The versatility of N-alkyl-methoxyamine bi-functional linkers for the preparation of glycoconjugates

Article abstract of DOI:10.1007/s10719-017-9785-4

The application of N-glycosyl-N-alkyl-methoxyamine bi-functional linkers for the synthesis of a variety of glycoconjugates is described. The linker contains a specific functional group, such as an amine, azide, thiol, or carboxylic acid, which can be used for conjugation methodologies that include amide ligation, sulfonylation, copper-mediated Huisgen cycloaddition or thiol-maleimide coupling. In this way, glycoconjugates equipped with biotin, a fluorescent reporter, or a protein were efficiently synthesised, thus demonstrating the versatility of this type of oxyamine linker for the construction of glycoconjugate probes.

Full text of DOI:10.1007/s10719-017-9785-4

Glycoconj J
DOI 10.1007/s10719-017-9785-4
ORIGINAL ARTICLE
The versatility of N-alkyl-methoxyamine bi-functional
linkers for the preparation of glycoconjugates
Stefan Munneke1 & Emma M. Dangerfield1 & Bridget L. Stocker1
&
Mattie S. M. Timmer1
Received: 12 May 2017 /Revised: 5 July 2017 /Accepted: 6 July 2017
#
Springer Science+Business Media, LLC 2017
Abstract The application of N-glycosyl-N-alkyl-
methoxyamine bi-functional linkers for the synthesis of
a variety of glycoconjugates is described. The linker con-
tains a specific functional group, such as an amine, azide,
thiol, or carboxylic acid, which can be used for conjuga-
tion methodologies that include amide ligation,
sulfonylation, copper-mediated Huisgen cycloaddition or
thiol-maleimide coupling. In this way, glycoconjugates
equipped with biotin, a fluorescent reporter, or a protein
were efficiently synthesised, thus demonstrating the ver-
satility of this type of oxyamine linker for the construc-
tion of glycoconjugate probes.
strategies for the conjugation of carbohydrates to molecules
such as fluorescent reporters [3, 4], biotin [5], dendrimers [6],
microarrays1 and proteins [9, 10]. While many complex gly-
cans have been prepared de novo, with the functionalised
linkers being incorporated during the synthesis of a protected
glycan,2 the use of carbohydrates from natural sources re-
quires conjugation strategies that are compatible with unpro-
tected sugars. To this end, a linker is often attached to the free
glycan and subsequently reacted, via the linker’s handle, with
the molecule of choice. Strategies that employ this concept
include Kochetkov amination [14], to prepare glycosylamines
that can be further functionalised, the use of oximes/
hydrazides or oxyamines [15], and reductive amination,3 al-
though the last methodology affects the structural integrity of
the reducing end carbohydrate.
.
.
.
Keywords Oxyamine Glycoconjugate Linker
.
Oligosaccharide Probe
Of these general ligation strategies, we were interested in
exploring and extending the scope of oxyamine methodology.
There are numerous elegant examples of the use of oxyamines
in the synthesis of glycoconjugates, as reported by the groups
of Peri [18], Boons [19], Blixt [20], Carrasco [21], Nitz [22]
and Jensen [23], whereby N-methyloxyamine linkers of ‘Type
A’ (Fig. 1) were described. In some instances however, the
yield and efficiency of the linker preparation could be im-
proved, and moreover, a general strategy that would allow
for the installation of the functional group of choice on the
linker terminus would be advantageous. Accordingly, we re-
cently developed a highly efficient strategy for the synthesis of
‘Type B’ oxyamine linkers that contained azide (1a), amine
(1b and 1c), thiol (1d and 1e) and carboxy (1f and 1 g)
bioorthogonal handles (Type B, Fig. 1) [24]. The linkers
showed good hydrolytic stability, which was comparable to
Introduction
A staggering number of proteins and lipids are glycosylated.
[1, 2] This has spurred interest in studying the biological ac-
tivities of glycoconjugates and the need to find efficient
Electronic supplementary material The online version of this article
(doi:10.1007/s10719-017-9785-4) contains supplementary material,
which is available to authorized users.
* Bridget L. Stocker
bridget.stocker@vuw.ac.nz
* Mattie S. M. Timmer
mattie.timmer@vuw.ac.nz
1 For example: [7, 8]
1
2 For example see: [11–13].
3 For example, see: [16, 17]
School of Chemical and Physical Sciences, PO Box 600,
Wellington, New Zealand

Glycoconj J
Fig. 1 ‘Type A’ N-methyl-
oxyamine and ‘Type B’ O-
methyloxyamine linkers 1a–g,
and representative examples of
probes that can be incorporated
using these linkers
the ‘Type A’ glycoconjugates [25], however the scope and
limitation of these ‘Type B’ oxyamine linkers for the prepara-
tion of glycoconjugates was not thoroughly explored. To this
end, we herein provide insight into the application of the dif-
ferent N-oxyamine linkers, with a focus on the ability to con-
jugate the linkers to other molecules using chemoselective
ligation methodologies that include amide formation [26,
27], sulfonylation [28], copper-mediated cycloaddition [29]
and thiol-maleimide conjugation [30]. In this way,
glycoconjugates equipped with biotin, a fluorescent reporter,
or a protein were efficiently synthesised, thus demonstrating
the versatility of the ‘Type B’ oxyamine linkers for the con-
struction of glycoconjugate probes.
MeOH/Et2O then provided conclusive evidence for the for-
mation of the β-pyranose configuration [31], which supported
our earlier NMR analysis that revealed a J1´,2′ of 9.8 Hz and an
HBMC between H-1′ and C-5′ [24]. The application of the
azide functionalised oxyamine linker 1a was then demonstrat-
ed by reacting neoglycoside 2 with propargyl alcohol under
copper-mediated azide-alkyne cycloaddition conditions [29]
to give triazole 3 in 79% yield. Here, propargyl alcohol was
used as a model substrate to demonstrate proof-of-principle
for the orthogonality of oxyamine linker 1a. Given the ease of
this copper-mediated ‘click’ reaction, it is envisioned that
oxyamine linker 1a will thus serve as a useful tool for the
conjugation of glycans to other functionalised alkynes.
Next, we sought to prepare different classes of
glycoconjugates using the amine-functionalised oxyamine
linkers 1b and 1c. First, N-acetylglucosamine was coupled
to the short-chain bi-functional amine linker 1b, followed by
conjugation of the resulting neoglycoside to D-biotin under
standard peptide coupling conditions [26, 27]. The conjuga-
tion reaction with D-biotin proceeded smoothly under the me-
diation of N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-
yl)uronium hexafluorophosphate (HBTU) to give the biotinyl-
ated glycoconjugate 4 in 71% yield. The applications of such
biotinylated glycans include streptavidin/biotin affinity chro-
matography and ELISA using streptavidin functionalised mi-
croarrays [32, 33]. In addition to the use of the amine-
functionalised linkers for amide bond formation, these linkers
can also be used for the synthesis of sulfonamides, as demon-
strated by the synthesis of a dansylated fluorescent
glycoconjugate. Here, GlcNAc was conjugated to oxyamine
1c and the resulting glycoconjugate reacted with dansyl chlo-
ride (5) under basic conditions to give sulfonamide 6 in 77%
Results and discussion
To synthesise the ‘Type B’ oxyamine linkers, acrolein was treat-
ed with either a functionalised thiol or with NaN3, and the
resulting Michael adduct was condensed with methoxyamine
and then reduced with NaCNBH3 to yield the oxyamines 1a,
1c, 1e–g in a three-step one-pot synthesis, as previously reported
[24]. The amine functionalised oxyamine 1b containing the
shorter linker was prepared via Staudinger reduction of azide
1a, while the synthesis of oxyamine 1d was achieved using
thioacetate as the nucleophile and involved the deacetylation
of the intermediate imine prior to the reduction step to avoid
S-to-N acyl migration [24]. In this way, the oxyamine linkers
were synthesised in 3–4 steps and in high yield (51–96%).
