Negatively Charged Yellow-Emitting 1-Aminopyrene Dyes for Reductive Amination and Fluorescence Detection of Glycans

Article abstract of DOI:10.1002/anie.201908063

1-Aminopyrenes with three ω-hydroxylated N-alkylsulfonamido or alkylsulfonyl residues in positions 3, 6, and 8 were prepared, O-phosphorylated, and applied for reductive amination of oligosaccharides. The dyes (?≈20 000 m^?1 cm^?1) with six negative charges (pH≥8) and low m/z ratios enable labeling and fluorescence detection of reducing sugars (glycans) related to the most structurally and functionally diverse class of natural products. Under excitation with a 488 nm laser, the new glycoconjugates emit yellow light of about 560 nm, outperforming (with respect to brightness and faster electrophoretic mobilities) the corresponding APTS derivatives (benchmark dye with green emission in conjugates).

Full text of DOI:10.1002/anie.201908063

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Accepted Article
Title: Negatively charged yellow-emitting 1-aminopyrene dyes for
reductive amination and fluorescence detection of glycans
Authors: Vladimir N Belov, Elizaveta A Savicheva, Guyzel Yu
Mitronova, Laura Thomas, Marvin J Böhm, Jan Seikowski,
and Stefan W Hell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201908063
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10.1002/anie.201908063
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Negatively charged yellow-emitting 1-aminopyrene dyes for
reductive amination and fluorescence detection of glycans
Elizaveta A. Savicheva,a Guyzel Yu. Mitronova,a Laura Thomas,a,b Marvin J. Böhm,a,c Jan Seikowski,d
Vladimir N. Belov,a* and Stefan W. Hella
Abstract: 1-Aminopyrenes with three -hydroxylated N-
alkylsulfonamido or alkylsulfonyl residues in positions 3, 6 and 8 were
prepared, O-phosphorylated and applied for reductive amination of
oligosaccharides. The dyes (ε ~ 20000 M-1 cm-1) with six negative
charges (pH ≥ 8) and low m/z ratios enable labeling and fluorescence
detection of reducing sugars (glycans) related to the most structurally
and functionally diverse class of natural products. Under excitation
with a 488 nm laser, the new glycoconjugates emit yellow light of
about 560 nm, outperforming (in respect of brightness and faster
electrophoretic mobilities) the corresponding APTS derivatives
(benchmark dye with green emission in conjugates).
hydrodynamic radius.5 A high throughput analysis of fluorescent
glycan derivatives is performed on commercial DNA sequencers
equipped with a CGE-LIF module.5c The fluorescent label (R3-NH2
in Scheme 1) applicable in CGE-LIF must have an amino group
suitable for reductive amination, high electrophoretic mobility and
“brightness” (which is the product of the fluorescence quantum
yield and absorption coefficient at the excitation wavelength).
H2N R3
H
R2
N
O
OH
O
R3
(R1O)n
R1O
OH
OH
-H2O
H
R2
(H+)
R1 = H or carbohydrate
R3 = dye/linker
reducing
agent
(H+)
Glycosylation is an enzymatically driven and highly diverse
transformation of proteins, lipids or other noncarbohydrates. The
products of glycosylation (glycoconjugates) have a new chemical
bond formed between a carbohydrate (glycan; donor) and other
molecule (acceptor). Glycoconjugates represent one of the most
structurally and functionally diverse class of natural products
involved in fundamental biochemical processes in living matter.1
Only few specific functions of these complex and carbohydrate-
rich molecules have been well understood so far.2 Further
progress in glycomics and glycobiology depends on the advances
in analytic techniques applicable to complex carbohydrates.
Carbohydrates do not absorb visible light, and for the sensitive
detection by emission they need to be labeled with a fluorescent
tag.3 Capillary gel electrophoresis (CGE) is an important method
H
H
R2
N
R2
N
R2
N
R3
R3
R3
H
H
3
1
2
Scheme 1. Reductive amination of mono and oligosaccharides.
