Rigid-core fluorescent oligothiophene-S,S-dioxide isothiocyanates. Synthesis, optical characterization, and conjugation to monoclonal antibodies

Article abstract of DOI:10.1021/jo026298o

In this paper we report the synthesis of a new class of fluorescent thiophene-based isothiocyanates containing a 3,5-disubstituted dithieno [3,2-b:2′,3′-d] thiophene-4,4-dioxide moiety as the rigid core, using the palladium-catalyzed cross-coupling reaction of aryl stannanes with aryl bromides (Stille coupling). By changing the molecular structure through the progressive addition of thienylene or phenylene units, light emission from blue to orange was obtained. Photoluminescence quantum yields ranged from 0.65 to 0.90 for blue and green light emitters to 0.10-0.35 for yellow and orange ones. Optically and chemically stable fluorescent bioconjugates were prepared by spontaneous reaction of the isothiocyanates with monoclonal antibodies anti-CD3 and anti-CD8 in slightly basic solutions.

Full text of DOI:10.1021/jo026298o

Rigid -Cor e F lu or escen t Oligoth iop h en e-S,S-d ioxid e
Isoth iocya n a tes. Syn th esis, Op tica l Ch a r a cter iza tion , a n d
Con ju ga tion to Mon oclon a l An tibod ies
Giovanna Sotgiu,† Massimo Zambianchi,† Giovanna Barbarella,*,† Fabio Aruffo,†
Franco Cipriani,‡ and Alfredo Ventola‡
Consiglio Nazionale Ricerche-ISOF, Via Gobetti 101, 40129 Bologna, Italy, and BIO-D,
Str. Provinciale per Casamassima Km. 3 Valenzano, Bari, Italy
barbarella@area.bo.cnr.it
Received August 7, 2002
In this paper we report the synthesis of a new class of fluorescent thiophene-based isothiocyanates
containing a 3,5-disubstituted dithieno[3,2-b:2′,3′-d]thiophene-4,4-dioxide moiety as the rigid core,
using the palladium-catalyzed cross-coupling reaction of aryl stannanes with aryl bromides (Stille
coupling). By changing the molecular structure through the progressive addition of thienylene or
phenylene units, light emission from blue to orange was obtained. Photoluminescence quantum
yields ranged from 0.65 to 0.90 for blue and green light emitters to 0.10-0.35 for yellow and orange
ones. Optically and chemically stable fluorescent bioconjugates were prepared by spontaneous
reaction of the isothiocyanates with monoclonal antibodies anti-CD3 and anti-CD8 in slightly basic
solutions.
In tr od u ction
and fluorescence detection techniques are becoming more
and more important in environmental studies.5
Today, there is great interest in fluorescent organic
compounds because of their potential in very different
fields, from electroluminescent devices and flat panel
display technology to biolabeling and measurements of
cells by flow cytometry or microscopy.1
We have recently reported not only that thiophene
oligomers exhibit great potential as active layers in thin-
film electroluminescent devices and lasers2a,b but that
they can also be modified in such a way as to become
attractive, photostable, fluorescent markers for bio-
polymers.2c,d
Fluorescence detection allows many clinical and re-
search applications aimed to identify specific populations
of cells and monitor metabolic processes.3 It is now largely
used in monitoring antigen-antibody interactions, in
nucleic acid labeling, in genomic studies, and in hysto-
logical tests.3 Furthermore, immunochemical methods
Currently, fluorescence techniques are limited by the
chemical nature of fluorescent markers. Commercially
available fluorophores are either small organic molecules
with great membrane permeability or high-weight natu-
ral compounds obtained from microalgae or even mix-
tures of compounds emitting light through a complex
pattern of intermolecular interactions, all requiring a
different chemistry to bind biomolecules.3,4
The growing importance of fluorescence detection
techniques has prompted the search for new classes of
compounds with improved optical properties and tunable
fluorescence frequencies that are easy to functionalize,
to allow the standardization of labeling procedures and
make multilabeling experiments more accessible. Re-
cently, several studies have been reported on modified
fluoresceins6 and cyanines,7 antracene and squaraine
derivatives,8,9 conjugated polymers,10 and probes able to
detect ions that play a metabolic role.11
† Consiglio Nazionale Ricerche-ISOF.
On these grounds, we have started research work
aimed at synthesizing new classes of thiophene-based
‡ BIO-D.
(1) (a) Kohle, A.; Wilson, J . S.; Friend, R. H. Adv. Mater. 2002, 14,
701. (b) Hide, F.; Diaz-Garcia, M. A.; Schwartz, B. J .; Heeger, A. J .
Acc. Chem. Res. 1997, 30, 430. (c) Miyata S., Nalwa, H. S., Eds. Organic
electroluminescent materials and devices; Gordon and Breach Publish-
ers: Amsterdam, 1997. (d) Fluorescent and luminescent Probes for
Biological Activity. A Practical Guide to Technology for Quantitative
Real-Time Analysis, 2nd ed.; Mason, W. T., Ed.; Academic Press: New
York, 1999.
(2) (a) Gigli, G.; Inganas, O.; Anni, M.; Vittorio M. M.; Cingolani
R.; Barbarella G. G.; Favaretto, L. Appl. Phys. Lett. 2001, 78, 1493.
(b) Zavelani-Rossi, M.; Lanzani, G.; De Silvestri, S.; Anni, M.; Gigli,
G.; Cingolani, R.; Barbarella, G.; Favaretto, L. Appl. Phys. Lett. 2001,
79, 4082. (c) Barbarella, G.; Zambianchi, M.; Pudova, O.; Paladini, V.;
Ventola, A.; Cipriani F.; Gigli, G.; Cingolani, R.; Citro, G. J . Am. Chem.
Soc. 2001, 123, 11600. (d) Barbarella, G. Chem.sEur. J . 2002, 8, 5072.
(3) Handbook of Fluorescent Probes and Research Chemicals, 7th
ed.; Molecular Probes Inc.: Eugene, OR, 1999.
(4) Oldham, P. B.; McCarrol, M. E.; McGown, L. B.; Warner, I. M.
Anal. Chem. 2000, 72, 197R.
(5) Dankwardt, A.; Hock, B. Chemosphere 2001, 45, 523.
(6) Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J . Org.
Chem. 1997, 62, 6469.
(7) Lin, Y.; Weissleder, R.; Tung, C. H. Bioconjugate Chem. 2002,
13, 605.
(8) Ihmels, H.; Meiswinkel, A.; Mohrschladt, C. J . Org. Lett. 2000,
2, 2865.
(9) Oswald, B.; Patsenker, L.; Duschl, J .; Szmacinski, H.; Wolfbeis,
O.; Terpetschnig, E. Bioconjugate Chem. 1999, 10, 925.
(10) Song, X.; Wang, H.; Shi, J .; Park, J . W.; Swanson, B. I. Chem.
Mater. 2002, 14, 2342.
(11) Hirano, T.; Kikuci, K.; Urano, Y.; Nagano, T. J . Am. Chem.
Soc. 2002, 124, 6555.
10.1021/jo026298o CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/23/2003
1512
J . Org. Chem. 2003, 68, 1512-1520

Oligothiophene-S,S-dioxide Isothiocyanates
SCHEME 1. Syn th esis of Isoth iocya n a te 14 Sta r tin g fr om Com m er cia l 3-Br om oth iop h en ea
a
Reagents and conditions: (i) phenylmagnesium bromide, Et2O; (ii) (1) NBS, CH2Cl2/CH3COOH; (2) Br2, CH2Cl2; (iii) n-BuLi, Et2O;
(iv) n-BuLi, SCl2, Et2O; (v) n-BuLi, CuCl2, Et2O; (vi) mCPBA, CH2Cl2; (vii) NBS, DMF; (viii) 9, Pd(AsPh3)4, toluene; (ix) NBS, CH2Cl2/
CH3COOH; (x) 12, Pd(PPh3)4, toluene; (xi) NaSCN, acetone.
fluorescent markers. Our strategy consists of building
thiophene-based structures containing the isothiocyanate
functionality, -NdCdS, capable of forming a stable
linkage with biomolecules containing -NH2 amino groups.3
In experiments with bovine serum albumin and mono-
clonal antibodies we have been able to demonstrate that
oligothiophene isothiocyanates bind spontaneously to
proteins and form very stable fluorescent bioconjugates
that display unaltered biological activity.2c
Scheme 1 reports the synthesis of 3,5-diphenyldithieno-
[3,2-b:2′,3′-d]thiophene-4,4-dioxide (DTTOPh, 7), starting
from commercial 3-bromothiophene, and isothiocyanate
14 bearing this moiety as the inner rigid core.
