Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-N-acetyl galactosaminidases through droplet-based screening

Article abstract of DOI:10.1039/d0ob02484h

As part of a search for a substrate for droplet-based microfluidic screening assay of α-N-acetylgalactosaminidases, spectral and physical characteristics of a series of coumarin derivatives were measured. From among these a new coumarin-based fluorophore, Jericho Blue, was selected as having optimal characteristics for our screen. A reliable method for the challenging synthesis of coumarin glycosides of α-GalNAc was then developed and demonstrated with nine examples. The α-GalNAc glycoside of Jericho Blue prepared in this way was shown to function well under screening conditions.

Full text of DOI:10.1039/d0ob02484h

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COMMUNICATION
Synthesis and evaluation of sensitive coumarin-based fluorogenic
substrates for discovery of α-N-acetyl galactosaminidases through
droplet-based screening
Hong-Ming Chen†, Seyed Amirhossein Nasseri†, Peter Rahfeld, Jacob F. Wardman, Maurits Kohsiek
and Stephen G. Withers*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
As part of a search for a substrate for droplet-based microfluidic analytical tool for the analysis of O-glycan structures and in
screening assay of -N-acetylgalactosaminidases, spectral and determining their attachment points to proteins. It is quite
physical characteristics of a series of coumarin derivatives were probable that distinctly different enzyme classes will be
measured. From among these a new coumarin-based fluorophore, required for these two applications.
Jericho Blue, was selected as having optimal characteristics for our As part of our quest to identify such enzymes in a high-
screen. A reliable method for the challenging synthesis of coumarin throughput manner from within large metagenomic libraries,
glycosides of α-GalNAc was then developed and demonstrated with we required access to suitable fluorogenic α-N-
nine examples. The α-GalNAc glycoside of Jericho Blue prepared in acetylgalactosaminide substrates to be used in screening via
this way was shown to function well under screening conditions.
‘water-in-oil’ droplet methodologies[7]. While we have
previously utilized plate-based approaches for such screening
efforts, droplet-based microfluidics enables higher-throughput
screening compared to plate-based assays (on the approximate
scales of >105 clones per day compared to <104 respectively).
Indeed, the capacity of microfluidic generated droplets for high-
throughput screenings has enabled rapid and expansive
N-Acetyl-galactosamine (GalNAc) linked as an α-glycoside plays
two especially important roles in nature. In one instance, it is
found linked to a serine or threonine residue as the first sugar
attached to proteins within mucin-type O-glycans, where it is
typically decorated with galactose, N-acetylglucosamine, or
another GalNAc to form the core structures of mammalian O-
glycans. Its other common occurrence is as the terminal sugar
on the A-antigen found on red blood cells and tissues of
individuals with A blood type. In this case it is attached α-1,3 to
the galactose of the H-antigen, fucose-α-1,2-galactose, which
sits at the end of glycan chains on membrane-bound glycolipids
directed evolution[8,9] and enzyme discovery campaigns[10,11]
.
Our past experiences in plate-based screening for other
enzymatic activities have shown that coumarin glycosides, or
more specifically the glycosides of 7-hydroxycoumarin
derivatives (known as umbelliferones), are useful screening
substrates.[12,13] Of these the 4-methylumbelliferones are the
best known. Umbelliferone derivatives have many
characteristics that make them attractive fluorophores for
screening purposes. Their aromatic moieties are readily
synthesised and, once attached to the anomeric centres of
sugars via the 7-hydroxyl, form compact substrates that are
virtually non-fluorescent. However, once the glycosidic linkage
has been hydrolyzed, they are highly fluorescent in their
deprotonated, anionic form. For our screening purposes, we
thus needed to incorporate hydroxycoumarins with pKa values
low enough that they would exist primarily in their fluorescent,
anionic form when released in screening assays that are
typically performed at neutral pH values.
or glycoproteins[1,2]
.
