Click glycosylation of peptides through cysteine propargylation and CuAAC

Article abstract of DOI:10.1016/j.bmc.2014.09.056

'Click' glycosylation of cysteine-containing peptides were carried out in good yield by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC). For that peptides were functionalized though direct propargylation of the cysteine residue allowing their use in CuAAC with suitable free or protected azido sugars of gluco, manno and galacto configuration. Among these free and protected glycopeptides a series of 'glycoRGD' peptides were obtained and submitted to in vitro platelet aggregation tests, showing that the pseudoglycosylation of the adhesion sequence lowers the IC₅₀ value and thus could improve the in vivo pharmacokinetic properties.

Full text of DOI:10.1016/j.bmc.2014.09.056

Bioorganic & Medicinal Chemistry 22 (2014) 6672–6683
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
‘Click’ glycosylation of peptides through cysteine propargylation and
CuAAC
a,b,
⇑
a,b,c
, Raphaël Hensienne , Christine Vala a,b,
a,b
Sandrine Lamandé-Langle
, Charlotte Collet
a,b
a,b,c
a,d
a,d
a,d
Françoise Chrétien , Yves Chapleur
, Amel Mohamadi , Patrick Lacolley , Véronique Regnault
a
Université de Lorraine, F-54500 Vandoeuvre-les-Nancy, France
CNRS, UMR 7565, F-54506 Vandoeuvre les Nancy, France
CHU de Nancy, Nancyclotep, 3 Rue du Morvan, F-54500 Vandoeuvre les Nancy, France
INSERM U1116, 9 av. de la forêt de Haye, F-54500 Vandoeuvre-les-Nancy, France
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2014
Revised 19 September 2014
Accepted 25 September 2014
Available online 27 October 2014
‘Click’ glycosylation of cysteine-containing peptides were carried out in good yield by Copper(I)-catalyzed
Azide–Alkyne Cycloaddition (CuAAC). For that peptides were functionalized though direct propargylation
of the cysteine residue allowing their use in CuAAC with suitable free or protected azido sugars of gluco, man-
no and galacto configuration. Among these free and protected glycopeptides a series of ‘glycoRGD’ peptides
were obtained and submitted to in vitro platelet aggregation tests, showing that the pseudoglycosylation of
the adhesion sequence lowers the IC50 value and thus could improve the in vivo pharmacokinetic properties.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Glycoconjugate
Click chemistry
CuAAC
Peptide
RGD
1
. Introduction
The glycosylation of proteins is an important process which
and compatible with a fluorine-18 (18F) radiolabelling step using
harsh conditions which will be done before the coupling. This pre-
cludes the use of the efficient, but reversible cysteineAmaleimide
9
controls the conformation and/or the biological properties of the
biomolecule. This important process is rather difficult to mimic
by chemical methods owing to the difficulty to selectively establish
bond formation as a coupling method. The high nucleophilicity
of the thiol function can be exploited to prepare S-alkylated deriv-
1
0
atives. For example, the introduction of a propargyl group on a
cysteine residue would allow its use in click triazole formation
with a suitable azido sugar which could be labeled with short-lived
fluorine-18. This possibility of exploiting S-propargyl-cysteine
derivatives for connection to a sugar has not been used until
1
glycosidic linkages on a multifunctional peptide backbone. Alter-
nate strategies of linking biomolecules have been searched for and
rapid and efficient methods have been reported.2 The concept of
click chemistry has been proposed has a powerful tool to link
,3
4
11
two partners using a rapid and often side-product free reaction.
recently. An elegant solution has been proposed by Dondoni to
Nevertheless, all these approaches need some selective and effi-
cient modifications of the partners for example of the peptide. This
concept has been widely and successfully used in carbohydrate
connect 1-thiosugars with cysteine-containing peptides using a
click ene reaction. The use of strained alkynes as a partner in a cop-
per free click cycloaddition was not primarily considered given the
5
12
chemistry. The click version of the Huisgen cycloaddition has
easiness of the S-propargylation of cysteine. Following our paper
attracted considerable attention allowing the connection between
sugar and amino-acids or more complex constructions.
on our preliminary results in the formation of neoglycoconjugates
using S-propargyl cysteine derivatives we report here more
6
7
13
In connection with our search for a fast sugar–peptide ligation
process, usable with labeled sugars, we reasoned that biologically
examples and some results showing the influence of the glycocon-
jugation on the biological properties of the conjugates.
8
significant peptides or proteins often contain a cysteine residue not
embedded in a disulfide bond. With the aim of an application for
PET imaging, the conjugation method has to be regioselective
2
2
. Results and discussion
.1. Synthesis of neoglycopeptides
⇑
Corresponding author. Tel.: +33 3 83 68 47 77; fax: +33 3 83 68 47 80.
E-mail address: Sandrine.langle@univ-lorraine.fr (S. Lamandé-Langle).
The synthetic strategy developed and shown on Scheme 1,
requires a glucidic partner equipped with an azide group suitable
http://dx.doi.org/10.1016/j.bmc.2014.09.056
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
6673
cysteine
residue
OH
OR
Peptide
S
CuAAC
O
O
Peptide
HO
HO
i
AcO
AcO
HS
S-propargylation
R'
O
OH
O
OAc
O
N3
N3
RO
RO
2, 4, 8
9
-11: R = Tr
12-14: R = H
OR
O
ii
R'
O
N
3
F
RO
RO
Peptide
iii
O
S
RO
RO
OR
O
OR
O
N
N
3
N
N
1
1
5-17: R = Ac
8-20: R = H
iv
O
Scheme 1. Synthetic strategy for ‘click’ glycosylation of peptides.
R
R
1
1OR2
R
1
2
O
2
O
R O
2
O
R O
2
R O
N
3
2
R O
2
R O
for a Copper(I)-catalyzed Azide–Alkyne Cycloaddition (CuAAC).
The introduction of the azide on the sugar and the propargyl on
the peptide and not the opposite was decided for different reasons.
Indeed, propargyl bromide is quickly introduced on the cysteine by
a simple alkylation reaction in aqueous medium. Introduction of an
azido group on cysteine would require the preparation of a less
reactive and more sensitive azido bromo ethylene derivative.
N
3
R O
O
N
3
2
OR2
R O
O
2
1
1
2
3: R 11 = OH, R2 = Ac
14: R1 = OH, R = Ac
12: R
= OH, R = Ac
1
1
1
5: R = F, R2 = Ac
8: R = F, R2 = H
16: R = F, R2 = Ac
17: R1 = F, R = Ac
2
1
9: R1 = F, R2 = H
20: R1 = F, R2 = H
1
Scheme 2. Reagents and conditions: (i) (1) TrCl, pyridine, DMAP (cat), rt. (2) Ac
rt, 1 h, 60–84%; (ii) AcOH/H O 3:1, 80 °C, 1 h, 38–93%; (iii) DAST, diglyme, 110 °C,
1 h, 25–70%; (iv) Na , MeOH, rt, 1 h30, 76–95%.
2
O,
2
0
2
-Azido ethyl glycosides were chosen as carbohydrate compo-
nents. The primary azide function was supposed to be reactive
and the two carbon link between the sugar and the peptide in
the final product was a good compromise in terms of flexibility
and length. Moreover, 2-azido ethyl glycosides are easily prepared
by known methods, mainly by boron trifluoride-etherate catalyzed
glycosylation of the peracetylated sugars with bromoethanol or
Fischer glycosylation with the same, and subsequent nucleophilic
substitution in the presence of sodium azide in hot DMF. To eval-
uate the scope and limitations of the reaction, four pyranosides
derivatives of gluco, manno and galacto configuration having differ-
ent anomeric configurations were selected as sugar components.
Compounds 1, 3 and 5 were prepared according to known
Br
HS
peptide
S
peptide
(
i) or (ii)
O
NH
2
O
H
H
N
R2
R1
N
O
HOOC
N
H
COOH
O
S
S
2
3 (81%)
2
2
1: R1 = Boc, R2 = Me (55%)
1
2
2: R = R = H (66%)
COOH
NH
2
O
H
N
H
H
H N
2
N
N
COOH
N
H
14
15
methods to provide compounds b-gluco 1,
a
-manno 3 and
b-galacto 5 in 65%, 65% and 60% yield, respectively. Compound
of -gluco configuration was synthesized by a Fisher glycosyla-
NH
O
O
S
O
O
7
k
OH
HN
O
24 (85%)
7
a
NH
tion using 2-bromoethanol followed by peracetylation with acetic
anhydride in pyridine. Substitution with azide was carried out in
hot DMF as before to give 716 in 10% yield over the three steps.
The four compounds were quantitatively deprotected to the free
HN
O
O
HN
HN
O
NH
NH
2
S
2
5 (82%)
HN
1
6
17 7k
16
sugars 2, 4,
6
and 8 by Zemplen deacetylation (Fig. 1).
1
8
2
H N
O
In the context of peptides labelling for PET application 6-flu-
oro sugars were envisioned as prosthetic groups thus the coupling
8
O
S
O
O
O
H
N
H
N
H
N
H
N
of the later with peptides was investigated. These compounds were
prepared from 2-azido ethyl glycosides 2, 4 and 8 by a one-pot pro-
tection of the primary alcohol with trityl chloride followed by per-
acetylation. Intermediates 12, 13 and 14, were then obtained after
2
H N
CONH2
S
N
H
N
N
H
N
H
O
H
N
O
O
O
N
26 (79%)
NH
H
2
removal of the trityl group carried out in a 3:1 AcOH–H O solu-
1
9
tion. Finally these compounds were fluorinated with DAST and
deprotected to provide fluoro compounds 18, 19 and 20 in good
yields (Scheme 2).
Scheme 3. Reagents and conditions: (i) DMF, Cs
h.
2
3 2 4
CO ; (ii) H O, MeOH, NH OH, rt,
3
With these azido sugars in hand, the S-alkylation of several cys-
teine-containing peptide models was investigated. The first exper-
iment involved Boc-OMe-cysteine which was reacted with
propargyl bromide in DMF in the presence of cesium carbonate7c
giving the propargylated protected cysteine 21 in 55% yield
(Scheme 3). Deprotection of 21 by basic hydrolysis followed by
acidic treatment provided the known free aminoacid 227b in 66%
yield. Although, efficient, this method used a solid base which
complicated the work-up and explained the modest yield observed
in the alkylation step. Moreover, it proved difficult to carry out on
peptides with functional groups.
OR
RO
RO
OR
O
N3
O
RO
RO
RO
O
N3
OR
O
1
2
R = OAc (65%)
R = H
3 R = OAc (65%)
4 R = H
OR
O
OR OR
N3
RO
RO
O
N3
O
RO
OR
O
OR
A more direct method for thiol propargylation was searched for
using glutathione as a model of free tripeptide. When the later was
treated at room temperature with one equivalent of freshly dis-
tilled propargyl bromide in a 1:1 methanol–water mixture and
5
6
R = OAc (60%)
R = H
7 R = OAc (10%)
8 R = H
Figure 1. Deprotected and peracetylated azido-carbohydrates.

6
674
S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
8
R6
O
ammonia as base, S-alkylation took place in 3 h giving derivative
3 in 85% yield. An almost pure compound was obtained bearing a
O
H
N
R5
2
OAc
R
R
4
1
single propargyl group as seen from proton NMR spectrum of the
crude. The signal of the propargylic proton was observed at
.60 ppm, chemical shifts of the methylene group of the propargyl
at 3.29 ppm in H NMR and at 18.9 ppm in C NMR established the
formation of a S-propargyl derivative. Mass spectra confirmed the
formation of the expected product (Scheme 3).
The propargylation of the cysteine residue of biologically signif-
icant peptides was next explored. Integrin receptors recognize
polypeptide domains containing the Arg-Gly-Asp (RGD) amino acid
sequence, which is present in several extracellular matrix pro-
teins such as vitronectin. The glycosylation of these peptides has
been reported and proved useful to target tumor cells and for
imaging of tumor angiogenesis. Two peptides containing the
RGD adhesion sequence were chosen, the commercially available
Arg-Gly-Asp-Cys (RGDC) and a cyclic form c(RGDfC).
A nine amino acid peptide Cys-Gln-Trp-Ala-Val-Gly-His-Leu-
Met (CQWAVGHLM) derived from bombesin was designed by add-
ing a cysteine residue at the N-terminus. This peptide includes the
active sequence of gastrin-releasing peptide (GRP) that exhibits
high affinity and specificity to the GRP receptor (GRPr). These
receptors are overexpressed in a variety of cancers, including small
cell lung cancer (SCLC), prostate, breast, pancreas and gastrointes-
O
R
3
S
AcO
O
3
R
2
2
N
N
1
13
N
1
4
2
5
6
2
2
2
3
7 R , R = H, R , R = OAc, R = Boc, R = Me
2
4
1
3
5
6
8 R , R = H, R , R = OAc, R = Boc, R = Me
9 R , R = H, R , R = OAc, R5 = Boc, R = Me
1
3
2
4
6
1
4
2
3
5
6
0 R , R = H, R , R = OAc, R , R = H
31 R , R = H, R , R = OAc, R , R = Me
32 R , R = H, R , R = OAc, R , R = H
2
4
1
3
5
6
1
3
2
4
5
6
2
0
NH
2
H
N
HOOC
OAc
N
H
COOH
O
R4
R
1
21
S
O
R
3
AcO
O
R
2
N
3
N
N
1
4
2
3
3
3 R , R = H, R , R = OAc
4 R , R = H, R , R = OAc
35 R , R = H, R , R = OAc
2
4
1
3
1
3
2
4
Figure 2. Peracetylated glycocysteines and glycoglutathiones.
catalyst in the presence of 20% mol of sodium ascorbate as
reducing agent but in water (Table 2). The reaction was difficult
to monitor by tlc, but it appeared that the color change of the
medium was a good indicator of reaction completion. The work-
up consisted on removal of copper ions with a chelating resin
and freeze-drying. As for protected sugar the major isomer (1,4 tri-
azole) was accompanied by variable amount (less than 10%) of
another yet unidentified triazole, not the 1,5-triazole, as seen from
2
2
tinal tumors.
S-propargylation of commercially available RGDC, c(RGDfC) and
CQWAVGHLM proceeded uneventfully using the above described
methodology and gave the expected peptides 24, 25 and 26,
respectively, in good purity and yield by simple evaporation
Scheme 3). H NMR of these crude products showed almost one
species in up to 90% purity. Only one signal corresponding to the
propargylic proton was observed around 2.65 ppm ±0.5 ppm
depending on the peptide. Mass spectrometry showed complete
disappearance of starting materials and the exact mass of the
desired products 24, 25 and 26.
1
(
13
NMR of the crude. The expected 1,2,3-triazoles 36–51 were
obtained in good yields (Fig. 3) after purification by LH20 gel filtra-
tion. High resolution mass spectrometry confirmed the structure of
the adduct as well as NMR data.
