Investigation of protecting group for sialic acid carboxy moiety toward sialylglycopeptide synthesis by the TFA-labile protection strategy

Article abstract of DOI:10.1016/j.tet.2021.132423

The sialylation using sialyl donor with 2,6-dimethylbenzyl (DMBn) ester at C-1 carboxy group rapidly proceeded with good α-selectivity to yield sialyl-N-acetylgalactosaminylated 9-fluorenylmethoxycarbonyl (Fmoc) serine unit. The unit was used for the solid-phase peptide synthesis (SPPS) of the sialyl glycopeptide. The DMBn group was removed during the final deprotection by the trifluoroacetic acid (TFA) treatment keeping the sialyl bond intact and the desired sialyl glycopeptide was successfully obtained. This protecting group strategy provided easier access to sialyl glycoamino acids for sialyl glycopeptides synthesis.

Full text of DOI:10.1016/j.tet.2021.132423

Tetrahedron 97 (2021) 132423
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Investigation of protecting group for sialic acid carboxy moiety toward
sialylglycopeptide synthesis by the TFA-labile protection strategy
Shun Ito, Yuya Asahina*, Hironobu Hojo**
Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The sialylation using sialyl donor with 2,6-dimethylbenzyl (DMBn) ester at C-1 carboxy group rapidly
Received 7 July 2021
Received in revised form
18 August 2021
Accepted 21 August 2021
Available online 24 August 2021
proceeded
with
good
a-selectivity
to
yield
sialyl-N-acetylgalactosaminylated
9-
fluorenylmethoxycarbonyl (Fmoc) serine unit. The unit was used for the solid-phase peptide synthesis
(SPPS) of the sialyl glycopeptide. The DMBn group was removed during the final deprotection by the
trifluoroacetic acid (TFA) treatment keeping the sialyl bond intact and the desired sialyl glycopeptide was
successfully obtained. This protecting group strategy provided easier access to sialyl glycoamino acids for
sialyl glycopeptides synthesis.
Keywords:
Sialic acid
© 2021 Elsevier Ltd. All rights reserved.
Glycopeptide
One-pot deprotection
TFA-labile protection strategy
2,6-dimethylbenzyl (DMBn) group
1. Introduction
independently reported the synthesis of oligosialosides containing
a
-2,8 linkage, one of the most difficult glycosidic linkages, with
A glycoprotein often bears a sialic acid at the reducing terminus
of its sugar chain. Sialic acid plays numerous roles in biological
processes [1], such as in the control of immune cell activation [2],
cell attachment [3], and glycoprotein half-life determination [4]. To
study the role of sialic acid in glycoproteins, synthetic efforts have
been undertaken to prepare samples of homogeneous sialyl
glycopeptide [5]. Sialic acid retains C-1 carboxy group, C-2
quaternary-substituted carbon, C-3 deoxy carbon, and C-7 to C-9
glycerol chain (Fig. 1). This unique structure leads to challenges in
chemical sialylation, because 1) the electron-withdrawing carboxy
group destabilizes the oxocarbenium ion, which makes the sialyl
donor less reactive; 2) the bulky anomeric carbon and glycerol
moiety block the approach of a glycosyl acceptor, which prevents
an efficient sialylation reaction; and 3) the deoxy C-3, next to the
anomeric position, cannot be used for a neighboring group partic-
ipation. To overcome these problems, many methods have been
established [6]. For example, modification of the C-5 acetamido
group successfully improves sialylation efficiency and stereo-
selectivity [6b,d,e,f]. Notably, Ando's and Tanaka's group
complete -selectivity using bicyclic sialyl donors [7]. These de-
velopments allow flexible access to a range of sialosides.
a
However, in the chemical synthesis of glycopeptides containing
a sialic acid, acid instability of the sialyl linkage causes a problem.
The sialyl linkage is generally unstable under acidic conditions
compared with the glycosidic bond of a hexose/hexosamine [8]; it is
easily cleaved by hydrolysis due to intramolecular protonation by
the carboxy group [9]. This acid instability conflicts with the SPPS,
because Fmoc-SPPS necessitates an acidic treatment using tri-
fluoroacetic acid (TFA) for the final deprotection step. To avoid the
unwanted cleavage of the sialyl linkage, protection of the carboxy
group by ester i.e. methyl ester has been used to make the sialyl
bond inert [5aꢀc]. However, saponification using strong bases are
required for the deprotection of these esters, which can cause the b-
elimination and racemization of the amino acid residues.
* Corresponding author.
** Corresponding author.
E-mail addresses: asahina@protein.osaka-u.ac.jp (Y. Asahina), hojo@protein.
osaka-u.ac.jp (H. Hojo).
Fig. 1. Structure of sialic acid.
https://doi.org/10.1016/j.tet.2021.132423
0040-4020/© 2021 Elsevier Ltd. All rights reserved.

S. Ito, Y. Asahina and H. Hojo
Tetrahedron 97 (2021) 132423
Additionally, this synthetic route cannot be incorporated in prep-
aration of a peptide thioester that is a key intermediate of the
peptide condensation method, i.e., the thioester method [10], and
the native chemical ligation method [11], to assemble a long
polypeptide chain.
From this background, we developed a novel protection strategy
using TFA-labile protecting groups, i.e., 4-methylbenzyl (MBn)
group and 4-methoxybenzyl (MPM) group, for the synthesis of
glycopeptides (Fig. 2) [12]. In this strategy, sugar hydroxy groups
and the carboxy group were protected by TFA-labile protecting
groups and the glycoamino acid derivative was then assembled
during the carbohydrate synthesis. In the subsequent stage of
peptide synthesis, the glycoamino acid derivative was applied in
the SPPS based on Fmoc chemistry. At the end of SPPS, the glyco-
peptidyl resin was treated with a TFA cocktail to remove all peptide
and sugar protecting groups. Thus, the fully deprotected glyco-
peptide was obtained in one step, avoiding the troublesome basic
treatment. Surprisingly, in this protecting system, the sialic acid
linkage almost withstood the TFA treatment [12b]. However, in this
Fig. 3. Reaction mechanisms of the side reaction during previous sialylation.
traps the oxocarbenium cation, generating the amidated byproduct
5 via the formation of an intramolecular asymmetric anhydride
followed by hydrolysis. Therefore, we concluded that the C-1 pro-
tecting group must be more stable under acidic conditions than
MPM group, but still removable by TFA treatment at the final
deprotection of the SPPS. Namely, accurate choice of the protecting
group considering sialylation and TFA treatment is important. From
this hypothesis, an alternative TFA-labile protecting group was
examined for the carboxy group of the sialic acid.
previous study, the yield of the
a2,6-sialylation reaction and a-
selectivity were moderate. The efficiency of this sialylation reaction
should be improved to assemble more complex sialylglycoamino
acids, containing not only the
a2,6 linkage but also the more
challenging 2,3 linkage for the synthesis of glycoproteins. In this
a
study, we report the properties of sialyl donor bearing TFA-labile
protecting group in sialylation and improvement of sialylation us-
ing this methodology.
