Phototriggered Dynamic and Biomimetic Growth of Chlorosomal Self-Aggregates

Article abstract of DOI:10.1021/jacs.8b13056

Supramolecular polymerizations mimicking native systems, which are step-by-step constructions to form self-aggregates, were recently developed. However, a general system to successively and spontaneously form self-aggregates from monomeric species remains

Full text of DOI:10.1021/jacs.8b13056

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Communication
Phototriggered dynamic and biomimetic
growth of chlorosomal self-aggregates
Shogo Matsubara, and Hitoshi Tamiaki
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13056 • Publication Date (Web): 09 Jan 2019
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Phototriggered dynamic and biomimetic growth of chlorosomal self-
aggregates
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Shogo Matsubara, and Hitoshi Tamiaki*
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Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
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Supporting Information Placeholder
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(Fig. 1a, left),7,9,12 and the aggregates are tube-like (a 5–30
ABSTRACT: Supramolecular polymerizations mimick-
ing native systems, which are step-by-step construction to
form self-aggregates, were recently developed. However,
a general system to successively and spontaneously form
nm diameter) or lamellar structures.10,11,13 Chlorosomal self-
aggregates were able to be prepared in vitro by self-assembly
of BChl-c, d, or e isolated from natural chlorosomes and
their model molecules such as Zn-HM (Fig. 1a, middle).8,14–
self-aggregates from monomeric species remains chal-
18 However, directly forming a tube-like structure as in a nat-
lenging. Here we report a photoinduced supramolecular
ural chlorosome is difficult because of the spontaneous for-
polymerization system as a biomimetic formation of chlo-
rophyll aggregates which are the main light-harvesting
antennas in photosynthetic green bacteria, called “chloro-
somes”. In this system, inert chlorophyll derivatives were
mation of amorphous particle-like aggregates. The predom-
inant preparation of tube-like chlorosomal aggregates was
required to stand aggregates in a solution for over 1 week as
described in previous reports.14–18 These studies indicated
UV-irradiated to gradually produce active species through
that the conventional method as shown in Fig. 1b preferen-
deprotection. Such active monomers spontaneously as-
tially induced the formation of kinetically trapped metasta-
ble aggregates, whereas tube-like aggregates are thermody-
namically stable.
sembled to form fiber-like chlorosomal self-aggregates in
a similar manner as a dynamic growth of natural chloro-
somal self-aggregates. The study would be useful for elu-
cidation of the formation process of the chlorosomal ag-
gregates and construction of other supramolecular struc-
tures in nature.
Supramolecular polymerization has been investigated in the
last decade for the step-by-step construction of biological
systems.1–5 Most of the supramolecular aggregates were
quickly prepared by dispersing a large amount of self-assem-
bling species (active species) in poor solvents at controlled
temperature and concentration. Particularly, the preparation
of aggregate nuclei known as seeds is important for sponta-
neous supramolecular polymerization.1,3,4 Supramolecular
structures in nature are gradually formed by assembling a
small amount of spatiotemporally biosynthesized active spe-
cies in which seeds are produced in limited amounts.
Here, we report a spatiotemporally controlled supramo-
lecular polymerization system whose conditions are similar
to those used in biosystem preparation; that is based on the
protection of active species and photoinduced deprotection
of the inactive species. Temporally inactive species prepared
by photoremovable protection of active species were illumi-
nated with light to generate self-assembling species (photo-
deprotection) to form its self-aggregates, where an active
monomer was gradually supplied as in native chlorosome
systems. In this study, we focused on constructing a special
light-harvesting antenna in a green photosynthetic bacterium
called “chlorosome”6–13 and mimicked its natural biological
supramolecular system.
Figure 1. Schematic chlorophyll self-assembly. (a) Chemi-
cal structures of natural BChl-c, d, and e molecules (left),
zinc Chl derivative possessing the 3-hydroxymethyl group
Zn-HM as a synthetic model (middle), and its protected
compounds with a photoremovable group Zn-PPGs (right).
(b) Self-assembly of synthetic Zn-HM to form metastable
aggregates. (c) Photoinduced deprotection of Zn-PPGs and
successive self-assembly of Zn-HM to directly form stable
nanofibers with a high-order J-aggregate.
To directly construct tube-like chlorosomal aggregates,
we propose a biomimetic system that gradually supply self-
assembling chlorophyll (Chl) molecules (Fig. 1c) by photol-
ysis of Chl derivatives (Fig. 1a, right) possessing a
A single chlorosome is constructed from J-aggregates of
bacteriochlorophyll-c, d, or e (BChl-c, d, or e) molecules
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photoremovable protecting group. Photoremovable protec-
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green),15,23 demonstrating that Zn-DNB was photo-depro-
tected by UV-irradiation to produce Zn-HM which self-as-
sembled to form chlorosomal self-aggregates (AggUV-1). The
result of reverse-phase high performance liquid chromatog-
raphy (RP-HPLC)-mass spectrometry (MS) analysis also in-
dicated that Zn-DNB was deprotected to generate Zn-HM.
