Generation of a glycosylated asparagine residue through chemoselective acylation of a glycosylhydrazide

Article abstract of DOI:10.1016/j.carres.2020.108022

Herein, we report the first selective anomeric N-acylation of a glycosylhydrazide. We show that this transformation can be harnessed to generate amino acid building blocks including FmocAsn(GlcNAc)OH (1), a residue that has been previously shown to be a c

Full text of DOI:10.1016/j.carres.2020.108022

Journal Pre-proof
Generation of a glycosylated asparagine residue through chemoselective acylation of
a glycosylhydrazide
Katie A. Rykaczewski, Kate E. Sabourin, Paul J. Goo, Lydia H. Griggs, Saumya Jain,
Paxton A.M. Reed, Joseph M. Langenhan
PII:
S0008-6215(20)30097-5
DOI:
https://doi.org/10.1016/j.carres.2020.108022
CAR 108022
Reference:
To appear in: Carbohydrate Research
Received Date: 13 February 2020
Revised Date: 23 April 2020
Accepted Date: 26 April 2020
Please cite this article as: K.A. Rykaczewski, K.E. Sabourin, P.J. Goo, L.H. Griggs, S. Jain, P.A.M.
Reed, J.M. Langenhan, Generation of a glycosylated asparagine residue through chemoselective
acylation of a glycosylhydrazide, Carbohydrate Research (2020), doi: https://doi.org/10.1016/
j.carres.2020.108022.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

1

Generation of a glycosylated asparagine
residue through chemoselective acylation of a
glycosylhydrazide
Katie A. Rykaczewski, Kate E. Sabourin, Paul J. Goo, Lydia H. Griggs, Saumya Jain, Paxton A. M.
Reed, Joseph M. Langenhan,*,†
†Department of Chemistry, Seattle University, Seattle, WA 98122
langenha@seattleu.edu
*To whom correspondence should be addressed: langenha@seattleu.edu
Abstract:
Herein, we report the first selective anomeric N-acylation of a glycosylhydrazide. We show that
this transformation can be harnessed to generate amino acid building blocks including
FmocAsn(GlcNAc)OH (1), a residue that has been previously shown to be a competent
reagent in the solid-phase peptide synthesis of N-linked glycopeptides.
1

1. Introduction
Post-translational N-linked glycosylation can significantly influence protein solubility, folding, and
function.1 Unfortunately, our understanding of the glycoproteome is limited in part by difficulties
associated with synthesizing homogeneous N-linked glycoproteins. In response, scientists have
developed a range of chemical, chemoenzymatic, and biosynthetic methods to synthesize N-
glycosylated proteins, peptides, and amino acids.2 Many approaches employ
success,3 although these intermediates can be prone to mutarotation, dimerization, and hydrolysis
during their generation and use. Several groups have investigated -glycosylamine surrogates that
circumvent some of these limitations while still reacting with activated aspartic acid side chains to form
-N-glycosylasparigines.4 We hypothesized that
-glycosylhydrazides might be advantageous
glycosylamine surrogates, reacting selectively with activated aspartic acid residues to form
glycosylasparigines following N-N bond cleavage (Fig. 1). -Glycosylhydrazides are relatively stable to
β-glycosylamines with
β
β
β
β-
β
-N-
β
hydrolysis,5 do not dimerize, and we imagined they could prove more nucleophilic than the
corresponding amines due to the alpha effect.6 Somewhat similar acylations are well precedented, for
example the acylation of semicarbazides to form azapeptides.7 Moreover, analogous
can be acylated selectively on the anomeric nitrogen atom, even in the presence of unprotected sugar
hydroxyl groups.8 Thus, we set out to determine if we could utilize
-glycosylhydrazides to generate
Fmoc-protected -N-glycosylasparigine building blocks (i.e., FmocAsn(GlcNAc)OH (1) and O-
β-glycosylamines
β
β
peracetylated FmocAsn(GlcNAc)OH (2)) that have been previously shown3a,9 to be competent reagents
in the solid-phase peptide synthesis of N-linked glycopeptides.
Fig. 1. Two anomeric N-acylation strategies for the generation of β-N-glycosylasparigines.
2. Results and discussion
We chose synthetic targets 1 and 2 because they bear the core N-acetylglucosamine residue found
in nearly all protein N-glycans. As a first step toward the validation of our novel strategy (Fig. 1), we set
out to synthesize β-N-acetylglucosylhydrazides and to identify conditions for their selective anomeric N-
acylation. Unprotected, unactivated GlcNAc (5) reacted with benzoyl and p-toluenesulfonyl hydrazides
2

