Facile and robust methods for the regioselective acylation of: N -acetylneuraminic acid

Article abstract of DOI:10.1039/c8nj02795a

The stereoselective synthesis of sialic acid glycoconjugates is still a challenge in the field. Surprisingly, little is known on the regioselective O-substitution of sialic acids. Consequently, the effect of O-protecting groups and/or regioselectively pro

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The role of atropisomers on the photo-reactivity and fatigue of
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Facile and robust methods for the regioselective acylation of N-acetylneuraminic acid
Melanie Shadrick, Charlene Yu, Scott Geringer, Sean Ritter, Alexanndra Behm , Abby Cox,
Matt Lohman, and Cristina De Meo *
Department of Chemistry Southern Illinois University Edwardsville
1 Hairpin Dr., Edwardsville, IL 62025, USA
Abstract
The stereoselective synthesis of sialic acid glycoconjugates is still a challenge in the field.
Surprisingly, little is known on the regioselective O-substitution of sialic acids. Consequently, the
effect of O-protecting groups and/or regioselectively protected building blocks in sialylations,
remains practically unexplored. O-Picoloyl protecting groups have emerged as novel substituents
that have a profound effect on sialylations. Recently, high stereoselectivities were obtained by
introducing picoloyl groups at the C-4 and C-7/C-8 positions. However, to understand the
relationship between the position of the picoloyl group and its exact effect in sialylations, a
convenient access to a wider range of regioselectively picoloylated building blocks is needed.
Reported herein is a new method that provides an accessible route to a wide array of
regioselectively acylated building blocks. The regioselective introduction of picoloyl groups at
various O-positions was achieved either by controlled direct picoloylation or by applying a
modified ReSET methodology.
Keywords: sialic acid, N-acetylneuraminic acid, regioselective acylation, protecting groups
Introduction
N-Acetylneuraminic acid is the most common member of the sialic acid family, as derivatives
of neuraminic acid. The biological functions of this unique carbohydrate are numerous, from being
mediator of influenza cell attack, to being essential in the development of embryonic cells.1,2 The
studies of efficient methods to synthesize sialic acid containing glycoconjugates have made a
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tremendous improvement for the synthesis of these unique molecules.3-5 However, when
comparing sialic acid chemistry to more investigated monosaccharides such as glucose and
galactose, it is clear that sialylations are still very challenging, and not so much is known on the
effects of O-protecting groups at C-4 and the glycerol chain. Protecting group manipulations are
the essential core of carbohydrate synthesis, with their effect not just limited to ‘protect’, but also
to influence the reactivity and stability of the carbohydrate moiety in glycosylation reactions.
Methodologies centered on discoveries or improvements in protecting groups for carbohydrates
have been the focus of research for many decades.6 Development of regioselective methods to
protect strategic hydroxyl groups have been having high priority. However, in sialic acid chemistry,
the only exploited investigation has been related to the N-5 position.7,8 The lack of an extensive
study of O-protecting groups limits the methodologies offered in sialylation reactions. Recently,
we reported the effect of C-4 protecting groups such as tert-butyl dimethyl silyl (TBDMS)9 and
picoloyl (Pico).10 For the latter, we proved a synergistic effect between Picoloyl group and triflic
acid in controlling the stereoselectivity of the reaction. More recently, a C-7,8-di-Pico sialyl donor
showed excellent stereocontrol in sialylations.11 To the growing importance of picoloylated
building blocks, there is also a lack of straightforward methodologies for their regioselective
introduction. Little is known of how the number of picoloyl groups or their relative positions might
influence the general outcome of sialylations. As mentioned above, the field itself lacks a
comprehensive investigation of O-protecting group manipulations, in part due to the structural
complexity of N-acetylneuraminic acid. Regioselective acetylations have been in general
characterized by several synthetic steps and protecting group manipulations12,13 On the other side,
regioselective benzoylations are easier to be performed,14 however benzoyl groups seem to be
deactivating and β-directing.9 Herein we disclose the synthesis of an array of novel
regioselectively functionalized building blocks bearing picoloyl groups at several positions. The
methodologies investigated herein can also be extended to other protecting groups.
