Chemoselective bioconjugation of triazole phosphonites in aqueous media

Article abstract of DOI:10.1002/chem.201404690

Readily accessible and versatile phosphonite building blocks with improved stability against hydrolysis were used for the efficient metal-free functionalization of peptides and proteins in aqueous buffers at low micromolar concentrations. The application of this protocol to the immobilization of a Rasa1-SH2 domain revealed high binding affinity to the human T-cell protein ADAP and supports the applicability of triazole phosphonites for protein modifications without harming their function.

Full text of DOI:10.1002/chem.201404690

DOI: 10.1002/chem.201404690
Communication
&
Bioconjugation
Chemoselective Bioconjugation of Triazole Phosphonites in
Aqueous Media
M. Robert J. Vallꢀe,[a] Paul Majkut,[a] Dagmar Krause,[a] Michael Gerrits,[c] and Christian
P. R. Hackenberger*[a, b]
is still of high interest to ensure high conversions and for the
Abstract: Readily accessible and versatile phosphonite
building blocks with improved stability against hydrolysis
attachment of diverse functional modules to biopolymers.
Our group has recently introduced the Staudinger-phosphite
were used for the efficient metal-free functionalization of
reaction as a metal-free chemoselective modification reaction
peptides and proteins in aqueous buffers at low micromo-
for azido-containing biomolecules.[8] While phosphites are easy
lar concentrations. The application of this protocol to the
to synthesize using standard phosphoramidite chemistry, it is
immobilization of a Rasa1-SH2 domain revealed high bind-
often difficult to conjugate a single functional module to the
ing affinity to the human T-cell protein ADAP and sup-
biomolecule due to an unselective hydrolysis of the phosphori-
ports the applicability of triazole phosphonites for protein
midate.[9] To achieve a monofunctionalization of azides, we re-
modifications without harming their function.
cently introduced arylphosphonites in Staudinger reactions.
These reagents underwent faster reaction rates than phos-
phites; however, their synthesis involves several steps, which
The chemoselective modification of peptides and proteins has
become an extremely active field of research,[1] since modified
biomolecules are very promising drug candidates as well as
important tools to elucidate biological function. With many
functional groups present in a natural biological environment,
bioorthogonal modification at specific positions is of special in-
terest.[1c] In recent years, many very potent reactions have
been developed and applied in this area, in particular the Cu-
catalyzed[2] and the strain-promoted[3] alkyne–azide cycloaddi-
tion (CuAAC and SPAAC, respectively), the inverse electron
demand Diels–Alder reaction with tetrazines,[4] and Staudinger
ligations,[5] as well as metal-catalyzed modification protocols
such as olefin cross metathesis[6] or the Suzuki–Miyaura cross
coupling reaction.[7] Although all of these reactions have
become very powerful tools for the chemoselective modifica-
tion of biomolecules, all of them have their limitations, too.
Obstacles, among others, include slow reaction rates, the intro-
duction of large sterically demanding scaffolds at the conjuga-
tion site, the requirement of often non-removable toxic metal
catalysts, and the instability and therefore difficult accessibility
of the reaction partner(s) in buffered solvents due to their high
intrinsic reactivity. To address the latter issue, the identification
and engineering of new chemoselective modification reactions
complicates the incorporation of complex functional mod-
ules.[10] Subsequently, the synthetic accessibility of phosphon-
ites equipped with fluorophores, labels, poly(ethylene glycol)
groups (PEGs), or carbohydrates was greatly enhanced with
the use of borane-protected alkyne phosphonite 1 for the
metal-free modification of small azido-containing molecules
and azido polyglycerol in organic solvents.[11] The alkyne phos-
phonite 1 contains two chemical reporter units, namely the
alkyne and, after its deprotection, the phosphonite moiety. In
the first step, the alkyne reacts, in a CuAAC reaction, with an
azide carrying the functional module (label, fluorophores).
After copper removal and borane deprotection, a reactive tri-
azole phosphonite is generated, which allows a final metal-free
Staudinger reaction with a second azido molecule (Scheme 1).
Scheme 1. Sequential azide–azide coupling using alkyne phosphonite 1.[11]
[a] Dr. M. R. J. Vallꢀe, Dr. P. Majkut, D. Krause, Prof. Dr. C. P. R. Hackenberger
Leibniz-Institut fꢁr Molekulare Pharmakologie (FMP)
Robert-Roessle-Str. 10, 13125 Berlin (Germany)
In our previous publication, we demonstrated the principle
applicability of Staudinger reactions with triazole phosphonites
in water. A water-soluble benzyl azide was treated at a concen-
tration of 10 mm with 25 equivalents of diethyl triazole phos-
phonite in water and 15 vol% DMSO. Full conversion of the
azide was monitored by HPLC-UV and the desired phosphona-
midate product was isolated in 74% yield.[11] This result served
as a promising starting point to further develop modular and
E-mail: hackenbe@fmp-berlin.de
[b] Prof. Dr. C. P. R. Hackenberger
Humboldt Universitꢂt zu Berlin, Department Chemie
Brook-Taylor-Strasse 2, 12489 Berlin (Germany)
[c] Dr. M. Gerrits
RiNA GmbH, Volmerstraße 9, 12489 Berlin (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201404690.
