Chemical diversification of sialic acid glycosides by stereospecific, chemoselective deamination

Article abstract of DOI:10.1002/anie.201303781

Late bloomer: Nitrosation of peracetylated sialic acid glycosides followed by treatment with sodium trifluoroethoxide and then a suitable nucleophile enables the late-stage modification of these glycosides with stereospecific replacement of the acetamido

Full text of DOI:10.1002/anie.201303781

Angewandte
Chemie
DOI: 10.1002/anie.201303781
Sialic Acids
Chemical Diversification of Sialic Acid Glycosides by Stereospecific,
Chemoselective Deamination**
Chandrasekhar Navuluri and David Crich*
The sialic acids are nine-carbon sugars based on the 2-keto-3-
deoxy-d-glycero-d-galactononulosonic acids, whose a-glyco-
sides adorn the nonreducing termini of many N-glycoproteins
and gangliosides and are the monomeric units of the a-(2!
8)- and a-(2!9)-oligosialic acids.[1] The recognition of these
sialyl glycoconjugates by various lectins and the trimming of
sialyl residues by sialidase enzymes play important roles in
many human disease states, and have stimulated interest in
the synthesis of libraries of modified sialyl glycoconjugates
and their deployment in the search for improved diagnostics
and therapeutics.[1,2] The difficulties inherent in sialic acid
chemistry and particularly in the stereocontrolled synthesis of
a-sialosides[3] have, however, restricted the preparation of
such libraries to enzymatic approaches, which are limited by
the range of substrates accepted.[4] We describe here a method
for the chemical synthesis of sialyl glycoside libraries that
combines recent progress in stereocontrolled a-sialoside
synthesis[5] with a mild oxidative deamination process to
enable late-stage modification of preassembled glycosides,
thereby extending the range of accessible diversity.
Focused libraries of specific classes of oligosaccharides
and/or glycoconjugates are arguably best accessed by the late-
stage modification of preassembled substances, combining
synthetic efficiency with the ability to introduce targeted
diversity with a minimum of synthetic effort. This strategy
Scheme 1. Chemical synthesis of sialoside libraries with late-stage
requires a reliable, robust methodology for glycoside syn-
thesis and a mild, efficient methodology for their subsequent
modification, thus differing from the current enzymatic
cascade approach to sialyl glycoside libraries, which demands
the synthesis of a different precursor for every eventual
member.[4] The introduction of the 4-O,5-N-oxazolidinone-
protected sialyl donors and their more readily deprotected N-
acetyl derivatives has removed many of the obstacles of
chemical a-sialoside synthesis (Scheme 1, upper part),[5]
thereby opening the door to the modification of sialyl
glycosides as a means of entry into libraries, provided that
suitably mild conditions for the subsequent modification can
be identified. We reasoned that the modified oxidative
deamination of neuraminic acid glycosides described by
Schreiner and Zbiral would be a suitable reaction for such
modifications if conditions could be found to extend the range
modification of preassembled glycosides.
of nucleophiles (NuH) beyond the previously employed
systems based on acetic and hydrazoic acid (Scheme 1,
lower part).[6]
Oxidative deamination of the peracetylated N-acetylneur-
aminic acid (NeuAc) methyl ester was achieved by Schreiner
and Zbiral with nitrosyl acetate, giving the N-nitroso
adduct,[6a] whereas in our laboratory we prefer to use the
more convenient, commercial nitrosyl tetrafluoroborate
(NOBF4) for this purpose.[6b] Subsequent steps involve
selective removal of the acetyl group from the N-nitro-
soacetamide with sodium trifluoroethoxide to give a diazo
derivative of NeuAc that is then substituted by the incoming
nucleophile. Participation by the 4-O-acetate is invoked to
explain both the regio- and stereoselectivity of the process.[6a]
As NOBF4 is known to activate thioglycosides toward
glycosylation,[7] we tested the compatibility of the oxidative
deamination conditions with thioglycosides. In the event,
treatment of a solution of thioglycoside 6 in dichloromethane
with NOBF4 in the presence of pyridine (Py) at ꢀ108C gave
a solution of the presumed N-nitroso derivative 7, which was
briefly exposed to trifluoroethanol (TFE) and sodium iso-
propoxide in isopropanol at the same temperature before
[*] Dr. C. Navuluri, Prof. Dr. D. Crich
Department of Chemistry, Wayne State University
5101 Cass Avenue, Detroit, MI 48202 (USA)
E-mail: dcrich@chem.wayne.edu
[**] We thank the NIH (GM 62160) for support of this work, and Dr. P. K.
Kancharla for experimental assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201303781.
