Chemoselective Staudinger-phosphite reaction of symmetrical glycosyl-phosphites with azido-peptides and polygycerols

Article abstract of DOI:10.1039/c2ob25207d

In this paper we present the synthesis of glyco-phosphoramidate conjugates as easily accessible analogs of glyco-phosphorous esters via the Staudinger-phosphite reaction. This protocol takes advantage of synthetically accessible symmetrical carbohydrate phosphites in good overall yields, in which ethylene or propylene linkers can be introduced between phosphorous and galactose or lactose moieties. The phosphites were finally applied for the chemoselective reaction with azido-peptides and polyazido(poly)glycerols.

Full text of DOI:10.1039/c2ob25207d

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PAPER
Chemoselective Staudinger-phosphite reaction of symmetrical
glycosyl-phosphites with azido-peptides and polygycerols†‡
Verena Böhrsch,§a,b Thresen Mathew,§a Maximilian Zieringer,a M. Robert J. Vallée,a Lukas M. Artner,a
Jens Dernedde,c Rainer Haaga and Christian P. R. Hackenberger*a
Received 27th January 2012, Accepted 18th May 2012
DOI: 10.1039/c2ob25207d
In this paper we present the synthesis of glyco-phosphoramidate conjugates as easily accessible analogs of
glyco-phosphorous esters via the Staudinger-phosphite reaction. This protocol takes advantage of
synthetically accessible symmetrical carbohydrate phosphites in good overall yields, in which ethylene or
propylene linkers can be introduced between phosphorous and galactose or lactose moieties. The phosphites
were finally applied for the chemoselective reaction with azido-peptides and polyazido(poly)glycerols.
The covalent attachment of single or oligomeric carbohydrates
represents a functional natural modification for proteins and
(phospho-)lipids, responsible for the regulation of molecular rec-
ognition, immune response, pathogen interaction and intracellu-
lar transport.1 In addition to the commonly occurring linkages of
Asn (N-linked) or Ser/Thr amino acids (O-linked glycosylation)
to the anomeric center of saccharides, glycans can also be linked
to phosphoresters, which often play a distinct role as virulence
factors in pathogens (e.g. leishmania), several bacteria and
yeasts.2 Additionally, glyco-phosphordiesters are components of
capsular polysaccharides (CPS) of Gram negative and Gram
positive bacteria, in which mono- or oligosaccharides can be
connected via interglycosidic phosphodiesters (e.g. in Neisseria
meningitidis type A or Haemophilus influenzae type C), via
glycerol phosphates (in N. meningitidis type Z) or via alditol
phosphates (in H. influenzae type A).3
synthesise and could furthermore lead to higher biological
activity and greater stability against enzymatic cleavage.2,4 It is
important to note that in particular glycosyl-phosphite precursors
have found considerable attention as key intermediates in the
synthesis of glyco-phosphates and glycoconjugates.5
Recently, our group has contributed to these methodologies
by using the Staudinger-phosphite reaction6 between azides
and phosphites to deliver phosphoramidate-linked glycoconjuga-
tes.4c,7 Specifically, α- or β-linked azido-monosaccharides were
reacted with protected Ser-phosphitylated peptides on solid
support, which yielded glyco-phosphoramidates under very good
retention of the anomeric linkage and in good overall isolated
yields as easily accessible N-analogs of glyco-phosphorester-
linked glycopeptides, which proved to be stable under physio-
logical conditions (Scheme 1A).8
In the current paper, we intended to broaden the applicability
of the Staudinger-phosphite reaction to the synthesis of phospho-
rester-linked glycoconjugates by employing easily accessible
azido-peptides and polymers as core substrates for the reaction
with symmetrical glycosyl-phosphite derivatives (Scheme 1B).
This reversion in the functionalities of the starting materials fea-
tures several potential advantages: Due to the ease of azide-
incorporation in many functional (bio-)polymeric core structures
by synthetic9 as well as biochemical10 protocols, a chemoselec-
tive glycan-conjugation by the Staudinger-phosphite reaction
would become possible. Thereby, additional acidic protecting
group manipulations, which are necessary for the protection of
other nucleophiles present in the synthesis of phosphite-contain-
ing core structures, could be avoided. Second, the use of sym-
metrical phosphites results in the attachment of two glycans to
the core, which points towards the generation of multivalent
scaffolds for recognition studies in a straightforward manner.