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efficient phosphonite reagents for the chemoselective modifi-
cation of peptides and proteins in aqueous buffer systems.
The crucial parameters to ensure high conversions for the
Staudinger-phosphonite reaction in aqueous systems are water
solubility[12] and stability against hydrolysis and oxidation. Al-
though arylphosphonites display sufficient oxidation stability
due to the sp2-hybridzed CÀP bond, we recently found that ex-
changing methoxy substituents on phosphorus with diethyl-
ene glycol monomethyl ethers led to an increase in solubility
and hydrolytic stability.[10] Following this concept, we set out
to synthesize the diethylene glycol chain-substituted alkyne
phosphonite 2 and, in addition, an electron-poor benzyl-substi-
tuted alkyne phosphonite 3 (Scheme 2), to which a hexaethyl-
ene glycol chain was attached for increased water solubility.
Table 1. CuAAC of alkyne phosphonites with azido compounds.
Alkyne
phos-
phonite
R’
Triazole
phosphonite–
borane
Yield
[%]
1
2
3
4-BH3
5-BH3
6-BH3
89
68
71
3
7-BH3
71
ite 3 to deliver biotinylated triazole phosphonite 7-BH3, for pro-
tein tagging or immobilization. The complete removal of
copper was established by inductively coupled plasma mass
spectrometry (ICP–MS) for triazole phosphonites 6-BH3 and
7-BH3. For the removal of the borane protecting group,
borane-protected triazole phosphonites 4-BH3, 5-BH3, and
6-BH3 were heated in benzene or toluene to 508C with 1,4-di-
azabicyclo[2.2.2]octane (DABCO) as a base. After 24 h, 31P NMR
spectroscopy of the crude reaction mixture indicated full de-
protection to the phosphonites 4–6.
Scheme 2. Synthesis of alkyne phosphonites 2 and 3.
We started the synthesis from phosphorus trichloride and sub-
stituted in the first step two chlorides with the corresponding
alcohols in the presence of triethylamine. In the second step,
an alkyne–Grignard reagent was added, followed by in situ
protection of the phosphonite with borane. This one-pot pro-
tocol yielded alkyne phosphonites 2 and 3 in good yields of 48
and 62% (Scheme 2). The borane protection guarantees the
stability of the phosphonite during intermediate purification
by column or HPLC chromatography and additionally ensures
the sequential coupling protocol as mentioned before.
Next, we probed the hydrolysis rate of the three differently
substituted triazole phosphonites 4, 5, and 6 by 31P NMR spec-
troscopy in Tris buffer (pH 8.2) with 10 vol% DMSO (Figure 1;
see the Supporting Information for experimental procedure).
As an additional comparison we also measured the hydrolytic
stability of the two previously published aryl phosphonites (8
and 9).[10] In our measurements we observed that the half-life
time of the ethoxy-substituted phosphonite 4 was almost dou-
bled by the incorporation of the p-mOEG6-m-nitrobenzyl sub-
stituents in phosphonite 6. Furthermore, the most stable phos-
phonite was the mOEG2 substituted derivative 5. Most impor-
tantly, all three triazole phosphonites 4, 5 and 6 revealed a sig-
nificant increase in stability against hydrolysis as compared to
the previously published aryl phosphonites 8 and 9, which hy-
drolyzed to over 90% within minutes.[10]
Having three different borane-protected alkyne phosphon-
ites (1, 2, and 3) in hand, the CuAAC reaction was carried out
with different azido compounds. For the hydrolysis and the re-
activity study, azido hexaethylene glycol monomethyl ether
(mOEG6) was chosen as the first azide reagent to be attached
to the alkyne phosphonites 1, 2, and 3 for sufficient water sol-
ubility for all triazole phosphonite reagents. CuAAC was per-
formed in tetrahydrofuran with tetrakis(acetonitrile)copper(I)
hexafluorophosphate as copper(I) source (Table 1). Quantitative
formation of borane-protected triazole phosphonites 4-BH3,
5-BH3, and 6-BH3 was confirmed by 31P NMR in all cases. The
crude reaction mixtures were purified by column chromatogra-
phy for 4-BH3 or preparative high-performance liquid chroma-
tography for the other phosphonites to yield the desired tri-
azole phosphonite–boranes in good yields (Table 1). Addition-
ally, a biotin-OEG7-azide was attached to the alkyne phosphon-
The Staudinger reaction of triazole phosphonites 4, 5, and 6
was subsequently tested with a model peptide 10, containing
an N-terminal p-azidophenylalanine as well as basic (lysine and
