Chromogenic carbamate and acetal substrates for glycosaminidases

Article abstract of DOI:10.1080/07328303.2011.610543

The enzyme Dispersin B is a medicinally relevant β-hexosaminidase with potential for use in the treatment of biofilm-related infections. Here we describe the synthesis and evaluation of a p-nitrophenyl glycosyl carbamate and a p-nitrophenyl glycosyl aceta

Full text of DOI:10.1080/07328303.2011.610543

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Chromogenic Carbamate and Acetal
Substrates for Glycosaminidases
Anthony Chibba a , Somnath Dasgupta a , Nandadeva Yakandawala b
Srinivasa Madhyastha b & Mark Nitz a
,
a Department of Chemistry, 80 St. George Street , University of
Toronto , Toronto , ON , M5S 3H6
b Kane Biotech, Inc. , Winnipeg , MB R3T 6C6 , Canada
Published online: 14 Dec 2011.
To cite this article: Anthony Chibba , Somnath Dasgupta , Nandadeva Yakandawala , Srinivasa
Madhyastha & Mark Nitz (2011) Chromogenic Carbamate and Acetal Substrates for Glycosaminidases,
Journal of Carbohydrate Chemistry, 30:7-9, 549-558, DOI: 10.1080/07328303.2011.610543
To link to this article: http://dx.doi.org/10.1080/07328303.2011.610543
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Journal of Carbohydrate Chemistry, 30:549–558, 2011
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C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2011.610543
Chromogenic Carbamate
and Acetal Substrates
for Glycosaminidases
Anthony Chibba,1 Somnath Dasgupta,1 Nandadeva
Yakandawala,2 Srinivasa Madhyastha,2 and Mark Nitz1
1Department of Chemistry, 80 St. George Street, University of Toronto,
Toronto, ON, M5S 3H6
2Kane Biotech, Inc., Winnipeg, MB R3T 6C6, Canada
The enzyme Dispersin B is a medicinally relevant β-hexosaminidase with potential for
use in the treatment of biofilm-related infections. Here we describe the synthesis and
evaluation of a p-nitrophenyl glycosyl carbamate and a p-nitrophenyl glycosyl acetal
as improved substrates of Dispersin B. The p-nitrophenyl glycosyl carbamate shows
substantially improved analytical signal in continuous assays and superior kinetics
(kcat/Km 6.1 mM−1 s−1) when compared to the benchmark substrate 4-nitrophenyl N-
acetyl-β-D-glucosaminide (kcat/Km 0.021 mM−1 s−1) in measuring Dispersin B activity.
The improvements observed are compared to the activity of jack bean β-hexosaminidase
toward these substrates.
Keywords Glycosaminidase; Dispersin B; Substrate; Chromophore
INTRODUCTION
Bacterial biofilms have been estimated to be a factor in 65% of human in-
fections.[1] One emerging treatment strategy for biofilm-related infections is
to enzymatically degrade the extracellular matrix supporting the biofilm.[2]
Dispersin B (DspB) is a candidate enzyme for treatment of a wide variety
of biofilm-related infections as it specifically hydrolyzes the 1→6-linked β-
N-acetylglucosamine exopolysaccharide found in many bacterial biofilms.[3–5]
DspB is a family 20 glycoside hydrolase, which includes exo-hexosaminidases
that use the 2-acetamido substituent as a neighboring participating group in
glycoside hydrolysis.[6] The exo-activity of DspB and specificity of the enzyme
has been confirmed by HPLC studies on synthetic oligosaccharides.[7,8]
Received May 31, 2011; accepted July 31, 2011.
Address correspondence to Mark Nitz, Department of Chemistry, 80 St. George Street,
University of Toronto, Toronto, ON, M5S 3H6. E-mail: mnitz@chem.utoronto.ca
549

