Site-Selective Acylations of α- And β-Hydroxyamides in Complex Molecules: Application of Template-Driven Acylation to Disaccharides and a Glycopeptide

Article abstract of DOI:10.1021/acs.orglett.1c00612

Site-selective acylations of α-and β-hydroxyamides in complex polyols are described. The combination of a pyridine aldoxime ester and Zn(OTf)2 facilitates the acylation of two types of N-glycolyl disaccharides, namely, Gal-GlcNGc and Neu5Gc-Gal, both of which are partial structures of polysaccharides responsible for biological actions, with highly site-selective modifications achieved. Furthermore, biotinylation, one of the most important techniques in chemical biology, is used to site-selectively acylate the β-hydroxyl group in a glycopeptide.

Full text of DOI:10.1021/acs.orglett.1c00612

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Letter
Site-Selective Acylations of α- and β‑Hydroxyamides in Complex
Molecules: Application of Template-Driven Acylation to
Disaccharides and a Glycopeptide
Yasuhiro Nishikawa,* Shione Toda, Takami Matsui, Hanae Takada, Kohei Takemoto, and Osamu Hara*
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ABSTRACT: Site-selective acylations of α-and β-hydroxyamides in complex
polyols are described. The combination of a pyridine aldoxime ester and Zn(OTf)2
facilitates the acylation of two types of N-glycolyl disaccharides, namely, Gal-
GlcNGc and Neu5Gc-Gal, both of which are partial structures of polysaccharides
responsible for biological actions, with highly site-selective modifications achieved.
Furthermore, biotinylation, one of the most important techniques in chemical
biology, is used to site-selectively acylate the β-hydroxyl group in a glycopeptide.
β-hydroxyamides 1β, probably because the methyl group in 2β
arbohydrates and glycosides are important biological
Cmolecules involved in dynamic biological phenomena, (R3 in Figure 1A) amplifies the proximity effect by positioning
the ester moiety toward the targeted hydroxyl group in the
transition state.6 In nature, the α-hydroxyamide structure has
been observed in a variety of N-glycolylsaccharides.7 For
example, N-glycolylneuraminic acid (Neu5Gc), a representa-
tive sialic acid analogue, is not expressed at significant levels on
healthy human tissue but is expressed on tumor tissue as
glycans; hence Neu5Gc is a potential biomarker for the early
diagnosis of cancer and cancer therapy.8,9 Whereas we
examined the site-selective chemical modifications of N-
glycolyl monosaccharides, there were insufficient examples
that demonstrated the utility of our system (Figure 1B).5
Complex polyols, such as natural polysaccharides, can exhibit
unpredictable behavior that interferes with functionalization,
which is ascribable to the disposition of the hydroxyl groups in
the steric environment, including higher order structures.
Developing a robust site-selective modification method
requires insight gained by examining new methodologies in
complex systems. Herein we report the metal-template-driven
acylations of more complex polyol compounds, namely,
disaccharides for α-hydroxyamides and an N-linked glycopep-
tide for a β-hydroxyamide.
