Dienophile-Modified Mannosamine Derivatives for Metabolic Labeling of Sialic Acids: A Comparative Study

Article abstract of DOI:10.1002/cbic.201700002

Sialic acids play an important role in numerous cell adhesion processes, and sialylation levels are known to be altered under certain pathogenic conditions, such as cancer. Metabolic glycoengineering with mannosamine derivatives is a convenient way to introduce non-natural chemical reporter groups into sialylated glycoconjugates, offering the opportunity to label sialic acids by using bioorthogonal ligation chemistry. The labeling intensity depends not only on the rate of the ligation reaction but also on the extent to which the natural sialic acids are replaced by the modified ones; that is, the incorporation efficiency. Here, we present a comparative study of eight mannosamine derivatives featuring terminal alkenes as chemical reporter groups that can be labeled by an inverse-electron-demand Diels–Alder (DAinv) reaction. The derivatives differed in chain length as well as the type of linkage (carbamates, amides, and a urea) that connects the terminal alkene to the sugar. As a general trend, increasing chain lengths resulted in higher DAinv reactivity and, at the same time, reduced incorporation efficiency. Carbamates were better accepted than amides with the same chain length; nevertheless, the latter resulted in more intense cell-surface staining, visible by live-cell fluorescence microscopy. A urea derivative was also shown to be accepted.

Full text of DOI:10.1002/cbic.201700002

Accepted Article
Title: Dienophile-Modified Mannosamine Derivatives for Metabolic
Labeling of Sialic Acids: A Comparative Study
Authors: Jeremias E. G. A. Dold, Jessica Pfotzer, Anne-Katrin Späte,
and Valentin Wittmann
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ChemBioChem
10.1002/cbic.201700002
Dienophile-Modified Mannosamine Derivatives for Metabolic Labeling
of Sialic Acids: A Comparative Study
Jeremias E. G. A. Dold, Jessica Pfotzer, Anne-Katrin Späte, and Valentin Wittmann*[a]
In memory of Professor Werner Reutter
[
a] J. E. G. A. Dold, J. Pfotzer, Dr. A.-K. Späte, Prof. Dr. V. Wittmann
University of Konstanz
Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB)
78457 Konstanz (Germany)
Fax: (+49) 7531-88-4573
E-mail: mail@valentin-wittmann.de
1
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ChemBioChem
10.1002/cbic.201700002
Abstract
Sialic acids play an important role in numerous cell adhesion processes and sialylation levels
are known to be altered under certain pathogenic conditions such as cancer. Metabolic
glycoengineering with mannosamine derivatives is a convenient way to introduce non-natural
chemical reporter groups into sialylated glycoconjugates offering the opportunity to label sialic
acids using bioorthogonal ligation chemistry. The labeling intensity not only depends on the rate
of the ligation reaction but also on the extent to which the natural sialic acids are replaced by
the modified ones, i.e. the incorporation efficiency. Here we present a comparative study of
eight mannosamine derivatives featuring terminal alkenes as chemical reporter groups that can
be labeled by an inverse-electron-demand Diels-Alder (DAinv) reaction. The derivatives differ in
chain length as well as the type of linkage (comprising carbamates, amides, and a urea) that
connects the terminal alkene to the sugar. As a general trend, increasing chain lengths result in
higher DAinv reactivity and at the same time reduced incorporation efficiency. Carbamates are
better accepted than amides with the same chain length; nevertheless do the latter result in
more intense cell-surface staining visible in life-cell fluorescence microscopy. Finally, a urea
derivative was shown to be accepted.
2
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ChemBioChem
10.1002/cbic.201700002
Introduction
Carbohydrates are a highly important class of biomolecules and heavily involved in
posttranslational protein modification. Of special interest are sialic acids that are positioned at
the outer end of cell-surface glycan structures. They are involved in many recognition
processes, for example during the inflammatory process or the adhesion of influenza viruses to
host cells.[1] Labeling of sialic acids can be conveniently achieved by metabolic
glycoengineering (MGE).[2] In this approach, mannosamine derivatives with an unnatural
chemical reporter group are added to the culture medium of cells. After uptake the
mannosamine derivatives are metabolized and incorporated into glycoproteins in the form of
sialic acids.[3] The chemical reporter group can, subsequently, be reacted in a bioorthogonal
[
4]
ligation reaction allowing the visualization of the sialic acid derivative.
Various derivatives of N-acetylmannosamine (ManNAc) but also of other sugars with different
reporter groups connected over different linkages have been used for MGE in the past.[2] Azides
and alkynes, for example, can be visualized through the Staudinger ligation[5] and/or azide-
alkyne cycloaddition[6] (also known as click reaction). More recently, alkenes have been
introduced as chemical reporter groups for MGE.[7] They can undergo inverse-electron-demand
Diels-Alder (DAinv) reactions[8] with 1,2,4,5-tetrazines (Scheme 1) that are fast, irreversible, and
do not need toxic heavy metals as catalysts allowing applications even within living cells.[9]
Terminal alkenes, besides their application in MGE, have proven to be useful in a number of
studies with proteins[10] and nucleic acids.[11] Their advantages are that they are small, very
stable, and easy to handle. However, their reactivity is strongly dependent on the electron
density of the double bond. As expected for a LUMOdien-HOMOdienophile-controlled reaction,
electron-rich alkenes react especially fast.[12]
The labeling efficiency of sialic acids on the surface of cells is not only dependent on the
reaction rate of the bioorthogonal ligation reaction but also on the acceptance of the unnatural
ManNAc derivative by the biosynthetic machinery of the cell. If only a small proportion of the
natural sialic acids (the most common representative in human cells being N-acetylneuraminic
acid, Neu5Ac[1a]) is replaced by the synthetic derivative, even a fast labeling reaction does not
guarantee a high labeling intensity. Although the degree, by which Neu5Ac is replaced by the
modified sialic acid, i.e. the incorporation efficiency, has such a high impact on the success of
MGE, it has been studied only occasionally,[13] and further investigations would be of high
interest. Here, we report a comparative study of the incorporation efficiencies of the series of
mannosamine derivatives depicted in Figure 1. The compounds do not only differ in the length
of the acyl side chain, that is terminated by an alkene serving as dienophile, but also in the type
3
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ChemBioChem
10.1002/cbic.201700002
of linkage to the sugar. Beside the previously reported carbamate and amide linkage we also
expand the repertoire of mannosamine derivatives by a urea derivative.
Scheme 1. A) DAinv reaction between a 1,2,4,5-tetrazine and a terminal alkene. B) Tetrazine derivatives
1
and 2 used in the current study.
Figure 1. Mannosamine derivatives with terminal alkenes employed for MGE featuring (A) carbamate, (B)
amide, and (C) urea linkages.
4
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ChemBioChem
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Results and Discussion
DMB labeling
To determine the proportion of sialic acids that were modified through MGE (i.e., the
incorporation efficiency), we added different mannosamine derivatives equipped with terminal
alkenes to the culture medium of HEK 293T cells (Scheme 2). After two days the cells were
treated with 3 M acetic acid at 80 °C for 90 min. Under these conditions, the sialic acids are
cleaved off and can subsequently be labeled by treatment with 1,2-diamino-4,5-
methylenedioxybenzene (DMB).[14] DMB selectively reacts with α-keto carboxylic acids such as
Neu5Ac forming a quinoxaline fluorophore. This fluorogenic reaction thus allows to detect the
only small proportion of α-keto acids within the complex mixture of the cell lysate by RP-HPLC
using a fluorescence detector (λex = 373 nm, λem = 448 nm).[13b] The incorporation efficiency IE
was calculated from the integrals I of the RP-HPLC signals of DMB-labeled Neu5Ac (INeu5Ac) and
the respective DMB-labeled modified sialic acid (INeu5R) according to the formula IE = INeu5R
–1
(INeu5R + INeu5Ac) .
