Synthesis of novel triazole-derived glycopeptides as analogs of α-dystroglycan mucins

Article abstract of DOI:10.1016/j.carres.2018.11.004

α-Dystroglycan (α-DG) mucins are essential for maintenance of the structural and functional stability of the muscle fiber and, when hypoglycosylated, they are directly involved in pathological processes such as dystroglycanopathies. Thus, this work report

Full text of DOI:10.1016/j.carres.2018.11.004

Accepted Manuscript
Synthesis of novel triazole-derived glycopeptides as analogs of α-dystroglycan
mucins
Marcelo Fiori Marchiori, Giulia Pompolo Iossi, Leandro Oliveira Bortot, Marcelo Dias-
Baruffi, Vanessa Leiria Campo
PII:
S0008-6215(18)30559-7
https://doi.org/10.1016/j.carres.2018.11.004
CAR 7628
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 26 September 2018
Revised Date: 5 November 2018
Accepted Date: 5 November 2018
Please cite this article as: M.F. Marchiori, G.P. Iossi, L.O. Bortot, M. Dias-Baruffi, V.L. Campo, Synthesis
of novel triazole-derived glycopeptides as analogs of α-dystroglycan mucins, Carbohydrate Research
(2018), doi: https://doi.org/10.1016/j.carres.2018.11.004.
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ACCEPTED MANUSCRIPT

1
ACCEPTED MANUSCRIPT
Synthesis of novel triazole-derived glycopeptides as analogs of α-dystroglycan mucins
1
1
1
1
Marcelo Fiori Marchiori, Giulia Pompolo Iossi, Leandro Oliveira Bortot, Marcelo Dias-Baruffi,
1
,2*1
Vanessa Leiria Campo
1
. Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP. Av. do Café S/N, CEP 14040-903,
Ribeirão Preto - SP, Brazil.
2
. Barão de Mauá University Centre. 423 Ramos de Azevedo Street, Jardim Paulista, CEP 14090-
180, Ribeirão Preto - SP, Brazil.
ABSTRACT
α-Dystroglycan (α-DG) mucins are essential for maintenance of the structural and functional
stability of the muscle fiber and, when hypoglycosylated, they are directly involved in pathological
processes such as dystroglycanopathies. Thus, this work reports the synthesis of the novel 1,2,3-triazole-
derived glycosyl amino acids αGlcNAc-1-O-triazol-2Manα-ThrOH (1) and Gal-β1,4-αGlcNAc-1-O-
triazol-2Manα-ThrOH (2), followed by solid-phase assembly to get the corresponding glycopeptides
NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH (3) and NHAcThrVal[Gal-β1,4-αGlcNAc-1-
triazol-2Manα]ThrIleArgGlyOH (4) as analogs of α-DG mucins. The glycosyl amino acids 1 (72%) and 2
(35%) were synthesized by Cu(I)-assisted 1,3-dipolar azide-alkyne cycloaddition reactions (CuAAC)
between the azide-glycosyl amino acid αManN -FmocThrOBn (5) and the corresponding alkyne-
3
functionalyzed sugars 2’-propynyl-αGlcNAc (6) and 2’-propynyl-Gal-β1,4-αGlcNAc (7), followed by
hydrogenation reactions. Subsequently, glycopeptides 3 (23%) and 4 (12%) were obtained by solid phase
synthesis, involving sequential couplings of Fmoc-protected amino acids or the glycosyl amino acids 1
and 2, followed by cleavage from resin, N-acetylation and O-deacetylation (NaOMe) reactions. Lastly,
enzymatic galactosylation of glycopeptide 3 with bovine β-1,4-GalT showed that it was not a substrate for
this enzyme, which could be better elucidated by docking simulations with β-1,4-GalT.
Keywords: α-Dystroglycan; Mucin; Glycopeptide; Triazole; Synthesis; β-1,4-Galactosyltransferase
1
Corresponding author. Tel.: +55 16 33154285; fax: +55 16 33154879; +55(16) 3603-6600
e-mail: vlcampo@fcfrp.usp.br

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ACCEPTED MANUSCRIPT
1
. Introduction
Dystroglycan (DG) is a core component of the dystrophin-glycoprotein (DGC) complex, which
binds the cytoskeleton (actin) to extracellular matrix proteins (ECMs), via dystrophin and sarcoglycans,
and is essential to maintain the structural and functional stability of the muscle fiber, enabling its
successive cycles of contraction and relaxation, besides being expressed in other tissues such as epithelial
and neural [1-3]. Dystroglycan (DG), encoded by the DAG1 gene, is biosynthesized as a precursor
molecule that is further cleaved into α- and β-dystroglycan subunits (post-translational modification). β-
Dystroglycan (β-DG) is a transmembrane protein that interacts with dystrophin through its intracellular
domain, whereas α-dystroglycan (α-DG) is located outside the membrane and binds directly to
extracellular matrix proteins including laminin, which is mostly characterized, agrin, perlecan and
neurexin [4,5].
α-DG is a highly glycosylated protein composed by globular N- and C-terminal domains that are
connected by a mucin-type domain, constituted by sequences rich in Ser/Thr, which represents potential
sites for extensive O-glycosylation (Fig. 1A). Among the main peptide sequences identified in α-DG
mucins is the DPVPGKPTVTIR sequence, whereas the major O-linked glycans found in α-DG mucins
are represented by the tetrasaccharide structural motif Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα-O-Ser/Thr,
containing the peculiar O-mannose units (Fig. 1B), followed by the less expressed Galβ1-3GalNAcα-O-
Ser/Thr, which is the most common core of other mammal mucins [4-7]. The α-DG tetrasaccharide
biosynthesis is triggered by the action of distinct genes that encode the glycosyltransferases protein O-
mannosyltransferases 1 and 2 (POMT1 and POMT2), and protein O-linked-mannose β-1,2-N-
acetylglucosaminyltransferase 1 (POMGnT1), which catalyze the mannose and N-acetylglucosamine
transfer reactions, respectively, as well as the LARGE glucosyltransferase and fukutin-related protein
(FKRP), whose functions are not fully elucidated [7-9].

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ACCEPTED MANUSCRIPT
O
O
O
HO OH
R= HO
HO
O
C
N
αMan
O
O
O
DPVPGKPTVTIR α-DG tandem repeat MUC
R
OH
CO H
2
HO
AcHN
OH OH
OH
OH
O
O
O
O
O
O
HO
HO
OH
AcHN
HO
HO
O
HO
Neu5Acα2-3Galβ1-4GlcNAclβ1-2Manα-O-Ser/Thr
OSer/Thr
Figure 1. (A) General representation of α-DG mucins; (B) Chemical structure of the tetrasaccharide motif
of α-DG.
Thus, abnormalities in the biosynthesis process of the above-mentioned O-mannosyl tetrasaccharide
leads to hypoglycosylation of α-DG, disrupting its binding to the extracellular matrix, which in turn may
result in various phenotypes of dystroglycanopathies known as congenital muscular dystrophies (CMDs),
such as Walker-Warburg syndrome (WWS), Muscle-eye-brain disease (MEB) and Limb-Girdle muscular
dystrophy (LGMD), which are manifested from the neonatal period and are generally characterized by
progressive muscular degeneration, starting with muscle weakness and evolving to loss of movement and
respiratory failure [7-12]. Despite their seriousness, so far there is no available treatment nor efficient
diagnostic strategies directed to these dystroglycanopathies. Therefore, the synthesis of O-linked glycans
found in α-DG mucins may represent a powerful tool to get synthetic substrates for glycosyltransferases
involved in α-DG biosynthesis, which may guide the deeper investigation of their functions, favoring the
development of effective therapeutic and diagnostic strategies towards CMDs.
The synthesis of α-DG tetrasaccharide core Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα-O-Ser/Thr
under different synthetic methods have been previously reported [13,14], as well as the synthesis of α-DG
mucin glycopeptides bearing this tetrasaccharide core along with others containing the trisaccharide
(Galβ1-4GlcNAcβ1-2Manα-O-Ser/Thr) and disaccharide (GlcNAcβ1-2Manα-O-Ser/Thr) intermediate
cores [15,16]. In a distinct approach, a phospho-O-mannosyl trisaccharide glycopeptide derived from α-
DG was also synthesized and applied for investigation of the enzyme factors that regulate O-glycans

4
ACCEPTED MANUSCRIPT
extension in α-DG [17]. Within this context, in a continuous seek for novel α-DG-derived glycans, we
asked whether or not the replacement of the β-1,2-glycosidic linkage between mannose and N-
acetylglucosamine sugar units found in α-DG by O-triazole linkage could represent an efficient strategy
to get peculiar α-DG mucins analogs as possible substrates for dystroglycan-related glycosyltransferases,
as well as tools for the generation of antibodies against distinct glycoforms involved in the pathology of
CMDs, which is an actual clinical demand. It is noteworthy the advantageous physicochemical properties
of the triazole group, which is not susceptible to hydrolysis, oxidation or reduction, besides acting as a rigid
link. Moreover, triazole derivatives can be promptly obtained by Cu(I)-catalyzed 1,3-dipolar azide-alkyne
cycloaddition (CuAAC) reactions from suitable azide and alkyne functionalized precursors [18].
Thus, upon these considerations, here we report the synthesis of the novel 1,2,3-triazole-derived
glycosyl amino acids αGlcNAc-1-O-triazol-2Manα-ThrOH (1) and Gal-β1,4-αGlcNAc-1-O-triazol-
2
Manα-ThrOH (2) (Fig. 2A), followed by solid-phase assembly to get the corresponding glycopeptides
NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH (3) and NHAcThrVal[Gal-β1,4-αGlcNAc-1-
triazol-2Manα]ThrIleArgGlyOH (4) (Fig. 2B) as analogs of α-DG mucins. Attempt for enzymatic
galactosylation of glycopeptide 3 by bovine β-1,4-galactosyltransferase (β-1,4-GalT), as well as its
docking simulations with β-1,4-GalT enzyme, will also be reported herein.

