Simplified beta-glycosylation of peptides

Article abstract of DOI:10.1016/j.tet.2018.04.082

A simple and effective activating system for S-phenyl thioglycosides, namely N-iodosuccinimide and catalytic copper(I) triflate, promotes beta-O-glycosylation at the serine and threonine hydroxyls of “mono-,” di-, and tripeptides. The same activator combination promotes carboxamide beta-N-glycosylation of asparagine-containing mono-, di, and tri-peptides, as well as a nucleoside carboxamide and a sulfonamide. An important feature of the copper(I) triflate method is that undesired amide O-glycosylation is largely circumvented. For both sets of biologically important acceptor sites (HO– and –CONH₂), a beta-GlcNAc-equivalent donor is demonstrated to provide the linkages efficiently. Streamlined deprotection sequences have been developed based on global hydrogenolysis that lead smoothly to the parent glycopeptides. The core glycopeptide region for biological protein N-glycosylation, represented by N⁴-(β-N-acetyl-D-2-glucosaminyl)-Asp-Gly-Thr-OH, has been prepared in solution, purified, and characterized as the fully deprotected (mono)glycosylated tripeptide.

Full text of DOI:10.1016/j.tet.2018.04.082

Tetrahedron 74 (2018) 2891e2903
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Simplified beta-glycosylation of peptides
Yonglian Zhang, Spencer Knapp*
Department of Chemistry & Chemical Biology, Rutgers The State University of New Jersey, 610 Taylor Rd., Piscataway, NJ, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and effective activating system for S-phenyl thioglycosides, namely N-iodosuccinimide and
catalytic copper(I) triflate, promotes beta-O-glycosylation at the serine and threonine hydroxyls of
“mono-,” di-, and tripeptides. The same activator combination promotes carboxamide beta-N-
glycosylation of asparagine-containing mono-, di, and tri-peptides, as well as a nucleoside carbox-
amide and a sulfonamide. An important feature of the copper(I) triflate method is that undesired amide
O-glycosylation is largely circumvented. For both sets of biologically important acceptor sites (HOe and
eCONH2), a beta-GlcNAc-equivalent donor is demonstrated to provide the linkages efficiently. Stream-
lined deprotection sequences have been developed based on global hydrogenolysis that lead smoothly to
the parent glycopeptides. The core glycopeptide region for biological protein N-glycosylation, repre-
Received 16 March 2018
Received in revised form
24 April 2018
Accepted 25 April 2018
Available online 27 April 2018
Keywords:
Lewis acid
Hydrogenolysis
Trichloroacetamide
Glycoprotein
Glycopeptide
sented by N4-(
and characterized as the fully deprotected (mono)glycosylated tripeptide.
© 2018 Published by Elsevier Ltd.
b-N-acetyl-D-2-glucosaminyl)-Asp-Gly-Thr-OH, has been prepared in solution, purified,
1. Introduction
the various amide and carbonyl-bearing protecting groups, which
can undergo wasteful carbonyl-O-glycosylations29e31 in competi-
tion with reaction at the desired acceptor site, namely serine/
threonine eOH or asparagine eCONH2. Thus, the chemical yields of
these glycosylation reactions can be unacceptably low. The devel-
opment of a simple and efficient process for O- and N- glycosylation
of peptides that need not be optimized for each new specific case
would benefit multistep preparations, especially those limited by
availability of material, that cannot themselves be taken through
extensive optimization. Part of this optimization should include
mild and efficient deprotection protocols.
In addressing an analogous situation in nucleoside glycosyla-
tion, namely, how can glycosylation be carried out at an acceptor
site that is not necessarily the most reactive one, we recently found
a practical solution.32 This features identifying an activator/Lewis
acid combination that promotes reversibility of the initial glyco-
slation at a competing acceptor site, such as an amide carbonyl, and
allows a slower but more permanent glycosylation at the desired
site. In this paper we apply similar reasoning to develop procedures
for peptide O- or N-glycosylation, and further sharpen the process
by streamlining the deprotection steps.
Enzyme mediated O- and N-glycosylation of proteins, particu-
larly at the side chain residues of serine, threonine, and asparagine,
is an important biological process that determines the properties,
function, and fate of the resulting glycoproteins.1e4 To take a recent
example: cryo-EM and other evidence indicates that single N-gly-
cosylated Asn154 of the Zika virus glycoprotein shell may function
to enable attachment of the virus to host cells.5e7 The analogous
but simpler glycosylated peptides and amino acids, including the
blood group antigens,8,9 have their own distinctive biological
roles.10,11 The chemical preparation of N-glycopeptides12e14 has
developed along two parallel lines, with notable successes for both:
(1) N-acylation of glycosylamines with activated aspartic acid de-
rivatives,15,16 and (2) direct O- and N-glycosylation of protected
peptides with activated glycosyl donors.17e21 In either case, elabo-
ration of the glycosylated amino acid or small glycopeptide into a
more complex glycopeptide can often be achieved by well pre-
cedented solution or resin-supported peptide coupling meth-
ods.22e28 The direct glycosylation approach, while more versatile in
principle, and more similar to the biological process itself, is limited
by the difficulty of carrying out a chemical glycosylation on a
complex acceptor with multiple Lewis basic sites. These can include
2. Results and discussion
2.1. Beta-glycosylation of serine hydroxyl
Thioglycoside donors have advantages for glycosylation that
* Corresponding author.
E-mail address: spencer.knapp@rutgers.edu (S. Knapp).
https://doi.org/10.1016/j.tet.2018.04.082
0040-4020/© 2018 Published by Elsevier Ltd.

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Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
include their simplicity and ease of preparation, their chemical
stability, and the versatility of the various activation methods.33e36
The beta-GlcNAc donor 1, with phenylthio as the activatable leaving
group, and trichloroacetamido as the participating group at C-2,37
was evaluated as a glycosylation donor with commercial N-Cbz
serine methyl ester (2) as the protected amino acid (“monopep-
tide”) acceptor. A qualitative screen of acids (triflic acid, TMS-
triflate) and metal triflates38e40 in combination with N-iodosucci-
nimide (NIS) led to the selection of copper(I) triflate, commercially
available as a 2:1 toluene complex, as an effective catalytic pro-
moter (91% yield, Scheme 1). This yield compares favorably with
the best literature methods for serine beta O-GlcNAc-ylation.41e46
The best promoter for nucleoside O-glycosylation, namely, indiu-
m(III) triflate,32 was not as effective for the peptide example.
Although we have no direct evidence, it is possible that Cu(I) is
better than the other promoters because it effectively isomerizes
mis-glycosylated imidate-type byproducts.
By application of the same reaction conditions, with 1 and also
with the analogous galacto donor47 4, to the glycosylation of several
other protected serine and threonine acceptors, the glycopeptides
in Fig. 1 were prepared in the yields shown. H-1 NMR spectroscopic
analysis (J at the anomeric H) indicates that products have exclu-
sively the beta glycosyl stereochemistry. The original acceptor
atoms are highlighted with red boxes.
Similarly, a dipeptide and a tripeptide acceptor were converted
to glycopeptides 11e14 by using donors 1 and 4 (Fig. 2). Yields are
somewhat reduced compared with the monopeptides, but are
nevertheless acceptable for acceptors with this number of
competing Lewis basic sites. The acceptor atoms are highlighted
with red boxes.
The use of hydrogenolyzable protecting and precursor groups on
both the donor and acceptor moieties enables a one step depro-
tection protocol, as shown in Scheme 2. No fewer than eight indi-
vidual hydrogenolytic steps (as indicated by the red arrows) occur
during the palladium hydroxide mediated deprotection of 6, which
leads to the hydrochloride of the GlcNAc serine derivative48 15 in
91% overall yield. Examination of the H-1 and C-13 NMR spectra
indicates that hydrogenolysis is complete. There are, for example,
no signals attributable to a mono-benzyl or a chloroacetamido
byproduct. Likewise, no stereoisomers, such as might have been
produced had partial epimerization occurred at the serine alpha
carbon, are observed. The same efficient procedure provides gly-
copeptides 16e19 from the respective glycosylation products
(Scheme 2). Because of the presence of aqueous HCl during
Fig. 1. O-Glycosylation of serine hydroxyls.
Scheme 1. Prototypical serine O-glycosylation.
Fig. 2. Dipeptide and tripeptide serine O-glycosylations.

Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
2893
Scheme 2. Global deprotection of glycopeptides.
hydrogenolysis, the glycopeptides 15e19 are obtained as their hy-
drochloride salts.49 The pyranoside H-1/H-2 vicinal coupling con-
stants (J ¼ 6.6e8.4 Hz) confirm the beta anomeric stereochemistry.
A notable feature of the mild final hydrogenolysis step is that
competing beta elimination from the threonine derivatives,13
which can occur under acidic or basic conditions, is avoided.
Fig. 3. Carboxamide beta-N-glycosylations.
products shown in Fig. 3. The application to more complex dipep-
tide and tripeptide acceptors also worked, but the yields were
reduced (see 24 and 25). N-Glucosylation of the carboxamide group
of protected ribavirin was carried out to give 26. The combination of
the GlcN donor 23 (bearing N-Troc) with asparagine-containing
mono-, di, and tripeptide acceptors gave the respective N-glyco-
peptides 27, 28, and 29. Glycosylation of tripeptide acceptors in
particular can be quite challenging,19 so the reduced yields are
nevertheless acceptable. The Troc protecting group, 2,2,2-
trifluoroethoxycarbonyl, is effective at directing beta glucosamini-
dation while also maintaining reactivity of the donor.50 A sulfon-
amide acceptor51 was also N-glycosylated with this donor (see 30).
The N-glucosaminidated asparagine peptides in Fig. 3 (27, 28,
and 29) feature the important biological linkage between aspara-
gine and the pentasaccharide of N-linked glycoproteins. In order to
establish a deprotection protocol for an elaborated glycopeptide,
the preparation of the core N-glucosamidated tripeptide portion of
natural glycoproteins,13 namely 33, was undertaken (Scheme 4).
Related or protected versions of 33 have been reported.16,17,52 The
asparagine derivative 27 was converted to an Asp-Gly-Thr tripep-
tide version (31) by deprotection53 of the allyl ester and then
coupling to the appropriately protected dipeptide amine. The
deprotection sequence began with conversion of N-Troc to N-acetyl
(green boxes), which was accomplished by zinc treatment and then
acetylation54 to provide 32. Global hydrogenolytic deprotection of
the tripeptide at six sites (red arrows) gave the free glycopeptide 33
in excellent yield. The pyranoside H-1/H-2 vicinal coupling con-
stant (J ¼ 8.0 Hz) confirms the beta anomeric stereochemistry.
2.2. Beta-N-glycosylation of asparagine carboxamide
N-Glycosylation of the carboxamide group in asparagine de-
rivatives comes with the particular challenge of avoiding or
reversing O-glycosylation at the amide carboxyl oxygen. The cop-
per(I) triflate procedure proved effective in this regard. Thus, pro-
tected asparagine 21 reacted with the beta-glucopyranosyl donor
20 to give glycopeptide 22 in 82% yield without significant O-
glycosylation interference (Scheme 3).
The Cu(I) glycosylation method proved to be successful for
various carboxamide acceptors, as exemplified by the glycosylated
2.3. Other acceptor substrates
Acceptor substrates for glycosylation (34e43) that are not
explicitly shown above are displayed in Fig. 4. Their provenance is
described in the Experimental Section.
Scheme 3. Prototypical beta-N-glycosylation of asparagine carboxamide.

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Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
3. Conclusion
The use of catalytic Cu(I) triflate as a co-activator of simple
thioglycosides is featured in the beta-O-glycosylation of eleven
serine-containing mono-, di-, and tripeptides, and the N-glycosyl-
ation of seven asparagine-containing mono-, di-, and tripeptides as
well as two miscellaneous amides. Yields are competitive or better
compared with prior methods, suggesting that the role of Cu(I) in
these reactions may include facilitating the isomerization of in-
termediates that are wrongly carbonyl-O-glycosylated. The selec-
tion of hydrogenolyzable protecting and precursor groups (O-
benzyl ethers and esters, N-Cbz, and N-trichloroacetyl) enables
efficient global deprotection to provide the parent glycopeptides,
including an asparagine N-GlcNAc-ylated tripeptide that represents
the concensus core of natural N-glycoproteins. Limitations of this
method include the decreasing yields with increasing acceptor
peptide length, and the inapplicability of hydrogenolysis to sulfur
containing peptides. Catalytic copper(I) promotion of glycosylation
holds promise for further application to alpha selective or more
elaborated glycosyl donors as well as additional complex acceptors
with competing Lewis basic sites.
4. Experimental section
4.1. General glycosylation procedure
A 10 mL vial containing the peptide acceptor (0.10 mmol), cop-
per(I) triflate (commercial toluene complex, 10.5 mg, 0.02 mmol),
activated 4 Å molecular sieves (150 mg), the glycosyl donor
(0.15 mmol) and 1,2-dichloroethane (1 mL) was stirred at 0 ꢀC
(external ice bath) for 15 min. N-iodosuccinimide (45 mg,
0.20 mmol) was added, and the reaction mixture was allowed to
stir at 0 ꢀC or room temperature for 2 h as needed for disappearance
Scheme 4. Preparation of the tripeptide glycoprotein core.
of donor. The reaction mixture was filtered through a 0.45 mM PTFE
syringe filter, and the cake was rinsed with dichloromethane
(2 ꢁ 5 mL). The organic solution was washed with 10 mL of satu-
rated aq sodium bisulfite, dried over anhydrous sodium sulfate, and
concentrated. The residue was purified by using a pre-packed silica
gel column or preparative silica thin layer chromatography plate to
afford the glycosylation product.
4.2. O-glycosylations
4.2.1. N-((2S,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)
methyl)-2-(phenylthio)tetrahydro-2H-pyran-3-yl)-2,2,2-
trichloroacetamide 137
A solution of commercial 1,3,4,6-tetraeO-acetyl-2-deoxyl-2-
[(trichloroacetyl)amino]-b-D-glucopyranose (5.5 g, 11.2 mmol) in
dry dichloromethane (100 mL) was treated sequentially with thi-
ophenol (2.3 mL, 22.4 mmol) and boron trifluoride etherate (4.3 mL,
33.6 mmol). The mixture was stirred for 15 h, and then was
quenched with saturated aq sodium bicarbonate (100 mL). The
organic phase was dried and then concentrated, and the residue
was purified by silica gel column chromatography, eluting with 4:6
ethyl acetate/hexane, to give the thioglycoside triacetate (4.1 g, 68%
yield) as a white solid.
A solution of above thioglycoside triacetate (4.0 g, 7.37 mmol) in
anhydrous methanol (80 mL) was treated with sodium methoxide
(40 mg, 0.74 mmol). The solution was stirred for 2 h, and then was
þ
neutralized with Dowex-H
resin. The resin was removed by
filtration, and the filtrate was concentrated to give 3.07 g of the triol
as a white solid. A solution of this material in dry dimethylforma-
mide (40 mL) was stirred at ꢂ10 ꢀC, and then was treated sequen-
tially with benzyl bromide (5.2 mL, 43.92 mmol) and sodium
hydride (60% in oil, 1.77 g, 43.92 mmol).55 The reaction mixture was
Fig. 4. Additional acceptor substrates used in this study.

Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
2895
stirred under nitrogen below 0 ꢀC for 2 h, and then was quenched
with 2 mL of methanol. Ethyl acetate (150 mL) was added, and the
organic solution was washed sequentially with water (100 mL) and
brine (100 mL), dried, and then concentrated. The residue was
purified by flash chromatography, eluting with 5:95 ethyl acetate/
hexane, to give 1 (3.62 g, 72% yield) as a white powder: 1H NMR
trimethylsilyl trifluoromethanesulfonate (5.5 mL, 30.4 mmol). The
resulting dark red solution was stirred for 18 h, and then was
poured into a vigorously stirred solution of aq sodium bicarbonate
(10.0 g in 30 mL water). After 30 min of stirring, the layers were
separated, and the aqueous layer was extracted with dichloro-
methane (100 mL). The combined organic extract was dried and
then concentrated. The residue was purified by flash chromatog-
raphy, eluting with 1:4 ethyl acetate/hexane, to provide 9.0 g of the
thioglycoside as a brown solid.
(400 Hz, CDCl3)
d
7.56 (d, 2 H, J ¼ 1.7 Hz), 7.20e7.54 (m, 18 H), 6.89
(d, 1 H, J ¼ 10.5 Hz), 5.13 (d, 1 H, J ¼ 12.5 Hz), 4.78 (dd, 1 H, J ¼ 6.6
and 13.4 Hz), 4.68 (d, 1 H, J ¼ 13.4 Hz), 4.64 (d, 1 H, J ¼ 11.1 Hz), 4.60
(d, 1 H, J ¼ 9.9 Hz), 4.56 (d, 1 H, J ¼ 14.9 Hz), 4.10 (dd, 1 H, J ¼ 10.3
and 11.9 Hz), 3.82 (dd, 1 H, J ¼ 2.9 and 13.5 Hz), 3.77 (dd, 1 H, J ¼ 4.9
and 13.5 Hz), 3.57e3.70 (m, 1 H), 1.57 (water peak); 13C NMR
A solution of above thioglycoside (9.0 g, 16.58 mmol) in anhy-
drous methanol (100 mL) was treated with sodium methoxide
(0.5 M in methanol, 830 mL, 1.66 mmol). The reaction was stirred at
(125 MHz, CDCl3)
d
161.6, 138.3, 137.9, 137.7, 133.2, 132.2, 129.2,
room temperature under nitrogen for 2 h, and then was quenched
with Dowex-Hþ resin. The reaction mixture was filtered, and the
filtrate was concentrated. A stirred solution of the residue in
anhydrous dimethylformamide (100 mL) was cooled to ꢂ10 ꢀC.
Benzyl bromide (3.3 mL, 27.6 mmol) and sodium hydride (60% in
mineral oil,1.11 g, 27.6 mmol) were added sequentially. After 2 h the
reaction was carefully quenched with 1 mL of methanol. Ethyl ac-
etate (150 mL) was added, and the organic solution was washed
sequentially with water (2 ꢁ 100 mL) and brine (100 mL), and then
dried and concentrated. The residue was purified by flash chro-
matography, eluting with 1:9 ethyl acetate/hexane, to give 4 (4.9 g,
128.7, 128.6, 128.5, 128.4, 128.1, 128.05; 128.0, 127.9, 127.8, 127.7,
92.6, 84.7, 81.5, 79.4, 78.4, 75.5, 74.9, 73.6, 69.0, 56.9; LC-ESI-MS
[MþNa]þ calcd for C35H34Cl3NO5SNa 709.11, found 709.28.
4.3. Methyl ((benzyloxy)carbonyl)-L-serinate 2
This acceptor is available commercially.
4.3.1. Methyl N-((benzyloxy)carbonyl)-O-((2R,3R,4R,5S,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)
tetrahydro-2H-pyran-2-yl)-L-serinate 3
43% yield) as
a
white powder: 1H NMR (400 MHz, CDCl3)
By following the general procedure, gluco donor 1 (103.1 mg,
0.15 mmol) was combined with commercial methyl ((benzyloxy)
carbonyl)-L-serinate (acceptor 2, 25.3 mg, 0.10 mmol) at room
temperature for 2 h. After work-up, purification by thin-layer
chromatography with 6:4 hexane/ethyl acetate as the eluant
afforded 3 (75.5 mg, 91%) as a white powder: 1H NMR (400 MHz,
d
7.51e7.53 (m, 2 H), 7.18e7.37 (m, 18 H), 6.82 (d, 1 H, J ¼ 7.4 Hz),
5.28 (d, 1 H, J ¼ 10.2 Hz), 4.88 (d, 1 H, J ¼ 11.4 Hz), 4.67 (d, 1 H,
J ¼ 11.2 Hz), 4.56 (d, 1 H, J ¼ 11.2 Hz), 4.52 (d, 1 H, J ¼ 11.2 Hz), 4.51
(d,1 H, J ¼ 11.2 Hz), 4.45 (d,1 H, J ¼ 11.2 Hz), 4.26 (dd,1 H, J ¼ 2.7 and
10.5 Hz), 4.06 (d, 1 H, J ¼ 2.1 Hz), 3.88e3.95 (m, 1 H), 3.67e3.76 (m,
3 H), 1.64 (water peak); 13C NMR (125 MHz, CDCl3)
d 161.7, 138.5,
CDCl3)
d
7.27e7.35 (m 18 H), 7.18e7.20 (m, 2 H), 6.98 (d, 1 H,
137.9, 137.4, 132.8, 132.4, 129.0, 128.8, 128.7, 128.5, 128.3, 128.2,
128.17, 128.02, 128.0, 127.9, 127.8, 127.6, 92.6, 84.5, 78.5, 74.6, 73.7,
72.7, 72.5, 68.6, 53.8; LC-ESI-MS [MþNa]þ calcd for
J ¼ 7.6 Hz), 5.70 (d, 1 H, J ¼ 8.2 Hz), 5.07e5.13 (m, 2 H), 4.84 (d, 1 H,
J ¼ 7.6 Hz), 4.77 (d, 1 H, J ¼ 10.3 Hz), 4.75 (d, 1 H, J ¼ 10.1 Hz), 4.68 (d,
1 H, J ¼ 11.2 Hz), 4.50e4.60 (m, 4 H), 4.30 (dd, 1 H, J ¼ 3.0 and
10.4 Hz), 4.05 (t, 1 H, J ¼ 8.9 Hz), 3.80 (dd, 1 H, J ¼ 2.9 and 10.0 Hz),
3.69e3.74 (m, 6 H), 3.57e3.60 (m, 2 H), 1.85 (water peak); 13C NMR
C
35H34Cl3NO5SNa 708.11, found 708.29.
4.3.3. Methyl N-((benzyloxy)carbonyl)-O-((2R,3R,4R,5S,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)
(125 MHz, CDCl3)
d 170.2, 161.9, 156.1, 137.9, 137.7, 136.2, 128.5,
128.44, 128.42, 128.2, 128.1, 127.9, 127.8, 127.7, 99.4, 92.4, 79.5, 77.9,
75.0, 74.8, 74.6, 73.5, 69.1, 68.6, 67.1, 57.5, 54.2, 52.7; HR-ESI-MS
[M þ H]þ calcd for C41H44Cl3N2O10 829.2056, found 829.2055.
tetrahydro-2H-pyran-2-yl)-L-threoninate 5
By following the general procedure, gluco donor 1 (103.1 mg,
0.15 mmol) was combined with threonine acceptor 34 (26.7 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 7:3 hexane/ethyl acetate as the
eluant afforded 5 (73.4 mg, 87%) as a white powder: 1H NMR
4.3.2. N-((2S,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)
methyl)-2-(phenylthio)tetrahydro-2H-pyran-3-yl)-2,2,2-
trichloroacetamide 447
(400 MHz, CDCl3)
d 7.26e7.35 (m 18 H), 7.19e7.21 (m, 2 H), 7.04 (d,
Triethylamine (16.5 mL, 116.0 mmol) was added to a stirred
suspension of D-galactosamine hydrochloride (12.5 g, 58.0 mmol)
1 H, J ¼ 7.6 Hz), 5.75 (d, 1 H, J ¼ 8.7 Hz), 5.09e5.16 (m, 2 H), 4.91 (d,
1 H, J ¼ 8.2 Hz), 4.78e4.83 (m, 2 H), 4.69 (d, 1 H, J ¼ 10.1 Hz), 4.60 (d,
1 H, J ¼ 10.1 Hz), 4.55 (d, 1 H, J ¼ 12.0 Hz), 4.48 (d, 1 H, J ¼ 12.1 Hz),
4.40e4.42 (m, 1 H), 4.35 (dd, 1 H, J ¼ 2.6 and 8.8 Hz), 4.14 (t, 1 H,
J ¼ 9.2 Hz), 3.71e3.76 (m, 2 H), 3.68 (s, 3 H), 3.42e3.52 (m, 2 H), 2.17
(water peak), 1.22 (d, 3 H, J ¼ 6.0 Hz); 13C NMR (125 MHz, CDCl3)
in methanol (100 mL) at 0 ꢀC. Trichloroacetyl chloride (6.5 mL,
58.0 mmol) was then added by drops. The suspension was warmed
to room temperature and stirred for 48 h. The reaction mixture was
filtered through a plug of Celite, washed with MeOH, and then
concentrated. The residue was dissolved in pyridine (100 mL) and
cooled to 0 ꢀC. Acetic anhydride (50 mL) was added by drops, and
the solution was allowed to warm to room temperature and stirred
for 16 h. The reaction mixture was filtered through a Celite
plug, concentrated, and dissolved in ethyl acetate (150 mL).
The organic solution was washed sequentially with 1 M hydro-
chloric acid (3 ꢁ 50 mL), saturated aqueous sodium bicarbonate
(3 ꢁ 50 mL), and brine (100 mL), and then dried and concentrated.
The residue was purified by flash chromatography eluting with
1:3 ethyl acetate/hexane to provide 15.0 g of the 2-deoxy-2-
trichloroacetamidogalactopyranose tetra-acetate.
d
170.8, 161.9, 156.8, 138.0, 138.0, 137.9, 136.4, 128.59, 128.56, 128.5,
128.48, 128.2, 128.1, 128.0, 127.9, 127.86, 127.8, 127.78, 97.3, 92.5,
79.7, 78.4, 75.0, 74.8, 74.5, 73.7, 68.7, 67.1, 57.8, 58.6, 52.5, 17.4; HR-
ESI-MS [M þ H]þ calcd for C42H46Cl3N2O10 843.2213, found
843.2208.
4.3.4. Benzyl N-((benzyloxy)carbonyl)-O-((2R,3R,4R,5S,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)
tetrahydro-2H-pyran-2-yl)- -serinate 6
L
By following the general procedure, gluco donor 1 (103.1 mg,
0.15 mmol) was combined with serine acceptor 35 (32.9 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 7:3 hexane/ethyl acetate as the
eluant afforded 6 (81.6 mg, 90%) as a white powder: 1H NMR
A
stirred solution of the 1,3,4,6-tetra-O-acetyl-2-deoxy-2-
trichloroacetamido- -galactopyranose described above (15.0 g,
b-D
30.4 mmol) in dry dichloromethane (100 mL) was treated sequen-
tially and dropwise with thiophenol (4.1 mL, 39.6 mmol) and
(400 MHz, CDCl3) d 7.25e7.33 (m, 23 H), 7.18e7.20 (m, 2 H), 6.93 (d,

