Synthesis of 6,6′-Bis(O-4-arylethynylbenzoyl)-α,α-Trehaloses and Their Utilization as Fluorescent Probes for Cellular Imaging

Article abstract of DOI:10.1002/ejoc.201800424

A series of 6,6′-bis(O-4-arylethynylbenzoyl)-α,α-trehaloses (aryl groups; 1a: 1-pyrenyl, 1b: 9-anthryl, 1c: phenyl) were synthesized and fully characterized. Their photophysical properties were investigated by UV/Vis/fluorescence spectroscopy, and were rationalized by TDDFT calculations. Moreover, the emission maxima of 1a and 1b in THF/H₂O considerably shifted to a longer wavelength region with increasing H₂O fraction owing to the formation of excimers. The pyrenyl derivative 1a was efficiently taken into HeLa CD4⁺ human cervical cancer cells and emitted bright green fluorescence.

Full text of DOI:10.1002/ejoc.201800424

A Journal of
Accepted Article
Title: Synthesis of 6,6'-Bis(O-4-arylethynylbenzoyl)-α,α-Trehaloses and
Their Utilization as Fluorescent Probes for Cellular Imaging
Authors: Kurumi Kobayashi, Rumiko Saito, Kaori Udagawa, Naoko
Miyano-Kurosaki, Naoto Asano, Tetsuo Iwanaga, Naozumi
Teramoto, Toshiaki Shimasaki, and Mitsuhiro Shibata
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800424
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800424
Supported by

European Journal of Organic Chemistry
10.1002/ejoc.201800424
Synthesisof6,6’-Bis(O-4-arylethynylbenzoyl)-α,α-TrehalosesandTheirUtilizationasFluorescentProbesfor
CellularImaging
[a]
[b]
[b]
[b]
[c]
Kurumi Kobayashi, Rumiko Saito, Kaori Udagawa, Naoko Miyano-Kurosaki, Naoto Asano, Tetsuo
[c]
[a]
,[a]
,[a]
Iwanaga, NaozumiTeramoto, ToshiakiShimasaki,* andMitsuhiroShibata*
[a]
DepartmentofAppliedChemistry,FacultyofEngineering
ChibaInstituteofTechnology
Narashino,Chiba275-0016,Japan
E-mail:tshima@sun.it-chiba.ac.jpandmitsuhiro.shibata@p.chibakoudai.jp
DepartmentofLifeScience,FacultyofAdvancedEngineering
ChibaInstituteofTechnology
[b]
Narashino,Chiba275-0016,Japan
[c]
DepartmentofChemistry,FacultyofScience
OkayamaUniversityofScience
1-1Ridaicho,Kita-ku,Okayama700-0005,Japan
Received###00,2018
----------------------------------------------
-
GraphicalAbstract
6,6’-Bis(O-4-arylethynylbenzoyl)-α,α-trehaloses 1a, 1b
and 1c (aryl = 1-pyrenyl, 9-anthryl and phenyl,
respectively) were synthesized, and their photophysical
properties were examined by UV-vis and fluorescence
spectroscopies in anhydrous or hydrous THF solutions.
Especially, the pyrenyl derivative 1a acted as an excellent
fluorescentprobeforcytofluorometricimaging.
1
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
Abstract
A series of 6,6’-bis(O-4-arylethynylbenzoyl)-α,α-trehaloses (aryl groups; 1a: 1-pyrenyl, 1b: 9-anthryl, 1c:
phenyl) were synthesized and fully characterized. Their photophysical properties were investigated by
UV-vis/fluorescence spectroscopies, and were rationalized by TDDFT calculations. Moreover, the emission maxima
of 1a and 1b in THF/H O considerably shifted to a longer wavelength region with increasing H O fraction
2
2
+
owing to the formation of excimers. The pyrenyl derivative 1a was efficiently taken into HeLa CD4 human
cervical cancer cells and emitted bright green fluorescence.
Keywords:Fluorescentprobe,Cellularimaginganalysis,Trehalose,Fusedaromatics,Photophysicalproperties
1.Introduction
Fused aromatic compounds possessing π-conjugated substituents have been gathering much attention as
1
2
photo functional materials such as organic solar cells, organic light-emitting diodes (OLEDs) and organic
3
4
field-effect transistors (OFETs) due to their high fluorescent quantum yields. The extension of
π-conjugation can be attained by fusing aromatic rings and/or introducing π-bridges. The elongation of
π-conjugation generally provides electron-rich aromatics with a highly fluorescent character. For example,
Rashatasakhon and co-workers reported a novel C symmetrical truxane derivative with three ethynylpyrene
3
5
pendants in 2014. This π-electron abundantly truxane derivative displayed the good emission properties
with high quantum yield even in aqueous THF, and it was successfully applied to the sensing of picric acid
with a detection limit of 0.15 ppm. Thus, the aromatic compounds having large π-conjugation can be used
6
for the chemical sensors by using their highly fluorescent character. Moreover, Vicente and co-workers
recently reported the synthesis and spectroscopic and cellular properties of aryl-fused
7
4
,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs).
In this paper, they reported that
2
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
[α]phenanthrene-fused BODIPYs strongly absorb and emit in a near-IR region (641−701 nnm), and can be
applied to bio-imaging agents in human HEp2 cells. Thus, the compounds having fused aromatic structure
8
and/or π-bridge are good candidates of fluorescent probes as well as organic electronic functional materials.
As examples of the cellular imaging by combining sugars and fluorescent aromatic compounds, Laughlin,
Baskin and co-workers reported in vivo imaging of membrane-associated glycans as follows: Zebrafish
embryos were treated with peracetylated N-azidoacetylgalactosamine; subsequently, the embryos were
reacted with a difluorinated cyclooctyne containing Alexa Fluor 555 (a derivative of
3,6-diaminoxanthene-4,5-disulfate) cadaverine as a fluorophore; enabling the visualization of glycans in vivo
9
at subcellular resolution during development. Also, Hsu and co-workers reported that the azide-alkyne
click chemistry of 3-azido-7-hydoxycoumarin with the cellular glycans, in which peracetylated alkynyl
fucose and N-acetylmannosamine were incorporated as fluorescent molecules, is an effective method for the
10
labeling and visualization of glycoconjugates in cells. Barattucci, Sciortino, Puntoriero and coworkers
reported that a dimethylamino-substituted oligo(phenylene-ethynylene) glucoside acts as an efficient
11
biocompatible fluorescent cell probe. Moreover, Ribagorda, Barattucci and co-workers reported that the
dimethylamino-oligo(phenylene-ethynylene) glucoside derivatives are applicable to photosensitizers in
photodynamic therapy by using their photophysical properties such as high quantum yield, singlet oxygen
12
production, bio-compatibility, stability, easy cell-internalization and very good response.