arginine), polar (serine and glutamine), and acidic (glutamic
acid) amino acid residues. Model peptide 13, with a final con-
centration of 1 mm, underwent reaction with all three different
triazole phosphonites (4, 5, and 6) under the previously investi-
gated buffer conditions at pH 8.2 (Scheme 3). In accordance
with their hydrolysis rate, 25 equivalents of diethyl phosphon-
ite 4 were required to gain full conversion of the azido pep-
tide 10, whereas with the more stable phosphonites 5 and 6,
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bility of the triazole phosphonite independently of the polarity
of the molecule that is attached to it by CuAAC. To quantify
the Staudinger-phosphonite reaction by mass spectrometry,
the desired phosphonamidate peptide product was synthe-
sized in acetonitrile with an azido peptide containing two
D3-alanine moieties. The deuterated phosphonamidate peptide
14-D6 was added to the crude reaction mixture in a known
quantity, which allows quantification by ESI-HRMS. The inte-
grals of the obtained HRMS spectra suggest that full conver-
sion to the phosphonamidate 15 is achieved with an azido
peptide concentration of 50 mm and at least 250 equivalents of
di-o-mOEG6-m-nitrobenzyl triazole phosphonite 6 without any
side-products detected. Nevertheless, it is unlikely that no re-
duction of the p-azidophenylalanine to the corresponding
p-aminophenylalanine occurred, as observed before. When the
amount of phosphonite was decreased to 100 equivalents, the
conversion dropped to a still very good 86%. Lowering the
concentration of azido peptide 14 to 25 mm was accompanied
by a decrease in the formation of phosphonamidate 15 (34–
81%). Thus, higher concentrations of di-o-mOEG6-m-nitroben-
zyl triazole phosphonite 6 (500 equivalents) were necessary to
reach a conversion of 85%. With a concentration of 25 mm
azido peptide 4 and only 100 equivalents of di-o-mOEG6-m-ni-
trobenzyl triazole phosphonite 6, phosphonamidate formation
was observed to be around 50% (Table 2).
Figure 1. Hydrolysis rate of triazole phosphonites 4, 5, and 6 and aryl phos-
phonites 8 and 9 in Tris/HCl buffer (pH 8.2 w. 10 vol% DMSO), determined
by 31P NMR spectroscopy.
To address the applicability of the Staudinger-phosphonite
reaction for the modification of functional proteins, we decid-
ed to conjugate the biotin phosphonite 7 to a protein domain
for a qualitative determination of protein–protein interactions.
Specifically, we focused on the interaction of SH2 domains
with tyrosine phosphorylated proteins as we demonstrated
Table 2. Staudinger-phosphonite reaction at low concentrations.
Scheme 3. Modification of azido peptide 10 with different triazole phos-
phonites.
only 15 equivalents were necessary to fully convert the model
peptide 10. The desired phosphonamidate peptide prod-
ucts 11, 12, and 13 could be isolated in good yields of 45–
65% after HPLC purification. The only peptidic byproduct de-
tected was the expected reduction product of the azide to the
corresponding amine, which is formed during the hydrolysis of
the phosphorimidate intermediate of the Staudinger reac-
tion[11] and which was isolated in an average yield of 14Æ2%.
After the successful aqueous Staudinger-phosphonite reac-
tions with peptides at concentrations of 1 mm, we turned our
attention to transformations with phosphonite 6 at lower con-
centrations to elucidate suitable reaction conditions for protein
modifications at lower concentrations. We focused on 6, be-
cause it showed a superior performance in the previous experi-
ment as compared to 4 and its OEG6 chains ensure water solu-
Azido peptide
[mM]
Phosphonite 6
[equiv]
DMSO
[%]
Phosphonamidate
yield [%]
50
50
50
50
25
25
25
25
500
250
250
100
500
250
250
100
10
10
5
10
5
10
5
5
100
100
100
86
85
81
77
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Figure 2. Pull-down of ADAP with immobilized Rasa1-N by biotin conjugation with phosphonite 7. A) Coomassie stain of the SDS-PAGE with the pull-down
samples; B) excitation of the SDS-PAGE at 633 nm to monitor ADAP fluorescence; C) Western blot of the pull-down samples, detection of biotinylation with
a Streptavidin-peroxidase conjugate. Abbreviations used: UP=unbound protein (Rasa1-N) prior to pull-down; F=pull-down flow-through; W=wash fraction;
S=strip fraction releasing bound ADAP, Rasa1-N and streptavidin (St).