The CuAAC reaction was tested first with the three protected
carbohydrates 1, 3 and 5 and the three S-propargyl derivatives
2
1, 22 and 23. The reaction was performed using standard proce-
dures in a 1:1 tert-butyl alcohol–water mixture in the presence
of sodium ascorbate as the reducing agent and with copper(II) ace-
tate as the catalyst. The results of these cycloadditions are summa-
rized in Table 1. As seen from this Table, the reactions proceeded
under very mild conditions giving high yields of the expected
Table 2
Click coupling of free azido-carbohydrates and S-propargylated peptides
R
O
HO
HO
+
S
peptide
peptide
OH
O
1
,4-triazoles as the only products (Fig. 2). Interestingly the reaction
N3
medium became immediately light green on addition of copper
acetate to the alkyne and azide mixture and turned blue within
R
O
S
1
–2 h at which time the reaction was completed as seen by tlc
HO
Cu(OAc)
2
HO
from sugar consumption.
O
OH
N
N
Na ascorbate
The reaction between free and water soluble carbohydrates 2, 4,
N
2
H O
6, 8, 18, 19 and 20 and peptides 23, 24, 25 and 26 was performed as
before at room temperature using 10% mol of Cu(OAc)
2
as the
Entry Azide
Alkyne
component
Product Time
(h)
Yield
(%)
component
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2
2
4
4
6
23
24
23
24
23
24
23
23
24
23
24
25
26
23
24
26
36
43
37
44
38
45
39
40
46
41
47
49
50
42
48
51
1
2
1
2
1
1
2
2
2
2
1
2
2
2
2
2
66
71
62
63
55
52
67
80
68
55
67
59
58
69
57
60
Table 1
Click coupling of peracetylated azido-carbohydrate and S-propargylated cysteine and
glutathione
Entry Azide
component
Alkyne
component
Product Time
(h)
Yield
(%)
6
8
1
2
3
4
5
6
7
8
9
1
21
22
23
21
22
23
21
22
23
27
30
33
28
31
34
29
32
35
2
2
2
2
2
2
2
1
1
73
70
83
90
75
85
73
62
60
18
18
19
19
19
19
20
20
20
1
1
3
3
3
5
5
5

S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
6675
R1
R
R
1
R1
R1
R
S
HO
R
HO
HO
S
R
O
O
O
HO
HO
HO
HO
S
HO
HO
O
S
O
N
O
OH
N
HO
N
O
N
O
HO
N
N
N
N
1
N
N
3
4
4
4
4
5
6 R = OH, R = glutathione
N
N
1
1 R = F, R = glutathione
7 R1 = OH, R = glutathione
0 R = F, R = glutathione
4 R = OH, R = RGDC
6 R = F, R = RGDC
1
3
39 R = OH, R = glutathione
42 R = F, R = glutathione
48 R = F, R = RGDC
51 R = F, R = CQWAVGHLM
1
1
3 R = OH, R = RGDC
1
38 R = OH, R = glutathione
45 R = OH, R = RGDC
1
4
4
4
1
1
7 R = F, R = RGDC
1
1
1
9 R = F, R = c(RGDfC)
1
1
1
0 R = F, R = CQWAVGHLM
R = glutathione :
NH
R = RGDC :
2
O
COOH
H
N
NH
2
O
H
N
H
N
H
HOOC
N
H
COOH
H
2
N
N
COOH
R = c(RGDfC) :
O
OH
N
H
O
NH
O
O
O
H
O
N
NH
H
2
N
O
R = CQWAVGHLM :
O
O
HN
O
HN
O
O
O
O
H
N
H
N
H
N
H
HN
H
2
N
N
CONH
2
NH
N
H
N
H
NH
N
NH
2
H
N
O
O
O
O
HN
S
N
N
H
H
Figure 3. Free glycopeptides.
HOOC
43
44
45
1
1
20
00
O
H
N
NH
2
H
N
RGD
H
N
RGDC
HOOC
S
NH
2
N
H
cRGDfV
cRGDfC
46
O
O
NH
47
48
4
9
3
5
8
6
0
0
N
N
N
O
HO
HO
OH
O
HO
O
40
20
0
OH
OH
53
N
N
N
S
NH
O
H
N
O
0
50
100
150
200
H
2
N
N
H
N
H
N
COOH
Peptide concentration (µM)
H
NH
2
O
COOH
Figure 5. Effects of peptides on ADP-induced platelet aggregation. Platelet aggre-
gation in human citrated PRP was triggered by 2.5 M ADP and monitored by
turbidimetric aggregometry. PRP was stimulated by active peptides at concentra-
tions varying from 12.5 to 200 M. Inhibition of platelet aggregation was measured
l
Figure 4. Bis glycopeptide.
l
at the maximal aggregation response. The results are the mean ± SEM of
independent experiments with PRP from different donors.
7
From these results, it is clear that the structure of the starting
-azido ethyl glycosides and its anomeric configuration had no
2
influence on the course of the click Huisgen reaction. The presence
of a fluorine atom at position 6 of the sugar also did not have an
effect on the course of the cycloaddition. All the glycopeptides
and fluoroglycopeptides were obtained in good yields. The
efficiency of the process was confirmed by the CuACC reaction of
a bis 6-azido sugar with two equivalents of propargylated peptide
RGDC 24. Thus, using the same methodology, the bis-6-azido
to their specificity to
a
IIbb3 integrins,25 was measured and com-
pared to reference peptides, that is, linear ones (the minimal recog-
nition sequence RGD and RGDC as structural reference) and cyclic
peptides (c(RGDfV) and c(RGDfC)). All compounds inhibited the
aggregation of platelets in citrated platelet-rich plasma (PRP)
induced by 2.5 lM ADP. As shown in Figure 5, the concentra-
2
3
trehalose 52 was reacted with 24 to give the expected bis
glycopeptide 53 (Fig. 4) in 65% yield.
tion–response curves of c(RGDfV) and c(RGDfC) peptides and all
glycopeptides except 49 were shifted upwards compared to those
of RGD or RGDC peptides. IC50 values for the maximum aggregation
response are listed in Table 3.
2
.2. Biological evaluation
To evaluate the influence of the sugar moiety on the biological
2.3. Discussion
activity of our conjugates, RGD-containing glycoconjugates, which
are potential ligands of integrins,24 seemed appropriate. Thus these
compounds were evaluated and their activities compared to those
of the native peptide. The ability of the eight RGD-containing
glycopeptides to inhibit ADP-induced platelet aggregation, thanks
From these results it appears that S-propargyl-cysteine deriva-
tives including highly functional peptides are easily available in a
highly regiospecific manner by treatment of free peptides with
propargyl bromide in basic medium. This simple derivatization

6
676
S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
Table 3
IC50 of glycopeptides and reference peptides in ADP-induced platelet aggregation
sequence in cyclic peptides. The process is applicable to fluoro sug-
ars paving the way to F labeled constructions suitable for PET
18
Entry
Peptide/glycopeptide
Structure
IC50
(
lM)
application. We are currently exploring this approach.
1
2
3
4
5
6
7
8
9
Linear reference
Linear reference
Cyclic reference
Cyclic reference
45
44
43
46
47
48
49
53
RGD
RGDC
c(RGDfV)
c(RGDfC)
OH-b-gal-RGDC
319
322
72
65
23
47.6
44
23.5
66
26
147.5
76.5
4. Experimental
4
.1. General
OH-
OH-b-gluc-RGDC
F- -man-RGDC
F-b-gluc-RGDC
F- -gluc-RGDC
F-b-gluc-c(RGDfC)
Trehalose-(RGDC)
a
-man-RGDC
Solvents and liquid reagents were purified and dried according
to recommended procedures. TLC analyses were performed using
standard procedures on Kieselgel 60 F254 plates or RP-18 F254 plates
a
10
11
12
a
(
Merck). Compounds were visualized using UV light (254 nm) and
a methanolic solution of sulfuric acid and charred. Column chro-
matography was performed on silica gel SI 60 (63–200 m)
Merck). Purification of free compounds was achieved by gel filtra-
2
l
(
can be performed in the presence of carboxylic, amino and guani-
dino groups. 2-Azido ethyl glycosides reacted efficiently with these
alkynylated peptides in a click Huisgen reaction, whatever the sugar
and the anomeric configuration (b-gluco,
-gluco). The reaction proceeds well in water in short reaction times
and high yields and tolerates the presence of a fluorine atom on the
sugar. The biological effect of peptides on IIbb3-mediated platelet
aggregation has been examined. As expected, the IC50 values of
linear references RGD or RGDC were higher than that of cyclic refer-
ences c(RGDfV) and c(RGDfC), in agreement with the idea that cyclic
RGD peptides are more effective inhibitors of platelet aggregation
than linear RGD peptides. It is noteworthy that the RGDC sequence
had the same inhibitory activity than RGD (compare entries 1 and
tion on LH20 using water or water/methanol mixture as eluent.
Melting points were determined with a Tottoli apparatus and are
uncorrected. Optical rotations were measured on an Anton-Paar
MCP 300 polarimeter. H, C NMR and F spectra were recorded
on a Bruker spectrometer DPX250 (250 MHz, 62.9 MHz and
a
-manno, b-galacto and
1
13
19
a
2
35 MHz, respectively) or DRX400 (400 MHz and 100.6 MHz,
a
1
13
respectively). For complete assignment of H and C signals,
1
1
1
13
two-dimensional H, H COSY and H, C correlation spectra were
recorded. Chemical shifts (d) are given in ppm relative to the sol-
vent residual peak. The following abbreviations are used for multi-
plicity of NMR signals: s = singlet, d = doublet, t = triplet,
q = quadruplet, m = multiplet, b = broad signal and app = apparent
multiplicity. The given J values refer to apparent multiplicities
and do not represent the true coupling constants. The protons
and carbons of the ethyl arm are numbered 7 and 8 in the NMR
description. Mass and HRMS spectra were recorded on a Bruker
microTOFQ apparatus and MALDI spectra on a Bruker Ultraflex
apparatus.
2
). The same holds for cyclic peptides (compare entries 3 and 4).
This confirmed that introduction of a cysteine residue had not sig-
nificant effect on activity. Interestingly all glycopeptides were more
potent inhibitors than the RGD sequence. They can be divided in
three groups according to their IC50 values. The first group encom-
passes peptides 47 and 53 (entries 9 and 12) with the lowest inhib-
itory activity, the second group of peptides 44 and 43 (entries 6 and
The following known compounds are not described:
S-Propargyl-N-[(1,1-dimethylethoxy)carbonyl]-L-cysteine
methyl ester (21).
7
) with an intermediate inhibitory activity and the third group com-
10c
prises peptides 45, 46 and 48 (entries 5, 8 and 10) with the highest
inhibitory activity. The cyclo-fluorinated glycopeptide 49 displayed
the lowest inhibitory activity (entry 11). Linear fluorinated glyco-
peptides 46 and 48, with IC50 values in the low micromolar range
were the most potent inhibitors for this series of ADP-induced
platelet aggregation and have thus been identified as potential can-
10b
S-Propargyl-L-cysteine hydrochloride (22).
23
Bis-6-azido trehalose (52).
4
.2. Preparation of 2-azidoethyl 2,3,4-tri-O-acetyl-6-O-trityl-
-mannopyranoside (9), 2-azidoethyl 2,3,4-tri-O-acetyl-6-O-tri-
tyl-b- -glucopyranoside (10) and 2-azidoethyl 2,3,4-tri-O-acetyl
6-O-trityl- -glucopyranoside (11)
a-
D
D
3
didates to develop a radiotracer for in vivo imaging of b integrins.
-
a-D
Moreover, all linear glycopeptides have a higher inhibitory activity
than cyclic peptides in this assay. These findings suggest that addi-
tion of sugar moieties on linear forms strongly modifies either the
physicochemical properties or induces beneficial conformational
modifications of the peptide as in the cyclic form. Interestingly,
introduction of a fluorine atom on glycopeptides (entries 8 and 9)
did not change significantly IC50 values compared to the non-
fluorinated glycopeptides (entries 6 and 7).
To a solution of compound 2,16 417 or 816 (35 mmol, 8.6 g) in
pyridine (200 mL) was added at room temperature TrCl (63 mmol,
7.6 g) and a catalytic amount of DMAP (3.5 mmol, 430 mg). The
solution was stirred at room temperature until completion (TLC
monitoring) then Ac O (45 mL) was added and the reaction was
1
2
stirred for 3 h. Water was then added (150 mL) and extracted with
EtOAc (3 ꢀ 100 mL). The organic layer was washed with HCl 1 M
(
3
150 mL), water, saturated NaHCO solution (150 mL) and brine
3
. Conclusion
(100 mL). The organic layer was dried over MgSO , filtered and
4
the solvent was removed under reduced pressure. The crude prod-
In conclusion, the propargylation of cysteine residues of simple
uct was purified by column chromatography (silica gel, Hex/EtOAc
amino-acids or complex peptides is an easily performed reaction to
get S-propargyl thioethers which can be regarded as a click chem-
istry reaction. The S-propargyl function reacts well with azido
ethyl glycosides and provides an easy and fast ligation method of
sugars with cysteine containing peptides. We found almost no
limitation in the structure of the sugar and in sensitive functions
present on complex peptides such as guanidine, carboxylate or pri-
mary amide. In vitro studies of ADP-induced platelet aggregation
inhibition show that introduction of a carbohydrate moiety on lin-
ear RGD peptides strongly enhances inhibitory activity. This simple
structural modification is as efficient as inclusion of the RGD
60:40) to obtain 9, 10 or 11.
4.2.1. 2-Azidoethyl 2,3,4-tri-O-acetyl-6-O-trityl-a-D-mannopy
ranoside (9)
Yield: 62%; R 0.4 (Hex/EtOAc 60:40); white solid; mp = 58 °C;
f
ꢁ
1
[
a
]D +49.3 (1.0, CHCl ); IR: film (m, cm ): 3059, 2932, 2105,
3
1
1748, 1371; H NMR (CDCl , 400 MHz) d 1.76 (s, 3H, OAc), 1.99
3
(s, 3H, CH ), 2.16 (s, 3H, CH ), 3.19 (dd, 1H, J
3
3
6,6
= 10.5 Hz,
0
0
J5,6 = 5.5 Hz, H6), 3.23 (dd, 1H, J5,6
2H, H8), 3.72 (ddd, 1H, J7,7 = 10.5 Hz, J = 6.5 Hz, J7,8
H7), 3.95–4.00 (m, 2H, H5, H7 ), 4.94 (d, 1H, J = 1.5 Hz, H1),
0
= 2.5 Hz, H6 ), 3.41–3.55 (m,
0
0
= 4.0 Hz,
7,8
0
1
,2

S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
6677
5
6
2
4
.27–5.37 (m, 3H, H2, H3, H4), 7.21–7.35 (m, 9H, H-Ar), 7.46 (dd,
H, J = 8.5 Hz, J = 1.0 Hz, H-Ar); 13C NMR (CDCl
, 100.6 MHz) d
1.0 (CH ), 21.1 (CH ), 21.3 (CH ), 50.8 (C-8), 62.8 (C-6), 66.8 (C-
), 67.1 (C-7), 69.6 (C-3 or C-2), 70.1 (C-3 or C-2), 71.0 (C-5), 87.0
5.40 (dd, 1H, H3); 13C NMR (CDCl
3
(CH ), 21.2 (CH ), 50.7 (C-8), 61.6 (C-6), 66.7 (C-4), 67.3 (C-7),
3
, 100.6 MHz) d 21.1 (CH
3
), 21.1
3
3
3
3
3
69.0 (C-3), 69.8 (C-2), 71.4 (C-5), 98.1 (C-1), 170.2 (C@O), 170.4
+
(C@O), 171.3 (C@O); MS m/z (ESI) 398 (M+Na) ; Anal. Calcd for
(
1
Cq Tr), 97.9 (C-1), 127.4 (3C-Ar), 128.2 (6C-Ar), 129.1 (6C-Ar),
44.1 (Cq-Ar), 169.8 (C@O), 170.4 (C@O), 170.5 (C@O); HRMS
14 21 3 9
C H N O : C: 44.80; H: 5.63; N: 11.19, found: C: 44.02; H:
5.64; N: 10.71.
