Synthesis of new ribosylated Asn building blocks as useful tools for glycopeptide and glycoprotein synthesis

Article abstract of DOI:10.1016/j.tetlet.2009.04.124

We performed the first synthesis of new Asn derivatives bearing α- or β-ribose as pure anomers, linked by an N-glycosidic bond, on the side chain of the Asn residue orthogonally protected for Fmoc/^tBu SPPS, by an efficient five-step strategy with a global yield of 73% starting from d-ribose. These building blocks are obtained in a large scale and can be useful tools for glycopeptide and glycoproteins synthesis.

Full text of DOI:10.1016/j.tetlet.2009.04.124

Tetrahedron Letters 50 (2009) 4151–4153
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of new ribosylated Asn building blocks as useful tools for glycopeptide
and glycoprotein synthesis
M. Angeles Bonache a,b, Francesca Nuti a,b, Alexandra Le Chevalier Isaad a,b, Feliciana Real-Fernández a,b
,
Mario Chelli a,b, Paolo Rovero a,c, Anna M. Papini a,b,
*
a Laboratory of Peptide & Protein Chemistry & Biology (PeptLab), Polo Scientifico e Tecnologico, University of Florence, via della Lastruccia 13, I-50019 Sesto Fiorentino (FI), Italy
b Dipartimento di Chimica Organica ‘Ugo Schiff’ and CNR-ICCOM, Polo Scientifico e Tecnologico, University of Florence, via della Lastruccia 13, I-50019 Sesto Fiorentino (FI), Italy
c Dipartimento di Scienze Farmaceutiche, Polo Scientifico e Tecnologico, University of Florence, Via Ugo Schiff 6, I-50019 Sesto Fiorentino (FI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We performed the first synthesis of new Asn derivatives bearing a- or b-ribose as pure anomers, linked by
an N-glycosidic bond, on the side chain of the Asn residue orthogonally protected for Fmoc/tBu SPPS, by
Received 6 April 2009
Revised 24 April 2009
Accepted 29 April 2009
Available online 3 May 2009
an efficient five-step strategy with a global yield of 73% starting from D-ribose. These building blocks are
obtained in a large scale and can be useful tools for glycopeptide and glycoproteins synthesis.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
N-Glycosylated Asn for SPGPS
a- and b-ribofuranose Asn
Triazine-based coupling reagent
Glycoproteins are fundamental for a variety of biological events,
that is, folding, fertilization, neuronal development, hormone
activities, immune surveillance, and inflammatory responses.
However, investigating these processes is a challenge by the fact
that naturally expressed glycoproteins often emerge as heteroge-
neous mixtures. In addition, post-translational modifications
(PTMs) such as alterations of sugars on the amino acid side chains
may in part lead to higher structural and functional protein diver-
sity. Two main forms of protein glycosylation are naturally found:
O- and N-glycosylation. The majority of N-linked motifs are
Asn(GlcNAcb) and O-linked forms are usually Ser/Thr(GlcNAcb)
of glycoproteins in mammalian cell culture habitually leads to mix-
tures of glycoforms.
In light of the heterogeneity for each glycan, which limits the
isolation, purification, and manipulation of natural glycoproteins,
the development of an efficient synthetic chemistry for the pro-
tein–sugar assembly is essential to advance in understanding the
biological role of glycoconjugates. The synthesis of glycopeptides
requires a combination of synthetic methods from both carbohy-
drate and peptide chemistry. To determine their activity relation-
ships not only sugars but also control of their configuration and
of the linkage with amino acids are needed. Several reviews cover
strategies for the preparation of N-linked and O-linked glyco-
aminoacids.7
Solid-Phase GlycoPeptide Synthesis (SPGPS) based on building
block approach is an efficient method to obtain glycosylated pep-
tides bearing different sugar moieties on the side chains of differ-
ent amino acids. SPGPS requires an excess of building block units
to achieve high yields, so it is necessary to prepare them in high
quantity. For this reason it is fundamental to develop convenient
synthetic pathways to glycosylated building blocks and efficient
glycopeptide solid-phase synthetic pathways. In our laboratory
several glycosylated building blocks orthogonally protected for
SPGPS were developed in large scale, for example, Glc, Gal, Man,
GlcbGlc, GlcNAc, and glycomimetics, on the side chains of Asp,
Ser, Glu, Thr, and HyPro.8 We identified Asn(b-Glc) as minimal epi-
tope for Multiple Sclerosis (MS) autoantibody recognition, after
evaluating in competitive and SP-ELISA a CSF114-type glycopep-
or Ser/Thr(GlcNAc
observed1 including Asn(GlcNAc
Asn(Rha),5 Ser/Thr(Man ),6 or even C-linked Trp(Man
a
). Although other unusual linkages are also
),2 Asn(Glc),3 Asn(GalNAc),4
).
a
a
a
Biosynthesis of the glycan portion is not DNA mediated as in the
case of proteins; the glycan structure is subjected to competition
between various enzymes and their substrates. Moreover, N-glu-
cosylation, a PTM not common in eukaryotic proteins, has been de-
tected only in bacterial glycoproteins.3 This variable post-
translational processing of glycans results in expression of an
assortment of possible glycan structures called ‘glycoforms’, glyco-
proteins that possess the same protein backbone but differ in the
oligosaccharide components and sites of glycosylation. Expression
* Corresponding author. Tel.: +39 055 457 3561; fax: +39 055 457 3584.
E-mail address: annamaria.papini@unifi.it (A.M. Papini).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.04.124

