Practical synthesis of maleimides and coumarin-linked probes for protein and antibody labelling via reduction of native disulfides

Article abstract of DOI:10.1039/b904060a

The cellular tracking, detection and sensing of protein or antibody movement are important aspects to advance our understanding of biomolecular interactions and activity. Antibodies modified with fluorescent dyes are also valuable tools, especially in immunology research. We describe here a proof-of-principle study of a new water-soluble coumarin probe with a maleimide thiol-reacting unit to fluorescently tag biomolecules. Highlights include: (1) a convenient water-based preparation of N-substituted maleimides, (2) a one-pot preparation of activated maleimido-esters, and (3) a bio-conjugation protocol for the selenol-promoted reduction of native disulfide bonds and the 'site-specific' labelling of antibodies with no significant loss of activity.

Full text of DOI:10.1039/b904060a

www.rsc.org/obc
Volume 7 | Number 17 | 7 September 2009 | Pages 3345–3612
ISSN 1477-0520
FULL PAPER
Hong Y. Song et al.
PERSPECTIVE
Practical synthesis of maleimides and Murat Sunbul and Jun Yin
coumarin-linked probes for protein
and antibody labelling via reduction of by enzymatic posttranslational
native disulfides modification
Site specific protein labeling

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Practical synthesis of maleimides and coumarin-linked probes for protein and
antibody labelling via reduction of native disulfides†
Hong Y. Song,a,b Mun H. Ngai, Zhen Y. Song, Paul A. MacAry, Jonathan Hobley and Martin J. Lear*
a
c
c
b
a
Received 26th February 2009, Accepted 9th June 2009
First published as an Advance Article on the web 3rd July 2009
DOI: 10.1039/b904060a
The cellular tracking, detection and sensing of protein or antibody movement are important aspects to
advance our understanding of biomolecular interactions and activity. Antibodies modified with
fluorescent dyes are also valuable tools, especially in immunology research. We describe here a
proof-of-principle study of a new water-soluble coumarin probe with a maleimide thiol-reacting unit to
fluorescently tag biomolecules. Highlights include: (1) a convenient water-based preparation of
N-substituted maleimides, (2) a one-pot preparation of activated maleimido-esters, and (3) a
bio-conjugation protocol for the selenol-promoted reduction of native disulfide bonds and the
‘site-specific’ labelling of antibodies with no significant loss of activity.
Introduction
azide-alkyne cycloaddition (CuAAC), also known as Huisgen
1
,3-dipolar cycloaddition, has become a prime example that is
Due to the rapid development of fluorescent probes and digital
fluorescence microscopy, the design of new and versatile fluo-
rescent water-soluble molecular probes is of high demand in
the biotechnological, environmental, and biomedical disciplines,
especially for studying the dynamic movement and interac-
6
widely used in bio-conjugation work. Despite these virtues, the
‘
site-specific’ pre-installation of azido or terminal alkynyl func-
tionality into biomolecules is still a challenge, as well as overcom-
ing the potential cytotoxicity of copper-catalysts. A noteworthy
advance by the Bertozzi group is the development of copper-
free ‘click chemistry’ using a highly strained and electron-deficient
difluorinated cyclooctyne (DIFO), although the 10-step synthesis
1
tions of proteins. In contemporary biomolecular research, ‘site-
specific’ protein labelling plays an important role as a method
to manipulate or visualize individual proteins. Tagged proteins
are typically generated to study protein function, movement,
isolation, interaction, and cellular localization to their targeted
7
or current commercial unavailability may limit its wide use.
8
In our study towards molecular imaging and ligand targeting,
we chose to focus on a direct (one-step) functionalization of
a biomolecule, which can be performed under physiological
conditions without the requirement of metal promoters. To this
end, we selected to advance cysteine–maleimide coupling as a
labelling stratagem. We thus report here practical methods to
make N-substituted maleimides and illustrate the use of a new
coumarin-based fluorescent probe that was bioconjugated to
an antibody #130 (specific for the human leukocyte antigen-
2
biomolecular entities. The green fluorescent protein (GFP), for
example, has been used most prominently as a marker or tag
to observe the localization and fate of endogenous proteins
in cells. Its large size (238 amino acids) and environmental
dependability can, however, limit its use, especially for laboratories
3
lacking the necessary biochemical machinery or techniques.
Therefore, new small-molecule fluorescent probes, which can be
conveniently, covalently and chemoselectively attached to proteins,
that combine all priorities of minimal invasiveness, wide photo-
physical varieties, ‘site-specificity’, ease-of-use and no dissociation
9
A2, HLA-A2) via sulfhydryl groups (cysteine) generated by
the selenol-promoted reduction of native disulfide bonds. These
methods are chemoselective, robust and rapid, and provide a
practical entry into the ‘site-specific’ labelling of thiol-bearing
biomolecules.
4
concerns, are still highly desirable.