Scheme 2. DMB labeling of modified sialic acids obtained by MGE.
Synthesis of reference compounds
To be able to identify the DMB-labeled modified sialic acids by RP-HPLC, we first synthesized a
series of reference compounds. Starting from the deacetylated mannosamine derivatives, an
aldolase reaction[15] delivered the respective sialic acids which were subsequently derivatized
with DMB (Scheme 3). We were pleased that the aldolase reaction could be performed
successfully with all derivatives even the bulky ManNHeoc (10) and the urea-linked ManNBeac
(
18). The formulas and chromatograms of the DMB-labeled products are shown in the
Supporting Information (Figures S2–S9).
5
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ChemBioChem
10.1002/cbic.201700002
Scheme 3. Synthesis of reference compounds 19–26 followed by DMB-labeling and subsequent RP-
HPLC analysis with fluorescence detection. F = fluorescence intensity, t = time
MGE with carbamate-modified mannosamines
Previously, we employed the series of carbamate-modified mannosamine derivatives depicted
in Figure 1A (Ac
4
ManNAloc (3), Ac
4
ManNBeoc (5), Ac
4
ManNPeoc (7), Ac ManNHeoc (9)) in
4
MGE.[7g] We found that an increase of the length of the acyl side chain from 5 to 8 atoms is
beneficial for the rate of the DAinv reaction as indicated by the increasing second-order rate
constants k
2
given in Table 1 (left panel). Interestingly, Ac ManNBeoc (5) gave by far the best
4
staining intensity on the cell surface within this series of sugars. We hypothesized that the
length of the side chain has opposite effects on the DAinv reactivity and the incorporation
efficiency.[7g] Although we expected Ac
4
ManNAloc (3) to be incorporated most efficiently, its low
DAinv reaction rate obviously prevented any staining. Ac
4
ManNBeoc (5) seemed to have the
best compromise between DAinv reactivity and incorporation efficiency leading to the most
intense staining.[7g] Since the varying incorporation efficiencies were a hypothesis and we could
not exclude that they are influenced by other factors beside the size of the side chain, we now
determined the incorporation efficiencies of the whole series of these sugars by DMB labeling.
As can be seen in Table 1 (left panel), there is a clear correlation between the incorporation
efficiency and the length of the side chain. Ac
4
ManNAloc (3) has by far the highest incorporation
efficiency (50 %). With increasing chain length 15 % (Ac
4
ManNBeoc, 5), 3.7 % (Ac ManNPeoc,
4
7
), and 0.3 % (Ac
4
ManNHeoc, 9) of the cellular sialic acids are replaced by the non-natural
derivatives.
6
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ChemBioChem
10.1002/cbic.201700002
Table 1. Incorporation efficiencies IE in MGE experiments and second-order rate constants k of the
2
investigated mannosamine derivatives. Left panel: carbamate derivatives, middle panel: amide
derivatives, right panel: urea derivative.
Chain
length[a] Ac
Carbamate
Amide
Ac ManNR
Urea
Ac ManNR
IE [%]
k
2
[M–1s–1][b]
IE [%]
k
2
[M–1s–1][b]
IE [%]
k
2
[M–1s–1][b]
4
ManNR
4
4
4
R = Btl (11)
R = Ptl (13)
R = Hxl (15)
62 ± 1.7 0,0011 ± 0.0001[c]
31 ± 5.6 0.021 ± 0.001[7b]
5
6
7
8
R = Aloc (3)
50 ± 1.2 0.0015 ± 0.0001[7g]
R = Beoc (5) 15 ± 1.3 0.014 ± 0.003[7g]
R = Peoc (7) 3.7 ± 1.0 0.038 ± 0.005[c]
R = Heoc (9) 0.3 ± 0.2 0.074 ± 0.0013[7g]
8.3 ± 1.3 0.041 ± 0.0008[7b] R = Beac (17)
6.6 ± 1.0 0.029 ± 0.001[c]
[a]
Length of the acyl side chain including the carbonyl C atom. [b] Second-order rate constants were
determined in all cases for reaction of the water-soluble deacetylated mannosamine derivatives with Tz-
PEG-OH (1) in acetate buffer (pH 4.8). [c] For experimental details see Figure S1.
MGE with amide-modified mannosamines
Since the natural N-acetylmannosamine contains an amide moiety, also amide-linked alkenes
are of high interest for MGE. Previous experiments with amide-linked mannosamine derivatives
showed that they are accepted by the cells and can be visualized with the DAinv reaction.[7b] To
find out whether amide-linked alkene-modified mannosamines show the same trend regarding
the DAinv reactivity and incorporation efficiency, we investigated the series of compounds
depicted in Figure 1B. To this end, Ac
published for the preparation of Ac ManNPtl (13) and Ac
second-order rate constant k for the reaction of ManNBtl (12) with Tz-PEG-OH (1) was
4
ManNBtl (11) was synthesized by the procedure
ManNHxl (15)[7b] (Scheme S1). The
4
4
2
–1 –1
determined to be 0.0011 ± 0.0001 M s (Figure S1). Comparison with the published values of
ManNPtl (14) and ManNHxl (16)[7b] revealed that also for the amide-linked alkenes the reaction
rate increases with growing chain length (Table 1, middle panel).
Next, we investigated the incorporation efficiencies of the amide-linked sugars by DMB-labeling
experiments. We observed that the alkene-modified mannosamines show the same trend as the
series of the carbamate-modified sugars. The incorporation efficiency decreases with growing
chain length from 62 % for Ac
4
ManNBtl (11) to 31 % for Ac
4
ManNPtl (13) and 8.3 % for
Ac ManNHxl (15). This indicates that also the amide series shows an opposite effect of the
4
chain length on the DAinv reactivity and on the incorporation efficiency.
To determine which derivative leads to the highest staining intensity on the cell surface, we
examined metabolically labeled cells by confocal fluorescence microscopy. Cells were cultivated
for 2 days with sugar and subsequently incubated with Tz-biotin (2) and then with streptavidin-
7
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ChemBioChem
10.1002/cbic.201700002
AlexaFluor-555 and Hoechst 33342. The microscopy images are shown in Figure 2. Although
Ac ManNBtl (11) has a very low DAinv reactivity after deacetylation, it leads to a decent staining
probably due to its efficient metabolic incorporation. Ac ManNPtl (13) and Ac ManNHxl (15)
lead to a comparable cell surface staining that is much more intense. Obviously, the higher
incorporation efficiency of Ac ManNPtl (13) and the higher DAinv reactivity of Hxl derivative
compensate each other, explaining the comparable staining intensity.
