Electrochemically active cross-linking reaction for fluorescent labeling of aliphatic alkenes

Article abstract of DOI:10.1002/chem.201103630

Although cross-linking reactions serve as a valuable tool for the integration of two or more functionalities or properties, the application of electrochemical synthesis to cross-linking reactions is restricted due to the difficulty of mass transfer. Thus, the primary purpose of this research is to explore electrochemical cross-linking systems to construct a fluorescent probe, triggered by the formation of a covalent linkage. The second purpose is to apply the probe to insoluble targets. Towards these goals, a combination of electrochemically active phenol derivatives and aliphatic alkenes were employed to form polycyclic compounds. Several of the dihydrobenzofuran derivatives formed through [3+2] cyclization reactions exhibited fluorescence. Furthermore, this approach allowed the effective modification of alkene-modified silica gel with electrochemically active species, which enables the construction of fluorescent probes that are triggered by C-C bond formation. Copyright

Full text of DOI:10.1002/chem.201103630

DOI: 10.1002/chem.201103630
Electrochemically Active Cross-Linking Reaction for Fluorescent Labeling of
Aliphatic Alkenes
Shokaku Kim,[a] Kumi Hirose,[b] Jumpei Uematsu,[a] Yuzuru Mikami,[a] and
Kazuhiro Chiba*[a]
Abstract: Although cross-linking reac-
tions serve as a valuable tool for the in-
tegration of two or more functionalities
or properties, the application of elec-
trochemical synthesis to cross-linking
reactions is restricted due to the diffi-
culty of mass transfer. Thus, the pri-
mary purpose of this research is to ex-
plore electrochemical cross-linking sys-
tems to construct a fluorescent probe,
triggered by the formation of a covalent
linkage. The second purpose is to apply
the probe to insoluble targets. Towards
these goals, a combination of electro-
chemically active phenol derivatives
and aliphatic alkenes were employed
to form polycyclic compounds. Several
of the dihydrobenzofuran derivatives
formed through [3+2] cyclization reac-
tions exhibited fluorescence. Further-
more, this approach allowed the effec-
tive modification of alkene-modified
silica gel with electrochemically active
species, which enables the construction
of fluorescent probes that are triggered
À
Keywords: C C coupling · electro-
À
by C C bond formation.
chemistry · luminescence · poly-
cycles · solid-phase synthesis
Introduction
ity, therefore, typical electro-organic approaches suffer from
in situ generation of unstable intermediates trapped within
the solid supports. Therefore, the development of electro-
chemically active cross-linkers for the modification of in-
soluble targets, nonconductive samples, and biomolecules re-
mains a significant challenge.
Cross-linking is a valuable method for the integration of two
or more functionalities or properties to facilitate the effec-
tive design and fabrication of bioconjugates and nanomateri-
als.[1] In particular, cross-linking reactions that use photoac-
tive linkers[2] or click-chemistry[3] for fluorescent labeling
offer versatile strategies to manufacture biomolecular
probes, which allow surface modification through stable co-
valent bonds. Although electro-organic synthetic methods
have produced a wide variety of useful intermediate species
under mild conditions,[4] their application to practical cross-
linking is restricted due to the difficulty of mass transfer,
that is, the unstable reactive intermediate generated from
the electrode must be transported to another location. To
date, attempts in solid-phase electro-organic synthesis have
been limited mostly to immobilization of substrates on an
electrode[5] or indirect electro-organic synthesis.[6] Further-
more, solid-phase syntheses often require excess amounts of
reagents to complete the target reactions due to low reactiv-
We have recently accomplished intermolecular carbon–
carbon bond-formation reactions with aliphatic alkenes via
anodically generated cation intermediates to afford a variety
of polycyclic systems.[7] Furthermore, less reactive alkenes
could be coupled with electrochemically active substrates,
which can then be trapped in a thermomorphic solution
phase, spatially separated from the electrode, by using cyclo-
hexane/nitromethane as a medium.[8] These solution-phase
reaction systems effectively accelerated intermolecular inter-
actions between the less reactive olefins and unstable cation
intermediates. We hypothesized that this approach would
allow selective, direct, and stable elaboration of alkene-
modified insoluble targets with electrochemically active spe-
cies. The electrochemically active cross-linking system could
also enable the construction of a fluorescent probe, trig-
gered by the formation of a covalent linkage (Figure 1). This
strategy addresses the time-consuming, multistep post-pro-
[a] Dr. S. Kim, J. Uematsu, Y. Mikami, Prof. Dr. K. Chiba
Laboratory of Bio-organic Chemistry
Tokyo University of Agriculture and Technology
3-5-8 Saiwai-cho, Fuchu
AHCTUNGTREGcNNUN essing problems associated with fluorescent labeling, such
Tokyo 183-8509 (Japan)
Fax : (+81)42-367-5761
E-mail: chiba@cc.tuat.ac.jp
[b] K. Hirose
Toppan Forms Co. Ltd.
1-2-6 Owada-cho, Hachioji
Tokyo 192-0045 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103630.
Figure 1. Schematic view of an electrochemically active cross-linking re-
action for the attachment of fluorescent labels to aliphatic alkenes.
6284
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Chem. Eur. J. 2012, 18, 6284 – 6288

