Indium mediated allylation in peptide and protein functionalization

Article abstract of DOI:10.1039/c1cc12926k

Indium-mediated allylation has been used in the site-selective functionalization of N-terminal aldehydes of peptides and proteins. This is the first demonstration of indium-mediated C-C bond formation in protein labelling studies under mild and environmentally friendly conditions.

Full text of DOI:10.1039/c1cc12926k

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COMMUNICATION
Indium mediated allylation in peptide and protein functionalizationw
Jenefer Alam, Thomas H. Keller and Teck-Peng Loh*
Received 18th May 2011, Accepted 27th June 2011
DOI: 10.1039/c1cc12926k
Indium-mediated allylation has been used in the site-selective
functionalization of N-terminal aldehydes of peptides and proteins.
This is the first demonstration of indium-mediated C–C bond
formation in protein labelling studies under mild and environ-
mentally friendly conditions.
Among them, the indium-mediated allylation discovered by
Araki, Chan and Li is one of the most popular, because of its
ability to be carried out in aqueous media under very mild
reaction conditions.7 In view of this, the reaction has been
used by our group to functionalize water-soluble compounds
such as carbohydrates.8 In connection with our interest in the
functionalization of proteins,9 we envisaged that indium-mediated
allylation reaction could be used in the chemical functionalization
of biomolecules.
Protein bioconjugation has evolved as a powerful technique
for the study of biological processes at a molecular level. Despite
its well-known application the chemistry available for such
transformation is rather narrow. One of the reasons is the limited
use of metal catalyzed reactions which can play a significant role
in expanding the tools available for protein modifications. The
requirement of aqueous media, with a narrow pH, tempera-
ture window and low concentrations (below 100 mM) to preserve
the structure and function of the biomolecule has made metal-
mediated protein functionalization a challenging process. Yet
several reports have successfully demonstrated the use of metals
in this area.1
In this communication, we describe a novel application
of indium-mediated allylation in aqueous media through the
site-selective functionalization of peptides and proteins. This
reaction furnishes a stable adduct and also expands the scope
of metal-mediated protein functionalization by the addition
of a new metal, indium (In). This was achieved through a
dual approach where initially the reactive aldehyde site at
the N-terminal was generated followed by indium-mediated
allylation reaction to label peptides and proteins.
In our initial model studies periodate oxidation of N-terminal
serine was used to furnish the N-terminal aldehyde in protected
dipeptide 1a and tetrapeptide 1b.10 We found that under
optimum reaction conditions, when the dipeptide aldehyde
2a was vigorously stirred with 2 equivalents of indium powder
and 3 equivalents of allyl bromide 4 in 0.025 M sodium
phosphate buffer (pH 7.0) for 18 h at room temperature,
the allylation product 3a was obtained in 56% isolated yield
(over 2 steps). Under the same conditions the reaction
with protected 2b proceeded efficiently with comparable yield
(53%) (Scheme 1).
In their pioneering work, Bonmafous et al. have used Ni, Mn,
Fe, Pd and Ru in the study of protein–protein interactions.2
Francis and coworkers have used rhodium carbenoids to
modify the indole functionality of tryptophan residues resulting
in a mixture of N-alkylated and C-alkylated products. The
same group also demonstrated the use of p-allylpalladium
complexes in the alkylation of phenolate oxygens of surface-
accessible tyrosines.3 Tachibana and colleagues have used Heck
coupling to modify genetically incorporated p-iodophenylalanine.4
Different variations of Cu(I)-catalyzed [3+2] cycloaddition
introduced by Sharpless and coworkers have been used to
couple genetically introduced terminal alkynes with azides.5
Elaborate genetic manipulation processes for the introduction
of coupling partners, non-selective labeling leading to product
mixtures, low conversion and the use of low pH limit the use
of many of these strategies, thus the search for new metal
mediated reactions is still important.
Metal-mediated allylation reactions are one of the most
successful nucleophilic addition reactions to carbonyls leading
to versatile homoallylic alcohols, which serve as building
blocks in the synthesis of biologically important compounds.6
Scheme 1 Indium mediated allylation of peptide aldehydes. Reaction
conditions: (i) peptide 1a, or 1b, 2 equiv. NaIO4, 10 mM sodium phosphate
buffer (pH 7.0), dark, 10 min–2 h; (ii) aldehyde 2a, or 2b, 3 equiv. of allyl
bromide 4, 2 equiv. of indium powder, 25 mM sodium phosphate buffer
Division of Chemistry and Biological Chemistry, School of Physical
and Mathematical Sciences, 1 Nanyang Walk, Block 5 Level 3,
Nanyang Technological University, Singapore 637371, Singapore.