to form closed ring glycosylhydrazides 610 and 7a predominately in the desired
β-pyranose form (Fig.
2). We explored two tactics for the generation of O-peracetylated glycosylhydrazides: acetylating
glycosylhydrazides 6 and 7a, and condensing protected reducing sugar 411 with hydrazides. In both
instances, only low yields of product were generated in the form of complex mixtures containing
β-
pyranose, -pyranose, and open-chain hydrazone isomers. Because it is not possible to resolve
α
isomeric glycosylhydrazide mixtures due to rapid equilibration between isomeric forms,12 we narrowed
our focus to the synthesis of 1 through derivatives 6 and 7a.
OAc
O
AcO
AcO
H
H2N
R
OAc
O
N
OAc
O
R
N
(NH4)2CO3
N
H
NHAc
OAc
OH
H
AcO
AcO
AcO
AcO
OAc
OH
THF/MeOH
85 %
AcOH, H2O
EtOH
low yield,
impure
NHAc
NHAc
AcO
AcO
N
3
4
R
N
H
NHAc
H2N
R
OH
OH
N
H
N
O
H
O
Ac2O, pyr.
low yield
HO
HO
HO
HO
R
OH
N
AcOH, H2O
EtOH or DMF
H
NHAc
NHAc
5
6: R = SO2pTol (81 %, 1:10 ( / ))
7a: R = Bz (96 %, 1:5 ( / ))
Fig. 2. Generation of glycosylhydrazides; attempts to generate O-peracetylated glycosylhydrazides
resulted in inseparable isomeric mixtures.
We next attempted to couple hydrazides 6 and 7a with a model carboxylic acid using COMU under
standard conditions (Fig. 3).13 Hydrazide 6 provided no traces of desired product 8, only mixed isolates
containing 6′-O-acylated and other mono-O-acylated products. Hydrazide 7a did result in a small trace
of 9a, but O-acylated products were again predominant. We next screened a representative coupling
agent from each remaining major class (BOPCl, EDCI/HOBt, EEDQ, T3P, EEDQ) using HPLC to
estimate product and byproduct formation for the conversion of 7a to 9a. These agents provided at
most traces of desired product 9a, as well as varying amounts of O-acylated material (data not shown).
O
OH
O
OH
O
R
R
HN
NH
HN
N
HO
HO
OMe
COMU, DIEA, iPrOH
OH
HO
HO
mono-O-
acylated
products
+
AcHN
AcHN
O
OMe
8: R = SO2pTol (0 %)
9a: R = Bz (trace)
R = SO2pTol (18 %)
R = Bz (9 %)
6: R = SO2pTol
7a: R = Bz
Fig. 3. Attempted anomeric N-acylation of glycosylhydrazides using a uronium-type coupling agent.
Other coupling agents provided similar results (data not shown).
Inspired by a report that unprotected O-glycosides of N-hydroxy-N-arylamines can be selectively N-
acylated using acyl chlorides,8b we attempted the selective anomeric N-acylation of glycosylhydrazides
3

under similar conditions (Fig. 4), reasoning that an “over activated” agent like an acyl chloride might be
required to acylate anomeric hydrazide nitrogen atoms. When we treated 6 and 7a with 4-
methoxyphenylacetyl chloride and NaHCO3 in dioxane, 6 provided only mixtures of O-acylated
products, but 7a provided a 55 % isolated yield of the desired N-acylated product (9a). The utilization of
alternative solvents (THF, CH2Cl2) and bases (DIEA, NMM, TMAP, DMAP) resulted in lower yields. To
our knowledge, our successful generation of 9a represents the first selective anomeric N-acylation of a
glycosylhydrazide, producing a novel class of glycoside products that could ultimately prove useful not
only in amino acid synthesis, but also efforts to glycorandomize natural products.14
O
OH
O
OH
O
R
R
HN
NH
HN
N
HO
HO
OMe
Cl
HO
HO
NaHCO3, 4A sieves
diox.
AcHN
AcHN
O
OMe
8: R = SO2pTol (0 %)
9a: R = Bz (55 %, 1:32 ( / ))
6: R = SO2pTol
7a: R = Bz
Fig. 4. Successful anomeric N-acylation of a glycosylhydrazide using an acyl chloride.
Given this positive result, we turned our attention to the formation of a suitably protected aspartic
acid chloride derivative for use in N-acylations. In our hands, treating FmocAsp(OH)OtBu and
FmocAsp(OH)OBn with thionyl chloride or oxalyl chloride led to the corresponding aspartic anhydrides.
However, treating FmocAsp(OtBu)OPfp with thionyl chloride and TFA resulted in FmocAsp(Cl)OPfp
(11) as previously reported.15 We treated hydrazide 7a with 11 in the presence of NaHCO3 in dioxane.
Gratifyingly, we obtained the desired N-acylated glycoside (10a), in a 57 % yield. To determine if this
methodology was compatible with other sugars to enable the synthesis of rare glycoforms,16 we worked
to generate a few additional representative glycosylhydrazides. While we were unable to cleanly
synthesize significant quantities of disaccharide glycosylhydrazides, we were able to make and
selectively N-acylate a glucosylhydrazide (10b), a xylosylhydrazide (10c), and a galactosylhydrazide
(10d) in modest to low yields (Table 1). The isomer ratios of N-acylated products 10a-d mirrored those
of hydrazides 7a-d, providing 10a as a 90:10 mixture (β/α) and 10b-d exclusively in the β-pyranose
form. We observed no evidence for mutarotation of compounds 10a-d, consistent with the instability of
the imminum-like ion that would be required for mutarotation to occur. The small change in anomer
ratio observed in the formation of 10a is likely due to fractionation during chromatography. Analogous
Fmoc-protected, Pfp-activated amino acids are suitable for solid-phase peptide synthesis,17 and thus
the generation of 10a-d provides novel building blocks for the generation of non-natural glycopeptide
derivatives. We plan to explore the utility of such building blocks in future work.
4