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Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
1 H, J ¼ 7.6 Hz), 5.71 (d, 1 H, J ¼ 8.3 Hz), 5.16e5.18 (m, 2 H),
5.06e5.12 (m, 2 H), 4.84 (d, 1 H, J ¼ 7.7 Hz), 4.78 (d, 1 H, J ¼ 5.0 Hz),
4.75 (d, 1 H, J ¼ 5.0 Hz), 4.67 (d, 1 H, J ¼ 11.0 Hz), 4.58 (d, 1 H,
J ¼ 11.4 Hz), 4.54 (d, 1 H, J ¼ 11.4 Hz), 4.47 (d, 1 H, J ¼ 12.1 Hz), 4.35
(dd, 1 H, J ¼ 2.9 and 10.1 Hz), 4.06 (t, 1 H, J ¼ 9.0 Hz), 3.81 (dd, 1 H,
J ¼ 3.0 and 10.1 Hz), 3.68e3.72 (m, 2 H), 3.46e3.55 (m, 2 H), 1.64
5.68 (d, J ¼ 8.7 Hz, 1 H); 5.06e5.15 (m, 3 H); 4.93 (d, J ¼ 8.2 Hz, 1 H);
4.85 (d, J ¼ 11.3 Hz), 4.63 (d, 1 H, J ¼ 11.3 Hz), 4.54 (d, 1 H,
J ¼ 11.3 Hz), 4.49 (d,1 H, J ¼ 11.3 Hz), 4.38e4.42 (m,1 H), 4.34 (d,1 H,
J ¼ 11.3 Hz), 4.33 (d, 1 H, J ¼ 11.3 Hz), 4.16 (dd, 1 H, J ¼ 2.6 and
11.0 Hz), 3.99 (d, 1 H, J ¼ 2.2 Hz), 3.57e3.70 (m, 2 H), 3.66e3.53 (m,
2 H), 1.61 (water peak), 1.18 (d, 3 H, J ¼ 6.3 Hz); 13C NMR (125 Hz,
CDCl3): 170.1, 161.9, 156.8, 138.4, 137.9, 137.5, 136.4, 135.5, 128.7,
128.6, 128.5, 128.3, 128.26, 128.2, 128.14, 128.1, 128.02, 128.0, 127.89,
127.8, 127.7, 97.1, 92.6, 77.0, 74.9, 74.4, 73.5, 73.5, 72.6, 72.3, 68.1,
(water peak); 13C NMR (125 MHz, CDCl3)
d 169.7, 162.0, 156.1, 138.0,
137.9, 137.8, 136.3, 135.5, 128.7, 128.6, 128.58, 128.5, 128.49, 128.4,
128.2, 128.19, 128.1, 128.0, 127.97, 127.94, 127.9, 127.88, 127.8, 99.4,
92.5, 79.6, 78.1, 77.5, 75.1, 75.0, 74.7, 73.7, 69.3, 68.7, 67.4, 67.2, 57.9,
54.4, 29.8; HR-ESI-MS [M þ H]þ calcd for C47H48Cl3N2O10 905.2369,
found 905.2358.
67.2, 67.0, 58.9, 55.9, 17.2; HR-ESI-MS [M
48H50Cl3N2O10 919.2526, found 919.2520.
þ
H]þ calcd for
C
4.3.8. Benzyl O-((2R,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6-
4.3.5. Benzyl N-((benzyloxy)carbonyl)-O-((2R,3R,4R,5S,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)
((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)tetrahydro-2H-
pyran-2-yl)-N-(tert-butoxycarbonyl)- -serinate 10
L
tetrahydro-2H-pyran-2-yl)-
L
-threoninate 7
By following the general procedure, galacto donor 4 (103.1 mg,
0.15 mmol) was combined with serine acceptor 37 (29.5 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 7:3 hexane/ethyl acetate as the
eluant afforded 10 (76.8 mg, 88%) as a white powder: 1H NMR
By following the general procedure, gluco donor 1 (103.1 mg,
0.15 mmol) was combined with threonine acceptor 36 (34.3 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 7:3 hexane/ethyl acetate as the
eluant afforded 7 (79.1 mg, 86%) as a white powder: 1H NMR
(400 MHz, CDCl3)
d 7.24e7.38 (m, 24 H), 7.12e7.16 (m, 1 H), 6.87 (d,
(400 MHz, CDCl3)
d
7.24e7.35 (m, 23 H), 7.17e7.20 (m, 2 H), 6.89 (d,
1 H, J ¼ 7.0 Hz), 5.38 (d, 1 H, J ¼ 8.6 Hz), 5.23 (d, 1 H, J ¼ 12.6 Hz), 5.11
(d,1 H, J ¼ 12.6 Hz), 4.91 (d,1 H, J ¼ 11.2 Hz), 4.89 (d,1 H, J ¼ 11.2 Hz),
4.66 (d, 1 H, J ¼ 11.3 Hz), 4.58 (d, 1 H, J ¼ 11.2 Hz), 4.52 (d, 1 H,
J ¼ 11.3 Hz), 4.43e4.47 (m, 2 H), 4.36 (dd, 1 H, J ¼ 2.5 and 9.8 Hz),
4.22 (dd, 1 H, J ¼ 2.2 and 11.0 Hz), 4.03 (d, 1 H, J ¼ 2.0 Hz), 3.57e3.75
(m, 6 H), 1.67 (water peak), 1.41 (s, 9 H); 13C NMR (125 Hz, CDCl3)
1 H, J ¼ 7.5 Hz), 5.73 (d, 1 H, J ¼ 8.9 Hz), 5.19 (d, 1 H, J ¼ 12.4 Hz),
5.11e5.14 (m, 2 H), 4.89 (d, 1 H, J ¼ 8.1 Hz), 4.79 (t, 2 H, J ¼ 10.6 Hz),
4.66 (d, 1 H, J ¼ 11.0 Hz), 4.57 (d, 1 H, J ¼ 11.0 Hz), 4.45e4.48 (m,
2 H), 4.36e4.40 (m, 1 H), 4.10 (t, 1 H, J ¼ 9.9 Hz), 3.63e3.70 (m, 2 H),
3.33e3.39 (m, 2 H), 1.71 (water peak), 1.21 (app d, 2 NH, J ¼ 6.3 Hz);
13C NMR (125 MHz, CDCl3)
d
170.2, 161.9, 156.8, 138.0, 138.0, 137.9,
d 170.1, 162.1, 155.5, 138.4, 137.8, 137.5, 135.6, 128.7, 128.6, 128.41,
136.4, 135.6, 128.6, 128.59, 128.55, 128.5, 128.4, 128.39, 128.2,
128.16,128.1,128.0,127.94,127.9,127.8,127.78,127.7, 97.1, 92.5, 79.6,
78.4, 75.1, 75.0, 74.8, 74.5, 73.6, 68.6, 67.3, 67.1, 58.9, 58.7, 17.4; HR-
ESI-MS [M þ H]þ calcd for C48H50Cl3N2O10 919.2526, found
919.2521.
128.37, 128.13, 128.12, 128.1, 128.0, 127.9, 127.8, 99.4, 92.6, 80.1, 77.3,
75.0, 73.7, 72.6, 72.4, 69.6, 68.3, 67.2, 55.9, 53.9, 28.4; HR-ESI-MS
[M þ H]þ calcd for C44H50Cl3N2O10 871.2526, found 871.2525.
4.3.9. Benzyl N-(((benzyloxy)carbonyl)-
((2R,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-
(2,2,2-trichloroacetamido)tetrahydro-2H-pyran-2-yl)- -serinate 11
L-valyl)-O-
4.3.6. Benzyl N-((benzyloxy)carbonyl)-O-((2R,3R,4R,5R,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)
L
By following the general procedure, gluco donor 1 (103.1 mg,
0.15 mmol) was combined with Val-Ser acceptor 38 (42.8 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 6:4 hexane/ethyl acetate as the
eluant afforded 11 (80.4 mg, 80%) as a white powder: 1H NMR
tetrahydro-2H-pyran-2-yl)- -serinate 8
L
By following the general procedure, galacto donor 4 (103.1 mg,
0.15 mmol) was combined with serine acceptor 37 (32.9 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 7:3 hexane/ethyl acetate as the
eluant afforded 8 (76.1 mg, 84%) as a white powder: 1H NMR
(400 MHz, CDCl3)
d
7.84 (d, 1 H, J ¼ 8.8 Hz), 7.14e7.37 (m, 23 H),
7.14e7.16 (m, 2 H), 6.75 (d, 1 H, J ¼ 7.4 Hz), 5.37 (d, 1 H, J ¼ 8.1 Hz),
5.18 (s, 2 H), 5.11 (d, 1 H, J ¼ 12.5 Hz), 5.05 (d, 1 H, J ¼ 12.5 Hz),
4.19e4.77 (m, 2 H), 4.66 (d, 1 H, J ¼ 10.7 Hz), 4.59 (d, 1 H,
J ¼ 12.1 Hz), 4.52 (d, 1 H, J ¼ 12.1 Hz), 4.49 (d, 1 H, J ¼ 12.1 Hz), 4.36
(dd, 1 H, J ¼ 1.4 and 11.3 Hz), 3.98 (dd, 1 H, J ¼ 9.1 and 18.0 Hz),
3.87e3.90 (m, 2 H), 3.76 (t, 1 H, J ¼ 8.6 Hz), 3.66e3.71 (m, 2 H),
3.24e3.27 (d, 1 H, J ¼ 9.1 Hz), 2.00e2.05 (m, 1 H), 0.98 (d, 3 H,
(400 MHz, CDCl3)
d 7.26e7.37 (m, 23 H), 7.16e7.18 (m, 2 H), 6.82 (d,
1 H, J ¼ 7.0 Hz), 5.66 (d, 1 H, J ¼ 8.3 Hz), 5.20 (d, 1 H, J ¼ 12.5 Hz), 5.14
(d, 1 H, J ¼ 12.6 Hz), 5.10 (d, 1 H, J ¼ 12.4 Hz), 5.05 (d, 1 H,
J ¼ 12.3 Hz), 4.87e4.91 (m, 2 H), 4.65 (d, 1 H, J ¼ 11.3 Hz), 4.57 (d,
1 H, J ¼ 11.3 Hz), 4.47e4.52 (m, 2 H), 4.43 (d,1 H, J ¼ 11.5 Hz), 4.42 (d,
1 H, J ¼ 11.5 Hz), 4.33 (dd, 1 H, J ¼ 2.7 and 10.1 Hz), 4.17 (dd, 1 H,
J ¼ 2.5 and 11.0 Hz), 4.02 (d, 1 H, J ¼ 2.3 Hz), 3.73e3.78 (m, 2 H),
3.56e3.64 (m, 3 H), 1.61 (water peak); 13C NMR (125 MHz, CDCl3)
J ¼ 6.7 Hz), 0.97 (d, 3 H, J ¼ 6.7 Hz); 13C NMR (125 Hz, CDCl3)
d 171.2,
169.0, 162.0, 156.8, 138.1, 138.0, 137.8, 136.0, 135.1, 128.8, 128.7,
128.6, 128.5, 128.44, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7,
127.3, 101.9, 93.1, 81.9, 78.0, 75.2, 75.1, 74.6, 73.5, 68.9, 68.6, 67.8,
67.2, 61.5, 57.1, 53.1, 30.5, 19.4, 18.7; HR-ESI-MS [M þ H]þ calcd for
d
169.7, 162.1, 156.1, 138.3, 137.8, 137.5, 136.2, 135.4, 128.6, 128.57,
128.53, 128.5, 128.4, 128.3, 128.2, 128.1, 128.09, 128.0, 127.9, 127.8,
127.79, 127.6, 99.4, 92.5, 77.3, 74.9, 73.7, 73.6, 72.5, 72.4, 69.2, 68.3,
67.3, 67.1, 55.7, 54.3; HR-ESI-MS [M þ H]þ calcd for C47H48Cl3N2O10
905.2369, found 905.2352.
C53H57Cl3N3O10 1004.3035, found 1004.3225.
4.3.10. Benzyl N-(((benzyloxy)carbonyl)-
((2R,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-
(2,2,2-trichloroacetamido)tetrahydro-2H-pyran-2-yl)- -serinate 12
L-valyl)-O-
4.3.7. Benzyl N-((benzyloxy)carbonyl)-O-((2R,3R,4R,5R,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(2,2,2-trichloroacetamido)
L
tetrahydro-2H-pyran-2-yl)-
L
-threoninate 9
By following the general procedure, galacto donor 4 (103.1 mg,
0.15 mmol) was combined with Val-Ser acceptor 38 (42.8 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 6:4 hexane/ethyl acetate as the
eluant afforded 12 (78.4 mg, 78%) as a white powder: 1H NMR
By following the general procedure, galacto donor 4 (103.1 mg,
0.15 mmol) was combined with threonine acceptor 36 (34.3 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 7:3 hexane/ethyl acetate as the
eluant afforded 9 (78.2 mg, 85%) as a white powder: 1H NMR
(400 MHz, CDCl3)
d
7.50 (d,1 H, J ¼ 8.4 Hz), 7.26e7.37 (m, 25 H), 6.75
(400 MHz, CDCl3)
d
7.20e7.35 (m, 25 H), 6.84 (d, 1 H, J ¼ 7.1 Hz, 1 H);
(d, 1 H, J ¼ 7.4 Hz), 5.39 (d, 1 H, J ¼ 8.2 Hz), 5.16 (s, 2 H), 4.96 (s, 1 H),

Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
2897
4.90 (d, 1 H, J ¼ 11.4 Hz), 4.64 (d, 1 H, J ¼ 11.5 Hz), 4.56e4.60 (m,
2 H), 4.48 (d, 1 H, J ¼ 11.6 Hz), 4.40e4.46 (m, 2 H), 4.32 (d, 1 H,
J ¼ 10.8 Hz), 4.21 (dd, 1 H, J ¼ 8.8 and 18.8 Hz), 3.97 (t, 1 H,
J ¼ 7.2 Hz), 3.93 (s, 1 H), 3.85 (dd, 1 H, J ¼ 2.0 and 10.7 Hz), 3.69 (dd,
1 H, J ¼ 2.0 and 10.8 Hz), 3.60 (t, 1 H, J ¼ 8.4 Hz), 3.53 (t, 1 H,
J ¼ 5.3 Hz), 3.36 (t, 1 H, J ¼ 6.1 Hz), 2.64 (broad, 1 H), 2.03e2.08 (m,
1 H), 1.27 (broad, 1 H), 0.95e0.98 (d, J ¼ 6.7 Hz, 6 H); 13C NMR
aq hydrochloric acid. The reaction was purged 3 times with a
balloon filled with hydrogen gas, and then stirred under positive
hydrogen pressure for 12 h. The reaction was then filtered through
Celite and concentrated to give the known48 glycosylated serine
product 15 (25.2 mg, 91% yield) as its hydrochloride: 1H NMR
(400 MHz, DMSO)
d 8.34 (trace of phthlate contaminant), 7.92 (d,
1 H, J ¼ 6.3 Hz), 4.43 (d, 1 H, J ¼ 6.6 Hz), 4.16 (s, 1 H), 4.05 (dd, 1 H,
J ¼ 2.9 and 9.1 Hz), 3.48e3.96 (broad, 4 H), 3.95 (d, 1 H, J ¼ 8.4 Hz),
3.68 (d, 1 H, J ¼ 9.5 Hz), 3.47 (dd, 1 H, J ¼ 3.6 and 9.1 Hz), 3.39 (t, 1 H,
J ¼ 7.4 Hz), 3.30 (t, 1 H, J ¼ 6.8 Hz), 3.07e3.11 (m, 2 H), 1.84 (s, 3 H);
(125 Hz, CDCl3)
d 171.2, 169.1, 162.2, 156.6, 138.4, 137.9, 137.6, 136.1,
135.2, 128.7, 128.62, 128.6, 128.5, 128.4, 128.35, 128.3, 128.24, 128.2,
128.0, 127.9, 127.8, 127.5, 101.4, 93.1, 74.7, 73.6, 73.55, 72.2, 71.8,
68.6, 68.4, 67.6, 66.9, 61.1, 54.7, 53.0, 30.8, 19.4, 18.4; HR-ESI-MS
[M þ H]þ calcd for C53H57Cl3N3O11 1004.3053, found 1004.3033.
13C NMR (125 MHz, CD3OD)
d 167.3102.2, 78.1, 75.6, 71.8, 66.9, 62.4,
56.7, 54.2, 23.0; HR-ESI-MS [M þ H]þ calcd for C11H21N2O8
309.1292, found 309.1292.
4.3.11. Benzyl N-(((benzyloxy)carbonyl)-
L-valyl)-O-
((2R,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-
(2,2,2-trichloroacetamido)tetrahydro-2H-pyran-2-yl)-L-
serylglycinate 13
By following the general procedure, gluco donor 1 (103.1 mg,
0.15 mmol) was combined with Val-Ser-Gly acceptor 39 (48.5 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 6:4 hexane/ethyl acetate as the
eluant afforded 13 (61.6 mg, 58%) as a white powder: 1H NMR
4.4.2. O-((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)- -threonine 16
L
Hydrogenolysis of 7 (79.1 mg, 0.086 mmol) by following the
same procedure as for 15 gave the known23 glycosylated threonine
product 16 (25.9 mg, 93% yield) as its hydrochloride: 1H NMR
(400 MHz, CD3OD)
d
4.45 (d, 1 H, J ¼ 8.2 Hz), 4.25 (t, 1 H, J ¼ 6.0 Hz),
3.92 (d, 1 H, J ¼ 11.8 Hz), 3.83 (d, 1 H, J ¼ 5.0 Hz), 3.62e3.69 (m, 2 H),
3.44e3.47 (m, 1 H), 3.30 (overlap with CD3OD, 2 H), 2.00 (s, 3 H),
(400 MHz, CDCl3) d 7.26e7.34 (m, 23 H), 7.19e7.20 (m, 2 H), 7.09 (m,
1.35 (d, J ¼ 5.8 Hz, 3 H); 13C NMR (125 MHz, CD3OD)
d 169.6, 101.0,
1 H), 6.78 (d, 1 H, J ¼ 5.3 Hz), 5.43 (d, 1 H, J ¼ 6.3 Hz), 5.14 (s, 2 H),
5.08e5.11 (m, 2 H), 4.77e4.81 (m, 4 H), 4.68e4.70 (m, 2 H),
4.55e4.57 (m, 2 H), 4.46 (d, 1 H, J ¼ 9.4 Hz), 3.48e4.07 (m, 5 H), 3.89
(dd, 1 H, J ¼ 3.5 and 14.7 Hz), 3.70e3.81 (m, 6 H), 3.55 (d, 1 H,
J ¼ 6.5 Hz), 2.05e2.09 (m, 1 H),1.86 (broad, 1 H), 0.84e0.91 (m, 6 H);
77.8, 75.2, 74.1, 71.8, 62.3, 58.5, 57.1, 22.9, 18.4; HR-ESI-MS [M þ H]þ
calcd for C12H23N2O8 323.1449, found 323.1447.
4.4.3. O-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-L-serine 17
13C NMR (125 Hz, CDCl3)
d
171.7, 169.6, 169.5, 162.2, 156.7, 137.9,
137.8, 136.2, 135.3, 128.8, 128.7, 128.64, 128.6, 128.5, 128.4, 128.1,
128.04, 128.0, 127.97, 127,9, 100.6, 92.7, 80.4, 78.1, 75.2, 74.9, 74.8,
73.6, 68.9, 68.6, 67.3, 61.0, 57.8,52.6, 41.5, 30.8,19.4,18.0; HR-ESI-MS
[M þ H]þ calcd for C54H60Cl3N4O12 1061.3268, found 1061.3246.
Hydrogenolysis of 8 (71.6 mg, 0.079 mmol) by following the
same procedure as for 15 gave the known9 glycosylated serine
product 17 (21.3 mg, 87% yield) as its hydrochloride: 1H NMR
(400 MHz, CD3OD)
d
4.41 (d, 1 H, J ¼ 8.4 Hz), 4.09e4.13 (m, 2 H),
3.96e4.01 (m, 3 H), 3.71e3.84 (m, 2H), 3.58 (dd, 1 H, J ¼ 2.9 and
4.3.12. Benzyl N-(((benzyloxy)carbonyl)-L-valyl)-O-
10.6 Hz), 3.54 (dd, 1 H, J ¼ 4.6 and 6.9 Hz), 2.00 (s, 3 H); 13C NMR
((2R,3R,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-
(125 MHz, CD3OD)
d 170.1, 168.3, 102.5, 77.0, 72.8, 69.6, 66.8, 62.6,
(2,2,2-trichloroacetamido)tetrahydro-2H-pyran-2-yl)-
L
-
60.7, 53.5, 23.1; HR-ESI-MS [M þ H]þ calcd for C11H21N2O8
serylglycinate 14
309.1292, found 309.1292.
By following the general procedure, galacto donor 4 (103.1 mg,
0.15 mmol) was reacted with Val-Ser-Gly acceptor 39 (48.5 mg,
0.10 mmol) at room temperature for 2 h. After work-up, purification
by thin-layer chromatography with 6:4 hexane/ethyl acetate as the
eluant afforded 14 (64.8 mg, 61%) as a white powder: 1H NMR
4.4.4. O-((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)- -threonine 18
L
Hydrogenolysis of 9 (78.2 mg, 0.085 mmol) by following the
same procedure as for 15 gave the known9 glycosylated threonine
product 18 (23.6 mg, 86% yield) as its hydrochloride: 1H NMR
(400 MHz, CDCl3)
d
7.25e7.37 (m, 25 H), 7.11 (d, 1 H, J ¼ 4.4 Hz), 6.99
(t, 1 H, J ¼ 4.2 Hz), 6.73 (d, 1 H, J ¼ 5.4 Hz), 5.28 (d, 1 H, J ¼ 6.4 Hz),
5.14 (s, 2 H), 5.09 (d, 1 H, J ¼ 9.7 Hz), 5.04 (d, 1 H, J ¼ 9.6 Hz), 4.89 (d,
1 H, J ¼ 9.1 Hz), 4.81 (d, 1 H, J ¼ 5.6 Hz), 4.66 (d, 1 H, J ¼ 9.2 Hz), 4.58
(d, 1 H, J ¼ 9.1 Hz), 4.51 (d, 1 H, J ¼ 9.2 Hz), 4.49 (d, 1 H, J ¼ 9.1 Hz),
4.44 (d, 1 H, J ¼ 9.4 Hz), 4.12 (dd, 1 H, J ¼ 3.7 and 8.1 Hz), 4.01e4.08
(m, 5 H), 3.96 (dd, 1 H, J ¼ 4.2 and 15.9 Hz), 3.64e3.71 (m, 4 H),
2.07e2.13 (m, 1 H), 1.69 (broad, 1 H), 0.95 (d, 3 H, J ¼ 5.3 Hz), 0.89 (d,
(400 MHz, CD3OD)
d
4.40 (d, 1H, J ¼ 8.3 Hz), 4.20 (t, 1 H, J ¼ 6.3 Hz),
3.91 (dd, 1 H, J ¼ 8.6 and 10.0 Hz), 3.70e3.81 (m, 4 H), 3.57 (dd, 1 H,
J ¼ 2.3 and 10.8 Hz), 3.50e3.53 (m, 1 H), 1.98 (s, 3 H), 1.32 (d, 1 H,
J ¼ 5.3 Hz); 13C NMR (125 MHz, CD3OD)
d 174.6, 169.5, 101.4, 76.9,
73.9, 72.4, 69.6, 62.6, 58.7, 54.0, 22.9, 18.4; HR-ESI-MS [M þ H]þ
calcd for C12H23N2O8 323.1449, found 323.1449.
3 H, J ¼ 5.3 Hz); 13C NMR (125 Hz, CDCl3)
d 171.5, 169.6, 169.5, 162,4,
156.6, 138.4, 137.8, 137.5, 136.3, 135.3, 128.8, 128.7, 128.68, 128.63,
128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 100.5, 92.8, 78.0,
77.4, 74.8, 73.7, 73.7, 72.3, 72.0, 68.3, 67.3, 67.2, 60.7, 55.3, 52.6, 41.6,
31.1, 19.4, 17.9; HR-ESI-MS [M þ H]þ calcd for C54H60Cl3N4O12
1061.3268, found 1061.3242.
4.4.5. N-(L-valyl)-O-((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-
6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)- -serine 19
L
Hydrogenolysis of 11 (80.4 mg, 0.080 mmol) by following the
same procedure as for 15 gave 19 (21.9 mg, 67% yield) as its hy-
drochloride: 1H NMR (400 MHz, CD3OD)
d
5.47 (d, 1 H, J ¼ 0.8 Hz),
4.65 (t, 1 H, J ¼ 4.6 Hz), 4.44 (d, 1H, J ¼ 8.4 Hz), 4.10 (dd, 1 H, J ¼ 6.8
and 10.6 Hz), 3.90 (dd, 1 H, J ¼ 4.3 and 9.2 Hz), 3.87 (d, 1 H,
J ¼ 12.4 Hz), 3.74 (d, 1 H, J ¼ 6.0 Hz), 3.60e3.68 (m, 2 H), 3.40e3.44
(m, 1 H), 3.32 (d, 1 H, J ¼ 0.9 Hz), 2.16e2.24 (m, 1 H), 1.99 (s, 3 H),
4.4. Deprotection procedures
4.4.1. O-((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-
L
-serine 15
1.08 (t, 3 H, J ¼ 7.5 Hz); 13C NMR (125 MHz, CD3OD)
d 174.4, 172.2,
A solution of 81.5 mg (0.090 mmol) of glycopeptide 6 in 3.0 mL
of a 5:1 t-butanol/water mixture was treated with 40.0 mg of
palladium hydroxide (20 wt % on activated carbon) and 20 mL of 1 N
169.6, 102.9, 78.1, 75.6, 72.0, 69.1, 62.6, 59.8, 57.1, 54.0, 31.6, 23.1,
18.8, 18.0; HR-ESI-MS [M þ H]þ calcd for C16H30N3O9 408.1977,
found 408.1978.

