Tyrosine specific sequential labeling of proteins

Article abstract of DOI:10.1016/j.bmcl.2013.09.002

We report (a) on the synthesis of a long-wavelength fluorescent coumarin containing an allyloxy acetate moiety, (b) the synthesis of two linkers containing an allyloxy acetate and an alkyne or azide function, respectively, and (c) the selective modification human serum albumin by a sequential method involving Pd(II) catalyzed modification of the phenolic side chain of tyrosine residues with an alkyne bearing linker and a subsequent azide-alkyne click reaction with an azide functionalized long-wavelength emitting coumarin dye. The method is likely to be applicable to various kinds of azido-modified fluorophores, and the Pd(II)-catalyzed modification of the tyrosines may also be used to introduce other kinds of tags. With these reagents, tyrosine specific modulation of proteins and peptides becomes possible either directly or in a sequential manner.

Full text of DOI:10.1016/j.bmcl.2013.09.002

Accepted Manuscript
Tyrosine specific sequential labeling of proteins
Gergely B. Cserép, András Herner, Otto S. Wolfbeis, Péter Kele
PII:
S0960-894X(13)01065-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.09.002
BMCL 20854
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
10 August 2013
30 August 2013
3 September 2013
Please cite this article as: Cserép, G.B., Herner, A., Wolfbeis, O.S., Kele, P., Tyrosine specific sequential labeling
of proteins, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.09.002
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Tyrosine specific sequential labeling of
proteins
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Gergely B. Cserép, András Herner, Otto S. Wolfbeis and Péter Kele
O
O
N
N
N
1
.
O
N
N
OH
Pd(OAc)2, TPPTS
pH= 7.4, rt., 1h
Protein
O3S
N3
2
.
Cu(I), TBTA
rt., 12h
Protein
O
O
SO3
O
O
N
N

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Tyrosine specific sequential labeling of proteins
±
a
± a
b
a, 
Gergely B. Cserép , András Herner , Otto S. Wolfbeis and Péter Kele
a
"
Lendület" Laboratory of Chemical Biology, Research Centre for Natural Sciences, Institute of Organic Chemistry, Hungarian Academy of Sciences,
Pusztaszeri út 59-67, H-1025 Budapest, Hungary
b
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We report (a) on the synthesis of a long-wavelength fluorescent coumarin containing an
allyloxy acetate moiety, (b) the synthesis of two linkers containing an allyloxy acetate and an
alkyne or azide function, respectively, and (c) the selective modification human serum albumin
by a sequential method involving Pd(II) catalyzed modification of the phenolic side chain of
tyrosine residues with an alkyne bearing linker and a subsequent azide-alkyne click reaction
with an azide functionalized long-wavelength emitting coumarin dye. The method is likely to
be applicable to various kinds of azido-modified fluorophores, and the Pd(II)-catalyzed
modification of the tyrosines may also be used to introduce other kinds of tags. With these
reagents, tyrosine specific modulation of proteins and peptides becomes possible either directly
or in a sequential manner.
Keywords:
Tyrosine tagging
Chemical reporter
Sequential labeling
Click chemistry
2
009 Elsevier Ltd. All rights reserved.
Bioorthogonal chemistry
In vitro and in vivo fluorescence imaging of biological species
is superior due to its sensitivity, excellent temporal and spatial
resolution, and its potential application to multicolor imaging.
Fluorescent tags with longwave excitation and emission maxima
are particularly useful for biolabeling because their excitation
does not generate the strong autofluorescence of biomatter that is
observed under shortwave (UV or blue) excitation. Covalent
labeling is expected to proceed rapidly, to give quantitative yields
in aqueous solutions at near-neutral pH values, while not to affect
any reactive sites of the biomolecule at the same time. Chemical
reactions that meet all these criteria are collected under the term
reactions are probably best represented by the robust Cu(I)-
catalyzed azide-alkyne cycloaddition, which is probably the most
widely used “click” reaction.
1
0,11
Most proteins are randomly labeled, usually at the -amino
group of lysine, the thiol group of cysteine, or after manipulation
7,12
1
-4
of N- and C-termini. Selective modification of tyrosine (Tyr)
residues is particularly attractive because Tyr is a fairly rare
amino acid. Moreover, surface-accessible tyrosines are rather
scarce in natural proteins so selective tagging of Tyr can lead to
13
14
site-specific labeling. Tilley et al. and Ma’s group employed
Pd(II)-catalyzed reaction to label the phenolic side chain of Tyr
“
bioorthogonal chemistry”. Orthogonal modification of
15
with allyloxy acetates. The group of Barbas have reported on
biomolecules is of great interest in chemical research, lately.
Reactions that exclusively take place between two functional
groups while not interfering with others are especially desirable
in the labeling schemes of biomolecules. Up to now, a set of
bioorthogonal schemes were delineated and applied e.g. for the
the use of a stable formylbenzene diazonium salt to modify
proteins through the phenolic side-chain of tyrosines.
Besides direct modification of Tyr side chains with e.g. a
fluorescent label, the use of allyloxy-acetates offers the
possibility to design bifunctional chemical reporters that combine
Tyr selective and “clickable” moieties as well. Selective
manipulation with such chemical reporters with subsequent
5-9
fluorescent tagging of proteins, glycans and nucleotides. Metal-
free bioorthogonal transformations are preferred in case of in vivo
labeling in order to avoid the use of toxic metal ion catalysts (for
example by making use of the strain-promoted cycloaddition of
further modification give rise to sequential approaches, a method
8
6,16-19
azides and alkynes ), while in case of in vitro labeling, metal ion
that holds many benefits and numerous applications.
For
catalyzed methods are also adequate. Such metal catalyzed
example, well-accessible Tyr residues may be modified with such
—
——

