Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites

Article abstract of DOI:10.1039/b926818a

Unsymmetrical phosphites react efficiently in a Staudinger reaction with p-azido-phenylalanine, which can be applied for the biotinylation of proteins, thereby expanding the scope of the chemoselective Staudinger-phosphite reaction of aryl azides with symmetrical phosphites to the corresponding phosphoramidates. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b926818a

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Site-specific functionalisation of proteins by a Staudinger-type reaction
using unsymmetrical phosphiteswz
ab
Verena Bohrsch,y Remigiusz Serwa,ya Paul Majkut,a Eberhard Krausec and
¨
Christian P. R. Hackenberger*a
Received (in Cambridge, UK) 21st December 2009, Accepted 19th February 2010
First published as an Advance Article on the web 11th March 2010
DOI: 10.1039/b926818a
Unsymmetrical phosphites react efficiently in a Staudinger
reaction with p-azido-phenylalanine, which can be applied for
the biotinylation of proteins, thereby expanding the scope of the
chemoselective Staudinger-phosphite reaction of aryl azides with
symmetrical phosphites to the corresponding phosphoramidates.
The development of methods that allows the decoration of
biopolymers with functional probes serving either to visualise
biological processes or to alter specifically their behaviour
holds mutual interest for biologists and chemists.1–5 Various
biochemical methods for the installation of an unnatural
chemical reporter on biological molecules have been
developed,6–9 which could be addressed in a chemo-selective
transformation.4,10–12 Most commonly, azides are employed
as chemical reporters in such bioorthogonal reactions. The
formation of triazoles as a result of a [3+2]-cycloaddition
between azides and alkynes was shown to be very efficient,10,12
but it usually requires the presence of cytotoxic Cu(I) catalysts,
thus limiting its in vivo applicability.4,13 A strain-promoted
triazole formation was introduced as a metal-free alternative,
which however inevitably introduces a large unnatural linkage
between the macromolecules and the functional module, that
may influence biological properties.14–16 The Staudinger ligation,
which is based on the reaction between azides and phosphines,
has also shown great potential in the labelling of biomolecules
in vivo,11,17 although sometimes the oxidation of phosphines
limits its utility.18 Recently, we have introduced the
Staudinger-phosphite reaction as an alternative, chemoselective
transformation of azides, which utilises the reactivity of air-
stable phosphites, and can be performed in water at room
temperature.19 Another advantage of the reaction is the
phosphoramidate linkage, which mimics natural occurring
phosphoric esters. In addition to the previously published
modifications of polypeptide azides 1 with symmetrical
phosphites 2, we report herein the first examples of
Staudinger-phosphite ligation between an azido amino acid
and easily accessible unsymmetrical phosphites 4 carrying only
Scheme 1 Functionalisation of azido-proteins 1 with symmetrical 2
and unsymmetrical 4 phosphites.
one functionalised substituent (Scheme 1). The obvious
advantage of using unsymmetrical ligation partners results
from the molecular economy, especially for complicated
functionalities, and the steric properties. The presence of three
bulky substituents on the phosphorous may hinder quantitative
conversion of shielded azido groups. The proximity of two
identical functionalities also may cause interactions, e.g. an
autoquench in the case of flourescence dyes. However, a
potential pitfall is associated with it, lying in the possible
and undesired hydrolysis of a functional substituent in
phosphorimidate intermediate 5, leading to an unfunctionalised
side product 6. Depending on the choice of substituents
(R and R0), different fractions of phosphoramidate products
6 and 7 are expected to be obtained.
Although the coupling between azides and unsymmetrical
phosphites has been applied in the preparation of small
molecules in organic solvents,20–25 including unnatural mono-
and dinucleotides,22–24 the utilisation of the transformation as
means for modification of macromolecules in aqueous
media has so far not been investigated. Furthermore, the
exact distribution of phosphoramidate products of these
transformations was often not addressed nor quantified.
