Enabling Wittig reaction on site-specific protein modification

Article abstract of DOI:10.1039/c2cc35738k

An efficient aqueous Wittig reaction was enabled on protein bioconjugation for the first time. By investigating the reaction on small molecules, peptides, and proteins, a site-specific reaction targeting aldehyde tag was presented. A variety of functional groups could be introduced into the protein of interest.

Full text of DOI:10.1039/c2cc35738k

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Enabling Wittig reaction on site-specific protein modificationw
Ming-Jie Han, De-Cai Xiong and Xin-Shan Ye*
Received 8th August 2012, Accepted 24th September 2012
DOI: 10.1039/c2cc35738k
An efficient aqueous Wittig reaction was enabled on protein
bioconjugation for the first time. By investigating the reaction on
small molecules, peptides, and proteins, a site-specific reaction
targeting ‘‘aldehyde tag’’ was presented. A variety of functional
groups could be introduced into the protein of interest.
The Wittig reaction, discovered in 1954 by Georg Wittig,12
is one of the most important approaches to construction of
C–C bonds. Many efforts have been made to improve the
reaction. Recently, it was found that water can serve as an
effective solvent13 in Wittig reaction. Inspired by this, we wish
to investigate the possibility of this reaction on labelling the
aldehyde functional group at the N-terminus of proteins.
To determine the viability of Wittig reaction on proteins, the
protected dipeptide 1 was firstly chosen as the substrate for the
preliminary model reactions. As displayed in Table 1, dipeptide 1
was oxidized with sodium periodate (NaIO4) in H2O, which was
followed by Wittig reaction with different ylides using H2O–
t-BuOH as solvent. It is delightful that the reactions proceeded
very well, various functional groups were introduced, and the
reaction yields were good. For instance, alkene (entry 4) and
alkyne (entry 5) functionalities, which could be further function-
alized by olefin metathesis14 or [3+2] cycloadditions of azides
and alkynes,6,7 were introduced smoothly through this approach.
Compound 3f with the fluoro-substituted functional group
(entry 6) that may have important applications in NMR, MRI,
and position emission tomography (PET) techniques15 was
obtained in 92% isolated yield. Encouragingly, the ylides 2g
and 2h with two substituents efficiently reacted with dipeptide 1
to produce compounds 3g and 3h (entries 7 and 8), respectively,
implying that the peptide and protein could be conjugated by two
different functional groups in a single process.
The site-selective modification of proteins provides a powerful
means to understand the protein dynamics and functions in living
systems,1 to improve the pharmacokinetics of protein-based drugs,2
and to create new materials.3 Lysine, cysteine, tryptophan and
tyrosine, as the endogenous amino acids, are often chosen as the
functionalized residues of proteins.4 To this end, bioorthogonal
reactions that occur selectively and rapidly under physiological
conditions are important tools for protein modification. Although a
number of bioorthogonal reactions such as the Staudinger ligation
of azides and triarylphosphines,5 the copper(I)-catalyzed6 or strain-
promoted7 1,3-dipolar cycloadditions of azides and alkynes, the
photo-induced cycloaddition reaction between tetrazoles and
alkenes8 have been developed, the discovery of new bioorthogonal
reactions is still in great demand. A major challenge in this respect is
the dearth of unique bioorthogonal reactions that can be employed
to functionalize chemical reporters in proteins of interest. The
aldehyde functionality has been always an attractive chemical
reporter for bioconjugation reactions. Several strategies have been
developed for incorporating an aldehyde functional group into
proteins. When treated with periodate, the N-terminal Ser or
Thr residues of protein would generate an aldehyde,9 which
could be further labelled with hydrazide or aminooxy reagents,
but only the oxime adducts are stable.4c The generated aldehyde
would also react with tryptophan, known as the Pictet–Spengler
reaction, though it suffered from slow reaction kinetics.4c
Francis and co-workers developed a novel biomimetic trans-
amination to introduce the aldehyde functional group at the
N-terminus by PLP (pyridoxal-5-phosphate) oxidation.10 Remark-
ably, the aldehyde reporter was also introduced at an interior
position of the protein by installing the 6-amino acid motif
(CXPXR) which was recognized by the formylglycine-generating
enzyme.11 In this report, our efforts are focused on the bioconjuga-
tion of the aldehyde reporter.
Then, the Wittig reaction was further applied to peptide
modification. The hexapeptide 4a (H2N-Ser-Leu-Lys-Phe-Tyr-
Gln-OH) with the N-terminal Ser residue was treated with NaIO4.
Without further purification, the generated aldehyde was sub-
sequently subjected to the reaction with ylide 2h in H2O–t-BuOH
(1 : 1) at room temperature. And the reaction was completed
within 30 min, yielding the only desired product 4b (Fig. 1) as
detected by LC-MS/MS. Similarly, the reaction of pentapeptide 5a
(H2N-Ser-Val-Thr-Arg-Ala-OH) with ylide 2h under the same
conditions provided 5b as the only product. Our results demon-
strated that the Wittig reaction is highly chemoselective and can be
used to modify peptides in a site-selective manner, which implies the
potential for applications in more complex protein modification.
To explore the potential of the Wittig reaction for protein
modification, we tested this method on the chemokine inter-
leukin-8 (IL-8) (8–79) which has a serine residue at the
N-terminus. As reported in the previous literature,16 IL-8
(8–79) (30 mM) was oxidized by NaIO4 (2 eq.) for 0.5 h at
room temperature to generate the N-terminal aldehyde success-
fully. Without further purification, the mixture was treated with
State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Xue Yuan Road No. 38,
Beijing 100191, China. E-mail: xinshan@bjmu.edu.cn;
Fax: +86 10 82802724; Tel: +86 10 82805736
w Electronic supplementary information (ESI) available: Full experi-
mental details and compound characterization. See DOI: 10.1039/
c2cc35738k
c
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Chem. Commun., 2012, 48, 11079–11081 11079

