Fluorescence Assisted Capillary Electrophoresis of Glycans Enabled by the Negatively Charged Auxochromes in 1-Aminopyrenes

Article abstract of DOI:10.1002/anie.202013187

A compact and negatively charged acceptor group, N-(cyanamino)sulfonyl, is introduced for dye design and its influence on the absorption and emission spectra of the “push–pull” chromophores is demonstrated with 1,3,6-tris[(cyanamino)sulfonyl]-8-aminopyrene. The new sulfonamides, including O-phosphorylated (3-hydroxyazetidine)-N-sulfonyl, are negatively charged electron acceptors and auxochromes. 1-Aminopyrenes decorated with the new sulfonamides have three or six negative charges (pH ≥8), low m/z ratios, high mobilities in an electric field, and yellow to orange emission. We labeled maltodextrin oligomers by reductive amination, separated the products by electrophoresis, and demonstrated their high brightness in a commercial DNA analyzer and the distribution of the emission signal among the detection channels.

Full text of DOI:10.1002/anie.202013187

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: New FACE for glycans: Fluorescence Assisted Capillary
Electrophoresis enabled by the negatively charged auxochromes
in 1-aminopyrenes
Authors: Vladimir N Belov, Elizaveta A Savicheva, Jan Seikowski,
Jeannette I Kast, Christoph R Grünig, and Stefan W Hell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013187
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10.1002/anie.202013187
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RESEARCH ARTICLE
New FACE for glycans: Fluorescence Assisted Capillary
Electrophoresis enabled by the negatively charged auxochromes
in 1-aminopyrenes
Elizaveta A. Savicheva,[a] Jan Seikowski,[b] Jeannette I. Kast,[c] Christoph R. Grünig,[c] Vladimir N.
Belov,*[a,b] and Stefan W. Hell[a]
[a]
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry (MPIBPC), Am Fassberg 11, 37077 Göttingen, Germany, +49 551-201-
2530, vbelov@gwdg.de
[b]
[c]
Facility for Synthetic Chemistry, MPIBPC
Microsynth AG, Schützenstrasse 15, CH-9436, Balgach, Switzerland
Supporting information for this article is given via a link at the end of the document.
Abstract: A compact and negatively charged acceptor group – N- and high mobility in the electric field) and chemically stable. At
(cyanamino)sulfonyl – is introduced to dye design and its influence on present, reductive amination is the most widely employed
the absorption and emission spectra of the “push-pull” chromophores derivatization method applicable to glycans released
is exemplified by 1,3,6-tris-[(cyanamino)sulfonyl]-8-aminopyrene. The (enzymatically or chemically cleaved) from the protein matrix9.
new sulfonamides, including O-phosphorylated (3-hydroxyazetidine)- 8-Aminopyrene-1,3,6-trisulfonic acid (APTS, Figure 1) is up to
N-sulfonyl, are negatively charged electron-acceptors and now the best reagent10 which can be used in CE with fluorescence
auxochromes. 1-Aminopyrenes decorated with new sulfonamides or MS (MALDI TOF) detection.11 However, the overall
have three or six negative charges (pH ≥ 8), low m/z ratios, high performance of APTS as a single fluorescent tag, with one
mobilities in electric field and yellow to orange emission. We labelled emission colour, improvable brightness and three negative
maltodextrin oligomers via reductive amination, separated the charges is limited. Other negatively charged dyes were developed
products by electrophoresis, demonstrated their high brightness in a for CE-LIF and CE-MS analysis of glycans, but their structures are
commercial DNA analyzer and distribution of the emission signal either unknown12, or they emit in the blue spectral region and
among the detection channels.