Thus, it is
expected that the compounds prepared by combining fluorescent aromatic compounds with sugars are
promising fluorescent probes for cellular imaging.
Trehalose has two glucose moieties linked together by an α,α-1,1’-glycoside bond between those
13
anomeric carbons, therefore it is a non-reducing disaccharide. Trehalose exists abundantly in nature and it
is widely used in food and cosmetics. As an example of glycolipids possessing trehalose moieties,
maradolipid, which is a dissymmetrically 6,6’-di-O-acylated trehalose, was isolated from C. elegans.
3
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
Maradolipid is also known to show diverse immune activities, and it was chemically synthesized by Kulkarni
14
and Knölker independently.
Glycolipids play an important role to maintain the stability of the cell
membrane and to facilitate cellular recognition, which is crucial to the immune response and in the
connections that allow cells to connect to one another to form tissues. For instance, very recently, Timmer
and Stocker synthesized 6,6’-bis(O-fatty acyl)trehaloses with linear and iso-branched alkyl chains, and
15
determined that these trehalose diesters activate macrophages in a Mincle-dependent manner.
Figure 1. Structure of 6,6’-bis(O-4-arylethynylbenzoyl)-α,α-trehaloses 1a−1c.
As described above, the biological compatibility of trehalose and intense fluorescent character of fused
aromatic compounds led us to design a series of 6,6’-bis(O-4-arylethynylbenzoyl)-α,α-trehaloses 1a−1cpossessing
1
-pyrenyl, 9-anthryl andandphenyl as aryl groups(Figure 1). Especially, it is expected that pyrenyl and anthryl
derivatives 1a and 1b are excellent candidates of fluorescent probes for cellular imaging, considering that
-phenylethynylpyrene and 9-phenylethylanthracene exhibits the intense fluorescence and very high quantum
1
16
yield. In this study, aryl-substituted trehaloses 1a−1c were successfully synthesized, and their structural and
photophysical properties were investigated in detail. Furthermore, based on the photophysical properties,
+
pyrenyl derivative 1a was examined as a fluorescent probe for in vitro imaging of HeLa CD4 human cervical
cancercells. Consequently,1aactedasanexcellentfluorescentprobeforcytofluorometricimaging.
4
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
Synthesis.
Scheme 1. Synthesis of 6,6’-bis(O-4-arylethynylbenzoyl)-α,α-trehaloses 1a−1c. Reagents and conditions:
o
(
a) trimethylsilyl chloride, Et N/CH Cl (1:1 v/v), 0 C, 24 h; (b) K CO (0.5 mol eq.), CH Cl /MeOH (1:3
3 2 2 2 3 2 2
o
v/v), 0 C, 1h; (c) 4-iodobenzoic acid (3.0 mol eq.), DCC (3.0 mol eq.), DMAP (0.3 mol eq.), CH Cl , rt, 24 h;
2
2
(d) a: 1-ethynylpyrene (4.0 mol eq.), b: 9-ethynylanthracene (4.0 mol eq.), c: ethynylbenzene (4.0 mol eq.),
PdCl (PPh ) (0.2 mol eq.), CuI (0.2 mol eq.), THF/NEt (3:1 v/v), rt, 48 h; (e) TBAF (7.2 mol eq.), THF, rt,
2
3 2
3
1h.
DCC = N,N'-dicyclohexylcarbodiimide, DMAP = 4-dimethylaminopyridine, TBAF =
tetrabutylammonium fluoride.
The synthetic route for bis(O-arylethynylbenzoyl)trehaloses 1a, 1b and 1c is outlined in Scheme 1.
14b
2
,3,4,2',3',4'-hexakis(O-trimethylsilyl)trehalose 3 was synthesized according to a known procedure via the selective
deprodectionreactionoftrimethylsilyl(TMS)groupsofper(O-trimethylsilyl)trehalose2inanoverallyieldof37%from
trehalose. Dihydroxy compound 3 was esterificated by 4-iodobenzoic acid to afford a bis(iodophenyl) derivative 4
(
81%). Thekeyintermediate4 was coupled with threekinds ofaryl acetylenes (Ar=1-pyrenyl, 9-anthryl and phenyl)
prepared from thecorrespondingaryl halides, to givecompounds5a−5cin amodest to good yield (5a: 61%, 5b: 69%,
c: 87%). At last, six TMS groups of 5a−5c were de-protected by tetrabutylammonium fluoride (TBAF) in THF to
generate objective compounds 1a−1c (1a: 52%, 1b: 40%, 1c: 47%). The arylethynyl-substituted trehaloses 1a−1c
5
1
13
werefullycharacterizedbyusingvariousanalyticaltechniquessuchas Hand CNMR,FT-IRspectroscopiesandhigh
5
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
resolutionmassspectrometry.
Molecularstructure.
Bis(O-arylethynylbenzoyl)trehaloses 1a−1c were soluble in high-polar solvents and cyclic ethers such as
DMSO, DMF, dimethylacetoamide (DMA), dioxane and THF, while insoluble in CH Cl , CHCl , methanol,
2
2
3
ethanol, isopropylalcohol, acetone, EtOAc, diethylether and hexane. Although we tried the recrystallization
of 1a−1c with dioxane or THF, we could not get their single crystals. Therefore, the structural properties of
compounds 1a−1cweredetermined by DFT calculations at the B3LYP/6-31G(d) level of theory.
Figure 2. Calculated structures of a) compound 1a; b) compound 1b; c) compound 1c at the
B3LYP/6-31G(d) level of theory. The lower structures represent the top view and the upper structures
represent side view, respectively.
