Selective Triazenation Reaction (STaR) of Secondary Amines for Tagging Monomethyl Lysine Post-Translational Modifications

Article abstract of DOI:10.1002/anie.202013997

Lysine monomethylation (Kme) is an impactful post-translational modification (PTM) responsible for regulating biological processes and implicated in diseases, thus there is great interest in identifying these methylation marks globally. However, the progr

Full text of DOI:10.1002/anie.202013997

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Triazenation Unable Selective Reaction of Secondary Amines
(TrUSS) for Tagging Monomethyl Lysine PTMs
Authors: Monika Raj, Ogonna Nwajiobi, Sriram Mahesh, and Xavier
Streety
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013997
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
Selective Triazenation Reaction (STaR) of Seconary Amines for
Tagging Monomethyl Lysine PTMs
Ogonna Nwajiobi,[a]# Sriram Mahesh,[b]# Xavier Streety[b] and Monika Raj*,[a]
Abstract: Lysine monomethylation Kme is an impactful post-
translational modification PTM responsible for regulating
biological processes and implicated in diseases thus there is
great interest in identifying these methylation marks globally.
However, the progress in this area has been challenging
because the addition of a small methyl group on lysine leads to
negligible change in the bulk, charge and hydrophobicity. Here,
we report an empowering chemical technology Selective
Triazenation Reaction “STaR” of secondary amines using arene
diazonium salts to achieve highly selective, rapid, and robust
that monomethylation of lysine does not add a substantial steric
bulk.25 Second, the lysine monomethylation does not neutralize
their positive charge and does not significantly change their
hydrophobicity (Figure 1a).25 Moreover, the methyl group is too
small to be captured by specific antibodies. Thus, it is very
difficult to develop a highly efficient enrichment strategy.26
The current methods for detecting Kme PTMs involve the use of
antibodies25,27 and methyl binding domains.28-29 However, these
affinity reagents suffer from serious drawbacks and are unable
to completely detect all the Kme sites because they are unable
to distinguish between different lysine methylation states (mono,
di- or tri-), adjacent PTMs can negatively affect antibody
recognition, cross-reactivity with off target antigens, and batch to
batch irreproducibility.30-32 Moreover, they cannot be used to
identify unknown PTMs due to their high sequence specificity.
tagging of Kme peptides from
a complex mixture under
biocompatible conditions. Although the resulting triazene-linkage
with Kme is stable, we highlight the efficient decoupling of
triazene-conjugate to afford unmodified starting components
under mild conditions when desired. Our work establishes a
unique chemoselective “traceless” bioconjugation strategy for
selective enrichment of Kme PTMs.
Recently,
small
molecules
and
peptides
mimicking
chromodomains have been reported for identifying trimethyl
lysine Kme3 PTMs but tools to selectively detect Kme are still
lacking.33-34 Another widely used approach for detecting Kme
utilizes mass spectrometry (MS). Although powerful but MS
analysis is expensive, time-consuming and can be complicated
by analytical challenges including low natural abundance of Kme
PTMs in complex mixtures. Also, the change in the mass by
monomethylation is identical to the substitutions of some amino
acids thus leading to the false identification.25,35 Together, none
of these methods are suitable for selective tagging of Kme in a
Pan specific manner due to the lack of empowering chemical
technology.
A game changer for studying lysine monomethylation would be
the discovery of novel chemoselective reaction for secondary
amines36 thus capable of tagging and identification of Kme
PTMs in a pan specific manner, independent of the peptide
sequence and presence of other reactive functional groups. As
such, we sought to pursue triazenation for selective tagging of
Kme. Many coupling methods exist that bring chemical units
together, but the methods that can be reversed easily to
generate the unchanged units are lacking. We report a method,
termed Selective Triazenation Reaction “STaR” that allows
chemoselective Kme tagging and enrichment from a complex
mixture followed by chemically triggered decoupling to generate
the native unchanged coupling partners in a “Traceless” manner
under mild conditions.
