Effective one-pot multienzyme (OPME) synthesis of monotreme milk oligosaccharides and other sialosides containing 4-: O -acetyl sialic acid

Article abstract of DOI:10.1039/c6ob01706a

A facile one-pot two-enzyme chemoenzymatic approach has been established for the gram (Neu4,5Ac₂α3Lac, 1.33 g) and preparative scale (Neu4,5Ac₂α3LNnT) synthesis of monotreme milk oligosaccharides. Other O-acetyl-5-N-acetylneuraminic

Full text of DOI:10.1039/c6ob01706a

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Effective one-pot multienzyme (OPME) synthesis of monotreme
milk oligosaccharides and other sialosides containing a 4-O-acetyl
Received 00th January 20xx,
Accepted 00th January 20xx
sialic acid†
Hai Yu,a‡ Jie Zeng,ab‡ Yanhong Li,a Vireak Thon,ac Baojun Shi,ad and Xi Chen*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract: A facile one-pot two-enzyme chemoenzymatic approach has been established for gram (Neu4,5Ac2α3Lac) and
preparative-scale (Neu4,5Ac2α3LNnT) synthesis of monotreme milk oligosaccharides. Other O-acetyl-5-N-acetylneuraminic
acid (Neu4,5Ac2)- or 4-O-acetyl-5-N-glycolylneuraminic acid (Neu4Ac5Gc)-containing α2–3-sialosides have also been
synthesized in preparative scale. Used as an effective probe, Neu4,5Ac2α3GalβpNP was found to be a suitable substrate by
human influenza A viruses but not bacterial sialidases.
Japanese dace,9 South American pit viper,10 rabbit,11 guinea-
pig.6 Neu4,5Ac2-containing oligosaccharides have been found
to be the dominant components of the acidic milk
Introduction
Sialic acids commonly presented as the terminal residues on
mammalian oligosaccharides or glycoconjugates are key
recognition components of bacterial adhesins, viral surface
proteins, selectins, Siglecs, and other sialic acid-binding
proteins involved in infection, inflammation, cancer metastasis,
and immunoregulation.1-4 Sialic acids present tremendous
structural diversity in nature and more than 50 members have
been identified so far. These include N-acetylneuraminic acid
(Neu5Ac), the non-human sialic acid N-glycolylneuraminic acid
(Neu5Gc), 2-keto-3-deoxy-nonulosonic acid (Kdn), their
derivatives with single or multiple O-acetylation and less
frequent 8-O-methylation, 8-O-sulfation, 9-O-lactylation, or 9-
O-phosphorylation.1-4
oligosaccharides (MOSs) of monotremes echidnas and
platypus.12-14 Both Neu4,5Ac2 and Neu4Ac5Gc have been
found in horse glycoproteins.5 The presence of Neu4Ac5Gc-
GM3 ganglioside in human colon cancer tissues was detected
using purified antibodies.15 Neu4Ac5Gc (
in
2) has also been found
α
2–8-linked polysialic acids of glycoproteins from
unfertilized kokanee salmon egg16 and in the serum of guinea
pig in trace amounts.17
HO
HO
HN
OH
OH
OH
OH
HO
O
HO
-
-
CO2
CO2
O
O
HN
HO
AcO
AcO
O
Neu4,5Ac2 (1)
Neu4Ac5Gc (2)
O-Acetylation is the most frequently occurring modification
of sialic acids. Single or multiple O-acetylation at positions 4, 7,
8, and/or 9 of sialic acid has been identified and the
presentation varies among different types of species, organs,
and cells.1-4 Comparing to 9-O-acetylation which is the most
common sialic acid O-acetylation, 4-O-acetylated sialic acids
(Fig. 1) including 4-O-acetyl-N-acetylneuraminic acid
Fig. 1 Structures of naturally occurring 4-O-acetyl-N-acetylneuraminic acid (Neu4,5Ac2,
1) and 4-O-acetyl-N-glycolylneuraminic acid (Neu4Ac5Gc, 2).
4-O-Acetylation of sialic acids provides resistance or
decreased sensitivity of sialosides to various sialidases.6, 18, 19
This property is believed to contribute to the inhibitory activity
of Neu4,5Ac2-rich horse and guinea pig α2-macroglobulins
against hemagglutination and infectivity of several human
(Neu4,5Ac2,
(Neu4Ac5Gc,
1) and 4-O-acetyl-N-glycolylneuraminic acid
) are less common.5 Neu4,5Ac2
(1) has been
18, 19
influenza viruses.6,
On the other hand, Neu4,5Ac2 is
2
selectively recognized by the hemagglutinin-esterase (HE)20, 21
of mouse hepatitis virus strain S (MHV-S), a type of murine
coronavirus, and infectious salmon anemia virus (ISAV).22
Nevertheless, the significance of naturally existing 4-O-
acetylated sialic acids is not well understood partly due to the
lack of access to sufficient amounts of the corresponding
sialosides.
found in horse,6 donkey,7 Australian monotreme Echidna,5, 8
To our knowledge, the synthesis of sialosides containing 4-
O-aceylated sialic acids by either chemical or enzymatic
methods has not been reported. Here we present a facile
chemoenzymatic approach for preparative and gram-scale
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Organic & Biomolecular Chemistry
synthesis of monotreme milk oligosaccharides including of the isopropylidene group using HOAc/H2O followed by
Neu4,5Ac2-containing
α
2–3-linked
sialyl
sialyl
lactose debenzylation using catalytic hydrogenation produced the
) in a 78% yield.
(Neu4,5Ac2
(Neu4,5Ac2
containing
α
α
α
3Lac)
and
lacto-N-neotetraose target Neu4Ac5Gc (2
3LNnT). Other Neu4,5Ac2- or Neu4Ac5Gc-
2–3-linked sialosides representating common
terminal sialosides in mammals are also synthesized.
Results and discussion
We envisioned that the desired 4-O-acetyl-sialic acid-
containing sialosides could be chemoenzymatically synthesized
using an effective one-pot two-enzyme sialylation system
containing a cytidine 5'-monophosphate-sialic acid (CMP-Sia)
synthetase (CSS) and a sialyltransferase (ST).23 To test the
feasibility, the corresponding 4-O-acetyl-sialic acids were
Scheme 2 Chemoenzymatic synthesis of Neu4Ac5Gc (2).
Sialyltransferase-catalyzed reactions use CMP-sialic acids
which can be potentially accessed by a suitable CMP-Sia
synthetase (CSS, EC 2.7.7.43). The tolerance of using
chemically synthesized. As shown in Scheme 1, Neu4,5Ac2 (
was synthesized from N-acetylneuraminic acid (Neu5Ac, ) by
esterification of the C1-carboxyl group with benzyl bromide to
provide benzyl ester with a 90% yield. Protection of at C8
1)
3
Neu4,5Ac2 (1) as a potential substrate was tested for several
4
4
bacterial CSSs including those from Neisseria meningitidis
(NmCSS),26 Pasteurella multocida (PmCSS), Haemophilus
ducreyi (HdCSS), as well as two NmCSS mutants (NmCSS_S81R
and NmCSS_Q163A).27 Small-scale reactions carried out in Tris-
and C9 by adding 2,2-dimethoxypropane in the presence of a
catalytical amount of p-toluenesulfonic acid produced partially
protected
acetylation of
room temperature provided the key intermediated
yield. Final deprotection of the O-isopropylidene group in
5
in an excellent 92% yield. Regioselectively
at O-4 using acetic anhydride in pyridine at
in a 81%
by
5
HCl buffer (100 mM, pH 7.1) at 37
layer chromatography (TLC) and mass spectrometry (MS)
analyses indicated that all CSSs tested could use Neu4,5Ac2 (
efficiently (Fig. S1, ESI). These results were quite exciting as a
°C for 15 h followed by thin-
6
6
1)
treating with 80% acetic acid and removal of the benzyl ester
group by catalytic hydrogenolysis with H2 in the presence of
†
previous report showed that CSSs purified from calf brain,
bovine, and equine submaxillary glands could not use
Pd/C in methanol resulted in Neu4,5Ac2 (1) in a 82% yield. The
obtained product has a proton nuclear magnetic resonance (1H
NMR) spectrum consistent with that reported.24 The 4-O-acetyl
modification caused a downfield shift of Neu5Ac H-4 signal.
Neu4,5Ac2 (1) as a substrate for the synthesis of the
corresponding CMP-sialic acid.28 NmCSS was chosen for further
studies and for preparative-scale and gram-scale syntheses.
Scheme 1 Chemical synthesis of Neu4,5Ac2 (1).
Scheme 3 One-pot two-enzyme (OP2E) synthesis of sialosides containing Neu4,5Ac2 (1)
or Neu4Ac5Gc (2). Enzymes: NmCSS, Neisseria meningitidis CMP-sialic acid synthetase;
PmST3, Pasteurella multocida α2–3-sialyltransferase 3. The pH of the reaction was
controlled at 7.1 to minimize de-O-acetylation during the enzymatic reaction. Yields
were calculated for purified products based on the amounts of acceptors (limiting
reagents) used.
