N-terminal labeling of proteins by the Pictet-Spengler reaction

Article abstract of DOI:10.1016/j.bmcl.2008.07.033

The Pictet-Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an α-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet-Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet-Spengler reaction.

Full text of DOI:10.1016/j.bmcl.2008.07.033

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4550–4553
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Bioorganic & Medicinal Chemistry Letters
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N-terminal labeling of proteins by the Pictet–Spengler reaction
*
*
Tsubasa Sasaki, Koichiro Kodama, Hiroaki Suzuki, Seketsu Fukuzawa , Kazuo Tachibana
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The Pictet–Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was
performed in the following two steps: (1) conversion of the N-terminal glycine residue to an -keto alde-
hyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with trypt-
amine analogues by the Pictet–Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular
dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pic-
tet–Spengler reaction.
Received 18 June 2008
Revised 9 July 2008
Accepted 10 July 2008
Available online 15 July 2008
a
Keywords:
Pictet–Spengler reaction
Myoglobin
Ó 2008 Elsevier Ltd. All rights reserved.
N-terminal
Biotin
Transamination reaction
Keto aldehyde
Functionalization of proteins is increasingly important for biol-
which was tautomerized and hydrolyzed to yield
a-keto alde-
1
,2
ogy studies. A variety of chemistries have been used for protein
functionalization, but some characteristics of the reactions often
prevent their use in a wider variety of applications. For instance,
hydes. The transamination reaction proceeded without affecting
7
the e-amino groups of the lysine residues.
We have applied the Pictet–Spengler reaction to the N-terminal
labeling of horse heart myoglobin, a 153 amino acid residue heme-
binding protein with an N-terminal glycine (Scheme 1). Trypto-
phan methyl ester and tryptamine were employed as the coupling
partners of 2. The N-terminally activated myoglobin 2 was incu-
bated with either tryptophan methyl ester (50 mM) or tryptamine
(50 mM) in phosphate buffer (pH 6.5) at 37 °C for 18 h. The result-
ing products were desalted and subjected to MALDIÀMS. The sin-
gly charged ion peaks at m/z 16952.0 and 17151.4 corresponded to
3
the use of copper ions for Huisugen cycloaddition and transi-
4
tion-metal catalyzed cross-coupling reactions may cause oxida-
tive modifications of proteins.5 The diene group, a chemical tag
for the Diels–Alder reaction, forms a sulfide bond with the thiols
6
of cysteine residues. Condensation products of an aldehyde (e.g.,
7
,8
9
oxime and hydrazone ) suffer from hydrolysis in aqueous solu-
tion. Therefore, there is a continuing demand for alternatives to
the existing reactions.
The Pictet–Spengler reaction is a cyclization reaction, by which
-(2-aminoethyl)indole or b-arylethylamine undergoes Mannich-
the theoretical mass values for 2 (
respectively (Fig. 1A). In addition, the singly charged ion peaks at
D
0.5 Da) and 3 (
D
À0.1 Da),
3
type ring formation with an aldehyde (Scheme S1). A stable C–C
bond can be formed on amino acid residues, but the typical Pic-
tet–Spengler reaction requires strong Bronsted acids.10 For in-
stance, Li et al. used trifluoroacetic acid (1%, v/v in water) as the
acid catalyst for chemoselective ligation of unprotected peptides.
Their conditions cannot be applied to protein modification, since
proteins are denatured under acidic conditions.
m/z 16952.6 and 17094.0 agreed with the theoretical mass values
for 2 (
D
1.1 Da) and the modified myoglobin 4 (
D
À1.5 Da), respec-
tively (Fig. 1B). The results showed that the Pictet–Spengler reac-
tion proceeded successfully with myoglobin. Reaction product 3
was then analyzed by UV/vis absorption spectroscopy. The UV/
vis spectra of the wild-type myoglobin 1 and the modified myoglo-
bin 3 overlapped each other, except for the absorption peak at
280 nm (Fig. 2A). The peak absorption wavelength of tetrahydro-
1
1
Several groups have developed methods for introducing an
7,8
12
aldehyde into targeted positions of proteins.
Gilmore et al. converted an N-terminal glycine residue to an alde-
hyde by a transamination reaction: the -amino group was con-
densed with pyridoxal 5-phosphate to form an iminium cation,
For example,
b-carboline is around 280 nm, suggesting that reaction product
3 contained a 1,2,3,4-tetrahydro-b-carboline group.
a
Reaction product 3 was digested with Achromobacter protease I
(Lys-C), and the Lys-C liberated peptides were subjected to MAL-
DIÀMS to detect the N-terminal peptide (K1-peptide; the region
from residue 1 to K16) of 3. The singly charged ion peaks at m/z
*
Corresponding authors. Tel./fax: +81 3 5841 8059.
1
817.5 and 2017.1 agreed with the calculated mass values for the
E-mail addresses: seketsuf@chem.s.u-tokyo.ac.jp (S. Fukuzawa), ktachi@chem.
s.u-tokyo.ac.jp (K. Tachibana).
K1-peptides of 2 ( 1.6 Da) and 3 ( 2.1 Da), respectively (Fig. S1).
D
D
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.033

T. Sasaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4550–4553
4551
Scheme 1. N-terminal labeling of myoglobin by the Pictet–Spengler reaction.
Figure 1. MALDIÀMS spectra of the Pictet–Spengler reaction products condensed
with (A) tryptamine and (B) tryptophan methyl ester. The calculated average mass
for 2, the modified myoglobin 3, and
respectively.
4 is 16951.5, 17151.5, and 17095.5,
Figure 2. (A) UV/vis and (B) CD spectra of the wild-type myoglobin 1 (1) and the
modified myoglobin 3 (2). The absorbance at around 410 nm is due to extinction
coefficient of heme.
This result suggested that the N-terminal residue was modified by
the Pictet–Spengler reaction. The K1-peptide of 3 was further puri-
fied by ODS-HPLC, using aqueous acetonitrile with 0.1% (v/v) triflu-
oroacetic acid, and was analyzed by MALDIÀMS and MALDI
postsource decay (PSD) fragmentation13 to confirm the amino acid
two findings: (1) the observed mass values for the K1-peptide of
3 agreed with the theoretical one when the K1-peptide was not
purified by ODS-HPLC (Fig. S1), and (2) the UV absorption peak
of 3 was observed at around 280 nm (Fig. 2A). Aromatization of tet-
rahydro-b-carboline shifts the UV absorption peak to higher values
of the resonant wavelength at around pH 6.5. The 1,2,3,4-tetra-
hydro-b-carboline residue might be spontaneously oxidized during
HPLC purification or laser desorption ionization.
sequence. The singly charged ion peak at m/z 2010.9 (D0.9 Da)
1
4
agreed with the calculated mass values for the K1-peptide contain-
ing a methyl b-carboline-3-carboxylate residue 6 (Figs. 3 and S2),
suggesting the aromatization of the 1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylate residue. The ion peaks observed in the MAL-
DIÀPSD spectrum from the precursor ion of m/z 2011.8 were
assigned along the amino acid sequence of 6 (Fig. 3). These results
The modified myoglobin 3 was analyzed by circular dichroism
(CD) spectroscopy. The CD spectral profiles of 3 and the wild-type
myoglobin 1 were superimposable (Fig. 2B), which showed that
the tertiary structure of the protein maintained in its native confor-
supported the conclusion that the N-terminal
a-keto aldehyde was
1
5
functionalized by the Pictet–Spengler reaction.
The 1,2,3,4-tetrahydro-b-carboline residue was not aromatized
during the reaction. This view was supported by the following
mation throughout the reaction. On the other hand, the incubation
of the wild-type myoglobin 1 at 70 °C was accompanied by reduc-
tions in the intensities of the negative bands at 208 and 222 nm