4
4
4
4
Figure 2. HEK 293T cells were grown with 100 µM Ac
4
ManNBtl (11), Ac
4
ManNPtl (13), Ac ManNHxl (15),
4
or DMSO only (negative control) for 48 h. Cells were incubated with 1 mM Tz-biotin (2) for 6 h followed by
incubation with streptavidin-AlexaFluor-555. Nuclei were stained with Hoechst 33342. Scale bar: 30 µm
Comparison of amide- and carbamate-linked mannosamines
Having determined the reaction rates and incorporation efficiencies of the two series of sugars,
a comparison of the influence of the type of linkage becomes possible. In Table 1 compounds
with the same length of the acyl side chain are arranged in the same row. Comparison of
Ac
Ac
4
ManNAloc (3) with Ac
4
ManNPtl (13), which both have a chain length of 5 atoms, and of
4
ManNBeoc (5) with Ac
4
ManNHxl (15) with a chain length of 6 atoms reveals that the
incorporation efficiencies of the carbamate-modified sugars are more than 1.6 times that of the
amide-modified sugars. In contrast, the amide-linked derivatives with the same chain length
–1 –1
feature higher DAinv reaction rates. For the Aloc derivative k is 0.0015 M s whereas for the
2
8
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ChemBioChem
10.1002/cbic.201700002
–1
–1
is 0.014 M–1s–1 whereas for the
Ptl derivative k
2
is 0.021 M s and for the Beoc derivative k
2
–1 –1
Hxl derivative k
2
is 0.041 M s . In summary, replacement of the CH group in α-position of the
2
carbonyl group by the heteroatom oxygen increases metabolic incorporation and reduces DAinv
reactivity due to the high electronegativity of the oxygen atom. Thus, we wondered whether
substitution of the oxygen with a less electronegative NH group could improve DAinv reactivity
while retaining the higher incorporation efficiency. Accordingly, we synthesized the
corresponding urea derivative Ac
4
ManNBeac (17) with the same chain length as that of
Ac ManNBeoc (5) and Ac ManNHxl (15).
4
4
MGE with a urea-modified mannosamine
The synthesis of Ac ManNBeac (17) is shown in Scheme 4. But-3-en-1-amine (27) was
4
activated with N,N′-disuccinimidyl carbonate (DSC) and then reacted with mannosamine
hydrochloride (29) followed by peracetylation.
Scheme 4. Synthesis of Ac ManNBeac (17).
4
So far, a mannosamine derivative carrying a urea moiety has not yet been tested for MGE.
Therefore, we treated HEK 293T cells with Ac ManNBeac (17) for 2 days and determined the
4
proportion of urea-modified sialic acids by DMB labeling. As shown in Table 1, right panel, the
incorporation efficiency is 6.6 %. Interestingly, this number is much lower than that of
Ac
4
ManNBeoc (3, 15 %) and even than that of Ac ManNHxl (15, 8.3 %). The second-order rate
4
constant for the DAinv reaction of ManNBeac (18) with Tz-PEG-OH (1) was determined to be
–
1 –1
k
k
2
2
= 0.029 M s . As expected, this represents an improvement over ManNBeoc (6,
–1
–1
–1 –1
= 0.014 M s ) but does not reach the performance of ManNHxl (16, k = 0.041 M s ).
2
Next we employed the three sugars Ac
17) with the same acyl chain length of 6 atoms in MGE followed by confocal fluorescence
microscopy. As can be seen in Figure 3, Ac ManNHxl (15) which combines the highest reaction
4
ManNBeoc (5), Ac
4
ManNHxl (15) and Ac
4
ManNBeac
(
4
9
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ChemBioChem
10.1002/cbic.201700002
rate with a medium incorporation efficiency results in the brightest staining. Ac
featuring the slowest reaction rate still shows a distinct staining ascribed to its superior
incorporation efficiency. Surprisingly, Ac ManNBeac (17) shows hardly any staining, although its
incorporation efficiency is almost as high as that of Ac ManNHxl (15). Possibly, its reaction rate
is not high enough to overcome the low incorporation efficiency.
4
ManNBeoc (5)
4
4
Figure 3. HEK 293T cells were grown with 250 µM Ac
4
ManNBeoc (5), Ac
4
ManNHxl (15), Ac ManNBeac
4
(
17) or DMSO only for 48 h. Cells were incubated with 1 mM Tz-biotin (2) for 6 h, followed by incubation
with streptavidin-AlexaFluor-555. Nuclei were stained with Hoechst 33342. Scale bar: 30 µm
Conclusion
MGE with mannosamine derivatives is a simple means to introduce non-natural functional
groups into sialylated glycoconjugates. This offers the opportunity to label cellular sialic acids
using biorthogonal ligation chemistry. The approach bears great potential for the diagnosis of
cell-surface sialic acid levels which are known to be altered under certain pathogenic conditions,
such as cancer. Prerequisite for quantification, however, is knowledge on the extent to which
the natural sialic acids are replaced by the modified ones and the reactivity of the introduced
functional group in the subsequent labeling reaction. We have studied a whole series of
1
0
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ChemBioChem
10.1002/cbic.201700002
mannosamine derivatives that are equipped with terminal alkenes that can undergo a DAinv
reaction. We compared different lengths of the side chains of the mannosamine derivatives and
three different types of linkages for attachment of the side chain: carbamates, amides, and a
urea. Both in the series of amides and carbamates the incorporation efficiency increases with
decreasing chain length. The butenoyl moiety in Ac
modification within all compounds tested and leads to the highest incorporation efficiency of
2 %. When comparing carbamates and amides with the same chain length, carbamates are
4
ManNBtl (11) represents the smallest
6
generally better accepted than amides. The length of the side chain has an opposite effect on
the rate constants of the DAinv reactions of the terminal alkenes. Higher reactivity is observed in
all cases with longer side chains. In each series a certain chain length leads to an optimal
balance between incorporation efficiency and reactivity visible by a maximal labeling intensity of
glycoconjugates on the surface of engineered cells. In the carbamate series Ac
has been shown to give the best staining.[7g] In the amide series Ac
ManNPtl (13) and
Ac ManNHxl (15) give the best staining. The shorter butenoyl group leads to a less intense
4
ManNBeoc (5)
4
4
staining, and the same can be expected for longer chains than the hexenoyl group. Finally, we
evaluated the suitability of a new type of mannosamine derivative featuring a urea linkage for
MGE. Ac ManNBeac (17) has a reasonable incorporation efficiency of 6.6 %. However, its
4
overall labeling intensity on the surface of engineered cells was rather low. Our comparative
study thus provides an overview of scope and limitations of terminal-alkene-modified
mannosamine derivatives which is of high interest for the development of future applications.
Experimental Section
General methods: All chemicals were purchased from Sigma-Aldrich, Apollo, and Carbosynth
and used without further purification. AlexaFluor-555-labeled streptavidin and Hoechst 33342
were purchased from Invitrogen. Sialic acid aldolase was purchased from Carbosynth
(
MS110801004). Reactions were monitored by TLC on silica gel 60 F254 (Merck) with detection
under UV light (λ = 254 nm). Additionally, acidic ethanolic p-anisaldehyde solution or basic
KMnO solution followed by gentle heating were used for visualization. Ac
ManNAloc (3),[7g]
ManNAloc (4),[7g] Ac ManNBeoc (5),[7g] ManNBeoc (6),[7g] Ac ManNPeoc (7),[7b] ManNPeoc
ManNHeoc (9),[7g] ManNHeoc (10),[7g] Ac ManNPtl (13),[7b] ManNPtl (14),[7b]
ManNHxl (15),[7b] and ManNHxl (16)[7b] were synthesized according to published procedures.
