Synthesis, characterization, photophysical properties, and biological labeling studies of a series of luminescent rhenium(I) polypyridine maleimide complexes.

Article abstract of DOI:10.1021/ic010602z

We report the synthesis, characterization, and photophysical properties of a series of rhenium(I) polypyridine maleimide complexes [Re(N-N)(CO)(3)(py-3-mal)](CF(3)SO(3)) [N-N = 1,10-phenanthroline, phen (1), 2,9-dimethyl-1,10-phenanthroline, 2,9-Me(2)-phen (2), 3,4,7,8-tetramethyl-1,10-phenanthroline, 3,4,7,8-Me(4)-phen (3), 4,7-diphenyl-1,10-phenanthroline, 4,7-Ph(2)-phen (4), 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, 2,9-Me(2)-4,7-Ph(2)-phen (5), 2,2'-biquinonine, biq (6); py-3-mal = N-(3-pyridyl)maleimide]. The X-ray crystal structure of complex 2 has been investigated. Upon excitation, the complexes exhibit intense and long-lived photoluminescence in fluid solutions at 298 K. The emission wavelengths range from 514 to 654 nm, and the emission lifetimes fall in the microsecond time scale. The luminescence is assigned to originate from a metal-to-ligand charge-transfer MLCT [dpi(Re) --> pi*(diimine)] triplet excited state. As the maleimide group can react with the sulfhydryl group to form a stable thioether moiety, these complexes have been used as thiol-specific luminescent labels for a thiolated oligonucleotide, glutathione, and bovine serum albumin and human serum albumin. The photoluminescence properties of the labeled biological species have also been investigated.

Full text of DOI:10.1021/ic010602z

Inorg. Chem. 2002, 41, 40−46
Synthesis, Characterization, Photophysical Properties, and Biological
Labeling Studies of a Series of Luminescent Rhenium(I) Polypyridine
Maleimide Complexes
,†
Kenneth Kam-Wing Lo,* Wai-Ki Hui,† Dominic Chun-Ming Ng,† and Kung-Kai Cheung‡
Department of Biology and Chemistry, City UniVersity of Hong Kong, Tat Chee AVenue,
Kowloon, Hong Kong, P.R. China, and Department of Chemistry, The UniVersity of Hong Kong,
Pokfulam Road, Hong Kong, P.R. China
Received June 6, 2001
We report the synthesis, characterization, and photophysical properties of a series of rhenium(I) polypyridine maleimide
complexes [Re(N−N)(CO)3(py-3-mal)](CF SO ) [N−N ) 1,10-phenanthroline, phen (1), 2,9-dimethyl-1,10-phenan-
3
3
throline, 2,9-Me2-phen (2), 3,4,7,8-tetramethyl-1,10-phenanthroline, 3,4,7,8-Me4-phen (3), 4,7-diphenyl-1,10-
phenanthroline, 4,7-Ph2-phen (4), 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, 2,9-Me2-4,7-Ph2-phen (5), 2,2′-
biquinonine, biq (6); py-3-mal ) N-(3-pyridyl)maleimide]. The X-ray crystal structure of complex 2 has been
investigated. Upon excitation, the complexes exhibit intense and long-lived photoluminescence in fluid solutions at
298 K. The emission wavelengths range from 514 to 654 nm, and the emission lifetimes fall in the microsecond
time scale. The luminescence is assigned to originate from a metal-to-ligand charge-transfer MLCT [dπ(Re) f
π*(diimine)] triplet excited state. As the maleimide group can react with the sulfhydryl group to form a stable
thioether moiety, these complexes have been used as thiol-specific luminescent labels for a thiolated oligonucleotide,
glutathione, and bovine serum albumin and human serum albumin. The photoluminescence properties of the labeled
biological species have also been investigated.
Introduction
specific fluorescent labeling reagents. Of particular interest
is N-(1-pyrenyl)maleimide since its excimer fluorescence
properties enable the label to be utilized as a probe for
proximity and conformational changes of proteins.8
On the other hand, labeling of biological species with
fluorescent markers is a very common procedure nowadays.9
However, designs of biological labels rely essentially on
The maleimide moiety possesses an activated carbon-
carbon double bond and is well-known to undergo specific
alkylation reactions with the sulfhydryl group to form stable
thioethers.1 The maleimide functional group has been linked
to lipids for anchoring of polypeptides to hydrophobic
membranes,2 anticancer drugs for conjugation to thiolated
carrier proteins3 and redox-active agents for providing
electrochemical properties to cysteine-containing oligo-
peptides and metalloproteins.4 Fluorescent organic com-
pounds such as fluorescein,5 eosin,6 and acridine7 have also
been functionalized with a maleimide group to produce site-
(4) (a) Di Gleria, K.; Hill, H. A. O.; Wong, L. L. FEBS Lett. 1996, 390,
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P.; Hill, H. A. O.; Wong, L. L.; Fu¨lo¨p, V. J. Am. Chem. Soc. 1998,
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* To whom all correspondence should be addressed. E-mail: bhkenlo@
cityu.edu.hk.
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Biol. Chem. 1993, 268, 22181-22187.
† City University of Hong Kong.
‡ The University of Hong Kong.
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867-870.
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82, 4600-4604.
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7797-7810. (b) Winters, R. A.; Zukowski, J.; Ercal, N.; Matthews,
R. H.; Spitz, D. R. Anal. Biochem. 1995, 227, 14-21. (c) Sedgwick,
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40 Inorganic Chemistry, Vol. 41, No. 1, 2002
10.1021/ic010602z CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/12/2001

Rhenium(I) Polypyridine Maleimide Complexes
Chart 1. Structures of Diimine Ligands of Complexes 1-6
fluorescent organic dyes while relatively less attention has
been paid on inorganic transition metal complexes.10 Despite
being commonly used, organic labeling reagents have several
disadvantages such as short emission lifetimes, strong pH
dependence, self-quenching effects, and low photostability.
