Amine-selective bioconjugation using arene diazonium salts

Article abstract of DOI:10.1021/ol5016509

A novel bioconjugation strategy is presented that relies on the coupling of diazonium terephthalates with amines in proteins. The diazonium captures the amine while the vicinal ester locks it through cyclization, ensuring no reversibility. The reaction is highly efficient and proceeds under mild conditions and short reaction times. Densely functionalized, complex natural products were directly coupled to proteins using low concentrations of coupling partners.

Full text of DOI:10.1021/ol5016509

Letter
pubs.acs.org/OrgLett
Amine-Selective Bioconjugation Using Arene Diazonium Salts
Stefan Diethelm, Michael A. Schafroth, and Erick M. Carreira*
Laboratorium fur Organische Chemie, ETH Zurich, HCI H335, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland
̈
̈
̈
S
* Supporting Information
ABSTRACT: A novel bioconjugation strategy is presented that relies on the coupling of diazonium terephthalates with amines
in proteins. The diazonium captures the amine while the vicinal ester locks it through cyclization, ensuring no reversibility. The
reaction is highly efficient and proceeds under mild conditions and short reaction times. Densely functionalized, complex natural
products were directly coupled to proteins using low concentrations of coupling partners.
Our interests in developing chemoproteomic reagents to
assess ligand−receptor interactions4 have led us to examine
novel bioconjugation strategies for protein modification. In
particular, we sought mildly electrophilic reagents that could
react with amino groups of proteins in aqueous media.5 We
reasoned that aryl diazonium salts could serve this purpose
because of their stability in water and their inherent reactivity
profile.6 The early reports on the reactivity of arene diazoniums
(3) toward single amino acids highlighted the potential
application of these electrophiles for the derivatization of
lysine, histidine, tryptophan, and tyrosine residues. However,
only their reaction with tyrosines has found application in
bioconjugate chemistry on the basis of their ability to form
diazo adducts such as 4 by electrophilic aromatic substitution
(Scheme 2A).7
To the best of our knowledge, the electrophilicity of arene
diazoniums toward lysine-ε-NH2 to furnish triazenes has never
been exploited for bioconjugation. This can likely be attributed
to two key reasons. First, lysine-ε-NH2’s on protein surfaces are
largely found as protonated ammonium salts (pKa 10.5),
rendering them poorly reactive under physiological conditions.
Second, it has been appreciated for some time that the triazene
adducts formed between amines and arene diazoniums are
susceptible to a variety of decomposition reactions, most
notably substitution by water or other nucleophiles (Scheme
2B).8 With these challenges in mind, we decided to embark on
the design of a diazonium reagent that would serve as a new
tool for targeting amines in proteins.
he chemical modification of biomolecules has gained
Ttremendous attention in the past few years. In particular,
the covalent coupling of two or more bioactive components, a
process generally termed bioconjugation,1,2 has allowed for a
myriad of applications in biological chemistry and pharmaceut-
ical sciences, including the investigation of native biomolecules
or the construction of new functional entities, such as
biomolecule-based materials. Despite the development of
numerous approaches, various challenges remain to be
addressed.2b,3 In particular, there is a need for the identification
of new bioconjugation reactions that combine efficiency along
with site-selective targeting and short reaction times as well as
the use of low substrate concentration. Herein, we report a new
approach for the selective modification of amino groups on
native proteins using newly designed diazonium terephthalate
ester 1 as a bioconjugation reagent (Scheme 1). The approach
allows for the attachment of highly functionalized components
to proteins, resulting in the formation of stable benzo[d]-
[1,2,3]triazin-4(3H)-one products 2. The reaction proceeds
rapidly under mild conditions at physiological pH and with low
substrate concentration.
Scheme 1. Amine-Selective Bioconjugation of Proteins with
Diazonium Salt 1
In laying the groundwork, several challenges would have to
be addressed, including the well-known potential for cross-
reactivity of the reagent with tyrosines (Scheme 2A) as well as
Received: June 8, 2014
© XXXX American Chemical Society
A
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Letter
formation between acid 11 and N-benzyl amine. 12 was
subjected to diazotization using NaNO2 in the presence of p-
toluenesulfonic acid at ambient temperature (15 min)
providing a solution of diazonium salt 13, which was used
directly for bioconjugation.
