Design and synthesis of a highly efficient labelling reagent for incorporation of tetrafluorinated aromatic azide into proteins

Article abstract of DOI:10.1016/j.tet.2019.01.002

Chemical labelling can significantly extend the structures and functions of proteins for advanced applications. Herein, a highly efficient bench-stable reagent diazo-azide was designed and synthesized for the incorporation of tetrafluorinated aromatic azi

Full text of DOI:10.1016/j.tet.2019.01.002

Tetrahedron 75 (2019) 888e893
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of a highly efficient labelling reagent for
incorporation of tetrafluorinated aromatic azide into proteins
,
,
,
,
d
,
, d
Dejun Ma c 1, Xueying Kang a 1, Yasi Gao a, Jiqin Zhu a **, Long Yi a *, Zhen Xi b
a State Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing,
100029, China
b State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, National Pesticide Engineering Research Center (Tianjin),
Nankai University, Tianjin, 300071, China
c State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China
d Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical labelling can significantly extend the structures and functions of proteins for advanced ap-
plications. Herein, a highly efficient bench-stable reagent diazo-azide was designed and synthesized for
the incorporation of tetrafluorinated aromatic azides into proteins via the diazonium coupling. The
diazo-azide-labelled proteins could be further functionalized via the nonhydrolysis Staudinger reaction
to achieve fluorescence labelling, PEGylation and biotinylation. The whole protein labelling processes
were catalysis-free and could be finished within several hours under the mild conditions. To this end, we
have prepared thickened viral nanoparticles with controllable diameters.
Received 12 November 2018
Received in revised form
1 January 2019
Accepted 3 January 2019
Available online 6 January 2019
Keywords:
© 2019 Elsevier Ltd. All rights reserved.
Staudinger reaction
Diazonium salts
Tetrafluorinated aromatic azide
PEGylation
Biotinylation
1. Introduction
proteins is the key step for the bioconjugation. For addressing this
issue, site-specific labelling approaches including the genetic
Strategies for the covalent incorporation of chemicals into pro-
teins are of paramount importance in the fields of chemical biology
and biomaterials [1e5]. Chemical modifications can be used for
manipulating protein functions, protein immobilization or conju-
gation to other molecules, and also increasing protein stability. To
this end, biorthogonal reactions are developed for the efficient
modification of proteins under mild conditions [4]. As usual, the
biorthogonal groups can be firstly incorporated into proteins, and
the second-step biorthogonal reaction provides the efficiency and
flexibility of installing versatile labels into proteins for various ap-
plications [5].
encoding methods and chemo-enzymatic tagging strategies are
developed [1a]. While for simple operation, direct bond-forming
approaches with natural amino acid residues are used, especially
in the case of large-scale preparation. Usually, the thiol, amine and
carboxyl groups of native amino acid residues can be directly
modified [5a]. Furthermore, other sub-abundant residues like
tyrosine (Tyr) and histidine (His) are also used to incorporate bio-
rthogonal groups into proteins [6].
Diazonium reagents have been reported to be highly efficient to
Tyr modification [6e]. However, such strategies are normally based
on in situ production of diazonium ions under acid conditions,
which may affect the structure of proteins and bring difficulty in
controlling the stoichiometry of the reaction. Recently, several
bench-stable diazonium reagents (Scheme 1) were designed and
synthesized for the direct incorporation of aldehyde, ketone or
alkyne groups into proteins, and for protein crosslinking [6d,7].
These reagents can be easily prepared from commercial chemicals,
which should be useful for the large-scale production of labelled
protein materials. As a continuation of our work, herein we report a
new diazonium salt called diazo-azide containing the
However, how to incorporate the biorthogonal groups into
* Corresponding author. State Key Laboratory of Organic-Inorganic Composites,
Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang
District, Beijing, 100029, China.
** Corresponding author.
E-mail addresses: zhujq@mail.buct.edu.cn (J. Zhu), yilong@mail.buct.edu.cn
(L. Yi).
1
The authors contributed equally to this work.
https://doi.org/10.1016/j.tet.2019.01.002
0040-4020/© 2019 Elsevier Ltd. All rights reserved.