In this regard, rhenium(I) polypyridine complexes are very
promising candidates for biological labeling in view of their
remarkable luminescence properties.11-16 Recently, utilization
of luminescent rhenium(I) polypyridine complexes as a
nucleic acid13a and uracil-dimer13b photocleavage agent and
a DNA metallointercalator14 has been reported. The strong
photooxidizing properties of these complexes have also been
employed in studying electron tunneling in metalloproteins.15
In addition, luminescent rhenium(I) complexes have also
been used as anisotropy probes for protein hydrodynamics.16
We anticipate that the photoluminescence of rhenium(I)
polypyridine complexes can be exploited in the development
of luminescent biological labeling reagents for the following
reasons. First, the use of various diimine ligands for the
rhenium(I) complexes allows control on the metal-to-ligand
charge-transfer (MLCT) emission energy and thereby offers
a series of multicolor luminescent labeling reagents. Also,
large Stokes’ shifts are usually observed for these complexes,
which can minimize self-quenching effects commonly en-
countered in multiple labeling of biomolecules with fluo-
rescent organic dyes.17 Moreover, the long emission lifetimes
of rhenium(I) polypyridine complexes, compared to those
of organic fluorophores, could be applied in time-resolved
detection techniques that can provide improved sensitivity.18
We report here the synthesis and characterization of a
series of new rhenium(I) polypyridine complexes containing
a maleimide moiety, [Re(N-N)(CO)3(py-3-mal)](CF3SO3)
[N-N ) 1,10-phenanthroline, phen (1), 2,9-dimethyl-1,10-
phenanthroline, 2,9-Me2-phen (2), 3,4,7,8-tetramethyl-1,10-
phenanthroline, 3,4,7,8-Me4-phen (3), 4,7-diphenyl-1,10-
phenanthroline, 4,7-Ph2-phen (4), 2,9-dimethyl-4,7-diphenyl-
1,10-phenanthroline, 2,9-Me2-4,7-Ph2-phen (5), 2,2′-bi-
quinonine, biq (6); py-3-mal ) N-(3-pyridyl)maleimide]. The
structures of the diimine ligands are shown in Chart 1. The
X-ray crystal structure of complex 2 has also been investi-
gated. The photophysical properties of these luminescent
complexes and their conjugation to different biological
molecules with the sulfhydryl group have also been studied.
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L.; Nair, R.; Johnson, M. L.; Nowaczyk, K. Method Enzymol. 2000,
323, 473-509. (h) Lewis, F. D.; Helvoigt, S. A.; Letsinger, R. L.
Chem. Commun. 1999, 327-328. (i) Krider, E. S.; Rack, J. J.; Frank,
N. L.; Meade, T. J. Inorg. Chem. 2001, 40, 4002-4009.
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2285-2297. (f) Stufkens, D. J. Comments Inorg. Chem. 1992, 13,
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J. N.; DeGraff, B. A. Inorg. Chem. 1993, 32, 5629-5632. (h) Stufkens,
D. J.; Vlcek, A. Coord. Chem. ReV. 1998, 177, 127-179.
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Rillema, D. P. Inorg. Chem. 1995, 34, 2875-2882. (d) Lucia, L. A.;
Abboud, K.; Schanze, K. S. Inorg. Chem. 1997, 36, 6224-6234.
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Chem. Soc., Dalton Trans. 1997, 2067-2072. (b) Kunkely, H.; Vogler,
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Experimental Section
Materials and Reagents. All solvents were of analytical reagent
grade and purified according to literature procedures.19 Re(CO)5-
Cl, 3-aminopyridine, maleic anhydride, and all the diimine ligands
were purchased from Aldrich and were used without purification.
A thiolated universal M13 reverse sequencing primer, M13-R [5′-
HS(CH2)6-AACAGCTATGACCATG-3′] (Sigma-Genosys), glu-
tathione, reduced form (Sigma), bovine serum albumin (Calbio-
chem), and human serum albumin, fraction V (Calbiochem), were
used as received. All buffer components were of molecular biology
grade and used without purification.
(15) (a) Connick, W. B.; Di Bilio, A. J.; Hill, M. G.; Winkler, J. R.; Gray,
H. B. Inorg. Chim. Acta 1995, 240, 169-173. (b) Winkler, J. R.; Di
Bilio, A. J.; Farrow, N. A.; Richards, J. H.; Gray, H. B. Pure Appl.
Chem. 1999, 71, 1753-1764. (c) Di Bilio, A. J.; Crane, B. R.; Wehbi,
W. A.; Kiser, C. N.; Abu-Omar, M. M.; Carlos, R. M.; Richards, J.
H.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2001, 123, 3181-
3812.
N-(3-Pyridyl)maleamic Acid. Maleic anhydride (4.42 g, 45.1
mmol) in 20 mL of ice-cold THF was added to 3-aminopyridine
(3.54 g, 37.6 mmol) in 20 mL of ice-cold THF. The mixture was
stirred for 1 h at 4 °C. The pale yellow solid formed was collected
by filtration and washed with THF and air-dried (6.72 g, yield )
(16) (a) Guo, X. Q.; Castellano, F. N.; Li, L.; Szmacinski, H.; Lakowicz,
J. R.; Sipior, J. Anal. Biochem. 1997, 254, 179-186. (b) Guo, X. Q.;
Castellano, F. N.; Li, L.; Lakowicz, J. R. Anal. Chem. 1998, 70, 632-
637. (c) Dattelbaum, J. D.; Abugo, O. O.; Lakowicz, J. R. Bioconjugate
Chem. 2000, 11, 533-536.
(17) Hirschfeld, T. Appl. Opt. 1976, 15, 3135-3139.
(18) Sammes, P. G.; Yahioglu, G. Nat. Prod. Rep. 1996, 1-28.
(19) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: New York, 1988.
Inorganic Chemistry, Vol. 41, No. 1, 2002 41

Lo et al.
1
94%). H NMR (300 MHz, DMSO-d6, 298 K, TMS): δ 10.57 (s,
used. 3 was isolated as pale yellow crystals. Yield: 312.8 mg, 58%.