previously that, upon tyrosine phosphorylation, the human
T-cell protein ADAP binds to Rasa1 (RasGAP, Ras-GTPase acti-
vating protein) with extraordinarily high affinity at an equilibri-
um rate constant Kd of 100 nm.[13] The main role of Rasa1 is to
inactivate the G-protein Ras p21[14] through its C-terminal
RasGAP domain, while its N-terminus contains numerous mod-
ular binding domains (two SH2 domains, one SH3, one PH,
one C2) that allow multiple concerted interactions.[15] The inter-
action to phosphorylated ADAP is mediated primarily through
the Rasa1’s N-terminal SH2-domain (Rasa1-N, residues 181–
272).
In conclusion, we have reported the synthesis of different
alkoxy-substituted alkyne and triazole phosphonites, which
were successfully applied for the modification of peptides and
proteins in aqueous buffers. The stability of triazole phosphon-
ites against hydrolysis was significantly improved by using
OEG or electron-deficient benzyl-substituted phosphonites.
Furthermore, azido peptides were transformed at lower phos-
phonite concentrations to reveal the chemoselective modifica-
tion of azides in aqueous systems. Upon expansion of the
Staudinger-phosphonite reaction to applications on the pro-
tein level, we could show that phosphonites could successfully
be used for the site-specific immobilization of an SH2 domain
(Rasa1-N), in which the so-functionalized Rasa1-N binds to its
partner ADAP in a phosphorylation-dependent manner. These
results demonstrate the applicability of triazole phosphonites
as promising chemoselective modification reagents for pro-
teins in aqueous systems.
To generate the N-terminal SH2 domain of Rasa1 (Rasa1-N)
as an azido protein, we relied on the high-yielding cell-free un-
natural protein synthesis[13,16] combined with an amber sup-
pressor-based orthogonal system,[17] to deliver a p-azidophenyl-
alanine residue to position 2 of Rasa1-N. The Staudinger-phos-
phonite reaction with biotin-phosphonite 7 for a later immobi-
lization of Rasa1-N on Streptavidin-coated magnetic beads[18]
was performed in 50 mm Tris/HCl, 150 mm NaCl, pH 8.0. Subse-
quent incubation and pull-down with fluorescence-labeled
phosphorylated (ADAP-P) and non-phosphorylated ADAP
(ADAP-OH)[13] was conducted to elucidate phosphorylation de-
pendent binding and to probe whether the reaction conditions
employed for the Staudinger-phosphonite reaction interfere
with the proteins folding state and function. Fluorescence la-
beling of both ADAP-P and ADAP-OH was carried out by
SNAP-tag fusion.[13]
Acknowledgements
The authors acknowledge support from the Deutsche For-
schungsgemeinschaft (SFB 765 and SPP 1623), the Fonds der
Chemischen Industrie, the Einstein Foundation, and the Boeh-
ringer–Ingelheim Foundation (Plus 3 award). Prof. Dr. Christian
Freund (Freie Universitꢂt Berlin) is acknowledged for helpful
discussions in the course of these experiments.
Our results show that Rasa1-N could successfully be modi-
fied with phosphonite 7, as proven by western blot (Fig-
ure 2C). Biotinylated protein could only be detected in the
strip fractions of the pull-down, implying that the converted
protein was completely immobilized on the solid support.
Bound biotinylated and unbound non-converted Rasa1-N
could be detected in comparable amounts in the respective
fractions suggesting a conversion rate of about 50% (Fig-
ure 2A). Most importantly, the immobilized protein was able to
selectively bind ADAP-P but not ADAP-OH (Figure 2B), show-
ing that the folding state of the SH2 domain was intact, which
supports the general utility of the Staudinger-phosphonite re-
action for the modification of functional proteins.
Keywords: bioorthogonal · chemoselectivity · phosphonites ·
protein conjugation · Staudinger reaction
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Published online on && &&, 0000
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COMMUNICATION
Readily accessible and versatile phos-
phonite building blocks showed im-
proved stability against hydrolysis and
proved to be efficient reactants with
azides in metal-free Staudinger reac-
tions for the functionalization of pep-
tides and proteins in aqueous buffers at
low micromolar concentrations (see
scheme).
&
Bioconjugation
M. R. J. Vallꢀe, P. Majkut, D. Krause,
M. Gerrits, C. P. R. Hackenberger*
&& – &&
Chemoselective Bioconjugation of
Triazole Phosphonites in Aqueous
Media
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