Functionalization of peptides and proteins by Mukaiyama aldol reaction

Article abstract of DOI:10.1021/ja102733a

The scope of the catalyst-free water-based Mukaiyama aldol reaction was explored through its application to the site-selective functionalization of N-terminal aldehydes of peptides and proteins. Various functional groups were introduced under mild and env

Full text of DOI:10.1021/ja102733a

Published on Web 06/29/2010
Functionalization of Peptides and Proteins by Mukaiyama Aldol Reaction
Jenefer Alam,†,‡ Thomas H. Keller,‡ and Teck-Peng Loh*,†
DiVision of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang
Technological UniVersity, Singapore 637371, and NoVartis Institute for Tropical Diseases, 10 Biopolis Road,
#05-01 Chromos, Singapore 138670
Received April 1, 2010; E-mail: teckpeng@ntu.edu.sg
corresponding aldol products at ambient temperature without any Lewis
Abstract: The scope of the catalyst-free water-based Mukaiyama
aldol reaction was explored through its application to the site-
selective functionalization of N-terminal aldehydes of peptides and
proteins. Various functional groups were introduced under mild
acid activation.5 Because of the mild reaction conditions, this method
seemed to be perfectly suited for the funtionalization of proteins.
Scheme 1. Mukaiyama Aldol Condensation of Peptide Aldehydesa
and environmentally friendly conditions, with the first demonstra-
tion of aldol C-C bond formation in protein labeling studies. The
efficiency and speed achieved in protein labeling can be of special
interest in chemical biology studies.
The chemical modification of proteins has become an important
tool for the study of complex biological systems in cells. The
availability of such techniques is crucial for the discovery of new
therapeutic agents, as it is very difficult to design modulators of
protein function if an understanding of the basic biological
mechanism is lacking. Furthermore, this may provide understanding
of protein function in chemical genetic studies.
a Conditions: (i) peptide 1a, 1b, or 1c, 2 equiv of NaIO4, 10 mM sodium
phosphate buffer (pH 7.0), dark, 10 min-2 h; (ii) aldehyde 2a, 2b, or 2c,
2 equiv of ketene acetal 4, 10 mM sodium phosphate buffer (pH 7.0), rt,
24 h. b Column-purified yield of Mukiayama aldol product after two steps.
c Isomeric ratios determined by 13C NMR spectroscopy: 3a (50:50), 3b (60:
40), 3c (53:47).
Ideally, protein functionalization should proceed rapidly with
high yield, without perturbation of the tertiary structure, and with
selectivity for one site in the amino acid chain. These requirements
pose extreme challenges for protein chemists, and a number of
different strategies have been investigated. In principle two hurdles
have to be overcome: (1) a unique site has to be introduced into a
protein, and (2) methodology for derivatizing the unique site under
mild aqueous conditions needs to be available. A number of groups
have developed methods for introducing unnatural amino acids into
proteins,1 and Cu(I)- or strain-promoted Huisgen azide-alkyne [3
+ 2] cycloadditions, Staudinger ligations, and anaerobic cross-
coupling reactions catalyzed by transition metals have been explored
for the targeted functionalization of these modified proteins
Francis and co-workers2 have developed a versatile oxidative
deamination reaction that can be used to introduce an N-terminal
aldehyde group into proteins. The most straightforward options for
selective modification of an aldehyde are the formation of imines
and oximes. However, only oximes are stable under physiological
conditions,3 while available alternatives such as the carbon-carbon
bond-forming Pictet-Spengler reaction4 are slow and suffer from
low conversion.
Initial model studies were undertaken with protected dipeptide
1a, tetrapeptide 1b, and unprotected heptapeptide 1c, each contain-
ing serine at the N-terminus. NaIO4 oxidation of the N-terminal
serine would provide an aldehyde at that site.6 After some
optimization of the reaction conditions, we found that when the
dipeptide aldehyde 2a generated by periodate oxidation was
vigorously stirred with 2 equiv of (1-methoxy-2-methylpropeny-
loxy)trimethylsilane (4) in 10 mM sodium phosphate buffer (pH
7.0) for 24 h at room temperature, the Mukaiyama aldol product
3a was obtained in 55% isolated yield (for two steps) (Scheme 1).
Further model reactions of this particular silyl ketene acetal with
protected peptide aldehyde 2b and unprotected peptide aldehyde
2c in buffer showed that the reaction proceeded efficiently, with
the yield of product improving to 80% for the longer, unprotected
peptide (Scheme 1).
Further examination of the reaction of peptide aldehydes with a
variety of ketene acetals provided Mukaiyama aldol products with
yields ranging from 11 to 55%. In general, alkyl-substituted ketene
acetals proved to be more suitable for this application, yielding
results comparable to those for compound 4 (Table 1, entry 1).
The results in Table 1 also suggest that this method is well-suited
for the introduction of substituents containing alkenes and alkynes
(Table 1, entries 4-6), which are functional groups that can be
further modified using olefin metathesis7 or Cu(I)-promoted Huisgen
[3 + 2] cycloadditions.8 Furthermore, entry 8 in Table 1 indicates
that polyethylene glycol-conjugated proteins9 could be synthesized
using this method. Since the reaction seemed to perform better on
more complex peptides, these results were very encouraging for
the functionalization of proteins (Scheme 1).
In this paper, we describe a novel method for site-directed
functionalization of peptides and proteins using the Mukaiyama
aldol condensation in aqueous media. This reaction furnishes a
carbon-carbon bond and therefore leads to a stable product.
Furthermore, the mild conditions and the high reactivity of the
reagent provide rapid and clean transformations.
In recent years, our group has demonstrated that silyl ketene acetals
can react with various reactive aldehydes in water to afford the
† Nanyang Technological University.
‡ Novartis Institute for Tropical Diseases.
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9546 J. AM. CHEM. SOC. 2010, 132, 9546–9548
10.1021/ja102733a  2010 American Chemical Society

