Self-assembly with fluorescence readout in a free base dipyrrin-polymer triggered by metal ion binding in aqueous solution

Article abstract of DOI:10.1039/c9nj01787a

A hydrophobic dipyrrin has been attached to a heterotelechelic amphiphilic random copolymer as part of a single-polymer-single-cargo strategy. The polymer-dipyrrin ("Pod-dipyrrin") exhibits an extended conformation in the organic solvent N,N-dimethylforma

Full text of DOI:10.1039/c9nj01787a

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Self-assembly with fluorescence readout in a free
base dipyrrin–polymer triggered by metal ion
binding in aqueous solution†
Cite this:DOI: 10.1039/c9nj01787a
Rui Liu,
Pothiappan Vairaprakash
and Jonathan S. Lindsey
*
A hydrophobic dipyrrin has been attached to a heterotelechelic amphiphilic random copolymer as part
of a single-polymer–single-cargo strategy. The polymer–dipyrrin (‘‘Pod-dipyrrin’’) exhibits an extended
conformation in the organic solvent N,N-dimethylformamide (DMF) but in aqueous solution forms a
unimeric nanoparticle (i.e., a foldamer) as indicated by dynamic light-scattering (DLS) data (hydrodynamic
diameter Dh = 140 nm versus 15 nm in the two solvents, respectively). Treatment of the Pod-dipyrrin with
Zn(II) in aqueous solution causes formation of a bis(Pod-dipyrrinato)Zn(II) complex as evidenced by a (1)
slight change in absorption spectrum, (2) B50-fold increase in fluorescence quantum yield (Ff from
0.0035 to 0.17), and (3) profound increase in Dh (from 15 nm to 260 nm). The Dh value for the bis(Pod-
dipyrrinato)Zn(II) complex is only slightly smaller in water (260 nm) than in DMF (310 nm), where a more
extended conformation is expected. The increase in fluorescence of the bis(Pod-dipyrrinato)Zn(II) complex
is readily observed by visual inspection (i.e., naked-eye readout). The conversion of a compact folded
unimer (15 nm) to a more extended dimer (260 nm) triggered by metal ion addition denotes a
conformationally malleable, environmentally sensitive molecular architecture. The self-assembly process
can be reversed upon treatment with a metal-ion sequestering agent such as EDTA. The facile synthesis
of a fluorogenic architecture that undergoes profound spontaneous change in molecular morphology
upon binding an exogenous agent in water under physiological conditions may open a number of
opportunities for sensory-mechanical studies in the life sciences.
Received 8th April 2019,
Accepted 13th May 2019
DOI: 10.1039/c9nj01787a
rsc.li/njc
We recently reported a new and simpler approach for
aqueous solubilization of neutral or moderately polar chromo-
Introduction
The absorption and fluorescence spectra of organic chromo- phores.2 The approach – extending extensive work by the
phores make for diverse applications in the life sciences yet the groups of Sawamoto3,4 and Zimmerman5,6 – entails covalent
tailoring of chromophores for solubility in aqueous media attachment of the chromophore to one terminus of an amphi-
often presents significant challenges. Absorption and fluores- philic random copolymer, thereby affording a single-polymer–
cence in the visible region inevitably entail
a sizeable single-chromophore construct. The polymer ‘‘folds’’ around the
p-chromophore, at least for intense p–p* transitions. The vast unadorned chromophore and enables the resulting ‘‘pod-
majority of dyes contain a chromophore that bears intrinsic chromophore’’ to dissolve in aqueous solution. This approach
charge,1 which also facilitates solubilization in aqueous media. separates the chromophore design and aqueous solubilization
For chromophores that lack intrinsic charge (e.g., carotenoids, into two separate spheres – chromophore cargo and polymer
perylenes, quinones, tetrapyrroles),1 the challenge of aqueous package – with covalent joining of the two entities in the final
solubilization is acute. While solubilizing substituents can synthetic step. To date, the polymer that we have employed
often be incorporated, in many cases the size of the p-system (F-Ph)2 is a polyacrylate/polyacrylamide containing three types
requires construction of a daunting superstructure to impart of pendant groups and distinct functional groups at the two
adequate aqueous solubility.
backbone termini (i.e., heterotelechelic). The three types of
pendant groups: a methoxy-terminated oligoethylene glycol
(PEG9; polar but nonionic), a sulfonate-terminated short alkyl
chain (ionic), and a lauryl (hydrophobic) group are incorpo-
rated from three corresponding monomers via living radical
polymerization. The chain-transfer agent in the polymerization
ultimately provides the two terminal functional groups, which
Department of Chemistry, North Carolina State University, Raleigh,
North Carolina 27695-8204, USA. E-mail: jlindsey@ncsu.edu
† Electronic supplementary information (ESI) available: Results upon complexa-
tion of Pod-Dipyrrin with various ZnX2; and NMR spectra for all new compounds.
See DOI: 10.1039/c9nj01787a
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irrevocably incarcerated in the inner space of the folded
polymer but does have exposure, or at least excursions, to the
outside milieu.
Here, we report our results concerning the metal binding of
a single dipyrrin in the polymer. We first describe the synthesis
of a maleimide-containing free base dipyrrin, then attach the
dipyrrin to the thiol group in the heterotelechelic amphiphilic
random copolymer described above. The resulting single-
polymer–single-dipyrrin architecture (Pod-Dipyrrin) was examined
for binding of divalent zinc and copper ions. The formation of the
bis(dipyrrinato)metal(II) complex would entail intermolecular
interaction of two pods rather than interaction with a single
pod as in the Pod-Rhodamine study. The fluorogenic response
here also is completely distinct from the common process where
amine-based photoinduced electron transfer is suppressed
upon metal binding, thereby turning on fluorescence. The
studies have led to new insights concerning fluorescence of
the free base dipyrrin, intrinsic properties of the pod architec-
ture, and morphological changes of the polymer–dipyrrin entity
upon binding metal ions.
Results and discussion
1. Dipyrrin molecular design
Chart 1 General design of the heterotelechelic amphiphilic random
copolymer for encapsulation of a covalently attached hydrophobic fluor-
ophore in aqueous solution. For F-Ph, m : n : p = 1 : 1 : 5 ratio with overall
molecular weight of 40 kDa (m, n B 20 and p B 100).
Free base dipyrrins bearing diverse substituents react readily
with various metal ions (such as Zn2+, Mg2+, Ni2+, Co2+, Cu2+, Pd2+,
In3+, Ga3+, etc.) to form the corresponding bis(dipyrrinato)metal(II) (or
tris(dipyrrinato)metal(III)) complexes. The bis(dipyrrinato)metal(II)
complexes absorb strongly in the blue-green region (e B 50 000–
100 000 Mꢀ1 cmꢀ1). Although first reported by Fischer in 1924,8 the
complexes were long considered to be non-fluorescent and were
little investigated.9 Replacement of the phenyl group at the
dipyrrin 5-position with the sterically encumbering mesityl
group increased the fluorescence quantum yield of the corres-
ponding bis(dipyrrinato)Zn(II) complexes by a factor of 60, from
Ff = 0.006 of the 5,50-phenyl substituted complex I to 0.36 of the
5,50-mesityl substituted complex II (Chart 2).10 The very large
increase in fluorescence intensity makes the resulting complexes
viable for use as legitimate fluorescent dyes. Accordingly, for the
in this case are carboxylic acid and dithiobenzoate groups
(Chart 1). The success relied on identification of the
appropriate ratios of the three monomers (m : n : p = 1 : 1 : 5
ratio of PEG9, lauryl and sulfonate-terminated units), affording
polymer F-Ph2 of size 40 kDa where m, n B 20 and p B 100.