Consequently, we report our recent studies for the synthesis of
symmetrical mono- and disaccharide-phosphites (galactose and
Over the last years, several protocols have been developed for
the synthesis of homogeneously glycosylated peptides, since
their isolation from natural sources proves to be very difficult.
These strategies aim to obtain either glycopeptides containing
natural or unnatural linkages, while the latter are often easier to
aInstitut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3,
14195 Berlin, Germany. E-mail: hackenbe@chemie.fu-berlin.de
bBeuth Hochschule für Technik Berlin, Fachbereich Life Sciences &
Technology, Labor für Biochemie, Seestr. 64, 13347 Berlin, Germany
cCharité – Universitätsmedizin Berlin, Institut für
Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, CBF,
Hindenburgdamm 30, 12200 Berlin, Germany
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
‡Electronic supplementary information (ESI) available: Synthetic proto-
cols for the synthesis of glycosyl-phosphites, Staudinger-phosphite reac-
tions with peptides and PG, SPR measurements, characterization of the
synthesised compounds. See DOI: 10.1039/c2ob25207d
§These authors contributed equally to the work.
This journal is © The Royal Society of Chemistry 2012
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Scheme 1 Staudinger-phosphite reaction for the synthesis of glycoconjugates. (A) Reaction of glycosylazides with protected phosphitylated pep-
tides;8 (B) Reaction of symmetrical glycosyl-phosphites with azido core structures.
Scheme 2 Synthesis of symmetrical galactose phosphites 1–3.
lactose) with different linker lengths, their Staudinger-phosphite
reactions with scaffolds like polyazido(poly)glycerols or unpro-
tected azido-peptides as well as the elucidation of the carbo-
hydrate-functionalized polymers in first lectin-binding
interactions.
For the synthesis of glycosyl-phosphite 1 peracetylated
galactose was selectively deprotected at the anomeric glycosidic
position to 5 with hydrazinium acetate at room temperature.11
Phosphite 1 was obtained via reaction with stoichiometric
amounts of PCl3 and triethyl amine at 0 °C in THF as a insepar-
able mixture of α,β-anomers. Separation of the diastereomers
failed due to low stability of the P(III)-compound. The identity
of the crude phosphite
1
was confirmed by 31P-NMR,
Results and discussion
IR and HRMS and it was directly used without further
purification in the reaction with polymeric azides later (see ESI‡
for details).
In order to achieve glycosyl-phosphites 2 and 3 with a defined
stereochemistry at the anomeric position, peracetylated galactose
was converted into the anomeric bromide by treatment with
HBr–AcOH and subsequently glycosylated with ethylenediol or
propylenediol under mercury bromide catalysis delivering
β-linked glycans 6 and 7 in 60% and in 75% yield.12
Synthesis of the symmetrical glycosyl phosphites
At the outset of our studies, we focused on the development of a
synthetic protocol for symmetrical glycosyl-phosphites, allowing
the linkage between the carbohydrate and the phosphorous to be
varied. Therefore, in addition to the direct connection of the
anomeric position to the phosphorous in galactose 1, ethylene
and propylene spacers were probed in galactoses 2 and 3
(Scheme 2).
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After isolation alcohols 6 and 7 were dried under vacuum and
treated with freshly distilled PCl3 and Et3N in anhydrous THF or
Et2O at 0 °C. The reaction was monitored both by 31P-NMR and
by TLC. Finally, the galactosyl-phosphites 2 and 3 were obtained
in good yields of 55% and 60% (overall yield 41% and 48%). In
contrast to phosphite 1, compounds 2 and 3 were stable during
purification by column chromatography (see ESI‡ for details).
