Click chemistry to functionalise peptidomimetics

Article abstract of DOI:10.1016/j.tetlet.2006.03.149

Sharpless modified Huisgen's [2+3] cycloaddition of azide and acetylenic derivatives was employed as an efficient and simple method to conjugate azabicycloalkane amino acids, mimics of a homoSer-Pro dipeptide, with biologically relevant partners.

Full text of DOI:10.1016/j.tetlet.2006.03.149

Tetrahedron Letters 47 (2006) 3697–3700
Click chemistry to functionalise peptidomimetics
Daniela Arosio,a,b Matteo Bertoli,a Leonardo Manzonib,c, and Carlo Scolastico
a,b,
*
*
a
`
Dipartimento di Chimica Organica e Industriale, Universita Degli Studi di Milano, Via Venezian 21, I-20133 Milano, Italy
bCentro Interdisciplinare Studi Bio-molecolari e Applicazioni Industriali (CISI), Via Fantoli 16/15, I-20138 Milano, Italy
cC.N.R.—Istituto di Scienze e Tecnologie Molecolari (ISTM), Via Venezian 21, I-20133 Milano, Italy
Received 24 November 2005; revised 21 March 2006; accepted 22 March 2006
Available online 12 April 2006
Abstract—Sharpless modified Huisgen’s [2+3] cycloaddition of azide and acetylenic derivatives was employed as an efficient and
simple method to conjugate azabicycloalkane amino acids, mimics of a homoSer-Pro dipeptide, with biologically relevant partners.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
molecules have been employed in the synthesis of a small
library of cyclic pseudopentapeptides containing the
RGD sequence (Fig. 1). Many members of this library
revealed themselves to be active and selective ligands
for avb3 and avb5 integrins.6
Biologically active peptidomimetics have often turned
out to be good drug candidates. These molecules are
usually characterised by high affinity for specific recep-
tors and, in contrast to their natural analogues, can be
metabolically stable towards endogenous protease, as
well as possess greater oral bio-availability and more
rapid excretion.1
Recently our research group has reported an efficient
and simple synthesis of functionalised azabicycloalkane
amino acids, mimics of a homoSer-Pro dipeptide
(Fig. 2), where the key step is an intramolecular nitrone
cycloaddition reaction on 5-allyl- or 5-homoallylproline
that was found to be completely regio- and stereoselec-
tive.7 These mimics present heteroalkylic side chain end-
ing with hydroxy group that can be easily transformed
into other suitable functional groups (i.e., azide, amine,
etc.) or directly used for conjugation.
The possibility to functionalise peptidomimetics with
molecules of biological interest makes these substances
even more attractive. The functionalisation of peptido-
mimetics with lipophilic or hydrophobic appendages
may improve peptide-receptor affinity by interacting
with hydrophobic or hydrophilic pockets. Moreover it
is also possible to conjugate peptidomimetics with other
biologically active molecules,2 with small molecular
probes (a fluorescent dye or an affinity tag) or with
radiolabelled molecules.3
Our approach to the synthesis of bio-conjugates is based
on the so called ‘click chemistry’.8 In particular we
adopted the Sharpless modified Huisgen’s [2+3] cyclo-
addition of azide and acetylene to give 1,2,3-triazoles.9
This reaction presents many advantages: it is
In the course of our studies on peptide secondary struc-
ture mimics, we have synthesised several 6,5- and 7,5-
fused-2-oxo-1-azabicyclo[X.3.0]alkane amino acids.4
These structures can be regarded as conformationally
restricted substitutes for Ala-Pro and Phe-Pro dipeptide
units and, if their conformations meet certain criteria,
they can be used to replace the central (i + 1 and
i + 2) residue of b-turns.5 In particular some of these
CO
CO2H
N
n
N
Arg
Gly
n
O
O
R
NHPG
R
NH
Asp
n = 1, 2
R = H, Alk., Bn
Keywords: Peptidomimetics; Conjugates; Click chemistry; [2+3] Cyclo-
addition.
*
Figure 1. Azabicyclo[X.Y.0]alkane amino acids and cyclic pseudopen-
Corresponding authors. Tel.: +39 02 50320906; fax: +39 02 50320919
tapeptides containing the RGD sequence.
(L.M.); e-mail: leonardo.manzoni@istm.cnr.it
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.149

