N-benzylgalactonoamidines as potent β-galactosidase inhibitors

Article abstract of DOI:10.1016/j.tet.2011.10.048

A series of N-benzylgalactonoamidines was synthesized to probe their inhibitory ability during the hydrolysis of o-nitrophenyl-β-d- galactopyranoside by β-galactosidase (Aspergillus oryzae). All compounds are characterized as potent competitive inhibitors with inhibition constants (K_i) in the low nanomolar range (12-48 nM). The structure of the inhibitors mimics the bond-lengthening during the hydrolysis and the aromatic aglycon of the substrate. The electronic nature of the substituent in p-position of the aglycon influences the overall inhibitory ability most when compared to the unsubstituted parent compound.

Full text of DOI:10.1016/j.tet.2011.10.048

Tetrahedron 68 (2012) 47e52
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
N-Benzylgalactonoamidines as potent
b
-galactosidase inhibitors
*
Rami Kanso, Elizabeth A. Yancey, Susanne Striegler
Auburn University, Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn, AL 36849, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-benzylgalactonoamidines was synthesized to probe their inhibitory ability during the hy-
Received 4 September 2011
Received in revised form 6 October 2011
Accepted 12 October 2011
Available online 20 October 2011
drolysis of o-nitrophenyl-b-D-galactopyranoside by b-galactosidase (Aspergillus oryzae). All compounds
are characterized as potent competitive inhibitors with inhibition constants (Ki) in the low nanomolar
range (12e48 nM). The structure of the inhibitors mimics the bond-lengthening during the hydrolysis
and the aromatic aglycon of the substrate. The electronic nature of the substituent in p-position of the
aglycon influences the overall inhibitory ability most when compared to the unsubstituted parent
compound.
Keywords:
Inhibitor
Amidine
Ó 2011 Elsevier Ltd. All rights reserved.
b
-galactosidase
1. Introduction
Considerably less attention has been given to account for the
lengthening of the glycosidic bond during the glycoside hydrolysis,
Emerging evidence suggests that the mechanistic pathways for
glycoside hydrolyses are distinctively different within the glyco-
sidase family.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 difficult and tied to experimental evaluation.1e3 While
many natural and rationally designed compounds are described
as TSAs for the hydrolysis of glycosides in excellent reviews,3e11
which might play an important role for the interaction of an in-
hibitor with the active site in the enzyme. We and others16,20 hy-
pothesized that the aglycon moiety of a substrate might have
additional or different interactions with the active site in a glyco-
sidase during the transition state that can only be identified by
inhibitors that account for the bond-lengthening during the re-
action. The spacer length was shown to influence the inhibitory
ability of glucono- and mannonoamidines to some extent.1,20 The
flexibility of the chosen aliphatic aglycons, however, renders a cor-
relation between their structure in an active site and the observed
inhibitory activity ambiguous.
only
a few of these have binding affinities in the sub-
micromolar concentration range accounting for such TSA-like
character.1
About 20 years ago, glyconoamidines were identified as com-
petitive inhibitors and putative TSAs due to their similarity in
structure, shape, and charge to the half-chair oxocarbenium ion
anticipated during the transition state of most enzymatically
promoted hydrolyses of glycosides.12e15 However, only a few no-
table examples of the inhibitors in this compound class display
inhibition of glycosidases in the nanomolar range.1 Efforts to im-
prove the inhibitory ability of glyconoamidines of unsubstituted
gluco- and benzylmannoamidines16e18 focused on the in-
corporation of supplementary electrostatic interaction to the
glyconoamidine core by using aliphatic aglycons with and without
additional N- or O-binding motifs,1 and the introduction of hy-
drophobic groups.19
In order to verify or reject our hypothesis, we designed a series
of N-substituted benzylgalactonoamidines as a first step toward the
development of potent TSAs of glycosidases and glycosyl-
transferases. The targeted compounds account for both the bond-
lengthening during the hydrolysis and the hydrophobic aromatic
character of the aglycon in a model substrate, such as o-nitro-
phenyl-b-
D-galactopyranoside (1). Substrate
1
is routinely
employed to assess the activity of glycosidases and glycosyl-
transferases. The methylene group in the benzyl moiety of the
substituted N-benzylgalactonoamidines accounts for the antici-
pated bond-lengthening in the transition state during the hydro-
lysis of 1.16 The influences of the electronic properties of the
substituents of the aromatic aglycon in the designed inhibitors
were additionally characterized. Both an electron-withdrawing
fluorine and an electron-donating methyl group were in-
corporated at various positions of the aglycon of N-benzylgalacto-
* Corresponding author. Tel.: þ1 334 844 6954; fax: þ1 334 844 6959; e-mail
noamidines.
