Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins

Article abstract of DOI:10.1016/j.bmcl.2004.03.078

Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O⁶-alkylguanine-DNA alkyltransferase (AGT) with O⁶-benzylguanine derivatives. Here we report two complementary routes for the synthesis of O⁶-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for various applications in functional proteomics.

Full text of DOI:10.1016/j.bmcl.2004.03.078

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2725–2728
Synthesis and characterization of bifunctional probes for
the specific labeling of fusion proteins
Maik Kindermann, India Sielaff and Kai Johnsson*
ꢀ
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fꢀedꢀerale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
Received 19 October 2003;revised 17 March 2004;accepted 26 March 2004
Abstract—Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently
introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O6-
alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. Here we report two complementary routes for the
synthesis of O6-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes
and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for
various applications in functional proteomics.
Ó 2004 Elsevier Ltd. All rights reserved.
Studying proteins often relies on providing the protein
of interest with a unique property that allows either for
purification from complex mixtures or for character-
ization in functional assays in vivo or in vitro. Usually,
the unique property is genetically encoded by expressing
the protein of interest as a fusion with an additional
polypeptide such as the hexa-histidine tag for protein
purification or with an autofluorescent protein for
studying protein function in living cells.1;2 Alternatively,
the protein of interest can be chemically labeled with a
synthetic probe that possesses the desired unique prop-
erty.3;4 While a wide variety of synthetic probes with
interesting properties for the studying of protein func-
tion are available, the specific labeling of the protein of
interest remains a challenge.3 We have recently intro-
duced a method that allows for the specific labeling of
fusion proteins of O6-alkylguanine-DNA alkyltransferase
(AGT) with synthetic probes and which is well suited for
applications in vivo and in vitro.5 The specific labeling
of AGT is based on its reaction with cell-permeable
O6-benzylguanine (BG) derivatives of type 1, leading to
the transfer of an appropriate label to a reactive cysteine
of AGT (Fig. 1A). The velocity of the reaction between
AGT and 1 is not significantly influenced by the nature
of the label, allowing the transfer of a wide variety of
different probes to AGT fusion proteins. Furthermore,
we have recently shown that AGT fusion proteins can be
specifically and covalently immobilized on surfaces dis-
playing BG.6 An important extension of this approach
for a variety of applications would be the use of BG
derivative of type 2 (Fig. 1B), leading to the labeling of
AGT fusion proteins with two different probes.7 For
example, the use of BG derivative 3 (Fig. 1C) would lead
to the labeling of AGT fusion proteins with the spec-
troscopic probe fluorescein and the affinity label biotin,
two labels that are employed in a variety of functional
assays. BG derivatives 4 and 5 (Fig. 1C) would lead to
the labeling with two different affinity probes: dinitro-
phenol and biotin in the case of 4, and digoxigenin and
biotin in the case of 5. A possible application for
bifunctional probes such as 4 or 5 would be their use in
tandem affinity purifications of proteins out of complex
mixtures using the immobilized corresponding antibody
and avidin.8
As starting point for the synthesis of bifunctional BG
derivatives we chose 6 and 7, in which orthogonally
protected L
-lysine derivatives 99–11 or 10 are linked to
the previously described BG derivative 8 (Fig. 2).
Bifunctional labels of the type 2 can then be synthesized
by successive couplings of the desired probes to one of
the two deprotected amino functionalities of 6 (Scheme
1) or 7 (Scheme 2). Two lysine derivatives with different
combinations of orthogonal protection groups were
prepared as the synthesis of bifunctional labels with
Keywords: Substrate analogs;Proteomics;Protein labeling.
* Corresponding author. Tel.: +41-21693-9356;fax: +41-21693-9365;
e-mail: kai.johnsson@epfl.ch
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.078