Before photoirradiation, a single HPLC peak of Zn-DNB
was observed at 13.5 min (Fig. 2c, red). During irradiation,
an arising 10.5-min peak was assigned to Zn-HM by com-
parison with the authentic sample (Fig. 2c, black and blue).
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tion has been utilized in various systems including fluores-
cence emission imaging and drug delivery systems.19–22 Be-
cause hydrogen bonding between a hydroxy group at the 31-
position and a keto carbonyl group at the 13-position as well
as coordination of the hydroxy group with a central metal are
essential for construction of in vivo and in vitro chlorosomal
J-aggregates.8,9,11,13 Chl derivatives protected at the 31-hy-
droxy group Zn-PPGs (Zn-ONB, Zn-DNB, and Zn-
NDBF) cannot assemble to form their chlorosomal self-ag-
gregates. Zn-PPGs were irradiated with UV-light to gener-
ate Zn-HM, which could self-assemble to form chlorosomal
aggregates.
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Figure 2. Photoinduced self-assembly of chlorophyll deriv-
atives. (a) UV-vis absorption and CD spectral change of Zn-
DNB (20 µM) in 1%(v/v) EtOH-hexane with UV-irradiation
(red to blue). (b) UV-vis absorption and CD spectra of Zn-
HM (20 µM) in tetrahydrofuran (THF; black) and 1%(v/v)
EtOH-hexane (green). (c) HPLC profiles of Zn-HM (upper),
Zn-DNB before UV irradiation (middle), and Zn-DNB after
UV irradiation for 28 min (lower); HPLC conditions: Cos-
mosil 5C18AR-II 3.0f × 150 mm, acetonitrile : acetone : pyr-
idine : H2O = 75.6 : 18.9 : 0.5 : 5, 1.0 mL/min.
Zn-DNB possessing a dinitrobenzyl group21,22 showed the
highest deprotection yield in the three Zn-PPGs as shown in
Supplementary Information. The photoinduced self-aggre-
gation behaviour of Zn-DNB was investigated using UV-
visible (vis) absorption and circular dichroism (CD) spectra.
Zn-DNB was dissolved in a 1%(v/v) EtOH-hexane solution
to give two sharp peaks called Soret and Qy absorption
bands at 426 and 654 nm, respectively (Fig. 2a, upper, red).
UV-irradiation (250–280 nm) to the Zn-DNB solution trig-
gered the appearance of red-shifted Soret and Qy bands at
450–500 and 735 nm, respectively, where CD signals were
visible (Fig. 2a, gray and blue). The resulting absorption
peaks and CD signals are comparable to those of Zn-HM
self-aggregates (AggHM) in 1%(v/v) EtOH-hexane (Fig. 2b,
Figure 3. AFM images of (a) AggHM just after preparation
and (b) 1 week, and (c) those of AggUV-1 just after UV-
irradiation for 20 min and (d) after standing the solution for
1 day on an HOPG substrate; all the scale bars are 1 µm. (e)
Height profiles along yellow, green, red, and blue lines in
Figs. 3b and 3d. (f) Schematic tube-like monolayer (left) and
bilayer structures (right) of Zn-HM self-aggregates.
To observe the structural morphology of self-aggregates
AggHM and AggUV-1, we analyzed their solids prepared by
drop-casting the sample solutions on a highly oriented
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pyrolytic graphite (HOPG) substrate by atomic force micros-
for 1 day on an HOPG substrate; all the scale bars are 1 µm.
(g) Height profiles along solid (just after preparation; left)
and broken red, green, and blue lines (after 1 day; right) in
Figs. 4a/b, 4c/d, and 4e/f.
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copy (AFM) measurements. Just after preparation, AggHM
was the principal nanoparticles present with 300–600
(NPlarge) and 100–200 nm (NPmedium) diameters (Fig. 3a), and
nanofibers (NFs) were partially observed after standing in
the solution for 1 week (Fig. 3b). This transformation indi-
cated that NP and NF were thermodynamically metastable
and stable aggregates, respectively, which agrees with pre-
vious studies.15 One more month later, numerous NFs were
observed (Fig. S11a), showing that metastable NPs slowly
transformed into more stable NFs. In contrast to AggHM, Ag-
gUV-1 just after preparation (20-min illumination) formed
small nanoparticles (NPsmall) with a <50 nm diameter (Fig.