cannot be excited with 488 nm light. For example, Cascade Blue
[(3,6,8-trisulfo-1-pyrenyloxy)acetic acid hydrazide] is much more
reactive in reductive amination than APTS, was successfully
applied for labelling of glycans, and the conjugates were
separated by CE-LIF13, but its fluorescence cannot be excited with
488 nm light (the absorption maximum at 400 nm). The bright and
negatively charged fluorescent dyes suitable for reductive
amination of carbohydrates are rare. Rhodamines and cyanines
Introduction
Glycosylation – an attachment of a carbohydrate to another
biomolecule – is an important transformation studied in biology,
pharmaceutical science and clinical chemistry1. Glycans – oligo-
and polysaccharides with glycosidic bonds between monomeric
units – participate in the formation of glycoproteins, cellular
homeostasis, immune regulation and diseases2. Further progress
in glycomics and glycobiology depends on the advances in
analytic techniques applicable to sugars, including mass
decolorize
in
reduction
with
borohydrides14;
2,9-
diaminoacridines15 and 2-aminoacridones16 have been applied
successfully, but proved to be rather “dark”; with large Stokes
shifts, low absorption and low fluorescence quantum yields. The
positively charged aminopyrene sulfonamides APTMP and
APTDP (Figure 1) have been reported very recently17. They may
be used in CE with the “reversed” polarity of the electric field
(Agilent). We prepared negatively charged aminopyrene sulfone
PSU and sulfonamide PSN dyes (Figure 1) as brighter
alternatives of APTS having multiple negative charges and higher
mobility in an electric field18. One of the motivations was to reduce
or even eliminate the cross-talk with APTS detection window
(~520 nm in a DNA sequencing equipment). The conjugates of
PSU and PSN dyes have absorption and emission maxima at ca.
500 nm and 560 nm, respectively, six negative charges (pH ≥ 8),
and low m/z ratios. Due to higher absorption at 488 – 506 nm, the
conjugates of PSU and PSN dyes are ca. 3 times brighter than
APTS derivatives, the excitation is performed with the 488 nm or
503 nm light. The new dyes provide different selectivity profile for
isomeric carbohydrates. The aminopyrene core remains an
attractive scaffold for fluorescent dyes whose emission is red-
shifted, after N-alkylation occurred in the reaction with
carbohydrates. Here we report a very compact negatively
,
spectrometry (MS)3,4 chromatographic methods5, capillary
electrophoresis (CE)6, and their combinations7. In most cases,
derivatization of glycans is necessary, because native
carbohydrates do not absorb light and their ionization abilities are
poor. Labelling with bright and well-detectable fluorescent tags
facilitates glycan analysis. Capillary gel electrophoresis with laser
induced fluorescence detection (CGE-LIF) is widely used as a
very sensitive and powerful technique for separation and
detection8. In this method, the enzymatically or chemically
released glycans are labelled with a fluorescent tag, which is
charged, and its conjugates move in an electric field. The high
throughput analysis of fluorescent glycan derivatives is performed
on commercial DNA sequencers equipped with a CGE-LIF
module with a 488 nm (argon) or 505 nm lasers7. For the sensitive
detection, the sugar – dye conjugate must strongly absorb at 488
– 505 nm and emit light with high fluorescence quantum yield.
Additionally, the fluorescent dyes must have a reactive group for
binding with glycans, be multi-charged (to provide low m/z ratio
1
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10.1002/anie.202013187
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RESEARCH ARTICLE
-
charged N-(cyanamino)sulfonyl residue and a more powerful
more powerful acceptor than SO3 . The acidity of NH-proton is
enhanced due to the neighbouring effects of two strong electron-
acceptor residues – SO2 and CN – and delocalization of the
negative charge between two nitrogen atoms:
acceptor
 O-phosphorylated (3-hydroxyazetidine)-N-sulfonyl
group, establish their electronic properties, introduce them into 1-
aminopyrenes, study the optical spectra and mobility of the new
dyes and their conjugates with carbohydrates.
To prove these assumptions, we prepared aminopyrene PCN with
three N-cyanosulfonamide groups. A simple two-step synthesis
involves APTS as a starting material and the corresponding tris-
chlorosulfonyl aminopyrene as an intermediate (Scheme 1). The
new PCN dye has absorption and emission maxima at 454 nm
and 531 nm, respectively (Table 1). A bathochromic shift of 30 nm
increases the absorption of the PCN dye (and its conjugates with
carbohydrates) at 488 nm, so that the detection ability of PCN dye
is 3-4 times higher than that of APTS.
