Development and applications of a near-infrared dye-benzylguanine conjugate to specifically label SNAP-tagged proteins

Article abstract of DOI:10.1039/c7ob01698k

Near-infrared (NIR) fluorescent probes are advantageous over visible ones, for they can avoid the interference from the short-wavelength background emission in biological systems. However, there are a very limited number of NIR probes that can specifically label target proteins in living cells. In this work, a series of long-wavelength dyes (N-NIR, S-NIR, and K-NIR) analogous to the novel Changsha NIR family are synthesized conveniently through a new approach that is different from the previously reported one. These three dyes have similar conjugation structures but exhibit tunable photophysical properties. N-NIR and S-NIR have large extinction coefficients over 100000, and high fluorescence quantum yields. Although NIR absorption and emission of K-NIR are inferior to the former two, it emits in a much longer wavelength region. And all the three dyes can easily pass through the cell membranes to obtain the high-resolution NIR fluorescence images. Furthermore, N-NIR is chosen as the NIR fluorophore to develop a protein-labeling reagent PYBG-D, since it demonstrates the highest fluorescence quantum yield of up to 0.4 (in methanol). PYBG-D is efficiently synthesized through Sonogashira coupling between bromo-substituted N-NIR and alkyne-substituted benzylguanine (PYBG). The conjugate PYBG-D proves to be a specific and efficient label for O⁶-alkylguanine-DNA alkyltransferase (SNAP-tag) that fused to target proteins in living cells, which contributes to high resolution NIR fluorescence images under a laser confocal microscope.

Full text of DOI:10.1039/c7ob01698k

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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Development and Applications of a Near-infrared dye-
Benzylguanine Conjugate to Specifically Label SNAP-tagged
Proteins
Received 00th January 20xx,
Accepted 00th January 20xx
Xinbo Song, a,§ Hui Bian,a,§ Chao Wang,a Mingyu Hu,a Ning Li a and Yi Xiao*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Near-infrared (NIR) fluorescent probes are advantageous over visible ones, for they can avoid the interference from
the short-wavelength background emission in biological systems. However, there are a very limited number of NIR probes
that can specifically label target proteins in living cells. In this work, a series of long-wavelength dyes (N-NIR, S-NIR, and K-
NIR) analogue to the novel Changsha NIRs family are synthesized conveniently through a new approach different from
previously reported one. These three dyes have similar conjugation structures but exhibit tunable photophysical
properties. N-NIR and S-NIR have large extinction coefficients over 100,000, and high fluorescence quantum yields.
Although NIR absorption and emission of K-NIR are inferior to the former two, it emits at much longer wavelength region.
And all the three dyes can easy pass through the cell membranes to obtain the high-resolution NIR fluorescent images.
Further, N-NIR is chosen as the NIR fluorophore to develop protein-labeling reagent PYBG-D, since it demonstrates highest
fluorescence quantum yield up to 0.4 (in methanol). PYBG-D is efficiently synthesized through a Sonogashira coupling
between bromo-substituted N-NIR and alkyne-substituted benzylguanine (PYBG). The conjugate PYBG-D proves to be a
specific and efficient label of for O6-alkylguanine-DNA alkyltransferase (SNAP-tag) that fused to target proteins in living
cells, which contributes to high resolution NIR fluorescence images under
a laser confocal microscope.
been developed recently. Their photophysical properties of
Scheme 1. Synthetic procedures for ChangSha dyes
Introduction
Fluorescence labeling has made a significant contribution to
the advances in cell and molecular biology.1-9 A variety of
fluorescent dyes and probes have been developed to label
different subcellular organelles, such as endoplasmic
reticulum,10 lysosome,4,11 and mitochondrion.12 However,
most of them have the absorption and emission in the visible
range (400-650 nm). The fluorescent dyes or probes with
absorption and emission in the near-infrared (NIR) region (650-
900 nm) are relatively few. When compared to fluorescent
dyes or probes with absorption and emission in the visible
range, the NIR fluorescent dyes or probes have the advantages
of minimum photodamage to biological samples, deep tissue
penetration, and minimum interference from background
auto-fluorescence by biomolecules in the living systems.13-15
Up to now, the common used NIR fluorophores belong to
cyanine dyes, such as Cy5 and Cy7.16-20 But their
photostabilities are unsatisfactory and their fluorescence
quantum yields are suboptimal.21 Some NIR-fluorescent dyes
include Si-rhodamines,22-23 Bodipys,24-26 and squaraines,27 have
these new NIR fluorophores are enhanced to some extent. But
their structures are complex and their synthetic methodologies
are complicated, which make their further derivatization to
obtain probes very challenging. Meanwhile, some NIR dyes
with large conjugation skeletons have poor biological
compatibility. Thus, the number of NIR probes is limited and
their practical applicability needs to be further improved.
There is still an urgent demand to develop more efficient NIR
dyes. In 2011, Lin’s group reported a class of novel NIR
fluorophores named Changsha NIRs (scheme 1A).28 Some of
them exhibit excellent NIR fluorescence which is
recommendable for biological imaging/sensing. However, so
far, only a few derivatives or analogues of Changsha NIRs have
a.State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2
Linggong Road, Dalian 116024, P.R. China E-mail: xiaoyi@dlut.edu.cn
§These authors contributed equally.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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been developed by Lin or others.28 And their applications are Changsha family. Here, to avoid the preparation of
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DOI: 10.1039/C7OB01698K
heterocyclic aldehydes, we decide to utilize easily obtained
confined to small-molecule chemosensors.
Specifically labelling of a target protein in living cells is critical intermediates for condensations, inspired by the syntheses of
for chemical biology and cell biology. In 2003, a powerful some cyanine dyes.37-39 As shown in scheme 1b, new
technology, named SNAP-tag, developed by Johnsson’s group, condensations are successful in the syntheses of different
utilizes the human DNA repair protein O6-alkyguanine-DNA positively hetorocyclic Changsha analogs. The reaction
alkyltransferase (AGT, known as SNAP-tag) to irreversibly conditions are mild and the yields of new dyes are similar to
transfer the alkyl group from O6-[4-aminomethyl-benzyl] Lin’s
approach
using
Fishers’
aldehyde.
guanine derivatives, which opens up the possibility to label a And we also expand the application scope of Changsha NIRs to
single AGT fusion protein with dyes or other functional specifically protein labelling by using a derivative in the design
molecules.29 From then on, there are some dye-alkyguanine of a substrate of genetically encoded SNAP protein tag.
conjugates that have developed and used for SNAP-tag
labelling. But, to our knowledge, extremely few NIR label of
SNAP-tag has been reported.30 7, 31-32
Results and discussion
In this work, we propose a new synthetic approach to the
derivatives/analogues of Changsha NIRs that is practical to
obtain more candidates promising in NIR imaging (scheme 1B).