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D. Arosio et al. / Tetrahedron Letters 47 (2006) 3697–3700
chloride to give the corresponding mesylate, which was
subsequently converted to the azide with NaN3 in
DMF at 80 ꢁC (Scheme 1).
CO2tBu
CO2tBu
N
N
NH2
N
HO
HO
O
O
O
1
HO
HO
2
NH2
The 1,3-dipolar cycloaddition between pseudopeptides 6
or 8 and the 1-O-propargyl 2,3,4,6-tetra-O-acetyl-b-D-
glucose 910 was performed using Cu(OAc)2 and Na
ascorbate as catalysts in t-BuOH/H2O 1:111,12 (Scheme
2). The reaction proceeded overnight at room tempera-
ture and the desired products were isolated, after purifi-
cation by flash chromatography, in good yields.
CO2tBu
CO2tBu
N
O
4
3
NH2
NH2
Figure 2. Functionalised azabicycloalkane amino acids mimics of a
To increase the solubility of the sugar in the reaction sol-
vents and to avoid a deprotection step, the reaction was
also performed in the same conditions, using the un-
protected glucose 1013 (Scheme 2). The cycloaddition
products were isolated, after purification by flash
chromatography, in slightly lower yields.
homoSer-Pro dipeptide.
chemo- and regioselective, it is performed in mild condi-
tions and is usually characterised by high yields.
In this letter, we report the studies towards the conjuga-
tion of the pseudodipeptide homoSer-Pro with entities
of biological interest.
The conjugation of biotin and fluorescein were also
attempted. In both cases, a spacer was introduced
between the pseudodipeptide and the molecular tag.
For this purpose, spacer 1514 was coupled with propar-
gylamine and the Boc protecting group was removed
(Scheme 3) to give 17, which was finally conjugated to
biotin using HBTU and DIPEA.
2. Results and discussion
To apply the click chemistry 1,3-dipolar cycloaddition
reaction, it is first necessary to introduce either an azide
or an acetylenic moiety on the azabicycloalkane scaf-
folds. It proved to be synthetically convenient to intro-
duce an azide group onto the scaffold and to
functionalise the biological molecules to be conjugated
with an alkyne. As proof of concept, we chose in parti-
cular to conjugate a sugar (glucose), a fluorescent dye
(fluorescein) and an affinity probe (biotin).
Conjugation to fluorescein, to give 20, was performed by
adding commercially available 5(6)-carboxyfluorescein-
N-hydroxysuccinimide ester 18 to a basic solution of
17 (Scheme 3).
The click reaction between biotin conjugated with the
functionalised spacer 21 and pseudopeptides 6 or 8
was performed under the same conditions reported
above, yielding the conjugation products 22 and 24,
respectively, in 50–72% yield (Scheme 4).
After protection of the free amine with CbzCl, the
hydroxy group was first treated with methansulfonyl-
CO2tBu
CO2tBu
N
n
N
n
b, c
a
CO2tBu
N
HO
n
N3
O
HO
O
O
NHCbz
NHCbz
NH2
5 80%
7 81%
2
3
n=2
n=1
6 90%
8 86%
Scheme 1. Introduction of the azide group on to the azabicycloalkane scaffolds. Reagents and conditions: (a) CbzCl, TEA, CH2Cl2, rt, 18 h; (b)
MsCl, TEA, CH2Cl2, rt, 45 min; (c) NaN3, DMF, 80 ꢁC, 18 h.
OR
O
RO
RO
a
CO2tBu
CO2tBu
O
N
N
n
n
OR
N3
N
N
OR
R = Ac
R = H 10
9
O
N
O
NHCbz
O
NHCbz
RO
O
RO
OR
11 n= 2, R = Ac 84%
12 n= 2, R = H 72%
13 n= 1, R = Ac 80%
6 n=2
8 n=1
14 n= 1, R = H
74%
Scheme 2. 1,3-Dipolar cycloaddition between pseudopeptides 6 or 8 and the 1-O-propargyl-2,3,4,6-tetra-O-acetyl-b-D-glucose. Reagents and
conditions: (a) 9 or 10, Na-ascorbate, Cu(OAc)2, t-BuOH/H2O 1:1, rt, 18 h.

D. Arosio et al. / Tetrahedron Letters 47 (2006) 3697–3700
3699
O
O
O
HO
N
H
O
NHBoc
NHBoc
3
15
a
O
O
O
N
N
O
3
H
H
16
b
O
O
O
O
HN
S
N
H
O
N
H
O
NH2
d
O
O
3
NH
O
17
HO
19
N
O
O
O
c
O
HN
S
O
O
OH
NH
18
O
N
H
N
H
O
N
3
H
HO
O
O
21
O
O
O
O
N
N
O
N
H
O
3
H
H
OH
20
HO
O
O
Scheme 3. Conjugation of fluorescein and biotin to the linker. Reagents and conditions: (a) propargylamine, HBTU, DIPEA, CH2Cl2, rt, 18 h (97%);
(b) TFA (50%), CH2Cl2, rt, 1 h; (c) 18, TEA, THF, rt, 22 h (78% over two steps); (d) 19, HBTU, DIPEA, CH2Cl2, rt, 20 h (60% over two steps).
R
CO2tBu
a
N
CO2tBu
n
N
n
N
N
N3
N
O
O
NHCbz
NHCbz
6 n=2
22 n=2, R= Linker-Biotin
50%
23 n=2, R= Linker-Fluorescein 92%
8 n=1
24 n=1, R= Linker-Biotin
25 n=1, R= Linker-Fluorescein 91%
72%
O
HN
O
O
O
O
NH
R=
R=
N
H
N
H
O
N
3
H
S
O
O
O
O
N
H
N
O
N
O
3
H
H
OH
HO
O
O
Scheme 4. 1,3-Dipolar cycloaddition between pseudopeptides 6 or 8 and the functionalised tags. Reagents and conditions: (a) 20 or 21, Na-ascorbate,
Cu(OAc)2, t-BuOH/H2O 1:1, rt, 20 h.
The same reaction performed between 6 or 8 and the
fluorescein tag 20, gave the final compounds 23 and 25
in higher yields (91–92%).
The click reactions between 6 or 8 and the biotinylated
tag 21 (Scheme 4) were also attempted using CuSO4 as
source of copper, but no increase in yield was observed.