A structureeactivity relationship between the
address: Susanne.striegler@auburn.edu (S. Striegler).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.048

48
R. Kanso et al. / Tetrahedron 68 (2012) 47e52
electronic properties of the aromatic aglycon and the inhibitor
performance was elaborated.
determination of the kinetic parameters in the presence of absence
of N-benzylgalactonoamidines, we thus determined the exact
molar mass of the enzyme, re-assessed its purity, and the de-
termined concentration of the supplied protein in the freeze-dried
powder batch.
2. Results and discussion
2.1. Synthesis of N-benzylgalactonoamidines
2.3. Determination of the molar mass of the enzyme
The synthesis of the N-benzylgalactonoamidines followed
The molar mass of the enzyme was determined by microchip gel
electrophoresis under reducing and non-reducing conditions in the
presence of a protein ladder for the 10e260 kDa range. The peak
migration times of the samples were normalized using internal size
markers and compared to the peak migration in the protein ladder.
a strategy we reported recently.21 In brief, 2,3,4,6-tetra-O-benzyl-
D-
galactothionolactam (2) was obtained as the main product in a 10g-
scale synthesis from 2,3,4,6-tetra-O-benzyl- -galactonolactam (3)
D
and Lawesson’s reagent (Scheme 1).22 In situ activation of 2 with
Meerwein’s salt and coupling of the resulting galactoiminothioether
(4) with dried N-benzylamine provided N-benzyl-2,3,4,6-tetra-O-
The molar mass of the tetrameric b-galactosidase (A. oryzae) used in
this study was determined as 86.8 kDa; no other protein that would
interfere with the planned analysis was detected (see
Supplementary data).23 Due to the known limited stability of the
enzyme in its pure form and its observed higher activity when
immobilized,24 the dextrin-stabilized enzyme was used as received.
The exact protein concentration in the supplied freeze-dried
powder was determined by the BCA assay using a known concen-
tration of bovine serum albumin (BSA) as standard.
benzyl-
D
-galactono-amidine (5) after chromatographic purifica-
tion.21 Quantitative O-debenzylation of 5 yielded N-benzyl-
D-gal-
actonoamidine (6) by hydrogenation in the presence of Pd/C and
TFA at ambient temperature in 100e150 mg scale reactions.21
OBn
OBn
BnO
BnO
BnO
BnO
H
N
H
N
(i)
(ii)
O
S
OBn
OBn
3
2
2.4. Determination of the enzyme activity
The hydrolytic activity of b-galactosidase (A. oryzae) was then
OBn
N
OBn
BnO
BnO
BnO
BnO
determined employing 1 as substrate in 50 mM acetate buffer at pH
H
N
5 and 30 ꢁC under steady state conditions (Fig. 1).
(iii)
(iv)
SEt
N
R
OBn
OBn
OH
4
HO
HO
5
O
NO2
O
OH
OH
OH
HO
HO
HO
H
N
1
N
HO
N
H
N
Fig. 1. Structure of the substrate (1).
R
R
OH
OH
The absorbance recorded for the product formation was trans-
formed into concentration by using the extinction coefficient of o-
6
R = H
6a R = o-Me
6b R = m-Me
6d R = o-F
6e R = m-F
nitrophenolate obtained separately from
a calibration curve
6c
6f
R = p-Me
R = p-F
(
3
¼816ꢂ56 cmꢀ1 Mꢀ1) under identical conditions. Initial rates of
400
Scheme 1. Synthesis of N-benzylgalactonoamidines. Reaction conditions: (i) Law-
esson’s reagent, benzene, reflux, 2 h, 93%; (ii) Meerwein salt, CH2Cl2, 0 ꢁC, 2 h; (iii)
ReC6H5CH2NH2, 0 ꢁC to rt, 16 h, 80% over two steps; (iv) H2, Pd/C, TFA, EtOH, rt 1 atm,
16 h, quantitatively.
the hydrolysis were obtained from plots of product formation over
time. The apparent MichaeliseMenten constant (Km¼2.8ꢂ0.1 mM)
and the rate constant (kcat¼4100ꢂ20 minꢀ1) were determined by
plotting the initial rates corrected for the enzyme concentration
(3ꢃ10ꢀ8 M) versus the substrate concentration (2e22 mM), and
fitting the hyperbolic data by non-linear regression applying the
MichaeliseMenten model (Fig. 2a). All experiments were con-
ducted in triplicate, and the resulting data averaged. The obtained
kinetic parameters are in good agreement with to those reported
for the same enzyme and substrate under similar conditions by
others (pH 4.5, Km¼1 mM).16
The subsequent syntheses of N-(2-methylbenzyl)-
actonoamidine (6a), N-(3-methylbenzyl)- -galactono-amidine (6b),
N-(4-methylbenzyl)- -galactonoamidine (6c), N-(2-fluorobenzyl)-
-galactonoamidine (6d), N-(3-fluorobenzyl)- -galactonoamidine
(6e), N-(4-fluorobenzyl)- -galactonoamidine (6f), were conducted
D-gal-
D
D
D
D
D
likewise using the corresponding methyl- and fluorobenzylamines
(Scheme 1). In contrast to previous reports on other glycoamidines,20
all compounds isolated herein are stable in aqueous solution at
ꢀ18 ꢁC for at least six months. Despite all attempts, compound 6e
was not obtained in sufficient purity, and was therefore neither fully
2.5. Inhibition of the enzyme activity
characterized nor evaluated as a potential inhibitor for
dases (see Supplementary data).