3c). Interestingly, transformation into NF with an accompa-
nying decrease in NPsmall was observed after standing for 1
day (Fig. 3d), and more NFs were observed after 1 week (Fig.
S11c); this transformation was much faster than that of Ag-
gHM. The diameter of these NFs was 5 or 10 nm (Fig. 3e).
Based on previously reported data for self-aggregates of Zn-
HM,15 these NFs were tube-like monolayer or bilayer aggre-
gates (Fig. 3f).
AggHM was formed by assembling a large number of Zn-
HM molecules simultaneously, while AggUV-1 was gradually
constructed from deprotected Zn-HM by UV-irradiation.
The aforementioned different behaviours of AggHM and Ag-
gUV-1 were attributed to the supply rate of self-assembling
Zn-HM (active spices). To control the rate, the light inten-
sity was changed and the structural morphology of the ob-
tained self-aggregates was investigated by AFM. UV-
irradiation of a Zn-DNB solution with a 2-fold higher light
intensity more rapidly generated Zn-HM aggregates (Ag-
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gUV-2) than the aforementioned photoirradiation experiment
(Figs. S10c and d). AggUV-2 just after 10-min irradiation
formed slightly larger NPs than the corresponding ones in
AggUV-1, to form NPmedium with a 200–300 nm diameter (Fig.
4a). NFs were partially observed in AggUV-2 after 1 day and
showed lower transformation into NFs (Fig. 3b) compared
to AggUV-1. After standing for one more week, numerous
NFs were observed in AggUV-2 (Fig. S11d), and their trans-
formation into NF from NP was faster than that of AggHM
.
UV-irradiation with 0.5-fold intensity for Zn-DNB re-
sulted in slower formation of self-aggregates of Zn-HM (Fig.
S10). Just after 40 min of irradiation, the aggregates(AggUV-
0.5) partially produced NFs (Fig. 4c). Furthermore, reducing
UV-irradiation to a light intensity of 0.25-fold afforded Zn-
HM much more slowly (Fig. S10a) and the aggregates (Ag-
gUV-0.25) were primarily NF forms just after 80-min illumina-
tion (Fig. 4e). After standing for an additional day, numerous
NFs were observed in AggUV-0.5 and AggUV-0.25 (Figs. 4d and
4f). A rapid supply of Zn-HM resulted in promoting the for-
mation of metastable NPs and inhibiting formation of the
thermodynamically stable NFs, but a slow supply promoted
the formation of NFs and inhibited that of NPs. The for-
mation of NFs was largely dependent on the rate of Zn-HM
production. We demonstrated that the spatiotemporal con-
trol of active species resulted in formation of fiber (tube)-
like aggregates mimicking a biological supramolecular sys-
tem such as a chlorosome.
The optical properties of AggUV-1 prepared by photo-
deprotection of Zn-DNB to Zn-HM in 1%(v/v) EtOH-hex-
ane were nearly the same as those of AggHM produced by the
quick addition of hexane to an EtOH solution of Zn-HM
(Figs. 2a and b). This indicated that the local supramolecular
structures were identical. Thermodynamically stable NFs
were likely formed by gradually elongating a small J-aggre-
gate (J-aggregate block), which functioned as a nucleus or
seed for NFs (Fig. 4). In contrast, metastable NPs (NPsmall
,
NPmedium, and NPlarge) were formed by van der Waals and hy-
drophobic interactions between J-aggregate blocks, similar
to a polycrystalline made of a large number of single crystals,
which was prepared by rapid crystal growth.24,25
Figure 4. AFM images of (a) AggUV-2, (c) AggUV-0.5, and (e)
AggUV-0.25 just after irradiation with a UV-light of double,
half, and quarter power comparing to the preparation of Ag-
gUV-1 for 10, 40, and 80 min, respectively, and (b) AggUV-2
,
(d) AggUV-0.5, and (f) AggUV-0.25 after standing in the solution
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Page 4 of 6
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ORCID
2
Hitoshi Tamiaki: 0000-0003-4797-0349
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Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENT
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We thank Prof. Manabu Abe of Hiroshima University for his
valuable advice about photo-removable protecting groups.
This work was supported by JSPS KAKENHI Grant Num-
bers JP18J21058 and JP17H06436.
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Figure 5. Assembly pathways toward nanofibers and nano-
particles. Pathways I and II were characterized by NP and
NF formations, respectively.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on
the ACS Publications website.
General information, synthesis procedures, spectral data,
Figure S1–S11 (PDF).
AUTHOR INFORMATION
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Corresponding Author
tamiaki@fc.ritsumei.ac.jp.
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