+
35 3 9
m/z (ESI) calcd for C33H N O Na [M+Na] 640.2271, found:
6
40.2336.
4.3.2. 2-Azidoethyl 2,3,4-tri-O-acetyl-b-D-glucopyranoside (13)
Yield: 93%; R
f
0.2 (Hex/EtOAc 60:40); white solid; mp = 112 °C;
ꢁ1
4
.2.2. 2-Azidoethyl 2,3,4-tri-O-acetyl-6-O-trityl-b-D-glucopyrano-
[
a
]
D
ꢁ42.0 (1.0, CH
2
Cl
1218; H NMR (CDCl
2CH ), 3.29 (ddd, 1H, J8,8
2
); IR: film (
, 250 MHz) d 2.01 (s, 3H, CH
m
, cm ): 3457, 2924, 2105, 1755,
), 2.05 (s, 6H,
= 5.0 Hz, J8,7 = 3.5 Hz, H8),
1
side (10)
Yield: 60%; R
+9.8 (1.0, CH
H NMR (CDCl , 400 MHz) d 1.75 (s, 3H, CH
3
3
f
0.45 (Hex/EtOAc 60:40); white solid; mp = 68 °C;
3
0
= 13.5 Hz, J8,7
0
ꢁ1
0
[
a
]
D
2
Cl
2
); IR: film (
m
, cm ): 2881, 2104, 1757, 1219;
), 2.00 (s, 3H, CH ), 2.07
= 10.5 Hz, J6,5 = 5.0 Hz, H6), 3.28 (dd,
3.40–3.78 (m, 5H, H5, 2H6, H7, H8 ), 4.04 (ddd, 1H, J
7
0
,7 = 10.5 Hz,
1
0
0
0
= 3.5 Hz, H7 ), 4.63 (d, 1H, J1,2 = 8.0 Hz, H1), 4.97–5.09 (m,
3
3
3
J
7
,8
1
3
(
1
s, 3H, CH
3
), 3.12 (dd, 1H, J6.6
0
2H, H4, H2), 5.27 (app t, 1H, J2,3 = J3,4 = 9.5 Hz, H3); C NMR (CDCl
3
,
0
H, J
6
0
,5 = 2.5 Hz, H6 ), 3.30–3.35 (m, 1H, H8), 3.50–3.62 (m, 2H, H5,
100.6 MHz) d 20.5 (CH ), 20.6 (CH ), 20.6 (CH ), 50.5 (C-8), 61.2 (C-
3
3
3
0
H8 ), 3.78 (ddd, 1H, J7,7
0
= 11.0 Hz, J7,8 = 8.0 Hz, J7,8
0
= 3.0 Hz, H7),
6), 68.4 (C-7), 68.6 (C-4), 71.2 (C-2), 72.7 (C-5), 74.2 (C-3), 100.6 (C-
0
4
.05–4.15 (m, 1H, H7 ), 4.61 (d, 1H, J1,2 = 7.5 Hz, H1), 5.05–5.22
1), 169.4 (C@O), 170.1 (C@O), 170.2 (C@O); MS m/z (ESI) 398
+
(
m, 3H, H2, H3, H4), 7.20–7.34 (m, 10H, H-Ar), 7.42–7.48 (m, 5H,
(M+Na) ; Anal. Calcd for C14
H
21
N
3
O
9
: C: 44.80; H: 5.63; N: 11.19,
1
3
H-Ar); C NMR (CDCl
3
, 100.6 MHz) d 20.4 (CH
3
3
), 20.6 (CH ), 20.7
found: C: 45.19; H: 5.75; N: 10.88.
(
CH ), 50.6 (C-8), 61.9 (C-6), 68.2 (C-7), 68.7 (C-4), 71.3 (C-2),
3
7
1
3.1 (C-5), 73.5 (C-3), 86.6 (Cq Tr), 100.8 (C-1), 127.1 (3C-Ar),
4.3.3. 2-Azidoethyl 2,3,4-tri-O-acetyl-a-D-glucopyranoside (14)
27.8 (6C-Ar), 128.7 (6C-Ar), 143.5 (3Cq-Ar), 168.9 (C@O), 169.5
Yield: 38%; R
mp = 78 °C; [
2928, 2109, 1751, 1370, 1226, 1039; H NMR (CDCl
2.02 (s, 3H, CH ), 2.06 (s, 3H, CH ), 2.07 (s, 3H, CH
= 13.5 Hz, J8,7
,7 = 7.0 Hz, J
1H, J7,7
f
0.44 (CycloHex/EtOAc 40:60); white solid;
+
ꢁ1
(
C@O), 170.4 (C@O); MS m/z (ESI) 640 [M+Na] ; Anal. Calcd for
a
]
D
+134.3 (0.5, CHCl
3
); IR: film (
m
, cm ): 3483,
1
C
33
H
35
N
3
O
9
: C: 64.17; H: 5.71; N: 6.80. Found: C: 64.29; H: 5.80;
3
, 400 MHz) d
3
), 3.39 (ddd,
N: 6.61.
3
3
1
H, J8,8
0
0
0
= 6.0 Hz, J8,7 = 3.5 Hz, H8), 3.48 (ddd, 1H,
0
4
.2.3. 2-Azidoethyl 2,3,4-tri-O-acetyl-6-O-trityl-
oside (11)
Yield: 84%; R
mp = 117 °C; [
107, 1754, 1224, 1043; H NMR (CDCl
CH ), 2.02 (s, 3H, CH ), 2.11 (s, 3H, CH
= 10.5 Hz, J6,5 = 5.0 Hz, H6), 3.23 (dd, 1H, J
a-
D-glucopyran-
J
8
8
0
,7
0
= 3.5 Hz, H8 ), 3.56–3.61 (m, 1H, H6), 3.62 (ddd,
0
= 10.5 Hz, H7), 3.71 (bdd, 1H, J6,6
0
= 12.5 Hz, J
0
,5 = 7.5 Hz,
6
0
0
f
0.45 (CycloHex/EtOAc 60:40); white solid;
H6 ), 3.81–3.86 (m, 1H, H5), 3.87 (ddd, 1H, H7 ), 4.85 (dd, 1H,
ꢁ1
a]
D
+108.8 (0.5, CHCl
3
); IR: film (
m
, cm ): 2934,
J2,3 = 10.0 Hz, J2,1 = 3.5 Hz, H2), 5.02 (app t, 1H, J4,3 = J4,5 = 10.0 Hz,
1
13
2
3
, 400 MHz) d 1.76 (s, 3H,
H4), 5.14 (d, 1H, H1), 5.56 (app t, 1H, H3); C NMR (CDCl
3
,
3
3
3
), 3.14 (dd, 1H,
100.6 MHz) d 20.6 (2CH ), 20.7 (CH ), 50.4 (C-8), 61.0 (C-6), 67.3
3
3
0
J
6,6
0
6
0
,5 = 2.0 Hz, H6 ),
(C-7), 68.8 (C-4), 69.6 (C-3), 69.7 (C-5), 70.8 (C-2), 96.0 (C-1),
3
(
.43 (ddd, 1H, J8,8
0
= 13.5 Hz, J8,7
0
= 6.0 Hz, J8,7 = 3.5 Hz, H8), 3.53
170.0 (C@O), 170.4 (C@O), 170.6 (C@O); HRMS m/z (ESI) calcd for
0
+
ddd, 1H, ,7 = 7.0 Hz,
= 10.5 Hz, H7), 3.95–4.02 (m, 2H, H5 and H7 ), 4.95 (dd, 1H,
2,3 = 10.0 Hz, J2,1 = 4.0 Hz, H2), 5.12 (app t, 1H, J4,3 = J4,5 = 10.0 Hz,
J
8
0
J
8
0
,7
0
= 3.5 Hz, H8 ), 3.70 (ddd, 1H,
C
14
21
H N
3
O
9
Na [M+Na] 398.1170, found: 398.1172.
0
J
J
7,7
0
4.4. Preparation of 2-azidoethyl 6-deoxy-6-fluoro-2,3,4-tri-O-
acetyl- -mannopyranoside (15), 2-azidoethyl 6-deoxy-6-fluoro
-2,3,4-tri-O-acetyl-b- -glucopyranoside (16) and 2-azidoethyl
6-deoxy-6-fluoro-2,3,4-tri-O-acetyl- -glucopyranoside (17)
H4), 5.22 (d, 1H, H1), 5.48 (app t, 1H, H3), 7.25 (bt, 3H, J = 7.0 Hz,
a-D
1
3
HAr), 7.31 (bt, 6H, J = 7.0 Hz, H-Ar), 7.45 (bd, 6H, H-Ar); C NMR
CDCl , 100.6 MHz) d 20.5 (CH ), 20.7 (CH ), 20.7 (CH ), 50.4 (C-
), 62.0 (C-6), 67.0 (C-7), 69.0 (C-4), 69.1 (C-5), 70.4 (C-3), 70.9
D
(
8
3
3
3
3
a-D
(
(
C-2), 86.6 (Cq-Tr), 95.8 (C-1), 127.0 (3C-Ar), 127.8 (6C-Ar), 128.7
To a solution of sugar 12, 13 or 14 (0.8 mmol, 300 mg) in dig-
lyme (6 mL) was added DAST (0.21 mL, 1.6 mmol). The reaction
was heated at 110 °C during 45 min then cooled to 0 °C and meth-
anol (1 mL) was carefully added. The reaction mixture was concen-
trated under reduced pressure. The residue was diluted with
6C-Ar), 143.6 (3Cq-Ar), 169.3 (C@O), 170.2 (C@O), 170.4 (C@O);
+
HRMS m/z (ESI) calcd for C33
6
H
35
N
3
O
9
Na [M+Na] 640.2271, found:
40.2277.
4
.3. Preparation of 2-azidoethyl 2,3,4-tri-O-acetyl-
-glucopyran-
-glucopyrano-
a
-
D
-manno-
CH
2
Cl
2
(10 mL), washed with saturated NaHCO
3
solution (10 mL).
pyranoside (12), 2-azidoethyl 2,3,4-tri-O-acetyl-b-
oside (13) and 2-azidoethyl 2,3,4-tri-O-acetyl-
D
The organic layer was dried over MgSO
under reduced pressure. The crude product was purified by column
4
, filtered and concentrated
a
-D
side (14)
chromatography (silica gel, Hex/EtOAc 70:30) to afford 15, 16 or
1
7.
To a solution of AcOH/H
or 11 (25 mmol, 15.4 g) and the reaction was stirred at 80 °C for 1 h
0 min. After completion of the reaction, the solvent was evapo-
2
O 3:1 (230 mL) was added sugar 9, 10
4.4.1. 2-Azidoethyl 6-deoxy-6-fluoro-2,3,4-tri-O-acetyl-a-D-man
nopyranoside (15)
3
rated and co-evaporated three times with toluene. The crude prod-
uct was purified by chromatography (silica gel, Hex/EtOAc 50:50 to
Yield: 70%; R
CHCl ); IR: film (
H NMR (CDCl , 250 MHz) d 2.02 (s, 3H, CH
.17 (s, 3H, CH
= 4.0 Hz, H8), 3.52 (ddd, 1H, J
3.65–3.73 (m, 1H, H7), 3.91 (ddd, 1H, J7,7
f
0.38 (Hex/EtOAc 60:40); oil; [
m
a
]
D
+42.9 (1.0,
, cm ): 2942, 2107, 1753, 1372, 1247, 1221;
), 2.09 (s, 3H, CH ),
ꢁ
1
3
1
3
0:70) to obtain 12, 13 or 14.
3
3
3
2
3
), 3.45 (ddd, 1H, J8,8
0
= 13.0 Hz, J8,7 = 6.0 Hz,
0
4
.3.1. 2-Azidoethyl 2,3,4-tri-O-acetyl-
a
-
D
-mannopyranoside (12)
+44.8 (1.0,
J
8,7
0
8
0
,7
0
= 7.0 Hz, J
8
0
,7 = 4.0 Hz, H8 ),
0
Yield: 75%; R
CHCl ); IR: film (
NMR (CDCl , 400 MHz) d 1.99 (s, 3H, CH
s, 3H, CH
f
0.15 (Hex/EtOAc 50:50); oil; [
a]
D
0
= 10.5 Hz, H7 ), 4.00–
ꢁ1
1
3
m
, cm ): 3469, 2107, 1752, 1639, 1371;
H
4.12 (m, 1H, H5), 4.42–4.51 (bdd, 2H, JH,F = 47.0 Hz, J6,5 = 4.5 Hz,
2H6), 4.90 (d, 1H, J1,2 = 1.5 Hz, H1), 5.30 (dd, 1H, J2,3 = 3.5 Hz, H2),
3
3
), 2.08 (s, 3H, CH ), 2.14
3
(
3
), 3.35–3.55 (m, 2H, H8), 3.60–3.70 (m, 3H, 2H6, H7),
5.31 (app t, 1H, J3,4 = J4,5 = 10.0 Hz, H4), 5.38 (dd, 1H, H3); 13
C
),
0
3
.80–3.95 (m, 2H, H5, H7 ), 4.89 (d, 1H, J1,2 = 1.5 Hz, H1), 5.25
3 3 3 3
NMR (CDCl , 100.6 MHz) d 21.1 (CH ), 21.1 (CH ), 21.3 (CH
(
app t, 1H, J4,5 = J4,3 = 10.0 Hz, H4), 5.28 (dd, 1H, J2,3 = 3.5 Hz, H2),
50.7 (C-8), 65.9 (d, JC,F = 6.5 Hz, C-4), 67.5 (C-7), 69.2 (C-3), 69.7

6
678
S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
(
(
2
C-2), 70.0 (d, JC,F = 19.0 Hz, C-5), 81.9 (d, JC,F = 176.0 Hz, C-6), 98.1
4.5.2. 2-Azidoethyl 6-deoxy-6-fluoro-b-
Yield: 76%; oil; [
ꢁ92.1 (0.76, H
2924, 2108, 1346, 1288; H NMR (D
D
-glucopyranoside (19)
19
ꢁ1
C-1), 170.2 (C@O), 170.2 (C@O), 170.5 (C@O); F NMR (CDCl
3
,
a]
D
2
O); IR: KBr (
O, 400 MHz) d 3.15–3.25 (m,
m, cm ): 3393,
1
35 MHz) d ꢁ231.6; HRMS m/z (ESI) calcd for C14 FNa
H
20
N
3
O
8
2
+
[
M+Na] 400.1132, found: 400.1131.