b-galactosi-
The kinetic experiments were then repeated in the presence of
three different concentrations (0.2e0.5 mM) of compounds 6aed
and 6f to characterize their inhibitory activity during the enzymatic
hydrolysis of 1. Typically, the value of the apparent Michae-
liseMenten constant ðKm0 Þ increases, while the apparent rate con-
stant remains uneffected in the presence of 6aed and 6f. The
2.2. Evaluation of N-benzylgalactonoamidines as inhibitors
for -galactosidase (Aspergillus oryzae)
b
To assess the inhibitory ability of the synthesized N-benzylga-
lactonoamidines 6aed and f, -galactosidase from Aspergillus ory-
zae (A. oryzae) was employed. The commercially available protein is
supplied as a purified, lyophilized powder, stabilized on dextrin
with a molar mass in a range between 45 and 113 kDa. Prior to the
hyperbolic
data,
LineweavereBurk,
EadieeHofstee,
and
b
HaneseWoolf plots obtained from the kinetic data in the presence
of 6c are depicted as representative examples for all others visu-
alizing the observed competitive inhibition during the hydrolysis of
1 (Fig. 2aed).

R. Kanso et al. / Tetrahedron 68 (2012) 47e52
49
3. Conclusion
In summary, six new N-benzylgalactonoamidines (6aef) were
synthesized. Their inhibitory ability on the hydrolysis of 2-
nitrophenyl-b-D-galactopyranoside (1) by b-galactosidase (A. ory-
zae) in 50 mM acetate buffer at pH 5 was explored. All compounds
are characterized as very potent competitive inhibitors with in-
hibition constants in the low nanomolar range (12e48 nM), which
renders them among the most potent glycosidase inhibitors known
up to date. The electronic nature of the substituent in the p-position
of the aromatic aglycon influences the overall inhibitory ability in
opposite directions when compared to the unsubstituted parent
compound. Current studies focus on the evaluation of compound 6c
as transition state analog, the determination of the inhibitory se-
lectivity of 6, 6aed, and 6f among glycosidases, and screens of all
inhibitors as antiviral drugs.
4. Experimental section
Fig. 2. Inhibitory effect of 6c on the enzymatic hydrolysis of 1 as (a) hyperbolic data,
(b) LineweavereBurk, (c) HaneseWoolf, and (d) EadieeHofstee plot; data for I¼0
are shown in black; I¼0.2 M (red), I¼0.3 M (green), I¼0.5 M (blue); data points not
considered during data fitting (gray).
4.1. Instrumentation
mM
m
m
m
1H and 13C NMR spectra were recorded on a Bruker AV400
(400.2 MHz for 1H, and 100.6 MHz for 13C). Chemical shifts ( ) in 1H
d
NMR are expressed in parts per million and coupling constants (J)
in hertz. Signal multiplicities are denoted as s (singlet), d (doublet),
t (triplet), q (quartet), and m (multiplet). Deuterated chloroform
and deuterium oxide were used as solvents, and chemical shift
values are reported relative to the residual signals of these solvents
Competitive inhibition constants (Ki) were estimated from the
apparent MichaeliseMenten constants in the presence and absence
of 6, 6aed, and 6f, and are given as an average of three independent
experiments for each concentration (Table 1). All evaluated N-
benzylgalactonoamidines showed remarkably high potency as
competitive inhibitors of the enzymatic hydrolysis of 1 with in-
hibition constants (Ki) in the low nanomolar range (12e48 nM)
rendering compounds 6, 6aed, and 6f among the most potent
competitive inhibitors for glycosidases known to date.1 The result is
even more significant when compared to the inhibition ability
previously reported for N-benzylmannonoamidine;16 the inhibitor
(CDCl3:
d
d
¼7.29 for 1H and
d
¼77.0 for 13C; D2O:
d
¼4.80 for 1H and
¼49.0 for 13C after addition of a few drops of CH3OD). The MS
analysis of the samples was performed using an Ultra Performance
LC Systems (ACQUITY, Waters Corp., Milford, MA, USA) coupled
with a quadrupole time-of-flight mass spectrometer (Q-TOF Pre-
mier, Waters) with electrospray ionization (ESI) in both ESI-MS and
ESI-MS/MS modes operated by the Masslynx software (V4.1). Each
sample in 50% aqueous acetonitrile was directly injected into the
ESI source at a flow rate of 50 mL/min. The ion source voltages were
set at 3 kV for positive and negative ion mode acquisitions, re-