To explore the versatility of the oxyamine linkers, we first
conjugated azide functionalised linker 1a to GlcNAc to give
neoglycoside 2 (Scheme 1). Crystallisation of 2 from

Glycoconj J
OMe
HN
HO
OH
N3
OH
OH
OH
OMe
N
OMe
N
1a
O
O
O
HO
HO
HO
HO
HO
HO
N
OH
N3
N
N
NHAc
NHAc
NHAc
NH4OAc buffer
87%
CuSO4
ascorbic acid
EtOH, 79%
2
3
2
OMe
O
HN
NH2
1b
NH4OAc buffer
81%
NH
HN
H
1)
OH
OH
H
OMe
O
H
N
O
HO
HO
HO
HO
N
OH
S
2) D-Biotin, HBTU
Et3N, DMF, 84%
3
NHAc
NHAc
O
4
OMe
HN
S
NH2
3
2
1c
OH
1)
NH4OAc buffer
81%
OH
OMe
O
H
N
O
HO
HO
HO
HO
O
N
S
OH
S
2)
Cl
NHAc
3
2
NHAc
NMe2
O
O
S
O
5
NMe2
6
NaHCO3, H2O, THF
77%
OMe
HN
HO
OH
NH2
OH
HO
HO
O
OH
1b
O
OH
O
O
HO
O
OMe
N
1)
2)
NH4OAc buffer
88%
O
OH
O
H
N
H
N
OH
O
O
AcHN
OH
O
OH
AcHN
OH
O
FITC
THF, rt
O
S
OH
HO
OH
OH
HO
7
2.5 h, 92%
8
O
HO
Scheme 1 Conjugation of azide- and amine-functionalised neoglycosides
yield. The synthesis of fluorescently labelled glycans was fur-
ther demonstrated by the preparation of a fluorescein isothio-
cyanate (FITC) labelled LewisX glycan 8. LewisX is a ligand
for several immune receptors, including DC-SIGN [34], and is
involved in numerous biological processes including cancer
metathesis and viral infection.4 To synthesise a LewisX-chem-
ical probe, LewisX (7) [38] was coupled to bi-functional amine
linker 1b and treated with FITC under mild reaction condi-
tions to give the fluorescein derivative 8 in excellent (92%)
yield. The ease of this synthesis illustrates the power of the
oxyamine-linker methodology for the preparation of more
complex chemical probes. Fluorescent glycoconjugates have
a number of applications, particularly in the study of glycan
uptake and cellular trafficking by confocal microscopy or flow
cytometry. As the physical-chemical properties of the
glycoconjugates can affect cellular trafficking [3, 4], it is ad-
vantageous to have the choice of either oxyamine linker 1b or
1c for the attachment of the fluorophore.
Glycans containing thiol-functionalised linkers have a num-
ber of applications that include use in gold-plated microarrays
[39] or as nucleophiles for Michael-addition [40]. Accordingly,
we next attempted to conjugate the thiol-functionalised
oxyamine 1d to GlcNAc to afford the corresponding thiol-
functionalised glycoconjugate. Unfortunately, this reaction did
not yield the target glycoside but instead appeared to give a
diastereomeric mixture of thiazinanes 9, which presumably
forms through a 6-endo-trig cyclization of an intermediate
oxyimminium ion with the pendant thiol group (Scheme 2).
Analysis by HRMS (m/z for [C12H25N2O6S]+ calc. 325.1428,
obs. 325.1445), as well as TLC [Rf = 0.32; 10:2:2:1,
CH2Cl2:EtOH:MeOH:NH3 (aq, 33%), v:v:v:v], clearly showed
1
the formation of a new compound, however, as H and 13C
NMR data for 9 suffered from significant peak broadening, even
when using different solvents [D2O, CD3OD, pyridine-d5 or
DMSO-d6] and varying temperatures (rt – 60 °C).
Accordingly, the mixture of thiazinanes was acetylated (→10),
and NMR data was obtained which allowed for the conclusive
assignment of the structures of the two thiazine diastereoisomers
(see SI for further details).
4 For some representative papers see: [35–37]

Glycoconj J
Scheme 2 The formation of N-
methoxy-thiazinanes 7 which,
after per-acetylation (→10), could
be confirmed by NMR analysis
To prevent the formation of thiazinanes, the thiol
functionalised linker 1e, which contains an extended linker
chain, was then used for the formation of glycoconjugates via
maleimide-mediated conjugation (Scheme 3). To this end, the
maleimide acceptor was prepared via Boc protection of
ethylenediamine (11), condensation of the remaining amine with
maleic anhydride to give a carboxy-amine that underwent ring-
closure to form the maleimide, and Boc deprotection to yield
functionalised amine 12 [41]. Conjugation of 12 to D-biotin via
an HBTU-mediated peptide coupling then gave biotin-
functionalised maleimide 13 in 92% yield. The conjugation of
thiol-functionalised oxyamine 1e with GlcNAc occurred
smoothly and following coupling with biotin-functionalised
maleimide 13 in water at room temperature gave the target gly-
coside 14 in good (60%) yield over the two steps after purifica-
tion by size exclusion chromatography. An advantage of this
approach for the formation of biotinylated glycoconjugates is
the ease of the thiol-maleimide conjugation, as mild conditions
are preferable when using small quantities of glycans.
Accordingly, we envisioned this protocol to have wide applica-
tion for the conjugation of glycans from both synthetic and
biological sources.
Finally, the versatility of the thiol-functionalised oxyamine
linker was further demonstrated by the formation of a glyco-
protein (Fig. 2a). For our study, the commercially available bi-
f u n c t i o n a l c r o s s - l i n k e r s u c c i n i m i d y l - 4 - [ N -
maleimidomethyl]cyclohexane-1-carboxylate (SMCC) 15 was
used for the maleimide functionalisation of the lysine residues
in Bovine Serum Albumin (BSA). The functionalised protein
was then coupled to thiol-functionalised neoglycoside 16 to
give glycoprotein 17. Literature studies for the conjugation of
glycans to proteins are often limited in experimental detail
relating to glycan loading. Thus, we investigated a variety of
reaction conditions and monitored the progress of the reactions
using MALDI-TOF analysis to determine the average molecu-
lar weight of the protein after SMCC conjugation and subse-
quent glycosylation. From these studies it was determined that
the reactions proceeded more efficiently when being performed
in 10% MeCN in PBS, so as to prevent denaturisation of the
protein while fully dissolving the SMCC cross-linker, and that
optimal glycan coupling to the SMCC-BSA conjugate occurred
after 60 min. Further time course and concentration studies then
concluded that the most efficient glycoconjugation was
achieved when BSA (10 mg/mL) was reacted with the
SMCC cross linker 15 (180 equiv.) for 60 min before the addi-
tion of excess neoglycoside 16 (360 equiv.), and incubation of
the glycoprotein at room temperature for a further 60 min
(Fig. 2b). This led to an average of nine glycans per BSA.