This foldamer approach proved viable with 8 classes of
chromophores including those that are neutral and hydrophobic
(bacteriochlorin, BODIPY, chlorin, coumarin, perylene, phthalo-
cyanine) or intrinsically charged (cyanine, rhodamine).2 The
presence of one and only one chromophore per pod resulted
in absorption and fluorescence features of the pod-chromophore
in aqueous solution that were essentially identical to those of the
hydrophobic chromophore in an organic solvent (e.g., toluene;
N,N-dimethylformamide, DMF). In addition, the fluorescence
quantum yield (Ff) was little changed for the pod-chromophore
in water versus the chromophore alone in organic media, thus
exhibiting none of the insidious diminution of fluorescence
brightness7 so often encountered upon placing an organic
chromophore in aqueous media.
One objective in developing the single-polymer–single-
chromophore foldamer approach was to embed the chromo-
phore in the inner confines of the polymer, insulated from the
surrounding environment. One chromophore examined was a
fluorogenic rhodamine–lactam that upon exposure to metal
ions is known to undergo ring-opening, thereby eliciting
fluorescence. Upon treatment of the Pod-Rhodamine with
various metal ions, the fluorescence turn-on phenomenon was
observed.2 This result implies that the fluorophore is not
Chart 2 Free base dipyrrin and two bis(dipyrrinato)Zn(II) complexes.
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free base dipyrrin here, we chose an analogous system wherein di-carbonylated products were obtained in yields of 11% and
the 5-aryl group is equipped with 2,6-dimethyl groups and a 10%, respectively. Upon use of Pd(OAc)2/Xantphos/Cs2CO3 in
tether at the 4-position. The presence of an amino or alkoxy an atmosphere of CO,21,22 the desired product dipyrromethane
group at the p-position of the 5-phenyl group is known to cause 3 was obtained in 61% yield. Treatment of 3 with p-chloranil in
quenching of the BODIPY analogues11 (presumably via internal THF at room temperature for 24 h afforded 4 in 69% yield. The
charge transfer), hence we chose to join the tether to the aryl oxidation could be completed within 1 h by sonicating the
p-position via a carbonyl group.
reaction mixture instead of stirring at room temperature for
24 h. The removal of the tert-butoxycarbonyl group of 4 and
introduction of the maleimido moiety were carried out in a
2. Synthesis of the bioconjugatable dipyrrin
The known aryl aldehyde 4-iodo-2,6-dimethylbenzaldehyde (1)12,13 single flask process. Thus, treatment of 4 with trifluoroacetic
was dissolved in pyrrole (serving as reactant in excess and solvent) acid in CH2Cl2 for 12 h caused removal of the tert-butoxycarbonyl
and treated with trifluoroacetic acid at room temperature group as confirmed by TLC analysis. The resulting residue
in a one-flask synthesis14,15 (Scheme 1). The corresponding after the removal of the volatile substances was treated with
dipyrromethane 2 was obtained in 60% yield without aqueous/ 6-maleimidohexanoic acid and O-(benzotriazol-1-yl)-N,N,N0,N0-
organic extraction. The aminocarbonylation of 2 with N-(tert- tetramethyluronium hexafluorophosphate (HBTU) in CH2Cl2
butoxycarbonyl)-1,6-diaminohexane was attempted using Mo(CO)6 containing N,N-diisopropylethylamine (DIEA) for 2 h. Workup and
as a solid source of carbon monoxide,16–20 but the mono- and chromatography afforded dipyrrin 5 in 77% yield. Finally, treatment
Scheme 1 Synthesis of dipyrromethanes 2 and 3; dipyrrins 4 and 5; and bis(dipyrrinato)Zn(II) complex 6.
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of the dipyrrin 4 with zinc acetate afforded bis(dipyrrinato)Zn(II)
1
complex 6. Each new compound was characterized by H NMR
and 13C NMR spectroscopy as well as accurate mass determina-
tion by electrospray ionization mass spectrometry (ESI-MS). The
1H NMR spectrum of the free base dipyrrins (4, 5) showed peaks
due to all protons, with the exception of that of the pyrrole N–H
in the dipyrrin moiety. This feature has been encountered
previously with other free base dipyrrins.9
The absorption and fluorescence properties of the
bis(dipyrrinato)Zn(II) complex 6 were examined in toluene
(Fig. 1). Coordination complex 6 has sharp absorption (fwhm
35 nm) and fluorescence (fwhm 32 nm) bands compared to
those of free base dipyrrin 4 in toluene. The bis(dipyrrinato)Zn(II)
complex 6 exhibits Ff = 0.16, to be compared with 0.0011 of the
free base dipyrrin 4. For these and all subsequent measure-
ments, the fluorescence standard is a BODIPY-hydrazide
(Ff = 0.97).2 Self-assembly of the two free base dipyrrins into
a bis(dipyrrinato)Zn(II) complex equipped with 2,6-dimethyl-
aryl moieties affords a substantially brighter chromophore.
The fluorescence spectra of matched samples of 6 and II in
toluene were found to be essentially identical, although the
Ff value of 6 (0.18) was exactly half that of II (0.36)10 using II
as the standard. The complexes differ only in the nature of the
p-aryl substituent (amido versus methyl); hence the amido
substituent apparently causes diminution of the fluorescence
intensity.
Fig. 2 Absorption and fluorescence spectra (normalized) of 6 in toluene
(black line), dichloromethane (blue line), methanol (red line), acetonitrile
(orange line), and DMF (magenta line).
Table 1 Spectral properties of 6 in organic solvents
Dielectric labs lem Stokes
fwhm
Entry Solvent
constant (nm) (nm) shift (nm) lem (nm) Ffa
1
2
3
4
5
Toluene
CH2Cl2
Methanol
Acetonitrile 36.6
DMF 38.3
2.4
9.1
32.6
488 503 15
486 498 12
30
27
35
34
27
0.18
0.079
0.032
0.050
0.041
485 494
482 493 11
487 496
9
9
a
All yield data were obtained with compound II as a reference
(Ff = 0.36).10
The absorption and fluorescence spectral properties of the
bis(dipyrrinato)Zn(II) complex 6 also were examined in organic
solvents of a range of polarity. The solvents range from the
nonpolar toluene to the quite polar DMF. The spectra are
displayed in Fig. 2. The absorption maximum varies little
(from 482–488 nm) although slightly greater variation occurs
in the position of the fluorescence maximum (493–503 nm),
giving a Stokes shift range of 9–15 nm (Table 1). The Ff values
decline with increasing polarity, from Ff = 0.18 in toluene to
0.041 in DMF, although the spectra shapes are little changed
with solvent polarity.
3. Synthesis of the polymer–dipyrrin conjugate
Synthesis of Pod-Dipyrrin follows the procedure employed
previously2 (Scheme 2). Treatment of polymer F-Ph in DMF
containing triethylamine and ethanolamine liberated the free
thiol, which was reacted in situ with dipyrrin-maleimide 5. The
crude reaction mixture in DMF was dialyzed to remove any free
fluorophore, which has been readily achieved previously with
diverse chromophores.2 The conjugation and purification pro-
cess required B4 days, whereupon Pod-Dipyrrin was obtained
as a yellow friable powder.
4. Characterization of the Pod-Dipyrrin
The Pod-Dipyrrin was examined by 1H NMR spectroscopy in
D2O at room temperature. Characteristic peaks were observed
for the polymer constituents but not of the dipyrrin moiety,
which is not surprising given that the latter is present on one
terminus of the polymer and represents only B1.5% of the
mass of the construct. While the extent of conjugation cannot
be readily established by NMR analysis, evidence presented
below supports the interpretation that the conjugation has
gone to completion. We turned to studies of the photophysical
properties of Pod-Dipyrrin in water. The absorption spectrum
of Pod-Dipyrrin (Fig. 3A, black solid line) in aqueous
solution shows a narrower absorption peak (full-width-at-half-
maximum, fwhm 39 nm) compared to that of the hydrophobic
benchmark 5 (fwhm 80 nm) in toluene (Fig. 3B, red solid line).