In order to accomplish this task, we decided to investigate two main strategies depicted in
Scheme 1. Following the general trend of relative reactivity of hydroxyl groups, it might be
possible to achieve controlled acylation of polyol 1 that would lead to a C-9 monoacylated product,
followed by a diacylated product at C-4,9 (Strategy 1, Scheme 1). On the other hand, Gervay’s
group reported a C4/9 inverted reactivity upon acetylation of persilylated sialyl moiety.15 The
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methodology, called Regioselective Silyl Exchange Technology (ReSET) gave on microwave
assisted reaction a reactivity sequence as C4>C9>C8>C7, and moderate yields of 28% (C-4 acetyl);
31% (C-4,9 diacetyl) and 32% (C-4,8,9 triacetyl). Hence, we decided to investigate if a silyl-acetyl
exchange reaction from 2 can also be feasible without the assistance of microwave and still lead
to similar order of reactivity and yields (Scheme 1, Strategy 2).
Scheme 1. Two major strategies for regioselective acylations
Results and Discussion
Strategy 1: Synthesis of 9-pico and 4,9 dipico thiosialosides
For our first regioselective picoloylation from polyol 1, we targeted O-9 mono-picoloylated
derivative 3 as product. A one-step acylation directly of C-9 is very appealing, and would
dramatically cut many synthetic steps. Our previous picoloylation of sialic acid was done in the
presence of N,N’-Dicyclohexylcarbodiimide (DCC) or 1-Ethyl-3-(3-dimethylaminopropyl
carbodiimide (EDC), depending on the reactivity of the substrate and the positions targeted, while
dichloromethane was the solvent of choice. In the presence of a 4,7-diol, DCC was able to
introduce the Pico group at both positions, while EDC only introduced to the most reactive position,
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i.e. C-4.10 Thus, we decided to test both coupling agents, and to use pyridine as the solvent of
choice, due to the poor solubility of 1 in dichloromethane. Moreover, using pyridine would offer
a better regiocontrol by being an acid scavenger and slowing down the reaction, as well as giving
the opportunity to acetylate the picoloylated product 3 in situ. Thus, the treatment of polyol 1 with
picolinic acid and DCC as the coupling reagent gave a mixture of 9-monopicoloylated product 3
as well as the 4,9-dipico product along with some unreacted starting material. Upon purification,
the desired product was isolated with yields ranging from 40 to 50%. Higher yield was observed
when EDC was used as the coupling reagent, resulting in a complete consumption of the starting
material and a commendable yield of 82% of the desired target 3 (Scheme 2). We believe that the
observed difference between the two coupling agents (EDC and DCC) is mainly due the onset of
side reaction from DCC that limit this reagent, unless DMAP is used as coactivator of the acyl
group. Acetylation of 9-Pico derivative 3 gave sialoside 4 in 75% yield. The synthesis of
thiosialoside 4 was therefore accomplished in only two steps. Our previous synthesis of the same
building block comprised six steps of protecting groups manipulations, including a C7-to-C9 acyl
migration (unpublished results).
Encouraged by these results, we proceeded in testing the feasibility of a C-9 picoloylation
followed by an in-situ acetylation. Thus, upon complete conversion of 1 into product 3, acetic
anhydride was added and the reaction was stirred overnight. TLC analysis of the crude showed
completion of the reaction and after column chromatography, the desired product 4 was obtained
in 52% yield over two steps (Scheme 2). This yield is comparable with the individual picoloylation
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and acetylation previously reported, and therefore can be a valuable option when acetyl or similar
group are planned to be introduced.
We then proceeded to investigate whether di-picoloylated target 5 can also be straightforwardly
synthesized from the common polyol precursor 1 (Scheme 3). In this case, EDC-mediated reaction
resulted in a sluggish, partial addition of picoloyl in position C-4. Conversely, treatment of polyol
1 with picolinic acid in the presence of DCC and DMAP, gave the desired target 5. Typical for
all DCC-mediated reactions, complete removal of side products required the development of
several troubleshooting scenarios and combinations of thereof. Finally, we settled upon the
precipitation from dichloromethane and filtration that allowed to remove DCC and its side
products. The filtrate was then separated by column chromatography to yield 5 in 75% yield.
Acetylation of 5 gave thiosialoside 6 in 72% yield (Scheme 3). Similarly to its mono-picoloylated
precursor, in situ 4,9 di-picoloylation of 1 followed by acetylation was successful, yielding
compound 6 in 75% yield (Scheme 3).
Strategy 2: Synthesis of 4-Pico, 7-Pico and 7,8-di-Pico thiosialosides.