D. Ma et al. / Tetrahedron 75 (2019) 888e893
889
(Fig. 1). The structure of 1 was characterized by 1H NMR, 13C NMR,
31P NMR and 19F NMR spectra. The high chemical shift values (8.90
and 8.34 ppm) in 1H NMR implied the existence of the positively
charged diazonium moiety. The 31P NMR and 19F NMR spectra
implied the coexistence of hexafluorophosphate and fluorinated
aromatic moieties. Though we were not successful in directly
obtaining HRMS of 1, the high resolution mass spectra (HRMS) of 1d
and the reaction solution of 1 and Tyr were successfully charac-
terized (see ESI).
The new reagent 1 exhibited the good solubility in PBS buffer
(20 mM, pH 7.4, containing 2% DMSO) (Fig. S1). The UVeVis spectra
(Fig. 2, Fig. S2) of the reaction solution of 1 with Tyr-containing
small molecule (benzyl-Tyr) indicated that the reaction could fin-
ish within 10 min with a kobs up to 4.94 ꢁ 10ꢂ3 sꢂ1 (the k2 ¼ kobs
/
Scheme 1. (a) Schematic illustration of protein Tyr modification via diazonium salt
reagents; (b) chemical structures of our labelling reagents with various biorthogonal
reactive groups.
[benzyl-Tyr] ¼ 24.7 Mꢂ1sꢂ1), implying that the reaction between 1
and Tyr was highly efficient. The expected product 2 from 1 and
benzyl-Tyr was further confirmed by HRMS (Fig. S3). Considering
the possible degradation of the diazonium salts in usage, we also
studied the stability of 1 in aqueous buffer and in solid state. The
results (Figs. S4 and S5) showed that 1 was gradually degraded
within 2 h in PBS buffer, which was much slower than the reaction
time between 1 and Tyr; while 1 was relatively bench-stable at
room temperature for 7 days with slight decomposition. Therefore,
the solid reagent 1 could be stored and transported for extensive
applications in protein labelling.
tetrafluorinated aromatic azide moiety for highly efficient labelling
of generic proteins and tobacco mosaic virus (TMV) under the mild
conditions.
On the other hand, we recently indicated that multi-fluorinated
aromatic azide could significantly enhance the reaction rate for the
Staudinger reaction [8], and discovered that the phosphanimine
intermediates were water-stable for the o,o‘-difluorinated and
o,o‘,m,m‘-tetrafluorinated aromatic azides [8,9], where we named
such reactions as nonhydrolysis Staudinger reactions (NSR). To this
end, we envision that the o,o‘,m,m‘-tetrafluorinated aromatic azide
could be incorporated into proteins via diazonium reagents for the
following NSR labelling.
In this work, we designed and synthesized diazo-azide (For
convenience, we numbered it as 1 in bold) for the incorporation of
tetrafluorinated aromatic azides into proteins and viral nano-
particles. Based on the NSR, proteins labelling of functional groups
(such as fluorophore, polyethyleneglycol (PEG) and biotin) could be
achieved within several hours under mild conditions. This work is
hopeful for further extending the scope of biorthogonal groups in
Tyr modification via diazonium reagents in the fields of protein
labelling.
To determine the selectivity of 1, we continued to detect the
reactivity of reagent 1 toward various amino acids (Figs. S6eS8).
Through comparing the absorbance at 400 nm, we could judge the
formation of azo bonds between 1 and amino acids [7]. The results
showed that 1 could selectively react with Tyr and His within
30 min, which was in accordance with that of diazo-tetrazine [7c].
The strong electron-withdrawing effect of the sulfonyl group in 1
increased the reactivity of diazonium, resulting in similar re-
activities toward Tyr and His (Fig. S8). Hence, the reagent 1 could be
used to label Tyr and His residues of proteins.