A. Chibba et al.
550
The substrate specificity of DspB leads to challenges in the development
assays to monitor DspB activity. DspB has poor activity toward common aryl
glycoside substrates, which makes assays with these substrates in dilute so-
lutions or complex biological media challenging. For example, the most com-
mon hexosaminidase substrate is 4-nitrophenyl N-acetyl-β-D-glucosaminide
(pNP-GlcNAc), which, upon enzymatic cleavage of the glycosidic bond, releases
4-nitrophenolate (pNP). Jack bean β-hexosaminidase, another family 20 exo-
hexosaminidase, has a Kcat/Km value of 13 mM−1 s−1 for pNP-GlcNAc, while
the DspB enzyme has a value of only 0.021 mM−1 s−1
.
In an attempt to find an improved DspB substrate, we have synthesized a
glycosyl acetal 1 and a glycosyl carbamate 2, which may better mimic the 1→6
linkage in the native DspB substrate. Both substrates present less steric bulk
around the glycosidic linkage than aryl glycosides but are still competent to
release a chromophore upon glycosidic bond cleavage (Fig. 1).
Substrates containing p-nitroanilide are commonly used in peptide-based
substrates for proteases; however, this is the first example of a p-nitroanilide-
containing glycosidase substrate. Glycosylcarbamates have been synthesized
as prodrugs and as enzymatically cleavable protecting groups, but kinetic anal-
yses of these substrates have not been reported.[9–12] Although many alkyl gly-
cosyl acetals have been synthesized, their activity as glycosidase substrates
has not been reported.
Figure 1: Carbamate and glycosyl acetal hexosaminidase substrates.

Glycosaminidases
551
RESULTS/DISCUSSION
Synthesis of Substrates
Synthesis of the target compound 1 (Sch. 1) began with known 3,4,6-
triacetyl-2-deoxy-2-phthalimido-D-glucose (3).[13] 1-Chloromethoxy-4-nitro-
benzene (8) was readily synthesized in two steps from p-nitrophenol and
chloromethylthioether (Sch. 2). Compound 3 was alkylated using (8) in
acetonitrile with catalytic NaI and K2CO3 as a base.[14] This gave solely
β-glycoside product in moderate yield (40%) (Sch. 1). The phthalimido group
of 4 was then removed with a solution of ethylenediamine in butanol and
the resulting aminol was acetylated with acetic anhydride in pyridine to give
compound 5 in a 78% yield. The final deprotection of 5 to give 1 was carried
out in the presence of NaOMe to afford compound 1 in 90% yield.
Scheme 1: (a) NaI, K2CO3, 8, CH3CN. (b) i. NH2CH2CH2NH2, butanol, 120◦C. ii. Py, Ac2O. (c)
MeOH, NaOMe.
Scheme 2: (a) ClCH2SCH3, NaI, NaH, DMF. (b) SOCl2, DCM.
Compound 2 was synthesized in four steps from commercially available N-
acetylglucosamine. Following peracetylation and selective anomeric deacetyla-
tion with benzyl amine,[15] the free hemiactal 9 was treated with p-nitrophenyl
isocyanate in toluene in the presence of a catalytic amount of triethylamine
(Sch. 3). The resulting protected glycosyl carbamate (10) precipitated directly
from the reaction mixture, giving only the β-carbamate. These results are con-
sistent with observations by Scheeren et al., where glucose derivatives were re-
acted with various alkyl and aryl isocyanates, yielding only the β-carbamates
in the presence of a catalytic base.[16] Deprotection of the remaining acetyl
groups was challenging given the acid and base sensitivity of the glycosyl car-
bamate 10. Zemplen conditions yielded a return of N-acetyl-glucosamine and