The previous synthetic approach of Changsha analogues by Lin
et al, which utilizes a readily available aldehyde, e.g. Fisher’s
aldehyde, to condense with another methylene intermediate
activated by pyrylium, as demonstrated in Scheme 1a.
However, we found that this approach might be not so
practical if we hope to get other type of heterocylic Changsha
analogs. This is because, as key intermediates, corresponding
heterocyclic (especially the positively charged ones e.g.
quinolinium and benzothiazolium) aldehydes are difficult to
prepared. The limited source of positively charged heterocylic
Design and Synthesis of NIR-fluorescence DYES: N-NIR, S-NIR, K-
NIR, and PYBG-D.
It is known that the absorption and emission wavelengths of
traditional rhodamine dyes are highly dependent on the
substituents on the amino group. For instance, rhodamines 6G
and B emit at 551 and 572nm.33 And the absorption and
emission wavelengths of traditional cyanine dyes can be
tunable by using different substituents, such as indolium,
thiazolium and quinolinium, this is because these moieties
exert distinct electronic effects within the chromophores.20,34-
36 On the basis of this consideration, we design NIR dyes N-NIR
S-NIR, and K-NIR, for the sake to find out which is the best
,
candidate
for
NIR
imaging.
aldehydes could have restricted the expansion of the novel
Scheme 2. Synthetic procedures for N-NIR, S-NIR, and K-NIR.
Condition: (I) 3-methyl-2-butanone, acetic acid, 90oC, 10h. (II) iodomethane, acetonitrile, reflux, 12h. (III) sodium hydroxide, water, 50oC, 2h. (IV) POCl3, DMF, 0oC for 45min, add d,
stir the mixture at 0oC for 20 min, then stir at 25oC for 12h. (V) triethyl orthoformate, acetic acid, 140oC, overnight. (VI) iodomethane, acetonitrile, reflux, 12h. (VII) iodomethane,
acetonitrile, reflux, 12h. (VIII) e, acetic anhydride, 150oC, 0.5h. (IX) e, acetic anhydride, 150oC, 0.5h. (X) 3-dimethylamino phenol, toluene, under N2, reflux, 3h; cool to 50oC; NaOH,
90oC, 6h; cool to 25oC, HCl. (XI) cyclohexanone, concentrated H2SO4, cool to 0oC; then add j in portions, 90oC, 1.5h; cool to 0oC, the mixture is added to water; add HClO4. (XII) a: k
and d, acetic anhydride, 50oC, 1h. b: k and g, CH2Cl2, CH3OH, acetic anhydride, 50oC, 1h. c: k and i, CH2Cl2, CH3OH, acetic anhydride, 50oC, 1h.
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The syntheses of N-NIR,
S-NIR, and K-NIR are illustrated in at 50oC for 1h), which give the target products S-NIR, and K-
scheme 1. Based on the retrosynthesis analysis, we design a NIR with satisfactory yields. This new approach is efficient and
multi-step synthetic route to N-NIR as outlined. Fisher’s versatile, which should be helpful to expand the diversity of
aldehyde d is synthesized according the literature,34-36 and the analogues to Changsha NIR familiy.
starting materials o-phthalic anhydride is commercial available. In later investigation of spectral properties, we confirm that N-
Compound
j
and
k
are prepared according to the reported NIR is more promising than the other two dyes. So, we choose
condenses with aldehyde d N-NIR as the fluorophore, and modify it by introducing a
procedures.4,28 Intermediate
k
o
under very mild conditions (in acetic anhydride at 50 C within bromo group to obtain Br-N-NIR that can act as a key scaffold
15 min) to give N-NIR as a bright green solid. The synthetic for synthesis of various NIR labels for different targets. Since in
approach (using to aldehyde for condensation reaction) of N- this work we want to label the cells through SNAP-tag fused
NIR is similar to Lin’s Changsha NIRs, but we find this approach proteins, a Sonogashira coupling between Br-N-NIR and a
is not feasible in the preparation of S-NIR, and K-NIR. As a terminal alkyne substituted O6-Benzylguaninethe (PYBG,
solution, we suggest a new approach that adopts g and i taking synthesized according our previous work 5), which produces a
place of aldehyde as condensation components. Intermediate novel conjugate PYBG-D as specific substrate and label of
k condensates with compound g or i under very mild SNAP-tag.
conditions (a mixture of CH2Cl2, CH3OH, and acetic anhydride
Scheme 3. Synthetic procedures for PYBG-D.
Condition: (I) 3-dimethylamino phenol, toluene, under N2, reflux, 3h; cool to 50oC; NaOH, 90oC, 6h; cool to 25oC, HCl. (II) cyclohexanone, concentrated H2SO4, cool to 0oC; then add
j in portions, 90oC, 1.5h; cool to 0oC, the mixture is added to water; add HClO4. (III) k, acetic anhydride, 50oC, 1h. (IV) NaH, 0oC, 15min; propargyl bromide, 0oC→25oC, 1h. (V) 1-
methyl-pyrrolidin, DMF, 50oC, 24h. (VI) 4-[2-propynylmethoxy]-benzyl alcohol, NaH, DMF, 0oC, 10min, then 5 4-dimethylaminopyridine and m are added, 25oC, 1h. (VII) under N2,
PPh3,
Pd[P[C6H5]3]4,
CuI,
DMF
and
trimethylamine,
85oC,
4h
Optical Properties.
The absorption and emission profiles of N-NIR
,
S-NIR, and K-
NIR in different solvents are shown in Figure 1 and the
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corresponding photophysical data are recorded in Tables 1. quantum yield of K-NIR reveals the non-radiatVivieew AdrteiccleaOynlionef
Interestingly, K-NIR exhibit remarkably longer (80-90nm) excitation energy, which is possibly dDuOeI:t1o0.1t0h3e9/Cn7oOnBp01la6n98aKr
absorption and emission than N-NIR and S-NIR. For instance, conjugated structure. The peri-H of quinoline should impose
in CH2Cl2, the absorption and emission of K-NIR are peaked at the steric hindrance effect to the adjacent CH=CH bond; thus,
790 and 810 nm, respectively. The longer wavelength the nonplanar chromophoric system will undergo further
absorption and emission can be ascribed to the fact that K-NIR motions (e.g. twist or torsion) to consume the excitation
possesses a larger conjugated skeleton. If we simply define the energy. Actually, the similar phenomena (weak fluorescence)
conjugated length as the distance between eletron-donating had also been observed to some 4-quinoline cyanine
dimethylamino nitrogen to the positively charged quaternary dyes.37,38,39 The water solubility of N-NIR and S-NIR is tested by
ammonium nitrogen, we just number the atoms and clearly fluorescence studies at different concentrations in ddH2O (SI
know that K-NIR have longer conjugated size. The molar Figure 15 and16). Both of N-NIR and S-NIR are dispersive in
extinction coefficients (ε up to 100000−120000 M−1cm−1) of N- ddH2O at low concentration(S-NIR, <5uM) and aggregate when
NIR and S-NIR are much larger than K-NIR. Also, N-NIR and S- increase the concentration. However, the fluorescence of K-
NIR emit remarkably stronger fluorescence than K-NIR
.