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addition of either acetic acid, thioacetic acid, or the hydrogen
fluoride/pyridine complex, resulting in the formation of the 5-
desacetamido acetoxy, acetylthio, and fluoro NeuAc deriva-
tives 8, 9, and 10, respectively, in good yield. Fluoride 10 was
however accompanied by 11% of the isopropoxy analogue 11
(Scheme 2).[8] The survival of the thioglycoside moiety is
a result of the use of pyridine, which captures the nitrosonium
ion as the N-nitrosopyridinium complex[9] 12 (Scheme 2) and
moderates its reactivity. While the formation of the 2-keto-3-
deoxy-d-glycero-d-galactononulosonic acid (KDN) deriva-
tive 8 recalls earlier studies[6] with acetic acid as nucleophile,
the formation of the acetylthio and fluoro derivatives
demonstrates the ability of the method to incorporate
a wider range of nucleophiles. The formation of the isopropyl
ether 11 as by-product alongside fluoride 10 could be avoided
by using preformed sodium trifluoroethoxide as base in
conjunction with 18-crown-6 ([18]C-6) and excluding isopro-
panol from the reaction mixture, giving fluoride 10 in 63%
yield. The formation of 11 as by-product also suggested that
conditions could be found for the use of simple alcohols as
nucleophiles in the oxidative deamination protocol. Thus,
after some experimentation we found that deacetylation with
sodium trifluoroethoxide, followed by the addition of prop-
argyl alcohol and tetrafluoroboric acid to protonate the
intermediate diazo compound, gave the propargyl ether 13 in
66% yield (Scheme 2). Next, a series of a-sialosides was
prepared from the donor 1 on activation with N-iodosucci-
nimide (NIS) and trifluoromethanesulfonic acid (TfOH) in
a mixture of dichloromethane and acetonitrile at ꢀ788C[5c]
(Table 1, entries 1–5). Alternatively, for more complex
acceptors based on the thioglycoside motif, the Wong sialyl
phosphate type donor[5e] 14 was employed with activation by
trimethylsilyl trifluoromethanesulfonate at ꢀ788C in
dichloromethane and acetonitrile (Table 1, entries 6 and 7).
In each case, the oxazolidinone then was removed under
Zemplen conditions and any acetate esters reinstalled with
acetic anhydride and pyridine (Scheme 1 and Table 1). Nitro-
sylation was achieved with NOBF4 in the presence of pyridine
and the intermediate N-nitrosoamides were substituted under
the conditions reported in Table 1.
We applied the protocol to the synthesis of KDN glyco-
sides incorporating a(1!6)-galactopyranoside, a(1!6)-glu-
copyranoside, and a(1!3)-galactopyranoside linkages, and
demonstrated the compatibility of the protocol with glyco-
sidic bonds and benzyl ethers (Table 1, entries 1–3). The
employment of thioacetic acid as nucleophile provided the
novel disaccharide 28 (Table 1, entry 4), which may be viewed
either as a 5-acetylthio-5-desacetamido derivative of a NeuAc
glycoside or as a 5-acetylthio-5-deoxy analogue of a KDN
glycoside. As selective deacetylation of thioacetates in the
presence of acetates is facile, thioacetate derivatives such as
28 enable further regioselective diversification at the 5-
position by alkylation of thiols and formation of disulfides, or
by the thiol-ene and yne click[10] and other processes.[11] As 5-
mercapto analogues of NeuAc and/or KDN have not
previously been accessible through the use of sialyl trans-
ferases,[4b,c] this example extends the range of accessible a-
sialoside derivatives modified at the 5-position and demon-
strates the versatility of the chemical approach. The forma-
tion of a 5-fluoro derivative of NeuAc and/or KDN is
illustrated in entry 5 (Table 1). The applicability of the
method to thioglycoside-containing substrates, whether of
the arylthio or alkylthio classes, is reiterated in entries 6 and 7
(Table 1). The possibility of diversification at the 5-position of
the sTn antigen (Table 1, entry 6) is especially noteworthy as
it has been previously demonstrated that modification of this
antigen at the amide can lead to analogues that display
improved antigenicity.[12] The oxidative deamination may be
applied concomitantly to two sialic acid residues in an a(2!
9)-linked disaccharide (Table 1, entry 7), representing the
first chemical synthesis of a KDN disaccharide of this class.
Such modifications of polysialic acids are of interest in view of
recent approaches to a(2!9)-linked polysialic acids because
of their potential as antibacterial agents and synthetic
vaccines.[13]
Scheme 2. Compatibility with a thioglycoside and introduction of acetoxy, acetylthio, and fluoride groups.
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Table 1: Stereoselective synthesis of a-sialosides and oxidative deamination.
