Chemoselective and Site-Selective Lysine-Directed Lysine Modification Enables Single-Site Labeling of Native Proteins

Article abstract of DOI:10.1002/anie.202000062

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single-site labeling of a high-frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent-accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys-selective electrophile connected by a spacer. Consequently, it enables the irreversible single-site labeling of a Lys residue independent of its place in the reactivity order. The user-friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site-selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe-tagged proteins. Besides, the methodology provides access to antibody-drug conjugate (ADC), which exhibits highly selective anti-proliferative activity towards HER-2 expressing SKBR-3 breast cancer cells.

Full text of DOI:10.1002/anie.202000062

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Accepted Article
Title: Chemoselective and Site-Selective Lysine-Directed Lysine
Modification Enables Single-Site Labeling of Native Proteins
Authors: Srinivasa Rao Adusumalli, Dattatraya Gautam Rawale,
Kalyani Thakur, Landa Purushottam, Neelesh C. Reddy,
Neetu Kalra, Sanjeev Shukla, and Vishal Rai
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202000062
Angew. Chem. 10.1002/ange.202000062
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10.1002/anie.202000062
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COMMUNICATION
Chemoselective and Site-Selective Lysine-Directed Lysine
Modification Enables Single-Site Labeling of Native Proteins
Srinivasa Rao Adusumalli,a# Dattatraya Gautam Rawale,a# Kalyani Thakur,a Landa Purushottam,a
Neelesh C. Reddy,a Neetu Kalra,b Sanjeev Shukla,b Vishal Rai*a
Abstract: The necessity for precision labeling of proteins emerged
during the efforts to understand and regulate their structure and
function. It demands selective attachment of tags such as affinity
probes, fluorophores, and potent cytotoxins. Here, we report a method
that enables single-site labeling of a high-frequency Lys residue in the
native proteins. At first, the enabling reagent forms stabilized imines
with multiple solvent-accessible Lys residues chemoselectively.
These linchpins create the opportunity to regulate the position of
specific residues.[11] Also, the labeling driven by ligand-protein
interaction[12] or the unique nature of His[13] or Lys residue[14]
inspired confidence for precision technologies. However, the off-
target reactions in protein modification resulting in
a
heterogeneous mixture of products remains significant
a
concern.[15] Primarily, it is due to the non-trivial task of identifying
a single residue amongst a range of nucleophilic residues. Further,
the diverse microenvironment and solvent accessibility of
proteinogenic residues enhance the complexity. Besides, it has
been a mammoth challenge to develop a modular method that
can go beyond the most reactive residue for its precise
conjugation. In this perspective, we recently developed a
methodology that enabled the single-site modular labeling of a His
in proteins.[16]
second Lys-selective electrophile connected by
a
spacer.
Consequently, it enables the irreversible single-site labeling of a Lys
residue independent of its place in the reactivity order. The user-
friendly protocol involves a series of steps to deconvolute and address
chemoselectivity, site-selectivity, and modularity. Also, it delivers
ordered immobilization and analytically pure probe-tagged proteins.
Besides, the methodology provides access to antibody-drug
conjugate (ADC), which exhibits highly selective anti-proliferative
activity towards HER-2 expressing SKBR-3 breast cancer cells.
Here, we report the linchpin directed modification (LDMK-K
)
technology that allows precise labeling of a single Lys (K), a high-
frequency residue. The selectivity remains uncompromised even
in the presence of N-terminus amine and up to nineteen Lys
residues in structurally diverse proteins. The approach is modular
and facilitates the attachment of tags in different domains of a
protein. Besides, a 19F NMR probe, biotin, and fluorophore can be
attached to deliver analytically pure single-site tagged protein.
The LDMK-K renders an antibody-drug conjugate (ADC,
trastuzumab-emtansine) linked through an amide bond at well-
defined Lys residues (K169 and K395). Interestingly, the LDMK-K
derived ADC offers remarkable activity and selectivity for the anti-
proliferation of HER-2 expressing cells. Besides, it compares well
with the FDA approved drug, Kadcyla.
