 (20 mM), Tris-HCl buffer (pH 8.5), NmCSS, Pd2,6ST, 37 C;
(e) NBS, acetone, H2O. Enzymes used: EcGalK, Escherichia coli galacto-
kinase; BiGalHexNAcP, Bifidobacterium infantis D-galactosyl-1–3-N-ac-
etyl-D-hexosamine phosphorylase; NmCSS, Neisseria meningitidis CMP-
sialic acid synthetase; Pd2,6ST, Photobacterium damselae 2–6-sialyl-
transferase.
Chemoenzymatic Synthesis of Disialyl Galactobiose
DSGalB (7). Gal1–3Gal (galactobiose, GalB) exists in nature
as a part of galactan structures in some bacteria and plants.29 It
also constitutes galacto-oligosaccharide (GOS) structures that
are currently used as prebiotics.30,31 Structurally, galactobiose
differs from Gal1–3GalNAc (Ta) only at the carbon-2 substi-
tution of the monosaccharide at the reducing end (OH of the Gal
in GalB versus NHAc of the GalNAc in Ta). It will be interest-
ing to test whether a single substitution in the core disaccharide
structure of DSTa tetrasaccharide (6) affects its NEC preventing
efficiency.
A similar thioethyl -glycoside acceptor strategy for the
production of disialyl glycan DSTa (6) was used for the synthe-
sis of DSGalB (7). GalSEt (17)32 was synthesized chemically
and used for producing Gal1–3GalSEt (18) using a one-pot
four-enzyme (OP4E) galactosylation system containing
EcGalK, BLUSP, PmPpA, and Campylobacter jejuni 1–3-ga-
lactosyltransferase (CjCgtB)33 (Scheme 5). To our delight, the
monosaccharide galactoside GalSEt (17) was a suitable accep-
tor for CjCgtB. A moderate 50% yield was obtained for the
OP4E galactosylation reaction. The relatively low yield was
due to GalSEt (17) being a less optimal acceptor than Gal-
NAcOR in GM2 or GD2 ganglioside oligosaccharides.34
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The Journal of Organic Chemistry
(DSGalB, 7) have been synthesized using a novel chemoen-
1
2
3
4
5
6
7
8
zymatic method involving disialylation of -thioethyl glyco-
sides followed by removal of the thioethyl group. The effects of
all six compounds synthesized have been studied in an estab-
lished neonatal rat NEC model. The glycan size, sialyl linkages,
the distance of two sialic acid residues, and sialic acid forms
have been indicated as important factors for the NEC-
preventing effects of disialyl glycans. The study helps to refine
the structure requirement of the NEC-preventing effect of dis-
ialyl glycans and provides important dose-dependent infor-
mation for using DSLNT analogs as potential therapeutics for
NEC prevention in preterm infants. Longer disialyl glycans and
glycans with more sialic acid residues can be explored further
for potentially improved NEC-preventing effect.
9
10
11
12
Figure 2. In vivo efficacy testing in preclinical rat model of NEC. A. Com-
13
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17
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paring all synthesized glycans at a concentration of 300 M. Number of
animals per group: dam fed (DF): 22; formula fed (FF): 14; HMO: 20;
DSLNnT: 18; DS'LNT: 17; Neu5GC-DS'LNT: 15; DS'LNnT: 13; DSTa:
11; DSGalB: 9.
4. EXPERIMENTAL SECTION
Materials and general methods. Chemicals and reagents
were purchased from commercial sources and used without fur-
ther purification unless stated otherwise. 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance-800 NMR spectrom-
eter and Bruker Avance-400 NMR spectrometer. High resolu-
tion electrospray ionization (ESI) mass spectra were obtained
using Thermo Electron LTQ-Orbitrap Hybrid MS at the Mass
Spectrometry Facility in the University of California, Davis.
Silica gel 60 Å (230–400 mesh, Sorbent Technologies) was
used for flash column chromatography. Thin-layer chromatog-
raphy (TLC, Sorbent Technologies) was performed on silica gel
plates using anisaldehyde sugar stain or 5% sulfuric acid in eth-
anol stain for detection. Gel filtration chromatography was per-
formed using a column (100 cm × 2.5 cm) packed with Bio-Gel
P-2 Fine resins (Bio-Rad). Lac, Gal, and GalNAc were pur-
chased from Fisher Scientific, Neu5Ac was from Atomole Sci-
entific, UTP and CTP were from Chemfun Medical Technology
Co., and ATP was from Beta Pharm Inc. Lacto-N-tetraose
(LNT) was from Elicityl (Crolles, France). Recombinant en-
zymes Bifidobacterium longum strain ATCC55813 N-
acetylhexosamine-1-kinase (BLNahK),18 Pasteurella multo-
cida N-acetylglucosamine uridyltransferase (PmGlmU),19 Pas-
teurella multocida inorganic pyrophosphatase (PmPpA),20
Neisseria meningitidis 1–3-N–acetylglucosaminyltransferase
(NmLgtA),21 Escherichia coli galactokinase (EcGalK),22
Bifidobacterium longum UDP-sugar pyrophosphorylase
(BLUSP),23 Neisseria meningitidis 1–4-galactosyltransferase
(NmLgtB),20 Neisseria meningitidis CMP-sialic acid synthetase
(NmCSS),13 Photobacterium damselae 2–6-sialyltransferase
(Pd2,6ST),14 Pasteurella multocida 2–3-sialyltransferase 1
M144D mutant (PmST1 M144D),24 Bifidobacterium infantis D-
galactosyl-1,3-N-acetyl-D-hexosamine phosphorylase (BiG-
alHexNAcP),26 and Campylobacter jejuni β13-galactosyl-
transferase (CjCgtB)33 were expressed and purified as described
previously. HMOs used were obtained previously.9 Major com-
ponents are 2’-fucosyllactose (2’-FL), lacto-N-fucopentaose 1
(LNFP1), lacto-N-tetraose (LNT), lacto-N-fucopentaose 2
(LNFP2), 3’-sialyllactose (3’-SL), lacto-N-neotetraose (LNnT),
3-fucosyllactose (3FL), sialyllacto-N-tetraose c (LSTc), sialyl-
Dose-dependent responses to DSLNnT (2) and DS’LNT (3)
were tested at five different concentrations (30, 100, 300, 1,000,
and 2,000 M). These concentrations represent the range of
DSLNT (1) in 10-fold diluted HMO sample to 7-fold concen-
trated HMO sample. As shown in Fig. 3, DSLNnT effects plat-
eaued at high concentrations between 300 and 2,000 M with-
out reaching the maximum beneficial effects of HMOs. The ef-
fects of DS’LNT, however, continued to improve with higher
concentrations, reaching similar protective effects as HMOs.