Product of
glycosylation
Product of
oxazolidinone removal
Nucleophile
(conditions)
Product of
substitution
AcOH
(TFE, NaOiPr)
1
2
3
15, 81%, a-only from 1
16, 84%, a- only from 1
20, 77%
21, 88%
25, 51%
26, 48%
AcOH
(TFE, NaOiPr)
AcOH
(TFE, NaOiPr)
17, 83%, 10:1 b:a, from 1
22, 76%
27, 54%
28, 67%
29, 63%
AcSH
(TFE, NaOiPr)
4
5
17
22
HF·NEt3
(NaTFE,
[18]C-6)
17
22
AcOH
(TFE, NaOiPr)
6
7
18, 86%, a- only, from 14
23, 91%
30, 49%
31, 42%
AcOH
(Bu4NOAc, TFE, NaOiPr)
19, –,[a] from 14
24, 74%, a-only[b]
[a] Not isolated. [b] Overall yield from 14. Ada=adamantyl, Bn=benzyl, Phth=phthaloyl, R=CH(OAc)CH(OAc)CH2OAc.
anhydrous pyridine (10 equiv) and cooled to ꢀ108C. After stirring for
10 min, powdered NOBF4 (4 equiv) was added in one portion to the
mixture. The resultant light-green solution was stirred at ꢀ108C for
3 h, then diluted with CH2Cl2, and washed sequentially with cold HCl
(1n), cold saturated aqueous NaHCO3, and brine. Drying (Na2SO4)
and concentration below 108C gave the nitrosated sialosides, which
were carried forward without further purification.
General procedure for deamination with acetic or thioacetic acid
as nucleophile: A solution (0.1m) of the nitrosyl sialoside and
trifluoroethanol (1.5 equiv) in anhydrous CH2Cl2 under Ar was
treated at ꢀ108C with freshly prepared sodium isopropoxide in
isopropanol (0.2n, 1.2 equiv). The resulting mixture was stirred for
exactly 2 min, then treated with a cold solution of glacial acetic acid
(20 equiv) or thioacetic acid (20 equiv) in CH2Cl2 (1m). After stirring
for 5 min, the reaction mixture was warmed to 08C and quenched
with saturated aqueous NaHCO3. The organic layer was washed with
cold brine, dried (Na2SO4), concentrated, and purified by column
chromatography on silica gel (eluent: hexane/ethyl acetate = 1:1) to
afford the deaminated sialosides.
Application of the oxidative deamination protocol to
a GM3 trisaccharide and a branched tetrasaccharide is
demonstrated beginning from trisaccharide 32, acetylation
of which gave the derivative 33, while glucosylation afforded
the tetrasaccharide 34 (Scheme 3). Both substances were
treated with NOBF4 and pyridine and then exposed to sodium
trifluoroethoxide followed by acetic acid, giving the KDN
analogues 35 and 36, respectively, in good yield.
The combination of N-acetyloxazolidinone-directed a-
sialidation with oxidative deamination provides rapid entry to
NeuAc and/or KDN analogues modified at the 5-position.
Suitable nucleophiles for this process include acetate, thio-
acetate, fluoride, and simple alcohols. The ability to chemi-
cally synthesize sialic acid glycosides in this manner and then
affect late-stage modification is potentially very flexible and
should enable access to many derivatives with a minimum of
synthetic effort. Modifications are not limited to those
incorporated in substrates for sialyl transferases.
General procedure for deamination with HF and alcohol
nucleophiles: 18-Crown-6 (0.22 mmol) and sodium 2,2,2-trifluoroeth-
oxide (54 mg, 0.2 mmol) were dissolved in anhydrous CH2Cl2
(0.5 mL) under Ar, cooled to ꢀ108C, and added to the nitrosyl
sialoside (0.1 mmol) in anhydrous CH2Cl2 (1 mL) at ꢀ108C under Ar.
After 2 min, either the alcohol (2 mmol in 2 mL CH2Cl2) immediately
followed by HBF4·Et2O (0.4 mmol) or HF·pyridine (2 mmol) was
Experimental Section
General procedure for nitrosation of sialosides: A stirred solution
(0.1m) of sialoside in anhydrous CH2Cl2 under Ar was treated with
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Keywords: deamination · diversification · glycosylation ·
.
nitrosation · sialic acids
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Scheme 3. Application of the oxidative deamination reaction to a GM3
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added to the reddish reaction mixture. The mixture was stirred for
5 min, diluted with CH2Cl2 (5 mL), and then quenched with saturated
aqueous NaHCO3 (5 mL). The organic layer was washed with brine
(5 mL), dried (Na2SO4), concentrated, and purified by column
chromatography on silica gel (eluent: hexane/ethyl acetate = 1:1) to
afford the deaminated sialosides.
Received: May 2, 2013
Revised: July 17, 2013
Published online: && &&, &&&&
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Sialic Acids
C. Navuluri, D. Crich*
&&&& — &&&&
Chemical Diversification of Sialic Acid
Glycosides by Stereospecific,
Chemoselective Deamination
Late bloomer: Nitrosation of peracety-
lated sialic acid glycosides followed by
treatment with sodium trifluoroethoxide
and then a suitable nucleophile enables
the late-stage modification of these gly-
cosides with stereospecific replacement
of the acetamido group. This method
should enable access to many glycoside
derivatives with a minimum of synthetic
effort.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!