4
4
4
4
(
8),[7b] Ac
4
4
Ac
4
Preparative flash column chromatography (FC) was performed with an MPLC-Reveleris X2
system from Grace. NMR spectra were recorded at room temperature with Avance III 400 and
Avance III 600 instruments from Bruker. Chemical shifts are reported relative to solvent signals
(CDCl
3
: δ
H
= 7.26 ppm, δ
C
= 77.16 ppm; CD
3
OD: δ
H
= 4.87 ppm, δ
C
= 49.00 ppm; D
2
O: δ = 4.73
H
1
1
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ChemBioChem
10.1002/cbic.201700002
ppm). Signals were assigned by first-order analysis; when feasible, assignments were
1
1
1
13
supported by two-dimensional H, H and H, C correlation spectroscopy (COSY, HSQC, and
HMBC). Numbering of structures is given in the supporting information. High resolution mass
spectra (HRMS) were obtained with a micrOTOF II instrument from Bruker Daltonics. Semi-
preparative high performance liquid chromatography (HPLC) was conducted on a LC-20A
prominence system (high-pressure pumps LC-20AT, auto sampler SIL-20A, column oven CTO-
20AC, diode array detector SPD-M20A, ELSD-LT II detector, controller CBM-20A, software LC-
solution) from Shimadzu under the following conditions. Column: Eurosphere 100 C18 from
-
1
Knauer (16 x 250 nm), flow: 9 mL min ; mobile phase: gradient of acetonitrile with 0.1 % formic
acid (solvent B) in water with 0.1 % formic acid (solvent A). Analytical RP-HPLC-MS was
performed on a LCMS2020 prominence system (pumps LC-20AD, column oven CTO-20AC,
UV/Vis detector SPD-20A, RF-20A prominescence fluorescence detector (λex = 372 nm, λem
56 nm), controller CBM-20A, ESI detector, software LC-solution) from Shimadzu under the
following conditions. Column: EC125/4 Nucleodur C18 from Macherey-Nagel, flow: 0.4 mL min–
1;
mobile phase: gradient of acetonitrile with 0.1 % formic acid (solvent B) in water with 0.1 %
=
4
formic acid (solvent A). UV/Vis absorption was measured with a Cary 50 instrument from Varian
and software Cary WinUV scanning kinetics. Confocal fluorescence microscopy was performed
with a Zeiss LSM 880 instrument equipped with a 40x 1.4 NA Plan-Apochromat oil immersion
objective and a GaAsP-detector array for spectral imaging. Analysis of the obtained data was
performed using image J software version 1.45.
Sialic acid aldolase reaction (general procedure 1, GP1): In a polypropylene vial the
deacetylated mannosamine derivative is dissolved in phosphate buffer (100 mM, pH 7.18) to
give a concentration of approx. 0.1 M. Sodium pyruvate (15 equiv) and sialic acid aldolase are
added and the mixture is gently shaken at room temperature until completion of the reaction.
The mixture is lyophilized and the sialic acid derivative is purified by RP-HPLC.
2
,5-Dioxopyrrolidin-1-yl but-3-en-1-ylcarbamate (28): But-3-en-1-amine (27, 0.45 g, 6 mmol)
was dissolved in dry MeCN (15 mL) under nitrogen atmosphere. N,N´-Disuccinimidyl carbonate
2.7 g, 10.5 mmol) and NEt (2.13 g, 21 mmol) were added. The mixture was stirred overnight at
(
3
room temperature. The solvent was removed and the product was purified by FC (silica, ethyl
acetate 20%-80% in petroleum ether for 30 min). Product 28 was obtained as a white solid
1
(
0.714 g, 56%). TLC: R
f
= 0.59 (petroleum ether/ethyl acetate 1:1); H NMR (400 MHz, CDCl
3
),
δ = 6.00 (t, J = 5.8 Hz, 1H; NH), 5.72 (ddt, J = 17.1, 10.2, 6.8 Hz, 1H; H-3´), 5.15 – 4.97 (m, 2H;
H-4´), 3.25 (q, J = 6.7 Hz, 2H; 2H-1´), 2.76 (s, 4H; 2H-3, 2H-4), 2.27 (m, 2H; 2H-2´) ppm; 13C
NMR (101 MHz, CDCl ), δ = 170.4 (C2, C5), 151.5 (NHC=O), 134.3 (C3´), 117.65 (C4´), 41.1
C1´), 33.5 (C2´), 25.5 (C3, C4) ppm.
2
3
(
1
2
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ChemBioChem
10.1002/cbic.201700002
2
-(3-(But-3-en-1yl)ureido)-2-desoxy-D-mannopyranose (ManNBeac, 18):
Mannosamine
hydrochloride (29, 471 mg, 2.2 mmol) was suspended in dry MeOH (10 mL), neutralized with
.5 M NaOMe solution (3.37 mL), and stirred for 1 h at room temperature under nitrogen. A
0
solution of 28 (500 mg, 2.73 mmol) in dry MeOH (8 mL) was added and the reaction mixture
was stirred overnight at room temperature. The solvent was evaporated and the product was
purified by FC (silica, MeOH 0%-10% in ethyl acetate for 30 min, to yield ManNBeac (18) as a
yellow solid (180.6 mg, 29%). This material was used for kinetic measurements. Full
characterization was carried out after acetylation as described in the following procedure.
1
,3,4,6-Tetra-O-acetyl-2-(3-(but-3-en-1yl)ureido)-2-deoxy-D-mannopyranose
(
Ac ManNBeac, 17): Mannosamine hydrochloride (29, 549 mg, 3 mmol) was suspended in dry
4
MeOH (18 mL), neutralized with 0.5 M NaOMe solution (3.6 mL), and stirred for 1 h at room
temperature under nitrogen. A solution of 28 (635.3 mg, 2.99 mmol) in dry MeOH (6 mL) was
added and the reaction mixture was stirred overnight at room temperature. After complete
removal of the solvent the residual syrup was dissolved in dry pyridine (18 mL), treated with
acetic anhydride (3.8 mL), and stirred overnight at room temperature. The solvent was
evaporated, and the remainder was dissolved in CH
2
Cl
2
. After washing with KHSO
4
(10 %), sat.
NaHCO , and brine, the organic layer was dried over MgSO
3
4
and concentrated. The product
was purified by FC (silica, ethyl acetate 20%-50% in petroleum ether in 40 min) to yield
1
Ac
MHz, CDCl
H-3’), 5.30 (dd, J = 10.2, 4.4 Hz, 1H; H-3), 5.24 – 5.05 (m, 3H; 2H-4’, H-4), 5.00 (d, J = 8.8 Hz,
4
ManNBeac as a white solid (143.5 mg, 11%) as an anomeric mixture (α/β 9:1). H NMR (400
3
), α-anomer: δ = 6.03 (d, J = 1.8 Hz, 1H; H-1), 5.77 (ddt, J = 17.0, 10.1, 6.7 Hz, 1H;
1
H; NH), 4.51 – 4.42 (m, 1H; H-2), 4.35 – 4.19 (m, 1H; H6a), 4.10 – 3.95 (m, 2H; H-6b, H-5),
.26 (t, J = 6.7 Hz, 2H; 2H-1’), 2.28 (q, J = 6.9 Hz, 2H; 2H-2’), 2.17 (s, 3H; OAc), 2.08 (s, 3H;
3
OAc), 2.05 (s, 3H; OAc), 2.02 (s, 3H; OAc) ppm; 13C NMR (101 MHz, CDCl
), α-anomer: δ =
70.8 (C=O), 170.2 (C=O), 169.9 (C=O), 168.6 (C=O), 157.5 (NC=O), 135.3 (C3´), 117.6 (C4´),
2.6 (C1), 70.3 (C-5), 69.3 (C4), 65.8 (C3), 62.4 (C6), 50.4 (C2), 39.9 (C1´), 34.2 (C2´), 21.0
: 445.1817
3
1
9
(
OAc), 20.9 (OAc), 20.9 (OAc), 20.8 (OAc) ppm; HRMS: m/z calcd for C19
H
28
N
2
O
4
+
[
M+H] ; found: 445.1810.
