Sulfonyl azide-mediated norbornene aziridination for orthogonal peptide and protein labeling

Article abstract of DOI:10.1039/c4cc04117h

We describe a new bioconjugation reaction based on the aziridination of norbornenes using electron-deficient sulfonyl azides. The reaction enables to attach various useful tags to peptides and proteins under mild conditions. This journal is

Full text of DOI:10.1039/c4cc04117h

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Sulfonyl azide-mediated norbornene aziridination
for orthogonal peptide and protein labeling†
Cite this: Chem. Commun., 2014,
50, 12568
Michael J. Gattner, Michael Ehrlich and Milan Vrabel*‡
Received 29th May 2014,
Accepted 27th August 2014
DOI: 10.1039/c4cc04117h
www.rsc.org/chemcomm
We describe a new bioconjugation reaction based on the aziridination nitrile imines.8a,b,e Unfortunately, the high reactivity of such systems
of norbornenes using electron-deficient sulfonyl azides. The reaction is often accompanied by reduced stability and an increased tendency
enables to attach various useful tags to peptides and proteins under toward side reactions.9 Moreover, synthetic access to these reagents
mild conditions.
often represents a considerable challenge. It is therefore desirable to
develop a methodology that not only enables robust protein labeling
Bioconjugation reactions substantially extend our ability to but also utilizes easily available starting materials.
chemically manipulate proteins. Numerous chemical strategies
Based on our experience using norbornenes as highly reactive
for the attachment of synthetic molecules to proteins have been compounds in combination with nitrile oxides, nitrile imines and
developed.1 Early approaches focused on native functional tetrazines10 we searched for reagents that do react with norbornenes,
groups present on endogenous amino acids.2 The main draw- but also meet the criteria for better synthetic accessibility. Here we
back of this approach is the lack of specificity since multiple show that aziridination of norbornenes using electron-deficient
copies of each amino acid are present in the primary protein sulfonyl azides represents an excellent balance between reactivity,
structure. Unique recognition elements can be introduced into stability and availability of the starting materials. We demonstrate
the protein structure to improve the selectivity of the ligation. that this reaction can be used for efficient labeling of norbornene-
One possibility is to add a specific amino acid sequence to the containing peptides and proteins.
target protein that can be recognized by either an appropriate
Inspired by the pioneering studies on norbornene aziridination
protein modifying enzyme3 or by specific chemical probes.4 by Franz et al.,11 we decided to investigate whether this reaction
Another approach uses the biosynthetic machinery of the cell can proceed in an aqueous environment and can be used for
for incorporation of unnatural amino acids containing artificial biomolecule labeling. Sulfonyl azides have been previously
functional groups.5 We and others have used this approach for successfully applied as reagents for detecting thiocarboxylates in
the incorporation of unnatural amino acids into proteins and bacterial proteomes12 and as ligation agents for thioacid-containing
have shown that the introduced functionality can be efficiently peptides and proteins.13 These studies clearly indicate that sulfonyl
tagged by orthogonal chemical reactions.6 The right choice of azides are compatible with natural systems. To investigate the
the reacting functional groups and the proper ligation technique reactivity of sulfonyl azides with norbornene under aqueous
plays a crucial role here.7 Among other suitable functional conditions we first performed a model reaction. We reacted
groups that enable efficient protein labeling various derivatives sulfonyl azide 1 with norbornene in water/acetonitrile (1: 1) at
of cyclooctyne, cyclooctene and cyclopropene have gained special moderate temperature overnight (Scheme 1 and Scheme S1, ESI†).
attention in the field.8 An extraordinary fast kinetic was observed After purification using semipreparative HPLC we obtained the
especially in combination with tetrazines in inverse electron- desired aziridine 2 in 78% yield. We also isolated the corresponding
demand Diels–Alder reactions or in dipolar cycloadditions with sulfonamide 3 (18%) as a by-product formed by the nucleophilic
attack of a water molecule on the aziridine and subsequent ring
opening. 2D-NMR analysis of 2 showed that only the exo-aziridine
product was formed in the reaction (see ESI†). The reaction mecha-
nism leading to the formation of the desired aziridine can involve
the initial formation of a triazole intermediate followed by the
extrusion of nitrogen. Alternatively, the aziridine formation parallels
that of epoxidation involving a concerted addition of the azide to the
double bond with a concomitant loss of nitrogen.11a No direct
Department of Chemistry, Ludwig-Maximilians-University, Butenandtstrasse 5-13,
81377 Munich, Germany
† Electronic supplementary information (ESI) available: Synthesis, experimental
details, NMR, mass spectrometry and additional analysis data. See DOI: 10.1039/
c4cc04117h
‡ Current address: Institute of Organic Chemistry and Biochemistry, Academy of
´
Sciences of the Czech Republic, Flemingovo nam. 2, 166 10, Prague, Czech
Republic. E-mail: vrabel@uochb.cas.cz; Tel: +420-220183317.