Table 1. Anomeric N-acylation of a β-glycosylhydrazides using FmocAsp(Cl)OPfp.
Sugar
GlcNAc
Glc
Substrate
β
-Pyr/
α
-pyra
Product
10a
Yield (%)
β-Pyr/α
-pyra
7a
7b
7c
7d
85:15
100:0
100:0
100:0
57
35
55
29
90:10
100:0
100:0
100:0
10b
Xyl
10c
Gal
10d
aIsomer ratios calculated via 1H NMR integration.
To generate our target, FmocAsn(GlcNAc)OH (1), we turned our attention to N-N bond cleavage
(Fig. 5). Literature precedents suggested that SmI2 in the presence of a proton source might cleave the
N-N bond of 10a to provide a solid-phase peptide synthesis-ready building block.18 However, our initial
19
attempts to react 10a using SmI2 in THF/MeOH resulted in complex product mixtures that appeared
to contain N-N cleaved product that had undergone transesterification to the corresponding methyl
ester. Attempts to avoid transesterification through the use PfpOH as a proton source resulted in low
yields of impure material. Based on these findings, we decided to hydrolyze the Pfp group of 10a20 and
to instead work with Fmoc amino acid 12. We were gratified to find that reacting 12 with SmI2 in
THF/MeOH successfully resulted in the formation of product 1a (51 % yield, 92:8 β/α).
In conclusion, glycosylhydrazides can be used to generate an Fmoc-protected N-glycosylasparigine
building block (i.e., 1) that has seen previous use in solid-phase peptide synthesis (overall yield: 23 %
over 4 steps). Our strategy involved the first selective anomeric N-acylation of a glycosylhydrazide; this
transformation can be used to provide new non-natural building blocks (9a, 10a-d) and may provide
inspiration for the generation of other derivatives containing novel glycosidic linkages.
5

Fig. 5. Hydrolysis and N-N bond cleavage provide an Fmoc-protected
block.
β-N-glycosylasparigine building
3. Experimental Section
3.1 General methods
Proton nuclear magnetic resonance (1H NMR) spectra and gcosy spectra were recorded in
deuterated solvents on a Bruker Avance III 400 MHz spectrometer. Chemical shifts are reported in
parts per million (ppm,
δ
) relative to tetramethylsilane (0.00) for d-chloroform, or the residual protic
1
solvent peak for other solvents. H NMR splitting patterns with observed first order coupling are
designated as singlet (s), doublet (d), triplet (t), or quartet (q). Splitting patterns that could not be
interpreted or easily visualized are designated as multiplet (m). The presence of rotamers was
confirmed using a NMR-based chemical-exchange experiment.21 Anomers assignments were deduced
using H-1 coupling constants; anomer and rotamer populations were estimated using 1H NMR
integration. Carbon nuclear magnetic resonance (13C NMR) spectra were recorded on a Bruker Avance
III 400 MHz spectrometer. Mass spectra (MS) were obtained using electrospray ionization (ESI).
Analytical thin layer chromatography (TLC) was carried out on TLC plates pre-coated with silica gel 60
(250 µm layer thickness). Visualization was accomplished using either a UV lamp or potassium
permanganate stain (2 g KMnO4, 13.3 g K2CO3, 2 mL 2M NaOH, 200 mL H2O). Flash column
chromatography was performed on 40-60 μm silica gel (230-400 mesh). Solvent mixtures used for
TLC and flash column chromatography are reported in v/v ratios. Commercially available reagents and
solvents were used without further purification. Compound 6 was generated as previously described.10
1-(2-Acetamido-2-deoxy-
D-glucopyranosyl)-2-benzoylhydrazine (7a). Benzoylhydrazide (2.46 g,
18.2 mmol) and N-acetyl- -glucosamine (2.0 g, 9.1 mmol) were dissolved in EtOH/H2O (3:1, 12 mL)
D
and 2 drops of AcOH were added. The resulting mixture was stirred at 60 °C for 48 h then
concentrated followed by co-evaporation with toluene then DCM/EtOH. The crude product mixture was
purified by SiO2 column chromatography eluting with 5:1 DCM/MeOH to provide X (TLC Rf = 0.10 in 5:1
DCM/MeOH) as an off white foam (2.95 g, 96 % yield). The product was comprised of an inseparable
mixture of
β-pyranoside (85 %) and
α
-pyranoside (15 %) isomers. HRMS (ESI, TOF) m/z (M + H)
6