Polybrominated thiophene 3 was prepared in two steps.
First, the R positions were brominated using N-bromo-
succinimide in CH2Cl2/CH3COOH, and then the â posi-
tion was brominated using Br2 in CH2Cl2. Subsequent
treatment with BuLi in ethyl ether led to 3-bromo-4-
phenylthiophene 4 in good yield. This procedure was
found to be much less expensive than the direct introduc-
tion of the phenyl group in one of the â positions through
the Grignard reaction on 3,4-dibromothiophene.14 Also,
disulfide 5 was not prepared according to well-established
procedures,12,15a but was synthesized in a more expedient
way by the action of SCl2 on 4. The subsequent steps of
dithienothiophene formation (v-ix, Scheme 1), namely,
selective sulfur oxidation, selective bromination at one
of the terminal positions, and cross-coupling of the bromo
derivative with thienyl stannanes occurred with yields
that were lower than those obtained in the preparation
of 3,5-dimethyldithieno[3,2-b:2′,3′-d]thiophene-4,4-diox-
ide (15, DTTOMe) and its derivatives.12a Probably, this
was the result of both the electronic and steric effects of
the phenyl substituents on the different reaction steps.
In particular, for steric reasons, the rate of the Stille
coupling in the presence of the bulky phenyl groups of
DTTOPh must be lower than in the presence of the
methyl substituents of DTTOMe. Low cross-coupling
rates favor the self-coupling of the reagents and bromine-
In this paper we report the synthesis of oligothiophene
isothiocyanates containing a 3,5-disubstituted dithieno-
[3,2-b:2′,3′-d]thiophene-4,4-dioxide moiety (DTTO), which
has been demonstrated to be a very efficient light
emitter.12a By means of the Stille coupling,13 we have
synthesized oligomers containing the DTTO moiety and
functionalized with the NCS group at one or both
terminal positions. The size of the oligomers was in-
creased by the systematic addition of thienylene or
phenylene rings, to tune the fluorescence frequency of
the molecule.
Finally, we show that the newly synthesized isothio-
cyanates react spontaneously with monoclonal antibod-
ies, forming bioconjugates that are chemically and opti-
cally stable in phosphate buffer solutions.
(12) (a) Barbarella, G.; Favaretto, L.; Sotgiu, G.; Antolini, L.; Gigli,
G.; Cingolani, R.; Bongini, A. Chem. Mater 2001, 13, 4112. (b)
Barbarella, G.; Favaretto, L.; Sotgiu, G.; Zambianchi, M.; Fattori, V.;
Cocchi, M.; Cacialli, F.; Gigli, G.; Cingolani, R. Adv. Mater. 1999, 11,
1375-1379.
Resu lts
(I) Syn th esis of Rigid -Cor e Oligoth iop h en e-S,S-
Dioxid e Isoth iocya n a tes. The synthetic patterns fol-
lowed for the preparation of rigid-core isothiocyanates via
the Stille coupling are shown in Schemes 1-3.
(13) Stille, J . K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508.
(14) Montheard, J . P.; Delzant, J . F.; Gazard, M. Synth. Commun.
1984, 14, 3, 289.
(15) De J ong, F.; J anssen, M. J . J . Org. Chem. 1971, 36, 1645.
J . Org. Chem, Vol. 68, No. 4, 2003 1513

Sotgiu et al.
SCHEME 2. Syn th esis of Isoth iocya n a tes 17, 20, 23, 26, a n d 31 Sta r tin g fr om Dith ien oth iop h en e 15a
a
Reagents and conditions: (i) LDA, ClSi(CH3)2CH2Cl, THF; (ii) NaSCN, acetone; (iii) NBS, CH2Cl2/CH3COOH; (iv) 12, Pd(AsPh3)4,
toluene; (v) 21, Pd(AsPh3)4, toluene; (vi) 24, Pd(AsPh3)4, toluene; (vii) 27, Pd(AsPh3)4, toluene.
SCHEME 3. Syn th esis of Isoth iocya n a te 35 fr om Tetr a m er 3212a ,a
a
Reagents and conditions: (i) NBS, CH2Cl2; (ii) 12, Pd(AsPh3)4, toluene; (iii) NaSCN, acetone.
tin exchange reactions,16 leading to a variety of undesired
byproducts, lowering the yields, and making more dif-
ficult the purification of the targeted oligomer.
Scheme 2 shows the synthesis of isothiocyanates 17,
20, 23, 26, and 31 containing DTTOMe12a as the rigid
core.
Compound 17, an intensely blue fluorescent compound,
was obtained by direct functionalization of DTTOMe with
chloro(chloromethyl)dimethylsilane following selective
lithiation at one of the terminal positions and subsequent
conversion of chlorine to NCS using sodium thiocyanate
in acetone. The other isothiocyanates were obtained via
the Stille coupling of mono- or dibrominated rigid cores
with the appropriate thienyl or phenyl stannanes fol-
lowed by conversion of chlorine to the isothiocyanate
functionality.
Scheme 3 shows the synthesis of compound 35, con-
taining the isothiocyanate functionality at both terminal
positions, obtained from the highly fluorescent tetramer
32.12a
The formation of dibromo derivative 33 was almost
quantitative, and the subsequent Stille coupling occurred
in good yield, as well as the conversion of the terminal
chlorine to -NCS at both positions.
(II) Op tica l P r op er ties. Table 1 shows the molar
extinction coefficients (ꢀ), the maximum absorption and
emission wavelengths (λmax and λPL), and the photolumi-
nescence quantum efficiencies (PLQEs) of the newly
synthesized isothiocyanates together with those of 32 and
15, used as reference compounds. Table 2 shows the effect
of solvent changes on the optical properties of isothio-
cyanates 31 and 35. Figure 1 shows the fluorescence
colors of the newly synthesized isothiocyanates under
excitation with a UV lamp at λexc ) 364 nm.
The conversion to -NCS of the chlorine atom of -Si-
(CH3)2CH2Cl attached to a phenyl group, for the prepara-
tion of compounds 26 and 31, occurred in rather low
yields (48% and 55%, respectively, compared to 98% and
88% conversion in compounds 20 and 23).
Table 1 shows that the λmax values of the isothiocyan-
ates vary within a range of 70 nm, and the λPL values
within a range of 115 nm, with the fluorescence colors
spanning from deep blue to orange (Figure 1).
(16) Hassan, J .; Se´vignon, M.; Gozzi, C.; Schulz, E.; Lemaire, M.
Chem. Rev. 2002, 102, 1359.
1514 J . Org. Chem., Vol. 68, No. 4, 2003

Oligothiophene-S,S-dioxide Isothiocyanates
TABLE 1. Mola r Extin ction Coefficien ts (E, cm -1 m ol-1),
Ma xim u m Absor p tion (λm a x, n m ) a n d Em ission (λP L, n m )
Wa velen gth s, a n d P h otolu m in escen ce Qu a n tu m
Efficien cies (P LQEs, %) of Rigid -Cor e Isoth iocya n a tes 14,
17, 20, 23, 26, 31, a n d 35 a n d of th e Refer en ce Com p ou n d s
15 a n d 32
compd
ꢀ
λmax
λPL
∆(λPL - λmax
)
PLQE
17
26
31
20
14
23
35
15b
32b
8050
15400
23400
12700
18500
21900
12100
7290
368
383
405
401
438
425
422
364
400
451
483
505
514
540
550
566
457
545
83 (5000)a
100 (5320)
100 (4920)
113 (5480)
102 (4480)
125 (5160)
144 (6030)
93 (5300)
0.65
0.70
0.90
0.35
0.20
0.10
0.10
0.77b,c
0.85b,c
16900
145 (6600)
a
b
Values in parentheses are in cm-1
.