NIR is too weak to be detected accurately in water, so, it’s not
Actually, the fluorescence quantum yields (Φf) of N-NIR and S- meaningful to study K-NIR’s relationship between
NIR are up to 0.4 and 0.21 in CH3OH, which are big values concentration
and
fluorescence
intensity.
among the known NIR functional dyes. The low fluorescence
Figure 1. A: Normalized absorption spectra of N-NIR. B: normalized fluorescence spectra of N-NIR. C: Normalized absorption spectra of S-NIR. D: normalized
fluorescence spectra of S-NIR. E: Normalized absorption spectra of K-NIR. F: normalized fluorescence spectra of K-NIR.
4 | J. Name., 2012, 00, 1-3
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Table 1. Photophysical properties of N-NIR, S-NIR, and K-NIR.
View Article Online
DOI: 10.1039/C7OB01698K
Dyes
Solvent
λabs (nm)
710
ε (M-1 cm-1)
108000
130000
129000
120000
118000
119800
122000
120000
115000
108000
λem (nm)
731
Φf
CH2Cl2
0.38[a]
0.40[a]
0.40[a]
0.36[a]
0.37[a]
0.15[a]
0.21[a]
0.18[a]
0.15[a]
0.10[a]
CH3OH
CH3CH2OH
CH3CN
DMSO
697
720
696
718
N-NIR
695
718
710
732
CH2Cl2
718
737
CH3OH
CH3CH2OH
CH3CN
DMSO
707
728
708
727
S-NIR
703
728
717
739
Not
detected
CH2Cl2
790
765
775
768
778
35650
65360
60000
28000
8000
812
801
798
802
812
Not
detected
CH3OH
CH3CH2OH
CH3CN
DMSO
Not
detected
K-NIR
Not
detected
Not
detected
[a] Reported 3, 5-bi (p–methoxy) phenyl-1, 7-bi (p-bromo) phenyl aza-BODIPY (Φf 0.42, in tolunene) is used as standard.13
Cell imaging applications of N-NIR, S-NIR, and K-NIR.
To confirm the applicability of N-NIR S-NIR, and K-NIR in living
cells, we attempt to label RAW cells and 7702 cells. These dyes
exhibit good cell-membrane permeability and can fast stain
RAW cells and 7702 cells, which is favourable for the high
fluorescence and there is weak fluorescence in the cytoplasmic
matrix. We do not know the reason why K-NIR can label the
nucleus, but we suspect that the structure of quinolone causes
its targetability to nucleus, because some reported dyes can
label nuclei which contain the structure of quinolone.37
Colocalization study of N-NIR and S-NIR with Rh-123
(Commercial mitochondrial probe) have been conducted. As
demonstrated in Figure 4, N-NIR showed red fluorescence in
channel 1 (Figure 4A). At the same time, Rh123 showed bright-
green fluorescence in channel 2 (Figure 4B). The merged image
(Figure 4C) indicates that the two channel images overlapped
very well, confirming that N-NIR can localize in the
mitochondria of living cells. The changes in the intensity profile
,
resolution NIR fluorescent images, as shown in Figure 2. There
is strong fluorescence in the cytoplasmic matrix and there is
very weak fluorescence in the nucleus (Even no fluorescence).
The place where N-NIR and S-NIR aggregated is like organelle
in the cells. K-NIR is also used to label RAW cells. It can pass
though cytomembrane to label the cells. However, its
localization is not same with N-NIR or S-NIR. As is shown in
Figure
3, K-NIR can enter the nucleus to show strong
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of linear regions of interest (ROIs) (Rh123 and N-NIR costaining) colocalization investigation confirmed that N-NVIiRew cAratincle sOtnalinine
tends toward synchronization (Figure 4D). From the intensity mitochondria specially. The similar resuDltOiIs: 1o0b.1t0a3i9n/eCd7OfBr0o1m698SK-
correlation plots (Figure 4E), a high Pearson’s coefficient and NIR
(Figure
5A
,
5B
,
5C,
5D
,
and
5E).
overlap coefficient of 0.915, is obtained. The above
Figure 2. MCF-7 cells are costained with N-NIR and S-NIR. A: 4 μM N-NIR (λex = 635 nm, λem = 650-750 nm, pseudo-color red). B: overlay of the fluorescence N-NIR and Hoechst
33342 (0.5 μM Hoechst 33342, λex = 405 nm, λem = 420-480 nm, pseudo-color green). C: 6 μM S-NIR (λex = 635 nm, λem = 650-750 nm) D: overlay of the fluorescence S-NIR and
hoechst33342 (0.5 μM Hoechst 33342, λex = 405 nm, λem = 420-480 nm).
Figure 3. RAW cells are costained with (A) 8 μM K-NIR (λex = 635 nm, λem = 650-750 nm) and (B) 0.5 μM Hoechst 33342, (λex = 405 nm, λem = 420-480 nm) . C: overlay of A, B and
brightfield .
Figure 4. 7702 cells are co-stained with 4 μM N-NIR (A: λex = 635 nm, λem = 650-750 nm) and 1 μM Rh-123 (B: λex = 488 nm, λem = 500-550 nm) ; C: Overlay of A, B and brightfield; D
Intensity profile of region of interest (ROI) cross 7702 cell; E: Correlation plot of N-NIR and Rh123.
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Figure 5. 7702 cells are co-stained with 4 μM S-NIR (A: λex = 635 nm, λem = 650-750 nm) and 1 μM Rh-123 (B: λex = 488 nm, λem = 500-550 nm) ; C: Overlay of A, B and brightfield; D
Intensity profile of region of interest (ROI) cross 7702 cell; E: Correlation plot of S-NIR and Rh123.
Design PYBG-D to label SNAP-tag proteins and gene encoding cells. analyzed by SDS-PAGE. As is shown in figure
6
, there is a strong
fluorescence band at 21 kDa ascribed to SNAP-tag, which
proves that PYBG-D labelling is successful.
To confirm the labelling reaction between PYBG-D and SNAP-
tag proteins, the covalent labeled SNAP-tag with PYBG-D are
Figure 6. SDS-PAGE analysis of PYBG-D labelled SNAP-tag. A: .The fluorescence image of the gel; B: The coomassie blue image of the same gel.