2898
Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
4.5. N-glycosylations
16.0 Hz), 2.78 (dd, 1 H, J ¼ 4.0 and 16.0 Hz); 13C NMR (125 MHz,
CDCl3)
d 172.5, 171.1, 156.4, 136.4, 131.7, 128.7, 128.3, 128.2, 118.8,
4.5.1. (2S,3R,4S,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-
67.2, 66.4, 50.9, 37.2; LC-ESI-MS [MþH]þ calcd for C15H19N2O5
2-(phenylthio)tetrahydro-2H-pyran-3-yl benzoate 2056
307.13, found 307.10.
A
solution of 33% HBr solution in acetic acid (3.18 mL,
53.1 mmol) was added dropwise to a solution of -glucopyranose
a-D
pentabenzoate (6.20 g, 8.85 mmol) in 50 mL of dry dichloro-
methane. The reaction mixture was stirred under nitrogen for 16 h,
and then diluted with 100 mL of dichloromethane. The organic
solution was washed sequentially with 100 mL each of water,
saturated aq sodium bicarbonate, and water. The organic phase was
dried and concentrated. A solution of the residue in 50 mL of
nitromethane was stirred with molecular sieves (4 Å, 1.50 g) under
nitrogen for 1 h. The flask was then covered with foil, and treated
4.5.3. (2R,3R,4S,5R,6R)-2-((S)-4-(allyloxy)-3-(((benzyloxy)
carbonyl)amino)-4-oxobutanamido)-4,5-bis(benzyloxy)-6-
((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl benzoate 22
By following the general procedure, gluco donor 20 (97.0 mg,
0.15 mmol) was combined with asparagine acceptor 21 (30.6 mg,
0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by thin-layer
chromatography with 6:4 hexane/ethyl acetate as the eluant
afforded 22 (70.95 mg, 82%) as a white powder: 1H NMR (400 MHz,
sequentially with
g
-collidine (1.50 mL, 11.36 mmol), dry methanol
CDCl3)
d
7.97 (d, 2 H, J ¼ 7.2 Hz), 7.58 (t, 1 H, J ¼ 7.2 Hz), 7.42e7.45
(0.34 mL, 8.9 mmol), and tert-butylammonium bromide (5.0 mmol,
1.62 g). After 16 h, triethylamine (0.4 mL) was added, the solid was
removed by filtration, and the filtrate was washed with 100 mL of
saturated aq sodium bicarbonate. The aqueous layer was further
extracted with dichloromethane (2 ꢁ 50 mL). The combined
organic extracted was washed with water, dried, and then
concentrated. The residue was debenzoylated and benzylated by
following the literature procedure.57 The resulting product was
purified by column chromatography on silica gel, eluting with 3:7
ethyl acetate/hexane, to give the known orthoester (3.57 g, 71%
yield) as a light yellow gel.
The orthoester described above (3.57 g, 6.28 mmol) was com-
bined with molecular sieves (4 Å, 1.5 g) and dry acetonitrile
(40 mL), and the resulting mixture was stirred under nitrogen for
30 min. Thiophenol (2.35 g, 21.3 mmol) and mercuric(II) bromide
(0.076 g, 0.211 mmol) were added sequentially, and the mixture
was heated at reflux for 2.5 h. The reaction mixture was cooled,
filtered, and then concentrated. A solution of the residue in
dichloromethane (20 mL) was washed sequentially with 1% aq so-
dium hydroxide (30 mL) and water (2 ꢁ 30 mL), dried, and then
concentrated. The residue was purified by column chromatography
on silica gel, eluting with 1:9 ethyl acetate/hexane, to give thio-
glycoside donor 20 (3.04 g, 75% yield) as a white powder: 1H NMR
(m, 2 H), 7.27e7.34 (m, 15 H), 7.10e7.17 (m, 5 H), 6.49 (d, 1 H,
J ¼ 9.2 Hz), 5.90 (d, 1 H, J ¼ 9.2 Hz), 5.54e5.61 (m, 1H), 5.22 (t, 1 H,
J ¼ 9.2 Hz), 5.13 (t, 1 H, J ¼ 9.2 Hz), 5.05e5.08 (m, 3H), 5.00 (d, 1 H,
J ¼ 10.6 Hz), 4.80 (d, 1 H, J ¼ 4.5 Hz), 4.77 (d, 1 H, J ¼ 4.5 Hz), 4.72 (d,
1 H, J ¼ 10.5 Hz), 4.65 (d, 1 H, J ¼ 10.5 Hz),4.47e4.55 (m, 4 H), 4.35
(dd, 1 H, J ¼ 5.8 and 13.1 Hz), 4.20 (dd, 1 H, J ¼ 5.8 and 13.5 Hz), 3.90
(t, 1 H, J ¼ 9.5 Hz), 3.86 (t, 1 H, J ¼ 9.5 Hz), 3.74e3.79 (m, 2 H), 2.85
(dd, 1 H, J ¼ 4.3 and 16.9 Hz), 2.69 (dd, 1 H, J ¼ 4.3 and 16.9 Hz), 1.64
(broad, 1 H); 13C NMR (125 MHz, CDCl3)
d 170.6, 170.4, 167.0, 156.2,
138.0, 137.8, 137.78, 136.4, 133.8, 131.5, 130.0, 129.1, 128.7, 128.6,
128.57, 128.56, 128.5, 128.2, 128.17, 128.21, 128.1, 128.06, 128.0,
127.94, 127.9, 118.4, 83.1, 78.5, 77.7, 77.4, 76.8, 75.7, 75.3, 73.8, 73.6,
68.1, 67.1, 66.1, 50.5, 37.8; HR-ESI-MS [M
49H51N2O11 843.3487, found 843.3464.
þ
H]þ calcd for
C
4.5.4. 2,2,2-Trichloroethyl ((2S,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-
((benzyloxy)methyl)-2-(phenylthio)tetrahydro-2H-pyran-3-yl)
carbamate 2319
A solution of the commercial
b-D-thioglycoside triacetate,
(2R,3S,4R,5R,6S)-2-(acetoxymethyl)-6-(phenylthio)-5-(((2,2,2-
trichloroethoxy)carbonyl)amino)tetrahydro-2H-pyran-3,4-diyl
diacetate, (2.5 g, 4.36 mmol) in methanol (50 mL) was treated with
sodium methoxide (24 mg, 0.44 mmol). The reaction mixture was
allowed to stir overnight, and then was neutralized with Dowex
50x8-100 acidic resin. The resin was removed by filtration, and the
filtrate was concentrated. The residue was dried azeotropically with
toluene (3 ꢁ 5 mL) and then was taken to the next step without
further purification.
(400 MHz, CDCl3)
d 8.03e8.05 (m, 2 H), 7.61e7.8 (m, 1H), 7.44e7.50
(m, 4 H), 7.19e7.36 (m, 13 H), 7.11e7.14 (m, 5 H), 5.29 (dd, 1 H, J ¼ 7.6
and 8.0 Hz), 4.82 (d, 1 H, J ¼ 9.2 Hz), 4.79 (d, 1 H, J ¼ 8.4 Hz), 4.73 (d,
1 H, J ¼ 8.8 Hz), 4.64 (d, 1 H, J ¼ 8.4 Hz), 4.56e4.61 (m, 3 H),
3.82e3.87 (m, 2 H), 3.73e3.78 (m, 2 H), 3.61e3.64 (m, 1 H), 1.56
(broad, 1 H); 13C NMR (125 MHz, CDCl3)
d 165.4, 138.4138.1, 137.8,
133.4, 133.1, 132.7, 130.1, 130.0, 129.0, 128.6, 128.6, 128.5, 128.4,
128.2, 128.2,127.9, 127.9, 127.8, 86.3, 84.5, 79.7, 75.5, 75.3, 73.7, 72.6,
69.2; LC-ESI-MS [MþNa]þ calcd for C40H38O6SNa 669.23, found
669.19.
A solution of the above triol (approximately 4.36 mmol) in
dimethylformamide (20 mL) was treated with 60% sodium hydride
suspension in mineral oil (697 mg, 17.44 mmol), and the resulting
mixture was stirred at 0 ꢀC for 30 min. Benzyl bromide (2.6 mL,
21.80 mmol) was added, and the mixture was stirred at room
temperature for 24 h. The reaction mixture was carefully quenched
with 100 mL of saturated aqueous ammonium chloride, and the
mixture was extracted with ethyl acetate (2 ꢁ 50 mL). The com-
bined extract was dried and then concentrated to a residue, which
was purified by column chromatography on silica gel, eluting with
1:9 ethyl acetate/hexane, to give the thioglycoside donor 23 (2.0 g,
4.5.2. Allyl ((benzyloxy)carbonyl)-
-asparaginate 2119
L
A stirred solution of N-(benzyloxycarbonyl)-L-asparagine (2.0 g,
7.51 mmol) in 35 mL dimethylformamide was treated with allyl
bromide (0.78 mL, 9.01 mmol) and sodium iodide (1.64 g,
11.0 mmol). After 24 h, the reaction mixture was partitioned be-
tween ethyl acetate (150 mL) and water (150 mL). The aqueous
layer was further extracted with ethyl acetate (2 ꢁ 50 mL), and the
combined organic extract was washed sequentially with 1.2 M aq
hydrochloric acid (100 mL), saturated aq sodium bicarbonate
(100 mL), and brine (100 mL), dried, and then concentrated. The
residue was purified by chromatography on silica gel eluting with
1:3 ethyl acetate/hexane to afford 21 (1.79 g, 78%) as a white
64% yield) as
a
white powder: 1H NMR (400 MHz, CDCl3)
d
7.52e7.54 (m, 2H), 7.21e7.35 (m, 18 H), 5.09 (d, 1 H, J ¼ 8.5 Hz),
4.95 (d, 1 H, J ¼ 10.5 Hz), 4.70e4.84 (m, 5 H), 4.61e4.63 (m, 2 H),
4.55 (d, 1 H, J ¼ 12.0 Hz), 3.89 (t, 1 H, J ¼ 8.5 Hz), 3.79 (d, 1 H,
J ¼ 11.0 Hz), 3.75 (dd, 1 H, J ¼ 4.0 and 11.5 Hz), 3.66 (t, 1 H,
J ¼ 9.0 Hz), 3.58 (broad, 1 H), 3.45e3.48 (m, 1 H); 13C NMR
powder: 1H NMR (400 MHz, CDCl3)
d
7.39e7.31 (m, 5 H), 6.03 (d,
(125 MHz, CDCl3) d 153.9, 138.4, 138.1, 132.2, 129.1, 128.6, 128.6,
1 H, J ¼ 4.0 Hz), 5.94e5.86 (m, 1 H), 5.57 (broad, 1 H), 5.45 (broad,
1 H), 5.32 (d, 1 H, J ¼ 16.0 Hz), 5.25 (d, 1 H, J ¼ 4.0 Hz), 5.13 (s, 2 H),
4.66 (d, 2 H, J ¼ 4.0 Hz), 4.64e4.62 (m, 1 H), 3.00 (dd, 1 H, J ¼ 4.0 and
128.5, 128.2, 128.1, 128.0, 127.95, 127.8, 127.7, 85.9, 82.4, 79.4, 78.6,
77.4, 75.4, 75.0, 74.6, 73.6, 69.1, 56.8; LC-ESI-MS [MþNa]þ calcd for
C36H36Cl3NO6SNa 738.12, found 738.29.

Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
2899
4.5.5. (2R,3R,4S,5R,6R)-4,5-bis(benzyloxy)-2-((S)-3-(((benzyloxy)
carbonyl)amino)-4-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-
yl)amino)-4-oxobutanamido)-6-((benzyloxy)methyl)tetrahydro-
2H-pyran-3-yl benzoate 24
By following the general procedure, gluco donor 20 (97.0 mg,
0.15 mmol) was combined with Asp-Val acceptor 40 (42.1 mg,
0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by thin-layer
chromatography with 6:4 hexane/ethyl acetate as the eluant
afforded 24 (53.7 mg, 56%) as a white powder: 1H NMR (400 MHz,
10 H), 7.11e7.19 (m, 8 H), 6.09 (s, 1 H), 5.51 (t,1 H, J ¼ 9.8 Hz), 5.39 (d,
1 H, J ¼ 6.4 Hz), 5.31 (d, 1 H, J ¼ 9.3 Hz), 4.96 (d, 1 H, J ¼ 5.7 Hz), 4.84
(d, 1 H, J ¼ 10.7 Hz), 4.80 (d, 1 H, J ¼ 10.7 Hz), 4.72 (d, 1 H,
J ¼ 11.2 Hz), 4.66 (d, 1 H, J ¼ 12.2 Hz), 4.58 (d, 1 H, J ¼ 10.7 Hz), 4.51
(d, 1 H, J ¼ 12.2 Hz), 4.37 (t, 1 H, J ¼ 6.8 Hz), 3.97 (t, 1 H, J ¼ 8.8 Hz),
3.92 (t, 1 H, J ¼ 9.3 Hz), 3.82 (dd, 1 H, J ¼ 2.4 and 10.7 Hz), 3.77 (d,
1 H, J ¼ 10.7 Hz), 3.71 (d, 1 H, J ¼ 9.3 Hz), 3.57 (dd, 1 H, J ¼ 7.3 and
10.7 Hz), 3.43 (dd, 1 H, J ¼ 7.3 and 10.7 Hz), 1.79 (q, 2 H, J ¼ 7.3 Hz),
1.73 (broad, 1 H), 1.65 (q, 2 H, J ¼ 7.3 Hz), 1.01 (t, 3 H, J ¼ 7.3 Hz), 0.90
(t, 3 H, J ¼ 7.3 Hz), 0.81 (s, 9 H), e0.09 (s, 6 H); 13C NMR (125 MHz,
CDCl3)
d
7.98 (d, 2 H, J ¼ 7.8 Hz), 7.56 (t, 1 H, J ¼ 7.3 Hz), 7.42 (t, 2 H,
J ¼ 7.8 Hz), 7.11e7.35 (m, 20 H), 6.99 (d, 1 H, J ¼ 8.7 Hz), 6.78 (d, 1 H,
J ¼ 9.2 Hz), 6.26 (d, 1 H, J ¼ 7.3 Hz), 5.23 (t, 1 H, J ¼ 9.2 Hz), 5.16 (t,
1 H, J ¼ 9.2 Hz), 5.07 (d, 1 H, J ¼ 12.0 Hz), 5.02 (d, 1 H, J ¼ 12.0 Hz),
4.80 (d, 1 H, J ¼ 10.6 Hz), 4.79 (d, 1 H, J ¼ 11.1 Hz), 4.74 (d, 1 H,
J ¼ 11.1 Hz), 4.65 (d, 1 H, J ¼ 12.1 Hz), 4.53 (d, 1 H, J ¼ 10.6 Hz), 4.48
(d, 1 H, J ¼ 12.1 Hz), 4.43 (m, 1H), 4.05 (m, 1H), 3.92 (dd, 1 H, J ¼ 8.6
and 9.2 Hz), 3.85 (dd, 1 H, J ¼ 8.6 and 9.2 Hz), 3.77 (dd, 1 H, J ¼ 2.4
and 10.6 Hz), 3.73 (d, 1 H, J ¼ 10.6 Hz), 3.61 (d, 1 H, J ¼ 9.6 Hz), 2.79
(dd, 1 H, J ¼ 3.9 and 16.0 Hz), 2.60 (dd, 1 H, J ¼ 5.8 and 16.0),
1.95e1.99 (m, 1H), 1.67 (broad, 1 H), 1.43 (s, 9H), 0.76 (d, 3 H,
J ¼ 7.3 Hz), 0.73 (d, 3 H, J ¼ 6.8 Hz); 13C NMR (125 MHz, CDCl3)
CDCl3) d 166.2, 158.7, 158.0, 138.1, 137.9, 137.8, 133.5, 130.0, 129.3,
128.6, 128.55, 128.5, 128.4, 128.1, 128.07, 128.0, 127.96, 127.9, 127.8,
118.0, 101.9, 93.6, 89.8, 84.6, 83.3, 82.5, 78.2, 77.7, 77.4, 77.0, 75.6,
75.2, 73.8, 73.4, 68.3, 63.4, 29.5, 29.2, 25.9, 18.3, 8.5, 7.8, 0.1, ꢂ5.3;
HR-ESI-MS [M þ H]þ calcd for C53H67N4O11Si 963.4570, found
963.4539.
4.5.8. Allyl N2-((benzyloxy)carbonyl)-N4-((2R,3R,4R,5S,6R)-4,5-
bis(benzyloxy)-6-((benzyloxy)methyl)-3-(((2,2,2-trichloroethoxy)
carbonyl)amino)tetrahydro-2H-pyran-2-yl)- -asparaginate 27
L
By following the general procedure, gluco donor 23 (107.5 mg,
0.15 mmol) was combined with asparagine acceptor 21 (30.6 mg,
0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by thin-
layer chromatography with 6:4 hexane/ethyl acetate as eluant
afforded 27 (55.7 mg, 61%) as a white powder: 1H NMR (400 MHz,
d
171.9, 170.5, 170.2, 167.0, 156.3, 138.0, 137.83, 137.8, 136.2, 133.6,
130.1, 129.3, 128.6, 128.5, 128.4, 128.2, 128.15, 128.1, 128.0, 127.99,
127.95,127.9, 127.86, 83.3, 81.9, 78.5, 77.4, 76.7, 75.6, 75.2, 73.7, 73.6,
68.2, 67.2, 57.7, 51.4, 37.4, 31.2, 28.1, 18.8, 17.6; HR-ESI-MS [M þ H]þ
calcd for C55H64N3O12 958.4485, found 958.4455.
CDCl3)
d
7.40e7.17 (m, 20 H), 6.89 (d, 1 H, J ¼ 8.3 Hz), 5.98 (d, 1 H,
J ¼ 9.2 Hz), 5.87e5.79 (m, 1 H), 5.26 (dd, 1 H, J ¼ 1.5 and 16.9 Hz),
5.18 (dd, 1 H, J ¼ 1.5 and 10.7 Hz), 5.13 (d, 1 H, J ¼ 12.1 Hz), 5.08 (d,
1 H, J ¼ 12.1 Hz), 4.84e4.89 (m, 2 H), 4.79 (d, 1 H, J ¼ 10.6 Hz), 4.76
(d, 1 H, J ¼ 12.1 Hz), 4.64e4.70 (m, 3 H), 4.57e4.59 (m, 2 H), 4.55
(d, 1 H, J ¼ 10.6 Hz), 4.47 (d, 1 H, J ¼ 7.6 Hz), 3.81 (t, 1 H, J ¼ 9.2 Hz),
3.69e3.76 (m, 2 H), 3.59 (dd, 1 H, J ¼ 7.2 and 9.2 Hz), 3.41e3.48 (m,
2 H), 2.86 (dd, 1 H, J ¼ 4.3 and 16.9 Hz), 2.68 (dd, 1 H, J ¼ 4.4 and
4.5.6. tert-Butyl (6S,9S,12S)-9-(2-(((2R,3R,4S,5R,6R)-3-
(benzoyloxy)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-
2H-pyran-2-yl)amino)-2-oxoethyl)-1,1,1-trichloro-6,12-diisopropyl-
4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate 25
By following the general procedure, gluco donor 20 (97.0 mg,
0.15 mmol) was combined with Val-Asp-Val acceptor 41 (56.2 mg,
0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by thin-layer
chromatography with 6:4 hexane/ethyl acetate as the eluant
afforded 25 (39.6 mg, 36%) as a white powder: 1H NMR (400 MHz,
16.9 Hz), 1.87 (broad, 2 H); 13C NMR (125 MHz, CDCl3)
d 170.8,
156.2, 156.0, 137.8, 137.5, 136.4, 131.7, 129.1, 128.8, 128.6,
128.55, 128.23, 128.2, 128.1, 127.9, 118.6, 95.4, 80.12, 80.1, 78.4,
77.4, 76.7, 75.1, 74.9, 74.6, 73.8, 68.1, 67.2, 66.3, 55.8, 50.6, 37.8; HR-
ESI-MS [M þ H]þ calcd for C45H49Cl3N3O11 912.2427, found
912.2420.
CDCl3)
d
7.99 (d, 2 H, J ¼ 9.6 Hz), 7.56e7.63 (m, 2 H), 7.43 (t, 2 H,
J ¼ 9.5 Hz), 7.26e7.33 (m, 7 H), 6.94e7.16 (m, 7 H), 6.95 (d, 1 H,
J ¼ 10.5 Hz), 5.17e5.23 (m, 2 H), 4.72e4.80 (m, 3 H), 4.67 (d, 1 H,
J ¼ 9.9 Hz), 4.64 (d, 1 H, J ¼ 10.0 Hz), 4.52 (d, 1 H, J ¼ 13.8 Hz), 4.48
(d, 1 H, J ¼ 15.2 Hz), 4.05 (dd, 1 H, J ¼ 6.8 and 10.0 Hz), 3.90e3.97 (m,
2 H), 3.82 (t, 1 H, J ¼ 11.7 Hz), 3.75 (broad, 1 H), 3.63 (d, 1 H,
J ¼ 12.0 Hz), 2.70 (dd, 1 H, J ¼ 3.6 and 16.0 Hz), 2.54 (dd, 1 H, J ¼ 7.4
and 16.0 Hz), 2.43 (broad, 2 H), 2.06e2.10 (m, 1H), 1.93e1.98 (m,
1 H), 1.43 (s, 9 H), 0.93 (d, 3 H, J ¼ 8.4 Hz), 0.87 (d, 3 H, J ¼ 8.4 Hz),
0.77 (d, 3 H, J ¼ 8.6 Hz), 0.74 (d, 3 H, J ¼ 8.6 Hz); 13C NMR (125 MHz,
4.5.9. tert-Butyl N2-((benzyloxy)carbonyl)-N4-((2R,3R,4R,5S,6R)-
4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-(((2,2,2-
trichloroethoxy)carbonyl)amino)tetrahydro-2H-pyran-2-yl)-
L-
asparaginyl- -valinate 28
L
By following the general procedure, gluco donor 23 (107.5 mg,
0.15 mmol) was combined with Val-Asp acceptor 40 (42.1 mg,
0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by thin-layer
chromatography with 6:4 hexane/ethyl acetate as the eluant
afforded 28 (46.3 mg, 45%) as a white powder: 1H NMR (400 MHz,
CDCl3)
d 172.5, 170.9, 170.3, 169.7, 167.1, 154.8, 138.0, 137.8, 133.6,
130.2, 129.5, 128.6, 128.57, 128.5, 128.2, 128.1, 128.0, 127.96, 127.9,
95.6, 83.2, 81.9, 78.5, 77.7, 77.4, 76.7, 75.7, 75.2, 74.8, 73.8, 73.8, 73.6,
68.3, 60.5, 57.9, 49.9, 37.2, 31.4, 31.1, 28.2, 19.3, 18.8, 17.7, 17.6; HR-
ESI-MS [M
1097.3801.
þ
H]þ calcd for C55H67N4O13 1097.3843, found
CDCl3)
d 7.28e7.40 (m, 18 H), 7.18e7.19 (m, 2 H), 7.02 (d, 1 H,
J ¼ 8.2 Hz), 6.43 (d, 1 H, J ¼ 7.3 Hz), 5.13 (d, 1 H, J ¼ 12.0 Hz), 5.09 (d,
1 H, J ¼ 12.0 Hz), 4.90 (d, 1 H, J ¼ 12.1 Hz), 4.86 (dd, 1 H, J ¼ 8.2 and
9.2 Hz), 4.85 (d, 1 H, J ¼ 12.0 Hz), 4.80 (d, 1 H, J ¼ 11.1 Hz), 4.62e4.70
(m, 4 H), 4.57 (d, 1 H, J ¼ 11.1 Hz), 4.48 (broad, 1H), 4.47 (d, 1 H,
J ¼ 12.1 Hz), 4.28 (dd, 1 H, J ¼ 4.9 and 8.7 Hz), 3.80 (t, 1 H, J ¼ 9.2 Hz),
3.67e3.76 (m, 2 H), 3.60 (m, 1 H), 3.47e3.49 (m, 2 H), 2.78 (dd, 1 H,
J ¼ 3.4 and 16.4 Hz), 2.58 (dd,1 H, J ¼ 5.8 and 16.4 Hz), 2.05e2.10 (m,
1 H), 1.44 (s, 9 H), 0.82 (d, 6 H, J ¼ 6.8 Hz); 13C NMR (125 MHz,
4.5.7. (2R,3R,4S,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-
2-(1-((3aR,4R,6R,6aR)-6-(((tert-butyldimethylsilyl)oxy)methyl)-
2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1H-1,2,4-triazole-
3-carboxamido)tetrahydro-2H-pyran-3-yl benzoate 26
By following the general procedure, gluco donor 20 (97.0 mg,
0.15 mmol 1.5 equiv) was combined with protected ribavirin
acceptor 42 (42.6 mg, 0.10 mmol) at 0 ꢀC for 2 h. After work-up,
purification by thin-layer chromatography with 8:2 hexane/ethyl
acetate as the eluant afforded 26 (50.1 mg, 52%) as a white powder:
CDCl3)
d 172.0, 170.6, 170.4, 156.1, 137.9, 137.8, 137.5, 136.2, 129.0,
128.8, 128.7, 128.6, 128.5, 128.3, 128.2, 128.17, 128.1, 127.9, 95.5, 82.0,
80.0, 78.5, 77.4, 76.7, 75.14, 75.1, 74.6, 73.8, 68.2, 67.3, 58.0, 55.9,
51.4, 37.6, 31.3, 28.2, 19.0, 17.7; HR-ESI-MS [M þ H]þ calcd for
1H NMR (400 MHz, CDCl3)
J ¼ 9.3 Hz), 7.55 (t, 1 H, J ¼ 7.3 Hz), 7.39e7.42 (m, 2 H), 7.27e7.37 (m,
d
7.96 (d, 2 H, J ¼ 7.8 Hz), 7.87 (d, 1 H,
C51H62Cl3N4O12 1027.3424, found 1024.3408.