Reference 12a. c Absolute
values measured using an integrating sphere.
TABLE 2. Effect of Solven t Ch a n ges on th e Op tica l
P r op er ties of Isoth iocya n a tes 31 a n d 35
b
b
compd
solvent
ꢀa
λmax
λPL
∆(λPL - λmax)
31
CH2Cl2
22700
90700
48700
22700
17700
18400
402
402
402
421
435
435
505
511
504
573
591
591
103 (5070)c
109 (5310)
102 (5030)
151 (6300)
156 (6070)
156 (6070)
CarbTweend
PBSe
35
CH2Cl2
CarbTweend
PBSe
a
b
In cm-1 mol-1
.
In nm. c Values in parentheses are in cm-1
.
F IGURE 2. Normalized absorption and photoluminescence
spectra of isothiocyanates 31 (top) and 35 (bottom), 10-5 M in
methylene chloride.
d
Carbonate (0.5 M) buffer solution containing 0.05% Tween 20,
pH 9.5. e Phosphate buffer solution, pH 7.2.
Oligothiophene-S,S-dioxides are amphiphilic com-
pounds, and their optical properties are more sensitive
to solvent variations than those of conventional thiophene
oligomers.18 High sensitivity to solvent is also displayed
by the isothiocyanates, as shown by the results reported
in Table 2 for 31 and 35.
The comparison of the optical characterististics of 31
and 35 in an organic solvent such as methylene chloride
with those in aqueous solvents such as CarbTween and
PBS shows that there is a remarkable increase in molar
extinction coefficients in aqueous solvents, whereas the
λmax and λPL values of both compounds are affected to a
much lesser extent. Indeed, while absorption and emis-
sion wavelengths of 31 are the same, within experimental
error in all solvents, those of 35 are increased by no more
than 14-18 nm in aqueous solvents.
F IGURE 1. Fluorescence of 10-5 M solutions of rigid-core
isothiocyanates under excitation with a UV lamp at λexc ) 364
nm.
The molar extinction coefficient increases as does the
oligomer size, with the highest value, 23400 cm-1 mol-1
,
being that of isothiocyanate 31. The differences between
absorption and emission wavelengths are quite large for
all compounds, being in the range 4480-6030 cm-1. As
an example, Figure 2 shows the normalized absorption
and emission spectra of isothiocyanates 31 (top) and 35
(bottom).
(III) Con ju ga tion w ith Mon oclon a l An tibod ies.
The newly synthesized isothiocyanates were conjugated
to either anti-CD3 (purified from mouse ascitic fluid) or
anti-CD8 antibodies.
The PLQE values of isothiocyanates 17 and 26 were
measured using 15 as the reference compound, whereas
the reference for 31, 20, 14, 23, and 35 was compound
32. For both 15 and 32, the absolute photoluminescence
efficiency values, measured by using an integrating
sphere, were indeed available12a (Table 1).
Although the measurements of PLQE values are
always affected by large experimental errors,17 the trend
of PLQEs in Table 1 shows clearly that, as the emission
wavelength increases, the photoluminescence quantum
yield decreases.
Anti-CD3 and anti-CD8 are IgG1 isotype-specific an-
tibodies which react with 30/32 kDa and 22-30 kDa CD8
and CD3 antigens, respectively, expressed on human T
cells having cytotoxic activity.19 Both are used for diag-
nostic purposes in flow cytometry and immunohistochem-
istry, and their conjugates with fluorescein isothiocyanate
(isomer 1, FITC) are commercially available.
(18) Lanzani, G.; Cerullo, C.; De Silvestri, S.; Barbarella, G.; Sotgiu,
G. J . Chem. Phys. 2001, 115, 1623.
(19) (a) Bisping, G.; Lugering, N.; Lutke-Brintrup, S.; Pauels, H.
G.; Schurmann, G.; Domschke, W.; Kucharzi, T. Clin. Exp. Immunol.
2001, 123, 15. (b) Zhang, J .; Gupta, A.; Dave, R.; Yimen, M.; Zerhouni,
B.; Saha, K. Nat. Med. 2001, 7, 65. (c) Dougall, D. S.; Lamouse-Smith,
D. S.; McCarthy, S. A. Cell. Immunol. 2000, 205, 1.
(17) (a) Eaton, D. Pure Appl. Chem. 1988, 60, 1107. (b) Fery-Forgues,
S.; Lavabre, D. J . Chem. Educ. 1999, 76, 1260.
J . Org. Chem, Vol. 68, No. 4, 2003 1515

Sotgiu et al.
F IGURE 4. Photoluminescence spectrum of the bioconjugate
of isothiocyanate 35 with antibody anti-CD8 in a 15:1 molar
ratio (red trace) compared to that of a 20 times more diluted
solution of the bioconjugate of FITC with the same antibody
in a 6:1 molar ratio (green trace).
i.e., in the experimental conditions described above. The
photoluminescence spectra were obtained by irradiation
at the maximum absorption wavelength of the fluoro-
phores.
F IGURE 3. Top: Absorption spectrum of isothiocyanate 14
alone (red trace) and bound to antibody anti-CD3 (green trace)
in PBS (pH 7.2). Bottom: Photoluminescence spectrum of the
bioconjugate.
To obtain the spectra of Figure 4, with comparable
photoluminescence intensities for the two bioconjugates,
it was necessary (a) to use a solution with a fluorophore
to antibody molar ratio that was 6:1 for FITC and 15:1
for 35 and (b) to have a solution of FITC/antibody anti-
CD8 conjugate that was 20 times more diluted than that
of the 35/anti-CD8 antibody bioconjugate. As shown in
the figure, the photoluminescence spectrum of the bio-
conjugate of 35 with anti-CD8 antibody is markedly
broader than that of the bioconjugate of FITC.
The conjugation was carried out according to standard
modalities, by dissolving the isothiocyanates in anhy-
drous dimethyl sulfoxide and then reacting them with
the antibodies in carbonate buffer solution (pH 9.5). The
mixtures were incubated for 3 h at ambient temperature,
and then the bioconjugates were separated from the
unbound fluorophores by filtration through a desalting
column that enabled the transfer of pure bioconjugate
in PBS solution (pH 7.2). Appropriate tests (indirect
method on flow cytometry using a second-step antibody
conjugated with FITC) showed that the biological activity
of the antibodies was entirely preserved in all cases. The
lifetimes of the solutions of bioconjugates in PBS re-
mained unaltered for many weeks, the limit being that
of the stability of the antibody. Values of the fluorophore
to protein molar ratios up to 30:1 were attained without
precipitation of the antibody.
Figure 3 shows the absorption spectra of isothiocyanate
14 alone (red trace) and conjugated to antibody anti-CD3
(green trace). It is seen that the absorption spectrum of
the bioconjugate shows only some broadening compared
to the absorption spectrum of unbound 14. A similar
broadening of the absorption band of the bioconjugates
(small for 17 and large for 35), relative to that of unbound
fluorophores, was observed for all oligothiophene isothio-
cyanates,2c including compound 31, whose bionconjugate
with antibody anti-CD8 is described in ref 2d.
Discu ssion
The use of thiophene oligomers as fluorescent markers
for biopolymers is recent, and the synthesis of only a few
oligothiophene isothiocyanates has been reported so far.2c
The data presented in this paper confirm that our
strategy of introducing the -NCS functionality into the
aromatic skeleton of a preformed oligomer by means of
commercial chloro(chloromethyl)dimethylsilane2c is very
useful and allows for a large variety of fluorescent
oligothiophene isothiocyanates to be prepared.