For the synthesis of 2 and 3, other electrophilic P(III) sources like
P(NiPr2)3 were also tested; however, the instability of the phos-
phites at elevated temperature, which is required for the phos-
phite synthesis with P(NiPr2)3 presented itself as a limiting factor
in this reaction. The pure phosphite reagents 2 and 3 could be
handled on air and stored in the freezer for at least eight weeks
without any signs of decomposition, while showing sensitivity
in solution especially under elevated temperature or in solvents
containing traces of acid (e.g. chloroform).
the glyco-phosphoramidate (Scheme 3b). Peptide 10 was
obtained by standard SPPS, coupling para-azido-Phe as the last
amino acid.13 This experiment already revealed a very good con-
version of the azido-peptide 10 to the peptidic phosphoramidate
11 of 86% in DMSO at room temperature, as determined by
HPLC-MS with a deuterium-labeled peptide as internal standard
(see ESI‡).14 In order to further demonstrate the chemoselectiv-
ity of the Staudinger-phosphite reaction6,13 another fluorescence-
labeled peptide 12 with an NBD-group attached to the ε-amino
group of Lys prepared, in which the NBD-Lys was introduced as
Fmoc-protected building block. As illustrated in Fig. 1, the reac-
tion resulted in a clean product formation and a quantitative
transformation of the azide peptide 10 to the phosphoramidate
13 without any side product formation.
Staudinger-phosphite reaction of multifunctional dendritic
azides
Reaction of glycosyl phosphites with peptides
After the successful transformation of azido-amino acids and
peptides, we next turned our attention to the reaction of azido-
polymers with glycosyl-phosphites. The bisfunctionality of the
resulting phosphoramidate products renders the reaction very
attractive for the generation of polymeric scaffolds for multiva-
lency binding studies. We therefore probed the applicability of
the Staudinger-phosphite reaction on multifunctional dendritic
core structures, in which the latter have been demonstrated pre-
viously as an excellent core structure for the presentation of
multivalent ligands.15
Initially, we tested the reaction of a small phosphite with poly-
glycerol (PG) azide (10 kDa, degree of functionalisation (DF) =
20%, 2.7 mmol N3-groups per g) that was obtained from multi-
functional dendritic glycerols,16 in which the terminal hydroxyl
groups were transformed into azides by mesylation followed by
nucleophilic substitution with sodium azide. Reaction of this
polymer with P(OMe)3 at 40 °C in wet DMF resulted in a quan-
titative conversion to the phosphoramidate as detected by NMR
and IR (data not shown).
The reactivity of the phosphites was first probed with small
organic azides. Thus, phosphites 2 and 3 were reacted with (3-
azidopropyl)benzene at 45 °C for 50 h. After deprotection, the
corresponding phosphoramidates 8 and 9 were obtained in 94%
and 96% isolated yield (Scheme 3a). For later applications on
the modification of azido-containing peptides, the reactivity of
amino acids bearing alkyl or aryl azides in the side chain was
then tested. Fmoc-protected para-azido-Phe was reacted with the
phosphite 3 in DMSO. To our delight the conversion to the
dibranched phosphoramidate was complete after 6 h at 28 °C as
monitored by HPLC-MS (see Scheme S1 in ESI‡). In contrast,
the reaction of 3 with the aliphatic Fmoc-protected ε-azido-Lys
demanded elevated temperature (40 °C) and longer reaction
times (24 h) for full conversion.
In addition to the reaction with single azido-containing amino
acids, we probed the Staudinger-phosphite reaction of glycosyl-
phosphites with unprotected azido-peptides. First, we investi-
gated the conversion of the aryl-azido-containing peptide 10 to
Scheme 3 (a) Synthesis of glycosyl phosphoramidates 8 and 9 via Staudinger-phosphite reaction of 2 and 3; (b) Reaction of glycosyl-phosphite 3
with azido-peptides. Pap = p-azidophenylalanine, NBD = 4-amino-7-nitrobenzo-2-oxa-1,3-diazole.
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Fig. 1 HPLC-fluorescence traces of the reaction of 3 with peptide 12 (see also Scheme 3b).
Scheme 4 Synthesis of lactose phosphite 4.