The narrower absorption feature may stem from restricted
conformational motion of the dipyrrin unit in Pod-Dipyrrin
Fig. 1 Electronic spectra in toluene at room temperature. Absorption
(blue solid line, lmax = 488 nm) and fluorescence (lexc 460 nm, blue
dashed line, lmax = 504 nm) of bis(dipyrrinato)Zn(II) complex 6, and
absorption (black solid line, lmax = 435 nm) and fluorescence (lexc
390 nm, black dashed line, lmax = 505 nm) of free base dipyrrin 4.
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Scheme 2 Synthesis of Pod-Dipyrrin.
due to the folded state of the amphiphilic polymer in aqueous
solution, whereas the dipyrrin chromophore of hydrophobic
benchmark 5 has a fuller range of motion in toluene. The
motions include butterfly flexing10,23 of the dihedral planes of
the pyrroles with respect to each other, and the rotation of the
aryl group about its p-substituted axis. Pod-Dipyrrin in water
has a weak and broad fluorescence peak similar to that of 5 in
toluene.
The Pod-Dipyrrin was examined by dynamic light-
scattering (DLS) spectroscopy in aqueous solution at concen-
trations ranging from 10–1 mg mLꢀ1, the approximate lower
limit of the DLS instrument. Essentially identical size distri-
butions were observed over this concentration range indicat-
ing the integrity of the folded architecture (Fig. 4A). On the
other hand, the Pod-Dipyrrin showed a profound difference
in molecular size in DMF versus water (Fig. 4B). In DMF the
size distribution is peaked at a hydrodynamic diameter Dh
=
140 nm, whereas the distribution peak is 15 nm in water. The
results are consistent with folding to form a compact unimer
in aqueous solution from a more extended and unstructured
set of conformations in the organic solvent DMF. The absorp-
tion spectrum of Pod-Dipyrrin (Fig. 4C) also is much narrower
in water (fwhm 39 nm) than in DMF (fwhm 79 nm). These
absorption spectral changes are similar to those for Pod-
Dipyrrin in water versus 5 in toluene (Fig. 3B). The data are
fully consistent with expectations concerning folding in aqu-
eous solution on the basis of prior data of the same polymer
bearing other families of chromophores.2
Fig. 3 (A) Absorption (solid line) and fluorescence (lexc 460 nm, dashed
line) spectra of Pod-Dipyrrin in water; and (B) absorption (red solid line)
and fluorescence (lexc 390 nm, red dashed line) spectra of free base
dipyrrin 5 in toluene.
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Fig. 5 Spectral and DLS data in water at room temperature. (A) Absorp-
tion spectra of Pod-Dipyrrin at 1 mg mLꢀ1 (black line), Pod-Dipyrrin
treated with Zn(II) (blue line), and Pod-Dipyrrin treated with Zn(II) then
EDTA (red line); (B) fluorescence spectra of Pod-Dipyrrin (black dashed
line), Pod-Dipyrrin treated with Zn(II) (blue dashed line), and Pod-Dipyrrin
treated with Zn(II) then EDTA (red dashed line); (C) DLS data of Pod-
Dipyrrin at 1 mg mLꢀ1 (black line), Pod-Dipyrrin treated with Zn(II) (blue
line), and Pod-Dipyrrin treated with Zn(II) then EDTA (red line). In all
treatments, Zn(II) refers to Zn(OAc)2 (440 equiv.).
Fig. 4 Properties of Pod-Dipyrrin at room temperature. (A) DLS data in
1 M aqueous NaCl at various concentrations: (A) 10 mg mLꢀ1, average
diameter 14.8 nm; 5.0 mg mLꢀ1, 13.4 nm; 2.5 mg mLꢀ1, 14.8 nm; and
1.25 mg mLꢀ1, 14.5 nm. (B) DLS data in DMF (blue line) and water (black
line) at 1.0 mg mLꢀ1, and (C) absorption in DMF (blue line) and water (black
line) at 1.0 mg mLꢀ1
.
5. Self-assembly of the Pod-Dipyrrin via metal ion chelation in
water
We have demonstrated that a related polymer–fluorophore is water (Fig. 5A). The fluorescence quantum yield (Ff = 0.17)
able to bind metals in aqueous solution.2 Here, we tested the also increased profoundly (B50-fold) compared to that of
ability of Pod-Dipyrrin to form dimeric pod assemblies owing to Pod-Dipyrrin (Ff = 0.0035 in water) (Fig. 5B). Such changes in
formation of the di(pyrrinato)metal(II) complexes upon the absorption and fluorescence are attributed to the formation of
introduction of divalent metal ions (Zn(II) and Cu(II)). A solution the bis(dipyrrinato)Zn(II) complex, which must then join two pod
of Pod-Dipyrrin in aqueous solution was treated with excess architectures; the resulting assembly is termed (Pod-Dipyrrin)2Zn(II).
Zn(OAc)2 (440 equiv.) at room temperature. Examination by The aforementioned spectral changes are visibly evident: (1) the free
absorption spectroscopy after 5 minutes revealed a bathochro- base dipyrrin in solution is yellow whereas the bis(dipyrrinato)Zn(II)
mic shift (12 nm) compared to the monomeric Pod-Dipyrrin in complex is light yellow; and (2) upon illumination, no fluorescence
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Table 2 Photophysical properties of diverse constructs in organic or
aqueous media
labs lexc lem fwhm
Entry Compound
Solvent (nm) (nm) (nm) lem (nm) Ffa
1
2
3
4
5
6
7
4
5
6
Toluene 435 390 505 34
Toluene 434 390 518 54
Toluene 488 460 504 32
Water 474 460 526 61
0.0011
0.0006
0.16
0.0035
0.0021
0.11
Pod-Dipyrrin
Pod-Dipyrrin
(Pod-Dipyrrin)2Zn(II) DMF
DMF
430 390 530 80
486 460 496 27
(Pod-Dipyrrin)2Zn(II) Water 487 470 498 43
0.17
a
All yield data obtained with BDPY-hydrazide as a reference (Ff = 0.97).2,24
is visible from the free base dipyrrin whereas the
bis(dipyrrinato)Zn(II) complex exhibits a green glow. The
fluorescence properties of the Pod-Dipyrrin species in water
are shown in Table 2 and can be compared with those of
Pod-Dipyrrin (in DMF) and 5 in toluene. The spectral properties
of the (Pod-Dipyrrin)2Zn(II) are described further in the Discus-
sion section.
The free base Pod-Dipyrrin and the assembled
(Pod-Dipyrrin)2Zn(II) were examined by DLS spectroscopy. The
free base Pod-Dipyrrin is unimeric in water with a peak in the
DLS distribution at 15 nm (Fig. 5C). On the other hand, the
(Pod-Dipyrrin)2Zn(II) exhibits large very assemblies of 260 nm
in dimension; such large assemblies are unexpected because
the simplest architecture would be simply twice the size of a
single Pod-Dipyrrin. The data imply the (Pod-Dipyrrin)2Zn(II)
must unfold upon formation of the bis(dipyrrinato)Zn(II) motif.
Upon treatment of the (Pod-Dipyrrin)2Zn(II) assembly in
aqueous solution with excess EDTA (3.2 equiv. per the total
quantity of Zn(II)), several distinct changes occurred: (1) the
absorption features reverted to those of the unimeric free base
Pod-Dipyrrin (Fig. 5A), reflecting a change in color from light
green to yellow; (2) the fluorescence diminished markedly and
visibly, to a slightly lower level than for the Pod-Dipyrrin alone
(Fig. 5B); and (3) DLS data revealed the reversion from the large
assembly to give the unimeric folded architecture characteristic
of Pod-Dipyrrin (Fig. 5C). The combined evidence of absorption
shifts, fluorescence brightness and size changes reveal a rever-
sible conversion of Pod-Dipyrrin to (Pod-Dipyrrin)2Zn(II) in
water accompanied by a profound mechanical change and
fluorescence readout: fluorescence turn-up upon addition of
Zn(II) and turn-down upon removal of Zn(II).