Chloro(chloromethyl)dimethylsilane can be directly
incorporated into the molecular skeleton at one of the
terminal positions following lithiation, as in the case of
compound 17, or by means of the Stille coupling with a
thienyl or phenyl stannane already functionalized with
the -Si(CH3)2CH2Cl moiety, as, for example, in the case
of compounds 14 (Scheme 1) and 31 (Scheme 2). Also, it
can be incorporated at both the terminal positions, as in
compound 35 (Scheme 3). Doubly functionalized oligo-
mers are easier to prepare than those containing the
-NCS group at only one of the terminal positions; thus,
they are more convenient in terms of reaction yields,
purification procedures, and overall costs.
Figure 4 shows the photoluminescence spectrum of the
conjugate of isothiocyanate 35 with antibody anti-CD8
(red trace) compared to the photoluminescence spectrum
(green trace) of the same antibody bound to FITC.3 The
fluorescence quantum efficiency and the molar extinction
coefficient of FITC, which emits an intense green light
When the -Si(CH3)2CH2Cl group is bound to a thienyl
ring, the conversion of chlorine into -NCS occurs in good
yield upon treatment with NaSCN in acetone. However,
when it is bound to a phenyl ring, the conversion is much
more difficult, as in the case of compounds 26 and 31.
upon photoexcitation, are 0.9 and 80000 cm-1 mol-1 3
,
respectively, and are then much higher than those of the
orange-emitting isothiocyanate 35 reported in Table 1.
Both bioconjugates were prepared by the same modality,
1516 J . Org. Chem., Vol. 68, No. 4, 2003

Oligothiophene-S,S-dioxide Isothiocyanates
Probably, this is due to the strongly stabilizing para
position of the -Si(CH3)2CH2Cl group in these com-
pounds, and future work should take into account phenyl
derivatives bearing the silylated group in the meta
position to increase the yields.
The versatility of the Stille reaction for the formation
of the aryl-aryl bond,16 combined with the possibility of
introducing the isothiocyanate functionality in the last
formation steps of the molecular skeleton, allows for a
great variety of fluorescent isothiocyanates to be pre-
pared. Those reported in this papersfluorescing from
deep blue to orange (Figure 1)sare only a few of the
many which could be synthesized using the rigid
dithienothiophene core DTTO.
The conjugation of oligothiophene isothiocyanates to
monoclonal antibodies occurs spontaneously at basic pH
by reaction of the -NdCdS functionality with the ꢀ-NH2
groups of the lysine residues to form thioureas (-NH-
C(S)-NH-).2c This is also the case for the isothiocyanates
described in this paper.
The bionconjugates formed by the isothiocyanates of
Schemes 1-3 show absorption spectra that are similar
to those of the unbound markers except for some broad-
ening of the low-energy band (see, for example, Figure
3), more or less accentuated depending on the molecular
structure of the marker. The fact that the smallest
broadening is observed for the shorter isothiocyanate, 17,
while the largest is observed for the longer one, 35,
suggests that the effect might be due to an increased
number of conformations of the marker once it is bound
to the antibody.
The PLQEs of the isothiocyanates of Table 1 are up to
2 orders of magnitude greater than those containing one
nonrigid thienyl-S,S-dioxide moiety.2c,12a,b
Isothiocyanate 35 was synthesized to check whether
the greater proximity of the fluorophore to the antibody,
caused by the attachment to the antibody with both ends
of the molecule, would lead to fluorescence quenching or
would alter the biological activity of the antibody. We
found that the biological activity of the anti-CD8 antibody
marked with 35 remained unaltered and that the result-
ing bioconjugate gave rise to an intense, orange, fluores-
cence signal, which is shown in Figure 4.
To gain a better insight into the efficiency of this
marker, we have compared the photoluminescence spec-
trum of the 35/anti-CD8 antibody conjugate with the
photoluminesce spectrum of the bioconjugate of FITC
with the same antibody, prepared using parallel modali-
ties.
The PLQE value of isothiocyanate 31 (0.9) is close to
that of FITC, the most studied and currently used
fluorescent marker in diagnostics.
Compound 31 emits an intense green light similar to
that of FITC. However, contrary to FITC, whose fluores-
cence decay time is only a few minutes,3 the fluorescence
of 31 is persistent. For example, solutions maintained
without any caution in the presence of air and light, at
ambient temperature, display unaltered optical charac-
teristics after one year.
Isothiocyanates 17 and 26, which emit blue light, are
also characterized by high photoluminescence efficiencies
(0.65 and 0.70), whereas the other isothiocyanates, emit-
ting yellow and orange light, display much lower fluo-
rescence efficiencies (from 0.10 to 0.35).
Figure 4 shows that it is possible to obtain photo-
luminescence spectra of comparable intensities. This was
achieved by diluting the solution of the conjugate with
FITC 20 times relative to that of 35. Moreover, the
fluorophore:antibody molar ratio was 6:1 for FITC and
15:1 for 35. Considering the differences in fluorescence
frequency and quantum yield between 35 and FITC, we
believe that the comparison of Figure 4 is very encourag-
ing.
Figure 4 shows also that the 35/anti-CD8 antibody
conjugate displays a photoluminescence signal which is
significantly broader than that of the corresponding
bioconjugate with FITC. A narrow photoluminescence
signal is important for multilabeling experiments, and
clearly, the synthesis of oligothiophene isothiocyanates
requires further tailoring to achieve more appropriate
signal widths.
On the other hand, oligothiophene isothiocyanates are
just beginning to be studied as fluorescent markers for
biopolymers, and the overall data reported in this paper
indicate that their level of efficiency can rapidly be
improved by way of chemical synthesis.
Nevertheless, even at the present level of efficiency
oligothiophene isothiocyanates are of great interest for
their optical stability and color tunability.
The decrease of PLQE on increasing fluorescence
frequencies is a commonly observed trend in organic and
inorganic fluorescent compounds. In the specific case of
oligothiophene isothiocyanates this is probably due to a
variety of different effects whose investigation is beyond
the aim of the present study. Nevertheless, we can
speculate that increased exciton delocalization and in-
creased conformational freedom (the oligomers emitting
yellow and orange light are the longer ones) should favor
competitive nonradiative processessprobably intra-
molecular, such as internal conversion (IC) and inter-
system crossing (ISC)18,21sand reduce the overall quan-
tum efficiency.
In the past few years the development of monoclonal
antibodies has greatly increased the capability of detect-
ing cells in situ for specific antigens in many applications,
including cancer detection. Monoclonal antibodies marked
with fluorescent compounds are detected by flow cytom-
etry, a technique that allows the number of cells to be
measured by their light-scattering characteristics and
their phenotype through fluorescence measurements.
This application provides information on the cell size and
morphology of intracellular and superficial antigens, and
many other important biochemical and clinical param-
eters.
Con clu sion
(20) (a) Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J .
Org. Chem. 1997, 62, 6469. (b) Adamczyk, M.; Grote, J . Synth.
Commun. 2001, 31, 2681.
The oligothiophene-S,S-dioxide isothiocyanates, whose
synthesis and conjugation to monoclonal antibodies are
reported in this paper, represent an improvement with
respect to the oligothiophene isothiocyanates already
(21) (a) Belijonne, D.; Cornil, J .; Friend, R. H.; J anssen, R. A.;
Bredas, J . L. J . Am. Chem. Soc.1996, 118, 6453. (b) Bredas, J . L.;
Cornil, J .; Beljonne, D.; Dos Santos, D. A.; Shuai, Z. Acc. Chem. Res.
1999, 32, 267.
J . Org. Chem, Vol. 68, No. 4, 2003 1517

Sotgiu et al.
7.48 (s, 2H), 7.39 (m, 2H); 13C NMR (CDCl3, TMS/ppm) δ
described. Our results confirm the great potential of
oligothiophene isothiocyanates as fluorescent markers,
owing to their good optical properties, chemical and
optical stability, easy color tunability, and facile modali-
ties to bind biomolecules.
