Bioorthogonal metabolic glycoengineering of human larynx carcinoma (HEp-2) cells targeting sialic acid

Article abstract of DOI:10.3762/bjoc.6.24

Sialic acids are located at the termini of mammalian cell-surface glycostructures, which participate in essential interaction processes including adhesion of pathogens prior to infection and immunogenicity. Here we present the synthesis and bioorthogonal metabolic incorporation of the sialic acid analogue N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex) into the cell-surface glycocalyx of a human larynx carcinoma cell line (HEp-2) and its fluorescence labelling by click chemistry.

Full text of DOI:10.3762/bjoc.6.24

Bioorthogonal metabolic glycoengineering of human
larynx carcinoma (HEp-2) cells targeting sialic acid
Arne Homann1, Riaz-ul Qamar1, Sevnur Serim1, Petra Dersch2
and Jürgen Seibel*1
Full Research Paper
Open Access
Address:
Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
1University of Würzburg, Department of Organic Chemistry, Am
Hubland, 97074 Würzburg, Germany and 2Helmholtz-Centre for
Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany
doi:10.3762/bjoc.6.24
Received: 01 December 2009
Accepted: 17 February 2010
Published: 08 March 2010
Email:
Jürgen Seibel* - seibel@chemie.uni-wuerzburg.de
Guest Editor: T. K. Lindhorst
* Corresponding author
© 2010 Homann et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
bioorthogonal metabolic glycoengineering; click chemistry; sialic acid
Abstract
Sialic acids are located at the termini of mammalian cell-surface glycostructures, which participate in essential interaction processes
including adhesion of pathogens prior to infection and immunogenicity. Here we present the synthesis and bioorthogonal metabolic
incorporation of the sialic acid analogue N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex) into the cell-surface glycocalyx of a
human larynx carcinoma cell line (HEp-2) and its fluorescence labelling by click chemistry.
Introduction
The surface of eukaryotic cells is heavily covered with glycan processes [4]. Recently, it was reported that neuraminic acid
structures of various types forming the individual, dynamic analogues enter the cell by pinocytosis and are incorporated into
glycocalyx of each cell type. These glycolipids and glyco- the cellular glycosylation machinery by active transporter
proteins often carry sialic acids, in humans N-acetylneuraminic systems [5]. In other mammals N-glycolylneuraminic acid
acid (Neu5Ac, 1, Scheme 1), at their terminal position which (Neu5Gc, 2, Scheme 1) corresponds to Neu5Ac 1 found in
mediate cell-cell recognition and signal transduction processes humans. Although the human gene for the synthesis of Neu5Gc
involved in infection, inflammation or tumor formation [1]. 2 is inactive, small amounts of Neu5Gc 2 are also found in the
Recent studies have shown that the surface of a T-cell line human metabolism presumably dietary derived from carbo-
(Jurkat), Chinese hamster ovary (CHO) cells, cervical adenocar- hydrate salvage pathways [5,6]. The efficient uptake and in-
cinoma (HeLa) cells as well as many other cell types can be corporation of sialic acid modified in positions C-5 and C-9 into
labelled with bioorthogonal, that is metabolically inert, func- human B-lymphoma cells (BJA-B), Jurkat and others including
tionalized carbohydrates both in vitro and in vivo [2,3]. Acet- primary cells has been demonstrated [3,7]. The sialic acid modi-
ylated monosaccharides, for example 2-azidoacetylamino-2- fications influence the interaction with sialic acid binding
deoxy-1,3,4,6-tetraacetyl-β-D-glucopyranoside (Ac4GlcNAz, immunoglobulin-like lectin (Siglec)-2 and infection processes
16), are believed to permeate the cell membrane by diffusion of BJA-B cells by the B-lymphotrophic papovavirus [8]. It was
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Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
further shown that the uptake and incorporation of alkynylated (HEp-2) cells by metabolic glycoengineering. The synthesis of
N-acetylmannosamine (1,3,4,6-tetraacetyl-N-(4- N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex, 3) was
pentynoyl)mannosamine) into six different kinds of cells was achieved by a previously described route [9]. The Petasis coup-
more efficient than the incorporation of its azido derivative ling was performed starting from D-arabinose (4), the
(1,3,4,6-tetraacetyl-N-azido-acetylmannosamine) [3]. In the secondary amine 5 and dibutyl vinyl boronic acid ester 6. In situ
current study, metabolic glycoengineering of human larynx hydrolysis of the bis(4-methoxyphenyl)methyl group with a
carcinoma (HEp-2) cells with N-(1-oxohex-5-ynyl)neuraminic catalytic amount of trifluoroacetic acid (TFA), followed by
acid (Neu5Hex, 3) is demonstrated. The bioorthogonal modifi- N-acylation with the activated ester 7 led to the alkyne 8 in a
cation, that is the introduction of hexyne, was carried out at the yield of 75% based on D-arabinose. A [3+2] cycloaddition reac-
sialic acid acetyl residue at position C-5 which is prone to tion between N-tert-butyl nitrone 9 and 8 and subsequent base-
mammalian evolution processes [5,6].