Introduction
Lysine methylation is one of the most important Post-
translational modifications (PTMs) of proteins because it
regulates various biological processes including cell growth,
division, gene expression, and DNA/RNA binding.1-5 Due to
lysine’s unique structure, it has the ability to undergo mono, di,
and tri methylation, and different levels of methylation give rise
to different functions and localization within a cell.6 PTMs
regulate cellular processes by influencing the interactions of
proteins with other proteins, nucleic acids, and lipids. We
focused our attention particularly on monomethylation of lysine
Kme which occurs on both histones and non-histone proteins.7-11
Monomethylated lysine (Kme) PTMs have been implicated not
only in transcriptional activation and silencing but the aberrant
PTM levels have been linked to numerous diseases and
disorders such as heart disease, cancer, and diabetes.12-19
Accordingly, there is a great interest in mapping out the global
role of monomethylated lysine (Kme) PTMs in development and
diseases. However, tools to detect monomethylated lysines Kme
are limited compared to other PTMs20-22 because the addition of
a methyl group leads to a negligible alteration in protein’s or
peptide’s physicochemical properties.23-24 The first challenge is
This strategy is well suited for pan specificity because of the
small size of the reagents required for STaR. There are currently
no known “Pan-specific” reagents for tagging Kme. These
innovative tools for detecting PTMs would augment existing
detection methods and expand the chemical tool kit available for
epigenetics research.
[a]
O. Nwajiobi, 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
S. Mahesh, X. Streety
Address: Department of Chemistry and Biochemistry,
Auburn University, Auburn, Alabama 36849, United States
O.N. and S.M. equally contributed in this project.
[b]
#
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
a. Lysine Methylation States
Me
+H2N
Me
Me NH+
Me
Me
+H3N
N+
Me
H
N
H
N
H
N
H
N
N
N
H
N
N
H
H
H
O
O
KMe
O
O
Lys
KMe2
KMe3
No Change in charge
Negligible alteration on physiochemical properties
Hydrophobicity, Steric bulk
H-Bond Donor Capacity
b.Previous Arene Diazonium Reactions
N N
3
+
N2 X-
HN
H2N
3
R
R = EWG, ERG
peptide
H2N
HN
N N
peptide
phosphate buffer, pH 7.0
+
N2 X-
OH
OH
N
N
R
R = EWG
R
H2N
peptide
peptide
phosphate buffer, pH 7.4
c.This work: Selective Triazenation Reaction (STaR) for Selective Tagging of Secondary Amines
+
N2 X-
Chemoselective (all side chains of
amino acids tolerated including lysine,
Kme2 and Kme3)
Stable Triazene Products
Pan Specific Reagent
N N
N
H2N
H2N
HN
R
3
3
3
3
R = ERG
Monomethyl Lysine Enrichment
Traceless Tagging
phosphate buffer, pH 7.0
TFA (10%) in H2O
H2N
peptide
H2N
peptide
Figure 1. Selective Triazenation Reaction “STaR” a, Different methylation states of lysine and challenges with selective tagging of monomethyl lysine. b,
Previous reactions with arene diazonium salts. Arene diazonium salts react with primary amines generating a reversible adduct. Selective tagging of tyrosine to
form azo coupling product with electron deficient arene diazonium salts under physiological conditions. c, One-step, chemoselective approach for labeling of
secondary amines using electron rich arene diazonium salts to give a stable triazene adduct under physiological conditions. Traceless cleavage of the triazene-
coupling adduct under acidic conditions.
diazonium ions are capable of forming triazenes with primary
Results and Discussion
amines but has never been pursued as a bioconjugation
method because of its reversible nature (Figure 1b).37-38 The
Development of chemoselective STaR
azo-coupling of arene diazonium ion with tyrosine is very well
known for bioconjugation but it requires more electrophilic
arene diazonium ion with electron withdrawing groups for
carrying out reaction under physiological conditions (pH 7.4)
(Figure 1b).39-42 The less electrophilic counterparts are only
able to react with Tyr at a significantly elevated pH (9.0).43-44
Inspired by observation of the facile reaction of the arene
diazonium ion with secondary amine to form
a stable
triazene,37-38 we reasoned that STaR might serve as an
attractive starting point, owing to the flexibility of introducing
various functionalities on the arene diazonium ion. Arene
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
Moreover, it has been reported that reaction with Cys is low
yielding under physiological conditions (pH 7.5) specially with
electron rich diazonium ions.37 Drawing inspiration from these
observations, we rationalized that identification of less
electrophilic arene diazonium ions with electron donating
substituents could form the basis of a novel chemoselective
reaction for secondary amines (Figure 1c).