Therefore, we decided to investigate the relative propensities
of simple substituents to be expelled during the hydrolysis of
phosphorimidate 5. We synthesised unsymmetrical model
phosphites that contained two methyl groups in addition to
a short (4a), long (4b), branched (4c) or functionalised (4d)
hydrocarbon chain, as well as phenyl (4e) and benzyl (4f)
substituents. All of these compounds were prepared in moderate
to good yields from the easily accessible phosphoramidate 8
a Institut fur Chemie und Biochemie, Freie Universitat Berlin, Takustr. 3
14195 Berlin, Germany. E-mail: hackenbe@chemie.fu-berlin.de
¨
¨
b Beuth Hochschule fur Technik Berlin, Fachbereich Life Sciences &
¨
Technology, Labor fur Biochemie, Seestr. 64, 13347 Berlin, Germany
¨
c Leibniz-Institut fur Molekulare Pharmakologie, Robert-Rossle-Str. 10
13125 Berlin, Germany
¨
¨
w This paper is dedicated to Prof. Jurgen H. Fuhrhop on the occasion
of his 70th birthday.
z Electronic supplementary information (ESI) available: Experimental
procedures and analytical data. See DOI: 10.1039/b926818a
y These authors contributed equally to this work.
ꢀc
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Table 1 The preparation of unsymmetrical phosphites
Phosphite
ROH
Isolated yield (%)
4a
4b
4c
EtOH
DecOH
iPrOH
89
94
90
Scheme 2 The biotinylation of amino acid 13 in Tris buffer (pH 8.2).
more readily. The results obtained for the transformation of
alkyl- and phenyl-substituted phosphites are in agreement
with the predicted trend. However, the observation of the
benzyl group (4f) leaving with unexpected ease suggests the
involvement of an alternative mechanism, in which heterolysis
of a C–O bond to form a stabilised carbocation occurs as the
first step. This result is in agreement with a previous
report that questioned the common belief for general Arbuzov
reactions to proceed via SN2 mechanism.32
4d
49a
4e
4f
PhOH
BnOH
93
87
a
For details on isolation and storage of the biotin phosphite 4d,
see ESIz.
and alcohols 9a–f via tetrazole-mediated substitution at a
phosphorous atom (Table 1).26–28 Unsymmetrical phosphites
4a–f were reacted with N-Fmoc-p-azido-phenylalanine 10 in
aqueous DMSO to ensure the solubility of all phosphites 4,
and the ratio between dimethylphosphoramidate 11 and a
mixed phosphoramidate 12 was monitored using LC-UV.29
From the results compiled in Table 2, it can be seen that alkyl
substituents of phosphites 4a–d (entries 1–4) are less prone to
hydrolysis than methyl groups, whereas phenyl (4e) and benzyl
groups (4f) (entries 5 and 6) are relatively good leaving groups.
Phosphorimidate hydrolysis has been discussed in the litera-
ture. Based on 18O-labelling experiments, it was suggested that
the hydrolysis proceeds via nucleophilic attack partially at the
sp3 hybridised a-carbon (if present) and partially at the
phosphorous atom.30,31
Additionally, to confirm that the distribution of products 11
and 12 does not depend on the stage of conversion, azide 10
was incubated with phosphite 4e, and the reaction was
monitored at intervals of one hour. The product distribution
was consistent throughout the course of transformation,
suggesting that secondary reactions of phosphoramidates do
not occur in the system (ESIz). Since the ultimate advantage
of a chemoselective Staudinger-phosphite reaction is the
possibility for its conduction in aqueous media, water soluble
p-azido-phenylalanine 13 was reacted with the biotin
phosphite 4d in Tris buffer without the addition of co-solvents
(Scheme 2). Biotinylated phosphoramidate 14 was the major
product in this transformation, and the fraction of 14 with
respect to phosphoramidate byproduct 15 (86 : 14) was even
greater than that observed in the buffer containing DMSO
for the Fmoc protected amino acid (Scheme 2). When the
transformation was repeated on a slightly larger scale,
biotinylated amino acid 14 was isolated in good yield (41%)
and its identity was confirmed by HRMS and NMR.
