Site-Selective Labeling of Native Proteins by a Multicomponent Approach

Article abstract of DOI:10.1002/chem.201605938

Chemical functionalization of proteins is an indispensable tool. Yet, selective labeling of native proteins has been an arduous task. The limited success of chemical methods allows N-terminus protein labeling, but the examples with side-chain residues are rare. Herein, we surpass this challenge through a multicomponent transformation that operates under physiological conditions in the presence of a protein, aldehyde, acetylene, and Cu-ligand complex. The methodology results in the labeling of a single lysine residue in nine distinct proteins.

Full text of DOI:10.1002/chem.201605938

A Journal of
Accepted Article
Title: Site-selective labeling of native proteins by a multicomponent
approach
Authors: Maheshwerreddy Chilamari, Landa Purushottam, and Vishal
Rai
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To be cited as: Chem. Eur. J. 10.1002/chem.201605938
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Site-selective labeling of native proteins by a multicomponent
approach
Maheshwerreddy Chilamari[a], Landa Purushottam[a] and Vishal Rai*[a]
Abstract: Chemical functionalization of proteins is an indispensable
tool. Yet, selective labeling of native proteins has been an arduous
task. The limited success of chemical methods allows N-terminus
protein labeling, but the examples with side-chain residues are rare.
focal challenge revolves around the tenet that difference in
nucleophilicity of multiple copies of an amino acid residue is
significantly low. In addition, the pKa of Lys residues can vary
considerably owing to its microenvironment.[13] Recently, we
reported labeling of Nα-NH2 with high degrees of chemo- and
site-selectivity (Scheme 1a).[12a] In this case, the site-selectivity
is compromised upon elevating the reaction time or
stoichiometry of electrophile in an attempt to increase the
conversions. Limitation of our previous method and other related
protocols[12c-d] was primarily due to the absence of a regulatory
tool that can allow control over reactivity without compromising
the selectivity.
Here, we surpass this challenge through
a multicomponent
transformation that operates under physiological conditions in the
presence of protein, aldehyde, acetylene, and Cu-ligand complex.
The methodology results in labeling of a single Lysine residue in
nine distinct proteins.
We argued that an apt electrophile (EA) can react in a fast
reversible step chemoselectively and render latent electrophiles
(Scheme 1b). Subsequently, the stoichiometry of nucleophile
(NuB) in a rate determining step can serve as a desired
regulatory tool. It is an imperative pre-requisite to block the Nα-
NH2 group reversibly. This will create an opportunity to
investigate whether one Nɛ-NH2 can be labeled selectively
amongst multiple Lys residues. Besides, if the electrophile (EA)
itself can decimate the nucleophilicity of Nα-NH2, additional site-
selective protection, and bio-orthogonal deprotection steps can
be avoided. In this perspective, here we report a chemo- and
site-selective coupling where a primary amine of protein (NuA),
formaldehyde (EA) and phenylacetylene (NuB) assemble to result
in single-site labeling of the protein. The formaldehyde blocks
the Nα-NH2 in the form of imidazolidinone through neighboring
group participation of backbone amide. Additionally, it creates
the latent electrophile with Lys side-chain residues in a
reversible reaction. The phenylacetylene serves as the
nucleophile after activation by the copper catalyst. The
methodology offers excellent selectivity at low micromolar
concentrations in physiological conditions. A single Nɛ-NH2
residue is labeled selectively in the presence of all the
nucleophilic proteinogenic amino acids, Nα-NH2, and multiple
copies of Nɛ-NH2. The methodology operates efficiently with a
structurally diverse set of nine proteins. The mild operational
conditions allow enzymes to retain their activity post-modification.
Attachment of tags to the proteins draws wide attention as they
provide utility platforms for biologics, biomaterials, and
biophysics.[1] Technology that can deliver single-site labeling of
endogenous proteins would be ideal to meet these requirements.
