Evaluating N-benzylgalactonoamidines as putative transition state analogs for β-galactoside hydrolysis

Article abstract of DOI:10.1039/c4ob00153b

Experimental evidence is provided for p-methylbenzyl-d-galactonoamidine to function as a true transition state analog for the enzymatic hydrolysis of aryl-β-d-galactopyranosides by β-galactosidase (A. oryzae). The compound exhibits inhibition constants in the low nanomolar concentration range (12-56 nM) for a selection of substrates. Along these lines, a streamlined synthetic method based on phase-transfer catalysis was optimized to afford the required variety of new aryl-β-d-galactopyranosides. Last, the stability of the galactonoamidines under the assay conditions was confirmed. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00153b

Organic &
Biomolecular Chemistry
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Evaluating N-benzylgalactonoamidines as putative
transition state analogs for β-galactoside
hydrolysis†
Cite this: Org. Biomol. Chem., 2014,
12, 2792
Qiu-Hua Fan, Susanne Striegler,* Rebekah G. Langston and James D. Barnett
Experimental evidence is provided for p-methylbenzyl-D-galactonoamidine to function as a true transition
state analog for the enzymatic hydrolysis of aryl-β-D-galactopyranosides by β-galactosidase (A. oryzae).
The compound exhibits inhibition constants in the low nanomolar concentration range (12–56 nM) for a
selection of substrates. Along these lines, a streamlined synthetic method based on phase-transfer cataly-
sis was optimized to afford the required variety of new aryl-β-D-galactopyranosides. Last, the stability of
the galactonoamidines under the assay conditions was confirmed.
Received 21st January 2014,
Accepted 7th March 2014
DOI: 10.1039/c4ob00153b
www.rsc.org/obc
Introduction
Emerging evidence suggests that the glycosidase family
includes multiple mechanistic pathways for glycoside hydroly-
ses.1 Given this observation, the prediction of whether or not a
given compound is a true transition state analog (TSA) for the
cleavage of a glycosidic bond by the glycosidase of interest
remains speculative and must be confirmed by experimental
evaluation. Such investigation becomes particularly valuable
when a putative TSA is not only to be identified as a very potent
enzymatic inhibitor, but to be used subsequently for the prepa-
ration of catalytic material such as functional enzyme mimics.
As a prerequisite, a potential TSA must behave as a competitive
inhibitor, and not as a non-competitive or mixed inhibitor, to
ensure interaction inside the active site rather than fortuitous
binding outside the active site of an enzyme.2
To experimentally categorize a compound as a TSA, kinetic
experiments with either mutants of the targeted enzyme or
various structurally related derivatives of the original substrate
are sufficient.2 The correlation of the inhibition constant (Ki)
versus the catalytic efficiency (kcat/Km) for a variety of substrates
should reveal a value close to 1 for a linear correlation on a
logarithmic scale if the compound is a true transition state
analog of the reaction; the value of the linear correlation will
be very different from 1, if not.2–5
Chart 1 Structures of aryl-D-galactonoamidines (1a–g).
hydrolysis of commercially available 2-nitrophenyl-β-D-galacto-
pyranoside (2a) as a model reaction.6
The previous studies disclosed 1a–e and 1g as competitive
inhibitors with inhibition constants (Ki) in the low nanomolar
concentration range (12–48 nM).6 Not all of these compounds
may fulfill the experimental requirements for a TSA, but they
may resemble fortuitous binders in the active site of β-galacto-
sidase (A. oryzae) instead. Prior to the preparation of catalytic
microgels from such aryl-D-galactonoamidines, it is thus
imperative to experimentally select a compound with true
transition state-like character for the hydrolysis of glycosidic
bonds.
Along these lines, we report here our preliminary results to
identify N-arylgalactonoamidine 1d as a putative transition state
analog for the enzymatic hydrolysis of β-D-galactopyranosides.
We additionally provide analytical data for 1f and evidence for
the stability of the N-arylgalactonoamidines under the experi-
mental conditions. We furthermore report the synthesis
and full characterization of 12 new aryl-β-D-galactopyranosides
(2b–g and 2i–n) as substrates for the spectroscopic evaluation
of β-galactosidases.
We previously examined the inhibitory effects of aryl-
D-galactonoamidines (1a–e, g) (Chart 1) on the enzymatic
119 Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville,
Results and discussion
AR 72701, USA. E-mail: susanne.striegler@uark.edu
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of all new aryl-tetra-O-acetyl-β-D-galactopyranosides 4b–g, 4i–r, aryl-β-D-galacto-
pyranosides 2b–g, 2i–r and galactonoamidine 1f. See DOI: 10.1039/c4ob00153b
In order to determine the transition state-like character of the
galactonoamidines 1a–f, β-galactosidase (A. oryzae) was used
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With a variety of substrates available, we then developed
kinetic assays in 96-well plate format to monitor the product
formation of the enzymatic glycoside hydrolysis in the pres-
ence or absence of galactonoamidines 1a–g using UV/Vis spec-
troscopy at 405 nm. The protein concentration of the
commercially available β-galactosidase (A. oryzae) was pre-
viously determined by a BCA assay.6 Conversion of the
measured absorbance into product concentration was achieved
with calibration curves for the molar absorptivity for each sub-
strate under the given experimental conditions. The high
absorption coefficient of 2-chloro- and 2-fluoro-4-nitrophenyl-
β-D-galactopyranoside renders these two substrates superior to
commercially available 2a and 2h, especially for assays requir-
ing acidic conditions. Compounds 2l–o, 2q,r were not used for
evaluating the enzymatic hydrolysis by UV/Vis spectroscopy
due to the low absorptivity of the resulting phenolates under
the reaction conditions and the accompanying uncertainty of
the data. Galactopyranoside 2p was found to show surprising
low water solubility under the used condition limiting its
usage in this assay as well.
Scheme 1 Synthesis of aryl-β-D-galactopyranosides substrates 2b–g
and 2i–r.
as model catalyst.6,7 Unfortunately, the spectroscopic evalu-
ation of β-galactosidases with commercial substrates is limited
to 2-nitro- and 4-nitrophenyl-β-D-galactopyranoside 2a and 2h,
respectively. In need of structurally related aryl-β-D-galactopyra-
nosides, we initially synthesized and characterized 15 new aryl-
β-D-galactopyranosides 2b–g and 2i–r (Scheme 1) taking advan-
tage of synthetic procedures toward related compounds
described previously by Thiem et al.8
Along these lines, we reacted a selection of commercially
available phenols with peracetylated α-galactosyl bromide (3)
using a tetrabutylammonium bromide-catalyzed glycosylation
in alkaline solution (Scheme 1, Table 1).8 Chromatographic
purification of the obtained tetraacetylaryl-β-D-galactopyrano-
sides 4b–g, i–r over silica gel and subsequent deacetylation
with ammonia in methanol afforded the target glycosides
2b–g, i–r in moderate to good yields (60–85%).
The rate of the reaction was corrected for the enzyme con-
centration and the uncatalyzed reaction, and then plotted
versus the substrate concentration. By applying a non-linear fit
to the resulting hyperbolic data, the catalytic rate constant
kcat [min−1] and the substrate affinity KM [mM] were dis-
cerned utilizing the Michaelis–Menten model (Table 2). In the
presence of galactonoamidines, apparent Michaelis–Menten
constants (K′m) and rate constants (k′ ) were determined in a
cat
similar fashion and allowed the calculation of the inhibition
constant (Ki) for each substrate (Table 2).