1
calculated for C15H22N3O6 340.1503, observed 340.1501.
MHz): 7.82 (m, 2H), 7.55 (m, 1H), 7.48 (m, 2H), 4.09 (d, J = 9.5, 1H), 3.80 (dd, J = 9.4, 9.4, 1H), 3.49
(dd, J = 10.2, 8.2, 1H), 3.29 (X of ABX, 1H) 3.25 (m, 1H), 3.86 (A of ABX, J = 11.9, 2.1, 1H), 3.62 (B of
ABX, J = 11.9, 6.1, 1H), 2.07 (s, 3H); 13C NMR (MeOD-d4), 100 MHz):
173.7, 167.2, 132.7, 131.6,
128.3, 126.8, 90.0, 77.9, 74.9, 70.9, 61.6, 53.3, 21.6.
-pyranoside: 1H NMR (MeOD-d4, 400 MHz):
β-pyranoside: H NMR (MeOD-d4, 400
δ
δ
α
δ
7.82 (m, 2H), 7.55 (m, 1H), 7.48 (m, 2H), 4.68 (d, J = 4.8, 1H), 4.07 (m, 1H), 3.97 (m, 1H), 3.92 (m,
1H), 3.74 (m, 1H), 3.30 (m, 1H), 3.69 (m, 1H), 2.09 (s, 3H).
Attempted peracetylation of 7a. Hydrazide 7a (153 mg, 452 μmol) was dissolved in pyridine
(0.9 mL) and cooled to 0 °C, then acetic anhydride (0.25 mL, 2.6 mmol) was added. The
resulting mixture was stirred at rt for 12 h then quenched with MeOH and concentrated. The
crude product mixture was purified by SiO2 column chromatography eluting with 15:1
DCM/MeOH to provide an impure, inseparable mixture (0.032 g) of O-peracetylated isomers
(TLC Rf = 0.16 and 0.23 in 15:1 DCM/MeOH); open-chain hydrazide, β-pyranoside, and α-
pyranoside (~ 15 : 6 : 1). Open-chain hydrazide: 1H NMR (MeOD-d4, 400 MHz): δ 7.92 –
7.45 (m, 5H), 7.80 (m, 1H), 5.72 – 5.62 (m, 1H), 5.42 (dd, J = 7.6, 3.5, 1H), 5.16 (m, 1H), 4.92
(m, 1H), 4.28 (m, 1H), 4.10 (m, 1H), 2.17 – 1.92 (m, 9H). β-pyranoside: 1H NMR (MeOD-d4,
400 MHz): δ 7.92 – 7.45 (m, 5H), 5.23 (dd, J = 10.3, 9.4, 1H), 5.00 (dd, J = 10.0, 9.6, 1H), 4.36
(d, J = 9.7, 1H), 4.27 (m, 1H), 4.10 (m, 1H), 4.08 (m, 1H), 3.80 (m, 1H), 2.17 – 1.92 (m, 9H). α-
pyranoside: 1H NMR (MeOD-d4, 400 MHz): δ 7.92 – 7.45 (m, 5H), 5.38 (dd, J = 11.4, 9.2,
1H), 4.07 (dd, J = 10.2, 9.2, 1H), 4.75 (d, J = 4.9, 1H), 4.55 (m, 1H), 4.33 (m, 1H), 4.31 (m,
1H), 4.12 (m, 1H), 2.17 – 1.92 (m, 9H).
1-(2-Acetamido-2-deoxy-
(9a). Hydrazide 7a (87.1 mg, 256
mg) were suspended in dioxane (7.3 mL) and stirred 5 min. 4-Methoxyphenylacetyl chloride (51.0
333 mol) was added. After 2 h, EtOH (120 L, 2.07 mmol) was added dropwise. The reaction mixture
D
-glucopyranosyl)-1-(4-methoxyphenylacetyl)-2-benzoylhydrazine
mol), NaHCO3 (43.1 mg, 512 mol), and 4 Å molecular sieves (91
L,
μ
μ
μ
μ
μ
was stirred 4 h, filtered, then concentrated. The crude product mixture was purified by SiO2 column
chromatography eluting with 9:1 DCM/MeOH to provide 9a (TLC Rf = 0.27 in 9:1 DCM/MeOH) as a
white solid (69.1 mg, 55 % yield). HRMS (ESI, TOF) m/z (M + H) calculated for C24H30N3O8 488.2027,
observed 488.2025. The product was comprised of an inseparable mixture of -pyranoside (97 %) and
-pyranoside (3 %) isomers. 8.05 –
-pyranoside: 1H NMR (MeOD-d4, 400 MHz) (rotamers, 92:8):
β
α
β
δ
7.47 (m, 5H), 7.24 – 6.81 (m, 4H), 5.89 (d, J = 10.4, 0.03H), 5.56 (d, J = 9.9, 0.97H), 3.84 - 3.52 (m,
2H), 3.81 (m, 1H), 3.76 (s, 3H), 3.54 (m, 2H), 3.52 (m, 1H), 3.39 (m, 1H), 3.11 (dd, 1H, J = 9.8, 8.8),
1.93 (s, 0.3), 1.78 (s, 2.7 H); 13C NMR (MeOD-d4), 100 MHz):
131.0, 129.8, 128.8, 127.3, 125.9, 113.7, 82.2, 79.6, 74.2, 70.7, 61.7, 54.3, 52.6, 39.1, 21.1.
δ 174.6, 174.4, 167.0, 158.8, 132.9,
α-
7