However, in the absence of enabling methods, the engineered
biosynthetic pathways have led the way. The gateway to such
protein conjugates is through nonsense suppression
technique,[2] chemoenzymatic transformations,[3] and ligations.[4]
In addition to unnatural amino acids, engineering Cys at the
desired position of protein or antibody sequence has led to their
chemoselective labeling.[5,6] These methods have evolved with
time, but they do not operate with native proteins and are limited
to systems regulated by exogenous gene expression.[2] On the
other hand, native protein modification has been largely
dependent on chemoselective transformations.[7] The low-
frequency residues such as Cys, Tyr and Trp are utilized to limit
the number of labeled sites.[7,8] The naturally abundant
proteinogenic residues with high average relative accessibility
such as Lys are off-target as they lead to a heterogeneous
mixture.[9] For instance, five Lys residues of bovine serum
albumin undergo acetylation to result in a mixture of proteins
within 3% conversion.[10] The task of site-selective labeling of
native proteins has proved daunting, yet its immense importance
has encouraged efforts in this direction.[11] In this perspective, N-
terminus α-amine (Nα-NH2) has been a confidence instilling
target due to its high reactivity.[12] However, it also obviates the
additional challenge associated with the modification of a Lys
residue (Nɛ-NH2) in presence of free Nα-NH2.
Protein is a multifunctional organic molecule that offers an
array of nucleophilic residues on its surface. The pre-requisite of
the labeling method involve the development of a chemical
transformation that would be effective in near neutral aqueous
buffer at ambient temperature. Besides, Lys residue (Nɛ-NH2)
has to compete with the other nucleophilic functionalities
(chemoselectivity) and all Nɛ-NH2 residues (site-selectivity). The
Scheme 1. Native protein modification through latent electrophile
The coupling reaction involving amines, aldehyde, and alkyne
(A3 coupling) result in propargyl amines that serve as synthetic
intermediate and natural product fragment.[14] Metal catalysis
has often led to the success of this three-component reaction.[15]
However, there are multiple challenges for its translation into a
protein compatible transformation. (a) The success of A3
coupling is dependent on the use of secondary amine as one of
[a]
Dr. Vishal Rai, Maheshwerreddy Chilamari, Landa Purushottam
Organic and Bioconjugate Chemistry Laboratory (OBCL)
Department of Chemistry
Indian Institute of Science Education and Research Bhopal
Bhauri, Bhopal 462 066, India
E-mail: vrai@iiserb.ac.in
Supporting information for this article is given via a link at the end of
the document.
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the components. Unfortunately, the primary amines proffered by
proteins constitute the class of highly challenging substrates. (b)
This multi-component process requires high reaction
concentration (0.3 M to solvent free)[16] and organic solvents.
Both the parameters are detrimental for experiments with protein
as it results in their aggregation and denaturation. (c) The
aerobic aqueous conditions required by proteins poison several
catalytic systems applied in these reactions.[17] (d) The presence
of numerous binding sites in the protein can lead to substantial
metal-protein binding.[18]
from their adducts by competitive substitution. The structure of
the labeled RNase A is unperturbed after the transformation (CD,
Figure S4 in Supporting Information). Next, the product (4a or
6a) is vortexed with proteolytic enzymes. It was surprising to
note the severely diminished activity of trypsin and α-
chymotrypsin. The control experiments (Figure S5 in Supporting
Information) confirmed that presence of CuCl compromises the
proteolytic activity of enzymes and hinders RNase A (1a)
digestion.
