Vy M Tran1, Balagurunathan Kuberan. 1. Departments of Medicinal Chemistry and †Bioengineering, University of Utah , Salt Lake City, Utah 84112, United States.
Abstract
Biosynthesis and functions of glycosaminoglycan (GAG) chains are complex and remain elusive. To better understand the factors that regulate the biosynthesis and functions, fluorophore-tagged xylosides carrying two different linkages between fluorophore and xylose residue were synthesized and evaluated for their ability to prime GAG chains such as heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) in various cell lines. These in vitro studies resulted in the identification of fluorophore-tagged xylosides that prime high molecular weight GAG chains. Primed GAG chains carrying a fluorophore group has several advantages for studying the factors that regulate the biosynthesis, analyzing intact fine structures at low detection limits, and setting the stage for studying structure-function relations of GAG chains of cellular origin.
Biosynthesis and functions of glycosaminoglycan (GAG) chains are complex and remain elusive. To better understand the factors that regulate the biosynthesis and functions, fluorophore-tagged xylosides carrying two different linkages between fluorophore and xylose residue were synthesized and evaluated for their ability to prime GAG chains such as heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) in various cell lines. These in vitro studies resulted in the identification of fluorophore-tagged xylosides that prime high molecular weight GAG chains. Primed GAG chains carrying a fluorophore group has several advantages for studying the factors that regulate the biosynthesis, analyzing intact fine structures at low detection limits, and setting the stage for studying structure-function relations of GAG chains of cellular origin.
Proteoglycans modulate
numerous pathophysiological functions such
as development, angiogenesis, axonal growth, anticoagulation, cancer
progression, microbial pathogenesis, and so forth.[1−4] The quantity and quality of GAG
structures, made by various cells, are dynamically regulated in a
spatiotemporal manner during the development of an organism and during
the normal aging of an organism, as well as during the progression
of several pathological conditions.[5,6] Profiling and
deciphering dynamic changes in GAG structures will provide new avenues
to diagnose disease states and may thwart those conditions with novel
therapeutics.[7] Most of these structural
changes have been deduced using radiolabeled monosaccharides and sulfate
as biosynthetic precursors in various cellular systems. However, these
radiolabeled precursors cannot be used in organisms as they pose toxicity
and other challenges. Several β-xyloside derivatives have been
shown to act as acceptors and substitute for core proteins in vivo
as well as in vitro in the production of core protein free GAG chains.[8−14] Fluorophore-tagged xylosides that are able to prime GAG chains will
be an excellent tool to study the structure–function relationship
in vivo. Commercial 4-methyl-umbelliferyl-β-d-xyloside
(UMB-O-xyloside) has been shown until now to function
as acceptor for the elongation of GAG chains; however, UMB-O-xylosides prime mostly CS chains or small oligossacharides.[15−17] Earlier studies examined several other fluorophore-tagged xylosides
for studying the mechanism of GAG biosynthesis and GAG priming activity.[18] These studies have shown successful internalization
of fluorophore-conjugated xylosides into the para- and perinuclear
regions of the cells. However, these molecules were unfortunately
not found to initiate GAG biosynthesis suggesting that either these
fluorophore xylosides failed to reach GAGOSOMES where GAG biosynthetic
enzymes reside within the complex Golgi apparatus or biosynthetic
enzymes could not recognize these fluorophore-conjugated xylosides
after these molecules reach GAGOSOMES. It has been known that both
the structure of the (fluorophore) aglycone and the type of linkage
between the (fluorophore) aglycone and xyloside can affect the priming
activity of xylosides.[9,10,18] Our efforts are, therefore, focused on the synthesis of expanded
repertoire of fluorophore-tagged xylosides, based on developments
in our lab and other laboratories, and screening of these novel xylosides
for their ability to prime GAG chains in a given cellular system and
provides novel avenues to profile and elucidate cellular GAG signatures
in a robust manner, and assist in establishing cell-specific GAG–protein
interactions.
Experimental Section
General Synthetic Procedures
All chemical reactions
were carried out under a nitrogen atmosphere in oven-dried glassware
using standard techniques. 1H and 13C NMR spectra
were obtained on a Bruker 400-MHz spectrometer. Chemical shifts are
relative to the deuterated solvent peak or the tetramethylsilane (TMS)
peak at (δ 0.00) and are in parts per million (ppm). High-resolution
mass spectrometry (HRMS) was performed using a Finnigan LCQ mass spectrometer
in either positive or negative ion mode. Thin layer chromatography
(TLC) was done on 0.25-mm-thick precoated silica gel aluminum sheets.