All compounds 1a–g are very potent inhibitors that hamper
the enzymatic hydrolysis of the selected substrates 2a–k in the
low nanomolar concentration range (8–63 nM). To evaluate
their potency as putative transition state analogs, we correlated
the catalytic efficiency of the uninhibited enzymatic catalysis
for each substrate (2a–k) to the corresponding inhibition con-
stant of galactonoamidines 1a–g. A reasonable correlation with
a value close to 1 is observed for 1d, while the correlations
obtained for 1b, 1c (Fig. 1) and for 1e and 1g (Fig. 2) clearly
result in scattered data, a linear factor (a) very different from 1,
and an overall very poor correlation factor (R2) for the fit of the
data. A reasonable correlation with a value close to 1 is also
observed for 1f (Fig. 2) and 1a (data not shown), but the corre-
lation factors for the fit of the data are not as good as for 1d.
Consequently, we propose 1d, p-methylbenzyl-D-galactonoami-
dine, as a true transition state analog for the enzymatic hydro-
lysis of β-D-galactopyranosides by β-galactosidase (A. oryzae).
Subsequently, the stability of 1d under various pH con-
ditions was studied prior to further investigation of the inhi-
bition of other glycosidases or the preparation of biomimetic
catalysts derived therefrom. Galactonoamidine 1d was found
stable for a minimum of 3 h, when exposed to nanopure water
at 30, 50 or 72 °C. Likewise, no evidence for compound
decomposition was observed by HPLC assays relying on a
carbohydrate-discriminating Na+-RMN column as stationary
phase and nanopure water as eluent, when exposing 1d to
Table 1 Arylglycosides 2a–n and the molar absorptivity of their aryl
aglycon in 50 mM acetate buffer at pH 5 and 30 °C
b
Entry
S
R2
R3
R4
R5
ε405
1
2
3
4
5
6
7
8
2aa
2b
2c
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
F
Cl
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NO2
NO2
Me
H
H
Me
F
Cl
Br
H
H
NO2
NO2
NO2
Me
Cl
H
H
MeO
NO2
NO2
H
H
H
H
H
F
Me
H
H
H
H
H
H
H
H
H
H
450
1083
851
936
1047
495
538
223
3145
5595
152
104
56
2d
2e
2fc
2g
2ha
2ic
2kc
2l
9
10
11
12
13
14
15
16
17
2m
2n
2o
2p
2q
2r
H
NO2
NO2
Me
H
182
2678
288
209
H
Me
a Commercially available. b ε [M−1 cm−1
] is the molar extinction
coefficient of the phenolate in 50 mM acetate buffer at pH 5 and 30 °C.
c Known.8,11
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Table 2 Kinetic parameters for the evaluation of 1a–g as putative transition state analogs
kcat
KM
[mM]
Ki [1a] ×
Ki [1b] ×
Ki [1c] ×
Ki [1d] ×
Ki [1e] ×
Ki [1f] ×
Ki [1g] ×
Entry
S
[min−1
]
10−9 [M]
10−9 [M]
10−9 [M]
10−9 [M]
10−9 [M]
10−9 [M]
10−9 [M]
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
2e
2f
2g
2h
2i
4590
4808
4658
4138
3882
3886
4209
3238
3670
2381
1.27
1.49
1.24
0.96
1.10
1.78
4.27
0.84
0.92
0.70
20.8
22.9
18.4
14.6
15.9
25.7
37.1
16.1
14.8
21.1
14.8
9.7
9.7
24.6
22.7
20.6
22.8
22.6
28.8
45.2
26.7
26.2
29.4
23.3
14.1
13.3
11.6
16.5
24.6
55.7
15.1
11.5
8.0
17.3
22.9
12.0
8.7
24.6
11.1
28.0
20.9
12.2
16.1
16.4
22.3
18.1
13.3
14.2
30.3
58.1
22.8
13.7
20.3
18.6
11.1
11.0
8.7
11.4
62.7
31.0
14.6
15.0
10.1
7.6
10.3
17.1
22.8
17.0
22.2
23.6
10
2k
Fig. 1 Double-logarithmic correlation between the catalytic efficiency
(kcat/KM) and the inhibition constant (Ki) with linear fit y = ax + b for 11
nitrophenyl-β-D-glycosides in the presence of 1b (black triangle), a =
0.42, R2 = 0.201; 1c (red circle), a = 0.44; R2 = 0.766; 1d (green penta-
gon); a = 1.07; R2 = 0.946.
Fig. 2 Double-logarithmic correlation between the catalytic efficiency
(kcat/KM) and the inhibition constant (Ki) with linear fit y = ax + b for 11
nitrophenyl-β-D-glycosides in the presence of 1e (blue triangle), a =
0.40, R2 = 0.204; 1f (green diamond), a = 0.95; R2 = 0.869; 1 g (purple
pentagon); a = 0.67; R2 = 0.625.
acetate buffer at 30 °C for the same amount of time. However,
the glyconoamidine stability under alkaline conditions is
debated in the literature,9–14 and insufficient stability of 1d in
conditions previously used for the preparation of microgels,
i.e. 50 mM CAPS buffer at pH 10.5 and 72 °C, was noted.
Recent preliminary results disclose sufficient stability of 1d in
5 mM CAPS buffer at pH 10.5 and 0 °C or 5 mM TAPS buffer at
pH 9 and 10 °C. As the preparation of enzyme mimics at elev-
ated pH is envisioned, related studies are ongoing.
p-methylbenzyl-D-galactonoamidine 1d (Ki = 8–56 nM) was
identified here as a putative TSA for the hydrolysis of β-galacto-
pyranosides by β-galactosidase (A. oryzae). The results obtained
indicate a strong stereoelectronic effect of the p-substituted
position of the aromatic aglycon in the selected aryl-D-galacto-
noamidines that influences both the binding affinity of the
inhibitors 1a–g and their ability to function as transition state
analogs during the enzymatic hydrolysis of β-D-galactopyrano-
sides. The presented results provide the foundation for the
evaluation of galactonoamidine 1d as an inhibitor and a puta-
tive TSA of other glycosidases, and their transition state-like
features, and its use as TSA during preparation of macromole-
cular enzyme mimics targeting the hydrolysis and synthesis of
glycosidic bonds.
Conclusions
The preparation of functional enzyme mimics based on a tem-
plate resembling a transition state analog-like structure ideally
includes an experimental evaluation of the compound prior to
its use to critically evaluate its potential. Along these lines, a
kinetic approach was used that relies on the correlation of the
inhibition constants (Ki) of the target compound to the cataly-
tic efficiency (kcat/KM) in the presence of various structurally
Experimental
Instrumentation
related substrates. Overall, 7 galactonoamidines, all previously 1H and 13C NMR spectra were recorded on a 400 MHz Bruker
identified as competitive inhibitors, were evaluated and magnet with Z gradient and 5 mm broadband head using
2794 | Org. Biomol. Chem., 2014, 12, 2792–2800
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Topspin 2.1 software. IR spectra were obtained on a Perkin- The pH meter was calibrated before each set of readings
Elmer Spectrum 100 FT-IR spectrophotometer with Perkin- (3-point calibration).
Elmer spectrum express software versions 1.01.00. High resolution
All commercially obtained chemicals had reagent grade
mass spectrometry data were obtained in the state-wide mass quality or better and were used as received, if not noted other-
spectrometry facility at Arkansas University on a Bruker wise. Per-acetylated galactosyl bromide was prepared as
ultrOTOF-Q quadrupole time-of-flight (qQ-TOF) mass spectro- described.15,16 All phenols 2-nitrophenol, 3-nitrophenol,
meter equipped with an electrospray ionization source or the 4-nitrophenol, 4-fluoro-2-nitrophenol, 4-chloro-2-nitrophenol,
Mass Spectrometry Facility at Georgia State University, Atlanta, 4-bromo-2-nitrophenol, 3-methyl-2-nitrophenol, 4-methoxy-
GA. Combustion data were obtained from Atlantic Microlab, 2-nitrophenol, 4-methyl-2-nitrophenol, 5-fluoro-2-nitrophenol,
Atlanta, GA. UV/Vis data were recorded on a FilterMax F5 5-methyl-2-nitrophenol, 4-methyl-3-nitrophenol, 4-chloro-3-
Multi-Mode Microplate Reader from Molecular Devices using nitrophenol, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenol,
96 well, medium-binding microlon Elisa-plates from Greiner 2-chloro-4-nitrophenol, 2-fluoro-4-nitrophenol and 3-iodo-4-
Bio-one. Lyophilization was performed on a FreeZone 1 liter nitrophenol were sublimed, recrystallized from water or
benchtop freeze dry system from Labconco. Melting points purified by column chromatography on silica gel using ethyl
were recorded on a Mel-Temp melting point apparatus, and acetate and hexane as eluent prior to the determination of
the values are uncorrected. Nanopure water at a resistance of their extinction coefficients.