catalyzed ring-opening and hydrolysis afforded N-(1-oxohex-5-
ynyl)neuraminic acid (Neu5Hex, 3) in 38% yield (Scheme 2).
Metabolic glycoengineering of human larynx
carcinoma (HEp-2) cells by incorporation of N-(1-
oxohex-5-ynyl)neuraminic acid (Neu5Hex, 3)
The metabolic labelling of human larynx carcinoma (HEp-2)
cell surfaces was carried out in order to study and characterize
the influence of sialic acid in cell signalling and cell-cell inter-
actions. HEp-2 cells were investigated because of their meta-
bolic capability to incorporate 2-azidoacetylamino-2-deoxy-
(1,3,4,6)-tetraacetyl-β-D-glucopyranoside (Ac4GlcNAz, 16).
The internalization of this acetylated monosaccharide was
described previously as a diffusion process through the
membrane of eukaryotic cells [3]. Neu5Hex (3) is a new
substrate for metabolic glycoengineering which is proposed to
be incorporated into the cell surface glycan structures. It was
Scheme 1: The natural forms of sialic acids, human N-acetyl-
neuraminic acid (Neu5Ac, 1) and mammalian N-glycolylneuraminic
acid (Neu5Gc, 2). N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex, 3) is
used for bioorthogonal metabolic labelling of human larynx carcinoma
(HEp-2) cells.
Results and Discussion
Synthesis of the sialic acid analogue N-(1-oxohex-5- shown that carbohydrates in growth media contribute to altera-
ynyl)neuraminic acid (Neu5Hex, 3)
tions in glycosylation patterns in human cells [8,10]. The
The bioorthogonality of N-(1-oxohex-5-ynyl)neuraminic acid bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-
(3) was exploited to incorporate it into human larynx carcinoma acetylmannosamine kinase (GNE) is the key enzyme in sialic
Scheme 2: Synthesis of N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex 3).
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Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
acid biosynthesis. The inhibitory effect of the sialic acid [13]. Ac4GlcNAz 16 or Neu5Hex 3, respectively, were incu-
concentration towards the UDP-N-acetylglucosamine bated with HEp-2 cells. Ac4GlcNAz 16 is believed to enter the
2-epimerase/N-acetylmannosamine kinase (GNE) by allosteric cell by diffusion through the membrane, to undergo deacetyla-
effects is known [11]. Recently, the regulation of UDP-GlcNAc tion in the cytoplasm and then incorporated into the cell surface
2-epimerase/ManNAc kinase expression on the transcriptional glycoproteins and glycolipids. Alternatively, it is metabolically
level by DNA methylation was demonstrated [12] and a genetic converted to Neu5Az [14].
feedback regulation for this process was proposed (Scheme 3)
Scheme 3: Metabolic pathway of Ac4GlcNAz and the genetic control of Neu5Ac 1 synthesis by feedback inhibition. The accumulation of Neu5Hex 3 is
proposed by incubation of 3 with the target cell line as the synthesis of Neu5Ac 1 is down-regulated [11,13]. UDP: uridine diphosphate; GNE: UDP-N-
acetylglucosamine 2-epimerase/N-acetylmannosamine kinase; ATP: adenosine triphosphate; PEP: phosphoenolpyruvate; CTP: cytidine triphosphate;
PPi: pyrophosphate; DNA: deoxyribonucleic acid; mRNA: messenger ribonucleic acid.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
Scheme 4: Proposed metabolic pathway of Neu5Hex 3 based on known mechanisms of Neu5Gc 2 uptake [5]. TGN: trans-Golgi network, CMP:
cytidine monophosphate, Sia: sialic acid.