2
,5-Dioxopyrrolidin-1-yl but-3-enolate (30): But-3-enoic acid (534 mg, 6.21 mmol) and N-
hydroxysuccinimide (1.0 g, 8.69 mmol) were dissolved in dry THF (18 mL) under nitrogen
atmosphere. Dicyclohexyl carbodiimide (1.54 g, 7.45 mmol) was suspended in dry THF (18 mL)
and added to the reaction mixture. The mixture was stirred over night at room temperature and
controlled by TLC. The solvent was removed and the product was purified by FC (silica,
petroleum ether/ ethyl acetate 2:1) to yield 30 as a white solid (867 mg, 76 %). TLC: R = 0.53
f
1
(
petroleum ether/ethyl acetate 1:1); H NMR (400 MHz, CDCl3): δ = 5.91 (ddt, J = 17.0, 10.2,
6.7 Hz, 1H; H-3´), 5.43 – 5.20 (m, 2H; 2H-4´), 3.38 (dt, J = 6.7, 1.5 Hz, 2H; 2H-2´), 2.82 (s, 4H;
1
3
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ChemBioChem
10.1002/cbic.201700002
2
1
H-3, 2H-4) ppm; 13C NMR (101 MHz, CDCl
20.6 (C4´), 35.5 (C2´), 25.6 (C3, C4) ppm.
3
): δ = 169.0 (C2, C5), 166.5 (C1´), 127.4 (C3´),
1
,3,4,6-Tetra-O-acetyl-2-((but-3-enoyl)amino)-2-deoxy-D-mannopyranose (Ac
4
ManNBtl,
11): Mannosamine hydrochloride (29, 983 mg, 4.56 mmol) was dissolved in dry MeOH (28 mL)
under nitrogen atmosphere. 0.5 M NaOMe solution (9.1 mL) was added and the mixture was
incubated at room temperature for 1.5 h. 30 (867 mg, 4.74 mmol) was dissolved in dry MeOH
(28 mL), added to the reaction mixture, and stirred for 16 h at room temperature. After complete
removal of the solvent the residual syrup was dissolved in dry pyridine (12 mL), treated with
acetic anhydride (4.2 mL), and stirred overnight at room temperature. The solvent was removed
and the product was purified by FC (silica, CH
2
Cl
2
/ methanol 5:1) to yield 11 as a white solid
(290 mg, 25%) as an anomeric mixture (α/β 2:1). A small amount of the mixture was purified by
semi-preparative RP-HPLC (36%-39% B in 20 min) to yield mainly the α-anomer (t = 11.8 min).
R
1
TLC R
f
= 0.2 (CH
2
Cl
2
/MeOH 5:1); α-Anomer: H NMR (400 MHz, CDCl
3
): δ = 6.02 – 5.90 (m,
2
H; NH, H-3´), 5.85 (d, J = 1.7 Hz, 1H; H-1), 5.35 – 5.24 (m, 2H; 2H-4´), 5.14 – 5.00 (m, 2H; H-
, H-4), 4.75 (ddd, J = 9.1, 3.9, 1.7 Hz, 1H; H-2), 4.25 (dd, J = 12.5, 4.9 Hz, 1H; H-6a), 4.10 (dd,
3
J = 12.5, 2.5 Hz, 1H; H-6b), 3.79 (ddd, J = 9.5, 4.9, 2.5 Hz, 1H; H-5), 3.09 (dt, J = 7.1, 1.3 Hz,
13
2
H; 2H-2´), 2.09 (m, 6H; 2OAc), 2.05 (s, 3H; OAc), 2.00 (s, 3H; OAc) ppm; C NMR (101 MHz,
CDCl ): δ = 171.2 (C=0), 170.6 (C=0), 170.2 (C=0), 169.7 (C=0), 168.4 (C=0), 131.2 (C3´),
20.1 (C4´), 90.7 (C1), 73.5 (C5), 71.5 (C3), 65.3 (C4), 61.9 (C6), 49.7 (C2), 41.6 (C2´), 24.8 –
0.2 (m; 4C(O)CH ): δ = 171.2 (C=O), 170.6
) ppm; β-Anomer: 13C NMR (101 MHz, CDCl
3
1
2
3
3
(
C=O), 170.2 (C=O), 169.7 (C=O), 168.4 (C=O), 131.0 (C3’), 120.1 (C4’), 90.8 (C1), 70.3 (C5),
9.1 (C3), 65.4 (C4), 62.0 (C6), 49.40 (C2), 41.5 (C2’), 21.0 – 20.8 (m, 4C(O)CH3) ppm; HRMS:
m/z calcd for C18 25NO10: 416.1551 [M+H]+; found: 416.1532.
6
H
2
-((But-3-enoyl)amino)-2-desoxy-D-mannopyranose (ManNBtl, 12): Ac
4
ManNBtl (11, 31 mg,
0.075 mmol, 0.035 M) was dissolved under nitrogen atmosphere in dry MeOH/N,N-
dimethylethylamine (4.5:1). The mixture was stirred at room temperature for 7 days and the
solvent was removed to yield ManNBtl as a yellow solid (18 mg, 97%). This material was used
without further purification for kinetic measurements.
N-(Allyloxycarbonyl)-neuraminic acid (Neu5Aloc, 19): ManNAloc (4, 78 mg, 0.3 mmol),
sodium pyruvate (490 mg, 4.46 mmol), and sialic acid aldolase (2 U) were reacted in phosphate
buffer (3 mL) for 7 days according to GP1. Semi-preparative RP-HPLC (3%-5% B in 20 min, t =
R
6.8 min) gave Neu5Aloc (19) as a white solid (30 mg, 38%) as an anomeric mixture (α/β 1:10).
1
TLC: R
f
= 0.2 (EtOAc/MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (400 MHz, D O), β-
2
Anomer: δ = 6.03 (m, 1H; H-2’), 5.56 – 5.18 (m, 2H; 2H-3’), 4.65 (m, 2H; 2H-1’), 4.11 (m, 2H; H-
4, H-6), 4.02 – 3.87 (m, 1H; H-5), 3.87 – 3.63 (m, 3H; H-9a, H-9b, H-8,), 3.62 – 3.38 (m, 1H; H-
13
7
), 2.36 (dd, J = 13.0, 4.9 Hz, 1H; H-3eq), 1.93 (“t”, J = 12.3 Hz, 1H; H-3ax) ppm; C NMR (101
1
4
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ChemBioChem
10.1002/cbic.201700002
MHz, D
2
O), β-Anomer: δ = 173.7 (C1), 158.3 (NC=O), 132.8 (C2´), 117.3 (C3´), 95.4 (C2), 70.6
(C6), 70.4 (C8), 68.3 (C7), 66.9 (C4), 66.0 (C1´), 63.3 (C9), 53.5 (C5), 39.0 (C3) ppm; HRMS:
-
m/z calcd for C13
H
21NO10: 350.1082 [M-H] ; found: 350.1083.