A. Chibba et al.
552
Scheme 3: (a) pNP-NCO, cat TEA, Tol, rt, 91%. (b) NH2NH2•H2O, MeOH, rt, 48%.
the p-nitrophenyl isocyanate, likely from deprotonation of the carbamate nitro-
gen followed by elimination of the hemiacetal. HCl in methanol also afforded
N-acetyl-glucosamine. Careful addition of 3.3 equivalents of hydrazine mono-
hydrate in methanol and monitoring the reaction over 6 h yielded the final
product 2 along with some carbamate cleavage product. Crude NMR analysis
revealed 62% yield of the desired product 2 by comparison of the integration of
anomeric signals; however, 48% was isolated via flash chromatography.
Substrate Activity
Initial enzymatic cleavage of substrate 1 was followed by absorbance spec-
troscopy. No increase in A405 indicative of glycosyl bond cleavage was observed
under standard assay conditions (5 mM 1, 0.1 µM DspB, 50 mM phosphate, 100
mM NaCl, pH 6) over a 30-min incubation. Further experiments were carried
out to determine if the glycosidic bond was being cleaved but the acetal failed
1
to fragment. Products of the enzyme incubation were analyzed by H NMR
and showed that no glycosidic bond cleavage was occurring over the course of
the incubation. Similar assays using substrate 1 were also carried out with
the jack bean β-hexosaminidase (1 5 mM, jack bean β-hexosaminidase 25 nM,
sodium citrate buffer 50 mM, pH 5.5). No cleavage of the substrate was ob-
1
served spectrophotometrically or by H NMR analysis. To determine if com-
pound 1 is an inhibitor of the enzymes, crude assays were carried out with
pNP-GlcNAc (0.5 mM) in the presence or absence of 1 (5 mM). No difference in
the pNP-GlcNAc hydrolysis rate in the presence or absence of 1 was observed,
suggesting the enzymes showed little or no recognition of 1.
It is surprising that no affinity for substrate 1 toward DspB or jack bean
β-hexosaminidase was observed. Although it is unclear as to the reason for the
reduced recognition of substrate 1 by the enzymes, we hypothesized that this
may be due to a strong conformational bias in the substrate, preventing recog-
nition. From the 1H NMR spectra of 1 the acetyl protons resonate at 1.46 ppm,
significantly upfield from the region of 1.9 to 2.1 ppm commonly observed for
acetyl protons in N-acetylglucosamine. This suggests that the anisotropic ef-
fect of the aryl ring may be sufficiently close to the acetyl proton to perturb
their resonance. 2-Phenethyl glycosides have been crystallized and subjected
to modeling; these compounds show the phenyl ring folded back toward the

Glycosaminidases
553
Table 1: Kinetic characterization of substrate 2 and comparison with pNPGlcNAc
Enzyme/
Substrate
KM
(mM)
kcat
kcat/KM
Literature kcat/KM
(s−1
)
(mM−1 s−1
)
(mM−1 s−1
)
DisB/2
DisB/pNP-GlcNAc
Jack bean/2
2.3 0.2
14.0 0.1
1.0 0.1
6.1
0.021
51
—
0.02[3]
—
46
2
0.43 0.02
22
2
Jack bean/pNPGlcNAc 0.58 0.03
7.9 0.3
13.6
35.6[18]
pyranoside,[17] and similar shifts in neighboring acetyl protons have been ob-
served in other large flexible aromatic glycosides, such as naphthylmethyl N-
acetyl glucosamine glycosides.[18] Unfortunately, our hypothesis could not be
supported by NOE experiments as no through-space interactions between the
aryl protons and the acetyl group were observed.
Analysis of substrate 2 with DspB and jack bean β-hexosaminidase yielded
promising results under the same conditions as substrate 1. With DspB a 20-
fold improved Km and a 200-fold improved kcat/Km were observed when com-
pared to the benchmark pNP GlcNAc substrate (Table 1). Interestingly, when
substrate 2 was studied with the jack bean β-hexosaminidase, only a three-
fold improvement in kcat was observed and the Km remained similar to pNP-
GlcNAc substrate. The values of Km and kcat for pNP-GlcNAc and jack bean
β-hexosaminidase agree well with previous literature values.[19]
The improved Km for 2 observed for DspB supports our hypothesis that
reduced steric congestion around the glycosidic linkage would improve recog-
nition of the substrate. As the jack bean β-hexosaminidase is promiscuous
with respect to the recognition of the aglycon, no substantial differences in Km
between substrate 2 and pNP-GlcNAc were observed. Both enzymes showed
improved turnover of the glycosylcarbamates in comparison to pNPGlcNAc,
demonstrating that glycosyl carbamates are good substrates for family 20 gly-
coside hydrolases.
The improved analytical signal obtained for substrate 1 over pNP-GlcNAc
is shown in Figure 2 upon hydrolysis with DspB. The increase in analytical
signal results from both the enzyme having improved kinetics as well as p-
nitroaniline having a higher extinction coefficient under the assay conditions
than p-nitrophenol. The combination of improved kinetics and improved ex-
tinction coefficient gives an overall sensitivity enhancement of approximately
35 times under the standard assay conditions (5 mM substrate).
In conclusion, we have demonstrated that the p-nitrophenyl glycosyl ac-
etal (1) is a poor substrate for two family 20 glycosidases. Further experiments
are required to determine the reason these glycosides are so poorly recognized
but the conformation of these substrates is likely playing a role. Glycosyl car-
bamates have been shown to be effective substrates for DspB and jack bean