FULL PAPER
as washing and purification, and reduces false signals that
arise from nonspecific adsorption or undesired cleavage of
a fluorophore in the detection environment.
cence characteristics; compound 3d provided a particularly
high quantum yield (F=0.65). The increased conjugation of
compound 3d was assumed to be caused by a structure in
which two alkene molecules are united with the two hydrox-
yl groups at the para positions of benzaldehyde. Next, we in-
vestigated the reactions of various alkenes with 1b
(Table 2). No reaction was observed when allylbenzene was
Herein, we describe an electrochemically active cross-
linking reaction for fluorescent labeling of aliphatic alkenes.
We are particularly interested in the possibility that the elec-
trochemically active species could react with less reactive
aliphatic alkenes bound to an
Table 1. Anodic intermolecular [3+2] cycloaddition reactions between various phenols 1 and 2-methyl-2-
butene (2).[a]
insoluble target to form fluores-
cent molecules.
Results and Discussion
Phenol
Product
Yield
[%][b]
lmax abs
lmax fl
Ff[d]
We have previously developed
intermolecular [4+2], [3+2],
and [2+2] cyclization reactions
with unactivated alkenes to
provide polycyclic compounds.
Initially, we examined the fluo-
rescent properties of the poly-
cyclic products, consistent with
the concept described above.
As a result, several dihydroben-
zofuran derivatives produced
by the [3+2] cyclization reac-
tion were found to exhibit fluo-
rescence; these results are sum-
marized in Table 1. The reactiv-
ity of various phenol derivatives
was investigated by using 2-
[nm][c]
[nm][c]
1
2
1a (R1 =H)
3a
3b
85
90
306
349
405
430
0.04
1b (R1 =Ac)
1c (R1 =CHO)
1d
0.25
0.26
0.65
3
4
3c
3d
52
71
367
395
454
495
[a] All reactions were performed in a lithium perchlorate/nitromethane electrolyte solution. [b] Yields were
1
determined by H NMR spectroscopy. [c] Measured in chloroform, abs=absorbance, fl=fluorescence. [d] Cal-
methyl-2-butene (2) to synthe- culated figures relative to the quantum yield of quinine sulfate dehydrate.
size dihydrobenzofurans 3. In
Table 2. Anodic intermolecular [3+2] cycloaddition reactions between various alkenes and 2-hydroxy-5-
the case of p-methoxyphenol
(1a) and 2-hydroxy-5-methoxy-
ACHTUNGTRENNUNGacetophenone (1b), the expect-
methoxyacetophenone.[a]
Alkene
Product
Yield
[%][b]
lmax abs
lmax fl
Ff[d]
[nm][c]
[nm][c]
ed cycloaddition reactions pro-
ceeded to afford dihydrobenzo-
furans 3a and 3b in excellent
yields (Table 1, entries 1 and 2).
In contrast, when aldehydes 1c
and 1d were used, the desired
products 3c and 3d were ob-
tained in lower yields (Table 1,
entries 3 and 4). It was assumed
that the lower amount of phe-
noxonium cation generated was
due to oxidation of the alde-
hyde group. Moreover, the al-
dehyde group could destabilize
the phenoxonium cation be-
cause the carbonyl carbon atom
does not neutralize a partial
positive charge as effectively as
the acetyl group. With the ex-
ception of compound 3a, all
1
2
3
4
–
5
>99
350
–
430
–
0.19
–
n.d.[e]
84
351
432
0.25
4
5
6
6
–
7
92
348
–
433
–
0.31
–
n.d.
80
350
432
0.22
[a] All reactions were performed in a lithium perchlorate/nitromethane electrolyte solution. [b] Yields were
determined by H NMR spectroscopy. [c] Measured in chloroform. [d] Calculated figures relative to the quan-
1
compounds exhibited fluores- tum yield of quinine sulfate dehydrate. [e] None detected.
Chem. Eur. J. 2012, 18, 6284 – 6288
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6285