Enzymatic cleavage of terminal α-GalNAc residues is
accomplished by exo-acting α-N-acetyl-galactosaminidases (α-
GalNAcase (EC 3.2.1.49)) that are found primarily in five of the
sequence-based CAZy families: GH27, GH36, GH109, GH129 and
most recently GH31[3,4]. Access to an expanded range of α-
GalNAcases with different specificity could be of value in several
applications. One of these is in the enzymatic conversion of A-
antigens to H-antigens on red blood cells as a means of creating
universal donor O-type red blood cells; a topic that has
attracted considerable interest[4–6]. The other would be as an
The second consideration in the design of substrates is specific
for droplet-based screening methods, which involve the
creation of small (<1 nL) sized ‘droplets’ of water within an oil
matrix. Each droplet functions as a small reaction vessel
containing enzyme and the fluorogenic glycoside. It is therefore
Department of Chemistry, University of British Columbia
2016 Main Mall, Vancouver, B.C., Canada
E-mail: withers@chem.ubc.ca.
† These authors have contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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COMMUNICATION
Journal Name
hydroxyl as well as the fluorescence
intensity of the molecule under assay
Table 1: Spectroscopic and chemical properties of coumarin derivatives
R8
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DOI: 10.1039/D0OB02484H
conditions. The fluorescence intensity is
itself determined by the inherent
fluorescence intensity of the molecule along
with its pKa. Importantly, the pKa of the
phenol also correlates to the leaving group
ability of the coumarin derivative and thus
the reactivity/sensitivity of the substrate.
High reactivity increases the probability that
enzymes from metagenomic sources, which
are often expressed at low levels, may be
detected. However, making the substrate
too reactive runs the risk of high background
fluorescence in the screen from
spontaneous hydrolysis of the substrates,
masking the activity of these minimally
expressed enzymes. Therefore, the phenolic
pKa should be as low as possible but just
high enough that the glycoside is stable.
As noted in Table 1, the derivatives with a
carboxylic acid group as R3 are generally
more fluorescent than their 4-methyl
counterparts. This is particularly so if the
carboxyl group is esterified. However, as
HO
O
O
R6
R3
R4
Relative fluor.
a
b
#
Compound
R3
R4
R6
R8
λex/λem
pKa
brightness c
0.082
0.077
0.12
1
2
3
4
5
6
7
8
Umbelliferone
MU
6-FMU
6,8-F2MU
6-ClMU
TFMU
H
H
H
H
H
H
Me
Me
Me
Me
CF3
CF3
H
H
H
H
H
H
H
H
H
H
-
H
H
F
H
H
H
F
H
H
F
H
H
H
H
Cl
Cl
H
H
F
365/455
365/450
360/450
360/450
365/450
390/505
390/505
390/450
400/450
395/450
410/450
390/450
410/450
390/450
405/450
390/450
405/450
484/519
7.6
7.7
6.6
4.9
6.1
7.1
4.3
7.3
6.5
5.7
4.8
5.7
4.9
6.0
5.2
4.7
3.7
6.2
F
0.10
0.14
0.049
0.036
0.26
0.68
0.35
0.58
0.32
0.49
0.26
0.45
0.31
0.54
1
Cl
H
F
H
H
Cl
Cl
H
H
F
H
H
6,8-F2TFMU
3-CU
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
CO2H
CO2Me
-
9
3-CUOMe
6-Cl-3-CU
6-Cl-3-CUOMe
8-Cl-3-CU
8-Cl-3-CUOMe
JB
JBOMe
PB
PBOMe
Fluorescein
10
11
12
13
14
15
16
17
18
F
F
F
-
F
-
[a] Measured for 100 µM solutions of each compounds in PBS buffer containing 5% DMSO.
[b] pKa of the phenol; calculated based on measured absorbance of 50 µM solutions of the compounds in
buffers with various pH values, each containing 5% DMSO.