Table 1. Spectral properties of the dyes and their conjugates
with glucose (dye-G) in aqueous solutions (pH 7.3)
Fluor.
Absorption
λmax, nm (ε)a
Emission
λmax, nm (Φfl)b
Dye
lifetime

, ns
APTSc
APTS-G6
PCN
424 (20600)
455 (17160)
454 (23900)
484 (29000)
471 (18000)
496 (30000)
476 (19000)
505
500 (0.95)
511 (0.92)
531 (0.93)
544 (0.92)
544 (0.91)
558 (0.91)
543 (0.92)
564 (0.90)
542 (0.92)
558 (0.95)
-
d
5.1
5.6
5.5
5.6
5.7
5.9
5.8
5.8
5.8
PCN-G
PSN
PSN-G
PAZ
PAZ-G
PSU
Figure 1. Commercial APTS dye, cationic pyrene APTDP, negatively charged
PSN and PSU reported previously; new tris-(N-cyanosulfonamide) PCN and
triple O-phosphorylated tris-(3-hydroxyazetidine)sulfonamide PAZ dyes.
477 (19600)
506
PSU-G
[a] molar extinction coefficient, M−1⋅cm−1; [b] absolute values of the fluorescence
quantum yields; [c] data from ref. 20; [d] data from ref. 10a
Results and Discussion
We also prepared
a new PAZ dye with three O-
Design of new dyes
phosphorylated (3-hydroxyazetidine)sulfonamide groups
(Figure 1). We expected that this dye would have smaller
hydrodynamic radius and provide higher mobility in
The cationic pyrene APTDP, PSN and PSU (Figure 1) have the
powerful acceptor groups (sulfonamide or alkyl sulfone) in “active”
positions of 1-aminopyrene. These groups shift the absorption
and emission maxima to the red and make the new dyes brighter
than APTS17,18. As one of the most intriguing, promising and least
studied, we found the compact N-cyanosulfonamide SO2NHCN
group and introduced it into the aminopyrene. This substituent
was reported in the context of medicinal chemistry19, and its
impact on spectra and electric charge remained unknown. It was
not clear, if this group will be negatively charged at basic pH and
have electron-acceptor properties. Nevertheless, we expected
that due to the presence of cyano group, SO2NHCN would be a
conjugates than its acyclic analogue PSN
.
The
electrophoretic mobility is inversely proportional to
hydrodynamic radius21. Sulfonamides PSN and PAZ differ
only by 6 hydrogen atoms, but the azetidine-containing
molecule of PAZ has 3 cycles and, therefore, enjoys much
less “conformational freedom” than the linear sulfonamides
PSN and PSU
.
2
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Scheme 1. (A) Synthesis of the new dyes; (B) reductive amination of glucose with new dyes.
Due to the presence of sulfonyl groups (into which the lone pair
on nitrogen may delocalize), the inversion barrier of nitrogen in N-
sulfonyl azetidines is possibly lower than 30 kJ/mol observed for
azetidine itself or N-alkylated derivatives. This became visible in
the sum of the bond angles centered at the nitrogen atom (334°
for N-methylsulfonylazetidine)22, which is higher than the value for
azetidine (320°). We measured 1H-NMR spectra of 3-hydroxy-N-
(3-/4-fluorophenyl)sulfonyl azetidines at +25°… -100 °C in
diethylether-d10, and observed no broadening or signal splitting,
which indicates the free inversion of the nitrogen “pyramide” in 3-
hydroxy-N-arylazetidines and the absence of syn-anti isomerism
in PAZ.
In conjugates with glucose (G), PAZ-G demonstrated a small (+6
nm) red-shift of the emission maximum (Table 1) compared to
PSN-G and PSU-G. This feature is important, as it further reduces
the cross-talk with APTS detection window. For all dyes, the
Stokes shifts are rather large (60-77 nm), and the fluorescence
quantum yields high (>0.9). This combination is valuable and rare,
because large Stokes shifts are often associated with reduced
emission efficiencies. The large Stokes shifts allow more freedom
in choosing the excitation wavelength and detection window,
which is an important condition for the envisaged cross-talk
reduction between colour channels in DNA analyser (APTS on
the one hand, and PSN/PSU/PAZ on the other hand).