K. Chiba et al.
used, whereas a high yield of 99% was obtained when sty-
rene was used (Table 2, entries 1 and 2). A causal relation-
ship between the reaction yield and conjugation of the elec-
tron-rich aromatic ring with the unsaturated moiety was ob-
served. In addition, it was confirmed that one of the carbon
atoms of the carbon–carbon double bond must be fully sub-
stituted (Table 2, entries 3–6) to stabilize the reaction inter-
mediate. The phenoxonium cation derived from the phenol
was subjected to nucleophilic attack of the alkene, which
generated a bi- or trivalent cation. However, the stability of
the bivalent cation was low and the reaction did not pro-
ceed. The compounds produced exhibited similar levels of
fluorescence.
The utility of this protocol was evaluated by testing a vari-
ety of phenols and aliphatic alkenes. As shown in Table 3,
the [3+2] cyclization reactions proceed effectively to afford
the desired products in good to high yields, without side re-
actions or serious decomposition of the functional groups,
even when p-alkoxyphenols substituted with electron-with-
À
À
À
À
drawing groups ( NO2, CO2H, CO2Me C(O)NHiPr) at
the 2-position were used. However, the naphthalene-1-ol de-
rivative 9 f was produced in moderate yield, although the
cation species derived from 8 f is more stable than those de-
rived from phenols 1a–d and 8a–e. Interestingly, the highly
hydrophobic alkenes 11a–e, which were derived from citro-
nellol or citroneic acid, were successfully coupled with
phenol 1b in high yields in a polar electrolyte solution.
Table 3. Anodic intermolecular [3+2] cycloaddition reactions between various phenols and alkenes.[a]
Phenol
Alkene
Product
Yield
[%][b]
1
2
3
4
5
8a R1 =NO2, R2 =Me
8b R1 =H, R2 =Ph
2
2
2
2
2
9a R1 =NO2, R2 =Me
70
92
85
70
91
9b R1 =H, R2 =Ph
8c R1 =CO2H, R2 =Me
8d R1 =CO2Me, R2 =Me
8e R1 =C(O)NHiPr, R2 =Me
9c R1 =CO2H, R2 =Me
9d R1 =CO2Me, R2 =Me
9e R1 =C(O)NHiPr, R2 =Me
6
7
8 f
2
9 f
10
54
1b
>99
8
9
10
11
12
1b
1b
1b
1b
1b
11a R3 =CH2OH
11b R3 =CO2H
12a R3 =CH2OH
12b R3 =CO2H
94
91
88
88
80
11c R3 =OAc
12c R3 =OAc
11d R3 =CH2OC(O)C5H11
11e R3 =C(O)NHiPr
12d R3 =CH2OC(O)C5H11
12e R3 =C(O)NHiPr
3
À
À
13
14
1b
1b
12 f R =C(O) Phe OMe
88
87
3
À
À À
12g R =Fmoc Gly O
3
À
À
À À
15
1b
12h R =Fmoc Tyr
N
83
1
[a] All reactions were performed in a lithium perchlorate/nitromethane electrolyte solution. [b] Yields were determined by H NMR spectroscopy.
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Chem. Eur. J. 2012, 18, 6284 – 6288