N-(But-3-en-1-yl-oxycarbonyl)-neuraminic acid (Neu5Beoc, 20): ManNBeoc (6, 20 mg, 0.09
mmol), sodium pyruvate (110 mg, 1.38 mmol), and sialic acid aldolase (2 U) were reacted in
phosphate buffer (800 µL) for 7 days according to GP1. Semi-preparative RP-HPLC (5%-25% B
in 20 min, t
R
= 12.5 min) gave Neu5Beoc (20) as a white solid (3.4 mg, 13%) as an anomeric
1
mixture (α/β 1:10). TLC: R
f
= 0.3 (EtOAc/MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (600
MHz, D
2
O), β-Anomer: δ = 5.82 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H; H-3’), 5.14 – 5.03 (m, 2H; H-4’),
.29 (m, 2H; H-4, H-6), 3.94 (t, J = 9.9 Hz, 2H; 2H-1’), 3.79 (dd, J = 11.9, 2.7 Hz, 1H; H-9a),
.70 (ddd, J = 9.2, 6.5, 2.7 Hz, 1H; H-8), 3.62 – 3.52 (m, 3H; H-7, H-5, H-9b), 2.34 (q, J = 6.4
4
3
Hz, 2H; 2H-2’), 2.18 (dd, J = 13.3, 4.9 Hz, 1H; H-3eq), 1.78 (“t”, J = 12.3 Hz, 1H; H-3ax) ppm;
13C NMR (151 MHz, D
O), β-Anomer: δ = 176.1 (C1), 158.6 (C=O), 134.8 (C3´), 117.0 (C4´),
6.1 (C2), 70.4 (C6), 68.4 (C8), 67.28 (C7), 64.5 (C1´), 63.25 (C9), 53.5 (C5), 39.2 (C3), 32.9
2
9
-
(
C2´) ppm; HRMS: m/z calcd for C14H23NO10: 364.1238 [M-H] ; found: 364.1244.
N-(Pent-4-en-1-yl-oxycarbonyl)-neuraminic acid (Neu5Peoc, 21): ManNPeoc (8, 28 mg, 0.10
mmol), sodium pyruvate (158 mg, 1.44 mmol), and sialic acid aldolase (2 U) were reacted in
phosphate buffer (0.98 mL) for 14 days according to GP1. Semi-preparative RP-HPLC (5%-15%
B in 30 min, t
R
= 16.6 min) gave Neu5Peoc (21) as a white solid (5.6 mg, 15%) as an anomeric
1
mixture (α/β 1:12.5). TLC: R
MHz, D
’), 4.13 – 3.97 (m, 4H; 2H-1’, H-4, H-6), 3.89 – 3.78 (m, 1H; H-9a), 3.73 (ddd, J = 9.3, 6.6, 2.6
Hz, 1H; H-8), 3.67 – 3.55 (m, 3H; H-9b, H-5, H-7), 2.24 (dd, J = 13.0, 4.9 Hz, 1H; H-3eq), 2.12
“q”, J = 7.3 Hz, 2H; 2H-3’), 1.83 (“t”, J = 12.3 Hz, 1H; H-3ax), 1.72 (“p”, J = 7.1 Hz, 2H; 2H-2’)
f
= 0.28 (EtOAc/MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (600
2
O), β-Anomer: δ = 5.88 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H; H-4’), 5.10 – 4.98 (m, 2H; 2H-
5
(
13
ppm; C NMR (151 MHz, D
14.9 (C5’), 95.7 (C2), 70.5 (C6), 70.3 (C8), 68.4 (C7), 67.1 (C4), 65.1 (C1’), 63.2 (C9), 53.4
C5), 39.1 (C3), 29.3 (C3’), 27.4 (C2’) ppm; HRMS: m/z calcd for C15
25NO10: 378.1395 [M-H]-;
found: 378.1392.
2
O / CD OD), β-Anomer: δ = 174.9 (C1), 158.8 (NC=O), 138.5 (C4’),
3
1
(
H
N-(Hex-5-en-1-yl-oxycarbonyl)-neuraminic acid (Neu5Heoc, 22): ManNHeoc (10, 30 mg, 0.1
mmol), sodium pyruvate (162 mg, 1.47 mmol), and sialic acid aldolase (2.5 U) were reacted in
phosphate buffer (1 mL) for 8 days according to GP1. Semi-preparative RP-HPLC (5%-20% B
in 20 min, t
R
= 20 min) gave Neu5Heoc (22) as a white solid (5 mg, 14%) as an anomeric
1
mixture (α/β 1:14). TLC: R
f
= 0.32 (EtOAc /MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (600
MHz, D
2
O), β-Anomer: δ = 5.94 (ddt, J = 17.0, 10.2, 6.6 Hz, 1H; H-5’), 5.14 – 5.01 (m, 2H; 2H-
’), 4.21 – 4.01 (m, 4H; H-4, H-6, 2-H-1’), 3.88 (dd, J = 12.0, 2.7 Hz, 1H; H-9a), 3.79 (ddd, J =
.2, 6.5, 2.7 Hz, 1H; H-8), 3.72 – 3.62 (m, 3H; H-5, H-7, H-9b), 2.31 (dd, J = 13.1, 5.0 Hz, 1H;
6
9
1
5
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ChemBioChem
10.1002/cbic.201700002
H-3eq), 2.13 (“q”, J = 7.2 Hz, 2H; 2H-4’), 1.90 (“t”, J = 12.3 Hz, 1H; H-3ax), 1.68 (quint, J = 7.0
Hz, 2H; 2H-2’), 1.55 – 1.45 (m, 2H; 2H-3’) ppm; 13C NMR (151 MHz, D
O), β-Anomer: δ =
74.35 (C1), 158.9 (NC=O), 139.6 (C5’), 114.43 (C6’), 95.6 (C2), 70.6 (C6), 70.4 (C8), 68.4
2
1
(C7), 67.0 (C1’), 65.7 (C9), 63.3 (C4), 53.5 (C5), 39.1 (C3), 32.6 (C4’), 27.7 (C2’), 24.4 (C3’)
-
ppm; HRMS: m/z calcd for C16
H
27NO10: 392.1551 [M-H] ; found: 392.1550.
N-(But-3-enoyl)-neuraminic acid (Neu5Btl, 23): ManNBtl (12, 70.7 mg, 0.28 mmol), sodium
pyruvate (474 mg, 4.3 mmol), and sialic acid aldolase (8 U) were reacted in phosphate buffer
(
2.9 mL) for 20 days according to GP1. Semi-preparative RP-HPLC (3%-5% B in 20 min, t =
R
7.4 min) gave Neu5Btl (23) as a white solid (20 mg, 21%) as an anomeric mixture (α/β 1:14).
1
TLC: R
f
= 0.27 (EtOAc /MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (400 MHz, D O), β-
2
Anomer: δ = 5.95 (ddt, J = 17.1, 10.1, 6.9 Hz, 1H; H3’), 5.35 – 5.21 (m, 2H; 2H-4’), 4.17 – 4.04
(
m, 2H; H-4, H-6), 3.96 (t, J = 10.2 Hz, 1H; H-5), 3.86 (dd, J = 11.8, 2.7 Hz, 1H; H-9a), 3.77
ddd, J = 9.0, 6.3, 2.5 Hz, 1H; H-8), 3.63 (dd, J = 11.8, 6.3 Hz, 1H; H-9b), 3.60 – 3.50 (m, 1H; H-
(
7
1
1
), 3.13 (d, J = 6.8 Hz, 2H; 2H-2’), 2.34 (dd, J = 13.0, 4.9 Hz, 1H; H-3eq), 1.90 (“t”, J = 12.3 Hz,
13
H; H-3ax) ppm; C NMR (101 MHz, D O), β-Anomer: δ = 175.3 (C1), 173.5 (C1’), 130.9 (C3’),
2
19.3 (C4’), 95.4 (C2), 70.4 (C6), 70.2 (C8), 68.3 (C7), 66.6 (C4), 63.2 (C9), 52.1 (C5), 40.7
-
(
C2’), 39.0 (C3) ppm; HRMS: m/z calcd for C13
H
21NO
9
: 334.1133 [M-H] ; found: 334.1139.