The followings were selected as candidates for the acid-labile
protecting group of the carboxy group: tert-butyl, methoxymethyl
(MOM), 2,4-dimethylbenzyl (DMBn), and 2,4,6-trimethylbenzyl
(TMBn). The sialylation reaction using GalNAc acceptor 2 (1.0 eq),
having a free 6-OH, with the donors (1.2 eq) was then performed
using NIS-TfOH activation system (Table 1). The result obtained
following the previous study is shown in entry 1 as a reference
[12b]. For the tert-butyl group, the donor 6 could not be completely
consumed during the reaction and consequently, the disaccharide
yield was low (entry 2). The reason for incomplete activation
seemed to be the high steric hindrance of the tert-butyl group at C-
1, which prevented the approach of the iodonium ion. When the
MOM group was employed (entry 3), the desired sialylation reac-
2. Results and discussion
Prior to this study, the side reaction of the previously reported
sialylation [12b] was re-investigated to understand the reason for
the moderate yield (Fig. 3). The glycosylation reaction GalNAc 2
with sialyl donor 1 was performed using a combination of triflic
acid (TfOH) and N-iodosuccinimide (NIS) as an activator in aceto-
nitrile or propionitrile [13]. Disaccharide 3 was obtained as 49%
a-
isomer and 8% -isomer. Additionally, the intermolecular migrated
b
byproduct, 4-methoxybenzylated acceptor 4 (2%), and C-2 amido-
glycoside 5 (13%) with free carboxy group were isolated. Therefore,
the mechanism of the side reaction was proposed as shown in
Fig. 3, wherein the sulfur atom should react with the iodonium ion
to yield desired product 3; however, if the sulfur atom attacks the
benzylic position of the neighboring MPM group, the sulfonium ion
leaves the anomer position and the nitrile solvent immediately
tion proceeded partially to give product 12 (a: 38%, b: 5%), but the
amidated byproduct 5 was obtained in 25% yield. Moreover, 17
bearing the MOM group on its C-6 alcohol was also obtained in 31%
yield [14]. Employing the TMBn group resulted in a yield that was
comparable to entry 1 (13, a: 40%, b: 8%, entry 4), but the amidation
reaction could not be completely suppressed (5, 17%), which indi-
cated that the TMBn group is also too acid-sensitive under glyco-
sylation conditions. In the next trial using the DMBn group (entry
5), the product yields were comparable (14, a: 42%, b: 8%) to the
those of the reference reaction. Notably, in this reaction, the for-
mation of amidated byproduct 5 was reduced to 5% and the
migration of the DMBn group to acceptor 2 was not detected. To
improve the yield and
performed in propionitrile under lower temperature (entry 6,
-78 ꢁC), which gave a significantly better result (
: 74%, : 9%) with
a-selectivity, the glycosylation reaction was
a
b
17% of unreacted donor 9 [15]. Further improvement for the yield
was investigated by using more reactive sialyl donor 10, in which
the aglycone was substituted to isopropyl group from methyl group
of 9 [16]. As a result, donor 10 was completely consumed and the
desired disaccharide 14 was obtained in yields of 84% for the
sialoside and 6% for the -sialoside (entry 7). Therefore, the
a
a
-
-
b
selectivity was also improved to 14:1 and amidation product 5 was
reduced to a trace amount. 2,3 Sialylation was also demonstrated
a
using 3-OH free Gal derivative 15 as a glycosyl acceptor (entry 8). As
a result, the desired disaccharide 16 was obtained with complete
Fig. 2. Previous synthesis of sialyl glycoprotein using TFA-labile protection strategy
[12b].
2

S. Ito, Y. Asahina and H. Hojo
Tetrahedron 97 (2021) 132423
Table 1
Study of the protecting groups for the sialic acid carboxy moiety..
stereoselectivity and excellent yield (94%,
a
only). Based on these
silica gel chromatography. The desired
a
-isomer was obtained in
results, the DMBn group was selected as the best protecting group
for the sialic acid carboxy group.
In the next stage, removal of the DMBn group in the final
deprotection reaction was validated via the Fmoc SPPS and treat-
ment with the TFA cocktail. Prior to the peptide synthesis, the Tn
60% yield of a-isomer with an isomeric ratio of 6:1 a/b, minimizing
the problematic side reactions. Subsequently, the azido group was
reduced using zinc powder in the presence of acetic acid and the
generated amine was reacted with acetic anhydride to obtain
diacetamide 22. After deprotection of the allyl ester using Pd(0)
catalyst with phenylsilane as a hydride donor, glycoamino acid 23
was successfully obtained. Throughout this synthetic route, the
DMBn group was completely stable.