[c] The slope of the graph of fluorescence vs concentration of compound. Measured at maximum wavelengths
of each compound in PBS buffer. Values are reported relative to Fluorescein. An estimated 10% error should
be considered for these values due to different purity grades of compounds from different sources.
crucial that the hydrolysed fluorophore should not be able to
shown below, since the charged carboxyl group is needed to
keep the fluorophores within the droplet, the esterified
versions cannot be used for our purposes. Nevertheless, for
purposes other than droplet-based assays, the esterified
versions have an advantage.
Finally, as these fluorophores need to fit into the enzyme active
site, the smaller they are the better. Thus, even though
chlorinated derivatives generally have a lower pKa and are more
fluorescent compared to their fluorinated analogues (for
instance see entries 3 vs 5, and 12 vs 14), the latter are
preferred. Indeed, we have previously observed some families
diffuse to the oil layer or, even worse, exchange between
droplets. The mechanism behind droplet-droplet exchange is
thought to be driven by encapsulation and transport of the
fluorophore between droplets in surfactant formed micelles[14]
and increasing the polarity of the fluorophore has been shown
to limit both partitioning into the oil and micellar transport
(albeit the mechanistic underpinnings of this have not been fully
established).[15] As demonstrated within this text, the partial
negative charge present on the phenolic oxygen of coumarins
of low pKa is not sufficient to retain the molecule in the water
droplets. This is further corroborated by other studies using
similar fluorophores in various oil systems[14]. Our approach to
this problem was to use hydroxycoumarins containing a
charged carboxylate moiety that will both block entry into the
oil and limit micellar transport.
of
glycosidases
that
do
not
hydrolyze
6-
chloromethylumbelliferone analogues (unpublished data).
Based upon the factors discussed above, we chose to initially
synthesise the α-GalNAc glycoside of the coumarin derivative
known as Pacific Blue (1) shown in Figure 1. However, as
discussed in more detail in the SI, during the course of synthesis
we found out that the protected glycoside of Pacific Blue is too
unstable to withstand the reaction conditions required to
access the final product 1. Not only did this make the current
synthetic route infeasible but also it indicated that the final
product would be too unstable to function as a useful screening
reagent for assays that typically run overnight. Presumably the
phenol, with its low pKa of 3.7 (as the methyl ester), is just too
good a leaving group.
Thus, in order to select a suitable fluorophore, we surveyed the
many different coumarins available from commercial sources or
reported in the literature. However, we found that a systematic
study of their properties has not been done, resulting in some
contradictory values being found in different sources[12,16–19]
.
We therefore examined, and report here, the spectroscopic and
chemical characteristics of a collection of coumarin-based
fluorophores that are relevant in the selection of an optimal
substrate for screening purposes (Table 1). These include their
excitation and emission wavelengths, their phenolic pKa values
and their relative fluorescence brightness. These data will be
very valuable for any similar study in deciding which one of the
many coumarin derivatives best matches the specific assay
being developed.