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Fig. 1 Reduction of the sulfonyl azide 1 in the presence of cysteine. The
new peak (10.3 min) was identified as the corresponding sulfonamide by
LC-MS (calc. for C10H11N2O5S [M À H]À: 271.0389, found: 271.0393).
Scheme 1 Model reaction of norbornene with sulfonyl azide 1. Yields:
78% of 2 and 18% of 3.
experimental evidence that would support or disprove any of protein labeling itself. However, considering the relatively high
these mechanisms was observed during our experiments. A more concentrations of cysteine and glutathione in cells, this side
detailed study is required to address this issue.
reactivity may hinder the use of this chemistry for in vivo
To gain more insight into the reaction kinetics we measured applications. To examine a possible reactivity of the aziridine
the bimolecular rate constant of the reaction in water/acetonitrile product with other nucleophiles such as amines or thiols we
(9 : 1) to simulate more relevant conditions required for its use on incubated 2 in 50 mM TRIS or in 50 mM cysteine solution. We
biomolecules. To ensure sufficient water solubility of the norbornene again observed only slow hydrolysis to form the corresponding
substrate we used endo-5-norbornene-2-methanol in these experi- sulfonamide 3. These experiments demonstrate that the aziridine
ments. The measurements were performed under pseudo first order moiety is stable toward these nucleophiles. Also, sulfonyl azides can
conditions using an excess of norbornene and were performed in react with nucleophilic double bonds including indole and N-methyl
triplicate (for details see ESI†). The second order rate constant was indole.15 To investigate whether this reaction takes place in
determined to be k = 1.7 Â 10À3 Æ 0.21 Â 10À3 MÀ1 sÀ1. This value tryptophan residues (Trp) we incubated compound 1 (0.5 mM)
is comparable to the rate constants of the strain-promoted azide– with Trp (0.5 mM). We did not observe any detectable reaction
alkyne cycloaddition reaction of the first generation cyclooctyne after two days (Fig. S8, ESI†). These additional experiments
derivatives that are commonly used for biomolecule labeling indicate that sulfonyl azides can be used for peptide and protein
applications.14 These promising results prompted us to further labeling in the presence of these native functional groups.
examine the reaction as a potentially new methodology for
modification of biomolecules.
To evaluate the reaction on biomolecules we next synthesized a
norbornene-containing peptide: AFDXKDKPAA, where X = endo
To investigate the stability of sulfonyl azides and products norbornene-containing amino acid 4. The peptide was incubated
formed in their reaction with norbornenes, we first incubated 1 at 50 mM final concentration with sulfonyl azide 1 (2.5 mM) in water/
and the isolated products 2 and 3 in a 50 mM MES buffer at acetonitrile (9 : 1) and the reaction was followed by MALDI-TOF
pH 5.5 (MES = 2-(N-morpholino)ethanesulfonic acid) and in spectrometry. The analysis of the reaction mixture showed complete
50 mM TRIS buffer at pH 8.5 (TRIS = 2-amino-2-hydroxymethyl- conversion of the starting peptide to the aziridinated product within
propane-1,3-diol). HPLC analysis of the mixtures did not show 35 hours (Fig. 2 and Fig. S3, ESI†). The hydrolysis of the aziridine to
any reaction or decomposition even after a prolonged incubation the corresponding sulfonamide was in this case observed only after
time (Fig. S6, ESI†). Only slow hydrolysis of aziridine 2 to the prolonged incubation time (72 hours, see Fig. S4, ESI†).
corresponding sulfonamide 3 was detected. The observed slow
Encouraged by these results we next moved to proteins. Using
hydrolysis of the originally formed aziridine in water is in fact the pyrrolysine amber suppression system we introduced the endo
not an obstacle for biomolecule labeling since the desired tag norbornene amino acid 4 into E. coli thioredoxin (Trx) and human
will stay attached to its target.