pyranoside: 1H NMR (MeOD-d4, 400 MHz) (rotamers, 59:41):
δ 8.05 – 7.47 (m, 5H), 7.24 – 6.81 (m,
4H), 6.24 (d, J = 10.4, 0.41H), 5.93 (d, J = 9.9, 0.59H), 3.84 - 3.52 (m, 2H), 3.81 (m, 1H), 3.76 (s, 3H),
3.54 (m, 2H), 3.52 (m, 1H), 3.39 (m, 1H), 3.11 (dd, 1H, J = 9.8, 8.8), 1.93 (s, 0.3), 1.78 (s, 2.7 H).
1-(D-Glucopyranosyl)-2-benzoylhydrazine (7b). Benzoylhydrazide (2.3 g, 16.8 mmol) and D-
glucose (2.0 g, 11.2 mmol) were dissolved in EtOH (8 mL) and 12 drops of AcOH were added. The
resulting mixture was stirred at 80 °C for 3 h then cooled and filtered. The filtrate was washed with hot
EtOH (50 mL), then cold EtOH (50 mL), and the resulting white solid 7b (TLC Rf = 0.14 in 5:1
DCM/MeOH) was used without further purification (3.1 g, 93 % yield). The product was 100 %
pyranoside. HRMS (ESI, TOF) m/z (M + H) calculated for C13H19N2O6 299.1238, observed 299.1237.
1H NMR (MeOD-d4, 400 MHz):
7.82 (m, 2H), 7.57 (m, 1H), 7.48 (m, 2H), 4.01 (d, J = 8.9, 1H), 3.91
β-
δ
(m, 1H), 3.63 (m, 1H), 3.42 (dd, 1H, J = 9.2, 9.2), 3.35 – 3.20 (m, 3H); 13C NMR (D2O, 100 MHz):
δ
173.3, 135.2, 134.7, 131.5, 130.0, 92.3, 79.7, 79.0, 73.5, 72.3, 63.6.
1-(D-Xylopyranosyl)-2-benzoylhydrazine (7c). Benzoylhydrazide (1.33 g, 9.77 mmol) and D-
xylose (0.98 g, 6.51 mmol) were dissolved in EtOH and 4 drops of AcOH were added. The resulting
mixture was stirred at 78 °C for 3 h then concentrated. The solid residue was suspended in EtOH and
filtered. The filtrate was washed with hot EtOH (20 mL), then cold EtOH (40 mL), and the resulting solid
7c (TLC Rf = 0.27 in 5:1 DCM/MeOH) was used without further purification (1.01 g, 58 % yield). The
product was 100 %
(m, 2H), 3.97 (d, 1H, J = 8.9), 3.90 (dd, 1H, J = 11.7, 5.9), 3.48 (m, 1H), 3.38 (t, 1H, J = 8.7), 3.21 (m,
2H); 13C NMR (MeOD-d4 + DMSO-d6), 100 MHz):
170.0, 134.0, 133.0, 129.6, 128.5, 93.2, 78.3,
β δ 7.81 (m, 2H), 7.55 (m, 1H), 7.47
-pyranoside. 1H NMR (MeOD-d4, 400 MHz):
δ
72.6, 71.2, 68.4. HRMS (ESI, TOF) m/z (M + H) calculated for C12H17N2O5 269.1132, observed
269.1131.
1-(D-Galactopyranosyl)-2-benzoylhydrazine (7d). Benzoylhydrazide (896 mg, 6.6 mmol) and D-
galactose (790 mg, 4.4 mmol) were dissolved in 3:1 EtOH/H2O (3.3 mL) and 4 drops of AcOH were
added. The resulting mixture was stirred at 60 °C for overnight then cooled and filtered. The filtrate was
washed with hot EtOH (25 mL) and the resulting white solid 7d (TLC Rf = 0.26 in 5:1 DCM/MeOH) was
used without further purification (769 mg, 59 % yield). The product was 100 %
β
-pyranoside. HRMS
1
(ESI, TOF) m/z (M + H) calculated for C13H19N2O6 299.1238, observed 299.1236. H NMR (D2O, 400
MHz):
δ 7.76 (m, 2H), 7.63 (m, 1H), 7.53 (m, 2H), 4.16 (d, 1H, J = 8.9), 3.93 (m, 1H), 3.81 (m, 4H),
3.61 (dd, 1H, J = 9.4); 13C NMR (D2O + d6-DMSO), 100 MHz):
δ 173.1, 135.1, 134.6, 131.4, 129.9,
93.2, 78.8, 75.8, 71.5, 71.0, 64.0.
1-(2-Acetamido-2-deoxy-D L-asparagine
-glucopyranosyl)-1-(Nα-(fluoren-9-ylmethoxycarbonyl)-
pentafluorophenyl ester))-2-benzoylhydrazine (10a). Hydrazide 7a (352 mg, 1.04 mmol), NaHCO3
8

(350 mg, 4.20 mmol), and 4 Å molecular sieves (356 mg) were suspended in dioxane (15 mL) and
stirred 5 min. Chloride 10 (560 mg, 1.04 mmol) was added, followed 1 h later by another addition of 10
(560 mg, 1.04 mmol). After 2 h, EtOH (120
μL, 2.07 mmol) was added. The reaction mixture was
stirred 20 min., filtered, then concentrated. The crude product mixture was purified by SiO2 column
chromatography eluting with 15:1 DCM/MeOH to provide 9a (TLC Rf = 0.44 in 9:1 DCM/MeOH) as a
white solid (495 mg, 57 % yield). HRMS (ESI, TOF) m/z (M + H) calculated for C40H36F5N4O11
843.2295, observed 843.2283. The product was comprised of an inseparable mixture of β-pyranoside
1
(90 %) and
85:15):
α
-pyranoside (10 %) isomers.
β
-pyranoside: H NMR (MeOD-d4, 400 MHz) (rotamers,
δ
8.07 – 7.46 (m, 8H), 7.40 – 7.22 (m, 5H), 5.90 (d, J = 9.8, 0.15H), 5.62 (d, J = 10.1, 0.85H),
5.03 (m, 1H), 4.51 – 4.31 (m, 2H), 4.23 (m, 1H), 3.90 (m, 1H), 3.81 (m, 1H), 3.61 (m, 1H), 3.52 (m,
1H), 3.41 (m, 1H), 3.25 (m, 1H), 3.16 (m, 1H), 2.99 (m, 1H), 1.99 – 1.91 (m, 3H); 13C NMR (MeOD-d4),
100 MHz):
δ
173.0, 171.1, 168.2, 164.4, 155.9, 143.7, 140.8, 132.7, 131.7, 129.0, 127.7, 127.2, 127.1,
1
127.0, 125.2, 120.2, 79.6, 73.7, 70.4, 66.0, 61.0, 54.0, 52.5, 49.9, 46.6, 33.9, 22.4.
NMR (MeOD-d4, 400 MHz) (rotamers, 54:46):
α
-pyranoside: H
δ
8.07 – 7.46 (m, 8H), 7.40 – 7.22 (m, 5H), 6.04 (d, J =
4.0, 0.54H), 6.28 (d, J = 4.0, 0.46H), 5.03 (m, 1H), 4.51 – 4.31 (m, 2.54H), 4.23 (m, 1.46H), 4.01 –
2.87 (m, 7H), 1.99 – 1.91 (m, 3H).
1-(
D
-Glucopyranosyl)-1-(Nα-(fluoren-9-ylmethoxycarbonyl)-
L-asparagine
pentafluorophenyl
ester))-2-benzoylhydrazine (10b). Hydrazide 7b (315 mg, 1.06 mmol), NaHCO3 (355 mg, 4.22
mmol), and 4 Å molecular sieves (362 mg) were suspended in dioxane (15 mL) and stirred 10 min.
Chloride 11 (570 mg, 1.06 mmol) was added, followed 1.5 h later by another addition of 11 (570 mg,
1.06 mmol). After 2 h, EtOH (123 μL, 2.11 mmol) was added. The reaction mixture was stirred 20 min.,
filtered, then concentrated. The crude product mixture was purified by SiO2 column chromatography
eluting with 15:1 DCM/MeOH to provide 10b (TLC Rf = 0.08 in 15:1 DCM/MeOH) as a white solid (295
mg, 35 % yield). HRMS (ESI, TOF) m/z (M + H) calculated for C38H33F5N3O11 802.2030, observed
1
802.2019. The product was 100 %
β
-pyranoside. H NMR (MeOD-d4, 400 MHz) (rotamers, 10:1):
δ
8.03 – 7.25 (m, 13H), 5.67 (d, 0.9H, J = 9.1), 5.49 (d, 0.1H, J = 9.0), 5.01 (m, 1H), 4.52 – 4.21 (m, 3H),
3.85 (m, 1H), 3.72 (m, 1H), 3.55 (t, 1H, J = 9.1), 3.44 (m, 1H), 3.37 (m, 1H), 3.27 (m, 1H), 3.20 (t, 1H, J
= 9.1), 3.06 (m, 1H); 13C NMR (MeOD-d4), 100 MHz):
δ 172.2, 169.2, 168.3, 156.3, 144.1, 141.2,
133.6, 131.0, 129.3, 128.6, 128.1, 127.5, 125.7, 120.6, 83.0, 80.0, 76.1, 70.1, 70.0, 66.5, 61.3, 55.4,
50.0, 47.0, 35.3.
1-(D L-asparagine pentafluorophenyl
-Xylopyranosyl)-1-(Nα-(fluoren-9-ylmethoxycarbonyl)-
ester))-2-benzoylhydrazine (10c). Hydrazide 7c (380 mg, 1.41 mmol), NaHCO3 (475 mg, 5.66
mmol), and 4 Å molecular sieves (485 mg) were suspended in dioxane (20 mL) and stirred 10 min.
Chloride 11 (764 mg, 1.41 mmol) was added, followed 1.5 h later by another addition of 11 (764 mg,
1.41 mmol). After 1.5 h, EtOH (170 μL, 1.82 mmol) was added. The reaction mixture was stirred 20
9