Chromatograms were observed under short and long wavelength UV light,
and were visualized by heating plates that were dipped in a solution
of Von’s reagent containing ammonium (VI) molybdate tetrahydrate
(12.5 g) and cerium (IV) sulfate tetrahydrate (5.0 g) in 10% aqueous
sulphuric acid (500 mL). Flash column chromatography was performed
using silica gel 60 (230–400 mesh) and employed a stepwise
solvent polarity gradient, correlated with TLC mobility, and were
run under pressure of 5–7 psi. HPLC was used to purify final
products using C18 column (VYDAC 2.2 cm × 25 cm) with solvent
A (25 mM formic acid) and solvent B (95% acetonitrile) at a flow rate
of 5 mL/min in a linear gradient over 120 min starting with 0% B.
N-(β-d-xylopyranosyl) Azide
(5)
2,3,4-Tri-O-acetyl-β-d-xylopyranosyl azide S1 (0.1 mmol) was taken in
dry methanol and was treated with freshly prepared 0.5 M solution
of CH3ONa (0.1 mL) in dry methanol at room temperature
for 3 h. Neutralization with H+ resin followed by concentration
at reduced pressure gave a syrupy liquid, which was purified by silica
flash column chromatography to give the title compound 5.
Propargyl UMB Derivative (11)
To the solution
(10 mL) of UMB derivative (10a and 10b)
(1 mmol) in acetone was added potassium carbonate (3 mmol). The reaction
mixture was stirred for 30 min at room temperature. Propargyl bromide
(3 mmol) was then added and the mixture was stirred overnight. The
reaction mixture was concentrated. The resulting crude material was
dissolved in ethyl acetate, washed with water and saturated sodium
chloride solution, dried over Na2SO4, and rotary
evaporated under reduced pressure. The residue was purified by column
chromatography to give the compound 11.
Synthesis
of Fluorophore-Tagged Xylosides with Amide Linkages
(2, 3, 4, 7, 8, and 9)
N-(β-d-xylopyranosyl) aminoacetamide 1 or N-(β-d-xylopyranosyl) amine 6 (0.1 mmol)
was dissolved in dry DMF (10 mL). Di-isopropylethylamine (0.1 mmol)
was added. The whole mixture was stirred for 30 min before adding
the commercially available activated fluorescent reagents (Dansyl
chloride, FITC and N-hydroxysuccinimidyl-1-pyrene
butyrate) (1 mmol). The reaction mixture was stirred for 4 h and purified
by C18-HPLC column.
Synthesis of Fluorophore-Tagged Xylosides
with Click Linkages
(13a and 13b)
To a solution of
alkyne (1 mmol) and azide (1 mmol) in DMF and water (4:1.3) solvent
mixture were added sodium ascobate (0.8 mmol) followed by Cu2SO4·5H20 (0.4 mmol) at room temperature,
and the mixture was stirred for 12 h or until disappearance of one
of the starting materials as indicated by TLC. At the end of the reaction
as confirmed by TLC analysis, the solvent of the reaction mixture
was evaporated using rotary-evaporator under reduced pressure. The
reaction mixture was purified by flash chromatography columns as described
above. The purified acetylated product (0.1 mmol) was taken in dry
methanol and was treated with freshly prepared 0.5 M solution of CH3ONa (0.1 mL) in dry methanol at room temperature for 3 h.
Neutralization with H+ resin followed by concentration
at reduced pressure gave a syrupy liquid, which was purified on HPLC
using C18 column to give the desired deprotected xyloside derivatives.
All xylosides were characterized using Bruker 400 MHz NMR spectrometer,
and structural data are furnished below:
Screening of Fluorophore-Tagged
Xylosides in Cell Culture
The priming of the xylosides in
xylosyl transferase-deficient CHO
cell line pgsA-745 was performed as described in
our earlier communication. Briefly, 1 × 105 cells
were plated per well, containing the appropriate complete growth medium,
in a 24-well plate and incubated at 37 °C in a humidified incubator
for 24 h to reach a confluency of about 50%. The cells were then washed
with sterile PBS and replaced with 450 μL appropriate medium
containing 10% dialyzed FBS. A serial dilution of the primers at 100×
the final concentration was prepared and 5 μL of appropriate
100× primer was added to various wells to yield final concentrations
respectively. 50 μCi 6-3H-glucosamine was then added
to each well for radiolabeling the GAG chains synthesized. The 24-well
plates were placed in the incubator for 24 h before the addition of
6× Pronase solution (100 μL) followed by incubation at
37 °C overnight.