A. Chibba et al.
554
Figure 2: Hydrolysis of substrate 2 (ꢀ) monitored at 410 nm and hydrolysis of pNP-GlcNAc (ꢁ)
monitored at 405 nm over time in the presence of 50 nM DisB, 50 mM phosphate (pH 5.8),
and 50 mM NaCl.
β-hexosaminidase. DspB displays substantially improved kinetics toward the
glycosyl carbamate 2 over the standard pNPGlcNAc substrate.
EXPERIMENTAL
General
Flash chromatography was performed on Silica-P Flash Silica Gel 60
(40–63 µm particle size, Silicycle). Reactions were monitored by TLC using
Silica Gel 60 F254 (EMD Science) with detection by quenching of fluores-
cence and/or by visualization with phosphomolybdic acid in ethanol (0.5% w/v),
methanolic H2SO4 (10% v/v), or ninhydrin in ethanol (0.2% w/v). Reagents
were obtained from Sigma-Aldrich or Acros Organics and were used without
further purification. Jack bean β-hexosaminidase was obtained from Sigma-
Aldrich as a solution concentration of 1.5 mg/mL. A molecular weight of
100 KDa was used to calculate the jack bean β-hexosaminidase concentration.
Dispersin B (42 KDa, ε = 58670 M−1 cm−1) was obtained from Kane Biotech. 1H
and 13C NMR spectra were recorded at 25◦C with a Mercury 300 MHz or a Var-
ian 400 MHz. High-resolution mass spectra were obtained from an ABI/Sciex
QStar mass spectrometer (ESI). Absorption spectra were measured on a Schi-
madzu UV-2401PC spectrophotometer.

Glycosaminidases
555
Synthesis
4-Nitrophenoxymethoxy 2-phthalimido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-glucopyranoside (4)
3,4,6-Triacetyl-2-deoxy-2-phthalimido-D-glucose (3) (500 mg, 1.15 mmol)
was dissolved in acetonitrile (5 mL) and to it K2CO3 (238 mg, 1.72 mmol) was
added followed by the addition of 4-nitrophenoxymethoxychloride (430 mg,
2.3 mmol) and NaI (258 mg, 1.72 mmol). The reaction was stirred for ap-
proximately 16 h at rt. TLC analysis showed generation of a new spot
(EtOAC:n-pentane, 1:2, Rf 0.5). Solvent was evaporated and the crude mix-
ture was diluted with dichloromethane (25 mL) and washed with water
(50 mL); the organic phase was separated, dried over MgSO4, filtered,
and evaporated to syrup in vacuo. The crude product thus obtained was
purified by flash chromatography eluting with (EtOAC:n-pentane, 1:3) to
furnish 4-nitrophenoxymethoxy 2-phthalimido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranoside 4 (269 mg, 40%) as colorless amorphous solid. 1H NMR
(CDCl3, 300 MHz) δ: 7.68–7.53 (m, 6H), 6.79–6.72 (m, 2H), 5.82 (dd, 2H, J =
9.2 Hz, J = 9.2 Hz,), 5.71 (d, 1H, J = 8.6 Hz), 5.44–5.37 (m, 2H), 5.18 (t, 1H,
J = 9.1 Hz), 4.39–4.28 (m, 2H), 4.22 (dd, 1H, J = 2.1 Hz, 10.2 Hz), 3.97–3.92
(m, 1H), 2.13 (s, 3H), 2.02 (s, 3H), 1.81 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ: 170.6 (2), 170 (2), 169.4, 160.1, 141.6, 134 (2), 125.3 (2), 123.3 (4), 115.2
(2), 94.1, 88.1, 72.2, 70.1, 68.5, 61.7, 54, 20.7, 20.6, 20.4. HRMS calcd for
C27H26N2O13Na(M+Na)+: 609.1333; found 609.1337.
4-Nitrophenoxymethoxy 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-glucopyranoside (5)
Compound 4 (200 mg, 0.34 mmol) was dissolved in butanol (5 mL) under
nitrogen and to it ethylenediamine (1 mL) was added. The solution was heated
to 120◦C for 20 h, after which time the solvent was evaporated and the crude
oil was dried under vacuum. To the crude oil, pyridine (3 mL) and acetic anhy-
dride (2 mL) were added and the solution was stirred at rt overnight. The sol-
vent was evaporated and the crude mixture was diluted with dichloromethane
(25 mL) and washed with 1N HCl (25 mL), water (50 mL), and sodium bicar-
bonate solution (25 mL); the organic phase was separated, dried over MgSO4,
filtered, and evaporated to syrup in vacuo. The crude product thus obtained
was purified by flash chromatography eluting with (EtOAC:n-pentane, 1:1)
to furnish 4-nitrophenoxymethoxy 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
glucopyranoside 5 (132 mg, 78%) as colorless oil. 1H NMR (CDCl3, 300 MHz) δ:
8.16–8.11(m, 2H), 7.07–7.02 (m, 2H), 5.44 (d, 1H, J = 7.6 Hz,), 5.34 (d, 1H, J =
7.6 Hz), 5.23 (t, 1H, J = 8.9 Hz), 5.13–5.05 (m, 2H), 4.85 (d, 1H, J = 8.5 Hz),
4.23 (dd, 1H, J = 4.5 Hz, 12 Hz), 4.12 (dd, 1H, J = 2.3 Hz, 10 Hz), 3.97–3.93
(m, 1H), 3.70–3.66 (m, 1H), 2.04 (s, 3H), 1.96 (s, 3H), 1.94 (s, 3H), 1.46 (s, 3H).