1H, H2), 3.38–3.58 (m, 5H, 2H8, H5, H4, H3), 3.71–3.78 (m, 1H,
0
H7), 3.90–3.97 (m, 1H, H7 ), 4.44 (d, 1H, J1,2 = 7.5 Hz, H1), 4.62
1
3
4
.4.2. 2-Azidoethyl 6-deoxy-6-fluoro-2,3,4-tri-O-acetyl-b-
D-gluco
(dd, 2H,
100.6 MHz) d 50.9 (C-8), 68.6 (d, JC,F = 7.0 Hz, C-4), 69.0 (C-7),
73.3 (C-2), 74.8 (d, C,F = 17.5 Hz, C-5), 75.8 (C-3), 82.2 (d,
J
H,F = 47.5 Hz,
J
6,5 = 2.0 Hz, 2H6);
2
C NMR (D O,
pyranoside (16)
Yield: 70%; R
ꢁ29.3 (1.0, CH
f
0.4 (Hex/EtOAc 60:40); white solid; mp = 105 °C;
J
ꢁ1
19
[a
]
D
2
Cl
219, 1038; H NMR (CDCl
H, CH ), 2.04 (s, 3H, CH
2
); IR: film (
, 400 MHz) d 1.99 (s, 3H, CH
), 3.28 (ddd, 1H,
m
, cm ): 3488, 2944, 2106, 1756,
), 2.03 (s,
= 13.5 Hz,
J
C,F = 169.0 Hz, C-6), 102.8 (C-1); F NMR (D
4
-MeOH, 235 MHz) d
1
1
3
3
3
ꢁ235.6 (td, JF,6 = JF,6
0
= 47.5 Hz, JF,5 = 24.0 Hz); HRMS m/z (ESI) calcd
+
0
8 14 3 5
for C H N O FNa [M+Na] 274.0815, found: 274.0827.
3
3
J
8,8
J
8,7
0
= 5.0 Hz, J8,7 = 3.5 Hz, H8), 3.43–3.82 (m, 5H, H5, 2H6, H7,
0
0
H8 ), 4.05 (ddd, 1H, J
7
0
,7 = 10.5 Hz, J
7
0
,8
0
= 3.5 Hz, H7 ), 4.62 (d, 1H,
4.5.3. 2-Azidoethyl 6-deoxy-6-fluoro-
a
-
D
-glucopyranoside (20)
+91.8 (0.2, H O); IR:
O,
= 3.0 Hz,
J
(
1,2 = 8.0 Hz, H1), 5.00 (dd, 1H, J4,3 = 9.5 Hz, J4,5 = 5.0 Hz, H4), 5.02
Yield: 95%; white solid; mp = 70 °C; [
a
]
D
2
13
ꢁ1
1
dd, 1H, J2,3 = 9.5 Hz, H2), 5.24 (app t, 1H, H3); C NMR (CDCl
3
,
KBr (
m
, cm ): 3426, 2933, 2113, 1281, 1026; H NMR (D
2
1
00.6 MHz) d 20.5 (2CH
3
) 20.6 (CH
3
), 50.5 (C-8), 68.0 (d, JC,F
=
400 MHz) d 3.48 (ddd, 1H, J8,8
0
= 13.5 Hz, J8,7 = 6.0 Hz, J8,7
0
6
.9 Hz, C-4), 68.5 (C-7), 71.0 (C-2), 72.6 (C-3), 72.7 (d, JC,F = 19.5 Hz,
H8), 3.53 (dd, 1H, J4,5 = 10.0 Hz, J4,3 = 9.5 Hz, H4), 3.59 (dd, 1H,
0
C-5), 81.3 (d, JC,F = 175.0 Hz, C-6), 100.5 (C-1), 169.3 (C@O), 169.4
J2,3 = 9.5 Hz, J2,1 = 4.0 Hz, H2), 3.58–3.65 (m, 1H, H8 ), 3.73 (ddd,
1
9
(
C@O), 170.2 (C@O); F NMR (CDCl
3
, 235 MHz) d ꢁ231.3 (td,
1H, J7,7
0
= 11.0 Hz, J7,8 = 3.0 Hz, H7), 3.77 (app t, 1H, H3), 3.89
0
JF,6 = JF,6
0
= 47.0 Hz,
J
F,5 = 19.0 Hz); HRMS m/z (ESI) calcd for
(dddd, 1H, JH,F = 29.0 Hz, J5,6
0
= 3.5 Hz, J5,6 = 2.0 Hz, H5), 3.92 (ddd,
+
0
C
14
20
H N
3
O
8
FNa [M+Na] 400.1132, found: 400.1128.
1H, J
7
0
,8
0
= 7.5 Hz, H7 ), 4.70 (ddd, 1H, JH,F = 48.0 Hz, J6,6
H6), 4.76 (ddd, 1H, JH,F = 47.0 Hz, H6 ), 5.00 (d, 1H, H1); C NMR
0
= 11.0 Hz,
0
13
4
.4.3. 2-Azidoethyl 6-deoxy-6-fluoro-2,3,4-tri-O-acetyl-
a-D-gluco
2
(D O, 100.6 MHz) d 50.3 (C-8), 66.8 (C-7), 68.3 (d, JC,F = 7.0 Hz, C-
pyranoside (17)
4), 70.7 (d, JC,F = 17.5 Hz, C-5), 71.1 (C-2), 72.7 (C-3), 82.1 (d,
1
9
Yield: 65%; R
f
0.34 (CycloHex/EtOAc 60:40); white solid;
J
C,F = 168.0 Hz, C-6), 98.4 (C-1); F NMR (D
ꢁ235.2 (td, JF,6 = JF,6 = 48.0 Hz, JF,5 = 29.0 Hz); HRMS m/z (ESI) calcd
for C FNa [M+Na] 274.0815, found: 274.0805.
4
-MeOH, 235 MHz) d
ꢁ
1
mp = 83 °C; [
a
]
D
+128.0 (0.2, CHCl
3
); IR: film (
, 400 MHz) d 2.00
), 3.44 (ddd, 1H,
8,7 = 3.5 Hz, H8), 3.48 (ddd, 1H,
m
, cm ): 2947,
0
1
+
2
109, 1753, 1369, 1223, 1037; H NMR (CDCl
3
8 14 3 5
H N O
(
s, 3H, CH
= 13.5 Hz,
,7 = 7.0 Hz, J
3
), 2.04 (s, 3H, CH
3
), 2.06 (s, 3H, CH
3
J
J
8,8
0
J
8,7
0
= 6.5 Hz,
= 3.5 Hz, H8 ), 3.63 (ddd, 1H, J7,7
J
4.6. General method for the propargylation of peptide
0
0
0
0
0
= 10.5 Hz, H7),
8
8
,7
0
3
.87 (ddd, 1H, H7 ), 4.04 (ddt, 1H, JH,F = 23.0 Hz, J5,4 = 10.0 Hz,
To a solution of commercial peptide (0.075 mmol) in water
(0.25 mL) and MeOH (0.25 mL), was added a 25% solution of NH4-
OH (0.45 mL) at 0 °C. For the propargylation of CQWAVGHLM,
the peptide was diluted in 1.25 mL of water and 1.25 mL of MeOH.
J
5,6 = 3.5 Hz, H5), 4.47 (dd, 2H, JH,F = 47.0 Hz, J
6
0
,5 = J6,5 = 3.5 Hz, H6
0
and H6 ), 4.86 (dd, 1H, J2,3 = 10.0 Hz, J2,1 = 3.5 Hz, H2), 5.04 (app t,
1
H, J4,3 = J4,5 = 10.0 Hz, H4), 5.13 (d, 1H, H1), 5.51 (app t, 1H, H3);
1
3
C NMR (CDCl
3
, 100.6 MHz) d 20.6 (CH
3
), 20.6 (CH
3
), 20.7 (CH
3
),
=
A freshly distilled solution of propargyl bromide (9.9 mg,
5
1
9
4
0.3 (C-8), 67.4 (C-7), 68.1 (d, JC,F = 7.0 Hz, C-4), 68.4 (d, JC,F
0.082 mmol) in MeOH (0.25 mL) was then introduced. The reaction
was stirred during 3 h at room temperature. The solution was con-
centrated under vacuum, water was then added and the solution
was freeze-dried to provide 23, 24, 25 or 26.
9.5 Hz, C-5), 69.9 (C-3), 70.5 (C-2), 81.3 (d, JC,F = 174.5 Hz, C-6),
1
9
5.9 (C-1); F NMR (CDCl
3
, 235 MHz) ꢁ232.2 (td, JF,6 = JF,6
0
=
7.0 Hz, JF,5 = 23.0 Hz); HRMS m/z (ESI) calcd for C14
20 3 8
H N O
FNa
+
[
M+Na] 400.1132, found: 400.1143.
4
.6.1. (
(23)
Yield: 81%; white solid; [
400 MHz) d 2.07 (dd, 2H, J = 15.0 Hz, J = 7.5 Hz, Hb Glu), 2.45 (m,
L-Arginyl glycyl-L-a-aspartyl-2-S-propyn-1-yl)L-cysteine
4
.5. Preparation of 2-azidoethyl 6-deoxy-6-fluoro-
pyranoside (18), 2-azidoethyl 6-deoxy-6-fluoro-b- -glucopyran-
-glucopyrano-
a-D-manno-
D
a]
D
ꢁ22.9 (1.5, H
O); 1H NMR (D
O,
2 2
oside (19) and 2-azidoethyl 6-deoxy-6-fluoro-
a-D
side (20)
2H, H-
c
Glu), 2.60 (app t, 1H, J = 2.5 Hz, H-alkyne), 2.92 (dd, 1H,
0
J = 14.5 Hz, J = 9.0, H-b Cys), 3.18 (dd, 1H, J = 5.0 Hz, H-b Cys),
To a solution of sugar 15, 16 or 17 (1.11 mmol, 420 mg) in 8 mL
of anhydrous methanol, was added a catalytic amount of sodium.
3.29 (m, 2H, H-
c
Cys), 3.70 (m, 3H, H-
a
Glu, H-
O, 100.6 MHz) d 18.9 (C-
Glu), 32.7 (C-b Cys), 43.4 (C- Gly), 52.8 (C-
a Glu), 72.5 (CH-alkyne), 80.3 (C-alkyne), 171.8
a
Gly), 4.65 (dd,
1
3
1H, H-
(C-b Glu), 31.4 (C-
Cys), 54.1 (C-
a
Cys); C NMR (D
2
c
Cys), 26.2
The reaction was stirred during 2 h then neutralized by a resin
c
a
a
Ò
(
Amberlite IR-120). The reaction mixture was filtered of and the
solvent was removed under reduced pressure to give 18, 19 or 20.
(C@O), 173.9 (C@O), 174.9 (C@O), 176.2 (C@O); HRMS m/z (ESI)
ꢁ
calcd for C13
H
18
N
3
O
6
S [MꢁH] 344.0922, found: 344.0925.
4
.5.1. 2-Azidoethyl 6-deoxy-6-fluoro-
Yield: 83%; oil; [ +46.6 (1.0, MeOH); IR: film (
931, 2107, 1443, 1285; H NMR (D
a-D-mannopyranoside (18)
ꢁ1
a]
D
m
, cm ): 3392,
4.6.2. Arg-Gly-Asp-Cys(S-propargyl) (24)
2 2
Yield: 85%; white solid; [ +1.0 (2.0, H O,
O); 1H NMR (D
1
2
2
O, 400 MHz) d 3.49 (ddd, 1H,
a
]
D
J
J
8,8
0
= 13.5 Hz,
= 6.5 Hz, J
J
8,7 = 6.5 Hz,
J
8,7
0
= 3.5 Hz, H8), 3.55 (ddd, 1H,
250 MHz) d 1.68 (m, 2H), 1.92 (m, 2H), 2.55 (dd, 1H, J = 8.0 Hz,
J = 16.0 Hz), 2.62–2.64 (m, 1H, H-alkyne), 2.70 (dd, 1H,
J = 5.0 Hz, J = 16.0 Hz), 2.99 (dd, 1H, J = 14.0, J = 7.0 Hz, Hz),
0
0
0
0
0
,7 = 3.0 Hz, H8 ), 3.72 (ddd, 1H, J7,7
0
= 11.0 Hz, H7 ),
8
,7
8
3
.77–3.83 (m, 1H, H4), 3.79–3.88 (m, 1H, H5), 3.84–3.89 (m, 1H,
H3), 3.91 (ddd, 1H, H7), 4.00 (dd, 1H, J2,3 = 3.0 Hz, J1,2 = 1.5 Hz,
3.10–3.35 (m, 5H), 3.75–4.25 (m, 3H), 4.38–4.48 (m, 2H); 13
C
1
3
H2), 4.61–4.84 (m, 2H, 2H6), 4.93 (d, 1H, H1); C NMR (D
2
O,
00.6 MHz) d 50.5 (C-8), 65.7 (d, JC,F = 7.0 Hz, C-4), 68.9 (C-7),
0.2 (C-2), 70.6 (C-3), 71.9 (d, C,F = 17.5 Hz, C-5), 82.6 (d,
NMR (D
2 2 2 2
O, 100.6 MHz) d 18.9 (CH ), 23.4 (CH ), 28.0 (CH ), 33.1
1
7
(CH ), 38.3 (CH
2
2
), 40.4 (CH ), 42.3 (CH
2
2
), 51.6 (CH), 52.8 (CH),
J
54.4 (CH), 72.4 (CH-alkyne), 80.4 (C-alkyne), 156.8 (C@N), 170.2
1
9
JC,F = 168.0 Hz, C-6), 100.5 (C-1);
F NMR (D
2
O, 235 MHz) d
(C@O), 170.7 (C@O), 172.5 (C@O), 176.3 (C@O), 177.8 (C@O);
+
+
ꢁ
234.4; HRMS m/z (ESI) calcd for
74.0815, found: 274.0804.
C
8
H
14
N
3
O
5
FNa [M+Na]
HRMS m/z (ESI) calcd for C18
30
H N
7
O
7
S [M+H] 488.1927, found:
2
488.1955.

S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
6679
ChelexÒ 100 resin (Na form 1 g) was added and the mixture was
stirred until the solution turned colourless and the resin turned
blue. The resin was filtered off and the residue was freezed-dried.
For the glutathione derivatives, the residue was purified on Sepha-
+
4
.6.3. Cyclo(Arg-Gly-Asp-
Yield: 82%; white solid; 1H NMR (D
m, 3H), 1.84–1.95 (m, 1H), 2.57 (dd, 1H, J = 16.0 Hz, J = 7.0 Hz),
.70 (dd, 1H, J = 16.0 Hz, J = 7.5 Hz), 2.70 (s, 1H, H-alkyne), 2.86
dd, 1H, J = 14.0 Hz, J = 9.0 Hz), 2.98 (dd, 1H, J = 14.5 Hz,
J = 5.5 Hz), 3.00–3.06 (m, 1H), 3.08 (dd, 1H, J = 13.5 Hz, J = 7.0 Hz),
D
-Phe-Cys)(S-propargyl) (25)
2
O, 400 MHz) d 1.49–1.77
(
2
(
Ò
dex LH20 with a water/methanol 90:10 mixture as eluent.
