Convenient synthesis of multivalent zinc(II)-dipicolylamine complexes for molecular recognition

Article abstract of DOI:10.1016/j.tetlet.2012.11.103

A pair of novel dipicolylamine ligands bearing isothiocyanate groups were used as conjugation reagents to prepare multivalent molecules with anionic recognition capability. The isothiocyanates were reacted with two classes of dendritic scaffolds bearing primary amines, squaraine rotaxanes, and PAMAM dendrimers, and the products were converted into water soluble zinc(II) coordination complexes. The multivalent squaraine rotaxanes exhibit high fluorescence quantum yields in water and are very well suited for biological imaging applications.

Full text of DOI:10.1016/j.tetlet.2012.11.103

Tetrahedron Letters 54 (2013) 861–864
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Convenient synthesis of multivalent zinc(II)–dipicolylamine complexes
for molecular recognition
⇑
Shuzhang Xiao, Serhan Turkyilmaz, Bradley D. Smith
Department of Chemistry and Biochemistry, 236 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN 46556, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A pair of novel dipicolylamine ligands bearing isothiocyanate groups were used as conjugation reagents
to prepare multivalent molecules with anionic recognition capability. The isothiocyanates were reacted
with two classes of dendritic scaffolds bearing primary amines, squaraine rotaxanes, and PAMAM dendri-
mers, and the products were converted into water soluble zinc(II) coordination complexes. The multiva-
lent squaraine rotaxanes exhibit high fluorescence quantum yields in water and are very well suited for
biological imaging applications.
Received 6 November 2012
Revised 21 November 2012
Accepted 23 November 2012
Available online 2 December 2012
Keywords:
Zinc dipicolylamine
Multivalent
Ó 2012 Elsevier Ltd. All rights reserved.
Squaraine rotaxane
Fluorescence
Dendrimer
There is a need for molecular probes that can selectively target
phosphorylated biomolecules in various types of biomedical sam-
ples. Supramolecular chemists have explored a wide array of or-
ganic and inorganic structures.1 For operation in aqueous
solution, metal cation coordination complexes are especially effec-
tive because they have the ability to form strong coordination
bonds with the phosphate oxygens. One of the most prominent
families of synthetic phosphate receptors uses zinc(II)–dipicolyl-
amine (Zn(II)–DPA) coordination complexes as the molecular rec-
ognition units.2 We have contributed to this topic by
demonstrating that Zn(II)–DPA complexes can selectively target
membrane surfaces that are rich in anionic phospholipids.2c This
discovery has high biomedical relevance since several cell systems
associated with disease, such as bacterial cells and apoptotic mam-
malian cells, have anionic surfaces. We have developed a number
of fluorescent Zn(II)–DPA probes and used them to image bacterial
infection and cell death in vitro and in vivo.3
are coated with Zn(II)–DPA complexes,4,5 but we wanted to pre-
pare smaller structures using organic scaffolds bearing primary
amines.6 Thus, a logical conjugation choice was reaction of the pri-
mary amines with isothiocyanates to form the corresponding thio-
ureas.7 This reaction typically proceeds under convenient
conditions with good yields and does not require a catalyst or cou-
pling agent. Additionally, isothiocyanates are easily prepared and
can be stored for extended periods of time. These reasons lead us
to synthesize DPA isothiocyanate 1 and BDPA isothiocyanate 2
(Chart 1) and react them with dendritic structures having multiple
branches terminating in primary amines.
We chose to modify two classes of dendritic scaffolds; squa-
raine rotaxanes, and PAMAM dendrimers. Squaraine rotaxanes,
which were developed in our laboratory,8 are interlocked mole-
cules each comprising a highly fluorescent squaraine dye encapsu-
lated inside
a tetralactam macrocycle. The steric protection
provided by the surrounding macrocycle inhibits decomposition
To date, our probe designs have incorporated one or two Zn(II)–
DPA recognition units, and we have observed a general trend that
the divalent systems exhibit a higher and more selective affinity
for anionic membranes over near-neutral membranes.3 These re-
sults have motivated us to investigate higher order multivalent
systems. To access these compounds, we needed to develop conve-
nient synthetic methods for attaching multiple copies of dipicolyl-
amine (DPA) or bis(dipicolylamine) (BDPA) ligands to a molecular
scaffold. There are several literature reports of nanoparticles that
N
N
N
N
N
N
N
N
N
NCS
O
NCS
3
1
2
⇑
Corresponding author. Tel.: +1 574 631 8632; fax: +1 574 631 6652.
E-mail address: smith.115@nd.edu (B.D. Smith).
Chart 1.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.11.103