Encouraging with above results, we further employed 1 for the
protein covalent labelling. After the first-round labelling, the 1-
labelled proteins could be used for the second-round labelling
through the NSR for fluorescence labelling, PEGylation or bio-
tinylation (Scheme 2). Firstly, after labelling with 1 (1 mM), the
resulted 1-labelled BSA was purified and treated with different
concentrations (0.1 mM or 0.5 mM) of dye 3 for 2 h at room tem-
perature to generate a fluorescently labelled 1-3-BSA, which was
identified by 12% SDS-PAGE (Fig. 3). In the absence of 1, the native
2. Results and discussion
The synthesis route of
1
was shown in Fig. 1. 4-
acetamidobenzene-1-sulfonyl chloride was treated with N-boc-
ethylenediamine and DIPEA in DMF to get a white solid 1a, which
then underwent the deprotection under concentrated HCl at 50 ꢀC
to get 1b. The activated 4-azido-2,3,5,6-tetrafluorobenzoic acid
product 1c could be selectively reacted with the alkylamine group
rather than the aromatic amine group in 1b. Mixture of 1b and 1c at
pH 9e10 led to 1d, which was further treated with HNO2 and then
HPF6 at 0 ꢀC to produce a yellowish precipitate 1 in good yield
Fig. 2. (a) Reaction of 1 and benzyl-Tyr to form the azo product 2; (b) time-dependent
Fig. 1. The synthesis route for the labelling reagent 1. Conditions: 1) N-boc-ethyl-
enediamine, DIPEA, DMF, 73.9%; 2) concentrated HCl, 50 ꢀC; 3) NHS, EDC, DMF, 75.8%;
4) NaHCO3 buffer (200 mM, pH 9e10), 89.9%; 5) HCl, NaNO2, then HPF6, 49.6%.
absorbance spectra of
absorbance at 400 nm of 1 (20
solid line represented the best fitting by a single exponential function.
1
(20
m
m
M) with benzyl-Tyr (200
M) with or without benzyl-Tyr (200
m
M); (c) time-dependent
mM). The black

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D. Ma et al. / Tetrahedron 75 (2019) 888e893
hardly labelled by 4 or 5, implying the present strategy could be
used for the highly-efficient protein PEGylation.
In the next application, we used this strategy to modify the
native virus nanoparticles [11]. Virus-based nanoparticles could be
used as templates for the controllable synthesis of inorganic
nanomaterials [12]. However, the native sizes and shapes of rod-
shape viruses are limited, which may limit the flexibility as tem-
plates in the construction of one-dimensional (1D) nano-
composites. Therefore, we hoped to develop controllable strategies
for tuning the size and surface groups of native viruses. Herein we
chose to label TMV, of which the exterior tyrosine residues could be
accessible by 1 and 6 for the biotinylation of nanorods (Fig. 5a).
Direct mixture of 1 and TMV solution produced 1-TMV, which was
further mixed with 6 (1 mM) at room temperature for 2 h to pro-
duce 1-6-TMV. Due to the possible damage of hydration layer
around TMV surfaces by the labelling, 1-6-TMV was prone to pre-
cipitate in aqueous buffer and could be purified by simple centri-
fugation (Fig. 5b). We further verified that the precipitate was
indeed the TMV nanorods by SDS-PAGE analysis (Fig. S11).
Compared with native TMV capsid protein (TMV-CP), the molecular
weight of the biotinylated TMV CP was increased up to about 1 kDa,
which could be visualized by the slight band shift in PAGE gel
(Fig. 5c) and also mass spectrum assay (Fig. S12). Compared with
the TMV labelling efficiency (about 50%) of Diazo-Tetrazine [7c], the
present strategy could achieve better labelling efficiency of TMV-CP
with totally band shift in contrast with the control (Fig. 5c).
To detect whether this labelling reaction affected the nanorod
structure of TMV, we performed the transmission electron micro-
scope (TEM) tests. As shown in Fig. 5d, the rod morphology was not
disrupted by the labelling, and meanwhile the diameter of 1-6-TMV
Scheme 2. Schematic illustration of protein labelling via the NSR and chemical
structures of the labelling reagents 3e6.
Fig. 3. The fluorescent labelling of BSA proteins using 3. The 12% SDS-PAGE gel for
fluorescence labelling of proteins was firstly imaged by fluorescence scanning (a) and
then stained with Coomassie Brilliant Blue (b). Lane 1: unmodified BSA; lane 2: un-
modified BSA with 0.5 mM dye 3; lane 3: 1-labelled BSA with 0.1 mM dye 3; lane 4: 1-
labelled BSA with 0.5 mM dye 3; lane 5: 1-labelled BSA; M: protein marker (116, 66.2,
45, 35, 25 kDa).