Neu5Hex may enter the cell by the previously described pino- addition [15,16]. The appropriate functionalized fluorescent
cytosis processes (Scheme 4) or by an, as yet, unknown intern- detection molecule and the conditions for the click reaction
alization mechanism [5]. It is believed that Neu5Hex enters the (CuSO4, sodium ascorbate and tris[(1-benzyl-1H-1,2,3-triazol-
nucleus and enhances the genetic feedback control of the GNE 4-yl)methyl]amine, TBTA) were applied in dimethylsulfoxide
coding gene which blocks the synthesis of natural Neu5Ac (DMSO) (Scheme 5). After one hour, the cells were analyzed
[11,13]. Alkyne- or azide-functionalized carbohydrates in the by microscopy (phase contrast) at the appropriate wavelength
glycocalyx are specifically addressed by complementary func- for fluorescence imaging. Although the incubation of HEp-2 in
tionalized fluorescence agents 9-[2-carboxy-4-[(2-propyn-1- DMSO and in the presence of copper ions is cytotoxic, the
ylamino)carbonyl]phenyl]-3,6-bis(dimethylamino)xanthylium, fluorescence in the labelled glycocalyx was clearly detectable.
alkynylated TAMRA or benzoic acid 2-[6-(3-azidopro- In order to analyze the natural background fluorescence of
panyloxy)-3-oxo-3H-xanthen-9-yl] 3-azidopropanyl ester, HEp-2, one sample was incubated without any additional carbo-
azido-fluorescein (14).
hydrates. The cells were analyzed by fluorescence microscopy
(580 nm for TAMRA staining and at 525 nm for fluorescein).
For the metabolic labelling of eukaryotic cells, HEp-2 cells At either wavelength, the negative control does not show any
were incubated in Dulbecco's modified Eagle's medium significant background fluorescence (Figure 1). In both the
(DMEM) with 10% fetal calf serum (FCS). At 80% confluence Neu5Hex fed HEp-2 and the incubation with Ac4GlcNAz a
they were split into 6-well plates with DMEM containing the clear staining of the cellular glycocalyx at the expected
functionalized carbohydrates (Ac4GlcNAz 16 or Neu5Hex 3, 25 wavelengths was observed.
µM, 48 h). HEp-2 cells were harvested with a cell scraper, not
trypsin, in order to preserve the partially protein-coupled Conclusion
glycocalyx. To highlight the successful incorporation of the Sialic acids are prominent sugars which are located in the
azide and alkyne functionalities into the glycocalyx of HEp-2 terminal position on cell-surface glycans. Although it has been
cells, the fluorescence labelling reaction was performed known for many years that sialic acids are involved in myriads
according to a modified protocol of the [3+2] triazole cyclo- of interaction processes including viral infections such as the
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Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
Scheme 5: Labelling of alkynylated neuraminic acid by azido-fluorescein.
tyne (DIFO) and cell-surface azido-glycans introduced recently
has been proven to be suitable for in vivo labelling [14,17,18].
Experimental
2-azidoacetylamino-2-deoxy-1,3,4,6-tetraacetyl-β-D-gluco-
pyranoside (16) was synthesized as described previously [9]
N-(1R,2R,3S,4R)-Hex-5-yonic acid (2,3,4,5-tetrahy-
droxy-1-vinyl-pentyl)-amide (8)
A solution of D-arabinose (1.09 g, 5.73 mmol, 4), 4,4’-dimeth-
oxybenzhydrylamine (1.39 g, 5.73 mmol, 5) and vinyl boronic
acid dibutyl ester (2.51 g, 11.46 mmol, 6) in aqueous ethanol
(60 mL, ethanol/H2O = 4:1) was stirred at 50 °C for 72 h. TFA
(1.79 ml) was added and the reaction mixture stirred for a
further 16 h. The solvent was evaporated and the residue
dissolved in MeOH (30 mL). Sodium bicarbonate (974 mg,
11.59 mmol) and 5-hexynoic acid, 2,5-dioxo-1-pyrrolidinyl
ester [15] (950 mg, 8.5 mmol) was added and the solution
stirred for 1 h at room temperature. The solids were removed by
Figure 1: Top left: HEp-2 cells incorporated with Ac4GlcNAz 16,
labelled with alkynylated TAMRA at 580 nm. Bottom left: HEp-2 cells
with incorporated NeuNHex 3, labelled with azido-fluorescein 14, at
525 nm. On the right: background and unspecific staining controls at
the same wavelengths as the corresponding pictures on the left.