5
ACCEPTED MANUSCRIPT
(
A)
(B)
OH
O
RO
HO
OAc
OAc
AcO
AcO
OAc
AcHN
N
O
O
O
AcO
AcO
O
AcO
O
AcO
AcHN
AcHN
O
O
N
N
NH2
NH
HN
O
N
N
HO
HO
HO
N
N
N
N
O
AcO
AcO
AcO
AcO
AcO
AcO
O
O
HO
O
O
O
H
H
H
O
O
N
N
N
AcHN
N
N
OH
H
H
OH
OH
O
O
O
FmocHN
FmocHN
O
O
HO OH
1
2
3 R= H
4 R=
O
HO
OH
Figure 2. (A) Chemical structures of the 1,2,3-triazole-derived glycosyl amino acids αGlcNAc-1-O-
triazol-2Manα-ThrOH 1 and Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2. (B) Chemical structures
of glycopeptides 3 and 4 as analogs of α-DG mucins.
2
2
. Results and Discussion
.1. Synthesis
The target glycopeptides NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH 3 and
NHAcThrVal[Gal-β1,4-αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH 4 were obtained by solid-phase
assembly, which required the prior synthesis of the 1,2,3-triazole-derived glycosyl amino acids αGlcNAc-
1
-O-triazol-2Manα-ThrOH 1 and Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2 as building blocks for
obtaining α-DG mucins analogs.
2
.1.1. Synthesis of 1,2,3-triazole-derived glycosyl amino acids
The chemical synthesis of the natural disaccharide (GlcNAcβ1-2Manα-O-Ser/Thr) and trisaccharide
(Galβ1-4GlcNAcβ1-2Manα-O-Ser/Thr) cores of α-DG is not straightforward since it demands extensive
synthetic routes involving previous selective functionalization reactions of donor and acceptor sugars,
followed by laborious glycosylation reactions using specific catalysts under inert conditions. In this

6
ACCEPTED MANUSCRIPT
regard, the use of CuAAC reactions to obtain the novel 1,2,3-triazole-derived glycosyl amino acids
αGlcNAc-1-O-triazol-2Manα-ThrOH 1 and Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2 as analogs
of α-DG mucins may circumvent these drawbacks, considering the direct and selective functionalization
of donor and acceptor sugars with alkyne and azide groups, which are prompt to the subsequent fast and
highly selective CuAAC reactions to afford regioselective 1,4-disubstituted triazole in replacement to β-
1
,2-glycosidic linkage.
Therefore, to get the 1,2,3-triazole-derived glycosyl amino acids 1 and 2 it was necessary the
previous synthesis of the azide-glycosyl amino acid αManN -FmocThrOBn (5) and the alkyne-based
3
sugars 2’-propynyl-αGlcNAc (6) and 2’-propynyl-βGal-1,4-αGlcNAc (LacNAc) (7) as their direct
precursors.
Firstly, the glycosylation reaction between the per-O-acetyl-2-azido-2-deoxy-mannosyl acetate (8)
and the protected amino acid FmocThrOBn (9), utilizing BF .Et O as catalyst and anhydrous
3
2
1
9
dichloromethane as solvent, afforded the azide-glycosyl amino acid αManN -FmocThrOBn 5 (40%)
3
with α configuration after a 17 h reaction time (Scheme 1A). Compound 5 was characterized by IR
-1
1
spectra [2100 cm (N )] and H NMR analysis, which showed a characteristic doublet of sugar H-1 at δ
3
4
.70 (J1,2 2,8 Hz), as well as amino acid related signals at δ 1.32 (CH Thr) and δ 7.8-7.1 (NFmoc and
3-
+
OBn). HRESI-MS analysis of 5 showed the characteristic adduct 767.2536 [M+Na] .
Subsequently, the synthesis of the alkyne-based sugar 2’-propynyl-αGlcNAc 6 was initiated by
glycosylation reaction of per-O-acetyl 2-azido-2-deoxy-glucosyl acetate (10) with p-thiocresol reagent,
utilizing BF .Et O as catalyst in anhydrous dichloromethane, giving the glycosyl donor GlcN STol (11)
3
2
3
as an anomeric mixture (α:β 1:0.3, 51%) [20]. Due to difficulties to separate these anomers, this mixture
of 11 was nextly employed to the glycosylation reaction with propargyl alcohol, in the presence of
NIS/TMSOTf as a catalytic system in anhydrous dichloromethane, furnishing 2’-propynyl-GlcN (12) as
3
anomeric mixture (α:β 1:0.35, 65%) [21], which was submitted to the reductive acetylation of the azido
group (zinc powder in THF/ acetic anhydride/ acetic acid) to give the final α anomer alkyne-
functionalyzed 2’-propynyl-αGlcNAc 6 (68%) after chromatographic purification (Scheme 1B) [22]. The

7
ACCEPTED MANUSCRIPT
1
H NMR spectra of compound 6 showed characteristic signals of propargyl group (δ 2.52, J 2.5 Hz, CH)
and NHAc (δ 5.8, J 9.0 Hz). HRESI-MS analysis of 6 showed the characteristic adduct 408.1268
+
[
M+Na] . Regarding the preparation of the alkyne-based sugar 2’-propynyl-Gal-β1,4-αGlcNAc 7, the first
step involved the treatment of the lactose azido-nitrate (13) with NaOAc/ AcOH for conversion of the
anomeric NO to OAc, followed by glycosylation of the obtained per-O-acetyl 2-azido-2-deoxy-lactosyl
2
acetate (14) (80%) with p-thiocresol reagent, under upon described conditions, to give the glycosyl donor
LacN STol (15) as an inseparable anomeric mixture (α:β 1:0.5, 60%) [20]. This mixture of 15 was then
3
applied to the glycosylation reaction with propargyl alcohol (NIS/TMSOTf) to afford the 2’-propynyl-
LacN (16) as anomeric mixture (α:β 1:0.35, 65%) [21], which was treated with zinc powder and CuSO4
3
(1M) in THF/ acetic anhydride/ acetic acid for reductive acetylation of the azido group, giving the final
alkyne-functionalyzed 2’-propynyl-Gal-β1,4-αGlcNAc 7 (35%) (Scheme 1C) [22]. As described for 6,
1
the H NMR spectra of compound 7 showed characteristic propargyl (δ 2.47, J 2.5 Hz, CH) and NHAc (δ
5
.68, J 9.0 Hz) signals, whilst the HRESI-MS analysis of 7 showed the characteristic adduct 696.2116
+
[
M+Na] .
N3
O
AcO
AcO
AcO
OH
N3
(
A)
AcO
AcO
AcO
+
O
OBn
a
O
OAc
FmocHN
O
OBn
3
7.4%
FmocHN
8
9
5
O
(B)
OAc
OAc
O
OAc
O
OAc
a
b
c
68%
O
AcO
AcO
AcO
AcO
O
AcO
AcO
AcO
AcO
OAc
STol
AcHN
N3
51.1%
N3
65 %
N3
O
O
10
11
12
6
(C)
OAc
OAc
OAc
O
AcO
AcO
OAc
AcO
AcO
OAc
AcO
AcO
OAc
O
O
O
a,b
O
c,d
O
O
O
O
NO
STol
2
AcO
AcO
AcO
AcO
AcO
AcO
N3
AcHN
4
8%
over 2 steps
N3
22.8%
over 2 steps
O
1
3
15
7

8
ACCEPTED MANUSCRIPT
Scheme 1. (A) Synthesis of azide-glycosyl amino acid αManN -FmocThrOBn 5. Reagents and
3
conditions: (a) BF .Et O, p-thiocresol, anhydrous DCM, RT, 24 h. (B) Synthesis of the alkyne-based
3
2
sugar 2’-propynyl-αGlcNAc 6. Reagents and conditions: (a) BF .Et O, p-thiocresol, anhydrous DCM,
3
2
RT, 24 h; (b) Propargyl alcohol, NIS/ TMSOTf, anhydrous DCM, RT, 3 h; (c) Zn, CuSO4,
THF/Ac O/AcOH, 1 h. (C) Synthesis of the alkyne-based sugar 2’-propynyl-αLacNAc 7; Reagents and
2
conditions: (a) NaOAc/ AcOH, 100°C, 24 h; (b) BF .Et O, p-thiocresol, anhydrous DCM, RT, 24 h; (c)
3
2
Propargyl alcohol, NIS/ TMSOTf, anhydrous DCM, RT, 3 h; (d) Zn, CuSO , THF/Ac O/AcOH, 1 h.
4
2
Once the azide-glycosyl amino acid αManN -FmocThrOBn 5 and the alkyne-functionalyzed sugars
3
2
’-propynyl-αGlcNAc 6 and 2’-propynyl-Gal-β1,4-αGlcNAc 7 were obtained, the syntheses of protected
,2,3-triazole-derived glycosyl amino acids αGlcNAc-1-O-triazol-2Manα-ThrOBn (17) and Gal-β1,4-
1
αGlcNAc-1-O-triazol-2αMan-ThrOBn (18) were performed by Cu(I)-catalyzed 1,3-dipolar azide-alkyne
cycloaddition reactions (CuAAC), using the catalytic system CuSO / sodium ascorbate and DMF as
4
solvent, in a microwave reactor [18,23]. Thus, after 30 min. of microwave irradiation at 100 °C (150 W)
the triazole-derived glycosyl amino acids (17) and (18) were obtained in corresponding yields of 72% and
3
5%, with regioselective formation of 1,4-disubstituted triazoles (Scheme 2). In order to improve the
CuAAC reaction yield for the structurally more complex compound 18, extended microwave irradiation
time (60 min.) was applied, which did not lead, however, to increased yield, but only to degradation of
1
starting materials. The H NMR spectra of compounds 17 and 18 showed a characteristic CH-triazole
singlet around δ 8.0, besides characteristic signals of COCH groups (δ 2.2-1.8) and CH Thr (δ 1.36).
3
3
+
HRESI-MS analyses of 17 and 18 showed the corresponding characteristic adducts of 1030.4066 [M+H]
+
and 1418.4845 [M+H] . The protected glycosyl amino acids 17 and 18 were then submitted to
hydrogenation reactions (H / Pd-C 10%) for removal of benzyl ester groups, affording the final
2
αGlcNAc-1-O-triazol-2Manα-ThrOH 1 (82%) and Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2
(97%) as prompt building blocks for solid-phase synthesis of glycopeptides 3 and 4 (Scheme 2). HRESI-