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M. Kindermann et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2725–2728
(A)
protein of
protein of
interest
interest
- Guanine
AGT
S
AGT
S
O
Label
N
+
Label
N
N
H
N
NH2
1
Label 1
HN
H
O
H
N
Label 2
(B)
(C)
O
N
H
O
O
O
O
N
N
N
H
2
N
NH2
Label 1:
Label 2:
OH
O
3
O
HN
NH
H
H
O
O
H
N
H
N
S
O
COOH
O
O
O
NO2
4
O
HN
H
NH
H
H
N
NO2
S
O
O
O
HO
O
5
HN
NH
H
H
H
O
O
H
N
H
N
O
O
H
OH
S
H
O
Figure 1. Labeling of AGT fusion proteins with synthetic molecules. (A) Mechanism of labeling reaction;(B) general structure of bifunctional BG
derivatives;(C) bifunctional BG derivatives 3–5 used for the labeling of AGT fusion protein.
different probes might require different conditions for
deprotection, although for the synthesis of the bifunc-
tional derivatives presented here a single set of protect-
ing groups would have been sufficient. Compounds 6
and 7 were synthesized by condensing 8 with the cor-
responding orthogonally protected lysine derivatives 9
or 10 (Schemes 1 and 2). Concerning a-azido-e-N-
Fmoc-lysine 9, the use of an azide to mask the a-amino
group of lysine was described earlier and 9 was prepared
from commercially available Fmoc-lysine using pub-
lished procedures.10 For the synthesis of 3, the a-azido
group of 6 was reduced under neutral conditions with
trimethylphosphine in aqueous 1,4-dioxane, yielding the
corresponding amine 11 (Scheme 1). Compound 11 was
reacted with N-(+)-biotin-6-amino-caproic acid N-
succinimidyl ester to give 12, followed by Fmoc depro-
tection with diethylamine to yield intermediate 13.
Fluorescein-5(6)-carboxamidocaproic acid N-succin-
imidyl ester was reacted with the free e-amino group of
13, leading to the bifunctional substrate 3. Synthesis of
the bifunctional labels 4 and 5 started from the fully
protected 7, which was prepared by condensing the
commercially available a-N-Fmoc-e-N-Dde-lysine 10
with 8 under standard peptide coupling conditions
(Scheme 2).12 Fmoc deprotection with diethylamine
followed by coupling of N-(+)-biotin-6-aminocaproic
acid N-succinimidyl ester yielded intermediate 15. The
Dde-protecting group was cleaved by using a 4%
hydrazine solution, yielding intermediate 13 with a pri-
mary amine for further derivatizations. For the synthesis
of 4, 13 was coupled to 2,4-dinitrofluorobenzene. The
reaction of digoxigenin-3-O-methylcarbonyl-e-amino-

M. Kindermann et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2725–2728
2727
NH2
O
O
O
O
N
O
N
N
N
H
8
NH2
O
O
N3
H
O
H
HO
O
O
N
H
O
NH
HO
O
N
H
O
10
9
Figure 2. Structures of (8)–(10).
H
R1
H
N
O
R2
O
O
O
O
N
O
a
e
N
N
3
+
9
8
N
H
NH2
R1 = N3
R2 = NH-Fmoc
R2 = NH-Fmoc
6
b
c
d
R1 = NH2
11
R1 = NH-Biotin R2 = NH-Fmoc
12
13
R2 = NH2
R1 = NH-Biotin
Scheme 1. Synthesis of the bifunctional BG derivative 3. Reagents and conditions: (a) EDC, HOBT (1 M in NMP) (2.5 equiv), DMF, 2 h, 57%;
(b) 1,4-dioxane/H2O (8:1), trimethylphosphine (1 M in THF), 1 h, 95%;(c) N-(+)-biotin-6-amino-caproic acid N-succinimidyl ester, DIPEA, DMF,
40 min, 71%;(d) diethylamine, DMF, 40 °C, 1.5 h, quant.;(e) fluorescein-5(6)-carboxamidocaproic acid N-succinimidyl ester, DIPEA, DMF, 40 min,
67%.
H
R1
H
N
O
R2
O
O
4
O
O
N
e
f
a
O
N
N
+
10
8
N
H
NH2
5
R1 = NH-Fmoc R2 = NH-Dde
R1 = NH2 R2 = NH-Dde
7
b
14
15
c
R1 = NH-Biotin R2 = NH-Dde
R1 = NH-Biotin R2 = NH2
d
13
Scheme 2. Synthesis of the bifunctional BG derivatives 4 and 5. Reagents and conditions: (a) EDC, HOBT (1 M in NMP) (2.5 equiv), DMF, 2 h,
64%;(b) diethylamine, DMF, 40 °C, 1.5 h;(c) N-(+)-biotin-6-amino-caproic acid N-succinimidyl ester, DIPEA, DMF, 2 h, 37% over two steps;
(d) hydrazine (4% in DMF), 20 min, then quenching with acetone;(e) 2,4-dinitrofluorobenzene, triethylamine, 45 °C, 2 h, 75%;(f) digoxygenin-
3-oxymethyl-carbonyl)-6-aminocaproic acid N-hydroxysuccinimide ester, triethylamine, 40 °C, 2 h, 45%.
caproic-acid-N-hydroxy-succinimide with the free ami-
no group of 13 yielded compound 5.
To demonstrate that the bifunctional probes 3–513 are
substrates of AGT fusion proteins and that after the