HPLC (Figure 2, Supplementary Figure 1). The azo-coupling
product was not observed with tyrosine even after 24 h
(Figure 2). Similar reactivity and high selectivity towards
monomethyl lysine was observed with another diazonium ion,
4-carboxybenzenediazonium (4CDz) on reaction with peptide
GAKmeF
under
optimized
conditions
(Figure
2,
Supplementary Figure 2). To characterize the triazene-
coupling products with 4-MDz, reactions with proline methyl
ester, 2-(methylamino) ethanol and a peptide (NHMe)Ala-
Phe-OMe containing secondary amines were carried out on a
large scale under the reaction conditions. The resulting
products were isolated and triazenation of secondary amines
was confirmed by NMR (1H and 13C) (Supplementary Figure
3). In contrast, no product was observed with alanine, thus
reconfirms literature reports that triazene-coupling product
with primary amine is reversible in nature (Supplementary
Figure 4).37-38
-
N2+BF4
a.
NH
Me
N
MeO
N
N
3
pH 7
+
3
4MDz-AKmeF
A
A
F
Kme
OMe
4MDz
F
Kme
OH
OH
N2+BF -
4
N
pH 7
N
+
OH
OH
H2N
H2N
Chemoselectivity of STaR
OMe
4MDz-Tyr
Tyr
O
OMe
O
To evaluate the chemoselective nature of the STaR, we
carried out reaction of 4MDz with various reactive amino
acids such as Ala, Asp, His, Asn, Pro, Arg, Ser, Trp and Tyr
under optimized reaction conditions. The reactions were
monitored for 30 mins, 2 hours and 24 hours by LCMS. The
data showed the formation of triazene-coupling product with
proline only (Figure 3a, Supplementary Figure 5). Further,
reaction of 4-MDz with unmethylated lysine and various
methylation states of lysine (Kme, Kme2 and Kme3)
confirmed high chemoselectivity of STaR for the monomethyl
lysine Kme (Figure 3b, Supplementary Figure 6). Next, we
carried out STaR on peptides (GDAKF, GYARF, GWAHF,
and GSANF, and DRVYIHPF) with reactive amino acids but
we did not observe any modification of peptides under the
optimized conditions (Figure 3c, Supplementary Figure 7).
b.
100
80
60
40
20
0
4MDz-AKmeF
4MDz-Tyr
5
10 15 20 25 30 60
Time
Figure 2. High reactivity of secondary amines toward electron rich
aryl diazonium ions 4MDz relative to Tyr at physiological conditions.
a, The coupling reaction of 4MDz with peptide AKmeF and Tyr. b,
Observed rate of coupling reaction of 4MDz with AKmeF and Tyr at 0.6
mM concentrations in 100 mM phosphate buffer (pH 7, 25 oC). Each time
point represents an average of three independent experiments.
Pan specificity of STaR
Having established the optimal conditions, we sought to
demonstrate the Pan specificity of STaR by carrying out
reactions of 4MDz and 4CDz with various peptides of
different sizes and amino acid composition containing
monomethylated lysine at different positions. As outlined in
Figure 3c, peptides bearing aromatic and aliphatic amino
acids next to the monomethyl lysine were fully tolerated in
this protocol by yielding the corresponding triazene-coupling
products with excellent conversions (90-98%, Figure 3c,
HRMS-Supplementary Figure 8). The reactions with a peptide
GEPGIAGFKmeGEQGPK (collagen fragment) bearing
reactive amino acids such as Glu, Gln and Lys did not
interrupt or influence the triazenation process, and afforded a
desired triazene-coupling product with Kme in good
conversion (85%, Figure 3c). Interestingly, the peptide
GKmeAKmeF with two monomethyl lysines at the alternate
position showed the double modification due to the formation
of triazene-coupled product with both monomethyl lysines
(89%, Figure 3c, HRMS-Supplementary Figure 8). Together,
To evaluate the reactivity of secondary amines toward less
electrophilic arene diazonium ions, we started our initial
investigation by carrying out
a
reaction between 4-
methoxybenzenediazonium ion (4MDz) and monomethyl
lysine amino acid. The detailed optimization studies revealed
that the reaction between the monomethyl lysine amino acid
(0.6 mM) and 4MDz (0.6 mM) proceeds most efficiently in the
presence of Na2CO3 (0.6 mM) in phosphate buffer (100 mM,
pH 7) at room temperature, resulting in the formation of a
stable triazene-coupling product with 99% conversion in 30
mins. Next, we monitored the triazene reaction between
4MDz (0.6 mM) with monomethyl lysine peptide AKmeF (0.6
mM) after regular intervals of time and compared it to the
corresponding reaction with tyrosine (0.6 mM) under
optimized conditions (phosphate buffer (100 mM), pH 7). The
formation of the chromophoric triazene-coupling product was
detected using spectrophotometry (increased absorption at
330-390 nm) and HPLC. The results showed 93% conversion
to triazene-coupling adduct in just 5 min as analyzed by
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
a.