9
ACCEPTED MANUSCRIPT
+
MS analyses of 1 and 2 showed the corresponding characteristic adducts of 1040.3441 [M+H] and
+
1
350.4281 [M+Na] .
OAc
O
AcO
AcO
AcHN
O
(A)
N3
N
OAc
AcO
AcO
AcO
N
O
O
N
AcO
AcO
AcO
AcO
AcO
+
a
O
O
AcHN
7
2%
O
OBn
O
FmocHN
O
OR
6
5
FmocHN
O
1
7 R= OBn
b
8
2%
1 R= OH
(
B)
OAc
AcO
AcO
OAc
O
O
O
AcO
AcO
N3
O
AcHN
OAc
AcO
AcO
AcO
AcO
AcO
OAc
O
O
O
O
AcO
a
+
N
AcO
O
AcHN
N
N
3
5%
O
OBn
FmocHN
AcO
AcO
AcO
O
O
7
5
O
OR
FmocHN
O
1
8
R= OBn
b
7%
9
2 R= OH
Scheme 2. (A) Synthesis of 1,2,3-triazole-derived glycosyl amino acid αGlcNAc-1-O-triazol-2αMan-
ThrOH 1. Reagents and conditions: (a) Na ascorbate, CuSO , DMF, microwave heating (100 °C, 150 W,
4
3
0 min); (b) H /Pd-C 10%, MeOH, AcOH, 0.5 h. (B) Synthesis of 1,2,3-triazole-derived glycosyl amino
2
acid Gal-β1,4-αGlcNAc-1-O-triazol-2αMan-ThrOH 2. Reagents and conditions: (a) Na ascorbate, CuSO4,
DMF, microwave heating (100 °C, 150 W, 30 min); (b) H /Pd-C 10%, MeOH, AcOH, 0.5 h.
2

1
0
ACCEPTED MANUSCRIPT
2
.1.2. Synthesis of glycopeptides
Moving towards more complex glycopeptides 3 and 4 as analogs of α-DG mucins, the orthogonal
Fmoc-based solid-phase peptide synthesis (SPPS) was carried out, utilizing commercial Wang resin pre-
loaded with Fmoc-Gly (0.65 mmol/g resin) as the solid support [22,24]. As the first step, N-Fmoc groups
were removed with 50% morpholine in DMF, followed by coupling of the amino acids building blocks in
the presence of the coupling reagents PyBOP/HOBt and N,N-diisopropylethylamine (DIPEA), in DMF.
Therefore, the synthesis of glycopeptides 3 and 4 comprised five rounds of coupling-deprotection
with sequential incorporation of Fmoc-Arg(Pbf)OH, FmocIleOH, αGlcNAc-1-O-triazol-2Manα-ThrOH 1
or Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2, FmocValOH and FmocThr(tBu)OH, affording the
resin-bound protected glycopeptides (19) and (20) (Scheme 3). The obtained peptide coupling rates varied
from 66% to 95%, as judged by spectrophotometrically detected dibenzofulvene released from the
product [22, 24]. Subsequently, the obtained glycopeptides 19 and 20 were submitted to N-acetylation
reactions (Ac O/ pyridine), followed by cleavage from resin in the presence of an aqueous mixture of
2
trifluoracetic acid and triisopropylsilane (TFA:TIPS:H O, 90:5:5), with simultaneous removal of Pbf and
2
OtBu protecting groups of the corresponding Arg and Thr amino acids. As the last step, removal of the
sugar acetyl groups using NaOMe in MeOH furnished crude glycopeptides 3 and 4, which were analysed
and purified by C18 reverse phase column, being obtained in corresponding yields of 23% and 12%. The
1
NMR H spectra of glycopeptides 3 and 4 showed a characteristic CH-triazole singlet around δ 8.0,
signals of sugars at δ 5.0 (H-1) and δ 1.9 (NHAc), as well as methylene and methyl signals of amino acids
+
between δ 3.12- δ 1.06. ESI-MS analyses also showed characteristic adducts for 3 ([M + H] 1134.5639)
+
and 4 ([M + H] : 1296.6166).

1
1
ACCEPTED MANUSCRIPT
OAc
O
RO
AcO
AcHN
O
N
N
N
NH-Pbf
NH
HN
O
AcO
O
AcO
AcO
But-O
O
O
a, b, a, c,
a, d, a, e,
a, f, a
O
O
H
N
H
N
H
O
N
Fmoc-HN
H
2
N
N
N
O
O
H
H
O
O
O
O
Fmoc-Gly Wang Resin
AcO OAc
19
R= H
20 R=
O
AcO
OAc
OH
O
RO
HO
AcHN
O
N
N
N
NH
NH
2
HN
HO
HO
HO
O
g, h, i
HO
O
O
O
O
H
H
H
N
N
N
AcHN
N
H
N
OH
H
O
O
O
HO OH
HO
3
R= H
4 R=
O
23%
OH
1
2%
Scheme 3. Solid phase synthesis of glycopeptides 3 and 4. Reagents and conditions: (a) 50% morpholine
in DMF; (b) Fmoc-Arg(Pbf)OH, PyBOP, HOBt, DIPEA; (c) FmocIleOH, PyBOP, HOBt, DIPEA; (d)
αGlcNAc-1-O-triazol-2Manα-ThrOH 1 or Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2, PyBOP,
HOBt, DIPEA; (e) FmocValOH, PyBOP, HOBt, DIPEA; (f) FmocThr(tBu)OH, PyBOP, HOBt, DIPEA;
(
g) Ac O/ pyridine; (h) TFA:TIPS:H O (90:5:5); (i) NaOMe/ MeOH.
2
2
2
.1.3. Enzymatic galactosylation of glycopeptide 3
Despite the preference of the enzyme bovine β-1,4-galactosyltransferase (β-1,4-GalT) to
galactosylate β-configured GlcNAc-acceptors, previous studies involving galactosylation of the α-
configured αGlcNAc-ThrOH, in the presence of β-1,4-GalT, showed that it was possible to get the

1
2
ACCEPTED MANUSCRIPT
desired galactosylated product αLacNAc-ThrOH at high enzyme concentration (2U/ mL) and longer
reaction time (96 h) [24]. Therefore, it prompted us to investigate the capacity of glycopeptide 3 to act as
substrate for bovine β1,4-GalT, despite bearing the unnatural α-linked glycosyl amino acid αGlcNAc-1-
O-triazol-2Manα-ThrOH. Accordingly, galactosylation reaction of glycopeptide 3 was carried out
utilizing the β-1,4-GalT enzyme, the donor substrate UDP-galactose (UDP-Gal) and MnCl as cofactor,
2
for 120 h at 37 °C (Scheme 4), and the obtained mixture was subsequently purified by gel filtration
chromatography. However, the ESI-MS analysis of the isolated products did not confirm the structure of
the expected galactosylated glycopeptide. This result led us to investigate the theoretical interactions
between the glycopeptide 3 and β-1,4-GalT, which might have impaired the galactose transfer to 3.
O
HO OH
OH
HO
HO
OH
OH
O
O
NH
HO
HO
O
O
HO
O
HO
OH
O
P
O-
O
P
O-
N
O
AcHN
N
OH
AcHN
O
O
O
O
O
O
N
UDP-Gal
OH OH
N
N
NH
NH
2
N
N
NH
NH
2
HN
O
a
HN
O
HO
HO
HO
HO
HO
HO
O
X
O
HO
O
HO
O
O
O
O
O
H
H
H
H
H
H
N
N
N
N
N
N
AcHN
N
N
OH
AcHN
N
N
OH
H
H
H
H
O
O
O
O
O
O
3
4
Scheme 4. Enzymatic galactosylation of glycopeptide 3.
2
.2. Molecular modeling studies
Docking simulations were performed using the crystallographic complex consisted of the bovine β-
2
+
1
1
,4-Galactosyltransferase 1 (β1,4GalT1) enzyme complexed with UDP-Gal and Mn cofactor (PDB ID
TVY) [25]. The docking protocol was validated by reproducing the experimentally observed position of
Chitobiose, which is a known substrate for β1,4GalT1, at the acceptor site (PDB ID 1TW5) (Fig. S19).
Both natural glycopeptide NHAcThrVal[GlcNAcβ1-2Manα]ThrIleArgGlyOH and its analog 3 were
docked to the β1,4GalT1–Mn–UDPGal complex using this validated protocol, and their interaction modes
were compared.