Purification and Quantification of GAGs
The entire
contents of the wells were transferred to a microcentrifuge tube and
subjected to centrifugation at 16 000×g for 5 min. The supernatant was transferred to a fresh tube and half-a-volume
of 0.016% Triton X-100 was added. The diluted supernatant was loaded
on to a DEAE-sepharose column (0.2 mL) pre-equilibrated with 10 column
volumes of wash buffer (20 mM NaOAc buffer (pH 6.0) containing 0.1
M NaCl and 0.01% Triton X-100) and the column was washed with 20 column
volumes of wash buffer. The bound HS/CS was eluted using 6 column
volumes elution buffer (20 mM NaOAc (pH 6.0) containing 1 M NaCl).
The amount of GAG primed by various xylosides was determined by quantifying
the 3H-radioactivity incorporated in the purified HS/CS
eluate. 50 μL of the various eluates was diluted with 5 mL of
scintillation cocktail and triplicate samples were measured using
a scintillation counter for total radioactivity.
Analysis
of Primed GAG Chains
The chain length of the
primed GAG was determined by measuring the migration time on two tandem
G2000SWXL (Tosoh, 7.8 mm × 30 cm) size exclusion columns using
the HPLC Hitachi system with an inline radiodetector or fluorescent
detector. The solvent containing phosphate (100 mM KH2PO4, 100 mM NaCl, pH 6) was used as an eluent. The average molecular
weight was determined by measuring the migration time of GAG chains
in comparison to those of polystyrene sulfonate standards examined
under similar conditions.The HS/CS composition of the primed
GAG chains was determined by digesting the GAG chains with heparitinase
I/II/III or chondroitinase ABC enzymes. The solution containing GAGs
was diluted to 0.2 M NaCl, followed by the addition of heparitinase
or chondroitinase ABC buffer and 5 mU of heparitinase I/II/III or
chondroitinase ABC enzyme. The reaction mixture was incubated at 37
°C for 2 h, the solution was then loaded on to two tandem G2000
SWXL columns (7.8 mm × 30 cm) and analyzed with the aid of an
inline radiometric detector using phosphate buffer (100 mM KH2PO4, 100 mM NaCl, pH 6) as an eluent. The percentage
of HS/CS was determined based on the percentage area of undigested
and digested GAG peaks.
Results and Discussion
Several fluorophore-tagged xylosides were synthesized and examined
to determine whether these fluorophore-tagged xylosides can elongate
GAG chains. These fluorophore-tagged xylosides offer prospects to
further our understanding of factors that regulate GAG biosynthesis
as well as new knowledge on the role of GAG chains in various signaling
events associated with pathophysiological processes.
Synthesis of Fluorophore-Tagged
Xylosides
Several studies
proved that stimulation of GAG chains is affected not only by hydrophobic
aglycones of xylosides, but also by their glycosidic linkages.[8,12−14,19] Therefore, several
fluorophore-tagged xylosides with amide and triazole in the glycosidic
linkage were synthesized in this study. N-(2,3,4-Trihydroxyl-β-xylopyranosyl)
acetamide 1 was synthesized from xylosyl azide as outlined
in Supporting Information (Scheme S1).[20] Fluorophore-tagged xylosides (2, 3, and 4) were synthesized from corresponding
commercially available activated fluorescent reagents (dansyl chloride,
FITC, and N-hydroxysuccinimidyl-1-pyrene butyrate)
by reacting with the xyloside 1 that contains the reactive
amine group (Scheme 1).
Scheme 1
Synthesis of Fluorophore-Tagged
Xylosides with Amide Linkages
It is known that the linker between xylose and the aglycone
moiety
(fluorophore-tag in this case) may dramatically influence the priming
activity. Therefore, a second strategy was devised in which xylose
was differentially attached to fluorescent tags by reacting xylosyl
azide 5 with triple bond containing amine groups using
click chemistry as shown in Scheme 2.[21] Xyloside 6 with a triazoyl linkage
is prepared, which contains the reactive amine group for conjugating
with activated fluorescent reagents to obtain the fluorophore-tagged
xylosides with triazoyl linkages (7, 8,
and 9).