Indeed, the chemically-controlled or ‘specific’ labelling of a
particular biomolecule within a complex biological environment
without disrupting proper functioning is an ongoing challenge. In
Design-wise, a fluorescent probe typically includes a fluo-
rophore, a linker, and a reacting unit that can conjugate chemose-
lectively to a particular functionality on a biomolecule. Our choice
2
001, Sharpless and co-workers identified a number of heterocycle-
forming reactions called ‘click chemistry’, which can respond
to this challenge. Amongst these reactions, the Cu(I)-catalyzed
10
of 7-dimethylaminocoumarin-4-acetic acid as the fluorophore
5
stemmed from several favourable characteristics: it possesses
desirable photophysical properties, such as a large Stokes shift
and visible excitation and emission wavelengths (lex = 370 nm, e =
a
Department of Chemistry, and Medicinal Chemistry Program of the Life
Sciences Institute, National University of Singapore, 3 Science Drive 3,
Singapore 117543. E-mail: chmlmj@nus.edu.sg; Fax: +65-67791691;
Tel: +65-65163998
-1
-1
11
2
2 000 M cm , lem = 459 nm, U = 0.1–0.4), lacks biological
8
1
2
1
3
activity , is small and is water soluble; it does not localize
within cells and can be readily removed by media washing or
dialysis. By further possessing immunoaffinity-fluorescent (IAF)
properties, one additional facet of this fluorophore is that it allows
biomolecule (typically protein) identification and isolation by
b
Institute of Materials Research and Engineering, A*STAR, 3 Research
Link, Singapore 117602
c
Immunology Programme, Centre for Life Sciences, National University of
Singapore, 28 Medical Drive, Singapore 117456
1
13
†
Electronic supplementary information (ESI) available: H and C spectra
8
co-immunoprecipitation with its own specific antibody partner.
for all compounds. See DOI: 10.1039/b904060a
3
400 | Org. Biomol. Chem., 2009, 7, 3400–3406
This journal is © The Royal Society of Chemistry 2009

a
While a short polyethylene glycol (PEG) chain functions as both
a linker and a hydrophilic group, the maleimide functionality
was chosen as a thiol reacting unit over iodoacetamides, since
it requires smaller molar quantities of reagent, less reaction time
Table 2 One-pot preparation of maleimido-carboxylic NHS-esters
14
and is more effective at alkylating thiols at physiological pHs.
b
Results and discussion
Entry
-X-
Product
Yield (%)
1
2
3
-CH
-CH
-(CH
-C H
6
2
CH
CH
2
2
-
3a
3b
3c
3d
61
73
80
90
Practical synthesis of N-alkylated maleimides and
maleimidocarboxylic NHS-esters
2
2
CH -
c
2
)
5
-
4
4
-
Maleimide-incorporated probes are used extensively in biology
and chemistry. Commercially available maleimide derivatives
are not cheap (Sigma-Aldrich: 2b S$600/g; 2c S$1155/g; 3a
S$223/25mg), while the syntheses reported have shortcom-
ings, such as harsh conditions, the use of large amounts of
a
2
Maleic anhydride 1 (0.03 mol), H N-X-COOH (0.03 mol), DMF (30 mL),
b c
NHS (0.037 mol) and DCC (0.063 mol). Isolated yield. The product was
obtained by DCM extraction.
15
toxic reagents, and low yields. In our study, we found that
N-substituted maleimides and their corresponding N-hydroxy
succinimide (NHS) esters could be readily prepared from maleic
anhydride (S$ 49/Kg) in high yield and in a practical fashion.
Table 1 summarizes our results to synthesize maleimide derivatives.
Typically, maleic anhydride 1 was mixed with an a, b, g or
related amino acid in acetic acid and the mixture was stirred at
room temperature for 2 to 8 hours until a mono-coupled amide
intermediate precipitated out. Filtration, followed by dissolution
at reflux in water, then gave the maleimides 2 as a precipitate in
good yields (68% to 87%).
Synthesis of coumarin probe 6. Having developed practical
methods to generate activated NHS-esters 3, we next designed
and prepared a hydrophilic fluorescent probe 6 (Scheme 1).
The coumarin carboxylic acid derivative 4 (an immunoaffinity-
8
fluorescent label, IAF) was prepared by the Von Pechmann
16
reaction, by mixing freshly purified m-dimethylaminophenol and
diethyl 1,3-acetonedicarboxylate with zinc chloride in anhydrous
ethanol, refluxing the mixture for 15 h, and hydrolyzing with
lithium hydroxide to provide the carboxylic acid 4. Coupling of
4
with N-Boc-1,8-diamino-3,6-dioxaoctane gave the coumarin-
linked derivative 5. Various conditions were attempted for the
coupling of coumarin 5 with the activated ester 3a. Optimal results
were obtained by N-Boc removal with TFA followed by N-methyl
morpholine (NMM) promoted coupling in DMF. This afforded
the fluorescent probe 6 in 88% yield over 2 steps.
Due to a need to make activated ester versions of 2, we
next developed a one-pot method to sequentially couple maleic
anhydride, an amino acid and N-hydroxylsuccinamide to generate
3
(Table 2). Here, maleic anhydride and the amino acid were
◦
reacted in DMF at room temperature for 2 hours, cooled to 0 C,
DCC and NHS were added, and the mixture was then allowed to
warm to room temperature and was stirred overnight. The solid
was filtered off, and the filtrate was poured onto ice, producing a
white to pale yellow solid. The solid was collected and dried to
obtain the desired NHS-ester 3 in good to excellent yield (61%
to 90%). Alternatively, after pouring onto ice, the solid could be
collected by dissolving in CH
2
2
Cl , washed and extracted, dried
over Na SO and concentrated to provide 3.
2
4
a
Table 1 Convenient synthesis of N-alkylated maleimides
Scheme 1 Reagents, conditions, and yields: (a) N-Boc-1,8-diamino-
b
Entry
-R
Product
Yield (%)
3
,6-dioxaoctane, EDC·HCl, DMAP, CH
2 2
Cl , rt, 6 h, 79%; (b) TFA,
CH CN, rt, 0.5 h, 100%; (c) 3a, NMM, DMF, rt, 5 h, 88%.