Fig. 6 All data were collected in water at room temperature. (A) Absorp-
tion spectra of Pod-Dipyrrin at 1 mg mLꢀ1 (black solid line), Pod-Dipyrrin
treated with Cu(II) (red solid line), and Pod-Dipyrrin treated with Cu(II) then
EDTA (red solid line); (B) fluorescence spectra of Pod-Dipyrrin (black
dashed line), Pod-Dipyrrin treated with Cu(II) (red dashed line), and Pod-
Dipyrrin treated with Cu(II) then EDTA (red dashed line); (C) DLS data of
Pod-Dipyrrin at 1 mg mLꢀ1 (black solid line), Pod-Dipyrrin treated with
Cu(II) (red solid line), and Pod-Dipyrrin treated with Cu(II) then EDTA (red
solid line). In all treatments, Cu(II) refers to CuCl2 (600 equiv.).
The self-assembly experiment with Pod-Dipyrrin was also
carried out with Cu(II) in water. Upon treatment with 600 equiv.
of CuCl2, the absorption spectrum shows a bathochromic shift copper–EDTA complex (green) is associated with the Pod-
(17 nm) (Fig. 6A), the fluorescence was strongly quenched Dipyrrin and causes quenching of the free base dipyrrin. The
(Fig. 6B), and the DLS data showed conversion of the compact combined evidence of absorption, fluorescence brightness and
unimer (Dh = 15 nm) to a large assembly (Dh = 230 nm) (Fig. 6C), size changes reveal a reversible self-assembly of Pod-Dipyrrin in
all of which signal the formation of (Pod-Dipyrrin)2Cu(II). water with fluorescence turn-off upon addition of Cu(II) and
Treatment with excess EDTA (2.3 equiv. per the total quantity turn-on upon removal of Cu(II).
of Cu(II)) caused reversion to the absorption spectrum and
The observed size distribution of (Pod-Dipyrrin)2Zn(II) (260 nm)
unimeric size of the Pod-Dipyrrin. The fluorescence increases in water matches that of two unfolded F-Ph (130 nm ꢁ 2) polymers
but only slightly, without full recovery of the fluorescence in DMF, but is far larger than expected for the simple dimerization
expected of the Pod-Dipyrrin; one interpretation is that the of two intact unimers (15 nm ꢁ 2) in water. To explore such large
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Fig. 7 Absorption (solid line) and fluorescence (dashed line) spectra of
(Pod-Dipyrrin)2Zn(II) in DMF at room temperature.
size distributions, the assembly of Pod-Dipyrrin was carried out in
DMF. Treatment of Pod-Dipyrrin in DMF with Zn(OAc)2 resulted in
formation of the corresponding (Pod-Dipyrrin)2Zn(II) as seen by the
absorption spectrum (Fig. 7). Examination by DLS spectroscopy
shows that the free base Pod-Dipyrrin in DMF exhibits Dh = 140 nm
(Fig. 8A) whereas the (Pod-Dipyrrin)2Zn(II) exhibits a much larger
Dh = 310 nm (Fig. 8B). Finally, treatment of polymer F-Ph with
Zn(OAc)2 in water resulted in no noticeable size change (Fig. 8C), as
expected for a polymer that lacks a dipyrrin unit altogether.
To probe the self-assembly process, Pod-Dipyrrin in aqueous
solution was titrated with zinc acetate. The addition of 1 equiv.
gave two peaks upon DLS spectroscopic examination, including
that of the uncomplexed unimer (Dh = 13 nm) and that for the
putative dimer at Dh = 200 nm (Fig. 9). Treatment with 10 equiv.
caused loss of the unimer peak and only the putative dimer
peak was observed. No further change was observed with
100 equiv. In this titration, no peaks intermediate between that
of unimer and dimer were observed.
Discussion
Fig. 8 DLS data at room temperature. (A) Pod-Dipyrrin in DMF before
(black solid line) and after (blue solid line) treatment of Zn(II); (B) Pod-
Dipyrrin in water before (black dashed line) and after (blue dashed line)
treatment of Zn(II); (C) F-Ph in water before and after treatment with Zn(II).
In all treatments, Zn(II) refers to Zn(OAc)2 (440 equiv.).
The development of foldamers enables diverse molecular
entities to be packaged in a relatively hydrophobic enclosure
for use in aqueous solution. The distinct feature of the strategy
we are developing enables packaging of a single item of
molecular cargo per polymer chain. The idea behind individual
packaging of a single molecular item was developed to preclude
quenching of fluorophores and deleterious contact with water
while achieving use in water. A limiting view is that the
fluorophore or other hydrophobic cargo item is ensconced in
with the self-assembly process leading to the bis(dipyrrinato)-
metal complexes from the foldamer–dipyrrin.
1. Spectra and photophysics of dipyrrins
the interior of the self-assembled foldamer. Yet the results The chemistry of dipyrrins25,26 is rapidly growing particularly
herein show that the cargo item – the dipyrrin – can bind with because of the facile synthesis and versatile coordination
metal ions in aqueous solution, and in so doing cause sub- features that can be exploited to create supramolecular
stantial structural change of the foldamer package. Hence the structures.27 The dipyrrinatoboron complexes (i.e., BODIPYs)28,29
foldamer is not like a rigid marble but is highly malleable and are the best known but the bis(dipyrrinato)metal complexes have
poised for morphological alteration. In the following sections, assembly features that the former lack. The fluorescence properties
we first discuss the spectra and photophysics of dipyrrins. We of dipyrrinato–metal complexes have been comprehensively
then discuss experiments that bear on the environmental reviewed,11,27,30–32 including the turn-on fluorescence that
polarity of the polymer-bound bis(dipyrrinato)Zn(II) complex. accrues upon exposure of a free base dipyrrin to a suitable
We finish by considering the mechanical change associated metal salt, termed chelation-enhanced fluorescence (CHEF).
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rather than by intersystem crossing.34,41 Obvious mechanisms
for internal conversion include rotation of the pyrrolic planes
from coplanarity with respect to each other, and proton tunnel-
ing from the pyrrolic unit to the neighboring imino nitrogen.34
A very recent study points instead, however, to the presence of a
conical intersection of the S1 and S0 potential energy surfaces;
such an intersection is less accessible energetically upon coor-
dination of the nitrogen atoms. If the latter view holds, the
diminished non-radiative decay upon metal coordination to the
dipyrrin is less about structural rigidification and absence of
proton transfer than shifting of molecular orbital energies.43
Here, three observations concerning the fluorescence of the
Pod-Dipyrrin system warrant discussion. First, the Pod-Dipyrrin
exhibits very low fluorescence in DMF (Ff = 0.0021) but
B1.7-fold increase upon foldamer assembly in water, albeit
to a still very low value (Ff = 0.0035) (Fig. 1 and 3). The dipyrrin
in the foldamer undergoes a striking spectral change in the
unfolded state in DMF (broad, unstructured absorption) to the
folded and compact unimer in water (more sharp absorption)
(Fig. 4C). The origin of such spectral change is unclear, as the
dipyrrin in the foldamer in aqueous solution (versus that in the
extended conformation in DMF) is in a more hydrophobic
environment, may be somewhat constrained conformationally,
and perhaps is hydrogen-bonded where (1) the backbone N–H
serves as the hydrogen-bond donor, (2) the amide carbonyl
serves as a hydrogen-bond acceptor, and/or (3) the lone
carboxylic acid at the polymer terminus serves as a proton
donor. Such hydrogen bonds to and from the dipyrrin would
likely supplant the known intramolecular hydrogen bond of the
dipyrrin44 and hence are only conjectural; a general rigidifica-
tion of the dipyrrin owing to the constrained environment of
the assembly may also contribute. Evidence for assembly of the
compact unimer stems not only from the DLS data but also,
inter alia, from 2D 1H NMR NOESY analysis of polymer F-Ph in
DMSO-d6 or D2O.2 The Ff value of a cyanine dye also increased
(by 2.5-fold) upon encapsulation in the pod architecture.2
Second, complexation with Zn(II) to form (Pod-Dipyrrin)2Zn(II)
causes the fluorescence intensity to be turned up markedly in
DMF (Ff = 0.11) and for the compact unimer in water (Ff = 0.17)
(Fig. 5B), resembling that of the dipyrrin lacking the attached
foldamer (Fig. 1, Table 1); on the other hand, the already low
fluorescence quantum yield of the free base dipyrrin is further
turned down (to the level of noise) by complexation with Cu(II) to
form (Pod-Dipyrrin)2Cu(II) (Fig. 6A and B). The absorption spec-
Fig. 9 All data were collected in water at room temperature. DLS data
(left panel) and photographs of the Pod-Dipyrrin solution under ultraviolet
illumination (right panel). (A) Pod-Dipyrrin; (B) Pod-Dipyrrin treated with
1.0 equivalent of Zn(II); (C) Pod-Dipyrrin treated with 10 equivalents of
Zn(II); and (D) Pod-Dipyrrin treated with 100 equivalents of Zn(II). In all
treatments, Zn(II) refers to Zn(OAc)2.