including various intercellular signal transduction processes on
the surfaces of cells.1 The key structural feature of this
carbohydrate family is the polyol function, which plays
complex biological-activity roles through hydrogen-bonding
interactions. The ability to chemically modify specific hydroxyl
groups in the polyol structure is a powerful tool for exploiting
the functions of this class of compound for biological research
and pharmaceuticals.2 In synthetic chemistry, however, the
desirable site-selective manipulation of a specific functional
group among multiple functional groups has been a formidable
challenge that often requires protection−deprotection sequen-
ces. Therefore, much effort has been dedicated to achieving
direct methods for the site-selective functionalization of
nonprotected polyols.3 Whereas most methods have been
developed for the selective functionalizations of relatively
simple molecules, such as diols or triols, their applications to
more complex molecules, such as natural products and
bioactive compounds, are quite limited.4 As a pioneering
work in this field, Miller and coworkers reported the
regioselective acylations of erythromycin A with peptide-
based organocatalysts.4h As another example, Kawabata and
coworkers reported the regioselective acylation of digitoxin
with a chiral DMAP-based catalyst.4f In this context, we
successfully site-selectively acylated α- and β-hydroxyamides in
polyols 1α and 1β by combining Lewis acids and 2-pyridyl
oxime esters 2 as mild acylating reagents (Figure 1A).5 The
formation of a metal complex preferentially coordinated by two
bidentate molecules, namely, α- and β-hydroxyamide 1 and 2,
facilitates the selective acylation of 1 in preference to other
hydroxyl groups due to the proximity effect. Methyl-
substituted Cu(I)−ketoxime ester complexes effectively acylate
In previous studies, N-glycolylglucosamine (1a) and N-
glycolylneuraminic acid (1b) were acylated in contrasting
chemical yields (Figure 1B). Prior to investigating the site-
Received: February 20, 2021
Published: March 18, 2021
https://doi.org/10.1021/acs.orglett.1c00612
© 2021 American Chemical Society
Org. Lett. 2021, 23, 2715−2719
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a
Scheme 1. Control Experiments
a
Yields were determined by 1H NMR analysis of the unpurified
b
reaction mixtures. Isolated yield.
Figure 1. Site-selective acylations of α- and β-hydroxyamides using
the metal-template strategy.
Scheme 2. Site-Selective Acylation of Disaccharide 1g
selective acylations of disaccharides with the N-glycolylglucos-
amine and N-glycolylneuraminic acid units, which are partial
polysaccharide structures, we examined the origin of the low
yield observed during the acylation of 1b to gain insight into
the reaction mechanism in our system. To evaluate the effect of
the 1,2,3-triol in 1b, we carried out a control experiment using
α-hydroxyamide 1c and glycerol as a model 1,2,3-triol
(Scheme 1a). An intermolecular competition reaction using
1.5 equiv of the acylating reagent 2a in the presence of 0.2
equiv of Zn(OTf)2 resulted in the complete acylation of 1c to
afford 3cA in 97% yield along with small amounts of 3dA and
3dB, which reveals that the α-hydroxyamide is much more
rapidly acylated than the 1,2,3-triol. On the contrary, a similar
control experiment, in which α-hydroxyacetic acid was used
instead of glycerol, led to the incomplete acylation of 1c (51%
yield; Scheme 1b).10 These results indicate that the low yield
obtained from the acylation of 1b is ascribable to the presence
of the carboxylic acid moiety, probably due to ionic
interactions with Zn(OTf)2 that inhibit the formation of the
desired metal template. In fact, the direct acylation of the
methyl ester 1f of N-glycolylneuraminic acid (1b) under
otherwise identical conditions produced monoacylate 3fA in
higher yield than the product obtained by acylating 1b
(Scheme 1c).
α-hydroxyl group during acylation. Accordingly, we inves-
tigated the acylation of 1g under slightly modified standard
1
conditions by analyzing the reaction mixture by H NMR
1
spectroscopy. The H NMR spectrum of the reaction mixture
was almost identical to that of pure 3gA, which indicates that
the acylation of 1g proceeded in a highly site-selective
manner.12 Overall, monoacylated 3gA was obtained in 63%
isolated yield after high-performance liquid chromatography
(HPLC) purification. This result suggests that the reactive
primary alcohols in the two carbohydrates do not interfere
with our α-acylation method.