1
3
ACCEPTED MANUSCRIPT
The results from docking simulations showed that the GlcNAc portion of both natural and triazole-
glycopeptides can interact with the acceptor site of β1,4GalT1, in the same way as Chitobiose. According
to the most likely complexes calculated by molecular docking, the GlcNAc portion of the natural
glycopeptide makes hydrogen bonds with residues Tyr-289, Gly-316, Asp-318 and Arg-359.
Additionally, there are hydrophobic interactions between the acetyl group of the sugar and the side chains
of residues Phe-360 and Ile-363. The amino acid Trp-314 also composes the binding site and makes
extensive CH-π interactions with the aliphatic ring of GlcNAc (Fig. 3A). This indicates that the GlcNAc
portion of both natural substrate and triazole analog can be correctly recognized by the enzyme, and that
the transfer reaction can proceed to generate the galactosylated product in both cases. In the case of the
natural glycopeptide, our docking results show that its Man portion can make one hydrogen bond with
residue Arg-359 (Fig. 3A). On the other hand, the Man portion of the triazole-disaccharide analog can
make one hydrogen bond with Gln-288 and the triazole ring makes stacking interactions with Tyr-286
(Fig. 3B).
Although the correct orientation of the GlcNAc portion of the glycopeptide 3 indicate that it can act
as acceptor substrate for β1,4GalT1, the galactosylated product was not detected in enzymatic reaction
using this triazole-glycopeptide as substrate. The catalytic cycle of β1,4GalT1 involves conformational
changes from an open and inactive state to a closed and active state. Such conformational change happens
in the short and long loops, comprising residues 313-315 and 345-365, and is initiated by substrate
2
+
binding to the acceptor site. The closed state is further stabilized by the binding of Mn and UDP-Gal to
the donor site. After catalysis, the enzyme must release the product and UDP in order to return to its open
state and restart the catalytic cycle. In this context, it was observed that mutations that stabilize the closed
state inhibit enzymatic activity due to the formation of a dead-end complex, which is locked in the closed
state during the phase of product release [25]. Our docking calculations support the hypothesis that
additional interactions established by the triazole moiety of glycopeptide 3, specifically stacking with
residue Tyr-286 (Fig. 3B), may stabilize the closed state of β1,4GalT1 and form a dead-end complex that
effectively inhibits its enzymatic activity.

1
4
ACCEPTED MANUSCRIPT
Figure 3. Interactions between the acceptor site of β1,4GalT1 and A) The natural α-DG glycopeptide
NHAcThrVal[GlcNAcβ1-2Manα]ThrIleArgGlyOH or B) α-DG triazole glycopeptide
NHAcThrVal[GlcNAcα1-O-triazol-2Manα]ThrIleArgGlyOH 3, as calculated by molecular docking.
Hydrogen, Nitrogen and Oxygen atoms are colored in white, blue and red, respectively. Carbon atoms are
colored as follows: protein residues in gray, the GlcNAc portion of the disaccharides in green, the Man
portion of the disaccharides in yellow and the CH -triazole moiety in cyan. Hydrogen bonds are indicated
2
by dashed lines and hydrophobic interactions are indicated by transparent spheres.
For the sake of clarity, the peptide chains of both glycopeptides are not shown.
3
. Conclusions
In summary, we obtained the novel 1,2,3-triazole-derived glycosyl amino acids αGlcNAc-1-O-
triazol-2Manα-ThrOH 1 and Gal-β1,4-αGlcNAc-1-O-triazol-2Manα-ThrOH 2, along with the
corresponding glycopeptides NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH and
3
NHAcThrVal[Gal-β1,4-αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH 4 as analogs of α-DG mucins. In
this regard, efficient synthetic routes led to the azide-glycosyl amino acid αManN -FmocThrOBn 5 and
3
the alkyne-functionalyzed sugars 2’-propynyl-αGlcNAc 6 and 2’-propynyl-Gal-β1,4-αGlcNAc 7 as
prompt building blocks for further CuAAC reactions, which proved to be effective to furnish the 1,2,3-
triazole-derived glycosyl amino acids 1 and 2 with regioselective formation of 1,4-disubstituted triazoles.
Despite the feasibility of glycopeptide 3 to act as a substrate for bovine β1,4-GalT, the
galactosylated glycopeptide 4 could not be obtained by enzymatic reaction with β1,4-GalT. This fact

1
5
ACCEPTED MANUSCRIPT
could be elucidated by docking simulations with β-1,4-GalT, which showed that additional stacking
interactions between the triazole moiety of glycopeptide 3 and the Tyr-286 residue might have interrupted
the catalytic cycle of β-1,4-GalT, leading to its inhibition.
4
4
. Experimental
.1. General methods
All chemicals were purchased as reagent grade and used without further purification, unless
otherwise noted. Reactions were monitored by thin layer chromatography (TLC) on precoated silica gel
plates (Whatman, AL SIL G/UV, aluminium backing). Compounds were visualized under UV light (254
nm) and/or by dipping and heat with ethanol-sulfuric acid (95:5, v/v) visualization solution.
Chromatographic purifications were carried out on Flash silica gel (40-63µm). Nuclear magnetic
resonance spectra were recorded on Bruker Avance DRX 300 (300 MHz), DPX 400 (400 MHz) or DPX
5
00 (500 MHz) spectrometers. Chemical shifts (δ) are reported in parts per million (ppm) downfield from
tetramethylsilane. Assignments were made with the aid of two-dimensional NMR spectra (COSY and
HMQC) recorded on the same instruments. Mass spectrometric analyses were performed on a Bruker
Daltonics MicroOTOF II ESI-TOF mass spectrometer, and accurate mass electrospray ionization mass
spectra (ESI-HRMS) were obtained using positive or negative ionization modes.
4
4
.2. Synthesis
.2.1. N-(Fluoren-9-ylmethoxycarbonyl)-(2-azide-2-deoxy-3,4,6-tri-O-acetyl-α-
D
-mannosyl)-L-
threonine benzyl ester 5
Peracetylated mannosyl azide 8 (167.6 mg, 0.45 mmol, 1.0 equiv) and the previously synthesized
FmocThrOBn 9 (96.6 mg, 0.22 mmol, 0.5 equiv) were solubilized in anhydrous dichloromethane (4.7
mL) and treated with BF .Et O (110.8 µL, 0.90 mmol, 2.0 equiv). The mixture was stirred for 17 h and
3
2
then concentrated under reduced pressure. The purification was performed by flash column
chromatography (toluene/EtOAc 9:1, v/v) and the product 5 was obtained in 37.4% yield (125.1 mg,
1
0
.168 mmol). H NMR (300 MHz, CDCl ): δ 7.81 (d, 2H, J 7.2 Hz, CH Fmoc arom.), 7.68 (d, 2H, J 7.7
3

1
6
ACCEPTED MANUSCRIPT
Hz, CH Fmoc arom.), 7.46-7.31 (m, 9 H, CH Fmoc arom, CH OBn arom), 5.67 (d, 1H, J 9.7 Hz, NH
Thr), 5.32 (d, 2H, J 12.1 Hz, CH OBn,), 5.25 (d, 1H, J 11.0 Hz, H-3), 5.15 (d, 1H, J3,4 11.0 Hz, H-4),
2
3,4
4
.63 (broad s, 1H, H-1), 4.53 (dd, 1H, J 2.1 Hz, J 9.7 Hz, CHα-Thr), 4.50-4.44 (m, 2H, CH Fmoc), 4.38-
2-
4
.28 (m, 2H, CH-Fmoc, CHβThr), 4.20 (d, 1H, J5,6a 6.3 Hz, H-6a), 4.12-4.08 (m, 1H, H-6b), 3.98 (d, 1H,
J5,6a 6.3 Hz, H-5), 3.44 (broad s, 1H, H-2), 2.14, 2.09, 2.08 (3 s, 9H, 3xCOCH ), 1.37 (d, 3H, J 6.3 Hz,
3
1
3
CH Thr). C NMR (75 MHz. CDCl ): δ 170.6 (CO Fmoc); 170.0; 169.8; 169.5 (COCH ), 156.6 (CO
3
3
3
OBn), 143.7; 141.3; 137.8; 135.3 (C quat. Fmoc Ph), 135.1 (C quat.OCH Ph), 129.0; 128.9; 128.8; 128.6;
2
1
28.5; 128.2; 128,1, 127.8; 127.1; 125.3; 125.1; 120.0 (CH Ph), 99.3 (C-1), 70.7 (C-3), 69.1 (C-4), 67.6
(
C-5), 67.2 (OCH Fmoc), 66.2 (OCH -OBn), 62.3 (C-6), 61.3 (C-2), 58.6 (CHαThr), 47.2 (CH Fmoc),
2
2
+
2
1.4; 20.6; 20.5 (COCH ), 18.0 (CH Thr). ESI-HRMS: calcd. for C H N NaO [M + Na] : 767.2535,
3 3 38 40 4 12
found: 767.2536.
4
.2.2. Tolyl 2-azido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-D-glucopyranoside 11
To a solution of peracetylated glucosyl azide 10 (300 mg, 0.83 mmol, 1.0 equiv) in anhydrous
dichloromethane (4.0 mL) was added 4-methylbenzenethiol (p-thiocresol) (103.8 mg, 0.83 mmol, 1.0
equiv) and BF .Et O (309 µL; 2.5 mmol, 3.0 equiv). The mixture was stirred for 23 h at room temperature
3
2
and followed by TLC. The reaction was then quenched with triethylamine, concentrated in vacuo and
purified by silica gel column chromatography (gradient toluene 100% → toluene/EtOAc 8:2, v/v) to give
1
the product 11 as an inseparable anomeric mixture (α:β 1:0.3) in 51.1% yield. H NMR (300 MHz,
CDCl ), α anomer: δ 7.39 (d, 2H, Jorto 8.1 Hz, CH STol arom.), 7.24 (d, 2H, Jorto 8.1 Hz, CH STol arom.),
3
5
.57 (d, 1H, J1,2 5.6 Hz, H-1), 5.37-5.34 (m, 1H, H-3), 5.10-5.00 (m, 1H, H-4), 4.62 (dd, 1H, J5,6a 5.0 Hz,
J5,6b 10.2 Hz, H-5), 4.30 (d, 1H, J5,6a 5.1 Hz, H-6a), 4.09-4.01 (m, 2H, H-2, H-6b), 2.34 (s, 3H, CH3-
1
3
thiocresol), 2.10, 2.06, 2.05 (3 s, 12H, 3x CH CO). C NMR (75 MHz. CDCl ): δ 171.5, 170.6, 170.1,
3
3
1
69.5 (CH CO), 132.7 (CH STol), 129.9 (CH Stol), 86.8 (C-1), 71.3 (C-3), 68.6 (C-4), 68.3 (C-5), 62.0
3