A. Chibba et al.
556
13C NMR (75 MHz, CDCl3) δ: 171, 170.7, 169.8, 161.6, 142.5, 125.7 (2), 116.1
(2), 97.3, 89.1, 72.2, 72.0, 68, 61.7, 53.9, 22.8, 20.7, 20.6 (2). HRMS calcd for
C21H26N2O12Na(M+Na)+: 521.1383; found 521.1386.
4-Nitrophenoxymethoxy 2-acetamido-2-deoxy- β-D-glucopyranoside (1)
Compound 5 (100 mg, 0.2 mmol) was dissolved in MeOH (5 mL) followed
by addition of NaOMe (0.5 M in MeOH, 0.1 mL). The solution was allowed
to stir at rt for 6 h. Then the solution was neutralized with Amberlite IR
120 H+ resin and filtered. The filtrate was evaporated to afford pure compound
4-nitrophenoxymethoxy 2-acetamido-2-deoxy- β-D-glucopyranoside 1 (67 mg,
1
90%) as amorphous white solid. H NMR (MeOD, 300 MHz) δ: 8.24–8.19 (m,
2H), 7.21–7.15 (m, 2H), 5.55 (d, 1H, J = 7.6 Hz), 5.46 (d, 1H, J = 7.6 Hz), 4.75
(d, 1H, J = 8.5 Hz), 3.89 (dd, 1H, J = 1.7 Hz, 10.4 Hz), 3.72–3.63 (m, 2H),
3.47–3.41 (ddd, 1H, J = 8.5 Hz, 1.5 Hz, 8.7 Hz), 3.36–3.27 (m, 3H), 1.49 (s,
3H). 13C NMR (75 MHz, MeOD) δ: 173.5, 163.6, 143.7, 126.6 (2), 117.6 (2), 99.3,
90.5, 78.4, 75.5, 72, 62.8, 57, 22.7. HRMS calcd for C15H20N2O9Na(M+Na)+:
395.1067; found 395.1064.
4-Nitrophenyl carbamoyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-glucopyranoside (10)
Compound 9 (200 mg, 0.58 mmol) was dissolved in toluene (10 mL) fol-
lowed by the addition of a catalytic amount of triethylamine (0.05 mL) and p-
nitrophenyl isocyanate (190 mg, 1.16 mmol). The solution was stirred at rt for
6 h, at which point a yellow solid had precipitated. The solution was chilled in
an ice bath and pure 4-nitrophenyl carbamoyl 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-glucopyranoside was isolated by filtration (253 mg, 86% yield) 1H
NMR (CDCl3, 400 MHz) δ: 8.22 (d, 2H, J = 9.2 Hz), 7.51 (d, 2H, J = 9.2 Hz),
5.61 (d, 1H, J = 8.8 Hz), 5.58 (bd, 1H, J = 5.2 Hz), 5.13–5.05 (m, 2H), 4.45 (d,
1H, J = 8.5 Hz), 4.35 (dd, 1H, J = 4.5 Hz, 12 Hz), 4.15 (dd, 1H, J = 2.3 Hz,
12 Hz), 3.86–3.72 (m, 1H), 2.06 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H).
13C NMR (75 MHz, CDCl3) δ: 171.4, 170.6, 169.8, 161.2, 148.5, 141.2, 130.7 (2),
116.1 (2), 102.3, 89.2, 73.1, 71.9, 68, 61.4, 53.7, 22.4, 20.8, 20.6 (2). HRMS calcd
for C21H26N2O12Na(M+Na)+: 534.1374; found 534.1379.
4-Nitrophenyl carbamoyl 2-acetamido-2-deoxy-β-D-glucopyranoside (2)
Compound 10 (200 mg, 0.4 mmol) was stirred as a slurry in MeOH (10 mL)
and to it was added hydrazine monohydrate (0.05 mL, 1.3 mmol). After 4 h, the
reaction mixture was evaporated under reduced pressure, and the remaining
residue was purified by flash chromatography eluting with MeOH:DCM (1:20)
to afford 4-nitrophenyl carbamoyl 2-acetamido-2-deoxy-β-D-glucopyranoside 2
(80 mg, 48% yield) as a faint yellow powder. 1H NMR (MeOD, 400 MHz) δ: 8.22
(d, 2H, J = 9.2 Hz), 7.51 (d, 2H, J = 9.2 Hz), 5.61 (d, 1H, J = 8.8 Hz), 3.95 (m,