Corresponding author. Tel.: (+36)1-372-2500 / ext. 1610; fax: (+36)1-372-2909. E-mail address: kele.peter@ttk.mta.hu (P. Kele)
±
These authors contributed equally to the work

chemical linkers in a first step. This reporter tag is then further
reacted in a subsequent step with a suitable label, for example
certain cell-permeable fluorophores bearing complementary
To evaluate the possibility of the sequential tyrosine-labeling
concept, we have designed and synthesized new, “clickable”
fluorescent dyes as well (15a,b; Scheme 3). Synthesis of these
2
0-22
24
functions.
“clickable dyes started from salicylaldehyde 11. Wittig reaction
with phosphonium salt resulted in coumarin 12, which was
subjected to Vilsmeyer-Haack formylation to furnish 13.
Condensation reaction with betains 14a,b resulted in the
We report here on the design and synthesis of a longwave
emitting fluorescent allyloxy acetate functionalized coumarin
displaing a large Stokes shift (1). We are also presenting two
generally applicable bioorthogonalized chemical reporters (2 and
3
clickable labels 15a,b. These fluorophores have a purple color
and display a Stokes shift (i.e., the difference between emission
and excitation maxima) of ~150 nm, with excitation and emission
maxima of 564 and 714 nm, respectively. Its purple color and
dark-red fluorescence make these dyes particularly suitable for
imaging applications as they are less interfered by biological
auto-fluorescence. The large Stokes shift, in turn, makes the
probe more resistant to interferences by scattered light.
3
) that combine the versatility of Tyr-specific labeling via an
allyloxy-acetates moiety and the excellent performance of azide-
alkyne click reaction-based fluorescent tagging (Figure 1).
O
N
N+
(
a)
O
9
SO Na
SO3-
3
8
CHO
O
+
(b)
Figure 1. Structures of the Tyr-specific fluorescent coumarin (1) and the two
bifunctional chemical linkers (2, 3) used for sequential labeling.
N
O
The synthesis of 2 and 3 is outlined in Scheme 1. The
procedure was adapted from Tilley's work and started with the
10
1
3
O
N+
transformation of the primary alcohols 4a and 4b to aldehydes
5
a, 5b via Swern oxidation, followed by a Wittig reaction with
O
freshly prepared phosphorane to from the desired acrylate
derivatives. These intermediates were then reduced to allyl
alcohols with excess amount of DIBAL-H. The bromine
substituent of 6b was exchanged for iodine and subsequently for
azide. In the last step, acylation of the hydroxy group by acetic
anhydride gave the desired products in moderate to good yields.
SO3-
N
O
O
1
Scheme 2. Synthesis of the fluorescent tag 1. Reagents and conditions: (a) 7,
MeCN:DMF 1.6:1, 55 °C; (b) 10, cat. piperidine, EtOH, 40 °C (yields: 9:
5
3%, 1: 33%).
Fluorescent label 1 was prepared as shown in Scheme 2. N-
alkylation of 4-methylpyridine-3-sulfonate (whose sulfo group
With these compounds in hands, we have selected compounds
and 15a to fluorescently modulate human serum albumin
HSA) as a model protein in a sequential manner. First, HSA was
modified with bifunctional chemical reporter 2, in the presence of
Pd catalyst at room temperature under similar conditions as
described in the work of Tilley et al. (pH 7.4 phosphate buffer,
improves water solubility) with
7 gave the zwitterionic
2
(
intermediate 9, which was then condensed with 3-
formylcoumarin (10). Both the alkylation and the condensation
2
3
step usually require higher temperature, however, due to the
sensitive nature of the allyloxy acetate moiety, the temperature
was kept below 60 °C in this case.
0
.2 eq. catalyst, 5 eq. 2).
CHO
(
a)
BrPh P
COOMe
3
N
O
O
N
OH
11
12
N+
R
1
4a R = N3
CHO
O
SO -
3
14b R = CCH
(
b)
N
O
(
c)
1
3
N+
R
Scheme 1. Synthesis of chemical linkers 2 and 3. Reagents and conditions:
SO -
3
N
O
O
5a,b
(
a) oxalyl chloride, DMSO, DCM, -78 °C; (b) appropriate phosphorane,
DCM, rt., Ar-atm.; (c) DIBAL-H, DCM, -78 °C; (d) NaI, acetone, Δ; (e)
NaN , acetone, Δ; (f) Ac O, cat. DMAP, pyridine:DCM 1:1; rt. (yields: 5a:
1
3
2
9
4
2%, 5b: 78%, 6a: 86%, 6b: 98%, 6c: 76%, 6d: 40%, 2: 80%, 3: 60%, 7:
Scheme 3. Synthesis of clickable fluorophores. Reagents and conditions: (a)
6%).
DBU, DMSO, Δ; (b) POCl , DMF, 60 °C; (c) cat. piperidine, EtOH, Δ
3
(
yields: 12: 63%, 13: 32%, 15a: 80%, 15b: 48%).