1
7
ACCEPTED MANUSCRIPT
(
C-6), 61.7 (C-2), 20.9 (CH STol), 20.4-18.8 (3xCH CO). ESI-HRMS: calcd. for C H N NaO S [M +
3 3 19 23 3 7
+
Na] : 460.4560, found: 460.1147.
4
.2.3. 2'-propynyl-2-azide-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranoside 12
To a solution of glycosyl donor GlcN STol 11 (84 mg, 0.19 mmol, 1.1 equiv) and N-
3
iodosuccinimide (NIS) (9.0 mg, 0.26 mmol, 1.5 equiv) in anhydrous dichloromethane (2.5 mL) was
added propargyl alcohol (30 µL, 0.52 mmol, 3.0 equiv). The mixture was then treated with TMSOTf (3.2
µL, 0.019 mmol, 0.1 equiv) and stirred for 3 h at room temperature. The resulting mixture was diluted
with dichloromethane, washed with both saturated NaHCO 10% and sodium thiosulphate solutions,
3
dried over MgSO and purified by silica gel column chromatography (gradient toluene 100% →
4
1
toluene/EtOAc 8:2, v/v) to afford the product 12 in 65 % yield (46.9 mg, 0.124 mmol). H NMR (300
MHz, CDCl ): α anomer: δ 5.50 (dd, 1H, J3,4 8.8 Hz, J4,5 10.6 Hz, H-4), 5.38 (d, 1H, J3,4 8.8 Hz, H-3),
3
5
4
3
7
2
.23 (d, 1H, J1,2 3.6 Hz, H-1), 4.34 (d, 2H, J 2.4 Hz, CH -prop.), 4.16-4.07 (m, 3H, H-5, H-6a, H-6b),
2
.43 (dd, 1H, J 3.6 Hz, J 10.6 Hz, H-2), 2.52 (t, 1H, J 2.4 Hz, CH-prop.), 2.11, 2.10, 2.05 (3 s, 12H,
1
,2
2,3
1
3
x CH CO). C NMR (75 MHz. CDCl ): δ 171.7, 170.7, 170.0, 169.2 (CH CO), 96.3 (C-1), 71.1 (C-3),
3
3
3
8.2 (C prop.), 75.4 (CH prop.), 68.0 (C-4), 68.3 (C-5), 62.0 (C-6), 61.7 (C-2), 55.3 (CH prop.), 23.1,
2
+
0.7, 20.5 (CH CO). ESI-HRMS: calcd. for C H KN O [M + K] : 409.0876, found: 409.1624.
3
15 20
3
8
4
.2.4. 2'-propynyl-2-acetamide-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside 6
To a solution of compound 12 (72.4 mg; 0.196 mmol, 1.0 equiv) in a mixture of THF/acetic
anhydride/acetic acid 3:2:1 v/v/v (3.0 mL ) was added zinc dust (77 mg, 1.18 mmol, 6.0 equiv) and 1M
CuSO solution (40 µL). After stirring for 2 hours at room temperature the reaction mixture was filtered
4
through Celite and purified by silica gel column chromatography (gradient EtOAc/hexane
1
:1→EtOAc/hexane 7:3, v/v), affording compound 6 (α anomer) in 68% yield (51.2 mg, 0.133 mmol).
1
H NMR (300 MHz, CDCl ): δ 5.77 (d, 1H, J
8.9 Hz, NHAc), 5.26-5.20 (m, 1H, H-3), 5.38 (t, 1H, J3,4
3
2,NH
J4,5 9.6 Hz, H-4), 5.03 (d, 1H, J1,2 3.7 Hz, H-1), 4.40 (td, 1H, J1,2 3.7 Hz, J2,3 = J2,NHAc 10.1 Hz, H-2),
=

1
8
ACCEPTED MANUSCRIPT
4
.27 (t, 2H, J 2.4 Hz, CH -prop.), 4.23 (d, 1H, J 4.2 Hz, H-6a), 4.10 (dd, 1H, J5,6b 2.3 Hz, J6a/6b 12.3
2 5,6a
Hz, H-6b), 4.00 (ddd, 1H, J5,6b 2.3 Hz, J5,6a 4.2 Hz, J4,5 9.7 Hz, H-5), 2.49 (t, 1H, J 2.4 Hz, CH-prop.)
13
2
.10 (s, 3H, CH CONH), 2.03, 2.02, 1.97 (3 s, 12H, 3x CH CO). C NMR (75 MHz. CDCl ): δ 171.7,
3 3 3
1
70.7, 170.0, 169.2 (CH CO), 96.3 (C-1), 71.1 (C-3), 78.2 (C prop.), 75.4 (CH prop.), 68.0 (C-4), 68.3
3
(
C-5), 62.0 (C-6), 55.3 (CH prop.), 51.4 (C-2), 23.1 (CH CONH), 20.7-20.5 (3xCH CO). ESI-HRMS:
2
3
3
+
calcd. for C H NO Na [M + Na] : 408.1265, found: 408.1268.
1
7
23
9
4
.2.5.
Tolyl-3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-
D
-galactopyranosyl)-2-azido-2-deoxy-1-
thio-D
-glucopyranoside 15
To a solution of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-
deoxy-1-thio- -glucopyranosyl nitrate 13 (500 mg, 0.75 mmol, 1.0 equiv.) in acetic acid (17 mL) was
added sodium acetate (225 mg, 2.74 mmol, 3.6 equiv) and the reaction mixture was stirred for 24 h at
00°C. The resulting mixture was then diluted with ethyl acetate, washed with saturated NaHCO , dried
D-galactopyranosyl)-2-azido-2-
D
1
3
over MgSO and purified by silica gel column chromatography (hexane/EtOAc, 3:7 v/v) to afford the
4
compound 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-azido-2-deoxy-1-thio-D-
glucopyranosyl acetate 14 (396.93 mg, 0.6 mmol, 80%) as an amorphous solid. To a solution of
compound 14 (300 mg, 0.45 mmol) in anhydrous dichloromethane were added p-thiocresol (56 mg, 0.45
mmol, 1.0 equiv) and BF .Et O (167.7 µL, 1.33 mmol, 3.0 equiv), and the reaction mixture was stirred for
3
2
1
2 h at room temperature. The reaction mixture was quenched with triethylamine and purified by silica
gel column chromatography (EtOAc/Hexane 1:1 v/v) to give compound 15 as an inseparable anomeric
1
mixture (α:β 1:0.5) in 60% yield (197.2 mg, 0.27 mmol). H NMR (300 MHz, CDCl ), α anomer: δ 7.38
3
(
d, 2H, Jorto 7.5 Hz, CH STol arom.), 7.13 (d, 2H, Jorto 7.0 Hz, CH STol arom.), 6.26 (d, 1H, J 3.7 Hz, H-
1,2
1
), 5.38 (d, 1H, J3’,4’ 2.0 Hz, H-4’), 5.25-5.16 (m, 2H, H-2’, H-3), 5.13 (dd, 1H, J2’,3’ 10.1 Hz, J3’,4’ 2.0 Hz,
H-3’), 5.02-4.93 (m, 3H, H-6b, H-1’, H-6a,), 4.90-4.84 (m, 1H, H-5’), 4.23-4.07 (m, 2H, H-6b’, H-6a’),
3
3
.74 (t, 1H, J 9.5 Hz, H-4), 3.65-3.58 (m, 1H, H-5), 3.48 (dd, 1H, J1,2 3.8 Hz, J2,3 10.5 Hz, H-2), 2.35 (s,
1
3
H, CH -thiocresol), 2.19-2.02 (6s, 18H, 6xCOCH ). C NMR (75 MHz. CDCl ): δ 169.3-167.8
3
3
3