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M. Kindermann et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2725–2728
with bifunctional labels of type 2, we incubated 5 with
cell extract of E. coli expressing GST–AGT and exam-
ined the specificity of the labeling by probing Western
blots of the sample with an anti-digoxigenin antibody
(Fig. 3). This experiment shows that the bifunctional
probes can be used for the specific labeling of AGT
fusion proteins in complex mixtures.
Figure 3. (A) AGT–GST fusion protein (2 lM) was labeled with the
respective bifunctional probe (15 min, rt). Lane 1: GST–AGT labeled
with 3 (25 lM), developed with neutravidin;lane 2: GST–AGT with-
out label as negative control, developed with neutravidin;lane 3: GST–
AGT labeled with 5 (65 lM), developed with neutravidin;lane 4: GST–
AGT without label, developed with neutravidin;lane 5: GST–AGT
labeled with 4 (150 lM), developed with neutravidin;lane 6: GST–
AGT without label, developed with neutravidin;lane 7: GST–AGT
labeled with 3, developed with anti-fluorescein antibody;lane 8: AGT–
GST without label, developed with anti-fluorescein antibody;lane 9:
GST–AGT labeled with 3, developed with anti-digoxygenin antibody;
lane 10: GST–AGT labeled with 4, developed with anti-DNP anti-
body;lane 11: GST–AGT without label, developed with anti-DNP
antibody;lane 12: GST–AGT labeled with 5, developed with anti-
digoxygenin antibody;lane 13: GST–AGT without label, developed
with anti-digoxygenin antibody;(B) lane 1: cell extract of E. coli
expressing GST–AGT fusion protein, labeled with substrate 5, Western
probed with anti-digoxygenin antibody;lane 2: cell extract of E. coli
expressing only GST after incubation with bifunctional probe 5,
Western probed with anti-digoxygenin antibody;lane 3: SDS gel from
cell extract, stained with Coomassie.
In summary, we present here two routes for the facile
preparation of bifunctional BG derivatives of the type 2,
which can be used for the selective labeling of AGT
fusion proteins. The developed chemistry should allow
the synthesis of BG derivatives displaying various
combinations of interesting probes, including cross-
linkers, photosensitizers or spectroscopic probes, and
should make bifunctional BG derivatives useful tools for
applications in functional proteomics.
Acknowledgements
This project was funded by the Swiss National Science
Foundation (2100-067983). I.S. was supported by a
fellowship of the Fonds der Chemischen Industrie
(FCI). We thank Nils Johnsson for valuable advice and
discussions.
reaction the probes can be recognized by other proteins,
we incubated 3–5 with a fusion protein of AGT with
glutathione S-transferase (GST–AGT) and analyzed the
labeling by probing Western blots of these samples with
appropriate antibodies or streptavidin (Fig. 3). In these
experiments, 30 lL of a solution of the fusion protein
(2 lM) were incubated with the bifunctional probes
(25 lM for substrate 3, 150 lM for substrate 4 and
65 lM for substrate 5) for 15 min at room temperature
before the reaction was analyzed by Western blotting.
As in earlier experiments, the labeling of the fusion
protein was nearly quantitative (data not shown).5 The
corresponding data show that all bifunctional probes
are accepted as substrates of GST–AGT and that each
individual probe can be recognized by either specific
antibodies or streptavidin. Furthermore, GST–AGT
labeled with biotin and fluorescein using the bifunc-
tional substrate 3 was not recognized by an anti-digox-
iginin antibody (Fig. 3, lane 9). We also measured the
absorption and emission spectra of GST–AGT after
incubating the fusion protein with 3 and removing
excess label through gel filtration. The measured max-
ima for absorption and emission of the labeled protein
of 498 and 526 nm, respectively, are in agreement with
the corresponding values of fluorescein. To demonstrate
the specificity of the reaction of AGT fusion proteins
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