18.2 mΩ was obtained from a ThermoScientific Barnstead
β-Galactosidase [3.2.1.23] from Aspergillus oryzae was
E-pure™ water purification system. The stability assays were obtained from Sigma-Aldrich as lyophilized powder stabilized
performed on an HPLC system from Shimadzu equipped with on dextrin and stored at −18 °C. A BCA assay confirmed that
SCL-10Avp system controller, 2 LC-20AD analytical pumps, the supplied batch contains 10% of protein.6 The molar
DGU-20A3R three channel online degassers, SIL-20A UFLC weight of the tetrameric protein was determined as 347.2 kDa
autosampler with 96 well capability, CTO-20A/prominence (86.8 kDa per unit) by microchip electrophoresis.6
column oven and ELSD-90LT light scattering and LC solution
software, version 1.25 from Shimadzu for data recording and
analysis.
Synthetic procedures
Synthesis of inhibitors: general procedure yielding N-benzyl-
D-galactonoamidines. The galactonoamidines 1a–g were pre-
Materials and methods
pared as described previously.6,7 Sufficient analytical data for
Chemical shifts (δ) in NMR data are expressed in parts per 1f were obtained after repetitive purification of the precursor
million (ppm) and coupling constants (J) in Hz. Signal multi- compound as described previously, and are summarized
plicities are denoted as s (singlet), d (doublet), t (triplet), q below.
(quartet) and m (multiplet). Deuterated chloroform, acetone-
3-Fluorobenzyl-D-galactonoamidine
(1f). m-Fluorobenzyl-
d6, DMSO-d6 and deuterium oxide were used as solvents. 2,3,4,6-tetra-O-benzyl-β-D-galactonoamidine6 (64.0 mg,
Chemical shift values are reported relative to the residual 0.1 mmol) and 128.0 mg 10% Pd on charcoal (0.06 mmol,
signals of these solvents (CDCl3, δH 7.29, δC 77.0; acetone-d6, 0.6 eq.) were stirred under hydrogen in 7.5 mL ethanol in the
δH 2.05, δC 29.8; DMSO-d6, δH 2.50, δC 39.5; D2O: δH 4.80 δC presence of 1.5 mL (19.5 mmol) trifluoroacetic acid at ambient
29.8 after addition of a few drops of acetone-d6. High resolu- temperature for 24 h. The mixture was filtered through a pad
tion mass spectrometry data were obtained from samples that of celite, and the celite was washed three times with 2 mL
were mixed with acetonitrile containing 0.1% formic acid and methanol. The combined filtrates were concentrated under
injected into the source via syringe pump operating at 3 µL h−1
.
reduced pressure, and the resulting residue was lyophilized
The dry gas temperature was 180 °C, the dry gas flow was yielding 1f as a very hygroscopic, colourless foam in 95% yield
5 L min−1, and the nebulizing gas pressure was 1 bar. The (27.0 mg, 0.095 mmol); Rf 0.25 (SiO2, MeOH); δH (D2O) 7.38
remaining instrument parameters were optimized to obtain (dd, 14.8, 7.5, 1H), 7.00–7.16 (m, 3H), 4.55–4.68 (m, 3H), 4.26
maximum signal for ions between 100–1000 amu. IR data were (br. s., 1H), 3.95 (dd, 10.0, 2.0, 1H), 3.70–3.78 (m, 2H), 3.66
obtained as thin films on KBr discs or as KBr pellets (ν in (dd, 13.3, 9.8, 1H); δC (D2O + MeOH-d4) 165.5, 163.9 (1JC,F
=
cm−1) with a resolution of 0.5 cm−1
.
245.0 Hz), 137.3 (3JC,F = 7.3 Hz), 131.8 (3JC,F = 8.1 Hz), 124.0
Column chromatography was carried out using silica gel 60 (4JC,F = 2.9 Hz), 116.2 (2JC,F = 21.3 Hz), 115.0 (2JC,F = 22.7 Hz),
from Silicycle® (40–63 µm, 230–240 mesh). Thin layer chrom- 71.6, 68.0, 67.5, 60.9, 58.4, 45.6 (4JC,F = 2.0 Hz; HR-ESI MS
atography (TLC) was performed using silica gel TLC plates found: 285.1248, calculated for C13H18FN2O4, [M + H]+:
from SORBENT Technologies, 200 µm, 4 × 8 cm, aluminum 285.1251.
backed, with fluorescence indicator F254 and detection by UV
Synthesis of substrates: general procedure yielding aryl-tetra-
light or by charring with an ethanolic vanillin-sulfuric acid O-acetyl-β-D-galactopyranosides.8 The syntheses of the title
reagent and subsequent heating of the TLC plate.
compounds were achieved by phase-transfer-catalyzed glycosy-
All pH values were obtained using a Beckman Φ 250 pH lation of phenols with peracetylated galactosyl bromide.15,16
meter equipped with a refillable ROSS combination pH elec- Chromatographic purification of the raw material on silica gel
trode from Orion with epoxy body and a 8 mm semi-micro tip. yielded the title compounds in moderate to good yields. The
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eluent consisted of cyclohexane–ethyl acetate mixtures if not 2.9 Hz), 127.4 (3JC,F = 11.0 Hz), 110.4 (2JC,F = 23.4 Hz), 107.0
noted otherwise.
(2JC,F = 27.1 Hz), 100.4, 70.3, 67.5, 66.7, 61.6, 20.5, 20.5, 20.4,
4-Methyl-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside 20.3 Calcd for C20H22FNO12 C, 49.29; H, 4.55; N, 2.87. Found:
(4b). Off-white solid; mp 188–190 °C; Rf 0.25 (SiO2, cyclo- C, 49.28; H, 4.63; N, 2.85.
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.58 (d, 1.5, 1 H),
5-Methyl-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside
7.40–7.17 (m, 2 H), 5.52 (dd, 10.5, 8.0, 1 H), 5.48–5.40 (m, 1 H), (4g). Off-white solid; mp 218–219 °C; Rf 0.25 (SiO2, cyclo-
5.09 (dd, 10.5, 3.4, 1 H), 5.02 (d, 8.1, 1 H), 4.25 (dd, 11.2, 6.9, hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.72 (d, 8.3, 1 H),
1 H), 4.16 (dd, 11.6, 6.3, 1 H), 4.09–3.95 (m, 1 H), 2.18 (s, 3 H), 7.15 (d, 1.0, 1 H), 7.03–6.91 (m, 1 H), 5.54 (dd, 10.6, 7.8, 1 H),
2.13 (s, 3 H), 2.06 (s, 3 H), 2.01 (s, 3 H); δC (CDCl3) 170.3, 5.46 (dd, 3.3, 1.0, 1 H), 5.13–5.04 (m, 1 H), 4.27–4.13 (m, 1 H),
170.2, 170.1, 169.4, 147.0, 141.2, 134.2, 134.1, 125.1, 120.1, 4.13–4.05 (m, 1 H), 2.18 (s, 3 H), 2.11 (s, 3 H), 2.07 (s, 3 H),
101.0, 71.2, 70.5, 67.8, 66.7, 61.3, 20.6, 20.6, 20.5, 20.4; Calcd 2.00 (s, 3 H); δC (CDCl3) 170.2, 170.1, 170.1, 169.3, 149.4,
for C21H25NO12 C, 52.17; H, 5.21; N, 2.90; found: C, 52.08; 145.3, 138.9, 125.2, 124.2, 120.0, 100.6, 71.4, 70.5, 67.8, 66.8,
H, 5.21; N, 2.92.