1
9
ACCEPTED MANUSCRIPT
(
COCH ), 132.7 (CH STol), 129.7 (CH Stol), 101.2 (C-1’), 85.7 (C-1), 74.3 (C-4), 71.0 (C-3’), 70.7 (C-
3
5
2
’), 69.2 (C-3), 69.1 (C-2’), 66.8 (C-5), 66.6 (C-4’), 62.4 (C-6), 62.1 (C-6’), 60.8 (C-2), 29.7 (CH STol),
3
+
+
1.1-20.5 (3xCH CO). ESI-HRMS: calcd. for C H N NaO S [M + Na] : 748.1994, found: 748.1996.
3
31 39
3
15
4
.2.6.
2’-propynyl-3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-azido-2-
deoxy-α-
D
-glucopyranoside 16
The lactosyl donor 15 (127 mg, 0.175 mmol, 1.0 equiv.) and N-iodosuccinimide (NIS) (45.3 mg,
0
.2 mmol, 1.15 equiv) were solubilized in anhydrous dichloromethane (3.5 mL), followed by addition of
propargyl alcohol (28.3 µL, 0.52 mmol, 3.0 equiv). The mixture was then treated with TMSOTf (1.55 µL,
.0175 mmol, 0.1 equiv) and stirred for 3 h at room temperature. The resulting mixture was diluted with
0
dichloromethane, washed with both saturated NaHCO 10% and sodium thiosulphate solutions, dried
3
over MgSO and purified by silica gel column chromatography (gradient toluene 100% → toluene/EtOAc
4
8
:2, v/v) to afford the product 16 as an anomeric mixture (α:β 1:0.35) in 65% yield (46.9 mg, 0.124
1
mmol). H NMR (300 MHz, CDCl ): α anomer: δ 6.19 (d, 1H, J 3.6 Hz, H-1), 5.35 (d, 1H, J3’,4’ 3.6 Hz,
3
1,2
H-4’), 5.33-5.27 (m, 2H, H-2’, H-3), 5.11 (dd, 1H, J2',3' 7.4 Hz, J3',4' 3.6 Hz, H-3'), 4.51 (d, 1H, J1’,2’ 7.9
Hz, H-1’), 4.19 (t, 2H, J 2.4 Hz, CH -prop.), 4.10-4.07 (m, 3H, H-5', H-6a', H-6b'), 4.04-4.03 (m, 3H, H-
2
5
2
1
2
, H-6a, H-6b), 3.93 (d, 1H, J3,4 9.7 Hz, H-4), 3.45 (dd, 1H, J1,2 3.6 Hz, J2,3 10.6 Hz, H-2), 2.47 (t, 1H, J
1
3
.4 Hz, CH-prop.), 2.11-1.99 (6s, 18H, 6xCOCH ). C NMR (75 MHz. CDCl ): δ 170.5-170.1 (COCH ),
3
3
3
01.3 (C-1’), 90.0 (C-1), 75.7 (C prop.), 75.6 (CH prop.), 74.1 (C-5’), 71.0 (C-3’), 70.7 (C-5), 70.2 (C-
’), 69.6 (C-3), 66.8 (C-4), 66.6 (C-4’), 62.1 (C-6’), 61.3 (C-6), 60.7 (C-2), 53.4 (CH prop.), 21.0-20.5
2
+
(
3xCH CO). ESI-HRMS: calcd. for C H N NaO [M + Na] : 680.1910, found: 680.1906.
3
27 35
3
16

2
0
ACCEPTED MANUSCRIPT
4
.2.7. 2’-propynyl-3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-2-
deoxy-α- -glucopyranoside 7
D
To a solution of compound 16 (112 mg, 0.16 mmol, 1.0 equiv.) in a mixture of THF/acetic
anhydride/acetic acid 3:2:1 v/v/v (3.0 mL) were added zinc dust (60 mg, 0.9 mmol; 6.0 equiv) and 1M
aqueous CuSO solution (30 µL), and the reaction mixture was stirred for 1 h at room temperature. The
4
mixture was filtered through Celite and purified by silica gel column chromatography (gradient
EtOAc/Hexane 7:3 v/v → EtOAc 100%) to afford compound 7 (α anomer) in 35% yield (37.4 mg, 0.055
1
mmol). H NMR (300 MHz, CDCl ): δ 5.62 (d, 1H, J 9.2 Hz, NH), 5.33-5.29 (m, 2H, H-4’, H-3), 5.12
3
(dd, 1H, J1',2'8.0 Hz, J2',3'10.4 Hz, H-2'), 4.96 (dd, 1H, J2’,3’ 10.5 Hz, J3’,4’ 3.4 Hz, H-3’), 4.92 (broad s, 1H,
H-1), 4.63-4.60 (m, 1H, H-2), 4.57 (d, 1H, J1’,2’ 8.0 Hz, H-1’), 4.31 (dd, 1H, J5,6b 1,8 Hz, J6a,6b 11.8 Hz,
H-6b), 4.23 (dd, 1H, J 2.4 Hz, J 11.8 Hz, CH -prop.), 4.23-4.16 (m, 2H, H-4, H-6b), 4.01 (dd, 1H, J
2
5,6a
7
.7 Hz, J6a,6b 11.2 Hz, H-6a), 3.97-3.94 (m, 1H, H-6a'), 3.86 (t, 1H, J5,6a/6b 6.9 Hz, H-5), 3.76 (t, 1H,
J5’,6a’/6b’ 9.6 Hz, H-5’), 2.45 (t, 1H, J 2.2 Hz, CH-prop.), 2.15 (s, 3H, CH CONH), 2.12-1.96 (18 H, 6 s,
3
13
6
xCOCH3). C NMR (75 MHz, CDCl ): δ 170.5-169.3 (COCH ), 100.8 (C-1’), 96.0 (C-1), 78.0 (C
3 3
prop), 75.7 (CH prop.), 74.4 (C-5’), 71.0 (C-3’), 70.6 (C-5), 69.9 (C-2’), 69.3 (C-3), 69.1 (C-4), 66.7 (C-
’), 62.7 (C-6’), 60.9 (C-6), 54.8 (CH prop.), 49.9 (C-2), 23.4 (CH CONH), 21.0-20.5 (3xCH CO). ESI-
4
2
3
3
+
HRMS: calcd. for C H NNaO [M + Na] : 696.2110, found: 696.2116.
2
9
39
17
4
.2.8.
N-(Fluoren-9-ylmethoxycarbonyl)-O-[(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-D-
glucopyranosyl)-(1→2)-(1,4-triazole-2-deoxy-3,4,6-tri-O-acetyl-α-D-mannosyl)-L-threonine benzyl
ester 17
A solution of αManN -FmocThrOBn 5 (44 mg, 0.06 mmol, 1.0 equiv), 2'-propynyl-2-acetamide-
3
3
,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside 6 (23 mg, 0.06 mmol, 1.0 equiv), sodium ascorbate (2
mg, 0.0125 mmol, 0.5 equiv.) and CuSO (1M, 6 µL, 0.006 mmol, 0.1 equiv) in DMF (0.5 mL) was
4
inserted into a microwave tube and nextly submitted to microwave irradiation for 30 min. (100 °C, 150
W). The reaction was followed by TLC (EtOAc) and after completion the reaction mixture was

2
1
ACCEPTED MANUSCRIPT
concentrated under reduced pressure and purified by silica gel column chromatography (gradient
EtOAc/hexane 7:3, v/v → EtOAc) to afford the desired αGlcNAc-1-O-triazol-2Manα-ThrOBn 17 (48.1
1
mg, 0.042 mmol) in a 72% yield. H NMR (300 MHz, CDCl ): δ 7.91 (s, 1H, H-triazol), 7.79 (d, 2H, J
3
7
.4 Hz, CH Fmoc arom.), 7.64 (d, 2H, J 7.3 Hz, CH Fmoc arom.), 7.45-7.29 (m, 9 H, CH Fmoc arom, CH
OBn arom), 6.12 (d, 1H, J 9.3 Hz, NHAc), 5.71 (d, 1H, J 9.3 Hz, NH Thr), 5.46 (dd, 1H, J1',2' 5.1 Hz, J2',3'
.9 Hz, H-2), 5.31-5.21 (m, 4H, H-3, H-4, H-3', H-4'), 5.19 (d, 1H, J1,2 5.1 Hz, H-1), 5.13 (d, 2H, J 12.8
Hz, CH OBn), 4.98 (d, 1H, J1,2 3.5 Hz, H-1'), 4.90 (d, 1H, J 12.5 Hz, CH -triazol), 4.66 (d, 1H, J 12.5
9
2
2a
Hz, CH -triazol), 4.55 (d, 1H, J 9.3 Hz, CHαThr), 4.46-4.23 (m, 6H, H-2', H-5, H-5', CH Fmoc,
2
b
2
CHβThr), 4.12 (dt, 4H, J5,6a/6b 4.6 Hz, J5',6a'/6b' 7.2 Hz, H-6a, H-6b, H-6a', H-6b'), 2.12, 2.11, 2.07, 2.05,
1
3
2
.03, 2.02, 2.00 (7 s, 21H, 7xCOCH ), 1.35 (d, 3H, J 6.2 Hz, CH Thr). C NMR (75 MHz, CDCl ): δ
3 3 3
1
71.2 (CO Fmoc), 170.7-169.3 (COCH ), 169.3 (CO OBn), 143.6-141.3 (Cquat. Fmoc Ph), 134.9 (C
3
quat.OCH Ph), 128.8-122.7 (CH Ph), 120.0 (CH-triazol), 98.4 (C-1), 96.8 (C-1’), 71.2 (C-3), 69.2 (C-3’),
2
6
8.8 (C-4), 68.3 (C-2), 68.2 (C-6), 67.8 (OCH OBn), 67.4 (OCH Fmoc), 65.2 (C-5’), 62.1 (CHβThr),
2 2
6
2
1
1.9 (C-6’), 60.9 (C-5), 60.6 (OCH -triazol), 60.4 (C-4’), 58.4 (CHαThr), 51.8 (C-2), 47.2 (CH Fmoc),
2
+
3.0 (CH CONH), 20.7-20.5 (COCH ), 17.7 (CH3Thr). ESI-HRMS: calcd. for C H N O [M + H] :
3
3
55 64
5
21
030.4088, found: 1030.4066.
4
.2.9.
N-(Fluoren-9-ylmethoxycarbonyl)-O-[(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-
D-
glucopyranosyl)-(1→2)-(1,4-triazole-2-deoxy-3,4,6-tri-O-acetyl-α-
D
-mannosyl)- -threonine 1
L
To a solution of compound 17 (14 mg, 0.012 mmol, 1.0 equiv) in MeOH (1.0 mL) was added
glacial AcOH (0.1 mL) and 10% Pd/C (4.5 mg). The reaction mixture was stirred and kept under H (1.5
2
atm) for approximately 30 min., being subsequently filtered through Celite, concentrated in vacuo and
purified by column chromatography (DCM/MeOH 9:1, v/v). The product 1 was obtained in 82% yield
1
(
10.3 mg; 0.009 mmol). H NMR (500 MHz, CDCl ): δ 7.95 (broad s, 1H, H-triazol), 7.77 (d, 2H, J 7.3
3
Hz, CH Fmoc arom.), 7.64 (dd, 2H, J 6.6 Hz, J 9.3 Hz, CH Fmoc arom.), 7.41-7.33 (m, 4 H, CH Fmoc