3
1
2
3
4
5
-CH
-CH
-CH
-CH
2
2
2
2
COOH
2a
2b
2c
2d
2e
83
87
85
76
68
CH
CH
2
COOH
CH COOH
COOH
2
2
The fluorescent probe 6 gave consistent excitation–emission
spectra (Fig. 1, lex 370 nm, lem 457 nm), which was found com-
CONHCH
2
-Cyclohexane
17
parable to the free coumarin acid 4 in ethanol. The fluorescence
data shows that 6 is suitable for biomolecule labelling and imaging
work.
a
Maleic anhydride 1 (0.1 mol), amine (0.1 mol), acetic acid (100 mL).
b
Isolated yield.
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 3400–3406 | 3401

Fig. 1 Fluorescence spectrum of coumarin 6 (normalised). Excitation
Fig. 2 Illustration of selenol-mediated reduction of antibody disulfide
bonds and labelling with fluorophore 6.
and emission spectra are shown by black (left) and blue (right) lines,
17
respectively. The spectra were taken with 10 mM of 6 in ethanol.
It has been reported that the use of selenol as a promoter
can rapidly reduce disulfide bonds in proteins and peptides while
retaining their affinity, activity and stability under non-denaturing
conditions. In our study, the selenol-mediated reduction of
antibody #130 was carried out over 5 min under ambient
and physiological-like conditions with dithiothreitol (DTT) or
tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Here, it was
important to employ selenolcystamine as a selenol precursor.
Following incubation with the coumarin probe 6 for 30 min,
the reductive and bioconjugative processes were quenched with
Labelling RGDC with coumarin probe 6. With the coumarin
fluorescent probe 6 in hand, labelling of a bioactive small peptide
bearing a cysteine sulfhydryl group was studied (Scheme 2a). To
this end, the arginine-glycine-aspartic acid (Arg-Gly-Asp; RGD)
ligand was selected, which has a high affinity for the integrin
24
a
v
b receptor that is up-regulated on tumor cell membranes
3
18
and plays an important role in metastasis. This peptide has
also been radio-labelled for PET imaging of tumors. Here, the
labelling was conveniently carried out with a 1 : 1 molar ratio
of coumarin probe 6 and Arg-Gly-Asp-Cys in methanol. The
reaction was completed quantitatively and cleanly within 1 hour.
After removal of solvent in vacuo, the conjugate 7 was verified by
19
20
2
-mercaptoethane-sulfonate (MESNA). Dialysation overnight in
a phosphate buffered saline solution then delivered the fluorescent-
tagged antibody material free from unreacted label 6.
21
LC-MS and HPLC (Scheme 2b). These results illustrate the clear
applicability of the coumarin reagent 6 as an efficient reagent to
label biomolecules with sulfhydryl groups.
In our study, a large excess of coumarin reagent 6 was used.
The yield of coumarin labelling of the antibody was estimated
spectroscopically to be ~15 : 1 (Fig 3). This was established by
correlating numerically the experimental spectra of pure coumarin
to both that of the pure antibody and that of the coumarin
labelled antibody. From the pure spectra, a calculated coumarin
labelled antibody spectrum was derived, which could be fitted
to the experimental spectrum of the labelled antibody in terms
of the spectral shape. Thus, by integrating within the antibody
absorption region of ~280 nm, a molar ratio of coumarin to
antibody of 15 : 1 was found to correlate the calculated spectrum
25
to that found experimentally. Compared to the Ellman’s assay,
which is generally used to evaluate the number of thiol groups
generated by reduction and attachment of thiol-reactive probes,
this method is simple and direct, with no additional antibody
required and no need for gel-filtration. For this calculation
-
1
-1
standard extinction coefficients were used: 22 000 M cm
Scheme 2 Labeling of Arg-Gly-Asp-Cys with fluorophore 6.
Antibody-labelling, cell culture and cell-sorting study
We next applied the strategy to fluorescently label an antibody
(
(
antibody #130) specific to the human leukocyte antigen A2
HLA-A2). Antibody #130 is derived from a murine monoclonal
22
hybridoma, produced in BALB/c mice utilizing a HLA-A2
23
monomer as an immunogen. The antibody was labelled via the
Fig. 3 UV absorption spectra of coumarin, antibody, coumarin labelled
sulfhydryl groups liberated by disulfide reduction (Fig. 2).
antibody and calculated coumarin labelled antibody.
3
402 | Org. Biomol. Chem., 2009, 7, 3400–3406
This journal is © The Royal Society of Chemistry 2009

1
1
-1
(
for pure coumarin ) and 210 000 M cm-1 (for pure antibody,
during fluorescence detection at high light intensities. To mitigate
photobleaching of the coumarin, lower excitation intensities are
recommended. In addition, the amount of fluorophore detected
indicated up to half of the disulfide linkages within the antibody
were reduced, which could lead to reduced binding ability and
activity. Here we found the antibody #130 to still bind well to
the cell surface antigen after the reduction process. Care should,
however, be given to avoid excess disulfide reduction. It should
be noted that this technique is limited to cell surface antibody-
labelling as it cannot efficiently label fixed cells nor selectively
label cytosolic material.
cf. Pierce Biotechnology catalogue).
