Efficient synthesis of a new bifunctional Cu(I) chelator

Article abstract of DOI:10.1016/j.tetlet.2015.04.079

Abstract Herein we report an efficient eight-step synthesis of N¹-(3-(methylthio)propyl)-2-((3-(methylthio)propyl)thio)benzene-1,4-diamine (1) from 6-nitrobenzo[d]thiazole. Incorporation of a nitro rather than a hydroxyl functionality off the a

Full text of DOI:10.1016/j.tetlet.2015.04.079

Tetrahedron Letters 56 (2015) 4434–4437
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Efficient synthesis of a new bifunctional Cu(I) chelator
⇑
Justin O. Massing, Roy P. Planalp
Department of Chemistry, University of New Hampshire, Durham, NH 03824, United States
a r t i c l e i n f o
a b s t r a c t
Herein we report an efficient eight-step synthesis of N1-(3-(methylthio)propyl)-2-((3-
(methylthio)propyl)thio)benzene-1,4-diamine (1) from 6-nitrobenzo[d]thiazole. Incorporation of a nitro
rather than a hydroxyl functionality off the aromatic ring serves to enhance conversion during thia- and
aza-conjugate additions. Ultimate reduction of the nitro substituent affords a bifunctional Cu(I) chelate in
gram-scale quantities for subsequent sensing applications.
Article history:
Received 17 February 2015
Revised 16 April 2015
Accepted 20 April 2015
Available online 25 April 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Cu(I)
Ligand design
Bifunctional
Indicator
Conjugate addition
As the third most abundant metal in biology, copper is inti-
mately involved in the transport and activation of molecular oxy-
gen.1 This role is due in large part to its facile redox nature.
However, the ability to access multiple oxidation states also ren-
ders this metal potentially toxic, with an increase in oxidative
and nitrosative stress being linked with cancer2 and neurodegener-
ation.3 Monitoring this metal in a biological context is therefore
necessary to elucidate the relationship between disruptions in
metal-ion homeostasis and pathophysiology. The most attractive
method for doing so is through the design and development of
ligands that result in a measurable change in fluorescence upon
metal-ion binding.4
Fahrni and co-workers have developed a series of fluorescent
pyrazolines exhibiting increased fluorescence in the presence of
Cu(I) through inhibition of photoinduced electron transfer (PET)
(Fig. 1).5 Chelation of Cu(I) serves to lower the ligand’s HOMO,
thereby deactivating the PET process and restoring fluorescence.
The authors quickly accessed these probes by first synthesizing a
bifunctional N1S3 ligand (2) that then underwent an aldol conden-
sation with 4-acetylbenzonitrile. Subsequent condensation with
one of five fluoro-substituted phenylhydrazine derivatives
afforded the corresponding N1S3-functionalized pyrazolines (3).
This ligand is extremely selective for Cu(I) over other interfering
metal ions given the soft sulfur donors6 and the propensity of this
ligand to form a tetrahedral coordination environment, thus stabi-
lizing Cu(I) relative to Cu(II).5 Despite their elegant synthesis, these
probes suffered from poor solubility in aqueous media in addition
to high-energy excitation (346–391 nm) and emission profiles
(432–486 nm) unsuitable for biological imaging.
To improve tissue penetration, Chang and coworkers recently
reported a near-infrared fluorescent sensor for monitoring biolog-
ical Cu(I) also using PET.7 They achieved this by developing a sim-
ilar bifunctional N1S3 ligand (4) that was then covalently attached
to cyanine 7 to afford 5 (Fig. 1). Although this account demon-
strated in vivo visualization of labile Cu(I), these ‘turn-on’
approaches toward metal-ion monitoring are incapable of provid-
ing quantitative measurements. Thus, sensors capable of modulat-
ing the ratio of multiple emission bands (ratiometric) are preferred
in that they generate quantitative information.8 However, few
ratiometric sensors for copper exist, while most suffer from poor
water solubility9 and irreversible sensing.10
Using a modular motif, we previously demonstrated ratiometric
fluorescent metal-ion sensing for quantifying Cu(II) concentrations
in environmental water samples.11–13 This was achieved by
copolymerizing a bifunctional ligand with poly(N-isopropylacry-
lamide) (PNIPAm) and a fluorophore pair capable of engaging in
Förster resonance energy transfer (FRET).12,13 Temperature-re-
sponsive PNIPAm undergoes a structural transformation (random
coil to collapsed globule) at 32 °C, known as its lower critical solu-
tion temperature (LCST). This temperature may be systematically
raised and lowered by increasing hydrophilic and hydrophobic
interactions, respectively.14 For instance, incorporating a charge-
neutral ligand results in LCST depression, whereas metal-ion bind-
ing introduces charge that causes the polymer to expand when
temperature is held constant. This change in shape results in mod-
ulation of FRET efficiency, which may then be correlated with
metal concentration (Fig. 2).12,13 The highly modular nature of this
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.tetlet.2015.04.079
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