Scheme 2
Synthesis of Fluorophore-Tagged Xylosides
with Triazoyl Linkages
The commercial, well-known UMB-O-xyloside
primes
mostly short chains of GAG chains or oligosaccharides in various cell
types.[15,17] Click chemistry was used to conjugate fluorescent
UMB derivatives 10a and 10b to the xylose
unit. Fully acetylated xyloside was reacted with UMB derivatives containing
a triple bond and these UMB-click-xylosides were deprotected under
Zemplen condition to obtain the final products 13a and 13b, as outlined in Scheme 3. All final
products were purified on a reverse phase C18 column using HPLC as
described in the Experimental Section, followed
by structural analysis using NMR and MS.
Scheme 3
Synthesis of Umbelliferyl
Xylosides with Triazoyl Linkages
Screening of Fluorophore-Tagged Xylosides
The priming
activity of these novel xylosides may perhaps be attributed to the
presence of a very hydrophobic fluorescent group, helping their transport
across the cell surface and Golgi membranes. At the beginning, the
priming ability of fluorophore-tagged xylosides are investigated using
a mutant Chinese hamster ovary (CHO) cell line, pgsA-745, which lacks active xylosyltransferase enzyme.[22] This cell line does not make HS, CS, or DS chains, as the
assembly of these GAG chain types requires the xylosylation of core
proteins by xylosyltransferase. It requires the exogenous supply of
β-xylosides to produce GAG chains such as HS, CS, and DS, and
is thus a convenient cellular system to ascertain the quantity of
the primed GAG chains by exogenously supplied fluorophore-tagged xylosides.
Neither dansyl group attached xylosides (2 and 7) nor fluorescein attached xylosides (3 and 8) primed any detectable GAG chains. It may perhaps be due
to the presence of charged amine (in dansyl moiety) and carboxyl (in
fluorscein moiety) groups preventing the uptake of xylosides across
the cell membrane. This is in accordance with Johnsson et al. who
found out that the dansyl group attached xylosides were unable to
prime any detectable amount of GAG chains.[18] Fluorophore-tagged xylosides without a charged group are next chosen
to be synthesized. Fluorophore-tagged xylosides (4, 9, 13a, and 13b), in which xylose
residue is attached to 1-pyrene butyrate and UMB derivatives, were
synthesized. Pyrene containing xyloside with amide linkages 4 was not able to prime GAG chains, while the pyrene containing
xyloside with triazoyl linkage 9 was able to prime at
various concentrations. It is interesting to observe that the pyrene
containing xyloside with triazoyl linkage 9 can prime
GAG chains, but the pyrene containing xyloside with amide linkage 4 cannot prime GAG chains. It is predicted that the triazoyl
ring may increase the diffusion rate and direct the primer to Golgi
compartments.Next, UMB-click-xylosides 13a and 13b were compared to the commercial UMB-O-xylosides that prime mostly GAG chains with a short chain length.
The priming activities of the UMB-click-xylosides 13a and 13b and the commercial xyloside were compared at
various concentration (50 μM, 100 μM, 300 μM, 600
μM, and 1 mM) (Figure 1). It is interesting
to note that priming activity of the UMB-click-xylosides 13a and 13b was concentration-dependent but the UMB-O-xyloside was not. We also tested the priming activity
of UMB-click-xylsoides 13a and 13b in endothelial
cells (BLMVEC). Both UMB-click-xylosides primed GAG chains very well
in BLMVEC cells at various concentrations.
Figure 1
Priming activity of UMB-click-xylosides
(13a, 13b) and UMB-O-xylosides
(control) in pgsA-745 cell line. CHO cells were treated
with fluorophore-tagged
xylosides at 50 μM, 100 μM, 300 μM, 600 μM,
and 1 mM in the presence of 3H(100 μCi) as described
in the Experimental Section. The GAG chains
were purified by anion exchange chromatography and quantitated using
a liquid scintillation counter. The results were the average of two
independent experiments.
Priming activity of UMB-click-xylosides
(13a, 13b) and UMB-O-xylosides
(control) in pgsA-745 cell line. CHO cells were treated
with fluorophore-tagged
xylosides at 50 μM, 100 μM, 300 μM, 600 μM,
and 1 mM in the presence of 3H(100 μCi) as described
in the Experimental Section. The GAG chains
were purified by anion exchange chromatography and quantitated using
a liquid scintillation counter. The results were the average of two
independent experiments.