Conclusions
In conclusion, we have demonstrated the versatility of the
Type B oxyamine linkers by preparing several
glycoconjugates. The formation of these glycoconjugates oc-
curred in good yield through the use of chemical ligation
reactions that include amide formation, sulfonylation,
copper-mediated azide-alkyne cycloaddition and thiol-
maleimide conjugations. In this way, representative biotinyl-
ated and fluorescent reporter groups were conjugated to
GlcNAc, the more complex (FITC) labelled LewisX chemical
probe was prepared, and a glycosylated protein was also syn-
thesised using an optimised protein conjugation protocol.
Given the ease and high-yielding synthesis of the ‘Type B’
Scheme 3 Application of thiol
oxyamine linker 1e to form a
biotinylated glycoconjugate

Glycoconj J
Fig. 2 a The synthesis of BSA-
glycoprotein 17. b SMCC
A
B
conjugation and glycan ligation
was monitored via MALDI-TOF
analysis. Conjugation of cross-
linker SMCC 15 was monitored at
each time point. At each time
point, an aliquot of the reaction
mixture was taken, glycoside 16
(360 equiv.) added and incubated
for 60 min, and then glycan
loading determined
20
15
10
5
SMCC conjugation
GlcNAc ligation
0
0
30
60
90
120
Cross-linking time (min)
oxyamine linkers and our proof-of-concept studies for the
formation of several classes of glycoconjugates, we envision
that these ‘Type B’ linkers will thus find wide application in
the synthesis of many other glycoconjugates using glycans
from both synthetic and natural sources.
glucosamine (34 mg, 0.15 mmol) and 3-azido-1-
methoxyaminopropane hydrochloride (1a) (195 mg,
1.50 mmol) were reacted in an AcOH/NH4OAc buffer
(1.5 mL, 2.0 M, pH 4.5, freshly prepared) at rt. for 24 h.
Purification by reverse phase chromatography (C18, H2O/
MeOH, 100/0 → 60/40, v/v) afforded neoglycoside 2
(44.6 mg, 0.13 mmol, 87%) as a white solid. Rf = 0.64 (silica,
CH2Cl2/EtOH/MeOH/NH3 (aq. 35%), 10/2/2/1, v/v/v/v);
αD20.3 = −19 (c = 0.1, MeOH); IR (film) 3369, 3296, 3253,
3003, 2953, 2943, 2889, 2099, 2067, 1735, 1647, 1605, 1575,
1567, 1487, 1446, 1419, 1377, 1349, 1308, 1253, 1079, 1020,
Experimental
General procedure for the chemical ligation of
methoxyamine linkers with carbohydrates To a solution
of carbohydrate (0.15 mmol) in an AcOH/NH4OAc buffer
(1.5 mL, 2.0 M, pH 4.5, freshly prepared), oxyamine linker
(1.5 mmol, 10 equiv., HCl salt) [25] was added and the reac-
tion mixture was stirred at rt. for 24 h. The crude mixture was
then directly loaded onto a size exclusion column (Bio-Gel
P-2, 1200 × 18 mm) and eluted with a 0.1 M aq. NH4HCO3
solution. Lyophilisation of the product fractions afforded the
neoglycoside. Alternatively, the neoglycoside can be purified
by directly loading the crude reaction mixture onto a reverse
phase column (C18) and/or by purification by silica gel col-
umn chromatography to afford the neoglycoside.
828 cm−1 1H NMR (500 MHz, D2O) δ 4.33 (d,1H,
;
J1′,2′ = 9.8 Hz, H-1′), 3.89 (dd, 1H, J1′,2′ = J2′,3′ = 9.6 Hz,
H-2′), 3.88 (dd, 1H, J5′,6′ = 1.5 Hz, J6a′,6b′ = 12.3 Hz, H-6a′),
3.73 (dd, 1H, J5′,6′ = 5.3 Hz, J6a′,6b′ = 12.3 Hz, H-6b′), 3.51
(dd, 1H, J2′,3′ = J3′,4′ = 8.7 Hz, H-3′), 3.50 (s, 3H, OCH3) 3.46–
3.34 (m, 4H, H-4′, H-5′, CH2–3), 3.09–2.98 (m, 2H, CH2–1),
2.04 (s, 3H, CH3 Ac), 1.88–1.74 (m, 2H, CH2–2); 13C NMR
(125 MHz, D2O) 173.9 (C = O), 90.0 (C-1′), 77.4 (C-5′), 75.5
(C-3′), 69.6 (C-4′), 61.0 (OCH3), 60.8 (C-6′), 52.3 (C-2′), 49.0
(C-3), 48.7 (C-1), 25.7 (C-2), 22.1 (CH3 Ac); HRMS(ESI) m/z
for [C12H24N5O6]+ calcd.: 334.1721, obsd.: 334.1719.
N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-N-(3-
azidopropyl)-O-methylhydroxylamine (2) N-Acetyl-
(1-(3-(Methoxy(2-acetamido-2-deoxy-β-D-glucosyl)amino)
propyl)-1H-1,2,3-triazol-4-yl)methanol (3) To a solution of

Glycoconj J
azide 2 (11.8 mg, 35 μmol) and propargyl alcohol (0.1 mL) in
ethanol (1 mL), a solution of L-ascorbic acid (6.2 mg,
35 μmol) and CuSO4 (1 mg) were added and the reaction
mixture was stirred for 3 d at rt. The crude mixture was fil-
tered, the residue washed with H2O (2 mL), and the combined
solvents concentrated in vacuo. Purification by reverse phase
chromatography (C18, H2O/MeOH, 100/0 → 90/10, v/v)
yielded triazole 3 (10.7 mg, 79%) as a colourless oil.
Rf = 0.53 (silica, CH2Cl2/EtOH/MeOH/NH3 (aq. 33%), 5/2/
2/1, v/v/v/v); αD19 = −0.82 (c = 0.1, MeOH); IR (film) 3350,
2934, 2874, 1643, 1555, 1442, 1378, 1319, 1227, 1104, 1054,
53.7 μmol), Et3N (12 μL) and HBTU (30.0 mg, 79 μmol)
were added and the reaction mixture was stirred for 2 h at rt.
To the reaction mixture, H2O (2 mL) was added and the white
precipitate was filtered and washed with water (2 × 1 mL).
The residue was then purified by C-18 reverse phase chroma-
tography (eluted with H2O), concentrated in vacuo, followed
by silica gel flash column chromatography (CH2Cl2/EtOH/
MeOH/NH3(aq. 33%), 15/2/2/1 → 10/2/2/1, v/v/v/v) to afford
glycoconjugate 4 (17.6 mg, 84%) as a colourless oil. Rf = 0.58
(CH2Cl2/EtOH/MeOH/NH3 (aq. 33%), 5/2/2/1, v/v/v/v);
αD20.0 = +3.1 (c = 0.2, MeOH); IR (film) 3343, 2927, 2857,
1671, 1652, 1637, 1559, 1542, 1457, 1437, 1395, 1375, 1317,
1
1033, 1021 cm−1; H NMR (500 MHz, CD3OD) δ 7.99 (s,
1
1H, triazole), 4.73 (s, 2H, C = C-CH2), 4.58–4.45 (m, 2H,
CH2 H-3), 4.33 (d, 1H, J1′,2′ = 9.8 Hz, H-1′), 3.91–3.85 (m,
2H, H-2′, H-6a′), 3.73 (dd, 1H, J5′,6b′ = 5.4 Hz,
J6a′,6b′ = 12.4 Hz, H-6b′), 3.53 (dd, 1H, J2′,3′ = 8.7 Hz,
J3′,4′ = 9.7 Hz, H-3′), 3.50 (s, 3H, OCH3), 3.42 (t, 1H,
J2′,3′ = J4′,5′ = 8.7 Hz, H-4′), 3.40–3.35 (m, 1H, H-5′), 3.03–
2.89 (m, 2H, H-1), 2.24–2.10 (m, 2H, H-2), 2.06 (s, 3H, CH3
Ac); 13C NMR (125 MHz, CD3OD) δ 173.9 (C = O Ac),
146.7 (Cq C = C), 124.0 (CH = C), 90.1 (C-1′′), 77.4 (C-5′
′), 75.4 (C-3′′), 69.6 (C-4′′), 61.2 (OCH3), 60.8 (C-6′′), 54.5
(CH2 allylic), 52.3 (C-2′′), 48.4 (C-1′), 48.2 (C-3′), 27.0
(C-2′), 22.1 (CH3 Ac); HRMS(ESI) m/z calcd. For
[C15H28N5O7]+: 390.1983, obsd.: 390.1986.