+
-
R
N2 BF4
HN
Na CO
2
3
NaP pH 7.0,
peptide
+
o
N
25 C, 30 min
N
N
O
R
peptide
unprotected peptides
R = OMe; 4MDz
R = COOH; 4CDz
O
b.
100
100
80
80
60
40
20
0
60
40
20
0
K
Kme Kme2 Kme3
methylated lysine
A
D
H
N
P
R
S
W
Y
amino acid
c.
A
A
Y
D
K
R
G
G
F
F
A
F
F
A
Kme
A
P
Kme
F
G
4MDz, 0%
4CDz, 0%
4MDz, 0%
4CDz, 0%
4MDz, 93%
4CDz, 90%
4MDz, 98%
4CDz, 95%
4MDz, 95%
4CDz, 94%
A
W
A
S
F
G
G
G
H
Kme
G
N
G
F
F
K
G
A
Kme
Kme
4MDz, 89%
4CDz, 94%
F
4MDz, 91%
4CDz, 90%
4MDz, 0%
4CDz, 0%
4MDz, 0%
4CDz, 0%
V
R
F
H
P
E
E
G
Kme
G
G
Y
P
F
G
Kme2
A
G
D
P
K
K
I
G
F
Kme
Q
G
I
G
4MDz, 0%
4CDz, 0%
4MDz, 90%
4CDz, 95%
4MDz, 85%
d.
4MDz-PAF
[M+Na]
489.22
+
-
N2 BF4
F
4MDz-AKmeF
[M+Na]
Kme
G
A
P
A
A
Kme
F
Kme
G
F
576.29
4MDz-GKmeAKmeF
[M+Na]
A
F
633.31
Kme
4MDz-GAKmeF
[M+Na]
909.46
4MDz
OMe
NaP, pH 7.0
30 min
F
Kme
G
A
P
A
A
Kme
F
Kme
F
G
F
A
Kme
500 550 600 650 700 750 800 850 900 950
`
Figure 3. Secondary amine polypeptide modification using electron rich aryl-diazoniums. a, The general reaction scheme for secondary amine triazene
formation. The reaction showed high chemoselectivity of 4MDz for proline as quantified by LC/MS among a panel of reactive amino acids. b, The reaction showed
high chemoselectivity of 4MDz for monomethyl lysine Kme among a panel of other methylated states of lysine as quantified by LC/MS. c, Chemoselective
triazenation of peptides showed tolerance of amino acid side chains with 4 MDz and 4CDz. d, Selective tagging of all the peptides in a complex mixture using 4-
MDz as analyzed by LCMS. Conditions for a-c: peptide/amino acid (0.6 mM), 4MDz or 4CDz (1 equiv., 0.6 mM), Na2CO3 (1 equiv., 0.6 mM), phosphate buffer
(NaP, 100 mM) at pH 7.0 at room temperature. The addition of Na2CO3 changed pH to 8.3. For peptide GKmeAKmeF with two Kme, double equivalents of 4MDZ
and Na2CO3 were added. The samples were incubated for 30 min in a and b, for 2 h in c and d. Yields of products were analyzed by LCMS.
these results confirmed the high chemoselectivity and pan
specificity of the STaR toward secondary amines. To
and GKmeAKmeF was treated with 4MDz and resulting
solution was analyzed by LC-MS (Figure 3d, Supplementary
Figure 9). We observed complete labeling of all the peptides.
These experiments demonstrate the high pan specificity of
determine the high selectivity of STaR in
a complex
environment, a mixture of peptides PAF, AKmeF, GAKmeF
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
a.
b.
NH2
NH2
Kme
ABDz
NaONO, HCl, H2O
NaP, pH 7.0, 30 min
G
P
G
A
H2N
P
F
Kme
F
A
F
A
A
ABDz
NaONO
F
Kme
A
A
F
Kme
Kme
F
HOBt, EDCl
DIEA, THF
HCl, H2O
NaP, pH 7.0
HN
O
O
HO
O
[M+Na]
770.3
[M+Na]
827.3
N3
HO
CuAAC
NaP, pH 7.0,
ABDz
PAF
4
ABDz
ABDz
Kme
ABDz
GAKmeF
AKmeF
[M+Na]
683.2
N
[M+H]
748.3
F
F
G
G
K
Kme
F
F
A
F
A
G
G
Kme
90%
Kme
91%
[M+H]
805.3
G
G
[M+H]
661.3
N
N
82%
G
F
P
A
A
Kme
F
G
K
Kme
A Kme
Kme2
G
Kme
0
A
Kme
F
650
700
750
800 850
90%
81%
+
-
d.
c.