Neu4Ac5Gc (
D-mannosamine hydrochloride (ManNH2
Scheme 2, N-(2-Benzyloxyacetyl)-D-mannosamine
obtained by treating ManNH2 HCl ( ) with 2-benzyloxyacetyl
2
) was chemoenzymatically synthesized from
HCl, ). As shown in
was
⋅
7
8
⋅
7
chloride in the presence of NaHCO3 in CH3CN and water. P.
multocida sialic acid aldolase (PmNanA)25-catalyzed aldol
A coupled enzymatic assay was used to evaluate several
bacterial sialyltransferases in using CMP-Neu4,5Ac2 and CMP-
reaction of
8
with sodium pyruvate at 37
°C for 48 h produced
the corresponding sialic acid derivative
Neu4Ac5Gc synthesized in situ by NmCSS from Neu4,5Ac2 (
and Neu4Ac5Gc ( ), respectively, as potential donor substrates.
Sialyltransferases tested include 2–3-sialyltransferases from
1)
9
in an excellent
2
quantitative yield. The introduction of 4-O-acetyl group was
α
Pasteurella multocida (PmST1,29 PmST2,30 PmST331) and
carried out similarly to that described above for the synthesis
PmST1_M144D mutant;32
and Psp26ST;34 as well as
α
2–6-sialyltransferases Pd26ST33
2–3/8-sialyltransferase CstII.35, 36
of Neu4,5Ac2 (1). Briefly, selective benzoylation of 9 with
benzyl bromide formed benzyl ester 10 in a 90% yield. Treating
10 with 2,2-dimethoxypropane produced 8,9-isopropylidene-
protected 11 in a 96% yield. Selective acetylation of benzoate
11 with Ac2O and pyridine produced the corresponding 4-O-
acetyl derivative 12 in a good yield (81%). Subsequent removal
α
Among these sialyltransferases, only PmST3 showed good
activity as shown by TLC and MS studies. PmST3 was
previously characterized as
a
monofunctional
α2–3-
sialyltransferase encoded by Pm1174 gene in Pasteurella
2 | J. Name., 2013, 00, 1-11
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multocida strain Pm70. It had broad acceptor substrate could not use fucosylated oligosaccharides, such as Lex, as
specificity and could use oligosaccharides, glycolipids, and acceptor substrates. It was delightful to identify the unique
37
glycopeptides as acceptors.31,
PmST3 could use efficiency of PmST3, among various bacterial sialyltransferases,
1–4-or 1–3-llinked in catalyzing the synthesis of sialosides containing 4-O-acetyl
oligosaccharides containing a terminal
β
β
galactose as acceptors, but had poor activity on using sialic acid.
galactosides containing a monosaccharide unit as acceptors. It
Table 1 One-pot two-enzyme (OP2E) preparative-scale synthesis of
amounts of acceptors (limiting reagents) used.
α
2–3-linked 4-O-acetylated sialosides. Yields were calculated for purified products based on the
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HO
HO
OH
O
HO
OH
O
OH
O
OH
O
OH
O
NmCSS and PmST3 were used in an efficient one-pot two-
38
(Scheme 3) for
GlcNAc, ATP, UTP
HO
HO
O
O
N3
O
N3
O
O
enzyme (OP2E) sialylation system23,
BLNahK, PmGlmU,
PmPpA, NmLgtA
94%
HO
HO
OH
NHAc
OH
Lc3
OH
OH
17
preparative-scale synthesis of target sialosides. To minimize
HO OH
O
OH
O
HO
OH
O
OH
Gal, ATP, UTP
loss of the 4-O-acetyl group, enzymatic reactions were carried
O
HO
O
O
O
N3
HO
O
EcGalK, BLUSP, PmPpA, NmLgtB
99%
HO
OH
NHAc
OH
OH
out at 37
NmCSS, PmST3, Mg2+, cytidine 5'-triphosphate (CTP),
Neu4,5Ac2 or Neu4Ac5Gc, and galactoside acceptor.
Monotreme milk oligosaccharides Neu4,5Ac2 3Lac (24) and
Neu4,5Ac2 3LNnT (25) were readily produced from lactose
(Gal 4Glc or Lac, 13) and previously synthesized lacto-N-
neotetraose (Gal
and 78% yields, respectively (Table 1). Neu4,5Ac2
°C in a Tris-HCl buffer (100 mM, pH 7.1) containing
23
Scheme
4 Sequential one-pot multienzyme (OPME) synthesis of LNnTβProN3 (23).
a
Enzymes: BLNahK or NahK_ATCC55813, Bifidobacterium longum N-acetylhexosamine-
1-kinase; PmGlmU, Pasteurella multocida N-acetylglucosamine uridyltransferase;
PmPpA, Pasteurella multocida inorganic pyrophosphatase; NmLgtA, Neisseria
meningitidis β1–3-N–acetylglucosaminyltransferase; EcGalK, Escherichia coli
galactokinase; BLUSP, Bifidobacterium longum UDP-sugar pyrophosphorylase;
NmLgtB, Neisseria meningitidis β1–4-galactosyltransferase.
α
α
β
39
β
4GlcNAc
β
3Galβ
4Glc or LNnT, 14
)
in 82%
α
3LNT (26)
was also synthesized from commercially available lacto-N-
tetraose (Gal 3GlcNAc 3Gal 4Glc or LNT, 15) in a 65% yield.
Gal pNP (16) with a monosaccharide unit was a less efficient
acceptor and Neu4,5Ac2 3Gal pNP (27) was obtained in a 31%
yield (the yield could not be improved further by adding more
enzymes). Other 2–3-linked sialylated disaccharides
propyl azide aglycone 28 33 were also
synthesized from the corresponding
22 in 74–88% yields. To demonstrate
the efficiency of the OP2E system, monotreme milk
trisaccharide Neu4,5Ac2 3Lac (24) was further synthesized in a
gram scale (1.33 g) with a 71% yield.
longer propyl azide-containing oligosaccharide
4GlcNAc 3Gal 4Glc ProN3 or LNnT ProN3 23 was
synthesized from Lac
Although less efficient compared to the synthesis of their
Neu4,5Ac2-containing matching pairs, Neu4Ac5Gc-containing
β
β
β
β
α
2–3-linked sialosides with a propyl azide aglycone (35
were successfully obtained from Neu4Ac5Gc ( ) and the
corresponding disaccharides (17 22) or tetrasaccharide (23) in
–41)
α
β
2
–
α
51–71% yields using the OP2E sialylation reaction (Scheme 3).
This indicated that CMP-Neu4,5Ac2 generated in situ is a better
donor substrate than in situ generated CMP-Neu4Ac5Gc for
PmST3. Longer reaction time needed for the synthesis of
Neu4Ac5Gc-containing compounds also led to de-O-
acetylation which also contributed to lower sialylaton yields.
All sialoside products synthesized in preparative scale were
purified by gel filtration chromatography and semi-prep high-
performance liquid chromatography (HPLC) using a reverse-
phase C18 column. The structures were characterized by 1H
and 13C nuclear magnetic resonance (NMR) spectroscopy as
wells as high resolution mass spectrometry (HRMS).
containing
a
(
–
)
successfully
disaccharides29, 40, 41 17
–
α
A
Galβ
β
β
β
β
(
)
β
ProN3 (17) using a sequential two-step
one-pot multienzyme (OPME)38 process (Scheme 4) similar to
that reported previously for LNnT.39 Briefly, Lc3 trisaccharide
42
GlcNAcβ3Galβ4GlcβProN3 was synthesized from LacβProN3
Neu4,5Ac2
(1.33 g) using a CombiFlash® Rf 200i system with a ODS-SM
column (51 g, 50 M, 120 Å, Yamazen).
synthesized pNP-tagged
3Gal pNP (27) was used in a 384-well plate-based
α3Lac (24) synthesized in gram scale was purified
(
17) and GlcNAc in a 94% yield using a one-pot four-enzyme
(OP4E) GlcNAc activation and transfer system containing
ꢀ
Bifidobacterium longum N-acetylhexosamine-1-kinase (BLNahK,
NahK_ATCC55813),43
The
sialoside
Pasteurella
multocida
N-
Neu4,5Ac2
α
β
acetylglucosamine uridyltransferase (PmGlmU),44 Pasteurella
multocida inorganic pyrophosphatase (PmPpA),40 and
high-throughput colorimetric assay50 for substrate specificity
studies of the α2–3-sialidase activity of Pasteurella multocida
sialyltransferase 1 (PmST1),29 four commercially available
bacterial sialidases, and four human influenza A viruses.51
While all bacterial sialidases tested including PmST1 as well as
commercially available sialidases from Arthrobacter
ureafaciens (Prozyme), Clostridium perfringens (Prozyme),
Vibrio cholerae (Prozyme), and Streptococcus pneumoniae
(Prozyme) did not show any sialidase activity towards
Neisseria meningitidis
(NmLgtA).45-47 LNnT
ProN3 (23
and galactose in an excellent (99%) yield using a OP4E
β1–3-N–acetylglucosaminyltransferase
42
β
)
was then produced from Lc3
39
galactosylation system37,
containing Escherichia coli
galactokinase (EcGalK),48 Bifidobacterium longum UDP-sugar
pyrophosphorylase (BLUSP),49 PmPpA,40 and Neisseria
47
meningitidis
Synthesis of Neu4,5Ac2
23) and Neu4,5Ac2 ( ) was successfully achieved in a good 81%
β
1–4-galactosyltransferase (NmLgtB).40,
α
3LNnTβProN3 (34) from LNnTβProN3
Neu4,5Ac2α3GalβpNP (27) under the conditions used, human
(
1
influenza A viruses (presumably the surface neuraminidases)
could hydrolyze it (Fig. 2 black columns). As expected, all
bacterial sialidases and viruses tested could cleave non-O-
yield using NmCSS and PmST3 in one-pot (Scheme 3).
acetylated sialoside Neu5Ac
positive control (Fig. 2 white columns).