2900
Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
4.5.10. tert-Butyl N4-((2R,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-
((benzyloxy)methyl)-3-(((2,2,2-trichloroethoxy)carbonyl)amino)
tetrahydro-2H-pyran-2-yl)-N2-(((2,2,2-trichloroethoxy)carbonyl)-
(10 mg, 0.075 mmol), and 1-(3-dimethyl aminopropyl)-3-
ethylcarbodiimide hydrochloride (12 mg, 0.075 mmol). After 3 h,
the reaction mixture was partitioned between dichloromethane
(10 mL) and 1.2 M hydrochloric acid (10 mL). The aqueous layer was
further extracted with dichloromethane (2 ꢁ 5 mL). The combined
organic extract was washed sequentially with saturated aq sodium
bicarbonate (10 mL) and brine (10 mL), dried, and concentrated.
The residue was purified by preparative reverse phase medium
pressure chromatography, eluting with 10%e100% acetonitrile/
water. The fraction containing product were conbined and dried by
lypholizer to give the glycosylated tripeptide 31 (39 mg,
0.032 mmol, 84% over 2 steps) as a white powder: 1H NMR
L
-valyl)-L-asparaginyl-L-valinate 29
By following the general procedure, gluco donor 23 (107.5 mg,
0.15 mmol) was combined with the Val-Asp-Val acceptor 41
(42.1 mg, 0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by
thin-layer chromatography with 6:4 hexane/ethyl acetate as the
eluant afforded 29 (37.4 mg, 32%) as a white powder: 1H NMR
(400 MHz, CDCl3)
d
7.76 (d, 1 H, J ¼ 8.7 Hz), 7.18e7.40 (m, 15 H), 5.66
(d, 1 H, J ¼ 10.1 Hz), 4.79e4.91 (m, 4 H), 4.63e4.71 (m, 4 H), 4.56 (d,
1 H, J ¼ 13.5 Hz), 4.48 (d, 1 H, J ¼ 15.1 Hz), 4.24 (dd, 1 H, J ¼ 5.8 and
10.5 Hz), 4.09 (t, 1 H, J ¼ 7.1 Hz), 3.60e3.81 (m, 4 H), 3.46 (d, 1 H,
J ¼ 11.3 Hz), 2.72 (dd, 1 H, J ¼ 3.8 and 20.0 Hz), 2.54 (dd, 1 H, J ¼ 7.5
and 19.8 Hz), 2.06e2.18 (m, 2 H), 1.85 (broad, 2 H), 1.44 (s, 9 H), 0.98
(d, 3 H, J ¼ 8.4 Hz), 0.93 (d, 3 H, J ¼ 8.4 Hz), 0.85e0.86 (m, 6 H); 13C
(400 MHz, CDCl3)
d
7.13e7.34 (m, 28 H), 7.05 (d, 1 H, J ¼ 8.7 Hz), 6.97
(d, 1 H, J ¼ 8.0 Hz), 6.32 (d, 1 H, J ¼ 8.6 Hz), 5.49 (d, 1 H, J ¼ 8.3 Hz),
5.20 (d, 1 H, J ¼ 12.7 Hz), 5.07e5.15 (m, 2 H), 4.99 (t, 1 H, J ¼ 9.2 Hz),
4.89 (d, 1 H, J ¼ 12.2 Hz), 4.79e4.83 (m, 1 H), 4.75 (d, 1 H,
J ¼ 12.3 Hz), 4.72 (d, 1 H, J ¼ 12.3 Hz), 4.71 (d, 1 H, J ¼ 12.3 Hz), 4.60
(d, 1 H, J ¼ 12.3 Hz), 4.57 (d, 1 H, J ¼ 12.2 Hz), 4.51 (d, 1 H,
J ¼ 10.8 Hz), 4.49 (d, 1 H, J ¼ 12.4 Hz), 4.44 (d, 1 H, J ¼ 12.5 Hz), 4.37
(d, 1 H, J ¼ 12.3 Hz), 4.11 (dd, 1 H, J ¼ 6.6 and 16.8 Hz), 3.86 (dd, 1 H,
J ¼ 4.0 and 10.0 Hz), 3.60e3.76 (m, 4 H), 3.46e3.54 (m, 2 H), 2.91
(dd, 1 H, J ¼ 3.3 and 16.1 Hz), 2.72 (broad, 2 H), 2.55 (q, 1 H, J ¼ 7.6);
NMR (125 MHz, CDCl3) d 172.5,170.9,170.4,170.0, 156.1, 137.9, 137.8,
137.5, 129.1, 128.8, 128.6, 128.58, 128.2, 128.1, 128.0, 95.5, 82.0, 80.1,
80.0, 78.5, 77.4, 76.7, 75.3, 75.1, 74.8, 74.6, 73.8, 68.2, 60.6, 58.2, 55.8,
49.9, 37.1, 31.4, 31.2, 28.2, 19.4, 19.0, 17.9, 17.7; HR-ESI-MS [M þ H]þ
calcd for C51H66Cl6N5O13 1166.2783, found 1166.2765.
4.5.11. 2,2,2-Trichloroethyl ((2R,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-
((benzyloxy)methyl)-2-((4-methylphenyl)sulfonamido)tetrahydro-
2H-pyran-3-yl)carbamate 30
By following the general procedure, donor 23 (107.5 mg,
0.15 mmol) was combined with 4-methylbenzenesulfonamide
(17.1 mg, 0.10 mmol) at 0 ꢀC for 2 h. After work-up, purification by
thin-layer chromatography with 4:1 hexane/ethyl acetate as the
eluant afforded 30 (56.0 mg, 72%) as a white powder: 1H NMR
13C NMR (125 Hz, CDCl3)
d 171.9, 171.4, 170.3, 169.1, 156.0, 138.0,
137.8, 137.78, 137.3, 136.0, 135.2, 128.8, 128.75, 128.7, 128.6, 128.58,
128.56, 128.4, 128.3, 128.28, 128.2, 128.1, 128.0, 127.9, 127.89, 95.6,
81.2, 79.8, 78.2, 77.4, 76.7, 75.1, 74.7, 73.7, 73.3, 69.3, 68.3, 67.7, 67.6,
56.2, 52.7, 43.1; LC-ESI-MS [MþH]þ calcd for C61H65Cl3N5O14
1196.35, found 1196.42.
4.6.2. Benzyl O-benzyl-N-(tert-butoxycarbonyl)-L-serinate58
(400 MHz, CDCl3)
d
7.77 (m 2 H), 7.17e7.39 (m, 17 H), 6.03 (d, 1 H,
A stirred solution of commercial N-(tert-butoxycarbonyl)-O-
benzyl-L-threonine (2.00 g, 6.46 mmol) in dimethylformamide
(15 mL) at 0 ꢀC was treated sequentially with triethylamine
(0.90 mL, 6.46 mmol) and benzyl bromide (0.76 mL, 6.46 mmol).
After 12 h, the reaction mixture was diluted with ethyl acetate
(100 mL), and the solution was washed successively with aq citric
acid (100 mL), saturated aq sodium bicarbonate (100 mL), and brine
(100 mL), dried, and then concentrated in vacuo. The residue was
purified by chromatography on silica, eluting with 1:9 ethyl ace-
tate/hexane, to afford the ester (1.10 g, 85% yield) as colorless syrup:
1H NMR (400 MHz, CDCl3) 7.21e7.32 (m, 10 H), 5.43 (d, 1 H,
J ¼ 8.7 Hz), 5.23 (d,1 H, J ¼ 12.4 Hz), 5.14 (d, 1 H, J ¼ 12.4 Hz), 4.50 (d,
1 H, J ¼ 12.0 Hz), 4.43 (d, 1 H, J ¼ 12.1 Hz), 3.91 (dd, 1 H, J ¼ 2.6 and
9.4 Hz), 3.69 (dd, 1 H, J ¼ 2.9 and 9.3 Hz), 1.44 (s, 9 H); 13C NMR
(125 MHz, CDCl3) 170.4,155.4,137.4,135.4,128.4,128,3,128.2,128.0,
127.6, 127.5, 79.7, 73.1, 70.0, 67.0, 54.1, 28.2. LC-ESI-MS [MþH]þ
calcd for C22H28NO5 386.20, found 386.18.
J ¼ 8.9 Hz), 4.87 (d, 1 H, J ¼ 7.8 Hz), 4.83 (d, 1 H, J ¼ 11.6 Hz), 4.79 (d,
1 H, J ¼ 10.9 Hz), 4.75 (d, 1 H, J ¼ 12.0 Hz), 4.63e4.70 (m, 3 H), 4.58
(d,1 H, J ¼ 11.0 Hz), 4.43 (d,1 H, J ¼ 12.1 Hz), 4.31 (d,1 H, J ¼ 12.1 Hz),
3.63e3.69 (m, 2 H), 3.51e3.61 (m, 2 H), 3.38e3.43 (m, 2 H), 2.35 (s,
3 H); 13C NMR (125 MHz, CDCl3)
d 155.8, 143.4, 138.9, 138.0, 137.8,
137.5, 129.4, 128.9, 128.6, 128.54, 128.5, 128.1, 127.9, 127.8, 127.3,
95.3, 84.4, 80.5, 78.3, 76.5, 75.1, 74.9, 74.8, 73.8, 68.4, 56.1, 40.5, 21.6;
LC-ESI-MS [MþH]þ calcd for C37H39Cl3N2O8S 777.15, found 777.13.
4.6. Glycoprotein core tripeptide synthesis
4.6.1. Benzyl O-benzyl-N-N2-((benzyloxy)carbonyl)-N4-
((2R,3R,4R,5S,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)-3-
(((2,2,2-trichloroethoxy)carbonyl)amino)tetrahydro-2H-pyran-2-
yl)-L-asparaginylglycyl-L-serinate 31
A solution of glycosylated asparagine 27 (35.0 mg, 0.038 mmol)
and N-methylaniline (8.2 mg, 0.077 mmol) in tetrahydrofuran
(1 ml) was treated with tetrakis(triphenylphosphine)palladium(0)
4.6.3. Benzyl O-benzyl-N-glycyl-L-serinate trifluoroacetate salt
A solution of benzyl O-benzyl-N-(tert-butoxycarbonyl)-L-seri-
nate (see above; 1.10 g, 2.85 mmol) in trifluoroacetic acid (5 mL)
and dichloromethane (20.0 mL) was stirred at room temperature
for 3 h, and then concentrated. The residue was used for the next
reaction without further purification.
A stirred and cooled (ice bath) solution of the crude tri-
fluoroacetate salt described above (approximately 2.85 mmol) and
(tert-butoxycarbonyl)glycine (509 mg, 2.90 mmol) in dry dichloro-
methane and dry dimethylformamide (10:1, 28.0 mL) was treated
sequentially with 1-hydroxybenzotriazole (579 mg, 4.27 mmol),
N,N-diisopropylethylamine (1.25 mL, 8.55 mmol), and 1-(3-
(4.0 mg, 3.8 mmol). After 15 min, the reaction mixture was
concentrated, the residue was dissolved in 2 mL of 10:1 dimethyl
sulfoxide/water, and the resulting solution was purified by pre-
parative reverse phase medium pressure chromatography, eluting
with a gradient from 10% to 100% acetonitrile/water. The fractions
containing product were combined and dried by lypholizer to
afford 33 mg of the carboxylic acid containing a trace amount of
triphenylphosphine.
The preparation of the dipeptide coupling partner benzyl O-
benzyl-N-glycyl-L-serinate 2,2,2-trifluoroacetate salt is given
following this paragraph. A stirred and cooled (ice bath) solution of
the carboxylic acid described above (33 mg, 0.037 mmol) and
benzyl O-benzyl-N-glycyl-L-serinate 2,2,2-trifluoroacetate salt
(34 mg, 0.075 mmol) in dry dichloromethane and dry dime-
thylformamide (10:1, 1 mL) was treated sequentially with N,N-dii-
sopropylethylamine (15 mg, 0.113 mmol), 1-hydroxybenzotriazole
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(818 mg, 4.27 mmol). After 13 h, the reaction mixture was dilated
with dichloromethane (50 mL), and the organic solution was
washed with 1 M hydrochloric acid (50 mL). The aqueous layer was
further extracted with dichloromethane (2 ꢁ 50 mL), and the

Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
2901
combined organic extract was washed sequentially with saturated
aq sodium bicarbonate and brine, dried, and then concentrated. The
residue was purified by using a prepacked C-18 reverse-phase
column (80 g), eluting with 5%e100% acetonitrile/water. The frac-
tions containing product were combined and then dried by
lypholizer to afford the dipeptide (945 mg, 75% over two steps),
which was used as obtained for the deprotection step.
J ¼ 4.2 and 12.1 Hz), 3.48 (t, 1 H, J ¼ 8.4 Hz), 3.32 (s, 2 H), 2.90 (dd,
1 H, J ¼ 4.9 and 17.3 Hz), 2.76 (dd, 1 H, J ¼ 7.5 and 17.0 Hz), 1.96 (s,
3 H); 13C NMR (125 Hz, CD3OD)
d 174.5, 173.2, 171.2, 171.0, 169.8,
80.3, 79.8, 76.2, 71.9, 62.8, 62.7, 56.3, 56.2, 51.2, 43.4, 36.9, 22.9;
HR-ESI-MS [M þ H]þ calcd for C17H31N5O11 480.1936, found
480.1938.
The solution of above dipeptide in dichloromethane (10 mL) and
trifluoroactetic acid (2 mL) was stirred for 3 h, and then concen-
trated. The residue was purified by using a prepacked C-18 reverse
phase column (80 g), eluting with 5%e100% acetonitrile/water. The
fractions containing product were combined and then dried by
lypholizer to afford the dipeptide salt (970 mg, 74% over 3 steps): 1H
4.7. Additional acceptors
4.7.1. Methyl ((benzyloxy)carbonyl)-
L
-threoninate 34
This acceptor is available commercially.
4.7.2. Benzyl ((benzyloxy)carbonyl)-
-serinate 3559
L
NMR (400 MHz, CDCl3)
d
7.39e7.31 (m, 5 H), 6.03 (d, 1 H, J ¼ 4.0 Hz),
Esterification of N-Cbz-(L)-serine by following the same pro-
5.94e5.86 (m, 1 H), 5.57 (broad, 1 H), 5.45 (broad, 1 H), 5.32 (d, 1 H,
J ¼ 16.0 Hz), 5.25 (d, 1 H, J ¼ 4.0 Hz), 5.13 (s, 2 H), 4.66 (d, 2 H,
J ¼ 4.0 Hz), 4.64e4.62 (m, 1 H), 3.00 (dd, 1 H, J ¼ 4.0 and 16.0 Hz),
2.78 (dd, 1 H, J ¼ 4.0 and 16.0 Hz); 13C NMR (125 MHz, CDCl3)
cedure as for 36 (below) gave 35 (89% yield) as a white powder: 1H
NMR (400 MHz, CDCl3)
d 7.26e7.35 (m, 10 H), 5.73 (d, 1 H,
J ¼ 6.3 Hz), 5.22 (s, 2 H), 5.12 (s, 2 H), 4.48e4.50 (m, 1 H), 3.95e4.00
(broad, 2 H), 2.17 (broad, 1 H), 1.62 (broad, 1 H); 13C NMR (125 Hz,
d
171.0, 167.5, 138.9, 137.0, 129.5, 129.34, 129.3, 129.2, 128.8, 128.76,
CDCl3) d 170.5, 156.4, 136.2, 135.2, 128.7, 128.6, 128.6, 128.3, 128.3,
74.3, 70.4, 68.2, 54.4, 41.4; HR-ESI-MS [M þ H]þ calcd for
128.2, 67.6, 67.3, 63.3, 56.3; LC-ESI-MS [MþH]þ calcd for C18H19NO5
C
19H23N2O4 343.1652, found 343.1655.
330.35, found 330.36.
4.6.4. Benzyl N-N4-((2R,3R,4R,5S,6R)-3-acetamido-4,5-
4.7.3. Benzyl ((benzyloxy)carbonyl)-
L
-threoninate 3660
bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)-
A
solution of (2S,3R)-2-benzyloxycarbonylamino-3-
N2-((benzyloxy)carbonyl)-
32
L
-asparaginylglycyl-O-benzyl-
L
-serinate
hydroxybutanoic acid (1.0 g, 4.0 mmol) in DMF (15.0 mL) and
deionized water (5.0 mL) was treated with cesium carbonate
(644.5 mg, 2.0 mmol), stirred for 30 min, concentrated, and then
azeotropically dried with toluene (3 ꢁ 10 mL). A solution of benzyl
bromide (0.71 mL, 5.9 mmol) in DMF (10.0 mL) was then added.
After 24 h, water (15 mL) was added, and the mixture was extracted
with ethyl acetate (2 ꢁ 30 mL). The organic extract was dried over
anhydrous sodium sulfate and concentrated. The residue was pu-
rified by flash column chromatography, eluting with 1:3 ethyl ac-
etate/hexane, to give 36 (1.149 g, 84% yield) as a white powder: 1H
A
stirred solution of glycosylated tripeptide 31 (39 mg,
0.032 mmol) in acetic acid (1 mL) was treated with zinc dust
(43.0 mg). After 4 h. The suspension was filtered, the filter cake was
washed with acetic acid (1 mL), and the filtrate was concentrated. A
stirred solution of the residue in pyridine (1 ml) was treated with
acetic anhydride (33 mg, 0.32 mmol). After 24 h, the reaction
mixture was concentrated and then dissolved in dimethylsulfoxide
(1 mL). The solution was by reverse phase preparative medium
pressure chromatography, eluting with a gradient of 10%e100%
acetonitrile/water. The fractions containing product were conbined
and dried by lypholizer to give 32 (29 mg, 0.027 mmol, 85% over 2
NMR (400 MHz, CDCl3)
d 7.26e7.35 (m, 10 H), 5.60 (d, 1 H,
J ¼ 8.7 Hz), 5.17e5.24 (m, 2 H), 5.13 (s, 2 H), 4.37e4.39 (m, 2 H), 1.97
(broad, 1 H), 1.63 (broad, 1 H), 1.23 (d, 1 H, J ¼ 6.3 Hz); 13C NMR
steps) as a white powder: 1H NMR (400 MHz, CDCl3)
d
7.14e7.35 (m,
(125 MHz, CDCl3) d 171.2, 156.9, 136.2, 135.3, 128.6, 128.5, 128.5,
30 H), 7.02 (d, 1 H, J ¼ 7.9 Hz), 6.27 (d, 1 H, J ¼ 8.6 Hz), 5.39 (d, 1 H,
J ¼ 7.2 Hz), 5.17 (d, 1 H, J ¼ 12.2 Hz), 5.12 (d, 1 H, J ¼ 12.2 Hz), 5.11 (d,
1 H, J ¼ 12.2 Hz), 5.07 (d, 1 H, J ¼ 12.2 Hz), 4.75e4.82 (m, 4 H), 4.61
(d, 1 H, J ¼ 12.0 Hz), 4.56 (d, 1 H, J ¼ 12.0 Hz), 4.52 (d, 1 H,
J ¼ 12.2 Hz), 4.48 (d, 1 H, J ¼ 12.1 Hz), 4.45 (d, 1 H, J ¼ 12.0 Hz), 4.39
(d, 1 H, J ¼ 11.9 Hz), 4.08 (d, 1 H, J ¼ 16.9 Hz), 3.86e3.93 (m, 2 H),
3.66e3.77 (m, 5 H), 3.41e3.47 (m, 2 H), 2.91 (dd, 1 H, J ¼ 3.0 and
16.4 Hz), 2.52 (dd, 1 H, J ¼ 4.6 and 16.4 Hz), 2.00 (s, 3 H); 13C NMR
128.2, 128.0, 68.0, 67.4, 67.2, 59.5, 19.9; LC-ESI-MS [MþH]þ calcd for
C19H21NO5 344.14, found 344.03.
4.7.4. Benzyl (tert-butoxycarbonyl)-
L
-serinate 37
This acceptor is available commercially.
4.7.5. Benzyl ((benzyloxy)carbonyl)-L-valyl-L-serinate 38
A
solution of N-(benzyloxycarbonyl)-L-valine
(2.51 g,
(125 Hz, CDCl3)
d
171.5, 171.4, 170.1, 169.0, 168.5, 138.1, 138.0, 137.9,
10.0 mmol) and benzyl L-serinate (1.95 g, 10.0 mmol) in dry
dichloromethane and dry dimethylformamide (10:1, 100 mL) was
treated sequentially with 1-hydroxybenzotriazole (1.61 g,
12.0 mmol), N,N-diisopropylethylamine (3.80 mL, 24.0 mmol), and
1-(3-dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride
(2.29 g, 12.0 mmol) at 0 ꢀC. After stirring for 3 h, the reaction
mixture was poured into a mixture of dichloromethane (100 mL)
and 1.2 M aq hydrochloric acid (150 mL). The aqueous layer was
further extracted with two portions of dichloromethane (50 mL).
The combined organic extract was washed sequentially with
saturated aq sodium bicarbonate (100 mL) and brine (100 mL),
dried over magnesium sulfate, and concentrated. The residue was
purified by flash chromatography on silica gel, eluting with 3:97
methanol/dichloromethane, to afford 38 (3.35 g, 78% yield) as a
137.3, 136.1, 135.3, 128.9, 128.8, 128.7, 128.65, 128.6, 128.57, 128.54,
128.5, 128.4, 128.3, 128.28, 128.2, 128.1, 128.06, 128.0, 127.97, 127.9,
81.4, 78.2, 77.4, 76.8, 75.2, 74.4, 73.7, 73.2, 69.4, 68.3, 67.7, 52.7, 43.2,
23.1; LC-ESI-MS [MþH]þ calcd for C60H66N5O13 1064.47, found
1064.52.
4.6.5. N4-((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-L-asparaginylglycyl-L-
serine 33
A solution of protected tripeptide 32 (25 mg, 0.023 mmol) in
methanol (2 mL) was combined with palladium(II) hydroxide
(0.0046 mmol, 33 mg, 10% on carbon). A hydrogen atmosphere
was supplied with balloon. After 3 h, the suspension was filtered
and the filtrate concentrated to give the deprotected tripeptide
33 (10 mg, 91% yield) as a white powder: 1H NMR (400 MHz,
white powder: 1H NMR (400 MHz, CDCl3)
d 7.26e7.35 (m, 10 H),
6.87 (d, 1 H, J ¼ 7.2 Hz), 5.44 (d, 1 H, J ¼ 8.1 Hz), 5.18e5.24 (m, 2 H),
5.10 (d, 1 H, J ¼ 12.0 Hz), 5.04 (d, 1 H, J ¼ 12.0 Hz), 4.70e4.72 (m,
1 H), 3.90e4.02 (m, 3 H), 2.04e2.13 (m, 1 H), 1.69 (broad, 2 H), 0.97
(d, 3 H, J ¼ 6.6 Hz), 0.93 (d, 3 H, J ¼ 6.6 Hz); 13C NMR (125 Hz, CDCl3)
CD3OD)
d
5.00 (d, 1 H, J ¼ 8.0 Hz), 4.49 (t, 1 H, J ¼ 5.8 Hz), 4.23 (t,
1 H, J ¼ 5.8 Hz), 4.00 (m, 2 H), 3.90 (dd, 1 H, J ¼ 4.5 and 10.9 Hz),
3.84 (d, 2 H, J ¼ 12.2 Hz), 3.72 (t, 1 H, J ¼ 9.9 Hz), 3.64 (dd, 1 H,