The functional diversity of proteins and their impact on vital
biological processes inspire the development of methods for
precision engineering of native proteins.[1] Over the past few
decades, the pre-engineered protein driven selective labeling
allowed the investigation of several biological processes through
reporters, toxins, biophysical probes, and imaging probes.[2] Here,
the unnatural inserts,[3] enzymatic protocols,[4] engineered Cys,[5]
or a Cys in π-clamp[6] have been the prime enablers. Though
these methods are remarkable, they do not extend the precision
biophysical and biochemical tools to a vast repository of native
proteins. Besides, the demand for selective technologies
escalated due to the rapidly growing protein-based therapeutics.[7]
In this perspective, chemical methods for functionalization of
native proteins have drawn considerable attention.[8] We and
others have demonstrated selective methods for labeling of low-
frequency residues,[9] N-terminus α-amine,[10] or N-terminus
The success of the methodology emerges from a sequence of
1
transformations derived from the FK -spacer-FK2 reagents (Figure
1). At first, the functional group FK1 reacts chemoselectively with
all the accessible Lys residues in a rapid and reversible reaction.
It provides the linchpin to the LDMK-K reagent and regulates the
micro-concentration of FK2 near a proximal Lys residue. Finally,
the site-selectivity is governed through an intramolecular
irreversible reaction of FK2 and a Lys. It also ensures that the
design of spacer, and not the order of Lys reactivity, regulates the
site of conjugation. The engineered relative reactivity of FK1 and
2
Dr. S. R. Adusumalli,# D. G. Rawale,# K. Thakur, Dr. L. Purushottam, N.
C. Reddy, Dr. V. Rai
FK keeps the background reaction in control. Besides, the
[a]
intramolecular reaction determined site-selectivity ensures a
robust protocol that is forgiving towards the subtle perturbations
in the reaction conditions.
Department of Chemistry
Indian Institute of Science Education and Research Bhopal
Bhopal Bypass Road, Bhauri, Bhopal 462 066 (India)
E-mail: vrai@iiserb.ac.in; Homepage: https://home.iiserb.ac.in/~vrai
Dr. N. Kalra, Dr. S. Shukla
During the initial investigations, we realized that targeting a
unique Lys-Lys pair would present complications in addition to the
challenges mentioned above. Any two Lys residues in the vicinity
would have similar microenvironment and can react with FK1 with
equal efficacy (stage 1, Figure 1). Such a scenario would
decimate the chances of site-selectivity and contribute to
[b]
[#]
Department of Biological Sciences
Indian Institute of Science Education and Research Bhopal
Bhopal Bypass Road, Bhauri, Bhopal 462 066 (India)
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the
document.
1
heterogeneity. We argued that the methodology might work if (a) FK
can preferentially react with one Lys residue out of a Lys pair, and (b)
FK2 can block the access of FK1 to its neighbouring Lys.
1
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Figure 2. (a) Identification of FK2. % Conversions are estimated by ESI-MS.
aHeterogeneous labeling, also see Figures S18-S20. (b) LDMK-K reagent design
(4a-4f, 3-7 step synthesis).
1
2
After the selection of FK and FK , we synthesized LDMK-K
reagents 4a-4f with distinct spacers (for synthesis, see section 3b,
ESI). Subsequently, we vortexed the LDMK-K reagent 4a with the
model protein RNase A 1a. We were excited to note the formation
of mono-labeled RNase A 6a (34%, Figures 3 and S24) in overall
16 h. The late-stage modification of the protein conjugate with O-
hydroxylamine (5) results in >99% conversion. The last step also
ensures the convenient removal of LDMK-K reagents bound to
protein through linchpins. The enzymatic digestion of 6a followed
by peptide mapping confirmed the homogeneity and led to the
identification of labeled KETAAAKF (residues 1-8, m/z 1176.6
[M+H]+). The sequencing by MS-MS confirmed the site-selective
modification of K7 (Figure S24). The homogeneity of bioconjugate
6a supports that FK1 preferentially reacts with one amine out of a
pair. It is noteworthy that Nα-NH2 is the kinetically preferred site
for amine-selective electrophiles.[15] Even if it is blocked, K37 is
known to exhibit the highest reactivity amongst Lys residues.[15]
Moreover, the conservation of structure and enzymatic activity of
RNase A after the bioconjugation (6a) highlights the mild nature
of the protocol (Figures S55 and S56).