N2 CF3CO2
NH
0 h
10 h
N
100
F
A
Kme
3
3
+
80
60
40
20
0
TFA (10%)
5 min
F
A
Kme
A
F
Kme
A
F
Kme
OMe
Traceless cleavage
DIC
PPR
DIEA
TCEP
0
2
4
6
8
10 12 14 16 18 min
CuAAC
Figure 4. Chemoselective enrichment of secondary amine peptides using alkyne functionalized arene diazonium. a, Synthetic scheme for alkyne
functionalized aniline and selective tagging of monomethyllysine in varying peptides by in situ generation of alkyne-arene diazonium ABDz from a corresponding
aniline derivative. b, Chemoselective triazenation of all the peptides in a complex mixture by in situ generated alkyne-arene diazonium ABDz followed by click
chemistry with azide for further functionalization of triazene-coupling product. c, High stability of triazene-secondary amine adduct towards a variety of bases
(DIEA and PPR), coupling reagents (DIC), reducing reagent (TCEP) and click chemistry conditions. d, Traceless cleavage of triazene-coupling product 4MDz-
AKmeF under mild acidic conditions. LC-MS analysis of this reaction shows the production of the unmodified starting material, AKmeF.
STaR from a single peptide to a complex mixture under mild
physiological conditions.
Selective tagging of monomethyl lysine in the
complex mixture using STaR
Synthesis of arene diazonium affinity tags for
monomethyl lysine
As a further demonstration of the high selectivity of STaR for
monomethyl lysine, we attempted tagging multiple peptides in
the same solution to test the potential of our method for
enrichment in a complex mixture. The mixture of peptides
PAF, AKmeF, GAKmeF were allowed to react for 1 hour
under the optimized reaction conditions with alkyne-derivative
of diazonium ion ABDz. This was followed by copper(I)-
catalyzed alkyne-azide cycloaddition (CuAAC)45 with 5-
azidopentanoic acid in the reaction mixture for 2 hours. The
reaction products were analyzed by MS and the data showed
the tagging with ABDz and enrichment with 5-azidopentanoic
acid for all the peptides in the reaction mixture (Figure 4b,
Supplementary Figure 12).
The effective bioconjugation reaction for enrichment should
have the ability to attach affinity tags, thus we generated the
alkyne functionalized aniline derived from a single step by the
amidation of 4-aminobenzoic acid with propargyl amine
(Figure 4a, Supplementary Figure 10). We then converted the
alkyne-functionalized
aniline
to
alkyne-functionalized
diazonium ion, 4-aminobenzoic acid alkyne-diazonium ion
(ABDz) in situ by treatment with sodium nitrite
(Supplementary Figure 11). The STaR with ABDz showed
high chemoselectivity for monomethyl lysine on reaction with
peptides such as GAKmeF, AKmeF, GGKmeGKF,
GKmeAKmeF and Kme2GGKmeGKF under optimized
conditions (89-95%, Figure 4a, Supplementary Figure 11).
Traceless enrichment of monomethyl lysine
using STaR
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10.1002/anie.202013997
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RESEARCH ARTICLE
Finally, we tested the chemical stability of the triazene-
coupling product under different conditions. The triazene-
coupling product act as a protecting group for secondary
amines and tolerate various coupling reagents N, N′-
Diisopropylcarbodiimide (DIC) and bases such as N, N-
Diisopropylethylamine (DIEA) and piperidine (PPR) which is
required for Fmoc peptide synthesis (Figure 4c,
Supplementary Figure 13). It is also stable to a strong protein
disulfide reducing agent tris(2-carboxyethyl)phosphine
(TCEP) and click chemistry (CuAAC) conditions (Figure 4c,
Supplementary Figure 13). The triazene-linkage showed
reasonable stability to sodium dithionate and was stable for 1
hour (Supplementary Figure 14). This is in contrast to the
stability of the azo-coupling product with Tyr, which degrades
quickly in the presence of sodium dithionate to generate
modified tyrosine.43-44 The triazene-coupling product 4MDz-
Pro-OMe was incubated in water: ACN mixture for 24 h and
no decomposition of the triazene-coupling product was
observed.
explore if the conjugate can be decoupled in a traceless
manner. Indeed, a slight exposure of the triazene-modified
peptide 4MDz-AKmeF to acidic conditions (10% TFA in H2O)
led to its rapid cleavage in 5 mins to unchanged peptide
AKmeF, as observed by HPLC and MS analysis (Figure 4d,
Supplementary Figure 15).