If this were to be the case, substituents containing more
electrophilic a-carbons should be prone to an enhanced attack
by nucleophiles, and more stabilised alkoxides should be
expelled from the pentavalent phosphorous transition state
Table
compound 10
2
Reaction of the mixed phosphites 4a–f with model
Encouraged by these results, we applied 4d to the biotinyl-
ation of a model protein (16) containing a single p-azido-
phenylalanine residue, which was incorporated via unnatural
protein translation in E. coli lysate.19 Since 16 presented
limited water solubility,33 it was incubated with 4d in
phosphate buffer containing DMSO. In order to probe the
occurrence of non-specific binding between protein 16 and the
biotin module, the protein was incubated with the chemically
inert alcohol precursor to 4d, biotinol 9d. Modification of
protein 16 was performed overnight at ambient temperature
and was followed by SDS-PAGE separation and subsequent
transfer of the protein content onto a PVDF membrane. The
membrane was then subjected to incubation with a streptavidin
horseradish peroxidase conjugate, and the interactions of
streptavidin with biotin, locally present on the surface of the
membrane, were detected in a luminol-based luminescence
assay (Fig. 1A). As can be deduced from the figure, biotin
was incorporated into protein 16 (lanes 1 and 2) in the
presence of phosphite 4d. On the other hand, as expected,
incubation of the protein with biotinol (lane 3) or the reaction
buffer (lane 4) did not result in protein biotinylation. The
Entry Phosphite
R
11a (%) 12a (%) Errorb (%)
1
2
4b
4c
Dec
iPr
15
21
85
79
1
2
3
4d
23
77
1
4
5
6
4a
4e
4f
Et
Ph
Bn
29
50
72
71
50
28
1
2
2
10 (10 mM) was reacted with 4a–f (30 mM) in DMSO containing 10%
a
of Tris buffer (1 M, pH 8.2) for 6 h at RT. Conversion: fraction of
products calculated based on LC-UV analysis, see ESIz for details;
b
reactions were repeated at least three times; error = standard
deviation of the performed measurements.
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1 The biotinylation of azido-protein 16 in aqueous media.
(A) Luminescence image of streptavidin-recognised protein on a Western
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membrane was stained with Ponceau S (Fig. 1B) in order to
visualise the total protein content and ensure proper protein
transfer from the gel. Additionally, MS analysis performed on
the whole protein (upon incubation with 4d) revealed the
covalent nature of the linkage between the protein and the
biotin moiety (ESIz).
In conclusion, unsymmetrical phosphites appear to be
useful reagents for the modification of proteins via aqueous
Staudinger-phosphite based ligations. Thereby, we showed
that not only symmetrical but also unsymmetrical phosphites
can be used as efficient modifiers of aryl azides. With an
application of model reagents, it was presented that, with
the exception of benzyl groups, a general trend can be
observed for the ability of substituents on a phosphite to
depart from phosphorimidates generated as primary products
of the Staudinger-phosphite reaction. An unsymmetrical
phosphite substituted with two methyl groups and a biotin
moiety was prepared in good yield, and was successfully
applied as a reagent for biotinylation of a model azido-protein
in aqueous solution. The applicability of the method for the
biotinylation of proteins was confirmed by the strong affinity
of streptavidin to a site-specific biotin phosphoramidate protein
conjugate, as demonstrated in a Western blot assay. Thereby,
it appears that the Staudinger-phosphite ligation is a promising
metal-free alternative to commonly utilised bioconjugation
methods. Currently, we are further working on the preparation
of phosphites for modification of azido-proteins with different
labels and functionalities.
The authors acknowledge financial support from the
German Science Foundation (Emmy-Noether program,
HA 4468/2-1), SFB 765, BMBF, the Max-Buchner Stiftung
and the Fonds der chemischen Industrie (FCI). We also thank
Iris Claußnitzer, Michael Gerrits and Heike Stephanowitz for
their help.
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buffer upon storage. Addition of DMSO helped to solubilise the
protein.
Notes and references
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This journal is The Royal Society of Chemistry 2010
3178 | Chem. Commun., 2010, 46, 3176–3178