139.44, 135.87, 134.50, 131.43, 129.06, 127.92, 126.57, 121.27.
3,5-Dip h en yld ith ien o[3,2-b:2′,3′-d ]th iop h en e-4,4-d iox-
id e (7). A 15 mL solution of 3-chloroperbenzoic acid (0.67 g,
3.86 mmol) previously dried over magnesium sulfate was
added dropwise to a solution of 6 (0.31 g, 0.90 mmol) in 15
mL of dichloromethane. The mixture was stirred at room
temperature overnight before washing sequentially with 10%
KOHaq, 10% NaHCO3 aq, and brine. The organic layer was dried
over sodium sulfate, and the solvent was removed in vacuo.
The crude product was purified by flash chromatography
(aluminum oxide; pentane/ethyl acetate, 8:2) to afford 0.16 g
(46% yield) of the title compound as a deep yellow solid: mp
> 250 °C; MS m/e 380 (M•+); λmax (CH2Cl2) 371 nm; FTIR (neat)
Exp er im en ta l Section
Syn th esis of Ma ter ia ls. Organic solvents were dried by
standard procedures. Analytical thin-layer chromatography
(TLC) was carried out using 0.2 mm sheets of silica gel 60 F254
or 0.2 mm sheets of aluminum oxide 60 F254 neutral. Prepara-
tive thin-layer chromatography (PLC) was performed using
glass plates precoated with silica gel 60 F254 with a layer
thickness of 1 mm. Preparative column chromatography was
performed on glass columns of different sizes packed with silica
gel 60 (particle sizes 0.040-0.063 mm) or aluminum oxide 90
standardized (particle sizes 0.063-0.200 mm). Petroleum ether
refers to the fraction of bp 40-70 °C. The reactions that allow
the introduction of the isothiocyanate group were performed
in 2.5-5-10 mL conical V-Vials made from Wheaton-33 low
extractable borosilicate glass. 2-Methylthiophene, tetrakis-
(triphenylphosphine)palladium(0), tributyltin chloride, diiso-
propylamine, bromine, phenylmagnesium bromide, [1,3-bis-
(diphenylphosphino)propane]nickel(II) chloride, n-butyllithium,
N-bromosuccinimide, copper(II) dichloride, 3-bromothiophene,
1,4-dibromobenzene, sulfur dichloride, sodium thiocyanate,
(chloromethyl)dimethylchlorosilane, and 3-chloroperbenzoic
acid were commercial compounds. Pd(AsPh3)4 was prepared
in situ from commercial tris(dibenzylideneacetone)dipalladium-
(0)-chloroform adduct and triphenylarsine.22 Compounds 2,14
4,23 122d, 15,15 212d, and 3215 have already been described.
1
νSO 1301, 1150 cm-1; H NMR (CDCl3, TMS/ppm) δ 7.82 (m,
2
4H), 7.46 (m, 4H), 7.39 (m, 2H), 7.35 (s, 2H); 13C NMR (CDCl3,
TMS/ppm) δ 141.12, 139.15, 137.01, 131.90, 129.18, 128.90,
127.78, 124.21.
2-(5-Meth ylth ien -2-yl)-3,5-d ip h en yl-6-[5-d im eth yl(ch lo-
r om eth yl)silylth ien -2-yl]d ith ien o[3,2-b:2′,3′-d ]th iop h en e-
4,4-d ioxid e (13). A 67 mg (0.12 mmol) sample of 11 and 57
mg (0.12 mmol) of 122c were added to a 5 mL toluene solution
containing 14 mg (0.012 mmol) of tetrakis(triphenylphos-
phine)palladium(0). The mixture was refluxed for 6 h. After
evaporation of toluene the residue was purified by flash
chromatography (silica gel; pentane/ethyl acetate/dichloro-
methane, 8:1:1) to give 57 mg (yield 72%) of the title product
as an amorphous orange powder: mp > 250 °C; MS m/e 664
(M•+); 1H NMR (CDCl3, TMS/ppm) δ 7.52 (m, 4H), 7.38 (m,
3
3
6H), 7.13 (d, J ) 3.6 Hz, 1H), 7.02 (d, J ) 3.6 Hz 1H), 6.83
3
(d, J ) 3.6 Hz, 1H), 6.61 (m, 1H), 2.89 (s, 2H), 2.41 (s, 3H),
0.417 (s, 6H); 13C NMR (CDCl3, TMS/ppm) δ 142.96, 142.78,
142.46, 140.19, 137.83, 137.29, 135.87, 135.76, 133.82, 133.04,
133.01, 131.91, 131.57, 130.99, 130.89, 129.84, 129.75, 129.12,
128.88, 128.88, 128.75, 128.71, 127.70, 125.78, 29.69, 15.32,
-3.56.
The synthesis and the characterization of compounds 3-4,
8-11, 18, 24, 28-29, and 33 is reported in the Supporting
Information.
3-P h en yl-4-[(4-p h en ylth ien -3-yl)th io]th iop h en e (5). n-
Butyllithium (5.65 mL, 14.12 mmol, 2.5 M in hexane) was
added over 15 min to a solution of 4 (3.07 g, 12.84 mmol) in
20 mL of dry ether at -70 °C. After 1 h sulfur dichloride (0.66
g, 0.41 mL, 6.42 mmol) was added dropwise. The mixture was
allowed to warm to room temperature and stirred overnight
before quenching with water. The product was extracted with
ether (2 × 25 mL) and CH2Cl2 (2 × 25 mL). The combined
organic layers were dried over anhydrous sodium sulfate, and
then the solvent was removed by rotary evaporation. The
residue was purified by flash chromatography (aluminum
oxide; pentane/ethyl acetate, 9:1) to afford 1.18 g (yield 52%)
of the title product as a faint yellow solid: mp 82-83 °C; MS
2-(5-Me t h ylt h ie n -2-yl)-3,5-d ip h e n yl-6-[5-(d im e t h yl-
(isot h iocya n a t om et h yl)silyl)t h ien -2-yl]d it h ien o[3,2-b:
2′,3′-d]th ioph en e-4,4-dioxide (14). A 2.5 mL conical Wheaton
V-Vial was charged with 13 (42 mg, 0.063 mmol) and sodium
thiocyanate (51 mg, 0.63 mmol) in distilled acetone (2.0 mL).
The mixture was vigorously stirred at 200 °C for 5 h. After
being cooled at room temperature, the crude material was
filtered through a silica gel plug to remove the excess sodium
salt and then was purified by preparative thin-layer chroma-
tography to provide 24 mg (yield 55%) of the title product as
a polycrystalline orange solid: mp > 250 °C; MS m/e 687 (M•+);
λmax (CH2Cl2) 438 nm; FTIR (neat) νNCS 2150, νSO 1313, 1145
2
cm-1; 1H NMR (CDCl3, TMS/ppm) δ 7.52 (m, 4H), 7.38 (m, 6H),
1
m/e 350 (M•+); λmax (CH2Cl2) 230 nm; H NMR (CDCl3, TMS/
3
3
3
7.13 (d, J ) 3.6 Hz, 1H), 7.05 (d, J ) 3.6 Hz, 1H), 6.83 (d, J
) 3.6 Hz, 1H), 6.61 (m, 1H), 2.49 (s, 2H), 2.41 (m, 3H), 0.472
(s, 6H); 13C NMR (CDCl3, TMS/ppm) δ 142.96, 142.78, 142.54,
140.95, 137.46, 136.34, 136.19, 135.33, 134.16, 133.30, 133.06,
131.79, 131.54, 130.95, 130.76, 129.84, 129.75, 129.12, 128.93,
128.82, 128.74, 127.74, 125.81, 113.79, 18.59, 15.35, -2.65.
Anal. Calcd for C33H25NO2S6Si (688.04): C, 57.61; H, 3.66.
Found: C, 57.48; H, 3.55.