min., filtered, then concentrated. The crude product mixture was purified by SiO2 column
chromatography eluting with 15:1 DCM/MeOH to provide 10c (TLC Rf = 0.37 in 15:1 DCM/MeOH) as a
1
white foam (603 mg, 55 % yield). H NMR (MeOD-d4, 400 MHz) (rotamers, 76:24):
δ 7.99 – 7.24 (m,
13H), 5.57 (d, J = 9.1, 0.76H), 5.43 (d, J = 8.8, 0.24H), 3.19 (m, 0.76H), 3.47 (m, 0.24H), 5.01 (m, 1H),
3.34 (m, 1H), 3.05 (m, 1H), 3.95 – 3.16 (m, 4H), 4.37 (m, 2H), 4.24 (m, 1H); 13C NMR (DMSO-d6), 100
MHz) (rotamers):
δ 172.4, 169.1, 168.2, 144.1, 141.1, 133.6, 130.9, 129.3, 129.1, 128.5, 128.1, 127.5,
125.6, 85.0, 83.8, 76.3, 70.0, 69.7, 68.3, 55.4, 50.0, 49.1, 47.0, 35.2. HRMS (ESI, TOF) m/z (M + H)
calculated for C37H31F5N3O10 772.1924, observed 772.1912.
1-(D L-asparagine pentafluorophenyl
-Galactopyranosyl)-1-(Nα-(fluoren-9-ylmethoxycarbonyl)-
ester))-2-benzoylhydrazine (10d). Hydrazide 7d (224 mg, 0.75 mmol), NaHCO3 (253 mg, 3.00
mmol), and 4 Å molecular sieves (258 mg) were suspended in dioxane (10.7 mL) and stirred 10 min.
Chloride 11 (406 mg, 0.75 mmol) was added, followed 1.5 h later by another addition of 11 (406 mg,
0.75 mmol). After 2 h, EtOH (88
filtered, then concentrated. The crude product mixture was purified by SiO2 column chromatography
eluting with 15:1 DCM/MeOH to provide -pyranoside 10d (TLC Rf = 0.13 in 15:1 DCM/MeOH) as a
μL, 1.50 mmol) was added. The reaction mixture was stirred 20 min.,
β
white solid (177 mg, 29 % yield). HRMS (ESI, TOF) m/z (M + H) calculated for C38H33F5N3O11
1
802.2030, observed 802.2003. H NMR (MeOD-d4, 400 MHz) (rotamers, 5:1):
δ 8.02 – 7.23 (m, 13H),
5.62 (d, J = 8.8, 0.8H), 5.50 (d, J = 10.0, 0.2H), 5.04 (m, 1H), 4.46 – 4.33 (m, 2H), 4.25 (m, 1H), 3.99 –
3.45 (m, 5.2H), 3.62 (dd, J = 9.3, 9.0, 0.8H), 3.37 (m, 1H), 3.10 (m, 1H); 13C NMR (MeOD-d4), 100
MHz):
δ 172.3, 169.2, 168.3, 156.3, 144.1, 141.2, 133.6, 131.1, 129.2, 128.6, 128.1, 127.5, 125.6,
120.6, 83.8, 78.4, 73.0, 68.3, 67.3, 66.5, 60.8, 55.4, 50.0, 47.0, 35.3.
1-(2-Acetamido-2-deoxy-D L-
-glucopyranosyl)-1-(Nα-(fluoren-9-ylmethoxycarbonyl)-
asparagine))-2-benzoylhydrazine (12). Residue 10a (304 mg, 0.360 mmol) was dissolved in 4:2:1
AcOH/THF/H2O (2.8 mL) and heated at 60 °C overnight. The reaction mixture was concentrated then
co-evaporated with toluene. The crude product mixture was purified by SiO2 column chromatography
eluting with 5:1 DCM/MeOH + 0.5 % AcOH to provide 12 (TLC Rf = 0.09 in 5:1 DCM/MeOH + 0.5 %
AcOH) as a white solid (202 mg, 83 % yield). HRMS (ESI, TOF) m/z (M + H) calculated for C34H37N4O11
677.2453, observed 677.2450. The product was comprised of an inseparable mixture of
(90 %) and -pyranoside (10 %) isomers.
-pyranoside: 1H NMR (MeOD-d4, 400 MHz) (rotamers,
82:18): 8.08 – 7.46 (m, 8H), 7.43 – 7.20 (m, 5H), 5.88 (d, J = 9.9, 0.18H), 5.62 (d, J = 10.0, 0.82H),
3.87 (m, 0.82H), 3.81 (m, 0.18H), 4.62 – 4.12 (m, 4H), 3.93 – 3.09 (m, 5H), 3.04 (m, 1H), 2.83 (m, 1H),
2.07 – 1.91 (m, 3H); 13C NMR (MeOD-d4, 100 MHz):
174.8, 173.2, 167.2, 157.0, 143.8, 141.1, 132.6,
131.0, 128.7, 127.4, 127.3, 126.8, 124.9, 119.5, 82.1, 79.6, 74.1, 70.7, 66.7, 61.7, 52.6, 46.9, 34.7,
21.4. 8.08 – 7.46 (m, 8H), 7.43 –
-pyranoside: 1H NMR (MeOD-d4, 400 MHz) (rotamers, 55:45):
7.20 (m, 5H), 6.20 (d, J = 3.9, 0.55H), 5.99 (d, J = 3.3, 0.45H), 4.62 – 4.12 (m, 4H), 3.93 – 3.09 (m,
β-pyranoside
α
β
δ
δ
α
δ
10