It is noteworthy that the acidic cleavage generated original
coupling partners in an unperturbed manner. In contrast,
handful of the coupling reactions are reversible but the
products derived from decoupling are modified versions of the
original reactants.44 The remarkable speed and selectivity of
the STaR, and the ability to subsequently cleave the resulting
triazene-linkage in
unmodified starting
advantages of this conjugation strategy.
a
traceless manner to regenerate
materials, underscores unique
Enrichment of monomethyl lysine Kme from
complex mixture
Next, we established rapid, near-quantitative, and site-
specific enrichment of monomethyl lysine peptides from a
complex mixture of proteolytic fragments by STaR. Trypsin
digested proteolytic mixture spiked with various peptides PAF,
AKmeF and GAKmeF was incubated with ABDz for 1h,
followed by incubation with azide-functionalized resins under
click chemistry conditions for 2h (Figure 5). The resin was
then thoroughly washed with solvents to remove any
untrapped proteolytic fragments. The enriched peptides were
recovered by cleaving them from resin under acidic
conditions (10% TFA in DCM) for 10 mins followed by LCMS
analysis (Figure 5, Supplementary Figure 16). The results
showed the capturing of all the peptides containing secondary
amines from the complex mixture.
NH2
N
peptide
N
N
N
ABDz
HN
O
N
N
HN
AKmeF
1. NaONO, HCl, H2O
NaP, pH 7.0, 1 h
A
F
F
G
A
Kme
PAF
HN
O
2.
A
F
Kme
P
GAKmeF
N3
N
H
4
STaR for solid phase peptide capture of
monomethyl lysine and release for high purity
samples for proteomics
CuAAC, 2 h
Wash
TFA:DCM (1:9)
355.1
[M+Na]
PAF
The efficient characterization of monomethyl lysine containing
peptides from proteolytic fragments is essential but
unfortunately their detection in the complex mixture is highly
challenging. One way to selectively trap and detect
monomethyl lysine PTM with high efficiency from the complex
mixture is to covalently bind monomethyl lysine peptides
directly to the solid support followed by their detachment from
solid support in the traceless manner. Currently, there are no
methods available in literature to achieve this goal.
10 min
PAF
[M+K]
371.1
GAKmeF
[M+Na]
499.2
Cleavage of peptide
from resin
PAF
[M+H]
AKmeF
[M+H]
420.2
AKmeF
GAKmeF
Mass Analysis by
PAF
477.2
333.1
446.2
We felt that this could be achieved by carrying out STaR
directly on solid support. Toward this end, arene diazonium
ion-functionalized resin was synthesized in two steps from 5-
amino-2-chloro benzylalcohol (Supplementary Figure 17).46-47
The arene diazonium ion-functionalized resin was incubated
with mixture of peptides AKmeF, SVF, NAF and RAF
containing primary amines and other reactive side chains for
16 h followed by the extensive washing of beads with water
and various organic solvents such as ACN, DMF, MeOH and
DCM to remove non-covalently attached peptide fragments.
The trapped peptides were released from the resin under mild
acidic conditions using 10% TFA/DCM for 10 mins
LC/MS
Figure 5. Site selective enrichment of secondary amine peptides
from a complex mixture. Trypsin digested proteolytic fragments spiked
with secondary amine peptides were treated with alkyne functionalized
arene diazonium ion ABDz followed by trapping of triazene-coupling
adducts on azide-functionalized beads using click chemistry. The trapped
peptides on beads were analyzed by traceless cleavage from the beads
under acidic conditions using LCMS, confirmed trapping of secondary
amines containing peptides.
Even though the triazene-secondary amine linkage is stable
under physiologically relevant conditions, we aimed to
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
a.