emerging flu variants, their biological role on cell surfaces of filtration, the filtrate was dried and the residue purified by flash
different cell lines and at different development states remains chromatography on silica gel (eluted with CH2Cl2 and MeOH)
unclear. As new techniques for probing glycans have evolved to afford 8 as white solid in 75% yield. Rf = 0.34 (CH2Cl2/
only relatively recently, more information about the funda- MeOH, 7:1); [α]D20 : +19.8 (c 1, MeOH); 1H NMR (400 MHz,
mental biological functions of carbohydrate structures can be CD3OD) δ = 6.05 (ddd, J = 17.29, 10.53, 5.66 Hz, 1H, 1”-H),
obtained. Therefore we introduced metabolic glycoengineering 5.27 (td, J = 17.29, 1.52 Hz, 1H, 2”-H), 5.22 (td, J = 10.54, 1.52
of the human larynx carcinoma cell line HEp-2. The incorpor- Hz, 1H, 2”-H), 4.56 (m, 1H, 1-H), 3.84–3.50 (m, 5H, 5-H2,
ation and cell surface presentation of Ac4GlcNAc 16 as well as 4-H2, 3-H, 2-H), 2.50 (t, J = 7.26 Hz, 2H, 2’-H2), 2.30–2.20 (m,
the new substrate Neu5Hex 3 was successful. The copper-cata- 3H, 6’-H, 4’-H2), 1.90–1.70 (m, 2H, 3’-H2); 13C NMR: (100
lyzed [3+2] triazole formation (“click reaction”) proved very MHz, CD3OD) δ = 175.29 (CO), 137.04 (C-1”), 116.65 (C-2”),
useful for the cell surface labelling because of its bioorthogon- 84.12 (C-5’), 72.46, 72.11, 71.55 (C-4, C-3, C-2), 70.34 (C-6’),
ality. The incubation of HEp-2 cells with the sialic acid 64.94 (C-5), 55.11 (C-1), 35.89 (C-2’), 25.77 (C-3’), 18.58
analogue Neu5Hex 3 guarantees its direct incorporation into the (C-4’); MS (ESI): m/z [M+Na]+ calculated for
cell surface glycan patterns bypassing metabolic bottlenecks. C1 3 H2 0 NO5 [Na]+ , 294.14, found 294.1.
Furthermore, the described genetic feedback inhibition by sialic
acid leading to an accumulation of the fed Neu5Hex 3 ensures Synthesis of N-(hex-5’-ynoyl)neuraminic acid
an efficient integration into the cell surface glycocalyx. A draw- (1”S,2”R,3”S,4”R)-2-tert-butyl-5-(1”-(hex-5’-
back of the reaction parameters and compounds used for the ynoyl)amino-2’’,3’’,4’’,5’’-tetrahydroxy-pentyl)-
click reaction is the cytotoxicity of DMSO and copper. But this isoxazolidine-3-carboxylic acid ethyl ester
problem for in vivo labelling can be overcome by different reac- Polyhydroxy olefin (1.50 g, 5.53 mmol, 8) and nitrone (2.01 g,
tion conditions and different detection molecules. For example, 11.6 mmol, 9) in dioxane (100 mL) were stirred at 30 °C for
the strain-promoted click reaction with difluorinated cyclooc- 14 d. After complete conversion of the starting material as