4-Fluoro-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside H, 5.21; N, 2.90. Found: C, 51.89; H, 5.03; N, 2.92.
(4c). Off-white solid; mp 172–173 °C; Rf 0.28 (SiO2, cyclo-
2-Fluoro-4-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside.8
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.53 (dd, 7.6, 3.0, (4i). Off-white solid; mp 130–131 °C (lit.8 mp 134 °C); Rf 0.31
1 H), 7.41 (dd, 9.3 Hz, 4.5), 7.30–7.20 (m, 1 H), 5.51 (dd, 10.5, (SiO2, cyclohexane–ethyl acetate 3/2, v/v); δH (CDCl3)
61.6, 21.8, 20.6, 20.6, 20.5; Calcd for C21H25NO12 C, 52.17;
=
8.0, 1 H), 5.45 (dd, 3.4, 0.9, 1 H), 5.09 (dd, 10.5, 3.4, 1 H), 5.01 8.09–8.08 (m, 1 H), 7.37–7.19 (m, 1 H), 5.54 (dd, 10.5, 8.0, 1 H),
(d, 7.8, 1 H), 4.25 (dd, 11.2, 6.9, 1 H), 4.15 (dd, 11.4, 6.3, 1 H), 5.47 (dd, 3.4, 0.9, 1 H), 5.20–5.04 (m, 2 H), 4.29–4.02 (m, 3 H),
4.08–4.00 (m, 1 H), 2.21–2.16 (m, 3 H), 2.12 (s, 3 H), 2.05 (s, 3 2.19 (s, 3 H), 2.12–2.04 (m, 6 H), 2.02 (s, 3 H); δC(CDCl3) 170.2,
H), 2.00 (s, 3 H); δC (CDCl3) 170.2, 170.1, 170.0, 169.4, 157.5 170.0, 170.0, 169.2, 152.0 (1JC,F = 253.2 Hz), 149.7 (3JC,F
=
(1JC,F = 248.8 Hz), 145.4 (4JC,F = 2.9 Hz), 122.5 (3JC,F = 8.1 Hz), 11.0 Hz), 143.2, 120.3 (4JC,F = 3.7 Hz), 118.4, 112.9 (2JC,F = 23.4
120.5 (2JC,F = 22.7 Hz), 112.3 (2JC,F = 27.8 Hz), 101.2, 71.4, Hz), 100.1, 71.6, 70.3, 68.1, 66.6, 61.2, 20.6, 20.6, 20.5; Calcd
70.4, 67.8, 66.6, 61.2, 20.6, 20.6, 20.6, 20.5; Calcd for for C20H22FNO12 C, 49.29; H, 4.55; N, 2.87. Found: C, 49.26;
C20H22FNO12 C, 49.29; H, 4.55; N, 2.87. Found: C, 49.13; H, H, 4.61; N, 2.89.
4.65; N, 2.87.
2-Chloro-4-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside.8,17
4-Chloro-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside (4k). Off-white solid; mp 148–149 °C (lit.17 mp 147–149 °C); Rf
(4d). Off-white solid; mp 192–193 °C; Rf 0.31 (SiO2, cyclo- 0.30 (SiO2, cyclohexane–ethyl acetate = 3/2, v/v); δH (CDCl3)
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.79 (d, 2.5, 1 H), 8.30 (d, 2.5, 1 H), 8.17–8.11 (m, 1 H), 7.29–7.27 (d, 10.4 Hz,
7.49 (dd, 8.8, 2.5, 1 H), 7.34 (d, 8.8, 1 H), 5.53 (dd, 10.5, 8.0, 1 H), 5.62 (dd, 10.4, 7.8, 1 H), 5.50 (dd, 3.4, 0.9, 1 H), 5.18–5.08
1 H), 5.47 (d, 3.0, 1 H), 5.10 (dd, 10.6, 3.3, 1 H), 5.05 (d, 7.8, (m, 1 H), 4.31–4.08 (m, 2 H), 2.20 (s, 3 H), 2.10 (s, 3 H), 2.08
1 H), 4.25 (dd, 11.4, 7.1, 1 H), 4.16 (dd, 11.4, 6.1, 1 H), (s, 3 H), 2.03 (s, 3 H); δC (CDCl3) 170.3, 170.1, 170.1,
4.10–4.02 (m, 1 H), 2.19 (s, 3 H), 2.13 (s, 3 H), 2.07 (s, 3 H), 169.2, 157.3, 143.1, 126.2, 125.4, 124.8, 124.5, 123.5, 116.3,
2.02 (s, 3 H); δC (CDCl3) 170.2, 170.1, 170.1, 169.4, 147.8, 116.2, 99.9, 71.6, 70.3, 67.8, 66.6, 61.3, 20.7, 20.6, 20.6, 20.5.;
141.6, 133.5, 129.1, 125.1, 121.4, 100.8, 71.5, 70.4, 67.7, 66.6, Calcd for C20H22ClNO12: C, 47.68; H, 4.40. Found: C, 47.81;
61.3, 20.6, 20.6, 20.5; Calcd for C20H22ClNO12 C, 47.68; H, 4.30.
H, 4.40; N, 2.78. Found: C, 47.65; H, 4.42; N, 2.74.
4-Chloro-3-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside
4-Bromo-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside (4m). Off-white solid; mp 121–122 °C; Rf 0.36 (SiO2, cyclo-
(4e). Off-white solid; mp 198–199 °C; Rf 0.30 (SiO2, cyclo- hexane–ethyl acetate = 3/2); δH (CDCl3) 7.57 (d, 3.0, 1 H), 7.47
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.92 (d, 2.3, 1 H), (d, 8.8, 1 H), 7.17 (dd, 9.0, 2.9, 1 H), 5.55–5.44 (m, 2 H), 5.13
7.62 (dd, 8.8, 2.5, 1 H), 7.25 (d, 1.5, 1 H), 5.52 (dd, 10.4, 8.1, (dd, 10.5, 3.4, 1 H), 5.09 (d, 7.8, 1 H), 4.18 (dd, 6.2, 1.6, 2 H),
1 H), 5.45 (d, 2.5, 1 H), 5.08 (dd, 10.5, 3.4, 1 H), 5.03 (d, 8.1, 4.13 (dd, 6.3, 1.0, 1 H), 2.19 (s, 3 H), 2.09 (d, 0.5, 6 H), 2.02 (s,
1 H), 4.23 (dd, 11.4, 7.1, 1 H), 4.15 (dd, 11.4, 6.1, 1 H), 3 H); δC (CDCl3) 170.5, 170.1, 170.0, 169.2, 155.2, 148.0, 132.6,
4.09–4.00 (m, 1 H), 2.18 (s, 3 H), 2.11 (s, 3 H), 2.06 (s, 3 H), 122.4, 121.1, 113.5, 99.2, 71.7, 70.5, 68.2, 66.8, 61.7, 20.7, 20.6,
2.00 (s, 3 H); δC (CDCl3) 170.3, 170.1, 170.1, 169.3, 148.3, 20.5, 20.5; Calcd for C20H22ClNO12 C, 47.68; H, 4.40; N, 2.78.
141.9, 136.5, 127.9, 121.6, 115.9, 100.8, 71.5, 70.4, 67.7, 66.6, Found: C, 47.79; H, 4.51; N, 2.77.