α3Galβ
pNP50 which was used as a
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performed on silica gel plates (Sorbent Technologies) using
anisaldehyde sugar stain for detection. Gel filtration
chromatography was performed with a column (100 cm × 2.5
cm) packed with Bio-Gel P-2 Fine resins (Bio-Rad). D-Galactose
(Gal) and N-acetyl-D-glucosamine (GlcNAc) were from Fisher
Scientific. GalβpNP was from Sigma. N-Acetyl-D-mannosamine
(ManNAc) and N-acetylneuraminic acid (Neu5Ac) was from Inalco
(Italy). Adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate
(UTP), and cytosine 5'-triphosphate (CTP) were purchased from
Hangzhou Meiya Pharmaceutical Co. Ltd. Lacto-N-tetraose (LNT)
was from Elicityl (Crolles, France). Aspergillus oryzae β-
galactosidase was from Sigma (St. Louis, MO). Recombinant
enzymes Bifidobacterium longum strain ATCC55813 N-
acetylhexosamine-1-kinase (BLNahK or NahK_ATCC55813),43
Pasteurella multocida N-acetylglucosamine uridyltransferase
0.6
0.5
0.4
0.3
0.2
0.1
0
(A)
(B)
1.2
1
0.8
0.6
0.4
0.2
0
Fig. 2 Human influenza A virus (A) and bacterial sialidase (B) substrate specificity
studies using Neu4,5Ac2α3GalβpNP (27) as the substrate (black columns). Non-O- (PmGlmU),44 Pasteurella multocida inorganic pyrophosphatase
acetylated sialoside Neu5Acα3GalβpNP50 (white columns) was used as
a positive
(PmPpA),40
acetylglucosaminyltransferase (NmLgtA),45,
Neisseria
meningitidis
β1–3-N-
Escherichia coli
control. Human influenza A viruses (purified) used: A/PR8, A/Puerto Rico/34/8 H1N1;
A/Philips, A/Philippines/2/82/X-79 H3N2; A/Mem71, A/Memphis/71 H3N1; A/Udorn72,
A/Udorn/307/72 H3N2.
46
galactokinase (EcGalK),48 Bifidobacterium longum UDP-sugar
pyrophosphorylase (BLUSP),49 Neisseria meningitidis β1–4-
galactosyltransferase (NmLgtB),40 Pasteurella multocida sialic acid
aldolase (PmNanA),25 Neisseria meningitidis CMP-sialic acid
synthetase (NmCSS),26 Pasteurella multocida CSS (PmCSS),
Haemophilus ducreyi CSS (HdCSS), and two NmCSS mutants
(NmCSS_S81R and NmCSS_Q163A),27 and Pasteurella multocida
multifunctional α2–3-sialyltransferase 3 (PmST3)31 were expressed
and purified as described previously. Purified human influenza A
viruses A/Puerto Rico/34/8 H1N1 (A/PR8), A/Philippines/2/82/X-79
H3N2 (A/Philips), A/Memphis/71 H3N1 (A/Mem71), and
A/Udorn/307/72 H3N2 (A/Udorn72) were described previously.51
LacβProN3 (17), LacNAcβProN3 (18), disaccharides 19–22,
tetrasaccharide LNnT (14) were prepared as described previously.29,
In order to test whether the cleavage of Neu4,5Ac2 by
human influenza A viruses was caused by de-O-acetylation or
direct de-sialylation, Neu4,5Ac2α3Lac was incubated with
purified human influenza A virus strain A/Puerto Rico/34/8
H1N1 (A/PR8) followed by high-resolution mass spectrometry
analysis. Major product peak for Neu4,5Ac2 was seen and only
a trace amount of Neu5Ac was observed, indicating a direct
de-sialylation of Neu4,5Ac2 mechanism was used by human
influenza A viruses.
40, 41
Conclusions
Tetrasaccharide LNnTβProN3 (23) was chemoenzymatically
synthesized via sequential one-pot multienzyme (OPME)
In conclusions, we report here a convenient and efficient
glycosylation approach from LacβProN3 (17) as described below:
chemoenzymatic sialylation approach for synthesizing
α2–3-
linked sialosides containing a 4-O-acetylated sialic acid. PmST3,
but not other bacterial sialyltransferases, was identified as a
unique sialyltransferase that can catalyze the transfer of 4-O-
acetylated sialic acid. Using chemically or chemoenzymatically
Chemical synthesis of 5-N-acetyl-4-O-acetylneuraminic acid
(Neu4,5Ac2, 1)
Benzyl
5-acetamido-3,5-dideoxy-D-glycero-β-D-galacto-2-
synthesized Neu4,5Ac2 (1) or Neu4Ac5Gc (2), α2–3-linked
sialosides were readily obtained by a one-pot two-enzyme
nonulopyranosonate (4). To a stirring solution of Neu5Ac (3, 5 g,
16.2 mmol) in water (20 mL), 10% Cs2CO3 was added dropwisely to
adjust the pH value of solution to neutral. The reaction mixture was
then evaporated and dried for overnight using a vacuum pump. The
obtained solid was dissolved in N,N-dimethylformamide (20 mL),
and benzyl bromide (3 mL) was added dropwisely. The mixture was
stirred under argon for overnight at room temperature and then
(OP2E) system containing NmCSS and PmST3. Using
Neu4,5Ac2α3GalβpNP as an effective probe, substrate specific
studies showed that while Neu4,5Ac2 was not hydrolyzed by
bacterial sialidases tested, it was effectively removed by
human influenza A virus neuraminidases.
filtered. The filtrate was evaporated to produce
a syrup.
Crystallization using 2-propanol produced 4 (5.8 g, 90%). 1H NMR
(800 MHz, D2O) δ 7.59–7.41 (m, 5H), 5.27 (dd, J = 27.2 and 10.8 Hz,
2H), 4.06–4.03 (m, 2H), 3.89 (t, J = 10.4 Hz, 1H), 3.80 (dd, J = 12.8
and 3.2 Hz, 1H), 3.67 (m, 1H), 3.58 (dd, J = 12.0 and 6.4 Hz, 1H), 3.53
(d, J =8.8 Hz, 1H), 2.29 (dd, J = 12.8 and 5.6 Hz, 1H), 2.03 (s, 3H),
1.88 (t, J = 12.8 Hz, 1H); 13C NMR (200 MHz, D2O) δ 174.71, 170.47,
134.80, 132.95, 130.65, 129.03, 128.77, 128.21, 95.20, 70.30, 69.98,
68.19, 68.02, 66.53, 63.00, 51.92, 39.90, 21.94.
Experimental Section
Materials and methods. Chemicals were purchased and used as
received. NMR spectra were recored in the NMR facility of
University of California, Davis on a Varian Inova 400 (400 MHz for
1H, 100 MHz for 13C) and a Bruker Avance-800 NMR spectrometer
1
(800 MHz for H, 200 MHz for 13C). Chemical shifts are reported in
parts per million (ppm) on the δ scale. High resolution (HR)
electrospray ionization (ESI) mass spectra were obtained using a
Benzyl 5-acetamido-3,5-dideoxy-8,9-O-isopropylidene-D-glycero-β-
Thermo Electron LTQ-Orbitrap Hybrid MS at the Mass D-galacto-2-nonulopyranosonate (5). Compound 4 (3.1 g, 7.76
Spectrometry Facility in the University of California, Davis. Silica
gel 60 Å (230–400 mesh, Sorbent Technologies) was used for flash camphorsulfonic acid (50 mg) was added. The mixture was stirred
mmol) was dissolved in 2,2-dimethoxypropane (50 mL) and
column chromatography. Thin layer chromatography was
for overnight at room temperature. Then trimethylamine (100 ꢀL)
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was added and the mixture was stirred for 15 min before it was (40 mL) and Ac2O (15 mL) was added. The mixture was stirred for 1
concentrated. The residue was purified by a silica gel column
(EtOAc:MeOH = 15:1, by volume) to produce compound 5 (3.7 g,
h at room temperature and ethanol (30 mL) was added at 0 °C.
After being concentrated, the residue was purified using a 150 g
92%). 1H NMR (800 MHz, CDCl3) δ 7.59–7.41 (m, 5H), 5.29 (d, J = RediSep Rf silica column (EtOAc:MeOH = 10:1, by volume) on a
12.8 Hz, 1H), 5.22 (d, J = 12.0 Hz, 1H), 4.27–3.84(m, 7H), 2.33 (dd, J CombiFlash® Rf 200i system to produce compound 6 (9.1 g, 84%). A
= 12.8 and 4.8 Hz, 1H), 2.08 (s, 3H), 2.01 (t, J = 12.8 Hz, 1H), 1.44 (s, solution of compound 6 (8.58 g, 17.83 mmol) in 90% acetic acid (40
3H), 1.38 (s, 3H); 13C NMR (200 MHz, CDCl3) δ 173.97, 169.61, mL) was stirred at 60 °C for 2 h. The reaction mixture was
134.97, 128.48 (2C), 128.38, 128.00 (2C), 109.30, 95.43, 74.15, evaporated and the obtained syrup was dissolved in methanol (50
71.19, 69.82, 69.72, 67.36, 67.05, 53.09, 39.06, 26.70, 25.07, 22.52.
mL) and treated with H2 in the presence of 10% Pd/C for 2 h at
room temperature. The solution was filtered and concentrated. The
residue was purified using a ODS-SM column (51 g, 50 ꢀM, 120 Å,
Yamazen) on a CombiFlash® Rf 200i system and was eluted with a
gradient of 0–100% acetonitrile in water containing 0.05% formic
acid over 30 minutes with a 30 mL/min flow rate. The fractions
containing the pure product were collected and concentrated to
produce the final product 1 (5.2 g, 83%) as a white powder.
