Development of improved dual-diazonium reagents for faster crosslinking of tobacco mosaic virus to form hydrogels

Article abstract of DOI:10.1039/c9ra05630k

New bench-stable reagents with two diazonium sites were designed and synthesized for protein crosslinking. Because of the faster diazonium-tyrosine coupling reaction, hydrogels from the crosslinking of tobacco mosaic virus and the reagent DDA-3 could be p

Full text of DOI:10.1039/c9ra05630k

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Development of improved dual-diazonium
reagents for faster crosslinking of tobacco mosaic
Cite this: RSC Adv., 2019, 9, 29070
virus to form hydrogels†
a
bc
Dejun Ma, ‡b Zhuoyue Chen,‡a Long Yi
*
*
and Zhen Xi
New bench-stable reagents with two diazonium sites were designed and synthesized for protein
crosslinking. Because of the faster diazonium-tyrosine coupling reaction, hydrogels from the crosslinking
of tobacco mosaic virus and the reagent DDA-3 could be prepared within 1 min at room temperature.
Furthermore, hydrogels with the introduction of disulfide bonds via DDA-4 could be chemically
degraded by dithiothreitol. Our results provided a facile approach for the direct construction of virus-
based hydrogels.
Received 21st July 2019
Accepted 9th September 2019
DOI: 10.1039/c9ra05630k
rsc.li/rsc-advances
of TMV into the porous alginate hydrogels greatly reduced the
immunogenicity in mice, and the covalent incorporation of
Introduction
tobacco mosaic virus into poly (ethylene glycol) diacrylate
hydrogels increased the stiffness.10 Secondly, the hydrogels are
directly made through chemically crosslinking TMV virus
matrix, which was rarely explored. In our previous work, a TMV-
based hydrogel was prepared by crosslinking TMV with a dual-
diazonium reagent DDA-1 (Scheme 1).11 However, the virus-
based hydrogels should be made within at least 30 min at
37 ꢁC due to the relatively low diazonium-tyrosine (Tyr) coupling
reaction12 between DDA-1 and Tyr residues.
In this work, we designed and synthesized three new dual-
diazonium reagents (DDA-2, DDA-3, DDA-4) to investigate the
effects of substituents (CO, PO, SO2) on the crosslinking effi-
ciency and gelation conditions. To our delight, DDA-3/DDA-4
could quickly crosslink TMV matrix as hydrogels within 1 min at
room temperature. Moreover, the DDA-4-based hydrogel could
Hydrogels are widely used as biomedical materials in
manufacturing cell culture matrices,1 tissue engineering scaf-
folds,2 drug delivery systems3 and wound dressings4 due to their
unique properties, such as water retention capacity, soness,
controllability and biocompatibility.5 One of the main
approaches for preparing hydrogels is the chemical cross-
linking of hydrophilic polymers (natural or synthetic) to provide
desirable mechanical and chemical properties.6 As natural
hydrophilic polymers, plant viruses like tobacco mosaic virus
(TMV, 300 nm ꢀ 18 nm) exhibit a nanoscale size and abundant
amino acid residues on their surface of capsid proteins, which
facilitate facile chemical reactions to produce nanomaterials
with multiple functions.7 Furthermore, plant viruses could be
largely produced in gram-scale quantities and are also consid-
ered much safer to mammals than human-associated viruses
like adenovirus and lentivirus in drug delivery and tissue
engineering.8 Hence, using of plant viruses as building blocks to
assemble virus-based hydrogels is attractive in biomedical
applications.9
Recently, there are mainly two approaches to utilizing plant
viruses to construct hydrogels. Firstly, mixture of TMV with
hydrogels alters their properties. As reported, the incorporation
aState Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical
Technology, Beijing 100029, China. E-mail: yilong@mail.buct.edu.cn
bState Key Laboratory of Elemento-Organic Chemistry, Department of Chemical
Biology, National Pesticide Engineering Research Center (Tianjin), Nankai
University, Tianjin, 300071, China. E-mail: zhenxi@nankai.edu.cn; Tel: +86 22
23504782
cCollaborative Innovation Center of Chemical Science and Engineering, Nankai
University, Tianjin, 300071, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra05630k
Scheme 1 (a) Schematic illustration for crosslinking via TMV and DDA-
1 in our previous work; (b) the chemical structure of new dual-dia-
zonium reagents (DDA-2, DDA-3 and DDA-4).