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Table 1 The model reactions by using dipeptide 1a
Entry Ylide
Product
Yieldb Ratioc
1
77
3 : 1
2
3
4
5
74
90
84
85
16 : 5
2 : 1
Fig. 2 MALDI-TOF analysis of IL-8 modification through Wittig
reaction: (a) unmodified IL-8; (b) the oxidation product of IL-8;
(c) modified IL-8.
16 : 9
18 : 7
6
7
92
17 : 12
67
67
15 : 4
Fig. 3 The functionalization of myoglobin: (a) myoglobin after
oxidation; (b) myoglobin after modification by Wittig reaction.
8
E-only
To further evaluate the scope of Wittig reaction for protein
modification, myoglobin was selected as another protein model.
Since myoglobin has a glycine residue at the N-terminus, we used
the PLP oxidation method10 for the aldehyde formation. In the
same media as that used for modification of IL-8 (8–79), the
intermediate aldehyde reacted with ylide 2h at a concentration as
low as 15 mM at 37 1C for 0.5 h. After the removal of small
molecules, the reaction yielded 65% labelling of myoglobin
determined by ESI-MS analysis (Fig. 3). To confirm the site-
specific modification of protein, the modified myoglobin was
subjected to trypsin digestion. Analysis of the resulting peptide
fragments by MALDI-TOF-TOF showed that the Wittig
reaction indeed only modified the protein at the N-terminus.
Upon comparing the spectrum of modified myoglobin with
that of native myoglobin, it was found that the UV-Vis
absorbance of heme was unchanged (Fig. 4), providing good
evidence that the tertiary structure was undamaged through
the modification process.10a,17 The circular dichroism (CD)
spectra of modified and unmodified proteins were in sub-
stantial agreement, which demonstrated that the modifica-
tion through Wittig reaction did not destroy the secondary
a
Conditions: dipeptide 1 (10 mM), NalO4 (2 eq.), ylides (2 eq.),
b
c
H2O : t-BuOH = 1 : 1, rt, 1.5 h. Isolated yield. Isomeric ratios
(Z : E) determined by isolated products.
ylide 2h (50 eq.) at 37 1C for 0.5 h. As expected, only
the desired protein modification product was observed by
MALDI-TOF analysis (Fig. 2). Various solution systems
including buffers and co-solvents were screened, and H2O–
t-BuOH (4 : 1) was shown to be the best medium for this
transformation. It is noteworthy that the conjugation yielded
the complete conversion at a neutral and alkaline environ-
ment, no byproduct was detected at all.
Fig. 1 The structures of modified peptides by Wittig reaction.
11080 Chem. Commun., 2012, 48, 11079–11081
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Fig. 4 Evaluation of the influence on structure and function of
modified protein: (a) UV and (b) CD spectra of the modified and
unmodified myoglobin; (c) releasing and (d) storing oxygen function of
the modified and unmodified myoglobin.
structure of myoglobin. Furthermore, the visible spectra
(450–700 nm) of myoglobin were also measured under both
the oxidation and reduction conditions. The identical traces
indicated that its function of storing and releasing oxygen was
not influenced after the conjugation. Additionally, the colour
changes of solution also drew the same conclusion (see the ESIw).
In conclusion, the Wittig reaction was explored on the
protein modification for the first time. Various functional
groups could be chemoselectively and efficiently introduced
into peptides and proteins via Wittig reaction under mild
conditions, offering a new member to the family of bioorthogonal
reactions. Furthermore, this reaction could label proteins at a
specific site in a di-substituted manner, which would make two
different functionalities attached. Wittig reaction may become
an important approach to the functionalization of biomolecules.
Further applications of this reaction in bioconjugation are now
under investigation.
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This work was financially supported by the National Natural
Science Foundation of China (81172916) and ‘973’ grant from the
Ministry of Science and Technology of China (2012CB822100).
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 11079–11081 11081