In light of this result we reconsidered our strategy. Clearly, a
phenol of higher pKa was needed. The conceptually simplest
way to reduce the leaving group ability of the phenol was to
Fig. 1 Pacific Blue α-D-GalNAc (1) and Jericho Blue α-D-GalNAc (2)
The most important characteristics for our purposes are the pKa
of the 7-
CN
F
CO2H
CONH2
b)
OAc
O
a)
i
ii
AcO
AcO
OAc
O
OH
Si
AcO
AcO
O
HO
HO
F
F
F
O
F
F
iii
i
O
OAc
ii
F
F
3
O
F
4
N3
AcO
This journal is © The Royal Society of Chemistry 20xx
5
2 | J. Name., 2012, 00, 1-3
N
3F
N
3F
iii
N
3F
12
11
13
CHO
OBn
14
CN
OBn
CHO
OH
v
iv
iv
BnO
BnO
HO
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F
Si
O
OH
F
6
F
vi
8
7
HO
O
O

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COMMUNICATION
Scheme 1. Reagents and conditions: a) i. DCM, oxalyl chloride, DMF, rt; CHCl3, NH4OH, 0C. ii. POCl3, 80C. iii. DMF, BnOH, K2CO3, 105C. iv. DCM,
DIBAL in cyclohexane (1 M), -78C. v. MeOH, THF, H2, Pd/C, rt. vi. H2O, Meldrum’s acid, NH4OAc, rt. vii. MeOH, H2SO4, refluxed, 90%. b) i). DCM,
HF/Pyridine, 0C, 93%. ii). MeOH, NH3, 0C, 95%. iii). Pyridine, di-tert-butylsilylbistriflate, 0C-rt; Ac2O, rt, 81%. iv). DCM, Jericho Blue.TBA salt (19c),
4Å Molecular sieves, BF3Et2O, 0C. v) THF, AcOH, TBAF, rt; Pyridine, Ac2O, rt, 65%; THF, H2O, Ph3P, silica gel, 50C; Pyridine, Ac2O, rt, 72%; MeOH,
Na, rt; THF, H2O, LiOH, 0C, 69%
reduce the number of ortho fluorine atoms from two to one, as We initially based our approach on the method pioneered by
embodied in compound 2 (Figure 1). However, this fluorophore Kiso in which a di-tert-butylsilylene protecting group installed
has not been reported previously and we had to first synthesize across the
4
and
6 positions strongly directs α-
the requisite monofluorocoumarin derivative. Our approach to glycosylation.[22,23] This is thought to be the outcome of steric
the synthesis of 8-fluoro-7-hydroxycoumarin-3-carboxylic acid blocking of the top (beta) face of the galactoside by the bulky
started from the inexpensive, commercially available 2,3,4- silyl protecting group. For the glycosylation step, we first
trifluorobenzoic acid (3),[20] as shown in Scheme 1a. Treatment followed the method described by Kiso using the Mitsunobu
with oxalyl chloride in dichloromethane containing a catalytic reaction.[24] However, we were unable to achieve the described
amount of dimethylformamide gave the aryl chloride which, yields, quite likely due to the poor nucleophilicity of the low
upon treatment with ammonium hydroxide, afforded amide 4. pKa coumarin derivatives as well as the insolubility of the
This amide was then dehydrated by reaction with phosphorus coumarin derivatives in the appropriate solvents. After testing
oxychloride to afford the electron deficient nitrile 5 which, on a number of approaches and conditions, we shifted our strategy
treatment with benzyl alcohol and base underwent nucleophilic to one involving use of a glycosyl fluoride donor in which
aromatic substitution at the ortho and para positions to yield coupling was performed under catalysis from BF3·Et2O. Further,
bis-benzyl-substituted arene 6. Conversion of the nitrile to the to improve the solubility of the coumarins we elected to convert
aldehyde was achieved by reduction with DIBAL at -78°C and them to their tetrabutylammonium salts (see Scheme S3). The
subsequent hydrolysis of the resultant imine to give aldehyde 7. route followed is shown in Scheme 1b and allowed for
The two benzyl protecting groups were removed by successful formation of the Jericho Blue GalNAc glycoside.
hydrogenation (Pd/C), to yield the diol 8 in good (82%) yield, Further couplings were attempted with a range of coumarin
along with a small amount of the by-product formed from aglycones with yields typically around 70%, though two of the
reduction of the aldehyde, which was removed by flash column more electron-deficient phenols yielded only around 45%. A
chromatography. Reaction of compound 8 with Meldrum’s acid detailed discussion of this synthesis can be found in the SI.