Following antibody labelling with 6, CIR A2 cells expressing
HLA-A2 were incubated with the tagged antibody material for
0 min at 4 C, and analyzed by flow cytometric analysis (Fig. 4a).
The reaction sample showed significant binding to the target
protein HLA-A2, as detected by flow cytometry, which showed
a significant rightward-shift relative to the negative control. CIR
A2 cells with bound coumarin-antibody #130 were also visualized
under fluorescence microscopy under both light and dark-field
modes (Fig. 4b). Evaluation of the FACS dot plot of labelled CIR
A2 cells showed that 70.2% cells were labelled (Fig. 5).
◦
3
Conclusions
In conclusion, we have designed, synthesized and successfully
demonstrated the applicability of the optical imaging reagent
6
for bioconjugation studies. In particular, 6 can efficiently
label cysteine-bearing biomolecules on peptides or proteins, even
sulfhydyl groups of native disulfide bonds that were reductively
liberated by selenol-reagents. Here it is important to have pu-
rified material for fluorescent labelling. For specific labelling of
biological mixtures of compounds, GFP labelling and metabolic
3
engineering techniques would be recommended. As a model
8
study, the coumarin-based IAF-labelled Arg-Gly-Asp derivative
7
was successfully prepared, which may find use in monitoring
the progress and activity of cancer cells. We further verified that
21
reductive protocols still allowed antibody material to retain their
stability and function for additional labelling, biomolecular and
cellular studies. Lastly, convenient methods were developed for the
synthesis of N-substituted maleimides (2) and we anticipate our
one-pot synthesis of activated maleimide NHS esters (3) to find
wide synthetic use.
Fig. 4 (a) Flow cytometric analysis of CIR A2 cells after incubation with
coumarin 6 labelled antibody #130. A significant detection of fluorescence
was observed on CIR A2 cells, which express the HLA-A2 protein (blue).
CIR cells, which do not express HLA-A2, were used as a negative control
Experimental
(
red). (b) Fluorescence microscopy. There was a significant detection of
General methods
fluorescence on CIR A2 cells (bottom-right) compared to the negative
control CIR cells (top-right).
All chemicals were purchased from the Sigma-Aldrich Chemical
Co. or Alfa Aesar and were used without further purification,
unless indicated otherwise. Experiments were conducted at am-
bient temperature, unless noted otherwise. Analytical thin layer
chromatography (TLC) was performed using a Merck 60 F254 pre-
coated silica gel plate. Subsequent to elution, plates were visualized
using UV radiation (254 nm). Further visualization was possible
by staining with a KMnO
4
water solution. Silica gel 60 (230–
4
00 mesh ASTM) was used for flash column chromatography.
Melting points were determined on a Buchi Melting point B-540
apparatus and are uncorrected. Infrared spectra were recorded on
1
13
a Bio-Rad FTS 165 FTIR spectrometer. H and C NMR spectra
were recorded onaBruker ACF 300or 500MHzspectrometer. The
NMR samples were prepared in a CDCl
3
, DMSO-d
6
or MeOD-d
4
solution. Chemical shifts are reported as d in units of parts per
million (ppm), calibrated based on the different solvents used
(CDCl
3
d 7.26; DMSO-d
6
d 2.50; MeOD-d d 3.31). Multiplicities
4
Fig. 5 FACS dot plot of labelled CIR A2 cells (top-right segment
are given as: s (singlet); d (doublet); t (triplet) or m (multiplets).
Coupling constants (J) are presented in Hz. Mass spectra were
recorded on a Finnigan LCQ mass spectrometer, or a Shimadzu
LC-IT-TOF spectrometer. Analytical HPLC was carried out on
Shimadzu LC-IT-TOF system equipped with an autosampler,
comprises 70.16% of labelled cells).
In this study, the yield of the antibody labelling reaction was
high (~15 : 1 coumarin–antibody), which may in principle lead
to fluorophore quenching and non-linear concentration effects
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 3400–3406 | 3403

˚
General experimental procedure for the one-pot preparation of
maleimido-carboxylic NHS-esters
using reverse-phase Phenomenex Luna 5 mm C18(2) 100 A 150 ¥
.0 mm column. 0.1% TFA/H O and 0.1% TFA/acetonitrile were
3
2
-
1
used as eluents. The flow rate was 0.6 ml min . High resolution
mass (HRMS) spectra were obtained using Finnigan MAT95XP
GC/HRMS. Molecular biology reagents were obtained from
First Base. Solutions for molecular biology were sterilized by
autoclaving. Plastic-ware were sterilised by autoclaving or were
purchased sterile directly from the manufacturer. Buffer solutions
were made using 18 MX Milli-Q water (Millipore).