3
3
.17 (d, 1H, J = 6.0 Hz), 3.18 (d, 1H, J = 6.0 Hz), 3.18–3.24 (m, 2H),
.51 (d, 1H, J = 15.0 Hz), 4.22 (d, 1H, J = 15.0 Hz), 4.26 (dd, 1H,
4.7.1. N-[(1,1-Dimethylethoxy)carbonyl]-S-[1-[2-[(2,3,4,6-tetra-
O-acetyl-b-
cysteine methyl ester (27)
Yield: 73%; R 0.45 (Hex/EtOAc 20:80); oil; [
CHCl ); IR: film (
400 MHz) d 1.47 (s, 9H, H-Boc), 2.00 (s, 3H, CH
CH ), 2.04 (s, 3H, CH ), 2.11 (s, 3H, CH ), 2.97 (dd, 1H, J = 14.0 Hz,
J = 6.5 Hz), 3.04 (dd, 1H, J = 14.0 Hz, J = 6.0 Hz), 3.71 (ddd, 1H,
J = 10.0 Hz, J = 4.5 Hz, J = 2.5 Hz), 3.78 (s, 3H, OCH ), 3.85 (s, 2H),
D-glucopyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-L-
J = 9.0 Hz, J = 5.5 Hz), 4.38 (dd, 1H, J = 9.0 Hz, J = 5.5 Hz), 4.65 (dd,
H, J = 9.5 Hz, J = 6.5 Hz), 4.70 (app t, 1H, J = 7.0 Hz), 7.28 (bd, 2H,
J = 7.5 Hz, H-Ar), 7.32 (bt, 1H, J = 7.5 Hz, H-Ar), 7.39 (bt, 2H,
1
f
a
]
D
+1.7 (1.0,
, cm ): 3374, 1752, 1712; H NMR (CDCl
), 2.02 (s, 3H,
ꢁ1
1
3
m
3
,
1
3
J = 7.5 Hz, H-Ar); C NMR (DMSO-D
4.5 (CH ), 27.1 (CH ), 31.3 (CH ), 36.5 (CH
CH ), 43.6 (CH ), 50.6 (CH), 52.7 (CH), 54.2 (CH), 55.1 (CH), 72.7
CH-alkyne), 79.8 (C-alkyne), 127.2 (CH-Ar), 128.8 (2CH-Ar),
29.2 (2CH-Ar), 136.0 (Cq-Ar), 156.7 (C@N), 171.2 (C@O), 172.3
C@O), 172.4 (C@O), 172.5 (C@O), 172.6 (C@O), 173.3 (C@O);
6
, 100.6 MHz) d 18.6 (CH
2
),
3
2
(
(
1
(
2
2
2
2
), 37.1 (CH ), 40.5
2
3
3
3
2
2
3
3.96 (ddd, 1H, J = 11.0 Hz, J = 8.0 Hz, J = 3.5 Hz), 4.16 (dd, 1H,
J = 12.5 Hz, J = 2.5 Hz), 4.24 (m, 1H), 4.27 (dd, 1H, J = 12.5 Hz,
J = 4.5 Hz), 4.51 (d, 1H, J = 8.0 Hz), 4.52–4.58 (m, 2H), 4.60 (ddd,
1H, J = 15.0 Hz, J = 8.0 Hz, J = 5.0 Hz), 5.02 (dd, 1H, J = 10.0 Hz,
J = 8.0 Hz), 5.10 (app t, 1H, J = 10.0 Hz), 5.20 (app t, 1H,
+
37 8 7
HRMS m/z (ESI) calcd for C27H N O S [M+H] 617.2500, found:
6
17.2487.
4
.6.4. Cys-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH
2
(S-propargyl)
J = 10.0 Hz), 5.50 (d, 1H, J = 7.5 Hz), 7.57 (s, 1H, H-triazole); 13
C
),
),
(
26)
Yield: 79%; white solid; 1H NMR (D
m, 12H, 4CH ), 1.20 (d, 3H, J = 7.0 Hz, CH
.89–2.10 (m, 5H), 2.13 (s, 3H, CH ), 2.20–2.30 (m, 2H), 2.47–
.67 (m, 2H), 2.70 (s, 1H, H-alkyne), 3.05–3.37 (m, 8H), 3.80–4.08
NMR (CDCl
27.1 (CH ), 28.7 (CH
53.8 (CH), 62.1 (CH ), 68.1 (CH
3
, 100.6 MHz) d 21.0 (2CH
3
), 21.1 (CH
3
), 21.2 (CH
3
2
O, 250 MHz) d 0.82–1.05
), 1.52–1.72 (m, 3H),
2
3
-Boc), 34.5 (CH ), 50.5 (CH
2
2
), 53.0 (OCH
3
(
1
2
3
3
2
2
), 68.6 (CH), 71.3 (CH), 72.4 (CH),
3
72.9 (CH), 80.5 (C-Boc), 101.0 (CH-anomer), 123.7 (CH), 145.3 (C),
155.6 (C@O), 169.7 (C@O), 169.8 (C@O), 170.5 (C@O), 171.0
(
(
7
m, 3H), 4.26 (app t, 1H, J = 6.5 Hz), 4.31–4.51 (m, 4H), 4.64–4.77
m, 2H), 7.14–7.34 (m, 4H, H-Ar), 7.52 (d, 1H, J = 7.5 Hz, H-Ar),
(C@O), 172.0 (C@O); HRMS m/z (ESI) calcd for C28
[M+Na] 713.2310, found: 713.2351.
H
42
N
4
O
14SNa
+
.64 (d, 1H, J = 7.5 Hz, H-Ar), 8.54 (s, 1H, H-Ar); 13C NMR (D
-
6
DMSO, 100.6 MHz) d 14.6 (CH
CH ), 19.2 (CH ), 21.4 (CH ), 23.1 (CH
8.5 (CH), 28.6 (CH ), 29.7 (CH ), 30.6 (CH
CH ), 32.2 (CH ), 42.0 (CH ), 48.2 (CH ), 51.3 (CH), 51.7 (CH),
3
), 17.8 (CH
), 24.1 (CH), 27.8 (CH
), 31.2 (CH ), 31.5
3
), 17.9 (CH
3
), 18.8
4.7.2. N-[(1,1-Dimethylethoxy)carbonyl]-S-[1-[2-[(2,3,4,6-tetra-
O-acetyl- -mannopyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
-cysteine methyl ester (28)
(
2
(
3
2
3
3
2
),
a-D
2
2
2
2
L
2
2
2
2
Yield: 90%; R
CHCl ); IR: film (
f
0.46 (Hex/EtOAc 20:80); oil; [
a
]
D
+19.0 (1.0,
3
, 400 MHz)
ꢁ1
1
5
7
1
1.7 (CH), 52.1 (CH), 52.2 (CH), 52.3 (CH), 53.1 (CH), 57.6 (CH),
4.2 (CH-alkyne), 80.1 (C-alkyne), 109.8 (Cq-Ar), 111.2 (CH-Ar),
18.2 (CH-Ar), 118.5 (CH-Ar), 120.8 (CH-Ar), 123.6 (CH-Ar), 123.7
3
m
, cm ): 3370, 1748; H NMR (CDCl
), 2.06 (s, 3H, CH
), 2.97 (dd, 1H, J = 14.0 Hz, J = 7.5 Hz),
3.03 (dd, 1H, J = 14.0 Hz, J = 6.5 Hz), 3.60 (ddd, 1H, J = 10.0 Hz,
J = 5.0 Hz, J = 2.5 Hz), 3.78 (s, 3H, OCH ), 3.86 (s, 2H), 3.84–3.94
d 1.46 (s, 9H, H-Boc), 2.02 (s, 3H, CH
3
3
), 2.11 (s,
3H, CH ), 2.16 (s, 3H, CH
3
3
(
(
CH-Ar), 127.3 (Cq-Ar), 134.3 (2Cq-Ar), 136.0 (CH-Ar), 167.0
C@O), 168.7 (C@O), 170.3 (C@O), 170.5 (C@O), 171.1 (C@O),
3
1
71.3 (C@O), 172.1 (C@O), 172.2 (C@O), 173.1 (C@O), 173.7
(m, 1H), 4.05 (dd, 1H, J = 12.5 Hz, J = 2.5 Hz), 4.13–4.17 (m, 1H),
4.24 (dd, 1H, J = 12.5 Hz, J = 5.0 Hz), 4.53–4.65 (m, 3H), 4.80 (d,
1H, J = 1.0 Hz), 5.20 (dd, 1H, J = 3.0 Hz, J = 1.0 Hz), 5.22 (dd, 1H,
J = 10.0 Hz, J = 3.0 Hz), 5.26 (app t, 1H, J = 1.0 Hz), 5.53 (d, 1H,
+
(
C@O); HRMS m/z (ESI) calcd for
C
49
H
73
N
14
O
10
S
2
[M+H]
1
081.5070, found: 1081.5033.
1
3
4
.7. General method for the preparation of glycopeptides with
J = 8.0 Hz), 7.65 (s, 1H, H-triazole); C NMR (CDCl
21.1 (3CH ), 21.2 (CH ), 27.0 (CH ), 28.7 (CH -Boc), 34.4 (CH
0.1 (CH ), 53.0 (OCH ), 53.8 (CH), 62.6 (CH ), 66.1 (CH), 66.6
(CH ), 69.2 (CH), 69.5 (CH), 69.6 (CH), 80.5 (C-Boc), 97.9 (CH-ano-
3
, 100.6 MHz) d
acetylated carbohydrates
3
3
2
3
2
),
5
2
3
2
General procedure 1: Click reaction between protected cysteine
2
2
1 and acetylated sugars.
To a solution of the azido glycoside (100 mg, 0.12 mmol) and the
mer), 123.6 (CH-triazole), 145.7 (C-triazole), 155.6 (C@O), 170.0
(C@O), 170.4 (C@O), 170.4 (C@O), 171.0 (C@O), 172.0 (C@O);
42 4
HRMS m/z (ESI) calcd for C28H N
NaO14S [M+Na]+ 713.2310,
S-propargyl cysteine 21 (32 mg, 0.12 mmol) in a mixture of tBuOH/
O (1:1 1 mL) was added Cu(OAc) O (2.3 mg, 0.012 mmol) and
H
2
ꢂH
2 2
found: 713.2314.
sodium ascorbate (4.6 mg, 0.024 mmol). The mixture was vigor-
ously stirred at room temperature for 1 or 2 h. The reaction mixture
was then diluted with ethyl acetate (100 mL) and washed with a
saturated aqueous NaCl solution (2 ꢀ 5 mL), the organic layer was
4.7.3. N-[(1,1-Dimethylethoxy)carbonyl]-S-[1-[2-[(2,3,4,6-tetra-
O-acetyl-b- -galactopyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
-cysteine methyl ester (29)
Yield: 73%; R 0.43 (Hex/EtOAc 20:80); oil; [
CHCl ); IR: film (
400 MHz) d 1.47 (s, 9H, H-Boc), 2.00 (s, 6H, 2CH
CH ), 2.18 (s, 3H, CH ), 2.99 (dd, 1H, J = 14.5 Hz, J = 6.5 Hz), 3.05
(dd, 1H, J = 14.5 Hz, J = 5.0 Hz), 3.78 (s, 3H, OCH ), 3.85 (s, 2H),
D
L
dried over Na
2
SO
4
, filtered and concentrated under reduced pres-
f
a
]
D
ꢁ2.6 (1.0,
, cm ): 3374, 1752, 1712; H NMR (D O,
), 2.07 (s, 3H,
ꢁ1
1
sure. The crude product was purified by silica gel chromatography
3
m
2
(
80:20 Hex–EtOAc) to afford the pure compounds.
3
General procedure 2: Click reaction between free S-propargyl
3
3
derivatives (22 and 23) and acetylated sugars.
3
To a solution of the azido glycoside (100 mg; 0.12 mmol), the
propargyl derivative (0.12 mmol) and sodium ascorbate (4.6 mg,
3.90–3.99 (m, 2H), 4.13 (dd, 1H, J = 12.0 Hz, J = 7.0 Hz), 4.19 (dd,
1H, J = 12.0 Hz, J = 7.0 Hz), 4.26 (app dt, 1H, J = 11.0 Hz, J = 4.0 Hz),
4.47 (d, 1H, J = 8.0 Hz), 4.49–4.58 (m, 2H), 4.62 (app dt, 1H,
J = 15.0 Hz, J = 4.0 Hz), 5.01 (dd, 1H, J = 10.5 Hz, J = 2.5 Hz), 5.21
(dd, 1H, J = 8.0 Hz, J = 2.5 Hz), 5.41 (bd, 1H, J = 10.5 Hz), 5.51 (d,
0
2
.024 mmol) in a mixture of tBuOH/H O (1:1 1 mL) was added
Cu(OAc) O (2.3 mg, 0.012 mmol). The mixture immediately
ꢂH
2 2
turned to a light green cloudy solution. The mixture was vigorously
stirred at room temperature whereas the solution became limpid
and blue coloured. TLC showed disappearance of the starting sugar.
1
3
1H, J = 7.5 Hz), 7.58 (s, 1H, H-triazole);
100.6 MHz) d 20.9 (CH ), 21.1 (2CH ), 21.2 (CH
C
NMR (D
2
O,
3
3
3
), 27.1 (CH ), 28.7
2

6
680
S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
(
(
(
1
1
7
CH
3
-Boc), 34.6 (CH
), 67.3 (CH), 67.9 (CH
2
), 50.5 (CH
2
), 53.0 (OCH
3
), 53.8 (CH), 61.6
J = 2.0 Hz), 4.29 (m, 1H), 4.36 (dd, 1H, J = 12.5 Hz, J = 3.5 Hz), 4.55
(dd, 1H, J = 9.0 Hz, J = 5.0 Hz), 4.63 (m, 2H), 4.75 (d, 1H, J = 8.0 Hz),
4.89 (dd, 1H, J = 9.5 Hz, J = 8.0 Hz), 5.07 (app t, 1H, J = 10.0 Hz), 5.28
(dd, 1H, J = 10.0 Hz, J = 9.5 Hz), 7.97 (s, 1H, H-triazole); 13C NMR
CH
2
2
), 68.9 (CH), 71.0 (CH), 71.3 (CH), 80.5
C-Boc), 101.4 (CH-anomer), 123.7 (CH-triazole), 145.3 (C-triazole),
55.6 (C@O), 169.9 (C@O), 170.4 (C@O), 170.5 (C@O), 170.8 (C@O),
72.0 (C@O); HRMS m/z (ESI) calcd for C28
13.2310, found: 713.2336.
+
H
42
N
4
NaO14S [M+Na]
(D
25.6 (CH
CH ), 53.1 (CH), 54.5 (CH), 62.1 (CH
2
O, 100.6 MHz) d 20.4 (CH
3
), 20.4 (CH
), 33.1 (CH
), 68.4 (CH
2
3
), 20.5 (CH
), 43.7 (CH
), 68.5 (CH), 71.4
3
), 20.5 (CH
3
),
2
), 26.6 (CH ), 31.8 (CH
2
2
2
2
), 50.4
(
2
2
4
.7.4. S-[1-[2-[(2,3,4,6-Tetra-O-acetyl-b-
-cysteine (30)
ꢁ8.0 (1.1, H
), 2.03 (s, 3H, CH
), 2.99 (dd, 1H, J = 15.0 Hz, J = 7.5 Hz), 3.08 (dd,
D
-glucopyranosyl)oxy]-
(CH), 71.6 (CH), 73.2 (CH), 100.3 (CH-anomer), 124.7 (CH-triazole),
145.0 (C-triazole), 172.1 (C@O), 172.7 (C@O), 173.1 (C@O), 173.4
ethyl]-1H-1,2,3-triazol-4-yl]-
Yield: 70%; white solid; [
00 MHz) d 1.97 (s, 3H, CH
.12 (s, 3H, CH
L
a
]
D
2
O); 1H NMR (D
), 2.07 (s, 3H, CH
2
O,
),
(C@O), 174.1 (C@O), 174.3 (C@O), 175.2 (C@O), 176.5 (C@O); HRMS
+
4
2
1
4
1
3
3
3
m/z (ESI) calcd for C29
H
43
N
6
O
16S [M+H] 763.2451, found: 763.2454.