61.3, 20.6, 20.6, 20.5; Calcd for C20H22BrNO12 C, 43.81;
H, 4.04; N, 2.25. Found: C, 43.67; H, 4.14; N, 2.53.
3-Nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside (4n). Off-
white solid; mp 103–104 °C, Rf 0.27 (SiO2, cyclohexane–ethyl
5-Fluoro-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside.8 acetate = 2/1, v/v); δH (CDCl3) 7.97 (dt, 8.2, 1.0, 1 H), 7.90 (t,
(4f). Off-white solid; mp 158–159 °C (lit.8 159 °C), Rf 0.37 2.3, 1 H), 7.48 (t, 8.2, 1 H), 7.35–7.32 (m, 1 H), 5.53 (dd, 10.4,
(SiO2, cyclohexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.90 7.9, 1 H), 5.50 (d, 3.5, 1 H), 5.16–5.13 (m, 2 H), 4.22–4.14 (m,
(dd, 9.1, 5.8, 1 H), 7.14 (dd, 10.0, 2.7, 1 H), 6.96–6.82 (m, 1 H), 3 H), 2.20 (s, 3 H), 2.11 (s, 3 H), 2.10 (s, 3 H), 2.03 (s, 3 H) δC
5.57 (dd, 10.5, 8.0, 1 H), 5.47 (dd, 3.4, 0.9, 1 H), 5.18–4.97 (m, 2 (CDCl3) 170.6, 170.1, 170.0, 169.3, 157.0, 149.1, 130.2, 123.7,
H), 4.37–3.98 (m, 3 H), 2.18 (s, 3 H), 2.14–2.11 (m, 3 H), 2.09 118.2, 111.3, 99.2, 71.7, 70.6, 68.3, 66.9, 61.8, 20.7, 20.6, 20.6,
(s, 3 H), 2.01 (s, 3 H); δC (CDCl3) 170.2, 170.0, 169.9, 169.2, 20.6; Anal. Calcd for C20H23NO12 C, 51.18; H, 4.94; N, 2.98.
164.8 (1JC,F = 256.9 Hz), 151.3 (3JC,F = 11.7 Hz), 137.3 (4JC,F
=
Found: C, 51.46; H, 5.03; N, 3.05.
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3-Methyl-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside 2.31 (s, 3H); δC (DMSO-d6) 147.5, 140.0, 134.5, 131.3, 124.5,
(4o). Off-white solid; mp 167–168 °C; Rf 0.32 (SiO2, cyclo- 117.0, 101.2, 75.7, 73.4, 70.0, 68.0, 60.2, 19.6; Calcd for
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.30 (t, 7.8, 1H), C13H17NO8 C, 49.52; H, 5.43; N, 4.44; found: C, 49.48; H, 5.51;
7.15 (d, 8.3, 1H), 7.02 (d, 7.8, 1H), 5.42–5.53 (m, 2H), 5.08 (dd, N, 4.39.
10.5, 3.5, 1H), 4.98 (d, 8.0, 1H), 4.27 (dd, 11.5, 7.0, 1H), 4.17
4-Fluoro-2-nitrophenyl-β-D-galactopyranoside (2c). Off-white
(dd, 11.0, 6.3, 1H), 4.06 (t, 6.3, 1H), 2.31 (s, 3H), 2.19 (s, 3H), solid; mp 179–181 °C; Rf 0.36 (SiO2, ethyl acetate–methanol =
2.14 (s, 3H), 2.08 (s, 3H), 2.01 (s, 3H); δC (CDCl3) 170.3, 170.2, 9/1, v/v); δH (DMSO-d6) 7.86 (dd, 8.0, 2.7, 1 H), 7.67–7.50 (m,
170.1, 169.3, 147.8, 143.3, 131.1, 130.5, 125.8, 115.9, 100.9, 1 H), 7.50–7.32 (m, 1 H), 5.18 (d, 5.1, 1 H), 4.99 (d, 7.6, 1 H),
71.3, 70.5, 67.7, 66.7, 61.3, 20.6, 20.5, 20.5, 16.9; Calcd for 4.91 (d, 5.8, 1 H), 4.77–4.52 (m, 2 H), 3.69 (br. s., 1 H),
C21H25NO12 C, 52.17; H, 5.21; N, 2.90. Found: C, 52.35; 3.66–3.59 (m, 1 H), 3.59–3.44 (m, 3 H), 3.44–3.30 (m, 2 H); δC
H, 5.30; N, 2.89.
(DMSO-d6) 155.3 (1JC,F = 241.0 Hz), 146.1 (4JC,F = 2.2 Hz), 140.1
4-Methoxy-2-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside (3JC,F = 8.8 Hz), 120.8 (2JC,F = 23.4 Hz), 118.9 (3JC,F = 8.1 Hz),
(4p). Off-white solid; mp 157–158 °C; Rf 0.29 (SiO2, cyclo- 111.8 (2JC,F = 27.8 Hz), 101.6, 75.8, 73.3, 70.0, 68.0, 60.3; Calcd
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 7.33 (d, 9.3 Hz, for C12H14FNO8 C, 45.15; H, 4.42; N, 4.39. Found: C, 44.89;
1 H), 7.04 (dd, 9.1, 3.3, 1 H), 5.50 (dd, 10.5, 8.8, 1 H), 5.45 (dd, H, 4.47; N, 4.32.
3.3, 1.0, 1 H), 5.09 (dd, 10.5, 3.4, 1 H), 4.96 (d, 8.1, 1 H), 4.25
4-Chloro-2-nitrophenyl-β-D-galactopyranoside (2d). Off-white
(dd, 11.2, 6.9, 1 H), 4.15 (dd, 11.2, 6.4, 1 H), 4.04–3.96 (m, solid; mp 207–208 °C; Rf 0.38 (SiO2, ethyl acetate–methanol =
1 H), 3.83 (s, 2 H), 2.19 (s, 3 H), 2.14 (s, 3 H), 2.05 (s, 3 H), 2.01 9/1, v/v); δH (DMSO-d6) 8.02 (d, 2.5, 1 H), 7.71 (dd, 9.1, 2.5,
(s, 3 H); δc (CDCl3) 170.3, 170.2, 170.1, 169.6, 155.6, 142.8, 1 H), 7.45 (d, 9.1, 1 H), 5.20 (d, 5.3, 1 H), 5.04 (d, 7.6, 1 H), 4.92
123.0, 119.9, 109.1, 101.6, 71.2, 70.5, 67.9, 66.7, 61.2, 56.0, (d, 6.1, 1 H), 4.67 (t, 5.4, 1 H), 4.62 (d, 4.3, 1 H), 3.72–3.61 (m,
20.7, 20.6, 20.5; Calcd for C21H25NO13 C, 50.50; H, 5.05; 2 H), 3.59–3.36 (m, 4 H); δC (DMSO-d6) 148.4, 140.5, 133.6,
N, 2.80. Found: C, 50.60; H, 5.14; N, 2.78.