4
DSLNnT
3
2
1
0
10
100
1000
10000
concentration (μM)
4
DS’LNT
3
2
1
0
10
100
1000
10000
concentration (μM)
Figure 3. Dose-dependent studies of DSLNnT and DS’LNT in the estab-
lished neonatal rat NEC model. Each compound at each concentration was
orally administered to 15 neonatal rats.
3. CONCLUSIONS
lacto-N-tetraose
b (LSTb), and disialyl lacto-N-tetraose
(DSLNT).
In summary, we have herein shown the efficient production
of a group of disialyl glycans by chemoenzymatic or enzymatic
synthetic approaches. Among them, DSLNnT (2) and DS’LNT
(3) have been synthesized in gram-scales. Disialyl tetrasaccha-
rides including disialyl Ta (DSTa, 6) and disialyl galactobiose
One-pot two-enzyme gram-scale synthesis of Gal1–
3GalNAc (GNB). A reaction mixture in Tris-HCl buffer (100
mM, pH 6.9) in a total volume of 270 mL containing GalNAc
(3.00 g, 13.6 mmol), galactose (3.17 g, 17.6 mmol), ATP (9.73
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Page 6 of 11
NMR (800 MHz, D2O)  5.21 (d, J = 3.2 Hz, 0.4H), 4.72 (d, J
g, 17.6 mmol), MgCl2 (20 mM), EcGalK (30 mg), and BiG-
1
2
3
4
5
6
7
8
alHexNAcP (50 mg) was incubated in a shaker at 37 C by ag-
itating at 140 rpm. The reaction was monitored by mass spec-
trometry. When an optimal yield was achieved (after 1 day), the
enzyme reaction was terminated by incubating the reaction mix-
ture in a boiling water bath for 15 min and the mixture was cen-
trifuged to remove the precipitates. The supernatant was con-
centrated and purified by silica gel chromatography
(EtOAc:MeOH:H2O = 2:1:0.1 by volume) to produce crude di-
saccharide Gal1–3GalNAc as - and -anomers (4.6 g, over
90% purity). The crude product was further crystallized from
water:EtOH = 1:1 (20 mL, 60 C) to produce 1.63 g of GNB
crystal as the -anomer. The spectroscopic data coincided with
a previous report.26
= 8.0 Hz, 1H), 4.66 (d, J = 8.0 Hz, 0.6H), 4.43 (d, J = 8.0 Hz,
1H), 4.38 (d, J = 8.0 Hz, 1H), 4.18 (s, 1H), 3.99–3.28 (m, 37H),
2.72–2.69 (m, 2H), 2.03 (s, 6H), 2.02 (s, 3H), 1.73 (t, J = 12.0
Hz, 1H). 1.69 (t, J = 12.0 Hz, 1H). 13C NMR (151 MHz, D2O)
 174.9, 174.8, 174.8, 173.4, 103.8, 103.1, 103.1, 102.5, 100.2,
100.1, 95.5, 91.7, 83.6, 82.0, 79.7, 79.6, 75.2, 74.6, 74.5, 74.2,
73.6, 73.5, 73.2, 72.4, 72.4, 72.3, 71.7, 71.7, 71.5, 70.9, 70.4,
69.9, 69.7, 69.7, 69.3, 68.6, 68.4, 68.3, 68.3, 68.2, 68.1, 68.1,
63.4, 63.4, 62.6, 60.6, 60.2, 60.0, 54.4, 51.7, 51.7, 40.1, 40.0,
22.2, 22.0. HRMS (ESI) m/z calculated for C48H78N3O37 [M-H]-
1288.4320, found 1288.4315.
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13
14
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16
17
18
19
20
21
22
23
24
25
26
27
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34
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One-pot two-enzyme preparative-scale synthesis of
Neu5Gc2–6Gal1–3GlcNAc1–3(Neu5Gc2–6)Gal1–
4Glc (Neu5Gc-DS’LNT, 4). A reaction mixture in a total vol-
ume of 10 mL containing Tris-HCl buffer (100 mM, pH 8.5),
LNT (150 mg, 0.21 mmol), Neu5Gc (206 mg, 0.63 mmol), CTP
(335 mg, 0.63 mmol), MgCl2 (20 mM), NmCSS (3.0 mg), and
Pd2,6ST (5.0 mg) was incubated in a shaker at 37 C for 2 d.