In order to develop the applicability of PYBG-D, we choose the Next, COS-7 cells are transfected with pSNAPf Vector that
localization of the SNAP-tag to nucleus which is achieved by expresses diffusely in cytoplasm and nucleus. After staining
fusion of SNAP-tag to the C-terminus of the nuclear histone with PYBG-D, high resolution fluorescence images are also
H2B proteins (pSNAPf-H2B, NEB). After transiently transfected obtained (Figure 7C). Again, the cells without expressing SNAP
with the plasmid, the nuclear histone H2B targeted SNAP-tag to be labelled show no fluorescence due to the washing out of
proteins are obtained in live cells. And then PYBG-D is used to the unreacted PYBG-D (Figure 7D).
label nuclear H2B. Fluorescent imaging is performed after Then we choose the cytochrome c oxidase specifically located
washing out the un-reacted dye molecules, which rules out the in the mitochondrial inner membrane as the target protein for
influence of non-specific staining. As shown in Figure 7A and fusion with SNAP-tag. Such a mitochondria-targeting plasmid
7B, the transfected COS-7 cells stained with PYBG-D show pSNAPf-Cox8A is achieved by ligating the cytochrome c oxidase
specific labeling of the nucleus to produce a high resolution submit 8(Cox8A) gene fragment to the upstream of the SNAP
fluorescence image without staining the area outside the coding sequence in the commercially available pSNAPf vector
nucleus. Due to the incompletely transfection, a part of cells (NEB). And the recombinant pSNAPf-Cox8A plasmid is stably
donot express H2B-SNAP, and thus they exhibit no intracellular transfected in COS-7 cells for PYBG-D labelling and
fluorescence. This phenomenon just, from another side, fluorescence
reveals the high specificity of PYBG-D to label intracellular
SNAP tag fused proteins.
imaging.
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Figure 7. The transfected COS-7 cells are co-stained with 5 μM PYBG-D for 1 h; the cells are washed by three times, and then cells are cultured for 1 h (λex = 635 nm, λem = 650-750
nm). A: The transfected cells express pSNAPf-H2B; B: Overlay of A and bright field; C: The transfected cells express pSNAPf; D: Overlay of C and bright field.
Figure 8. The transfected COS-7 cells are stained with 5 μM PYBG-D for 1 h; the cells are washed by three times, and then cells are cultured for 1 h (A:λex = 635 nm, λem = 650-750
nm). And then the cells are stained with 0.5 μM Rh-123 for 5 min, washed three times(B: λex = 488 nm, λem = 500-550 nm) ; C: Overlay of A, B and bright field; D: Correlation plot of
PYBG-D and Rh123
As shown in Figure 8A, the cells transfected with pSNAPf-
COX8A plasmid and stained with PYBG-D show excellent
specific labelling of the mitochondria. To confirm the
Conclusion
subcellular localization of the dyes, a commercially available
mitochondrial dye (Rh123) is employed for the colocalization
In conclusion, we have synthesized a series of NIR fluorescent
dyes, i.e. N-NIR S-NIR, and K-NIR. By proposing a more
,
study. As shown in Figure
fluorescence in channel 1 (Figure 8B). At the same time PYBG-
showed red fluorescence in channel 2 (Figure 8A). The
8, Rh123 showed bright-green
versatile synthetic strategy, we expand the diversity of
analogues to the novel Changsha NIRs family. The new dyes
exhibit tunable emission in the NIR region. For example, in
dichloromethane their emission peaks are at 731, 737, and 812
nm respectively. Importantly, the fluorescent quantum yields
of N-NIR and S-NIR are high, compared with most fluorescent
dyes in the same spectral region. In chloromethane these
values are up to 0.4 and 0.2 respectively. In addition, the dyes
can readily pass through the cell membrane to stain different
subcellular areas. Interestingly, N-NIR and S-NIR can target the
mitochondria and their specific staining produce high
resolution images of mitochondria. K-NIR exhibits a tendency
to be localized in cell nuclei. Considering that N-NIR possesses
the highest fluorescence quantum yield up to 0.4, an aromatic
bromo is introduced to N-NIR to get Br-N-NIR, which makes
the further modifications feasible. Next, using Sonogashira
D
merged image (Figure 8C) indicates that the two channel
images overlapped very well, confirming that PYBG-D can
specially react with fusion proteins expressed in the
mitochondria of living cells. From the intensity correlation
plots (Figure 8D), a high Pearson’s coefficient and overlap
coefficient of 0.84, respectively, are obtained. And the stable
covalent labeling method can be used for the dynamic process
research of mitochondria even at mitochondrial membrane
potential decrease and mitochondrial dysfunction, which
cannot be well revealed by conventional mitochondrial
trackers relying on the mitochondrial membrane potentials.
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coupling reaction between Br-N-NIR and PYBG (a terminal Hyclone). The cells are cultured in a humidified atmosphere of
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alkyne substituted O6-benzylguanine developed by our group), 5% CO2/95% air at 37 °C (CO2 incubator, Thermo Scientific).
DOI: 10.1039/C7OB01698K
a new NIR substrate of SNAP-tag, i.e. PYBG-D, is efficiently Cells were transiently transfected with pSNAPf Vector, pSNAPf-
synthesized with high yield. In the cells transfected with H2B plasmid (NEB), or pSNAPf-COX8A plasmid (the
pSNAPf-H2B, pSNAPf, and pSNAPf-Cox8A, PYBG-D can readily construction was performed as we previously described26) by
and specifically label these SNAP-tag fused proteins of interest using Lipofectamine 2000 (Invitrogen) following the standard
to produce the high resolution NIR images. Thus, the NIR protocol. And stable transfected cells were established by
fluorescent dyes developed in this work demonstrate G418 (Geneticin, Invitrogen) selection. Cells were cultured in
recommendable application properties. To our knowledge, the 35 mm glass bottom dishes (Φ = 20 mm) for 24 h and used for
number of efficient NIR dyes is still very limited, and they have fluorescent imaging.
been rarely applied in genetically encoded fusion protein PYBG-D labels the cells.
labelling.7, 32 To this date, Changsha NIRs analogs/derivatives
The probe PYBG-D is dissolved in DMSO (1 mM) by vortexing.
have yet been adopted in this powerful technology. And we
The stock solution is mixed with culture medium (DMEM with
expect that our new dyes will become more valuable tools for
10% FBS) also by vortexing to obtain the labeling solution. Cells
fluorescent imaging and sensing in cell biology.
cultured in 35 mm glass bottom dishes are stained with the
PYBG-D solution (5 μM) at 37 °C for 60 min and washed with
PBS (3 × 1 mL). After incubation (1 h), the DMEM (10% FBS) is
replaced with fresh medium 30 min before fluorescent imaging.
Experimental
General methods
SDS-PAGE analysis of labeled SNAP-tag protein.