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D. Arosio et al. / Tetrahedron Letters 47 (2006) 3697–3700
3. Fichna, J.; Janecka, A. Bioconjugate Chem. 2003, 14,
3. Conclusions
3–17.
4. Belvisi, L.; Colombo, L.; Manzoni, L.; Potenza, D.;
Scolastico, C. Synlett 2004, 9, 1449–1471, and references
cited therein.
In conclusion, click chemistry has been established as an
efficient and simple method for the functionalisation of
azabicycloalkane amino acids, mimics of a homoSer-
Pro dipeptide with biologically relevant partners. The
compatibility of Cu(I)-catalysed azide–alkyne coupling
with many functional groups allows for the versatile
modular synthesis of pseudopeptide conjugates using a
variety of suitable functionalised molecule (drugs,
fluorophores, affinity tags, etc.). Moreover conjugation
occurs through the formation of a 1,2,3-triazole, the
presence of which could impart greater stability to enzy-
matic degradation.
5. (a) Belvisi, L.; Bernardi, A.; Manzoni, L.; Potenza, D.;
Scolastico, C. Eur. J. Org. Chem. 2000, 2563–2569; (b)
Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.; Scolas-
tico, C. Eur. J. Org. Chem. 1999, 389–400; (c) Gennari, C.;
Mielgo, A.; Potenza, D.; Scolastico, C.; Piarulli, U.;
Manzoni, L. Eur. J. Org. Chem. 1999, 379–388.
6. (a) Belvisi, L.; Bernardi, A.; Checchia, A.; Manzoni, L.;
Potenza, D.; Scolastico, C.; Castorina, M.; Cupelli, A.;
Giannini, G.; Carminati, P.; Pisano, C. Org. Lett. 2001, 3,
1001–1004; (b) Belvisi, L.; Riccioni, T.; Marcellini, M.;
Chiarucci, I.; Efrati, D.; Vesci, L.; Potenza, D.; Scolastico,
C.; Manzoni, L.; Lombardo, K.; Stasi, M. A.; Nico, B.;
Ribatti, D.; Presta, M.; Carminati, P.; Pisano, C. Mol.
Cancer Ther. 2005, 4, 1670–1680; (c) Belvisi, L.; Bernardi,
A.; Colombo, M.; Manzoni, L.; Potenza, D.; Scolastico,
C.; Giannini, G.; Marcellini, M.; Riccioni, T.; Castorina,
M.; LoGiudice, P.; Pisano, C. Biorg. Med. Chem. 2006, 14,
169–180.
The pseudopeptide conjugates will be investigated as
potential bioactive molecules (to be reported in due
course), but can also be employed to substitute natural
peptides in a more complex structure of biological
interest.
7. Manzoni, L.; Arosio, D.; Belvisi, L.; Bracci, A.; Colombo,
M.; Invernizzi, D.; Scolastico, C. J. Org. Chem. 2005, 70,
4124–4132.
8. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem.,
Int. Ed. 2001, 40, 2004–2021.
Studies are in progress to apply this methodology to a
small oligopeptide containing the homoSer-Pro dipep-
tide mimic.
9. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
10. Mereyala, H. B.; Gurrala, S. R. Carbohydr. Res. 1998,
307, 351–354.
Acknowledgements
We thank CNR and MIUR for financial support.
11. General procedure for click reaction: To a solution of
compound 6 or 8 (0.1 mmol) and an appropriate alkynyl
derivative (9, 10, 18, 19) (0.1 mmol) in H2O/t-BuOH 1:1
(500 lL), a solution of sodium ascorbate 0.9 M (44 lL,
0.04 mmol, 0.4 mol equiv) and a solution of Cu(OAc)2
0.3 M (67 lL, 0.02 mmol, 0.2 mol equiv) were added. The
reaction mixture was stirred at rt for ca. 18 h with TLC
monitoring. After the completion of the reaction, com-
pounds 11–14 were extracted with CH2Cl2 (2 mL · 3) and
the organic phase was washed with a saturated solution of
NaHCO3 (5 mL · 1) and subsequently with brine
(5 mL · 1). The organic phase, dried with Na2SO4, was
evaporated under reduced pressure. For compounds 22–
25, after the completion of the reaction, the solvent was
evaporated under reduced pressure. The crudes were
purified by flash chromatography on silica gel affording
the desired products.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2006.03.149.
References and notes
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