125.0, 124.2, 118.8, 101.1, 75.8, 73.3, 69.9, 67.9, 60.2; Calcd for
2-Methyl-4-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside C12H14ClNO8 C, 42.93; H, 4.20; N, 4.17. Found: C, 43.01; H,
(4q). Colorless solid; mp 128–129 °C, Rf 0.52 (SiO2, cyclo- 4.07; N, 4.03.
hexane–ethyl acetate = 1/1, v/v); δH (CDCl3) 8.07–8.05 (m, 2 H),
4-Bromo-2-nitrophenyl-β-D-galactopyranoside (2e). Off-white
7.07–7.04 (m, 1 H), 5.59 (dd, 10.5, 7.8, 1 H), 5.50 (d, 3.3, 1 H), solid; mp 207–208 °C; Rf 0.36 (SiO2, ethyl acetate–methanol =
5.15 (dd, 12.0, 4.0, 1 H), 5.13 (d, 8.0, 1 H), 4.26–4.13 (m, 3 H), 9/1, v/v); δH (DMSO-d6) 8.12 (d, 2.5, 1H), 7.83 (dd, 9.0, 2.5, 1H),
2.25 (s, 3 H), 2.20 (s, 3 H), 2.08 (s, 6 H), 2.03 (s, 3 H); δC 7.39 (d, 9.3, 1H), 5.20 (d, 5.3, 1H), 5.05 (d, 7.8, 1H), 4.92 (d,
(CDCl3) 170.3, 170.1, 170.0, 169.2, 159.5, 142.7, 129.0, 126.4, 6.0, 1H), 4.67 (t, 5.5, 1H), 4.62 (d, 4.5, 1H), 3.70 (t, 3.9, 1H),
123.1, 113.5, 98.8, 71.4, 70.5, 68.2, 66.7, 61.3, 20.7, 20.6, 20.6, 3.64 (t, 6.0, 1H), 3.45–3.60 (m, 3H), 3.40 (ddd, 9.3, 6.0, 3.3,
20.5, 16.2; Calcd for C21H25NO12 C, 52.17; H, 5.21. Found: C, 1H); δC (DMSO-d6) 148.7, 140.8, 136.4, 126.9, 119.1, 112.3,
52.18; H, 5.18.
3-Methyl-4-nitrophenyl-tetra-O-acetyl-β-D-galactopyranoside 37.91; H, 3.71; N, 3.68. Found: C, 37.93; H, 3.77; N, 3.65.
(4r). Off-white solid; mp 100–101 °C; Rf 0.28 (SiO2, cyclo-
5-Fluoro-2-nitrophenyl-β-D-galactopyranoside.8 (2f). Off-
101.1, 75.8, 73.3, 69.9, 67.9, 60.2; Calcd for C12H14BrNO8 C,
hexane–ethyl acetate = 3/2, v/v); δH (CDCl3) 8.21–7.86 (m, 1 H), white solid; mp 173–174 °C; Rf 0.45 (SiO2, ethyl acetate–metha-
7.06–6.77 (m, 1 H), 5.57–5.43 (m, 1 H), 5.22–5.02 (m, 1 H), nol = 9/1, v/v); δH (DMSO-d6) 8.00 (dd, 9.1, 6.1, 1 H), 7.32 (dd,
4.32–4.05 (m, 2 H), 2.63 (s, 3 H), 2.20 (s, 3 H), 2.08 (s, 3 H), 11.1, 2.5, 1 H), 7.04 (ddd, 8.8, 7.8, 2.5, 1 H), 5.23 (d, 5.3, 1 H),
2.03 (s,
3 H); δC (CDCl3) 170.3, 170.1, 170.0, 169.3, 5.10 (d, 7.6, 1 H), 4.94 (d, 6.1, 1 H), 4.70 (t, 5.6, 1 H), 4.63 (d,
159.6, 144.0, 136.9, 127.2, 120.1, 114.1, 100.0, 98.5, 71.4, 70.6, 4.5, 1 H), 3.70 (t, 3.8, 2 H), 3.64–3.45 (m, 3 H), 3.40 (ddd, 9.4,
68.3, 66.7, 61.4, 21.4, 20.7, 20.6, 20.6, 20.5; Calcd for 6.0, 3.3, 1 H); δC (DMSO-d6) 164.5 (1JC,F = 251.8 Hz), 151.8
C21H25NO12 C, 52.17; H, 5.21; N, 2.90. Found: C, 52.10; H, (3JC,F = 12.4 Hz), 136.6, 127.4 (3JC,F = 11.7 Hz), 108.7 (2JC,F
5.22; N, 2.91.
=
24.2 Hz), 104.7 (2JC,F = 27.8 Hz), 101.0, 75.9, 73.3, 69.9, 68.0,
General procedure yielding aryl-β-D-galactopyranosides. The 60.3; Anal. Calcd for C12H14FNO8: C, 45.15; H, 4.42. Found:
title compounds were obtained by deacetylation of 3–4 g C, 45.18; H, 4.80.
(6–8 mmol) aryl-tetra-O-acetyl-β-D-galactopyranosides 4a–r in
5-Methyl-2-nitrophenyl-β-D-galactopyranoside (2g). Off-white
50 mL of 7 N ammonia in methanol. The peracetylated glyco- solid; mp 150–151 °C; Rf 0.39 (SiO2, ethyl acetate–methanol =
sides were dissolved in the reagent, stirred at ambient temp- 9/1, v/v); δH (DMSO-d6) 7.75 (d, 8.3, 1H), 7.24 (s, 1H), 6.97 (dt,
erature over 24 h. The title compounds were subsequently 8.3, 1.0, 1H), 5.14 (d, 5.3, 1H), 5.02 (d, 7.8, 1H), 4.90 (d, 6.3,
isolated by filtration from the suspensions in very good to 1H), 4.68 (t, 5.5, 1H), 4.60 (d, 4.5, 1H), 3.70 (t, 4.0, 1H), 3.63 (t,
excellent yields (90–95%).8
6.0, 1H), 3.50–3.60 (m, 2H), 3.47 (dd, 10.8, 5.8, 1H), 3.36–3.43
4-Methyl-2-nitrophenyl-β-D-galactopyranoside (2b). Off- (m, 1H), 2.37 (s, 3H); δC (DMSO-d6) 149.9, 145.2, 137.8, 124.7,
white solid; mp 197–199 °C; Rf 0.36 (SiO2, ethyl acetate–metha- 122.1, 117.3, 101.0, 75.7, 73.4, 70.1, 68.0, 60.2, 21.4; Calcd for
nol = 9/1, v/v); δH (DMSO-d6) 7.65 (d, 1.5, 1H), 7.43 (ddd, 8.5, C13H17NO8 C, 49.52; H, 5.43; N, 4.44. Found: C, 49.28; H, 5.40;
2.3, 0.5, 1H), 7.30 (d, 8.5, 1H), 5.11 (d, 5.3, 1H), 4.97 (d, 7.8, N, 4.43.
1H), 4.87 (d, 6.0, 1H), 4.64 (t, 5.5, 1H), 4.56 (d, 4.5, 1H), 3.69 (t,
3.9, 1H), 3.43–3.63 (m, 4H), 3.39 (ddd, 9.3, 6.0, 3.3, 1H), white solid; mp 188–189 °C; Rf 0.39 (SiO2, ethyl acetate–metha-
2-Fluoro-4-nitrophenyl-β-D-galactopyranoside.8 (2i). Off-
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nol = 9/1, v/v); δH (DMSO-d6 8.20 (dd, 10.8, 2.8, 1H), 8.10 (ddd, 1 H), 4.87 (s, 1 H), 4.67–4.61 (m, 1 H), 4.57 (d, 4.3, 1 H), 3.78
9.2, 2.7, 1.4, 1H), 7.49 (t, 9.0, 1H), 5.36 (d, 5.5, 1H), 5.15 (d, 7.8, (s, 3 H), 3.68 (t, 3.9, 1 H), 3.60–3.35 (m, 5H); δC (DMSO-d6)
1H), 4.97 (d, 5.8, 1H), 4.68 (t, 5.5, 1H), 4.62 (d, 4.8, 1H), 3.72 (t, 153.4, 143.4, 140.7, 120.0, 118.9, 108.9, 102.0, 75.7, 73.3, 70.1,
4.0, 1H), 3.61–3.70 (m, 2H), 3.55 (dd, 11.0, 5.8, 1H), 3.47–3.52 68.0, 60.2, 56.0; Calcd for C13H17NO9 C, 47.13; H, 5.17; N, 4.23.