The reaction was monitored by mass spectrometry. When an
optimal yield was achieved, 10 mL of ethanol was added to the
reaction mixture and the resulting mixture was incubated at 4
C for 30 min. The precipitates were removed by centrifugation
and the supernatant was concentrated and purified by a Bio Gel
P-2 gel column (water was used as an eluent). Further purifica-
tion was achieved by silica gel chromatography
(EtOAc:MeOH:H2O = 4:3:2 by volume) and by Bio-Gel P-2
column (eluted with H2O) in the final step to produce
Neu5Gc2–6Gal1–3GlcNAc1–3(Neu5Gc2–6)Gal1–
4Glc hexasaccharide as white powder (4, 272 mg, 94%). 1H
NMR (800 MHz, D2O)  5.21 (d, J = 3.2 Hz, 0.4H), 4.72 (d, J
= 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz, 0.6H), 4.41 (d, J = 8.0 Hz,
1H), 4.37 (d, J = 8.0 Hz, 1H), 4.17 (s, 1H), 4.10 (s, 4H), 3.99–
3.28 (m, 37H), 2.73–2.70 (m, 2H), 2.01 (s, 3H), 1.74 (t, J = 12.0
Hz, 1H). 1.70 (t, J = 12.0 Hz, 1H). 13C NMR (201 MHz, D2O)
 175.6, 175.5, 174.8, 173.4, 173.4, 103.8, 103.1, 103.1, 102.5,
100.2, 100.0, 95.5, 91.7, 83.5, 81.9, 79.7, 79.6, 75.2, 74.6, 74.5,
73.6, 73.5, 73.2, 72.3, 72.1, 72.1, 71.8, 71.7, 71.5, 70.9, 70.4,
69.8, 69.7, 69.6, 68.6, 68.3, 68.2, 68.1, 68.1, 68.0, 67.9, 63.4,
62.5, 60.9, 60.5, 60.1, 60.0, 54.4, 51.4, 51.4, 40.1, 40.0, 22.1.
HRMS (ESI) m/z calculated for C48H78N3O39 [M-H]-
1320.4218, found 1320.4210.
One-pot two-enzyme preparative-scale synthesis of
Neu5Ac2–6Gal1–4GlcNAc1–3(Neu5Ac2–6)Gal1–
4Glc (DSLNnT, 2). A reaction mixture in a total volume of 50
mL containing Tris-HCl buffer (100 mM, pH 8.5), LNnT12
(1.05 g, 1.48 mmol), Neu5Ac (1.37 g, 4.43 mmol), CTP (2.35
g, 4.43 mmol), MgCl2 (20 mM), NmCSS (13 mg), and Pd2,6ST
(16 mg) was incubated in a shaker at 37 C for 2 d. The reaction
was monitored by mass spectrometry. When an optimal yield
was achieved, 50 mL of ethanol was added to the reaction mix-
ture and the resulting mixture was incubated at 4 C for 30 min.
The precipitates were removed by centrifugation and the super-
natant was concentrated and purified by a Bio Gel P-2 gel col-
umn (water was used as an eluent). Further purification was
achieved by silica gel chromatography (EtOAc:MeOH:H2O =
4:3:2 by volume) and by Bio-Gel P-2 column (eluted with H2O)
in the final step to produce Neu5Ac2–6Gal1–4GlcNAc1–
3(Neu5Ac2–6)Gal1–4Glc hexasaccharide as white powder
1
(2, 1.70 g, 96%). H NMR (800 MHz, D2O)  5.21 (d, J = 3.2
Hz, 0.4H), 4.71 (d, J = 8.0 Hz, 1H), 4.66 (d, J = 8.0 Hz, 0.6H),
4.43 (d, J = 8.0 Hz, 1H), 4.39 (d, J = 8.0 Hz, 1H), 4.17 (s, 1H),
4.00–3.28 (m, 37H), 2.70 (dd, J = 4.8 and 12.0 Hz, 1H), 2.66
(dd, J = 4.8 and 12.0 Hz, 1H), 2.04 (s, 3H), 2.02 (s, 6H), 1.72
(t, J = 12.0 Hz, 1H). 1.71 (t, J = 12.0 Hz, 1H). 13C NMR (151
MHz, D2O)  174.8, 173.4, 173.4, 103.4, 103.1, 103.1, 102.5,
100.1, 100.0, 95.5, 91.7, 82.0, 80.4, 79.6, 79.5, 74.5, 74.5, 74.1,
73.5, 73.1, 72.4, 72.4, 72.2, 72.2, 71.6, 71.6, 71.5, 70.9, 70.6,
69.8, 69.5, 69.5, 68.2, 68.2, 68.1, 68.0, 68.0, 63.3, 63.2, 62.5,
62.4, 60.1, 60.0, 59.9, 54.8, 51.7, 51.6, 40.0 39.9, 22.1, 21.9,
21.8. HRMS (ESI) m/z calculated for C48H78N3O37 [M-H]-
1288.4320, found 1288.4305.
One-pot two-enzyme preparative-scale synthesis of
Neu5Ac2–3Gal1–4GlcNAc1–3(Neu5Ac2–6)Gal1–
4Glc (DS’LNnT, 5).
One-pot two-enzyme preparative-scale synthesis of
DS’LNT
Neu5Ac2–6Gal1–3GlcNAc1–3(Neu5Ac2–
(a) One-pot two-enzyme preparative-scale synthesis of
GlcNAc1–3(Neu5Ac2–6)Gal1–4Glc (11). The reaction
mixture in a total volume of 15 mL containing Tris-HCl buffer
(100 mM, pH 8.5), Lc3 trisaccharide GlcNAc1–3Gal1–4Glc
(0.2 g, 0.367 mmol), Neu5Ac (0.17 g, 0.55 mmol), CTP (0.29
g, 0.55 mmol), MgCl2 (20 mM), NmCSS (4 mg), Pd26ST (5
mg) was incubated in a shaker with agitation (100 rpm) at 37
C for 24 h. The product formation was monitored by TLC (i-
PrOH:H2O:NH4OH = 5:2:1 by volume, and detected by p-
anisaldehyde sugar stain) and mass spectrometry. When an op-
timal yield was achieved, ethanol (15 mL) was added and the
mixture was incubated at 4 C for 30 min. The precipitates were
removed by centrifugation and the supernatant was concen-
trated and purified by a Bio-Gel P-2 gel column (water was used
as an eluate). Further purification was achieved by silica gel
chromatography (EtOAc:MeOH:H2O = 4:2:1 by volume) and
6)Gal1–4Glc (DS’LNT, 3). A reaction mixture in a total vol-
ume of 50 mL containing Tris-HCl buffer (100 mM, pH 8.5),
LNT (1.0 g, 1.41 mmol), Neu5Ac (1.31 g, 4.24 mmol), CTP
(2.23 g, 4.24 mmol), MgCl2 (20 mM), NmCSS (12 mg), and
Pd2,6ST (15 mg) was incubated in a shaker at 37 C for 2 d.
