Versatile approaches to sugar amino acid building blocks as precursors of glycopeptides

Article abstract of DOI:10.2533/chimia.2006.286

The synthesis of monosaccharide units functionalised with different amino acids is described herein. Sugar amino acid templates linked at position 6 of the sugar were prepared by various nucleophilic substitutions introducing a spacer of variable length o

Full text of DOI:10.2533/chimia.2006.286

FH – HES
286
CHIMIA 2007, 61, No. 5
doi:10.2533/chimia.2006.286
FH – HES Fachhochschule – Hautes Ecoles Spécialisées
Chimia 60 (2006) 286–288
© Schweizerische Chemische Gesellschaft
ISSN 0009–4293
Versatile Approaches to Sugar Amino
Acid Building Blocks as Precursors of
Glycopeptides
Christelle Jablonski-Lorin*a, Matthias Nolda, Arthur Bodenmüllerb, and Ernst Hungerbühler*a
Abstract: The synthesis of monosaccharide units functionalised with different amino acids is described herein.
Sugar amino acid templates linked at position 6 of the sugar were prepared by various nucleophilic substitutions
introducing a spacer of variable length or by traceless Staudinger ligation. Sugar amino acid templates attached
at the anomeric position and representing a class of precursors of glycopeptides were synthesized in three steps
via addition of a solution of trimethylsilylnitrate-trimethylsilylazide on a glycal, followed by subsequent reduction of
the sugar azide and coupling with diverse amino acids.
Keywords: Glycopeptides · Staudinger ligation · Sugar amino acid templates · Sugar azide
The growing recognition of the roles of
protein.[2] The hydrophilic character of the
linked at the C-6 position of the monosac-
carbohydrates in fundamental biological glycopeptides tested emerged as a determi- charide (Fig.: type I). We attempted the
processes and their potential application nant factor that improved dramatically the synthesis first via a classical approach in-
as new therapeutics has emerged in recent water solubility of the biomolecule. The
years. Several groups have reported the role of glycoproteins in molecular recogni- introduction of a spacer, cleavage of the
effects in the activity, stability and me- tion was also proved.[1]
protecting group and coupling with dif-
tabolism of glycosylated peptide drugs.[1,2]
In collaboration with our spin-off com- ferent amino acids. This approach is very
volving a nucleophilic substitution for the
Positive effects in the bioactivity, pharma- pany SynphaBase, we defined simplified versatile since it resulted in a high degree
cokinetics and immunogenicity of proteins glycopeptide targets suitable for further of freedom in the choice of the protected
were observed while the activity of certain molecular recognition study and for the sugar unit, the length and functionality of
protein-linked glycans were being studied preparation of bulk pharmaceutical rather the spacer and the protecting groups of the
and compared to the activity of the isolated than the synthetically complex, branched amino acids. Scheme 1 illustrates the mul-
oligosaccharides typical of eukaryotic pro- tistep synthetic route for the preparation of
teins. We focused our efforts on the synthe- the functionalised derivate of 1,2,3,4-tetra-
sis of monosaccharide units functionalised O-benzyl glucopyranoside (3).
with different amino acids. These sugar
A possible extension of this part of the
amino acid templates represent interest- project is the introduction of the spacer
ing precursors of glycopeptides and are into a secondary alcohol functionality of
required in large amounts and great vari- the sugar unit. As a first example, students
ety for the understanding of the biologi- at the FHNW are currently optimising
cal roles of protein-linked glycans. To our
knowledge, only a restricted number of The starting material 4 was produced on
such sugar amino acid scaffolds are com- a kilogram scale, intermediate products 5
the synthesis of 7a, 7b, 7c (Scheme 2).
*Correspondence: Prof. Dr E. Hungerbühlera
Tel.:+41 61 467 43 88
Fax: +41 61 467 44 57
E-Mail: ernst.hungerbuehler@fhnw.ch,
christelle.jablonski@fhnw.ch
aFachhochschule Nordwest Schweiz (FHNW)
Hochschule für Life Sciences
Institut für Chemie und Bioanalytik
Gründenstrasse 40
mercially available in mg amount and then and 6 were prepared on a 100 g scale. All
only at a high price. The chemical synthesis
the final compounds described herein were
of such targets is difficult as carbohydrates characterised by LCMS-MS (Agilent sys-
are highly functionalized with hydroxyl tem 1100), 1H- and 13C-NMR experiments
1
groups of similar reactivity.
(Bruker Avance 400 MHz), H-1H COSY
CH-4132 Muttenz
bSynphaBase AG
Our first interest was related to the and NOESY experiments are required to
elaboration of sugar amino acid templates confirm the structures.
Gründenstrasse 40
CH-4132 Muttenz