1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.62 (s, 2H, H2
and H9 of 3,4,7,8-Me4-phen), 8.69 (d, 1H, J ) 2.1 Hz, H2 of py-
3-mal), 8.67 (d, 1H, J ) 5.6 Hz, H6 of py-3-mal), 8.47 (s, 2H, H5
and H6 of 3,4,7,8-Me4-phen), 7.98-7.95 (m, 1H, H4 of py-3-mal),
7.48 (dd, 1H, J ) 8.5 and 5.6 Hz, H5 of py-3-mal), 7.08 (s, 2H,
maleimide H’s), 2.94 (s, 6H, Me at C4 and C7 of 3,4,7,8-Me4-
phen), 2.80 (s, 6H, Me at C3 and C8 of 3,4,7,8-Me4-phen). Anal.
Calcd for C29H22N4O8SF3Re: C, 41.98; H, 2.67; N, 6.75. Found:
C, 42.07; H, 2.58; N, 6.60. IR (KBr) (ν/cm-1): 2032 (s, CtO),
1921 (s, CtO), 1722 (s, CdO, maleimide), 1160 (m, CF3SO3-),
1033 (m, CF3SO3-). Positive-ion ESI-MS: m/z at 680, {[Re(3,4,7,8-
Me4-phen)(CO)3(py-3-mal)]}+.
[Re(4,7-Ph2-phen)(CO)3(py-3-mal)](CF3SO3) (4). The synthetic
procedure is similar to that for 1 except that [Re(4,7-Ph2-
phen)(CO)3(CH3CN)](CF3SO3) (515.3 mg, 0.65 mmol) was used.
4 was isolated as orange-yellow crystals. Yield: 385.1 mg, 64%.
1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 9.94 (d, 2H, J )
5.3 Hz, H2 and H9 of 4,7-Ph2-phen), 8.85-8.84 (m, 1H, H6 of
py-3-mal), 8.60 (d, 1H, J ) 1.8 Hz, H2 of py-3-mal), 8.29 (d, 2H,
J ) 5.6 Hz, H3 and H8 of 4,7-Ph2-phen), 8.25 (s, 2H, H5 and H6
of 4,7-Ph2-phen), 8.04-8.00 (m, 1H, H4 of py-3-mal), 7.77-7.69
(m, 10H, Ph of 4,7-Ph2-phen), 7.51 (dd, 1H, J ) 8.2 and 5.6 Hz,
H5 of py-3-mal), 7.07 (s, 2H, maleimide H’s). Anal. Calcd for
C37H22N4O8SF3Re: C, 48.00; H, 2.40; N, 6.05. Found: C, 48.13;
H, 2.67; N, 5.95. IR (KBr) (ν/cm-1): 2033 (s, CtO), 1923 (s,
CtO), 1722 (s, CdO, malemide), 1154 (m, CF3SO3-), 1031 (m,
CF3SO3-). Positive-ion ESI-MS: m/z at 776, {[Re(4,7-Ph2-phen)-
(CO)3(py-3-mal)]}+.
[Re(2,9-Me2-4,7-Ph2-phen)(CO)3(py-3-mal)](CF3SO3) (5). The
synthetic procedure is similar to that for 1 except that [Re(2,9-
Me2-4,7-Ph2-phen)(CO)3(CH3CN)](CF3SO3) (533.5 mg, 0.65 mmol)
was used. 5 was isolated as yellow crystals. Yield: 403.0 mg, 65%.
1H NMR (300 MHz, acetone-d6, 298 K, TMS): δ 8.33-8.29 (m,
2H, H2 and H6 of py-3-mal), 8.26 (s, 2H, H3 and H8 of 2,9-Me2-
4,7-Ph2-phen), 7.99 (s, 2H, H5 and H6 of 2,9-Me2-4,7-Ph2-phen),
7.98-7.95 (m, 1H, H4 of py-3-mal), 7.70-7.64 (m, 10H, Ph of
2,9-Me2-4,7-Ph2-phen), 7.47 (dd, 1H, J ) 5.9 and 8.2 Hz, H5 of
py-3-mal), 7.06 (s, 2H, maleimide H’s), 3.57 (s, 6H, Me of 2,9-
Me2-4,7-Ph2-phen). Anal. Calcd for C39H26N4O8SF3Re: C, 49.11;
H, 2.75; N, 5.87. Found: C, 49.37; H, 2.71; N, 5.99. IR (KBr)
(ν/cm-1): 2030 (s, CtO), 1922 (s, CtO), 1721 (s, CdO,
malemide), 1148 (m, CF3SO3-), 1031 (m, CF3SO3-). Positive-ion
ESI-MS: m/z at 804, {[Re(2,9-Me2-4,7-Ph2-phen)(CO)3(py-3-
mal)]}+.
1H, COOH), 8.76 (d, 1H, J ) 1.8 Hz, H2 of pyridine), 8.30 (dd,
1H, J ) 4.7 and 1.5 Hz, H6 of pyridine), 8.07 (ddd, 1H, J ) 8.2,
2.3 and 1.5 Hz, H4 of pyridine), 7.38 (ddd, 1H, J ) 8.2, 4.7 and
0.6 Hz, H5 of pyridine), 6.50 (d, 1H, J ) 12.0 Hz, NHCOCHd
CHCOOH), 6.34 (d, 1H, J ) 12.0 Hz, NHCOCHdCHCOOH). IR
(KBr) (ν/cm-1): 2526 (br, OH), 1703 (m, CdO), 1566 (m, CdC).
Positive-ion ESI-MS: m/z at 193, {M + H+}+.
N-(3-Pyridyl)maleimide, py-3-mal. Acetic anhydride (20 mL)
and ammonium acetate (3 g) were added to N-(3-pyridyl)maleamic
acid (1.0 g, 5.2 mmol), and the mixture was heated at 100 °C for
10 min. The solution was then cooled and added into 100 mL of
cold water. The solution was neutralized to pH 7 with NaHCO3,
and the product was extracted with ethyl acetate. The organic
solution was collected, washed with saturated NaHCO3, brine, and
water, and dried over anhydrous magnesium sulfate. The product
was purified by column chromatography (silica gel) using ethyl
acetate as the eluent (396 mg, yield ) 44%). 1H NMR (300 MHz,
acetone-d6, 298 K, TMS): δ 8.65 (m, 1H, H2 of pyridine), 8.58
(dd, 1H, J ) 4.9 and 1.6 Hz, H6 of pyridine), 7.84 (ddd, 1H, J )
8.2, 2.5 and 1.6 Hz, H4 of pyridine), 7.52 (ddd, 1H, J ) 8.2, 4.7
and 0.8 Hz, H5 of pyridine), 7.10 (s, 2H, maleimide H’s). IR (KBr)
(ν/cm-1): 1713 (s, CdO), 1583 (m, CdC). Positive-ion ESI-MS:
m/z at 175, {M + H+}+.