In the next step, the SPPS using glycoamino acid was carried out
according to Scheme 2. The model undecapeptide, the same
sequence in the previous study [12b], was a part of the tandem
repeat domain of mucin-1 (MUC1) SAPDTRPAPGS (underline shows
the glycosylated serine). The peptide chain was elongated from a
Rink amide resin by the Fmoc method using a microwave peptide
synthesizer. At the introduction of the N-terminal serine, glycosyl
serine 23 was manually loaded into the peptidyl resin using N,N-
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt)
antigen disaccharide 14 containing the
a2,6 sialyl linkage was
derivatized to Fmoc-glycoamino acid 23 (Scheme 1). Disaccharide
14 was converted to the glycosyl imidate 19 by treating with tetra-
n-butylammonium fluoride (TBAF), followed by 2,2,2-trifluoro-N-
phenylacetimidoyl chloride. The glycosylation reaction of the
Fmoc-serine allyl ester 20 [17] with imidate 19 was performed
using a catalytic amount of Lewis acid [18]. In the first trial, tri-
methylsilyl trifluoromethanesulfonate (TMSOTf), a general Lewis
acid in the imidate method, was employed; however, cleavage of
MPM groups on the sialic acid moiety and O-trimethylsilylation of
serine derivative was partially observed. Thus, triisopropylsilyl
trifluoromethanesulfonate (TIPSOTf), which is more bulky and
milder Lewis acid, was used to suppress these side reactions. The
diastereomeric mixture of crude disaccharide 21 was separated by
Scheme 1. Synthetic route of sialylglycoamino acid 23. Reaction conditions: (a) TBAF,
AcOH, THF, 0 ꢁC to r.t., 24 h, 94%; (b) 2,2,2-trifluoro-N-phenylacetimidoyl chloride,
Et3N, DMAP, CH2Cl2, 0 ꢁC to r.t., 30 min, 95%; (c) TIPSOTf, MS4Å, CH2Cl2, -20 ꢁC, 30 min,
Scheme 2. Synthetic scheme of glycopeptide 24 using sialylglycoamino acid 23. Re-
action condition: (a) Fmoc SPPS; (b) 23, DIC, HOBt, DMF, 50 ꢁC, 1 h; (c) TFA/i-Pr3SiH/
H2O/DODT 92.5:2.5:2.5:2.5, r.t., 2 h.
a
-isomer 60%,
b
-isomer 10%; (d) (1) Zn, AcOH, THF, 0 ꢁC to r.t., 30 min; (2) Ac2O, CH2Cl2,
r.t., 30 min, 83% (in two steps); (e) Pd(PPh3)4, PhSiH3, THF, r.t., 10 min, 95%.
3

S. Ito, Y. Asahina and H. Hojo
Tetrahedron 97 (2021) 132423
6 h followed by distillation; (2) reflux with CaH2 for 6 h followed by
distillation. THF was used for the reactions after distilling from
sodium dispersion and benzophenone [20]. Unless otherwise
noted, materials purchased from distributors were used without
any purification. The specific rotation values were determined at
20e25 ꢁC using a SEPA-300 polarimeter (Horiba Ltd., Kyoto). The
NMR spectra were recorded using a AV400 spectrometer at
400 MHz (1H NMR) and 100 MHz (13C{1H} NMR) or AV500 spec-
trometer at 500 MHz (1H NMR) and 125 MHz (13C{1H} NMR)
(Bruker corporation, MA). The chemical shifts are expressed in ppm
downfield from the signal for the internal trimethylsilane for the
solution in the deuterated solvents. Multiplicities are given as “s”
(singlet), “d” (doublet), “t” (triplet), and “m” (multiplet). Broad
peaks in NMR spectra are indicated as “br”. The SPPS was per-
formed using a Liberty Blue peptide synthesizer (CEM corporation,
NC). RP-HPLC was carried out on RP-18 GP II (4.6 ꢂ 150 mm) (Kanto
Chemical Co., Inc., Tokyo) using a linear increasing gradient of
MeCN in 0.1% TFA/H2O. Detection was by an absorbance measure-
ment at 220 nm. ESI-HRMS spectrum was measured using an ESI-
LTQ- Orbitrap XL (Thermo Fisher Scientific, Inc., MA). Experi-
mental procedures for the preparation of monosaccharide de-
rivatives were shown in the supplementary data.
Fig. 4. HPLC profile of deprotection reaction for glycopeptidyl resin. Asterisk is desialyl
glycopeptide. A linear gradient starting from 20% CH3CN at 0 min to 40% at 20 min was
applied.
at 50 ꢁC. The reaction was monitored by the ninhydrin test and
completed within 1 h. The peptidyl resin was then treated by a TFA
cocktail containing 2.5% water, 2.5% triisopropylsilane, and 2.5%
3,6-dioxa-1,8-octanedithiol (DODT) [19] for 2 h at room tempera-
ture to remove all protecting groups from the peptide and the
carbohydrate moiety. The deprotection profile of this peptide was
analyzed by reverse-phase (RP) HPLC followed by mass spectrom-
etry. The chromatogram is shown in Fig. 4. The fully deprotected
glycopeptide 24 was observed as the highest peak. Additionally, a
product with the DMBn group was not detected. Moreover, hy-
4.2. tert-Butyldiphenylsilyl {2,6-dimethylbenzyl [5-azido-3,5-
dideoxy-4,7,8,9-tetra-O-(4-methoxybenzyl)-D-glycero-
galacto-2-nonulopyranosyl]onate}- -(2 / 6)-2-azido-2-deoxy-
3,4-di-O-(4-methylbenzyl)- -D-galactopyranoside (14)
a-D-
a
b
drolysis of the
a2,6 sialyl linkage during the TFA treatment was
A stirred mixture of sialyl donor 10 (0.11 g, 0.12 mmol), tert-
butyldiphenylsilyl 2-azido-2-deoxy-3,4-di-O-(4-methylbenzyl)-
minimized, which was comparable with previous reactions
employing the MPM protecting group. These results proved that
the DMBn group is a convenient, compatible and stable protecting
group for not only the carbohydrate synthesis but also the TFA
treatment in the final deprotection step of glycopeptide synthesis.
b-
D-galactopyranoside (65 mg, 0.10 mmol) [12b], NIS (54 mg,
0.24 mmol) and dried MS3Å (0.36 g) in EtCN (3.6 mL) was cooled
to ꢀ78 ꢁC under an Ar atmosphere and then stirred for 30 min. After
1% TfOH/EtCN (0.21 mL, 24 mmol) was slowly added, the mixture
was stirred for 10 min at the same temperature. Sat. NaHCO3 aq.
and 10% Na2SO3 aq. were added to quench the reaction. The
mixture was diluted with EtOAc and then filtrated through Celite
pad. The organic layer was successively washed with H2O and
brine, dried over Na2SO4, filtrated, and concentrated under reduced
pressure. The residue was purified by gel filtration chromatography
(Biobeads SX-3 toluene/EtOAc 3:1) followed by silica gel column
chromatography (toluene/EtOAc 19:1) to afford disaccharide 14
3. Conclusion
In conclusion, we developed a novel TFA-labile protecting group,
DMBn group, for the sialic acid carboxy moiety in the synthesis of
glycopeptides. During carbohydrate synthesis, the DMBn group
almost suppressed the problematic side reactions, such as the
intermolecular migration reaction of the protecting group to the
glycosyl acceptor and the amidation reaction of the sialyl donor.