The reaction was monitored by mass spectrometry. When an
optimal yield was achieved, 50 mL of ethanol was added to the
reaction mixture and the resulting mixture was incubated at 4
C for 30 min. The precipitates were removed by centrifugation
and the supernatant was concentrated and purified by a Bio Gel
P-2 gel column (water was used as an eluent). Further purifica-
tion was achieved by silica gel chromatography
(EtOAc:MeOH:H2O = 4:3:2 by volume) and by Bio-Gel P-2
column (eluted with H2O) in the final step to produce
Neu5Ac2–3Gal1–3GlcNAc1–3(Neu5Ac2–6)Gal1–
1
4Glc hexasaccharide as white powder (3, 1.79 g, 95%). H
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The Journal of Organic Chemistry
finally Bio-Gel P-2 column (eluted with H2O) to produce Glc- = 8.0 Hz, 1H), 4.66 (d, J = 8.0 Hz, 0.6H), 4.54 (d, J = 8.0 Hz,
1
2
3
4
5
6
7
8
1H), 4.41 (d, J = 8.0 Hz, 1H), 4.18–3.26 (m, 38H), 2.74 (dd, J
= 4.8 and 12.0 Hz, 1H), 2.70 (dd, J = 4.8 and 12.0 Hz, 1H),
2.01 (s, 9H), 1.80 (t, J = 12.0 Hz, 1H). 1.72 (t, J = 12.0 Hz, 1H).
13C NMR (201 MHz, D2O)  174.9, 174.8, 174.8, 173.8, 173.5,
173.4, 173.4, 103.1, 102.8, 102.7, 102.4, 100.2, 99.7, 95.5, 91.7,
82.0, 79.6, 79.5, 77.9, 75.3, 75.0, 74.6, 74.5, 74.4, 73.6, 73.1,
72.8, 72.4, 72.1, 71.7, 71.6, 70.9, 70.6, 69.6, 69.3, 68.1, 68.0,
67.4, 63.4, 63.4, 62.6, 62.5, 62.4, 60.9, 60.9, 60.1, 60.0, 59.7,
55.1, 51.7, 51.6, 40.0, 40.0, 39.5, 39.5, 22.1, 22.0, 22.0. HRMS
(ESI) m/z calculated for C48H78N3O37 [M-H]- 1288.4320, found
1288.4318.
NAc1–3(Neu5Ac2–6)Gal1–4Glc as white powder (11,
0.30 g, 96%). 1H NMR (800 MHz, D2O)  5.20 (d, J = 3.2 Hz,
0.4H), 4.66 (d, J = 8.0 Hz, 0.6H), 4.65 (d, J = 8.0 Hz, 1H), 4.41
(d, J = 8.0 Hz, 1H), 4.19–3.28 (m, 25H), 2.68 (dd, J = 4.8 and
12.0 Hz, 1H), 2.01 (s, 6H), 1.72 (t, J = 12.0 Hz, 1H). 13C NMR
(201 MHz, D2O)  174.8, 173.4, 103.1, 102.8, 100.2, 95.5, 91.7,
82.0, 79.6, 79.5, 75.5, 74.6, 74.5, 73.6, 73.5, 73.1, 72.4, 71.7,
70.9, 69.9, 69.6, 68.2, 68.1, 63.3, 62.5, 60.4, 60.1, 55.5, 51.7,
40.0, 22.0, 21.9. HRMS (ESI) m/z calculated for C31H51N2O24
[M-H]- 835.2837, found 835.2833.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(b) One-pot four-enzyme preparative-scale synthesis of
Gal1–4GlcNAc1–3(Neu5Ac2–6)Gal1–4Glc (12). The re-
action mixture in a total volume of 10 mL containing Tris-HCl
buffer (100 mM, pH 8.0), tetrasaccharide 11 (0.15 g, 0.175
mmol), galactose (0.047 g, 0.261 mmol), ATP (0.144 g, 0.261
mmol), UTP (0.138 g, 0.261 mmol), MgCl2 (20 mM), EcGalK
(3 mg), BLUSP (4 mg), NmLgtB (3 mg), and PmPpA (2 mg)
was incubated in a shaker with agitation (100 rpm) at 30 C for
2 days. The product formation was monitored by TLC (n-
PrOH:H2O:NH4OH = 5:2:1 by volume, and detected by p-
anisaldehyde sugar stain) and mass spectrometry. When an op-
timal yield was achieved, ethanol (10 mL) was added and the
mixture was incubated at 4 C for 30 min. The precipitates were
removed by centrifugation and the supernatant was concen-
trated and purified by a Bio-Gel P-2 gel column (water was used
as an eluate). Further purification was achieved by silica gel
chromatography (EtOAc:MeOH:H2O = 4:2:1 by volume) and
finally Bio-Gel P-2 column (eluted with H2O) to produce
Gal1–4GlcNAc1–3(Neu5Ac2–6)Gal1–4Glc as white
Synthesis of Neu5Ac2–6Gal1–3(Neu5Ac2–6)Gal-
NAc (DSTa, 6).
(a) Synthesis of ethyl-3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-1-thio--galactopyranoside (13). D-Galactosa-
mine hydrochloride (3.0 g, 13.9 mmol) was dissolved in a solu-
tion of 1 M NaOH (aq) (1.02 g, 24.7 mmol) (24 mL). Phthalic
anhydride was added (3.1 g, 20.9 mmol) and the reaction mix-
ture was stirred at room temperature for overnight to form a
clear yellow aqueous solution which was washed with 3 
EtOAc (10 mL). The water was removed under reduced pres-
sure to produce 2-(2-carboxybenzamido)-2-deoxy-D-galacto-
pyranose as a yellow foam. The crude compound was dissolved
in pyridine (20 mL) and acetic anhydride (14 mL, 0.15 mol) was
added at 0 °C. The mixture was left to attain ambient tempera-
ture and was stirred for overnight. The reaction was quenched
with MeOH at 0 °C. Solvents were evaporated and co-evapo-
rated with toluene, and the brown suspension was dissolved in
EtOAc and washed with brine, HCl solution (1 M), brine, satd.