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CHIMIA 2007, 61, No. 5
More elaborate templates of type I were
also synthesized: Scheme 3 displays the
further linkage of a second amino acid to
the functionalised derivate of 1,2:3,4-di-
O-isopropylidene galactopyranoside (8),
spacer
sugar
aa1
OP
aa2
(
)
COOH
6
n
1
PO
as well as the final cleavage of protecting
group leading to the protected hydrophilic
Type I
glycopeptide 9. Whereas the coupling of
this second amino was performed manu-
ally, we envisaged exploring the great ad-
vantages of an automated synthesis of short
OP
6
peptides in solution phase. We are mostly
taking advantage of the Fmoc strategy for
the terminal NH of the amino acid allowing
sugar
aa 1
X
aa2
(
)
COOH
1
n
PO
the protection of the sugar with acid labile
groups and the further protection of the
amino acid with benzyl protecting groups.
We have also investigated a more direct
approach to our sugar amino acid templates
oftypeIviatracelessStaudingerligation.[3,4]
This novel reaction – compatible with di-
verse functional groups – for the formation
of amide bonds involved an azide func-
tion as in 10 and a well-engineered phos-
Type II
Fig. General structure
of the glycopeptide
targets
aa = amino acid, P = protecting group, X = O, N or C
O
OtBu
OH
O
O
ii/ 78%
iii/ 50%
O
i/ 55%
BnO
BnO
OBn
BnO
BnO
OBn
OBn
OBn
phine partner such as 11a or 11b (Scheme
4). The Staudinger ligation consists of
the nucleophilic attack of a phosphine on
an azide to give a phosphazide which is
spontaneously transformed into a reactive
azaylide. The subsequent hydrolysis results
in amide-linked products. The preparation
1 (prepared in three steps)
2
O
NH
O
O
BnO
BnO
OBn
COOH
NHFmoc
OBn
3
of 11a and 11b required six steps includ-
ing the purification of each intermediate
by chromatography and was optimised to
i/ t-butyl-bromoacetate, NaOH aq, Bu4NHSO4, rt, CH2Cl2
ii/ CF3COOH, CH2Cl2 then pentafluorophenol, EDC, rt, CH2Cl2
iii/ Fmoc-Lys-OH, N-methylmorpholine, rt, DMF
yield the functionalized phosphine in 57%
yield compared to 43% described in the lit-
Scheme 1. Example of the synthesis of glycopeptide of type I
erature.
The synthesis of glycopeptides 12a and
O
O
O
O
O
O
12b was accomplished in a straightforward
O
O
O
fashion:theazidosugar10[5] reactedwiththe
i/ 65%
iii/ 30-40%
O
HO
H
O
phosphino derivative 11a or 11b (Scheme
4). The coupling was achieved in moderate
ii/ >99%
O
O
ROOC
O
R2HN CO
O
O
O
to medium yields (22–47%), however the
high selectivity generally obtained in this
type of coupling encouraged us to evaluate
the Staudinger ligation for future synthesis
of glycopeptides of type I and II.
4
5 (R = ethyl), 6 (R = H)
7a (R2 = Lys-Fmoc)
7b (R2 = Asp-OBzl)
7c (R2 = Asp(OBzl)-OH)
i/ NaH, THF, reflux then BrCH2COOEt, 0 °C, 16 h
ii/ NaOH aq, reflux, 1 h
iii/ DIC, HOBt, THF, rt
Natural protein-linked glycans exist
only as glycoconjugates in which the sugar
and amino acid moieties are directly linked
Scheme 2. Glycopeptide templates derivatives from 1,2:4,6-di-O-isopropylidene glucofuranose
attheanomericcarbonofthesugar.Thelack
of stability of O- and N-glycosides during
in vivo enzymatic processes compared to
C-glycosides can explain the great current
interest in the preparation of C-glycopep-
tides mimetics.
Because of the natural pre-eminence of
glycopeptides linked at C-1, we also inves-
tigated the synthesis of sugar-amino acid
templates of type II (Fig.) with the anomeric
centre as point of attachment. In this case, 2-
azido-2-deoxy azido galactopyranose (14)
was readily accessible from the commer-
O
O
COOH
O
N
H
COONHBn
NHFmoc
NH
O
NHAc
NH
O
O
O
i/ 58%
ii/ 86%
O
O
O
O
O
O
8
9
Scheme 3. Example of
glycopeptide of type
I bearing two amino
acids
i/ H-Leu-NHBn, EDC, CH2Cl2, rt
ii/ tris-aminoethylamine, CH2Cl2, rt then Ac2O, Et3N, CH2Cl2, rt