‡ The authors contribute equally to the work.
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be efficiently degraded with reducing chemicals like dithio- which was indirectly veried that the obtained diazonium salt
threitol (DTT), which enhanced the biocompatibility and was DDA-2 (see the ESI†). As for DDA-3 and DDA-4, the high
1
biodegradability of such hydrogel.
chemical shi values in H NMR compared to their aromatic
amine feedstock also implied the existence of positively charged
diazo moieties. All dual-diazonium hexauorophosphates were
bench-stable and could be stored in the refrigerator (ꢂ20 ꢁC) for
at least two months.
Results and discussion
As shown in Scheme 2, DDA-2 was obtained by nitration,
reduction, diazotization and hexauorophosphoric acid
precipitation from the inexpensive methyl biphenyl phosphine
oxide. The nal product DDA-2 (white solid) was obtained aer
mixing 2 with the concentrated HCl, NaNO2 and HPF6 in one-
pot. DDA-3 (light yellow solid) could be prepared from 4,40-
diaminodiphenyl sulfone in one-pot synthesis. For DDA-4,
cystamine was rstly reacted with 4-acetamidobenzenesulfonyl
chloride to provide 3, which was then hydrolysed with the
concentrated HCl to give 4. Aer treatment of the concentrated
HCl, NaNO2 and HPF6 in one-pot, a yellow solid DDA-4 was
obtained with good yield.
We rstly studied the reaction of DDA-2 or DDA-3 with Tyr
using the UV-Vis spectrophotometry (Fig. 1). The time-
dependent absorption changes at 400 nm implied the forma-
tion of azo bonds11,12 and enabled us to follow the reaction
kinetics conveniently. When the dual-diazonium reagent and
Tyr were mixed under pseudo-rst-order conditions, the
absorption peak at 400 nm was monitored with time. The
pseudo-rst-order rate kobs was determined by tting the data
with a single exponential function. The linear tting between
kobs and Tyr concentrations gave the reaction rate (k2). DDA-2
showed much smaller k2 (18.39 Mꢂ1 sꢂ1) than that of DDA-3
(71.19 Mꢂ1 sꢂ1), implying that the substitution of CO group with
SO2 group greatly enhanced the reaction rate (Fig. 1, S1 and
S2†). Substituents linked to aromatic cycles could affect the
reactivity of diazonium with Tyr due to conjugate and other
effects. Herein, the Hammett equation was further used to
study the relationship between the para and meta substituents
These dual-diazonium reagents (DDA-2, DDA-3 and DDA-4)
were well characterized by 1H NMR, 13C NMR, 31P NMR (see the
1
ESI†). Compared with the chemical shi of 2 in H NMR (less
than 8 ppm), the high chemical shi (9.19, 8.86, 8.63 and 8.17
ppm) of DDA-2 implied the existence of a positively charged
diazonium moiety. Affected by phosphorus, the methyl group in
1
DDA-2 was split into two peaks in H NMR and 13C NMR. The
31P NMR implied the existence of two kinds of phosphorus, in
which the seven peaks were the result of the coupling of six
uorine nucleuses in hexauorophosphate. Since the dual-
diazonium salt was not successfully characterized by mass
spectrum, we tried to react DDA-2 with 4,40-methylenediphenol
1
to get 5. The structure of 5 was clearly conrmed by H NMR,
13C NMR spectra and high-resolution mass spectra (HRMS),
Fig. 1 Kinetic assay of dual-diazonium reagents with Tyr. (a) Azo-
coupling reaction of diazonium reagent with Tyr, R1 ¼ POCH3 (DDA-2),
R2 ¼ SO2 (DDA-3); (b) time-dependent UV-Vis absorbance spectra of
DDA-2 (20 mM) with Tyr (400 mM); (c) time-dependent absorbance at
400 nm of DDA-2 with Tyr; (d) time-dependent UV-Vis absorbance
spectra of DDA-3 (20 mM) with Tyr (300 mM); (e) time-dependent
Scheme 2 The synthesis route for the dual-diazonium reagents DDA- absorbance at 400 nm of DDA-3 with Tyr. The rate constants of the
2 (a), DDA-3 (b) and DDA-4 (c).
azo-coupling reactions are indicated in the inset.