4152
M. A. Bonache et al. / Tetrahedron Letters 50 (2009) 4151–4153
tide library based on glyco-amino acid diversity.9 The role of ribose
moiety has not been previously studied.
It is well known that ribose (Rib) is produced in vivo from glu-
cose and it plays a fundamental role in cellular energy metabolism
and cellular signaling. Mono (ADP-ribosyl) transferases transfer
single ADP-ribose to acceptor proteins.
Due to high interest in the glycan–protein linkages as N-(Asn)
glycosides, now we are interested to obtain new N-linked ribofura-
nose Asn derivatives. We report herein an efficient synthesis of two
O
N3
O
OAc Me3SiN3, SnCl4
AcO
AcO
CH2Cl2
96%
AcO
OAc
AcO
OAc
2
1
O
NH2
H2, Pd(OH)2
AcO
MeOH
6%
AcO
OAc
3
a
N -Fmoc protected asparagine building blocks bearing
a
- or b-
-Asn[(2,3-O-isopro-
-Asn(RibCMe2Ac )-OH, 9 ] and
-Asn[(2,3-O-isopropylidene,5-OAc)b-Rib]-OH [Fmoc-
Asn(RibCMe2Acb)-OH, 9b] (Fig. 1).
Most of the protected glycosylamines are prepared by hydroge-
-b-glycosyl azide using different catalytic re-
D-ri-
Scheme 1. Synthesis of 2,3,5-triacetyl-b-
D
-ribofuranosylamine.
bose, orthogonally protected for SPGPS: Fmoc-
pylidene,5-OAc) -Rib]-OH [Fmoc-
Fmoc-
L
a
L
a
a
1) Acetone,
O
L
L
-
O
OAc
OH
CuSO4, H2SO4
AcO
HO
2) Ac2O, py
68%
O
O
O
O
HO
OH
nation of an acety
L
4
5
agents.10 Applying the catalyst Pd(OH)2 to this synthetic strategy,
ribosylamine 3 was obtained in a very low yield (6%) (Scheme 1).
In order to improve the yield of the reduction reaction, we
investigated an alternative synthetic strategy, starting from ribose,
protected in C-2 and C-3 with a rigid isopropylidene group that
O
O
N3
N3
Me3SiN3, SnCl4
CH2Cl2
AcO
AcO
+
61%,
/ (2:3)
α β
O
O
6α
6β
avoids the opening of the sugar ring. The new building blocks 9
a
and 9b were synthesized starting from the 2,3-O-isopropylidene-
1,5-diacetate-protected ribosyl derivative 5, obtained by reaction
of 4 with acetone and subsequent acetylation with Ac2O in pyri-
dine.11 Azide moiety was introduced by reaction with Me3SiN3,
Scheme 2. Synthesis of the ribofuranosylazide 6
a
and 6b.
O
O
NH2
+
NH2
via SnCl4 catalysis, affording a mixture of the anomers 6a:6b
AcO
AcO
H2, Pd black
6β
(38:62). It was possible to easily separate the two anomers by
FCC and to characterize them as pure compounds (Scheme 2).
The next reduction was performed on the b-anomer in the pres-
ence of palladium black, obtaining in any case a mixture of amines
EtOH
99%
O
O
O
O
7α
7β
7a
:7b in good yield, as detected by 1H NMR spectrum.
Fmoc-Asp-OAll, DMTMM/BF4 MeCN
40%
Coupling reaction of the anomeric mixture of the ribosyl amines
7
a
:7b with Fmoc-L-Asp-OAll afforded the desired N-glycosidic
H
H
O
O
(R)
(R)
(S)
N
N
bond, by using the new efficient triazine-based coupling reagent
CO2R
CO2R
Fmoc
(S)
(TBCR).12
AcO
AcO
(R)
+
a
O
HN
O
HN
Allyl group protection was chosen for COOH because it is
O
O
O
O
Fmoc
orthogonal with the isopropylidene and acetyl groups on the hy-
droxyl functions of the ribosyl moiety. Activation of carboxylic
function by the TBCR is particularly efficient because of the forma-
tion of triazine ‘superactive ester’, as recently confirmed with the
synthesis of protected dipeptides and esters, sterically hindered
amino acids, manual and automated solid-phase peptide synthesis
(SPPS) of difficult peptide sequences, and head-to-tail constrained
8β: R = All
9β: R = H
8α: R = All
9α: R = H
Pd(PPh3)4, Ph3SiH
CH2Cl2, 97%
Scheme 3. Synthesis of the new building block N-linked ribofuranose Asn
derivatives 9 and 9b.
a
cyclopeptide analogs.13 The two pure anomers 8
a and 8b of the
a
is possible to observe the difference in chemical shift of the C1
d) shown by the -anomer (80.6 ppm) compared to the corre-
sponding value for the b-anomer (88.6 ppm) in accordance with
the literature.17 Subsequent removal of the allyl group with
Pd(PPh3)4 and triphenylsilane afforded the anomerically pure
new protected ribosylated N -Fmoc-asparagine allyl esters were
obtained after an efficient separation by FCC from the correspond-
ing anomeric mixture (3:2) (Scheme 3).
(D
a
By analyzing the 1H and 13C NMR spectra it is possible to assign
the configuration of the compounds 8a14 and 8b.15 The anomeric
building blocks 9a and 9b in a very good yield (Scheme 3).
configuration of 8
of H-1 that is usually bigger for the
sponding b-anomers.16 In fact H-1 and H-2 are less eclipsed in the
case of b-anomers. In our case, the -anomer shows J1,2 = 4.4 Hz
and the b-anomer J1,2 = 0. As Tam et al. described, the chemical
shift of H-1 is further downfield for the -anomers (d 5.74 ppm)
than for the corresponding b-anomers (d 5.57 ppm). Moreover, it
a
was assigned by the J1,2 spin coupling constant
In conclusion, we developed an efficient method to synthesize
a
-anomers than for the corre-
N-ribosylated Asn derivatives orthogonally protected for Fmoc/tBu
SPPS. In particular, we obtained two new pure anomers,
a and b, of
a
N-ribosylated Asn derivatives, independently by an efficient strat-
egy in five steps. These building blocks can be obtained in large
scale and can be useful tools for glycopeptides and glycoproteins
synthesis.
a
H
N
H
N
Acknowledgments
O
O
CO2H
Fmoc
CO2H
Fmoc
AcO
AcO
This work was supported by Ente Cassa Risparmio di Firenze
and FIRB Internazionalizzazione 2004 RBIN04TWKN, MIUR (Italy).
M.A.B. thanks the Alfonso Martín Escudero Foundation (Spain) for
her post-doctoral fellowship. We thank Dr. Marco Boccalini for
his assistance with NMR spectroscopy.
O
HN
O
HN
O
O
O
O
9α
9β
Figure 1. New building block N-linked ribofuranose Asn derivatives 9
a
and 9b.