BSA could not be fluorescently labelled by 3, indicating the speci-
ficity and effectiveness of the NSR ligation.
Accordingly, this labelling strategy was also used to introduce
PEG group into proteins. PEGylation was a gentle strategy to
improve the protein stability and reduce immunogenicity [10], and
could be used to directly indicate the protein labelling efficiency by
SDS-PAGE assay. The 1-labelled BSA was treated with phosphorus-
containing PEG2000 (4) or PEG4000 (5) [9a] to achieve PEGylation.
The SDS-PAGE result showed that PEGylations in both 1-4-BSA and
1-5-BSA can be observed because of 100% band shift in contrast
with the unmodified BSA. In the control, the unmodified BSA could
not be directly labelled by only 4 or 5 (Fig. S9), which is in accor-
dance with the above fluorescence labelling. As BSA contained so
many Tyr residues that the number of PEGylations in proteins was
unknown, we next chose proteins without His residues but with
two solvent-exposed Tyr residues (Tyr139 and Tyr2) such as the
capsid protein of tobacco mosaic virus (TMV-CP) [11]. As shown in
Fig. 4 and Fig. S10, an obvious gel shift was observed after the
labelling of 4 or 5 with 1-TMV while the unmodified TMV was
Fig. 5. The biotinylation labelling of TMV using 6. (a) The illustration of biotin-TMV (1-
6-TMV) with the thickened diameter for the exterior labelling of 1 and 6. D: the
diameter of TMV nanorods; (b) Images of 1-6-TMV precipitates after the labelling by 6
(1 mM); (c) The 12% SDS-PAGE gel assay of labelling products of TMV and azido-TMV.
Lane 1: the untreated TMV or azido-TMV; Lane 2: the untreated TMV or azido-TMV
treated with 6 (0.1 mM); Lane 3: the untreated TMV or azido-TMV treated with 6
(1 mM); Protein marker was 116, 66.2, 45, 35, 25, 18.4, 14.4 (kDa). (d) TEM images for
TMV (left) and labelled 1-6-TMV (right). Scale bar, 100 nm.
Fig. 4. The PEGylation labelling of TMV using 4 (a) and 5 (b) at various concentrations
(0.2, 1, 2, 5, mM). The 12% SDS-PAGE gel was stained with Coomassie Brilliant Blue. The
red and black triangle indicated the PEGylated TMV CP with multiple and one modi-
fication, respectively.

D. Ma et al. / Tetrahedron 75 (2019) 888e893
891
rods in the phosphate buffer (100 mM, pH 7.5) was increased from
16.3 1.6 nm to 21.8 3.5 nm (Fig. S13), which was consistent with
biotin-functionalized big disk-like structures having the greater
diameter than the unmodified TMV rods [11b]. Hence, this labelling
strategy could be efficiently used for the surface modification of
viruses without disrupting the nanostructures. Moreover, this
strategy of thickening viral particles would have potential to pro-
vide new nano-templates [12] with controllable diameters which
wide-type virus could not give.
mixture was diluted by water (50 mL) and extracted with EtOAc
(3 ꢁ 20 mL). The combined EtOAc solution was washed by brine
and dried by Na2SO4. After removing the solvent under reduced
pressure, the residue was purified by silica gel column chroma-
tography with CH2Cl2 to get 1c (107 mg, 75.8%). 1H NMR (400 MHz,
CDCl3)
d 2.91 (s, 4H).