J. O. Massing, R. P. Planalp / Tetrahedron Letters 56 (2015) 4434–4437
4435
Figure 1. Bifunctional N1S3 ligands and their incorporation within sensing scaffolds for fluorescently monitoring Cu(I).
Figure 2. Temperature-responsive PNIPAm incorporating a charge-neutral ligand exists as a collapsed globule above its LCST. This macromolecular structure facilitates facile
energy transfer between a donor and acceptor fluorophore pair. Binding of metal ions by this ligand, however, introduces charge along the polymer scaffold, thereby elevating
the LCST. This results in polymer expansion and a concomitant decrease in energy transfer. By physically separating metal recognition from fluorescence transduction we
have demonstrated ratiometric sensing in a modular fashion.
sensor permits selective sensing of the desired metal through
rational design of the bifunctional ligand only.
design. To our delight, this resulted in an 80% yield over three steps
as shown in Scheme 1 versus the previously reported 20% for the
analogous reactions done in two steps during the synthesis of 4.
Our synthesis commenced with deprotection of 6-ni-
trobenzo[d]thiazole by treatment with hydrazine monohydrate to
afford o-aminothiophenol 6,15 which was carried on immediately
to minimize disulfide formation (Scheme 1). In the event that sub-
stantial material was converted to the disulfide, addition of stoi-
chiometric amounts of dithiothreitol was sufficient to generate
the free thiol in situ during the subsequent thia-conjugate addi-
tion.16 Thia- and aza-conjugate additions with acrylic acid were
initially attempted in a single step, though this method proved
unsuccessful. While the thia-conjugate addition proceeded
smoothly in refluxing MeCN to afford a mixture of product and
unreacted acrylic acid, the aza-conjugate addition required H2SO4
as an additive in refluxing H2O. Upon cooling, pure diacid 7 precip-
itated out of solution as a yellow solid and was used without
Given our modular sensing platform, we proceeded to design a
ligand similar to 4 that instead incorporated a polymerizable acry-
lamide for ratiometric sensing of Cu(I). Unfortunately, synthesis of
4 suffered from low yields throughout, especially during conjugate
addition and Fischer esterification (Fig. 1). This observation is likely
attributed to undesirable dialkylation of the anilino nitrogen as
previously suggested.5 Fahrni and co-workers initially overcame
this obstacle by using benzothiazolinone to simultaneously mask
the nitrogen’s free valency and lower its pKa. However, despite effi-
cient monoalkylation, hydrolysis of the carbonyl and concomitant
S-alkylation in a single step was hampered by a 24% yield. We
therefore proposed starting with 6-nitrobenzo[d]thiazole as a
means to deactivate dialkylation during aza-conjugate addition.
Further, this functionality would allow easy access to the desired
acrylamide for subsequent incorporation within our indicator