2902
Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
d
171.9, 170.3, 157.0, 136.2, 135.2, 128.7, 128.6, 128.6, 128.4, 128.3,
4.7.8. tert-Butyl ((2,2,2-trichloroethoxy)carbonyl)-
asparaginyl- -valinate 41
Palladium-on-carbon (0.30 g, 10 wt%) was added to a solution of
N-(benzyloxycarbonyl)-L-asparaginyl-L-valine 40 (3.00 g,
L-valyl-L-
128.2, 67.6, 67.3, 62.9, 60.7, 54.8, 31.3, 19.3, 18.1; HR-ESI-MS [M þ
L
H]þ calcd for C23H29N2O6 429.2020, found 429.2025.
7.63 mmol) in methanol (76.0 mL). The reaction mixture was stirred
under a hydrogen atmosphere for 3 h, filtered through a pad of
Celite, and then concentrated. The residue was used for the next
reaction without further purification.
4.7.6. Benzyl ((benzyloxy)carbonyl)-L-valyl-L-serylglycinate 39
A solution of dipeptide ester 38 (1.2 g, 2.80 mmol) in methanol
(30 mL) was treated with 2 N sodium hydroxide (2.8 mL,
5.60 mmol) at room temperature. The mixture was stirred for 5 h
and then concentrated. The residue was partitioned between ethyl
acetate (50) mL and 2 N aqueous HCl (50 mL). The organic layer was
separated, dried over Na2SO4, and then concentrated to afford 1.1 g
of the crude dipeptide acid as a white solid that was used directly in
the next step.
A solution of the crude dipeptide acid (1.1 g, 2.80 mmol) and
benzyl glycinate hydrochloride (564 mg, 2.80 mmol) in dry
dichloromethane and dry dimethylformamide (10:1, 30 mL) was
treated sequentially with 1-hydroxybenzotriazole (402 mg,
3.0 mmol), N,N-diisopropylethylamine (1.0 mL, 6.0 mmol) and 1-(3-
A
stirred solution of the above residue (approximately
7.63 mmol) and ((2,2,2-trichloroethoxy)carbonyl)-L-valine (2.67 g,
9.16 mmol) in dry dichloromethane and dry dimethylformamide
(10:1,
76.0 mL)
was
treated
sequentially
with
1-
hydroxybenzotriazole (1.45 g, 10.7 mmol), N,N-diisopropylethyl-
amine (2.24 mL, 15.3 mmol) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (2.05 g, 10.7 mmol) at 0 ꢀC. After
13 h, the reaction mixture was partitioned between dichloro-
methane (100 mL) and 1 M aq hydrochloric acid (100 mL). The
aqueous layer was further extracted with dichloromethane
(2 ꢁ 50 mL), and the combined organic extract was washed
sequentially with saturated aq sodium bicarbonate and brine,
dried, and concentrated. The residue was purified on a C-18
reverse-phase column (80 g), eluting with 5%e100% acetonitrile/
water. The desired fractions were conbined and concentrated by
lypholizer to afford 41 (2.55 g, 59% yield over 2 steps) as a white
dimethyl
aminopropyl)-3-ethylcarbodiimide
hydrochloride
(763 mg, 3.0 mmol) at 0 ꢀC. After stirring for 3 h, the reaction
mixture was poured into a mixture of dichloromethane (100 mL)
and 1.2 M aqueous hydrochloric acid (100 mL). The organic layer
was separated and the aqueous layer was extracted with two
additional portions of dichloromethane (50 mL each). The com-
bined organic extract was washed sequentially with saturated aq
sodium bicarbonate (100 mL) and brine (100 mL), dried over
magnesium sulfate, and then concentrated. The residue was puri-
fied by flash chromatography on silica gel, eluting with 4:96
methanol/dichloromethane, to afford 39 (1.0 g, 74% yield) as a white
powder: 1H NMR (500 MHz, DMSO‑d6)
d
8.16 (d, 1 H, J ¼ 9.5 Hz),
7.79 (d,1 H, J ¼ 10.4 Hz), 7.74 (d,1 H, J ¼ 11.0 Hz), 7.33 (s,1 H), 6.93 (s,
1 H), 4.82 (d, 1 H, J ¼ 15.5 Hz), 4.72 (d, 1 H, J ¼ 15.5 Hz), 4.62 (m, 1 H),
3.99 (dd, 1 H, J ¼ 7.0 and 10.3 Hz), 3.88 (dd, 1 H, J ¼ 9.0 and 10.7 Hz),
2.49 (dd, 1 H, J ¼ 6.7 and 19.4 Hz), 2.40 (app q, 1 H, J ¼ 9.4), 1.99 (m,
1 H), 1.37 (s, 9 H), 0.81e0.89 (m, 12 H); 13C NMR (125 MHz,
powder: 1H NMR (400 MHz, DMSO‑d6)
d
8.30 (t,1 H, J ¼ 5.4 Hz), 7.89
DMSO‑d6) d 171.4, 170.9, 170.6, 170.2, 154.6, 96.1, 80.6, 73.5, 60.5,
(d, 1 H, J ¼ 7.8 Hz), 7.27e7.34 (m, 10 H), 5.20 (s, 2 H), 4.98e5.05 (m,
2 H), 4.86 (t, 1 H, J ¼ 5.6 Hz), 4.33e4.38 (m, 1 H), 3.89e3.95 (m, 3 H),
3.55 (t, 2 H, J ¼ 5.4 Hz), 1.96e2.01 (m, 1 H), 0.84 (d, 3 H, J ¼ 6.7 Hz),
60.0, 57.9, 51.7, 49.3, 37.1, 30.3, 30.0, 27.6, 19.1, 18.8, 18.1, 17.8; HR-
ESI-MS [M þ H]þ calcd for C21H36Cl3N4O7 561.1644, found 561.1650.
0.79 (d, 3 H, J ¼ 6.7 Hz); 13C NMR (125 Hz, DMSO‑d6)
d
171.1, 170.4,
4.7.9. 1-((3aR,4R,6R,6aR)-6-(((tert-butyldimethylsilyl)oxy)methyl)-
2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1H-1,2,4-triazole-
3-carboxamide 42
169.6, 156.2, 137.1, 135.9, 128.4, 128.3, 128.1, 127.9, 127.8, 127.6, 65.9,
65.5, 61.8, 60.1, 64.8, 40.8, 30.4,19.2,17.9; HR-ESI-MS [M þ H]þ calcd
for C25H32N3O7 486.2235, found 486.2244.
A solution of Ribavirin (2.44 g, 10.00 mmol) in 3-pentanone
(20 mL) was treated with trimethyl orthoformate (3.18 g,
30.00 mmol) and 4-methylbenzenesulfonic acid monohydrate
(0.38 g, 2.00 mmol). The mixture was stirred at 50 ꢀC for 2 h, and
then concentrated. A solution of the residue in 10 mL of dimethyl
sulfoxide was chromatographed with a pre-packed C-18 reverse
phase column (120 g), eluting with 1:19 acetonitrile/water. The
fractions containing product were combined and lyophilized to
afford the Ribavirin ketal (2.72 g, 87% yield) as a white powder: 1H
4.7.7. tert-Butyl ((benzyloxy)carbonyl)-L-asparaginyl-L-valinate
4019
A
stirred solution of N-(benzyloxycarbonyl)-L-asparagine
(1.88 g, 7.06 mmol) and L-valine tert-butyl ester hydrochloride
(1.48 g, 7.06 mmol) in dry dichloromethane and dry dime-
thylformamide (10:1, 70 mL) was treated sequentially with 1-
hydroxybenzotriazole (1.14 g, 8.48 mmol), N,N-diisopropylethyl-
amine (2.24 mL, 14.12 mmol) and 1-(3-dimethyl aminopropyl)-3-
ethylcarbodiimide hydrochloride (1.62 g, 8.48 mmol) at 0 ꢀC. After
3 h, the reaction mixture was partitioned between dichloro-
methane (50 mL) and 1.2 M aqueous hydrochloric acid (100 mL).
The aqueous layer was extracted with additional dichloromethane
(2 ꢁ 50 mL), and the combined organic extract was washed
sequentially with saturated aq sodium bicarbonate (50 mL) and
brine (50 mL), dried, and concentrated. The residue was purified by
silica gel flash chromatography eluting with 4:96 methanol/
dichloromethane to afford 40 (2.40 g, 82% yield) as a white powder:
NMR (400 MHz, CD3OD)
d
8.71 (s, 1 H), 6.21 (d, 1 H, J ¼ 1.5 Hz), 5.28
(dd, 1 H, J ¼ 1.5 and 6.3 Hz), 4.96 (dd, 1 H, J ¼ 2.0 and 6.3 Hz), 4.39
(m, 1 H), 3.62 (dd, 1 H, J ¼ 5.4 and 11.7 Hz), 3.56 (dd, 1 H, J ¼ 5.4 and
11.7 Hz), 1.78 (q, 2 H, J ¼ 7.8 Hz), 1.65 (q, 2 H, J ¼ 7.3 Hz), 0.98 (t, 3 H,
J ¼ 7.3 Hz), 0.89 (t, 3 H, J ¼ 7.8 Hz); 13C NMR (125 MHz, CD3OD)
d
163.2, 158.3, 146.7, 118.9, 95.3, 90.7, 86.5, 83.6, 63.3, 30.2, 30.1, 8.7,
8.0; LC-ESI-MS [MþH]þ calcd for C13H21N4O5 313.15, found 313.00.
A solution of the above ketal (2.72 g, 8.71 mmol) in 10 mL of
dimethylformamide was treated sequentially with imidazole
(1.19 g, 17.42 mmol) and tert-butyldimethylsilyl chloride (1.97 g,
13.06 mmol). The mixture was stirred at room temperature for 12 h,
then was diluted with ethyl acetate (100 mL). The solution was
washed sequentially with water (100 mL) and brine (100 mL), dried
over magnesium sulfate, and then concentrated. The residue was
purified by silica gel chromatography, eluting with 1:4 ethyl ace-
tate/hexane, to give 42 (3.53 g, 95% yield) as a white powder: 1H
1H NMR (400 MHz, CDCl3)
d 7.31e7.42 (m, 5H), 6.42 (d, 1 H,
J ¼ 7.2 Hz), 5.91 (s, 1 H), 5.47 (s, 1 H), 5.14 (s, 2 H), 4.58 (m, 1 H), 4.35
(dd, 1 H, J ¼ 4.3 and 8.7 Hz), 2.96 (dd, 1 H, J ¼ 3.4 and 16.0 Hz), 2.62
(dd, 1 H, J ¼ 6.8 and 16.0 Hz), 2.17 (m, 1 H), 1.46 (s, 9 H), 0.91 (d, 3 H,
J ¼ 6.8 Hz), 0.89 (d, 3 H, J ¼ 6.8 Hz); 13C NMR (125 MHz, CDCl3)
d
173.7171.2, 170.5, 156.3, 136.2, 128.4, 128.1, 128.0, 81.8, 66.9, 57.9,
NMR (400 MHz, CDCl3) d 8.37 (s, 1 H), 7.02 (s, 1 H), 6.55 (broad, 1 H),
51.6, 37.0, 31.1, 28.0, 18.9, 17.5; LC-ESI-MS [MþH]þ calcd for
6.05 (s, 1 H), 5.32 (d, 1 H, J ¼ 5.8 Hz), 4.83 (d, 1 H, J ¼ 6.3 Hz), 4.51
C
21H32N3O6 422.23, found 422.13.
(dd, 1 H, J ¼ 4.4 and 4.9 Hz), 3.74 (dd, 1 H, J ¼ 4.4 and 11.2 Hz), 3.64

Y. Zhang, S. Knapp / Tetrahedron 74 (2018) 2891e2903
2903
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(dd, 1 H. J ¼ 4.9 and 11.2 Hz), 1.78 (q, 2 H, J ¼ 7.3 Hz), 1.63 (q, 2 H,
J ¼ 7.3 Hz), 0.97 (t, 3 H, J ¼ 7.3 Hz), 0.89 (t, 3 H, J ¼ 7.3 Hz), 0.82 (s,
9 H), 0.03 (s, 6 H); 13C NMR (125 MHz, CDCl3)
d 163.2, 157.2, 144.0,
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4.7.10. p-Toluenesulfonamide 43
This acceptor is available commercially.
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Acknowledgments
We are grateful to Merck & Co. for financial support, and to
Junling Gao for analytical assistance.
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enumerated. These include a somewhat reduced reactivity compared with
Schmidt donors (see ref. 32), the sulfurous odor that can be associated with the
thiophenol starting material (but not the S-glycosides or the reaction prod-
ucts), and the necessity to synthesize the donor separately rather than use the
reducing sugar or the anomeric acetate directly.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2018.04.082.
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