Benzyl 5-acetamido-4-O-acetyl-3,5-dideoxy-8,9-O-isopropylidene-
D-glycero-β-D-galacto-2-nonulopyranosonate (6). To a solution of
compound 5 (2.0 g, 4.55 mmol) in pyridine (4 mL), acetic anhydride
(2 mL) was added. The mixture was stirred for 1 h at room
temperature and then ethanol (10 mL) was added. The solution was
evaporated to dryness and the residue was purified by a silica gel
column (EtOAc:Hexane = 2:1, by volume) to produce compound 6
(1.7 g, 81%). 1H NMR (400 MHz, CDCl3) δ 7.36–7.29 (m, 5H), 6.22 (d,
J = 8.0 Hz, 1H, NH), 5.36 (td, J = 10.8 and 5.6 Hz, 1H, H-4), 5.26 (d, J
= 12.4 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 4.54–3.82 (m, 7H), 2.23 (dd,
Synthesis of Neu4Ac5Gc (2)
N-(2-Benzyloxyacetyl)-D-neuraminic acid (9). Solid K2CO3 (14.4 g,
.
J = 12.8 and 4.8 Hz, 1H), 2.04 (t, J = 12.8 Hz, 1H), 2.03 (s, 3H), 1.97 (s, 104.3 mM) was added to a stirred mixture of ManNH2 HCl (7, 4.5 g,
3H), 1.34 (s, 3H), 1.28 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 172.78,
20.8 mM) in dry MeOH (100 mL). The mixture was stirred for 30 min
and benzyloxyacetyl chloride (5.8 g, 31.3 mM) was added
dropwisely. When the reaction was completed as monitored by TLC,
the mixture was concentrated and purified by flash chromatography
(DCM:MeOH = 10:1, by volume) to produce ManNGc analog 8 (5.8
g, 85%). Compound 8 (3.5 g, 10.7 mM) and pyruvate (4.11 g, 37.4
mM) were dissolved in water (35 mL) in a 50 mL centrifuge tube.
After the addition of an appropriate amount of PmNanA (52 mg),
the reaction mixture was incubated in an isotherm incubator for 48
h at 37 °C with gentle shaking. The reaction progress was monitored
172.23, 168.76, 134.85, 128.68 (2C), 128.62, 128.16 (2C), 109.19,
94.92, 74.17, 71.80, 69.81, 68.93, 67.89, 67.44, 51.91, 35.90, 27.00,
25.22, 23.04, 21.09.
5-Acetamido-4-O-acetyl-3,5-dideoxy-D-glycero-β-D-galacto-2-
nonulopyranosonate (1). A solution of compound 6 (1.1 g, 2.28
mmol) in 90% acetic acid was stirred for 2 h at 60 °C. The reaction
mixture was evaporated to produce a syrup which was dissolved in
methanol (20 mL) and treated with H2 in the presence of 10% Pd/C
for 2 h at room temperature. The solution was then filtered through
Celite and concentrated. The residue was purified by a silica gel
column (EtOAc:MeOH:H2O = 5:2:1, by volume) to produce a white
powder 1 (658 mg, 82%). 1H NMR (800 MHz, D2O) δ 5.27 (td, J =
11.2 and 4.8 Hz, 1H, H-4), 4.19 (d, J = 10.4 Hz, 1H, H-8), 4.15 (t, J =
10.4 Hz, 1H, H-5), 3.82 (dd, J = 12.0 and 2.4 Hz, 1H, H-9a), 3.74 (m,
1H, H-6), 3.60 (dd, J = 12.0 and 6.4 Hz, 1H, H-9b), 3.56 (d, J = 9.6 Hz,
1H, H-7), 2.32 (dd, J = 12.8 and 5.6 Hz, 1H, H-3eq), 2.04 (s, 3H), 1.99
(t, J = 11.2 Hz, 1H, H-3ax), 1.97 (s, 3H); 13C NMR (200 MHz, D2O) δ
174.39, 173.31, 173.29, 95.16, 70.00, 69.98, 69.91, 67.95, 63.02,
49.31, 35.95, 21.76, 20.22; HRMS (ESI) calculated for C13H20NO10
(M-H) 350.1093, found 350.1093.
by TLC (nPrOH:H2O:HOAc
= 5:2:1, by volume) and mass
spectrometer (MS). When the reaction was completed, ice-cold
ethanol (35 mL) was added and the reaction mixture was incubated
at 4 °C for 30 min. The mixture was centrifuged and the supernatant
was concentrated. The residue was purified by silica gel column
flash chromatography (EtOAc:MeOH:H2O = 6:2:1 then 4:2:1, by
volume) to produce the desired Neu5GcOBn 9 (4.4 g, quant.) as a
white solid. 1H NMR (800 MHz, D2O) δ 7.45–7.41 (m, 5H), 4.64 (s,
2H), 4.12 (s, 2H), 4.09 (m, 1H), 4.05 (d, J = 10.4 Hz, 1H), 3.96 (t, J =
10.4 Hz, 1H), 3.81 (dd, J = 12.0 and 3.2 Hz, 1H), 3.75 (m, 1H), 3.58
(dd, J = 12.0 and 6.4 Hz, 1H), 3.50 (d, J = 9.6 Hz, 1H), 2.21 (dd, J =
12.8 and 4.8 Hz, 1H), 1.82 (t, J = 12.8 Hz, 1H); 13C NMR (200 MHz,
D2O) δ 176.65, 173.01, 136.45, 128.72(2C), 128.56(2C), 128.48,
96.33, 73.20, 70.30, 69.88, 68.38, 68.33, 66.86, 63.10, 51.88, 39.37.
HRMS (ESI) m/z calcd for C18H24NO10 (M-H) 414.1400, found
414.1399.
Multigram scale synthesis of Neu4,5Ac2 (1). A multigram scale
synthesis of Neu4,5Ac2 (1) was also achieved using the procedure
described above. Briefly, Neu5Ac (10.1 g, 32.65 mmol) was
dissolved in water (50 mL) and 10% Cs2CO3 was added to adjust the
solution to neutral. After removal of the solvent and dried under
Benzyl
5-glycolylamido-3,5-dideoxy-D-glycero-β-D-galacto-2-
vacuum, the obtained solid was dissolved in dry DMF (60 mL), and nonulopyranosonate (10). To a stirring solution of Neu5GcOBn (9,
3.0 g, mmol) in water (25 mL), 10% CsCO3 was added dropwisely to
adjust pH to neutral. The solution was then evaporated and the
residue was dried for overnight by a vacum pump to produce the
cesium salt of Neu5GcOBn. The salt was dissovled in N,N-
dimethylformamide (40 mL), benzyl bromide (1.5 mL) was then
added to the solution. The mixutre was strired under argon for 2 h
at room temperature. After evaporation, the residue was purified
by silica gel column chromatography (EtOAc:MeOH:H2O = 6:2:1, by
volume) to produce compound 10. 1H NMR (400 MHz, CD3OD) δ
7.35–7.24 (m, 10H), 5.26 (m, 2H, PhCH2), 4.65 (m, 2H, PhCH2), 4.39–
3.53 (m, 9H), 2.36 (dd, J = 12.8 and 4.8 Hz, 1H), 2.17 (t, J = 12.8 Hz,
benzyl bromide (6 mL) was added at 0 °C. The mixture was stirred
at room temperature under argon for overnight and then filtered.
The filtrate was evaporated to a syrup which was crystallized using
2-propanol to produce 4 (11.1 g, 85%). Compound 4 (10.08 g, 25.23
mmol) was then dissolved in acetone (150 mL), and 2,2-
dimethoxypropane (20 mL) and camphorsulfonic acid (150 mg)
were added. The mixture was stirred at room temperature for 2
hours. Trimethylamine (500 ꢀL) was added and the mixture was
stirred for 15 min before it was concentrated. The obtained residue
was purified using a 150 g RediSep Rf silica column (EtOAc:MeOH =
5:1, by volume) on a CombiFlash® Rf 200i system to produce 5 (10.5
g, 95%). Compound 5 (9.89 g, 22.50 mmol) was dissolved in pyridine 1H); 13C NMR (100 MHz, CD3OD) δ 172.27, 170.95, 132.14, 131.50,
6 | J. Name., 2013, 00, 1-11
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127.86 (4C), 127.78, 127.70 (4C), 126.90, 97.80, 73.37, 71.18, 70.23, concentrated and passed through a Bio Gel P-2 gel filtration column
69.22, 68.92, 68.61, 67.31, 66.96, 53.05, 39.06.
