Synthesis of partially O-acetylated N-acetylneuraminic acid using regioselective silyl exchange technology

Article abstract of DOI:10.1021/ol502389g

Postglycosylation acetylation of sialic acid imparts unique roles to sialoglycoconjugates in mammalian immune response making structural and functional understanding of these analogues important. Five partially O-acetylated Neu5Ac analogues have been synt

Full text of DOI:10.1021/ol502389g

Letter
pubs.acs.org/OrgLett
Synthesis of Partially O-Acetylated N‑Acetylneuraminic Acid Using
Regioselective Silyl Exchange Technology
Simon S. Park and Jacquelyn Gervay-Hague*
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States
*
S Supporting Information
ABSTRACT: Postglycosylation acetylation of sialic acid imparts unique roles to sialoglycoconjugates in mammalian immune
response making structural and functional understanding of these analogues important. Five partially O-acetylated Neu5Ac
analogues have been synthesized. Reaction of per-O-silylated Neu5Ac ester with AcOH and Ac O in pyridine promotes
2
regioselective silyl ether/acetate exchange in the following order: C (2°) > C (1°) > C (2°) > C (anomeric). Subsequent
4
9
8
2
hydrogenolysis affords the corresponding sialic acid analogues as useful chemical biology tools.
1
n nature, sialic acids are found in more than 50 forms. These
important carbohydrates are nine carbon keto-aldonic acids
Table 1. Acetylated Sialic Acids: Natural Occurrence and
Structural Divergence
I
typically attached to the terminal ends of glycolipids and
glycoproteins in vertebrates and various pathogenic bacteria
2
(
Table 1). The most common form of sialic acid is Neu5Ac
3
(Table 1), which plays critical roles in many biological and
3
physiological functions such as signal transduction, cell−cell
recognition and growth, and immunology. The structures of
compd name
abbreviation
Neu5Ac
occurrence
4
5
5
-N-acetylneuraminic acid
V, E, Ps, Pz, F, B
sialoglycoconjugates are further diversified by O-acetylation
6
5-N-acetyl-4-O-acetylneuraminic acid Neu4,5Ac
2
V
V
(
Table 1). These derivatives are products of sialate O-
5
5
5
5
-N-acetyl-4,9-di-O-acetylneuraminic Neu4,5,9Ac3
acid
acetyltransferases (SOATs) that selectively O-acetylate at various
positions of Neu5Ac. O-Acetylation influences the biology of
mammalian cells by altering the ligand properties and
-N-acetyl-4,7,9-tri-O-
Neu4,5,7,9Ac4
V
V
acetylneuraminic acid
7,8
-N-acetyl-4,7,8,9-tetra-O-
acetylneuraminic acid
Neu4,5,7,8,9Ac5
degradation pathways of sialoglycoconjugates. In bacteria, O-
acetylation can lead to inhibition of the host immune response,
thereby serving as a masking system that enables pathogenic
-N-acetyl-7-O-acetylneuraminic acid Neu5,7Ac2
V, Pz, B
9
5-N-acetyl-9-O-acetylneuraminic acid Neu5,9Ac
2
V, E, Pz, F, B
functions.
Abbreviations used: V, vertebrates; E, echinoderms; Ps, protostomes
insects and mollusks); Pz, protozoa; F, fungi; B, bacteria.
Historically, it has been suggested that O-acetylation can
(
potentially serve as a clue to mammalian evolutionary
1
0
phenomena. However, to date, only sialate-4-O-acetyltransfer-
11
have proven useful in monitoring degradation products of
Neu5Ac lyase during sialoglycoconjugate isolation and other
ase (4-SOAT) has been identified in mammals, and isolation
and cloning 4-SOAT have not yet been successful.
12
biochemical assays.
There is sufficient evidence documenting the presence of 4-O-
acetyl containing Neu5Ac analogues (Table 1); however, full
characterization and biological understanding of these derivatives
is lacking and the limitations of current extraction methods make
synthesis of these analogues important. While naturally occurring
sialic acids found in mammalian cells are often conjugated to
other sugars, partially acetylated monomers have been isolated
from natural sources (Table 1). Moreover, synthetic standards
With growing interest in Neu5Ac analogues and glycoside
synthesis, methodologies that allow regioselective functionaliza-
tion of carbohydrates in an efficient manner are of great utility to
synthetic chemists. However, Neu5Ac contains several hydroxyl
Received: August 11, 2014
Published: September 23, 2014
©
2014 American Chemical Society
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dx.doi.org/10.1021/ol502389g | Org. Lett. 2014, 16, 5044−5047