Encouraged by these results, we challenged the methodology
with insulin 1b (two Nα-NH2 and one Nε-NH2). Here, the LDMK-K
reagents 4e and 4f render a negligible conversion of 12% and 9%,
respectively (Figures S40 and S41). It was exciting to note that
the reagent 4b results in mono-labeled insulin (6b, 48% conv.,
Figures 3 and S25). The reduction of the disulfide bridges and MS
indicates the presence of tag on chain B of 6b. The subsequent
digestion by α-chymotrypsin and MS-MS of the labeled peptide
confirms the single-site labeling of Nε-NH2 (K29, TPKT, residues
27-30) of insulin. Notably, the Nα-NH2 of chain B is the most
reactive primary amine and the preferential site of labeling in other
reports.[18] Next, we selected aprotinin 1c bearing a moderate
number of primary amines (one Nα-NH2 and four Nε-NH2). Initially,
the results were not promising with the reagents 4c-4f (4-18%
conv., Figures S42-S45). However, the LDMK-K reagent 4a results
in moderate conversion of mono-labeled aprotinin (Figure S46).
Figure 1. Hypothesis and key challenges in the LDMK-K technology for single-
site labeling of a Lys residue.
Our earlier investigations suggest that a Lys residue can deliver
exclusive reactivity without the interference of Lys placed next in
the order of reactivity.[14d] However, the successful implementation
of our hypothesis required a leap of faith. At first, we selected the
1
o-hydroxyaldehyde core to serve as FK based on our earlier
findings.[16] On the other hand, the FK2 is desired to deliver high
chemoselectivity for Lys in an irreversible reaction. Secondly, it
should exhibit a considerably slow intermolecular reaction in
1
comparison to FK . The second parameter is critical to eliminate
the possibilities of non-selective background reactions. We
decided to explore the acylating group as a potential FK2 owing to
its chemoselectivity with amines and generation of a stable C-N
bond. In this perspective, we identified six activated carbonyl
compounds 2a-2f to cover a landscape of leaving group abilities
(Figure 2a). Also, we selected RNase A (1a) as a model protein
(ten Lys Nε-NH2 and one N-terminus Nα-NH2 in 124 residues).
RNase A (1a) reacts rapidly with 2a and renders a heterogeneous
mixture of products with six-eleven labels in 24 h (Figures 2 and
S18). The high order labeling is apparent even within 1 h and rules
out 2a for further screening. Also, the reagents 2b and 2c led to
heterogeneous bioconjugation within 24 h (Figures 2, S19 and
S20). Finally, 2d-2f gave negligible conversions and rules out the
non-selective intermolecular background reaction (Figures S21-
S23). In contrast to the reagents 2a-c, the reagents 2d-2f show
minimal hydrolysis in the aqueous medium.[17] At the end, the
reagent 2f inspired the selection of FK2 due to better solubility than
the other two candidates (2d and 2e) along with the stability
towards hydrolysis (Figure S52).
2
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Figure 3. LDMK-K enables single-site labeling of proteins. ESI-MS estimates the overall % conversion for two steps (6a-6b, and 6d-6h, MALDI-ToF MS for 6c). PB:
phosphate buffer. Rx indicates the labels from oxime derivative of the LDM reagent and x varies as per the LDM reagent. For data, see Figures S24-S31.
At this stage, we were curious to see whether the change in the
probes (Figure 4). The FK1 can co-exist in two forms, cyclic imine
(7) and free aminoaldehyde (8). The ESI-MS of the labeled RNase
A 7/8 before oxime derivatization allows visualization of both the
species in equal stoichiometry (7:8 is ~1:1) (Figure S24b). It also
indicates that a Lys is appropriately placed to serve as a linchpin
for LDMK-K reagent. Once FK1 completes the job of linchpin, it also
offers a site for chemically orthogonal installation of diverse
probes. To achieve this, we synthesized the derivatives of O-
hydroxylamine 9a, 9b, and 9c (Figures S13-S15). The reaction
mixture containing 7/8 and unreacted RNase A 1a were treated
separately with 9a-9c (Figures S32b-S34b). The late-stage
tagging led to the installation of an NMR tag, affinity tag, and
fluorophore 10a-10c (Figures S32c-S34c). The six magnetically
equivalent 19F nuclei in the NMR probe labeled RNase A 10a
shows a sharp signal at -62.65 ppm by 19F NMR spectroscopy
(TFA, internal standard at -75.45 ppm, Figure S53). The
fluorophore installed RNase A 10c shows emission at 428 nm
upon excitation at 333 nm (Figure S54).
Next, we established a highly efficient immobilization and
purification protocol using the versatility of FK1 (Figure 4). The
RNase A (1a) forms 7/8 after single-site labeling using the LDMK-
K reagent 4a (step 1). The product (7/8) and unreacted RNase A
(1a) are mixed with hydrazide activated resin (11) in a protein
purification column (step 2). The unreacted RNase A (1a) is
recovered and product is immobilized on resin selectively through
K7 with excellent efficiency (12, >95%). Subsequently, the resin
is distributed in four tubes for transoximization with O-
hydroxylamine (5) and its derivatives 9a-c (step 3). The resin (11)
is recycled and centrifugal spin concentration delivers analytically
pure K7-tagged RNase A with hydroxylamine (6a) and three
different probes (10a-10c, Figures 4 and S32c-S34c). The parallel
installation of 19F NMR probe, biotin, and fluorophore could be
achieved with excellent efficiency (25%, 30%, and 28% overall
isolated yields).
1
length of the linker between FK and FK2 can lead to improved
reactivity. Interestingly, the LDMK-K reagent 4b results in 86%
mono-labeled aprotinin 6c (Figure 3 and S26b). The analysis of
the site of labeling in 6c (Nε-NH2, K41) involves peptide mapping
of its proteolytic digest followed by MS-MS of the modified peptide
(GGCRAKRNNF, residues 36-45, Figure S26c). We believe that
the length of the spacer and its capability to adapt the protein
surface-induced conformational rigidity is critical. Next, we
selected ubiquitin (1d) with one Nα-NH2 and seven Nε-NH2 groups.
The LDMK-K reagents 4a and 4c-4f led to 15, 10, 8, 13, and 5%
conversion, respectively (Figures S47-S51). To our delight, the
LDMK-K reagent 4b proved successful in rendering single-site (K6)
labeled ubiquitin 6d (35% conv., Figures 3 and S27).
After successful demonstration of LDMK-K with proteins bearing
moderate number of primary amines, we selected α-lactalbumin,
cytochrome C, and myoglobin with higher number of primary
amines (13-20). The vortexing of α-lactalbumin 1e (one Nα-NH2
and twelve Nε-NH2) with 4a renders mono-labeled product 6e
(37% conv., Figures 3 and S28). The digestion, peptide mapping,
and MS-MS confirmed Nε-NH2 of K16 as the exclusive site of
labeling (RELKDLKGY, residues 10-18, 6e). Next, we selected
cytochrome C 1f with nineteen Nε-NH2 for screening. The LDMK-K
reagent 4b results in the exclusive labeling of Nε-NH2 of K79 (28%
conv., IPGTKM, residues 75-80, 6f, Figures 3 and S29). We were
excited to note that a different Lys residue K72 (33% conv.,
LENPKKY, residue 68-74) can be labeled by the reagent 4d
(Figures 3 and S30). The two distinct labeled sites in cytochrome
C (K79 with 4b and K72 with 4d) establish the modularity. Finally,
we selected myoglobin 1g bearing one Nα-NH2 and nineteen Nε-
NH2. Here, 4b results in selective labeling of Nε-NH2 of K42 (26%
conv., TGHPETLEKF, residues 34-43, Figures 3 and S31).
After establishing the efficacy of LDMK-K in single-site labeling of
proteins, we developed a protocol for late-stage installation of
3
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10.1002/anie.202000062
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COMMUNICATION
In summary, we demonstrated a linchpin directed modification
(LDMK-K) for single-site labeling of Lys residue in proteins. In this
pursuit, the LDMK-K reagent efficiently regulates the
chemoselectivity,
site-selectivity,
and
modularity.
The
methodology operates with multiple proteins presenting a wide
range of structural complexity. It opens the gateway for labeling
several domains on the protein surface as Lys displays high
frequency and solvent accessibility. The easy to operate front-end
protocol proceeds under mild physiological conditions. Besides,
1
the FK creates the additional opportunity for ordered
immobilization or installation of the desired tag through late-stage
modification. It empowers the precise and parallel introduction of
an NMR tag, an affinity tag, and a fluorophore. The convenient
recovery of the unreacted native protein and recycling of
hydrazide activated resin renders a cost-effective purification
protocol. Also, LDMK-K enables the synthesis of ADC that exhibits
highly specific antiproliferative activity toward HER-2 positive
SKBR-3 breast cancer cells.
Figure 4. Label-tag-purify protocol for rendering analytically pure single-site
Acknowledgements
tagged protein with probe of interest.
This work is supported by SERB, DBT, DST, and IISER Bhopal.
S.R.A., D.G.R., K.T., L.P., and N.C.R. are recipient of a research
fellowship from CSIR, UGC, and IISER Bhopal. V.R. is a recipient
Chemical methods for precise antibody conjugation are
essential for therapeutics and diagnostics.7 We applied the LDMK-
technology for installation of fluorophore and payload on the
K
of
Swarnajayanti
fellowship
(DST/SJF/CSA-01/2018-19;
monoclonal antibody (mAb, trastuzumab). Initially, the reaction of
trastuzumab 13 with LDMK-K reagent 4a followed by the late-stage
installation of fluorophore 9c gave coumarin-tagged mAb 14. The
SDS-PAGE exhibits the fluorescent band for both heavy and light
chains (Figure S57b). Our in-house sensitivity booster
technology15 enabled an unambiguous analysis of the sites-of-
conjugation (K169-light chain and K395-heavy chain, Figure S59).
Alongside this, the drug 9e (DM1) was conveniently installed on
mAb 13 to render the ADC (15, Figure 5). Further, the results of
the MTT assay confirm the dose-dependent response of ADC (15)
in HER-2 overexpressing SKBR-3 breast cancer cells. The LDMK-
K ADC 15 showed 45% growth inhibition at 2 nM concentration in
comparison to 38% with Kadcyla (16) in SKBR-3 cells (Figure 5b).
Importantly, the LDMK-K ADC 15 did not show significant impact
on the growth in HER-2 negative MDA-MB-231 cells, thus ruling
out any non-specific binding to the cells (Figure 5c).
SB/SJF/2019-20/01).
Keywords: Bioconjugation • Protein modification • Lysine
labeling • Antibody-drug conjugate • ADC
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10.1002/anie.202000062
Angewandte Chemie International Edition
COMMUNICATION
Table of Contents
A chemical methodology finally addresses the undefeated challenge of modular and precision engineering of a Lys residue in a
native protein. The remarkable control over selectivity provides the first-ever approach to target Lys beyond the protein-defined
reactivity hotspot. This technology unfolds new gateways and renders analytically pure protein bioconjugates and homogeneous
antibody-drug conjugates.
Institute and/or researcher Twitter usernames: @vishal_iiserb, @iiserbhopal
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This article is protected by copyright. All rights reserved.