3
ppm) δ 7.42 (m, 4H), 7.33 (m, 6H), 7.25 (d, J ) 3.6 Hz, 2H),
7.01 (d, 3J ) 3.6 Hz, 2H); 13C NMR (CDCl3, TMS/ppm) δ
142.88, 135.45, 130.53, 128.66, 128.09, 127.44, 126.83, 123 80.
3,5-Dip h en yld ith ien o[3,2-b:2′,3′-d ]th iop h en e (6). n-Bu-
tyllithium (1.50 mL, 3.74 mmol, 2.5 M in hexane) was added
over 15 min to a solution of 5 (0.66 g, 1.87 mmol) in 10 mL of
dry ether at room temperature. The mixture was refluxed for
2 h and then was added via cannula to a solution of copper(II)
dichloride (0.55 g, 4.11 mmol) in 5 mL of dry ether at 0 °C.
The mixture was stirred overnight at room temperature before
quenching with water. The product was extracted with ether
(3 × 25 mL) and CH2Cl2 (1 × 25 mL), and the organic extracts
were dried over sodium sulfate. The solvent was removed by
rotary evaporation, and the residue was purified by flash
chromatography (aluminum oxide; pentane/ethyl acetate, 8:2)
to provide 0.31 g (yield 48%) of the title product as a yellow
powder: mp 153-154 °C; MS m/e 348 (M•+); λmax (CH2Cl2) 317
nm; 1H NMR (CDCl3, TMS/ppm) δ 7.79 (m, 4H), 7.50 (m, 4H),
2-Dim eth yl(ch lor om eth yl)silyl-3,5-d im eth yld ith ien o-
[2,3-d :3′,2′-b]t h iop h en e-4,4-d ioxid e (16). n-Butyllithium
(0.14 mL, 0.34 mmol, 2.5 M in hexane) was added dropwise
at -78 °C to a solution of diisopropylamine (34 mg, 0.066 mL,
0.34 mmol) in THF (2 mL). The mixture was allowed to warm
to 0 °C for 15 min and recooled to -78 °C. The above solution
was added dropwise to a mixture of 1512a (80 mg, 0.31 mmol)
and (chloromethyl)dimethylchlorosilane (48 mg, 0.044 mL, 0.34
mmol) in dry THF (10 mL) at -78 °C via cannula. The solution
was stirred at room temperature for 1 h and then heated to
reflux for 2 h before quenching with water. The aqueous layer
was extracted with ether, and the organic extracts were
sequentially washed with saturated NaHCO3 aq, brine, and
water. After the organic extracts were dried over sodium
sulfate, the solvent was removed by rotary evaporation, and
(22) Barbarella, G.; Zambianchi, M.; Sotgiu, G.; Bongini, A.
Tetrahedron 1997, 53, 9401.
(23) Rieke, R. D.; Kim, S. H.; Wu, X. J . Org. Chem. 1997, 62, 6921.
1518 J . Org. Chem., Vol. 68, No. 4, 2003

Oligothiophene-S,S-dioxide Isothiocyanates
the residue was purified by column chromatography (SiO2;
petroleum ether/dichloromethane, 8:2) to provide 39 mg (yield
35%) of the title product as a yellow solid: mp 123-125 °C;
MS m/e 362 (M•+); 1H NMR (CDCl3, TMS/ppm) δ 6.89 (q, 3J )
1.1 Hz, 1H), 2.98 (s, 2H), 2.47 (s, 3H), 2.41 (d, 3J ) 1.1 Hz,
3H), 0.50 (s, 6H); 13C NMR (CDCl3, TMS/ppm) δ 144.97, 143.19,
140.27, 139.88, 135.46, 135.21, 133.12, 125.15, 29.48, 14.18,
13.09, -3.37.
was washed with ethanol and hexane to provide 70 mg (yield
67%) of an amorphous red powder: mp 178-180 °C °C; MS
m/e 526 (M•+); λmax (CH2Cl2) 424 nm; FTIR (neat) νSO 1300,
2
1146 cm-1; 1H NMR (CDCl3, TMS/ppm) δ 7.26 (d, 3J ) 3.6 Hz,
3
3
1H), 7.23 (d, J ) 3.6 Hz, 1H), 7.14 (d, J ) 3.6 Hz, 1H), 7.05
(d, 3J ) 3.6 Hz, 1H), 6.88 (q, 4J ) 1.2 Hz, 1H), 2.96 (s, 2H),
2.54 (s, 3H), 2.42 (d, J ) 1.2 Hz, 3H), 0.48 (s, 6H) 13C NMR
4
(CDCl3, TMS/ppm) δ 143.81, 142.30, 141.81, 138.17, 136.26,
135.65, 135.60, 135.50, 133.12, 133.00, 132.19, 128.52, 127.24,
125.46, 125.04, 124.46, 30.33, 13.09, 12.80, -3.54.
2-Dim e t h yl(isot h iocya n a t om e t h yl)silyl-3,5-d im e t h -
yld ith ien o[3,2-b:2′,3′-d ]th iop h en e-4,4-d ioxid e (17). A mix-
ture of 16 (30 mg, 0.08 mmol) and sodium thiocyanate (65 mg,
0.80 mmol) in distilled acetone (1.5 mL) was placed in a 2.5
mL conical Wheaton V-Vial. The mixture was vigorously
stirred at 200 °C for 30 min and then cooled to room
temperature. A 10 mL portion of dry ether was added, and
the mixture was filtered through a silica gel plug to provide
26 mg (yield 81%) of the title product as a thick off-white
liquid: MS m/e 385 (M•+); λmax (CH2Cl2) 368 nm; FTIR (neat)
2-[(5-(5-Dim eth yl(isoth iocya n a tom eth yl)silyl)th ien -2-
yl)th ien -2-yl]-3,5-dim eth yldith ien o[3,2-b:2′,3′-d]th ioph en e-
4,4-d ioxid e (23). A 10 mL conical Wheaton V-Vial was
charged with 22 (63 mg, 0.12 mmol) and sodium thiocyanate
(194 mg, 2.40 mmol) in distilled acetone (9 mL). The mixture
was vigorously stirred at 200 °C for 2 h. After being cooled at
room temperature, the crude material was filtered through a
silica gel plug to remove the excess sodium salt. The solvent
was removed by rotary evaporation, and the residue was
washed with ether before recrystallization from toluene to
provide 58 mg (yield 88%) of the title product as an amorphous
red solid: mp 174-176 °C; MS m/e 549 (M•+); λmax (CH2Cl2)
425 nm; FTIR (neat) νNCS 2146, νSO2 1297, 1144 cm-1; 1H NMR
1
νNCS 2150, νSO 1034, 1152 cm-1; H NMR (CDCl3, TMS/ppm)
2
4
δ 6.92 (q, J ) 1.2 Hz, 1H), 2.53 (s, 2H), 2.48 (s, 3H), 2.42 (d,
4J ) 1.2 Hz, 3H), 0.57 (s, 6H); 13C NMR (CDCl3, TMS/ppm) δ
145.45, 143.67, 141.10, 140.64, 135.42, 133.88, 133.48, 125.84,
113.69, 29.94, 14.51, 13.35, -2.10. Anal. Calcd for C14H15
-
3
3
NO2S4Si (385.62): C, 43.61; H, 3.92. Found: C, 44.78; H, 3.79.
(CDCl3, TMS/ppm) δ 7.30 (d, J ) 3.6 Hz, 1H), 7.24 (d, J )
3.6 Hz, 1H), 7.18 (d, 3J ) 3.6 Hz, 1H), 7.08 (d, 3J ) 3.6 Hz,
Dim et h yl(ch lor om et h yl)silylt h ien -2-yl]-3,5-d im et h -
yld it h ien o[2,3-d :3′,2′-b]t h iop h en e-4,4-d ioxid e (19). To a
4
1H), 6.91 (q, J ) 1.2 Hz, 1H), 2.55 (s, 3H), 2.50 (s, 2H), 2.43
(d, 4J ) 1.2 Hz, 3H), 0.54 (s, 6H); 13C NMR (CDCl3, TMS/ppm)
δ 143.97, 143.12, 142.04, 137.93, 136.76, 135.67, 135.43,
134.11, 133.51,133.14, 132.45, 128.86, 127.43, 125.67, 125.08,
124.81, 113.86, 18.71, 13.11, 12.78, -2.58. Anal. Calcd for
flame-dried flask charged with
a 5 mL toluene solution
containing 0.011 mmol of Pd(Ph3As)4 formed in situ was added
18 (75 mg, 0.22 mmol), then the mixture was heated to reflux,
and 122c (105 mg, 0.22 mmol) was slowly added by syringe.