862
S. Xiao et al. / Tetrahedron Letters 54 (2013) 861–864
N
N
X
N
N
O
X
N
N
N
N
N
NH
O
N
NHBoc i.
ii.
HN
O
H2N
O
O
3
NHBoc
3
7
8
2+
N
N
O
NH
HN
N
N
N
iii.
N
O
1
O
NH2
3
3, X = R1
9
N
N
N
N
X
N
N
N
N
O
X
N
N
N
N
N
NH
O
HN
O
iii.
O
O
O
2
2+
N
N
O
O
NH
O
NH2
HN
N
10
O
N
N
O
X
Scheme 1. Reagents and conditions: (i) 2-chloromethyl pyridine, Na2CO3, MeCN,
reflux, 16 h, 80%; (ii) CH2Cl2, TFA, rt, 16 h, quant.; (iii) CS2, NEt3, THF, 0 °C, 30 min
followed by H2O2, THF, 0 °C, 40% (30% for 10).
N
N
X
N
N
4a, X = R1
4b, X = R2
4c, X = NH2
O
N
NH
O
B
A B
A
A
B
B
X
HN
O
A
X
A
B
B
A
A
O
O
B
B
A
A
B
HN
B
NH
A
A
2+
N
A
N
B
B
A
O
O
O
B
B
A
HN
A
N
B
A
NH
B
N
O
PAMAM G4
A
B
A
B
A
B
11
O
N
B
A
A
O
B
O
B
A
i., ii.
A
B
B
A
NH2
NH2
HN
NH
A
B
B
A
A
B
B
A
N
N
A
B
N
N
B
B A
A
A
O
X
B
N
N
X
N
O
5a, X = R1
5b, X = R2
HN
O
6, A = R1, B = R3
NH
O
O
2+
N
N
O
NH
HN
N
N
N
N
O
Zn2+ Zn2+
N
Zn2+
N
S
N
N
O
12
N
N
N
H
N
H
-
2 NO3
3
iii., iv.
-
R2
4 NO3
S
O
N
H
N
H
3
NH2
R3
R1
Scheme 2. Reagents and conditions: (i) N3(CH2)3NHBoc, CuSO4, sodium ascorbate,
CHCl3, H2O, rt, 16 h, 90%; (ii) TFA, CH2Cl2, 0 °C to rt, 16 h, quant.; (iii) 2, DMF, rt, 16 h,
40%; (iv) Zn(NO3)2, MeOH, 16 h, quant.
Chart 2.
of the squaraine by nucleophilic attack.9 Squaraine rotaxanes emit
a strong and narrow fluorescence in the deep red/near-infrared
region, and their high stabilities make them very well suited for
animal imaging studies as tracer dyes or as targeted probes for
molecular imaging.10 We chose to make multivalent versions, 3,
4a, and 4b for eventual study as fluorescence imaging probes. In
addition, isothiocyanate reagents 1 and 2 were then used to
convert PAMAM dendrimers into multivalent recognition mole-

S. Xiao et al. / Tetrahedron Letters 54 (2013) 861–864
863
Figure 1. Absorption and emission spectra for squaraine rotaxanes 4a (upper panel) and 4b (lower panel).
cules.11 Specifically, we modified commercially available G0 and
G4 PAMAM dendrimers and prepared the multivalent Zn(II)–DPA
complexes 5a, 5b, 6 (Chart 2).
in moderate yield. Isothiocyanate 2 was prepared in a similar fash-
ion (Scheme 1) starting with the known BDPA-butylamine 10.15
With isothiocyanates 1 and 2 in hand, we prepared squaraine
rotaxanes 3, 4a, and 4b bearing multiple copies of DPA or BDPA
A number of methods exist for preparing isothiocyanates.
Primary amines can be reacted with carbon disulfide (CS2) in
ammonium hydroxide solution and the resulting ammonium
dithiocarbonate salts are treated with lead nitrate or tosyl chloride
to give the corresponding isothiocyanates.12 Another method
involves the thermal fragmentation of 1,4,2-oxathiazoles.13 In our
hands, the reaction of primary amines with CS2 and subsequent
treatment with hydrogen peroxide proved to be a convenient
method.14 Preparation of isothiocyanate (1) required the synthesis
of precursor DPA-hexylamine, 9. Thus, monoprotected 1,6-
bisaminohexane was refluxed in acetonitrile with 2-(chloro-
methyl)pyridine in the presence of Na2CO3 to furnish the
N-protected DPA derivative 8 in good yield which was quantita-
tively deprotected using TFA in CH2Cl2 to give 9. Treatment of 9
with CS2 followed by H2O2 afforded the desired isothiocyanate 1
ligands. The bis(amine) 12 was prepared by conducting
a
copper-catalyzed cycloaddition reaction using the known bis-
alkyne 11 and N-tert-butoxycarbonyl-3-azidopropylamine and
then removing the Boc protecting groups using TFA in CH2Cl2.16
Reaction of 12 and 2 in the presence of NEt3 in DMF, followed by
complexation with Zn(NO3)2 in methanol gave the targeted squa-
raine rotaxane 3. Squaraine rotaxanes 4a and 4b were prepared
in a similar fashion by treating the known tetra(amine) 4c (Chart 2)
with 1 or 2 followed by complexation with Zn(NO3)2 (Scheme 2).17
Squaraine rotaxanes 3, 4a, and 4b exhibited high water solubility
and excellent solution phase stability even when exposed to light.
The spectral plots in Figure 1 show the characteristic narrow in-
tense absorption and emission bands, with no evidence for inter-
molecular aggregation. The photophysical properties listed in