[Re(phen)(CO)3(py-3-mal)](CF3SO3) (1). An anhydrous THF
solution of [Re(phen)(CO)3(CH3CN)](CF3SO3) (416.4 mg, 0.65
mmol) and py-3-mal (112.9 mg, 0.65 mmol) was refluxed under
nitrogen in the dark for 4 h. After which the solution was evaporated
to dryness and the yellow solid was dissolved in CH2Cl2 and
purified by column chromatography on silica gel. The product was
eluted with CH2Cl2/acetone (1:1 v/v). Slow diffusion of diethyl ether
vapor into a concentrated CH2Cl2/acetone solution of the complex
afforded [Re(phen)(CO)3(py-3-mal)](CF3SO3) as yellow crystals.
1
Yield: 286.6 mg, 57%. H NMR (300 MHz, acetone-d6, 298 K,
TMS): δ 9.88 (d, 2H, J ) 5.2 Hz, H2 and H9 of phen), 9.12 (d,
2H, J ) 8.2 Hz, H4 and H7 of phen), 8.70 (m, 1H, H6 of py-3-
mal), 8.63 (d, 1H, J ) 2.2 Hz, H2 of py-3-mal), 8.39 (s, 2H, H5
and H6 of phen), 8.35 (dd, 2H, J ) 8.5 and 5.2 Hz, H3 and H8 of
phen), 7.99 (m, 1H, H4 of py-3-mal), 7.53 (dd, 1H, J ) 8.2 and
5.5 Hz, H5 of py-3-mal), 7.05 (s, 2H, maleimide H’s). Anal. Calcd
for C25H14N4O8SF3Re: C, 38.81; H, 1.82; N, 7.24. Found: C, 38.66;
H, 1.80; N, 7.42. IR (KBr) (ν/cm-1): 2039 (s, CtO), 1929 (s,
CtO), 1720 (s, CdO, maleimide), 1158 (m, CF3SO3-), 1027 (m,
CF3SO3-). Positive-ion ESI-MS: m/z at 624, {[Re(bpy)(CO)3(py-
3-mal)]}+, and 450, {[Re(bpy)(CO)3]}+.
[Re(2,9-Me2-phen)(CO)3(py-3-mal)](CF3SO3) (2). The syn-
thetic procedure is similar to that for 1 except that [Re(2,9-Me2-
phen)(CO)3(CH3CN)](CF3SO3) (434.6 mg, 0.65 mmol) was used.
2 was isolated as yellow crystals. Yield: 354.3 mg, 68%. 1H NMR
(300 MHz, acetone-d6, 298 K, TMS): δ 8.86 (d, 2H, J ) 8.5 Hz,
H4 and H7 of 2,9-Me2-phen), 8.29-8.26 (m, 3H, H3 and H8 of
2,9-Me2-phen, and H2 of py-3-mal), 8.24-8.22 (m, 1H, H6 of py-
3-mal), 8.16 (s, 2H, H5 and H6 of 2,9-Me2-phen), 8.08-7.94 (m,
1H, H4 of py-3-mal), 7.43 (dd, 1H, J ) 8.0 and 5.2 Hz, H5 of
py-3-mal), 7.04 (s, 2H, maleimide H’s), 3.49 (s, 6H, Me of 2,9-
Me2-phen). Anal. Calcd for C27H18N4O8SF3Re: C, 40.45; H, 2.26;
N, 6.99. Found: C, 40.51; H, 2.30; N 6.87. IR (KBr) (ν/cm-1):
2032 (s, CtO), 1934 (s, CtO), 1723 (s, CdO, maleimide), 1154
(m, CF3SO3-), 1031 (m, CF3SO3-). Positive-ion ESI-MS: m/z at
652, {[Re(2,9-Me2-phen)(CO)3(py-3-mal)]}+.
[Re(biq)(CO)3(py-3-mal)](CF3SO3) (6). The synthetic procedure
is similar to that for 1 except that [Re(biq)(CO)3(CH3CN)](CF3-
SO3) (465.8 mg, 0.65 mmol) was used. 6 was isolated as orange
1
crystals. Yield: 309.3 mg, 56%. H NMR (300 MHz, acetone-d6,
298 K, TMS): δ 9.12 (d, 2H, J ) 8.8 Hz, H4 and H4′ of biq),
9.01 (d, 2H, J ) 8.8 Hz, H8 and H8′ of biq), 8.86 (d, 2H, J ) 8.8
Hz, H3 and H3′ of biq), 8.40 (d, 2H, J ) 8.0 Hz, H5 and H5′ of
biq), 8.35-8.29 (m, 2H, H7 and H7′ of biq), 8.09-8.03 (m, 3H,
H6 and H6′ of biq, and H4 of py-3-mal), 7.89 (d, 1H, J ) 2.5 Hz,
H2 of py-3-mal), 7.68-7.67 (m, 1H, H6 of py-3-mal), 7.41 (dd,
1H, J ) 8.2 and 5.5 Hz, H5 of py-3-mal), 6.94 (s, 2H, maleimide
H’s). Anal. Calcd for C31H18N4O8SF3Re: C, 43.82; H, 2.14; N,
6.59. Found: C, 43.80; H, 2.23; N, 6.38. IR (KBr) (ν/cm-1): 2031
(s, CtO), 1918 (s, CtO), 1721 (s, CdO, maleimide), 1145 (m,
CF3SO3-), 1032 (m, CF3SO3-). Positive-ion ESI-MS: m/z at 700,
{[Re(biq)(CO)3(py-3-mal)]}+.