spectively. The sampling cone was set at 37 V and the extraction
cone was at 3 V. In both modes the source and desolvation tem-
perature was maintained at 120 ꢁC and 225 ꢁC, respectively, with
the desolvation gas flow at 200 L/h. IR spectra of oils were recorded
as thin films on KBr discs; solids were measured as KBr pellets with
a resolution of 0.5 cmꢀ1 using a Shimadzu IR Prestige-21 FT-In-
was found to be inactive toward
showed inhibition ability toward mannosidases in the low micro-
molar concentration range (6e25
M).16 Distinctly different
b-galactosidase from A. oryzae and
m
mechanistic pathways for the hydrolysis of glycosides by the en-
zymes explored might account for this observation.1
Table 1
Competitive inhibition constants (Ki) for N-arylgalactonoamidines 6, 6aed, 6f
Entry
Inhibitor
KiꢂDKi [nM]
frared Spectrometer;
was performed using silica gel TLC plates from SORBENT Technol-
ogies, 200
n
in cmꢀ1. Thin layer chromatography (TLC)
1
2
3
4
5
6
6
21ꢂ0.1
19ꢂ0.4
21ꢂ0.1
12ꢂ0.1
16ꢂ0.1
48ꢂ1.7
6a
6b
6c
6d
6f
m
m, 4ꢃ8 cm, aluminum backed, with fluorescence in-
dicator F254 and detection by UV light or by charring with an
aqueous vanillinesulfuric acid reagent and subsequent heating of
the TLC plate. Column chromatography was carried out using silica
gel 60 from SilicycleÒ (40e63
m
m, 230e240 mesh) as stationary
phase or basic aluminum oxide (pH range 9.7ꢂ0.4, activity I),
32e63
m, surface area 150 m2 gꢀ1 from Sorbent Technologies. The
Notably, the substitution of the N-benzyl aglycon in the p-posi-
tion leads to a noteworthy alteration of the inhibitory ability of the
corresponding glycoamidine, which depends on the electronic na-
ture of the substituent. Incorporation of a methyl group increases
the electron density in the aromatic ring system of the aglycon and
decreases the inhibition constant by one half rendering the inhibitor
more potent (6c<6, Table 1, entries 1 and 4); the substitution in the
same position with an electron-withdrawing fluoro atom increases
the inhibition constant more than twofold rendering the inhibitor
less efficient than the unsubstituted parent compound (6<6f, Table
1, entries 1 and 6). Alterations of 6 with methyl- and fluoro-
substituents in the o- or m-position have a less pronounced effect
on the inhibitory ability of the resulting compounds, and will be
consequently not further pursued (Table 1, entries 2,3, and 5).
m
UV/vis spectra were recorded on a Cary WinUV, Vers. 3.0, Analysis
Suite with Suprasil standard cells (200e2000 nm) of 1 cm thickness
and 4.5 ml volume at 30 ꢁC over a range of 200e800 nm pH values
were measured using a Beckman
F 250 pH meter equipped with
refillable long Futura pH electrode of 0.7 mm thickness. The pH
meter was calibrated before each set of readings with standard
buffer solutions for pH 4, 7, and 10. Nanopure water was obtained
from an EASYpureÒ II water system from Barnstead (18.2 M
U/cm).
4.2. Chemicals
Benzylamine, o-methylbenzylamine, m-methylbenzylamine, p-
methylbenzylamine, o-fluorobenzylamine, m-fluorobenzylamine,

50
R. Kanso et al. / Tetrahedron 68 (2012) 47e52
and p-fluorobenzylamine were obtained from Acros, distilled in
vacuum and stored over molecular sieves 3 A prior to use;
dichloromethane (Pharmco-Aaper) and benzene (SigmaeAldrich)
were dried dynamically over neutral aluminum oxide from Acros or
basic aluminum oxide from EMD. o-Nitrophenol was obtained from
2863, 1653, 1492, 1087, 733, 699 cmꢀ1; ESI-TOF MS m/z calcd for
C41H41FN2O4 (MþH)þ: 645.3129; found: 645.3129.
ꢀ
4.3.5. 3-Fluorobenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(5e). Colorless oil (140 mg, 60%); dH (CDCl3) 7.49e7.30 (m, 21H,
PhH), 7.04e6.94 (m, 3H, PhH), 5.30 (d, 11.3, 1H, PhCH2), 4.98 (d, 11.4,
1H, PhCH2), 4.88 (d, 11.7, 1H, PhCH2), 4.80e4.60 (m, 6H, eCH, OCH2),
4.41e4.27 (m, 3H, eCH, NCH2), 4.03 (dd, 1.7, 9.7, 1H, eCH),
3.85e3.70 (m, 3H, eCH, eCH2), 1.35 (s, 1H, NH); dC (CDCl3) 164.0,
155.7, 142.0, 140.0, 138.3, 138.0, 137.7, 129.7 (d, 8.3, eCH),
128.6e127.4, 123.2, 114.5 (d, 20.4, eCH), 113.7 (d, 21, eCH), 82.0,
75.2, 74.4, 74.2, 73.7, 73.3, 71.1, 71.0, 59.5, 44.4; nmax (KBr, thin film)
3426, 3031, 2853, 1640, 1487, 1092, 744, 691 cmꢀ1; ESI-TOF MS m/z
calcd for C41H41FN2O4 (MþH)þ: 645.3129; found: 645.3126.