The calculated structures of compounds 1a−1c were shown in Figure 2. The calculated bond lengths of
two C≡C bonds (1.22 Å) in each compound were almost identical to a typical C≡C bond length. Two
ethynylene-bridged 1-pyrenyl and phenylene moieties of compound 1a were slightly twisted by 0.54 and
o
5
.52 . Also, the torsional angles between two ethynylene-bridged 9-anthryl and phenylene moieties of 1b
o
were 1.66 and 4.84 . In the case of compound 1c, the torsional angles between two pairs of benzene rings
6
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
o
were 1.10 and 0.91 , indicating that the steric hindrance is alleviated in comparison with 1a and 1b. Thus,
DFT calculation revealed that each terminal arylethynylphenyl moiety of 1a−1c is somewhat deviated from a
planar structure. These arylethynylphenyl moieties were located far apart from each other as shown at the
top of Figure 2. End-to-end distances between terminal two aryl groups for 1a, 1b and 1c were 34.6, 31.2
and 28.8 Å, respectively. On the other hand, the distances of OH−O for 1a (2.13−2.46 Å), 1b (2.13−2.47 Å)
and 1c (2.13−2.46 Å) in trehalose moiety were shorter than the sum of van der Waals radii (2.72 Å) of
hydrogen (1.20 Å) and oxygen (1.52 Å). Therefore, all hydroxy groups of trehalose for 1a, 1b and 1c can
be considered to form the intramolecular multi hydrogen bonds. Such intramolecular multi hydrogen bonds
strongly indicate that the trehalose takes the rigid conformation. These results of DFT calculations exhibit
that the hydrophobic and hydrophilic moieties for 1a−1c are located on the periphery and center of molecular
structure, respectively.
ElectronicAbsorptionSpectroscopy.
1
60
1a
1
b
c
1
1
20
6a
6
b
c
6
8
4
0
0
0
ε
ε
2
20
270
320
370
/ nm
420
470
λ
Figure 3. Electronic absorption spectra of bis(O-arylethynylbenzoyl)trehaloses 1a−1c and their reference
compounds 6a−6c in THF at 298 K.
a,b
Table1. UV-vis spectral data of 1a−1c and 6a−6c, and theoretical calculated lowest absorption maxima
7
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
abs
abs
λmax
ε
calcd λmax
composition of band of calcd
−1
−1
b
absb
[
nm] (eV)
238 (5.21)
86 (4.34)
02 (4.11)
70 (3.35)
95 (3.14)
[M cm ]
46,100
31,900
34,200
43,300
38,100
146,700
20,400
26,400
24,100
37,400
34,100
21,600
44,900
37,800
26,100
24,200
29,200
18,700
33,100
30,400
99,300
44,900
12,600
16,600
15,300
17,900
14,500
[nm] (f)
λmax
1a
407 (1.502)
H ‰ L+1, 55%
H−1 ‰ L, 42%
2
3
3
3
1b
263 (4.71)
443 (0.739)
H ‰ L+1, 64%
H−1 ‰ L, 35%
c
2
94 (4.22)
3
09 (4.01)
c
3
84 (3.23)
4
03 (3.08)
4
25 (2.92)
1c
220 (5.64)
319 (1.694)
387 (0.838)
H ‰ L+1, 61%
H−1 ‰ L, 37%
2
98 (4.16)
3
13 (3.96)
6a
232 (5.34)
H ‰ L, 97%
c
2
42 (5.12)
c
2
92 (4.25)
3
43 (3.62)
3
62 (3.43)
3
81 (3.25)
6b
262 (4.73)
431 (0.339)
294 (0.930)
H ‰ L, 99%
2
97 (4.18)
c
3
74 (3.32)
3
96 (3.13)
4
18 (2.97)
6c
280 (4.43)
H ‰ L, 97%
2
97 (4.18)
H−3 ‰ L+4, 3%
a
b
in THF. The data were afforded by time-dependent (TD) DFT calculations of 1a−1c and 6a−6c at the
B3LYP/cc-pVDZ // B3LYP/6-31G(d) level of theory using the optimized structures.; f = oscillator strength. H =
c
HOMO, L= LUMO. Peak as a shoulder.
To investigate the basic photophysical properties of 1a−1c, the UV/Vis absorption spectra of dilute
8
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
−5
solutions in THF (1 × 10 M) at 298 K were compared with those of 1-phenylethynylpyrene 6a,
9-phenylethynylanthracene 6b and phenylethynylbenzene 6c as reference compounds (Figure 3). Table 1
summarizes their experimental spectral data as well as time-dependent (TD) DFT results at the
B3LYP/cc-pVDZ // B3LYP/6-31G(d) . The absorption spectrum of dipyrenyl compound 1a was mainly
composed of three bands, among which the highest energy band was located at 238 nm. The second band
abs
for 1a had two peaks at 286 and 302 nm, and the third band (λmax ) also had two peaks at 370 and 395 nm.
The dianthryl compound 1b also exhibited a similar spectral pattern to that of 1a. The highest energy band
of 1b was located at 263 nm and the second one was at 309 nm with a shoulder at around 294 nm. The third
one was composed of two peaks at 403 and 425 nm, which was observed at a lower energy region than a
typical α-absorption band of anthracene by the π-conjugation through ethynylene bridges. On the other
hand, the spectrum of 1c was quite simple, and mainly two curves appeared at around 220 and 300 nm. Τhe
molar absorption coefficients (
.5timeslargerthanthoseofthecorrespondingreferencecompounds6aand6b(6a:26,100(232nm),6b:99,300 (262
nm)).
ε) for 1a and 1b (1a: 46,100 (238 nm), 1b: 146,700 (263 nm)) were approximately
1
The fact that the absorption terminals of 1a and 1b reached to a visible region means that visible light can
abs
be used for excitation when these compounds are introduced into cell. On the other hand, the longest λmax
of compound 1c appeared at 313 nm, which was located at a considerably shorter wavelength region than
those of 1a and 1b.
Fluorescence Spectroscopy.
9
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
1a
1b
1c
6a
6b
6c
250
350
450
550
650
Wavelength / nm
Figure 4. Fluorescence spectra of bis(O-arylethynylbenzoyl)trehaloses 1a−1c and their reference compounds
a−6c in THF at 298 K.
6
a
Table 2. Fluorescence data of 1a−1c.
abs
fl
−1b
d
λmax [nm]
λex [nm]
λmax [nm]
νST / cm
Φ
f
(in THF)
(in THF)
1
a
395
395
439
2,537
0.91
0.76
c
498
1b
425
425
446
63
1,108
1,931
3,047
4
1c
313
381
313
381
346
0.42
0.79
6a
395
930
1,916
775
4
11
432
53
313
22
6b
418
297
418
0.84
n.d.