As mentioned above, microwave-assisted acetylation of a polysilylated precursor, showed a rather
unusual order of regioselectivity, with the C-4 position being more reactive than the C-9.15 To
investigate the suitability of this method for obtaining our targets, we synthetized thiosialoside 2
by per-silylation of polyol 1. Thus, the treatment of 1 with trimethylsilyl chloride in the presence
of hexamethyldisilazane afforded the desired compound 2 in 69% yield (Scheme 4). It should be
noted that this yield is associated with a loss of material during the purification step, due to the
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lability of the TMS group. To minimize the losses, in subsequent syntheses of this building block,
no purification was performed, and crude per-silylated compound 2 was used directly.
To test the feasibility of regioselective acetylations, and to maximize the yields of each acetylated
product, several concentrations of acetic anhydride and DMAP were tested (Table 1, see Scheme
4 for chemical structures). The ratio between the two reagents and the reaction time were critical.
Thus, in the presence of 10 equiv. of acetic anhydride and 0.1 equiv. of DMAP, it was possible to
obtain C-4 monoacetylated thiosialoside 7 with a yield of 48% (entry 1, Table 1). By decreasing
the excess of acetic anhydride while concomitantly increasing the amount of DMAP it was
possible to synthesize 4,9-diacetylated compound 8 in 57% yield (entry 2).
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Table 1
Entry*
Ac2O eq
DMAP eq.
Yield %
Yield %
Yield %
Yield %
2
7
8
9
10
5
0.1
0.2
0.2
15
33
-
1
2
3
48
-
-
7
-
33
57
63#
20
22
* All the reactions were performed at rt, except entry 3, which required higher temperature (40 C);
recovered starting material
#peracetylated thiosialoside was also recovered in 14% yield.
Removal of the remaining TMS groups in 4,9-diacetyl 8 under mildly acidic conditions gave
intermediate 10, which was then picoloylated at positions C-7,8 to yield thiosialoside 11 in 67%
yield. The same target can also be obtained in comparable yields by performing regioselective
acetylation of 1 at C-4,9 followed by 7,8-picoloylation (Scheme 5).
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Acetylation of position C-8 to obtain 4,8,9-triacetate 9 from precursor 2 required a much larger
amount of acetic anhydride and an increase in temperature (40 °C). As a result of this strategic
adjustment, compound 9 was obtained in 63% yield (Table 1, entry 4). Similarly, the removal of
the remaining TMS group in 9 gave intermediate 12 in 82% yield. The introduction of picoloyl
was then performed, and 7-picoloylated sialoside 13 was obtained in 92% yield (Scheme 6).
To expand this methodology to other acyl groups, we tested if it was possible to introduce a
picoloyl group at C-4 directly from the persilylated precursor 2. Our previous synthesis of a C-
4 picoloylated thiosialoside is described in Scheme 7a. It required several protecting group
manipulations in order to regioselectively introduce picoloyl at C-4. When persilylated compound
2 was reacted with picolinic acid in the presence of EDC as coupling reagent, the C-4 picolylated
thiosialoside 14 was successfully obtained in 62% yield (Scheme 7b). Acetylation of 14 gave the
direct access to thiosialoside 16 in 77% yield, shortening the number of steps required for its
synthesis.10
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a) Org. Lett. 2017, 19, 2638-2641
O
c
O
O
CO2Me
SPh
O
CO2Me
SPh
a
2
HO
AcHN
HO
AcHN
O
b
O
87%
PicoO
HO
HO
HO
OH
CO2Me
SPh
O
AcHN
PicoO
b) This work
c (67% over 2 steps)
CO2Me
TMSO
TMSO
AcHN
OTMS
CO2Me
SPh
AcO
OAc
a
c
O
2
AcO
AcHN
SPh
O
77%
62%
PicoO
14
PicoO
16
a) Picolinic acid, EDC, py, 62%; b) Dowex, H+, MeOH; c) Ac2O, Py, rt
Scheme 7
Conclusions
A series of mono- and di-picoloylated thiosialosides was successfully synthesized by
regioselective acylations (picoloylations and acetylations) of polyol and persilylated precursors.
The methodology disclosed herein allows for obtaining valuable synthetic targets in a minimal
number of steps and, in some cases, in one pot. For instance, C-9 picoloylated thiosialoside 4 was
obtained in a two-step reaction, vs. the five-step reaction that was previously needed for this target.
The method also allows for the regioselective synthesis of 4,9-diacetylated and dipicolylated
building blocks, which are not accessible by other synthetic routes. We are confident that the
methodology herein described can be also expanded to other acyl groups.
ACKNOWLEDGMENT
This work was supported by the National Science Foundation (NSF, CHE-RUI#1465003) and by
the National Institutes of Health (NIH, GM120673). Dr. Winter (UM – St. Louis) is thanked for
HRMS determinations.
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