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S. Xiao et al. / Tetrahedron Letters 54 (2013) 861–864
Table 1
References and notes
Photophysical parameters for multivalent squaraine rotaxanes in water
1. For reviews of phosphate recognition see: (a) Kubik, S.; Reyheller, C.; Stuwe,
S. J. Inclusion Phenom. Macrocyclic 2005, 52, 137–187; (b) Hirsch, A. K. H.;
Fischer, F. R.; Diederich, F. Angew. Chem., Int. Ed. 2007, 46, 338–352; (c)
Blondeau, P.; Segura, M.; Perez-Fernandez, R.; de Mendoza, J. Chem. Soc.
Rev. 2007, 36, 198–210; (d) Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc.
Chem. Res. 2009, 42, 23–31; (e) Bazziacalupi, C.; Bencini, A.; Lippolis, V.
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T.; Sessler, J. L.; Anslyn, E. V. Chem. Rev. 2011, 111, 6603–6782.
2. For reviews on Zn(II)–DPA complexes as phosphate receptors see: (a)
Sakamoto, T.; Ojida, A.; Hamachi, I. Chem. Commun. 2009, 2, 141; (b) Kruppa,
M.; König, B. Chem. Rev. 2006, 106, 3520; (c) O’Neil, E. J.; Smith, B. D. Coord.
Chem. Rev. 2006, 250, 3068; (d) Kim, S. K.; Lee, D. H.; Hong, J.; Yoon, J. Acc. Chem.
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Suckow, M. A.; Achilefu, S.; Smith, B. D. J. Am. Chem. Soc. 2010, 132, 67–69; (b)
Smith, B. A.; Gammon, S. T.; Xiao, S.; Wang, W.; Chapman, S.; Ryan McDermott,
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5. Leevy, W. M.; Lambert, T. N.; Johnson, J. R.; Morris, J.; Smith, B. D. Chem.
Commun. 2008, 2331–2333.
6. For other ways of converting amines into DPA ligands, see: (a) DiVittorio, K. M.;
Johnson, J. R.; Johansson, E.; Reynolds, A. J.; Jolliffe, K. A.; Smith, B. D. Org.
Biomol. Chem. 2006, 4, 1966–1976; (b) Carolan, J. V.; Butler, S. J.; Jolliffe, K. A. J.
Org. Chem. 2009, 74, 2992–2996.
7. Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic Press: New York,
2008. p 170.
8. Gassensmith, J. J.; Baumes, J. M.; Smith, B. D. Chem. Commun. 2009, 6329–6338.
9. Arunkumar, E.; Forbes, C. C.; Noll, B. C.; Smith, B. D. J. Am. Chem. Soc. 2005, 127,
3288–3289.
10. (a) Johnson, J. R.; Fu, N.; Arunkumar, E.; Leevy, W. M.; Gammon, S. T.; Piwnica-
Worms, D.; Smith, B. D. Angew. Chem., Int. Ed. 2007, 46, 5528–5531; (b) Smith,
B. A.; Xie, B.-W.; van Beek, E. R.; Ivo Que, I.; Blankevoort, V.; Xiao, S.; Cole, E. L.;
Hoehn, M.; Kaijzel, E. L.; Löwik, C. W. G. M.; Smith, B. D. ACS Chem. Neurosci.
2012, 3, 530–537; (c) Cole, E. L.; Arunkumar, E.; Xiao, S.; Smith, B. A.; Smith, B.
D. Org. Biomol. Chem. 2012, 10, 5769–5773.
11. For examples of other multivalent systems based on the PAMAM scaffold see:
(a) Yordanov, A. T.; Kobayashi, H.; English, S. J.; Rejinders, K.; Milenic, D.;
Krishna, M. C.; Mitchell, J. B.; Brechbiel, M. W. J. Mater. Chem. 2003, 13, 1523–
1525; (b) Roy, R.; Baek, M.-G. Methods Enzymol. 2003, 362, 240–249; (c)
Howell, B. A.; Fan, D. Proc. R. Soc. A 2010, 466, 1515–1526; (d) Myung, J. H.;
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1–5.
Compound
kabs (nm)
kem (nm)
log
e
Uf
3
4a
4b
653
653
653
672
672
674
5.16
5.16
5.20
0.30
0.30
0.25
Table 1 include the impressive high quantum yields in water of
0.25–0.30. Taken together, the chemical and photophysical proper-
ties of squaraine rotaxanes 3, 4a, and 4b indicate that they are well
suited for both in vitro and in vivo biological imaging
applications.18
We also modified commercially available PAMAM G0 (4 pri-