We next demonstrated the site-selective acylation of
Neu5Gcα(2,6)Galβ methoxyphenyl glycoside (1h), a com-
mercially available disaccharide containing the Neu5Gc core
(Figure 2A). 1h corresponds to the partial structure of the
oligosaccharide attached to cetuximab, an epidermal growth
factor receptor inhibitor and monoclonal antibody produced
by murine myeloma cells, in which the terminal Neu5Gc in the
glycan is a potential immunogen.13 The site-selective chemical
Having obtained complementary results that reveal the
preferences of the substrates in our system, we turned our
attention to site-selectively acylating disaccharides. First,
disaccharide 1g, an N-glycolyl equivalent of Galβ(1−3)-
GlcNAc, the partial structure of the terminal oligosaccharides
in the blood group antigen, was prepared from a commercially
available disaccharide (Scheme 2).11 The site-selective
acylation of 1g was envisaged to be challenging because the
six other hydroxyl groups in 1g can compete with the targeted
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This template strategy was further applied as a site-selective
method for the acylation of β-hydroxyamides because the β-
hydroxyl groups in amides are very attractive targets for
chemical modification, as evidenced by the side chains of Ser
and Thr residues in peptides.14,15 The introduced acylating
motif, which modifies the molecule, must suit the requirements
for further applications, which is another important issue. We
focused our attention on the biotinylation chemical mod-
ification using our strategy because it is one of the most
common techniques used to conjugate a range of small
molecules and proteins for purification, detection, and various
biological assays that utilize strong interactions between
streptavidin and biotin.16,17 To the best of our knowledge,
biotin has yet to be site-selectively introduced in organic
chemistry, which is probably due to its hydrophilicity that
potentially inhibits site-selective control through hydrogen-
bonding interactions and Lewis-acid activation. Accordingly, a
new pyridylketoxime-type reagent, 2b, was prepared for site-
selective biotinylation.
We first applied the optimized reaction conditions used for
the acylation of glycopeptide 1i to the reaction of 2b, which led
to a low conversion of 1i and a 16% yield of the
monobiotinylated product 3iA with moderate site selectivity
(Table 1, entry 1).5b The acylation of 1i using a ketoxime
derivative of 2a under otherwise identical conditions provided
the corresponding acylate in 29% isolated yield; this low yield
is possibly ascribable to the chelating properties of the
tetrahydrothiophene and imidazolidone cores in biotin that
Table 1. Site-Selective Biotinylation of Glycopeptide 1i
Figure 2. (A) Site-selective acylation of disaccharide 1h containing
the Neu5Gc core. (B) Extracted-ion chromatogram of the reaction of
1h.
modifications of Neu5Gc-containing oligosaccharides may lead
to significant substrate-selective transformations for separation,
detection, and chemical modification. Although we expected a
low chemical yield from the acylation of 1h bearing a
carboxylic acid moiety, conditions that resulted in incomplete
acylation in the control experiment, we believed that the site-
selective modification of this class of substrate is highly
valuable, even in low yield, because accomplishing this is
challenging using the currently available methods. Disaccharide
1h was acylated under the same conditions used to acylate N-
glycolyl neuraminic acid (1f) to give 3hA. Preliminary HPLC
analysis of the reaction solution revealed low conversion of 1h,
and the NMR spectrum of the reaction mixture was unable to
provide site-selectivity information due to its complexity.
Accordingly, we used LC-MS followed by analysis of the
extracted ion chromatogram (XIC), which is a powerful tool
for detecting isomers bearing the same exact mass as the target
product that can reveal the site selectivity of the reaction.
Surprisingly, the XIC of the monoacylated product ([M + H]+
m/z 724.2458, red trace, Figure 2B), which is highly mass
accurate (<3 ppm), revealed the presence of essentially one
product. In addition, very small peaks corresponding to
diacylated products ([M + H]+ m/z 856.3033, blue trace)
were detected in the XIC, confirming that the reaction
proceeded in a highly site-selective manner. The prepared
monoacylated product was isolated by preparative HPLC using
almost the same gradient-elution method used during LC-MS
and was unambiguously determined to be 3hA by 2D NMR
spectroscopy.12
a
a
conc.
yield (%)
selectivity
b
entry CuOTf·1/2 toluene
(mM)
3iA
(3iA/isomers )
1
2
3
4
5
6
0.5 equiv
0.5 equiv
1.0 equiv
1.0 equiv
1.0 equiv
100
25
25
5
16
22
21
62:38
67:33
64:36
72:28
72:28
35:65
c
38 (31)
d
c
5
− (28)
f
conventional conditions
11
a
Yields and selectivities were determined by HPLC with UV
detection using an internal standard. Monobiotinylated products.