b-Alanine (2.7 g, 30 mmol) was added to a solution of maleic
anhydride (2.9 g, 30 mmol) in 30 mL of DMF and the mixture was
stirred for 2 h. The resulting solution was cooled in an ice bath
and N-hydroxysuccinimide (4.2g, 37mmol) was addedfollowed by
DCC (12.9 g, 63 mmol). After 5 min, the ice bath was removed and
the solution was vigorously stirred overnight. The white precipitate
(
urea) formed was filtered, washed with 5 mL of DMF and the
filtrate was poured onto ice. The white precipitate formed in the
water was filtered, washed with 10 mL of H O and dried in vacuo to
give succinimido 3-maleimido-propionoate 3a (4.5 g, 61%). The
General experimental procedure for the synthesis of N-alkylated
maleimides
2
30
product can also be isolated in good yield by CH
2
Cl
2
extraction
To a solution of maleic anhydride (9.8 g, 0.1 mol) in 100 mL
of glacial AcOH were added 7.5 g (0.1 mol) of glycine with
vigorous stirring. The resulting slurry was stirred for 8 h under
nitrogen at room temperature. The white precipitate was collected
◦
30
after pouring onto ice. mp 158–159 C (from H
2
O) (lit. 158–
◦
-1
1
(
7
2
61 C). IR nmax (pellet)/cm 1718. d
4H, s, -CH CH
-), 3.04 (2H, t, J = 7 Hz, -CH
-), 7.04 (2H, s, -CH=CH-). d
(125 MHz, DMSO-d
5.9, 29.5, 33.2, 135.1, 167.2, 170.4, 171.0. MS (ESI ): m/z calcd
H
(500 MHz, DMSO-d
6
) 2.79
2
2
2
-), 3.74 (2H, t, J =
Hz, -CH
2
C
6
)
by filtration, and washed with H
solid was dispersed in 200 mL of H
resultant clear solution was refluxed for another 30 min, then
cooled down to room temperature and most H O removed on
a rotary evaporator in vacuo. The white precipitate generated was
collected by filtration, washed with 20 mL of H O, and dried under
2
O (20 mL) twice. The white
+
2
O, and heated to reflux. The
+
for C11
H
10
N
2
6
O [M + Na] 289.0, found 289.0.
2
Succinimido 4-maleimidobutanoate (3b). Obtained from 4-
◦
aminobutyric acid in 73% yield. mp 120–122 C (from H
2
O)
2
3
1
◦
-1
(
lit. 123–129 C). IR nmax (pellet)/cm 1749, 1710. d
H
(500 MHz,
-), 2.72 (2H, t, J =
-), 3.49 (2H, t, J = 7.0 Hz,
(125 MHz, DMSO-d ) d 24.2,
6.4, 28.7, 37.1, 135.5, 169.5, 171.1, 172.0. MS (ESI ): m/z calcd
26
high vacuum to give 2-maleimidoacetic acid 2a in 83% yield. mp
DMSO-d
.6 Hz, -CH
NCH
), 6.99 (2H, s, -CH=CH-). d
6
) d 1.85 (2H, quin, J = 7.6 Hz, -CH
2
◦
27
◦
-1
1
3
)
1
10–111 C (from H
208, 1727, 1617. d
2
O) (lit. 112–113 C). IR nmax (pellet)/cm
(300 MHz, DMSO-d ) 3.67 (2H, s, -CH
(75 MHz, DMSO-d ) 40.3, 135.4,
NO [M + Na + H]
7
-
2
2
CO-), 2.80 (4H, s, -CH
2
H
6
2
-
2
C
6
, 6.08 (2H, s, -CH=CH-). d
C
6
+
+
+
67.5, 169.4. MS (ESI ): m/z calcd for C
6
H
5
4
+
for C12
H
12
N
2
6
O [M + Na] 303.1, found 303.0.
1
79.0, found 179.1.
32
Succinimido 4-maleimidohexanoate (3c) . Obtained from 6-
2
7
3
-Maleimidopropionic acid (2b) . Obtained from b-alanine in
◦
aminohexanoic acid in 80% yield. mp 79–80 C (from CH
2
2
Cl )
◦
26,27
◦
8
(
(
7% yield. mp 97–98 C (from H
pellet)/cm- 3334, 1710, 1569. d
2
O) (lit.
92–94 C). IR nmax
3
1
◦
-1
(
1
lit. 70–73 C). IR nmax (thin film)/cm 3624, 3459, 2942, 1815,
784, 1696. d (500 MHz, CDCl ) d 1.40 (2H, m, -CH -), 1.61 (2H,
quin, J = 7.6 Hz, -CH -), 1.76 (2H, quin, J = 7.6 Hz, -CH -), 2.58
2H, t, J = 7.6 Hz, -CH CO-), 2.81 (4H, s, -CH -), 3.51 (2H, t, J =
.9 Hz, -NCH (75 MHz, CDCl
-), 6.67 (2H, s, -CH=CH-). d
1
H
(300 MHz, DMSO-d
-), 6.04 (2H,
) 31.8, 35.2, 136.4, 167.7,
6
) 2.57
H
3
2
2H, t, J = 7 Hz, -CH
2
-), 3.00 (2H, t, J = 7 Hz, -CH
(75 MHz, DMSO-d
NO [M + H] 170.0, found
2
2
2
s, -CH=CH-). d
C
6
(
6
2
2
+
+
1
1
72.3. MS (ESI ): m/z calcd for C
7
H
7
4
2
C
3
)
69.9.
d 24.0, 25.5, 25.8, 28.0, 30.7, 37.4, 134.0, 168.3, 169, 170.8. MS
(ESI ): m/z calcd for C14
2
8
+
+
4
-Maleimidobutyric
acid
(2c) . Obtained
from
4-
O)
H
16
N
2
O [M + Na] 331.3, found 331.0.
6
◦
aminobutyric acid in 85% yield. mp 98–99 C (from H
2
30
Succinimido 4-maleimidobenzoate (3d) . Obtained from 4-
2
6
◦
-1
(
(
-
d
(
lit. 95–98 C). IR nmax (pellet)/cm 3401, 1707, 1572. d
300 MHz, DMSO-d ) 1.74 (2H, m, -CH
-), 2.33 (2H, t, J = 7 Hz,
CH -), 2.81 (2H, t, J = 8 Hz, -CH
-), 6.01 (2H, s, -CH=CH-).