4
1,848
1,721
2,614
6c
297
3
a
−5
b
c
d
Measured at 1 × 10 M in THF. Stokes shift. Peak as a shoulder.
determined by an integrating sphere system.
Absolute quantum yields were
As described above, it is well known that fused aromatic compounds such as pyrene and anthracene
derivatives generally show a fluorescent character. Actually, not only fused aromatic compounds1a and 1b,
but also monocyclic aromatic compound 1c exhibited intense fluorescence character in diluted THF solutions
1
0
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
−5
fl
(
1 × 10 M) as shown in Figure 4. The fluorescent maximum (λmax ) for compound 1a was observed as a
broad peak having no vibrational structure at 439 nm, which was in a longer wavelength region than that of
the reference compound 6a (411 nm). Furthermore, the emission band contained a shoulder peak at around
500 nm as shown in Figure 4. When the fluorescent spectrum was measured in more diluted concentration
−6
(
1 × 10 M), the shoulder was not appeared in spectral band (see Figure S22 and Table S1). Also, the total
−5
−6
spectral shapes of 1a between 1 × 10 M and 1 × 10 M were substantially different from each other.
Hence, we assigned that this shoulder peak shown in Figure 4 would be derived from an intermolecular
excimer (excited dimer) emission band. On the other hand, it is inferred that the excimer emission is not
formed for 1b, because there was little difference in the spectral shape with the vibrational structure between
fl
fl
1
b (λmax = 463 nm) and 6b (λmax = 453 nm). Additionally, the total spectral shape was also almost no
−5
−6
changed between 1 × 10 M and 1 × 10 M (see Figures 4 and S22). Although the Storks shift (ν ) of 1c
ST
−1
−1
−1
(
3,047 cm ) was relatively large compared with those of 1a (2,537 cm ) and 1b (1,931 cm ), it was
fl
observed still in a UV region ( max = 346 nm). Table 2 also summarizes absolute fluorescent yields (Φ) of
λ
f
1
a−1c and 6a−6c determined by an integrating sphere system. The Φ values of fluorescent probes 1a−1c
f
were 0.91 (1a), 0.76 (1b) and 0.42 (1c), these values were comparable to or higher than those (6a: 0.79, 6b:
0
.84. 6c: n.d.) of reference compounds 6a−6c with no hydrophilic moieties. The higher orders of Φ values
f
were 1a > 1b > 1c for 1a−c, and 6a > 6b > 6c for 6a−c.
11
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
(
a)
(b)
fw (vol%)
fw (vol%)
9
0
90
80
70
60
50
30
20
10
80
70
50
40
30
20
10
3
90
460
530
600
670
415
470
525
580
635
690
Wavelength / nm
Wavelength / nm
Figure 5. Fluorescence spectra of 1a (a) and 1b (b) in THF/water mixtures with different water fractions
fw) at 298 K.
(
Figure 6. The fluorescence photographs of 1a (a) and 1b (b) in THF/H O mixtures with different water
2
fractions (fw).
a
Table 3. Fluorescence data of 1a and 1b in THF/ H O mixtures with different H O fractions (f ).
2
2
w
−1b
fl
fl
fl
−1b
c
fl
fl
fl
c
fw
THF/H O)
λmax [nm] λmax
−
λ0 cm
Φf
λmax [nm] λmax
−
λ0 cm
Φf
(
for 1a
1a
for 1a
for 1b
1b
−
for 1b
2
100/0
439
−
0.91
443
0.76
4
63
443
65
−
90/10
440
51.8
−
0
−
4
92.9
1
2
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European Journal of Organic Chemistry
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8
0/20
0/30
0/40
0/50
0/60
0/70
0/80
0/90
445
449
449
457
465
307
507
507
897
−
−
444
65
445
65
446
65
447
65
50.8
92.9
102
−
−
4
7
4
92.9
152
6
−
−
4
92.9
202
5
−
−
4
92.9
302
4
1,274
2,412
2,658
0.72
−
449
467
453
0.45
−
491
92.1
498
3
497
506
509
467
92.1
322
2
3,016
3,033
−
470
−
d
5
43
3,089
1,206
1,598
3,350
d
1
0.31
468
00
48
0.02
5
5
a
−5
b
fl
fl
Measured at 1 × 10 M in THF with different H O fractions. λ0 isdλmax of 1a and 1b in THF.
2
c
Absolute quantum yields were determined by an integrating sphere system. Peak as a shoulder.
In the fluorescence spectra of compound 1a and 1b measured in THF/H O mixtures with different H O
2
2
fl
fractions (Figure 5), the values of λmax for 1a and 1b became longer with increasing H O fraction. The
2
fluorescent curves of 1a in THF/H O 40/60 and 1b in THF/ H O 30/70 were composed of two peaks at 465
2
2
and 491 nm, and 449 and 467 nm, respectively. When the ratio of THF/H O is 10/90 for 1a, the fluorescent
2
fl
fl
−1
band became a broad peak at 509 nm, and the value of λmax
− λ0 was 3,033 cm (Table 3). Thus, when
the water fraction was increased for 1a, the original fluorescent emission band graduallydisappeared, and the
shoulder band shifted to a longer wavelength region. It is inferred that the spectral change for 1a is derived
from the excimer, which is formed by the aggregation of hydrophobic 1-phenylethynylpyrenyl moieties of1a
17
by the addition of H O. Also, the longest emission band of 1b shifted to 548 nm in THF/H O 10/90, and
2
2
fl
fl
−1
the value of λmax
− λ0 was 3,350 cm . In order to play a role as a highly efficient cellular fluorescent
11
probe, the broad emission band is quite important. When 1a−1c are used as a fluorescent probe in a living
1
3
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
cell, it is also quite important to elucidate the water content-dependency of fluorescent spectra, because it is
necessary to generate fluorescent light in a H O-rich condition. Therefore, we measured fluorescent yields
2
(
Φ ) of 1a and 1b in the THF/H O ratios of 40/60 and 10/90 by an integrating sphere system. The Φ values
f
2
f
(
0.31 and 0.02) of 1a and 1b in THF/H O 10/90 were much smaller than those (0.72 and 0.45) in THF/H O
2
2
4
0/60. We confirmed that 1a exhibits a green color emission due to the excimer formation in THF/H O
2
1
0/90, whereas 1b almost did not emit light in the H O-rich condition, as is obvious from Figure 6.
2
ComputationalAnalysis.