NaHCO3 (aq), and brine again. The solution was dried over
MgSO4 and to remove residue solvent by treatment under re-
duced pressure to produce 1,3,4,6-tetra-O-acetyl-2-deoxy-2-
phthalimido-D-galactopyranoside as a yellow foam. The crude
compound (4.6 g, 9.6 mmol) was dissolved in dry CH2Cl2 (45
mL). At 0 °C, BF3·OEt2 (6.6 mL, 48 mmol) was added drop-
wisely and the mixture was stirred for 10 min under argon.
EtSH (2.2 mL, 29 mmol) was added and the reaction was stirred
at room temperature for 20 h. TLC analysis indicated comple-
tion of the reaction and quenching was done with Et3N at 0 °C.
After removal of the solvent, the crude residue was purified by
flash chromatography (EtOAc:Hexane = 2:3 by volume) to pro-
duce pure compound ethyl-3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-1-thio--galactopyranoside 13 as white foam (2.9
1
powder (12, 0.167 g, 94%). H NMR (800 MHz, D2O)  5.22
(d, J = 3.2 Hz, 0.4H), 4.68 (d, J = 8.0 Hz, 1H), 4.66 (d, J = 8.0
Hz, 0.6H), 4.47 (d, J = 8.0 Hz, 1H), 4.41 (d, J = 8.0 Hz, 1H),
4.19–3.28 (m, 31H), 2.70 (dd, J = 4.8 and 12.0 Hz, 1H), 2.02
(s, 6H), 1.72 (t, J = 12.0 Hz, 1H). 13C NMR (201 MHz, D2O) 
174.8, 174.8, 173.4, 173.4, 103.1, 102.8, 102.7, 100.2, 95.5,
91.7, 82.1, 79.6, 79.5, 78.0, 75.2, 74.6, 74.5, 74.4, 73.6, 73.1,
72.4, 72.4, 72.1, 71.7, 71.5, 70.9, 70.9, 69.9, 69.6, 69.6, 68.5,
68.4, 68.2, 68.1, 63.3, 62.5, 60.9, 60.1, 60.0, 59.7, 55.1, 51.8,
51.7, 40.0, 22.1, 22.0. HRMS (ESI) m/z calculated for
C37H61N2O29 [M-H]- 997.3365, found 997.3360.
(c) One-pot two-enzyme preparative-scale synthesis of
DS’LNnT Neu5Ac2–3Gal1–4GlcNAc1–3(Neu5Ac2–
6)Gal1–4Glc (5). To prepare DS’LNnT, a reaction mixture in
a total volume of 10 mL containing Tris-HCl buffer (100 mM,
pH 8.5), pentasaccharide 12 (100 mg, 0.098 mmol), Neu5Ac
(45 mg, 0.145 mmol), CTP (78 mg, 0.145 mmol), MgCl2 (20
mM), NmCSS (3.0 mg), and PmST1_M144D (3.0 mg) was in-
cubated in a shaker at 37 C for 24 h. The reaction was moni-
tored by TLC (n-PrOH:H2O:NH4OH = 4:2:1 by volume and de-
tected by p-anisaldehyde sugar stain) and mass spectrometry.
When an optimal yield was achieved, 10 mL of ethanol was
added to the reaction mixture and the resulting mixture was in-
cubated at 4 C for 30 min. The precipitates were removed by
centrifugation and the supernatant was concentrated and puri-
fied by a Bio Gel P-2 gel column (water was used as an eluent).
Further purification was achieved by silica gel chromatography
(EtOAc:MeOH:H2O = 4:3:2 by volume) and by Bio-Gel P-2
column (eluted with H2O) in the final step to produce
Neu5Ac2–3Gal1–4GlcNAc1–3(Neu5Ac2–6)Gal1–
4Glc hexasaccharide as white powder (5, 121 mg, 93%). 1H
NMR (800 MHz, D2O)  5.21 (d, J = 3.2 Hz, 0.4H), 4.68 (d, J
1
g, 6.05 mmol). Yield was 43.5% over 3 steps. H NMR (400
MHz, CDCl3)  7.92–7.82 (m, 2H), 7.81–7.71 (m, 2H), 5.85
(dd, J = 11.0 and 3.3 Hz, 1H), 5.53 (d, J = 3.2 Hz, 1H), 5.48 (d,
J = 10.5 Hz, 1H), 4.62 (t, J = 10.7 Hz, 1H), 4.27–4.08 (m, 3H),
2.80–2.60 (m, 2H), 2.21 (s, 3H), 2.07 (s, 3H), 1.86 (s, 3H), 1.23
(t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)  170.4, 170.3,
169.8, 168.0, 167.4, 134.4, 134.3, 131.6, 131.3, 123.7, 123.6,
81.7, 74.5, 68.8, 67.0, 61.7, 50.2, 24.6, 20.8, 20.7, 20.5, 15.0.