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Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
monitored by TLC, the solvent was removed at reduced pres- (1:1, 25 mL) and the reaction mixture stirred overnight. The
sure. The residue was purified by normal silica gel chromato- crude mixture was concentrated under reduced pressure, diluted
graphy (MeOH/CH2Cl2, 1:10 to 1:5) to afford the ester as with water and extracted with EtOAc (3 × 25 mL). The
colourless oil (2.06 g, 4.51 mmol) in 82% yield. Rf = 0.42 combined organic layers were dried, concentrated followed and
(CH2Cl2/MeOH, 7:1); [α]D20 = +7.2 (c 1, MeOH); 1H NMR purified by flash chromatography to afford the pure required
(400 MHz, CD3OD) δ = 4.66–4.61 (dt, J = 8.44, 1.56 Hz, 1H, product (1.1 g, 2.2 mmol) in 84% yield. Rf = 0.34 (EtOAc),
5-H), 4.14–4.08 (dq, J = 7.16, 1.54 Hz, 2H, OCH2CH3), 1H NMR (400 MHz, CDCl3), δ = 8.18 (dd, J = 7.80, 1.38 Hz,
3.91–3.87 (t, J = 8.52 Hz, 1H, 1’’-H), 3.86–3.47 (m, 5H, 5’’-H2, 1H), 7.65 (m, 2H, 4-H, 5-H), 7.25 (dd, J = 7.55, 1.23 Hz, 1H,
4’’-H, 3’’-H, 2”-H), 3.32–3.28 (dd, J = 8.70, 0.83 Hz, 1H, 3-H), 3-H), 6.90 (d, J = 2.44 Hz, 1H, 5’’’-H), 6.82 (d, J = 8.91 Hz,
3.23–3.20 (m, 1H, 6’-H), 2.65–2.57 (m, 1H, 4-Ha), 2.40–2.34 (t, 1H, 8’’’-H), 6.78 (d, J = 9.71 Hz, 1H, 1’’’-H), 6.68 (dd, J =
J = 7.18 Hz, 2H, 4’-H2), 2.18–2.06 (m, 3H, 4-Hb, 2’-H2), 8.91, 2.44 Hz, 1H, 7’’’-H), 6.47 (dd, J = 9.71, 1.97 Hz, 1H,
1.79–1.71 (m, 2H, 3’-H2), 1.20–1.15 (t, J = 7.12 Hz, 3H, 2’’’-H), 6.38 (d, J = 1.97 Hz, 1H, 4’’’-H), 4.10 (t, J = 5.95 Hz,
OCH2CH3), 1.05 (s, 9H, C(CH3)3); 13C NMR (75 MHz, 2H, OCH2), 4.01 (m, 2H, OCH2), 3.47 (t, J = 6.50 Hz, 2H,
CD3OD) δ = 177.07 (CONH), 174.22 (COO), 84.19 (C-5’), CH2N3), 2.99 (m, 2H, CH2N3), 2.03, 1.55 (2m, 4H, 2’’-H2,
77.34 (C-5), 72.15, 71.42, 70.63 (C-4’’, C-3’’, C-2”), 70.35 2’’’-H2). 13C NMR (101 MHz, CDCl3), δ = 185.56 (C-3’’’),
(C-6’), 65.27 (C-5’’), 62.50 (OCH2CH3), 62.23 (C-3), 61.21 165.23 (C-1’), 163.10 (C-6’’’), 158.70 (C-4a’’’), 154.12
(C(CH3)3, 53.71 (C-1’’), 39.07 (C-4), 35.92 (C-2’), 25.91 (C-5a’’’), 149.67 (C-9a’’’), 134.15 (C-2), 132.78, 131.31,
(C-4’), 25.91 (C(CH3)3), 25.85 (C-3’), 14.45 (OCH2CH3); MS 130.49, 130.11, 130.08, 129.72, 128.90 (C-3, C-4, C-5, C-6,
(ESI): m/z [M+Na]+ calculated for C21H36N2O8[Na]+ 467.2, C-1’’’, C-7’’’, C-8’’’), 130.27 (C-9’’’), 117.71, 114.91 (C-1,
found 467.2.
C-8a’’’), 113.55 (C-5’’’), 105.88 (C-2’’’), 100.93 (C-4’’’),
65.38 (C-1’’’’), 62.37 (C-1’’), 47.92 (C-3’’’’), 47.77 (C-3’’),
28.45 (C-2’’’’), 27.76 (C-2’’).