and ammonium acetate in water afforded the desired Pacific Having the substrates at hand, we moved on to demonstrate
Blue analogue, monofluorocoumarin 9, which we have named that the newly synthesized fluorophore would be useful in
Jericho Blue in honour of Jericho beach adjacent to the UBC screening metagenomic libraries in nano-droplets. First, we
campus in Vancouver. The overall yield (from 3 to 9) was a tested to see if the free fluorophore would leak out of the
respectable 57% with only one intermediate requiring water-in-fluorinated oil droplets and exchange with adjacent
purification by flash column chromatography, the rest being droplets. As depicted in Figure S1, only fluorophores that
accomplished by extraction and precipitation. We then contain a charged carboxylate are retained within the droplets
proceeded to characterize this novel fluorophore as reported in upon extended incubation. The retention of the fluorescence
Table 1. The 1.3-unit pKa difference between Jericho Blue and
Pacific Blue was thought to be sufficient to make the glycoside
stable enough for use in screens.
Next, we had to develop a reliable method for the α-
glycosylation reaction of GalNAc with the phenols. Such
syntheses of alpha-configured GalNAc derivatives are much
more difficult than the synthesis of the corresponding beta
glycosides, which benefit from neighbouring group
participation from the 2-acetamide. However, several methods
to effect stereo-controlled 1,2-cis glycosylations of this kind
have been developed over the years, with varying levels of
success. Such approaches have been nicely reviewed by
Demchenko[21]
.
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Fig 2. Droplets after incubation overnight (16 h) at 37oC, a) under bright light and b) UV light (365 nm). The droplets were loaded with enzyme-
active and inactive E. coli in a 1:1000 ratio. Using a fluorescence microscope c) a single droplet containing cleaved substrate can be identified,
showing clear separation from the neighboring droplets
Notes and references
and lack of crosstalk between droplets thus maintains the
genotype-phenotype linkage required for successful screening.
[1]
[2]
[3]
M. E. Taylor, K. Drickamer, Introduction to Glycobiology,
Oxford University Press., 2011.
A. Varki, Essentials of Glycobiology, Old Spring Harbor
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Coutinho, B. Henrissat, Nucleic Acids Res. 2013, 42, D490–
D495.
These leakage results are largely consistent with those
previously observed for similar 7-aminocoumarin derivatives in
the same perfluorinated oil system[15]. Next we performed a set
of mock screens where two types of E. coli cells were
encapsulated within aqueous droplets: those bearing an empty
plasmid and those with the plasmid for expression of an α-
GalNAcase at a ratio of 1000:1 respectively. The aqueous
droplets, suspended in perfluorinated oil, contained the
appropriate growth medium for E. coli cells along with 50 µM of
the substrate 2. As shown in Figure 2, we observed that
approximately 1 in each 1000 droplets were fluorescent, and
that the fluorescence remained stable over time for at least
three days with no observable leakage of fluorophore to the
neighbouring droplets. This experiment was repeated using the
less expensive and more readily available mineral oil and similar
results were observed (Figure S2). Therefore, substrates that
are based upon Jericho Blue appear to be ideal for droplet-
based screening of metagenomic libraries
[4]
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[6]
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In conclusion, we have synthesized a novel 3-carboxycoumarin,
Jericho Blue, that is tailored for water-in-oil droplet-based
screening of glycosidases. We have also devised a reliable
synthetic method for the challenging stereoselective α-
glycosylation of 2-acetamido sugars with these coumarin
derivatives. Finally, we demonstrated the applicability of
Jericho Blue glycosides for droplet-based screening approaches.
[8]
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Conflicts of interest
There are no conflicts to declare.
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We thank the Natural Sciences and Engineering Research
Council of Canada and the Canadian Institutes for Health
Research for financial support. We also thank Dr Ryan
Sweeney for early suggestions on the synthesis, Soroush
Nasseri for his kind gift of the microdroplet chips, and Dan
Seale and Dr. Sachia Traving for assistance in acquiring the
microscope images.
[14]
[15]
[16]
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OH
OH
HO
O
OH
OH
F
F
AcHN
HO
AcHN
O
O
O
O
O
O
O
O
O
+
OH
O
O
Synthesis of sensitive coumarin α-GalNAc
glycosides as substrates for droplet-based
screening of GalNAcases