mary amines) and PAMAM G4 (64 primary amines) dendrimers.
Reaction of PAMAM G0 with 1 or 2 in DMF overnight furnished
the corresponding thioureas in good yields (78% and 75%, respec-
tively) and complexation of these products with Zn(NO3)2 in meth-
anol afforded 5a and 5b as water soluble compounds. The reaction
of PAMAM G4 with 2 was much slower, and 1H NMR of product 6
indicated that only ꢀ50% of the peripheral amines had reacted
after stirring in DMF for one week. This sluggish reactivity is attrib-
uted to steric crowding at the surface of the dendrimer. Thus, there
appears to be a steric limit to the number of Zn(II)–BDPA units that
can be appended to the surface of high generation polyamine den-
drimers. This finding is not unexpected, and is not a major draw-
back since partially coated high generation dendrimers are
known to exhibit powerful multivalent molecular recognition ef-
fects.19,20 Indeed, preliminary membrane recognition experiments
that added the above multivalent Zn(II)–DPA compounds to dis-
persions of anionic liposomes composed of 1:1 phosphatidylcho-
line/phosphatidylserine induced crosslinking and precipitation of
the liposomes, with the higher order multivalent systems produc-
ing the most liposome precipitation. Conversely, no liposome pre-
cipitation was observed in control experiments using liposomes
composed of entirely zwitterionic phosphatidylcholine. In due
course we will describe the impressive ability of these multivalent
Zn(II)–DPA complexes to target the anionic membrane surfaces of
apoptotic mammalian cells and bacteria.
In summary, we prepared novel dipicolylamine isothiocyanates
1 and 2 and showed that they are convenient conjugation reagents
for attaching Zn(II)–DPA molecular recognition units to dendritic
scaffolds terminating in primary amines. The multivalent squa-
raine rotaxanes 3, 4a, and 4b exhibit high fluorescence quantum
yields in water and are very well suited for biological imaging
applications. We anticipate that compounds 1 and 2 will be
broadly useful for conjugation to a wide range of amine-containing
molecules, including peptides and proteins.
12. Wong, R.; Dolma, S. J. J. Org. Chem. 2007, 72, 3969–3971.
13. Kim, N. J.; Jung, K. S.; Lee, H. J.; Son, J. S. Tetrahedron Lett. 1997, 38, 1597–1598;
(b) O’Reilly, R. J.; Radom, L. Org. Lett. 2009, 11, 1325–1328.
14. Li, G.; Tajima, H.; Ohtani, T. J. Org. Chem. 1997, 62, 4539–4540.
15. Lakshmi, C.; Hanshaw, R. G.; Smith, B. D. Tetrahedron 2004, 60, 11307–
11315.
Acknowledgments
16. Gassensmith, J. J.; Arunkumar, E.; Lorna Barr, L.; Baumes, J. M.; Noll, B. C.;
Smith, B. D. J. Am. Chem. Soc. 2007, 129, 15054–15060.
We are grateful for funding support from NIH and the Walther
Cancer Foundation.
17. Xiao, S.; Fu, N.; Peckham, K.; Smith, B. D. Org. Lett. 2010, 12, 140–143.
18. For recent reviews of fluorescent sensors for biological applications see: (a)
Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155; (b) Panigrahi, M.;
Dash, S.; Patel, S.; Mishra, B. K. Tetrahedron 2012, 68, 781–805.
19. Incomplete derivatization of terminal amines in high generation PAMAM
dendrimers is precedented; see Ref. 11a.
Supplementary data
Supplementary data (synthetic procedures and spectral data)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.tetlet.2012.11.103.
20. For an example of enhanced multivalent recognition using partially coated
dendrimers, see Ref. 11d.