1a (400 mg,1.12 mmol) was dissolved in 15 mL concentrated HCl
and heated at 50 ꢀC overnight to get 1b, which was used directly in
following coupling. The pH of the HCl solution was adjusted to 4e5
by the saturated solution of NaOH at 0 ꢀC. Then NaHCO3 was added
to adjust the pH to 9e10, and 1c (446 mg, 1.34 mmol) in 20 mL DMF
was added. The resulted mixture was stirred at room temperature
for 4 h. The mixture was extracted with EtOAc (30 mL ꢁ 3). The
combined EtOAc solution was washed by water and brine and dried
by Na2SO4. After removing the solvent under reduced pressure, the
product was purified by silica gel column chromatography
(MeOH:CH2Cl2 ¼ 3%) to give 1d (435 mg, 89.9%). 1H NMR (400 MHz,
3. Conclusion
In summary, we have designed and synthesized a highly effi-
cient reagent diazo-azide for the direct incorporation of tetra-
fluorinated aromatic azides into proteins and TMV nanorods. In
combination with the nonhydrolysis Staudinger reaction [10], a
facile strategy could be developed for fluorescence labelling,
PEGylation and biotinylation et al. Our protein labelling procedure
was catalysis-free and could be finished within several hours under
the mild conditions. We also prepared the thickened viral particles
as new nano-templates with controllable diameters. This labelling
strategy based on the diazo-azide reagent would provide a useful
toolbox for protein bioconjugation in chemical biology and
biomaterials.
MeOD)
d
8.01 (s, 1H), 7.56 (t, J ¼ 5.6 Hz, 2H), 6.79e6.69 (m, 2H), 3.48
(t, J ¼ 6.5 Hz, 2H), 2.89 (s, 2H). 13C NMR (101 MHz, MeOD)
d 164.8,
160.3, 154.2, 146.2, 143.8, 143.1, 140.5, 130.0, 127.0, 123.2, 114.5,
113.0, 43.0, 41.0, 37.0. HRMS (ESI): m/z [MþH]þ calcd. For
C
15H13F4N6O3Sþ: 433.0700; found: 433.0712.
1d (432 mg, 1 mmol) was dissolved in 2 mL MeOH, and then
20 mL HCl (concentrated HCl:H2O ¼ 2:1) was added and cooled
down to 0 ꢀC. The water solution of NaNO2 (560 mg, 8 mmol) was
slowly added. After reaction for 30 min, 2 mL HPF6 was added and
stirred for 1 h. The precipitate was collected by filtration and
washed by ice-cold water and diethyl ether, yielding a yellowish
4. Experimental section
4.1. General information and methods
solid of
1
(292 mg, 49.6%). 1H NMR (400 MHz, DMSO‑d6)
All chemicals and solvents used for synthesis were purchased
from commercial suppliers and applied directly in the experiment
without further purification. The progress of the reaction was
monitored by TLC on pre-coated silica plates (Merck 60F-254,
d
9.12e8.99 (m, 1H), 8.90 (t, J ¼ 11.1 Hz, 2H), 8.51 (t, J ¼ 5.8 Hz, 1H),
8.34 (d, J ¼ 8.7 Hz, 2H), 3.40e3.28 (m, 2H), 3.09e2.97 (m, 2H). 13
C
NMR (101 MHz, DMSO‑d6)
d 157.3, 150.2, 144.2, 141.7, 141.3, 138.8,
134.1, 128.8, 121.2, 120.1, 115.6, 111.9, 41.7. 31P NMR (162 MHz,
250
mm in thickness), and spots were visualized by UV light. Merck
DMSO‑d6)
DMSO‑d6)
d
ꢂ144.19 (h, JP-F ¼ 710.6 Hz). 19F NMR (376 MHz,
silica gel (100e200 mesh) was used for general column chroma-
tography purification. 1H NMR and 13C NMR spectra were recorded
on a Bruker 400 spectrometer. Chemical shifts were reported in
parts per million relative to internal standard tetramethylsilane
d
ꢂ70.15 (d, JP-F ¼ 710.6 Hz), ꢂ142.59.
Reagent 3. A mixture of NBD-Cl (400 mg, 2 mmol) and 2-
(methylamino) ethan-1-ol (200 L, 2.5 mmol) was dissolved in
20 mL CH2Cl2, and then DIPEA (430 L, 2.5 mmol) was added. The
m
m
(Si(CH3)4 ¼ 0.00 ppm)
or
residual
solvent
peaks
reaction mixture and stirred at room temperature for 2 h. The
mixture was washed by water (3 ꢁ 25 mL) and brine, and dried by
Na2SO4. After removing the solvent under reduced pressure, the
residue was purified by silica gel column chromatography to give a
(DMSO‑d6 ¼ 2.50 ppm, MeOD ¼ 3.31 ppm). 1H NMR coupling con-
stants (J) were reported in Hertz (Hz), and multiplicity is indicated
as the following: s (singlet), d (doublet), t (triplet), dd (doublet
doublet), m (multiple). High-resolution mass spectra (HRMS) were
obtained on a XEVO-G2QTOF (ESI) (Waters, USA) or Bruker Apex IV
FTMS. The UVevisible spectra were recorded on a UV-6000
UVeVis-spectrophotometer (METASH, China).