Glycosaminidases
557
2H), 3.75 (dd, 1H, J = 1.7 Hz, 10.4 Hz), 3.62–3.31 (m, 3H), 2.04 (s, 3H). 13C
NMR (75 MHz, MeOD) δ: 173.5, 143.6, 141.7, 129.2 (2), 113.6 (2), 105.2, 90.5,
78.1, 75.2, 72.3, 62.4, 57.3, 22.4. HRMS calcd for C15H19N3O9Na(M+Na)+:
408.1062; found 408.1068.
Enzyme Assays
Enzyme reactions were carried out in solutions containing 50 mM phos-
phate (pH 5.8); 50 mM NaCl, 25 nM DspB, or 50 mM citrate (pH 5.5); 10 nM
jack bean β-hexosaminidase; and varying concentrations of 2 (0.1–2.5 mM)
at 25◦C. Due to substrate solubility constraints and background absorbance,
2.5 mM was used as a maximum concentration of substrate. Reactions were
monitored in real time using the increase of absorbance at 410 nm resulting
from the hydrolysis and release of pNA. Michaelis-Menten kinetic parame-
ters were obtained from direct curve fitting of the data. A slow but measur-
able background hydrolysis rate was also monitored and subtracted from the
enzyme-catalyzed reaction. A control experiment replacing DspB with bovine
serum albumin (BSA, 1 µM) showed no increase in reaction rate above back-
ground. The assay was also performed with varying concentrations of pNPGlc-
NAc (5—1 mM) as a substrate for comparison measuring absorbance at 405
nm, in the same concentration range; however, Michaelis-Menten kinetic pa-
rameters were obtained from double reciprocal plots (1/v vs. 1/S) using first-
order kinetics due to solubility limitations of pNPGlcNAc. All assays were
performed in triplicate. Under the assay conditions the extinction coefficients
of the chromophores were p-nitrophenol 7280 M−1 cm−1 at 405 nM and p-
nitroaniline 5450 M−1 cm−1 at 410 nM.
ACKNOWLEDGEMENT
The authors kindly acknowledge the Natural Science and Engineering Re-
search Council of Canada (NSERC) for funding this work.
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