Acros and recrystallized from Ethanol prior to use. b-Galactosidase
[3.2.1.23] from A. oryzae was obtained as lyophilized powder sta-
bilized on dextrin from SigmaeAldrich and stored at ꢀ18 ꢁC prior to
use. All other chemicals were reagent grade or better, and were
used as received.
4.3. General procedure for the synthesis of N-aryl-2,3,4,6-
tetra-O-benzyl-D-galactonoamidines 5aef
Meerwein’s salt (1.5 equiv) is added under argon to a solution of
2,3,4,6-tetra-O-benzyl- -galactothionolactam (2) (200 mg,
D
4.3.6. 4-Fluorobenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
0.362 mmol,1 equiv) in dry CH2Cl2 at 0 ꢁC. The resulting suspension
is stirred at 0 ꢁC for 2 h, and is used directly for the next step
without further purification. A solution of the corresponding ben-
zylamine (2 equiv) in dry CH2Cl2 (2 mL) is then added under argon
at 0 ꢁC. The reaction mixture is allowed to warm to ambient tem-
perature and is stirred overnight.
(5f). Colorless oil (90 mg, 39%); dH (CDCl3) 7.48e7.30 (m, 20H, PhH),
7.22e7.15 (m, 2H, PhH), 7.04e6.97 (m, 2H, PhH), 5.01 (d, 11.3, 1H,
PhCH2), 4.95 (d, 11.4,1H, PhCH2), 4.85 (d,11.8, 1H, PhCH2), 4.77e4.60
(m, 6H, eCH, OCH2), 4.38 (t, 1.9, 1H, eCH), 4.28 (q, 14.4, 2H, NCH2),
4.01 (dd, 1.7, 9.7, 1H, eCH), 3.85e3.68 (m, 3H, eCH, eCH2), 1.32 (s,
1H, NH); dC (CDCl3) 163.1, 155.8, 139.0, 138.4, 138.0, 137.7, 129.4 (d,
7.6, eCH), 128.6e127.4, 115.1 (d, 21.4, eCH), 81.9, 75.3, 74.5, 74.1,
73.7, 71.1, 71.0, 59.5, 44.3; nmax (KBr, thin film) 3430, 3027, 2859,
1646, 1503, 1085, 744, 694 cmꢀ1; ESI-TOF MS m/z calcd for
C41H41FN2O4 (MþH)þ: 645.3129; found: 645.3128.
4.3.1. 2-Methylbenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(5a). Yellow oil (90 mg, 43%); dH (CDCl3) 7.57e7.27 (m, 24H, PhH),
5.12 (d, 11.4, 1H, PhCH2), 5.04 (d, 11.5, 1H, PhCH2), 4.95 (d, 11.8, 1H,
PhCH2), 4.86 (d, 11.4, 1H, PhCH2), 4.80e4.69 (m, 5H, eCH, OCH2),
4.50e4.32 (m, 3H, eCH, NCH2), 4.12 (dd,1.4, 9.7,1H, eCH), 3.99e3.82
(m, 3H, eCH, eCH2), 2.37 (s, 3H, eCH3),1.43 (br s,1H, NH); dC (CDCl3)
155.7, 139.1, 138.4, 138.1, 137.7, 137.0, 136.4, 130.1e125.8, 81.9, 75.3,
74.4, 74.0, 73.7, 73,3, 71.2, 71.0, 59.6, 42.9, 18.9; nmax (KBr, thin film)
3432, 3018, 2914, 2858,1642,1455,1091, 739, 694 cmꢀ1; ESI-TOF MS
m/z calcd for C42H45N2O4 (MþH)þ: 641.3375; found: 641.3375.
4.4. General procedure for the O-debenzylation of N-aryl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidines; synthesis of N-
aryll- -galactonoamidines 6aef
D
The perbenzylated amidine (40e80 mg) and Pd/C (80e160 mg,
200% w/w) were stirred in 5 mL of ethanol in the presence of TFA
(1 mL, 13.5 mmol) under hydrogen at room temperature. After 16 h,
a TLC analysis on silica gel with cyclohexane/EtOAc (1/1; v/v) as
eluent showed complete transformation of the starting material
toward a more polar spot at the baseline. The reaction mixture was
diluted with EtOH (10 mL) and filtered on Celite. The filtrate was
concentrated under reduced pressure at 37 ꢁC and dried in vacuum
for 16 h at room temperature.