Figure 7. Calculated FMO orbitals for 1a−1c and their transition properties at the B3LYP/cc-pVDZ //
B3LYP/6-31G(d) level of theory.
The time-dependent (TD) DFT and single point calculations using global minimum optimized structures
for compounds 1a−1c and 6a−6c were performed at the B3LYP/cc-pVDZ // B3LYP/6-31G(d) level of theory
to obtain some insights into the electronic properties. As shown in Figure 7, the HOMO and LUMO
1
4
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
orbitals of each compound were spread on the aromatic and ethynylene moieties. Especially, the LUMO
orbital of each compound was also located at a carbonyl group beyond the connected ethynylene bridge.
abs
The fact that λmax values of 1a−1c were a little longer than those of reference compounds 6a−6c can be
explained by a decrease of the HOMO−LUMO gap due tothe LUMO stabilization.
abs
The transition properties of the longest λmax for compounds 1a−1c were elucidated by TDDFT
calculations at the B3LYP/cc-pVDZ level. As described in Table 1 and Figure 7, the absorption maxima of
compounds 1a−1c are based on the transitions from HOMO−1 to LUMO and from HOMO to LUMO+1.
abs
The calculated longest λmax of 9-ethynylanthryl compound 1b was 443 nm, which was in good agreement
abs
with the experimental value. The fact that the calculated longest λmax (407 nm) of 1-ethynylpyrenyl
compound 1a was shorter by 36 nm than that of 1b is caused by the lowering of the LUMO level of 1b. On
the other hand, the HOMO−LUMO gap of ethynylphenyl compound 1c was larger than those of 1a and 1b,
reflecting that 1c have no fused aromatic rings.
Cellular Imaging Study.
Figure 8. (a) Bright field image, (b) Fluorescence image (490 nm excitation, 520−530 nm emission range),
+
(
c) Overlay image of HeLa CD4 cells in presence of 1a (15.0 µM).
1
5
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
Figure 9. (a) Bright field image, (b) Fluorescence image (490 nm excitation, 520−530 nm emission range),
+
(
c) Overlay image of HeLa CD4 cells in presence of 1a (37.5 µM).
Based on the experimental and calculated results of photophysical properties for compounds 1a−1c, we
+
performed an uptake study into HeLa CD4 human cervical cancer cells using 1a as a green color fluorescent
probe. The cells were cultivated on D-MEM basal media with 10µLof 1a (37.5 µM in DMSO/PBS 80/20),
o
1
0% FBS and penicillin/streptomycin at 37 C under air condition including 5%CO for 24 h. The pyrenyl
2
+
compound 1a was successfully taken into the HeLa CD4 cells (Figure 9 (a) and (b)), even when cells are
incubated at low concentrations of up to 15.0 µM (Figure 8 (a) and (b)). It was demonstrated that the
+
pyrenyl compound 1a in the HeLa CD4 cells emitted bright green luminescence when excited by 490 nm
visible light. The luminescence was the same color as the luminescence (520−530 nm) of 1a in THF/H O
2
10/90. Moreover, in order to examine the bio-compatibility of fluorescent probe 1a, we performed the cell
viability tests by using DMSO/PBS (80/20) solutions of 1a as shown in Figure S23. Consequently, the
averages of cell viabilities in various concentrations were 48−64% after 24 h, and were almost the same as the
case of only DMSO/PBS (60%). These results strongly suggest that the modest cell viabilities are caused by
the toxicity of DMSO. Thus, we clarified that pyrenyl derivative 1a was efficiently taken toward the HeLa
+
CD4 cells, and served as a green color cell imaging probe.
Conclusions.
In summary, we synthesized a series of new aryl-substituted trehaloses 1a−1c via the TMS-protection of hydroxy
1
6
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
groupsoftrehalose,selectivedeprotection,esterificationoftwoprimaryhydroxygroupsandSonogashiracross-coupling
reactions. The structural features of 1a−1c were investigated by DFT calculations at the B3LYP/6-31G(d) level of
theory. TheUV-visspectralanalysisfor1a−1crevealedthatpyrenylandanthrylderivatives1aand1bhaveabsorption
maxima in a visible region, while phenyl derivative1c does not. The compounds 1a−1c emitted intense fluorescence
at 313−415 nm in diluted THF solutions, and had high quantum yields (1a: 0.91, 1b: 0.76, 1c: 0.42).
Although 1b almost did not emit light in THF/H O 10/90 (Φ = 0.02), 1a emitted a relatively intense
2
f
fluorescence character even in THF/H O 10/90 (Φ = 0.31). It was demonstrated that pyrenyl derivative 1a
2
f
+
acts as a fluorescent probe having bright green emission, when it was introduced into the HeLa CD4 cells.
The investigation of the second generation trehalose-based fluorescent probes is underway in our laboratory.
Experimental Section.
General experimental methods
All of the chemicals and solvents were purchased from commercial sources and used without further
purification unless otherwise stated. Column chromatography and plug filtrations were performed on silica
gel 60. TLC was performed on aluminum sheets that were coated with silica gel 60 F ; visualization was
254
performed with a lamp (
using the attenuated total reflectance (ATR) method. UV/Vis spectra were recorded in a quartz cell (width:
0 mm). The UV/Vis and fluorescence spectra were measured in a cuvette of 1 cm at 298 K. The
λ = 254 or 365 nm). Melting points are uncorrected. IR spectra were recorded by
1
abs
absorption maxima (λmax ) are reported in nm, and the relative intensities or molar extinction coefficients are
given in parentheses. Shoulders are denoted as sh. Absolute quantum yields were determined by using a
calibrated integrating-sphere system in THF and THF/H O. Spectroscopic grade THF and H O was
2
2
deaerated under ultrasound irradiation for 10 min just prior to use. NMR spectra were measured in CDCl3
and DMSO d . Chemical shifts (δ) are given in ppm relative to tetramethylsilane (TMS). Coupling
6
constants (J) are given in Hz. The apparent resonance multiplicity is described as s (singlet), d (doublet), t
1
7
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
(triplet), q (qualtet), quin (quintet), sep (septet) and m (multiplet). HRMS was performed for solutions in
DMSO/MeCN on APCI mass spectrometers. All the theoretical calculations were performed by using the
18
Gaussian program package. The geometry optimizations were performed by the Becke’s three-parameter
19
20,21
22
hybrid functional (B3), the Lee, Yang, and Parr (LYP) correlation,
and the 6-31G(d) Pople basis set.