The 500 (1H) MHz NMR and 100 (13C) MHz NMR spectra are
registered at room temperature on a Bruker 500 MHz
spectrometer using perdeuterated solvents as internal
standard. Chromatographic purification is conducted with
silica gel. All solvent mixtures are given as volume/volume
ratios. UV spectra are recorded on TU-1901 UV-Vis
Spectrophotometer and fluorescence spectra are recorded on
a F-7000 spectrometer. Relative quantum efficiencies of
fluorescence of compounds are obtained by comparing the
areas under the corrected emission spectrum of the test
sample in diluted solvents with a reported aza-BODIPY
derivative in toluene which has a quantum efficiency of 0.42
according to the literature.[1] Non-degassed, spectroscopic
grade toluene and a 10 mm quartz cuvette are used. Dilute
solutions (0.01 < A < 0.05) are used to minimize reabsorption
effects. Quantum yields are determined using the equation (1):
The method of labeling purified SNAP-tag proteins (20 μM)
with PYBG-D (10 μM) is the same as that of labeling cells. Then
labeled SNAP-tag proteins (10 μM, 8 μL) are analyzed by 15%
SDS-PAGE. After electrophoresis, the fluorescence of gel is
imaged with a UV transilluminator (GBOX-HR, SYNGENE) with
an ethidium bromide filter. Then the gel is visualized by
staining with Coomassie Blue.
Preparation of a
A solution of phenylhydrazine hydrochloride (30 g, 206mmol)
and 3-methyl-2-butanone (27 ml, 248 mmol) in 500 ml AcOH
are heated to 90oC for 10h. The mixture is distilled under
reduced pressure to yield dark solid. The water (150 ml) is
added and adjust pH to 10. The mixture is extracted with ether,
dried with MgSO4, and concentrated in vacuo to yield a red oil
1
(24 g, 151 mmol, yields 72%). H NMR (400 MHz, CDCl3) δ 7.56
(d, J = 7.6 Hz, 1H), 7.33 (dd, J = 7.5, 1.2 Hz, 1H), 7.32 – 7.29 (m,
1H), 7.22 (dd, J = 7.6, 7.1 Hz, 1H), 2.30 (s, 3H), 1.32 (s, 6H).
Preparation of b, f, and h
(sample)
(standard)
ФF
F(standard)) equation (1)
= Ф(standard) × (Abs
× F(sample)) / (Abs(sample)
×
Methyl iodide (1.5 eq) is added to the solution of 2,3,3-
Where Ф(standard) is the reported quantum yield of the standard,
Abs is the absorbance at the excitation wavelength, F is the
integrated emission spectra.
trimethylindolenine,
2-methylbenzothiazole,
or
4-
methylquinoline in acetonitrile. And the reaction mixture is
heated at reflux for 12 h. The resulting solid is filtered under
vacuum, washed with Et2O and dried to afford pure indolium b,
f, and h. b 1H NMR (400 MHz, DMSO) δ 7.84 – 7.79 (m, 1H),
7.75 – 7.70 (m, 1H), 7.56 – 7.48 (m, 2H), 3.88 (s, 3H), 2.67 (s,
3H), 1.43 (s, 6H). f 1H NMR (400 MHz, DMSO) δ 8.44 (d, J = 8.1
Hz, 1H), 8.29 (d, J = 8.5 Hz, 1H), 7.92 – 7.86 (m, 1H), 7.80 (t, J =
7.7 Hz, 1H), 4.20 (s, 3H), 3.17 (s, 3H). h,1H NMR (400 MHz,
DMSO) δ 9.37 (d, J = 6.0 Hz, 1H), 8.58 – 8.47 (m, 2H), 8.28 (ddd,
J = 8.6, 7.0, 1.2 Hz, 1H), 8.07 (dd, J = 8.7, 7.4 Hz, 2H), 4.59 (s,
3H), 3.01 (s, 3H).
Culture cells.
7702 cells and RAW cells are obtained from Institute of Basic
Medical Sciences (IBMS) of Chinese Academy of Medical
Sciences (CAMS). All cell lines are maintained under standard
culture conditions (atmosphere of 5% CO2 and 95% air at 37 °C)
in RPMI 1640 medium or DMEM medium, supplemented with
10% FBS (fetal bovine serum). The cells is used after the cells in
the exponential phase of growth on 35 mm glass bottom
culture dishes (Φ 20 mm) for 1 day.
Preparation of d34-36
Cells transfected.
A mixture of b (15 g, 50 mmol) and sodium hydroxide (2.3 g, 58
COS-7 cells are purchased from Cell Bank of Type Culture
Collection of Chinese Academy of Sciences. The cells are all
maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco) supplemented with 10% fetal bovine serum (FBS,
mmol) is stirred vigorously at 50oC in 100 ml water for 2 h and
then extracted with dichloromethane. The organic phase is
dried (MgSO4) and then concentrated under reduced pressure
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Journal Name
to obtain the product
c
which is used for the next step without at 90 °C for 1.5 h, cooled down, and poured onto ice (300 g),
View Article Online
DOI: 10.1039/C7OB01698K
further purification. POCl3 (7.5 mL) is dropped to 24 ml cold and HClO4 is added to the solutions and the resulting
DMF under stirring and the mixture is stirred below 10 °C for precipitate is filtered off and washed with cold water (100 mL).
45min. After adding a mixture of
c (12.98 g ) in DMF to the Compound k obtained as a bluish green is used for the next
reaction vessel at this temperature, the reaction solution is step without further purification.1H NMR (400 MHz,
stirred at room temperature for 10h. Then the reaction CDCl3:MeOD=2:1) δ 8.33 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 7.5 Hz,
solution is poured into ice water and pH is adjust to 10 by 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.26 – 7.13 (m, 3H), 6.98 (d, J = 1.9
addition of saturated sodium hydroxide solution. The resulting Hz, 1H), 3.36 (s, 6H), 3.15 (t, J = 6.1 Hz, 2H), 2.32 (t, J = 5.8 Hz,
solution is heated to 60oC for a few minutes and cooled to 2H), 2.02 (d, J = 5.8 Hz, 2H), 1.88 – 1.79 (m, 2H).13C NMR (100
room temperature. The product d is precipitated and filtered MHz, CDCl3:MeOD=2:1) δ 173.98 (s), 170.69 (s), 170.27 (s),
off giving 8.1 g (55%). Red crystals; mp 118 °C.
Preparation of e
163.24 (s), 161.46 (s), 138.21 (s), 137.14 (s), 135.60 (s), 134.32
(s), 133.92 (s), 133.36 (s), 132.23 (s), 125.91 (s), 122.00 (s),
121.40 (s), 99.52 (s), 44.56 (s), 33.38 (s), 28.97 (s), 25.20 (s),
24.79 (s).
A mixture of aniline (11 mL, 120 mmol), triethylorthoformate
(10 mL, 60 mmol) and acetic acid (0.2 ml, 3.3 mmol) is heated
at 140 ˚C overnight. The crude solid is triturated in cold n-
Preparation of N-NIR
pentane and filtered. Then it is washed with n-pentane and Compound d (50 mg, 0.25 mmol) and compound k (130mg,
dried in vacuo to give the crude product as a pale yellow solid 0.37mmol) are dissolved in acetic anhydride (5 mL). The
(9 g, yield: 77%) which is used for next step without further mixture is heated to 50
℃ for 1h. The solvent is removed under
purification.