APTS (Scheme 2) emerged as a unique dye for reductive
amination and detection of glycans.5,6,7 The fluorescence of APTS
derivatives is captured in the “green” color channel of the standard
DNA sequencers.5c Conjugates of APTS are excitable with an
argon laser (emission lines 488 nm and 514 nm).5c,5e However,
the performance of APTS as a fluorescent tag providing only one
emission color, moderate brightness and three negative charges,
is limited. To facilitate further progress in glycomics, glycobiology
and analytical chemistry of complex carbohydrates, we developed
bright fluorescent dyes with an aromatic amino group, multiple
negative charges and yellow emission.
for
analyzing glycoconjugates
including
glycoproteins,
glycopeptides and “released” (enzymatically cleaved from the
acceptors) N- or O-glycans.4 The net electrical charge is required
for separation of the analytes by CGE. The native carbohydrates,
except sialic or glucuronic acids, sulphated or phosphorylated
derivatives, are electroneutral and cannot be separated by their
mass to charge ratio (electrophoresis). Importantly, the features
of an “ideal” fluorescent label required for CGE – the electrical
charge, emissive properties and the reactive group – can be
incorporated in one fluorescence dye with (multiple) charges and
the amino group reacting with aldehyde residues in reducing
sugars (Scheme 1). Combined with laser induced fluorescence
detection (LIF), this method allows fast and very fine resolution of
analytes according to their mass to charge ratio (m/z) and
Design and synthesis of the new pyrene dyes. The high-
performing fluorescent tags applicable in the reductive amination
and CGE-LIF of glycans must have an amino group with pKa of
the conjugated acid in the range of 3–4 for the efficient reaction
(Scheme 1) at pH ~ 3, net charge of –3...–6 at pH = 8 (pH of the
buffer solution in CE) to provide high electrophoretic mobility,
solubility in aqueous buffers and stability against reduction with
boranes or borohydrides in a wide pH range (3–8). The absorption
at 488 nm (488) or 505 nm (505) determined the excitation
efficiency with an argon ion laser or solid state laser; high
brightness and the minimal cross-talk with the “APTS channel” in
the detector (low emission of conjugates at 520 nm) are also
required. These features are set by the reaction conditions in
Scheme 1, and the properties of the standard DNA sequencing
equipment used for the separation and detection of the
fluorescent glycan derivatives. APTS is a reference dye with
green emission in conjugates with oligosaccharides (see Figure 2
and Supporting Information). We designed new dyes with yellow
emission in conjugates, trying to minimize the interference with an
APTS detection window.
[a]
Elizaveta A. Savicheva, Dr. Guyzel Yu. Mitronova, Dr. Laura
Thomas, Marvin J. Böhm, Dr. Vladimir N. Belov, and Prof. Dr. S.
W. Hell, Department of Nanobiophotonics, Max Planck Institute
for Biophysical Chemistry (MPIBPC), Am Fassberg 11, 37077
Göttingen, Germany
Website: http://www.mpibpc.gwdg.de/abteilungen/200
Present address: Artesan Pharma GmbH, Germany
Present address: Institut für organische und biomolekulare
Chemie der Georg-August-Universität Göttingen, Germany
Jan Seikowski, Facility for Synthetic Chemistry, MPIBPC
E-mail: vladimir.belov@mpibpc.mpg.de
[b]
[c]
[d]
[*]
Supporting information for this article is given via a link at the end
of the article.
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Table 1. Spectral properties of the dyes and their m/z ratios
Absorption
λmax, nm
(ε, M-1 cm-1)
Emission
λmax, nm
(Φfl)a
Fluor.
lifetime ,
ns
Dye
(m/z)
Solvent
APTSb
424 (20600)
500 (0.95)
aq. PBS
-
(151)
c
APTS-G6
455 (17000)
471
511
H2O
H2O
-
6-H (144)
7-H
544 (0.88)
535 (0.96)
549 (0.97)
563 (0.85)
5.9
5.6
5.9
3.6
477 (22400)
493 (23000)
502
MeOH
MeOH
H2O
7-Me
9
502 (23400)
509 (19500)
550 (0.88)
563 (0.67)
MeOH
H2O
6.3
6.4
13
15
486 (21000)
477 (19600)
534 (0.80)d,e
542 (0.92)
MeOH
4.9
5.8
16 (137)
TEABf,g
Scheme 2. Triple O-phosphorylated 1-aminopyrene trisulfonamides: a) ClSO3H,
65°C, 3 h; b) 5, Et3N, MeCN, r. t., overnight; c) CF3CO2H, r. t., 1 h, then 1 M aq.