(mesomeric) effect of the group (transferred via -systems, d-
orbitals and their combination). Taft demonstrated that it is
possible to separate the polar (휎I) and resonance (휎R0 ) effects25a
.
The chemical shifts in 19F-NMR spectra relate to the electron
density on fluorine nuclei and are very sensitive to the nature of
meta- or para-substituents in the benzene ring. Therefore, the
chemical shifts in 19F-NMR spectra of meta- and para-substituted
fluorobenzenes allow to calculate 휎퐼 and 휎R0 values of the new
groups25. There is a good linear correlation between 19F shielding
parameters (derived from the values of 19F chemical shifts) and
the values of 휎I and 휎R0 . The inductive 휎I and resonance 휎R0
constants of the new substituents may be estimated by fitting the
measured 19F NMR shielding parameters to straight line plotted
for other groups with already known values of 휎I and 휎R0. Figure 2
illustrates this approach, provides the required values for the new
substituents (SO2NHCN and SO2NC3H5OH) and quantifies their
polar and resonance effects. A good correlation is observed with
R2 = 0.92 and 0.94 for parameters 휎I and 휎R0, respectively.
Knowing the values of 휎I and 휎R0, it is possible to calculate the
Hammett substituent constants 휎푝 and 휎푚 according to eq. 1 and
2:25
휎푝 = 휎I + 휎R0 (1); 휎푚 = 휎I +

휎R0; 0 < 훼 < 1 (2)
The physical sense of eq. 1 and 2 follows from the clear
separation of the polar and resonance effects: 휎푝 is simply the
sum of the parameters reflecting these two effects, while 휎푚
includes only a constant part of the resonance effect (due to less
Evaluation of electronic effects and their influence on
spectra
We considered the values of Hammett 휎푝 and 휎푚 constants for
the acceptor substituents “opposing” the donor amino group in 1-
aminopyrene and providing the red-shifts in the spectra. These
values, as well as the field and resonance parameters (휎I and 휎R0 ),
for SO2NHCN and SO2NC3H5OH (N-[3-hydroxyazetidine]sulfonyl)
groups were unknown23. The new substituents are interesting as
polar acceptor groups not only for 1-aminopyrene, but also for
other “push-pull” chromophores24. We assessed the donor-
acceptor properties of SO2NHCN and SO2NC3H5OH groups by
calculating their 휎I and 휎R0 values and included them into the row
of common electron-acceptor groups. The parameter 휎I reflects
the polar effect of the substituent – combination of the inductive
and field effects (transferred along -bonds and through space).
efficient conjugation with the meta-position). According to Taft25c
,
we applied α ≈ 0.5. We compared the calculated values of 휎푝 and
휎푚 for the new groups (SO2NHCN and SO2NC3H5OH) with the
known data for sulfonamides and sulfones (Table 2). Importantly,
N-(cyanamino)sulfonyl group turned out to be a stronger acceptor
than sulfonic acid, but weaker than unsubstituted sulfonamide. N-
(3-hydroxyazetidine)sulfonyl group is a stronger acceptor than
N,N-dimethylaminosulfonyl residue; though both are tertiary
sulfonyl amides.
The parameter 휎R0 is associated with
a pure resonance
3
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10.1002/anie.202013187
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RESEARCH ARTICLE
19F NMR method that allowed to determine the complete set of 휎
values with their predictive force.
Figure 2. Linear correlation of 19F NMR shielding parameters 훿퐻푚(푋) and
훥훿퐻푝(푋)−푚(푋) (ref. to fluorobenzene) with σI (A) and σ0R (B); 19F NMR
spectra were measured in this study; the known values of 휎I and 휎R0 are
taken from ref. 23.
Figure 3. Correlation of 휎푝 with the positions of absorption (A) and
emission (B) maxima of the free dyes and their conjugates with glucose.