(75 MHz, DMSO-d ) 23.0, 30.9, 38.8, 136.6, 167.9, 174.3. MS
H
◦
aminobenzoic acid in 90% yield. mp 195–197 C (from H
2
O)
(300 MHz,
-), 7.26 (2H, s, -CH=CH-), 7.70
(75 MHz,
6
2
3
0
◦
-1
(
lit. 205–207 C). IR nmax (pellet)/cm 1767, 1730. d
H
2
2
DMSO-d ) 2.91 (4H, s, -CH CH
2H, d, J = 9 Hz, Ar-H), 8.22 (2H, d, J = 9 Hz, Ar-H). d
6
2
2
C
6
(
C
+
+
ESI ): m/z calcd for C
8
H
9
NO
4
[M + H] 184.2, found 184.0.
DMSO-d
6
) 26.0, 123.3, 126.9, 131.3, 135.5, 138.3, 161.7, 169.8,
-
+
2
-(2-Maleimidoacetamido)acetic acid (2d). Obtained from gly-
170.8. MS (ESI ): m/z calcd for C15
H
10
N
2
O
6
[M - H] 313.0,
◦
cylglycine in 76% yield. mp 113–115 C (from H
2
O). IR nmax
found 312.9.
(
3
-
d
pellet)/cm- 3315, 1741, 1679, 1493. d
.61 (2H, s, -CH
-), 3.86 (2H, d, J = 6 Hz, -CH
CH=CH-), 8.66 (1H, t, J = 6 Hz, -NH-). d
(75 MHz, DMSO-
) 40.5, 41.2, 136.4, 166.7, 167.6, 171.1. MS (ESI ): m/z calcd for
1
H
(300 MHz, DMSO-d
6
)
N-Boc-1,8-diamino-3,6-dioxaoctane. A solution of (Boc)
2
O
2
2
-), 6.03 (2H, s,
(
2.206 g, 10.1 mmol) in CH Cl (50 mL) was slowly added to
2
2
C
a solution of 1,8-diamino-3,6-dioxaoctane (10.7 g, 72.3 mmol)
+
6
◦
in CH
2
Cl
2
(70 mL) at 0 C. The reaction mixture was stirred
+
C
8
H
8
N
2
O [M + Na] 235.0, found 235.0.
5
◦
from 0 C to room temperature for 3 h. The organic layer was
29
Maleimidocyclohexane (2e) . Obtained from amino-
successively washed with water (2 ¥ 60 mL), brine (60 mL),
◦
29
cyclohexane in 68% yield. mp 90–91 C (from H
2
O) (lit.
(300 MHz,
) 1.08–1.88 (10H, m, c-hexyl-H), 2.96 (1H, m, c-hexyl-
H), 6.04 (2H, s, -CH=CH-). d
(75 MHz, DMSO-d ) 24.1, 24.9,
0.7, 50.0, 136.5, 167.7. MS (ESI ): m/z calcd for C10
dried over anhydrous Na
2
SO and concentrated in vacuo to afford
4
◦
-1
8
6–88 C). IR nmax (pellet)/cm 2939, 1579, 1493. d
H
N-Boc-1,8-diamino-3,6-dioxaoctane (2.01 g, 80%) as a colourless
-
1
DMSO-d
6
oil. IR nmax (thin film)/cm 3540, 2869, 1712. d
CDCl ) 1.43 (9H, s, t-Bu), 1.76 (2H, br s, NH ), 2.68 (2H, br s,
-CH NH NHBoc), 3.53
-), 3.31 (2H, dt, J = 5.1, 5.1 Hz, -CH
(4H, dt, J = 5.1, 5.1 Hz, -OCH CH O-), 3.61 (4H, s, -OCH -),
H
(300 MHz,
C
6
3
2
+
3
H
13NO
2
2
2
2
+
[M + Na] 202.1, found 202.2.
2
2
2
3
404 | Org. Biomol. Chem., 2009, 7, 3400–3406
This journal is © The Royal Society of Chemistry 2009

5
7
.17 (1H, br s, NH). d
C
(75 MHz, CDCl
3
) 28.3, 40.2, 41.4, 70.02,
d
C
(125 MHz, DMSO-d
6
) 28.2, 38.7, 38.8, 38.9, 40.1, 68.9, 69.1,
+
0.08, 73.1, 79.0, 155.9. MS (ESI ): m/z calcd for C11
H
24
N
2
O
4
69.4, 69.5, 77.5, 97.4, 108.2, 108.9, 109.3, 125.9, 151.3, 152.8, 155.3,
155.5, 160.6, 167.9. HRMS (ESI ): m/z calcd for C24
Na] 500.2367, found 500.2389.
+
+
[M + H] 249.2, found 249.0.
H
35
N
3
7
O [M +
+
7
-Dimethylaminocoumarin-4-acetic acid (4). Coumarin 4 was
synthesized from m-dimethylaminophenol and diethyl 1,3-
N-(2-(2-(2-(2-(Coumarin-4-acetamido)ethoxy)ethoxy)ethyl-)-3-
17
acetonedicarboxylate as reported, but by using freshly purified
starting materials. m-Dimethylaminophenol (12 g, 87.5 mmol),
diethyl 1,3-acetonedicarboxylate (19.46 g, 96.2 mmol, 1.1 equiv.)