Scheme 2. Arene Diazonium Salts for Bioconjugation
In order to evaluate the chemistry of the aryl diazonium salt
13 as a reagent for amine-selective bioconjugation, we first set
out to conduct model studies using two tripeptides substrates
14 and 15 (H2N-VGS-CO2H and H2N-AYF-CO2H; Figure 1).
the instability of the triazene adducts formed from amines. We
hypothesized that the first of these issues might be circum-
vented by the careful selection of conjugation conditions and
reagent design. The problem of triazene instability could be
addressed by incorporating a suitably positioned reactive entity
proximal to the diazonium that would capture the triazene
intermediate and thereby preclude adduct decomposition
(Scheme 2C). We hypothesized that an electrophilic group
such as an ester could trap an unstable, initially formed triazene
intermediate 6 as the corresponding benzotriazinone 7.9
The study commenced with the preparation of an aniline as a
diazonium precursor incorporating a linker component
(Scheme 3). As outlined in Scheme 3, we chose commercially
Figure 1. Conjugation of tripeptide 14 (1.0 equiv) with 13 (2.0 equiv)
and tripeptide 15 (1.0 equiv) with 13 (0.9 equiv).
Diazonium tosylate 13 was allowed to react with tripeptide
H2N-VGS-CO2H (14) in pH 7.0 phosphate buffer (100 mM
NaH2PO4).11 After 2 h the desired benzotriazinone 16 could be
isolated in 56% yield after esterification, and its structure was
confirmed by 2D NMR analysis. Similarly, tyrosine containing
tripeptide H2N-AYF-CO2H (15) was reacted with 0.9 equiv of
13. Analysis of this reaction by LC-MS followed by NMR-
spectroscopic characterization of the isolated product 17
indicated selective coupling of the N-terminus of the peptide
substrate (see Supporting Information). Only a small amount
of tyrosine coupled product could be detected by LC-MS.
We next examined the use of aryl diazonium salt 13 for
amine-selective bioconjugation of proteins. Treatment of a 100
μM solution of myoglobin with diazonium salt 13 (1 mM) at
pH 7.0 led to modification of the protein substrate (Figure 2).12
ESI-MS analysis of the product after 30 min of reaction time
indicated the conjugation of up to six linker molecules to
myoglobin (Figure 2A).13 Most importantly, no byproducts
Scheme 3. Preparation of Diazonium Terephthalate 13
available anthrinilate ester derivative 8 as a starting point.10
Attachment of a hydrophilic spacer component was carried out
by peptide coupling of the carboxylic acid 8 with mono-Cbz
protected diethylene glycol bis(3-aminopropyl) ether, produc-
ing amide 9 in 99% yield. Hydrogenolytic cleavage of the
benzyl carbamate delivered amine 10. Its subsequent
condensation with succinic anhydride furnished carboxylic
acid 11 in 75% yield. Both, amine 10 and acid 11, could serve
as versatile platforms for the introduction of functional
components to the linker system. As a test substrate, we
prepared benzyl amide 12 in 83% yield by peptide bond
Figure 2. Diazonium bioconjugation of myoglobin. ESI-MS spectrum
(deconvoluted) of the product mixture (A) and unmodified
myoglobin (B).
B
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Letter
resulting from the reaction of electron-rich aromatic rings of
tyrosine, tryptophan, or histidine residues could be detected by
mass spectrometric methods.14 Moreover, no uncyclized
triazene intermediates (6) were observed.
We next examined various reaction parameters including
substrate concentration and reaction time (see Supporting
Information for more details). A prolonged reaction time of 2 h
led to a slight increase in the conversion to modified products
with up to nine linker molecules attached to myoglobin. When
5 equiv of 13 relative to protein were used, the conversion
slightly decreased (Table 1, entry 1). Using a more dilute
Table 1. Protein Bioconjugation Using Diazonium Reagent
a
13
unmod.