(water was used as an eluent). The fractions containing the product
were collected, concentrated, further purified by silica gel column
Benzyl
5-glycolylamido-3,5-dideoxy-8,9-O-isopropylidene-D- (EtOAc:MeOH:H2O = 5:2:1, by volume) and finally by a Bio-Gel P-2
column (eluted with H2O) to produce Lc3 trisaccharide
glycero-β-D-galacto-2-nonulopyranosonate (11). To a solution of
compound 10 (1.5 g, 2.97 mmol) in acetone (40 mL), 2,2- GlcNAcβ3Galβ4GlcβProN342 (0.625 g, 94%). 1H NMR (800 MHz, D2O)
dimethoxypropane (4 mL) and camphorsulfonic acid (15 mg) were
added. The mixture was stirred for 3 h at room temperature. Then
δ 4.67 (d, J = 8.0 Hz, 1H), 4.48 (d, J = 8.0 Hz,1H), 4.43 (d, J = 8.0 Hz,
1H), 4.15 (d, J = 3.2 Hz, 1H), 4.01–3.97 (m, 2H), 3.90 (d, J = 11.2
trimethylamine (200 ꢀL) was added and the solution was stirred for Hz,1H), 3.76–3.55 (m, 13H), 3.47–3.44 (m, 4H), 3.30 (d, J = 8.0
Hz,1H), 2.03 (s, 3H), 1.91 (m, 2H); 13C NMR (200 MHz, D2O) δ 174.85,
15 min. The reaction mixture was concentrated and the residue was
purified by a silica gel column (EtOAc:Hexane = 5:1, by volume) to
produce compound 11 (1.56 g, 96%) as a white solid. H NMR (800
102.81, 102.74, 101.99, 81.83, 78.20, 75.52, 74.76, 74.65, 74.23,
73.43, 72.66, 69.87, 69.54, 68.21, 67.24, 60.83, 60.34, 59.92, 55.53,
1
MHz, D2O) δ 7.43–7.37 (m, 10H), 7.17 (d, J = 8.8 Hz, 1H), 5.27 (d, J = 47.73, 28.10, 22.03. HRMS (ESI) m/z calculated for C23H40N4O16Na
12.0 Hz, 1H), 5.20 (d, J = 12.0 Hz, 1H), 4.60 (d, J = 12.0 Hz, 1H), 5.27 (M+Na) 651.2337, found 651.2337.
(d, J = 11.2 Hz, 1H), 4.49 (d, J = 6.4 Hz, 1H), 4.25–3.99 (m, 9H), 3.59
(t, J = 7.2 Hz, 1H), 2.36 (dd, J = 12.0 and 4.0 Hz, 1H), 2.15 (t, J = 12.0 One-pot
four-enzyme
3Gal 4Glc
preparative-scale
synthesis
of
Hz, 1H), 1.45 (s, 3H), 1.37 (s, 3H); 13C NMR (200 MHz, D2O) δ 172.51, Gal
β
4GlcNAc
β
β
β
ProN3 (LNnT ProN3, 23). The reaction
β
169.48, 136.54, 134.94, 128.61(2C), 128.58(2C), 128.45, 128.27, mixture in a total volume of 30 mL containing Tris-HCl buffer (100
128.01(2C), 127.99(2C), 109.15, 95.39, 74.59, 73.41, 71.23, 69.56, mM, pH 8.0), Lc3 trisaccharide (0.55 g, 0.875 mmol), galactose
68.96, 67.61, 66.93, 66.76, 52.44, 38.98, 26.97, 25.28.
5-Glycolylamido-4-O-acetyl-3,5-dideoxy-D-glycero- -D-galacto-2-
(0.204 g, 1.14 mmol), ATP (0.627 g, 1.14 mmol), UTP (0.626 g, 1.14
mmol), MgCl2 (20 mM), EcGalK (12.0 mg), BLUSP (10.0 mg), NmLgtB
(7.0 mg), and PmPpA (8 mg) was incubated in a shaker with
β
nonulopyranosonate (2). To a solution of 8,9-O-isopropylidene- agitation (100 rpm) at 37 °C for 5 hrs. The product formation was
Neu5GcOBn (11, 1.1 g, 2.02 mmol) in pyridine (4 mL), acetic monitored by TLC (n-PrOH:H2O:NH4OH = 5:2:1 and detected by p-
anhydride (2 mL) was added. The mixture was stirred for 1 h at anisaldehyde sugar stain) and mass spectrometry. When an optimal
room temperature and ethanol (10 mL) was added. The solution yield was achieved, ethanol (30 mL) was added and the mixture was
was evaporated to dryness and the residue was purified by a silica
gel column (EtOAc:toluene = 1:2, by volume) to produce compound
12 (0.96 g, 81%). A solution of compound 12 (0.8 g, 1.36 mmol) in
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 eluate). Further
90% acetic acid was stirred for 2 h at 60 °C. The reaction mixture purification was achieved by silica gel chromatography
was evaporated to give a syrup which was dissolved in methanol (EtOAc:MeOH:H2O = 5:3:1.5) and finally Bio-Gel P-2 column (eluted
with H2O) to produce Galβ4GlcNAcβ3Galβ4GlcβProN342 (23, 0.613 g,
99%). H NMR (800 MHz, D2O) δ 4.69 (d, J = 8.0 Hz, 1H), 4.48 (d, J =
(20 mL) and treated with H2 in the presence of 10% Pd/C for 2 h at
room temperature. The solution was filtered through Celite and
1
concentrated. The residue was purified by a silica gel column
(EtOAc:MeOH:H2O = 5:2:1, by volume) to produce a white powder 2
(0.31 g, 78%). 1H NMR (800 MHz, D2O) δ 5.36 (td, J = 11.2 and 4.8 Hz,
1H, H-4), 4.32 (d, J = 10.4 Hz, 1H, H-8), 4.23 (t, J = 10.4 Hz, 1H, H-5),
4.05 (d, J = 6.4 Hz, 2H, OCH2-), 3.82 (dd, J = 12.0 and 2.4 Hz, 1H, H-
9a), 3.75 (m, 1H, H-6), 3.61 (dd, J = 12.0 and 6.4 Hz, 1H, H-9b), 3.56
(d, J = 8.8 Hz, 1H, H-7), 2.35 (dd, J = 12.8 and 5.6 Hz, 1H, H-3eq),
2.04 (s, 3H), 2.01 (t, J = 12.8 Hz, 1H, H-3ax); 13C NMR (200 MHz, D2O)
δ 75.34, 173.25, 172.97, 95.06, 69.99, 69.75, 69.70, 67.86, 62.98,
7.2 Hz,1H), 4.47 (d, J = 8.0 Hz, 1H), 4.43 (d, J = 7.2 Hz, 1H), 4.15 (d, J
= 3.2 Hz, 1H), 4.01–3.92 (m, 4H), 3.85–3.57 (m, 19H), 3.53 (t, J =8.8
Hz, 1H), 3.46 (t, J = 7.2 Hz, 2H), 3.30 (d, J = 8.0 Hz,1H), 2.03 (s, 3H),
1.90 (m, 2H); 13C NMR (200 MHz, D2O) δ 174.80, 102.82, 102.73,
102.64, 101.99, 81.92, 78.20, 78.00, 75.23, 74.75, 74.65, 74.42,
74.23, 72.66, 72.37, 72.05, 70.84, 69.83, 68.42, 68.20, 67.24, 60.91,
60.83, 59.92, 59.72, 55.06, 47.73, 28.10, 22.05. HRMS (ESI) m/z
calculated for C29H50N4O21Na (M+Na) 813.2865, found 813.2865.
60.71, 48.97, 35.98, 20.22; HRMS (ESI) calculated for C13H20NO11 General procedures for one-pot two-enzyme (OP2E) preparative-
(M-H) 366.1042, found 366.1042.
scale synthesis of α2−3-linked sialosides containing a 4-O-acetyl
sialic acid. An acceptor (10 mM, 1.0 eq, chosen from 13–23), 4-O-
Chemoenzymatic synthesis
of
GlcNAcβ3Galβ4GlcβProN3 acetyl-sialic acid (1.2 eq, 1 or 2), and CTP (2.0 eq), MgCl2 (20 mM)
were dissolved in Tris-HCl buffer (100 mM, pH 7.1), containing
NmCSS (3 mg/mL), and PmST3 (5 mg/mL). The reaction mixture was
incubated at 37 °C in an incubator with agitation (100 rpm) for 14 h.