Table 1. Ligands to enable Cu-catalyzed A3 coupling
The re-discovery of A3 coupling was imperative for its
application to proteins in physiological conditions. Initially, we
employed a broad range of catalysts[15] [AuCl3, AuCl(PPh3),
RuCl3, InCl3, NiCl2, Ni(COD)2, and CdCl2] for examining their
efficiency with RNase
A
(73 µM), formaldehyde and
phenylacetylene in aqueous conditions (Table S1, Figure S1 in
Supporting information). Unfortunately, these catalysts were
found to be unsuccessful in resulting transformation to the
desired product. Next, we generated Cu(I) in-situ with CuSO4
and sodium ascorbate for regulated oxidation of Cu(I) to Cu(II)
(entry 1, Table 1). Here, RNase A reacted within 2 h but with
poor selectivity and resulted in mono- and bis-A3 coupling
products along with several unidentifiable compounds. Whereas
CuI led to no conversion (entry 2), CuCl as a catalyst resulted in
<5% conversion over a period of 2 d (entry 3). All the initial
attempts of catalytic transformations with proteins were either
marred with the lack of reactivity or selectivity. The experiments
with a model substrate, benzylamine (Scheme S1 in Supporting
Information), routed for the possibility of over-alkylation of amine
residues (5a/7a). Besides, formaldehyde can lead to the
formation of reactive intermediates primed for multiple reaction
pathways (Figure S2 in Supporting information).[19] Such
intermediates are also known to react with R, Y, H, W and Q
residues to result in protein-protein conjugates.[20] We observed
multiple unidentified adducts upon incubating protein with 50-
100 equivalents of formaldehyde. However, all of them were
found to be reversible during dialysis and do not inhibit or alter
the course of the reaction. On the other hand, adducts formed
with 200 equivalents HCHO require additional assistance of
hydroxylamine or acidic conditions (pH 4) for reversibility. The
interplay of HCHO concentration and reaction time allows a
window to operate without the interference from protein-protein
conjugates (Figure S3 in Supporting Information). Besides, the
alkyne is inert for proteinogenic nucleophiles in presence or
absence of a catalyst (Table S2 in Supporting Information). Next,
we argued that the poor reactivity might be culminating from the
binding of Cu with protein. The identification of protein-copper
complex (MALDI-ToF MS, Figure S1 in Supporting Information)
in reaction mixture supports this attribute. We hypothesized that
a suitable ligand might allow us to fine-tune the reactivity of
catalyst,[21] reduce its binding to the protein and favorably alter
the course of disproportionation. We identified CuCl and CuI for
the next stage screening. CuSO4/sodium ascorbate is excluded
owing to the potential alternative pathways[22] in aerobic
conditions and capabilities of ascorbate to alter the pKa of Nɛ-
NH2 unpredictably.[23]
Entry[b]
Catalyst
Time
%Conversion (4a or 6a)[c]
1
2
CuSO4/Na ascorbate
CuI
2 h
2 d
Complex mixture
0
3
4
CuCl
2 d
2 d
<5
<5
CuCl, 9 or 10 or 11 or
12 or 13 or 14
5
6
CuCl, 15
CuI, 9
14 h
3 d
3 d
3 d
3 d
3 d
3 d
3 d
>99
[d]
-
7
CuI, 10
CuI, 11
CuI, 12
CuI, 13
CuI, 14
CuI, 15
<5
7
8
9
30
38
60
90
10
11
12
[a] Phosphate buffer (pH = 7.8, 0.1 M):DMSO = 9:1. [b] RNase A (1a, 73 µM),
HCHO (2, 7.3 mM), phenylacetylene (3, 7.3 mM), CuI (7.3 mM), CuCl (7.3
mM) and ligand (29.2 mM). [c] Product is 4a or 6a. Reactions were monitored
by using MALDI-ToF MS. [d] Unidentified products.
These results shifted our focus to CuI catalyzed transformation.
The digestion of labeled protein (4a or 6a) with α-chymotrypsin
went smoothly in this case. The ɛ-amine of K31 residue (K31-Nɛ-
NHR, 6a) was identified by peptide mapping and MS-MS (Figure
1b) to be the exclusive site of modification. The site of the
modification remains unaltered in the presence of different
ligands (entries 9-12). It is exciting to note that the Nα-NH2 did
not participate in any irreversible process. We can attribute this
feature to the reversible formation of imidazolidinone that can be
traced by NMR or MALDI-ToF MS of a dipeptide Ala-Ala (Figure
S6 in Supporting information) and RNase A respectively (Figure
From
a
pool of ligands 9-15 (Table 1), 1, 10-
phenanthroline (15) resulted in excellent conversion with RNase
A (1a) in the presence of CuCl (>99% conversion, entry 5, Table
1) and CuI (90% conversion, entry 12). It was exciting to note
the formation of mono-labeled mono-alkylated product in both
the cases. The treatment of product (4a or 6a, entry 5, Table 1)
with hydroxylamine and EDTA removes formaldehyde and Cu
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1a).[12b] Phosphate buffer is the choice of solvent, and its
concentration (0.1 M) was selected to prevent the formation of
Cu-phosphate complexes. The co-solvent (DMSO) is required to
attain a homogenous solution. However, it is restricted to 10% in
the buffer so as to avoid protein denaturation and curb
competitive binding to CuI. The optimized ligand-Cu ratio of 4:1
resulted in an efficient transformation (Table S3 in Supporting
Information). The stoichiometry of formaldehyde, alkyne and
copper iodide (100 equivalents each) were optimized to achieve
high reactivity (Table S4 in Supporting Information). As
hypothesized, the stoichiometry of Cu-alkyne complex was
critical for enhancing the conversions without compromising the
selectivity (Table S4 in Supporting Information). Under the
optimized conditions, we do not observe the formaldehyde-
mediated labeling of Tyr.[24] Even though the reaction conditions
are aerobic, the desired multicomponent pathway outcompetes
protein surface throughout the process (Schiff base intermediate
and secondary amine product).