Table 2. Hammett 휎푝 and 휎푚 values for sulfonamides, sulfones,
and sulfonic acid
Figure 2 lists the 휎I and 휎R0 values for electron acceptors. This
data and휎푝, 휎푚 values in Table 2 indicate the trend and suggest
substituents useful for the design of the new “push-pull” dyes24. If
an electron-donor (“push”) group is directly attached to the
aromatic system, it can be opposed by one or several electron-
acceptor (“pull”) groups selected from sulfonic acid, N-
(cyanamino)sulfonyl, sulfonamide and alkylsulfone residues. Alkyl
sulfonates are the strongest acceptors, but they are too reactive.
The previous studies report the correlations between
photophysical properties (e.g., positions of absorption and/or
휎푝
0.35a
Substituent
휎푚
0.30a
-
SO3
SO2NHCN
SO2NH2
0.55b
0.49b
0.60a
0.53a
SO2NMe2
SO2Alk
0.65a
0.51a
0.72-0.77a
0.74b
0.60-0.66a
0.58b
SO2NC3H5OH
[a] data from ref. 23; [b] calculated values of the present work.
fluorescence maxima) and Hammett 휎푝 constants26,27
. We
evaluated the relationship between the positions of the absorption
and emission maxima of new dyes (and their conjugates) and
Hammett 휎푝 constants of the substituents present in the
structures (Figure 3). Remarkably, a better correlation was
observed for absorption (R2 = 0.97; 0.99) than for emission (R2 =
0.89; 0.93). All these results support the power and accuracy of
Reductive amination and detection of sugars
The reductive amination of sugars is a two-step process involving
the reversible formation of the Schiff base, which is then
irreversibly reduced to a stable secondary amine. The influence
of concentration, temperature, time28, the nature of acid29 and
reducing agent10d were optimized for APTS. Further
4
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RESEARCH ARTICLE
improvements such as evaporative reductive amination have
been published recently30. We found that the new compounds
PCN and PAZ are applicable for reductive amination of sugars
under water-free conditions (2-picoline-borane complex and
saccharides were well resolved and detectable up to G12. APTS
and PCN in lanes 1 and 2 have the same net electrical charge
(q=-3), but due to slightly higher molecular mass, PCN conjugates
move slower. PCN dye is fully ionized at pH 8.3. Due to much
malonic acid) reported previously for PSN and PSU dyes18. APTS, higher brightness, PCN dye is perspective as an alternative or
PCN and PSN were applied in recovery experiments with glucose
using classic protocol with NaBH3CN and citric acid31.
complementary reagent (in the sense of separation performance
and selectivity towards isomeric carbohydrates) to APTS.
Figure 4. Normalized absorption (dashed lines) and emission (solid lines)
spectra in aqueous buffer (pH 7.3) of conjugates with glucose prepared from
APTS, as well as new PCN and PAZ dyes.
The conjugates with glucose were prepared, isolated, and
characterized by 1H-NMR, HR-MS, UV/Vis and fluorescence
spectra (Table 1, Figure 4 and Supporting Information). As
mentioned above, the red shift in absorption and emission is
important, as it reduces the cross-talk with APTS detection
window (shown with a vertical line at 510 nm in Figure 4). The
absorption maxima of PCN and PAZ conjugates nicely match the
emission lines (488 nm or 505 nm) of the excitation lasers used
in DNA analysers. Higher absorption at 488 nm and 505 nm
improves the brightness of the new fluorescent tags by a factor of
3-4 (compared with APTS).
Recovery experiments were performed with glucose and three
dyes – APTS, PCN and PSN taken in 5 or 10-fold excess31. The
isolated and purified compounds APTS-G, PCN-G and PSN-G
were used as standards for determining the conversion degrees
by means of HPLC analysis. Using the reagents which are most
widely applied for labelling of glycans – NaBH3CN in THF and aq.
citric acid (40°C, 18 h)31 – we observed high recovery degrees of
90–120% for glucose and each of these dyes (see Supporting
Information for details).