[Re(3,4,7,8-Me4-phen)(CO)3(py-3-mal)](CF3SO3) (3). The syn-
thetic procedure is similar to that for 1 except that [Re(3,4,7,8-
Me4-phen)(CO)3(CH3CN)](CF3SO3) (452.8 mg, 0.65 mmol) was
Labeling of Single-Stranded DNA with Complex 1. In a typical
labeling reaction, complex 1 (0.7 mg, 0.90 µmol) in 50 µL
42 Inorganic Chemistry, Vol. 41, No. 1, 2002

Rhenium(I) Polypyridine Maleimide Complexes
anhydrous DMSO was added to a thiolated universal M13 reverse
sequencing primer M13-R (10 nmol) in 450 µL of 50 mM
potassium phosphate buffer pH 7.4. The mixture was stirred gently
in the dark at RT (room temperature) for 24 h. The solid residue
was removed by centrifugation. To the supernatant, 50 µL of
NaOAc (3.0 M, pH 5.2) and 900 µL of 2-propanol were added.
The labeled DNA was collected by centrifugation, and the pellet
was washed with 70% aqueous EtOH and twice with absolute EtOH
and then dried in vacuo. The labeled oligonucleotide was further
purified by RP-HPLC with CH3CN (5-50% over 50 min.) and
0.1 M triethylammonium acetate pH 7.0 as the mobile phase at a
and refinement by full-matrix least-squares using the software
package TeXsan21 on a Silicon Graphics Indy computer. One
crystallographic asymmetric unit consists of one formula unit. Two
of the O atoms in the anion were disordered and were placed at
three positions with O(7), O(7′), and O(8) having occupation
numbers of 0.75, 0.45, and 0.8, respectively. In the least-squares
refinement, 38 non-H atoms were refined anisotropically; the F and
O atoms of the anion were refined isotropically, and 18 H atoms
at calculated positions with thermal parameters equal to 1.3 times
that of the attached C atoms were not refined. H atoms bonded to
the disordered methyl C atom were not included in the calculation.
Convergence for 371 variable parameters by least-squares refine-
flow rate of 1 mL min-1
.
2
2
2
ment on F with w ) 4Fo /σ2(Fo ), where σ2(Fo ) ) [σ2(I) +
Labeling of Glutathione (γ-Glu-Cys-Gly) (GSH) with Com-
plex 1. Complex 1 (1.8 mg, 2.32 µmol) in 50 µL anhydrous DMSO
was added to glutathione (0.2 mg, 0.65 µmol) dissolved in 100 µL
of 50 mM potassium phosphate buffer, pH 7.4. The mixture was
stirred gently in the dark at RT for 24 h. The solid residue was
removed by centrifugation. The excess label was removed by
washing the aqueous solution repeatedly with ethyl acetate, and
the labeled peptide was further purified by HPLC.
2 2
(0.025Fo ) ] for 3636 reflections with I > 3σ(I) was reached at R
) 0.038 and Rw ) 0.050 with a goodness-of-fit of 1.94. (∆/σ)max
) 0.05 except for the disordered O atoms. The final difference
Fourier map was featureless, with maximum positive and negative
peaks of 0.96 and 0.76 e Å-3, respectively.
Photophysical Measurements. Electronic absorption and steady-
state emission spectra were recorded on a Hewlett-Packard 8452A
diode array spectrophotometer and a Spex Fluorolog-2 model F
111 fluorescence spectrophotometer, respectively. Unless specified,
all solutions for photophysical studies were degassed with no fewer
than four successive freeze-pump-thaw cycles and stored in a
10-cm3 round-bottomed flask equipped with a sidearm 1-cm
fluorescence cuvette and sealed from the atmosphere by a Rotaflo
HP6/6 quick-release Teflon stopper. Luminescence quantum yields
were measured by the optical dilute method22 using an aerated
aqueous solution of [Ru(bpy)3]Cl2 (Φ ) 0.028, excitation wave-
length at 455 nm)23 as the standard solution. The 355-nm output
(third harmonic) of a Quanta-Ray Q-switched GCR-150-10 pulsed
Nd:YAG laser was the excitation source for emission lifetime
measurements. Luminescence decay signals from a Hamamatsu
R928 photomultiplier tube were converted to potential changes by
a 50-Ω load resistor and then recorded on a Tektronix model TDS
620A digital oscilloscope.
Labeling of Bovine Serum Albumin (BSA) and Human
Serum Albumin (HSA) with Complex 1. Complex 1 (1.3 mg,
1.67 µmol) in 20 µL of anhydrous DMSO was added to BSA or
HSA (1.32 mg, 20 nmol) dissolved in 180 µL of 50 mM potassium
phosphate buffer, pH 7.4. The mixture was incubated in the dark
at RT for 12 h. The solid residue was removed by centrifugation.
The supernatant was diluted to 1 mL with 50 mM Tris-HCl, pH
7.4 and loaded onto a PD-10 column (Pharmacia) that had been
equilibrated with the same buffer. The first band that came out
from the column with intense yellow luminescence was collected,
and the solution was concentrated with a YM-30 centricon
(Amicon). The labeled protein was further purified by HPLC
equipped with a size-exclusion column. The mobile phase was 50
mM Tris-HCl, pH 7.4, at a flow rate of 1 mL min-1
.