After 1 h reaction, label 15a was added to the reaction mixture
along with a catalytic quantity of Cu(I), and the mixture was then
stirred overnight. The mixture was then subjected to size
exclusion chromatography using Sephadex G-25 to remove
unreacted linkers and labels from tagged HSA.
References and notes
1. Lin, Y.; Weissleder, R.; Tung, C. H. Bioconjugate Chem. 2002,
1
3, 605.
2
3
4
.
.
.
Cserép, G. B.; Enyedi, K. N.; Demeter, A.; Mező, G.; Kele, P.
Chem. Asian J. 2013, 8, 494.
Nagy, K.; Orbán, E.; Bősze, Sz.; Kele, P. Chem. Asian J. 2010, 5,
The excitation and emission spectra of the free dye and the
fluorescently modified HAS are shown in Figure 2. The spectra
show substantial differences: a) the emission maximum of the
labeled HSA is shortwave-shifted by 40 nm relative to that of the
free dye (from 714 nm to 673 nm); b) the excitation maxima
remained the same (564 nm), however, the shape of the band is
significantly altered. Such changes are in accordance with the
rules of solvatochromism and suggest that the overall
microenvironment of the label on the Tyr is less polar than the
bulk of the buffer.
7
73.
Link, M; Kele, P.; Achatz, D. E.; Wolfbeis, O. S. Bioorg. Med.
Chem. Lett. 2011, 21, 5538.
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.
Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48,
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Varga, B. R.; Kállay, M.; Hegyi, K.; Béni, Sz.; Kele, P. Chem.
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1
0. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
In order to exclude non-selective physical adsorption of the
fluorophore on the surface of the protein, we also performed the
experiments either without the chemical reporter, the Pd(II)
catalyst or the Cu(I) catalyst. In none of these cases did we
observe the formation of fluorescent HSA. This indicates that
covalent binding of the chemical linkers and subsequent click
labeling are mandatory for successful tagging of the Tyr residues
of HSA.
Angew. Chem. Int. Ed. 2002, 41, 2596.
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1
2. Hermanson, G. T. Bioconjugate Techniques, 1st ed., Academic
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Ed. 2009, 48, 344.
1
1
In conclusion, we have shown that the use of our new linkers
and appropriately functionalized fluorescent labels with a large
Stokes shift enables specific labeling of proteins directly at Tyr
units. The sequential tagging scheme reported here combines the
selectivity of the Pd(II) catalyzed reaction between an aromatic
hydroxy group and allyloxy acetates with the robustness of the
Cu(I) catalyzed azide-alkyne dipolar cycloaddition reaction. We
beleive that (a) the method is likely to be applicable to various
kinds of azido / alkyne-modified fluorophores, and that (b) the
Pd(II)-catalyzed modification of the tyrosine hydroxy group may
also enable the introduction of other kinds of tags, e.g. biotin or
nonfluorescent labels, provided that they carry a complementary
function.
1
7. Achatz, D. E.; Mező, G.; Kele, P.; Wolfbeis, O. S. ChemBioChem
2
009, 10, 2316.
1
1
8. Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272.
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Biomol. Chem. 2013, 11, 3297.
3. At temperatures above 60 °C the allyloxy-acetate moiety
decomposes and mainly forms a diene product (according to NMR
and HRMS), which is in accordance with Tilley’s observations.
4. Lemieux, G. A.; de Graffenried, C. L; Bertozzi C. R. J. Am. Chem.
Soc. 2003, 125, 4708.
2
2
2
2
Supplementary Material
Supplementary data
(experimental
procedures
and
characterization data for all new compounds associated with this
article can be found, in the online version, at http://dx.doi.org/
Figure 2. Excitation and emission spectra of the free dye 15a (grey) and the
labeled HSA protein (black) in water.
Acknowledgments
Financial support of the Hungarian Scientific Research Fund
(
“
(
OTKA, grant numbers K-100134 and NN-110214) and the
Lendület” Program of the Hungarian Academy of Sciences
LP2013-55/2013) is greatly acknowledged.