3
H, J = 15.0 Hz, J = 4.5 Hz), 3.91 (s, 2H), 3.90–3.95 (m, 1H), 4.00–
.05 (m, 1H), 4.13 (dd, 1H, J = 13.0 Hz, J = 2.0 Hz), 4.15–4.20 (m,
H), 4.28–4.32 (m, 1H), 4.36 (dd, 1H, J = 13.0 Hz, J = 4.0 Hz), 4.62
4.7.8.
L
-
c
-Glutamyl-S-[1-[2-[(2,3,4,6-tetra-O-acetyl-
a
-
D
-manno-
pyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
L-cysteinyl-glycine
(34)
(
5
m, 2H), 4.77 (d, 1H, J = 8.0 Hz), 4.87 (dd, 1H, J = 9.5 Hz, J = 8.0 Hz),
.07 (app t, 1H, J = 9.5 Hz), 5.28 (app t, 1H, J = 9.5 Hz), 7.98 (s, 1H,
Yield: 85%; white solid; [
250 MHz) d 1.91 (s, 3H, CH ), 1.98 (s, 3H, CH
2.06 (s, 3H, CH ), 2.40–2.45 (m, 2H), 2.75 (dd, 1H, J = 14.0 Hz,
a]
D
+6.7 (0.2, H
2
O); 1H NMR (D
), 2.01 (s, 3H, CH
2
O,
3
3
3
),
1
3
H-triazole); C NMR (D
0.1 (2CH ), 25.0 (CH ), 31.8 (CH
CH ), 67.9 (CH ), 68.2 (CH), 71.1 (CH), 71.3 (CH), 73.0 (CH), 99.9
CH-anomer), 124.6 (CH-triazole), 144.2 (C-triazole), 172.4 (C@O),
72.6 (C@O), 172.7 (C@O), 173.1 (C@O), 173.9 (C@O); HRMS m/z
2
O, 100.6 MHz) d 20.0 (CH
3
), 20.0 (CH
3
),
3
2
(
(
3
2
2
), 50.1 (CH ), 53.4 (CH), 61.8
2
J = 9.0 Hz), 2.95–3.00 (m, 2H), 3.10–3.17 (m, 2H), 3.60–4.20 (m,
8H), 4.14 (dd, 1H, J = 12.5 Hz, J = 4.0 Hz), 4.45 (dd, 1H, J = 9.0 Hz,
J = 5.0 Hz), 4.60 (m, 2H), 4.82 (d, 1H, J = 1.5 Hz), 4.94–5.17 (m,
2
2
1
3
1
3H), 8.01 (s, 1H, H-triazole); C NMR (D
(3CH ), 20.4 (CH ), 25.4 (CH ), 26.4 (CH ), 31.6 (CH
3.6 (CH ), 50.1 (CH ), 53.0 (CH), 54.3 (CH), 62.0 (CH
66.0 (CH ), 68.2 (CH), 69.4 (CH), 69.8 (CH), 96.7 (CH-anomer),
2
O, 62.9 MHz) d 20.3
), 33.2 (CH ),
), 65.6 (CH),
+
(
ESI) calcd for C22
33
H N
4
O
12S [M+H] 577.1810, found: 577.1847.
3
3
2
2
2
2
4
2
2
2
4
.7.5. S-[1-[2-[(2,3,4,6-Tetra-O-acetyl-
-cysteine (31)
+26.8 (0.74, H
), 2.11 (s, 3H, CH ), 2.13 (s, 3H, CH
), 3.03 (dd, 1H, J = 15.0 Hz, J = 7.5 Hz), 3.13 (dd,
a-D-mannopyranosyl)oxy]-
2
ethyl]-1H-1,2,3-triazol-4-yl]-
Yield: 75%; white solid; [
00 MHz) d 2.04 (s, 3H, CH
.19 (s, 3H, CH
L
125.3 (CH-triazole), 145.2 (C-triazole), 171.9 (C@O), 172.8 (C@O),
172.9 (C@O), 173.1 (C@O), 173.7 (C@O), 174.1 (C@O), 175.0
a]
D
2
O); 1H NMR (D
2
O,
),
4
2
1
1
4
1
1
3
3
3
(C@O), 176.3 (C@O); HRMS m/z (ESI) calcd for C29
[M+H] 763.2451, found: 763.2474.
H
43
N
6
NaO16
S
+
3
H, J = 15.0 Hz, J = 4.0 Hz), 3.20 (m, 1H), 3.95 (s, 2H), 3.96 (dd,
H, J = 7.5 Hz, J = 4.0 Hz), 3.98–4.06 (m, 2H), 4.10–4.13 (m, 1H),
.27 (dd, 1H, J = 12.5 Hz, J = 4.0 Hz), 4.70–4.75 (m, 2H), 4.95 (d,
H, J = 1.5 Hz), 5.11 (dd, 1H, J = 10.0 Hz, J = 3.5 Hz), 5.21 (app t,
H, J = 10.0 Hz), 5.26 (dd, 1H, J = 3.5 Hz, J = 1.5 Hz), 8.15 (s, 1H, H-
4.7.9.
L
-
c
-Glutamyl-S-[1-[2-[(2,3,4,6-tetra-O-acetyl-b-
D
-galacto-
pyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
L-cysteinyl-glycine
(35)
Yield: 60%; white solid; [
400 MHz) d 1.97 (s, 3H, CH ), 1.98 (s, 3H, CH
2.14–2.30 (m, 2H), 2.19 (s, 3H, CH ), 2.50–2.65 (m, 2H), 2.88 (m,
a]
D
ꢁ14.0 (1.0, H
2
O); 1H NMR (D
), 2.08 (s, 3H, CH
2
O,
1
3
triazole); C NMR (D
5.3 (CH ), 32.4 (CH
CH), 66.2 (CH ), 68.4 (CH), 69.5 (CH), 69.9 (CH), 96.9 (CH-anomer),
25.6 (CH-triazole), 145.0 (C-triazole), 172.9 (C@O), 173.0 (C@O),
73.1 (C@O), 173.3 (C@O), 173.9 (C@O); HRMS m/z (ESI) calcd for
2
O, 100.6 MHz) d 20.1 (2CH
3
), 20.2 (2CH
3
),
3
3
3
),
2
(
1
1
C
2
2
), 50.3 (CH ), 53.7 (CH), 62.1 (CH
2
2
), 65.7
3
2
1H), 3.02 (m, 1H), 3.93 (s, 2H), 4.01 (s, 2H), 4.08 (m, 1H), 4.13
(m, 1H), 4.19 (s, 3H), 4.25–4.30 (m, 1H), 4.55–4.58 (m, 2H), 4.60–
4.65 (m, 1H), 4.73 (d, 1H, J = 8.0 Hz), 4.97 (dd, 1H, J = 10.0 Hz,
J = 8.0 Hz), 5.16 (app t, 1H, J = 10.0 Hz), 5.42 (d, 1H, J = 10.0 Hz),
+
22
33 4
H N NaO12S [M+H] 577.1810, found: 577.1855.
1
3
7
.99 (s, 1H, H-triazole); C NMR (D
20.5 (CH ), 20.6 (CH ), 25.8 (CH ), 26.0 (CH
(CH ), 41.5 (CH ), 50.7 (CH ), 52.7 (CH), 53.0 (CH), 62.2 (CH
68.1 (CH ), 68.3 (CH), 69.6 (CH), 70.9 (CH), 71.3 (CH), 100.7 (CH-
2
O, 100.6 MHz) d 20.3 (2CH
), 31.3 (CH ), 33.3
),
3
),
4
.7.6. S-[1-[2-[(2,3,4,6-Tetra-O-acetyl-b-
-cysteine (32)
ꢁ5.0 (0.4, H
), 1.98 (s, 3H, CH
), 2.87–3.16 (m, 2H), 3.85–3.91 (m, 1H), 3.91 (s,
D-galactopyranosyl)oxy]-
3
3
2
2
2
ethyl]-1H-1,2,3-triazol-4-yl]-
Yield: 62%; white solid; [
00 MHz) d 1.96 (s, 3H, CH
.20 (s, 3H, CH
L
2
2
2
2
a]
D
2
O); 1H NMR (D
), 2.09 (s, 3H, CH
2
O,
),
2
4
2
2
4
2
5
3
3
3
anomer), 125.2 (CH-triazole), 144.7 (C-triazole), 172.9 (C@O),
172.9 (C@O), 173.0 (C@O), 173.2 (2C@O), 173.6 (C@O), 173.9
3
H), 4.12 (ddd, 1H, J = 11.0 Hz, J = 8.0 Hz, J = 4.0 Hz), 4.20 (s, 3H),
.30 (ddd, 1H, J = 11.0 Hz, J = 8.0 Hz, J = 4.0 Hz), 4.60–4.65 (m,
H), 4.74 (d, 1H, J = 8.0 Hz), 4.98 (dd, 1H, J = 10.0, Hz, J = 8.0 Hz),
.16 (dd, 1H, J = 10.0 Hz, J = 3.0 Hz), 5.43 (d, 1H, J = 3.0 Hz), 7.97
(C@O), 174.6 (C@O); HRMS m/z (ESI) calcd for C29
[M+H] 763.2451, found: 763.2510.
H
43
N
6
O
16
S
+
4.8. General method for the preparation of glycopeptides with
free carbohydrates
1
3
(
(
6
1
s, 1H, H-triazole); C NMR (D
CH ), 20.6 (CH ), 25.4 (CH ), 32.2 (CH
2.2 (CH ), 68.2 (CH ), 68.3 (CH), 69.6 (CH), 70.9 (CH), 71.3 (CH),
00.5 (CH-anomer), 125.0 (CH-triazole), 144.6 (C-triazole), 173.0
2
3
O, 100.6 MHz) d 20.3 (2CH ), 20.5
3
3
2
2
), 50.5 (CH ), 53.7 (CH),
2
2
2
To a solution of S-propargyl derivative (0.1 mmol), of the azido
sugar (0.1 mmol) and sodium ascorbate (4 mg, 0.02 mmol) in
water (2 mL) was added, at room temperature a solution of
(
2C@O), 173.0 (C@O), 173.6 (C@O), 173.8 (C@O); HRMS m/z (ESI)
+
calcd for C22
H
33
N
4
O
12S [M+H] 577.1810, found: 577.1857.
2
Cu(OAc) (2 mg, 0.01 mmol) in water (0.3 mL). The solution turned
immediately pale green. The reaction was stopped when the
intense blue color reappeared. Chelex resin (250 mg) was then
added to the blue solution and the suspension was stirred until
the solution turned colorless. The resin was filtered off and the
resulting solution was freeze-dried. Purification was achieved by
LH20 gel filtration. Elution with water provided pure triazole.
For c(RGDfC) and CQWAVGHLM only 1 mg of these peptides
was introduced, proportion of other reactive was respected. In
the case of CQWAVGHLM the reaction proceeded in 2 mL of a
water/MeOH (50:50) solution.
4
.7.7.
anosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
Yield: 83%; white solid; [
00 MHz) d 1.97 (s, 3H, CH ), 2.05 (s, 3H, CH
.26 (s, 3H, CH ), 2.50–2.55 (m, 2H), 2.85 (dd, 1H, J = 14.0 Hz,
L
-
c
-Glutamyl-S-[1-[2-[(2,3,4,6-tetra-O-acetyl-b-
-cysteinyl-glycine (33)
ꢁ11.0 (1.1, H O,
O); 1H NMR (D
), 2.12 (s, 3H, CH ),
D-glucopyr-
L
a]
D
2
2
4
2
3
3
3
3
J = 9.0 Hz), 3.04 (dd, 1H, J = 14.0 Hz, J = 5.0 Hz), 3.72 (d, 1H,
J = 17.0 Hz), 3.75–3.80 (m, 1H), 3.80 (d, 1H, J = 17.0 Hz), 3.89 (s,
2
H), 4.01 (ddd, 1H, J = 10.0 Hz, J = 3.5 Hz, J = 2.0 Hz), 4.12 (ddd, 1H,
J = 11.5 Hz, J = 7.0 Hz, J = 4.5 Hz), 4.18 (dd, 1H, J = 12.5 Hz,

S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
6681
4
1
.8.1.
,2,3-triazol-4-yl]-
Yield: 66%; white solid; [
00 MHz) d 2.15–2.20 (m, 2H), 2.50–2.55 (m, 2H), 2.88 (dd, 1H,
L
-
c
-Glutamyl-S-[1-[2-[(b-
-cysteinyl-glycine (36)
ꢁ20.4 (0.4, H
D
-glucopyranosyl)oxy]ethyl]-1H-
1H), 3.70–3.80 (m, 3H), 3.81–3.89 (m, 3H), 3.92–3.99 (m, 4H),
4.00–4.06 (m, 1H), 4.13–4.20 (m, 1H), 4.53–4.75 (m, 3H), 4.88
L
13
a
]
D
2
O); 1H NMR (D
2
O,
(bs, 1H), 8.10 (s, 1H, H-triazole); C NMR (D
2
O, 100.6 MHz) d
), 43.7 (CH ), 50.5
), 53.2 (CH), 54.6 (CH), 65.6 (d, JC,F = 7.3 Hz, CH), 66.2 (CH ),
70.2 (CH), 70.7 (CH), 71.9 (d, C,F = 17.5 Hz, CH), 82.6 (d,
C,F = 167.8 Hz, CH ), 100.2 (CH-anomer), 125.1 (CH-triazole),
145.0 (C-triazole), 172.2 (C@O), 174.3 (C@O), 175.3 (C@O), 176.4
4
25.6 (CH
(CH
2
2
), 26.6 (CH ), 31.8 (CH
2
), 33.2 (CH
2
2
J = 14.0 Hz, J = 8.0 Hz), 3.08 (dd, 1H, J = 14.0 Hz, J = 5.0 Hz), 3.27
dd, 1H, J = 14.0 Hz, J = 8.5 Hz), 3.36–3.55 (m, 3H), 3.69 (dd, 1H,
J = 13.0 Hz, J = 6.0 Hz), 3.75–3.95 (m, 6H), 4.15 (m, 1H), 4.33 (m,
H), 4.45 (d, 1H, J = 8.0 Hz, H-anomer), 4.56 (dd, 1H, J = 9.0 Hz,
2
2
(
J
J
2
2
1
3
19
J = 5.0 Hz), 4.70–4.75 (m, 1H), 8.08 (s, 1H, H-triazole); C NMR
O, 100.6 MHz) d 25.6 (CH ), 26.5 (CH ), 31.7 (CH ), 33.1 (CH ),
3.7 (CH ), 50.7 (CH ), 53.1 (CH), 54.4 (CH), 61.0 (CH ), 68.4
CH ), 69.9 (CH), 73.3 (CH), 75.9 (CH), 76.2 (CH), 102.7 (CH-ano-
mer), 125.1 (CH-triazole), 145.1 (C-triazole), 172.2 (C@O), 174.3
C@O), 175.2 (C@O), 176.6 (C@O); HRMS m/z (ESI) calcd for
(C@O); F NMR (D
2
O, 235 MHz) d ꢁ234.0; HRMS m/z (ESI) calcd
(
D
2
2
2
2
2
for C21 11S [M+H]+ 597.1990, found: 597.2012.