After this successful transformation, we synthesised lactose-
phosphite 4 as another symmetrical carbohydrate for lectin–
peanut agglutinin (PNA) binding studies, since lactose-conju-
gates were previously shown to exhibit significantly better
binding than the corresponding galactose derivatives.17 The
lactose-phosphite 4 could be obtained in 27% yield over two
steps, whereas longer reaction times for the introduction of the
propane linker were needed in comparison to the synthesis of
galactose-phosphite 3 (Scheme 4). About 10% of the alcohol 14
was still present in the purified phosphite 4, which however did
not disturb the later Staudinger reaction. Replacing HgBr2 with
Ag2CO3 in the glycosylation reaction led to a similar outcome of
the reaction.12
the carbohydrate used (Fig. 2 and 3). Since the addition of fresh
phosphites did not result in higher loadings, we assumed sterical
reasons for the different observed conversions, resulting in a
dense carbohydrate shell surrounding the polydendritic core. The
carbohydrate moieties were deprotected with NaOMe–MeOH to
yield approximately 2 mg of each desired compound. The depro-
tection was quantitative according to 1H-NMR and IR. Since the
glyco-polymers presented insufficient water solubility for
reliable lectin binding studies, two polyglycerols C and D with a
lower azide-substitution (C: core 7.7 kDa, DF
= 30%,
4.05 mmol N3-groups per g and D: core 10.6 kDa, DF = 32%,
4.32 mmol N3-groups per g) containing free hydroxy groups as
solubility mediators were prepared and submitted to the Staudin-
ger phosphite reaction with lactose phosphite 4. Although the
azide conversion was still incomplete, the deprotected products
possessed sufficient water solubility to be suitable for binding
studies, as we could demonstrate in a first test lectin binding
assay. In this study, the inhibitory effect of the glycan-conjugated
PGs on PNA binding to the immobilized Thomsen–Friedenreich
(TF) antigen was probed via a competitive surface plasmon res-
onance (SPR) binding assay and we obtained inhibition values
of about 60% with both polymers 4C and 4D (Fig. 3).
Staudinger-phosphite reactions of phosphites 1–4 with two
different PG azides A and B (A: core 7.7 kDa, DF 100%,
13.5 mmol N3-groups per g, and B: core 12.6 kDa, DF 98%,
13.2 mmol N3-groups per g) were performed in wet DMSO at
40 °C for 48 h, while adding the phosphites in two portions
(Fig. 2). The polymers were purified via dialysis and the loading
1
was determined by H-NMR. The conversions as determined by
NMR ranked from 23–91% with respect to the azido groups.
The conversion rates were mainly dependant on the linker and
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Fig. 2 Functionalisation of polyazido(poly)glycerols with phosphites 1–4. Reaction rates are given for the conversion of azides (for additional infor-
mation see the ESI‡).
Fig. 3 Binding studies of polylactose(poly)glycerols 4C and 4D. Reaction rates are given for the conversion of azides (for additional information
see the ESI‡).
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Summary and conclusion
Taken together we present here the synthesis of different sym-
metrical glycosyl-phosphites with three carbohydrate side chains.
These phosphite building blocks were applied in chemoselective
Staudinger-phosphite reactions with a variety of substrates, includ-
ing peptides and polyglycerol-azides. Generally, the reactions are
easy to conduct and proceeded in high conversions. The carbo-
hydrate phosphites 2–4 present themselves as easy to use reagents
for the introduction of carbohydrate residues on biomolecules or
on polymers as tools for delivery and recognition studies. Purifi-
cation after the short ligation/deprotection protocol can simply be
done via dialysis in water. Consequently, we believe that the
reagents will be particularly useful for researchers in the fields of
modern organic chemistry, material science and chemical biology
due to the simplicity of application to complex structures.
An advantage of our methodology towards other reactions
used in biochemistry labs such as copper catalysed click chem-
istry is, besides the avoiding of transition metals, the introduction
of two carbohydrate residues as analogs of glyco-phosphodie-
sters. Given the CPS-Structure of many pathogens and the multi-
valent recognition patterns of most glycan binding molecules,
this presents a distinct advantage in such studies, which will be
in the focus of future investigations.
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Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft
(SFB 765 – Multivalency as chemical organization and action
principle), the Emmy Noether Programm (HA 4468/2-2), the
Boehringer-Ingelheim Foundation (Plus 3 Programm), the Fonds
der Chemischen Industrie and the COST Action CM0802, for
the financial support and the Swiss National Science Foundation
for the postdoctoral fellowship to TM (PBEZP2-133274).
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