Table 2. Expanding the concept of site-selective native protein modification[a,b]
Cu-catalyzed
oxidation,
oxygenation,
and
Glaser-Hay
coupling.[25] The operational simplicity of the optimized process
is noticeable.
R = -CH2C≡CPh, [a] Phosphate buffer (pH = 7.8, 0.1 M):DMSO = 9:1. [b]
Protein (1, 73 µM), HCHO (2, 7.3 mM), phenylacetylene (3, 7.3 mM), CuI (7.3
mM) and 1, 10-phenanthroline (15, 29.2 mM). [c] Conversions based on
MALDI-ToF MS analysis. Site of modification is identified by protein
sequencing using MALDI-ToF MS. (refer Section 10 in SI).
In the pursuit to understand the generality of this methodology, it
was imperative to investigate proteins that can offer nucleophilic
residues with the diverse microenvironment. We selected
Ubiquitin (76 residues, Nα-NH2, seven Nɛ-NH2) that provides a
pair of highly reactive and competing Nɛ-NH2 residues (K48 and
K63). It was exciting to note that the reaction resulted in K48-Nɛ-
NHR (6b) with excellent chemo- and site-selectivity. Next, we
selected Lysozyme C (129 residues, Nα-NH2, six Nɛ-NH2) that
bears Nα-NH2 with reduced reactivity likely due to its coordination
with aptly positioned E7. A single site modification resulted in
K116-Nɛ-NHR (6c) without interference from competing
pathways within the reaction time. In particular, no Trp-
formaldehyde adduct is observed even in the presence of six
Trp residues. Subsequently, we selected two proteins with very
high Lys frequency. Myoglobin (153 residues, Nα-NH2, nineteen
Nɛ-NH2) results in site-selective modification of K147-Nɛ-NHR
(6d), whereas K5-Nɛ-NHR (6e) is the single site of modification
Figure 1. [a] Representative scheme for RNase A modification with MALDI-
ToF MS of RNase A (1a), imidazolidinone of RNase A, and mono-labeled
RNase A (6a). [b] MS-MS spectra of the modified peptide fragment (K*SRNL)
of RNase A after digestion with α-Chymotrypsin.
in Cytochrome
C
(Nα-NHCOCH3, nineteen Nɛ-NH2). The
efficiency of transient Nα-NH2 protection in Myoglobin is similar
to the protected N-terminus in Cytochrome C. To investigate the
competitive formation of Tyr-formaldehyde adducts, Subtilisin A
(286 residues, Nα-NH2, nine Nɛ-NH2) with thirteen Tyr residues
was considered adequate. It was pleasing to note the single-site
labeling of K264-Nɛ-NHR (6f) with the quantitative conversion.
RNase A served as a model protein as it offered a highly
reactive Nα-NH2 and ten competing Nɛ-NH2 groups. The
secondary structure of protein remains unaffected (CD
experiment, Figure S4 in Supporting Information) in the process
of site-selective labeling under the optimized conditions. It is
potentially due to the unperturbed charge distribution on the
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(123
residues,
Nα-NH2,
twelve
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In summary, we have developed a chemical methodology that
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can inhibit the binding of Cu catalyst to the protein while
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Keywords: Multicomponent reaction • Protein modification •
Chemoselectivity • Site-selectivity • A3 coupling reaction
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Entry for the Table of Contents
COMMUNICATION
Chemoselective and site-selective
coupling of Lysine residue, aldehyde,
and alkyne enable single-site native
protein labeling. The mild reaction
conditions do not perturb the structure
or activity of the protein.
Maheshwerreddy Chilamari, Landa
Purushottam, Vishal Rai*
Page No.1 – Page No.5
Site-selective labeling of native
proteins by a multicomponent
approach
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