+
N2 Cl-
[M+Na]
400.23
O
AKmeF
Cl
H2O:ACN
Na CO ,16 h
[M+H]
Trypsin
(1:1),
AKmeF
378.25
digestion
Myoglobin
Cytochrome C
Insulin
2
3
[M+K]
416.20
Proteolytic
fragments
1. ACN, H2O, MeOH, DMF
2. 10% TFA/DCM, 10 min
300 350 400
450
b.
peptide
N
+
N2 Cl-
O
N
N
O
GAKmeF
AKmeF
AKmeF
PAF
Cl
Cl
GAKmeF
ACN:Buffer (pH 7.0)
,1 h
AKmeF
AKmeF
Step
Step
1
2
Wash
TFA:DCM (1:9)
(1:1),Na2CO3
Wash
Wash
Step
AKmeF
GAKmeF
Cleavage of peptide
Step
1
PAF
Resin Reused
2
Resin Reused
from resin
400.23
AKmeF
[M+Na]
401.21
AKmeF
[M+Na]
355.17
[M+H]
GAKmeF
477.28
Step
Step
1
2
PAF
[M+Na]
[M+H]
AKmeF
379.23
AKmeF
[M+H]
378.25
PAF
[M+H]
333.19
AKmeF
[M+K]
416.20
PAF
[M+K]
372.14
[M+K]
AKmeF
417.19
AKmeF
Mass Analysis
LC/MS
350
400
350
400
400
450
O
c.
2.0
1.6
(NHMe)AVF
GAKmeF
GAKmeF
peptide
(NHMe)AVF
N
N
N
1.2
0.8
GAKmeF
(NHMe)AVF
Cl
0.4
0
Analysis by
LC-MS
(NHMe)AVF
GAKmeF
0
1
2
Time (h)
Figure 6. Selective enrichment of secondary amines from a complex mixture using arene diazonium-functionalized solid support. a, Selective
enrichment of monomethyl lysine peptide AKmeF from a complex mixture of proteolytic fragments using arene diazonium-functionalized solid support followed by
detachment under mild acidic conditions to give an unmodified peptide as analyzed by MS. b, Reusability of the arene diazonium-functionalized resin for the
selective enrichment of secondary amine peptides such as AKmeF, GAKmeF and PAF in three different cycles. The results showed reusability of resin for three
times without any significant loss of activity. c, Quantification of the peptide trapping by the arene diazonium-functionalized resin. Conditions for a-b: peptide (1
equiv.), resin (25 equiv.), Na2CO3 (2 equiv./peptide) H2O:ACN (1:1) at room temperature for 16 h incubation for a and 1 h for b, washing of resin with ACN, water,
MeOH, DMF and peptide detachment from resin by 10% TFA in DCM for 10 min. Analysis of the detached peptides by LCMS. For b, Repeat the above steps
three times with different peptides.
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
(Supplementary Figure 18). High mass intensity of the
unmodified monomethyl lysine peptide AKmeF was observed
after the cleavage from the resin without any further
purification. Masses of peptides without secondary amines
SVF, NAF, RAF were not observed. To determine the
compatibility of this method in a complex mixture, diazonium
ion-functionalized resin was incubated with trypsin digested
protein mixture spiked with monomethylated peptide AKmeF
for 16 h followed by excessive washings to remove untrapped
peptides from the resin (Figure 6a).
reagents and metals; 2) is chemoselective for monomethyl
lysine Kme rather than any other amino acid residues
including lysine and other methylation states of lysine Kme2
and Kme3; 3) exhibits high reaction kinetics and efficiently tag
monomethyl lysine even at low concentrations and in a
complex mixture; 4) systematically introduce multiple
functional groups including affinity tags by rational
substitution on the arene-diazonium ion, and 5) cleaves the
triazene-linkage to generate unchanged starting materials
“traceless manner” under mild acidic conditions. The potency
of the STaR for pan specificity is well demonstrated by the
selective labeling of all the monomethyl lysine peptides in a
complex mixture under physiological conditions as shown in
Figures 4-5. Another attractive feature of STaR is the
compatibility with existing bio-orthogonal conjugation
reactions such as azide-alkyne click reaction thus enable site-
specific labeling of proteolytic fragments with multiple affinity
tags for the enrichment of peptides from a complex mixture.
Moreover, this chemistry was used for binding of monomethyl
lysine peptides on a solid support in a covalent manner
leading to their efficient enrichment, quantification and
Finally, the trapped peptides were released under mild
conditions
(10%
TFA/DCM)
and
unambiguously
characterized by MS to confirm the enrichment of AKmeF
from a complex mixture (Figure 6a, Supplementary Figure 19).
We were able to develop the STaR to enrich and analyze the
monomethyl lysine peptides from a complex mixture by
capturing them on solid support and by reversing it in a
traceless manner to release highly pure unmodified peptides.