Additionally, the DMBn protection of the carboxy moiety signifi-
cantly improved the yield of the chemical sialylation reactions
(0.13 g, 84%) and
Anal. Calcd for C88H100N6O16Si: C, 69.27; H, 6.61; N, 5.51, found: C,
69.00; H, 6.42; N, 5.62. 1H NMR (CDCl3, 400 MHz):
7.71e7.67 (m,
b
-isomer (9.7 mg, 6%). [
a]
ꢀ26.6 (c 1.5, CHCl3).
D
d
(a2,6: 84%, a/b 14:1 and a2,3: 94%, a only) compared with that
4H, ArH), 7.31e7.28 (m, 5H, ArH), 7.25e7.22 (m, 5H, ArH), 7.17e7.07
(m, 11H, ArH), 7.03 (d, 2H, J ¼ 7.9 Hz, ArH), 6.97 (d, 2H, J ¼ 7.6 Hz,
ArH), 6.87e6.82 (m, 6H, ArH), 6.75 (d, 2H, J ¼ 8.6 Hz, ArH), 5.25 (d,
1H, J ¼ 12.0 Hz, ArCH2-), 5.08 (d, 1H, J ¼ 12.0 Hz, ArCH2-), 4.68 (d,
1H, J ¼ 11.2 Hz, ArCH2-), 4.58 (d, 1H, J ¼ 10.9 Hz, ArCH2-), 4.47e4.23
(m, 11H, H-1a, ArCH2-), 3.85e3.75 (m, 11H, H-7b, H-8b, -OCH3),
3.72e3.61 (m, 8H, H-2a, H-4a, H-6a, H-6b, H-9b, -OCH3), 3.54e3.43
(m, 3H, H-6a, H-5b, H-9b), 3.38e3.32 (m, 1H, H-4b), 3.20 (brt, 1H,
J ¼ 6.7 Hz, H-5a), 3.00 (dd, 1H, J ¼ 2.8 Hz, 10.4 Hz, H-3a), 2.55 (dd,
1H, J ¼ 4.5 Hz, 12.9 Hz, H-3beq), 2.33 (s, 3H, ArCH3), 2.28 (s, 9H,
achieved in a previous study using the MPM ester. In the peptide
synthesis, the DMBn group behaved as a stable protecting group for
the carboxylic acid. After the final deprotection using the TFA
cocktail, the DMBn group was removed completely, minimizing the
undesired cleavage of the sialic acid. These accomplishments
significantly increased the efficiency and the productivity of sia-
lylglycopeptide synthesis. Currently, the synthesis of more complex
sialyl sugar chains is ongoing in our laboratory.
4. Experimental section
ArCH3), 1.55 (brt, 1H, J ¼ 9.3 Hz, H-3bax), 1.04 (s, 9H, C(CH3)3). 13
C
{1H} NMR (CDCl3, 125 MHz):
d 167.4 (C-1b), 96.8 (C-1a), 37.4 (C-3b),
4.1. General
(3JC1b-H3bax ¼ 7.0 Hz).
Analytical thin-layer chromatography (TLC) was performed on
glass plates using TLC Silica gel 60 F254. Flash column chromatog-
4.3. 4-Methoxyphenyl {2,6-dimethylbenzyl [5-azido-3,5-dideoxy-
4,7,8,9-tetra-O-(4-methoxybenzyl)-D-glycero- -D-galacto-2-
nonulopyranosyl]onate}- -(2 / 3)-2,4,6-tri-O-(4-methylbenzyl)-
-D-galactopyranoside (16)
a
raphy was performed on silica gel (40e50
mm). CH2Cl2 and DMF
a
were dried with molecular sieves (MS) 3Å or 4Å before use. EtCN
was used for the glycosylation after two step purifications
described as follows: (1) reflux with diphosphorus pentoxide for
b
A stirred mixture of sialyl donor 10 (0.14 g, 0.15 mmol), glycosyl
4

S. Ito, Y. Asahina and H. Hojo
Tetrahedron 97 (2021) 132423
acceptor 15 (60 mg, 0.10 mmol), NIS (67 mg, 0.30 mmol) and dried
MS3Å (0.45 g) in EtCN (4.5 mL) was cooled to ꢀ78 ꢁC under an Ar
atmosphere and then stirred for 30 min. After 1% TfOH/EtCN
4.5. 2,2,2-Trifluoro-N-phenylacetimidoyl {2,6-dimethylbenzyl [5-
azido-3,5-dideoxy-4,7,8,9-tetra-O-(4-methoxybenzyl)-D-glycero-
D-galacto-2-nonulopyranosyl]onate}- -(2 / 6)-2-azido-2-deoxy-
3,4-di-O-(4-methylbenzyl)-D-galactopyranoside (19)
a-
a
(0.54 mL, 60 mmol) was slowly added, the mixture was stirred for
30 min at the same temperature. Sat. NaHCO3 aq. and 10% Na2SO3
aq. were added to quench the reaction. The mixture was diluted
with EtOAc and then filtrated through Celite pad. The organic layer
was successively washed with H2O and brine, dried over Na2SO4,
filetaraed, and concentrated under reduced pressure. The residue
was purified by gel filtration chromatography (Biobeads SX-3
toluene/EtOAc 3:1) followed by silica gel column chromatography
Product 18 (0.84 g, 0.65 mmol) in CH2Cl2 (7.0 mL) was stirred at
ꢁC under an Ar atmosphere. After 2,2,2-trifluoro-N-phenyl-
0
acetimidoyl chloride (0.15 mL, 0.98 mmol), triethylamine (0.18 mL,
1.3 mmol) and DMAP (8.0 mg, 65 mol) were successively added,
m
the mixture was stirred for 30 min at room temperature. H2O was
added to quench the reaction. The mixture was then diluted with
EtOAc. The organic layer was successively washed with H2O and
brine, dried over Na2SO4, filtrated, and concentrated under reduced
pressure. The residue was purified by silica gel column chroma-
tography (hexane/EtOAc 2:1) to afford product 19 (0.90 g, 95%).
Anal. Calcd for C80H86F3N7O16: C, 65.88; H, 5.94; N, 6.72, found: C,
(hexane/EtOAc 3:1) to afford disaccharide 16 (0.14 g, 94%)
a only.