Preparation of g
reduced pressure to give the crude product, which is purified
by silica gel flash chromatography using CH2Cl2 to
CH2Cl2/CH3OH (200:1 to 20:1) as eluent to afford N-NIR. H-
1
2,3-Dimethylbenzothiazolium iodide (f, 6 g, 20 mmol) and e (8
g, 40 mmol) are suspended in acetic anhydride (30 mL). The
suspension was stirred at 150 °C for 0.5 h. The reaction
mixture is cooled to room temperature. Then the mixture is
distilled under reduced pressure. Ethyl acetate is added to the
residue, and the precipitate is filtered and dried in vacuo to
give g as a dark red powder (4.5 g, 71%). 1H NMR (400 MHz
DMSO) δ 8.79 (d, J = 14.2, 1H), 8.31 (d, J = 22.0, 1H), 8.09
(d, J = 8.3, 1H), 7.80–7.52 (m, 7H), 5.68 (d, J = 13.7, 1H), 3.87 (s,
3H), 2.05 (s, 3H); HRMS (ESI) calc. for C18H17N2OS ([M − I]+)
309.1062, found 309.1073.
NMR (400 MHz, CD3OD) δ 8.70 (d, J = 14.4 Hz, 1H), 8.23 (d, J =
7.0 Hz, 1H), 7.74 (d, J = 4.5 Hz, 1H), 7.67 (d, J = 4.4 Hz, 1H), 7.59
– 7.50 (m, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.29 (dd, J = 24.4, 6.3 Hz,
3H), 6.77 (d, J = 12.9 Hz, 3H), 6.20 (d, J = 15.0 Hz, 1H), 3.65 (s,
3H), 3.16 (s, 6H), 2.69 (s, 2H), 2.36 (s, 2H), 2.12 (d, J = 65.5 Hz,
2H), 1.83 (s, 6H).13C NMR (126 MHz, MeOD) δ 175.52 (s),
164.64 (s), 156.93 (s), 155.59 (s), 144.34 (s), 143.70 (s), 142.40
(s), 137.30 (s), 133.95 (s), 132.33 (s), 130.86 (s), 130.66 (s),
130.46 (s), 129.85 (s), 129.03 (s), 126.40 (s), 123.38 (s), 122.33
(s), 116.56 (s), 114.95 (s), 113.39 (s), 111.96 (s), 100.74 (s),
97.14 (s), 50.59 (s), 40.40 (s), 31.66 (s), 28.66 (s), 27.88 (s),
21.70 (s). m/z (FTMS+p ESI): Calcd [M+]+ for C35H35N2O3:
531.2642, found: 531.2629.
Preparation of i
The method is similar to g. 1H NMR (400 MHz, DMSO) δ 9.15 (d,
J = 6.6 Hz, 1H), 8.88 (d, J = 14.0 Hz, 1H), 8.48 (d, J = 8.9 Hz, 1H),
8.37 – 8.33 (m, 1H), 8.27 (dd, J = 8.6, 7.3 Hz, 1H), 8.16 (s, 1H),
Preparation of S-NIR
8.09 – 8.03 (m, 2H), 7.93 – 7.86 (m, 1H), 7.71 – 7.61 (m, 2H), Compound
7.53 (d, J = 7.3 Hz, 1H), 6.03 (d, J = 14.0 Hz, 1H), 4.57 (s, 3H),
3.00 (s, 3H)
HRMS (ESI) calc. for C20H19N2O ([M - I]+) 303.1497,
found 303.1503.
Preparation of j
g (200 mg, 0.46 mmol) and compound k (318mg,
0.92mmol) are dissolved in a mixture of CH2Cl2 (25 mL), CH3OH
(12 mL), and acetic anhydride (1.5 mL). The mixture is stirred
for 1h at 50oC. The solvent was removed under reduced
pressure to give the crude product, which is purified by silica
gel flash chromatography using CH2Cl2 to CH2Cl2/CH3OH (200:1
to 20:1) as eluent to afford S-NIR (120mg, yields 50%). 1H-
NMR (500 MHz, MeOD) δ 8.21 (d, J = 13.8 Hz, 1H), 7.89 (d, J =
5.9 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.45 (dt, J = 7.0, 6.1 Hz, 4H),
7.30 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.72 (d, J = 9.0
Hz, 1H), 6.50 (dd, J = 9.1, 2.3 Hz, 1H), 6.39 (d, J = 2.1 Hz, 1H),
6.25 (d, J = 13.8 Hz, 1H), 3.63 (s, 3H), 2.86 (s, 6H), 2.49 (s, 2H),
2.36 (s, 1H), 2.23 (s, 1H), 1.73 (s, 1H), 1.60 (s, 1H). 13C NMR
(126 MHz, CDCl3) δ 159.37 (s), 157.66 (s), 145.98 (s), 137.97 (s),
133.98 (s), 133.59 (s), 132.66 (s), 132.07 (s), 129.19 (d, J = 8.2
Hz), 126.28 (s), 125.31 (s), 118.26 (s), 116.36 (s), 115.76 (s),
100.03 (s), 43.89 (s), 36.91 (s), 30.49 (s), 24.36 (s). m/z (TOF
LD+): Calcd [M+] for C32H29N2O3S+: 521.1899, found: 521.1888.
Preparation of K-NIR
;
A solution of 3-dimethylamino phenol (4.11 g, 30.0 mmol) and
phthalic anhydride (4.66 g, 31.5 mmol) in toluene (30 mL) is
refluxed under N2 for 3 h, and cooled to 50–60 °C. Then 30 mL
of 35% aqueous NaOH is added and heated at 90 °C for 6 h.