N-(Hex-5’-ynoyl)neuraminic acid (3)
Isoxazoline (2.50 g, 5.63 mmol) and NaOMe (0.74 mL of 5.4 M
solution in MeOH) in anhydrous MeOH (100 mL) were stirred Cultivation and metabolic labelling of HEp-2 cells
at room temperature overnight. Water (100 mL) was added and Human larynx carcinoma (HEp-2) cells were cultivated in
the solution stirred for further 24 h. The mixture was then neu- Dulbecco's modified Eagle's medium (DMEM) containing 10%
tralized with acidic ion exchange resin containing formate ions fetal calf serum (FCS) at 37 °C under a 5% CO2 atmosphere. At
(Amberlyte). The solvent was removed under reduced pressure 80% confluence the medium was discarded and the cells
and the crude product subjected for size exclusion chromato- washed with PBS buffer (Gibco). After the addition of 1.5 ml of
graphy with Biogel P2 (Bio-Rad) to afford pure 3 (934 mg) in a trypsin/EDTA mixture, the cells were detached for 5 min at
46% yield. Rf = 0.29 (CH2Cl2/MeOH, 5:2); [α]D20 = -19.04 (c 37 °C. They were supplied with 8.5 ml of fresh medium and
1, H2O); 1H NMR (300 MHz, CD3OD), β-anomer: δ = ppm split in a ratio of 1:10.
4.09–4.02 (m, 1H, 4-H), 4.03–4.00 (d, J = 10.74 Hz, 1H, 6-H),
3.87–3.81 (t, J = 10.29 Hz, 1H, H-5), 3.81–3.79 (dd, J = 11.47, For the metabolic labelling, HEp-2 cells were cultivated as
2.74 Hz, 1H, 9-Ha), 3.74–3.89 (m, 1H, 8-H), 3.64–3.60 (dd, J = described above. Subsequently, at 80% confluence they were
11.21, 5.60, 1H, 9-Hb), 3.52–3.49 (d, J = 9.35, 1H, 7-H), seeded into 6-well dishes and incubated in 2 ml of the medium
3.23–3.20 (m, 1H, 6’-H), 2.42–2.38 (t, J = 7.35 Hz, 2H, 4’-H2), described above. The medium contained 25 μM of the modified
2.26–2.20 (m, 4H, 4-Ha, 4-Hb, 2’-H2), 2.17–2.11 (dd, J = 12.83, carbohydrate to be incorporated (Ac4GlcNAz 16 or Neu5Hex
4.87, H-3eq), 1.86–1.80 (m, 3H H-3ax, 3’-H2); 13C NMR (75 3). The incubation time was 48 hours. The cells were detached
MHz, CD3OD), δ = 177.00 (2 × CONH), 173.49 (COOH), using a cell scraper in order to retain the glycocalyx. 150 μL
96.49 (C-1), 84.05 (C-5’), 72.03 (C-8), 71.55 (C-6), 70.08 from each well was transferred into an 8-well microscopy
(C-7), 70.03 (C-6’), 67.63 (C-4), 64.68 (C-9), 53.94 (C-5), cultivation slide and filled with 150 μL of the fresh medium.
40.94 (C-3), 35.67 (C-2’), 25.64 (C-3’), 18.49 (C-4’); MS The cells were cultivated at the described growth conditions
(ESI): m/z [M-H]- calculated for C14H21NO9[H]- 360.13, found until reattachment. The medium was discarded and the cells
360.2.
were washed several times with PBS buffer (Gibco). The
labelling reaction was performed in the dark with 2 mM of the
complementary labelling molecule 9-[2-carboxy-4-[(2-propyn-
1-ylamino)carbonyl]phenyl]-3,6-bis(dimethylamino)xanthyl-
ium, alkynylated TAMRA or azido-fluorescein 14) with 2 mM
Benzoic acid 2-[6-(3-azidopropanyloxy)-3-oxo-3H-
xanthen-9-yl] 3-azidopropanyl ester, azido-fluores-
cein (14)
Iodopropyl azide (210 mg, 26 mmol) was added to a solution of CuSO4, 10 mM sodium ascorbate and 2 mM Tris-[(1-benzyl-
fluorescein (1g, 2.6mmol) in a mixture of distilled THF/MeOH 1H-1,2,3-triazol-4-yl) methyl]amine (TBTA) in DMSO. After
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Beilstein Journal of Organic Chemistry 2010, 6, No. 24.
1 h each well was washed several times with DMSO/water (1:1)
and subsequently examined by fluorescence microscopy.
License and Terms
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Creative Commons Attribution License
Acknowledgements
We acknowledge partial support by the Deutsche Forschungs-
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
gemeinschaft (SFB 630).
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