2
2
ACCEPTED MANUSCRIPT
arom.), 5.46 (dd, 1H, J 2.4 Hz, J 8.6 Hz, H-2), 5.80-5.72 (m, 1H, H-3), 5.55-5.49 (m, 1H, H-3’), 5.24-
1
,2
2,3
5
1
4
.09 (m, 5H, H-1, H-1’, H-4, H-4’, CH -triazol), 4.88 (dd, 1H, J 3.5 Hz, J 7.4 Hz, CH-Fmoc), 4.63 (d,
2a
H, J 12.3 Hz, CH -triazol), 4.40-4.23 (m, 7H, H-2', H-5, H-5', CH Fmoc, CHαThr, CHβThr), 4.11 (d,
2
b
2
H, J6a/6b = J6a'/6b' 12.6 Hz, H-6a, H-6b, H-6a', H-6b'), 2.12-1.89 (m, 30H, 10xCOCH ), 1.36 (d, 3H, J 5.8
3
13
Hz, CH Thr). C NMR (125 MHz, CDCl ): δ 171.2 (CO Fmoc), 170.7-169.3 (COCH ), 143.6-141.3
3
3
3
(
(
(
(
Cquat. Fmoc Ph), 134.9 (C quat.OCH Ph), 127.5-122.7 (CH Ph), 123.1 (CH-triazol), 98.4 (C-1), 96.8
2
C-1’), 91.4 (C-2), 77.3 (C-3), 70.4 (C-3’), 69.4 (C-4), 69.1 (C-2'), 68.6 (C-6), 68.6 (OCH Fmoc), 67.8
2
C-5’), 65.3 (CHβThr), 61.9 (C-6’), 61.7 (C-5), 60.6 (OCH -triazol), 60.4 (C-4’), 60.1 (CHαThr), 47.7
2
CH Fmoc), 22.4 (CH CONH), 20.7-20.5 (COCH ), 18.3 (CH Thr). ESI-HRMS: calcd. for
3
3
3
+
C H N O [M + H] : 1040.3619, found: 1040.3441.
48
58
5
21
4
.2.10. 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-
glucopyranoside-1-O-(methyl-1H-1,2,3-triazol-4-yl)-2-deoxy-3,4,6-tri-O-acetyl-α-
mannopyranosyl-N-(fluoren-9-ylmethoxycarbonyl)- -threonine benzyl ester 18
D-galactopyranosyl)-2-acetamido-2-deoxy-α-D-
D
-
L
A solution of αManN -FmocThrOBn 5 (42.4 mg, 0.057 mmol, 1.0 equiv.), 2’-propynyl-Gal-β1,4-
3
αGlcNAc 7 (38.4 mg, 0.057 mmol, 1.0 equiv.), sodium ascorbate (5.6 mg, 0.028 mmol, 0.5 equiv.) and
CuSO (1M, 5.7 µL, 0.0057 mmol, 0.1 equiv) in DMF (0.5 mL) was inserted into a microwave tube and
4
nextly submitted to microwave irradiation for 30 min (100 °C, 150 W). The reaction was followed by
TLC (EtOAc) and after completion the reaction mixture was concentrated under reduced pressure and
purified by silica gel column chromatography (EtOAc) to afford the desired Gal-β1,4-αGlcNAc-1-O-
1
triazol-2Manα-ThrOBn 18 (28.3 mg, 0.019 mmol, 35%). H NMR (500 MHz, CDCl ): 7.88 (s, 1H, H-
3
triazol), 7.77 (d, 2H, J 7.3 Hz, CH Fmoc arom.), 7.64 (d, 2H, J 7.3 Hz, CH Fmoc arom.), 7.43-7.27 (m,
9
H, CH Fmoc arom, CH OBn arom), 5.70 (d, 1H, J 9.0 Hz, NHAc), 5.66 (d, 1H, J 8.8 Hz, NH Thr), 5.44
dd, 1H, J3'',4'' 4.8 Hz, J2'',3'' 9.8 Hz, H-3''), 5.34-5.30 (m, 3H, H-4'', H-3, H-4), 5.26 (d, 2H, J 11.9 Hz,
CH OBn), 5.18-5.08 (m, 4H, H-2, H-3', H-4', H-4''), 5.04 (broad s, 1H, H-1), 4.95-4.94 (dd, 1H, J 3.4
(
2
1',2'
Hz, J2',3' 10.5 Hz, H-2'), 4.89-4.84 (m, 2H, CH -triazol, H-1'), 4.66 (d, 1H, J 12.8 Hz, CH -triazol), 4.57
2a
2b

2
3
ACCEPTED MANUSCRIPT
(d, 1H, J1'',2'' 8.0 Hz, H-1''), 4.51-4.37 (m, 4H, H-2'', CHFmoc, CHαThr, CHβ-Thr), 4.31-4.23 (m, 3H, H-
5
, CH Fmoc), 4.19-4.14 (m, 4H, H-6a’, H-6b’, H-6ª’’, H-6b’’), 4.12-4.08 (m, 1H, H-6b), 3.99 (dd, 1H,
2
J5,6a 7.7 Hz, J6a,6b 11.1 Hz, H-6a), 3.86 (t, 1H, J5’’,6a’’/6b’’ 6.5 Hz, H-5’’), 3.78 (t, 1H, J5’,6a’/6b’ 9.5 Hz, H-5’),
1
3
2
.15-1.96 (m, 30H, 10xCOCH ), 1.34 (d, 3H, J 6.2 Hz, CH Thr). C NMR (125 MHz, CDCl ): δ 171.2
3 3 3
(
CO Fmoc), 170.7-169.3 (COCH ), 169.3 (CO OBn), 143.6-141.3 (Cquat. Fmoc Ph), 134.9 (C quat.
3
OCH Ph), 128.8-120.1 (CH Ph), 122.9 (CH-triazol), 101.0 (C-1''), 98.7 (C-1), 98.3 (C-1’), 92.6 (C-2),
2
7
5
7.8 (C-6''), 75.6 (C-6), 74.2 (C-5’), 70.9 (C-2), 69.9 (C-3), 70.6 (C-6’), 69.9 (C-4''), 69.0 (C-3’), 68.8 (C-
''), 68.6 (C-4), 68.3 (C-3''), 67.8 (OCH OBn), 67.4 (OCH Fmoc), 61.5 (CHβThr), 60.8 (C-5), 61.1
2
2
(OCH -triazol), 60.7 (C-4’), 61.1 (CHαThr), 51.3 (CH Fmoc), 49.9 (C-2''), 23.3 (CH CONH), 20.6-20.4
2 3
+
(
COCH ), 17.7 (CH Thr). ESI-HRMS: calcd. for C H N O [M + H] : 1418.4933, found: 1418.4845.
3
3
67 80
5
29
4
.2.11. 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-2-deoxy-α-D-
glucopyranoside-1-O-(methyl-1H-1,2,3-triazol-4-yl)-2-deoxy-3,4,6-tri-O-acetyl-α-
D-
mannopyranosyl-N-(fluoren-9-ylmethoxycarbonyl)- -threonine 2
L
To a solution of compound 18 (28 mg, 0.02 mmol, 1.0 equiv) in MeOH (3.0 mL) was added glacial
AcOH (0.1 mL) and 10% Pd/C (15 mg, 0.06 mmol, 3.0 equiv). The reaction mixture was stirred and kept
under H (1.5 atm) for approximately 2 hours, being subsequently filtered through Celite, concentrated in
2
vacuo and purified by column chromatography (DCM/MeOH 9:1, v/v). The product 2 was obtained in
1
9
7
5
7% yield (26.7 mg; 0.019 mmol). H NMR (500 MHz, CD OD): δ 8.23 (s, 1H, H-triazol), 7.80 (d, 2H, J
3
.4 Hz, CH Fmoc arom.), 7.71 (t, 2H, J 7.1 Hz, CH Fmoc arom.), 7.41-7.31 (m, 4 H, CH Fmoc arom.),
.57 (dd, 1H, J1,2 4.9 Hz, J2,3 10.0 Hz, H-2), 5.37-5.34 ( m, 2H, H-3, H-4''), 5.31 (t, 1H, J ' = J3',4' 10.0
2',3
Hz, H-3'), 5.24-5.21 (m, 1H, H-4'), 5.12 (dd, 1H, J3'',4'' 3.5 Hz, J2'',3'' 10.2 Hz, H-3''), 5.05-5.01 (m, 2H, H-
', H-2''), 4.74-4.71 (m, 2H, H-1, H-2'), 4.68 (d, 1H, J1'',2'' 8.9 Hz, H-1''), 4.52-4.46 (m, 2H, CHαThr,
CHβThr), 4.37-4.32 (m, 3H, H-5'', CH Fmoc), 4.28-4.24 (m, 3H, H-6a'', H-6b'', CHFmoc), 4.21 (d, 2H, J
4
2
1
2.5 Hz, CH -triazol), 4.13-4.07 (m, 4H, H-6a, H-6b, H-6a', H-6b'), 3.97 (d, 2H, J
J5',6a' 6.6 Hz H-5,
5,6a = ,
2