The resulting mixture was poured into H2O, acidified with HCl,
and allowed to stand at room temperature for 2 h. The
suspension is filtered. Then the solid is recrystallized from a
mixture of water and methanol, and dried to afford the
1
desired product (5.8 g, 69%). H NMR (400 MHz, CDCl3) δ 8.12
(dd, J = 27.4, 8.1 Hz, 2H), 7.97 (s, 1H), 7.58 (s, 1H), 6.91 (d, J =
9.8 Hz, 1H), 6.14 – 6.03 (m, 2H), 3.06 (s, 6H).
Preparation of k28
Freshly cyclohexanone (6.6 mL, 63.7 mmol) is added to
concentrated H2SO4 drop-wisely (7.0 mL) and then cooled
down to 0 °C. Then, compound j (32 mmol) is added in
portions with vigorous stirring. The reaction mixture is heated
The synthetic process is similar to that of compound S-NIR. It is
purified by silica gel flash chromatography using CH2Cl2 to
PAGE 2
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ARTICLE
CH2Cl2/CH3OH (200:1 to 20:1) as eluent to afford K-NIR (130 58 mmol). The mixture is heated at 85 °C for 6 hours, then the
View Article Online
1
DOI: 10.1039/C7OB01698K
mg, yields 55%). H NMR (500 MHz, CDCl3:MeOD=2:1) δ 8.58 temperature is elevated to 120 °C and kept for 5 hours. The
(d, J = 14.3 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.17 (s, 1H), 8.06 (d, solvent is evaporated in vacuo and the resident is dissolved in
J = 7.9 Hz, 1H), 7.91 (t, J = 7.8 Hz, 1H), 7.81 (d, J = 8.9 Hz, 1H), 100 mL methanol. Thionyl chloride (6 mL) was added in drops
7.69 (t, J = 7.7 Hz, 1H), 7.55 (d, J = 4.8 Hz, 1H), 7.05 (d, J = 7.4 within 30 min in ice baths. Then the mixture is heated under
Hz, 1H), 6.92 (d, J = 14.4 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 6.59 (s, reflux for 6 hours. After the methanol is removed, the reaction
1H), 6.25 (d, J = 9.0 Hz, 1H), 4.06 (s, 3H), 2.91 (s, 6H), 2.67 (td, J mixture is washed with a large amount of water for three
= 15.7, 9.2 Hz, 2H), 2.45 (ddd, J = 89.9, 15.3, 8.2 Hz, 2H), 1.94 – times. The aqueous solution is extracted with dichloromethane
1.67 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 176.54 (s), 164.08 (50 mL×3). The combined organic extracts are dried over
(s), 158.74 (s), 156.43 (s), 154.75 (s), 152.61 (s), 146.58 (s), MgSO4 and the solvent is evaporated. The resident is purified
143.02 (s), 142.19 (s), 138.16 (s), 137.34 (s), 133.65 (s), 133.19 by silica gel column chromatography using dichloromethane
(s), 132.63 (s), 132.21 (s), 131.67 (s), 130.85 (s), 129.25 (s), and n-hexane (1.5:1) as eluent to obtain pale-yellow oil. The oil
1
128.93 (s), 125.61 (s), 120.98 (s), 117.80 (s), 114.04 (s), 113.61 is recrystalized in methanol to obtain the solid. H NMR (400
(s), 112.47 (s), 100.58 (s), 46.06 (s), 43.68 (s), 28.81 (s), 24.51 MHz, CDCl3) δ 8.20 (d, J = 1.9 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H),
+
(s). m/z (TOF LD+): Calcd [M+]+ for C34H31N2O3 : 515.2335, 7.76 (dd, J = 8.1, 2.0 Hz, 1H), 7.69 (dd, J = 8.4, 2.0 Hz, 1H), 7.55
found: 515.2322.
(d, J = 1.9 Hz, 1H), 7.29 (d, J = 2.7 Hz, 1H), 6.90 (dd, J = 9.1, 1.3
Hz, 2H), 6.19 (d, J = 2.4 Hz, 2H), 6.11 (ddd, J = 9.3, 7.1, 2.5 Hz,
2H), 3.75 (s, 3H), 3.74 (s, 2H), 3.07 (s, 4H), 3.07 (s, 6H).13C NMR
(100 MHz, CDCl3) δ 197.59 (S), 196.80 (S), 165.69 (S), 165.18 (S),
165.11 (S), 156.07 (S), 156.03 (S), 142.25 (S), 139.38 (S), 135.06
(S), 134.02 (S), 133.99 (S), 133.21 (S), 132.37 (S), 131.83 (S),
130.90 (S), 130.66 (S), 129.47 (S), 127.58 (S), 127.18 (S), 123.25
(S), 110.15 (S), 110.00 (S), 104.16 (S), 104.06 (S), 97.81 (S),
52.63 (S), 52.48 (S), 39.99 (S).The solid (1.5 g, 4.0 mmol) is
dissolved in methanol (20 mL), 10% aq. NaOH (0.2 g, 5 mmol)
and refluxed for 3 h. The methanol is evaporated. Water (30
mL) is added and the solution is acidified to pH 1.5 with 5% aq.
H2SO4. The pale-yellow precipitate is filtered, washed with
Preparation of l5
It is synthesized according to the literature procedures.5 1,4-
benzenedimethanol (3 g, 21.71 mmol ) is dissolved in 10 mL
dry DMF, and NaH (1g) is added in small portions over 10 min
at 0°C . The mixture is stirred for 15min. Propargyl bromide
(2.32 g 19.54 mmol) is added and the mixture is stirred for 1h
at 25°C . The mixture is quenched with water, extracted with
CH2Cl2, and purified by flash column chromatography with
CH2Cl2 to get 4-[prop-2-ynyloxymethy]-benzyl alcohol (2.68g,
15.21mmol, 70%).
Preparation of m 29
It is synthesized according to the literature procedures.29 6-
chloro-guanine (0.5g , 5.9 mmol)) is dissolved in 15 ml DMF at
50°C. After cooling to room temperature, 1-methyl-pyrrolidin
(1.4 ml, 13.2 mmol) is added and the reaction mixture is stirred
for 24 h. Then 2 ml of acetone is added to complete
precipitation. The solid is filtered, washed with ether and dried
in vacuo, yielding 0.55 g product ( 2.16 mmol, 71%).
Preparation of PYBG
water and dried in vacuo to give r
. 1H NMR (400 MHz, CDCl3) δ
8.24 (d, J = 1.9 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.78 (dd, J = 8.1,
2.0 Hz, 1H), 7.69 (dd, J = 8.4, 2.0 Hz, 1H), 7.53 (d, J = 1.9 Hz, 1H),
7.26 (d, J = 8.1 Hz, 1H), 6.89 (dd, J = 9.1, 1.8 Hz, 2H), 6.20 –
6.16 (m, 2H), 6.15 – 6.07 (m, 2H), 3.07 (s, 5H), 3.07 (s, 6H). 13C
NMR (100 MHz, CDCl3) δ 197.29 (s), 196.46 (s), 169.22 (s),
168.58 (s), 165.23 (s), 156.18 (s), 156.15 (s), 142.75 (s), 139.82
(s), 135.67 (s), 134.14 (s), 133.96 (s), 132.59 (s), 132.42 (s),
4-[2-Propynylmethoxy]-benzyl alcohol (662 mg, 7.33 mmol) is 131.09 (s), 129.63 (s), 128.04 (s), 126.46 (s), 123.24 (s), 110.12
dissolved in 3 mL dry DMF, and NaH (65 mg, 2.83 mmol) is (s), 109.96 (s), 104.27 (s), 104.18 (s), 97.80 (s), 40.00 (s).