6H), 3.04 (m, 1H), 2.83 (m, 1H), 2.07 – 1.91 (m, 3H).
Nα-(Fluoren-9-ylmethoxycarbonyl)-Nγ-(2-acetamido-2-deoxy-
D-glucopyranosyl)-L-asparagine
(1). Residue 12 (49.9 mg, 0.074 mmol) was dissolved in MeOH (0.74 mL) under nitrogen. A 0.1 M
solution of SmI2 in THF (1.1 mL, 0.111 mmol) was added dropwise. After 5 min. the reaction mixture
was exposed to the atmosphere. After the color had faded from dark blue to yellow, the mixture was
concentrated. The material was suspended in 1:1 AcOH/H2O (2 mL) and loaded onto a 6 mL Resprep
C18 disposable solid phase extraction cartridge that had been preconditioned with MeOH then H2O.
Using a vacuum manifold, the cartridge was washed with H2O, 0.1 M aq. HCl, and H2O (1 column
volume each) then dried for 5 min. The cartridge was then eluted with 2:1 CHCl3/MeOH + 0.5 % AcOH
to provide 1 (TLC Rf = 0.08 in 2:1 CHCl3/MeOH + 0.5 % AcOH; mp = 211 °C (decomp.)) as a white
solid (20.9 mg, 51 % yield). The product was comprised of an inseparable mixture of
β-pyranoside (92
%) and -pyranoside (8 %) isomers. δ
α
β
-pyranoside: 1H NMR (AcOD-d4, 400 MHz) (rotamers, 8:2):
7.82 – 7.27 (m, 8H), 5.16 (d, J = 9.7, 1H), 4.76 (m, 0.8H), 4.60 (m, 0.2H), 4.56 – 4.20 (m, 3H), 4.06 –
3.50 (m, 6H), 2.92 (m, 1.6H), 2.75 (m, 0.4H), 2.01 (s, 2.4H), 2.06 (s, 0.6H); 13C NMR (AcOD-d4, 100
MHz):
δ
175.6, 174.3, 172.1, 157.0, 143.8, 141.2, 127.6, 127.0, 125.0, 119.8, 78.9, 77.3, 74.2, 70.0,
-pyranoside: 1H NMR (AcOD-d4, 400 MHz): δ δ 7.82 – 7.27
67.2, 60.8, 54.5, 50.3, 46.9, 37.1, 21.6.
α
(m, 8H), 5.62 (d, J = 2.7, 1H), 4.76 (m, 0.8H), 4.60 (m, 0.2H), 4.56 – 4.20 (m, 3H), 4.06 – 3.50 (m, 6H),
2.92 (m, 1.6H), 2.75 (m, 0.4H), 2.01 (s, 2.4H), 2.06 (s, 0.6H). HRMS (ESI, TOF) m/z (M + H) calculated
for C27H32N3O10 558.2082, observed 558.2086.
Associated Content
1H, gcosy, and 13C NMR spectra.
Author Information
Corresponding author
*Phone: 206-296-2368. Fax: 206-296-5786. E-mail: langenha@seattleu.edu.
Acknowledgements
We thank Martin Sadilek and Loren Kruse for acquiring HRMS data. Research reported in this
publication was supported by the National Institute of General Medical Sciences of the National
Institutes of Health under award number R15GM109839-01A1. The content is solely the responsibility
of the authors and does not necessarily represent the official views of the National Institutes of Health.
11