23
TD calculations were performed by using the correlation consistent cc-pVDZ basis set of Dunning.
Synthesis of compounds 1a−1c, 3, 4, 5a−5c and 6a
2
,3,4,2',3',4'-Hexakis(O-trimethylsilyl)-α,α-trehalose(3)
,3,4,2',3',4'-Hexakis(O-trimethylsilyl)-α,α-trehalose 3 was synthesized according to the reported manner.
Allspectraldatawerecompletelyidenticalwiththereporteddata.
14b
2
1
-(Phenylethynyl)pyrene(6a)
-(Phenylethynyl)pyrene 6a was synthesized according to the previous literature. All spectral data were
completelyidenticalwiththereporteddata.
24
1
6,6'-Bis(O-4-iodobenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)-α,α-trehalose(4)
Undera nitrogen atmosphere, DCC (1.98 mL, 8.82mmol) and DMAP(110.2 mg. 0.880 mmol)was added into a
solution of 2,3,4,2',3',4'-hexakis(O-trimethylsilyl)-α,α-trehalose 3 (2.28 g, 2.94 mmol) and 4-iodobenzoic acid
(
2.19 g, 8.82 mmol) in CH Cl (40 mL) at room temperature. After stirring for 48h, the whitepowderwas removed
2 2
by filtration, and the solvent was removed under reduced pressure. The residual mixture was purified by flash
column chromatography on silica gel (n-hexane/AcOEt, 19:1) to give
,6'-bis(O-4-iodobenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose 4 as a white powder. Yield,
6
α,α
1
8
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
o
1
2
.95g(2.39mmol),81%;mp,139.5−140.5 C; HNMR(500MHz,CDCl )δ0.12(18H,s),0.14(18H,s),0.18(18H,
3
s),3.48(2H,dd,J=3.1and9.4Hz),3.61(2H,t,J=8.8Hz),3.96(2H,t,J=8.9Hz),4.11(2H,dt,J=3.2and9.2Hz),
4.27(2H,dd,J=3.7and12.0Hz),4.56(2H,dd,J=2.5and12.1Hz),4.95(2H,d,J=3.1Hz),7.75(4H,d,J=8.7Hz),
13
7
.82 (4H, d, J = 8.7 Hz); C NMR (125 MHz, CDCl ) δ 0.25, 0.89, 1.07, 63.9, 70.8, 71.9, 72.7, 73.6, 94.5, 100.9,
3
−1
1
29.5, 131.1, 137.8, 165.9; IR (ATR, cm ) ṽ = 750, 831, 868, 899, 966, 1007, 1063, 1072, 1101, 1144, 1169, 1248,
1
263, 1393, 1587, 1722, 2905, 2959; HRMS (APPI): m/z calcd for C H I O Si Na: 1257.1883; found: 1257.1893
4
4 76 2 13 6
+
[
(M+Na) ].
6
,6'-Bis(O-4-pyren-1-ylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)-
Under a nitrogen atmosphere, 1-ethynylpyrene (1.29 g, 4.32 mmol) was added into a solution of
,6'-bis(O-4-iodobenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose 4 (1.33 g, 1.08 mmol),
PdCl (PPh ) (151.3 mg, 0.216 mmol) and CuI (41.0 mg, 0.216 mmol) in triethylamine (27 mL) and THF (38 mL),
α,α-trehalose(5a)
6
α,α
2
3 2
and the mixture was stirred at room temperature for 48 h. After volatile materials were removed under reduced
pressure, the residue was dissolved in CH Cl (50 mL). The organic layer was washed with aqueous H O (10 mL×
2
2
2
3
), and dried over Na SO . The crude mixture was purified by column chromatography on silica gel
2 4
(
n-hexane/EtOAc, 7:1). Furtherpurification was alsocarried out bycolumn chromatographyon silicagel(toluene)to
give 6,6'-bis(O-4-pyren-1-ylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose 5a as a yellow
solid. Yield, 952.1 mg (0.663 mmol), 61%; mp, 219.5−220.5 C; H NMR (500 MHz, CDCl )δ0.18 (18H, s), 0.20
α,α
o
1
3
(18H,s),0.22(18H,s),3.57(2H,dd,J=3.1and9.3Hz),3.70(2H,t,J=9.0Hz),4.03(2H,t,J=8.9Hz),4.18(2H,dt,
J=2.6and9.4Hz),4.35(dd,J=3.8and11.8Hz),4.65(dd,J=2.4and11.8Hz),5.06(2H,d,J=3.0Hz),7.80(4H,d,
13
J = 8.2 Hz), 7.99−8.03 (4H, m), 8.08−8.21 (16H, m),8.62 (2H, d, J = 9.1 Hz); C NMR (125 MHz, CDCl ) δ 0.31,
3
0.95, 1.11, 64.0, 70.9, 72.0, 72.8, 73.6, 91.9, 94.3, 94.5, 117.0, 124.2, 124.4, 124.5, 125.3, 125.7, 125.8, 126.3, 127.2,
−1
1
28.4, 128.5, 129.4,129.7,129.8, 131.0,131.2,131.6(2C),132.0, 166.0;IR(ATR, cm )ṽ=715,748, 764,837,870,
1
9
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
964, 1011, 1072, 1097, 1171, 1250, 1263, 1408, 1607, 1724, 2342, 2903, 2955, 3042; HRMS (APCI): m/z calcd for
+
C H O Si :1431.5383;found:1431.5374[(M+H) ].
