Staudinger-phosphonite reactions for the chemoselective transformation of azido-containing peptides and proteins

Article abstract of DOI:10.1021/ol2020175

Site-specific functionalization of proteins by bioorthogonal modification offers a convenient pathway to create, modify, and study biologically active biopolymers. In this paper the Staudinger reaction of aryl-phosphonites for the chemoselective functiona

Full text of DOI:10.1021/ol2020175

ORGANIC
LETTERS
2011
Vol. 13, No. 20
5440–5443
Staudinger-Phosphonite Reactions for the
Chemoselective Transformation of Azido-
Containing Peptides and Proteins
ꢀ
€
M. Robert J. Vallee, Paul Majkut, Ina Wilkening, Christoph Weise, Gregor Muller, and
Christian P. R. Hackenberger*
€
Institut fu€r Chemie und Biochemie, Freie Universitat Berlin, Takustr. 3, 14195 Berlin,
Germany
hackenbe@chemie.fu-berlin.de
Received July 26, 2011
ABSTRACT
Site-specific functionalization of proteins by bioorthogonal modification offers a convenient pathway to create, modify, and study biologically
active biopolymers. In this paper the Staudinger reaction of aryl-phosphonites for the chemoselective functionalization of azido-peptides and
proteins was probed. Different water-soluble phosphonites with oligoethylene substituents were synthesized and reacted with unprotected azido-
containing peptides in aqueous systems at room temperature in high conversions. Finally, the Staudinger-phosphonite reaction was successfully
applied to the site-specific modification of the protein calmodulin.
The selective transformation of a particular functional
group inthe presence of additional chemicalfunctionalities
by chemoselective reactions is an important tool in organic
synthesis1 as well as chemical biology.2 In addition to
simplifying synthetic routes for the synthesis of natural
products, these reactions allow the site-specific modifica-
tion of proteins by selectively conjugating functional modu-
les to proteins, which carry bioorthogonal reporters.3 In
combination with the many advances for the introduction
of bioorthogonal functionalities into biopolymers,4 this
concept has proven to be especially useful in the area of
proteomic research, in particular for elucidating the role of
posttranslational modifications in key biological processes
such as cellular recognition and signal transduction.3,5
Over the past years many bioorthogonal reactions have
been identified and applied to the transformation of azides
which can be easily introduced into biopolymers.2,6 Pro-
minent examples include 1,3-dipolar cycloadditions, such
as the Cu(I)-catalyzed reaction of azides with alkynes
(“click reaction”).6,7 To address the toxicity of the Cu-
catalyst, reactions with highly reactive strained alkynes8
and Staudinger reactions with phosphines9 have been
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r
10.1021/ol2020175
Published on Web 09/29/2011
2011 American Chemical Society

developed, which can be applied even for modifications in
vivo.10 Nevertheless, there is still a great demand for the
development of new metal-free reactions in aqueous buf-
fers, for which the azide reaction partner is easily acces-
sible, allowing a straightforward conjugation of various
functional modules to biomolecules to gain deeper insight
into their function and operational mode.
Recently, we have identified the Staudinger-phosphite
reaction as a chemoselective transformation of azides
under physiological conditions.11,12 In the current study,
we introduce phosphonites as another type of P(III)-
reagents for the chemoselective functionalization of azido-
biopolymers. Our main motivation was to employ the high
intrinsic reactivity of phosphonites in Staudinger reac-
tions, which have been mainly used for transformations
in organic solvents in the past.13 Additionally, phospho-
nites have the potential to transfer a single functional
module or label that is attached to the carbon chain at
phosphorus to an azido-containing biopolymer.12b How-
ever, since alkyl-phosphonites 1 appeared to oxidize ra-
pidly, we focused on aryl-substituted analogues 2, in which
the sp2-hybridized carbon at phosphorus accounts for a
higher stability upon air exposure (Scheme 1).
transformations from the literature,13,14 an analogous
reaction with a water-soluble unprotected arylazido-peptide
3a delivered only a very moderate conversion of the peptide
(see Scheme 1 and Table 2, entry 1) as monitored by HPLC-
MS (see SI). Nevertheless, a phosphonamidate-peptide
obtained from 2a did not show signs of decomposition
under physiological (pH 7.6À8.2, 26 h) or HPLC conditions
(1% AcOH in AcCN/H2O, 3 h), indicating the stability of
the Staudinger-phosphonite conjugates (see SI).
Table 1. Synthesis of Water-Soluble Phosphonites
Scheme 1. Oxidation of Phosphonites and Staudinger-Phos-
phonite Reaction of Peptides
a Substituent in meta position. b Substituent in para position. c HPLC
purified.
In our investigations, the reactivity of phosphonites 2
with azides was probed. First, we used the commercially
available dimethyl phenylphosphonite (2a), which is how-
ever only partially soluble in water. Although reactions of
benzyl azide in organic solvents proceed in high yields to
the corresponding phosphonamidate (see Supporting In-
formation (SI)), which is in accordance with similar
To enhance the water solubility of the phosphonite, we
decided to synthesize aryl-phosphonites 2bÀh, in which
different oligo(ethylene glycol)(OEG)-substituents were at-
tached to the aromatic ring and the phosphorus atom. For
2bÀe, which contain the OEG-moiety only at the aromatic
ring, meta- or para-bromophenol was reacted with tosylated
tri- or tetraethylene glycol to yield 5aÀc (see SI). The
phosphorus atom was then introduced by halogenÀ
lithium exchange and reaction with either trimethyl phos-
phite to yield 6aÀcor diethyl chlorophosphite for 6d in yields
between 44 and 79% (Table 1). It is important to note that
the phosphonites were obtained as borane adducts in a one-
pot process to allow easy purification and prolonged storage.
Thereby, borane-protected phosphonites could be stored at
4 °C for more than a year without signs of decomposition.
Removal of the borane group was accomplished by heating
(10) (a) Hong, V.; Steinmetz, N. F.; Manchester, M.; Finn, M. G.
Bioconjugate Chem. 2010, 21, 1912–1916. (b) Laughlin, S. T.; Baskin,
J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664–667.
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(11) (a) Serwa, R.; Wilkening, I.; del Signore, G.; Muhlberg, M.;
Claußnitzer, I.; Weise, C.; Gerrits, M.; Hackenberger, C. P. R. Angew.
Chem., Int. Ed. 2009, 48, 8234–8239. (b) Boehrsch, V.; Serwa, R.;
Majkut, P.; Krause, E.; Hackenberger, C. P. R. Chem. Commun. 2010,
46, 3176–3178.
(12) For reviews on the Staudinger reaction, see: (a) Gololobov,
Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981, 37, 437–
472. (b) Koehn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43,
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3106–3116. (c) Schilling, C. I.; Jung, N.; Biskup, M.; Schepers, U.; Brase,
S. Chem. Soc. Rev. 2011, 40, 4840–4871.
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Petrovskii, P. V. Zhurnal Obshchei Khimii 1987, 58, 1967–1973.
(b) Malenko, D M.; Kasukhin, L. F.; Ponomarchuk, M. P.; Gololobov,
Yu. G. Zhurnal Obshchei Khimii 1979, 50, 1950–1957.
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Commun. 2011, 47, 349–351.
Org. Lett., Vol. 13, No. 20, 2011
5441