Organic Letters
Letter
groups with similar reactivities that are challenging tocontrol, and
there is evidence that intramolecular hydrogen bonding creates
an ether silylation method reported by Sweeley and co-workers,
using hexamethyldisilazane (HMDS) and chlorotrimethylsilane
(TMSCl) in pyridine, successfully afforded 2 in 85% yield
13
further complexity. To avoid these issues, traditional chemical
methods have utilized multiple protection−deprotection steps,
and although enzymatic approaches do not require protecting
group manipulations these methods are applicable to a limited
21
(Scheme 1).
ReSET studies were initiated by diluting 2 in dry acetic
anhydride and pyridine and 3 equiv of glacial acetic acid
(≥99.85%) were added. The reaction mixture was stirred at rt
overnight to afford a distribution of acetylated Neu5Ac analogues
(3−6) of which 6 was the major product (Table 2, entry 1).
Delighted with this result, we then attempted to reduce the
reaction time by subjecting the reaction mixture to microwave
irradiation in a commercial CEM-microwave reactor at 60 °C and
30 W power for 30 min, which afforded 3−6 in a slightly lower
overall yield (Table 2, entry 2). Reducing the amount of acetic
acid to 2 equiv and heating the reaction to 70 °C with 40 W power
for 30 min gave 3−6 in the most even distribution (Table 2, entry
14
number of substrates.
Only a few chemical syntheses of partially O-acetylated
Neu5Ac have appeared in the literature. In 1990, Hasegawa
and co-workers first reported the preparation of Neu4,5(Ac)2
using isopropylidene protection of the C and C of Neu5Ac
8
9
15
thioglucosides followed by kinetically controlled acetylation.
More recently, Clarke and co-workers synthesized a series of
12
monoacetylated Neu5Ac with an improved adaptation of the
Hasegawa approach using free Neu5Ac instead of preparing
Neu5Ac thioglucosides. The overall yields of both approaches
were comparable.
3
). To increase the scale of the reaction, the amount of 2 was
Previously in our laboratory, selective acetylation of aldose
sugars was achieved using regioselective silyl-exchange technol-
nearly doubled and set up with 2 equiv of acetic acid at 58 °C and
3
0W power for 18 min to afford 3−6 with noticeably increased
1
6,17
ogy (ReSET).
Readily available per-O-silylated sugars were
amounts of 5 and 6 (Table 2, entry 4). Likewise, we were to able
to optimize for the production of 3 and 4 by reducing the amount
of acetic acid to 1 equiv while running the reaction at 55 °C and
dissolved in pyridine and acetic anhydride, and upon addition of
acetic acid the silyl protecting groups exchanged with acetate in a
predictable manner, depending upon the structure of the aldose.
Although Neu5Ac is a keto-aldonic sugar rather than an aldose,
we were hopeful that the methodology would prove equally
successful. With growing interest in step economy synthese-
3
0 W power (Table 2, entry 5). Optimizing conditions for the
production of compound 4 was especially important because it is
a precursor to analogue 15 (see Scheme 3). To further shorten
the synthesis, attempts were made to directly apply ReSET to 1;
however, per-O-acetylated Neu5Ac was the only product
observed after 10 min. This result illustrates the importance of
the silyl protecting groups in achieving regioselective exchange.
Each ReSET product was analyzed by heteronuclear multiple
bond correlation (HMBC) and heteronuclear single quantum
coherence (HSQC) NMR experiments to determine the position
of the acetyl protecting groups. The HMBC NMR experiments
were critical to observe the correlation between the sugar
backbone C−H protons to the carbonyl carbon of the acetyl
protecting groups to determine the position of the acetyl
protecting group (Figure 1). A four-bond HMBC NMR
experiment was performed to observe correlation between
methyl protons of the acetate to the sugar carbon to characterize
1
8a−c
s,
we endeavored to apply ReSET toward the synthesis of
partially O-acetylated Neu5Ac natural products.
The research began with sialic acid benzyl ester formation
using K CO and BnBr in DMF to afford 1 in 85% yield (Scheme
2
3
1). Esterification minimized solubility issues associated with the
Scheme 1. Benzylation and Silylation of Neu5Ac
6
since theanomeric carbon of Neu5Ac does notbear aproton for
three-bond HMBC.
Once the products of the reactions were identified, we were
able to determine the order of acetate exchange using TLC data
that had been collected during the course of the reaction. The first
spot to form below the starting material (2) was 3 then 4 and 5.
The last spot to form on the TLC was compound 6. The C9,
bearing the primary OTMS group, was expected to be the first to
Neu5Ac carboxylic acid. After benzyl ester formation, our focus
turned to the preparation of per-O-TMS Neu5Ac benzyl ester
(
2). Attempts were made to prepare 2 using published
19,20
17
protocols;
however, we found that Neu5Ac benzyl ester
exchange as observed in our previous work with aldohexoses;
was only partially silylated under these conditions. Gratifyingly,
instead, the secondary hydroxyl group (C ) next to the NHAc
4
Table 2. Various Conditions of ReSET To Afford 3-6
entry
scale (mg)
time (min)
T (°C)
power (W)
AcOH (equiv)
3 (%)
4 (%)
5 (%)
6 (%)
1
2
3
4
5
113
207
234
470