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of benzene derivatives and the reaction rate. In this work, the tests. The results showed that 10 mM DDA-3 could completely
Hammett parameter s13 was obtained by using two kinds of solidify TMV matrix (2.5 mg mLꢂ1) in 1 min at 25 ꢁC. While for
similar structural substituents (Fig. S3†).14 By comparing the DDA-2, the complete gelation should cost at least 15 min under
reactivity of the three diazonium salts, we found that the similar conditions (Fig. S9†). Therefore, we further used DDA-3
crosslinking rate of the diazonium cations and Tyr was higher in following studies. The dose-dependent tests also suggested
with increasing electron withdrawing of the substituent: DDA-3 that 1.25 mM DDA-3 and 1.25 mg mLꢂ1 TMV matrix were the
> DDA-1 > DDA-2. Remarkably, the slope of the linear t was minimum concentration for gelation within 30 min (Fig. 3, S10
positive, demonstrating that the electron-withdrawing substit- and S11†). The high concentration of crosslinker and TMV
uents accelerated the reaction, which was consistent with the matrix like 10 mM DDA-3 and 5 mg mLꢂ1 TMV matrix also
kinetic results. This study might help us to construct more provided the high solidication. These results implied that
efficient multi-diazonium reagents in the future.
improved dual-diazonium reagents could be used for faster
To compare the efficiency of dual-diazonium reagents (DDA-1, crosslinking of tobacco mosaic virus as hydrogels, which
DDA-2, DDA-3) in protein labelling, we rstly used these reagents further simplify the gelation method.
to crosslink the capsid proteins from tobacco mosaic virus (TMV
To further probe the crosslinking efficiency, we subsequently
CP) at 25 ꢁC. The SDS-PAGE assay revealed that DDA-3 had higher determined the rheological characters of the hydrogels formed
crosslinking efficiency than that of DDA-1 and DDA-2 at room by TMV and DDA-2 or DDA-3 from the solution phase to gel
temperature. When the concentration of DDA-3 reached at least phase. As shown in Fig. 4a, the viscosity of both hydrogels
1.25 mM, the percentage of crosslinked capsid proteins could be started to increase aer 100 s, while the hydrogel with DDA-3
more than 85%. At such concentration, DDA-1 and DDA-2 could showed higher viscosity (about 2.5-fold) than that with DDA-2,
just achieve 30–50% protein crosslinking (Fig. 2).
implying that DDA-3 had the higher gelation efficiency. Both the
Subsequently, we used these reagents to crosslink TMV virus hydrogels were solidied aer 400 s. Moreover, we also analysed
at 25 ꢁC and 37 ꢁC. Based on SDS-PAGE analysis, we also found the storage modulus of TMV hydrogels (Fig. 4b) and found that
ꢁ
that the crosslinking efficiency of DDA-3 at 37 C were slightly the hydrogel based on DDA-3 had much faster gelation speed
higher than that at 25 ꢁC while the crosslinking efficiency of with a relatively higher storage modulus than that based on
DDA-1 at 25 ꢁC was signicantly lower than that at 37 ꢁC DDA-2. These results indicate that the improved reagent DDA-3
(Fig. S4–S6†). Furthermore, time-dependent crosslinking of could be used for faster crosslinking of TMV as hydrogels than
TMV also suggested that the reaction could nish within 10 min DDA-2.
for DDA-3 (Fig. S7†) and not nish until 60 min for DDA-1
We further used scanning electron microscope (SEM) to
ꢁ
(Fig. S8†) at 25 C. These results clearly demonstrated that the analyze the structure of TMV hydrogels from 10 mM DDA-2 or
substitution of CO group with SO2 group in the linker reagent DDA-3 and 5 mg mLꢂ1 TMV. SEM results showed multiple virus
greatly enhanced the speed and efficiency of protein cross-
linking and TMV crosslinking at room temperature.
Encouraging with the above results, we then employed these
reagents to crosslink TMV virus matrix to construct hydrogels.