[a]
D
ꢀ22.6 (c 1.0, CHCl3). Anal. Calcd for C87H97N3O18: C, 70.95; H,
6.64; N, 2.85, found: C, 70.98; H, 6.82; N, 2.83. 1H NMR (CDCl3,
400 MHz): 7.27 (d, 2H, J ¼ 8.6 Hz, ArH), 7.18e7.01 (m, 19H, ArH),
d
6.95 (d, 2H, J ¼ 9.1 Hz, ArH), 6.91 (d, 2H, J ¼ 7.8 Hz, ArH), 6.81e6.76
(m, 10H, ArH), 5.34 (d, 1H, J ¼ 12.0 Hz, ArCH2-), 5.30 (d, 1H,
J ¼ 12.0 Hz, ArCH2-), 4.95 (d, 1H, J ¼ 11.2 Hz, ArCH2-), 4.81 (d, 1H,
J ¼ 10.4 Hz, ArCH2-), 4.64 (d, 1H, J ¼ 7.6 Hz, H-1a), 4.58 (d, 2H,
J ¼ 10.8 Hz, ArCH2-), 4.52e4.33 (m, 7H, ArCH2-), 4.28 (d, 1H,
J ¼ 11.6 Hz, ArCH2-), 4.05 (d, 1H, J ¼ 10.6 Hz, ArCH2-), 4.02 (d, 1H,
J ¼ 10.6 Hz, ArCH2-), 3.94e3.72 (m, 18H, H-2a, H-3a, H-4a, H-6b, H-
7b, H-8b, -OCH3), 3.65 (s, 3H, -OCH3), 3.57 (brdd, 1H, J ¼ 1.9 Hz,
10.7 Hz, H-9b), 3.50e3.35 (m, 5H, H-6a, H-6a0, H-4b, H-5b, H-9b’),
3.27 (t, 1H, J ¼ 6.3 Hz, H-5a), 2.50 (dd, 1H, J ¼ 3.9 Hz, 13.5 Hz, H-
3beq), 2.37 (s, 6H, ArCH3), 2.32 (s, 3H, ArCH3), 2.30 (s, 3H, ArCH3),
2.22 (s, 3H, ArCH3),1.84 (dd,1H, J ¼ 11.8 Hz,13.2 Hz, H-3bax). 13C{1H}
65.75; H, 6.09; N, 6.68. 1H NMR (CDCl3, 400 MHz):
d 7.24e7.09 (m,
17H, ArH), 7.05e6.96 (m, 5H, ArH), 6.86e6.74 (m, 10H, ArH), 5.36
(brs, 1H, H-1a) 5.23 (d, 1H, J ¼ 12.0 Hz, ArCH2-), 5.14 (d, 1H,
J ¼ 12.0 Hz, ArCH2-), 4.70 (d, 1H, J ¼ 11.0 Hz, ArCH2-), 4.60 (d, 1H,
J ¼ 10.7 Hz, ArCH2-), 4.48e4.22 (m, 10H, ArCH2-), 3.98e3.93 (m, 1H,
H-2a), 3.88e3.84 (m, 2H, H-6a, H-7b), 3.80e3.33 (m, 22H, H-4a, H-
5a, H-6a0, H-4b, H-5b, H-6b, H-8b, H-9b, H-9b’, -OCH3), 3.14 (brd,
1H, J ¼ 9.4 Hz, H-3a), 2.69 (dd, 0.15H, J ¼ 4.3 Hz, 12.6 Hz, H-3beq),
2.61 (dd, 0.85H, J ¼ 4.4 Hz, 13.1 Hz, H-3beq), 2.33 (s, 3H, ArCH3), 2.29
(s, 6H, ArCH3), 2.27 (s, 3H, ArCH3), 1.66 (brt, 0.15H, J ¼ 12.1 Hz, H-
3bax), 1.55 (brt, 0.85H, J ¼ 11.4 Hz, H-3bax isomerA). 13C{1H} NMR
(CDCl3, 100 MHz): 37.6 (C-3b, isomerA), 37.2 (C-3b, isomerB).
NMR (CDCl3, 100 MHz):
d 168.2 (C-1b), 102.8 (C-1a), 34.4 (C-3b),
(3JC1b-H3bax ¼ 7.1 Hz).
4.6. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-dimethylbenzyl [5-
azido-3,5-dideoxy-4,7,8,9-tetra-O-(4-methoxybenzyl)-D-glycero-
D-galacto-2-nonulopyranosyl]onate- -(2 / 6)-2-azido-2-deoxy-
-D-galactopyranosyl}-L-serine allyl
a-
a
3,4-di-O-(4-methylbenzyl)-
a
4.4. 2,6-Dimethylbenzyl [5-azido-3,5-dideoxy-4,7,8,9-tetra-O-(4-
methoxybenzyl)-D-glycero- -D-galacto-2-nonulopyranosyl]onate-
-(2 / 6)-2-azido-2-deoxy- 3,4-di-O-(4-methylbenzyl)-D-
galactopyranoside (18)
ester (21)
a
a
A stirred mixture of glycosyl donor 19 (0.38 g, 0.26 mmol), N-(9-
fluorenylmethoxycarbonyl)-L-serine allyl ester (0.11 g, 0.31 mmol)
[16], and dried MS4Å (0.78 g) in CH2Cl2 (7.8 mL) was cooled
to ꢀ20 ꢁC under an Ar atmosphere and then stirred for 30 min. After
Disaccharide 14 (1.4 g, 0.94 mmol) in THF (5.0 mL) containing
AcOH (0.54 mL, 9.4 mmol) was stirred at 0 ꢁC. After 1 M n-tetra-
butylammonium fluoride (5.6 mL) was slowly added, the mixture
was stirred for 19 h at room temperature. After concentration, the
residue was purified by silica gel column chromatography (toluene/
EtOAc 7:1) to afford product 18 (1.1 g, 94%). Anal. Calcd for
1% TIPSOTf/CH2Cl2 (0.35 mL, 13 mmol) was slowly added, the reac-
tion mixture was stirred for 30 min at the same temperature. Sat.
NaHCO3 aq. was added to quench the reaction. The mixture was
diluted with EtOAc and then filtrated through Celite pad. The
organic layer was successively washed with H2O and brine, dried
over Na2SO4, filtrated, and concentrated under reduced pressure.