+1
+2
+3
+4
+5
+6
b
c
entry
protein
(%)
(%) (%) (%) (%) (%) (%)
d
1
100 μM A
10 μM A
16
0
29
6
26
12
39
12
21
13
17
18
29
22
27
21
8
21
7
3
18
0
0
14
0
de
,
2
3
4
5
6
100 μM B
100 μM C
100 μM D
100 μM E
6
20
10
12
4
e
e
4
21
21
25
13
13
19
8
0
6
0
12
a
Reagents and conditions: protein, diazonium reagent 13 (1 mM), pH
b
Figure 3. Natural product derivatives tested in the bioconjugation of
myoglobin.
7.0 buffer (100 mM NaH2PO4), 23 °C, 2 h. A: myoglobin; B:
lysozyme; C: cytochrome c; D: ribonuclease a; E: α-chymotrypsi-
nogen. Determined through the relative ratio of peak intensities in the
c
Table 2. Diazonium Bioconjugation of Myoglobin (100 μM)
Using Different Natural Product Incorporating Aniline
Substrates
ESI-MS spectra of the product mixture. unmod. = unmodified; +1 =
singly modified protein, etc. Only 500 μM of 13 employed. Minor
amounts of +7 and +8 conjugates were also observed.
d
e
a
unmod
b
+1
+2
+3
+4
+5
+6
solution of protein substrate (10 μM) full conversion to
modified products could still be achieved using 500 μM of 13
(entry 2). We next tested various other protein substrates for
the reaction with the arene diazonium 13. As outlined in Table
1, lysozyme (B), cytochrome c (C), ribonuclease a (D), and α-
chymotrypsinogen (E) were all modified with excellent
efficiency (entries 3−6).
Having an efficient bioconjugation protocol in hand, we next
turned to evaluating its compatibility with a range of
conjugation partners (Figure 3). We ultimately opted for the
direct attachment of highly functionalized components to
proteins. In particular, bioactive natural products would
represent interesting targets for the conjugation to proteins,
thereby creating novel molecules with potentially interesting
bioactivities.15,16 Accordingly, various linkers incorporating
secondary metabolites of different substance classes were
prepared starting from either amine 10 or acid 11.17 Conjugates
containing carboxylic acid derivatives such as pantothenic acid
(18), biotin (19), the terpenoid plant hormones abscisic acid
(20) or gibberellic acid (21), and the steroid cholic acid (22)
were synthesized in a single step from the natural products by
peptide coupling to amine 10. Moreover, the antibiotic
lincomycin was coupled to 11, furnishing 23. Finally, we
synthesized deacetoxy colchicine conjugate 24.
entry
substrate
(%)
(%) (%) (%) (%) (%) (%)
c
1
1 mM 18
1 mM 19
1 mM 20
1 mM 21
2 mM 22
2 mM 23
1 mM 24
5
7
11
18
9
20
23
16
16
27
24
21
23
22
21
20
19
23
15
19
16
22
20
5
13
9
6
4
c
2
c
3
2
14
13
1
8
c
4
2
8
11
0
5
6
20
7
28
18
21
15
12
9
4
c
7
21
7
3
a
Reagents and conditions: myoglobin (100 μM), diazonium reagent
b
(1 mM), pH 7.0 buffer (100 mM NaH2PO4), 23 °C, 2 h. Determined
through the relative ratio of peak intensities in the ESI-MS spectra of
the product mixture. unmod. = unmodified; +1 = singly modified
c
protein, etc. Minor amounts of +7 and +8 conjugates were also
detected.
Additionally, electron-rich and -deficient olefins were compat-
ible with the reagent system (entries 3 and 4), and even the
highly electron-rich aromatic ring of colchicine derivative 24
was not affected (entry 7). In all cases conjugates resulting from
the selective coupling of the protein’s amino groups were the
only products observed. As before, no side reactions involving
other functional groups could be detected by MS-analysis.