red solid 3a (138 mg, 29%). 1H NMR (400 MHz, MeOD)
d 8.54 (d,
J ¼ 9.1 Hz, 1H), 6.44 (d, J ¼ 9.1 Hz, 1H), 4.33 (s, 2H), 3.95 (t, J ¼ 5.5 Hz,
2H), 3.62 (s, 3H). 2-(Diphenylphosphanyl) benzoic acid (123 mg,
0.41 mmol), EDC (84 mg, 0.44 mmol) and DMAP (54 mg,
0.44 mmol) were dissolved by CH2Cl2, and then 3a (80 mg,
0.34 mmol) was added. The mixture was stirred at room temper-
ature under nitrogen protection overnight. After removing the
solvent under reduced pressure, the resulting residue was purified
by silica gel column chromatography with CH2Cl2 to get an orange
4.2. Synthesis
Reagent 1. 4-acetamidobenzene-1-sulfonyl chloride (100 mg,
0.43 mmol) was dissolved in DMF (5 mL), and then N-boc-ethyl-
enediamine (81.3
m
L, 0.51 mmol) and DIPEA (72.8
mL, 0.43 mmol)
foam 3 (146 mg, 87%). 1H NMR (400 MHz, CDCl3)
d 8.36 (d,
were added. The mixture was stirred at room temperature over-
night. The mixture was diluted by deionized water (50 mL) and
extracted with EtOAc (3 ꢁ 20 mL). The combined EtOAc solution
was washed by brine and dried by Na2SO4. After removing the
solvent under reduced pressure, the residue was purified by silica
gel column chromatography (MeOH:CH2Cl2 ¼ 5%) to give a white
J ¼ 8.9 Hz, 1H), 7.78 (s, 1H), 7.34 (dt, J ¼ 10.8, 6.1 Hz, 8H), 7.21 (dd,
J ¼ 10.4, 4.9 Hz, 4H), 6.91 (s, 1H), 6.04 (d, J ¼ 9.0 Hz, 1H), 4.52 (t,
J ¼ 5.3 Hz, 2H), 4.37 (s, 2H), 3.35 (s, 3H). 13C NMR (101 MHz, CDCl3)
d
166.4, 145.42 (s), 144.7, 140.97 (s), 140.7, 137.5, 135.2, 134.6, 134.0,
133.8,133.3,133.1,132.6,130.5,128.9,128.6,128.4,123.2,102.0, 62.2,
54.2, 41.9. 31P NMR (162 MHz, DMSO‑d6)
d
ꢂ4.37. HRMS (ESI): m/z
solid 1a (113 mg, 73.9%). 1H NMR (400 MHz, MeOD)
d
7.84e7.73 (m,
[MþH]þ calcd. for C28H24N4O5Pþ: 527.1479; found: 527.1488.
Reagent 6. D-Biotin (293 mg, 1.2 mmol) was dissolved in 5 mL
DMF, and then DMAP (183 mg, 1.5 mmol), EDC (288 mg, 1.5 mmol),
2-(diphenylphosphanyl) phenol (278 mg, 1 mmol) were added. The
mixture was stirred for 4 h under N2 gas. Then the mixture was
diluted by deionized water (50 mL) and extracted with EtOAc
(3 ꢁ 20 mL). The combined EtOAc solution was washed by brine
4H), 3.12 (t, J ¼ 6.3 Hz, 2H), 2.93 (t, J ¼ 6.4 Hz, 2H), 2.18 (s, 3H), 1.43
(s, 9H).