1266, 1110, 1078, 1053, 1033 cm−1; H NMR (500 MHz,
D2O) δ 4.58 (dd, 1H, J7,8 = 4.7 Hz, J6,7 = 7.9 Hz, H-7), 4.39
(dd, 1H, J5,6 = 4.5 Hz, J6,7 = 7.9 Hz, H-7), 4.30 (d, 1H, J1′′,2′
′ = 9.8 Hz, H-1′′), 3.90–3.84 (m, 2H, H-6a′′, H-2′′), 3.71 (dd,
1H, J5′′,6′′ = 5.4 Hz, J6a′′,6b′′ = 12.5 Hz, H-6b′′), 3.49 (t, 1H, J3′
′,4′′ = J4′′,5′′ = 8.9 Hz, H-3′′), 3.47 (s, 3H, OCH3), 3.42–3.34 (m,
2H, H-4′′, H-5′′), 3.34–3.28 (m, 1H, H-5), 3.28–3.22 (m, 1H,
H-3a′), 3.22–3.15 (m, 1H, H-3b′), 3.03 (m, 3H, CH2–1′,
H-8a), 2.75 (d, 1H, J8a,8b = 13.1 Hz, H-8b), 2.23 (t, 2H,
J1,2 = 7.2 Hz, CH2–1), 2.02 (s, 3H, CH3NAc), 1.78–1.50 (m,
6H, CH2–2′, CH2–2, CH2–4), 1.44–1.34 (m, 2H, CH2–
3); 13C NMR (125 MHz, D2O) δ 176.6 (C = O amide),
174.0 (C = O acetamide), 165.3 (C = O carbamide),
90.0 (C-1′′), 77.3 (C-5′′), 75.5 (C-3′′), 69.6 (C-4′′),
62.0 (C-6), 61.0 (OCH3), 60.8 (C-6′′), 60.1 (C-7), 55.3
(C-5), 52.2 (C-2′′), 48.7 (C-1′), 39.6 (C-8), 37.0 (C-3′),
35.4 (C-1), 27.7 (C-3), 27.6 (C-4), 25.9 (C-2′), 25.0
(C-2), 22.1 (CH3 Ac); HRMS(ESI) m/z calcd. For
[C22H39N5O8SNa]+: 556.2414, obsd.: 556.2416.
N-(3-(methoxy[2-acetamido-2-deoxy-β-D-glucopyranosyl]
-amino)propyl)-D-biotinamide (4) N-Acetyl-glucosamine
(34.0 mg, 0.15 mmol) and 3-(methoxyamino)propan-1-amine
hydrochloride 1b (211 mg, 1.50 mmol) were reacted in an
AcOH/NH4OAc buffer (1.5 mL, 2.0 M, pH 4.5, freshly pre-
pared) at rt. for 24 h. Purification by size exclusion chroma-
tography (Bio-Gel P-2) afforded N-(2-acetamido-2-deoxy-β-
D - g l u c o p y r a n o s y l ) - N - ( 3 - a m i n o p r o p y l ) - O -
methylhydroxylamine (28.3 mg, 0.092 mmol, 81%). Rf = 0.16
(silica, CH2Cl2/EtOH/MeOH/NH3 (aq. 35%), 5/2/2/1, v/v/v/
v); αD20.3 = −21 (c = 0.1, MeOH); IR (film) 3401, 3375, 3322,
3311, 3291, 3278, 2938, 2885, 2727, 2058, 1651, 1567, 1490,
5-(dimethylamino)-N-(2-((3-(methoxy(2-acetam
ido-2-deoxy-β-D-glucopyranosyl)amino)-propyl)-
thio)ethyl)naphthalene-1-sulfonamide (6) N-
Acetylglucosamine (12.0 mg, 54.2 μmol) and oxyamine 8
(89.1 mg, 0.54 mmol) were reacted in an AcOH/NH4OAc
buffer (1.5 mL, 2 M, freshly prepared, pH 4.5) at rt. for
72 h. Purification by reverse phase chromatography (C18,
H2O/MeOH, 100/0 → 90/10, v/v), followed by silica gel flash
column chromatography (CH2Cl2/EtOH/MeOH/NH3 (aq.
33%), 10/2/2/1 → 5/2/2/1, v/v/v/v) afforded
2 - ( 3 - [ m e t h o x y - ( 2 - a c e t a m i d o - 2 - d e o x y - β - D -
glucopyranosyl)amino]propylthio)ethan-1-amine as a
1
1459, 1437, 1376, 1315, 1257, 1108, 1023, 633 cm−1; H
NMR (500 MHz, D2O) δ 4.41 (d, 1H, J1′,2′ = 9.9 Hz, H-1′),
3.92 (dd, 1H, J5′,6′ = 0.9 Hz, J6a′,6b′ = 12.5 Hz, H-6a′), 3.85
(dd, 1H, J1′,2′ = J2′,3′ = 9.8 Hz, H -2′), 3.75 (dd, 1H,
J5′,6′ = 4.9 Hz, J6a′,6b′ = 12.5 Hz, H-6b′), 3.55 (dd, 1H,
J2′,3′ = J3′,4′ = 9.1 Hz, H-3′), 3.52 (s, 3H, OCH3), 3.45–3.40
(m, 2H, H-4′, H-5′), 3.12–3.01 (m, 4H, CH2–1, CH2–3), 2.06
colourless oil (16.1 mg, 81%). Rf = 0.53 (CH2Cl2/EtOH/
20.3
(s, 3H, CH3 Ac), 1.93 (p, 2H, J1,2 = J2,3 = 7.1 Hz, CH2–2); 13
C
MeOH/NH3 (aq. 33%), 5/2/2/1, v/v/v/v); αD
= −25
NMR (125 MHz, D2O) δ 174.1 (C = O Ac), 90.5 (C-1′), 77.4
(C-5′), 75.2 (C-3′), 69.6 (C-4′), 61.1 (OCH3), 60.8 (C-6′), 52.3
(C-2′), 47.9 (C-1), 37.6 (C-3), 24.5 (C-2), 22.1 (CH3 Ac);
HRMS(ESI) m/z calcd. For [C12H26N3O6]+: 308.1816, obsd.:
308.1820. To a solution of N-(2-acetamido-2-deoxy-β-D-
glucopyranosyl)-N-(3-aminopropyl)-O-methylhydroxylamine
(12.0 mg, 39.1 μmol) in DMF (0.2 mL), D-biotin (13.0 mg,
(c = 0.1, MeOH); IR (film) 3287, 2938, 2056, 1690, 1650,
1559, 1434, 1375, 1316, 1131, 1080, 1034, 947, 632 cm−1; 1H
NMR (500 MHz, D2O) δ 4.34 (d, 1H, J1′,2′ = 9.7 Hz, H-1′),
3.93–3.85 (m, 2H, H-2′, H-6a′), 3.89 (t, 1H,
J1′,2′ = J2′,3′ = 9.7 Hz, H-2′), 3.73 (dd, 1H, J5′,6′ = 5.3 Hz,
J6a′,6b′ = 12.4 Hz, H-6b′), 3.56–3.47 (m, 4H, H-2′, OCH3),
3.46–3.37 (m, 2H, H-4′, H-5′), 3.07–3.00 (m, 2H, CH2–1),

Glycoconj J
2.85 (t, 2H, J4,5 = 6.6 Hz, CH2–5), 2.71–2.54 (m, 4H, CH2–3,
CH2–4), 2.04 (s, 3H, CH3 Ac), 1.81 (m, 2H, CH2–2). 13C
NMR (125 MHz, D2O) δ 173.9 (C = O), 90.1 (C-1′), 77.5