Free base dipyrrins are at best only very weakly fluorescent,30–32 tral features of the (Pod-Dipyrrin)2Zn(II) differ slightly in water
although distinctions between one extreme of ‘‘complete lack versus DMF (compare Fig. 5A and 7); both the significant spectral
of fluorescence’’33 and no fluorescence even at 1.2 K,34 the changes of the dipyrrin and slight spectral changes of the
common report of no detectable fluorescence (Ff o 5 ꢁ 10ꢀ4 35
;
bis(dipyrrinato)Zn(II) unit in DMF and water will require further
r0.001;11,32,36–38 r10
;
ꢀ4 39), and the other extreme of a mea- investigation.
surable albeit tiny fluorescence (Ff = 8.1 ꢁ 10ꢀ4 40
;
2 ꢁ 10ꢀ3 41
;
Third, treatment of (Pod-Dipyrrin)2M(II), where M = Cu or
0.01–0.02;42) may stem in part from different structural features Zn, with EDTA in each case failed to give full reversion to the
of the dipyrrin in question. Some studies concerned the HBr expected (limited) fluorescence of the free base dipyrrin (Fig. 5B
salt of the free base dipyrrin, which may remain partially and 6B). A tentative interpretation is that the M(II)–EDTA
complexed in solution and contribute to the observed proper- complex may associate with the Pod-Dipyrrin and cause
ties. Regardless, the photodynamics of free base dipyrrins were quenching of the free base dipyrrin. Exploring the photophysics
recognized early on to be dominated by internal conversion of the dipyrrins, particularly upon metal complexation,45–49
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remains an area of active investigation, and gaining a deep under- over a range of concentrations but is more extended in the
standing will require further experimental and theoretical work.
organic solvent DMF (Dh = 140 nm) (Fig. 4A and B). In DMF, the
dimeric (Pod-Dipyrrin)2Zn(II) remains intact as evidenced by
absorption and fluorescence spectroscopy (Fig. 7) yet exhibits a
size approximately twice that of the monomer (Dh = 310 versus
2. Polarity-dependent fluorescence probe in the self-
assembled polymer
The fluorescence quantum yields of bis(dipyrrinato)Zn(II) com- 140 nm). While we cannot rule out assemblies due to multimer
plexes bearing a- and/or b-alkyl substituents are known to be aggregation, the cleanliness of the DLS data (Fig. 4A, B, 5C, 8
dependent on solvent polarity.50,51 Such complexes are fluor- and 9) upon adding zinc acetate is consistent with (1) complete
escent in a non-polar solvent such as toluene, but weakly or reaction of F-Ph with 5 to form the Pod-Dipyrrin construct
essentially non-fluorescent in modestly polar organic solvents (no signal corresponding to a monomer remained upon adding
(e.g., dichloromethane). The diminution in fluorescence is excess zinc acetate), and (2) a monomer – dimer system
attributed to an energetically accessible and non-emissive (no signals corresponding to intermediate or larger assemblies were
excited-state charge-transfer process.50,51 The benchmark observed). In DMF, both Pod-Dipyrrin and (Pod-Dipyrrin)2Zn(II)
bis(dipyrrinato)Zn(II) complex 6 also exhibited a decrease in architectures are expected to be extended with exploration of diverse
Ff value with increasing solvent polarity, but the effect was of conformational landscapes. On the other hand, in water, treatment
lesser magnitude over a greater range of polarity: the decline with Zn(II) causes the folded, compact unimeric Pod-Dipyrrin to
was B4.5-fold in total over the increase in polarity from toluene partially unfold upon metal-ion binding and join with another
to DMF (Fig. 2 and Table 1). The benchmark bis(dipyrrinato)Zn(II) unfolded Pod-Dipyrrin to make a dimer. The change in size –
complex 6 lacks a- and b-alkyl substituents; alkyl substituents can from Dh = 15 nm to 260 nm indicates the unimer has largely
profoundly alter the energetics of pyrrole molecules52 and are unfolded. The (Pod-Dipyrrin)2Zn(II) has rather similar size in
similarly expected to influence the energetics of dipyrrin analogues. DMF (Dh B 310 nm) and water (Dh B 260 nm) consistent with a
The solvent-dependence of the fluorescence of bis(dipyrrinato)- largely extended conformation in both solvents, in contrast to
Zn(II) complexes can be exploited as an initial gauge of the polarity the distinct sizes for the free base Pod-Dipyrrin (Dh = 140 nm
of the ‘‘Pod’’. The Ff values of (Pod-Dipyrrin)2Zn(II) in water (0.17) versus 15 nm) in the same solvents in the absence of Zn(II)
and DMF (0.11) compared with 6 in toluene (0.18) and DMF complexation.
(0.041) indicate that in water (Tables
1
and 2), the
Toward the end of this study we returned to examine a
bis(dipyrrinato)Zn(II) complex of (Pod-Dipyrrin)2Zn(II) is in a minor feature of the absorption spectrum of the (Pod-
nonpolar environment comparable to that of toluene. The Dipyrrin)2Zn(II). The absorption spectrum shown in Fig. 5A
hydrophobic environment is attributed to the pendant lauryl exhibits a small shoulder (B517 nm) on the long-wavelength
groups and the polymer backbone in the folded assembly. This side of the main peak, which is not characteristic of typical
agrees with our previously reported several ‘‘Pod-fluorophores’’, bis(dipyrrinato)Zn(II) complexes as illustrated by 6 in diverse
which have Ff values in aqueous solution similar to those of their solvents (Fig. 1 and 2) and the (Pod-Dipyrrin)2Zn(II) in DMF
hydrophobic benchmarks in toluene.2 On the other hand, in DMF (Fig. 7). Illumination into the long-wavelength shoulder did not
the bis(dipyrrinato)Zn(II) complex of (Pod-Dipyrrin)2Zn(II) exhibits result in any distinguishable fluorescence apart from that of the
fluorescence intensity that reflects a modestly polar environment, (Pod-Dipyrrin)2Zn(II). Complexation of Pod-Dipyrrin with Zn(II)
but still less polar than that upon full exposure to bulk DMF. More containing other counterions (chloride, triflate, sulfate) at
in-depth analysis of the local environment will require examination 10 equiv. or 440 equiv. gave the (Pod-Dipyrrin)2Zn(II) with a clean
of solvatochromic chromophores that exhibit changes in spectra absorption spectrum lacking the long-wavelength shoulder and
alone or in conjunction with changes in fluorescence intensity.