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CHIMIA 2007, 61, No. 5
COOBn
O
NHR
NH
N3
O
O
O
COOBn
NHR
O
O
O
toluene, 70 °C
then H2O
O
+
Ph2P
S
O
O
O
O
10
11a (R = Boc), 11b (R = Cbz)
12a (R = Boc)
12b (R = Cbz)
Scheme 4. Traceless Staudinger ligation for the synthesis of glycopeptides 12a and 12b
OBn
O
OBn
O
BnO
BnO
BnO
BnO
OBn
O
BnO
BnO
ii/ and iii/
i/ 76%
H
N
R
10-30% over two steps
O
N3
15a,b
13
14
i/ Me3SiONO2, Me3SiN3, CH2Cl2, rt
ii/ Ph3P, THF:H2O
iii/ Boc-Leu-OH, THF or Fmoc-Ser(OtBu)-OH, THF
Scheme 5. Formation of the sugar azide 14 and coupling reaction to generate glycopeptides of type
II 15a and 15b
established during the Diploma project of
Stefan Habermacher and also for allowing the
publication of the core structures synthesised by
Matthias Nold; all the students of the FHNW
Muttenz involved in the project: Andreas
Schneider, Julien Heusser, Patrick Wehrli,
Roger Nützi and Mari Frrokaj. We would like
cially available 3,4,6-tri-O-benzyl galactal
using the method described by Reddy et
al.[6] (Scheme 5). A one-pot procedure for
the reduction of the azide to amino group
and subsequent coupling reaction with a
choice of simple protected amino acids
was successfully accomplished to generate also to thank Prof. G. Aspin for his help with
the language.
our first sugar-amino acid templates of type
II, 15a and 15b, with excellent selectivity
(only the β anomer was obtained) and in
moderate yield (20–40% over two steps).
We intend to pursue the project by de-
veloping the synthesis of more glycopep-
tides by the well-established methods de-
scribed in this article, since the strategies
developed afford the choice of a large va-
riety of sugar, spacer and protected amino
Received: March 27, 2007
[1] D. A. Cumming, Glycobiology 1991, 1,
115.
[2] J. F. Fisher, A. W. Harrison, G. L. Bundy,
K. F. Wilkinson, B. D. Rush, M. J. Ruwart,
J. Med. Chem. 1991, 34, 3140.
[3] E. Saxon, J. I. Armstrong, C. R. Bertozzi,
Org. Lett. 2000, 2, 2141.
[4] C. Grandjean, A. Boutonnier, C. Guerrei-
acids. The investigation of a three-compo-
ro, J. M. Fournier, L. A. Mulard, J. Org.
Chem. 2005, 70, 7123;A. Bianchi, A. Ber-
nardi, J. Org. Chem. 2006, 71, 4565.
nent Staudinger ligation also appears very
promising.[7]
[5] Special caution is recommended for the
Acknowledgements
safe handling of the azide 10 which can
Special acknowledgements for this work are
generate HN3.
addressed to: SynphaBase, our industrial partner
[6] B. G. Reddy, K. P. Madhusudanan, Y. D.
for the successful collaboration regarding the
Staudinger ligation project mostly performed
during the Diploma project work of Andreas
Gernandt, for the know-how regarding the
Vankar, J. Org. Chem. 2004, 69, 2630.
[7] K. J. Doores, Y. Mimura, R. A. Dwek, P.
M. Rudd, T. Elliott, B. G. Davis, Chem.
Commun. 2006, 1401.
nucleophilic additions on the 2-nitrogalactal