8
0 95 13 6
6
,6'-Bis(O-4-anth-9-ylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)-
Under a nitrogen atmosphere, 9-ethynylanthracene (770.0 mg, 2.81 mmol) was added into a solution of
,6'-bis(O-4-iodobenzoyl)-2,3,4,2',3',4'-hexakis-(O-trimethylsilyl)- -trehalose 4 (860.4 mg, 1.08 mmol),
PdCl (PPh ) (100.2 mg, 0.140 mmol) and CuI (30.1 mg, 0.140 mmol) in triethylamine (13 mL) and THF (20 mL),
α,α-trehalose(5b)
6
α,α
2
3 2
and the mixture was stirred at room temperature for 48 h. After volatile materials were removed under reduced
pressure, the residue was dissolved in CH Cl (50 mL). The organic layer was washed with aqueous H O (10 mL×
2
2
2
3
), and dried over Na SO . The crude mixture was purified by column chromatography on silica gel
2 4
(
n-hexane/EtOAc, 7:1). Furtherpurification was alsocarried out bycolumn chromatographyon silicagel(toluene)to
give 6,6'-bis(O-4-anth-9-ylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose 5b as a yellow
solid. Yield, 665.9 mg (0.480 mmol), 69%; mp, 145.5−146.5 C; H NMR (500 MHz, CDCl )δ0.19 (18H, s), 0.21
α,α
o
1
3
(18H,s),0.22(18H,s),3.57(2H,dd,J=3.0and9.3Hz),3.71(2H,t,J=9.0Hz),4.03(2H,t,J=8.9Hz),4.20(2H,d,
J=9.3Hz), 4.36(2H,dd,J=3.4and12.0Hz), 4.66(2H,d,J=12.0Hz),5.06(2H, d,J=2.9Hz),7.51(4H,t,J=8.2
Hz), 7.61 (4H, t,J=8.6 Hz), 7.85 (4H, d,J=7.7 Hz), 8.01 (4H, d,J=8.4 Hz), 8.16 (4H, d,J=7.9 Hz), 8.45 (2H, s),
13
8
.62(4H, d,J=8.0 Hz); CNMR(125MHz, CDCl )δ0.30, 0.94, 1.11, 63.9, 70.9, 72.0, 73.6, 76.7, 89.5, 94.6, 99.9,
3
−1
1
16.5, 125.8, 126.5, 126.9, 128.4 (2C), 128.8, 129.4, 129.8, 131.1, 131.5, 132.7, 165.9; IR (ATR, cm ) ṽ = 735, 766,
831, 866, 934, 968, 1013, 1074, 1107, 1153, 1171, 1248, 1404, 1603, 1717, 2195, 2361, 2899, 2957, 3057; HRMS
+
(
APCI):m/zcalcdforC H O Si :1383.5310;found:1383.5276[(M+H) ].
76 95 13 6
6
,6'-Bis(O-4-phenylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)-
α,α-trehalose(5c)
Under a nitrogen atmosphere, ethynylbenzene (183.4 mg, 1.80 mmol) was added into a solution of
2
0
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
6
,6'-bis(O-4-iodobenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)-
α
,α
-trehalose 4 (740.4 mg, 0.60 mmol),
PdCl (PPh ) (80.0mg,0.120mmol)andCuI(20.1mg,0.120mmol)intriethylamine(15mL)andTHF(21mL),and
2
3 2
the mixture was stirred at room temperature for 48h. After volatile materials were removed under reduced pressure,
the residue was dissolved in CH Cl (50 mL). The organic layer was washed with aqueous H O (10 mL× 3), and
2
2
2
dried overNa SO . Thecrudemixturewas purified bycolumn chromatographyon silicagel (n-hexane/EtOAc,7:1).
2
4
Further purification was also carried out by column chromatography on silica gel (toluene) to give
,6'-bis(O-4-phenylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose 5c as a white powder.
Yield,612.9mg(0.520mmol),87%;mp,154.5−155.5 C; HNMR(500MHz,CDCl )δ0.14(18H,s),0.16(18H,s),
6
α,α
o
1
3
0.19 (18H, s), 3.52(2H, dd,J=3.0and9.4Hz), 3.65 (2H, t,J=8.9 Hz), 3.99(2H, t,J=9.0Hz), 4.14(2H, dt,J=3.2
and9.1Hz),4.30(2H,dd,J=3.7and12.0Hz),4.59(2H,dd,J=2.3and12.0Hz),5.00(2H,d,J=3.1Hz),7.35−7.38
13
(
6H, m), 7.54−7.56 (4H, m), 7.61 (4H, d,J=8.6 Hz), 8.04(4H, d,J=8.6 Hz); CNMR (125 MHz, CDCl )δ 0.27,
3
0.90,1.08,63.9,70.8,72.0,72.7,73.6,88.6,92.5,94.6,122.6,128.1,128.4,128.8,129.3,129.6,131.5,131.7,165.9;IR
−1
(
ATR, cm ) ṽ = 756, 833, 868, 901, 943, 964, 1005, 1043, 1074, 1097, 1142, 1169, 1250, 1269, 1406, 1607, 1726,
+
2
340,2359,2907,2959;HRMS(APCI):m/zcalcdforC H O Si Na:1205.4576;found:1205.4581[(M+Na) ].
60 86 13 6
6
,6'-Bis(O-4-pyren-1-ylethynylbenzoyl)-
Under a nitrogen atmosphere, 1.0 M THF solution of tetrabutylammonium fluoride (1.15 mL, 1.15 mmol) was
added into solution of 6,6'-bis(O-4-pyren-1-ylethynylbenzoyl)-
,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose 5a (229.8 mg, 0.160 mmol) in THF (23 mL) and methanol
16 mL), and the mixture was stirred at room temperature for 2 h. The solvents were removed under reduced
α,α-trehalose(1a)
a
2
α,α
(
pressure, the crude mixture was purified by column chromatography on silica gel (toluene/EtOAc/methanol, 2:2:1).
Additionalpurificationwascarriedoutbycolumnchromatographyonflorisil(EtOActoEtOAc/methanol,6:1)togive
6,6'-bis(O-4-pyren-1-ylethynylbenzoyl)-α,α-trehalose 1a as a yellow solid. Yield,83.9mg(84.0µmol),52%;mp,
2
1
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
o
1
1
85−186 C; HNMR(500MHz,DMSOd )δ3.33−3.37(2H,m),3.43−3.47(2H,m),3.66−3.71(2H,m),4.18−4.21
6
(2H,m),4.39−4.43(2H,m),4.52(2H,d,J=10.7Hz),5.00(2H,d,J=3.8Hz),5.09(2H,d,J=5.9Hz),5.11(2H,d,J
=
4.6Hz),5.32(2H,d,J=5.2Hz),7.86(4H,d,J=8.2Hz),8.05(4H,d,J=8.2Hz),8.06(2H,d,J=7.4Hz),8.12(2H,
13
d, J = 9.0 Hz), 8.19−8.23 (6H, m), 8.27−8.30 (6H, m), 8.54 (2H, d, J = 9.1 Hz); C NMR (125 MHz, DMSO d ) δ
6
64.4, 69.9, 70.2, 71.5, 72.9, 91.3, 94.0, 94.3, 116.0, 123.3, 123.5, 124.7, 124.9, 126.08, 126.13, 126.7, 127.1, 128.56,
−1
1
28.62,128.99,129.6(2C),129.8,130.4,130.7,131.25,131.29,131.8,165.1;IR(ATR,cm )ṽ=718,766,845,984,
1016,1045,1074,1126,1175,1269,1377,1445,1605,1713,2342,2876,2945,2959,3312(br);HRMS(APPI):m/z
−
calcdforC H O Cl:1033.2632;found:1033.2679[(M+Cl) ].