also the expected unimeric behavior as assessed by DLS
spectroscopy (Fig. S1, see ESI†). The presence of the long-
wavelength shoulder is tentatively attributed to the effect
3. Self-assembly and mechanical change
Self-assembly processes afford larger architectures the struc- of the acetate counterion (although the molecular origin
tures of which are manifested by the spontaneous interactions remains unknown) and does not alter the conclusions concerning
of the individual constituents.53–58 While self-assembling archi- assembly and disassembly of the fluorogenic bis(dipyrrinato)Zn(II)
tectures offer numerous attractive features, some limitations complex. During the course of these ZnX2 complexation experi-
remained to be addressed. Common methods to characterize ments, two observations were made concerning the size of the
supramolecular assemblies include NMR spectroscopy, mass (Pod-Dipyrrin)2Zn(II) complexes in water (Fig. S1 and S2, ESI†):
spectrometry, and microscopy (if size permits), all of which can (1) the size decreased by 9–30 nm in going from 10 to 440 equiv. of
pose difficulties in interpretation, which is rendered more ZnX2, and (2) the use of 440 versus 10 equiv. of ZnCl2 or Zn(OAc)2
complex when a distribution of species is present in an resulted in slight broadening of the peak observed upon DLS
equilibrium mixture. Moreover, many self-assembly processes examination. The change in size distribution may reflect specific
need to be carried out in organic solution given that most of the effects of counterions as well as a general electrostriction, and
materials are hydrophobic, shutting off many applications in warrants further examination.
life sciences.
The size of the assembled complex (Pod-Dipyrrin)2Zn(II) in
The Pod-Dipyrrin in aqueous solution is folded, compact water or DMF is comparable to or larger than the discrete
and unimeric as evidenced by DLS interrogation (Dh = 15 nm) assemblies derived from metals and organic ligands bearing
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two or three free base dipyrrin ligands.59 In limited studies 52.0 mL, 750 mmol) was degassed with a stream of Ar for
where the Pod-Dipyrrin was titrated with Zn(II), no intermediates 20 min. Trifluoroacetic acid (82.0 mg, 54.0 mL, 700 mmol)
were observed; in other words, the assembly to give the was then added, and the solution was stirred under Ar at
(Pod-Dipyrrin)2Zn(II) was an all-or-nothing phenomena giving room temperature for 15 min. The solution was concentrated
the homoleptic product. In contrast, a dibenzo-annulated and chromatographed [silica, hexanes/CH2Cl2/ethyl acetate
1,9-dicarboethoxydipyrrin (L–H) embedded in a dendrimer (75 : 20 : 5)] to obtain a pale yellow solid (1.58 g, 60% yield):
1
for aqueous solubilization underwent complexation with zinc mp 162–163 1C; H NMR (400 MHz, CDCl3) d 2.06 (s, 6H), 5.90
acetate to give the heteroleptic complex MLX where M = Zn(II) (s, 1H), 5.96–5.98 (m, 2H), 6.17–6.20 (m, 2H), 6.68–6.70 (m, 2H),
and X = acetate; the architecture was proposed as a biosensor for 7.43 (s, 2H), 7.95 (s, 2H); 13C NMR (100 MHz, CDCl3) d 20.5,
detection of Zn(II).60
38.7, 93.2, 107.1, 109.0, 116.8, 130.5, 137.8, 138.4, 140.3; ESI-MS
obsd 377.0495, calcd 377.0509 [(M + H)+, M = C17H17IN2]; anal.
calcd for C17H17IN2: C, 54.27; H, 4.55; N, 7.45. Found: C, 54.09;
H, 4.44; N, 7.27.
Outlook
5-[4-(6-(tert-Butoxycarbonylamino)hexylaminocarbonyl)-2,6-
dimethylphenyl]dipyrromethane (3). Following an established
carbonylation procedure,21,22 a Schlenk flask containing samples
of 2 (188 mg, 500 mmol), Pd(OAc)2 (11.0 mg, 50.0 mmol),
Xantphos (29.0 mg, 50.0 mmol) and cesium carbonate (490 mg,
1.50 mmol) was evacuated and purged with carbon monoxide. A
solution of N-(tert-butoxycarbonyl)-1,6-diaminohexane (216 mg,
1.00 mmol) in toluene (5.00 mL) was purged with Ar (30 min)
and subsequently with carbon monoxide (30 min). The resulting
solution was transferred into the Schlenk flask containing the
solid materials under an atmosphere of CO. The reaction flask
was placed in a preheated oil bath and stirred for 2 h at 90 1C.
The resulting mixture was allowed to cool to room temperature.
The solid material was filtered off and washed with toluene. The
filtrate was concentrated, and the resulting residue was chroma-
tographed [silica, hexanes/ethyl acetate (2: 1)] to obtain a pale
yellow solid (150 mg, 61% yield): mp 55–58 1C; 1H NMR
(300 MHz, CDCl3) d 1.35–1.52 (m, 13H), 1.56–1.66 (m, 4H),
2.13 (s, 6H), 3.11 (q, J = 6.6 Hz, 2H), 3.43 (q, J = 6.6 Hz, 2H),
4.54 (br s, 1H), 5.93–5.95 (m, 2H), 5.97 (s, 1H), 6.17 (q, J = 3.0 Hz,
2H), 6.29 (br s, 1H), 6.68 (dd, J1 = 2.4 Hz, J2 = 4.2 Hz, 2H), 7.43 (s,
2H), 7.98 (s, 2H); 13C NMR (100 MHz, CDCl3) d 20.9, 26.1, 26.3,
28.5, 29.6, 30.1, 38.9, 39.7, 40.2, 79.2, 106.8, 108.6, 116.8, 128.0,
130.5, 133.1, 138.2, 141.5, 156.3, 167.6; ESI-MS obsd 515.2991,
calcd 515.2993 [(M + Na)+, M = C29H40N4O3]; labs (CH2Cl2)
406 nm.
In summary, the single-polymer–single cargo strategy with a
dipyrrin as the cargo has revealed fundamental features concern-
ing the conformational pliability of the amphiphilic polymer. The
presence of a divalent ion (Zn(II), Cu(II)) triggers profound con-
formational change of the polymer–dipyrrin architecture. The
molecular architecture of Pod-Dipyrrin may be unusual – and
with fecund possibilities in supramolecular chemistry – in that
mechanical change prompted by Zn(II) is accompanied by a
striking B50-fold increase in fluorescence. The change in fluores-
cence is visible by the naked eye although the change in absorp-
tion, while measurable by absorption spectroscopy, is far less
evident by visual inspection. A common and versatile approach
for metal ion sensing relies on binding to the lone pair of
electrons on an amino group, thereby suppressing photoinduced
electron transfer to an adjacent chromophore, thereby unleashing
fluorescence.61 The fluorogenic mechanism here, based on dipyrrin
dimerization, is complementary. The bis(dipyrrinato)metal assem-
bly process is robust and can be reversed by metal ion sequestra-
tion. Mechanosensory receptors, which sense mechanical changes
caused by pressure, vibration or electric fields, are well known in
biology.62 Given the importance of mobile Zn(II) as a signaling
molecule in neurophysiology,63–65 the ability to chelate Zn(II), detect
the chelation fluorogenically, and also exert a mechanical change
may enable the design of novel sensory-mechanical constructs for
fundamental manipulations in neurobiology.
5-[4-(6-(tert-Butoxycarbonylamino)hexylaminocarbonyl)-2,6-
dimethylphenyl]dipyrrin (4). Following an established procedure,9
a solution of 3 (98.0 mg, 200 mmol) and p-chloranil (62.0 mg,
250 mmol) in THF (5.00 mL) was sonicated in a laboratory sonica-
tion bath for 1 h. The solution was concentrated and chromato-
graphed [silica, hexanes/ethyl acetate (2 : 1)] to obtain a yellow solid
Experimental procedures
and characterization data
General methods
All chemicals obtained commercially were used as received
unless otherwise noted. Reagent-grade solvents (CH2Cl2, hexanes,
methanol, toluene, ethyl acetate) and HPLC-grade solvents
(toluene, CH2Cl2, hexanes) were used as received. THF was freshly
distilled from sodium/benzophenone ketyl and used immediately.