2
4
ACCEPTED MANUSCRIPT
1
3
H-5'), 2.11-1.91 (m, 30H, 10xCOCH ), 1.32 (d, 3H, J 6.5 Hz, CH Thr). C NMR (125 MHz, CD OD): δ
3
3
3
1
71.4 (CO Fmoc), 171.1-169.9 (COCH ), 144.0-141.3 (Cquat. Fmoc Ph), 127.5-119.6 (CH Ph), 124.2
3
(CH-triazol), 100.8 (C-1''), 98.6 (C-1), 98.2 (C-1’), 74.1 (C-5), 70.8 (C-3''), 70.1 (C-6), 70.0 (C-4'), 69.3
(
C-4), 68.8 (C-5'), 68.7 (C-2), 67.2 (C-3), 66.7 (CHαThr), 65.0 (C-3’), 62.5 (OCH Fmoc), 62.5 (C-6''),
2
6
2.3 (C-5'’), 61.0 (C-6’), 60.7 (C-4''), 60.2 (C-2''), 59.8 (C-2'), 59.7 (OCH -triazol), 49.8 (CHβThr), 47.0
2
(
CH Fmoc), 21.1 (CH CONH), 19.8-19.2 (COCH ), 17.8 (CH3Thr). ESI-HRMS: calcd. for
3
3
+
C H N NaO [M + Na] : 1350.4283, found: 1350.4281.
60
73
5
29
4
.2.12. General method for glycopeptide synthesis
The solid-phase synthesis of glycopeptides 3 and 4 were assembled manually using a fritted glass
reaction vessel. FmocGly-Wang resin (100 mg) was swollen in CH Cl for 2 h and then washed with
2
2
DMF (3×). Deprotection of the NFmoc group was performed using 50% morpholine-DMF followed by
filtration and washing with DMF (3x), whilst coupling reactions were carried out with 2.0 mol equiv of
Fmoc-amino acid and coupling reagents (PyBOP and HOBt) dissolved in DMF, and the base DIPEA. The
coupling times varied from 24 h for the amino acids to 48 h and 68 h for the glycosyl-amino acids 1 and
2
. The reaction mixtures were filtered after each coupling and the resin was washed three times with
DMF, CH Cl and MeOH. After drying, small aliquots of resin (1 mg) were treated with 50%
2
2
morpholine-DMF for removal of NFmoc group, with consequent formation of dibenzofulvene product,
allowing spectrophotometric detection of coupling reactions at 290 nm. After the last removal of NFmoc
group, N-acetylation reactions of glycopeptides were performed using Py and Ac O, followed by their
2
cleavage from resin using an aqueous mixture of trifluoracetic acid and triisopropylsilane
(
TFA:TIPS:H O, 90:5:5). The obtained solutions were then filtered, concentrated and treated with 1M
2
sodium methoxide solution in MeOH for removal of the sugar acetyl groups. The purification of the crude
glycopeptides 3 and 4 was carried out on Shimadzu Shim-PaK HPLC equipment using a semi-preparative
C18 reverse-phase column Shim-PacK CLC-ODS (M) column (250 x 10 mm), under gradient conditions
-1
(
A: 0.1% TFA/ H O, B: MeCN, 10-70% B, within 40 min.), at a flow rate of 3.0 mL min. , with UV
2

2
5
ACCEPTED MANUSCRIPT
detection (224 nm). Upon these conditions the retention times for glycopeptides 3 and 4 were 15 min. and
1
1 min., respectively.
4
.2.12.1. NHAcThrVal[αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH 3
Using the SPPS method previously described, followed by purification by HPLC, glycopeptide 3
1
was obtained as an amorphous solid (3.2 mg, 2.8 µmol, 23 %). Rt (224 nm)= 14.3 min. H NMR (500
MHz, D O): δ 8.10 (s, 1H, H-triazol), 4.99 (d, 1H, J 5.6 Hz, H-1), 4.46 (d, 1H, J 4.1 Hz, H-1’), 4.28-
2
1,2
1’,2’
4
.26 (m, 2H, 2x CHαThr), 4.20-3.96 (m, 11Η, CHαIle, CHαVal, CHβThr, CH Gly, H-4, H-4’, H-3, H-3’,
2
H-2, H-2’), 3.90 (d, 1Η, J 8.0 Hz, CHβThr), 3.79-3.76 (m, 3H, H-6a, H-6b, CH triazol), 3.69-3.66 (m,
2
a
3
H, H-6a’, H-6b’, CH triazol), 3.59-3.51 (m, 1H, H-5’), 3.37 (t, 1H, J 9.0 Hz, H-5), 3.08 (t, 3H, J 6.5
2b 5,6a
Hz, CHαArg, CH δArg), 1.96-1.87 (m, 15H, 2x NHAc, CH Thr, 2x CH Val), 1.78-1.53 (m, 8H,
2
3
3
CH βArg, CH γArg, CH γIle, CHβIle, CHβVal), 1.18-1.06 (m, 9H, CH Thr, 2x CH Ile). ESI-HRMS:
2
2
2
3
3
+
calcd for C H N O [M + H] : 1134.5637, found: 1134.5639.
4
6
80 13 20
4
.2.12.2. NHAcThrVal[Gal-β1,4-αGlcNAc-1-triazol-2Manα]ThrIleArgGlyOH 4
Using the SPPS method previously described, followed by purification by HPLC, glycopeptide 4
1
was obtained as an amorphous solid (3.4 mg, 2.4 µmol, 12 %). Rt (224 nm)= 9.2 min. H NMR (500
MHz, D O): δ 8.15 (s, 1H, H-triazol), 5.13 (s, 1H, H-4’’), 5.0 (d, 1H, J 4.7 Hz, H-1), 4.62 (d, 2H, J 12.3
2
1,2
Hz, CH triazol), 4.47 (d, 1H, J1’,2’ 3.9 Hz, H-1’), 4.35 (d, 1H, J1’’,2’’ 7.8 Hz, H-1''), 4.29 (dd, 1H, J1,2 5.7
2
Hz, J2,3 8.4 Hz, H-2), 4.25-4.22 (m, 2H, 2x CHαThr), 4.20-3.98 (m, 11Η, CHαIle, CHαVal, CHβThr,
CH Gly, H-4, H-4’, H-3, H-3’, H-2, H-2’), 3.88 (d, 1Η, J 10.6 Hz, CHβThr), 3.83-3.76 (m, 5H, H-3’’, H-
2
6
a, H-6b, H-6a’,H-6b’), 3.68-3.61 (m, 4Η, Η-4'', H-5'', H-6a'', H-6b''), 3.57-3.55 (m, 2H, H-5, H-5’), 3.43
(
dd, 1H, J1'',2'' 8.0 Hz, J2'',3'' 9.6 Hz, H-2''), 3.12-3.08 (m, 3H, CHαArg, CH δArg), 1.97-1.92 (m, 9H, 2x
2
NHAc, CH Thr), 1.75-1.24 (m, 14H, CH βArg, CH γArg, CH γIle, CHβIle, CHβVal, 2x CH Ile), 11.19-
3
2
2
2
3

2
6
ACCEPTED MANUSCRIPT
+
+
1
.07 (m, 9H, CH Thr, 2x CH Val). ESI-HRMS: calcd. for C H N O
52 90 13 25
[M + H] : 1296.6165, found:
3
3
1
296.6166.
4
.2.13. Enzymatic galactosylation of glycopeptide 3
The enzyme bovine β-1,4-galactosyltransferase (2.0 U/mL, EC 2.41.22, Sigma-Aldrich) was added
to a solution of glycopeptide 3 (1.4 mg, 1.26 µmol) and UDP-galactose (0.38 mg, 0.63 µmol), in HEPES
buffer (0.4 mL, 50 mM, pH 7.0, containing 100 mM MnCl ). The mixture was incubated for 120 h at 37
2
°
C, and was subsequently ultrafiltered against deionized water using a membrane of 3 kDa. The resulting
product was dissolved in water and purified by gel filtration chromatography (TOYOPEARL HW-405,
-1
8
5 cm x 15 mm) using 0.1% TFA in water, at a flow rate of 1.0 mL min.
4
.3. Molecular modeling studies
The initial structure of the natural glycopeptide Thr-Val-Thr[βGlcNAc-1,2αMan]-Ile-Arg-Gly from
α-dystroglycan was obtained from the GlycamWeb server [26]. Then, this structure was modified to
substitute βGlcNAc for αGlcNAc and to add the CH -triazole group between GlcNAc and Man with
2
PyMOL to yield the glycopeptide analog 3. The newly modified portion of the molecule was optimized at
the semi-empirical level using the PM7 hamiltonian considering water as the implicit solvent with
MOPAC [27] prior to docking.
The crystallographic structure of the bovine β-1,4-Galactosyltransferase 1 (β1,4GalT1) in which it
2
+
is complexing the UDP-Galactose (UDP-Gal) and the Mn cofactor was used (PDB ID 1TVY) [25]. The
M344H mutation was not reversed since it is not expected to alter the acceptor site. Hydrogens were
added with the open source version of PyMOL.
Docking calculations were performed with AutoDock Vina [28] with exhaustiveness parameter of
1
000. The search volume was focused on the active site and the docking protocol was validated by
reproducing the experimentally observed position of Chitobiose at the acceptor site as shown by the
crystallographic structure PDB ID 1TW5 [25]. No water molecules were necessary to reproduce the

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experimental data. Both glycopeptides were docked to the β1,4GalT1–Mn–UDPGal complex using the
validated protocol.
1
13
Supporting Information Available: The Supporting Information comprises the H NMR, C NMR,
HSQC and ESI-MS analyses of the main compounds, as well as a complementary figure related to
molecular modeling studies.
Acknowledgments
We acknowledge the financial support and fellowships from FAPESP (Fundação de Amparo à
Pesquisa do Estado de São Paulo, Proc. 2012/19390-0). We are grateful to Vinícius Palaretti for NMR
analyses and to José Carlos Tomaz for ESI-MS analyses.
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