After this addition the reflux was continued for 4 h, then the
solvent was removed by rotary evaporation, and the residue
was purified by column chromatography (silica gel; petroleum
ether/dichloromethane/ethyl acetate, 7:2:1) to afford 65 mg
(yield 67%) of the title product as a yellow solid: mp 156-158
C
22H19NO2S6Si (549.87): C, 48.06; H, 3.48. Found: C, 47.89;
H, 3.55.
2-[(4-Dim eth yl(ch lor om eth yl)silyl)p h en yl]-3,5-d im eth -
yld ith ien o[3,2-b:2′,3′-d ]th iop h en e-4,4-d ioxid e (25). 18 (137
mg, 0.41 mmol) dissolved in toluene (10 mL) was added to a
10 mL toluene solution containing 0.02 mmol of tetrakis-
(triphenylarsine)palladium(0) prepared in situ. The reaction
was heated to reflux, and then 24 (194 mg, 0.41 mmol) was
added dropwise by syringe. After this addition the reflux was
continued for 4 h. The solvent was removed by rotary evapora-
tion, and the remaining residue was purified by column
chromatography (SiO2; hexane/dichloromethane, 8:2) to pro-
vide 40 mg (yield 22%) of the title compound as a medium
°C; MS m/e 444 (M•+); λmax (CH2Cl2) 403 nm; FTIR (neat) νSO
2
1294, 1145 cm-1; H NMR (CDCl3, TMS/ppm) δ 7.29 (d, J )
3.2 Hz 1H), 7.24 (d, 3J ) 3.2 Hz 1H), 6.90 (q, 4J ) 1.2 Hz, 1H),
2.97 (s, 2H), 2.53 (s, 3H), 2.42 (d, 4J ) 1.2 Hz, 3H), 0.49 (s,
6H); 13C NMR (CDCl3, TMS/ppm) δ 143.83, 141.93, 140.20,
137.52, 135.97, 135.76, 135.57, 133.107, 132.61, 128.82, 127.94,
125.01, 30.24, 13.13, 12.74, -3.49.
1
3
2-[5-Dim et h yl(isot h iocya n a t om et h yl)silylt h ien -2-yl]-
3,5-d im e t h yld it h ie n o[3,2-b:2′,3′-d ]t h iop h e n e -4,4-d iox-
id e (20). A mixture of 19 (60 mg, 0.13 mmol) and sodium
thiocyanate (109 mg, 1.35 mmol) in distilled acetone (3 mL)
was placed in a 5 mL conical Wheaton V-Vial. The mixture
was vigorously stirred at 200 °C for 1 h before being cooled to
room temperature. A 10 mL portion of dry ether was added,
and then the mixture was filtered through a silica gel plug to
provide 62 mg (yield 98%) of the title product as a yellow-brown
solid: mp 134-136°C; MS m/e 467 (M•+); λmax (CH2Cl2) 401
1
yellow powder: mp 177-179 °C; MS m/e 438 (M•+); H NMR
3
3
(CDCl3, TMS/ppm) δ 7.63 (d, J ) 8 Hz, 2H), 7.43 (d, J ) 8
Hz, 2H), 6.90 (s, 1H), 2.98 (s, 2H), 2.48 (s, 3H), 2.44 (s, 3H),
0.46 (s, 6H); 13C NMR (CDCl3, TMS/ppm) δ 143.74, 142.43,
141.93, 136.93, 136.02, 134.39, 133.82, 133.23, 133.09, 128.63,
128.16, 124.79, 30.09, 13.15, 12.30, -4.51.
2-[(4-Dim et h yl(isot h iocya n a t om et h yl)silyl)p h en yl]-
3,5-d im e t h yld it h ie n o[3,2-b:2′,3′-d ]t h iop h e n e -4,4-d iox-
id e (26). A 2.5 mL conical Wheaton V-Vial was charged with
25 (36 mg, 0.08 mmol) and sodium thiocyanate (66 mg, 0.82
mmol) in distilled acetone (1.5 mL). The mixture was vigor-
ously stirred at 200 °C for 4 h. After being cooled at room
temperature, the crude material was poured into 10 mL of
ether and filtered through a silica plug to remove the excess
sodium salt. The solvent was evaporated, and the crude
product was recrystallized from 2-propanol to afford 18 mg
(yield 48%) of the title product as orange microcrystals: mp
156-157 °C; MS m/e 461 (M•+); λmax (CH2Cl2) 384 nm; FTIR
(neat) νNCS 2150, νSO2 1303, 1145 cm-1; 1H NMR (CDCl3, TMS/
nm; FTIR (neat) νNCS 2150, νSO 1032, 1146 cm-1 1H NMR
;
(CDCl3, TMS/ppm) δ 7.29 (d, 3J 2 ) 3.2 Hz 1H), 7.25 (d, 3J )
3.2 Hz 1H), 6.91 (q, 4J ) 1.2 Hz, 1H), 2.54 (s, 3H), 2.51 (s,
4
2H), 2.43 (d, J ) 1.2 Hz, 3H), 0.55 (s, 6H); 13C NMR (CDCl3,
TMS/ppm) δ 143.84, 142.01, 140.91, 136.41, 136.02, 135.63,
135.12, 133.14, 132.88, 129.11, 128.10, 125.17, 113.81, 29.68,
13.13, 12.77, -2.58. Anal. Calcd for C18H17NO2S5Si (467.75):
C, 46.22; H, 3.66. Found: C, 46.31; H, 3.74.
2-[(5-(5-Dim eth yl(ch lor om eth yl)silyl)th ien -2-yl)th ien -
2-yl]-3,5-d im eth yld ith ien o[3,2-b:2′,3′-d ]th iop h en e-4,4-d i-
oxid e (22). 18 (67 mg, 0.20 mmol) in 2 mL of toluene was
added to a 4 mL toluene solution containing 0.02 mmol of Pd-
(Ph3As)4 prepared in situ. The mixture was warmed to reflux,
and then 212c (112 mg, 0.20 mmol) dissolved in 2 mL of toluene
was added dropwise. After this addition the reflux was
continued for 16 h. After the mixture was cooled at room
temperature, the solvent was removed by rotary evaporation,
and then the crude material dissolved in 10 mL of dichlo-
romethane was filtered through Celite to remove the palladium
residue. After evaporation of the solvent, the resulting solid
3
3
ppm) δ 7.59 (d, J ) 8 Hz, 2H), 7.46 (d, J ) 8 Hz, 2H), 6.90
(q, 4J ) 1.2 Hz, 1H), 2.52 (s, 2H), 2.48 (s, 3H), 2.44 (d, 4J ) 1.2
Hz, 3H), 0.51 (s, 6H); 13C NMR (CDCl3, TMS/ppm) δ 143.77,
142.02, 141.99, 135.90, 135.47, 134.38, 134.22, 133.40, 133.10,
128.82, 128.38, 124.92, 114.03, 18.19, 13.13, 12.31, -3.67. Anal.
Calcd for C21H19NO2S4Si (461.72): C, 52.03; H, 4.15. Found:
C, 52.16; H, 4.28.
2-P h en yl-3,5-d im eth yl-6-[(4-d im eth yl(ch lr om eth yl)si-
lyl)ph en yl]dith ien o[3,2-b:2′,3′-d]th ioph en e-4,4-dioxide (30).
To a 5 mL toluene solution containing 0.006 mmol of Pd(Ph3-
J . Org. Chem, Vol. 68, No. 4, 2003 1519

Sotgiu et al.