Fluorescent Labeling of Aliphatic Alkenes
FULL PAPER
Table 4. Luminescence images of phenols and their respective dihydro-
benzofuran derivatives.[a]
Furthermore, the amino acid derivatives 11 f–h, which
were modified with lipophilic chains at each N-terminal, C-
terminal, and side-chain position, also reacted to give prod-
ucts 12 f–h. It was expected that these cross-linking reaction
systems could be used for the effective modification of lipo-
philic biomaterials with olefin regions, especially for the
modification of lipophilic peptide targets.
To study the selectivity of the functional groups in this re-
action system, electrochemical coupling reactions were con-
ducted with phenol 1b in the presence of an assortment of
terminal monosubstituted olefins and trialkyl-substituted
olefin 2 (Scheme 1). As expected, phenol 1b did not couple
Phenol
Dihydro-
Phenol
Dihydro-
benzofuran
benzofuran
1
2
1b
3b
3c
5
6
7
8c
9c
9d
9 f
1c
8d
8 f
3
4
1d
8a
3d
9a
[a] Photograph of each compound dissolved in chloroform taken under
black light (l=330–350 nm).
Scheme 1. Alkene selectivity in the LiClO4/MeNO2 reaction system.
with the terminal olefins and provided product 3b exclusive-
ly. In addition, the product of coupling with the amino
group of isopropyl amine was not detected in a competition
experiment with 2 under similar conditions (Scheme 2).
These results indicate that the phenoxonium cations anodi-
cally generated in the LiClO4/MeNO2 reaction system can
be selectively trapped with trialkyl-substituted olefins to
afford the cycloadduct as a fluorescent unit in high yields.
further modification and integration of target functionalities
or properties. These results indicate that this strategy can
allow effective opportunities to label fluorescent probes for
reducing false signals that arise from nonspecific adsorption
or undesired cleavage of a fluorophore in the detection en-
vironment.
Finally, based on these results, we attempted to synthesize
a fluorescent probe, constructed from unactivated 3,7-di-
methyl-6-octenoic acid and amino-modified silica, to con-
firm the lifetime of the phenoxonium cation (Scheme 3).
The size of the amino-silica particles was approximately 0.6–
0.7 mm and the amino group content was 0.4 mmolgÀ1. After
the reaction, the mixture was washed in water and the solid
phase was collected by filtration. Fluorescence on the silica
was observed by fluorescent microscopy (Figure 2). The re-
active site of the alkene cannot approach the electrode be-
cause the silica molecule is too large, thus, it was assumed
Scheme 2. Functional group selectivity in the LiClO4/MeNO2 reaction
system.
We next turned our attention to the determination of suit-
able combinations of phenol and cyclized product consistent
with the concept that the formation of a covalent linkage
can trigger fluorescence (Table 4). Thus, it is required that
the cycloadduct exhibit fluorescence but the phenol deriva-
tive does not. Neither 8a nor the respective cyclization
product 9a exhibit fluorescent properties (Table 4, entry 4),
whereas both starting material 8 f and product 9 f fluoresce
(Table 4, entry 7). On the other hand, phenols 1b–d and 8d
could act fluorescent probe precursors; the construction of
the fluorescent unit was triggered by the formation of a co-
valent linkage (Table 4, entries 1–3 and 6). In addition, the
presence of an ester bond (Table 4, entry 6) can allow for
Scheme 3. Anodic cycloaddition between 1b and 3,7-dimethyl-6-octenoic
acid tethered to the silica surface.
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K. Chiba et al.
the solution was washed with a 5% aqueous solution of NaHCO3, then
brine. The organic layer was dried over anhydrous MgSO4. After filtra-
tion and evaporation under reduced pressure, the residue was purified by
silica gel column chromatography (n-hexane/EtOAc) to give the cycload-
1
duct. The products yields were determined by H NMR spectroscopy.
Figure 2. Luminescence images of the silica surface obtained by fluores-
cent microscopy under a) incandescent and b) black light (l=330–
350 nm).
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science and
Technology.
that the phenoxonium cation was stable until reaction on
the silica surface. We have previously examined the lifetime
of the phenoxonium cation in the presence of aliphatic al-
kenes or styrene by using laser Raman spectroscopy on an
Au electrode. The microscopic laser Raman spectrum
showed a typical signal for the anodically generated phe-
noxonium cation at n˜ =1664 cmÀ1 (assigned to C=O) for
more than 30 s after the oxidation current was stopped,
even in the presence of methylenecyclohexane as an unacti-
vated nucleophilic alkene. Thus, this phenomenon could be
visually monitored.
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An electrochemically active cross-linking reaction was de-
veloped to attach fluorescent probes to aliphatic alkenes.
Several dihydrobenzofuran derivatives formed by [3+2] cyc-
lization reactions exhibited fluorescence properties. Further-
more, this approach allowed the effective and stable elabo-
ration of alkene-modified silica gel with electrochemically
active species, which enables the construction of fluorescent
probes, triggered by the formation of a covalent linkage.
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Experimental Section
General procedure: Phenol (0.1 mmol), alkene (0.3 mmol), and AcOH
(17.5 mmol) were added to LiClO4/CH3NO2 (3.0m, 20 mL). The reaction
cell was capped with a septum equipped with a carbon felt anode (20ꢁ
30 mm) and Pt cathode (10ꢁ10 mm). The electrolysis was performed at
the peak oxidation potentials of the substrates. After the reaction was
complete (2.5–3.0 F), the reaction mixture was poured into EtOAc and
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Received: November 18, 2011
Published online: March 20, 2012
6288
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Chem. Eur. J. 2012, 18, 6284 – 6288