(b) Synthesis of ethyl 2-deoxy-2-acetimido-1-thio--D-ga-
lactopyranoside (14). Ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-1-thio--D-galactopyranoside (13) (2.8 g, 5.17
mmol) was dissolved in dry MeOH (65 mL), and hydrazine
monohydrate (15 mL, 0.31 mol, 5 eq.) was added. The mixture
was refluxed under argon for 8 h. The reaction mixture was con-
centrated and co-evaporated 2  with EtOH (10 mL). The resi-
due was purified by silica gel flash chromatography
(NH4OH:MeOH:EtOAc = 1:5:30 by volume) to yield ethyl 2-
deoxy-2-amino-1-thio--D-galactopyranoside as a pale-yellow
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solid. The crude material was dissolved in MeOH, acetic anhy- 173.4, 104.5, 100.6, 100.4, 84.6, 80.1, 80.1, 77.2, 74.3, 73.1,
1
2
3
4
5
6
7
8
dride (1.55 mL, 15.3 mmol) was added at 0 °C. The reaction
mixture was stirred at room temperature for overnight. Upon
solvent evaporation under reduced pressure, the crude product
was purified by flash chromatography (EtOAc/MeOH/H2O =
60:10:1) to produce ethyl 2-deoxy-2-acetimido-1-thio--D-ga-
lactopyranoside as white foam (14, GalNAcSEt) (1.44 g).
72.7, 72.6, 72.3, 71.8, 71.6, 70.5, 69.5 68.4, 68.3, 68.2, 68.2,
68.1, 64.0, 63.2, 62.7, 59.3, 52.0, 51.9, 50.0, 40.2, 40.2, 24.7,
22.3, 22.0, 14.4. HRMS (ESI) m/z calculated for C38H62N3O26S
[M-H]- 1008.3348, found 1008.3351.
(e) Synthesis of Neu5Ac26Gal13(Neu5Ac26)Gal-
NAc (6). Tetrasaccharide thioglycoside 16 (100 mg, 0.099
mmol) was dissolved in acetone/water (5 mL, 50% v/v). The
reaction mixture was cooled down to 0 C and NBS (35 mg,
0.20 mmol) was added in one portion in the dark. After 30 min
of vigorous stirring, acetone was evaporated under reduced
pressure. The residue was purified by flash chromatography
(EtOAc:MeOH:H2O = 4:2:1 by volume) to produce the desired
1
Yield was 92.9% over two steps. H NMR (400 MHz, D2O) 
4.49 (d, J = 10.4 Hz, 1H), 3.90 (d, J = 3.3 Hz, 1H), 3.87 (t, J =
10.4 Hz, 1H), 3.75–3.59 (m, 3H), 2.75–2.56 (m, 2H), 1.96 (s,
3H), 1.16 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, D2O) 
174.6, 84.4, 78.9, 72.1, 67.9, 61.1, 51.3, 24.5, 22.2, 14.3.
9
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12
13
14
15
16
17
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26
27
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41
42
43
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45
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48
49
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(c) Synthesis of ethyl -D-galactopyranosyl-(1–3)-2-acet-
amido-2-deoxy--D-glucopyranoside (15). GalNAcSEt 14
(200 mg, 0.75 mmol), Gal (203 mg, 1.13 mmol), ATP (574 mg,
1.13 mmol) were dissolved in water in a 50 mL centrifuge tube
containing Tris-HCl buffer (100 mM, pH 6.8) and MgCl2 (20
mM). After the addition of appropriate amount of EcGalK (5.4
mg), BiGalHexNAcP (15 mg), water was added to bring the
volume of the reaction mixture to 30 mL. The reaction was car-
ried out by incubating the solution in an incubator shaker at 37
°C with agitating at 100 rpm. After 36 h, the reaction was
quenched by adding methanol (30 mL) and incubating at 4 °C
for 30 min. The mixture was then centrifuged to remove precip-
itates. The supernatant was concentrated and purified by silica
gel chromatography (EtOAc:MeOH:H2O = 7:3:0.3 to 6:4:0.4
by volume) to produce a white solid. Then it was purified by
Bio-Gel P-2 gel filtration column (2.5  80 cm) to produce ethyl
-D-galactopyranosyl-(13)-2-acetamido-2-deoxy--D-glu-
copyranoside as white powder (15, Gal1–3GalNAcSEt) (199
1
disialyl glycan DSTa 6 as a white powder (90 mg, 94%). H
NMR (800 MHz, D2O)  5.18 (d, J = 3.2 Hz, 0.6H), 4.64 (d, J
= 8.0 Hz, 0.4H), 4.49 (d, J = 8.0 Hz, 0.6H), 4.42 (d, J = 8.0 Hz,
0.4H), 4.29–3.48 (m, 26H), 2.74–2.70 (m, 2H), 2.03 (s, 3H),
2.02 (s, 3H), 2.01 (s, 3H), 1.71–1.66 (m, 2H). 13C NMR (201
MHz, D2O)  174.9, 174.9, 174.9, 174.8, 174.5, 173.4, 173.4,
173.3, 104.4, 104.2, 100.5, 100.5, 100.3, 100.3, 95.1, 91.1, 79.2,
76.2, 73.4, 73.0, 72.6, 72.6, 72.5, 72.5, 72.3, 72.3, 71.7, 71.6,
71.4, 70.4, 70.4, 68.9, 68.6, 68.3, 68.2, 68.2, 68.1, 68.1, 68.1,
68.1, 68.1, 68.0, 64.2, 64.1, 63.1, 63.0, 62.6, 62.5, 62.5, 52.3,
51.9, 51.9, 51.7, 49.0, 40.2, 40.1, 40.1, 40.0, 22.2, 22.0, 21.9.
HRMS (ESI) m/z calculated for C36H58N3O27 [M-H]- 964.3263,
found 964.3241.
Synthesis of Neu5Ac2–6Gal1–3(Neu5Ac2–6)Gal
(DSGalB, 7).
(a) Synthesis of disaccharide thioglycoside 18. The reac-
tion mixture in a total volume of 10 mL containing Tris-HCl
buffer (100 mM, pH 8.0), GalSEt32 (17, 60 mg, 0.27 mmol),
galactose (0.072 g, 0.40 mmol), ATP (0.221 g, 0.40 mmol),
UTP (0.212 g, 0.40 mmol), MgCl2 (20 mM), EcGalK (2 mg),
BLUSP (3 mg), PmPpA (2 mg), and CjCgtB (3 mg) was incu-
bated in a shaker with agitation (100 rpm) at 37 C for 2 days.