M. A. Bonache et al. / Tetrahedron Letters 50 (2009) 4151–4153
4153
À
12. DMTMM/BF4
,
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
Supplementary data
tetrafluoroborate.
13. (a) Kamin´ ski, Z. J.; Kolesin´ ska, B.; Kolesin´ ska, J.; Sabatino, G.; Chelli, M.; Rovero,
P.; Błaszczyk, M.; Główka, M. L.; Papini, A. M. J. Am. Chem. Soc. 2005, 127,
16912–16920; (b) Kaminski, Z.; Papini, A. M.; Kolesin´ ska, B.; Kolesin´ ska, J.;
Jastrzabek, K.; Sabatino, G.; Bianchini, R. PCT/EP2005/055793 (2005).
Supplementary data (in the supplementary material experi-
mental procedures and characterization data of the products are
available) associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2009.04.124.
14. Fmoc-L-Asn(2,3-O-Isopropyliden-5-O-acetyl-a-D-ribofuranosyl)-OAll (8a
). 1H
NMR (400 MHz, CDCl3) d 7.73 (d, 2H, J = 6.8 Hz, Fmoc 4-H and 5-H), 7.59 (d, 2H,
J = 6.4 Hz, Fmoc 1-H and 8-H), 7.39–7.27 (m, 4H, Fmoc 2-H, 7-H, 3-H and 6-H),
6.59 (d, 1H, J = 9.2 Hz, 1-NH), 6.06 (d, J = 7.2 Hz, NHFmoc), 5.94–5.84 (m,
1H, = CH), 5.74 (dd, 1H, J = 4.4, J = 9.6 Hz, 1-H), 5.32 (d, 1H, J = 17.0 Hz, @CH2),
References and notes
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