Numbers in parentheses refer to isolated yields. Reaction was
performed at 40 °C. Reaction was performed with 1i and biotin N-
hydroxysuccinimide ester (1.2 equiv) in pyridine at 40 °C for 67 h.
See the SI for details.
b
c
d
f
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can hamper the formation of the appropriate metal template
between 1i, 2b, and CuOTf. During optimization of the
reaction conditions, we observed that dilution to 25 mM
slightly improved the yield of 3iA (entry 2). On the contrary,
increasing the amount of CuOTf did not affect the yield, which
was unexpected (entry 3). The reaction outcome was further
improved under highly dilute conditions, which produced 3iA
in 38% yield with small amounts of monobiotinylated isomers
and dibiotinylated byproducts (entry 4). We previously
reported that DMA was the best solvent in terms of both
the yield and the site selectivity. We reasoned that DMA, as a
chelating substrate, engages in the dynamic chelation−
dissociation process on the Cu center, with a β-hydroxyamide,
a ketoxime ester, and the ketoxime leaving after acylation. As a
result, the excess DMA present under dilute conditions may
provide opportunities to construct the desired metal complex
comprising the β-hydroxyamide and the ketoxime ester by
ejecting a ligand on the undesired but relatively strong metal
complex, thereby facilitating the site-selective reaction.
Unfortunately, a higher reaction temperature did not increase
the selectivity or the yield of the isolated product (entry 5).
Conventional conditions using the commercially available
biotin N-hydroxysuccinimide ester in pyridine produced
substantial amounts of regioisomers, with unreacted 1i
remaining (entry 6).18 These results clearly highlight that the
reactivities of the five hydroxyl groups in glycopeptide 1i can
be controlled in a desirable fashion to achieve the site-selective
reaction of a β-hydroxyl group, even during biotinylation.
In conclusion, we demonstrated the site-selective acylations
of α- and β-hydroxyamides in disaccharides and glycopeptides.
N-Glycolyl disaccharides present in the partial structures of
bioactive polysaccharides were acylated with excellent site-
selectivities using Zn(OTf)2 and pyridine aldoxime ester 2a as
acylating agents. In addition, site-selective biotinylation, one of
the most important techniques in chemical biology and related
fields, was accomplished during the site-selective functionaliza-
tion of the β-hydroxyl group in a glycopeptide bearing an
unprotected glucose for the first time. Further applications of
this metal-template strategy toward different classes of
substrates are currently being explored by our group.
Hanae Takada − Faculty of Pharmacy, Meijo University,
Nagoya, Aichi 468-8503, Japan
Kohei Takemoto − Faculty of Pharmacy, Meijo University,
Nagoya, Aichi 468-8503, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c00612
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partially supported by JSPS KAKENHI grant
number JP19K07007 (Y.N.) for Scientific Research (C). Y.N.
thanks Meijo University for financial support.
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ASSOCIATED CONTENT
* Supporting Information
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https://pubs.acs.org/doi/10.1021/acs.orglett.1c00612.
Arguelles, A. J.; DeMars, M. D., II; Zimmerman, P. M.; Sherman, D.
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AUTHOR INFORMATION
Corresponding Authors
■
Yasuhiro Nishikawa − Faculty of Pharmacy, Meijo University,
Nagoya, Aichi 468-8503, Japan; orcid.org/0000-0003-
1606-7288; Email: yasuhiro@meijo-u.ac.jp
Osamu Hara − Faculty of Pharmacy, Meijo University,
Nagoya, Aichi 468-8503, Japan; Email: oshara@meijo-
u.ac.jp
Authors
Shione Toda − Faculty of Pharmacy, Meijo University,
Nagoya, Aichi 468-8503, Japan
Takami Matsui − Faculty of Pharmacy, Meijo University,
Nagoya, Aichi 468-8503, Japan
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