4436
J. O. Massing, R. P. Planalp / Tetrahedron Letters 56 (2015) 4434–4437
Scheme 1. Efficient synthesis of a new amine-functionalized Cu(I) chelator.
Scheme 2. Model (12) and proposed copolymerizable compound (13), the latter of which will be incorporated within a water-soluble copolymer designed to fluorescently
sense bioavailable Cu(I).
further purification. Fischer esterification of this material in reflux-
ing EtOH afforded diester 8 upon workup in 80% yield over three
steps. This material was then reduced to the corresponding diol
(9) in 81% yield with LiAlH4 in anhydrous THF. Conversion of 9 to
10 in the presence of CBr4 and PPh3 was achieved in 55% yield, fol-
lowed by nucleophilic displacement of the corresponding alkyl
bromides with NaSMe to yield dithioether 11 in 89% yield.
Reduction of the nitro functionality with SnCl2ÁH2O under acidic
conditions generated the desired bifunctional N1S3 ligand (1) in
84% yield.
Comparison of the above eight-step synthesis with the previ-
ously reported seven-step synthesis of 4 demonstrates an increase
in the overall yield of a bifunctional Cu(I) chelator by eightfold
(Fig. 1). Though nearly twice as long as the synthesis of 2, our
approach to inhibiting overalkylation of the anilino nitrogen
afforded increased yields throughout. A number of intermediates
were also accessed along the way with potential for tuning the
indicating range of our sensor (7 and 9). Moreover, the bifunctional
handle has been replaced with an aromatic amine, though the N1S3
moiety remains unchanged from 4. As previously stated, our group
is interested in converting this amine handle into an acrylamide
(13) for subsequent copolymerization within a temperature-re-
sponsive polymer that functions as a fluorescent metal-ion indica-
tor (Scheme 2).11–13 This acrylamide functionality is necessary to
ensure random distribution within our PNIPAm copolymer owing
to similar kinetics of polymerization. To mimic the electronics
experienced following copolymerization of target acrylamide 13,
we have synthesized model ligand 12. Characterization of the
metal-binding abilities of 12 by potentiometric/spectrophotomet-
ric titrations and cyclic voltammetry is currently underway.
Further, 1 is predicted to display a heightened affinity for Cu(I) rel-
ative to 2 and 4 due to the increased electron-donating tendency of
the amino functionality, thus yielding greater sensitivity necessary
for biological sensing applications.
To conclude, we have designed and synthesized a new bifunc-
tional ligand selective for Cu(I) over other interfering metal ions
based upon previously reported N1S3 ligands. The overall yield
was increased substantially (eightfold) relative to the account of
Chang and coworkers by incorporating an electron-withdrawing
nitro functionality to minimize dialkylation during aza-conjugate
addition. Further, this strategy afforded enhanced yields through-
out when compared to the approach taken by Fahrni and co-work-
ers. Work is currently underway to demonstrate the sensing
capability of this ligand following incorporation within a tempera-
ture-responsive polymer capable of ratiometrically sensing Cu(I)
through macromolecular changes in conformation.
Acknowledgments
We gratefully acknowledge the National Science Foundation
(CHE-1012897) and the University of New Hampshire
Dissertation Year Fellowship to J.M. for financial support. J.O.M.
thanks M. Abdalrahman for assistance regarding submission of
samples for mass analysis.
Supplementary data
Supplementary data (experimental procedures and 1H and 13C
NMR spectra) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.tetlet.2015.04.079.

J. O. Massing, R. P. Planalp / Tetrahedron Letters 56 (2015) 4434–4437
4437
9. Goswami, S.; Maity, S.; Maity, A. C.; Maity, A. K.; Das, A. K.; Saha, P. RSC Adv.
2014, 4, 6300–6305.
References and notes
10. Yuan, L.; Lin, W.; Chen, B.; Xie, Y. Org. Lett. 2012, 14, 432–435.
11. Du, J.; Yao, S.; Seitz, W. R.; Bencivenga, N. E.; Massing, J. O.; Planalp, R. P.;
Jackson, R. K.; Kennedy, D. P.; Burdette, S. C. Analyst 2011, 136, 5006–5011.
12. Yao, S.; Jones, A. M.; Du, J.; Jackson, R. K.; Massing, J. O.; Kennedy, D. P.;
Bencivenga, N. E.; Planalp, R. P.; Burdette, S. C.; Seitz, W. R. Analyst 2012, 137,
4734–4741.
13. Osambo, J.; Seitz, W.; Kennedy, D.; Planalp, R.; Jones, A.; Jackson, R.; Burdette,
S. Sensors 2013, 13, 1341–1352.
14. Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496–2500.
15. Chedekel, M. R.; Sharp, D. E.; Jeffery, G. A. Synth. Commun. 1980, 10, 167–173.
16. Cleland, W. W. Biochemistry 1964, 3, 480–482.
1. Bertini, I.; Gray, H. B.; Stiefel, E. I.; Valentine, J. S. Biological Inorganic Chemistry:
Structure and Reactivity; University Science Books: Sausalito, CA, 2007.
2. Finkel, T.; Serrano, M.; Blasco, M. A. Nature 2007, 448, 767–774.
3. Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. Rev. Drug. Discov. 2004, 3, 205–214.
4. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C.
P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515–1566.
5. Chaudhry, A. F.; Mandal, S.; Hardcastle, K. I.; Fahrni, C. J. Chem. Sci. 2011, 2,
1016–1024.
6. Hancock, R. D.; Martell, A. E. Chem. Rev. 1989, 89, 1875–1914.
7. Hirayama, T.; Van de Bittner, G. C.; Gray, L. W.; Lutsenko, S.; Chang, C. J. Proc.
Natl. Acad. Sci. U.S.A. 2012, 109, 2228–2233.
8. You, Y.; Han, Y.; Lee, Y.-M.; Park, S. Y.; Nam, W.; Lippard, S. J. J. Am. Chem. Soc.
2011, 133, 11488–11491.