Et3N*H2CO3 (pH 8–9), overnight; d) CH3NHCH2CH2OH, aq. MeCN, r. t.,
overnight; e) (CF3CO)2O, CH2Cl2, Et3N, then CH3I, Cs2CO3, DMF, 70 °C, 40 min;
f) MeOH, NaHCO3; g) aq. NaOH, MeOH, r. t.; h) POCl3, (MeO)3PO, r. t., 3 h,
then aq. Et3N*H2CO3 buffer (pH 8–9), overnight.
[a] absolute values of the fluorescence quantum yields (if not stated otherwise);
[b] data from ref. [6b], abs. measured in H2O, emission - in aq. phosphate buffer
at pH =7.4; [c] conjugate with maltohexaose (G6), data from ref. [5b]; [e]
excitation at 375 nm; [e] Rhodamine 6G as a ref. dye with Φfl = 0.9; [f] aq.
Et3N*H2CO3, pH = 8–8.5; [g] fluorescein as a ref. dye with Φfl = 0.9 in 0.1 M
NaOH.
For achieving this goal, we converted the sulfonic acid residues
in APTS (Scheme 2) into more powerful electron acceptors –
sulfonamides. Sulfonamides (represented by dyes 6–9 in Scheme
2) have higher values of the Hammett σ-constants (σm = 0.53, σp
= 0.60 for SO2NH2)8 than ionized sulfonic acid residues in APTS
(σm = 0.05, σp = 0.09).9 The presence of an electron-donating (N-
alkyl)amino group and the acceptor groups in “active” positions (3,
6 and 8) of the pyrene system leads to the “push-pull” dyes10
emitting blue-green (APTS), green (6-R1, 7-H) or yellow (7-Me, 9)
light. The spectral properties of the dyes are given in Table 1 (see
also Supporting Information).
To provide multiple negative charges and high electrophoretic
mobility at pH 8, primary phosphates (R-OPO3H2) are preferred
over phosphonates,11a because their first and second pKa values
are in the range of 1.5-1.9 and 6.3-6.8, respectively.11b In the
electrophoresis buffer solution, one primary phosphate group
introduces two negative charges (phophonates are less acidic
and not fully ionized at pH 8). APTS6b (Scheme 2) was converted
into the relatively stable 1,3,6-tris(chlorosulfonyl)pyrene-8-amine
(4) and then to sulfonamides (6-tBu, 7-H) by reaction with the
corresponding amine (5 or CH3NHCH2CH2OH).
Direct phosphorylation of three hydroxyl groups in 7-H or 8
(reagent h in Scheme 2) followed by hydrolysis12 afforded dyes 6-
H (16%) or 9 bearing six negative charges at pH 8. To improve
the yield, we prepared O-phosphorylated N-(methylamino)ethanol
5, let it react with compound 4, isolated the intermediate 6-tBu and
then converted it into dye 6-H (83%).
Trying to increase the polarity of aminopyrene chromophore even
further, we introduced alkyl sulfonyl groups into positions 3, 6 and
8 of 1-aminopyrene and prepared dyes 13, 15, and 16 (Scheme
3). Alkyl sulfonyl substituents have higher values of the Hammett
σ-constants (σm = 0.56-0.66, σp = 0.68-0.77 for SO2Alkyl)8 than
sulfonamides (σm = 0.53, σp = 0.60 for SO2NH2).8 This indicates
that they are more powerful acceptors than sulfonamides
(compounds 6-R1, 7-R2 and 9 in Scheme 2).
We found that trifluoroacetyl residue is a better protecting group
for 1-aminopyrene than acetyl,13 because it is acid-stable, but can
be easily cleaved under mild basic conditions. Bromination of 1-
(trifluoroacetylamino)pyrene led to tribromide 10 (Scheme 3), an
important precursor to the functionally substituted aminopyrenes.
Palladium-catalyzed cross-coupling14 of tribromide 10 with 3-
mercapto-1-propanol afforded triol 11 with three alkyl aryl sulfide
residues. Oxidation of 11 with hydrogen peroxide in acetic acid in
the presence of sodium tungstate15 led to trisulfonyl derivative 12.