We rstly performed the time- and dose-dependent gelation
Fig. 2 The crosslinking of capsid proteins from tobacco mosaic virus
with three crosslinking reagents. The dual-diazonium reagent (DDA-1, Fig. 3 Dose-dependent TMV gelation. (a) The effect of the concen-
DDA-2 or DDA-3) was firstly dissolved in DMSO and then diluted to tration of DDA-3 on TMV gelation. The final concentration of DDA-3
a gradient concentration (100, 50, 25, 12.5, 6.25, 3.12, 1.56, mM). The was varying from 0–10 mM and the concentration of TMV matrix was
reaction was performed by mixing 9 mL TMV capsid proteins (2 mg mLꢂ1
)
2.5 mg mLꢂ1. (b) The effect of the concentration of TMV matrix on TMV
and 1 mL dual-diazonium reagent with various concentrations. The gelation. The final concentration of TMV matrix was varying from 0–
reaction was incubated at 25 ꢁC for 30 min. After reaction, 10 mL 10 mg mLꢂ1 and the concentration of DDA-3 was 10 mM. The gelation
sample for each treatment was directly used for 12% SDS-PAGE assay. time was kept within 5 min at room temperature.
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Fig. 4 Rheological characters of TMV hydrogels. (a) The viscosity of
TMV hydrogels with DDA-2 or DDA-3 was measured within 400 s at
25 ꢁC at a shear rate of 1 sꢂ1. (b) The storage modulus of TMV hydrogels
with DDA-2 or DDA-3 was measured within 4000 s at 25 ꢁC. The
scanning strain value and the oscillation frequency were set as 1% and
0.1 Hz, respectively.
Fig. 6 TMV hydrogel degradation with dithiothreitol (DTT). The capital
“BUCT” and “NKU” were made from TMV hydrogel crosslinked with
DDA-4 on the Petri dish. After gelation, the TMV hydrogel was
immersed with 20 mL DTT (500 mM) or 20 mL deionized water. The
gel degradation was imaged at different time (0 min, 1 h, 8 h).
rods in DDA-2 constructed hydrogel was crosslinked to form the
3D net structure, while a large number of sheets and less net
structure were observed in DDA-3 constructed hydrogel (Fig. 5).
Considering the importance of biocompatibility and degrad-
ability of hydrogels in applications, we also synthesized DDA-4
through introducing a disulde bond on the basis of DDA-3. The
DDA-4 has similar ability as DDA-3 for TMV gelation (Fig. S9†). We
then used the TMV hydrogels with DDA-4 to make the capital
“BUCT” and “NKU” in the dish (Fig. 6), and determined whether
the hydrogels could be degraded by reducing chemicals like DTT.
Aer the gelation, we immersed the gel in the DTT solution (500
mM) and observed the degradation of the gel within 8 h, sug-
gesting the disulde bond was gradually cleaved by DTT (Fig. 6 and
S12†). On the contrary, the same hydrogel in the dish was not
affected when no DTT was added. We believe that this kind of
disulde bond-based hydrogel could be disrupted by reducing
molecules in vivo, which might add its biocompatibility for
chemically controlled disassembly.15
Conclusions
To improve the reaction rate and gelation efficiency, we devel-
oped new bench-stable reagents with different substituents
(CO, PO, SO2) between two diazonium sites. Through
comparing the reaction rate constant and protein crosslinking
efficiency, we found that DDA-3 greatly improved the cross-
linking efficiency and also shortened the gelation time even at
room temperate, which was a great improvement compared
with DDA-1.11 The SDS-PAGE and SEM characterizations also
conrmed the virus crosslinking via the dual-diazonium
reagents. Furthermore, hydrogels with the introduction of
disulde bonds via DDA-4 could be chemically degraded by
biocompatible reducing molecules. We propose that the new
dual-diazonium reagents may provide a general approach to
preparing diverse functional hydrogels from other biocompat-
ible viruses for future biomedical applications.
Experimental
General
All chemicals and solvents used for synthesis were purchased from
commercial suppliers and applied directly in the experiments
without further purication. The progress of the reaction was
monitored by TLC on pre-coated silica plates (Merck 60F-254, 250
mm in thickness), and spots were visualized by UV light (254 nm).