The residue was purified by gel filtration chromatography (Bio-
beads SX-3 toluene/EtOAc 3:1) followed by silica gel column
chromatography (toluene/EtOAc 19:1) to afford product 21 (0.25 g,
60%) and
Calcd for C93H101N7O20: C, 68.24; H, 6.22; N, 5.99. found: C, 68.24;
H, 6.08; N, 6.01. 1H NMR (CDCl3, 400 MHz):
7.73 (d, 2H, J ¼ 7.4 Hz,
C
72H82N6O16: C, 67.17; H, 6.42; N, 6.53, found: C, 67.18; H, 6.62; N,
6.71. 1H NMR (CDCl3, 400 MHz):
7.30e7.09 (m, 15H, ArH),
7.03e6.98 (m, 4H, ArH), 6.87e6.79 (m, 8H, ArH), 5,28e5.18 (m, 2H,
ArCH2-), 5.08 (d, 0.67H, J ¼ 3.4 Hz, H-1a- isomer), 4.77e4.72 (m,
1H, ArCH2-), 4.62e4.36 (m, 10H, ArCH2-), 4.31 (d, 1H, J ¼ 10.9 Hz,
ArCH2-), 4.18 (d, 0.33H, J ¼ 8.0 Hz, H-1a- isomer), 4.11 (dd, 0.67H,
J ¼ 5.0 Hz, 7.7 Hz, H-5a- isomer), 3.88e3.71 (m, 19.34H, H-2a-
isomer, H-3a- isomer, H-4a- isomer, H-4a- isomer, H-6a-
complex, H-6b, H-7b, H-8b, H-9b- complex, -OCH3), 3.65e3.56
(m, 2.33H, H-2a-
isomer, H-6a0-
3.49 (t,1H, J ¼ 9.6 Hz, H-5b), 3.45e3.36 (m,1.33H, H-5a-
4b), 3.11 (dd, 0.33H, J ¼ 2.7 Hz, 10.3 Hz, H-3a-
(m, 1H, H-3beq), 2.34e2.33 (m, 9H, ArCH3), 2.28 (s, 3H, ArCH3),
1.64e1.56 (m, 1H, H-3bax). 13C{1H} NMR (CDCl3, 100 MHz):
96.2
(C-1a, isomer), 92.4 (C-1a, isomer), 37.6 (C-3b).
d
a
b
-isomer (44 mg, 10%). [
a]
þ20.2 (c 1.0, CHCl3). Anal.
D
b
a
a
d
a
a
b
a
,b
ArH), 7.58 (dd, 2H, J ¼ 2.9 Hz, 7.1 Hz, ArH), 7.38e7.34 (m, 2H, ArH),
7.29e7.21 (m, 8H, ArH), 7.16e7.09 (m, 8H, ArH), 7.07 (d, 1H,
J ¼ 7.2 Hz, ArH), 7.01 (d, 2H, J ¼ 7.8 Hz, ArH), 6.96 (d, 2H, J ¼ 7.5 Hz,
ArH), 6.85e6.81 (m, 6H, ArH), 6.77 (d, 2H, J ¼ 8.5 Hz, ArH),
5.93e5.83 (m, 1H, CH]CH2), 5.79 (d, 1H, J ¼ 8.5 Hz, FmocNH),
5.33e5.20 (m, 3H, CH]CH2, ArCH2-), 5.12 (d,1H, J ¼ 12.0 Hz, ArCH2-
), 4.79 (d, 1H, J ¼ 3.2 Hz, H-1a), 4.72 (d, 1H, J ¼ 10.8 Hz, ArCH2-),
a,b
b
a
,
b
complex, H-9b0-
a
,
b
b
complex),
isomer, H-
b
isomer), 2.66e2.60
d
b
a
4.63e4.55 (m, 5H, ArCH2-), 4.52e4.40 (m, 8H, Ser-aH, CH2eCH]
5

S. Ito, Y. Asahina and H. Hojo
Tetrahedron 97 (2021) 132423
CH2, Ar2CHCH2-, ArCH2-), 4.31 (d, 1H, J ¼ 10.8 Hz, ArCH2-),
-OCH3), 3.43 (dd, 1H, J ¼ 8.7 Hz, 12.6 Hz, H-6a’), 3.33e3.27 (m, 1H,
H-4b), 2,61 (dd, 1H, J ¼ 3.7 Hz, 12.5 Hz, H-3beq), 2.36 (s, 3H, ArCH3),
2.30 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 1.89 (s, 6H, ArCH3), 1.69 (brt,
4.28e4.18 (m, 3H, Ar2CH-, ArCH2-), 3.89e3.73 (m, 19H, Ser-bH, H-
2a, H-3a, H-4a, H-5a, H-6a, H-7b, H-8b, H-9b, eOCH3), 3.70e3.67
(m, 4H, H-6b, -OCH3), 3.61 (dd, 1H, J ¼ 4.7 Hz, 11.0 Hz, H-9b0),
3.54e3.46 (m, 2H, H-6a’, H-5b), 3.39e3.32 (m, 1H, H-4b), 2.66 (dd,
1H, J ¼ 4.2 Hz, 12.9 Hz, H-3beq), 2.33 (s, 3H, ArCH3), 2.29 (s, 6H,
ArCH3), 2.26 (s, 3H, ArCH3), 1.69e1.63 (m, 1H, H-3bax). 13C{1H} NMR
1H, J ¼ 12.5 Hz, H-3bax). 13C{1H} NMR (CDCl3, 100 MHz):
d 98.7 (C-
1a), 37.3 (C-3b).
4.9. Synthesis of the glycopeptide carrying Tn antigen (24)
Rink amide MBHA resin (60 mg, 20 mol) was subjected to
(CDCl3, 100 MHz):
d 167.5 (C-1b) 99.4 (C-1a), 37.1 (C-3b).
