Supporting information for:
Practical labeling methodology for cholinederived lipids and applications in live cell
fluorescence imaging
Caishun Li, Jessie A. Key, Feng Jia, Arpan Dandapat, Soo Hur, and
Christopher W. Cairo*
Alberta Glycomics Centre, Department of Chemistry, University of
Alberta, Edmonton Alberta, T6G 2G2, Canada
*To whom correspondence should be addressed. Tel.: 780 492 0377;
fax: 780 492 8231; e-mail: ccairo@ualberta.ca
SI1
Fluorescence microscopy
Figure SI1. Fluorescence microscopy imaging of live Jurkat T cells with
metabolically labelled choline and cyanine dyes. For each dye, cells were
observed after one of the following conditions: metabolic label and no dye (+-),
no metabolic label and dye (-+), and metabolic label and dye (++). Azido-choline
(5) (a), Alkyne- (3) (b), and 17-octadecynoic acid (9) (c) labelled cells were
probed with complementary fluorophore labels Alkynyl Cy5 (12) (a) and azide
Cy5 (14) (b, c), in the presence of CuSO4 (50 M), L-histidine (50 M) and
ascorbic acid (75 mmol) in PBS buffer for approximately 20 min. DIC images are
shown on the left, and TIRF images (ex 633 nm, em 670 nm) of the same field
are shown on the right. Scale bar represents 10 m.
SI2
Figure SI2. Fluorescence microscopy imaging of live Jurkat cells with
metabolically labelled choline and NBD dyes. For each dye, cells were observed
after one of the following conditions: metabolic label and no dye (+-), no
metabolic label and dye (-+), and metabolic label and dye (++). Azido-choline (5)
(a), Alkyne- (3) (b), and 17-Octadecynoic acid (9) (c) labelled cells were probed
with complementary fluorophore labels PA-NBD (SI14) (a) and NBD-N3 (SI13)
(b, c), in the presence of CuSO4, L-histidine and ascorbic acid in PBS buffer for
approximately 20 min. DIC images are shown on the left, and TIRF images (ex
488 nm, em 520 nm) of the same field are shown on the right. Scale bar
represents 10 m.
SI3
Figure SI3. Fluorescence microscopy imaging of live HeLa cells with
metabolically labelled azido choline (5) and PA-NBD (SI14). Azido-choline (5)
labelled cells were probed with the complementary fluorophore label, PA-NBD
(SI14), in the presence of CuSO4 (50 M), L-histidine (50 M) and ascorbic acid
(75 mmol) in PBS buffer for approximately 20 min. Cells were observed after one
of the following conditions: metabolic label and no dye (+-), no metabolic label
and dye (-+), and metabolic label and dye (++). DIC images are shown on the left,
and TIRF images (ex 488 nm, em 520 nm) of the same field are shown on the
right. Scale bar represents 10 m.
SI4
Figure SI4. Chemical adhesion of labeled polystyrene beads to cells. Jurkat
cells were metabolically labeled with compound 5, or untreated controls. Binding
to fluorescent polystyrene beads (1 m) was detected by flow cytometry. Control
cells incubated with unmodified beads (--), or beads modified with
propargylamine (+-) showed only background labeling. Metabolically labeled
cells incubated with unmodified beads were no different from the other controls (+); however, labeled cells with modified beads showed a substantial increase in
labeling (++). Bead binding to cells was determined by gating on fluorescence
intensity that was greater than the bulk of cell autofluorescence seen in untreated
cells (--). The percentage of cells detected within the gate is indicated in each
panel as a percentage of all cells detected.
SI5
Synthetic protocols
Scheme SI1. Synthesis of amine linkers used for probe synthesis. The 3azidopropylamine linker (12) was synthesized in one step by nucleophilic
displacement.(1) Boc-protected amino-oxy linker (14) was synthesized in
74% total yield over three steps.(2)
3-azidopropan-1-amine (13).