C O M M U N I C A T I O N S
Scheme 3. Mukaiyama Aldol Condensation of Myoglobin
Table 1. Mukaiyama Aldol Condensation with Silyl Ketene
Acetalsa
As a next step, we sought to apply the Mukaiyama aldol
functionalization to proteins. Horse heart myoglobin served as a
convenient model system.2
While the periodate method of aldehyde generation was conve-
nient for the model studies, we switched to the pyridoxal-5′-
phosphate (PLP, 21) oxidation method2 for our protein modification
reactions, since myoglobin has an N-terminal Gly that cannot be
oxidized with periodate. In the event, a 50 µM solution of the
protein was incubated with 10 mM 21 for 20 h at 37 °C in 25 mM
sodium phosphate buffer (pH 6.5). After removal of excess 21 with
NanoSep 10 filtration devices, the product solution was divided
into two portions. One portion was incubated with 25 mM biotin-
X-hydrazide10 (22) overnight to confirm the formation of the
aldehyde,10 while the other portion was incubated with 5 M silyl
ketene acetal 4 in 25 mM phosphate buffer (pH 7.0) (Scheme 3).
After overnight reaction, 60% conversion to the biotinylated product
23 (Scheme 3 and Figure 1b) was identified by liquid chromatogra-
phy-electrospray ionization mass spectrometry (LC-ESI-MS). So-
a Conditions: aldehyde 2a, 2 equiv of ketene acetal, 10 mM sodium
phosphate buffer (pH 7.0), rt, 24 h. b Column-purified yield of
Mukiayama aldol product. c Isomeric ratios determined by 13C NMR
spectroscopy.
In order to demonstrate the utility of the Mukaiyama adducts,
compound 12 was coupled to thioallyl fluorescent probe 19 under
the cross-metathesis conditions using the Hoveyda-Grubbs second-
generation catalyst as reported by Davis,6 providing compound 20
in 68% yield (Scheme 2).
Scheme 2. Functionalization of Mukaiyama Aldol Product 12a
Figure 1. Mukaiyama aldol modification of myoglobin. (a-c) LC-ESI-
MS data for (a) unmodified myoglobin and the (b) biotinylated (60%) and
(c) Mukaiyama aldol-modified (90%) protein products. (d) UV and (e) CD
traces of modified and unmodified myoglobin. (f) Steady-state kinetic
parameters for the peroxidase activities of unmodified and modified
myoglobin.
a Refer to the Supporting Information for details.
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that this newly developed reaction is well-suited for the function-
alization of proteins.
In conclusion, we have described here the application of an aldol
carbon-carbon bond formation for the functionalization of N-
terminal aldehydes of peptides and proteins. The method can be
used for the targeted introduction of a wide variety of functional
groups into specific N-terminal proteins under very mild conditions.
It has been demonstrated that in the case of myoglobin, the
functionalization of the protein can be carried out without disturbing
either the tertiary structure or, more importantly, the enzymatic
activity of the protein. The Mukaiyama aldol reaction of proteins
is very fast and highly efficient and as a consequence may be of
particular interest when the stability of the protein is a concern.
Acknowledgment. We thank Ms. Peggy Brunet-Lefeuvre
(Novartis Institute for Biomedical Research, Basel, Switzerland)
for protein mass spectrometry analyses. We gratefully acknowledge
the Nanyang Technological University and the Singapore Ministry
of Education Academic Research Fund Tier 2 (T207B1220RS) and
Novartis Institute for Tropical Diseases (doctoral fellowship to J.A.)
for financial support of this research.
Figure 2. Alkene-functionalized myoglobin 25.
dium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
analysis and Western blotting of the SDS-PAGE gel were used to