196
overnight
rt
60
70
58
55
no
30
40
30
30
3
3
2
2
1
4
5
11
13
20
8
20
22
17
32
16
43
24
28
46
11
30
30
18
45
11
13
28
31
5
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Organic Letters
Letter
center is not readily accessible. These experimental findings
further illustrate the remarkable balance between steric and
17
electronic effects of ReSET (Figure 2).
In targeting naturally occurring 7 and 8, our plan was to use
2
2,23
methanolysis to deprotect the TMS silyl ethers first
and then
remove the benzyl ester. However, upon methanolysis, we
observed slow reaction times in addition to transesterification. To
avoid these complications, 3−6 were subjected to hydrogenation
to first remove the benzyl ester. Fortuitously, the TMS groups
were also deprotected under these conditions. While 3 and 4
readily reacted in a mixture of ethyl acetate, methanol and water,
analogues 5 and 6 were sluggish in this solvent system. It is known
that protic solvents enhance hydrogenation in comparison to
aprotic organic solvents (e.g., ethyl acetate, acetonitrile), which
can coordinate with the palladium metal reducing hydrogen
adsorption. The combination of 2-propanol and methanol led
to increased efficiency for TMS deprotection of 5 requiring only 4
h compared to 19 h when reacted in an ethyl acetate/methanol/
water mixture. With this global deprotection protocol, we
Figure 1. Key HMBC signals for characterization.
24
was most reactive. Upon introduction of the C acetate, silyl
4
exchange next occurred at the primary C , as evidenced by
9
formation of 4 on the TLC. Once the C acetate was introduced,
9
the C was acetylated in favor of exchange of the anomeric ether.
8
obtained the naturally occurring Neu4,5(Ac) (7) in 92% yield,
2
Thus, the order by which regioselective silyl exchange occurred
Neu4,5,9(Ac) (8) quantitatively, and Neu4,5,8,9(Ac) (9) in
3
4
was as follows: C (2°) > C (1°) > C (2°) > C (anomeric). The
4
9
8
2
88% yield (Scheme 2).
C-7 TMS ether did not exchange under these conditions (Figure
).
2
Scheme 2. Deprotection of TMS and Bn Groups
Figure 2. Preferred silyl ether/acetate exchange of Neu5Ac: C (2°) > C9
4
(
1°) > C (2°) > C (anomeric).
8 2
Neu5Ac ReSET revealed completely different regioselectivity
1
6,17
than previous work with pyranose sugars.
In aldohexoses, the
primary C typically exchanges first followed by the anomeric C .
6
1
After C exchange, C is usually next to react then further
In pursuit of the synthesis of Neu4,5,7,8,9(Ac)5 (15),
1
2
exchange occurs ina sequential manner around the pyranose ring.
Witschi and co-workers also performed ReSET on N-acetyl
glucosamine (GlcNAc), which is an aldose sugar structurally
similar to Neu5Ac in terms of bearing an NHAc group. In that
compound 4 was selectively deprotected to expose the C and
7
C diol (11, Scheme 3). The anomeric silyl protecting group
8
remained in tact presumably due to steric hindrance. Subjecting
11 to 1.5 equiv acetic anhydride gave selective acetylation of C7
(12), while excess acetic anhydride gave 13 (Scheme 3). Upon
hydrogenolysis of 12, acyl migration from the 7-O-acetyl to the C8
position occurred affording compound 9. Attempts to avoid
migration using various catalysts including palladium (98%),
palladium hydroxide, platinum(IV) oxide, and Raney nickel were
unsuccessful. C to C acyl migration occurred under all
case, the first exchange also occurred at the primary C rather than
6
16
the anomeric position, which was proximal to the amide.
The presence of NHAc in 2 presumably pulls electron density
from the C O−Si bond, which allows for exchange to occur first
4
at C in favor of the primary C position. Moreover, the presence
4
9
of methylene protons at C assures a less sterically hindered
3
7
8
environment than what is found in common pyranose sugars.
Once C is acetylated, C is the next to react. Again, the electronic
conditions, suggesting the C-8 acetate is a thermodynamic sink.
Meanwhile, 13 was subjected to hydrogenation to remove the
anomeric silyl and benzyl groups to afford naturally occurring 15
in 92% yield. This route allowed for an alternative synthesis of 15,
9
8
effect of the C ester group makes the C O−Si bond most
9
8
susceptible to attack. The observation of C exchange in favor of
8
25
the anomeric silyl ether group indicates that the quaternary
which had been previously synthesized.
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Organic Letters
Letter
Scheme 3. Alternative Synthetic Route to Neu4,5,7,8,9(Ac)5
AUTHOR INFORMATION
Corresponding Author
■
*
Fax: (530) 754-6915. Tel: (530) 754-9557. E-mail:
jgervayhague@ucdavis.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work is supported by the National Institutes of Health, NIH
Grant No. R01GM090262. NSF CRIF program (CHE -
■
9
808183), NSF Grant No. OSTI 97-24412, and NIH Grant
No. RR11973 provided funding for the NMR spectrometers used
on this project. We thank Dr. Jerry Dallas (University of
California, Davis) for help with the long-range HMBC NMR
experiments and 2D NMR experiments.
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ASSOCIATED CONTENT
■
*
S
Supporting Information
General experimental procedures, NMR, and mass spectra. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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