(
(
0
maleimido)propionamide
(6). Boc-coumarin-carbamate
5
200 mg, 0.4 mmol) was dissolved in 3 mL of CH
C and 1 mL of TFA was added. After 30 min, as monitored
3
CN, cooled to
◦
and ZnCl
2
(14.31 g, 105 mmol) were dissolved in absolute ethanol
by TLC, no more starting material was observed, and the solvent
was removed in vacuo. The residue was dissolved in 5 mL of
DMF, followed by addition of 3-maleimidopropionoic acid 3a
111mg, 0.42 mmol) and 25 mL of NMM under nitrogen. The
mixture was stirred at room temperature for 5 h, then poured
(
50 mL). The reaction mixture was heated at reflux for 15 h.
The reaction mixture was cooled to room temperature and the
yellow precipitate formed was filtered. The filtrate was poured into
ice-water. The precipitate formed was filtered and air-dried. The
product was recrystallized to give ethyl 7-dimethylaminocoumarin
(
into 5 mL of ice water. The solution was acidified to pH 3~4,
-
1
as orange needles (15.26 g, 63%). IR nmax (pellet)/cm 2981, 1721,
600. d (300 MHz, CDCl ) 3.05 (1H, s, -N(CH ), 3.67 (2H, s,
CH CH ), 6.05 (1H, s,
-CO-), 4.17 (2H, q, J = 7.1 Hz, -OCH
Coum-CH-), 6.52 (1H, d, J = 2.7 Hz, Coum-CH-), 6.62 (1H, dd,
extracted three times with CH
was washed with water, brine, dried over anhydrous Na
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (MeOH–CH Cl , 1% to 4%) to give
2
Cl
2
. The combined organic layer
1
H
3
)
3 2
2
SO and
4
-
2
2
3
2
2
J = 8.9, 2.5 Hz, Coum-H), 7.40 (1H, d, J = 8.9 Hz, Coum-H). d
C
coumarin 3-(maleimido)propionamide 6 (186 mg, 88%) as a light
(
75 MHz, CDCl
3
) 14.0, 38.1, 40.0, 61.5, 98.2, 108.4, 108.9, 110.5,
-1
yellow sticky oil. IR nmax (thin film)/cm 3321, 2924, 1712, 1659,
1
C
25.2, 148.4, 152.8, 155.8, 161.6, 169.0. MS (FAB): m/z calcd for
1
647, 1612, 1531. d
H
(500 MHz, DMSO-d
-), 3.01 (6H, s, -N(CH ), 3.15 (2H, m, -CH
-), 3.36 (2H, m, -CH
-), 3.42 (2H, t, J = 6 Hz, -CH
CH -), 3.61 (2H, s,
-), 3.59 (2H, t, J = 8 Hz, -CH
-), 5.99 (1H, s, Coum-H), 6.54 (1H, d, J = 3 Hz, Coum-H),
6
) 2.33 (2H, t, J = 8 Hz,
-), 3.23 (2H, m,
-), 3.49
+
15
H
17NO [M + H] 276.1, found 276.1.
4
-
-
(
-
6
7
8
3
1
CH
)
2
3
2
2
Ethyl 7-dimethylaminocoumarin (10 g, 36.32 mmol) was dis-
CH
2
2
2
◦
solved in THF–H
2
O (3 : 1) (60 mL) and cooled to 0 C. 2 M LiOH
4H, s, -CH
CH
2
2
2
solution (36.3 mL, 72.64 mmol, 2.0 equiv.) was added dropwise.
The reaction mixture was stirred at room temperature for 0.5 h.
Water (60 mL) was added, the aqueous layer was extracted with
2
.70 (1H, dd, J = 3 Hz, 9 Hz, Coum-H), 6.99 (2H, s, -CH CH-),
.54 (1H, d, J = 9 Hz, Coum-H), 8.00 (1H, t, J = 5 Hz, -NH-),
(125 MHz, DMSO-d ) 34.4, 34.5,
=
Et O (3 ¥ 50 mL). The aqueous layer was acidified to pH 2 by
2
.29 (1H, t, J = 6 Hz, -NH-). d
C
6
a 2 M HCl solution. The precipitate formed was filtered and
air dried to give coumarin 4 as a yellow solid (8.20 g, 91%). IR
8.9, 39.1, 39.2, 40.5, 69.4, 69.5, 69.9, 70.0, 97.9, 108.7, 109.4,
09.7, 126.5, 135.0, 151.8, 153.2, 155.8, 161.2, 168.4, 170.0, 171.2.
n
max (pellet)/cm- 1706, 1621. d
1
H
(300 MHz, MeOD) 3.09 (1H, s,
-CO-), 6.08 (1H, s, Coum-
+
+
HRMS (ESI ): m/z calcd for C26
H
32
N
4
O [M + Na] 551.2112,
8
-
N(CH
3
)
2
), 3.81 (2H, d, J = 1 Hz, -CH
2
found 551.2101.