(Lc3βProN3) and Galβ4GlcNAcβ3Galβ4GlcβProN3 (LNnTβProN3, 23)
One-pot four-enzyme preparative-scale synthesis of trisaccharide
GlcNAcβ3Galβ4GlcβProN3 (Lc3βProN3). A reaction mixture in a total The product formation was monitored by MS. The reaction was
volume of 40 mL containing Tris-HCl buffer (100 mM, pH 7.5), terminated by adding the same volume (10 mL) of ice-cold EtOH
LacβProN3 (17, 0.45 g, 1.06 mmol), N-acetylglucosamine (GlcNAc, followed by incubation at 4 °C for 30 min. The mixture was
0.304 g, 1.38 mmol), ATP (0.758 g, 1.38 mmol), UTP (0.756 g, 1.38 centrifuged to remove precipitates. The supernatant was
mmol), MgCl2 (20 mM), BLNahK (12.0 mg), PmGlmU (6.0 mg), concentrated and passed through a Bio-gel P-2 gel filtration column
NmLgtA (6.0 mg), and PmPpA (4.0 mg) was incubated in a shaker with water as the eluate to obtain the desired product. The
compound was further purified by a reverse-phase C18 column
with agitation (100 rpm) at 37 °C for 15 h and then 72 h at room
temperature. The product formation was monitored by TLC (Phenomenex, 10 ꢀm, 21.2 × 250 mm) with a flow rate of 10
(EtOAc:MeOH:H2O:HOAc = 4:2:1:0.2 by volume and detected by p- mL/min using a gradient elution of 0–100% acetonitrile in water
anisaldehyde sugar stain) and mass spectrometry (MS). When the containing 0.05% formic acid over 20 minutes [Mobile phase A: 0.05%
reaction was completed, the same volume (40 mL) of 95% ethanol formic acid in water (v/v); Mobile phase B: acetonitrile (v/v);
was added to the reaction mixture and the mixture was incubated Gradient: 0% B for 3 minutes, 0% to 100% B over 12 minutes, 100%
B for 2 minutes, then 100% to 0% B over 3 minutes]. The fractions
at 4 °C for 30 min. After centrifugation, the supernatant was
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containing the pure product were collected and concentrated to 78.12, 75.41, 75.02, 74.65, 74.22, 72.68, 72.26, 71.63, 71.04, 69.23,
provide the final purified product.
67.78, 67.35, 67.24, 62.41, 60.90, 59.92, 49.16, 47.74, 36.54, 28.11,
21.76, 20.21; HRMS (ESI) calculated for C28H45N4O20 (M-H) 757.2627,
1
Neu4,5Ac2 3Galβ4Glc (24). 56 mg, yield 82%; white solid. H NMR found 757.2633.
α
(800 MHz, D2O) δ 5.20 (d, J = 4.0 Hz, 0.4H, α-isomer), 4.94 (m, 1H),
4.64 (d, J = 8.0 Hz, 0.6H, β-isomer), 4.51 (d, J = 8.0 Hz, 1H), 4.12–
3.25 (m, 18H), 2.75 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 1.94 (s,
3H), 1.91 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O) δ 174.43,
173.42, 173.09, 102.49, 99.62, 95.65, 78.03, 75.41, 75.02, 74.68,
74.20, 73.67, 72.74, 71.63, 71.04, 69.23, 67.78, 67.05, 62.41, 60.91,
59.93, 49.68, 36.54, 21.75, 20.20; HRMS (ESI) m/z calcd for
C25H40NO20 (M-H) 674.2144, found 674.2149.
Neu4,5Ac2α3Galβ4GlcNAcβProN3 (29). 32 mg, yield 85%; white
solid. 1H NMR (800 MHz, D2O) δ 4.97 (m, 1H), 4.53 (d, J = 8.0 Hz, 1H),
4.501 (d, J = 8.8 Hz, 1H), 4.12 (dd, J = 10.4 and 3.2 Hz, 1H), 4.08 (t, J
= 10.4 Hz, 1H), 3.99–3.54 (m, 18H), 3.36 (m, 2H), 2.75 (dd, J = 12.8
and 4.8 Hz, 1H), 2.05 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H), 1.94 (t, J =
12.0 Hz, 1H), 1.82 (m, 2H); 13C NMR (200 MHz, D2O) δ 174.44,
174.38, 173.09, 172.78, 102.42, 101.02, 99.29, 78.17, 75.39, 74.97,
74.61, 72.36, 72.22, 71.36, 70.79, 69.24, 67.78, 67.39, 67.00, 62.52,
60.85, 59.90, 54.95, 49.10, 47.63, 36.34, 27.97, 22.02, 21.75, 20.19;
HRMS (ESI) calculated for C30H48N5O20 (M-H) 798.2893, found
798.2893.
Neu4,5Ac2α3Galβ4GlcNAcβ3Galβ4Glc (25). 34 mg, yield 78%;
1
white solid. H NMR (800 MHz, D2O) δ 5.21 (d, J = 4.0 Hz, 0.4H, α-
isomer), 4.97 (m, 1H), 4.69 (d, J = 8.8 Hz, 0.4H, α-isomer), 4.68 (d, J
= 8.0 Hz, 0.6H, β-isomer), 4.65 (d, J = 8.0 Hz, 0.6H, β-isomer), 4.55 (d,
J = 8.0 Hz, 1H), 4.43 (d, J = 8.0 Hz, 1H), 4.15 (d, J = 4.0 Hz, 1H), 4.13
(dd, J = 3.2 and 9.6 Hz, 1H), 4.09 (t, J = 9.6 Hz, 1H), 3.96–3.26 (m,
28H), 2.76 (dd, J = 4.8 and 12.0 Hz, 1H), 2.06 (s, 3H), 2.02 (s, 3H),
1.96 (s, 3H), 1.94 (t, J = 12.0 Hz, 1H). 13C NMR (200 MHz, D2O) α/β
isomers: δ 174.77, 174.45, 173.08, 172.92, 102.80, 102.77, 102.67,
102.40, 95.61, 81.92, 81.90, 78.25, 78.14, 77.88, 75.42, 74.99, 74.77,
74.68, 74.42, 74.23, 73.65, 72.35, 72.03, 71.44, 71.28, 71.00, 70.86,
70.00, 69.87, 69.84, 69.25, 68.21, 67.78, 67.39, 62.50, 60.87, 60.85,
59.94, 59.72, 55.06, 49.12, 46.55, 36.41, 22.05, 21.77, 20.20. HRMS
(ESI) m/z calculated for C39H63N2O30 (M-H) 1039.3466, found
1039.3442.
Neu4,5Ac2α3Galβ3GlcNAcβProN3 (30). 24 mg, yield 86%; white
solid. 1H NMR (800 MHz, D2O) δ 4.98 (m, 1H), 4.54 (d, J = 8.0 Hz, 1H),
4.48 (d, J = 8.0 Hz, 1H), 4.10–4.07 (m, 2H), 3.96–3.36 (m, 20H), 2.75
(dd, J = 12.0 and 4.8 Hz, 1H), 2.06 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H),
1.94 (t, J = 12.0 Hz, 1H), 1.83 (m, 2H); 13C NMR (200 MHz, D2O) δ
174.46 (2C), 173.12, 172.33, 103.23, 100.77, 98.92, 82.44, 75.53,
75.24, 74.85, 72.36, 71.28, 70.61, 68.96, 68.61, 67.77, 67.26, 67.03,
62.53, 60.80, 60.60, 54.32, 49.06, 47.66, 36.29, 27.97, 22.17, 21.78,
20.19; HRMS (ESI) calculated for C30H48N5O20 (M-H) 798.2893, found
798.2900.
Neu4,5Ac2α3Galβ3GlcNAcαProN3 (31). 21 mg, yield 80%; white
solid. 1H NMR (800 MHz, D2O) δ 4.93 (m, 1H), 4.84 (d, J = 4.0 Hz, 1H),
4.50 (d, J = 8.0 Hz, 1H), 4.10–4.05 (m, 3H), 3.93–3.44 (m, 19H), 2.76
(dd, J = 10.8 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H),
1.91–1.88 (m, 3H); 13C NMR (200 MHz, D2O) δ 174.42, 174.33,
173.40, 173.10, 103.20, 99.49, 96.97, 80.21, 75.61, 74.90, 72.19,
71.73, 71.48, 71.04, 69.01, 68.55, 67.73, 67.16, 64.82, 62.30, 60.87,
60.39, 52.36, 49.13, 48.04, 36.68, 27.84, 21.92, 21.76, 20.21; HRMS
(ESI) calculated for C30H48N5O20 (M-H) 798.2893, found 798.2895.
Neu4,5Ac2α3Galβ3GlcNAcβ3Galβ4Glc (26). 29 mg, yield 65%;
1
white solid. H NMR (800 MHz, D2O) δ 5.20 (d, J = 3.2 Hz, 0.4H, α-
isomer), 4.93 (m, 1H), 4.71 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 8.0 Hz,
0.6H, β-isomer), 4.50 (d, J = 8.0 Hz, 1H), 4.43 (d, J = 8.0 Hz, 1H), 4.13
(d, J = 3.2 Hz, 1H), 4.09 (dd, J = 3.2 and 9.6 Hz, 1H), 4.07 (t, J = 9.6 Hz,
1H), 3.94–3.26 (m, 27H), 2.75 (dd, J = 4.0 and 12.0 Hz, 1H), 2.05 (s,
3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.89 (t, J = 12.0 Hz, 1H). 13C NMR (200
MHz, D2O) β-isomer: δ 174.81, 174.41, 173.41, 173.09, 103.25,
102.78, 102.40, 99.45, 95,61, 81.99, 81.80, 78.16, 75.24, 75.08,
74.95, 74.78, 74.68, 74.23, 73.65, 72.19, 71.73, 71.03, 69.89, 68.95,
68.33, 67.74, 67.13, 62.30, 60.91, 60.85, 60.38, 59.93, 54.47, 49.14,
36.70, 22.17, 21.76, 20.21. HRMS (ESI) m/z calculated for
C39H63N2O30 (M-H) 1039.3466, found 1039.3487.