Structural Analysis of Primed GAG Chains in CHO Cells
The
GAG chains primed by these fluorophore-tagged xylosides 9, 13a, and 13b were further analyzed
for their molecular weights using size exclusion columns, as outlined
in the Experimental Section. The chain length
of GAG chains primed by fluorophore-tagged xyloside 9 in CHO cells was determined by measuring the migration time of GAG
chains in comparison to those of polystyrene sulfonate standards performed
under similar conditions on the size exclusion column and suggests
that GAG chains, primed by fluorophore-tagged xyloside 9 in CHO cells, have 27 KDa at 50 μM and 20 KDa at 100 μM
(Figure S1). However, the fluorophore-tagged
xyloside 9 was not sensitive enough; therefore, the structural
analysis of GAG chains primed by fluorophore-tagged xyloside 9 could not be studied using fluorescent detectors and relied
instead on radiometric detector.The UMB-O-xyloside
has been shown to function as acceptor for the elongation of GAG chains
by several groups. However, this well-known fluorophore-tagged xyloside
mostly prime CS with low MW chains.[15,17] By changing
the O linkage of the fluorophore-tagged xyloside
to click linkage, the fluorophore-tagged click-xylosides were predicted
to prime both HS and CS with higher MW chains as many other click-xylosides.
The results from priming activity analysis suggest that optimized
priming concentration of UMB-click-xylosides 13a and 13b is 300 μM. Therefore, the GAG chains primed in CHO
cells by these xylosides at 300 μM were further analyzed for
their molecular weight using the size exclusion column and HS/CS composition
using heparitinase I, II, and III. It is interesting to note that
both fluorophore-tagged click-xylosides13a and 13b primed GAG chains whose average molecular weight (42 kDa)
is higher than those primed by commercial UMB-O-xyloside
(4 kDa) (Figure 2). Furthermore, it is surprising
to note that fluorophore-tagged click-xylosides13a and 13b primed about 30% HS chains, whereas commercial UMB-O-xylosides primed less than 5% HS chains (Figure 3). Some minor peaks appeared beyond Vt in the size
exclusion column, and appearance of these late peaks may be attributed
to the interactions of unknown small molecules carrying hydrophobic
fluorophore with the column.
Figure 2
Size exclusion profiles of GAG chains primed
by fluorophore-tagged
xylosides (UMB-O-xyloside, 13a, and 13b) in pgsA-745 cells. GAG chains primed
by fluorophore-tagged xylosides at 300 μM concentration in pgsA-745 cells for 24 h. The primed GAG chains were then
purified and analyzed as described in the Experimental
Section. The elution profile of the GAG chains primed by UMB-O-xyloside (gray trace), by fluorophore-tagged xyloside 13a (red trace), and by fluorophore-tagged xyloside 13b (black trace).
Figure 3
HS/CS compositions of GAG chains primed by fluorophore-tagged xylosides
(UMB-O-xyloside, 13a, and 13b) in pgsA-745 cells. GAG chains primed by fluorophore-tagged
xylosides at 300 μM concentration in pgsA-745
cells for 24 h. The HS/CS composition of the primed GAG chains was
determined by digesting the GAG chains with heparitinase I, II, III.
The purified GAG chains were analyzed by size exclusion chromatography.
The elution profiles of the primed GAG chains without heparitinase
I, II, III (gray trace). The elution profiles of the primed GAG chains
with heparitinase I, II, III (black trace). (A) The elution profiles
of GAG chains primed by UMB-O-xyloside. (B) The elution
profile of GAG chains primed by fluorophore-tagged click-xyloside 13a. (C) The elution profiles of GAG chains primed by fluorophore-tagged
click-xyloside 13b.
Size exclusion profiles of GAG chains primed
by fluorophore-tagged
xylosides (UMB-O-xyloside, 13a, and 13b) in pgsA-745 cells. GAG chains primed
by fluorophore-tagged xylosides at 300 μM concentration in pgsA-745 cells for 24 h. The primed GAG chains were then
purified and analyzed as described in the Experimental
Section. The elution profile of the GAG chains primed by UMB-O-xyloside (gray trace), by fluorophore-tagged xyloside 13a (red trace), and by fluorophore-tagged xyloside 13b (black trace).HS/CS compositions of GAG chains primed by fluorophore-tagged xylosides
(UMB-O-xyloside, 13a, and 13b) in pgsA-745 cells. GAG chains primed by fluorophore-tagged
xylosides at 300 μM concentration in pgsA-745
cells for 24 h. The HS/CS composition of the primed GAG chains was
determined by digesting the GAG chains with heparitinase I, II, III.