6aÀd to 50 °C with DABCO delivering phosphonites 2bÀe
in 46À67% yields (Scheme 2).15 Subsequent conversion of
azido-peptide 3a with the obtained free phosphonites 2bÀe
in aqueous buffer at room temperature was again deter-
mined by HPLC-MS (Table 2). Peptide conversion with
phosphonites carrying methoxy or ethoxy groups at phos-
phorus was between 28 and 33% with 100 equiv. Only higher
amounts of phosphonite (500 equiv) led to a moderate
product formation. The best conversion rate of 70% was
measured for 2d (Table 2, entry 6) carrying a tetraethylene
glycol chain, which supports the importance of the phos-
phonite solubility in water for a successful transformation.16
Along those lines, phosphonite hydrolysis to a phosphinic
acid ester was observed by 31P NMR as another limiting
factor in the Staudinger-phosphonite reaction. Conse-
quently, we addressed these two issues by introducing OEG-
groups at phosphorus to further enhance the water solubility
and reduce the probability of nucleophilic attack at the
phosphorus.
the highest stability, in which solubilized phosphonite was
present after more than 24 min (Figure 1).16
In contrast to the previous conversion studies, 2fÀh
showed significantly higher conversion rates. Phosphonite
2f containing ethylene glycol chains at the phosphorus
doubled the conversion rate (Table 2, entry 8) as compared
to the methoxy derivative 2b (Table 2, entry 1). The best
conversions between 86 and 95% could be achieved with
500 equiv of 2g and 2h. It is also important to note that the
reaction appeared to be very fast and a conversion of 89%
could already be observed after 20 min. Additionally, we
could show that the deprotected phosphonite could be
used without additional purification, since similar conver-
sions were observed when crude phosphonite 2h was used
in the reaction (Table 2, entry 14).
Scheme 2. Deprotection of Borane-Protected Phosphonitesa
a R: Me, Et, or (CH2CH2O)xMe; x = 3 or 4.
For the synthesis of the second set of phosphonites 2fÀh
bis(diethylamino) phosphines 7aÀb were accessed first by
reaction of the lithiated aryl species with bis(diethylamino)
chlorophosphine (Table 1). The common reaction condi-
tions for the synthesis of phosphites using 1 equiv of
tetrazole were found to be inefficient. Reducing the amount
of tetrazole to 1 mol % and simultaneous heating of the re-
action mixture finally led to a protocol that proved to be
very suitable. Borane-protected phosphonites with one (6e)
or two ethylene glycol units (6f and 6g) at phosphorus were
achieved in good yields, further deprotected as stated before
and used in reactions with the azido-peptide 3a (Table 2).
31P NMR measurements revealed that replacing the
methoxy group at the phosphorus (in 2b) with a diethylene
glycol chain (in 2g) increased the solubilized amount of
phosphonite from 4% to 14%. The solubility was even
further increased by a tetraethylene glycol chain at the
aromatic system in 2h, which led to 45% dissolved phos-
phonite in buffer after 4 min. Dissolved 2b was almost
completely decomposed after less than 10 min, whereas 2g
already showed higher stability. Remarkably, 2h displayed
Figure 1. Hydrolysis and solubility of phosphonites. aValues
were obtained by 31P NMR in Tris/HCl buffer at pH = 8.2 with
disodium hydrogen phosphate as external standard. bNMR
measurements were started at the indicated time.
Since our investigations focused so far on the azido-
peptide conversion, we next probed if side reactions with
the other peptide functionalities occur or if other reaction
products than the expected phosphonamidates 4 are
formed. Therefore, we prepared a fluorophore-containing
peptide 3b by SPPS incorporation of an ε-NBD-lysine
building block into the model peptide. This fluorescent
marker allowed the identification and quantification of
peptidic byproducts. Subsequent treatment of the peptide
3b with 2h revealed that the only by-product formed during
this reaction was the corresponding amino-peptide (see SI).
Finally, we attempted to transfer the Staudinger-phos-
phonite reaction to the protein level. For this purpose we
chose calmodulin with a p-azidophenylalanine (Pap) at
position 2 as the model protein 8. Calmodulin is a 17 kD
calcium-binding protein found in all eukaryotes, which
(15) Deprotected phosphonites can be extracted with n-hexane (see
SI). This protocol can however lead to lower yields for derivatives with
many OEG-groups.
(16) For a related report on the stabilty and water solubility of phos-
phites, see: Serwa, R.; Majkut, P.; Horstmann, B.; Swiecicki, J.-M.;
Gerrits, M.; Krause, E.; Hackenberger, C. P. R. Chem. Sci. 2010, 1,
596–602.
(17) Haribabu, B.; Hook, S. S.; Selbert, M. A.; Goldstein, E. G.;
Tomhave, E. D.; Edelman, A. M.; Snyderman, R.; Means, A. R. EMBO J.
1995, 14, 3679–86.
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Org. Lett., Vol. 13, No. 20, 2011