E-mail: teckpeng@ntu.edu.sg; Fax: +65 6515 8229;
Tel: +65 6316 8899
b
(pH 7.0), rt, 18 h. Yield refers to column-purified yield of the allylation
product after two steps. cIsomeric ratios determined by 13C NMR
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc12926k
spectroscopy: 3a (50 : 50), 3b (60 : 40).
c
9066 Chem. Commun., 2011, 47, 9066–9068
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Table 1 Allylation of peptide aldehyde 2a with various allylic bromidesa
Yieldb,c
Scheme 2 Fluorescent tagging of peptide.
Entry Allylic bromide
Product
The utility of this reaction was demonstrated by the successful
coupling of rhodamine labeled fluorescent tag 19 to peptide 2a in
72% yield (Scheme 2).
56
1
50 : 50
Pyridoxal-50-phosphate (PLP, 21) mediated biomimetic
transamination reaction developed by Francis group was used
to generate the N-terminal aldehyde in proteins.11 This method
is versatile and can be used for more classes of proteins irres-
pective of their N-terminus amino acid.
50
2
3
4
21 : 24 : 23 : 22
Application of water-based indium-mediated allylation to a
protein molecule was illustrated with horse heart myoglobin.
The 153-amino acid protein has an N-terminal glycine residue.
To generate the N-terminal aldehyde, the procedure reported
by Francis group was adopted. Protein in 75 mM concen-
tration was reacted with 10 mM PLP for 20 h at 37 1C in
25 mM sodium phosphate buffer (pH 6.5). Following removal
of excess PLP with NanoSep 10 filtration devices, the product
solution was divided into two portions. One portion was
incubated with 25 mM biotin amidohexanoicacid hydrazide12
(22) overnight to trap the aldehyde and confirm the success of
transamination,12 while the other portion was incubated at
room temperature with indium (2 equiv.) and 2.85 mM allyl
bromide 4 (a solution in t-BuOH) (200 equiv.) in 25 mM
phosphate buffer (pH 7.0) after proper pipette mixing. 60%
conversion to the biotinylated product 23 was determined by
liquid chromatography–electrospray ionization mass spectro-
metry (LC-ESI-MS) after overnight reaction (Fig. 1a and b).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) analysis and Western blotting of the SDS-PAGE
gel were used to further confirm biotinylation.12 However to
our surprise there was no evidence of allylation product 24
(Scheme 3).
45
51 : 49
34
50 : 35 : 1 : 4
19
5
6
65 : 35
25
45 : 44 : 6 : 5
42
7
63 : 37
The unsatisfactory result could be ascribed to the poor
solubility of allyl bromide in water, together with a lack of
vigorous stirring. To address this problem in the first instance
the reaction was subjected to shaking on a laboratory rotisserie.
It is known that proteins will lose their stability upon stirring,13
hence the use of a cosolvent was explored. Thus the reaction was
repeated in a 50% tert-butyl alcohol/buffer system with gentle
shaking as this solvent system gave us positive results in our
Mukaiyama aldol methodology.9a This time trace amounts of
the product were obtained. After a number of experiments we
identified tert-butyl alcohol/water (40 : 60) as the best solvent,
with 54% conversion to the desired product (Scheme 3 and
Fig. 1c). The partial dissociation of the heme14 in the presence of
high tert-butyl alcohol concentration was addressed through
reconstitution experiments.12 The circular dichroism (CD) and
UV spectroscopy traces of the modified reconstituted protein
and unmodified protein were identical showing the success of
reconstitution experiments (Fig. 1d and e).
46
8
60 : 40
a
b
Reaction conditions: Scheme 1 step (ii). Yield refers to column-
c
purified yield of the allylation product. Isomeric ratios determined
by 13C NMR spectroscopy.
Further screening with a variety of allylic bromide reagents
with peptide aldehyde 2a provided allylation products with
yields ranging from 19 to 56% (Table 1). Yields comparable
to compound 4 were obtained with alkyl substituted allylic
bromides (Table 1, entries 1–3). Important functionalities
(amines, amino acids) were successfully introduced into the
peptides in the form of acrylate derivatives through this method
(Table 1, entries 5, 7 and 8). Furthermore, entry 8 in Table 1
indicates that peptide conjugated proteins could be synthesized
using this method. These results were very encouraging for the
metal mediated functionalization of proteins.