3-azidopropan-1-amine was prepared following a modification of the method
reported by Lewis et al.(1) Bromopropylamine (SI1) (1 g, 4.57 mmol, 1 equiv)
and sodium azide (0.89 g, 13.7 mmol, 3 equiv) were dissolved in water (15 mL)
and heated at 80 oC for 12 h. The reaction mixture was made alkaline using 5%
NaOH (25 mL), followed by toluene extraction (3 x 20 mL). Caution – this
material may be unstable and should be treated with care. The product was stored
in toluene as a stabilizer, yield was determined by 1H NMR (150 mg, 33%). 1H
SI6
NMR (300 MHz, CDCl3): δ 3.41 (t, 2H, 3J = 6.9 Hz), 2.84 (t, 2H, 3J = 6.9 Hz),
1.77 (q, 2H, 3J = 6.9 Hz).
Benzyl 2-bromoethylcarbamate (SI3).
Benzyl 2-bromoethylcarbamate was prepared following the procedure reported
by Carrasco et al.(2) N-Cbz-ethanolamine (SI2) (1.95 g, 10 mmol, 1 equiv) and
methanesulfonyl chloride (0.93 mL, 12 mmol, 1.2 equiv) were dissolved in
dichloromethane (30 mL), and triethylamine (1.8 mL, 13 mmol, 1.3 equiv) was
added dropwise and allowed to stir for 45 min at room temperature. Lithium
bromide (8.7 g, 100 mmol, 10 equiv) and acetone (30 mL) were then added, and
the reaction was allowed to stir at room temperature for approximately 12 h. The
solvent was removed in vacuo, and redissolved in ether (30 mL). The ether layer
was washed with water (40 mL), 0.1 M sodium bisulfite (20 mL), brine (20 mL)
and dried with magnesium sulphate. Solvent was then removed in vacuo resulting
in a yellow oil, which may solidify (2.296 g, 89%). 1H NMR (400 MHz, CDCl3):
δ 7.39 (m, 5H), 5.14 (s, 2H), 3.63 (m, 2H), 3.49 (m, 2H);
13
C NMR (100 MHz,
CDCl3): δ 156.4, 136.5, 128.8, 128.5, 128.4, 67.3, 43.0, 32.7; IR (microscope): ṽ
= 3310, 3062, 2972, 2949, 1682, 1543, 1453, 1273 cm-1; EI-HRMS calculated for
C10H12NO2Br: 257.0051; observed: 257.0055. Rf = 0.30 (1:6 EtOAc/hexanes).
Benzyl 2- (tert-butoxycarbonylaminooxy)ethylcarbamate (SI5).
SI7
Benzyl 2-bromoethylcarbamate (SI3) (1g, 3.89 mmol, 1 equiv), N-Boc-Nmethylhydroxylamine (SI4) (0.73 g, 5.5 mmol, 1.4 equiv), and DBU (0.87 mL,
5.8 mmol, 1.5 equiv) were dissolved in ether and allowed to stir overnight at room
temperature. The reaction mixture was then taken up into ethyl acetate (30 mL)
and washed with water (30 mL), 0.1 M sodium bisulfite (30 mL), and brine (30
mL). Magnesium sulphate was used to dry the organic layer, followed by
evaporation in vacuo resulting in a volatile yellow oil (1 g, 83%). 1H NMR (400
MHz, CDCl3): δ 7.39 (m, 5H), 5.13 (s, 2H), 3.88 (t, 2H, 3J = 4.7 Hz ), 3.45 (m,
2H), 1.49 (s, 9H);
13
C NMR (100 MHz, CDCl3): δ 157.7, 157.1, 136.9, 128.7,
128.3, 128.4, 82.4, 66.9, 39.4, 28.4; IR (microscope): ṽ = 3596, 3066, 2979, 2935,
1705, 1522, 1456, 1252, 1162, 1111 cm-1; EI-HRMS calculated for (M+H - Boc
C4H9 fragment) C11H14N2O5: 254.0903; observed: 254.09104. Rf = 0.65 (1:1
EtOAc/hexanes).
tert-butyl 2-aminoethoxycarbamate (15).