The reaction was quenched by adding 10 mL of ethanol and the
mixture was incubated at 4 C for 30 min. The mixture was then
centrifuged to remove precipitates. The supernatant was con-
1
mg, 62%). H NMR (400 MHz, D2O)  4.53 (d, J = 10.5 Hz,
1H), 4.37 (d, J = 7.7 Hz, 1H), 4.13 (d, J = 3.1 Hz, 1H), 4.00 (t,
J = 10.5 Hz, 1H), 3.82 (d, J = 3.4 Hz, 1H), 3.79 (dd, J = 10.4
and 3.1 Hz, 1H), 3.73–3.60 (m, 5H), 3.57 (dd, J = 7.8 and 4.5
Hz, 1H), 3.53 (dd, J = 10.0 and 3.6 Hz, 1H), 3.44 (dd, J = 9.8
and 7.8 Hz, 1H), 2.76–2.56 (m, 2H), 1.93 (s, 3H), 1.16 (t, J =
7.4 Hz, 3H). 13C NMR (101 MHz, D2O)  174.5, 104.8, 84.2,
80.9, 78.6, 75.0, 72.5, 70.6, 68.6, 68.2, 61.0, 61.0, 50.0, 24.4,
22.3, 14.3. HRMS (ESI) m/z calculated for C16H29NNaO10S
[M+Na]+ 450.1410, found 450.1422.
centrated and purified by flash
chromatography
(EtOAc:MeOH:H2O = 4:2:0.2 by volume) to produce disaccha-
ride 18 as white solid (52 mg, 50%). 1H NMR (600 MHz, D2O)
 4.48 (d, J = 7.7 Hz, 1H), 4.37 (d, J = 9.9 Hz, 1H), 4.07 (d, J =
3.1 Hz, 1H), 3.76 (d, J = 2.6 Hz, 1H), 3.67 (dd, J = 9.4 and 3.2
Hz, 1H), 3.64–3.36 (m, 9H), 2.68–2.53 (m, 2H), 1.12 (t, J = 7.4
Hz, 3H). 13C NMR (151 MHz, D2O)  104.2, 85.2, 83.2, 78.5,
75.0, 72.4, 71.0, 68.6, 68.6, 68.5, 60.9, 60.9, 24.0, 14.4.
(d) Synthesis of tetrasaccharide thioglycoside 16. Gal1–
3GalNAcSEt 15 (106 mg, 0.25 mmol), Neu5Ac (230 mg, 0.75
mmol), and CTP (392 mg, 0.75 mmol) were dissolved in water
in a 15 mL centrifuge tube containing Tris-HCl buffer (100
mM, pH 8.5) and MgCl2 (20 mM). After the addition of appro-
priate amounts of NmCSS (3 mg) and Pd2,6ST (4 mg), water
was added to bring the volume of the reaction mixture to 10 mL.
The reaction was carried out by incubating the solution in an
incubator shaker at 37 °C by agitating at 100 rpm. After 3 day,
the reaction was quenched by adding 10 mL of methanol and
the mixture was incubated at 4 °C for 30 min. The mixture was
then centrifuged to remove precipitates. The supernatant was
concentrated and purified by silica gel (EtOAc:MeOH:H2O =
4:2:0.8 by volume) and Bio-Gel P-2 gel filtration column (2.5
 80 cm) to produce a white solid (16, 249 mg, 93%). 1H NMR
(400 MHz, D2O)  4.49 (d, J = 10.5 Hz, 1H), 4.37 (d, J = 7.8
Hz, 1H), 4.10 (d, J = 3.1 Hz, 1H), 4.01 (t, J = 10.4 Hz, 1H), 3.89
(dd, J = 10.4 and 8.5 Hz, 1H), 3.85 (dd, J = 6.9 and 3.1 Hz, 1H),
3.83–3.69 (m, 8H), 3.68–3.47 (m, 13H), 3.42 (dd, J = 9.9, 7.7
Hz, 1H), 2.76–2.58 (m, 4H), 1.96 (s, 6H), 1.94 (s, 3H), 1.62 (t,
J = 12.1 Hz, 1H), 1.61 (t, J = 12.1 Hz, 1H), 1.18 (t, J = 7.4 Hz,
3H). 13C NMR (101 MHz, D2O)  175.1, 175.0, 174.5, 173.5,
(b) Synthesis of tetrasaccharide thioglycoside 19. Gal1–
3GalSEt 18 (20 mg, 0.052 mmol), Neu5Ac (64 mg, 0.21
mmol), and CTP (109 mg, 0.21 mmol) were dissolved in water
in a 15 mL centrifuge tube containing Tris-HCl buffer (100
mM, pH 8.5) and MgCl2 (20 mM). After the addition of appro-
priate amounts of NmCSS (2 mg) and Pd2,6ST (3 mg), water
was added to bring the total volume of the reaction mixture to 5
mL. The reaction was carried out by incubating the solution in
an incubator shaker at 37 C by agitating at 100 rpm. After 2
day, the reaction was quenched by adding 5 mL of methanol
and the mixture was incubated at 4 C for 30 min. After being
centrifuged for 30 min at 7,000  g in a Sorvall legend T/RT
bench-top centrifuge, the clear supernatant was transferred to a
100 mL round-bottom flask. The protein precipitates were
washed with 4 mL of deionized water. The mixture was then
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The Journal of Organic Chemistry
centrifuged to remove precipitates. The supernatant was con- apoptosis of villus enterocytes, crypt hyperplasia and misa-
1
2
3
4
5
6
7
8
centrated and purified by Bio-Gel P-2 gel filtration column (2.5
 80 cm) and flash chromatography (EtOAc:MeOH:H2O =
4:2:0.8 by volume) to produce compound 19 as a white solid
(38 mg, 84%). 1H NMR (800 MHz, D2O)  4.63 (d, J = 7.8 Hz,
1H), 4.50 (d, J = 9.9 Hz, 1H), 4.20 (d, J = 3.2 Hz, 1H), 3.95 (dd,
J = 10.1 and 8.5 Hz, 1H), 3.92 (d, J = 3.4 Hz, 1H), 3.92–3.77
(m, 9H), 3.76–3.61 (m, 10H), 3.60–3.53 (m, 4H), 2.82–2.68 (m,
4H), 2.03 (s, 3H), 2.02 (s, 3H), 1.70 (t, J = 12.2 Hz, 1H), 1.69
(t, J = 12.2 Hz, 1H), 1.28 (t, J = 7.3 Hz, 3H). 13C NMR (201
MHz, D2O)  174.9, 174.9, 173.2, 173.1, 103.8, 100.3, 100.2,
95.1, 85.4, 82.7, 77.0, 73.2, 72.6, 72.5, 72.3, 71.5, 71.4, 70.9,
68.6, 68.4, 68.2, 68.1, 68.0, 68.0, 63.9, 63.1, 62.6, 62.5, 51.9,
51.7, 40.1, 40.0, 24.3, 21.9, 21.9, 14.5. HRMS (ESI) m/z
calculated for C36H59N2O26S [M-H]- 967.3082, found 967.3078.