Deprotection of the amino group in compound 12 by heating with
aq. NaOH in methanol gave amine 15 (model compound).
Another model dye with N-methyl group (13) was prepared from
intermediate 11 by N-methylation of trifluoroacetylamino group
and mild hydrolysis of the amide group. Phosphorylation of 12
followed by hydrolysis led to aminopyrene 16 with three primary
phosphate groups attached to alkyl sulfonyl residues.The
phosphate groups in dyes 6-H, 9 and 16 are hydrolytically stable
within a broad range of pH: from 3 (reductive amination
conditions) to 8.3 (electrophoresis) and beyond.
The model pyrenes with N-methylamino groups (7-Me, 9 in
Scheme 2 and 13 in Scheme 3) are structurally similar to the final
products formed in Scheme 1 upon reductive amination of
carbohydrates; they allow to measure the red-shifts in the
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Figure 1. Absorption and emission spectra (aq. Et3N*H2CO3 buffer) of
conjugates with glucose prepared from dyes 6-H and 16.
Figure 1). The electron-donating capacity of the amino group as
a single donor is limited, and this can explain only the small red-
shift observed by transition from sulfonamides to alkyl sulfones.
The brightness of a glycan label is termed as a product of the
extinction coefficient (at 488 nm, as an excitation wavelength) and
the fluorescence quantum yield. The fluorescence quantum yields
of dyes 6-H and 16 (Table 1) are 0.88 and 0.92, respectively . For
APTS conjugates, the extinction coefficient at the maximum (455
nm) is 17000 M1 cm1, and the absorption at 488 nm is ca. 35%
of the maximal value.5b For all new N-alkylated pyrenes (7-Me, 9,
13, 6-H+G, 16+G) we can assume the extinction coefficient at 488
nm to be about 18000 M1 cm1. Therefore, the conjugates of the
new dyes are ca. 3 times brighter than APTS derivatives (under
excitation with the 488 nm laser).
Scheme 3. Triple O-phosphorylated 1,3,6-tris[(3-hydroxypropyl)sulfonyl]-
pyrene-8-amine 16: a) HS(CH2)3OH, DIPEA, Pd2(dba)3, XantPhos, DMF,
100 °C, overnight; b) aq. H2O2, Na2WO4, AcOH, r. t., overnight; c) aq. NaOH,
MeOH; d) POCl3, (MeO)3PO, r. t., then aq. Et3N*H2CO3 buffer (pH 8–9); e) CH3I,
Cs2CO3, DMF.
absorption and emission spectra and extinction coefficients (7-Me,
13 in Table 1). Importantly, dyes 7-Me, 9 and 13 did not participate
in the reductive amination of glucose (even under harsh
conditions). This result may be explained by the additional steric
hindrance pertinent to the planar iminium ion (Scheme 3), in which
two aromatic hydrogen atoms adjacent to the reaction center are
expected “to repulse” the N-methyl group.
Gel electrophoresis. We used APTS as a reference dye,
compounds 6-H and 16 as new reagents, and obtained their
conjugates with glucose (G) and oligomers (maltotriose G3 and
maltoheptaose G7), mannose (M) and oligomers [2-O-, 3-O- and
4-O-(-D-mannopyranosyl)-D-mannoses (M2-2O, M2-3O and M2-
4O), mannotriose (M3), mannotetraose (M4)], as well as 3'- and 6'-
sialyllactoses. Due to the presence of the strong acceptors –
sulfonamido or alkylsulfonyl groups – dyes 6-H and 16 undergo
reductive alkylation more reluctantly than APTS. The detailed
procedures for reductive amination16 and yields of the individual
conjugates17 are given in Supporting Information. Figure 1 shows
absorption and emission spectra of glucose conjugates prepared
from dyes 6-H and 16. The absorption spectra are very similar,
and the emission spectra are practically identical. Therefore, we
preferentially used dye 16 in reductive amination. Figure 2 shows
the gel electrophoresis results obtained with APTS and its
conjugates (lanes 1, 3, 5), as well as compound 16 and its
conjugates with reducing sugars (lanes 2, 4, 6). The conjugates
of dye 16 move faster than the corresponding conjugates of APTS.