References
1. Dalziel, M., Crispin, M., Scanlan, C.N., Zitzmann, N., Dwek, R.A. Science 2014, 343, 1235681.
2. Wang, L.-X.; Amin, M. N. Chem. Biol. 2014, 21, 51−66; Gamblin, D. P.; Scanlan, E. M.; Davis,
B. G. Chem. Rev. 2009, 109, 131−163; Unverzagt, C.; Kajihara, Y. Chem. Soc. Rev. 2013, 42,
4408−4420.
3. See for example: a) Bejugam, M.; Flitsch, S.L. Org. Lett. 2004, 6, 4001-4004; b) Likhosherstov,
L.M.; Novikova, O.S.; Derevitskaja, V.A.; Kochetkov, N.K. Carbohydr. Res. 1986, 146, C1; c)
Hackenberger, C.P.R.; O’Reilly, M.K.; Imperiali, B. J. Org. Chem. 2005, 70, 3574-3578; d) Du,
J.-J.; Gao, X.-F.; Xin, L.,-M.; Lei, Z.; Liu, Z.; Guo, J. Org. Lett. 2016, 18, 4828-4831.
4. See for example: a) Mizuno, M.; Muramoto, I.; Kobayashi, K.; Yaginuma, H.; Inazu, T.
Synthesis 1999, 162-165; b) Inazu, T.; Kobayashi, K. Synlett 1993, 869-871; c) He, Y.; Hinklin,
R.J.; Chang, J.; Kiessling, L.L. Org. Lett. 2004, 6, 4479-4482; d) Bianchi, A.; Russo, A.;
Bernardi, A. Tetrahedron: Asymmetry 2005, 16, 381-386; e) Doores, K.J.; Mimura, Y.; Dwek,
R.A.; Rudd, P.M.; Elliott, T.; Davis, B.G. Chem. Commun. 2006, 1401-1403; f) Hagiwara, M.;
Dohi, M.; Nakahara, Y.; Komatsu, K.; Asahina, Y.; Ueki, A.; Hojo, H.; Nakahara, Y.; Ito, Y. J.
Org. Chem. 2011, 76, 5229-5239.
5. Gudmundsdottir, A.V.; Nitz, M. Carbohydrate Res. 2007, 342, 749-752.
6. a) Jencks, W.P.; Carriuolo, J. J. Am. Chem. Soc. 1960, 82, 675-681; b) Edwards, J.O.;
Pearson, R.G. J. Am. Chem. Soc. 1962, 84, 16-24.
7. Chingle, R.; Proulx, C.; Lubell, W.D. Acc. Chem. Res. 2017, 50, 1541-1556.
8. a) Onodera, K.; Kitaoka, S. J. Org. Chem. 1960, 25, 1322-1325; b) Yoshioka, T.; Uematsu, T. J.
Chem. Soc. Perkin Trans I, 1985, 1261-1269.
9. Ying, L.; Li, R.; Zhang, J.; Lam, K.; Lebrilla, C.B.; Gervay-Hague, J. J. Comb. Chem. 2005, 7,
372-384.
10. Edgar, L.J.G.; Dasgupta, S.; Nitz, M. Org. Lett. 2012, 14, 4226-4229.
11. Wu, L.; Sampson, N.S. ACS Chem. Biol. 2014, 9, 468-475.
12. Langenhan, J.M.; Peters, N.R.; Guzei, I.A.; Hoffman, F.M.; Thorson, J.M. Proc. Natl. Acad. Sci.
2005, 102, 12305-12310.
13. El-Faham, A.; Funosas, R.S.; Prohens, R.; Albericio, F. Chem. Eur. J. 2009, 15, 9404-9416.
14. Langenhan, J.M.; Griffith, B.R.; Thorson, J.M. J. Nat. Prod. 2005, 68, 1696-1711.
15. Meldal, M.; Bock, K. Tetrahedron Lett. 1990, 31, 6987-6990.
16. Takeda, T.; Sugiura, Y.; Ogihara, Y.; Shibata, S. Can. J. Chem. 1980, 58, 2600-2603; Messner,
P. Glycocong. J. 1997, 14, 3; Schreiner, R.; Schnabel, E.; Wieland, F. J. Cell. Biol. 1994, 124,
1071-1081.
17. Christiansen-Brams, I.; Meldal, M.; Bock, K. J. Chem. Soc. Perkin Trans. I 1993, 1461-1471.
18. Saaby, S.; Bella, M.; Jorgensen, K.A. J. Am. Chem. Soc. 2004, 126, 8120-8121; Taylor, J.E.;
Daniels, D.S.B.; Smith, A.D. Org. Lett. 2013, 15, 6058-6061; Ding, H.; Friestad, G.K. Org. Lett.
12

2004, 6, 637-640;
19. Szostak, M.; Spain, M.; Procter, D.J. J. Org. Chem. 2012, 77, 3049-3059.
20. Bernady, K.F.; Floyd, M.B.; Poletto, J.F.; Weiss, M.J. J. Org. Chem. 1979, 44, 1438-1447.
21. Hu, D.X.; Grice, P.; Ley, S.V. J. Org. Chem. 2012, 77, 5198-5202.
13

•
•
Glycosylhydrazides can be selectively acylated at the anomeric nitrogen
This transformation can be harnessed to generate amino acid building blocks (e.g.,
FmocAsn(GlcNAc)OH).
1

Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: