One-Step Azolation Strategy for Site- and Chemo-Selective Labeling of Proteins with Mass-Sensitive Probes

Article abstract of DOI:10.1002/anie.202007608

The chemical modification of proteins in a site-selective manner leads to many advances in various scientific fields. The major challenges with conventional N-terminal bioconjugation techniques are the lack of universal sequence compatibility and poor mass-detection sensitivity of the resulting bioconjugates. This approach efficiently analyzes proteolytic fragments and native proteins in a complex mixture. Multiple chemical steps are usually required for the site-selective synthesis of bioconjugates with enhanced mass-detection sensitivity. We present a single-step, versatile strategy for the selective modification of protein N-termini with mass boosters. The chemical tag enhances the peptide detection by multiple orders thus leading to the unambiguous analysis of the resulting bioconjugates. We demonstrate that tagging proteolytic fragments with mass sensitivity probes in a complex mixture improves the detection of resulting bioconjugates.

Full text of DOI:10.1002/anie.202007608

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: One Step Azolation strategy for site- and chemo-selective
labeling of proteins with mass sensitive probes
Authors: Monika Raj and Kuei C. Tang
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.202007608
Link to VoR: https://doi.org/10.1002/anie.202007608

10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
One Step Azolation strategy for site- and chemo-selective
labeling of proteins with mass sensitive probes
Kuei C. Tang,[a] and Monika Raj*,[a]
Abstract: The chemical modification of proteins in a site-
bioconjugates is due to the blockage of the free N-terminus by
selective manner leads to many advances in various scientific
fields. The major challenges with conventional N-terminal
bioconjugation techniques are the lack of universal sequence
compatibility and poor mass detection sensitivity of resulting
bioconjugates. This approach efficiently analyze proteolytic
fragments and native proteins in a complex mixture. Multiple
chemical steps are usually required for the site-selective
synthesis of bioconjugates with enhanced mass detection
sensitivity. We present a single step, versatile strategy for the
selective modification of protein N-termini with mass boosters.
The chemical tag enhances the peptide detection by multiple
orders thus leads to the unambiguous analysis of resulting
bioconjugates. We demonstrate that tagging proteolytic
fragments with mass sensitivity probes in a complex mixture
improves the detection of resulting bioconjugates.
hydrophobic groups.22 This is a major issue while analyzing the
conjugated proteins in a proteomic mixture. Consequently, there
is a great need to develop new synthetic methodologies that can
circumvent the aforementioned limitations and provide an
efficient strategy for site-selective labeling of proteins with mass
sensitive probes and exhibit universal sequence compatibility.
The current method to increase the detection senstivity of
bioconjugates involve the addition of mass senstive probes but
in a non-selective manner.23-24 This leads to the formation of
heterogeneous mixture which makes the analysis difficult
(Figure 1b). Multiple methods are usually needed to achieve all
these goals, for example, one method is required for the site-
selective modification and a second method for the addition of
chemical tag that enhances mass sensitivity of the bioconjugate.
As a result, a simple, one-step method capable of achieving all
the abovementioned goals with the broad scope of structurally
and chemically varied peptides and proteins would be highly
beneficial. This would enable efficient analysis of the modified
peptides in a complex mixture and aid in the detection of low
abundant peptides.
Introduction
Modification of proteins at a single site with varying synthetic
molecules is of high significance in the field of chemical biology,
for efficient drug delivery, material science and synthesis of bio-
hybrid materials.1-5 Recently, there is a great increase in the
number of methods for site-selective labeling of proteins but is
limited by the requirement of particular amino acids at the N-
terminus.6-12 Such as N-terminal cysteines leading to the
formation of thiazolidines with aldehydes.13 Periodate oxidation
of N-terminal serine and threonine residues14 for oxime15,
hydrazone,16 Wittig,17 Aldol18 and Henry bioconjugation.19 N-
terminal tryptophans can be modified via Pictet-Spengler
reactions.20 Some of the methods also required multiple steps.15-
Results and Discussion
Design of azoles for N-terminal modification
To develop a single step site-selective modification of proteins
that enhances the mass sensitivity, we sought to use azolines
for bioconjugation. As a key design element, we hypothesized
that the nucleophilic attack of the N-terminus on 2-methyl thiol
azolines would lead to the formation of a stable C-N bond at the
site of conjugation under physiological conditions. The azoline
group has sp2 hybridized nitrogen thus increases the ionization
of peptides and enhance the mass sensitivity (Figure 1c). This
approach represents a rare example of selective N-terminal
azolation of proteins with improved mass sensitivity that might
16
A recent 2 pyridinecarboxaldehyde (2PCA) method provides
one-step approach for the N-terminal modification of proteins but
is unable to tag proteins with proline amino acid in the second
position, and doubly modify the peptide with glycine amino acid
at the N-terminus, thus lack universal sequence compatibility
(Figure 1a).21
Another major limitation with the current methods of N-terminal
labeling of proteins is the low mass detection sensitivity of the
resulting protein bioconjugates thus making it difficult to
characterize. The low mass sensitivity of the N-terminal labeled
offer
a general strategy for efficient synthesis of azoline
bioconjugates (Figure 1c).
Among several advantages, we recognized that the azoline
strategy would: 1) be chemoselective for the N-terminus rather
than any other amino acid residues including lysine; 2) enhance
the detection sensitivity of bioconjugates due to the high
ionization of azolines; 3) labels the N-terminus of proteolytic
[a]
K. Tang, Prof. Dr M. Raj
Present address: Department of Chemistry, Emory University,
Atlanta, Georgia 30322, United States
Email: monika.raj@emory.edu
Home page: https://raj.emorychem.science
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
Previous Bioconjugation Technique
a
H2N
H2N
O
H2N
H2N
3
3
N
Site-selective
N-terminal capping
Reduce ionization
3
3
H
A1
O
O
H
N
N
peptide
HN
H2N
peptide
N
Low mass senstivity
O
A2
A2
A1
O
b
Previous Mass Senstivity Booster Technique
_
O
Br
+
N
O
H2N
H2N
HN
HN
N
2
Non site-selective
5
3
3
3
3
Heterogenous mixture
Enhanced ionization
High mass senstivity
O
N
N
O
H2N
peptide
N
H
peptide
c This work: One-Step N-terminal azolation of proteins leading to enhanced mass senstivity
R
O
N
H2N
H2N
Chemoselective (all side chains of
amino acid tolerated including lysine)
Site-selective
H2N
H2N
R
S
3
3
3
3
O
= alkyne or azide
biotin
R
Homogenous mixture
Enhanced ionization
High mass senstivity
Universal sequence compatibility
+
N
N
peptide
H2N
peptide
H
N-terminal azolation
of peptides and proteins
phosphate buffer pH 7.5
H
d Azoline-containing bioactive compounds
O
O
O
S
S
N
N
O
O
H
O
H
N
N
N
N
N
O
H
N
N
O
HN
O
NH
HN
NH
O
HN
O
NH
N
N
O
N
N
H
N
O
S
O
O
S
O
Westiellamide
Bistratamide A
Patellamide A
Figure 1. On step azolation for selective N-terminal labeling of biomolecules. a. Limitations of the current N-terminal 2-PCA labeling method. b. Limitations of the
current mass booster techniques. c. Selective N-terminal labeling of biomolecules via azolation with mass boosters. d. Azoline-containing bioactive compounds.
Screening of Azoline: Universal Sequence
Compatibility
fragments in a complex mixture thus able to detect low
abundant peptides (Figure 1c). This strategy introduces 1,3-
azoline moiety into the bioconjugate, a privileged scaffold in
the medicinal chemistry and pharmaceutical industry (Figure
1d).25 Azoline moieties in the backbones of peptidic natural
products are important structural motifs that contribute to
diverse bioactivities such as protease inhibitor or cytotoxic
activity.26-28 Here in, we report the successful execution of
these ideas and present a rapid, N- terminal azolation for use
on a wide range of peptides and proteins under physiological
conditions.
We initiated our study by screening a variety of heterocyclic
2-methylthio azolines such as oxazoline 1a, imidazoline 1b
and thiazoline 1c with a model peptide FKVCF 2a containing
multiple nucleophilic residues such as N-terminus, lysine and
cysteine under physiological conditions (phosphate buffer pH
7.5, 10 mM) (Figure 2a). A treatment of peptide FKVCF 2a (4
mM) with excess of 2-methylthio oxazoline 1a, Ox1 (200 mM)
exclusively labeled the N-terminus to generate the oxazoline-
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10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
chain of lysine remained protonated under the physiological
conditions thus no labeling of lysine was observed.
modified peptide Ox1-FKVCF 3a as confirmed by MS/MS
analysis (Supplementary Figure 1).
The reaction of peptide FKVCF 2a (4 mM) with 2-methylthio
imidazoline 1b (200 mM) modified lysine side chain as
confirmed by MS/MS analysis (Figure 2a and Supplementary
Figure 1). This might be due to the ability of imidazole to act
as a base and deprotonate the lysine side chain. We have
confirmed this by carrying out the reaction with N-acetylated
2-methyl thioimidazoline (Ac-1b) that showed highly
selectivity for labeling the N-terminus (Supplementary Figure
2). Interestingly, the modification of the cysteine side chain
with both 1a and 1b was not observed under the reaction
conditions although it is highly nucleophilic under
physiological conditions. This is most likely due to the
reversibility of the oxazoline-cysteine and imidazoline-
cysteine conjugates which exhibit functionally similar
thioether as the starting material 1 and can react with N-
terminus or lysine to form stable products (Figure 2b). We
have confirmed the reactivity with amines by carrying out the
reaction of imidazoline-cysteine peptide conjugate (Im-Cys)
with Ac-Lys-OMe (Figure 2b, Supplementary Figure 3). The
reaction of a peptide FKVCF 2a (4 mM) with 2-methylthio
thiazoline 1c (200 mM) modified cysteine only as confirmed
by MS/MS (Supplementary Figure 4). Any modification of the
N-terminus and lysine side chain was not observed due to the
low electrophilicity of the 2-methylthio thiazoline 1c (Figure
2a). The formation of small amounts of a disulfide bond
between peptide FKVCF 2a was also observed during the N-
terminal oxazolation reaction. To simplify the analysis, a
peptide FVKAF 2b without cysteine was used for further
studies. 2-methylthio oxazoline 1a selectively modified the N-
terminus of the peptide FVKAF 2b and generated Ox1-
FVKAF conjugate 3b with high conversion (80%) under the
reaction conditions. The site of modification was confirmed by
MS/MS analysis of Ox1-FVKAF 3b (Supplementary Figure 5).
As further validation of the reaction site-selectivity, no
modification of the N-acetyl tripeptide Ac-AKF was observed
with 1a, Ox1 under the physiological conditions (pH 7.5) and
at high pH 8.5 (Supplementary Figure 6). These results
suggested that the reaction proceeds via nucleophilic attack
of the N-termini, which remains unprotonated at physiological
conditions pH 7.5 as compared to the protonated side chain
of lysine.
a.
NH2
N
SH
3
O
1a, X = O
V
3a
K
HN
C
NaP pH 7.5
F
F
NH2
NH
SH
3
N
NH
V
K
SH
C
F
F
3
1b, X = NH
2a
H
N
H2N
V
K
NaP pH 7.5
+
C
F
F
N
X
SMe
N
X = O, NH, S
NH
S
N
NH2
S
3
1c, X = S
V
K
NaP pH 7.5
C
H2N
F
F
b.
H
N
S
SH
Ac-Lys-OMe
NaP pH 7.5
N
AcHN
C
F
V
AcHN
C
F
Ac-Lys(Im)-OMe
V
c.
d.
100
80
100
80
60
40
60
40
20
0
20
0
D
XAF sequence X = any amino acid
APF
E
F
G M
P
T
Y
Figure 2. Site-selective polypeptide modification using azolines. a.
Screening of various azolines such as oxazoline 1a, imidazoline 1b and
thiazoline 1c to determine their chemoselectivity for peptides. b.
Reversibility of azoline-cysteine adduct with lysine. c. Universal sequence
Next, we evaluated the effect of the N-terminal amino acids
on oxazolation efficiency and chemoselectivity by screening a
panel of peptides with varying reactive amino acid residues at
the N-terminus. The incubation of peptides (XAF, 2c-2j X = D,
E, F, G, M, P, T, Y (6.25 mM) with 2-methylthio oxazoline 1a,
Ox1 (312.5 mM) in 10 mM phosphate buffer at pH 7.5 for 3 h
compatibility of peptides with azolination. Conditions for a–b:
4 mM
peptide, 200 mM azoline, 10 mM phosphate buffer at pH 7.5 and 37 °C.
Conditions for c: 6.25 mM peptide, 312.5 mM 1a, Ox1, 10 mM phosphate
buffer at pH 7.5 and 37 °C for 3 h.
High-performance liquid chromatography (HPLC) analysis
showed that 82% conversion to the N-terminal oxazoline-
modified peptide 3a was achieved in 2h. Lysine side chains
were shown to be unreactive towards 2-methylthio oxazoline
1a because the pKa of the side chain of lysine ~ 10.6 is
higher than pKa of the N-terminus ~ 9.6. Therefore, the side
yielded N-terminus Ox1-modified peptides 3c-3j in
a
quantitative manner as determined by LCMS (Figure 2c and
Supplementary Figure 7). No modifications of the reactive
side-chains were observed under the reaction conditions thus
reaction is highly selective for the N-terminus. The N-terminal
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10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
oxazolation of peptides GAF 2f and APF 2k with glycine at
the N-terminus and proline at the second position proceeded
smoothly to generate N-terminal oxazoline-modified peptides
(3f-3k) in a quantitative manner (Supplementary Figure 8).
This is in contrast to the 2PCA method resulting in the double
modification with glycine at the N-terminus and no
modification of a peptide with proline at the second position21.
These studies showed that oxazolation is independent of the
nature of amino acid sequence thus exhibit universal
sequence compatibility. Further, Ox1-GAF-OMe conjugate 3f-
OMe was synthesized on a large scale and characterized by
NMR (Supplementary Figure 9).
70% conversion achieved in just 10 min (Figure 3a and
Supplementary Figure 10). The rate studies of the
oxazolation between tripeptide GAF-OMe 2f-OMe (1 mM)
and 2-methylthio oxazoline 1a (1 mM) at pH 7.5 were
monitored over 60 min and showed that the reaction follows a
second-order rate constant (k = 1.66 X 10-2M-1S-1) (Figure 3a,
Supplementary Figure 11). The stability of protein conjugates
is a major concern for bioconjugation reactions. Therefore,
we evaluated the stability of oxazole-peptide conjugate Ox1-
GAF-OMe 3f-OMe in aqueous buffers at pH ranging from 3.5
to 10.5, for 48 h at 25 °C and 37 °C. HPLC analysis showed
high stability of Ox1-GAF-OMe 3f-OMe at varying pH
conditions (Figure 3b, Supplementary Figure 12). Taken
together, these studies establish that oxazolation is N-
terminal selective under physiological conditions, employs
mild conditions, exhibits fast kinetics, gives higher yields and
forms stable oxazoline-adducts thus has the potential to be
an effective bioconjugation strategy.
To gain a deeper understanding of reaction rates and the
products formed, time-course studies on a linear peptide
GAF-OMe 2f-OMe were undertaken. For this investigation,
quantitative monitoring was carried out by injecting samples
for HPLC analysis at regular time intervals. The oxazolation
between tripeptide GAF-OMe 2f-OMe (2 mM) and 2-
methylthio oxazoline 1a (100 mM) at pH 7.5 was monitored
over 60 min (Supplementary Figure 10).
Protein modification-reaction optimization
To determine the applicability of this reaction to proteins, we
started our initial investigation on protein myoglobin Mb.
Detailed optimization studies with myoglobin Mb revealed that
the most efficient conditions are 2-methylthio oxazoline 1a in
10 mM phosphate buffer at pH 7.5 and 25 oC for 12 h (Figure
4a and Supplementary Figure 13). Long reaction time is
needed for high labeling yield (70 -> 99%) but moderate
amounts of labeling are observed even in shorter reaction
time (6 h) (50-80%). The concentration of protein (0.75 mM –
3 mM) did not influence the extent of modification (Figure 4a,
Supplementary Figure 13). Excellent conversion (70%) to N-
terminal oxazoline-modified myoglobin Ox1-Mb was obtained
at the more neutral pH as confirmed by MS/MS analysis
(Figure 4a, Supplementary Figure 13). In contrast to the NHS
ester method, only the single modification product was
observed despite the presence of 17 lysine residues on Mb.
Low pH 6.5 reduced the conversion to modified protein (43%,
Figure 4a). An increase in pH 8.5 increased the % conversion
to modified protein (52%) but also resulted in the lower
selectivity, leading the small amounts of modification on the
side chain of lysine (30%) (Figure 4a, Supplementary Figure
13). The increase in the probe equivalents from 50 to 200
lead to the multiple modifications of protein Mb. High site-
selectivity for the N-terminus was observed with 50 equiv. of
the probe (Figure 4a).
a.
OMe
OMe
O
S
Buffer
(pH 7.5), 37
O
oC
G
A
F
G A
3f-OMe
F
+
N
N
H
N
2f-OMe
100
80
25
20
15
10
5
-1S-1
k = 0.0166M
60
40
20
Ox-conjugate
Ox-conjugate
10 15
Time (min)
0
0
0
0 5 10 15 20 25
Time (min)
5
b.
100
0 h
1 h
10 h
24 h
80
60
40
20
0
pH 3.5
pH 7.5
pH 10.5
Synthesis of functionalized Oxazoles for N-
terminal modification
Figure 3. Rate and stability studies of oxazolation. a. Rate studies for
synthesis of Ox-GAF-OMe conjugate 3f-OMe. b. The oxazolation of
tripeptide GAF-OMe 2f-OMe (1 mM) with 1a (1 mM) at pH 7.5 follows a
second order rate constant. b. pH stability of Ox-GAF-OMe conjugate 3f-
OMe at 25 °C.
Next, we synthesized 2-methylthio oxazolines with a variety
of functional groups to attach various synthetic groups on
proteins (Figure 4b). Compound I was synthesized in two
steps from serine methylester (Supplementary Figure 14).
From these data, it is clear that the initial rate of the formation
of Ox1-GAF-OMe conjugate 3f-OMe is considerably fast with
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10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
a
3 M
2 M
M
WT Mb
MS = 16950
2 M
M
2 M
M
M
4 M
M
3 M
O
S
2 M
M
pH 8.5, 3 mM
pH 6.5, 3 mM
pH 7.5, 3 mM
pH 7.5,1.5 mM
pH 7.5, 0.75 mM
N
200 equiv.
Ox1
M
N
150 equiv.
100 equiv.
Mb
O
Mb
M = 70
pH 7.5
50 equiv.
0 equiv.
16800 16850 16900 17000 17100 17200
15800 16100 16400 16700 17000 17300
b
Cl-
NH3
R
O
O
O
multiple
steps
N
S
O
H
+
CS2, Et3N
THF, 12 h
CH3I, K2CO3
N
N
O
MeO
MeO
MeO
S
S
DMF, 14 h
I
OH
O
c
O
17076
Ox3, R =
16950
17101
N3
Mb+
Ox3
R =
Ox2
Mb
Mb+
Ox2
N
17361
Mb+
H
O
16950
Ox4, R =
Mb+
2Ox3
Ox5
Mb
O
17201
O
H
N
O
H
16950
O
N
2
N
H
Mb
3
H
NH
Ox5
R =
S
16500
17200
14326
16500
17500
16500
17500
H
d
5929
Ins+
5943
8975
Ub+
12507
Cy+
Ins
lactalbumin
+
Ox3
Ox5
6081
+
Ox2
8563
Ox2
Ins
Ox4
5806
Ins
Ub
+
lactalbumin
2Ox4
14177
6000
6100
8000
10000
5500
6500
12300
13000
14200
14400
Figure 4. Site-specific attachment of oxazoline and its derivatives to a variety of different proteins. a. Optimization of protein myoglobin modification with oxazoline
1. b. Synthetic scheme for varying functionalized oxazoline derivatives such as alkyne, azide and biotin tags. c. Site-selective modifications of myoglobin Mb with
varying oxazoline derivatives. d. Site-selective modifications of a variety of different proteins with oxazoline derivatives. Conditions for c–d: 3 mM protein, 50-300
equiv. oxazoline, 10 mM phosphate buffer at pH 7.5 and 40 °C. The samples were incubated for 12 h in c and d.
The acylation with NHS ester provided an oxazoline for
attaching biotin (Ox5, Figure 4b and Supplementary Figure
14). Propargylation of compound I introduced alkyne group
on the oxazoline Ox4, and azidation of this compound
generated azide tag on oxazoline Ox3 (Figure 4b and
Supplementary Figure 14). Varying degree of modification of
Mb with different functional group handles is dictated by the
bulk of the handles. Smaller the size of the handle, higher
labeling efficiency was observed, 90% labeling of Myoglobin
(Mb) with Ox2 (Figure 4d and Supplementary Figure 15). Ox5
due to its bulky nature leads to reduced labeling of Mb (40%)
(Figure 4c and Supplementary Figure 15). The reaction of
myoglobin with 1b modified multiple lysine side chains as
analyzed by HRMS and MS/MS (Supplementary Figure 16).
Protein structure and function is retained after
modification
The UV studies on oxazoline-modified myoglobin Ox1-Mb
and unmodified myoglobin Mb indicated that the secondary
and tertiary structure of a protein is unaffected due to the
modification under the reaction conditions (Supplementary
Figure 17).
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10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
Myoglobin bioactivity assay
the peptides in a quantitative manner as analyzed by MS
(Figure 5 and Supplementary Figure 21). The ability to modify
all the N-termini of peptides in the complex mixture could be
highly valuable in proteomics studies.
The activity assay of Ox1-Mb for its ability to oxidize o-
phenylenediamine with hydrogen peroxide to 2,3-
diaminophenazine29 further confirmed that the installation of
an oxazoline at the N-terminus does not alter the activity of
Mb (Supplementary Figure 18). Together, these results
N
O
S
F
F
F
F
G
G A
A
A
F
F
O
S
Ox4
A
A
F
reaffirmed that the oxazolation of
a
protein selectively
F
V
A
V
NaP, pH 7.5
installed probe at a single-site while conserving its structure
and bioactivity.
A
A
L
F
F
F
F
L
A
A
A
A
452.19
Ox4-GAF
[M+Na]
466.20
Ox4-AAF
[M+Na]
Protein Scope for N-terminal Oxazolation
We carried out the oxazolation of a variety of proteins such as
cytochrome C, alpha-lactalbumin, ubiquitin and insulin (3 mM)
with varying functionalized oxazolines (50-300 equiv.). The
reactions afforded good to excellent yields (50-99%) with
excellent selectivity (Figure 4d and Supplementary Figure 19).
Notably, ubiquitin with seven lysine residues also generated
single modification product at the N-terminus. Insulin with two
N-termini generated double modification products due to
reactions at both the N-termini (Figure 4d and Supplementary
Figure 19). Varying degree of modification of proteins with
different functional group handles is dictated by the bulk of
the handles and accessibility to the N-terminus. Smaller the
size of the handle, higher labeling efficiency was observed,
>99% labeling of Cytochrome (Cy) with Ox2, >99% labeling
of Insulin (Ins) with Ox2 and Ox4, 90% labeling of Myoglobin
(Mb) with Ox2, and 90% labeling of Lactalbumin with Ox2
(Figure 4d and Supplementary Figure 19). Ox5 due to its
bulky nature leads to reduced labeling of proteins (40-70%)
(Figure 4d and Supplementary Figure 19). These examples
indicated that reaction is tolerant to a wide range of molecular
weights, three-dimensional structures and structurally critical
disulfide bonds. Selective modification of proteins in the
presence of cysteine is highly challenging due to the high
nucelophilicity of the cysteine thiol group at neutral pH. The
cross-reactivity with cysteine is commonly observed in
various N-terminal bioconjugation methods.30-32 No such
modification of cysteine was observed during the oxazolation
of protein with reduced insulin containing six free cysteines
(Supplementary Figure 20). The unique feature of our
approach is that it leads to the single-site N-terminal
modification of proteins in the presence of highly nucleophilic
cysteines under physiological conditions.
Ox4-LAF
[M+Na]
Ox4-AAF
Ox4-FAF
[M+Na]
542.23
508.25
Ox4-VAF
[M+Na]
494.24
[M+1]
Ox4-GAF
[M+1]
430.20
444.22
440
490
m/z
Figure 5. Selective N-terminal labelling of all the peptides by oxazoline
Ox4 in the complex mixture of a variety of peptides.
Oxazoline as Mass Sensitivity Booster
One of the major limitations with current methods of N-
terminal modification is the difficulty in the characterization of
resulting bioconjugates. MS is widely used to determine the
labeling and to detect the site of modification but
unfortunately unable to map a considerable part of the
peptides due to the poor ionization of the labeled peptide or
suppression from other ions. The poor ionization of N-
terminal labeled peptides is due to the blockage of the free N-
terminus with hydrophobic labels. This limitation is more
pronounced in the complex mixtures. Therefore it is essential
to develop methods that can enhance the detection of labeled
proteolytic fragments. One way to achieve this goal is the
addition of chemical tags that increases ionization of the
labeled peptides in the MS. Some strategies have been
reported for enhancing the ionization ability of peptides but
they are not selective and modify various amino acids thus
making the analysis difficult.23
Labeling of proteolytic fragments in the complex
mixture
To determine the applicability of oxazolation to
simultaneously tag the N-termini of multiple peptides and
proteins in a complex system, oxazolation was performed
with both Ox1 and Ox4 on a mixture of peptides with different
amino acids at the N-terminus (XAF (2e-2f, 2l-2n) X = F, G, A,
V, L). The results indicated full N-terminal oxazolation of all
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10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
a
N
O
H
+
S
X
O
N
X
O
H
H
Ox1
N
R
N
R
O
NaP, pH 7.5
O
H2N
N
N
H
N
H
H
O
O
O
R = H 2e, X = F; 2fOMe, X = G
, X = F; 3fOMe, X = G
3e
R= OMe 2lOMe, X = A; 2mOMe, X = V
3lOMe, X = A;3mOMe, X = V
100
100
80
60
40
20
0
100
80
100
3e
3e
M+H
3fOMe
399.16
452.22
3lOMe
391.19
419.22
3mOMe
+H
3lOMe
3fOMe
80
60
40
3mOMe
80
2mOMe
M+H
350.20
2lOMe
3lOMe
M+Na
60
M+H
M+Na
M+Na
413.17
60
40
20
0
3e
M+Na
474.20
2fOMe
M+Na
330.14
344.15
+Na
441.20
2e
M+Na
405.18
2mOMe
372.18
+Na
2lOMe
40
2lOMe
M+H
322.17
2fOMe
20
20
2e
0
0
350 390 460
450
500
320 390 460
350 400 450
N
b
KASEDLKKHGTVVLTALGGILKK
KGHHEAELKPLAQSHATKHKIPI
KYLEFISDAIIHVLHSKHPGDFGA
GLSDGEWQQVLNVWGKVE
ADIAGHGQEVLIRLFTGHPET
S
CNBr,
O
NaP, pH 7.5
LEKFDKFKHLKTEAEM
Ox1
peptide a
DAQGAM
peptide b
NaP, pH 7.5
TKALELFRNDIAAKYKELGFQG
peptide c
Mb
Ox1-labeled
proteolytic fragments
unlabeled proteolytic
fragments after cleavage
Ox1-peptide a
646.5892
peptide a
646.8323
443.2357
668.3522
1292.1619
1292.6641
629.3294
629.0804
1257.6553
1257.1599
1293.1794
465.5666
Ox1-peptide b
peptide b
465.8998
924.3876
697.8554
574.2813
846.3101
1394.6921
742.3583
743.0295
907.4221
646.5800
Ox1-peptide c
peptide c
646.8323
663.8363
861.7581
862.1034
678.3392 884.7761
885.4433
1292.1487
838.7664
1326.6669
1327.1757
1327.6577
658.7506
646.0753
1257.6292
m/z
m/z
Figure 6. Tagging peptides and proteolytic fragments with mass sensitivity boosters. a. Oxazoline tagging of N-terminal peptides increases the mass detection
sensitivity as compared to untagged peptides. b. Oxazoline tagging improves the detection of proteolytic fragments in a complex mixture. Proteolytic fragment a is
barely visible after cleavage of myoglobin (left MS trace). All the proteolytic fragments of myoglobin, peptides a, b and c are more ionized and clearly visible after
tagging with oxazoline (right MS trace).
The unique feature of our approach is that it not only
selectively labels N-terminus but also introduces the
oxazoline group containing a sp2 nitrogen thus enhances the
ionization of the labeled fragments. To determine the effect of
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10.1002/anie.202007608
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RESEARCH ARTICLE
and interference from lysine and highly nucleophilic cysteine
side chains was not observed. Although some N-terminal
methods are often dependent on the nature and sequence of
amino acids, no such effect was observed for this method.
The potency of the oxazolation is well demonstrated by the
selective labeling of a variety of peptides and proteins with
different amino acid compositions including reaction with
proteolytic fragments in complex mixtures as shown in
Figures 5 and 6. The N-terminal labeling methods often suffer
from low detection sensitivity of the resulting bioconjugates
due to the blockage of the free N-terminus by the
hydrophobic group and multiple derivatizations are needed
for the complete analysis. The N-terminal oxazolation
generated bioconjugates with remarkably high detection
sensitivity. This is due to the sp2-hybridized nitrogen of the
oxazoline, which enhances the ionization of the labeled
peptides. This strategy provides an excellent tool for the
unambiguous analysis of bioconjugates in a complex mixture.
One of the major advantages of our approach is that it carries
out the site-selective modification of the intact protein at the
N-terminus, increases its mass sensitivity without diminishing
its activity which is in contrast to other mass sensitive
boosters.
the oxazoline tag on mass sensitivity, we compared the
sensitivity of the multiple unlabeled peptides with methyl ester
protected C-termini and rich in hydrophobic groups (XAF-
OMe (2e, 2fOMe, 2lOMe-2mOMe), X = F, G, A, V) with
corresponding N-terminal oxazoline-labeled peptides (Ox-
XAFOMe, X = F, G, A, V 3e, 3fOMe, 3lOMe-3mOMe) by
mixing equal concentrations of these peptides followed by
analysis using MS (Figure 6a and Supplementary Figure 22).
Insights into the impact of the oxazoline moiety on the
detection sensitivity were revealed by MS analysis (Figure
6a). Peptides with oxazoline tags (Ox-XAF-OMe, 3e, 3fOMe,
3lOMe-3mOMe) showed remarkable improvement in the
signal enhancement irrespective of the unmodified sequence
of the peptides (Figure 6a).
Inspired by these results, we investigated the mass sensitivity
of proteins and their oxazoline-labeled bioconjugates. We
selected cytochrome C and myoglobin to test the potential of
oxazoline as a sensitivity booster. For this investigation,
cytochrome C and myoglobin was digested using cyanogen
bromide followed by reaction with 2-methyl-thio oxazoline 1a.
The proteolytic fragments before and after the N-terminal
oxazoline labeling were analyzed directly by MS without any
purification (Supplementary Figure 23 and Figure 24). For
unlabeled proteolytic fragments of cytochrome c, low
sensitivity was observed and some fragments remained
undetected (proteolytic fragment a, Supplementary Figure 23).
For the labeled proteolytic fragments of cytochrome c, all the
fragments were detected with significantly high mass
intensities (Supplementary Figure 23). We observed huge
change in the MS of the oxazoline-labeled and unlabeled
myoglobin proteolytic fragments. For unlabeled proteolytic
fragments of myoglobin, very poor sensitivity was observed
for all the fragments (proteolytic fragments a, b and c, Figure
6b, Supplementary Figure 24). For the labeled proteolytic
fragments of myoglobin, all the fragments were detected with
significantly high mass intensities (Figure 6b, Supplementary
Figure 24). These studies showed improved sensitivity of the
labeled proteolytic fragments in the complex mixture thus
highly significant and circumvent detection limitation of the
current N-terminal bioconjugation methods. The ability to
modify all the N-termini of digested proteolytic fragments in
the complex mixture could be highly valuable in proteomics
studies. We also showed that mass sensitivity of the N-
terminal modified myoglobin by Ox1 enhanced significantly as
compared to the unmodified intact myoglobin (Supplementary
Figure 25). Moreover, the N-terminal oxazolation method
could be used to detect the unique proteolytic fragments
obtained due to chemotherapy-induced cell death leading to
the discovery of novel biomarkers of cell death.
The unique feature about our approach as compared to other
methods for mass sensitivity booster is that it is highly
selective and generates only one modified fragment rather
than mixture of the multiple modified fragments generated by
the use of commercial mass sensitivity reagents due to their
non-selective nature. Analysis of the mass data with a
mixture of differently modified fragments is highly complicated.
The N-terminal oxazoline-modified peptides and proteins
exhibit high stability over
a range of pH conditions,
demonstrating the potential utility of this chemistry in the
conjugation of pharmaceutically active compounds and
biological probes. Considering the simple setup and
chemoselective nature of this bioconjugation reaction with
easily derivatized mass sensitivity boosters and its use in
efficient tagging of proteolytic fragments from a complex
mixture, we anticipate that this method will become a highly
useful tool in my scientific fields.
Experimental Section
See the supporting information for materials, instruments, reaction
procedures, azolation, stability study, mass sensitivity booster studies,
and products characterization by HRMS, HPLC, and NMR (PDF).
Acknowledgments
This research was supported by NIH (Grant No. 1R35GM133719-01)
granted to M.R. We thank Auburn University for the infrastructure. We
started this work at Auburn University.
Conclusion
Keywords: Site-Selective, Chemoselective, N-terminal
In summary, we have developed a novel oxazolation strategy,
a single-step approach for the selective labeling of proteins
with mass sensitive probes under physiological conditions.
This method is highly selective for N-terminus because of the
increased availability of deprotonated alpha-amino groups
Labeling, Oxazolation, Mass Sensitivity Boosters
[1] G. T. Hermanson, Bioconjugate Techniques, 1st ed., AcademicPress,
San Diego, CA, 1996.
[2] B. A. Griffin, S. R. Adams, R. Y. Tsien, Science 1998, 281, 269-272.
This article is protected by copyright. All rights reserved.

10.1002/anie.202007608
Angewandte Chemie International Edition
RESEARCH ARTICLE
[3] S. Zalipsky, Bioconjug. Chem. 1995, 6, 150-165.
[4] J. Rao, A. Dragulescu-Andrasi, H. Yao, Curr. Opin. Biotechnol. 2007,
18, 17-25.
[5] O. Boutureira, G. J. Bernardes, Chem. Rev. 2015, 115, 2174-2195.
[6] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science 1994, 266,
776-779.
[7] T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249-289.
[8] M. Raj, H. B. Wu, S. L. Blosser, M. A. Vittoria, P. S. Arora, J. Am. Chem.
Soc. 2015, 137, 6932-6940.
[9] A. O. Y. Chan, C. M. Ho, H. C. Chong, Y. C. Leung, J. S. Huang, M. K.
Wong, C. M. Che, J. Am. Chem. Soc. 2012, 134, 2589-2598.
[10] A. C. Obermeyer, J. B. Jarman, M. B. Francis, J. Am. Chem. Soc.
2014, 136, 9572-9579.
[11] D. Chen, M. M. Disotuar, X. C. Xiong, Y. X. Wang, D. H. C. Chou,
Chem. Sci. 2017, 8, 2717-2722.
[12] L. Purushottam, S. R. Adusumalli, U. Singh, V. B. Unnikrishnan, D. G.
Rawale, M. Gujrati, R. K. Mishra, V. Rai, Nat. Commun. 2019, 10.
[13] J. P. Tam, Q. Yu, Z. Miao, Biopolymers 1999, 51, 311-332.
[14] K. F. Geoghegan, J. G. Stroh, Bioconjug. Chem. 1992, 3, 138-146.
[15] A. Dirksen, T. M. Hackeng, P. E. Dawson, Angew. Chem. Int. Ed.
2006, 45, 7581-7584.
[16] A. Dirksen, S. Dirksen, T. M. Hackeng, P. E. Dawson, J. Am. Chem.
Soc. 2006, 128, 15602-15603.
[17] M. J. Han, D. C. Xiong, X. S. Ye, Chem. Commun. 2012, 48, 11079-
11081.
[18] T. S. Howard, R. D. Cohen, O. Nwajiobi, Z. P. Muneeswaran, Y. E.
Sim, N. N. Lahankar, J. T. Yeh, M. Raj, Org. Lett. 2018, 20, 5344-5347.
[19] S. Mahesh, V. Adebomi, Z. P. Muneeswaran, M. Raj, Angew. Chem.
Int. Ed. 2020, 59, 2793-2801.
[20] X. Li, L. Zhang, S. E. Hall, J. P. Tam, Tetrahedron Lett. 2000, 41,
4069-4073.
[21] J. I. MacDonald, H. K. Munch, T. Moore, M. B. Francis, Nat. Chem.
Biol. 2015, 11, 326-331.
[22] A. Bagag, J. M. Jault, N. Sidahmed-Adrar, M. Refregiers, A. Giuliani, F.
Le Naour, PLoS. One 2013, 8, e79033.
[23] R. Singudas, N. C. Reddy, V. Rai, Chem Commun. 2019, 55, 9979-
9982.
[24] M. Cydzik, M. Rudowska, P. Stefanowicz, Z. Szewczuk, J. Pept. Sci.
2011, 17, 445-453.
[25] Y. Goto, Y. Ito, Y. Kato, S. Tsunoda, H. Suga, Chem. Biol. 2014, 21,
766-774.
[26] E. W. Schmidt, J. T. Nelson, D. A. Rasko, S. Sudek, J. A. Eisen, M. G.
Haygood, J. Ravel, Proc. Natl. Acad. Sci. U S A 2005, 102, 7315-
7320.
[27] B. M. Degnan, C. J. Hawkins, M. F. Lavin, E. J. McCaffrey, D. L. Parry,
D. J. Watters, J. Med. Chem. 1989, 32, 1354-1359.
[28] M. R. Prinsep, R. E. Moore, I. A. Levine, G. M. Patterson, J. Nat. Prod.
1992, 55, 140-142.
[29] S. R. Adusumalli, D. G. Rawale, U. Singh, P. Tripathi, R. Paul, N.
Kalra, R. K. Mishra, S. Shukla, V. Rai, J. Am. Chem. Soc. 2018, 140,
15114-15123.
[30] X. Chen, K. Muthoosamy, A. Pfisterer, B. Neumann, T. Weil,
Bioconjug. Chem. 2012, 23, 500-508.
[31] V. Fernandez-Moreira, J. V. Alegre-Requena, R. P. Herrera, I.Marzo.
M. Concepcion Gimeno, RSC. Adv. 2016, 6, 14171–14177.
[32] H. G. Lee, G. Lautrette, B. L. Pentelute, S. L. Buchwald, Angew. Chem.
Int. Ed. 2017, 56, 3177-3181.
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Table of Contents
We report a novel one-step Azolation strategy for the site- and chemo-
selective labeling of the N-terminus with mass sensitive probes. This method
is tolerant to a variety of proteins and is applied for the N-terminal tagging of
proteolytic fragments in the digested complex mixture in one-pot, under mild
reaction conditions.
Kuei C. Tang, and Monika Raj*
Page No. – Page No.
One Step Azolation strategy for
site- and chemo-selective labeling
of proteins with mass sensitive
probes
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