4
552
T. Sasaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4550–4553
with biotinylated tryptophan 7 (Fig. 4B, lane 2). In addition, no bio-
tinylation was detected when myoglobin 2 was incubated with 7 in
the presence of methoxy amine (Fig. 4B, lane 1), which competi-
tively reacted with the aldehyde group on 2. However, undesired
biotinylation was detected after biotinylated tryptophan 7 was
incubated with the wild-type myoglobin 1, instead of using the
N-terminal activated myoglobin 2 as a substrate (Fig. S4). The myo-
globin 1 isolated from horse heart might contain additional alde-
hyde groups besides that at the N-terminus (e.g., lysine, arginine,
1
6
and/or proline residues), which underwent the Pictet–Spengler
reaction to yield the non-specifically biotinylated myoglobin. Oxi-
dized proteins containing aldehyde groups are known to accumu-
late in aging cells. However, the amount of oxidized myoglobin in
the reaction mixture is negligible; the MALDIÀMS spectrum indi-
cated that the molecular ion for the myoglobin containing two tet-
rahydro-b-carboline groups (17.4 kDa.) was weakly observed, as
compared with that for the modified myoglobin 3 (Fig. 1A), sug-
gesting that the Pictet–Spengler reaction occurred predominantly
at the N-terminal aldehyde.
In this study, the N-terminal residue of horse heart myoglobin
was modified by the Pictet–Spengler reaction. The protein sub-
strate is not restricted to myoglobin containing an N-terminal gly-
cine residue; our approach is generally applicable to recombinant
proteins. An N-terminal glycine residue can be generated by prote-
ase digestion of the cleavage site located between an N-terminal
affinity tag and a glycine residue.
Figure 3. MALDI-PSD fragment spectrum of the N-terminal peptide (K1) from 3.
Inset shows the chemical structure of the oxidized K1-peptide 6. The b (N-terminal)
and y (C-terminal) fragment ions are labeled in the spectrum. The monoisotopic
mass calculated from the sequence of 6 is given in Table S1.
(Fig. S3B), suggesting that the proportion of a-helix decreased, due
to heat denaturation. In addition, UV/vis absorption spectroscopy
indicated that the incubation of the wild-type myoglobin 1 at
3
-(2-Aminoethyl)indole was used as a substrate, and the Pictet–
Spengler reaction proceeded under nearly physiological conditions:
aqueous media, ambient temperature (<37 °C), and neutral pH (pH
7
0 °C reduced the intensity of the absorption peak at 410 nm against
that at 280 nm (A410/A280) (Fig. S3A); the relative amount of the apo
form of myoglobin 1 increased by heat denaturation.
6
.5). Electron-rich 3-(2-aminoethyl)indole is a preferred substrate
for the Pictet–Spengler reaction, as compared with b-arylethyl-
Biotinylation of myoglobin was performed as an application of
the Pictet–Spengler reaction to the site-specific functionalization
of proteins. The biotinylation unit 7 (10 mM), consisting of trypto-
phan, an ethylene oxide linker, and biotin (Schemes 4 and S2), and
the protein substrate 2 were mixed in phosphate buffer (pH 6.5)
and incubated at 37 °C for 18 h. The resulting product 5 was sub-
jected to SDS–PAGE and stained with fluorescent dyes to detect
myoglobin (Fig. 4A). The proteins fractionated on the SDS–PAGE
gel were also analyzed by Western blotting to detect biotin
1
7
amine. Therefore, tryptophan, with a free carboxy group, will be
a versatile coupling partner of aldehyde groups on proteins.
We used biotinylated tryptophan 7 to functionalize myoglobin
with biotin. In addition, labeling with a fluorescent group is possi-
ble when a modification unit with a fluorescent dye is used instead
of 7. An advantage of N-terminal labeling with a low molecular-
weight fluorescent group is that the activity of the modified pro-
teins is less severely affected than when the proteins are fused
with GFP (green fluorescent protein). The derivatization of the N-
terminal residue with a fluorescent group can be an alternative ap-
proach to the labeling of proteins with fluorescent groups. This is
possible by the Pictet–Spengler reaction and the following aroma-
tization of the tetrahydro-b-carboline residue, since b-carboline
derivatives (e.g., harmine, harmane, and norharmane) are fluores-
(Fig. 4B). The results indicated that myoglobin 2 was functionalized
1
4
cent. Thus, the application of the Pictet–Spengler reaction to pro-
tein functionalization will open up a new frontier of biotechnology.
Acknowledgments
We are grateful to Prof. Hiroshi Nishihara and Dr. Shoko Kume
of the University of Tokyo, for help in measuring MALDI mass spec-
tra and to Masaru Kinugawa of the University of Tokyo for help in
measuring NMR spectra. Thanks are also due to Prof. Shigeyuki
Yokoyama, Dr. Takuhiro Ito and Shinya Mimasu of the University
of Tokyo, for help in HPLC purification and UV/vis absorption mea-
surement and to Dr. Hiroshi Nakayama of RIKEN for valuable sug-
gestion. This work was partly supported by a Grant-in-Aid for the
Global COE program from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
Supplementary data
Figure 4. (A) Fluorescence detection of the protein moiety by staining with SYPRO
Tangerine, and (B) chemiluminescence detection of the biotin moiety with
streptavin-HRP. An asterisk indicates the dye front in a 15% SDS–PAGE gel. Inset
shows the chemical structure of modification unit 7.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.033.

T. Sasaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4550–4553
4553
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