4.3.2. 3-Methylbenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(5b). Yellow oil (140 mg, 55%); dH (CDCl3) 7.52e7.08 (m, 24H, PhH),
5.06 (d, 11.3, 1H, PhCH2), 4.99 (d, 11.4, 1H, PhCH2), 4.90 (d, 12.1, 1H,
PhCH2), 4.80 (d, 11.4, 1H, PhCH2), 4.75e4.66 (m, 5H, eCH, OCH2),
4.43 (t, 2.9, 1H, eCH), 4.35 (q, 14.3, 2H, NCH2), 4.08 (dd, 1.8, 9.7, 1H,
eCH), 3.93e3.77 (m, 3H, eCH, eCH2), 2.43 (s, 3H, eCH3), 1.39 (br s,
1H, NH); dC (CDCl3) 156.1, 139.0, 138.4, 138.0, 137.7, 128.5e127.4,
124.8, 81.7, 75.2, 74.5, 73.9, 73.6, 73.3, 71.0, 45.2, 21.3; nmax (KBr, thin
film) 3440, 2919, 2863,1650,1494,1092, 743, 694 cmꢀ1; ESI-TOF MS
m/z calcd for C42H45N2O4 (MþH)þ: 641.3375; found: 641.3389.
4.4.1. 2-Methylbenzyl- -galactonoamidine (6a). The hydrogenation
D
of 5a (80 mg, 0.125 mmol) yielded 6a as colorless oil (30 mg, 86%);
dH (D2O) 7.4e7.3 (m, 4H, PhH), 4.70e4.60 (m, 3H, eCH, eNCH2), 4.30
(t, 1H, 2.1, eCH), 3.99 (dd, 1H, 10.7, 2.4, eCH), 3.83e3.70 (m, 3H,
eCH, eCH2), 2.30 (s, 3H, eCH3); dC (D2O/MeOD) 165.0, 137.8, 132.3,
131.6, 129.5, 128.4, 127.3, 71.4, 67.8, 67.2, 60.8, 58.2, 44.8, 18.8; nmax
(KBr, thin film): 3425, 2921, 1670, 1199, 1134 cmꢀ1; HRMS (þTOF-
MS) m/z calcd for C14H21N2O4 (MþH)þ: 281.1501; found: 281.1501.
4.3.3. 4-Methylbenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(5c). Yellow oil (180 mg, 51%); dH (CDCl3) 7.43e7.24 (m, 20H, PhH),
7.35 (s, 4H, PhH), 4.98 (d, 11.4, 1H, PhCH2), 4.91 (d, 11.5, 1H, PhCH2),
4.82 (d,12.0,1H, PhCH2), 4.73 (d,11.4, 1H, PhCH2), 4.68e4.58 (m, 5H,
eCH, OCH2), 4.35 (t, 2.0, 1H, eCH), 4.25 (q, 14.1, 2H, NCH2), 4.0 (dd,
1.8, 9.7, 1H, eCH), 3.84e3.69 (m, 3H, eCH, eCH2), 2.38 (s, 3H, eCH3),
1.32 (br s, 1H, NH); dC (CDCl3) 155.9, 139.0, 138.4, 138.1, 137.7, 136.5,
136.2, 129.0e127.4, 81.8, 75.3, 74.5, 73.9, 73.7, 73,3, 71.2, 71.0, 59.6,
44.9, 21.1; nmax (KBr, thin film) 3431, 3023, 2930, 2878, 1653, 1498,
1091, 730, 696 cmꢀ1; ESI-TOF MS m/z calcd for C42H45N2O4
(MþH)þ: 641.3375; found: 641.3370.
4.4.2. 3-Methylbenzyl- -galactonoamidine (6b). The hydrogenation
D
of 5b (70 mg, 0.11 mmol) yielded 6b as a yellowish oil (30 mg,
quantitative) and analyzed by 1H NMR spectroscopy in D2O; dH
(D2O) 7.33e7.10 (m, 4H, PhH), 4.63e4.55 (m, 3H, eCH, eNCH2), 4.25
(t, 1H, 2.2, eCH), 3.95 (dd, 1H, 10.3, 2.4, eCH), 3.77e3.64 (m, 3H,
eCH, eCH2), 2.30 (s, 3H, eCH3); dC (D2O/CD3OD) 165.3, 140.4, 134.7,
130.1, 128.9, 125.3, 71.7, 68.0, 67.5, 61.0, 58.4, 46.2, 21.3; nmax (KBr,
thin film): 3420, 2924, 1676, 1201, 1139 cmꢀ1; HRMS (þTOF-MS) m/
z calcd for C14H21N2O4 (MþH)þ: 281.1501; found: 281.1490.