Figure 5. PAGE plates (migration from “north” to “south”, pH 8.3) show
maltodextrin ladder labelled with negatively charged aminopyrenes from Figure
1. The fluorescent spots under excitation with 365 nm light. Left pane: triple-
charged APTS and PCN (lanes 1 and 2, respectively) and PSN (lane 3) having
6 charges. Right pane: triple-phosphorylated pyrenes PSN, PAZ and PSU
(lanes 3, 4, and 5 respectively). Weak horizontal lines enable to compare the
positions of spots.
Conjugates of all phosphorylated dyes (Figure 5, lanes 3-5) with
6 negative charges move faster than APTS and PCN derivatives
having three negative charges (lanes 1, 2). The m/z ratio of PAZ
is not lower than m/z values of PSN and PSU (Figure 1), but PAZ
(lane 4) shows higher mobility in conjugates. It is visible for
heavier oligomers (G8–G12) on the right panel of Figure 5. Due
to the gel’s sieving effect, electrophoretic mobility of a labelled
sugar depends on the size (shape) of the molecule21. Azetidine-
containing PAZ dye has 3 cycles and therefore, it is more compact
and has less “conformational freedom” than linear sulfonamides
(PSN) or alkyl sulfones (PSU).
Desialysation study was performed for PCN dye. The loss of sialic
acid residues often occurs if glycans are kept for longer times in
acidic media at elevated temperatures28. Two commercially
available 3’- and 6’-sialyllactoses as well as lactose were labelled
with PCN. To show the separation power of the dye, we analyzed
the reaction mixtures by HPLC and PAGE. We measured the
peak areas of the sialylated and desialylated (lactose) conjugates
in HPLC. A slight degree of desialysation was observed (ca. 5%;
see Supporting Information for details).
PAGE electrophoresis
We labelled commercially available maltodextrin oligosaccharides
ladder (G2 to G15, Carbosynth) with new dyes PCN and PAZ,
previously reported PSN and PSU, as well as APTS. We used a
water-free protocol for reductive amination18, in which the excess
of free dye had been removed prior to electrophoresis by flash
chromatography on reversed phase (15C18AQ-F0025 cartridge).
Then the mixtures of conjugates were analyzed by PAGE
electrophoresis according to P. Jackson32 and separated into
individual fluorescent zones (Figure 5). The runs with APTS (q=-
3, SO3H) and PCN (q=-3, SO2NHCN) are shown in lanes 1 and 2,
and the runs with phosphorylated six-charged PSN, PSU and
PAZ dyes (q=-6, OPO(OH)2)  in lanes 3-5. The conjugates
migrate according to their charge-to-mass ratio, or, more strictly,
the charge-to-hydrodynamic volume21. The bands of individual
5
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RESEARCH ARTICLE
Capillary electrophoresis with laser-induced fluorescence Conclusion
detection (CE-LIF)
Commercial DNA sequencers represent standard devises for
glycan analysis7. The DNA analyzers are equipped with LIF
detectors and excitation sources. These devises offer the
possibility of multicolour detection33. There are 5 colour channels
– blue, green, yellow, orange and red – reserved for four
nucleotides and a reference (DNA ladder labelled with a red-
emitting dye). We used the ABI 3730 XL DNA Analyzer platform
having a CE-LIF unit and a laser diode emitting 503 nm light.
Figure 6 illustrates the CE-LIF results obtained with glucose
oligomers labelled with APTS (~1 pmol), PCN (~1 pmol) and PSU
(~0.1 pmol) dyes. The PCN dye provides ca. four-fold better
signal level than APTS, but somewhat higher cross-talk with the
“green” detection window. If the dye is used alone in the
application (this is now the case with APTS), the cross-talk with
any detection windows, except the red one, may be tolerated. The
PSU dye in the green detection channel provides the similar
signal level as PCN. However, nearly half of the emission “leaks”
into the yellow slot.