Due to higher net charge of dyes 6-H, 16 and their conjugates,
the distances between yellow bands related to them are shorter
than the distances between green bands of APTS conjugates
(Figures 2, S5 and S6). In case of mannobiose isomers, the
selectivity profile of dye 16 is different from that of APTS (Figure
2). Conjugates of M2-2O and M2-3O with APTS move as one spot
(lane 3, fourth spot from the top), and APTS+M2-4O moves slower
Spectral properties of dyes and conjugates. The
photophysical properties of APTS, its conjugate with
maltohexaose (APTS-G6) and the new pyrenes, are given in
Table 1. The dyes in Table 1 form two groups: compounds with a
primary amino group (APTS, 6-H, 7-H, 15 and 16) and dyes with
a secondary amino group (APTS-G6, 7-Me, 9, and 13). Pyrenes
of the first group absorb at 424 nm (APTS) to 486 nm (15).
Compounds of the second group relate to the products formed in
the course of reductive amination (Scheme 1); their absorption
maxima are red-shifted and found in the range from 455 nm
(APTS-G6) to 509 nm (13) (in aqueous solutions). For example,
N-methylation (6-H  9) shifted the absorption maximum to the
red by 31 nm, while the emission underwent bathofluoric shift of
“only” 19 nm. Thus, the Stokes shift reduced from 73 nm (6-H) to
61 nm (9). Importantly, within each group the spectrum of APTS
or APTS-G6 conjugate is separated from the other spectra of the
same group by 42–69 nm (absorption maxima) and by 38–54 nm
(emission maxima): new dyes absorb and emit at longer
wavelengths. This feature is important, as the glycan conjugates
of dyes 6-H and 16 are intended to have minimal emission in the
APTS detection window. Comparing sulfonamides (6-H, 7-Me)
with structurally related alkyl sulfones (13, 16) we observed the
red shift of only 6–9 nm in absorption, while the positions and
shapes of emission bands remained the same (Table 1 and
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biomolekulare Chemie, Georg-August-Universität, Göttingen, Germany) for
recording NMR and mass-spectra.
Keywords: fluorescent probes • glycoconjugates •
electrophoresis • chromophores • donor-acceptor systems
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Figure 2. Gel electrophoresis (migration from “north” to “south”, pH 8.3);
detection by emission (excitation at 365 nm). From down to top. Lane 1: APTS
(lowest; blue), APTS+G, APTS+G3, APTS+G7 (green). Lane 2: dye 16 (green),
16+G, 16+G3, 16+G7 (yellow). Lane 3: APTS, APTS+M, APTS+M2-
2O/APTS+M2-3O (unresolved), APTS+M2-4O, APTS+M3 and APTS+M4. Lane
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are moving faster than the corresponding APTS analogs.18
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“heavy” glycans undetectable with APTS due to very long
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limited brightness. An access to the DNA sequencer with a CGE-
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Acknowledgements
L. T. is grateful to Max Planck Institute for Dynamics of Complex Technical
Systems (Magdeburg) for financial support. We thank Dr. M. Fomin (MPIBPC)
for discussions and help with software. We thank J. Bienert (MPIBPC), Dr. H.
Frauendorf, Dr. M. John and co-workers (Institut für organische und
[17]
Yields were determined by integration of HPLC peak areas for the free
dyes and their conjugates at isosbestic points; see Table S1 in
Supporting Information.
[18] 6-H+G moves slower than APTS+G (Figure S5).
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COMMUNICATION
COMMUNICATION
Bright 1-aminopyrenes (ε ~
20.000 M-1cm-1) with six
negative charges (at pH ≥ 8)
and high mobility in the
E. A. Savicheva, G. Yu.
Mitronova, L. Thomas, M. J.
Böhm, J. Seikowski, V. N. Belov,*
and S. W. Hell
electric field are designed for
labeling and fluorescence
detection of glycans related to
the most structurally and
functionally diverse class of
natural products. Right and
left lanes: new and reference
dyes (lowest bands) and their
conjugates.
Page No. – Page No.
Negatively charged yellow-
emitting 1-aminopyrene dyes
for reductive amination and
fluorescence detection of
glycans
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