Merck silica gel (100–200 mesh) was used for general column
chromatography purication. 1H NMR and 13C NMR spectra were
recorded on a Bruker 400 spectrometer. Chemical shis are re-
ported in parts per million relative to internal standard tetrame-
thylsilane (Si(CH3)4 ¼ 0.00 ppm) or residual solvent peaks (DMSO-
d6 ¼ 2.50 ppm, 1H; 39.52 ppm, 13C). 1H NMR coupling constants (J)
are reported in hertz (Hz), and multiplicity is indicated as the
Fig. 5 Characterization of the TMV hydrogel with DDA-2 (a) and DDA-
3 (b) by SEM. Scale bar, 500 mm for full view and 500 nm for the red
rectangular box.
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following: s (singlet), d (doublet), t (triplet), q (quartet), td (triplet slowly added to the mixture at 0 ꢁC. Aer reaction for 1 h, 4 mL
doublet), dd (doublet of doublets), m (multiple). High-resolution 60% HPF6 was added and the mixture was stirred for 1 h. The
mass spectra (HRMS) were obtained on an Agilent 6540 UHD product was collected by ltration and washed with methanol
Accurate-Mass Q-TOFLC/MS. The UV-Visible spectra were recorded and dichloromethane, yielding a yellow solid of DDA-3 (2.01 g,
on a UV-6000 UV-VIS-NIR-spectrophotometer (METASH, China).
77.7%). 1H NMR (400 MHz, DMSO-d6) d 8.94 (d, J ¼ 8.8 Hz, 4H),
8.65 (d, J ¼ 8.8 Hz, 4H); 13C NMR (101 MHz, DMSO-d6) d 147.6,
134.3, 130.6, 122.9; 31P NMR (162 MHz, DMSO-d6) d ꢂ144.19 (h,
JP-F ¼ 711.3 Hz).
Synthesis of DDA-2
Methyl diphenyl phosphine oxide (4.89 g, 22.6 mmol) was dis-
solved in concentrated sulfuric acid (20 mL), and stirred under
ice-cooling to dissolve the solid. A mixture of concentrated
Synthesis of DDA-4
sulfuric acid (20 mL) and concentrated nitric acid (20 mL) was Cystamine dihydrochloride (0.99 g, 4.4 mmol) and 4-acet-
slowly added dropwise with a dropper. Aer the addition was amidobenzenesulfonyl chloride (2.48 g, 10.6 mmolꢁ) was dis-
completed, the ice bath was removed and the mixture was solved in 10 mL DMF. The mixture was stirred at 0 C; DIPEA
stirred at room temperature overnight. The reaction was (3.4 mL, 20.0 mmol) was added into 10 mL DMF and add in
monitored by TLC (CH2Cl2 : MeOH ¼ 19 : 1). The reaction batches with a syringe in portions at 0 ꢁC. The mixture was
mixture was poured into ice water and then the precipitated stirred at room temperature overnight. The solvent was evapo-
solid were collected by ltration. Aer being washed with rated under reduced pressure and the crude was diluted with
saturated aqueous NaHCO3 until neutral pH, the product was 50 mL dichloromethane. The product was collected by ltration
recrystallized with absolute ethanol, yielding a light-yellow solid and washed with dichloromethane to give 3 (1.76 g, 73.1%). 1H
of 1 (4.74 g, 68.5%). 1H NMR (400 MHz, DMSO-d6) d 8.61 (d, J ¼ NMR (400 MHz, DMSO-d6) d 10.32 (s, 1H), 7.78–7.67 (m, 2H),
12.0 Hz, 2H), 8.41 (d, J ¼ 8.1 Hz, 2H), 8.29 (dd, 2H), 7.85 (td, J ¼ 2.98 (q, J ¼ 13.1, 6.5 Hz, 1H), 2.66 (t, J ¼ 6.9 Hz, 1H), 2.08 (s, 1H);
7.9, 2.6 Hz, 2H), 2.31 (d, J ¼ 14.1 Hz, 3H); 13C NMR (101 MHz, 13C NMR (101 MHz, DMSO-d6) d 169.0, 142.8, 133.9, 127.7,
DMSO-d6) d 148.0, 147.8, 137.0, 136.7, 136.6, 136.0, 130.8, 130.7, 118.7, 41.7, 37.0, 24.1.
126.7, 126.7, 125.0, 124.9, 15.6, 14.9.
