Selective tryptophan modification with rhodium carbenoids in aqueous solution

Article abstract of DOI:10.1021/ja047272c

A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh₂(OAc)₄, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted indole products were observed in the reaction. Following initial small-molecule studies, the reaction was performed on two protein substrates. Both myoglobin and subtilisin Carlsberg were modified readily in aqueous solution, and the tryptophan selectivity of the reactions was confirmed through MS analyses of trypsin digest fragments. It was also demonstrated that myoblobin concentrations as low as 10 μM still led to appreciable levels of modification. Reconstitution experiments confirmed that myoglobin retained its ability to bind heme following modification. Copyright

Full text of DOI:10.1021/ja047272c

Published on Web 07/30/2004
Selective Tryptophan Modification with Rhodium Carbenoids in Aqueous
Solution
John M. Antos and Matthew B. Francis*
Department of Chemistry, UniVersity of California, Berkeley, California 94720-1460, and
Material Science DiVision, Lawrence Berkeley National Labs, Berkeley, California 94720
Received May 10, 2004; E-mail: francis@cchem.berkeley.edu
Scheme 1. Covalent Modification of Tryptophan Residues on
Proteins Using Metallocarbenes
The conjugation of small molecules to protein targets has
occupied a central role in the study of biological systems,1 resulting
in an ever increasing need for new reactions that can modify proteins
in a selective manner.2 Transition metal-based methods could
provide an exceptionally powerful set of tools for this purpose, as
many metal complexes can activate otherwise unreactive functional
groups with excellent selectivity. Many of these reactions exhibit
virtually complete functional group tolerance and have been used
successfully in aqueous solution.3 A particularly interesting feature
for bioconjugation is the ability to influence the reactivity of
transition metal complexes through proper selection of the metal
ion and ligand sphere. Such tuning could allow selective targeting
of one amino acid side chain over another, and the complex steric
environments provided by asymmetric ligands could provide a much
needed way to distinguish between several occurrences of a
particular residue.
Scheme 2. Modification of 3-Methylindole with Metallocarbenes in
Aqueous Media
To explore these possibilities, we have focused on the use of
metallocarbenoids (2) for bioconjugation, as these species could
react with many of the functional groups that are present on protein
surfaces.4 It was hypothesized that by tuning the reaction param-
eters, selectivity between these pathways could be achieved. We
have found that this is in fact the case, and as a result of these
studies a highly selective method for the modification of tryptophan
residues has emerged, Scheme 1. This reaction proceeds readily in
aqueous solution, and can modify protein substrates at concentra-
tions as low as 10 µM.
In preliminary studies, it was found that metallocarbenoids
derived from stabilized vinyl diazo compound5 3 can react with
3-methylindole (4) in a highly efficient manner6 despite the
anticipated reaction of the metallocarbene intermediate with water7
(Scheme 2). With only 4 equiv of diazo compound, a 1:1.4 mixture
of N-alkylated (5) and 2-alkylated (6) products was obtained in
51% combined yield, presumably through direct N-H insertion
and through the intermediacy of a cyclopropane intermediate,
respectively. The remainder of the diazo compound reacted with
water to form alcohol 7 as well as trace amounts of pyrazole 8
through a metal-independent electrocyclization pathway.8 As the
organic substrates possess little water solubility, a cosolvent was
used to achieve optimal reactivity. With the goal of developing
protein-compatible reaction conditions, we selected ethylene glycol
for this purpose, as studies have demonstrated that protein structures
are resistant to denaturation by this solvent.9 It was also found that
the addition of HONH2‚HCl dramatically enhanced the reactivity
of the catalyst, presumably by binding to the metal center and
stabilizing the reactive intermediates. The origin of this effect is
currently under study.
tolerate the large number of polar spectator groups that are present
on the surface of all proteins. The ability of the reaction to meet
these criteria was first tested on a 100 µM solution of horse heart
myoglobin in a 80:20 water/ethylene glycol mixture. The protein
solution was exposed to 100 equiv of 3 and 1 equiv of Rh2(OAc)4
at room temperature for 7 h, Figure 1a.10 After removal of the small-
molecule byproducts using gel filtration, the protein mixture was
analyzed using ESI-MS. Clean conversion to the singly and doubly
modified products was observed (Figure 1b), as would be expected
by the presence of two tryptophan residues in the amino acid
sequence. Based on ESI-MS analysis, approximately 60% conver-
sion was obtained for this reaction. Quantitative SDS-PAGE
analyses indicated excellent (>90%) protein recovery after the
reaction. Importantly, no appreciable conversion was observed in
the absence of the rhodium catalyst (Figure 1c), implicating the