further confirm biotinylation.10
Only a trace amount of the aldol product 24 was formed under
the conditions used for the peptides (Table 1). A likely explanation
for the unsatisfactory result is the poor solubility of the silyl ketene
acetal in water, together with a lack of stirring. Since it is known
that proteins lose their stability upon stirring,11 the use of a
cosolvent was explored.
Supporting Information Available: Additional experimental pro-
cedures and spectral data for reaction products. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
Ethylene glycol is known to be a suitable solvent for protein
functionalizations.12 However, in the case of the Mukaiyama aldol
reaction, water/ethylene glycol mixtures were not suitable. After a
number of experiments, we identified tert-butyl alcohol/water (17:
83) as the best solvent, with 90% conversion13 to the desired product
24 (Scheme 3 and Figure 1c) within 1 h.
(1) (a) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974–
6998. (b) Link, A. J.; Mock, M. L.; Tirrell, D. A. Curr. Opin. Biotechnol.
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The modified protein was characterized using ESI-MS (Figure
1c). The UV and circular dichroism (CD) spectroscopy traces of
the modified and unmodified proteins were identical, suggesting
that the reaction conditions did not modify the tertiary structure of
the myoglobin (Figure 1d,e). Rate constants k1 and k3 for the ping-
pong mechanism of peroxidase defined by Dunford14 were deter-
mined for both unmodified and modified myoglobin under steady-
state conditions. The results showed that under saturating
concentrations of peroxide, the modification did not have any effect
on the reaction of myoglobin with the substrate ABTS (Figure 1f).10
Finally, to confirm the site specificity of the modification, products
23 and 24 were subjected to tryptic digestion. Analysis of the
resulting peptide fragments by LC-ESI-MS showed that the
reactions had successfully modified the protein N-terminus.10
After the successful demonstration that the Mukaiyama aldol
reaction can be applied to proteins, the amount of silyl ketene acetal
needed for the reaction was optimized. Near-quantitative conversion
could be achieved with as little as 190 equiv (0.3 µL) of the substrate.10
Similar results were obtained with silyl ketene acetal 11. A 95%
conversion to the desired product 25 was seen (Figure 2), suggesting
(8) (a) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192–3193. (b) Beatty, K. E.;
Xie, F.; Wang, Q.; Tirrell, D. A. J. Am. Chem. Soc. 2005, 127, 14150–
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(9) Parveen, S.; Sahoo, S. K. Clin. Pharmacokinet. 2006, 45, 965–988.
(10) Refer to the Supporting Information.
(11) Chi, E. Y.; Krishnan, S.; Randolph, T. W.; Carpenter, J. F. Pharm. Res.
2003, 20, 1325–1336.
(12) Khmelnitsky, Y. L.; Mozhaev, V. V.; Belova, A. B.; Sergeeva, M. V.;
Martinek, K. Eur. J. Biochem. 1991, 198, 31–41.
(13) The optimized conditions for the Mukaiyama aldol reaction with myoglobin
use 190 equivalents of silyl ketene acetal. The greater efficiency of the
reaction relative to that of the peptides (Table 1) is likely due to the greater
excess of ketene acetal. We have shown that the yield of the Mukaiyama
aldol reaction with peptides can be improved if a larger excess of ketene
acetal is used (see the experimental conditions and comments in the
Supporting Information).
(14) Dunford, H. B. In Peroxidases in Chemistry and Biology; Everse, J., Everse,
K. E., Grisham, M. B., Eds.; CRC Press: Boca Raton, FL, 1991; Vol. II,
pp 1-24.
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