(C-5′), 75.5 (C-3′), 69.7 (C-4′), 61.1 (OCH3), 60.9 (C-6′), 52.3
(C-2′), 50.3 (C-1), 39.4 (C-5), 32.9 (C-3), 28.4 (C-4), 26.4
(C-2), 22.2 (CH3 Ac); HRMS(ESI) m/z calcd. For
[C14H30N3O6S]+: 368.1850, obsd.: 368.1864. To a solution
of 2-(3-[methoxy-(2-acetamido-2-deoxy-β-D -
glucopyranosyl)amino]propylthio)ethan-1-amine (10.9 mg,
29.7 μmol) in H2O (2 mL), NaHCO3 (15 mg, 179 μmol)
was added. Dansylchloride (12.0 mg, 44.5 μmol) was dis-
solved in distilled THF (1 mL) and added drop wise to the
reaction mixture. After 2 h the THF was concentrated in vacuo
and the remaining aqueous reaction mixture was purified by
reverse phase chromatography (C18, H2O/MeOH, 100/0 →
25/75, v/v) to give fluorescent glycoside 6 (13.8 mg, 77%);
Rf = 0.50 (CH2Cl2/MeOH, 8/1, v/v); αD17.8 = −14.4 (c = 0.5,
MeOH); IR (film) 3304, 3283, 3088, 2939, 2795, 1650, 1572,
1503, 1456, 1409, 1374, 1356, 1316, 1232, 1201, 1142, 1076,
(CH2Cl2:EtOH:MeOH:NH3(35% aq), 5:2:2:1, v/v);
αD19.5 = −9.3 (c = 0.2, MeOH); IR (film) 3341, 2925, 2852,
17,117, 1647, 1586, 1466, 1451, 1415, 1380, 1350, 1302,
1233, 1193, 1085, 1026, 968, 917 cm−1 1H NMR
;
(500 MHz, D2O) δ 5.12 (d, 1H, J1^’,2″’ = 3.9 Hz, H-1″’),
4.84 (q, 1H, J5^’, 6″’ = 6.7 Hz, H-5″’), 4.49–4.42 (m, 1H,
H-1′, H-1″), 4.05 (m, 2H, H-2′, H-6a’), 3.93–3.81 (m, 5H,
H-3″’, H-4″, H-4′, H-3′, H-6b’), 3.80 (d, 1H, J3^’, 4″’ = J4^’,
5″’ = 2.9 Hz, H-4″’), 3.76 (m, 3H, H-6a^, H-6b^, H-2″’), 3.64
(dd, 1H, J3^, 4″ = 3.2 Hz, J2^, 3″ = 9.9 Hz, H-3″), 3.60–3.55 (m,
1H, H-5″), 3.54–3.46 (m, 1H, H-5″), 3.54–3.46 (m, 5H,
OCH3, H-5′, H-2″), 3.11–2.92 (m, 4H, CH2–1, CH2–3), 2.02
(s, 3H, CH3 NAc), 1.95 (m, 2H, CH2–2), 1.17 (d, 3H,
J5^’,6″’ = 6.7 Hz, H-6″’); 13C NMR (125 MHz, D2O) 170.5
(C = O), 101.8 (C-1″), 98.7 (C-1″’), 90.5 (C-1′), 76.9 (C-5′),
76.1 (C-3′), 74.8 (C-5″), 73.2 (C-4′), 72.4 (C-3″), 71.8 (C-4″’),
70.9 (C-2″), 69.1 (C-3″’), 68.3 (C-4″), 67.6 (C-2″’), 66.7
(C-5″’), 61.4 (C-6″), 60.9 (OCH3), 59.7 (C-6′), 52.5 (C-2′),
47.2 (C-1), 37.5 (C-3), 24.5 (C-2), 22.1 (CH3 NAc), 15.2
(C-6″’); HRMS(ESI) m/z calcd. For [C24H46N3O15]+:
616.2923, obsd.: 616.2938. To a solution of N-(2-acetamido-
2- deo xy-3 - O - ( α -L - f u c o p y r a n o s y l ) - 4 - O - ( β - D -
galactopyranosyl)-β-D-glucopyranosyl)-N-(3-aminopropyl)-
O-methylhydroxylamine (12.0 mg, 0.0195 mmol) in H2O
(0.5 mL), was added NaHCO3 (9 mg, 0.107 mmol) followed
by fluorescein isothiocyanate isomer I (12.5 mg, 0.032 mmol)
in THF (0.25 mL). The reaction was stirred at room tempera-
ture for 2.5 h, then concentrated in vacuo and purified by
reverse phase chromatography (C18, H2O/MeOH, 100/0 →
10/90, v/v) to give fluorescent glycoside 8 (18 mg, 92%);
Rf = 0.08 (silica, CH2Cl2/MeOH/AcOH, 70/29/1, v/v/v);
αD21.1 = − 0.17 (C = 0.7, H2O); IR (film) 3356, 2929, 1656,
1576, 1505, 1464, 1391, 1329, 1212, 1170, 1111, 1081, 1039,
1
1037, 945, 793, 686, 625, 573, 554, 536 cm−1; H NMR
(500 MHz, CD3OD) δ 8.57 (d, 1H, J2,3 = 8.6 Hz, H-2), 8.34
(d, 1H, J3,4 = 8.7 Hz, H-4), 8.21 (d, 1H, J7,8 = 7.3 Hz, H-8),
7.62–7.56 (m, 2H, H-3, H-7), 7.28 (d, 1H, J6,7 = 7.6 Hz, H-6),
4.22 (d, 1H, J1′′,2′′ = 9.8 Hz, H-1′′), 3.85 (dd, 1H, J5′′,6a′
= 1.8 Hz, J6a′′,6b′′ = 12.0 Hz, H-6a′′), 3.78 (dd, 1H, J1′′,2′
′
′ = J2′′,3′′ = 9.9 Hz, H-2′′), 3.68 (dd, 1H, J5′′,6b′′ = 5.6 Hz, J6a′
′,6b′′ = 12.0 Hz, H-6b′′), 3.47 (s, 3H, OCH3), 3.41 (dd, 1H, J2′′,3′
′ = J3′′,4′′ = 9.0 Hz, H-3′′), 3.36–3.27 (m, 1H, H-4′′), 3.21 (ddd,
1H, J5′′,6a′′ = 1.9 Hz, J5′′,6b′′ = 5.4 Hz, J4′′,5′′ = 9.6 Hz, H-5′′),
3.02 (t, 1H, J4′,5′ = 7.2 Hz, H-5′), 2.98–2.85 (m, 8H, H-1′,
2 × N-CH3), 2.49–2.32 (m, 4H, H-3′, H-4′), 1.95 (s, 3H,
CH3 Ac), 1.69–1.59 (m, 1H, H-2′); 13C NMR (125 MHz,
CD3OD) δ 173.3 (C = O Ac), 153.2, 137.1, 131.32, 131.30
(C-1/4a/5/8a), 130.9 (C-2), 130.1 (C-8), 129.2, 124.3 (C-3/C-
7), 120.6 (C-4), 116.5 (C-6), 92.4 (C-1′′), 79.8 (C-5′′), 77.5
(C-3′′), 71.8 (C-4′′), 62.9 (C-6′′), 62.2 (OCH3), 54.1 (C-2′′),
51.4 (C-1′), 45.8 (N(CH3)2), 43.9 (C-5′), 32.4 (C-4′), 30.2
(C-3′), 28.5 (C-2′), 23.0 (CH3 Ac); HRMS(ESI) m/z calcd.