6
H34FN O
4
(
2
2
2
2
4.8.6.
syl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
Yield: 55%; white solid; [
ꢁ18.3 (0.5, H2O); H NMR (D
400 MHz) d 2.21 (q, 2H, J = 7.0 Hz), 2.54–2.60 (m, 2H), 2.91 (dd,
H, J = 15.0 Hz, J = 8.0 Hz), 3.18 (dd, 1H, J = 15.0 Hz, J = 4.5 Hz),
L
-c-Glutamyl-S-[1-[2-[(6-deoxy-6-fluoro-b-
D-glucopyrano-
L
-cysteinyl-glycine (41)
1
(
C
a
]
D
2
O,
+
21
H
33
N
6
Na
2
O
12S [MꢁH+2Na] 639.1673, found: 639.1672.
1
4
1
.8.2.
L
-
c
-Glutamyl-S-[1-[2-[( -mannopyranosyl)oxy]ethyl]-
a
-
D
3.28–3.33 (m, 1H), 3.48–3.68 (m, 4H), 3.65–3.85 (m, 3H), 3.91–
3.98 (m, 2H), 4.16–4.25 (m, 1H), 4.30–4.40 (m, 1H), 4.49 (d, 1H,
H-1,2,3-triazol-4-yl]- -cysteinyl-glycine (37)
Yield: 62%; white solid; [
L
a
]
D
ꢁ3.5 (0.4, H
2
O); 1H NMR (D
2
O,
J = 7.8 Hz), 4.56–4.62 (m, 1H), 4.72 (dd, 2H,
J = 2.0 Hz), 4.70–4.74 (m, 1H), 8.09 (s, 1H, H-triazole); C NMR
(D O, 100.6 MHz) d 25.7 (CH ), 26.6 (CH ), 31.8 (CH ), 33.2 (CH ),
50.8 (CH and CH), 53.2 (CH and CH), 68.7 (CH and CH), 73.3
(CH), 74.8 (d, JC,F = 17.4 Hz, CH), 75.8 (CH), 82.3 (d, JC,F = 165.5 Hz,
CH ), 102.9 (CH-anomer), 125.4 (CH-triazole), 145.9 (C-triazole),
172.2 (C@O), 174.3 (C@O), 175.3 (C@O), 176.5 (C@O); F NMR
(D
O, 235 MHz) d ꢁ235.2 (td, J = 47.0 Hz, J = 26.0 Hz); HRMS m/z
(ESI) calcd for C21
JH,F = 47.5 Hz,
13
4
1
1
4
00 MHz) d 2.10–2.15 (m, 2H), 2.48–2.53 (m, 2H), 2.82–2.86 (m,
H), 3.02–3.07 (m, 2H), 3.59 (app t, 1H, J = 9.5 Hz), 3.61–4.00 (m,
H), 4.10–4.15 (m, 1H), 4.54 (dd, 1H, J = 9.0 Hz, J = 5.0 Hz), 4.62–
O, 100.6 MHz)
), 43.7 (CH ), 50.5
), 65.8 (CH ), 66.7 (CH), 70.2
CH), 70.7 (CH), 73.1 (CH), 99.9 (CH-anomer), 125.0 (CH-triazole),
2
2
2
2
2
2
2
2
.67 (m, 2H), 8.03 (s, 1H, H-triazole); 13C NMR (D
2
d 25.6 (CH
2
), 27.1 (CH
2
), 31.8 (CH
2
), 33.1 (CH
2
2
2
19
(
(
CH
2
), 53.1 (CH), 54.6 (CH), 61.0 (CH
2
2
2
+
1
45.3 (C-triazole), 172.2 (C@O), 175.3 (C@O), 175.4 (C@O), 176.6
6
H34FN O11S [M+H] 597.1990, found: 597.2005.
ꢁ
(
C@O); HRMS m/z (ESI) calcd for
C
21
H
33
N
6
O
12
S
[MꢁH]
5
93.1883, found: 593.1905.
4.8.7.
osyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
2 2
Yield: 69%; white solid; [ +18.7 (0.05, H O); H NMR (D O,
L-
c-Glutamyl-S-[1-[2-[(6-deoxy-6-fluoro-
a-D-glucopyran-
L
-cysteinyl-glycine (42)
1
4
1
.8.3.
H-1,2,3-triazol-4-yl]-
Yield: 55%; white solid; [
00 MHz) d 2.10–2.15 (m, 2H), 2.50–2.55 (m, 2H), 2.84 (dd, 1H,
L
-c
-Glutamyl-S-[1-[2-[(
-cysteinyl-glycine (38)
ꢁ14.2 (0.6, H
a
-
D
-galactopyranosyl)oxy]ethyl]-
O); 1H NMR (D
O,
2
a]
D
L
400 MHz) d 2.21 (dd, 2H, J = 14.0 Hz, J = 7.5 Hz), 2.50–2.56 (m,
2H), 2.82–3.11 (m, 3H), 3.41 (dd, 1H, J = 10.0 Hz, J = 9.0 Hz), 3.52
(dd, 1H, J = 10.0 Hz, J = 3.5 Hz), 3.58 (app t, 1H, J = 9 Hz), 3.73–
3.84 (m, 3H), 3.87–3.93 (m, 2H), 4.00 (app dt, 1H, J = 11.0 Hz,
J = 3.5 Hz), 4.12 (ddd, 1H, J = 11.5 Hz, J = 8.0 Hz, J = 4.0 Hz), 4.39–
a]
D
2
4
J = 14.0 Hz, J = 8.0 Hz), 3.05 (dd, 1H, J = 14.0 Hz, J = 5.0 Hz), 3.48
dd, 1H, J = 9.0 Hz, J = 8.0 Hz), 3.60–3.80 (m, 8H), 3.88–3.93 (m,
H), 4.10–4.15 (m, 1H), 4.25–4.30 (m, 1H), 4.35 (d, 1H, J = 8.0 Hz,
H-anomer), 4.51 (dd, 1H, J = 9.0 Hz, J = 5.0 Hz), 4.70–4.75 (m, 1H),
(
3
4.76 (m, 5H), 4.92 (d, 1H, J = 3.5 Hz), 8.08 (s, 1H, H-triazole); 13
C
NMR (D
(CH ), 50.1 (CH
C,F = 6.5 Hz, CH), 70.6 (d, JC,F = 17.5 Hz, CH), 71.0 (CH), 72.6 (CH),
82.0 (d, JC,F = 167.0 Hz, CH ), 98.1 (CH-anomer), 129.7 (CH-tria-
zole), 145.3 (C-triazole), 171.9 (2C@O), 174.8 (2C@O); F NMR
(D
O, 235 MHz) d ꢁ235.2 (td, J = 47.5 Hz, J = 28.5 Hz); HRMS m/z
(ESI) calcd for C21
2
O, 100.6 MHz) d 25.2 (CH
2
2 2
), 26.1 (CH ), 31.3 (CH ), 32.8
8
2
.05 (s, 1H, H-triazole); 13C NMR (D
6.9 (CH ), 31.8 (CH ), 33.1 (CH ), 43.7 (CH
), 68.4 (CH ), 68.9 (CH), 70.9 (CH), 73.0
), 103.4 (CH-anomer), 125.2 (CH-triazole), 145.1
C-triazole), 172.2 (C@O), 175.0 (C@O), 175.3 (C@O), 176.6
2
O, 100.6 MHz) d 25.6 (CH
2
),
2
2
and CH), 52.8 (CH
2
), 66.2 (CH
2
and CH), 68.1 (d,
2
2
2
2
), 50.7 (CH ), 53.1
2
J
(
(
(
(
CH), 54.6 (CH), 61.3 (CH
CH), 75.5 (CH
2
2
2
19
2
2
2
ꢁ
+
C@O); HRMS m/z (ESI) calcd for
C
21
H
32
N
6
O
12
S
[Mꢁ2H]
6
H34FN O11S [M+H] 597.1990, found: 597.2004.
2
96.0905, found: 296.0920.
4
.8.8. Arg-Gly-Asp-Cys-S-[[1-[2-[(b-
1H-1,2,3-triazol-4-yl]methyl] (43)
Yield: 71%; white solid; [
D
-glucopyranosyl)oxy]ethyl]-
O); 1H NMR (D
O,
4
1
.8.4.
,2,3-triazol-4-yl]-
Yield: 67%; white solid; H NMR (D
J = 14.0 Hz, J = 7.5 Hz), 2.50–2.55 (m, 2H), 2.85–2.94 (m, 1H), 3.03–
L
-
c
-Glutamyl-S-[1-[2-[(
a-D-glucopyranosyl)oxy]ethyl]-1H-
L-cysteinyl-glycine (39)
a]
D
ꢁ9.6 (0.5, H
2
2
1
2
O, 250 MHz) d 2.16 (dd, 2H,
400 MHz) d 1.58–1.70 (m, 2H), 1.85–1.97 (m, 2H), 2.49–2.61 (m,
1H), 2.62–3.00 (m, 3H), 3.11–3.19 (m, 3H), 3.25–3.40 (m, 3H),
3.61 (dd, 1H, J = 12.5 Hz, J = 5.5 Hz), 3.80 (dd, 2H, J = 12.5 Hz,
J = 2.0 Hz), 3.88 (d, 2H, J = 16.5 Hz), 4.01–4.10 (m, 3H), 4.20–4.30
3
.10 (m, 1H), 3.34 (app t, 1H, J = 9.5 Hz), 3.47–3.60 (m, 3H), 3.62–
3
.67 (m, 1H), 3.75–3.80 (m, 3H), 3.87–4.00 (m, 3H), 4.08–4.16
(
2
m, 1H), 4.26–4.36 (m, 1H), 4.50–4.58 (m, 1H), 4.64–4.74 (m,
(m, 2H), 4.34 (d, 1H, J = 8.0 Hz), 4.53–4.62 (m, 3H), 8.03 (s, 1H, H-
1
3
13
H), 4.88 (d, 1H, J = 3.5 Hz), 8.08 (s, 1H, H-triazole); C NMR
O, 100.6 MHz) d 25.2 (CH ), 26.1 (CH ), 31.3 (CH ), 32.8 (CH ),
5.1 (CH ), 50.1 (CH ), 52.8 (CH), 54.6 (CH), 60.1 (CH ), 65.9
), 69.1 (CH), 71.1 (CH), 71.8 (CH), 72.8 (CH), 98.0 (CH-anomer),
24.9 (CH-triazole), 145.5 (C-triazole), 171.9 (C@O), 173.3 (C@O),
74.8 (C@O), 176.3 (C@O); HRMS m/z (ESI) calcd for C21
triazole); C NMR (D
2
O, 100.6 MHz) d 23.4 (CH
), 37.9 (CH ), 40.4 (CH ), 42.5 (CH
), 60.7 (CH ), 68.0 (CH), 69.5 (CH), 71.1 (CH), 72.9
2
), 25.5 (CH
2
),
(
D
2
2
2
2
2
28.0 (CH
2
), 33.4 (CH
2
2
2
2
), 50.4
4
2
2
2
(CH ), 51.7 (CH
2
2
2
(
CH
2
(CH), 75.6 (CH), 75.9 (2CH), 102.4 (CH-anomer), 125.2 (CH-tria-
zole), 145.3 (C-triazole), 156.7 (C@N), 170.7 (C@O), 171.6 (C@O),
1
1
H
35
N
6
O
12
S
172.5 (C@O), 176.2 (C@O), 178.5 (C@O); HRMS m/z (ESI) calcd for
+
+
[
M+H] 595.2028, found: 595.2052.
C
26
45
H N
10
O
13S [M+H] 737.2888, found: 737.2888.
4
.8.5.
osyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]-
Yield: 80%; white solid; [ +4.1 (0.5, H
L
-
c
-Glutamyl-S-[1-[2-[(6-deoxy-6-fluoro-
-cysteinyl-glycine (40)
O); 1H NMR (D
a
-
D
-mannopyran-
4.8.9. Arg-Gly-Asp-Cys-S-[[1-[2-[(
ethyl]-1H-1,2,3-triazol-4-yl]methyl] (44)
Yield: 63%; white solid; [ +4.1 (0.5, H
a
-
D
-mannopyranosyl)oxy]-
O); 1H NMR (D
O,
2
L
a
]
D
2
2
O,
a]
D
2
4
00 MHz) d 2.23 (q, 2H, J = 7.5 Hz), 2.55–2.62 (m, 2H), 2.93 (dd,
400 MHz) d 1.59–1.69 (m, 2H), 1.82–1.97 (m, 2H), 2.53 (dd, 1H,
1
H, J = 14.0 Hz, J = 9.5 Hz), 3.12 (dd, 1H, J = 4.5 Hz), 3.16–3.20 (m,
J = 15.5 Hz, J = 8.5 Hz), 2.62–2.70 (m, 1H), 2.75–2.97 (m, 2H),

6
682
S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
2
.98–3.05 (m, 1H), 3.14 (app t, 2H, J = 7.0 Hz), 3.51 (app t, 1H,
4.8.13. Arg-Gly-Asp-Cys-S-[[1-[2-[(6-deoxy-6-fluoro-
a
-
D
-gluco-
J = 9.5 Hz), 3.58 (dd, 2H, J = 13.0 Hz, J = 5.0 Hz), 3.67 (dd, 1H,
J = 12.0 Hz, J = 2.5 Hz), 3.76–3.92 (m, 6H), 3.99–4.10 (m, 3H),
pyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]methyl] (48)
1
Yield: 57%; white solid; [a] +12.4 (0.045, H O); H NMR (D O,
D 2 2
1
3
4
.25–4.32 (m, 1H), 4.54–4.62 (m, 3H), 8.01 (s, 1H, H-triazole);
NMR (D O, 100.6 MHz) d 23.4 (CH ), 25.5 (CH ), 28.0 (CH ), 33.6
CH ), 38.2 (CH ), 40.4 (CH ), 42.5 (CH ), 50.2 (CH ), 51.7 (CH ),
0.7 (CH ), 65.4 (CH), 66.4 (CH), 69.3 (CH), 69.9 (CH), 70.4 (CH),
2.8 (2CH), 99.6 (CH-anomer), 125.0 (CH-triazole), 145.0 (C-tria-
C
400 MHz) d 1.65–1.79 (m, 2H), 1.92–2.03 (m, 2H), 2.62 (dd, 1H,
J = 16.5 Hz, J = 9.0 Hz), 2.75 (dd, 1H, J = 16.5 Hz, J = 4.0 Hz), 2.89
(dd, 1H, J = 13.5 Hz, J = 7.0 Hz), 2.92–3.04 (m, 1H), 3.23 (app t,
2H, J = 6.5 Hz), 3.41 (app t, 1H, J = 9.5 Hz), 3.52 (dd, 1H,
J = 9.5 Hz, J = 3.5 Hz), 3.59 (app t, 2H, J = 9.5 Hz), 3.84–3.91 (m,
2H), 3.94–4.03 (m, 2H), 4.05–4.19 (m, 3H), 4.34–4.57 (m, 3H),
2
2
2
2
(
6
7
2
2
2
2
2
2
2
zole), 156.8 (C@N), 170.2 (C@O), 170.7 (C@O), 172.5 (C@O), 176.2
(
[
C@O), 177.1 (C@O); HRMS m/z (ESI) calcd for C26
M+H] 737.2888, found: 737.2903.