As)4 prepared in situ was added 50 mg (0.12 mmol) of 29
dissolved in 10 mL of toluene. The mixture was warmed to
reflux, and then 24 was slowly added by syringe. After this
addition the reflux was continued for 3 h, and then the solvent
was removed by rotary evaporation. The resulting residue was
purified by column chromatography (SiO2; hexane/dichloro-
methane, 8:2) to afford 26 mg (yield 43%) of the title compound
as a bright yellow solid: mp 127-129 °C; MS m/e 514 (M•+);
2,6-Di[3-m eth yl-5-(5-d im eth yl(isoth iocya n a tom eth yl)-
silyl)t h ien -2-yl)t h ien -2-yl]-3,5-d im et h yld it h ien o[3,2-b:
2′,3′-d ]th iop h en e-4,4-d ioxid e (35). A 10 mL conical Wheaton
V-Vial was charged with 34 (0.12 g, 0.15 mmol) and sodium
thiocyanate (0.12 g, 1.50 mmol) in distilled acetone (9 mL).
The mixture was vigorously stirred at 200 °C for 2 h. After
being cooled at room temperature, the crude material was
filtered through a silica gel plug to remove the excess sodium
salt. The solvent was removed by rotary evaporation, and the
residue was purified by preparative thin-layer chromatography
to provide 0.93 g (yield 71%) of the title product as an
amorphous red-orange solid: mp > 250 °C; MS m/e 870 (M•+);
1
λmax (CH2Cl2) 405 nm; H NMR (CDCl3, TMS/ppm) δ 7.64 (d,
3J ) 8 Hz, 2H), 7.44 (m, 7H), 2.99 (s, 2H), 2.50 (s, 3H), 2.48 (s,
3H), 0.46 (s, 6H); 13C NMR (CDCl3, TMS/ppm) δ 142.95, 142.88,
136.88, 134.38, 133.85, 133.40, 133.04, 132.57, 128.98, 128.85,
128.63, 128.59, 128.34, 128.12, 30.09, 12.36, 12.26, -4.52.
2-P h en yl-3,5-dim eth yl-6-[(4-dim eth yl(isoth iocyan atom -
et h yl)silyl)p h en yl]d it h ien o[3,2-b:2′,3′-d ]t h iop h en e-4,4-
d ioxid e (31). A 2.5 mL conical Wheaton V-Vial was charged
with 30 (21 mg, 0.041 mmol) and sodium thiocyanate (33 mg,
0.41 mmol) in 2.5 mL of distilled acetone. The mixture was
vigorously stirred at 200 °C for 2 h. After being cooled at room
temperature, the crude material was filtered through a silica
gel plug to remove the excess sodium salt. The solvent was
removed by rotary evaporation. No further purification was
necessary to afford 12 mg (yield 55%) of the title compound
as a yellow solid: mp 126-128 °C; MS m/e 537 (M•+); λmax (CH2-
λmax (CH2Cl2) 422 nm; FTIR (neat) νNCS 2150, νSO 1308, 1144
2
3
cm-1; 1H NMR (CDCl3, TMS/ppm) δ 7.26 (d, J ) 3.2 Hz, 2H),
3
7.22 (d, J ) 4 Hz, 2H), 7.07 (s, 2H), 2.51 (s, 4H), 2.40 (s, 6H),
2.23 (s, 6H), 0.55 (s, 12H); 13C NMR (CDCl3, TMS/ppm) δ
143.06, 142.05, 138.94, 137.29, 136.58, 134.33, 133.96, 133.76,
131.50, 127.32, 126.03, 125.45, 113.84, 29.77, 15.15, 12.49,
-2.41. Anal. Calcd for C36H34N2O2S9Si2 (871.43): C, 49.62; H,
3.93. Found: C, 49.72; H, 4.07.
Biocon ju ga tion Mod a lities. Monoclonal antibodies anti-
CD3 and anti-CD8 were first concentrated via ultrafiltration
under nitrogen on a micrometer membrane with a 10kDa
cutoff. Then they were transferred to a 0.05 M carbonate buffer
solution containing 0.05% Tween 20 (pH 9.5), with the use of
a buffer exchange column of Sephadex G25 equilibrated with
5 bed volumes of the same buffer. Fractions of 0.5 mL were
collected. To these solutions aliquots of oligothiophene-S,S-
dioxide isothiocyanates dissolved in DMSO (concentration 10
mg/mL) were added in the amount needed to reach the desired
protein:fluorophore molar ratios. The solutions were incubated
for 3 h at room temperature with stirring. Finally, the
conjugates were chromatographed on a desalting 1.0 mL GH25
column in PBS (pH 7.4).
1
Cl2) 405 nm; FTIR (neat) νSO 1144, 1302, νNCS 2149 cm-1; H
NMR (CDCl3, TMS/ppm) δ 72.60 (d, 3J ) 8 Hz, 2H), 7.45 (m,
7H), 2.52 (s, 2H), 2.50 (s, 3H), 2.48 (s, 3H), 0.52 (s, 6H); 13C
NMR (CDCl3, TMS/ppm) δ 143.34, 143.20, 142.27, 135.68,
134.68, 134.47, 133.85, 133.18, 132.77, 129.24, 129.10, 129.05,
128.92, 128.62, 114.28, 18.45, 12.505, -3.42. Anal. Calcd for
C
26H23NO2S4Si (537.82): C, 58.07; H, 4.31. Found: C, 57.99;
H, 4.27.
2,6-Di[3-m eth yl-5-(5-dim eth yl(ch lor om eth yl)silyl)th ien -
2-yl)]-3,5-d im et h yld it h ien o[3,2-b:2′,3′-d ]t h iop h en e-4,4-
d ioxid e (34). To a 10 mL toluene solution containing 0.05
mmol of tetrakis(triphenylarsine)palladium(0) prepared in situ
was added 33 (0.25 g, 0.41 mmol) in 10 mL of toluene. The
mixture was warmed to reflux, and then 12 (0.39 g, 0.82 mmol)
in 4 mL of toluene was added dropwise. After this addition
the reflux was continued for 5 h. The solvent was removed by
rotary evaporation, and the remaining residue was purified
by flash chromatography (SiO2; pentane/ethyl acetate/di-
chloromethane, 90:5:5) to provide 0.21 g (yield 62%) of the title
compound as an amorphous red-orange powder: mp > 250 °C;
MS m/e 826 (M•+); λmax (CH2Cl2) 422 nm; FTIR (neat) νSO2 1309,
1145 cm-1; 1H NMR (CDCl3, TMS/ppm) δ 7.25 (d, 3J ) 3.6 Hz,
Ab sor p t ion a n d P h ot olu m in escen ce Mea su r em en t s.
Absorption and photoluminescence spectra were recorded
using 10-5-10-6 M solutions in CH2Cl2 or aqueous solvents
(absorbance 0.1-0.2). The excitation wavelengths were those
of the maximum absorption wavelengths of the fluorophores.
Ack n ow led gm en t. Thanks are due to Dr. Mario
Benzi (CNR-ISOF) for his collaboration in the optical
investigations and Mr. A. Guerrini (CNR-ISOF) for
helpful discussions.
Su p p or tin g In for m a tion Ava ila ble: Synthesis and char-
acterization of compounds 3-4, 8-11, 18, 28-29, and 33 and
NMR spectra of compounds 9 and 24. This material is
available free of charge via the Internet at http://pubs.acs.org.
3
2H), 7.23 (d, J ) 3.6 Hz, 2H), 7.06 (s, 2H), 2.97 (s, 4H), 2.40
(s, 6H), 2.22 (s, 6H), 0.49 (s, 12H); 13C NMR (CDCl3, TMS/
ppm) δ 142.35, 142.04, 138.88, 137.68, 136.10, 135.33, 134.32,
134.08, 131.46, 127.09, 125.75, 125.33, 30.49, 15.17, 12.51,
-3.32.
J O026298O
1520 J . Org. Chem., Vol. 68, No. 4, 2003