4.3.4. 2-Fluorobenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(5d). Colorless oil (100 mg, 43%); dH (CDCl3) 7.44e7.2 (m, 22H,
PhH), 7.10e7.0 (m, 2H, PhH), 4.98 (d, 11.3, 1H, PhCH2), 4.92 (d, 11.4,
1H, PhCH2), 4.82 (d, 11.5, 1H, PhCH2), 4.74e4.58 (m, 6H, eCH, OCH2),
4.40e4.32 (m, 3H, eCH, NCH2), 3.99 (dd, 1.7, 9.7, 1H, eCH),
3.83e3.67 (m, 3H, eCH, eCH2), 1.31 (s, 1H, NH); dC (CDCl3) 162.3,
155.6, 139.0, 138.4, 138.1, 137.8, 130.1 (d, 4.4, eCH), 128.5e127.3,
123.9 (d, 2.9, eCH), 115.1 (d, 21.6, eCH), 81.8, 75.4, 74.4, 73.7, 73.3,
71.2, 71.0, 59.6, 38.5; nmax (KBr, thin film) 3440, 3058, 3028, 2915,
4.4.3. 4-Methylbenzyl- -galactonoamidine (6c). The hydrogenation
D
of 5c (40 mg, 0.063 mmol) yielded 6c as a colorless oil (17 mg,
quantitatively); dH (D2O) 7.26 (dd, 4H, 12.5, 8.2, PhH), 4.63e4.55 (m,
3H, eCH, eNCH2), 4.28 (t, 3H, 1.8, eCH), 3.97 (dd, 1H, 10.1, 2.4, eCH),
3.68e3.58 (m, 3H, eCH, eCH2), 2.32 (s, 3H, eCH3); dC (D2O/CD3OD)
165.2, 134.0, 131.7, 130.7, 128.4, 71.6, 68.0, 67.5, 61.0, 58.4, 46.0, 21.1;

R. Kanso et al. / Tetrahedron 68 (2012) 47e52
51
nmax (KBr, thin film) 3427, 2975, 1667, 1430, 1202, 1137 cmꢀ1; HRMS
(þTOF-MS) m/z calcd for C14H21N2O4 (MþH)þ: 281.1501; found:
281.1496.
infra). Typically, 2e4 mg of the inhibitors were dissolved in nano-
pure water yielding 6e15 mM aqueous solutions that were stored
at ꢀ18 ꢁC prior to use. Appropriate volumes of the aqueous in-
hibitor solutions were diluted into 1000 mL of buffer solutions to
4.4.4. 2-Fluorobenzyl-
D-galactonoamidine (6d). The hydrogenation
obtain 20e50 mM inhibitor stock solution, which were then used
of 5d (70 mg, 0.11 mmol) yielded 6d as a colorless oil (30 mg, 97%);
dH (D2O) 7.46e7.13 (m, 4H, PhH), 4.66 (s, 2H, PhCH2), 4.61 (d, 1H,
10.1, CH), 4.29 (d, 1H, 1.6, CH), 3.97 (dd, 1H, 10.2, 1.7, CH), 3.86e3.68
(3H, m, CH, CH2); dC (D2O/CD3OD) 165.2, 131.8, 130.6, 125.8, 116.8,
116.6, 71, 6, 68.0, 67.5, 61.1, 40.8; nmax (KBr, thin film) 3278, 2931,
1676, 1497, 1459, 1200, 1137 cmꢀ1; ESI-TOF MS m/z calcd for
C13H18N2O4F (MþH)þ: 285.1251; found: 285.1251.
for the kinetic analysis.
4.6.2. Kinetic assay. Initially, 10 mL of the inhibitor stock solutions
were added to 100e700
resulting solution was diluted with buffer solution to a total volume
of 990 L of the en-
L and equilibrated at 30 ꢁC. After 30 min, 10
mL aliquots of the substrate solution; the
m
m
zyme stock solution were added, the solutions mixed, and the
product formation followed spectrophotometrically at 400 nm for
15 min.
4.4.5. 3-Fluorobenzyl- -galactonoamidine (6e). The hydrogenation
D
of 5e (70 mg, 0.11 mmol) yielded 6e as a colorless oil in insufficient
purity as judged by 1H NMR spectroscopy (31 mg, quantitative);
ESI-TOF MS m/z calcd for C13H18N2O4F (MþH)þ: 285.1251; found:
285.1248.
4.6.3. Data analysis. The absorbance recorded for the product for-
mation was transformed into concentration by the extinction co-
efficient of o-nitrophenolate at pH 5; initial rates of the reaction
were obtained from plots of the product concentration over time.