Neu4,5Ac2α3Galβ3GalNAcβProN3 (32). 22 mg, yield 81%; white
solid. 1H NMR (800 MHz, D2O) δ 4.95 (m, 1H), 4.49 (d, J = 8.0 Hz, 1H),
4.48 (d, J = 8.0 Hz, 1H), 4.16 (d, J = 3.2 Hz, 1H), 4.09–4.06 (m, 2H),
4.16–3.59 (m, 16H), 3.52 (dd J = 9.6 and 8.0 Hz, 1H), 3.36 (m, 2H),
2.75 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.01 (s, 3H), 1.94 (s,
3H), 1.92 (t, J = 12.0 Hz, 1H), 1.83 (m, 2H); 13C NMR (200 MHz, D2O)
δ 174.62, 174.43, 173.10, 172.92, 104.46, 101.28, 99.22, 79.95,
75.49, 74.62, 74.58, 72.26, 71.49, 70.85, 68.86, 67.76, 67.75, 67.30,
66.88, 62.43, 60.82 (2C), 51.03, 49.10, 47.67, 36.46, 27.99, 22.16,
21.76, 20.19; HRMS (ESI) calculated for C30H48N5O20 (M-H) 798.2893,
found 798.2903.
1
Neu4,5Ac2
α3Gal
β
pNP (27). 13 mg, yield 31%; white solid. H NMR
(800 MHz, D2O) δ 8.27 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H),
5.30 (d, J = 8.0 Hz, 1H), 4.95 (m, 1H), 4.25 (dd, J = 9.6 and 4.0 Hz,
1H), 4.09 (t, J = 10.4 Hz, 1H), 4.04 (d, J = 2.4 Hz, 1H), 3.93–3.57 (m,
10H), 2.79 (dd, J = 12.0 and 4.8 Hz, 1H), 2.06 (s, 3H), 1.95 (s, 3H),
1.92 (t, J = 12.0 Hz, 1H); 13C NMR (200 MHz, D2O) δ 174.48, 173.37,
173.10, 163.03, 162.87, 161.61, 142.42, 126.03, 116.31, 99.69,
99.51, 75.43, 75.31, 72.28, 71.69, 71.02, 68.66, 67.79, 67.16, 62.43,
60.54, 49.17, 36.63, 21.82, 20.27; HRMS (ESI) calculated for
C25H33N2O17 (M-H) 633.1779, found 633.1807.
Neu4,5Ac2α3Galβ3GalNAcαProN3 (33). 18 mg, yield 74%; white
solid. 1H NMR (800 MHz, D2O) δ 4.94 (m, 1H), 4.89 (d, J = 4.0 Hz, 1H),
4.53 (d, J = 8.0 Hz, 1H), 4.32–3.43 (m, 22H), 2.75 (dd, J = 10.8 and
4.8 Hz, 1H), 2.06 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H), 1.91–1.88 (m, 3H);
13C NMR (200 MHz, D2O) δ 174.47, 174.43, 173.43, 173.10, 104.35,
99.55, 97.08, 77.27, 75.60, 74.65, 72.20, 71.73, 71.06, 70.52, 68.95,
68.47, 67.75, 67.29, 64.80, 62.35, 61.11, 60.86, 49.15, 48.57, 48.06,
36.65, 27.85, 21.93, 21.76, 20.21; HRMS (ESI) calculated for
C30H48N5O20 (M-H) 798.2893, found 798.2902.
Neu4,5Ac2α3Galβ4GlcβProN3 (28). 28 mg, yield 88%; white solid.
1H NMR (800 MHz, D2O) δ 4.98 (m, 1H), 4.51 (d, J = 8.0 Hz, 1H), 4.47
(d, J = 8.0 Hz, 1H), 4.11 (dd, J = 9.6 and 3.2 Hz, 1H), 4.07 (t, J = 10.4
Hz, 1H), 3.98–3.94 (m, 3H), 3.89–3.55 (m, 14H), 3.44 (t, J = 6.4 Hz,
2H), 3.29 (t, J = 8.8 Hz, 1H), 2.75 (dd, J = 12.8 and 4.8 Hz, 1H), 2.10 (s,
3H), 1.95 (s, 3H), 1.91 (t, J = 12.8 Hz, 1H), 1.89 (m, 2H); 13C NMR
(200 MHz, D2O) δ 174.44, 173.42, 173.09, 102.51, 102.02, 99.63,
8 | J. Name., 2013, 00, 1-11
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Neu4,5Ac2 3Gal 4GlcNAc 3Gal 4GlcβProN3 (34). 20 mg, yield
ARTICLE
4.48 (d, J = 8.0 Hz, 1H), 4.16–3.37 (m, 24H), 2.75 (dd, J = 12.0 and
4.8 Hz, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.90 (t, J = 12.0 Hz,
1H), 1.83 (m, 2H); 13C NMR (200 MHz, D2O) δ 175.34, 174.61, 173.48,
173.04, 104.49, 101.28, 99.44, 79.90, 75.49, 74.64, 74.63, 71.94,
71.73, 70.77, 68.85, 67.75, 67.70, 67.20, 66.87, 62.28, 60.86, 60.82,
60.70, 51.04, 48.85, 47.66, 36.78, 27.99, 22.17, 20.21; HRMS (ESI)
calculated for C30H48N5O21 (M-H) 814.2842, found 814.2872.
α
β
β
β
81%; white solid. 1H NMR (800 MHz, D2O) δ 4.93 (m, 1H), 4.67 (d, J =
8.0 Hz, 1H), 4.54 (d, J = 8.0 Hz, 1H), 4.47 (d, J = 8.0 Hz, 1H), 4.41 (d, J
= 8.0 Hz, 1H), 4.14 (d, J = 3.2 Hz, 1H), 4.11 (dd, J = 9.6 and 3.2 Hz,
1H), 4.07 (t, J = 10.4 Hz, 1H), 3.98–3.54 (m, 28H), 3.44 (t, J = 6.4 Hz,
2H), 3.29 (t, J = 8.0 Hz, 1H), 2.75 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s,
3H), 2.01 (s, 3H), 1.94 (s, 3H), 1.91 (t, J = 12.0 Hz, 1H), 1.89 (m, 2H);
13C NMR (200 MHz, D2O) δ 174.78, 174.44, 173.40, 173.08, 102.82,
102.69, 102.41, 101.98, 99.64, 81.93, 78.21, 77.85, 75.41, 75.02,
74.77, 74.65, 74.42, 74.23, 72.65, 72.27, 72.02, 71.62, 71.03, 69.83,
69.25, 68.20, 67.76, 67.36, 67.24, 62.41, 60.91, 60.84, 59.91, 59.70,
55.05, 49.15, 47.73, 36.53, 28.10, 22.04, 21.75, 20.20; HRMS (ESI)
calculated for C42H68N5O30 (M-H) 1122.3949, found 1122.3965.
Neu4Ac5Gcα3Galβ3GalNAcαProN3 (40). 11 mg, yield 52%; white
solid. 1H NMR (800 MHz, D2O) δ 5.02 (dt, J = 10.4 and 4.8 Hz, 1H, H-
4’’), 4.89 (d, J = 4.0 Hz, 1H), 4.53 (d, J = 8.0 Hz, 1H), 4.30 (dd, J = 11.2
and 4.0 Hz, 1H), 4.23 (d, J = 3.2 Hz, 1H), 4.15 (t, J = 10.4 Hz, 1H),
4.10–3.41 (m, 21H), 2.78 (dd, J = 12.8 and 4.8 Hz, 1H), 2.04 (s, 3H),
2.01 (s, 3H), 1.90 (t, J = 12.8 Hz, 1H), 1.88 (m, 2H); 13C NMR (200
MHz, D2O) δ 175.35, 174.45, 173.44, 173.05, 104.34, 99.51, 97.07,
77.24, 75.58, 74.64, 71.97, 71.75, 70.77, 70.51, 68.95, 68.47, 67.69,
67.27, 64.79, 62.31, 61.10, 60.85, 60.70, 48.85, 48.56, 48.05, 36.74,
27.84, 21.93, 20.22; HRMS (ESI) calculated for C30H48N5O21 (M-H)
814.2842, found 814.2874.
Neu4Ac5Gcα3Galβ4GlcβProN3 (35). 15 mg, yield 67%; white solid.
1H NMR (800 MHz, D2O) δ 4.98 (dt, J = 11.2 and 4.8 Hz, 1H, H-4’’),
4.52 (d, J = 8.0 Hz, 1H), 4.47 (d, J = 8.0 Hz, 1H), 4.16–3.55 (m, 21H),
3.44 (t, J = 6.4 Hz, 2H), 3.30 (t, J = 8.8 Hz, 1H), 2.78 (dd, J = 12.0 and
4.8 Hz, 1H), 2.05 (s, 3H), 1.92 (t, J = 12.0 Hz, 1H), 1.89 (m, 2H); 13C
NMR (200 MHz, D2O) δ 175.38, 173.45, 173.05, 102.51, 102.01,
99.61, 78.11, 75.39, 75.02, 74.64, 74.21, 72.68, 72.04, 71.67, 70.77,
69.24, 67.72, 67.35, 67.24, 62.39, 60.90, 60.71, 59.92, 48.88, 47.74,
36.65, 28.11, 20.23; HRMS (ESI) calculated for C28H45N4O21 (M-H)
773.2576, found 773.2500.