(m, 1H), 3.41–3.47 (m, 1H); δC (DMSO-d6) 150.8 (2JC,F
10.2 Hz), 150.5 (1JC,F = 249.6 Hz), 141.0 (3JC,F = 7.3 Hz), 121.1
=
Found: C, 46.97; H, 5.12; N, 4.19.
2-Methyl-4-nitrophenyl-β-D-galactopyranoside (2q). Colorless
(3JC,F = 2.9 Hz), 116.5, 112.4 (2JC,F = 23.4 Hz), 100.7, 75.9, 73.2, solid after chromatographic purification on silica gel (ethyl
69.9, 68.0, 60.2; Calcd for C12H14FNO8 C, 45.15; H, 4.42; acetate–CH2Cl2–MeOH = 5/5/2, v/v/v); mp 245–246 °C, Rf 0.22
N, 4.39. Found: C, 44.89; H, 4.56; N, 4.35.
(SiO2, ethyl acetate–methanol = 9/1, v/v); δH (DMSO-d6)
2-Chloro-4-nitrophenyl-β-D-galactopyranoside.8,17 (2k). Off- 8.10–8.06 (m, 2 H), 7.24 (d, 9.0, 1 H), 5.29 (dd, 5.1, 0.6, 1 H),
white solid; mp 210–211 °C (lit.5 mp 213–215 °C); Rf 0.33 5.00 (d, 7.5, 1 H), 4.94 (d, 5.0, 1 H), 4.68 (t, 5.1, 1 H), 4.59
(SiO2, ethyl acetate–methanol = 9/1, v/v); δH (DMSO-d6) 8.33 (d, (d, 4.5, 1 H), 3.72 (t, 3.8, 1 H), 3.68–3.63 (m, 2 H),
2.8, 1 H), 8.20 (dd, 9.3, 2.8, 1 H), 7.46 (d, 9.3, 1 H), 5.29 (d, 5.6, 3.58–3.42 (m, 2 H), 2.29 (s, 3 H); δC (DMSO-d6) 160.8, 141.1,
1 H), 5.18 (d, 7.8, 1 H), 4.97 (d, 5.8, 1 H), 4.74–4.59 (m, 2 H), 128.1, 125.7, 123.3, 113.9, 100.7, 75.7, 73.1, 70.2, 68.0, 60.3,
3.77–3.61 (m, 3 H), 3.59–3.39 (m, 3 H); δC (DMSO-d6) 157.9, 15.9; Calcd for C13H17NO8 C, 49.52; H, 5.43. Found: C, 49.44;
141.4, 125.5, 124.2, 122.2, 115.6, 100.6, 75.9, 73.3, 69.9, 68.0, H, 5.38.
60.2; Calcd for C12H14ClNO8 C, 42.93; H, 4.20; N, 4.17. Found:
C, 42.87; H, 4.16; N, 4.13.
3-Methyl-4-nitrophenyl-β-D-galactopyranoside (2r). Off-white
solid after chromatographic purification on silica gel (ethyl
4-Chloro-3-nitrophenyl-β-D-galactopyranoside (2m). Off- acetate–CH2Cl2–MeOH = 2/2/1, v/v/v); mp 184–186 °C; Rf 0.38
white solid, mp 176–178 °C, Rf 0.50 (SiO2, ethyl acetate–metha- (SiO2, ethyl acetate–MeOH = 9/1, v/v); δH (DMSO-d6) 8.04 (d,
nol = 5/1, v/v); δH (DMSO-d6) 7.10 (d, 2.8, 1 H), 7.69 (d, 9.0, 1 H), 7.09 (s, 1 H), 7.05 (dd, 9.0, 2.3, 1 H), 5.25 (d, 5.3,
9.2, 1 H), 7.36 (dd, 8.8, 2.8, 1 H), 5.27 (d, 5.2, 1 H), 4.95 (d, 1 H), 4.99 (d, 7.8, 1 H), 4.92 (d, 5.8, 1 H), 4.67 (t, 5.5, 1 H), 4.56
8.0, 1 H), 4.93 (d, 5.6, 1 H), 4.67 (t, 5.6, 1 H), 4.57 (d, 4.4, 1 H), (d, 4.5, 1 H), 3.71 (t, 3.8, 1 H), 3.66–3.61 (m, 1 H), 3.60–3.47
3.70 (t, 4.0, 1 H), 3.63 (t, 6.4, 1 H), 3.60–3.45 (m, 3 H), (m, 3 H), 3.44–3.40 (m, 1 H), 2.54 (s, 3 H); δC (DMSO-d6) 160.8,
3.43–3.38 (m, 1 H); δC (DMSO-d6) 156.4, 148.1, 132.2, 121.9, 142.5, 136.1, 127.1, 119.5, 114.3, 100.5, 75.7, 73.2, 70.0, 60.3,
116.9, 113.1, 101.1, 75.7, 73.1, 70.0, 68.0, 60.2; HRMS (ESI): 20.7; Calcd for C13H17NO8: C, 49.52; H, 5.43; N, 4.44. Found:
m/z Calcd for C12H14ClNO8 [M + Na]+: 358.0306; Found: C, 49.35; H, 5.57; N, 4.29.
358.0291.
Kinetic assays
3-Nitrophenyl-β-D-galactopyranoside (2n). Off-white solid
after chromatographic purification on silica gel (ethyl acetate–
Molar absorptivity of the phenols. Typically, 5 mg of a
CH2Cl2–methanol = 10/10/5); mp 175–177 °C, Rf 0.33 (SiO2, phenol were dissolved in 25 mL of 50 mM acetate buffer at pH
ethyl acetate–methanol, 10/1, v/v); δH (DMSO-d6) 7.87 (dd, 7.8, 5.0. Subsequently, 10–50 µL aliquots of the solution were
1.8, 1H), 7.82 (t, 2.3, 1H), 7.60 (t, 8.3, 1H), 7.49 (dd, 8.0, 1.8, diluted into 100 µL volumes in 96-well plates by addition of
1H), 5.27 (d, 5.0, 1H), 4.97 (d, 7.8, 1H), 4.93 (d, 5.5, 1H), 4.69 buffer yielding 0.1–0.7 mM phenol solutions. The solutions
(t, 5.5, 1H), 4.57 (d, 4.8, 1H), 3.71 (t, 3.8, 1H), 3.63–3.68 (m, were thoroughly mixed and then equilibrated at 30.0 0.1 °C
1H), 3.61 (dt, 7.5, 2.0, 1H), 3.56 (dd, 11.0, 6.0, 1H), 3.50 (dd, for 30 min prior to determination of their absorbance at
12.3, 5.3, 1H), 3.44 (ddd, 9.3, 5.8, 3.4, 1H); δC (DMSO-d6) 157.9, 405 nm. The obtained absorbance values were plotted versus
148.6, 130.7, 123.3, 116.7, 110.9, 101.2, 75.7, 73.1, 70.2, 68.1, known phenol concentrations. The linear fit of the data equals
60.3; Calcd for C12H15NO8 C, 47.84; H, 5.02; N, 4.65. Found: the product of extinction coefficient ε405 times the unknown
C, 47.68; H, 5.06; N, 4.57.
path length d for product absorbance in 96-well plates contain-
3-Methyl-2-nitrophenyl-β-D-galactopyranoside (2o). Off- ing 100 µL solutions in 50 mM acetate buffer at pH 5.0. For
white solid after chromatographic purification over silica gel each phenol, three independent experiments were performed
using ethyl acetate–CH2Cl2–MeOH = 10/10/4, v/v/v) as eluent; and the data averaged. The extinction coefficients summarized
mp 149–154 °C; Rf 0.52 (SiO2, ethyl acetate–methanol = 5/1, (Table 1) are corrected by the path length using the infor-
v/v); δH (DMSO-d6) 7.41 (t, 7.7, 1H), 7.22 (d, 8.5, 1H), 7.04 (dd, mation by the manufacturer for the correlation between path
7.7, 0.6, 1H), 5.11 (d, 5.5, 1H), 4.98 (d, 7.8, 1H), 4.88 (d, 1.0, length and cell filling volume.
1H), 4.66 (t, 5.5, 1H), 4.58 (d, 4.5, 1H), 3.68 (t, 3.6, 1H), 3.61 (t,
Enzyme stock solution. Typically, 3–5 mg of β-galactosidase
6.3, 1H), 3.54 (dd, 11.3, 6.0, 1H), 3.43–3.51 (m, 2H), 3.35–3.42 (A. oryzae) were dissolved in 5 mL of 50 mM acetate buffer
(m, 1H), 2.23 (s, 3H); δC (DMSO-d6) 148.2, 141.8, 131.0, 130.0, at pH 5.0. Subsequent dilution of 250 µL of this solution
123.6, 113.9, 101.0, 75.8, 73.4, 70.0, 68.0, 60.3, 16.3; HRMS into a 5 mL volume yielded a 3 × 10−8 M enzyme stock
(ESI): m/z Calcd for C13H17NO8 [M + Na]+: 338.0852; Found: solution.
338.0845.
Substrate stock solution. Typically, 7–10 mg substrate were
4-Methoxy-2-nitrophenyl-β-D-galactopyranoside (2p). Off- dissolved in 5.0 mL of 50 mM acetate buffer at pH 5.0 yielding
white solid; mp 199–200 °C; Rf = 0.52 (SiO2, ethyl acetate– 15–20 mM substrate stock solutions.
methanol = 5/1, v/v); δH (DMSO-d6) 7.41 (d, 3.0, 1 H), 7.36 (d,
Inhibitor stock solution. Typically, 50 µM inhibitor solu-
9.3, 1 H), 7.22 (dd, 9.3, 3.0, 1 H), 5.11 (d, 5.3, 1 H), 4.89 (d, 1.5, tions in nanopure water were kept frozen at −18 °C and
2798 | Org. Biomol. Chem., 2014, 12, 2792–2800
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thawed when needed. Serial dilution in nanopure water Then, 60 µL (2.83 mg, 10.11 µmol) of the amidine stock solu-
yielded 0.25–1.0 µM inhibitor stock solutions that were used tion described above were added respectively.
for the inhibited enzymatic substrate hydrolysis described
below.
Subsequently, 60 µL aliquots of the equilibrated solutions
were taken in regular time intervals at 0, 5, 10 15, 20, 25, 30,
Assay for enzymatic substrate hydrolysis. The substrate 40, 50, 60, 75, and 90 min. The aliquots were immediately sub-
stock solution was placed in 10–70 µL aliquots in 96-well merged into liquid nitrogen and stored at −18 °C prior to
plates and diluted with acetate buffer into solutions with an HPLC analysis. The equilibrating solutions were periodically
overall volume of 80 µL. These solutions were equilibrated at mixed thoroughly throughout the entire experiment time.
30.0 0.1 °C for 30 min. The enzymatic hydrolysis was then
Amidine quantification assay. All experiments were con-
initiated by addition of a 20 µL aliquot of the enzyme stock ducted on a Shimadzu HPLC with a Rezex-Carbohydrate Na+
solution followed by thorough mixing. The product formation (8%) column 300 × 7.8 mm and 50 × 7.8 mm guard column
was monitored at 405 nm using UV/Vis spectroscopy over the (Phenomenex) using nanopure water as eluent with a flow rate
initial 15 min of the reaction. A typical final substrate concen- of 0.4 mL min−1 at 80 °C. Immediately prior to analysis, the
tration in the assay was 5–40 µM, the overall aliquot volume frozen samples were thawed and subjected to analysis in 10 µL
was 100 µL.
aliquots. The elution was followed for 60 min. Galactono-
The absorbance recorded was plotted versus time in amidine 1d elutes at 10.6 min.
minutes. The rate of the reaction was determined at each sub-
strate concentration from the slope of the linear fit of the data
after conversion of the absorbance into product concentration
using the calibration curves described above. The rate of the
Acknowledgements
reaction was corrected for the enzyme concentration and the An undergraduate research fellowship of the University of
uncatalyzed reaction, and then plotted versus the substrate Arkansas Honors College to R.G.L. and support by the
concentration. By applying a non-linear fit of the resulting National Science Foundation (CHE-1244755, CHE-1305543) to
hyperbolic data, the catalytic rate constant kcat [min−1] and S. S. are gratefully acknowledged. This study was furthermore
the substrate affinity KM [mM] were discerned utilizing the supported by Grant Number P30 GM103450 from the National
Michaelis–Menten model. All experiments were conducted in Institute of General Medical Sciences (NIGMS) of the National
triplicate and the data were averaged.
Institutes of Health (NIH).
Assay for the inhibited enzymatic substrate hydrolysis. The
assay was prepared as described earlier, but 10 µL of the buffer
solution were substituted by 10 µL of the inhibitor stock solu-
tion. The enzymatic catalysis was initiated and monitored as
described above.
Notes and references
1 M.-P. Heck, S. P. Vincent, B. W. Murray, F. Bellamy,
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Apparent Michaelis–Menten constants (K′m) and rate con-
stants (k′ ) were determined by plotting the initial rates cor-
cat
rected for the enzyme concentration versus the substrate
concentration, and fitting the hyperbolic data by non-linear
regression. Typically, kinetic parameters for three different
inhibitor concentrations were determined. The inhibition con-
stant (Ki) for competitive inhibition was calculated from
eqn (1), where K′m and Km are Michaelis–Menten constants in
the presence and absence of the inhibitor:
2 P. A. Bartlett and C. K. Marlowe, Biochemistry, 1983, 22,
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3 M. A. Phillips, A. P. Kaplan, W. J. Rutter and P. A. Bartlett,
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6 R. Kanso, E. A. Yancey and S. Striegler, Tetrahedron, 2012,
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K′m ¼ Km ꢀ ð1 þ ð½Iꢁ=KiÞÞ
ð1Þ
7 R. Kanso and S. Striegler, Carbohydr. Res., 2011, 346, 897–
904.
All experiments were conducted in triplicate for each inhibi-
tor concentration, and the data were averaged.
8 L. Kröger and J. Thiem, Carbohydr. Res., 2007, 342, 467–
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9 Y. Bleriot, T. Dintinger, A. Genre-Grandpierre, M. Padrines
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Stability assays
Amidine stock solution. A 0.168 M stock solution of 1d
(30.69 mg, 0.109 mmol) in 0.650 mL of nanopure water was 10 Y. Bleriot, A. Genre-Grandpierre and C. Tellier, Tetrahedron
prepared and kept at ambient temperature. Aliquots of this Lett., 1994, 35, 1867–1870.
solution were used for all amidine stability assays described 11 B. Ganem, Acc. Chem. Res., 1996, 29, 340–347.
below.
Amidine stability assay. Solutions of 740 µL of nanopure
12 B. Ganem and G. Papandreou, J. Am. Chem. Soc., 1991, 113,
8984–8985.
water or of 50 mM acetate buffer at pH 5 were respectively ther- 13 Y. T. Pan, G. P. Kaushal, G. Papandreou, B. Ganem and
mostated in a modular heating block at 30, 50 or 72 °C for 1 h.
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Paper
Organic & Biomolecular Chemistry
14 M. K. Tong, G. Papandreou and B. Ganem, J. Am. Chem. 16 A. I. Vogel, B. S. Furniss, A. J. Hannaford, P. W. G. Smith
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2800 | Org. Biomol. Chem., 2014, 12, 2792–2800
This journal is © The Royal Society of Chemistry 2014