Electrospray ionization mass spectrometry (ESI-MS) data are
reported for the cationized molecular ion.
1
(68.0 mg, 69% yield): mp 58–60 1C; H NMR (400 MHz, CDCl3) d
1.36–1.55 (m, 15H), 1.66 (qt, J = 7.2 Hz, 2H), 2.18 (s, 6H), 3.16 (q, J =
6.4 Hz, 2H), 3.48 (q, J = 6.4 Hz, 2H) 4.58 (m, 1H), 6.32 (dq, J1 =
1.2 Hz, J2 = 4.8 Hz, 4H), 6.43 (br s, 1H), 7.53 (s, 2H), 7.63 (t, J =
1.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) d 20.2, 26.2, 26.4, 28.6,
29.7, 30.2, 39.8, 40.3, 79.2, 118.2, 125.8, 127.1, 134.6, 137.7, 139.6,
140.0, 144.0, 156.3, 167.8; ESI-MS obsd 491.3013, calcd 41.3017
[(M + H)+, M = C29H38N4O3]; labs (CH2Cl2) 429 nm.
Synthesis
5-(4-Iodo-2,6-dimethylphenyl)dipyrromethane (2). Following an
5-[4-(6-(6-Maleimidohexanoylamino)hexylaminocarbonyl)-2,6-
established procedure,21,22
a
solution of 4-iodo-2,6-dimethyl- dimethylphenyl]dipyrrin (5). A solution of 4 (49.0 mg, 100 mmol)
benzaldehyde (1,12,13 1.82 g, 7.00 mmol) in pyrrole (50.3 g, in CH2Cl2 (1.50 mL) was purged with Ar (for
5
min).
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Trifluoroacetic acid (1.50 mL) was added, and the mixture was examined with Pod-Dipyrrin at B20 mM (A B 1 in a 1 cm
stirred for 12 h at room temperature. The volatile components pathlength cuvette).
(solvent and trifluoroacetic acid) were removed by purging with Ar.
Fluorescence spectroscopy
The resulting residue was treated with HBTU (76.0 mg, 200 mmol),
6-maleimidohexanoic acid (32.0 mg, 150 mmol), CH2Cl2 (5.00 mL) Fluorescence spectra were recorded as described previously2
and N,N-diisopropylethylamine (500 mL) under Ar. The resulting at room temperature using BDPY-hydrazide in methanol
solution was stirred for 2 h at room temperature. The mixture was (Ff = 0.97)2,24 and II in toluene (Ff = 0.36)10 as quantitative
concentrated and chromatographed [silica, ethyl acetate (2 : 1)] to standards with correction for instrument sensitivity but with-
obtain a brown solid (45.0 mg, 77% yield): mp 44–46 1C; 1H NMR out correction for solvent refractive indices. Unless stated
(300 MHz, CDCl3) d 1.26–1.69 (m, 14H), 2.15–2.20 (m, 8H), 3.26 otherwise, samples were examined with Pod-Dipyrrin at
(q, J = 6.6 Hz, 2H), 3.46–3.53 (m, 4H), 5.69 (m, 1H), 6.31–6.35 (m, B2 mM in a 1 cm pathlength cuvette. The typical wavelength
4H), 6.45 (br s, 1H), 6.68 (s, 2H), 7.52 (s, 2H), 7.63 (s, 2H); 13C NMR of excitation of the (Pod-Dipyrrin)2Zn(II) was into the short-
(100 MHz, CDCl3) d 20.3, 25.4, 26.2, 26.3, 26.5, 28.5, 29.7, 29.8, 36.7, wavelength shoulder at B460 nm.
37.8, 39.2, 39.8, 118.3, 125.8, 127.2, 134.3, 134.6, 137.7, 139.7, 140.0,
Dynamic light scattering (DLS) measurements
144.0, 167.9, 171.0, 173.0; ESI-MS obsd 584.3231, calcd 584.3231
[(M + H)+, M = C34H41N5O4]; labs (CH2Cl2) 468 nm.
DLS analysis of the Pod-Dipyrrin was performed in cuvettes
Bis[5-(4-(6-(tert-butoxycarbonylamino)hexylaminocarbonyl)- with a Zetasizer Nano ZS instrument. A typical analysis was as
2,6-dimethylphenyl)dipyrrinato]zinc(II) (6). A solution of 4 follows: a sample was dissolved in HPLC-grade water contain-
(15 mg, 30 mmol) in CHCl3 (2.0 mL) was treated with ing 1.0 M NaCl (filtered with a 220 nm membrane) for analysis,
Zn(OAc)2ꢂ2H2O (33 mg, 150 mmol, 10 equiv.) in methanol in some cases with a preceding step of filtration through a
(0.5 mL). After stirring for 15 h at room temperature, the mixture 220 nm membrane. Illumination was performed at 632.8 nm at
was diluted with CHCl3, washed with water, dried with anhy- room temperature. Unless stated otherwise, samples were
drous sodium sulfate and chromatographed [silica, hexanes/ examined with Pod-Dipyrrin at B20 mM.
ethyl acetate, (1: 1)]. Decomplexation occurred during chromato-
graphy, and the starting material 4 was recovered. The reaction
Nuclear magnetic resonance (NMR) spectroscopy
was repeated in identical fashion with the recovered material. 1H NMR spectra were measured at room temperature.
The mixture was washed with water. The organic layer was
Metal coordination reactions
concentrated to dryness to obtain a red solid (13.6 mg, 85%
1
yield): mp 205 1C (discoloration); H NMR (400 MHz, CDCl3) d The number of equivalents of a metal salt in a coordination
1.40–1.54 (m, 30H), 1.68 (qt, J = 6.8 Hz, 4H), 2.27 (s, 12H), 3.16 reaction of a dipyrrin is described relative to the bis(dipyrrinato)-
(q, J = 6.4 Hz, 4H), 3.50 (q, J = 6.4 Hz, 4H), 4.58 (br s, 2H), 6.37 (d, metal complex; e.g., 5.5 mmol of Zn(OAc)2 and 0.025 mmol of Pod-
J = 4.4 Hz, 4H), 6.43 (br t, J = 4.8 Hz, 2H), 6.52 (d, J = 4 Hz, 4H), Dipyrrin corresponds to 440 equiv. of Zn(OAc)2.
7.49 (br s, 4H), 7.57 (br s, 4H); 13C NMR (100 MHz, CDCl3) d 20.3,
General method for metal coordination reactions of
Pod-Dipyrrin, illustrated with Zn(II) in water
1043.5079, calcd 1043.5096 [(M + H)+, M = C58H74N8O6Zn]; labs A solution of Pod-Dipyrrin in water (1.0 mg mLꢀ1) was mea-
26.2, 26.4, 28.7, 29.9, 30.3, 39.9, 40.4, 79.3, 117.8, 125.7, 131.1,
134.5, 137.6, 139.5, 141.5, 146.9, 149.8, 156.4, 167.9; ESI-MS obsd
(toluene) 487 nm, e488nm = 52900 Mꢀ1 cmꢀ1
.
sured directly, without dilution, by DLS and absorption spectro-
scopy in a 1 cm pathlength cuvette. Then a part of the solution
(B200 mL) was diluted (by B6-fold) by water and transferred
Pod-Dipyrrin
A solution of F-Ph2 (30 mg), 5 (0.53 mg, 0.90 mmol), and into another 1 cm pathlength cuvette. The resulting solution
ethanolamine (0.24 mL) in DMF (400 mL) was treated with measured by absorption and fluorescence spectroscopy. A
triethylamine (1 drop). The resulting mixture was stirred at room solution of Pod-Dipyrrin (1.0 mL, 1.0 mg mLꢀ1) was treated
temperature for 16 h. Then the mixture was diluted with 1.0 mL with Zn(OAc)2 (1.0 mg, 5.5 mmol, B440 equivalent of Pod-
of DMF. The resulting solution was transferred into a dialysis Dipyrrin). The resulting solution was allowed to sit for 5 min
membrane as described in a prior ESI†2 in detail. The solution and measured directly, without dilution, by DLS and absorption
was then dialyzed in DMF to remove the excess fluorophore. The spectroscopy in a 1 cm pathlength cuvette. Then a part of the
dialysis reservoir volume was replaced with fresh DMF four Zn(II)-containing Pod-Dipyrrin solution (B200 mL) was diluted
times over the course of B24 h. The resulting solution was dried (by B6-fold) by water and transferred into another 1 cm
under high vacuum at 50 1C, and the resulting solid was pathlength cuvette. The resulting solution was measured by
dissolved in DI water. The aqueous solution was then freeze- absorption and fluorescence spectroscopy. The Zn(II) contain-
dried to give a pink solid (26 mg). The characterization and ing Pod-Dipyrrin solution (0.8 mL, 1.0 mg mLꢀ1) was treated
properties of the construct are described in the Results section. with EDTA (4.0 mg, 14 mmol, B3.2 equivalent per total Zn(II)).