In summary, we have developed an efficient bioconjugation
protocol relying on the coupling of o-ester substituted
diazonium salts with amino groups on proteins and peptides.
The design of the reagent relies on the initial capture of amines
by an electrophilic diazonium salt followed by a locking
mechanism to form stable benzotriazinone adducts. The
reaction proceeds smoothly at physiological pH with short
reaction times. Highly functionalized substrates could be
attached to proteins using small concentrations of both
coupling partners. Moreover, the reported protocol is
As presented in Table 2, the aniline substrates depicted in
Figure 3 were subjected to the standard diazotization
conditions (NaNO2/p-TsOH) and the resulting aryl diazonium
tosylates were reacted with myoglobin as the coupling partner.
Notably, in all cases excellent conversion to modified products
was detected. In particular, various unprotected functional
groups were well tolerated under the reaction conditions
including alcohols (entries 1, 3−6), tertiary amines (entry 6),
thioethers (entries 2 and 6), and sugar derivatives (entry 6).
C
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Letter
(8) Similar observations have been reported previously: (a) Gescher,
A.; Threadgill, M. D. Pharmacol. Ther. 1987, 32, 191−205.
(b) Abdulhameed, A. R. L. Gazz. Chim. Ital. 1989, 119, 453−456.
characterized by preparative ease and is free of transition metal
additives. The reagent system offers further opportunities for
fine-tuning and enhancing its reactivity through substitution of
the aryl ring in 25.18 Investigations toward this end are
currently underway in our laboratory and will be reported in
due course.
(c) Fernan
́
dez-Alonso, A.; Bravo-Díaz, C. J. Phys. Org. Chem. 2007, 20,
547−553.
(9) Similar strategies have previously been employed to trap
triazenes: (a) LeBlanc, R. J.; Vaughan, K. Can. J. Chem. 1972, 50,
2544−2551. (b) Treppendahl, S.; Jakobsen, P. Acta Chem. Scand. B
1984, 38, 185−187. (c) Vaughan, K.; LaFrance, R. J.; Tang, Y.;
Hooper, D. L. Can. J. Chem. 1985, 63, 2455−2461.
(10) For an overview covering methods of diazonium generation
from anilines, see: O’Leary, P. In Science of Synthesis, Vol. 31b;
Ramsden, C. A., Ed.; Georg Thieme Verlag KG: Stuttgart, 2007; pp
1364−1384.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and full spectroscopic data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) 13 can be employed in concentrations of 50 mM or more in a
100 mM phosphate buffer.
(12) Myoglobin harbors a total of 19 lysine residues, all of which are
surface exposed.
AUTHOR INFORMATION
■
Corresponding Author
(13) When the reaction was allowed to run for 2 h a slightly better
degree of protein modification could be observed (see Supporting
Information for detail).
(14) Such byproducts would be characterized by a mass difference of
32 Da with respect to the desired ring closed product resulting from
amine-selective reaction.
(15) For a review on the bioconjugation of bioactive small molecules
to proteins, see: Veronese, F. M.; Morpurgo, M. Il Farmaco 1999, 54,
497−516.
(16) For a recent example on the bioconjugation of natural products
and small molecule drugs to proteins, see: Zhou, Q.; Gui, J.; Pan, C.-
M.; Albone, E.; Cheng, X.; Suh, E. M.; Grasso, L.; Ishihara, Y.; Baran,
P. S. J. Am. Chem. Soc. 2013, 135, 12994−12997.
(17) For details on the preparation of these linkers, see Supporting
Information.
(18) For the influence of substituent effects on the reactivity of
diazonium salts, see: Hanson, P.; Jones, J. R.; Taylor, A. B.; Walton, P.
H.; Timms, A. W. J. Chem. Soc., Perkin Trans. 2 2002, 1135−1150. See
also ref 8b.
*E-mail: carreira@org.chem.ethz.ch.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant of the Swiss National
Science Foundation (Project number 205320_150170). S.D.
acknowledges the Stipendienfonds Schweizerische Chemische
Industrie (SSCI).
REFERENCES
■
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