N-(Pent-4-enoyl)-neuraminic acid (Neu5Ptl, 24): ManNPtl (14, 100 mg, 0.38 mmol), sodium
pyruvate (631 mg, 5.74 mmol), and sialic acid aldolase (6 U) were reacted in phosphate buffer
(
3.9 mL) for 26 days according to GP1. Semi-preparative RP-HPLC (18%-20% B in 30 min, t =
R
4.8 min) gave Neu5Ptl (24) as a with solid (10 mg, 8%) as an anomeric mixture (α/β 1:7). TLC:
1
R
f
= 0.30 (EtOAc /MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (600 MHz, D O), β-Anomer: δ =
2
5
.85 (ddt, J = 16.5, 10.3, 6.2 Hz, 1H; H-4’), 5.15 – 4.98 (m, 2H, 2H-5’), 4.04 – 3.95 (m, 2H; H-4,
H-6), 3.89 (t, J = 10.2 Hz, 1H, H-5), 3.85 – 3.78 (m, 1H; H-9a), 3.73 (ddd, J = 9.2, 6.6, 2.6 Hz,
H; H-8), 3.56 (dd, J = 11.8, 6.7 Hz, 1H, H-9b), 3.51 (dd, J = 9.1, 1.2 Hz, 1H; H-7), 2.42 – 2.29
m, 4H; 2H-2’,2H-3’), 2.22 (dd, J = 13.0, 4.9 Hz, 1H; H-3eq), 1.81 (dd, J = 13.0, 11.5 Hz, 1H; H-
ax) ppm; 13C NMR (151 MHz, D
O), β-Anomer: δ = 176.8 (C1), 175.5 (C1’), 137.0 (C4’), 115.7
1
(
3
2
(C5’), 96.0 (C2), 70.3 (C8), 70.2 (C6), 68.5 (C7), 66.9 (C4), 63.2 (C6), 52.0 (C5), 39.2 (C3), 35.2
-
(
C2’), 29.3 (C3’) ppm; HRMS: m/z calcd for C14
H
23NO
9
: 348.1289 [M-H] ; found: 348.1292.
N-(Hex-5-enoyl)-neuraminic acid (Neu5Hxl, 25): ManNHxl (16, 32 mg, 0.12 mmol), sodium
pyruvate (203 mg, 1.84 mmol), and sialic acid aldolase (2 U) were reacted in phosphate buffer
(
1.25 mL) for 20 days according to GP1. Semi-preparative RP-HPLC (3%-5% B in 30 min, t =
R
19.7 min) gave Neu5Hxl (25) as a white solid (14 mg, 31%) as an anomeric mixture (α/β 1:7.6).
1
TLC: R
f
= 0.28 (EtOAc /MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (400 MHz, D O),
2
β-Anomer: δ = 5.94 (ddt, J = 17.1, 10.3, 6.6 Hz, 1H; H-5’), 5.20 – 5.04 (m, 2H; 2H-6’), 4.13 –
.02 (m, 2H; H-4, H-6), 3.99 (dd, J = 10.3, 9.3 Hz, 1H; H-5), 3.94 – 3.85 (m, 1H; H-9a), 3.85 –
4
1
6
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ChemBioChem
10.1002/cbic.201700002
3
.78 (m, 1H; H-8), 3.70 – 3.62 (m, 1H; H-9b), 3.57 (d, J = 9.0 Hz, 1H; H-7), 2.38 (t, J = 7.3 Hz,
H; 2H-2’), 2.29 (dd, J = 12.9, 4.8 Hz, 1H; H-3eq), 2.20 – 2.07 (m, 2H; 2H-4’), 1.90 (“t”, J = 12.1
2
13
Hz, 1H; H-3ax), 1.77 (quint, J = 7.4 Hz, 2H; 2H-3’) ppm; C NMR (151 MHz, D
2
O), β-Anomer: δ
=
6
177.7 (C1), 160.5 (C1’), 138.6 (C5’), 115.1 (C6’), 90.9 (C2), 70.3 (C6), 68.6 (C8), 67.0 (C7),
3.2 (C4), 59.3 (C9), 52.1 (C5), 39.3 (C3), 35.3 (C2’), 32.4 (C4’), 24.6 (C3’) ppm; HRMS: m/z
-
calcd for C15
H
25NO
9
: 362.1446 [M-H] ; found: 362.1449.
N-(But-3-en-1-yl-aminocarbonyl)-neuraminic acid (Neu5Beac, 26): ManNBeac (18, 41 mg,
.15 mmol), sodium pyruvate (245 mg, 2.22 mmol), and sialic acid aldolase (4 U) were reacted
in phosphate buffer (1.5 mL) for 15 days according to GP1. Semi-preparative RP-HPLC (3%-8%
0
B in 30 min, t
R
= 18 min) gave Neu5Beac (26) as a white solid (0.3 mg, 5%). TLC: R = 0.2
f
1
(EtOAc /MeOH/EtOH/H
2
O/AcOH 2:1:1:1:0.1); H NMR (400 MHz, D
2
O), β-Anomer: δ = 5.91 (m,
1H; H-3’), 5.26 – 5.09 (m, 2H; 2H-4’), 4.15 – 3.95 (m, 2H; H-4, H-6), 3.95 – 3.70 (m, 3H; H-9a,
H-5, H-8), 3.70 – 3.54 (m, 2H; H-7, H-9b), 3.31 (“t”, J = 6.8 Hz, 2H; 2H-1’), 2.64 (“t”, J = 6.6 Hz,
2
H; 2H-2’), 2.38 – 2.25 (m, 1H; H-3eq), 1.98 – 1.82 (m, 1H; H-3ax) ppm; HRMS: m/z calcd for
-
C H N O
14 24 2 9
: 363.1398 [M-H] ; found: 363.1456.
Kinetic measurements: Kinetic measurements were carried out as previously described
Figure S1).[7b] Briefly, solutions of ManNPeoc (8), ManNBtl (12), or ManNBeac (18) (each 10
(
mM) and Tz-PEG-OH (1) (10 mM) in acetate buffer (pH 4.8) were mixed in a quartz cuvette to
give final concentrations of each 5 mM. The reaction was monitored over time by measuring the
decrease of absorption at 522 nm which is characteristic for Tz-PEG-OH (1). Second-order rate
constants k
were determined by plotting [Tz-PEG-OH]–1 against the time, followed by analysis
2
via linear regression. The measurements were carried out in triplicates. The stability of Tz-PEG-
OH (1) was published before.
Cell growth conditions: HEK 293T (human embryonic kidney) cells were grown in Dulbecco’s
Modified Eagle’s Medium (DMEM) containing fetal bovine serum (FBS, 10%), penicillin
–
1
–1
(
100 U mL ) and streptomycin (100 U mL ). The cells were incubated at 37 °C and 5 % carbon
dioxide in a water-saturated incubator. The cells were diluted every third day by washing with
PBS buffer and detaching with trypsin and EDTA.