Crystal Structure Determination. Crystallographic data for
complex 2: [(C26H18N4O5Re)+(CF3SO3-)], formula weight )
801.72, monoclinic, space group P21/c (No. 14), a ) 10.460(3) Å,
b ) 17.673(3) Å, c ) 15.359(2) Å, â ) 95.91(2)°, V ) 2824.4(9)
Å3, Z ) 4, Dc ) 1.885 g cm-3, µ(Mo KR) ) 44.55 cm-1, F(000)
) 1560, T ) 301 K. A yellow crystal of dimensions 0.30 × 0.10
× 0.07 mm in a glass capillary was used for data collection at 28
°C on a Rigaku AFC7R diffractometer with graphite-monochro-
matized Mo KR radiation (λ ) 0.710 73 Å) using ω-2θ scans
with ω-scan angle (0.68 + 0.35 tan θ)° at a scan speed of 8.0 deg
min-1 [up to 6 scans for reflection with I < 15σ(I)]. Unit-cell
dimensions were determined on the basis of the setting angles of
25 reflections in the 2θ range of 28.4-32.5°. Intensity data (in the
range of 2θmax ) 52°; h, 0 to 12; k, 0 to 21; l, -18 to 18; 3 standard
reflections measured after every 300 reflections showed decay of
2.66%) were corrected for Lorentz and polarization effects, and
empirical absorption corrections were based on the ψ-scan of five
strong reflections (minimum and maximum transmission factors
0.774 and 1.000). A total of 6052 reflections were measured, of
which 5734 were unique and Rint ) 0.020. A total of 3636
reflections with I > 3σ(I) were considered observed and used in
the structural analysis. The space group was uniquely determined
on the basis of systematic absences, and the structure was solved
by Patterson methods and expanded by Fourier methods (PATTY20)
Results and Discussion
Synthesis and Characterization. The rhenium(I) poly-
pyridine maleimide complexes 1-6 were obtained in moder-
ate yields from the reactions of [Re(N-N)(CO)3(CH3CN)]-
(CF3SO3)11b and the ligand py-3-mal. The complexes were
characterized by 1H NMR, positive-ion ESI-MS, and IR and
gave satisfactory elemental analysis. The maleimide moiety
of the complexes is characterized by a singlet at ca. δ 7.0 in
1
the H NMR spectra and a strong absorption at ca. 1722
cm-1 in the IR spectra. The complexes are air-stable, and
the characterization data showed that their identities remained
the same in a period of months when stored at -20 °C in
the absence of moisture. However, it is well-known that the
maleimide group is generally susceptible to hydrolysis.
Crystal Structure Determination. Single crystals of
complex 2 were obtained by layering a concentrated acetone
solution of the complex with a mixture of diethyl ether and
petroleum ether. The perspective drawing of the complex
cation of 2 with atomic numbering is depicted in Figure 1.
Selected bond distances and angles are summarized in Table
(20) PATTY: Beurskens, P. T.; Admiral, G.; Beurskens, G.; Bosman, W.
P.; Garcia-Granda, S.; Gould, R. O.; Smits, J. M. M.; Smykalla, C.
The DIRDIF program system; Technical Report of the Crystallography
Laboratory, University of Nijmegen: Nijmegen, The Netherlands,
1992.
(21) TeXsan, Crystal Structure Analysis Package; Molecular Structure
Corp.: The Woodlands, TX, 1985 and 1992.
(22) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991-1024.
(23) Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697-2705.
Inorganic Chemistry, Vol. 41, No. 1, 2002 43

Lo et al.
Figure 2. Electronic absorption (a) and emission (b) spectra of complex
1 in CH2Cl2 at 298 K.
Table 2. Electronic Absorption Spectral Data for Complexes 1-6 at
298 K
complex
medium
CH2Cl2
λ )
abs/nm (ꢀ/dm3 mol-1 cm-1
1
258 (20 745), 276 (23 930), 296 sh (13 385),
324 sh (5865), 364 sh (3725)
Figure 1. Perspective view of the complex cation of 2 with atomic
numbering scheme. Hydrogen atoms have been omitted for clarity. Thermal
ellipsoids are shown at the 50% probability level.
CH3CN
CH2Cl2
CH3CN
276 (24 605), 318 sh (6880), 362 sh (3555)
284 (22 585), 324 sh (8320), 368 sh (2425)
284 (24 645), 306 sh (14 880), 318 sh (10 395),
364 sh (2390)
250 sh (19 815), 282 (23 165), 324 sh (8670),
370 sh (2860)
250 sh (21 025), 282 (23 830), 316 sh (10 205),
370 sh (2490)
294 (29 160), 332 sh (12 900), 380 sh (6545)
260 sh (25 610), 292 (40 215), 324 sh (16 340),
374 sh (7415)
260 (21 085), 300 (31 915), 334 sh (13 255),
378 sh (4550)
254 (20 605), 298 (30 225), 332 sh (11 710),
374 sh (3965)
270 (32 570), 300 sh (11 450), 364 (13 985),
382 (19 870), 408 sh (3255)
270 (39 240), 298 sh (13 610), 360 (15 855),
378 (18 670), 406 sh (3600)
2
3
Table 1. Selected Geometric Data for Complex 2
CH2Cl2
CH3CN
Bond Lengths (Å)
Re(1)-N(1)
Re(1)-N(3)
Re(1)-C(2)
O(4)-C(21)
C(22)-C(23)
2.199(7)
2.219(6)
1.918(10)
1.20(1)
Re(1)-N(2)
Re(1)-C(1)
Re(1)-C(3)
O(5)-C(24)
2.215(7)
1.916(9)
1.90(1)
1.19(1)
4
5
CH2Cl2
CH3CN
1.32(1)
CH2Cl2
CH3CN
CH2Cl2
CH3CN
Bond Angles (deg)
N(1)-Re(1)-N(2)
N(1)-Re(1)-C(1)
N(1)-Re(1)-C(3)
N(2)-Re(1)-C(1)
N(2)-Re(1)-C(3)
N(3)-Re(1)-C(2)
C(1)-Re(1)-C(2)
C(2)-Re(1)-C(3)
75.5(3)
95.8(3)
172.5(3)
93.8(3)
101.2(4)
95.5(3)
87.9(4)
82.1(5)
N(1)-Re(1)-N(3)
N(1)-Re(1)-C(2)
N(2)-Re(1)-N(3)
N(2)-Re(1)-C(2)
N(3)-Re(1)-C(1)
N(3)-Re(1)-C(3)
C(1)-Re(1)-C(3)
81.4(2)
101.0(3)
82.8(2)
176.3(3)
176.0(3)
91.6(4)
91.1(5)
6
intense absorptions at ca. 250-296 nm are assigned to
intraligand (IL) transitions [π f π* (bpy and py-3-mal)]
since similar absorption bands are observed for the uncoor-
dinated ligands. On the basis of previous spectroscopic
studies of rhenium(I) polypyridine complexes,11-16 the lower-
energy absorption shoulders of 1 at ca. 324-364 nm with
extinction coefficients in the order of 103 dm3 mol-1 cm-1
are assigned to spin-allowed metal-to-ligand charge-transfer
(MLCT) [dπ(Re) f π*(bpy)] transitions. Similar spectro-
scopic assignments are made for the other complexes in this
work. However, owing to the extended π-conjugation of the
ligands 4,7-Ph2-phen, 2,9-Me2-4,7-Ph2-phen, and biq, the
intraligand absorptions of 4-6 are extended to a lower energy
region (ca. 332-382 nm) and the 1MLCT [dπ(Re) f
π*(diimine)] transitions of these complexes occur at a further
lower energy (ca. 374-408 nm).