3 (1.16 g, 2.1 mmol) was dissolved in 40 mL concentrated
ꢁ
Tin II chloride (14.48 g, 64.2 mmol) was dissolved in 40 mL HCl, and the mixture was stirred at 60 C for 4 h. Then it was
concentrated HCl. 1 (3.11 g, 10.1 mmol) was added portionwise collected by ltration and the saturated solution of NaHCO3 was
with stirring and the reaction mixture was reuxed at 80 ꢁC for added until pH 7–8. Next, the mixture was extracted with EtOAc,
2 h. Aer the mixture had cooled to room temperature, the pH and the combined organic extracts were washed with brine,
of the mixture was adjusted to 9–10 by the saturated solution of dried over anhydrous Na2SO4, and evaporated under reduced
NaOH at 0 ꢁC. The mixture was extracted with EtOAc. The pressure. The product was puried by silica gel column chro-
organic phase was washed by brine and dried over anhydrous matography (CH2Cl2 : MeOH ¼ 95 : 5) to give 4 (823 mg, 84.7%).
Na2SO4. Aer removing the solvent under reduced pressure, the 1H NMR (400 MHz, DMSO-d6) d 7.41 (t, J ¼ 5.6 Hz, 1H), 7.29 (t, J
product was puried by silica gel column chromatography to ¼ 5.9 Hz, 1H), 6.63–6.59 (m, 1H), 5.93 (s, 1H), 2.92 (dd, J ¼ 13.7,
give 2 (2.15 g, 86.4%). 1H NMR (400 MHz, DMSO-d6) d 7.11 (td, J 6.2 Hz, 1H), 2.65 (t, J ¼ 7.0 Hz, 1H); 13C NMR (101 MHz, DMSO-
¼ 7.7, 3.6 Hz, 2H), 6.89 (d, J ¼ 13.1 Hz, 2H), 6.78 (dd, J ¼ 11.1, d6) d 152.6, 128.5, 125.2, 112.7, 41.8, 37.0. HRMS (ESI): m/z [M +
7.6 Hz, 2H), 6.67 (d, J ¼ 7.9 Hz, 2H), 5.31 (s, 4H), 1.81 (d, J ¼ H]+ calculated for C16H23N4O4S4+: 463.0597; found: 463.0597.
13.1 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) d 148.8, 148.6,
4 (523 mg, 1.1 mmol) was dissolved in 2ꢁ0 mL concentrated
136.2, 135.2, 129.1, 128.9, 117.1, 117.0, 116.4, 116.4, 115.2, HCl and 20 mL H2O and cooled down to 0 C. The water solu-
115.1, 16.4, 15.6; 31P NMR (162 MHz, DMSO-d6) d 28.63 (s).
tion of NaNO2 (483 mg, 7.0 mmol) was slowly added to the
2 (998 mg, 4.0 mmol) was dissolved in 30 mL cold concen- mixture at 0 ꢁC. Aer reaction for 1 h, 2 mL 60% HPF6 was
trated HCl and cooled down to 0 ꢁC. The water solution of added and the mixture was stirred for 1 h. The product was
NaNO2 (1.82 g, 26.4 mmol) was slowly added to the mixture at collected by ltration and washed with ice-cold water, yielding
1
0 ꢁC. Aer reaction for 30 min, 4 mL 60% HPF6 was added and a yellow solid of DDA-4 (741 mg, 86.3%). H NMR (400 MHz,
the mixture was stirred for 1 h. The product was collected by DMSO-d6) d 8.87 (d, 4H), 8.60 (t, J ¼ 5.6 Hz, 2H), 8.33 (d, 4H),
ltration and washed by ice-cold water, yielding a creamy-white 3.16 (dd, J ¼ 12.6, 6.4 Hz, 4H), 2.75 (t, J ¼ 6.7 Hz, 4H); 13C NMR
1
solid of DDA-2 (1.34 g, 59.8%). H NMR (400 MHz, DMSO-d6) (101 MHz, DMSO-d6) d 150.3, 134.1, 128.7, 120.0, 41.7, 37.1; 31
d 9.19 (d, J ¼ 11.5 Hz, 2H), 8.86 (d, J ¼ 8.2 Hz, 2H), 8.63 (t, 2H), NMR (162 MHz, DMSO-d6) d ꢂ144.19 (h, JP-F ¼ 711.3 Hz).