4-azido-2,3,5,6-tetrafluorobenzoic acid (100 mg, 0.43 mmol)
was dissolved in DMF (2 mL), and then 1-hydroxypyrrolidine-2,5-
dione (63.6 mg, 0.55 mmol) and EDC (81.5 mg, 0.43 mmol) were
added. The mixture was stirred at room temperature overnight. The

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D. Ma et al. / Tetrahedron 75 (2019) 888e893
and dried by Na2SO4. After removing the solvent under reduced
pressure, the residue was purified by silica gel column chroma-
tography (MeOH:CH2Cl2 ¼ 4%) to give a white foam 6 (477 mg,
stirred on ice for precipitation of virus particles. After 6 h of stirring
and centrifugation at 10,000ꢁg for 20 min, the precipitate was
resuspended in 5 mL phosphate buffer (0.01 M, pH 7.0). The virus
supernatant was collected by centrifugation at 10,000ꢁg for
20 min, which was further mixed with 0.2 g NaCl (4%, w/v) and 0.2 g
PEG6000 (4%, w/v) overnight on ice for the precipitation. After
centrifugation at 10,000ꢁg for 20 min, the precipitate was dis-
solved again with 2 mL phosphate buffer (0.01 M, pH 7.0) on ice and
stirred for 6 h. After the final centrifugation at 10,000ꢁg for 5 min,
the virus supernatant was collected and characterized by the
absorbance ratio of A280/A260 (0.84) and A260/A248 (1.09). The virus
concentration was calculated by the formula C (mg/mL) ¼ A260/3.1.
TMV was stored at 4 ꢀC for the following labelling.
94.6%). 1H NMR (400 MHz, DMSO‑d6)
d 7.48e7.37 (m, 7H), 7.29e7.17
(m, 6H), 6.79e6.73 (m, 1H), 6.45e6.39 (m, 2H), 4.31 (dd, J ¼ 7.5,
4.9 Hz, 1H), 4.12 (dd, J ¼ 7.7, 4.4 Hz, 1H), 3.05 (dt, J ¼ 8.5, 6.1 Hz, 1H),
2.83 (dd, J ¼ 12.4, 5.1 Hz, 1H), 2.59 (d, J ¼ 12.4 Hz, 1H), 2.27 (t,
J ¼ 7.3 Hz, 2H), 1.63e1.53 (m, 1H), 1.49e1.38 (m, 3H), 1.35e1.22 (m,
2H). 13C NMR (101 MHz, DMSO‑d6)
d 171.4, 163.2, 153.2, 135.7, 134.0,
133.6, 130.7, 129.7, 129.3, 126.8, 123.4, 61.5, 59.7, 55.8, 40.4, 33.6,
28.4, 24.5. 31P NMR (162 MHz, DMSO‑d6)
d
ꢂ17.55.
4.3. UVeVis spectrophotometry assay
PEGylation of TMV: 50
in 100 mM phosphate buffer) was mixed with the 50
m
L 4 or 5 (10 mM, 4 mM, 2 mM, 0.4 mM,
L 1-TMV
The solution was monitored by UVeVis spectrophotometry
from 250 nm to 600 nm in PBS buffer (20 mM, pH 7.4, containing 2%
DMSO). For solubility tests, 1 was dissolved in PBS buffer with
m
solution (2 mg/mL). The mixture was incubated at room tempera-
ture for 2 h. The 1-4-TMV and 1-5-TMV were validated by 12% SDS-
PAGE. The PAGE was stained by Coomassie brilliant blue R250 and
imaged.
different concentrations (50, 100, 150
(20 mM stock solution in DMSO) was added to 1998
solid reagent 1 was stored at room temperature for 1, 4 or 7 day(s),
and then 10 L 1 (20 mM in DMSO) was added to 1990 L buffer.
Reaction of Tyr-containing molecule (benzyl-Tyr) and 1: 2 L 1
(20 mM) and 4 L Benzyl-Tyr (100 mM in DMSO) were added to
1994 L PBS buffer, and the time-dependent spectra were moni-
tored immediately. The amino acid selectivity of the diazonium
reagent 1: 2 L 1 (20 mM) and different amino acids (Arg, Cys, Glu,
His, Lys, Met, Phe, Pro, Ser, Thr, or Tyr, 100 M final concentration)
were added to 1996 L PBS buffer. Each reaction was incubated for
30 min at room temperature. Reaction of 1 and BSA: 2 L 1 (20 mM)
and 200 L BSA (1 mM in H2O) were added to 1798 L PBS buffer.