1. The rhenium(I) center adopts a distorted octahedral
coordination geometry, and the carbonyl groups are arranged
in a facial orientation. The bond lengths and angles involving
the rhenium(I) center are normal compared to those observed
in related systems.12,13a,b,15a It is noteworthy that the dihedral
angle between the ideal ring plane of the 2,9-Me2-phen ligand
and the C(2)-Re(1)-C(3) plane is ca. 154.1°. Deviation
from 180° of the ideal case is probably a result of the steric
requirements of the methyl groups. The geometric data of
the maleimide group are similar to those of the unsubstituted
maleimide (C4H3NO2) molecule.24 Owing to the bulkiness
of the maleimide carbonyl groups, the maleimide ring
establishes a dihedral angle of ca. 25.7° with the pyridyl ring.
Photophysical Properties. The complexes are soluble in
common organic solvents such as dichloromethane, aceto-
nitrile, and acetone, giving yellow to orange solutions. The
electronic absorption spectral data for the complexes are
listed in Table 2. The electronic absorption spectrum of 1 in
CH2Cl2 at 298 K is shown in Figure 2 (spectrum a). The
Excitation of complexes 1-6 at λex > 350 nm gives rise
to intense and long-lived green to orange-red luminescence.
The photophysical data are listed in Table 3. As an example,
complex 1 emits at 530 nm (Figure 2, spectrum b) with a
lifetime of 2.30 µs in CH2Cl2 at 298 K. A shift of ca. 1.07
eV is observed from the absorption shoulder at ca. 364 nm
(24) Cox, P. J.; Parker, S. F. Acta Crystallogr. C 1996, 52, 2578-2580.
44 Inorganic Chemistry, Vol. 41, No. 1, 2002

Rhenium(I) Polypyridine Maleimide Complexes
Scheme 1. Proposed Reaction between the Complex Cation of 1 and
the Sulfhydryl Group of a Biomolecule
Table 3. Photophysical Data for Complexes 1-6 at 298 K
complex
medium
λ
em/nm
τo/µs
Φ
1
CH2Cl2
acetone
CH3CN
CH2Cl2
acetone
CH3CN
CH2Cl2
acetone
CH3CN
CH2Cl2
acetone
CH3CN
CH2Cl2
acetone
CH3CN
CH2Cl2
acetone
CH3CN
530
546
546
518
532
532
514
518
518
542
558
558
536
544
544
632
654
650
2.30
1.84
0.51
1.95 (5%), 0.19 (95%)
1.34
1.77
2.08 (6%), 0.13 (94%)
5.21
2.45
9.60 (70%), 1.64 (30%)
7.42
2.48
5.81 (15%), 1.06 (85%)
6.18
3.43
2
3
4
5
6
0.43
0.13
0.53
0.21
8.78 (15%), 1.55 (85%)
0.10
0.05
0.06
0.0020
to the emission maximum at ca. 530 nm. For 1-6, the
relatively long emission lifetimes (in the microsecond time
scale), together with large absorption-emission shifts,
indicate the phosphorescence nature of the emission. The
emission energies of the complexes follow the order 3 > 2
> 1 ≈ 5 > 4 > 6, which is in line with the π-accepting
ability of the diimine ligands. The emissions of the com-
plexes exhibit a red-shift upon changing the solvent from
less polar CH2Cl2 to more polar acetone and CH3CN. On
the basis of the dependence between the emission energies
of the complexes and the energy levels of the empty π*
orbitals of the diimine ligands, together with the observation
of solvatochromic behavior, the photoluminescence of the
complexes is assigned to associate with an MLCT [dπ(Re)
f π*(diimine)] triplet excited-state. Similar assignments
have been made in other luminescent rhenium(I) polypyridine
complexes.11-16 The luminescence quantum yields of the
complexes are comparable to those of related [Re(N-N)-
(CO)3(py)]+ complexes.11b,e,g,16a
saturated Tris buffer are in line with those of other ruthe-
nium(II)-labeled oligonucleotides.10h,i On the other hand,
since the substituents at the meta positions of the pyridine
ligand have only minor effects on the rhenium(I) diimine
luminophore, a change from the maleimide group to a
thioether is not expected to perturb significantly the MLCT
emission behavior of the complex. Therefore, the small red-
shifts and decreased lifetimes of the emissions of the free
labels from CH2Cl2 to CH3CN and to the labeled DNA in
Tris buffer appear to result from the increasing polarity of
the solvent environment (and the presence of highly polar
phosphates of the oligonucleotides). Such an observation of
solvatochromism has been observed for ReI polypyridine
MLCT emitters.11a,c-f,12b Nonetheless, given the strong pho-
tooxidizing behavior of rhenium(I) polypyridine complexes,15
the decreased emission lifetimes may be a result of oxidation
of guanines by the excited complexes.