Since the acidic cleavage regenerates the unmodified
diazonium ion functionalized resin, we attempted to reuse it
again to capture other set of monomethyl lysine peptide
fragments from a complex mixture. To test the reusability, we
first incubated the diazonium ion-functionalized resin with
peptides AKmeF and GAKmeF and Na2CO3 for 1 h followed
by washing and detachment under mild acidic conditions
(10% TFA in DCM in 10 mins). The MS analysis confirmed
the attachment of both AKmeF and GAKmeF peptides on the
resin (Figure 6b). This is followed by the incubation of the
cleaved resin with other peptide PAF to avoid any kind of
false results due to first peptide capturing. Again, MS analysis
after decoupling confirmed the attachment of PAF peptide on
the resin. We repeated this process third time with peptide
AKmeF and each time we observed the enrichment of the
secondary amine peptide using the same resin (Figure 6b,
Supplementary Figure 20). We were able to reuse the resin
thrice to enrich monomethyl lysine/secondary amine peptides
without any significant loss in the reactivity of the resin. We
next performed experiments to enrich secondary amine
containing peptides from a complex mixture of proteins using
purification from
a complex mixture, thus provides an
excellent tool for unambiguous analysis of monomethyl
Lysine PTMs. Considering the simple setup of this
bioconjugation reaction with easily derivatized arene-
diazonium ions, we anticipate that this method will become a
highly useful tool for selective functionalization of antibodies
and proteins. In general protocol for enrichment of Kme
peptides from proteolytic digested fragments, iodoacetamide
is added to block all the cysteines and also Kme peptides can
be selectively released from beads in a traceless manner
under mildly acidic conditions thus we do not anticipate any
interference from reactions with cysteine and tyrosine. Thus,
the STaR offers a chemical platform for identifying and
studying the role of monomethyl lysine Kme in whole
proteome, as well as
a starting point for therapeutic
interventions. Since, this reaction is highly selective for
secondary amines, it can be used for the bioconjugation of
proteins with proline at the N-terminus with a variety of
different moieties such as fluorophore, PEG, drug, affinity
reagents such as alkyne and biotin and for synthesis of
antibody drug conjugates. The work in these directions is
currently underway in our laboratory.
a
biotin functionalized probe followed by streptavidin
enrichment (Supplementary Figure 21). The trapped peptides
were released in a traceless manner under acidic conditions
from streptavidin beads and the reaction was analyzed by
LCMS (Supplementary Figure 21). The results indicated that
secondary amine containing peptides were enriched
selectively from a complex mixture. Next, the peptide trapping
was determined in a quantitative manner by incubating
peptides (NHMe)AVF and GAKmeF with arene-diazonium ion
resin followed by measuring the reduction in the peptide
concentration in solution using anisole as standard over a 2h
period at room temperature using LCMS. The results
indicated the trapping of 56% of peptides in 1h and 87% of
peptides in 2h (Figure 6c, Supplementary Figure 22).
Experimental Section
See the supporting information for materials, instruments, reaction
procedures, STaR, chemoselectivity studies, stability study, pan
specificity studies, enrichment studies, traceless cleavage and
products characterization by HRMS, HPLC, and NMR (PDF).
Acknowledgments
This research was supported by NSF (Grant No. CHE-1752654)
granted to M.R. We thank Auburn University for the infrastructure. We
started this work at Auburn University.
Conclusion
In summary, STaR provides a novel, single step secondary
amine selective label, capture and release strategy for
enrichment and identification of monomethyl lysine proteolytic
fragments from a complex mixture. The STaR 1) selectively
Keywords: Chemoselective, Panspecific, Monomethyl lysine
PTM Identification, PTM Enrichment, Traceless Cleavage
labels
secondary
amines/monomethyl
lysine
under
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10.1002/anie.202013997
Angewandte Chemie International Edition
RESEARCH ARTICLE
Table of Contents
We report a novel chemoselective Selective Triazenation Reaction “STaR” of
secondary amine for selective enrichment of monomethyl lysine Kme PTMs
from a complex protein mixture using diazonium ions under physiological
conditions followed by “traceless” release of Kme peptides under mild acidic
conditions.
Ogonna Nwajiobi, Sriram Mahesh,
Xavier Streety and Monika Raj*
Page No. – Page No.
Selective Triazenation Reaction
“STaR” of Secondar Amines for
Tagging Monomethyl Lysine PTMs
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