Benzyl 2-(tert-butoxycarbonylamino-oxy)ethylcarbamate (SI5) (0.93 g, 3
mmol, 1 equiv), was dissolved in chloroform (0.3 mL) and methanol (9 mL), and
10% Pd/C (0.74 g) was added. Triethylsilane (4.8 mL, 30 mmol, 10 equiv) was
then added dropwise over 1 h. The reaction mixture was filtered over celite and
dried in vacuo, giving an off-white solid (0.59 g, quant.) 1H NMR (400 MHz,
CDCl3): δ 9.03 (broad s, 1H), 8.20 (broad s, 2H), 4.21 (broad s, 2H), 3.39 (broad
s, 2H), 1.46 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 158.1, 82.5, 72.1, 38.3, 28.6,
SI8
28.5; IR (microscope): ṽ = 3411, 2980, 2937, 1713, 1609, 1479, 1455, 1166, 1111
cm-1; ES-HRMS calculated for C7H17N2O3: 177.1234; observed: 177.1236.
Scheme SI2. Synthesis of bifunctional cyanine-5 dye, 10. Substituted
cationic indoliums SI8 and SI10 were synthesized neat and carried forward
without purification to the condensation step with malonaldehyde dianilide
hydrochloride (SI11).
SI9
2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indolium (SI8).
2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indolium
was
prepared
following
a
modification of the method reported by Kiyose et al.(3) 1,4-butanesultone (SI7)
(0.37 mL, 3.65 mmol, 1.05 equiv) and 2,3,3-trimethyl-3H-indole (SI6) (0.56 mL,
3.5 mmol, 1 equiv) were heated neat for 3 h at 120 oC. The reaction mixture was
allowed to cool to room temperature and washed with ether and the resulting
purple-red sticky solid was then used without further purification. 1H NMR (400
MHz, CD3OD): δ 7.93 (m, 1H), 7.76 (m, 1H), 7.64 (m, 2H), 4.55 (t, 2H, , 3J = 8.0
Hz) 3.30 (s, 3H), 2.89 (t, 2H, 3J = 6.8 Hz), 2.14 (q, 2H, 3J = 8.4 Hz) 1.94 (q, 2H,
3
J = 7.6 Hz), 1.60 (s, 6 H);
13
C APT NMR (100 MHz, CD3OD): δ 196.9, 142.2,
141.4, 130.0, 129.4, 123.4, 115.5, 54.8, 51.3, 50.6, 50.4, 29.3, 27.5, 26.2, 25.2,
22.6, 22.1; IR (microscope): ṽ = 3445, 3051, 2969, 2930, 1647, 1483, 1186, 1038
cm-1; ES-MS calculated for C14H18NO2Na: 318.1; observed: 318.1.
SI10
1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-indolium (SI10).
1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-indolium was prepared following a
modification of the method used by Kiyose et al.(3) 2,3,3-trimethyl-3H-indole
(SI6) (1.6 mL, 10 mmol, 1 equiv) and 3-iodopropionic acid (SI9) (2.8 g, 14
mmol, 1.4 equiv) were combined and heated at 105 oC, neat. The reaction was
allowed to proceed for 2.5 h, and was then quenched with deionized water. A
DCM wash (2 x 20 mL) was performed, and the aqueous layer was lyophilized.
An orange, oily product (2.91 g) was obtained and used without further
purification. 1H NMR (400 MHz, Ac-D6): δ 8.11 (m, 1H), 7.88 (m, 1H), 7.66 (m,
2H), 4.93 (t, 2H, , 3J = 6.4 Hz) 3.29 (s, 3H), 3.12 (m, 2H) 1.69 (s, 6 H); APT 13C
NMR (125 MHz, Ac-D6): δ 198.5, 142.4, 141.5, 129.9, 129.4, 123.8, 116.1, 55.1,
49.0, 44.9, 31.5, 22.2, 15.4, 15.2; IR (microscope): ṽ = 3422, 3034, 2974, 1731,
1626, 1460, 1176 cm-1; ES-HRMS calculated for C14H18NO2: 232.1332;
observed: 232.1329.
SI11
2-((1E,3E,5Z)-5-(1-(2-Carboxyethyl)-3,3-dimethylindolin-2-ylidene)penta1,3-dienyl)-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium (10).