Photophysical Properties of Bioconjugates. Since the
maleimide group can react with the sulfhydryl group to form
a stable thioether, the rhenium(I) polypyridine maleimide
complexes in this work are designed to react specifically with
sulfhydryl-containing biomolecules (Scheme 1). As an
example, the thiolated M13 sequencing primer M13-R [5′-
HS(CH2)6-AACAGCTATGACCATG-3′] has been labeled
with complexes 1-6. Although these rhenium(I) complexes
are sparingly soluble in water, the DNA primers coupled
with 1-6 are very soluble in water and aqueous buffers and
exhibit intense and long-lived greenish yellow to orange
luminescence upon photoexcitation (Table 4). The emission
wavelengths are indistinguishable to those of the free labels
in CH3CN, and the trend of the emission energies of the
labeled primers, 1-M13-R-6-M13-R, is the same to that of
the free complexes. This finding is important because it is
our intention to use various diimine ligands to provide
multicolor labeling reagents for biological substrates. In view
of this observation, together with the microsecond time scale
emission lifetimes, the emissive states of these labeled
Other biomolecules, including a cysteine-containing pep-
tide, glutathione (GSH), and proteins such as bovine serum
albumin (BSA) and human serum albumin (HSA), have also
been labeled with complex 1. The UV-vis absorption spectra
of these bioconjugates showed low-energy absorption shoul-
ders at ca. 328 and 368 nm in Tris buffer, typical of 1MLCT
[dπ(Re) f π*(phen)] transitions. According to the absorption
spectral data, the rhenium:protein ratios have been deter-
mined to be ca. 4.0 and 4.8 for the conjugates 1-BSA and
1-HSA, respectively. Upon photoexcitation, the bioconju-
gates display intense and long-lived yellow luminescence in
buffer solutions (Table 5). The emission wavelengths are
indiscernible to that of the labeled oligonucleotide 1-M13-
3
R. The emission is also assigned to arise from an MLCT
[dπ(Re) f π*(phen)] excited state. The luminescence
quantum yields of these bioconjugates are similar in mag-
nitude to those of other biomolecules labeled with lumines-
cent rhenium(I) complexes.16a Concerning the emission
lifetimes, while the bioconjugate 1-GSH exhibits a single-
exponential decay, both 1-BSA and 1-HSA show double-
exponential decays with emission-lifetime components of ca.
1.1 and 0.2 µs. The observation of double-exponential and
oligonucleotides should bear
a high parentage of
3MLCT [dπ(Re) f π*(diimine)] character. The luminescence
quantum yields of 1-M13-R-6-M13-R in degassed and air-
Inorganic Chemistry, Vol. 41, No. 1, 2002 45

Lo et al.
Table 4. Photophysical Data for Labeled Oligonucleotides 1-M13-R-6-M13-R in 50 mM Tris-HCl, pH 7.4, at 298 K
degassed buffer
air-saturated buffer
labeled oligonucleotide
λem/nm
τo/µs
Φ
λ
em/nm
τ/µs
Φ
1-M13-R
2-M13-R
3-M13-R
4-M13-R
5-M13-R
6-M13-R
548
536
522
558
544
636
0.99 (18%), 0.31 (82%)
1.46 (47%), 0.31 (53%)
5.66 (67%), 0.74 (33%)
2.72 (52%), 0.51 (48%)
6.42 (58%), 0.88 (42%)
0.12
0.026
0.020
0.021
0.022
0.021
0.0012
548
536
522
558
544
636
0.61 (37%), 0.23 (63%)
1.29 (43%), 0.31 (57%)
2.76 (59%), 0.28 (41%)
2.07 (52%), 0.41 (48%)
4.39 (56%), 0.96 (44%)
0.12
0.019
0.016
0.011
0.016
0.013
0.0010
Table 5. Photophysical Data for the Bioconjugates 1-GSH, 1-BSA, and 1-HSA in 50 mM Tris-HCl, pH 7.4, at 298 K
degassed buffer
air-saturated buffer
τ/µs
bioconjugate
λem/nm
τo/µs
Φ
λ
em/nm
Φ
1-GSH
1-BSA
1-HSA
548
544
544
0.99
0.11
0.069
0.051
548
544
544
0.65
0.076
0.040
0.040
1.09 (49%), 0.19 (51%)
1.11 (48%), 0.20 (52%)
0.86 (47%), 0.18 (53%)
1.00 (43%), 0.26 (57%)
multiexponential emission decays is not uncommon for
luminescent inorganic complexes attached to biomolecules.16
On the other hand, the luminescence of the conjugates
1-GSH, 1-BSA, and 1-HSA is quenched by oxygen mol-
ecules owing to the triplet character of the emissive states.
However, it is interesting to note that for 1-GSH, the
emission quenching by oxygen is more significant (kq ) 2.12
× 109 dm3 mol-1 s-1) than the labeled serum albumins (1-
BSA, kq ) 1.23 × 109 dm3 mol-1 s-1; 1-HSA, kq ) 7.66 ×
108 dm3 mol-1 s-1). In view of the large difference in the
molecular sizes of glutathione and the serum albumins, it
appears that the labels coupled to BSA and HSA are well
protected in the interior of the proteins and a lower exposure
to the solvent surrounding could account for the less efficient
quenching by the oxygen molecules. A similar observation
has been noticed in other luminophore-labeled protein
systems.16 Nevertheless, the observations of emission life-
times in the microsecond time scale and generally high
luminescence quantum yields for the labeled DNA, peptide,
and serum albumins suggest that these rhenium(I) maleimide
complexes are promising candidates as thiol-specific labels
in time-resolved bioassays.
characterized, and their photophysical properties have been
studied. The complexes have been employed as site-specific
labels for biological species containing the sulfhydryl group.
The labeled substrates exhibit long-lived and intense photo-
3
luminescence typical of rhenium(I) polypyridine MLCT
emissions. It is envisaged that these new thiol-specific labels
can be applied in DNA hybridization assays, sequencing
experiments, polymerase chain reactions, immunoassays, and
probing the structures of biological substrates.
Acknowledgment. We thank the Hong Kong Research
Grants Council (Project No. CityU 1116/00P) and the City
University of Hong Kong for financial support. W.-K.H.
acknowledges the receipt of a postgraduate studentship
administered by the City University of Hong Kong. We are
grateful to Prof. Vivian W. W. Yam of The University of
Hong Kong for access to the equipment for photophysical
measurements and for her helpful discussion.
Supporting Information Available: Tables giving atomic
coordinates, anisotropic displacement parameters, and bond lengths
and bond angles for complex 2 in a CIF file. This material is
available free of charge via the Internet at http://pubs.acs.org.
Concluding Remarks
A series of new rhenium(I) polypyridine complexes
containing a maleimide moiety has been synthesized and
IC010602Z
46 Inorganic Chemistry, Vol. 41, No. 1, 2002