For [C26H41N4O8S2]+: 601.2360, obsd.: 601.2362.
1
1021, 915, 852, 809, 770, 670 cm−1; H NMR (500 MHz,
D2O) δ 7.58–7.38 (m, 2H, H-aromatic), 7.23–7.07 (m, 3H,
H-aromatic), 6.53–6.48 (m, 4H, H-aromatic), 5.10 (d, 1H, J1′
′′′,2′′′′ = 3.9 Hz, H-1′′′′), 4.82 (q, 1H, J5′′′′,6′′′′ = 6.6 Hz, H-5′′′′),
4.41 (d, 1H, J1′′,2′′ = 7.0 Hz, H-1′′), 4.37 (d, 1H, J1′′′,2′′
= 7.2 Hz, H-1′′′), 4.08 (s, 1H, H-2′′), 3.94 (d, J6a′′, 6b′
′
= 11.7 Hz, H-6a′′), 3.90–3.75 (m, 6H, H-3′′′′, H4′′′, H-4′′,
′
N-(2-Acetamido-2-deoxy-3-O-(α-L-fucopyranosyl)-4-O-
(β-D-galactopyranosyl)-β-D-glucopyranosyl)-N-(2-((3-(3-
(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-
xanthen]-5-yl)thioureido)propyl)-O-methylhydroxylamine
(8) To a solution of LewisX (5.6 mg, 10.6 μmol) in a
AcOH/NH4OAc buffer (0.5 mL, 2 M, freshly prepared,
pH 4.5), 3-(methoxyamino)propan-1-amine hydrochloride
(1b) (15.9 mg, 113.2 μmol) was added and the reaction mix-
ture was stirred at 40 °C for 35 h. The crude mixture was
directly loaded on a size exclusion column (Bio-Gel P-2,
1200 × 18 mm) and eluted with 0.1 M NH4HCO3 (aq).
Lyophilization of the product fractions afforded the
n e o g l y c o s i d e ( 5 . 7 m g , 8 8 % ) . R f = 0 . 1 0
H-3′′, H6b′′, and H-4′′′′), 3.74–3.57 (m, 6H, H-3a′, H-3b′,
H-6a′′′, H-6b′′′, H-3′′′, H-2′′′′), 3.55–3.44 (m, 6H, OCH3,
H-5′′, H-5′′′, H-2′′′), 3.02 (br s, 2H, H-1′), 2.04 (s, 3H, CH3
Ac), 1.91–1.81 (m, 2H, H-2′), 1.15 (d, 3H, J5′′′′,6′′′′ = 6.6 Hz,
H-6′′′′); 13C NMR (150 MHz, D2O) δ 180.5 (C = S), 174.0
(C = O), 173.8 (C = O NAc), 158.7 (C-quart), 158.6 (C-quart),
141.1 (C-quart), 138.1 (C-quart), 131.5 (C-aromatic), 131.3
(C-aromatic), 125.8 (C-aromatic), 124.6 (C-aromatic), 122.8
(C-aromatic), 112.4 (C-quart), 112.3 (C-quart), 103.6 (C-
quart), 103.5 (C-aromatic), 101.9 (c-1′′′), 98.7 (C-1′′′′), 90.0
(C-1′′), 77.0 (C-5′′), 76.3 (C-3′′), 74.8 (C-5′′′), 73.5 (C-4′′),
72.3 (C-3′′′), 71.8 (C-4′′′′), 70.9 (C-2′′′), 69.1 (C-3′′′′), 68.2
(C-4′′′), 67.6 (C-2′′′′), 66.6 (C-5′′′′), 61.4 (C-6′′′), 59.8 (C-6′

Glycoconj J
′), 52.4 (C-2′′), 48.7 (C-1′), 42.9 (C-3′), 25.9 (C-2′), 22.3 (CH3
Ac), 15.2 (C-6′′′′); HRMS(ESI) m/z calcd. For
[C45H57N4O20S]+ 1005.3281, obsd.: 1005.3278.
3H, CH3 -Ac), 2.02 (s, 3H, CH3 -Ac), 2.00 (s, 3H, CH3 -Ac),
1.97–1.84 (m, 5H, CH3 -Ac, H-2eq, H-2axisomer A), 1.49 (bd,
1H, H-2eqisomer B); 13C NMR (150 MHz, CDCl3, 50 °C) δ
171.2 (C = O), 170.8 (C = O), 170.53 (C = O), 170.46
(C = O), 170.3 (C = O), 170.2 (C = O), 169.9 (C = O), 169.8
(2 × C = O), 169.6 (C = O), 70.9, 70.5, 69.9, 69.4 (C-3′, C-4′
isomer A + B) 69.0 (C-5′ isomer A + B), 68.6 (C-1′ isomer B),
67.6 (C-1′ isomer A), 62.3 (C-6′ isomer A/B), 62.1 (C-6′ isomer
A/B), 59.8 (OCH3isomer B), 58.2 (OCH3isomer A), 52.9
(CH2NO isomer B), 51.6 (CH2NO isomer A), 51.5 (C-2′ isomer
A), 49.5 (C-2′ isomer B), 28.1 (CH2S isomer B), 26.8 (CH2S
isomer A), 23.4 (C-CH2-C isomer A/b), 23.2 (C-CH2-C isomer
A/b), 20.9, 20.8, 20.7 (CH3 Ac); TOCSY NMR (300 MHz, D2O,
50 °C) Isomer a δ 3.59 (excited, app. bd, J1ax,1eq = 14.6 Hz,
H-1 eq), 2.84 (app. bt, J1ax,1eq = J1ax,2ax = 13.1 Hz, H-1ax),
2.13 (app. bq, J1ax,2ax = J2ax,2eq = J2ax-3ax = 13.0 Hz, H-2 ax),
1.50 (app. bd, J2eq,2ax = 14.3 Hz, H-2 eq), 2.97 (app. bt,
J2ax,3ax = J3ax,3eq = 13.0 Hz, H-3ax), 2.64 (app. bd,
J3eq,3ax = 13.3 Hz, H-3 eq); HRMS(ESI) m/z calcd. For
[C20H33N2O10S]+: 493.1850, obsd.: 493.1856.