H
45
N
10
O
13
S
4.62–4.72 (m, 3H), 4.92 (d, 1H, J = 3.5 Hz), 8.06 (s, 1H, H-triazole);
+
13
C NMR (D
), 40.3 (2CH
(CH), 54.3 (CH), 66.2 (CH), 68.1 (d, JC,F = 7.0 Hz, CH), 70.6 (d,
C,F = 17.0 Hz, CH), 71.0 (CH), 72.6 (CH ), 81.9 (d, JC,F = 168.0 Hz,
CH ), 98.1 (CH-anomer), 125.0 (CH-triazole), 140.2 (C-triazole),
156.7 (C@N), 170.2 (C@O), 170.7 (2C@O), 172.5 (C@O), 176.2
2 2 2 2
O, 100.6 MHz) d 23.4 (CH ), 25.3 (CH ), 27.9 (CH ),
3
3.5 (CH
2
2
), 42.4 (CH ), 50.1 (CH
2
2
), 51.6 (CH), 52.8
4
.8.10. Arg-Gly-Asp-Cys-S-[[1-[2-[(b-
ethyl]-1H-1,2,3-triazol-4-yl]methyl] (45)
Yield: 52%; white solid; [
ꢁ14.8 (0.4, H
00 MHz) d 1.62–1.66 (m, 2H), 1.70–1.75 (m, 2H), 2.61 (dd, 1H,
J = 16.0 Hz, J = 8.0 Hz), 2.74 (dd, 1H, J = 16.0 Hz, J = 4.0 Hz), 2.86
dd, 1H, J = 7.0 Hz, J = 5.0 Hz), 2.96–3.00 (m, 1H), 3.18–3.29 (m,
D-galactopyranosyl)oxy]-
J
2
a]
D
2
O); 1H NMR (D
2
O,
2
4
1
9
(C@O);
2
F NMR (D O, 235 MHz) d - 235.0 (td, J = 47.0 Hz,
(
J = 28.0 Hz); HRMS m/z (ESI) calcd for C26H44FN10O
12S [M+H]+
3
4
1
H), 3.46–3.51 (m, 1H), 3.56–3.80 (m, 2H), 3.84–3.98 (m, 5H),
.06–4.15 (m, 3H), 4.26–4.38 (m, 4H), 4.64–4.70 (m, 2H), 8.04 (s,
739.2845, found: 739.2883.
1
3
H, H-triazole); C NMR (D
), 30.9 (CH ), 33.7 (CH ), 38.6 (CH
0.7 (CH ), 51.8 (CH), 54.2 (CH), 54.7 (CH), 61.3 (CH
2
O, 100.6 MHz) d 24.3 (CH
), 41.1 (CH ), 42.7 (CH
), 68.3 (CH
2
), 25.6
4.8.14. Cyclo(Arg-Gly-Asp-D-Phe-Cys)-S-[[1-[2-[(6-deoxy-6-fluoro-
(
CH
2
2
2
2
2
2
),
),
b-D-glucopyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]methyl] (49)
+
5
6
1
1
2
2
2
HRMS m/z (ESI) calcd for C35H52FN11O12S [M+H] 868.3423,
found: 868.3061, [M+H+Na] 445.6660, found: 445.6457.
2
+
8.9 (CH), 70.9 (CH), 73.0 (CH), 75.5 (CH), 103.4 (CH-anomer),
25.2 (CH-triazole), 145.2 (C-triazole), 157.1 (C@N), 171.4 (C@O),
72.9 (C@O), 176.7 (C@O), 178.0 (C@O), 178.3 (C@O); HRMS m/z
4.8.15. Cys-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH
deoxy-6-fluoro-b-D-glucopyranosyl)oxy]ethyl]-1H-1,2,3-triazol-
2
-S-[[1-[2-[(6-
+
(
ESI) calcd for
C
26
H
44
N10NaO13
S
[M+Na] 759.2708, found:
7
59.2703.
4-yl]methyl] (50)
57 15 2
C H86FN17O S
Na [M+Na]+
MS m/z (Maldi) calcd for
4
.8.11. Arg-Gly-Asp-Cys-S-[[1-[2-[(6-deoxy-6-fluoro-
a-D-manno-
1354.5812, found: 1354.6.
pyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]methyl] (46)
Yield: 68%; white solid; [ +4.6 (0.5, H
O); 1H NMR (D
00 MHz) d 1.58–1.69 (m, 2H), 1.85–1.96 (m, 2H), 2.53 (dd, 1H,
J = 16.5 Hz, J = 7.5 Hz), 2.66 (dd, 1H, J = 16.5 Hz, J = 5.0 Hz), 2.70
dd, 1H, J = 14.0 Hz, J = 8.5 Hz), 2.91 (dd, 1H, J = 14.0 Hz,
J = 5.0 Hz), 2.96–3.08 (m, 1H), 3.14 (app t, 2H, J = 7.0 Hz), 3.57–
.62 (m, 2H), 3.78–3.82 (m, 2H), 3.84–3.92 (m, 3H), 3.98–4.10
m, 3H), 4.27 (dd, 1H, J = 7.0 Hz, J = 5.0 Hz), 4.38–4.54 (m, 2H),
a]
D
2
2
O,
4.8.16. Cys-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH
deoxy-6-fluoro- -glucopyranosyl)oxy]ethyl]-1H-1,2,3-triazol-
4-yl]methyl] (51)
MS m/z (Maldi) calcd for
1354.5812, found: 1354.7.
2
-S-[[1-[2-[(6-
4
a-D
57 15 2
C H86FN17O S
Na [M+Na]+
(
3
(
4.8.17. Bis-(Arg-Gly-Asp-Cys-S)-6-(6-deoxy-
a-
D-trehalose)-bis-
4
.55–4.62 (m, 3H), 4.73 (d, 1H, J = 1.1 Hz), 8.02 (s, 1H, H-triazole);
[[1H-1,2,3-triazol-4-yl]methyl] (53)
1
3
O 80:20); 1H NMR,
C NMR (D
2
O, 100.6 MHz) d 23.4 (CH
2
), 25.5 (CH
2
), 27.9 (CH
2
),
Yield: 65%; white solid; R
2
(D O, 400 MHz) d 1.60–1.77 (m, 4H), 1.85–2.00 (m, 4H), 2.63 (dd,
f
0.18 (MeOH/H
2
3
3.6 (CH ), 38.3 (CH
2
2
), 40.3 (CH ), 42.5 (CH
2
2
), 50.1 (CH
2
), 51.6
(
6
J
(
(
CH), 52.8 (CH), 54.2 (CH), 65.2 (d, JC,F = 7.8 Hz, CH), 65.7 (CH),
9.7 (CH ), 70.2 (CH), 71.5 (d, C,F = 18.2 Hz, CH), 82.0 (d,
C,F = 169.0 Hz, CH ), 99.8 (CH-anomer), 124.7 (CH-triazole), 145.0
C-triazole), 156.8 (C@N), 170.1 (C@O), 170.7 (C@O), 172.5
C@O), 176.2 (C@O), 177.8 (C@O); 19F NMR (D
O, 235 MHz) d
234.0 (td, J = 47.0 Hz, J = 27.0 Hz); HRMS m/z (ESI) calcd for
2H, J = 16.0 Hz, J = 8.0 Hz), 2.76 (dd, 2H, J = 16.0 Hz, J = 5.0 Hz),
2.88 (dd, 2H, J = 14.0 Hz, J = 7.5 Hz), 2.99 (dd, 2H, J = 14.0 Hz,
J = 4.5 Hz), 3.22 (app t, 4H, J = 7.0 Hz), 3.26 (t, 2H, J = 9.0 Hz), 3.51
(dd, 2H, J = 10.0 Hz, J = 3.5 Hz), 3.77 (app t, 2H, J = 9.0 Hz), 3.88
(s, 4H), 3.98 (d, 2H, J = 17.0 Hz), 4.00–4.08 (m, 4H), 4.15 (d, 2H,
J = 17.0 Hz), 4.39 (dd, 2H, J = 7.0 Hz, J = 4.5 Hz), 4.53–4.63 (m, 2H),
4.57 (d, 2H, J = 4.0 Hz), 4.69 (dd, 2H, J = 8.0 Hz, J = 4.5 Hz), 4.75–
2
J
2
2
ꢁ
+
C
26
H44FN10
O
12S [M+H] 739.2845, found: 739.2831.
1
3
4
.84 (m, 2H), 7.98 (s, 2H, H-triazole); C NMR (D
d 23.6 (2CH ), 25.3 (2CH ), 28.7 (2CH ), 33.3 (2CH
40.4 (2CH ), 42.4 (2CH ), 50.8 (2CH ), 51.6 (2CH), 53.1 (2CH),
2
O, 100.6 MHz)
4
.8.12. Arg-Gly-Asp-Cys-S-[[1-[2-[(6-deoxy-6-fluoro-b-
D
-gluco-
2
2
2
2
), 38.3 (2CH ),
2
pyranosyl)oxy]ethyl]-1H-1,2,3-triazol-4-yl]methyl] (47)
Yield: 67%; white solid; [
ꢁ25.1 (0.3, H
O); 1H NMR (D
00 MHz) d 1.71-1,76 (m, 2H), 1.94–1.98 (m, 2H), 2.61 (dd, 1H,
J = 16.0 Hz, J = 8.0 Hz), 2.74 (dd, 1H, J = 16.0 Hz, J = 4.0 Hz), 2.86
dd, 1H, J = 7.0 Hz, J = 5.0 Hz), 2.95–3.00 (m, 1H), 3.18–3.29 (m,
2
2
2
a]
D
2
2
O,
54.4 (2CH), 70.4 (2CH), 70.6 (2CH), 70.9 (2CH), 72.6 (2CH), 93.1
(2CH-anomer), 125.2 (2CH-triazole), 145.0 (2C-triazole), 156.7
(2C@N), 170.9 (2C@O), 172.6 (2C@O), 176.3 (2C@O), 178.0
4
78 20 23 2
(2C@O); HRMS m/z (ESI) calcd for C48H N O S
[(M/2)+2H]2+
(
3
H), 3.43–3.60 (m, 3H), 3.86 (s, 2H), 3.97 (d, 1H, J = 10.0 Hz), 4.06–
.28 (m, 3H), 4.24–4.38 (m, 3H), 4.44 (d, 1H, J = 5.0 Hz), 4.60–4.74
684.2568, found: 684.2567.
4
(
(
m, 3H), 8.01 (s, 1H, H-triazole); 13C NMR (D
2
O, 100.6 MHz) d 23.7
), 38.5 (CH ), 40.7 (CH ),
), 53.2 (CH), 54.6 (CH), 68.5 (CH),
), 74.6 (CH), 75.7 (CH), 81.4 (CH), 83.0 (CH), 102.9 (CH-ano-
4.9. Platelet aggregation assay
CH
2
), 25.6 (CH
2.8 (CH ), 50.7 (CH
3.2 (CH
2
), 28.3 (CH
2
), 33.7 (CH
2
2
2
4
7
2
2
), 52.0 (CH
2
Venous blood was withdrawn from healthy volunteers who had
not taken anti-platelet or non-steroidal anti-inflammatory drugs
during the previous 10 days. Blood was collected in S-Monovette
tubes containing 0.106 M Trisodium citrate solution (Sarstedt).
Platelet-rich plasma (PRP) was prepared by centrifugation of whole
blood at 190g for 10 min at 20 °C. The remaining blood was again
centrifuged at 1750g for 10 min at 20 °C to obtain platelet-poor
2
Ò
mer), 125.2 (CH-triazole), 145.3 (C-triazole), 157.1 (C@N), 170.5
1
9
(
C@O), 171.1 (C@O), 172.9 (C@O), 176.7 (C@O), 178.2 (C@O);
F
NMR (D
m/z (ESI) calcd for C26
2
O, 235 MHz) d ꢁ235 (td, J = 46.0 Hz, J = 24.0 Hz); HRMS
ꢁ
H42FN10
O
12S [MꢁH] 737.2688, found:
7
37.2710.

S. Lamandé-Langle et al. / Bioorg. Med. Chem. 22 (2014) 6672–6683
6683
9
plasma (PPP). PRP was adjusted to 300 ꢀ 10 platelets/L by addi-
tion of autologous PPP.
Haddleton, D. M.; Geng, J.; Mantovani, G.; Lindqvist, J. Polym. Prepr. 2008, 49,
02; (g) Yoon, S.; Hwang, S. H.; Ko, W. B. J. Nanosci. Nanotechnol. 2008, 8, 3136;
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6
(
2
Platelet reactivity was measured by light transmission aggre-
gometry (SD Innovation Aggregometer, Frouard, France). A 300 lL
aliquot of PRP was preincubated with glycopeptides or reference
RGD, RGDC, c(RGDfV) or c(RGDfC) peptides (Bachem, Bubendorf,
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(
2
o) Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc.
002, 124, 14397.
Inhibition of platelet aggregation was measured at the maximal
aggregation response estimated quantitatively by measuring the
maximum curve height above baseline.
IC50 values were determined from concentration–response
curves fitted with a hyperbolic equation.
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Acknowledgments
1
We thank the European Commission for a fellowship to CV (Pro-
ject N°516.984), région Lorraine and Grand Nancy for a fellowship
to C.C. and B. Fernette and F. Dupire for recording NMR and mass
spectra, respectively. This work was partly supported by the
6173; (d) Kalai, T.; Hubbell, W. L.; Hideg, K. Synthesis 2009, 1336; (e) Bernardes,
G. J. L.; Chalker, J. M.; Errey, J. C.; Davis, B. G. J. Am. Chem. Soc. 2008, 130, 5052.
1. Lo Conte, M.; Pacifico, S.; Chambery, A.; Marra, A.; Dondoni, A. J. Org. Chem.
2010, 75, 4644.
2. (a) Kurra, Y.; Odoi, K. A.; Lee, Y.-J.; Yang, Y.; Lu, T.; Wheeler, S. E.; Torres-Kolbus,
J.; Deiters, A.; Liu, W. R. Bioconjugate Chem. 2014, 25, 1730; (b) Wang, S.; Yang,
X.; Zhu, W.; Zou, L.; Zhang, K.; Chen, Y.; Xi, F. Polymer 2014, 55, 4812; (c) Zhang,
X.; Zhang, Y. Molecules 2013, 18, 7145.
1
1
‘
Agence Nationale pour la Recherche’ [ANR-13-BSV1-0026-01].
Supplementary data
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Tetrahedron Lett. 2011, 52, 17–20.
Supplementary data associated with this article can be found, in
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