The resulting solution was allowed to sit for 5 min and
Absorption spectroscopy
measured directly, without dilution, by DLS and absorption
Absorption spectra were recorded at room temperature as spectroscopy in a 1 cm pathlength cuvette. Then a part of the
described previously.2 Unless stated otherwise, samples were EDTA treated Pod-Dipyrrin solution (B200 mL) was diluted
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3 M. Kamigaito, T. Ando and M. Sawamoto, Chem. Rev., 2001,
101, 3689–3745.
(by B6-fold) by water and transferred into another 1 cm
pathlength cuvette. The resulting solution was measured by
absorption and fluorescence spectroscopy.
4 M. Matsumoto, T. Terashima, K. Matsumoto, M. Takenaka
and M. Sawamoto, J. Am. Chem. Soc., 2017, 139, 7164–7167.
5 Y. Bai, H. Xing, G. A. Vincil, J. Lee, E. J. Henderson, Y. Lu,
N. G. Lemcoff and S. C. Zimmerman, Chem. Sci., 2014, 5,
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6 Y. Li, Y. Bai, N. Zheng, Y. Liu, G. A. Vincil, B. J. Pedretti,
J. Cheng and S. C. Zimmerman, Chem. Commun., 2016, 52,
3781–3784.
7 M. Taniguchi, G. Hu, R. Liu, H. Du and J. S. Lindsey, Proc.
S.P.I.E. BiOS, Reporters, Markers, Dyes, Nanoparticles, and
Molecular Probes for Biomedical Applications X, 2018, vol.
10508, p. 1050806.
Metal binding reactions of Pod-Dipyrrin with Cu(II) in water
Following the method describe for the reaction of Pod-Dipyrrin with
Zn(II), a solution of Pod-Dipyrrin in water (1.0 mL, 1.0 mg mLꢀ1) was
treated with CuCl2 (1.0 mg, 7.5 mmol, B600 equivalent). The
resulting solution was measured by DLS, absorption and
fluorescence spectroscopy. Then the resulting solution was
treated with EDTA (4.0 mg, 14 mmol, 2.3 equivalent per total
Cu(II)) and measured by DLS, absorption and fluorescence
spectroscopy.
8 H. Fischer and M. Schubert, Ber. Dtsch. Chem. Ges., 1924, 57,
610–617.
Metal binding reactions of F-Ph with Zn(II) in water
Following the method describe for the reaction of Pod-Dipyrrin with
9 L. Yu, K. Muthukumaran, I. V. Sazanovich, C. Kirmaier,
E. Hindin, J. R. Diers, P. D. Boyle, D. F. Bocian, D. Holten
and J. S. Lindsey, Inorg. Chem., 2003, 42, 6629–6647.
10 I. V. Sazanovich, C. Kirmaier, E. Hindin, L. Yu, D. F. Bocian,
J. S. Lindsey and D. Holten, J. Am. Chem. Soc., 2004, 126,
2664–2665.
Zn(II) in water, a solution of F-Ph in water (1.0 mL, 1.0 mg mLꢀ1
)
was measured by DLS spectroscopy and treated with Zn(OAc)2
(1.0 mg, 5.5 mmol). The resulting solution was then measured by
DLS spectroscopy.
Metal binding reactions of Pod-Dipyrrin with Zn(II) in DMF
11 E. V. Antina, R. T. Kuznetsova, L. A. Antina, G. B. Guseva,
N. A. Dudina, A. I. V’yugin and A. V. Solomonov, Dyes Pigm.,
2015, 113, 664–674.
12 S. Kajigaeshi, T. Kakinami, H. Yamasaki, S. Fujisaki and
T. Okamoto, Bull. Chem. Soc. Jpn., 1988, 61, 600–602.
13 R. W. Wagner, T. E. Johnson and J. S. Lindsey, J. Am. Chem.
Soc., 1996, 118, 11166–11180.
Following the method describe for the reaction of Pod-Dipyrrin
with Zn(II) in water, a solution of Pod-Dipyrrin in DMF (1.0 mL,
1.0 mg mLꢀ1) was measured by DLS, absorption and fluores-
cence spectroscopy. Then the mixture was treated with Zn(OAc)2
(1.0 mg, 5.5 mmol, B440 equivalent). The resulting solution was
measured by DLS, absorption and fluorescence spectroscopy.
14 B. J. Littler, M. A. Miller, C.-H. Hung, R. W. Wagner,
D. F. O’Shea, P. D. Boyle and J. S. Lindsey, J. Org. Chem.,
1999, 64, 1391–1396.
Conflicts of interest
15 J. K. Laha, S. Dhanalekshmi, M. Taniguchi, A. Ambroise and
J. S. Lindsey, Org. Process Res. Dev., 2003, 7, 799–812.
16 N.-F. K. Kaiser, A. Hallberg and M. Larhed, J. Comb. Chem.,
2002, 4, 109–111.
J. S. L. is cofounder of NIRvana Sciences, which develops
fluorophores for use in clinical diagnostics.
Acknowledgements
17 J. Georgsson, A. Hallberg and M. Larhed, J. Comb. Chem.,
2003, 5, 350–352.
The synthesis of compounds 2–6 was supported in 2011–2013
by the Photosynthetic Antenna Research Center (PARC), an
Energy Frontier Research Center funded by the U.S. Depart-
ment of Energy, Office of Science, Office of Basic Energy
Sciences, under Award No. DE-SC0001035. Extension of 5 to
form the Pod-Dipyrrin for fundamental studies was supported
by a grant (1R41GM131501) to NIRvana Sciences from the
NIGMS. Mass spectrometry measurements were carried out in
the Molecular Education, Technology, and Research Innovation
Center (METRIC) at NC State University.
¨
18 X. Wu, R. Ronn, T. Gossas and M. Larhed, J. Org. Chem.,
2005, 70, 3094–3098.
19 O. Lagerlund and M. Larhed, J. Comb. Chem., 2006, 8, 4–6.
20 B. Roberts, D. Liptrot, L. Alcaraz, T. Luker and M. J. Stocks,
Org. Lett., 2010, 12, 4280–4283.
´
21 C. Ruzie, M. Krayer, T. Balasubramanian and J. S. Lindsey,
J. Org. Chem., 2008, 73, 5806–5820.
22 J. R. Martinelli, D. A. Watson, D. M. M. Freckmann,
T. E. Barder and S. L. Buchwald, J. Org. Chem., 2008, 73,
7102–7107.
23 H. L. Kee, C. Kirmaier, L. Yu, P. Thamyongkit, W. J.
Youngblood, M. E. Calder, L. Ramos, B. C. Noll, D. F.
Bocian, W. R. Scheidt, R. R. Birge, J. S. Lindsey and
D. Holten, J. Phys. Chem. B, 2005, 109, 20433–20443.
24 https://www.lumiprobe.com/p/bodipy-fl-hydrazide, acces-
sion date 09/10/2018.
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