D-gluco-2-(1-Acetamido-2,3,4,5-tetrahydroxy-pentyl)-3-
methoxy-1,3-thiazinane (9) N-Acetylglucosamine (34 mg,
0.15 mmol) and 3-(methoxyamino)propane-1-thiol (1d)
(182 mg, 1.50 mmol) were reacted in an AcOH/NH4OAc
buffer (1.5 mL, 2.0 M, pH 4.5, freshly prepared) containing
5% TCEP at rt. for 72 h. Purification by reverse phase chro-
matography (C18, H2O/MeOH, 100/0 → 60/40, v/v) afforded
thiazinane 9 (44 mg, 89%) as an colourless oil. Rf = 0.32
(silica, CH2Cl2/EtOH/MeOH/NH3 (aq. 33%), 10/2/2/1, v/v/
v/v); IR (film) 3298, 2940, 1646, 1545, 1428, 1374, 1318,
1
1284, 1190, 1079, 1037, 951, 867, 844, 694, 632 cm−1; H
NMR (300 MHz, D2O, 50 °C) δ 4.85–4.62 (m, 2H), 4.63–
4.38 (m, 2H), 4.12–3.72 (m, 10H), 3.35–2.91 (m, 4H), 2.40–
2.13 (m, 5H), 2.11–1.77 (m, 1H); 13C NMR (150 MHz, D2O)
δ 174.1, 173.8, 72.2, 71.3, 69.3, 68.4, 68.0, 62.6, 62.3, 58.6,
58.0 53.5, 50.6, 27.2, 22.1, 22.0, 17.8; HRMS(ESI) m/z calcd.
For [C12H25N2O6S]+: 325.1428, obsd.: 325.1426.
1-(2-D-biotinamidoethyl)-1H–pyrrole-2,5-dione (13) To a
solution of 2-aminoethyl maleimide 1230 (159 mg, 1.13 mmol)
and D-biotin (363.4 mg, 1.31 mmol) in DMF (10 mL), HBTU
(516 mg, 1.36 mmol) and triethylamine (0.1 mL) were added
and the reaction mixture was stirred at rt. for 2 h. The crude
reaction mixture was concentrated and purified by reverse
phase column chromatography (C18, H2O/MeOH, 100/0 →
70/30, v/v) and size exclusion chromatography (Sephadex
LH-20, CH2Cl2/MeOH, 50/50, v/v) to give biotin
functionalised maleimide 13 as a white solid (380 mg,
1.04 mmol, 92%). Rf = 0.59 (silica, CH2Cl2/MeOH, 80/20,
v/v); αD20.6 = + 26.5 (c = 0.2, MeOH); IR (film) 3283, 3087,
2936, 2864, 1701, 1552, 1461, 1439, 1408, 1360, 1332, 1266,
D-gluco-2-(1-Acetamido-2,3,4,5-tetra-acetoxy-pentyl)-3-
methoxy-1,3-thiazinane (10) The mixture of thiazinanes 9
(12.0 mg, 37.0 μmol) was co-evaporated with pyridine:acetic
anhydride (3 mL, 2/1, v/v) and dissolved in acetic anhydride
(2 mL) and pyridine (1 mL) and stirred at rt. overnight. The
crude reaction mixture was concentrated and purified by silica
gel flash column chromatography (PE/EtOAc, 95/5 → 80/20, v/
v) to give diastereomeric thioaminals 10 (17.8 mg, 98%) as a
colorless oil. Rf = 0.38 (EtOAc); IR (film) 2951, 2847, 2816,
1742, 1668, 1514, 1429, 1370, 1313, 1211, 1033, 953, 916,
1
859, 845, 795, 729, 646, 633 cm−1; H NMR (300 MHz,
CDCl3, 50 °C) δ 6.06 (d, 1H, JNH,2′ = 9.8 Hz, NH isomer A),
5.64 (d, 1H, JNH,2′ = 10.1 Hz, NH isomer B), 5.50 (dd, 1H,
J3′,4′ = 2.0 Hz, J2′,3′ = 9.4 Hz, H-3′ isomer A), 5.43 (dd, 1H,
J3′,4′ = 2.0 Hz, J2′,3′ = 8.6 Hz, H-3′ isomer B), 5.43–5.37 (m, 2H,
H-4′ isomer A + B), 5.23–5.13 (m, 2H, H-5′ isomer A + B), 4.84
(dt, 1H, J1′,2′ = J2′,3′ = 5.5 Hz, J2′,NH = 10.0 Hz, H-2′ isomer B),
1
1169, 1101, 1055, 1033, 828, 763, 696 cm−1; H NMR
(500 MHz, CD3OD) δ 6.82 (s, 2H, HC = CH), 4.60 (s, 1H,
NH), 4.49 (dd, J6,7 = 4.9 Hz, J7,8 = 7.7 Hz, 1H, H-7), 4.67 (dd,
1H, J6,7 = 4.9 Hz, J5,6 = 7.8, Hz H-6), 3.61 (dd, 2H,
J1′,2′ = 5.5 Hz, CH2–1′), 3.36 (dd, 2H, J1′,2′ = 5.5 Hz, CH2–
2′), 3.24–3.17 (m, 1H, H-5), 2.93 (dd, 1H, J7-8a = 5.0 Hz, J8a-
8b = 12.8 Hz, H-8a), 2.70 (d, 1H, J8a-8b = 12.8 Hz, H-8b), 2.12
(dt, 2H, J1a,2 = 3.4 Hz, J1b,2 = 7.4 Hz, CH2–1), 1.79–1.66 (m,
1H, CH2–4a), 1.66–1.52 (m, 3H, CH2–2, CH2–4b), 1.50–1.35
(m, 2H, CH2–3); 13C NMR (125 MHz, CD3OD) δ 176.3
(C = O amide), 172.6 (2 × C = O maleimide), 166.1 (C = O
carbamate), 135.5 (C = C), 63.3 (C-6), 61.6 (C-7), 56.9 (C-5),
41.0 (C-8), 38.8 (C-2′), 38.4 (C-1′), 36.7 (C-1), 29.7 (C-3),
29.4 (C-4), 26.6 (C-2); HRMS(ESI) m/z calcd. For
[C16H23N4O4S]+: 367.1435, obsd.: 367.1429.