With the colour settings standard for a DNA analyser, the PSU
dye has a non-zero cross-talk with the “blue” detection window
(positive or negative signal; it depends on concentration of PSU
conjugates). The recalibration of the colour settings of a DNA
analyser is possible by using a new set of fluorescent dye
conjugates and creating a new matrix file33. For the PSU dye, that
would enable to collect more light, which is now distributed
between green and yellow detectors, and probably fully avoid the
cross-talk with the APTS detection window. If so, PSU or acridine
dyes15 could be used not only as brighter or “rapid” (swifter
moving) alternatives to APTS (which is now possible), but also for
creating new internal standards for glycan analysis based on
natural carbohydrates and compatible with APTS in one run.
We introduced a compact and negatively charged acceptor group
– N-(cyanamino)sulfonyl – to dye design and demonstrated its
influence on the absorption and emission spectra of the “push-
pull”
chromophores
(exemplified
by
1,3,6-tris-
[(cyanamino)sulfonyl]-8-aminopyrene). N-(Cyanamino)sulfonyl or
O-phosphorylated (3-hydroxyazetidine)-N-sulfonyl residues are
electron-acceptors and auxochromes. Three of them confer three
or six negative charges (pH ≥ 8) to the dye, provide low m/z ratios,
high mobilities in electric field and the red-shifted absorption and
emission spectra. Aminopyrenes were decorated with these
sulfonamides, conjugated with reducing sugars and compared
with the corresponding APTS derivatives, as references. We
found that the brightness of the new dyes is superior to APTS.
Conjugates of APTS, PCN and PSU dyes were analyzed by
means of CE-LIF in a DNA sequencer. An average peak width at
half maximum is ca. 30% smaller for PSU-conjugates, compared
with APTS- or PCN-labelled sugars: an advantage for an
analytical reagent. The PCN derivatives were detected in the blue
channel (the same as for APTS) with ca. 4-fold higher signal
intensity than APTS derivatives (at equal amounts); a low cross-
talk with a green detection window was detected. The signal of
PSU derivatives was equally distributed between the green and
yellow detectors. We expect that the new color calibration (which
is technically possible) will further improve the signal intensity by
collecting emission in one detection window; e.g., by a factor of 2
for PSU detection. By using new fluorescent dyes for glycans, we
plan to recalibrate the color settings of commercial DNA analyzers.
The new internal standards based on dye-glycan oligomers of
various lengths and detected separately from APTS-glycan
conjugates are highly needed for improving the precision of
glycan analysis. The “ideal” standards (not yet available) based
on natural glycans (not on DNA strands, like now) and co-
injectable with analytes (glycans labelled with APTS or other dye)
are expected to properly align the retention times, compensate
drift, and make the double calibration unnecessary. Thus, new
dyes superior to APTS or complementary to it (in emission,
electric charge and mobility) are expected to accelerate the
progress in glycomics.
Acknowledgements
The authors are grateful to Dr. Michael John, Dr. Holm Frauendorf and
their co-workers (Institut für Organische und Biomolekulare Chemie,
Georg-August-Universität Göttingen) for recording high resolution NMR
and mass-spectra. We gratefully acknowledge the excellent assistance of
Mr. Jürgen Bienert in HPLC analysis (MPI BPC).
Keywords: fluorescent probes • glycoconjugates •
electrophoresis • chromophores • donor-acceptor systems
Figure 6. CE-LIF of maltodextrin ladder labelled with APTS (1 pmol), PCN (1
pmol) and PSU (0.1 pmol) dyes. For structures, see Figure 1; for PAGE
electrophoresis, see Figure 5. X-axis: retention time (a.u.), y-axis – emission
intensity (a.u.). Blue, green and yellow detection channels are shown with
corresponding colours. The averaged relative peak widths at half maxima are
1.42, 1.35 and 1.00 for APTS, PCN and PSU conjugates, respectively.
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Entry for the Table of Contents
Negatively charged electron-acceptors and auxochromes? New sulfonamides make it possible! They reduce m/z ratios and carry the
load: 1-aminopyrenes bound with reducing sugars. The conjugates were separated and detected (sub pmol) by multicolor emission in
a DNA analyzer.
8
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