Pap2Ser mutant of calmodulin 10, which was incubated
with phosphonite 2h under the same conditions as before.
SDS-PAGE analysis of the crude reaction mixture shows
only one band for the unmodified calmodulin 10, which
demonstrates that phosphonite 2h reacted exclusively with
Table 2. Reactivity of Phosphonites in Buffer
Scheme 3. Chemoselective Staudinger-Phosphonite Reaction of
Azido-Calmodulin 8a
a SDS-Page stained with Coomassie; lane 1: Molecular Weight
Marker; lane 2: azido-calmodulin 8; lane 3: azido-calmodulin 8 reacted
with 2h to 9; lane 4: Pap2Ser mutant 10 of calmodulin; lane 5: Pap2Ser
mutant 10 of calmodulin reacted with 2h (see SI).
a Substituent in para position. b Reaction was performed with crude
deprotection product. c Reaction time: 20 min. d Peptide 3b was used:
62% product 4i and 21% amino-peptide was formed (see SI).
the azide and no unspecific interactions between the
phosphonite and the protein functionalities occurred.
In summary, we have developed the first example of a
Staudinger-phosphonite reaction for the chemoselective
transformation of azido-containing peptides and pro-
teins in aqueous systems. Several water-soluble phos-
phonites were synthesized and probed with respect to
their stability and performance in this bioorthogonal
transformation. Currently, we are developing new func-
tional phosphonites with higher stability against hydro-
lysis and higher solubility for metal-free functionaliza-
tions of different biopolymers.
plays an important role in many regulatory pathways by
controlling various kinases and phosphatases, most nota-
bly the CaM-Kinase family.17 The unnatural amino acid
was incorporated into the protein by in vitro translation in
RF1-deficient E. coli lysates using the Amber suppression
methodology.18
For the reaction with azido-calmodulin 8 we chose
phosphonite 2h, which gave the highest conversion rates
for peptides (Table 2). The crude protein reaction mixture
was first analyzed by gel electrophoresis (Scheme 3), in
which the modified calmodulin product 9 was observed by
a clear mobility shift. MALDI-TOF analysis further proved
the Staudinger-phosphonite reaction to be successful and
indicated an estimated conversion of 70% (see SI). To
additionally verify that no unspecific binding between
the phosphonite and the protein occurred, we prepared a
Acknowledgment. The authors acknowledge financial
support from the German Science Foundation within the
Emmy-Noether program (HA 4468/2-1) and the SFB 765,
the Boehringer-Ingelheim Foundation (“Plus 3 program”),
and the Fonds der Chemischen Industrie (FCI).
Supporting Information Available. Experimental pro-
cedures, compound characterization, and spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
€
(18) Gerrits, M.; Strey, J.; Claußnitzer, I.; von Groll, U.; Schafer, F.;
Rimmele, M.; Stiege, W. Cell-free Synthesis of Defined Protein Conju-
gates by Site-directed Cotranslational Labeling. In Cell-free Expression;
Kudlicki, T., Katzen, F., Bennett, R., Eds.; Landes Bioscience: Austin, TX,
2007; pp 166À180.
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