8.17 (t, J ¼ 7.6 Hz, 2H), 2.36 (d, J ¼ 14.2 Hz, 3H); 13C NMR (101
P
MHz, DMSO-d6) d 141.7, 141.6, 137.3, 136.4, 135.9, 135.1, 135.0,
132.0, 131.9, 118.0, 117.8, 15.5, 14.8; 31P NMR (162 MHz, DMSO-
d6) d 26.52 (s), ꢂ144.20 (h, JP-F ¼ 711.3 Hz).
UV-Vis spectrophotometry assay
All spectroscopic measurements were performed in phosphate-
buffered saline buffer (50 mM, pH 7.4, containing 5% DMSO) in
a 3 mL corvette with 2 mL solution. Compounds were dissolved
into DMSO to prepare the stock solutions with a concentration
Synthesis of DDA-3
4,40-Diaminodiphenyl sulfone (1.16 g, 4.6 mmol) was dissolved of 200 mM Tyr or 20 mM diazonium reagent.
in 20 mL concentrated HCl and 40 mL H2O and cooled down to Reaction of the diazonium reagent (DDA-2 or DDA-3) and
ꢁC. The water solution of NaNO2 (2.07 g, 29.6 mmol) was Tyr-containing molecule (H-Tyr-OBzl Tos): 2 mL DDA-2 or DDA-3
0
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and 4 mL Tyr was added to 1994 mL buffer. The progress of the
TMV virus crosslinking: TMV virus crosslinking was per-
reaction was monitored by UV-Vis spectrophotometry from 250 formed by mixing TMV virus with various concentrations of the
to 600 nm. dual-diazonium reagent (DDA-1, DDA-2, DDA-3). The dual-
Determination of the kinetic parameters of the reaction diazonium reagent was diluted to a gradient concentration
between the diazonium reagent (DDA-2 or DDA-3) and tyrosine: (100, 50, 25, 12.5, 6.25, 3.12, 1.56, mM). The reaction was per-
the reaction rate was measured under pseudo-rst order formed by mixing 9 mL TMV virus (1 mg mLꢂ1) and 1 mL the dual-
conditions, where 20 mM DDA-2 or DDA-3 was mixed with an diazonium reagent (100, 50, 25, 12.5, 6.25, 3.12, 1.56, mM). The
excess of tyrosine (0.1–1 mM) in PBS buffer (50 mM, pH 7.4, reaction was incubated at 25 ꢁC or 37 ꢁC for 30 min or 60 min.
containing 5% DMSO) and the absorption at 400 nm was
recorded per second.
Aer reaction, all samples were mixed with 5ꢀ SDS loading
buffer and used for 12% SDS-PAGE assay. The SDS-PAGE gel was
stained with Coomassie blue R250 and imaged with Quantity
One soware (Bio-Rad).
TMV and TMV CP purication
According to our previous procedure, TMV virus and TMV
capsid proteins were puried.12a
Construction of TMV hydrogel with the dual-diazonium
reagent
TMV purication: TMV-inoculated tobacco leaves were
ground fully and mixed in 25 mL 0.2 M phosphate buffer (pH
7.0) including 250 mL b-mercaptoethanol (1%, v/v) on ice. Aer
the ltration with two-layer screen cloth, the clear lysates were
mixed with n-butyl alcohol (8%, v/v) for 15 min. Aer centrifu-
gation at 10 000 ꢀ g for 20 min, the precipitate was dissolved
again with 2 mL 0.01 M phosphate buffer (pH 7.0) on ice and
stirred for 6 h. Aer centrifugation at 10 000 ꢀ g for 20 min, the
pellet was resuspended in 5 mL 0.01 M phosphate buffer (pH
7.0) for 2 h. Aer centrifugation at 10 000 ꢀ g for 20 min, the
supernatant was then added with NaCl (4%, w/v) and PEG6000
(4%, w/v) for overnight precipitation. Aer centrifugation at
10 000 ꢀ g for 20 min, the precipitate was resuspended in 2 mL
0.01 M phosphate buffer (pH 7.0) for 6 h. Aer centrifugation at
10 000 ꢀ g for 5 min, the nal virus suspension was obtained
and the virus purity was evaluated according to the indicated
ratio of A280/A260 (0.84) and A260/A248 (1.09). TMV concentration
was calculated according to C (mg mLꢂ1) ¼ A260/3.1. TMV
solution was stored at 4 ꢁC for the following test.