To confirm the site specificity of the modification, products
23 and 24 were subjected to tryptic digestion. The resulting
peptide fragments were then analyzed by LC-ESI-MS which
c
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N-terminal aldehydes of peptides and proteins through the
formation of a stable carbon–carbon bond allowing the protein
to be refolded without loss of structural stability. It equips the
protein with a terminal olefin handle that opens up a wide
avenue for their orthogonal modifications through ruthenium-
catalyzed metathesis.15 It complements existing methods of
introducing olefins into proteins which face the challenges of
non-selective reactions and undesired side product formation.16
The use of indium as the metal-mediator adds to the growing
repertoire of metal-mediated reactions in protein function-
alization encouraging protein chemists to further explore into
the untapped elements of the periodic table. We believe that this
method has the potential to be used for the efficient labeling of
proteins, with an array of functionalities thus aiding in the
study and manipulation of biological functions.
We gratefully acknowledge the Nanyang Technological
University and the Singapore Ministry of Education Academic
Research Fund 2010 (No. T2-2-067) and Novartis Institute
for Tropical Diseases (doctoral fellowship to J.A.) for the
financial support of this research.
Notes and references
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3 J. M. Antos and M. B. Francis, J. Am. Chem. Soc., 2004,
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4 K. Kodama, S. D. Fukuzawa, H. Nakayama, T. Kigawa,
K. Sakamoto, T. Yabuki, N. Matsuda, M. Shirouzu, K. Takio
and K. Tachibana, ChemBioChem, 2006, 7, 134.
5 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int.
Ed., 2001, 40, 2004.
Fig. 1 Allylation of myoglobin. (a–c) LC-ESI-MS data for (a) unmodified
myoglobin and the (b) biotinylated 60% and (c) allylation modified
(54%) protein products. (d) CD and (e) UV traces of modified
reconstituted and unmodified myoglobin.
6 W. R. Roush, in Comprehensive Organic Synthesis, ed. B. M. Trost,
C. H. Fleming and C. H. Heathcock, Pergamon Press, Oxford, 1991,
vol. 2, p. 1; Y. Yamamoto and N. Asao, Chem. Rev., 1993, 93, 2207.
7 C. J. Li and T. H. Chan, Organic Reactions in Aqueous Media,
John Wiley and Sons, New York, 1997; C. J. Li and T. H. Chan,
Tetrahedron, 1999, 55, 11149; S. Araki, S. Jin, Y. Idou and
Y. Butsugan, Bull. Chem. Soc. Jpn., 1992, 65, 1736.
8 T. P. Loh and G. L. Chua, Chem. Commun., 2006, 2739.
9 J. Alam, T. H. Keller and T. P. Loh, J. Am. Chem. Soc., 2010,
132, 9546; E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed.,
2009, 48, 6974; S. L. van Kasteren, H. B. Kramer, H. H. Jensen,
S. J. Campbell, J. Kirkpatrick, N. J. Oldham, D. C. Anthony and
B. G. Davis, Nature, 2007, 446, 1105; T. L. Foley and M. D.
Burkart, Curr. Opin. Chem. Biol., 2007, 11, 12.
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D. Chelius and T. A. Shaler, Bioconjugate Chem., 2003, 14, 205.
11 J. M. Gilmore, R. A. Scheck, A. P. Esser-Kahn, N. S. Joshi and
M. B. Francis, Angew. Chem., Int. Ed., 2006, 45, 5307.
12 Refer to the ESIw.
13 E. Y. Chi, S. Krishnan, T. W. Randolph and J. F. Carpenter,
Pharm. Res., 2003, 20, 1325.
14 Y. L. Khmelnitsky, V. V. Mozhaev, A. B. Belova, M. V. Sergeeva
and K. Martinek, Eur. J. Biochem., 1991, 198, 31–41.
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37, 3281; G. J. L. Bernardes, J. M. Chalker, J. C. Errey and
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Scheme 3 Indium mediated allylation of myoglobin.
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and P. G. Schultz, Angew. Chem., Int. Ed., 2005, 44, 34; D. A.
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showed that the reactions had successfully modified the protein
N-terminus.
In summary, we have described here the first demonstration
of indium-mediated allylation in the functionalization of
c
9068 Chem. Commun., 2011, 47, 9066–9068
This journal is The Royal Society of Chemistry 2011