The purified GAG chains were analyzed by size exclusion chromatography.
The elution profiles of the primed GAG chains without heparitinase
I, II, III (gray trace). The elution profiles of the primed GAG chains
with heparitinase I, II, III (black trace). (A) The elution profiles
of GAG chains primed by UMB-O-xyloside. (B) The elution
profile of GAG chains primed by fluorophore-tagged click-xyloside 13a. (C) The elution profiles of GAG chains primed by fluorophore-tagged
click-xyloside 13b.
Structural Analysis of Primed GAG Chains in Endothelial Cells
(BLMVEC)
The fluorophore-tagged xylosides did prime GAG chains
well in endothelial cells at various concentrations. At the optimal
concentration (300 μM), molecular weight of GAG chains primed
by fluorophore-tagged xyloside 13b is much higher than
those primed by commercial UMB-O-xyloside but the
molecular weight of GAG chains primed by fluorophore-tagged xyloside 13a is not higher than those primed by commercial UMB-O-xyloside. The highly electron withdrawing substituent
of −CF3 may result in longer GAG chains in BLMVEC
cells. These GAG chains primed by fluorophore-tagged xylosides 13a and 13b and commercial UMB-O-xyloside were digested with heparitinase I/II/III to determine the
percentage of HS. These fluorophore-tagged click-xylosides produced
a significant amount of HS than the commercial xyloside as shown in
Figure 4.
Figure 4
HS/CS compositions of GAG chains primed
by fluorophore-tagged xylosides
(UMB-O-xyloside, 13a, and 13b) in endothelial cells. GAG chains primed by fluorophore-tagged xylosides
at 300 μM concentration in BLMVEC cells for 24 h. The HS/CS
composition of the primed GAG chains was determined by digesting the
GAG chains with heparitinase I, II, and III. The purified GAG chains
were analyzed by size exclusion chromatography. The elution profiles
of the primed GAG chains before heparitinase I, II, III treatment
(gray trace). The elution profiles of the primed GAG chains after
heparitinase I, II, III (black trace). (A) The elution profiles of
GAG chains primed by UMB-O-xyloside. (B) The elution
profile of GAG chains primed by fluorophore-tagged click-xyloside 13a. (C) The elution profile of GAG chains primed by fluorophore-tagged
click-xyloside 13b.
HS/CS compositions of GAG chains primed
by fluorophore-tagged xylosides
(UMB-O-xyloside, 13a, and 13b) in endothelial cells. GAG chains primed by fluorophore-tagged xylosides
at 300 μM concentration in BLMVEC cells for 24 h. The HS/CS
composition of the primed GAG chains was determined by digesting the
GAG chains with heparitinase I, II, and III. The purified GAG chains
were analyzed by size exclusion chromatography. The elution profiles
of the primed GAG chains before heparitinase I, II, III treatment
(gray trace). The elution profiles of the primed GAG chains after
heparitinase I, II, III (black trace). (A) The elution profiles of
GAG chains primed by UMB-O-xyloside. (B) The elution
profile of GAG chains primed by fluorophore-tagged click-xyloside 13a. (C) The elution profile of GAG chains primed by fluorophore-tagged
click-xyloside 13b.
Conclusions
A small library of novel fluorophore-tagged
xylosides was synthesized
to evaluate their GAG-priming activity. Pyrene-click-xyloside 9 and UMB-click-xylosides 13a and 13b were able to participate in the stimulation of GAG biosynthesis.
These fluorophore-tagged xylosides containing the triazyol rings were
more stable than commercially available xylosides and predictably
have a longer in vivo half-life. Moreover, they were able to prime
a significant amount of HS chains and higher MW than a commercial
fluorophore-tagged O-xyloside. Therefore, these novel
fluorophore-tagged click-xylosides have the potential to profile and
elucidate cellular-specific GAG chains to define various dynamic interactions
in the complex systems and to offer prospects to further our understanding
of factors that regulate GAG biosynthesis as well as new knowledge
on the role of GAG chains in various signaling events associated with
pathophysiological processes.
Scheme 1
Scheme 2
Scheme 3
Figure 1
Figure 2
Figure 3
Figure 4