Trimethyl-1-(4-sulfobutyl)-3H-indolium (SI8), carried forward from above,
and malonaldehyde dianilide hydrochloride (SI11) (0.9 g, 3.5 mmol, 1 equiv)
were dissolved in acetic anhydride (15 mL) and allowed to stir at room
temperature for approximately 1 h. The solvent was then removed in vacuo and
the reaction mixture was then run through a short silica plug column (10:1
DCM/methanol) to enrich for the 3,3-dimethyl-2-((1E,3E)-4-(phenylamino)buta1,3-dienyl)-1-(4-sulfobutyl)-3H-indolium intermediate (0.46 g crude, Rf = 0.16
(9:1 DCM/methanol). The mixture (0.46g, 0.98 mmol, 1 equiv) was then
dissolved in acetic anhydride (5 mL) with 1-(2-carboxyethyl)-2,3,3-trimethyl-3Hindolium (SI10) (0.23 g, 0.98 mmol, 1 equiv) and DIPEA (0.17 mL, 0.98 mmol, 1
equiv) with heating at 120 oC for 3 h. The solvent was then removed in vacuo and
the resulting blue, oily, solid was washed with ether (3 x 40 mL). The crude
mixture was purified by C18 RP-HPLC using a 1% TFA in water/acetonitrile
gradient (acetonitrile 5% to 80%). Yield as determined by the integration of an
analytical scale RP-HPLC trace at 254 nm was 23%. 1H NMR (500 MHz,
CD3OD): δ 8.23 (q, 2H, 3J = 13.0 Hz), 7.49 (d, 1H, 3J = 8.5 Hz), 7.35-7.47 (m,
4H), 7.28 (t, 2H, 3J = 8.5 Hz), 7.22 (t, 1H, 3J = 6.0 Hz), 6.65 (t, 1H, 3J = 12.0 Hz),
6.41 (d, 1H, 3J = 14.0 Hz), 6.28 (d, 1H, 3J = 13.5 Hz), 4.36 (t, 2H, 3J = 7.5 Hz),
4.17 (t, 2H, 3J = 6.5 Hz), 2.88 (t, 2H, 3J = 7.0 Hz), 2.80 (t, 2H, 3J = 7.0 Hz), 1.97
(m, 4H), 1.71 (d, 12H, 3J = 6.5 Hz). APT 13C NMR (125 MHz, CD3OD): δ 175.7,
173.9, 156.2, 155.1, 143.4, 142.9, 142.4, 129.9, 129.6, 127.2, 126.6, 125.9, 123.4,
SI12
123.3, 112.5, 111.7, 105.4, 104.0, 51.7, 50.8, 50.3, 49.9, 45.0, 40.8, 32.6, 28.0,
27.9, 27.3, 23.5; IR (microscope): ṽ = 3359, 2998, 2715, 1682, 1492, 1457, 1205,
1338 cm-1; ES-HRMS calculated for C32H38N2O5SNa: 585.2394.; observed:
585.2385.
Scheme SI3. Reaction of the amino-oxy dye 17 with acetone to form an
oxime. To confirm the reactivity of the aminooxy-Cy5 dye, compound 17, it was
reacted with acetone and characterized.
2-((1E,3E,5Z)-5-(3,3-dimethyl-1-(3-oxo-3-(2-(propan-2-ylideneaminooxy)ethylamino)propyl)indolin-2-ylidene)penta-1,3-dienyl)-3,3-dimethyl-1-(4sulfobutyl)-3H-indolium (SI12).