Neu4Ac5Gcα3Galβ4GlcNAcβ3Galβ4GlcβProN3 (41). 10 mg, yield
62%; white solid. 1H NMR (800 MHz, D2O) δ 5.04 (dt, J = 10.4 and
4.8 Hz, 1H, H-4’’’), 4.70 (d, J = 8.8 Hz, 1H), 4.56 (d, J = 8.0 Hz, 1H),
4.49 (d, J = 8.0 Hz, 1H), 4.44 (d, J = 8.0 Hz, 1H), 4.18–3.57 (m, 33H),
3.46 (t, J = 6.4 Hz, 2H), 3.12 (t, J = 8.0 Hz, 1H), 2.80 (dd, J = 12.0 and
4.8 Hz, 1H), 2.07 (s, 3H), 2.03 (s, 3H), 1.91 (t, J = 12.0 Hz, 1H), 1.91
(m, 2H); 13C NMR (200 MHz, D2O) δ 174.78, 174.44, 173.40, 173.08,
102.82, 102.69, 102.41, 101.98, 99.64, 81.93, 78.21, 77.85, 75.41,
75.02, 74.77, 74.65, 74.42, 74.23, 72.65, 72.27, 72.02, 71.62, 71.03,
69.83, 69.25, 68.20, 67.76, 67.36, 67.24, 62.41, 60.91, 60.84, 59.91,
59.70, 55.05, 49.15, 47.73, 36.53, 28.10, 22.04, 21.75, 20.20; HRMS
(ESI) calculated for C42H68N5O31 (M-H) 1138.3898, found 1138.3938.
Neu4Ac5Gcα3Galβ4GlcNAcβProN3 (36). 17 mg, yield 71%; white
solid. 1H NMR (800 MHz, D2O) δ 5.02 (dt, J = 11.2 and 4.8 Hz, 1H, H-
4’’), 4.54 (d, J = 8.0 Hz, 1H), 4.51 (d, J = 8.0 Hz, 1H), 4.15 (t, J = 11.2
Hz, 1H), 4.13 (dd, J = 10.4 and 3.2 Hz, 1H), 4.07–3.55 (m, 20H), 3.37
(m, 2H), 2.80 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.03 (s, 3H),
1.95 (s, 3H), 1.93 (t, J = 12.0 Hz, 1H), 1.82 (m, 2H); 13C NMR (200
MHz, D2O) δ 175.38, 174.38, 173.45, 173.05, 102.45, 101.03, 99.63,
78.17, 75.39, 75.02, 74.62, 72.22, 72.04, 71.67, 70.77, 69.26, 67.73,
67.38, 67.04, 62.39, 60.91, 60.71, 59.90, 54.96, 48.88, 47.65, 36.64,
27.98, 22.05, 20.24; HRMS (ESI) calculated for C30H48N5O21 (M-H)
814.2842, found 814.2870.
Gram-scale OP2E synthesis of monotreme milk trisaccharide
Neu4,5Ac2α3Lac. Lactose (1.09 g, 3.05 mmol), Neu4,5Ac2 (1.07 g,
3.05 mmol), and CTP (1.93 g, 3.66 mmol) were dissolved in water
(30 mL) and the pH of the solution was adjusted to 7.1 using NaOH
solution (4 M). MgCl2 (20 mM), Tris-HCl buffer (100 mM, pH 7.1),
NmCSS (15 mg), and PmST3 (20 mg) were then added and the total
volume of the reaction mixture was brought up to 100 mL by adding
H2O. The reaction mixture was incubated in an incubator shaker
with shaking (100 rpm) at 30 °C for overnight. The reaction was
monitored by mass spectrometry (MS). Additional CTP (0.96 g) was
added to the reaction mixture and the pH was carefully adjusted to
pH 7.1 using NaOH solution (4 M). The reaction was allowed to
continue for another 20 h at room temperature until an optimal
yield was achieved. After adding ethanol (100 mL), the reaction
mixture was incubated at 4 °C for 1 h. The precipitates were
removed by centrifuge and the supernatant was concentrated. The
residue was purified using a ODS-SM column (51 g, 50 ꢀM, 120 Å,
Yamazen) on a CombiFlash® Rf 200i system. Mobile phase A: water;
Mobile phase B: acetonitrile; Gradient: 10% B for 6 minutes, 10% to
100% B over 12 minutes, 100% B for 6 minutes, then 100% to 80% B
over 1 minutes. The fractions containing the pure product were
collected and concentrated to produce the final purified product
Neu4,5Ac2α3Galβ4Glc (1.33 g, 71%) as a white powder. The identity
and the purity of the product were confirmed by NMR and HRMS.
Neu4Ac5Gcα3Galβ3GlcNAcβProN3 (37). 14 mg, yield 65%; white
solid. 1H NMR (800 MHz, D2O) δ 5.02 (dt, J = 11.2 and 4.8 Hz, 1H, H-
4’’), 4.54 (d, J = 8.0 Hz, 1H), 4.48 (d, J = 8.0 Hz, 1H), 4.15–3.34 (m,
24H), 2.78 (dd, J = 12.0 and 4.8 Hz, 1H), 2.05 (s, 3H), 2.02 (s, 3H),
1.90 (t, J = 12.0 Hz, 1H), 1.83 (m, 2H); 13C NMR (200 MHz, D2O) δ
175.34, 174.45, 173.43, 173.05, 103.31, 100.78, 99.43, 82.35, 75.54,
75.25, 74.95, 71.96, 71.75, 70.75, 68.94, 68.59, 67.67, 67.14, 67.04,
62.25, 60.89, 60.71, 60.59, 54.33, 48.85, 47.66, 36.79, 27.97, 22.18,
20.22; HRMS (ESI) calculated for C30H48N5O21 (M-H) 814.2842, found
814.2869.
Neu4Ac5Gcα3Galβ3GlcNAcαProN3 (38). 12 mg, yield 56%; white
solid. 1H NMR (800 MHz, D2O) δ 5.02 (dt, J = 10.4 and 4.8 Hz, 1H, H-
4’’), 4.85 (d, J = 3.2 Hz, 1H), 4.51 (d, J = 8.0 Hz, 1H), 4.15 (t, J = 10.4
Hz, 1H), 4.12–3.42 (m, 23H), 2.79 (dd, J = 12.8 and 4.8 Hz, 1H), 2.05
(s, 3H), 2.02 (s, 3H), 1.91 (t, J = 12.8 Hz, 1H), 1.89 (m, 2H); 13C NMR
(200 MHz, D2O) δ 175.35, 174.32, 173.43, 173.06, 103.19, 99.48,
96.98, 80.19, 75.60, 74.90, 71.96, 71.76, 71.48, 70.76, 69.02, 68.54,
67.67, 67.14, 64.83, 62.27, 60.87, 60.71, 60.39, 52.36, 48.85, 48.05,
36.78, 27.84, 21.93, 20.23; HRMS (ESI) calculated for C30H48N5O21
(M-H) 814.2842, found 814.2875.
Sialidase assays. Sialidase assays were carried out in duplicate at 37
°C for 40 min (for bacterial sialidases) or 30 min (for human
influenza A viruses) in a 384-well plate (Fisher Scientific, Chicago, IL)
in a final volume of 20 ꢀL containing Neu4,5Ac2α3GalβpNP (0.3 mM)
Neu4Ac5Gcα3Galβ3GalNAcβProN3 (39). 10 mg, yield 51%; white
solid. 1H NMR (800 MHz, D2O) δ 5.02 (m, 1H), 4.50 (d, J = 8.0 Hz, 1H),
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ARTICLE
Organic & Biomolecular Chemistry
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and Aspergillus oryzae β-galactosidase (12 ꢀg, 126 mU). The
amount of the β-galactosidase required to completely hydrolyze
GalβpNP (0.3 mM) within the time frame of the assay was
predetermined and confirmed by control assays with GalβpNP (0.3
mM). The reactions were stopped by adding 40 ꢀL of N-cyclohexyl-
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sialidase (1.5 mU) in sodium acetate buffer (100 mM, pH 5.5); C.
perfringens sialidase (3 mU) in MES buffer (100 mM, pH 5.0); V.
cholerae sialidase (1.5 mU) in sodium acetate buffer (100 mM, pH
5.5) containing NaCl (150 mM) and CaCl2 (10 mM); S. pneumoniae
sialidase (1.5 mU) in sodium acetate buffer (100 mM, pH 6.0);
PmST1 (10 ꢀg) in sodium acetate buffer (100 mM, pH 5.5). Viral
sialidase assays were carried out in a MES buffer (100 mM, pH 5.0)
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,
13.T. Urashima, H. Inamori, K. Fukuda, T. Saito, M. Messer and O.
T. Oftedal, Glycobiology, 2015, 25, 683-697.
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This work was supported by US NIH grants R01HD065122,
R01GM076360-04S1 (to X.C.), and U01CA199792 (to A.V.) as
well as scholarships from China Scholarship Council (to J.Z. and
B.S). Bruker Avance-800 NMR spectrometer was funded by
NSF grant DBIO-722538. We would like to thank Professor
Nicole Baumgarth at the University of California-Davis for
providing purified human influenza viruses as kind gifts.
H.Y., Y.L., and X.C. are co-founders of Glycohub, Inc., a
company focused on the development of carbohydrate-based
reagents, diagnostics, and therapeutics. Glycohub, Inc. played
no role in the design, execution, interpretation, or publication
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