H), 6.58 (1H, d, J = 3 Hz, Coum-H), 6.79 (1H, dd, J = 9, 3 Hz,
Labelling Arg-Gly-Asp-Cys (RGD-C) with coumarin 6. RGD-
C (5 mg, 0.01 mmol) and coumarin 6 (5.3 mg, 0.01 mmol) were
dissolved in 0.75 mL of MeOD-d4. The mixture was stirred at
Coum-H), 7.55 (1H, d, J = 9 Hz, Coum-H). d
C
(75 MHz, DMSO-
d ) 37.2, 39.8, 97.4, 108.1, 109.1, 109.6, 126.0, 150.2, 152.8, 155.4,
6
+
1
60.7, 170.8. MS (FAB): m/z calcd for C13
H
13NO [M + H] 248.1,
4
1
room temperature for 1 h. H NMR analysis showed that no more
found 248.0.
starting material was left, and pure product was verified by LC-MS
Boc 2-(2-(2-(coumarin-4-acetamido)ethoxy)ethoxy)ethyl-carba-
mate (5). To a solution of 7-dimethylaminocoumarin-4-acetic
+
+
and HRMS. MS (ESI ): m/z 979.4 ([M + 2H] , 100%). HRMS
+
32
+
(
9
ESI ): m/z: calcd for C41
H
59
N O15 S [M + 2H] 979.4064, found
11
acid
dioxaoctane (843 mg, 3.4 mmol) in 15 mL of anhydrous CH
were added EDC·HCl (680 mg, 3.6 mmoL) and DMAP (75 mg,
.6 mmoL) at 0 C. The mixture was stirred from 0 C to room
temperature for 6 h. After the reaction completed (as monitored by
TLC), it was diluted with 5% KHSO /ice (20 mL) and extracted
with CH Cl . The combined organic layer was washed with water,
% NaHCO , water and brine, dried over anhydrous Na SO and
4
(766 mg, 3.1 mmol) and N-Boc-1,8-diamino-3,6-
79.4043. HPLC showed quantitative labelling of RGD-C (cf.
Scheme 2b).
2
Cl
2
◦
◦
0
Antibody thiol liberation by selenol/DTT disulfide reduction and
fluorescent labelling using coumarin 6. The antibody #130 was
23
4
generated by a hybridoma technique as previously reported. To
a solution of antibody #130 in PBS (250 ml, 0.34 mg/mL), PBS
buffer (32 mL, 0.5 M with 1 mM EDTA, pH 7.4) and EDTA (3 mL,
0.1 M) were added, followed by selenocystamine solution (7.5 mL,
20 mM) and aqueous dithiothreitol (DTT) solution (7.5 mL,
40 mM). The mixture was stirred at room temperature for 5 min,
after which the coumarin probe 6 (80 mL, 10 mM) was added.
After 30 min of the labelling reaction, 2-mercaptoethanesulfonate
(MESNA) (15 ml, 0.1 M) was added to quench the reaction for
1 min. The reaction was dialyzed against 1 ¥ PBS overnight at
2
2
5
3
2
4
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (hexane–ethyl acetate, 1 : 8 to 0 : 1)
to give Boc-coumarin-carbamate 5 (1.12 g, 79%) as a light yellow
-
1
solid. IR nmax (thin film)/cm 3348, 3275, 2931, 1732, 1720.
(500 MHz, DMSO-d ) 1.36 (9H, s, -C(CH ), 3.01 (6H, s,
N(CH NH-), 3.23 (2H,
), 3.06 (2H, dt, J = 6 Hz, 6 Hz, -CH
NH-), 3.37 (2H, t, J = 6 Hz, -CH -),
-), 3.49 (4H, s, -CH CH -), 3.61 (2H, s,
CO-), 6.00 (1H, s, Coum-H), 6.54 (1H, d, J = 3 Hz, Coum-
d
-
H
6
3 3
)
3
)
2
2
dt, J = 6 Hz, 6 Hz, -CH
2
2
◦
6
3
.42 (2H, t, J = 6 Hz, -CH
2
2
2
4 C. Next, the reaction mixture (20 mL) was added to 1 ¥ 10 CIR
◦
CH
2
A2 cells for 30 min at 4 C, followed by three washes in 1 ¥ PBS
H), 6.71 (1H, dd, J = 3 Hz, 9 Hz, Coum-H), 6.73 (1H, m, -NH-),
before being analyzed separately via a CyanADP flow cytometric
machine (Dako) or via an Axioimager Z1 fluorescence microscope
7
.54 (1H, d, J = 9 Hz, Coum-H), 8.27 (1H, t, J = 6 Hz, -NH-).
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 3400–3406 | 3405

(
Zeiss). The same results and observations were obtained with
9 R. J. Moots, M. Matsui, L. Pazmany, A. J. McMichael and J. A.
Frelinger, Immunogenetics, 1991, 34, 141.
0 J. P. Goddard and J. L. Reymond, Trends Biotechnol., 2004, 22, 363;
N. C. Lim and C. Br u¨ ckner, Chem. Commun., 2004, 1094; S. R. Trenor,
A. R. Shultz, B. J. Love and T. E. Long, Chem. Rev., 2004, 104,
antibody #130 when reduced by selenocystamine/TCEP.
1
1
Acknowledgements
3
059; N. C. Lim, J. V. Schuster, M. C. Porto, M. A. Tanudra, L. Yao,
H. C. Freake and C. Br u¨ ckner, Inorg. Chem., 2005, 44, 2018; M. S. T.
Gon c¸ alves, Chem. Rev., 2009, 109, 190.
We thank the Singapore Bioimaging Consortium and A*STAR for
funding (SBIC 005/2005 and INN-006/2006) and a postdoctoral
scholarship (to H.Y.S.). We further thank the National University
of Singapore for a Graduate Scholarship (to M.H.N.). This work
was funded in part by the Competitive Research Program of
the National Research Foundation of Singapore (NRF-G-CRP
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406 | Org. Biomol. Chem., 2009, 7, 3400–3406
This journal is © The Royal Society of Chemistry 2009