ligned nuclei in the epithelium. If at least one pathology sign
was observed, a score of 0.5–1.5 was assigned depending on
severity. Two or three signs together resulted in a score of 2–3.
The maximum score of 4 was given in case of complete oblite-
ration of the epithelium with or without intestinal perforation.
The animal protocol was approved by The University of Cali-
fornia San Diego Institutional Animal Care and Use Committee
(IACUC), and complied with the Guide for the Care and Use of
Laboratory Animals.
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ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of the glycans
synthesized. This material is available free of charge via the Inter-
net at http://pubs.acs.org.
(c) Synthesis of Neu5Ac26Gal13(Neu5Ac26)Gal
(7). Tetrasaccharide thioglycoside 19 (18 mg, 0.019 mmol) was
dissolved in acetone/water (1 mL, 50% v/v). The reaction mix-
ture was cooled down to 0C and NBS (8 mg, 0.046 mmol) was
added in one portion in the dark. After 20 min of vigorous stir-
ring, acetone was evaporated under reduced pressure. The resi-
due was purified by flash chromatography (EtOAc:MeOH:H2O
= 4:2:1 by volume) to produce DSGalB (7) as a white powder
(15 mg, 87%). 1H NMR (800 MHz, D2O)  5.25 (d, J = 3.2 Hz,
0.4H), 4.61 (d, J = 8.0 Hz, 0.6H), 4.60 (d, J = 8.0 Hz, 1H), 4.25–
3.51 (m, 26H), 2.74–2.69 (m, 2H), 2.03 (s, 6H), 1.73–1.66 (m,
2H). 13C NMR (201 MHz, D2O)  175.0, 174.9, 174.9, 173.4,
173.4, 173.4, 173.3, 103.8, 100.4, 100.3, 100.3, 100.3, 96.0,
92.0, 88.9, 81.9, 78.9, 74.4, 73.3, 73.1, 73.1, 72.5, 72.5, 72.5,
72.5, 72.3, 72.3, 71.6, 71.6, 71.4, 70.9, 70.9, 70.8, 68.9, 68.8,
68.4, 68.3, 68.2, 68.1, 68.0, 67.4, 64.2, 64.1, 63.1, 63.0, 62.6,
62.5, 59.4, 51.9, 51.9, 51.7, 40.2, 40.1, 40.0, 21.9, 21.9. HRMS
(ESI) m/z calculated for C34H55N2O27 [M-H]- 923.2998, found
923.2991.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hyu@glycohubusa.com
*E-mail: xiichen@ucdavis.edu
Present Addresses
†These authors contributed equally.
‡Current address: Division of Chemistry and Chemical Engineer-
ing, California Institute of Technology, Pasadena, CA 91125, USA.
Notes
HY, YL, and XC are co-founders of Glycohub, Inc., a company
focused on the development of carbohydrate-based reagents, diag-
nostics, and therapeutics.
ACKNOWLEDGMENT
This work was supported by NIH grants R43HD087077 (to H.
Y.) and R01HD065122 (to X. C.) as well as a scholarship from
the China Scholarship Council (to X. Y.). The support of the
Family Larsson-Rosenquist Foundation (to L.B.) is gratefully
acknowledged.
In vivo Efficacy of Disialyl Glycans in Preventing Ne-
crotizing Enterocolitis (NEC) in a Preclinical Neonatal Rat
Model. In vivo efficacy was tested in an established NEC model
in neonatal rats as previously described.9 Single dose (300 M)
and dose-dependent (30, 100, 300, 1,000, and 2,000 M) stud-
ies were carried out in the same manner. The concentration of
HMOs used was 10 mg/mL, the same as that used previously.9,12
Briefly, pregnant time-dated Sprague-Dawley rats were in-
duced at term using Pitocin (1–2 U per animal). Immediately
after birth, neonatal rats were randomized into the different
study groups. Animals in the dam-fed (DF) group remained
with the dam. All other animals were separated from the dam,
housed in a temperature- and humidity-controlled incubator and
orally gavaged with a special rodent formula (0.2 mL; without
and with different oligosaccharides) twice daily. The formula
approximates the protein and caloric content of rat breast milk
and consists of 15 g Similac 60/40 (Ross Pediatrics, Columbus,
Ohio, USA) in 75 mL of Esbilac canine milk replacer (Pet-Ag,
Hampshire, Illinois, USA). All animals, dam-fed and gavaged,
were exposed to 10 min of hypoxia (5% O2, 95% N2) thrice
daily in a modular chamber. All animals were sacrificed 96 h
post-partum; their intestines were collected and inspected for
the presence of gross necrotic changes or pneumatosis intesti-
nalis. A 0.5 cm section of the terminal ileum was prepared for
H&E staining per standard protocols and scored blindly by two
investigators based on morphological changes that included ep-
ithelial sloughing, villus oedema, infiltration of neutrophils,
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