carbonic anhydrase (HCA) as model proteins.10c,16 The norbornene
Although sulfonyl azides have been previously used in the functionality was incorporated at positions Asn65 of Trx (Trx N65X)
biological context,12,13 to exclude possible side reactions with and His36 of HCA (HCA H36X) (for expression and purification
endogenous amino acids we performed additional experiments. details see the ESI†). We next synthesized sulfonyl azide derivatives
The electron deficient sulfonyl azides could react with nucleophilic 5 and 6 bearing a biotin tag or a dansyl fluorophore to examine the
groups on proteins (e.g. cysteines or lysines). Our stability study norbornene aziridination on proteins (Fig. 3). We first incubated a
showed that sulfonyl azides were not affected by amino groups 40 mM solution of Trx N65X with 50 equivalents of 5 overnight at
since the incubation of compound 1 in TRIS buffer that contains a 37 1C in 50 mM Tris (pH 7.5). The successful, almost quantitative
primary amino group did not show any reaction even at pH 8.5. labeling of Trx N65X with 5 was indicated by a gel shift of the
When we incubated 1 (0.5 mM) with cysteine (50 mM or 2.5 mM) protein band after SDS-PAGE and further confirmed by intact mass
the corresponding sulfonamide was formed as a result of azide spectrometry (Fig. 3A, estimated yield 95%).
reduction (Fig. 1, Fig. S6 and S7, ESI†). No nucleophilic substitution
In addition, after tryptic digestion and MS analysis we found
reaction was detected. This reaction again will not interfere with the expected mass of the peptide LNIDHXPGTAPK containing
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The proteins (40 mM final concentration) were incubated with
the fluorescent sulfonyl azide 6 (2 mM, 50 equiv.) in 50 mM
MES buffer, pH 5.5 at 37 1C overnight (Fig. 3B). SDS-PAGE and
subsequent fluorescence detection showed selective labeling of
the norbornene containing HCA H36X. Only after prolonged
incubation time (four days) we observed a weak fluorescent
signal in the reaction of the HCA wt protein, which could be
due to non-specific labeling. The origin of the non-specific
reaction was not clear since our investigations regarding possible
side reactions with endogenous amino acids did not show any
reaction. Moreover, the sulfonyl azides were stable under the
labeling conditions used (50 mM MES, pH 5.5). Similar non-
specific reaction was previously described in the literature, where
fluorescent sulfonyl azides were used for visualization of thiocar-
Fig. 2 Aziridination of norbornene-containing peptide AFDXKDKPAA (X =
norbornene amino acid 4) using sulfonyl azide 1. (A) Structure of norbornene
containing amino acid 4. (B) MALDI-TOF spectrum of the starting norbornene-
containing peptide (calc. mass: [M À H]À: 1279.7 Da). (C) MALDI-TOF spectrum boxylates in bacterial proteome.12 However, in this case also the
showing the formation of the aziridinated peptide (calc. mass: [M À H]À:
side reactivity could not be appropriately explained. Nevertheless,
the specific reaction with norbornene is much faster and there-
fore the side reaction can be eliminated by simply removing the
1549.7 Da). DMcalc. = 270.0 Da; DMfound = 270.1 Da; conditions: 50 mM
peptide, 2.5 mM sulfonyl azide 1, H2O : CH3CN = 9 : 1, 37 1C, 35 h.
excess reagent after the reaction. To verify the presence of the
the desired biotin modification at position X ([M + 2H]2+
=
desired fluorescent dansyl tag we digested the protein and
analyzed the peptide mixture using HPLC-MS. These data
demonstrated that the modification was present in the correct
position within the protein structure (peptide QSPVDIDTXTAK,
calc.
1188.0978, [M + 2H]2+obs. = 1188.0919, DM = 4 ppm). To further
evaluate the chemistry on proteins we used the wild type HCA
(HCA wt) and norbornene containing HCA H36X respectively.
X = norbornene amino acid 4 aziridinated by 6: [M + 2H]2+
1079.5082, [M + 2H]2+obs. = 1079.5039, DM = 4 ppm).
=
calc.
In summary, we show that norbornene aziridination using
electron-deficient sulfonyl azides can be used for orthogonal
peptide and protein labeling. The reaction proceeds efficiently
under mild conditions, does not require any catalysis and
is orthogonal to functional groups of native proteins. Since
norbornenes, sulfonyl azides and derivatives thereof are easily
accessible compounds the presented technology constitutes an
attractive alternative to currently used bioconjugation techniques
especially for in vitro applications. Further optimization and evalua-
tion of this chemistry toward its use for in vivo peptide and protein
labeling is ongoing.
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