Sugar stock solutions: The sugars were prepared as stock solutions (100 mM) in DMSO and
stored at –20 °C. They were freshly diluted into media on the day of the experiment.
MGE and fluorescence microscopy: Similar to previously described experiments,[7b] HEK
–2
2
93T cells (18,000 cells cm ) were seeded in 4-well ibiTreat µ-Slides (ibidi) Ph+ coated with
poly-L-lysine (0.0025%, 1 h, 37 °C). After one day, cells were incubated with the mannosamine
derivatives Ac ManNBtl (11), Ac ManNPtl (13), Ac ManNHxl (15), Ac ManNBeoc (5) or
4
4
4
4
1
7
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ChemBioChem
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Ac
DMSO only was added. Cells were washed twice with PBS and then incubated with Tz-biotin (2,
mM in media) for 6 h at 37 °C. After washing twice with PBS, the cells were incubated with
4
ManNBeac (17) (100 or 250 µM) for 48 h. As negative control the corresponding volume of
1
–1
–1
streptavidin-AlexaFluor-555 (6.6 µg mL ) and Hoechst 33342 (10 µg mL ) for 20 min at 37 °C.
After three washes with PBS, media (DMEM) was added for fluorescence microscopy.
Preparation of reference compounds for DMB labeling experiments: For the synthesis of
the DMB-labeled reference components the neuraminic acid derivative (Neu5Aloc (19),
Neu5Beoc (20), Neu5Peoc (21), Neu5Heoc (22), Neu5Btl (23), Neu5Ptl (24), Neu5Hxl (25) or
Neu5Beac (26), 0.032 mmol) was dissolved in 265 µL DMB solution (5.3 mM DMB · 2 HCl, 16
mM Na
2
S
2
O , 40 mM TFA in MilliQ water). The mixture was incubated for 2.5 h at 56 °C in a
4
thermomixer (300 rpm) in the dark. After the incubation the mixture was cooled on ice for 10 min
and neutralized with aqueous NaOH (0.5 M, 21 µL). The solution was analyzed by analytical
RP-HPLC-MS measurements. For fluorescence detection (excitation 372 nm, emission 456 nm)
a dilution was necessary (1:400).
DMB-Neu5Aloc (31): analytical RP-HPLC (10%-30% B in 40 min): t = 22.5 min; HRMS: m/z
R
+
calcd for C20
H
25
N
3
O
10: 468.1613 [M+H] ; found: 468.1613.
DMB-Neu5Beoc (32): analytical RP-HPLC (10%-30% B in 40 min): t
0 min): t = 36.3 min; HRMS: m/z calcd for C21
R
= 24.9 min, (10%-25% in
+
4
R
27 3
H N O
10: 482.1769 [M+H] ; found: 482.1748.
DMB-Neu5Peoc (33): analytical RP-HPLC (10%-30% B in 40 min): t
R
= 34.5 min; HRMS: m/z
= 41.0 min; HRMS: m/z
+
calcd for C22
H
29
N
3
O
10: 496.1926 [M+H] ; found: 496.1926.
DMB-Neu5Heoc (34): analytical RP-HPLC (10%-30% B in 40 min): t
R
+
calcd for C23
H
31
N
3
O
10: 510.2082 [M+H] ; found: 510.2059.
DMB-Neu5Btl (35): analytical RP-HPLC (10%-18% B in 85 min): t
R
= 29.3 min; HRMS: m/z
= 48.1 min; HRMS: m/z
+
calcd for C20
H
25
N
3
O
9
: 452.1664 [M+H] ; found: 452.1651.
DMB-Neu5Ptl (36): analytical RP-HPLC (10%-18%B in 85 min): t
R
+
calcd for C21
H
27
N
3
O
9
: 466.1820 [M+H] ; found: 466.1805.
DMB-Neu5Hxl (37): analytical RP-HPLC (10%-18% B in 85 min): t
min): t = 37.9 min; HRMS: m/z calcd for C22
R
= 75.5 min, (10%-25% in 40
+
R
H N O
29 3 9
: 480.1977 [M+H] ; found: 480.1953.
DMB-Neu5Beac (38): analytical RP-HPLC (10%-25% B in 40 min): t = 24.1 min; HRMS: m/z
R
+
calcd for C21
H
28
N
4
O
9
: 481.1881 [M-H] ; found: 481.2292.
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8
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ChemBioChem
10.1002/cbic.201700002
DMB labeling of sialic acids released from engineered cells: HEK 293T cells were seeded
450,000 cells in a 5 cm dish) and incubated for 24 h with 4 mL media. The medium was
exchanged with media (4 mL) containing 100 µM of the modified sugar (Ac ManNAloc (3),
ManNPtl (13),
ManNBeac (17)). As a solvent control the corresponding volume of
(
4
Ac
Ac
4
ManNBeoc (5), Ac
4
ManNPeoc (7), Ac
4
ManNHeoc (9), Ac
4
ManNBtl (11), Ac
4
4
ManNHxl (15), or Ac
4
pure DMSO was added. The cells were cultured for 48 h with or without added sugar. The cells
were trypsinated, resuspended in media and pelleted by centrifugation (5 min, 500 g). The
pellets were washed 3 times with PBS Buffer (5 mL) and transferred to Eppendorf tubes
(400,000 cells per tube). The cells were pelleted again (5 min 500 g) and the supernatant was
discarded. The cells were resuspended in AcOH (3 M, 300 µL), and the mixture was incubated
at 80 °C for 90 min in a thermomixer (300 rpm). The solution was diluted with MilliQ water (400
µL) and neutralized with a solution of NH (25%) in water (20 µL). The solvent was removed in a
3
SpeedVac, and the pellet was washed 3 times with EtOH (200 µL) and concentrated after every
step. Then the pellet was dissolved in 265 µL DMB-labeling solution (5.3 mM DMB · 2 HCl, 16
mM Na
S
2 2
4
O , 40 mM TFA in MilliQ water). The mixture was incubated at 56 °C for 2.5 h in a
thermomixer (300 rpm) in darkness. The mixture was cooled on ice for 10 min, neutralized with
aqueous NaOH (0.5 M, 25 µL) and analyzed by analytical RP-HPLC-MS (for gradients and
chromatograms see Figures S10–S22).
Acknowledgements
We are thankful to the late Professor Werner Reutter whose research laid the basis for the
current investigations. We will not forget the many inspiring discussions we had during our
collaboration. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 969
and SPP 1623), the University of Konstanz, and the Konstanz Research School Chemical
Biology. We thank Stefan Bitter for support with synthesis, the Bioimaging Center of the
University of Konstanz for providing the fluorescence microscopy instrumentation and the NMR
Core Facility for providing the NMR instrumentation.
Keywords: carbohydrates • Diels-Alder reaction • metabolic engineering • sialic acids • DMB
labeling
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ChemBioChem
10.1002/cbic.201700002
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Entry for the table of contents
Impact of chain length and linker moiety on glycan visualization: Eight different N-
acylmannosamine derivatives including carbamates, amides, and a urea were evaluated for
their suitability for metabolic glycoengineering. Terminal alkenes served as chemical reporter
groups for bioorthogonal ligation with an inverse-electron-demand Diels-Alder reaction. The
overall labeling efficiency on cell surfaces is determined by the metabolic acceptance of the
carbohydrate and its reactivity in the ligation reaction.
2
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