intermediacy of a rhodium carbenoid species. Subsequent reactions
have indicated that similar conversion can be achieved for 10 µM
solutions of myoglobin.11
To confirm the site selectivity of the reaction unambiguously,
the protein product mixture was digested with trypsin. Analysis of
the resulting peptides using MALDI-TOF MS indicated that the
reaction occurred only on the segment containing the two tryptophan
residues. Both the singly and doubly modified fragments were
further subjected to MS/MS analysis, which confirmed modification
at W7 and W14. The secondary ion spectrum for the doubly
modified fragment is shown in Figure 1d. In addition to the full
y-ion series expected for the doubly modified peptide, a diagnostic
i-ion for the modified tryptophan12 (9, m/z 465) can be observed.
Although the low pH of the reaction media (ca. 3.5) caused
dissociation of the heme and presumably changes in the tertiary
To modify tryptophan residues on protein substrates, two
additional criteria needed to be met. First, the reaction must be
viable at substrate concentrations of 100 µM and below. Second,
both the metallocarbenoid and the rhodium catalyst itself must
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C O M M U N I C A T I O N S
denaturation of the protein, which increases the solvent accessibility
of the tryptophan residue.
These initial studies indicate that transition metal-catalyzed
reactions are indeed capable of modifying native protein functional
groups with very high selectivity.14 The reaction described herein
represents one of the first methods for selective tryptophan
bioconjugation and one of the first examples of a metallocarbene-
based reaction in aqueous solution. Current efforts are focused on
increasing the pH of the reaction to broaden the substrate scope.
In light of the relatively low abundance of tryptophan residues
on protein surfaces, this technique offers a selective bioconjugation
strategy that complements more commonly used cys- and lys-based
reactions. This possibility is currently being explored for the
functionalization of introduced tryptophans on expressed proteins.
Furthermore, as tryptophan residues often occur in binding sites
and can serve as mediators of electron transfer,15 this reaction
provides a new tool to alter or block their participation in these
pathways.
Acknowledgment. We gratefully acknowledge the University
of California, Berkeley, the Nanoscale Science, Engineering, and
Technology Program (NSET), and the Center for New Directions
in Organic Synthesis. CNDOS is supported by Bristol-Myers Squibb
as a Sponsoring Member and Novartis Pharma as a Supporting
Member. J.M.A. was supported by a Berkeley Fellowship for
Graduate Study. We gratefully acknowledge Waters Inc. for access
to a Q-TOF Micro mass spectrometer, and Jacob M. Hooker for
his analysis expertise.
Supporting Information Available: Full experimental procedures
and characterization data for all compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 1. Modification of myoglobin with metallocarbenes. (a) A 100 µM
solution of horse heart myoglobin was exposed to 3 and Rh2(OAc)4 for 7
h. The two tryptophan residues are shown in green. (b) Following removal
of the small molecules via gel filtration, the sample was analyzed by ESI-
MS. Both singly and doubly modified protein products were identified in
the mass reconstruction. (c) In the absence of Rh2(OAc)4, no products were
obtained under otherwise identical conditions. (d) After digestion with
trypsin, MS/MS analysis of the doubly modified peptide fragment confirmed
modification of only the tryptophan residues. All assigned species agree to
within 0.1% of the expected mass values.
References
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Figure 2. Modification of subtilisin Carlsberg with rhodium carbenoids.
Conditions: 100 µM protein, 10 mM 3, 100 µM Rh2(OAc)4 and 75 mM
HONH2‚HCl (pH 1.5) in 80% water/20% ethylene glycol, rt, 7 h. Following
removal of the small molecules via gel filtration, the sample was analyzed
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as would be expected by the single tryptophan residue (shown in green).
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protein structure,13 subsequent reconstitution experiments confirmed
that the modified myoglobin was still competent to bind the heme
group.11
Attempts to modify subtilisin Carlsberg, a protein possessing a
single tryptophan residue, were unsuccessful under analogous
conditions. However, by lowering the pH of the reaction to 1.5,
clean conversion to a singly modified product was observed, Figure
2. Trypsin digest analysis again confirmed that the reaction occurred
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W113. The higher conversion at lower pH presumably results from
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(11) See Supporting Information for details and spectra.
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(14) The ability of the reaction to modify cysteine and methionine residues is
currently being evaluated.
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Y. Chem. ReV. 2003, 103, 2167-2201.
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