added in small portions over 10 min at 0oC. And then the Preparation of s28
mixture is stirred for 15 min at 0oC. 1-(2-Amino-7H-purin-6-yl)-
1-methyl-pyrrolidinium chloride (m, 300 mg, 1.18 mmol) and
4-dimethylaminopyridine (30 mg, 0.25 mmol) are added and
the mixture is stirred for 1h at 25°C . The mixture is quenched
with 0.5 mL water and purified by flash column
The synthetic process is similar to that of compound k.1H NMR
(400 MHz, CDCl3:MeOD=2:1) δ 8.45 (d, J = 1.9 Hz, 1H), 8.20 (d,
J = 8.4 Hz, 1H), 7.92 (ddd, J = 25.9, 7.4, 5.5 Hz, 2H), 7.40 (d, J =
1.8 Hz, 1H), 7.24 – 7.13 (m, 5H), 6.96 (dd, J = 4.1, 2.3 Hz, 2H),
3.36 (d, J = 3.8 Hz, 12H), 3.13 (dd, J = 17.4, 11.2 Hz, 4H), 2.38 –
2.26 (m, 4H), 2.02 (s, 4H), 1.89 – 1.79 (m, 4H).13C NMR (100
MHz, CDCl3:MeOD=2:1) δ 174.23(s), 169.36(s), 166.42(s),
163.18(s), 161.45(s), 140.13(s), 139.92(s), 138.60(s), 137.55(s),
137.13(s), 136.93(s), 135.24(s), 134.96(s), 133.90(s), 133.67(s),
128.45(s), 125.76(s), 122.38(s), 122.21(s), 121.35(s), 110.78(s),
99.66(s), 44.71(s), 33.42(s), 29.04(s), 25.17(s), 24.75(s).
Preparation of Br-N-NIR
chromatography (gradient: CH2Cl2/CH3OH 40:1→15:1) to yield
1
255 mg
n
(0.83 mmol, 72%). mp 111.1-112.9°C. H NMR (400
MHz, DMSO) δ 11.60 (s, 1H), 6.99 (s, 1H), 6.64 (d, J = 7.8 Hz,
2H), 6.50 (d, J = 7.8 Hz, 2H), 5.45 (s, 2H), 4.63 (s, 2H), 3.68 (s,
2H), 3.33 (s, 2H), 2.65 (s, 1H). 13C NMR (100 MHz, DMSO) δ
159.6 (s) , 137.4 (s), 136.2 (s) , 128.4 (s) , 127.8 (s) , 80.1 (s) ,
77.5 (s), 70.4 (s) , 66.4 (s) , 56.9 (s) . m/z (FTMS+p ESI): Calcd
[M+H]+ for C16H16N5O2: 310.1299, found : 310.1295.
Preparation of r
r is synthesized according to the literature procedures.40
4-Bromophthalic anhydride (q, 12 g, 53 mmol) is added to dry
120 mL toluene which contains 3-dimethylamino phenol (8.0 g,
The synthetic process is similar to that of compound N-NIR. It
is purified by silica gel flash chromatography using CH2Cl2 to
CH2Cl2/CH3OH (200:1 to 20:1) as eluent to afford Br-N-NIR
(120 mg, yields 50%). 1H NMR (400 MHz, CD3OD) δ 8.71 (d, J =
10.8 Hz, 1H), 8.22 (d, J = 7.2 Hz, 1H), 7.75 (d, J = 4.6 Hz, 1H),
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View Article 2O0nl1in3e,
13. Zhang, X. and Xiao, Y.; The Journal of Organic Chemistry
7.70 – 7.62 (m, 1H), 7.55 (d, J = 7.0 Hz, 1H), 7.44 (d, J = 7.0 Hz,
1H), 7.36 – 7.21 (m, 3H), 6.77 (d, J = 11.9 Hz, 3H), 6.21 (d, J =
15.3 Hz, 1H), 3.65 (s, 3H), 3.16 (s, 6H), 2.69 (s, 2H), 2.36 (s, 2H),
1.82 (s, 8H). 13C NMR (126 MHz, MeOD) δ 175.15 (s), 164.75 (s),
156.95 (s), 155.57 (s), 144.30 (s), 143.52 (s), 142.21 (s), 135.73
(s), 135.44 (s), 135.01 (s), 132.05 (s), 129.96 (s), 129.32 (s),
126.45 (s), 124.20 (s), 123.41 (s), 122.56 (s), 116.80 (s), 115.03
(s), 113.54 (s), 111.89 (s), 100.55 (s), 97.22 (s), 50.55 (s), 40.77
(s), 31.91 (s), 29.07 (s), 27.98 (s), 21.71 (s). m/z (TOF LD+):
78 (18), 9153-9160.1408
14. Kiyoseꢀ, K. and Naganoꢀ, T., ChemistDryOI–: 10A.1n03A9s/iCa7nOBJ0o1u6r9n8aKl
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3443-3480.1416
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1517-1549.1422
Calcd [M+]+ for C35H34BrN2O3 : 609.1747, 611.1727 , found :
+
19. Kobayashi, H. and Ogawa, M.; Chem. Rev. 2010, 110 (5),
2620-2640.1424
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2728.1428
609.1732, 611.1727.
Preparation of PYBG-D
A mixture of Br-N-NIR (35 mg, 0.058 mmol), PPh3 (5 mg, 0.02
21. Benson, R. C. and Kues, H. A., J. Chem. Eng. Data 1977, 22 (4),
379-383.1426
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1782.1432
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608.1245
24. Zhang, X. and Xiao, Y., The Journal of Organic Chemistry 2012,
77 (1), 669-673.1438
mmol),
n (20 mg, 0.064 mmol), Pd[P[C6H5]3]4 (4 mg, 0.004
mmol), CuI (2 mg, 0.01 mmol), and DMF/triethylamine (1.5
mL/0.5 mL) are placed in a 10 mL round bottomed flask with a
magnetic stirrer bar under a nitrogen atmosphere. The mixture
is heated at 85
column chromatography (gradient:CH2Cl2/CH3OH 40:1
℃
for 4 h. Then the mixture is purified by
8:1)
→
to yield 35 mg PYBG-D (0.058 mmol, 72%). m/z (TOF-LD+):
25. Bellier, Q. and Pégaz, S.; Org. Lett. 2011, 13 (1), 22-25.1440
26. Loudet, A.; Bandichhor, R.; Org. Lett. 2008, 10 (21), 4771-
4774.1261
Calcd [M+] for C51H48N7O5: 838.3701, found : 838.3717. The
resolutions of HNMR and 13CNMR spectra are not satisfactory
1
due to the extremely poor solubility of PYBG-D in CDCl3,
CD3OD, and DMSO-d6 etc. The total purity of the two
regioisomers (ratio ~1:1) measured by HPLC is 94.4%, as
recorded in supporting information (Figure S12).
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1472.1444
Acknowledgements
This work is supported by National Natural Science Foundation
of China (Nos. 21174022, 21376038, 21421005, and 21576040)
and National Basic Research Program of China (No.
2013CB733702).
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