To optimize conditions for hydrogel preparation, we tried
different concentrations of TMV matrix (0–10 mg mLꢂ1) and
DDA-3 (0–10 mM). TMV hydrogel was made by gently mixing
different concentrations of DDA-3 and TMV solution in a glass
vial at room temperature for 0–30 min to evaluate the gelation
condition.
For TMV hydrogel with 2.5 mg mLꢂ1 TMV matrix and 10 mM
DDA-2 or DDA-3, DDA-2 or DDA-3 was freshly prepared in DMSO
as 100 mM stock solution. 50 mL of DDA-2 or DDA-3 (100 mM)
was added to 325 mL of 0.1 M phosphate buffer (pH 7.0) to
prepare solution A; 62.5 mL of TMV solution (20 mg mLꢂ1) was
added to 62.5 mL of phosphate buffer to prepare solution B. The
reaction was performed by mixing solution A and solution B.
The gelation of TMV hydrogel was observed within 30 min at
room temperature. It was the same for DDA-4 constructed
hydrogel except that the concentration of DDA-4 and TMV
matrix were 2 mM and 2.5 mg mLꢂ1, respectively.
Scanning electron microscopy (SEM) characterization
Purication of TMV capsid proteins (TMV CP): TMV solution
was rstly diluted to 5 mg mLꢂ1 and then stirred with two TMV hydrogel was made by 5 mg mLꢂ1 TMV and 10 mM DDA-2
ꢁ
volumes of glacial acetic acid for 2 h at 4 C. Subsequently, an or DDA-3 at room temperature for 1 h. Then, the hydrogel was
equal volume of sterile water was added and all the liquid was rstly cold-dried and loaded onto the sample vessel. The sample
then transferred into the dialysis bag. Aer three to ve times of was imaged using a Hitachi S-4700 eld emission SEM.
water exchange, TMV CP precipitated. Aer centrifugation at
12 000 rpm for 30 min, TMV CP precipitate was obtained and
dissolved in 50 mL 0.1 M phosphate buffer (pH 7.0). The
concentration of TMV CP was calculated according to C (mg
mLꢂ1) ¼ A282/1.27. TMV CP was stored at 4 ꢁC for the following
test.
Rheology
All rheological measurements were performed using an
ar2000ex Rheometer from TA Instruments at 25 C. When the
ꢁ
crosslinking reagent and the TMV solution were mixed, the
mixture was quickly added to the mold to start testing before
the hydrogel was completely solidied. TMV hydrogel was made
by 2.5 mg mLꢂ1 TMV and 10 mM DDA-2 or DDA-3. Due to the
faster rate of DDA-3 crosslinking, we formulated this hydrogel
in ice-water bath conditions.
Crosslinking TMV CP and TMV virus with the dual-diazonium
reagent
TMV CP crosslinking: TMV CP crosslinking was performed by
mixing TMV CP with various concentrations of the dual-
diazonium reagent (DDA-1, DDA-2, DDA-3). The dual-
TMV hydrogel degradation
diazonium reagent was diluted to a gradient concentration To degrade virus hydrogel with chemical agents, TMV hydrogel
(100, 50, 25, 12.5, 6.25, 3.12, 1.56, mM). The reaction was per- was made according to the above procedure with DDA-4. The
formed by mixing 9 mL TMV CP (2 mg mLꢂ1) and 1 mL the dual- TMV hydrogel was degraded by dithiothreitol (DTT). We rstly
diazonium reagent (100, 50, 25, 12.5, 6.25, 3.12, 1.56, mM). carved the word “BUCT” and “NKU” on the Petri dish. The
hydrogel was made by transferring the mixture of 2.5 mg mLꢂ1
ꢁ
The reaction was incubated at 25 C for 30 min.
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TMV and 2 mM DDA-4 on the word groove for gelation at room
temperature. Aer gelation, 20 mL DTT (500 mM) as the test
group and 20 mL deionized water as the control group were
transferred into the Petri dish to immerse the TMV hydrogel.
The gel degradation was imaged at different time (0–12 h).
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Conflicts of interest
There are no conicts to declare.
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Acknowledgements
This work was supported by NSFC (21572019) and Beijing
Municipal Natural Science Foundation (2192038).
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RSC Adv., 2019, 9, 29070–29077 | 29077