SI13
2-((1E,3E,5Z)-5-(1-(2,2-dimethyl-4,10-dioxo-3,6-dioxa-5,9-diazadodecan-12yl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dienyl)-3,3-dimethyl-1-(4sulfobutyl)-3H-indolium (16) (5 mg, 0.009 mmol) was deprotected in situ with
1:1 TFA/DCM (2 mL) and exposed to excess acetone for one hour at room
temperature. The resulting oxime product was purified by normal phase flash
column chromatography (DCM/methanol) and the resulting product was a blue
solid (2.1 mg, 36%). 1H NMR (500 MHz, CD3OD): δ 8.23 (q, 2H, 3J = 15.5 Hz),
7.49 (d, 1H, 3J = 7.5 Hz), 7.45 (d, 1H, 3J = 7.5 Hz), 7.41 (d, 1H, 3J = 7.3 Hz), 7.37
(m, 2H), 7.28 (m, 2H), 7.22 (t, 1H, 3J = 7.4 Hz), 6.64 (t, 1H, 3J = 12.3 Hz), 6.41
(d, 1H, 3J = 13.8 Hz), 6.27 (d, 1H, 3J = 13.6 Hz), 4.35 (t, 2H, 3J = 6.8 Hz), 4.17 (t,
2H, 3J = 7.0 Hz), 3.89 (t, 2H, 3J = 5.6 Hz), 3.34 (t, 2H, 3J = 5.6 Hz), 2.89 (t, 2H, 3J
= 7.2 Hz), 2.80 (t, 2H, 3J = 6.7 Hz), 1.97 (m, 4H), 1.81 (s, 3H), 1.78 (s, 3H), 1.71
(d, 12H, 3J = 6.8 Hz). APT 13C NMR (125 MHz, CD3OD): δ 175.6, 173.9, 172.4,
157.1, 156.2, 155.2, 143.4, 142.8, 142.4, 129.9, 129.6, 127.1, 126.6, 125.9, 123.4,
123.3, 112.5, 111.9, 105.3, 104.1, 72.3, 51.7, 50.8, 50.4, 49.9, 49.6, 45.0, 41.5,
40.2, 34.6, 30.8, 28.1, 27.9, 27.3, 23.5, 21.7, 15.6; IR (microscope): ṽ = 3288,
3060, 2926, 2856, 2396, 1687, 1481, 1452, 1133, 1105, 1037, 1016 cm-1; ESHRMS calculated for C37H49N4O5S: 661.3418; observed: 661.3411. Rf = 0.79 (5:1
DCM/methanol).
SI14
Scheme SI4. NBD-based dyes used for additional experiments. NBD dyes
were synthesized as previously reported.(4, 5) Dyes were used to visualize the
corresponding reactive groups in labeled cells: alkynes from compound 3 were
labeled with azido-NBD (NBD-N3); azides from compound 5 were labeled with
propargylamino-NBD (PA-NBD); ketones from compound 8 were reacted with
aminooxy-NBD (NBDAO), or NBD-hydrazide (NBDH).
Spectroscopic characterization
Absorption
maxabs maxfluor
Coefficient
 Quantum Brightness
Compound (nm)
(nm)a
(cm-1 M-1)b
Yielda,c
( x)
4
643
663
19.1 x 10
0.21
4.0 x 104
10
4
642
662
16.0 x 10
0.21
3.4 x 104
12
643
662
6.5 x 104
0.15
1.0 x 104
14
4
644
662
12.9 x 10
0.15
1.9 x 104
17
Table SI1. Spectral properties of Cy5 compounds in methanol.
a. Excitation was at 590 nm.
b. Absorption coefficients were measured at 640 nm.
c. Cresyl violet was used as a quantum yield standard (in methanol.
SI15
Figure SI4: Absorbance and emission spectra of cyanine dyes in methanol
SI16
References
1.
2.
3.
4.
5.
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Am. Chem. Soc. 126: 9152-9153.
Carrasco, M. R., Alvarado, C. I., Dashner, S. T., Wong, A. J., and Wong, M. A. (2010)
Synthesis of aminooxy and n-alkylaminooxy amines for use in bioconjugation, J. Org.
Chem. 75: 5757-5759.
Kiyose, K., Aizawa, S., Sasaki, E., Kojima, H., Hanaoka, K., Terai, T., Urano, Y., and
Nagano, T. (2009) Molecular design strategies for near-infrared ratiometric fluorescent
probes based on the unique spectral properties of aminocyanines, Chemistry – A
European Journal 15: 9191-9200.
Key, J. A., and Cairo, C. W. (2011) Identification of fluorogenic and quenched
benzoxadiazole reactive chromophores, Dyes Pigm. 88: 95-102.
Key, J. A., Li, C., and Cairo, C. W. (2012) Detection of cellular sialic acid content using
nitrobenzoxadiazole carbonyl-reactive chromophores, Bioconjugate Chem. 23: 363-371.
SI17