m
4.7. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-dimethylbenzyl [5-
acetamido-3,5-dideoxy-4,7,8,9-tetra-O-(4-methoxybenzyl)-D-
peptide elongation using automate peptide synthesizer (Liberty
Blue, CEM) to give the resin, H-Ala-Pro-Asp(tBu)-Thr(tBu)-Arg(Pbf)-
Pro-Ala-Pro-Gly-Ser(tBu)-NH-resin. After compound 23 (65 mg,
glycero-
a-D-galacto-2-nonulopyranosyl]onate-a-(2 / 6)-2-
acetamido-2-deoxy 3,4-di-O-(4-methylbenzyl)-
a-D-
40 mmol), 1 M HOBt/DMF (60 mL), 0.50 M DIC/DMF (0.12 mL) was
galactopyranosyl}-L-serine allyl ester (22)
successively added to the resin at room temperature, the mixture
was vortexed for 1 h at 50 ꢁC. After acetyl capping by treating with
10% Ac2O and 5% DIEA in DMF for 3 min at room temperature, the
resin was successively washed with DMF (x3), CH2Cl2 (x3), ether
(x3), and dried under reduced pressure to give the resin, Fmoc-
Ser(Neu5Ac-GalNAc)-Ala-Pro-Asp(tBu)-Thr(tBu)-Arg(Pbf)-Pro-Ala-
Pro-Gly-Ser(tBu)-NH-resin. A part of the resin (5.0 mg) was treated
with TFA cocktail (TFA/TIS/H2O/DODT 92.5:2.5:2.5:2.5) for 2 h at
room temperature. The mixture was concentrated by N2 stream and
then precipitated with cold ether. The precipitate was washed with
ether (x3) and then dried. After the crude peptide was dissolved in
CH3CN aq. and then filtered, the solution was analyzed by RPHPLC.
The profile of the crude was shown in Fig. 4.
Product 21 (0.20 g, 0.12 mmol) in THF (2.0 mL) containing AcOH
(0.80 mL, 14 mmol) was stirred at room temperature. After
powdered Zn (0.47 g, 7.2 mmol) was added, the mixture was stirred
for 30 min at room temperature. Sat. NaHCO3 aq. was added to
quench the reaction. The mixture was diluted with EtOAc and then
filtrated through Celite pad. The organic layer was successively
washed with H2O and brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was dissolved in
CH2Cl2 (2.0 mL) containing Ac2O (0.23 mL, 2.4 mmol) and stirred for
30 min at room temperature. After the mixture was concentrated
under reduced pressure, the residue was purified by silica gel col-
umn chromatography (CHCl3:MeCN 9:1 containing 1% AcOH) to
afford product 22 (0.17 g, 83%). [
a
]
D þ38.3 (c 1.8, CHCl3). ESI-HRMS:
Declaration of competing interest
m/z 1690.7228, calcd for [C97H109N3O22þNa]þ 1690.7395. 1H NMR
(CD3OD, 500 MHz):
ArH), 7.34e6.96 (m, 23H, ArH), 6.86e6.73 (m, 8H, ArH), 5.85e5.79
(m, 1H, CH]CH2), 5.33 (d, 1H, J ¼ 12 Hz, ArCH2-), 5.26e5.09 (m, 3H,
d
7.74 (d, 2H, J ¼ 6.9 Hz, ArH), 7.61e7.57 (m, 2H,
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
CH]CH2, ArCH2-), 4.65e4.32 (m, 18H, Ser-aH, H-1a, H-2a,
CH2eCH]CH2, Ar2CHCH2-), 4.20e4.10 (m, 4H, H-5b, H-8b, Ar2CH-,
Acknowledgements
ArCH2-), 3.81e3.54 (m, 22H, Ser-bH, H-3a, H-4a, H-5a, H-6a, H-6b,
H-7b, H-9b, H-9b0, -OCH3), 3.49e3.44 (m,1H, H-6a’), 3.31 (br, 1H, H-
4b), 2.71 (dd, 1H, J ¼ 4.3 Hz, 13 Hz, H-3beq), 2.34 (s, 6H, ArCH3), 2.29
(s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 1.88 (s, 6H, Ac), 1.69 (t, 1H,
We deeply thank Dr. Yohei Miyanoiri and Dr. Shoko Shinya for
their kind help with NMR measurement and management for NMR
instruments at Institute for Protein Research, Osaka University. This
work was performed in part using the NMR spectrometers under
the Collaborative Research Program of Institute for Protein
Research, Osaka University. The HRMS measurements were per-
formed at the Analytical Instrument Facility, Graduate School of
Science, Osaka University. This work was supported by “SUNBOR
GRANT” (SUNTORY foundation for life sciences, Japan), and “JSPS
KAKENHI” (Japan Society for the Promotion of Science, Grant
Number 18K14336 and 21K05275).
J ¼ 12 Hz, H-3bax). 13C{1H} NMR (CDCl3, 100 MHz):
d 100.5 (C-1a),
38.7 (C-3b).
4.8. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-dimethylbenzyl [5-
acetamido-3,5-dideoxy- 4,7,8,9-tetra-O-(4-methoxybenzyl)-D-
glycero-a-D-galacto-2-nonulopyranosyl]onate-a-(2 / 6)-2-
acetamido-2-deoxy-3,4-di-O-(4-methylbenzyl)-
a-D-
galactopyranosyl}-L-serine (23)
A mixture of 22 (0.43 g, 0.26 mmol) and PhSiH3 (38
0.31 mmol) in THF (3.0 mL) was stirred at room temperature under
an Ar atmosphere. After (PPh3)4Pd (15 mg, 13 mol) was added, the
mixture was stirred for 10 min and then concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (CHCl3:MeOH 9:1 containing 1% AcOH) to afford
mL,
Appendix A. Supplementary data
m
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2021.132423.
References
product 23 (0.40 g, 95%). [
94H105N3O22: C, 69.31; H, 6.50; N, 2.58. found: C, 69.31; H, 6.64; N,
2.57. 1H NMR (CD3OD, 400 MHz):
a]
þ58.4 (c 1.0, CHCl3). Anal. Calcd for
D
C
[1] A. Varki, P. Gagneux, Ann. N. Y. Acad. Sci. 1253 (2012) 16e36.
[2] P.R. Crocker, S. Kelm, C. Dubois, B. Martin, A.S. McWilliam, D.M. Shotton,
J.C. Paulson, S. Gordon, EMBO J. 10 (1991) 1661e1669.
[3] (a) R.D. Cummings, D.F. Smith, Bioessays 14 (1992) 849e856;
(b) C. Foxall, S.R. Watson, D. Dowbenko, C. Fennie, L.A. Lasky, M. Kiso,
A. Hasegawa, D. Asa, B.K. Brandley, J. Cell Biol. 117 (1992) 895e902.
[4] G. Ashwell, J. Harford, Annu. Rev. Biochem. 51 (1982) 531e534.