mM). For stability tests, 2
mL 1
mL buffer, or the
Biotinylation of TMV: The labelling of TMV was performed to
m
m
firstly generate azido-TMV (1-TMV) by mixing 50
mL TMV (2 mg/
m
mL) and 50 L 1 (0.2 mM or 2 mM) in the phosphate buffer
m
m
(100 mM, pH 7.5). The reaction solution was incubated at room
temperature for 2 h and then treated with the ultrafiltration tube
(0.5 mL, 10 kDa cut-off) to remove the excess 1. The 1-TMV was
further diluted into 1 mg/mL with phosphate buffer for the
m
m
m
following biotinylation: 10 mL 6 (10 mM) was mixed with 90 mL 1-
m
TMV solution (1 mg/mL) and incubated for 2 h. The 1-6-TMV pre-
cipitate was isolated by centrifugation at 10,000ꢁg and further
validated by 12% SDS-PAGE.
m
m
m
Then the reaction process was monitored by UVeVis spectropho-
tometry at 400 nm.
4.6. Protein mass assay
4.4. Protein labelling
90 mL 1-6-TMV solution (1 mg/mL) was mixed with glacial acetic
acid and shaken overnight. After centrifugation at 12,000 rpm, the
supernatant was collected and buffer-changed with distilled water
for five times to reduce the salt concentration. The final aliquot was
tested by AutoflexIII LRF200-CID (Bruker Daltonics) for MALDI-TOF
assay.
The protein labelling by 1: the reaction was performed to get
azido-BSA (1-labelled BSA) by mixing 50
mL protein (2 mg/mL BSA)
with 50 L 1 (2 mM) in phosphate buffer (100 mM, pH 7.5). The
m
mixture was incubated at room temperature for 2 h and then
transferred into the ultrafiltration tube (0.5 mL, 10 kDa cut-off). The
excess 1 was removed after five times of phosphate buffer exchange
(100 mM, pH 7.5). The 1-labelled BSA was further diluted into 1 mg/
mL with phosphate buffer for the following labelling.
4.7. Transmission electron microscope (TEM) assay
The 1-6-TMV precipitate was resuspended in phosphate buffer
(100 mM, pH 7.5). A drop of the TMV suspension was loaded onto a
carbon-coated copper grid and removed with the filter paper after
3 min. The copper grid was then immersed with a 2% uranyl acetate
solution (pH 7.5) for 2 min, and the uranyl solution was finally
removed with the filter paper. The 1-6-TMV nanorods was
observed by the JEM-2100F transmission electron microscope at
200 kV. The images were processed with the ImageJ software.
Fluorescence labelling of BSA: 0.1 or 0.5
DMSO) was firstly mixed with 10 L DMSO and then added into
90 L 1-labelled BSA solution (1 mg/mL). The mixture was incu-
mL dye 3 (100 mM in
m
m
bated at room temperature for 2 h. The 1-3-BSA was validated by
12% SDS-PAGE. The PAGE was firstly imaged under UV light and
then stained by Coomassie brilliant blue R250.
PEGylation of BSA: 10
mL 4 or 5 (10 mM in DMSO) was added into
the 90 L 1-labelled BSA solution (1 mg/mL). The mixture was
m
incubated at room temperature for 2 h. The 1-4-BSA and 1-5-BSA
were validated by 12% SDS-PAGE. The PAGE was stained by Coo-
massie brilliant blue R250 and imaged.
Acknowledgments
This work was supported by NSFC (21572019) and Beijing
Municipal Natural Science Foundation (2192038).
4.5. TMV preparation for PEGylation and biotinylation
Appendix A. Supplementary data
TMV virus was extracted and purified according to our previous
method [7a]. Firstly, TMV-infected tobacco leaves were homoge-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2019.01.002.
nized in 25 mL phosphate buffer (0.2 M, pH 7.0, containing 250
mL
b-mercaptoethanol) on ice. After filtration by two-layer cloth, the
filtrate was added with 8% n-butyl alcohol and shaken for 15 min
for the elimination of chlorophyll, which would be followed by
centrifugation at 10,000ꢁg for 20 min. The supernatant was further
mixed with 1 g NaCl (4%, w/v) and 1 g PEG 6000 (4%, w/v) and
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