1
4.53 (bd, 1H, J1′,2′ = 4.5 Hz, JCH = 147 Hz, H-1′ isomer A),
4.40 (dt, 1H, J1′,2′ = J2′,3′ = 4.9 Hz, J2′,NH = 9.7 Hz, H-2′ isomer
A), 4.28 (dd, 1H, J5′,6a′ = 3.2 Hz, J6a′,6b′ = 12.4 Hz, H-6a′ isomer
B), 4.22 (dd, 1H, J5′,6a′ = 2.9 Hz, J6a′,6b′ = 12.5 Hz, H-6b′ isomer
A), 4.15–4.07 (m, 2H, H-6b′ isomer A + B), 3.90 (bd, 1H,
1
J1′,2′ = 5.2 Hz, JCH = 149 Hz, H-1′ isomer B), 3.68 (s, 3H,
OCH3isomer A), 3.63–3.52 (m, 1H, H-1eqisomer A), 3.49 (s,
3H, OCH3isomer B), 3.39 (dt, J1eq,2eq = J1eq,2ax = 4.3 Hz,
J1eq,1ax = 13.3 Hz, H-1eqisomer B), 2.95 (dt, 1H,
J2eq,3ax = 1.7 Hz, J2ax,3ax = J3eq,3ax = 13.0 Hz, H-3axisomer B),
2.83 (ddd, 1H, J1ax,2eq = 2.9 Hz, J1ax,2ax = 13.0 Hz, J1ax,-
1eq = 15.5 Hz, H-1axisomer B), 2.75–2.57 (m, 3H, H-3eqisomer
B, H-1axisomer A, H-3axisomer A, H-3eqisomer A), 2.18 (s, 3H,
CH3-Ac), 2.10 (s, 3H, CH3 -Ac), 2.04 (s, 3H, CH3 -Ac), 2.03 (s,
1-(2-D-biotinamidoethyl)-3-(3-(3-(methoxy[2-aceta
mido-2-deoxy- β -D-glucopyranosyl]-amino)
propylthio)propylthio)pyrrolidine-2,5-dione (14) N-

Glycoconj J
Acetylglucosamine (7.8 mg, 35.3 μmol) and
3-(3-(methoxyamino)propylthio)propane-1-thiol (1e)
(69.0 mg, 0.35 mmol) were reacted in a mixture of
AcOH/NH4OAc buffer (0.2 mL, 2 M, freshly prepared,
pH 4.5), ethanol (0.2 mL) and CH2Cl2 (0.1 mL) at 40 °C for
72 h. The crude reaction mixture was directly loaded on a size
exclusion column (Bio-Gel P-2, 600 × 10 mm) and eluted
with 0.1 M aq. NH4HCO3. Lyophilisation of the product frac-
tions afforded 1,2-bis(3-[3-(methoxy-[2-acetamido-2-
deoxy-β-D-glucopyranosyl]amino)propylthio]propyl)-
disulfane (9.0 mg, 64%) as a colorless oil. Rf
(Sulfhydryl) = 0.34 (10/2/2/1, CH2Cl2/EtOH/MeOH/
1.76–1.66 (m, 1H, H-4a), 1.62–1.50 (m, 3H, H-4b, CH2–2),
1.42–1.33 (m, 2H, CH2–3); 13C NMR (125 MHz, D2O) δ
182.0, 180.8 (2 × C = O pyrrolidine), 179.5 (C = O biotin),
176.3 (C = O NAc), 167.8 (C = O carbamate-biotin), 92.6
(C-1′′′′), 80.0 (C-4′′′′), 78.1 (C-3′′′′), 72.2 (C-5′′′′), 64.6
(C-6), 63.7 (OCH3), 63.4 (C-6′′′′), 62.8 (C-7), 57.8 (C-5),
54.9 (C-2′′′′), 52.9 (C-1′′′), 42.5 (C-1′′), 42.3 (C-8), 41.2
(C-1′), 39.1 (C-2′), 38.5 (C-2′′), 38.0 (C-1), 32.4 (C-6′′′),
32.3 (C-4′′′), 31.2 (C-3′′′), 30.6, 30.5 (C-5′′′, C-3), 30.2
(C-4), 28.9 (C-2′′′), 27.5 (C-2), 24.8 (CH3 Ac); HRMS(ESI)
m/z calcd. For [C31H53N6O10S3]+: 765.2980, obsd.: 765.2975.
NH3(aq, 33%)), Rf = (disulfide) = 0.17 (10/2/2/1, CH2Cl2/
Glycoprotein 17 To a solution of Bovine Serum Albumin
(1 mg, 0.015 μmol) in PBS (1 mL), SMCC cross-linker 15
(0.9 mg, 2.73 μmol) in MeCN (0.1 mL) was added and the
reaction mixture incubated at rt. To 10 μL of the crude reac-
tion mixture, neoglycoside 16 (2.2 mg, 5.45 μmol) in water
(10 μL) was added and the reaction mixture was allowed to
stir at rt. to obtain glycoprotein 17.
19.8
EtOH/MeOH/NH3(aq, 33%)); αD
= +3.5 (c = 0.1,
MeOH); IR (film) 3313, 3273, 3094, 3070, 2656, 2909,
1632, 1557, 1487, 1439, 1355, 1215, 1195, 1111, 1038,
1
1000, 986, 903, 755, 721, 691, 620, 604 cm−1; H NMR
(500 MHz, D2O) δ 4.34 (d, 2H, J1′,2′ = 9.8 Hz, H-1′), 3.92–
3.86 (m, 4H, H-6a′, H-2′), 3.74 (dd, 2H, J5′,6b′ = 5.4 Hz,
J6a′,6b′ = 12.4 Hz, H-6b′), 3.55–3.46 (m, 8H, OCH3, H-3′),
3.44–3.36 (m, 4H, H-4′, H-5′), 3.10–3.00 (m, 4H, CH2–1),
2.85 (t, 4H, J5,6 = 7.1 Hz, CH2–6), 2.70 (t, 4H,
J4,5 = 7.1 Hz, CH2–4), 2.68–2.57 (m, 4H, CH2–3), 2.04 (s,
6H, CH3NAc), 2.00 (p, 4H, J4,5 = J5,6 = 7.0 Hz, CH2–5), 1.86–
1.78 (m, 4H, CH2–2); 13C NMR (125 MHz, D2O) δ 173.8
(C = O), 90.0 (C-1′), 77.4 (C-5′), 75.5 (C-3′), 69.6 (C-4′), 61.1
(OCH3), 60.8 (C-6′), 52.3 (C-2′), 50.3 (C-1), 36.5 (C-6), 29.4
(C-4), 28.6 (C-3), 27.9 (C-5), 26.3 (C-2), 22.1 (CH3NAc);
HRMS(ESI) m/z calcd. For [C30H59N4O12S4]+: 795.3007,
obsd. : 795. 3018; HRMS(ESI) m/z calcd. For
[C15H31N2O6S2]+: 399.1618, obsd.: 399.1609. To a solution
of 1,2-bis(3-[3-(methoxy-[2-acetamido-2-deoxy-β-D-
glucopyranosyl]amino)propylthio]propyl)-disulfane (7.0 mg,
17.6 μmol) in H2O (1 mL), maleimide 13 (9.0 mg, 24.6 mmol)
was added, and the reaction mixture was stirred at rt. for 1 h.
The crude reaction mixture was purified using size exclusion
chromatography (Bio Gel P-2) to yield glycoconjugate 14
(12.6 mg, 16.5 μmol, 94%) as a colourless oil. Rf = 0.54
(CH2Cl2/EtOH/MeOH/NH3 (aq. 33%), 5/2/2/1, v/v/v/v); IR
(film) 3338, 3286, 2933, 2871, 2859, 1700, 1648, 1559,
1452, 1439, 1332, 1265, 1188, 1107, 1080, 1026, 969, 901,
847, 760,699 cm−1; 1H NMR (500 MHz, D2O) δ 4.60 (dd, 1H,
MALDI-TOF To a solution of cinnapinic acid (15 mg) in
MeCN:H2O (1 mL, 1/1, v/v), TFA (1 μL) was added and the
mixture was vortexed for 3 min and centrifuged for 10 min, to
obtain the matrix solution. A 1–2 μL aliquot of the glycopro-
tein (~1.0 mg/mL) was added to 20 μL of the matrix solution
and a pipette was used to mix the sample. Next, 2 μL of the
sample/matrix was spotted on a MALDI-plate, dried for 1 h,
and measured using MALDI-TOF.
Acknowledgments The authors would like to thank the Health
Research Council of New Zealand for financial support (Hercus
Fellowship, BLS).
Compliance with ethical standards
Conflicts of interest The authors declare that they have no conflicts of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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