6
2 46 13
6
,6'-Bis(O-4-anth-9-ylethynylbenzoyl)-
Under a nitrogen atmosphere, 1.0 M THF solution of tetrabutylammonium fluoride (1.44 mL, 1.44 mmol) was
addedintoasolutionof6,6'-bis(O-4-anth-9-ylethynylbenzoyl)-2,3,4,2',3',4'-hexakis(O-trimethylsilyl)- -trehalose
b(186.2mg, 0.140mmol)inTHF(20mL)andmethanol(14mL), andthemixturewasstirredat roomtemperature
for 2 h. The solvents were removed under reduced pressure, the crude mixture was purified by column
chromatography on florisil (EtOAc to EtOAc/methanol, 6:1) to give
,6'-bis(O-4-anth-9-ylethynylbenzoyl)- -trehalose 1b as an orange color solid. Yield, 40.6 mg (42.7 µmol),
1%;mp, 230−231 C; HNMR(500MHz, DMSOd )δ3.29−3.33(2H, m), 3.41−3.45(2H, m), 3.66 (2H, sep, J =
α,α-trehalose(1b)
α,α
5
6
α,α
o
1
3
6
4.6 Hz),4.18−4.21(2H,m),4.39(2H,q, J = 5.9 Hz),4.51(2H,d, J = 10.3 Hz),4.97(2H,d, J = 3.5 Hz),5.07(4H,
t, J = 5.6 Hz),5.29(2H,d, J = 5.6 Hz),7.58(4H,t, J = 7.9 Hz),7.68(4H,t, J = 6.8 Hz),7.98(4H,d, J = 8.3 Hz),
13
8
.08 (4H, d, J = 8.3 Hz), 8.14 (4H, d, J = 8.6 Hz), 8.56 (4H, d, J = 8.8 Hz), 8.70 (2H, s); C NMR (125 MHz,
DMSOd )δ64.5,69.9,70.2,71.5,72.9,88.7,94.0,99.9,115.1,125.9,126.1,127.2,127.7,128.7,128.9,129.0,129.6,
6
−1
1
30.7, 131.9, 132.0, 165.2; IR (ATR, cm ) ṽ = 725, 764, 841, 880, 945, 988, 1015, 1065, 1107, 1136, 1179, 1281,
1
329, 1406, 1605, 1697, 2361, 2914, 3418 (br); HRMS (APPI): m/z calcd for C H O Cl: 985.2632; found:
5
8 46 13
2
2
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
−
9
85.2666[(M+Cl) ].
6
,6'-Bis(O-4-phenylethynylbenzoyl)-
Under a nitrogen atmosphere, 1.0 M THF solution of tetrabutylammonium fluoride (1.99 mL, 1.99 mmol) was
added into a solution of 6,6'-bis(O-4-phenylethynylbenzoyl)-2,3,4,2',3',4'-hexakisO-(trimethylsilyl)- -trehalose
c(249.9 mg, 0.210 mmol)inTHF(30mL) andmethanol(21mL), andthemixturewas stirred atroom temperature
for 2 h. The solvents were removed under reduced pressure, the crude mixture was purified by column
chromatography on florisil (EtOAc to EtOAc/methanol, 6:1) to give
,6'-bis(O-4-phenylethynylbenzoyl)- -trehalose 1c as an orange color solid. Yield, 74.0 mg (98.6µmol), 47%;
α,α-trehalose(1c)
α,α
5
6
α,α
o
1
mp,204.5−205.5 C; HNMR(500MHz,DMSOd )δ3.24−3.31(2H,m),3.33−3.40(2H,m),3.61(2H,sep, J = 6.1
6
Hz), 4.10−4.14 (2H, m), 4.32−4.37 (2H, m), 4.43 (2H, d, J = 13.3 Hz), 4.88 (2H, d, J = 4.5 Hz), 5.02−5.04 (4H, m),
5.25(2H,d,J=6.7Hz),7.43−7.44(6H,m),7.58(4H,dd,J=4.7and7.3Hz),7.69(4H,d,J=10.7Hz),7.98(4H,d,J
13
=
10.7Hz); CNMR(125MHz,DMSOd )δ64.3,69.8,70.1,71.4,72.9,88.5,92.3,94.0,121.7,127.0,128.9,129.3,
6
−1
1
29.5 (2C), 131.6, 131.7, 165.1; IR (ATR, cm ) ṽ = 754, 804, 856, 935, 984, 1016, 1043, 1076, 1142, 1177, 1271,
1
406, 1441, 1607, 1715, 2365, 2941, 3298 (br); HRMS (APPI): m/z calcd for C H O Cl: 785.2006; found:
4
2 38 13
−
7
85.2043[(M+Cl) ].
Cellular Imaging Study
+
HeLa CD4 cells were grown on cultureslides (96-well plate)and treated with 10% fetal bovineserum, penicillin
o
and streptomycin at 37 C. Under 5% CO atmosphere, 10µL of 1a solution (0.75 to 37.5 µM in DMSO/PBS
2
+
+
(
80/20)) was added to the HeLa CD4 cells on D-MEM medium and incubated. After 90 min, the HeLa CD4
cells werewashed with D-MEM medium two times, thenthenew medium (100µL)was added, andincubated. After
8 h, the cells were washed with PBS (100µL) for two times. The cells were subsequently analyzed using a filter
1
2
3
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European Journal of Organic Chemistry
10.1002/ejoc.201800424
(>488nm)onaBZ-8000fluorescencemicroscope(KEYENCE,Japan).
Acknowledgements
We thank Mr. Takeshi Akimoto and Mr. Shinji Kawaguchi of Chiba Institute of Technology for measurements of
the NMR spectroscopy and high resolution mass spectrometry. We are also grateful to Prof. Dr. Shin-ichiro Kato of
TheUniversityofShigaPrefectureforusefuldiscussionswithrespecttothephotophysicalpropertiesoftrehalose-besed
fluorescentprobes.
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