[5] (a) Y. Nakahara, Y. Nakahara, Y. Ito, T. Ogawa, Tetrahedron Lett. 38 (1997)
7211e7214;
d
7.76 (d, 2H, J ¼ 7.6 Hz, ArH), 7.61
(t, 2H, J ¼ 7.5 Hz, ArH), 7.37e7.32 (m, 4H, ArH), 7.27e7.19 (m, 6H,
ArH), 7.12e7.06 (m, 9H, ArH), 6.99e6.96 (m, 4H, ArH), 6.86e6.81
(m, 6H, ArH), 6.74 (d, 2H, J ¼ 8.3 Hz, ArH), 5.34 (d, 1H, J ¼ 12.1 Hz,
ArCH2-), 5.24 (d, 1H, J ¼ 12.1 Hz, ArCH2-), 4.70 (d, 1H, J ¼ 3.4 Hz, H-
1a), 4.61 (d,1H, J ¼ 11.0 Hz, ArCH2-), 4.58 (d,1H, J ¼ 10.0 Hz, ArCH2-),
(b) K. Peilstocker, H. Kuntz, Synlett 6 (2000) 823e825;
4.53 (d, 1H, J ¼ 10.0 Hz, ArCH2-), 4.48e4.28 (m, 12H, Ser-
aH, H-2a,
(c) R. Okamoto, S. Souma, Y. Kajihara, J. Org. Chem. 73 (2008) 3460e3466;
(d) P. Wang, S. Dong, J.A. Brailsford, K. Iyer, S.D. Townsend, Q. Zhang,
R.C. Hendrickson, J. Shieh, M.A.S. Moore, S.J. Danishefsky, Angew. Chem. Int.
Ed. 51 (2012) 11576e11584;
Ar2CHCH2-, ArCH2-), 4.20 (d, 1H, J ¼ 11.6 Hz, ArCH2-), 4.16e4.09 (m,
3H, H-5b, H-6b, Ar2CH-), 3.81e3.70 (m, 16H, Ser- H, H-4a, H-6a, H-
7b, H-8b, H-9b, -OCH3), 3.62e3.57 (m, 6H, H-3a, H-5a, H-9b0,
b
6

S. Ito, Y. Asahina and H. Hojo
Tetrahedron 97 (2021) 132423
[10] H. Hojo, S. Aimoto, Bull. Chem. Soc. Jpn. 64 (1991) 111e117.
[11] P.E. Dawson, T.W. Muir, I.C. Lewis, S.B.H. Kent, Science 266 (1994) 776e779.
[12] (a) Y. Asahina, R. Fujimoto, A. Suzuki, H. Hojo, J. Carbohydr. Chem. 34 (2015)
12e27;
(b) N. Takeda, T. Takei, Y. Asahina, H. Hojo, Chem. Eur J. 24 (2018) 2593e2597;
(c) Y. Asahina, T. Kawakami, H. Hojo, Eur. J. Org Chem. (2019) 1915e1920.
[13] (a) O. Kanie, M. Kiso, A. Hasegawa, J. Carbohydr. Chem. 7 (1988) 501e506.
[14] Even at lower temperature (-78 ꢁC), the intermolecular migration of the MOM
group was observed to give 17 (53%).
(e) C. Unverzagt, Y. Kajihara, Chem. Soc. Rev. 42 (2013) 4408e4420.
[6] (a) Y. Ito, T. Ogawa, Tetrahedron Lett. 29 (1988) 3987e3990;
(b) A.V. Demchenko, G.J. Boons, Tetrahedron Lett. 39 (1998) 3065e3068;
(c) Y. Nakahara, H. Iijima, S. Sibayama, T. Ogawa, Tetrahedron Lett. 31 (1990)
6897e6900;
(d) C. De Meo, A.V. Demchenko, G.J. Boons, J. Org. Chem. 66 (2001)
5490e5497;
(e) C.-S. Yu, K. Niikura, C.-C. Lin, C.-H. Wong, Angew. Chem. Int. Ed. 40 (2001)
2900e2903;
(f) H. Ando, Y. Koike, H. Ishida, M. Kiso, Tetrahedron Lett. 44 (2003)
6883e6886;
[15] Use of additional TfOH (0.48 eq.) was examined, but the unreacted donor of
6% was still recovered.
(g) S. Escopy, S.A. Geringer, C. De Meo, Org. Lett. 19 (2017) 2638e2641;
(h) J. Chen, T. Hansen, Q.-J. Zhang, D.-Y. Liu, Y. Sun, H. Yan, J.D.C. Codee,
R.R. Schmidt, J.-S. Sun, Angew. Chem. Int. Ed. 58 (2019) 17000e17008.
[7] (a) H. Tanaka, Y. Nishiura, T. Takahashi, J. Am. Chem. Soc. 128 (2006)
7124e7125;
[16] M. Lahmann, S. Oscarson, Can. J. Chem. 80 (2002) 889e893.
[17] B.G. de la Torre, J.L. Torres, E. Bardají, P. Clapes, N. Xaus, X. Jorba, S. Calvet,
ꢀ
F. Albericio, G. Valencia, J. Chem. Soc., Chem. Commun. (1990) 965e967.
[18] (a) R.R. Schmidt, J. Michel, Angew. Chem. Int. Ed. 19 (1980) 731e732;
(b) B. Yu, H. Tao, Tetrahedron Lett. 42 (2001) 2405e2407.
[19] DODT is non-malodorous thiol and be used as an effective scavenger of cat-
ions. It can avoid use of unpleasantly smelled 1,2-ethanditiol, A. Teixeira,
W.E. Benckhuijsen, P.E. de Koning, A.R.P.M. Valentijn, J.W. Drijfhout, Protein
Pept. Lett. 5 (2002) 379e385.
(b) N. Komura, K. Kato, T. Udagawa, S. Asano, H. Tanaka, A. Imamura, H. Ishida,
M. Kiso, H. Ando, Science 364 (2019) 677e680;
(c) R. Koinuma, K. Tohda, T. Aoyagi, H. Tanaka, Chem. Commun. 56 (2020)
12981e12984.
[8] M. Ashwell, X. Guo, M. Sinnott, J. Am. Chem. Soc. 114 (1992) 10158e10166.
[9] M. Murakami, R. Okamoto, M. Izumi, Y. Kajihara, Angew. Chem. Int. Ed. 51
(2012) 3567e3572.
[20] R. Inoue, M. Yamaguchi, Y. Murakami, K. Okano, A. Mori, ACS Omega 3 (2018)
12703e12706.
7