Apparent MichaeliseMenten constants ðKm0 Þ and rate constants
ðk0catÞ were determined by plotting the initial rates corrected for the
enzyme concentration versus the substrate concentration, and fit-
ting the hyperbolic data by non-linear regression. All experiments
were conducted in triplicate and the data were averaged. Kinetic
parameters for three different inhibitor concentrations were de-
termined, and LineweavereBurk ([E]/rate vs 1/[S]), EadieeHofstee
(rate vs rate/[S]), and Hanes plots ([S]/rate vs [S]) constructed to
visualize and examine the inhibition type. The inhibition constant
(Ki) for competitive inhibition was calculated from Eq. 1, where Km0
and Km are MichaeliseMenten constants in the presence and ab-
sence of the inhibitor:
4.4.6. 4-Fluorobenzyl- -galactonoamidine (6f). The hydrogenation
D
of 5f (40 mg, 0.062 mmol) yielded 6f as a colorless oil (17 mg,
quantitative); dH (D2O) 7.34 (dd, 2H, 8.5, 5.4, PhH), 7.14 (t, 2H, 8.9,
PhH), 4.65e4.58 (m, 3H, eCH, eNCH2), 4.28 (t, 1H, 2.3, eCH), 3.97
(dd, 1H, 10.1, 2.4, eCH), 3.83e3.64 (m, 3H, eCH, eCH2); dC (D2O/
MeOD) 165.3, 130.4, 130.3, 117.0, 116.7, 71.7, 68.0, 67.5, 61, 0., 58.5,
45.6; nmax (KBr, thin film): 3378, 2922, 1673, 1202, 1138 cmꢀ1
;
HRMS (þTOF-MS) m/z calcd for C13H18N2O4F (MþH)þ: 285.1251;
found: 285.1245.
4.5. Assay for the activity of b-galactosidase (A. oryzae)
The activity of tetrameric
347.2 kDa, unit: 86.6 kDa) was determined in 50 mM buffered solu-
tions at pH5 using o-nitrophenyl- -galactopyranoside as substrate.
b-galactosidase (A. oryzae, MW
Km0 ¼ Km ꢃ ð1 þ ð½Iꢄ=KiÞÞ
Acknowledgements
(1)
b-D
4.5.1. Enzyme stock solution and concentration. Typically, 10 mg of
the lyophilized protein were dissolved in 10 mL of a 50 mM acetate
solution yielding a 3ꢃ10ꢀ8 M stock solution; the concentration was
determined by comparison of the absorbance at 280 nm to a cali-
bration curve prepared by the BCA assay using BSE as standard.
A CAREER award from the National Science Foundation to S.S.
(CHE-0746635) is gratefully acknowledged; the authors are also
indebted to Christopher Easley and Joonyul Kim for conducting the
gel electrophoresis experiments on the microchip.
Supplementary data
4.5.2. Substrate stock solution and kinetic assay. Typically, 10 mL of
a 33 mM substrate stock solution (100.0 mg, 0.323 mmol) were
1H, 13C NMR, IR, and MS spectra of all new compounds 5aef,
6aef; gel electrophoresis experiments on the microchip. Supple-
mentary data associated with this article can be found, in the online
version, at doi:10.1016/j.tet.2011.10.048.
prepared and 100e700
buffer solution, and thermostated for 30 min at 30 ꢁC. After addi-
tion of 10 L aliquots of the enzyme stock solution and thorough
mL aliquots were diluted into 990 mL with
m
mixing, the formation of o-nitrophenolate was followed spectro-
photometrically at 400 nm for 15 min.
References and notes
4.5.3. Data analysis. The absorbance recorded for the product for-
1. Heck, M.-P.; Vincent, S. P.; Murray, B. W.; Bellamy, F.; Wong, C.-H.; Mioskowski,
C. J. Am. Chem. Soc. 2004, 126, 1971e1979.
2. Galliene, E.; Gefflaut, T.; Bolte, J.; Lemaire, M. J. Org. Chem. 2006, 71,
894e902.
3. Schramm, V. L. Annu. Rev. Biochem. 2011, 80, 703e732.
4. Greig, I. R.; Williams, I. H. Chem. Commun. 2007, 3747e3749.
5. Pandey, G.; Dumbre, S. G.; Kapur, M. Proc. Indian Natl. Sci. Acad., Part A 2005, 71,
137e153.
6. Gloster, T. M.; Roberts, S.; Ducros, V. M. A.; Perugino, G.; Rossi, M.; Hoos, R.;
Moracci, M.; Vasella, A.; Davies, G. J. Biochemistry 2004, 43, 6101e6109.
7. Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102,
515e553.
mation was transformed into concentration by using the extinction
coefficient of o-nitrophenolate at pH 5 (
3
400
¼816ꢂ56 cmꢀ1
M
ꢀ1),
which was obtained from a calibration curve. Initial rates of the re-
action were obtained from plots of product concentration over time.
Apparent MichaeliseMenten constants (Km) and rate constants
(kcat) were determined by plotting the initial rates corrected for the
enzyme concentration versus the substrate concentration, and fit-
ting the hyperbolic data by non-linear regression. All experiments
were conducted in triplicate, and the resulting data averaged.
8. Wong, C.-H.; Provencher, L.; Porco, J. A., Jr.; Jung, S.-H.; Wang, Y.-F.; Chen, L.;
Wang, R.; Steensma, D. H. J. Org. Chem. 1995, 60, 1492e1501.
9. Ermert, P.; Vasella, A.; Weber, M.; Rupitz, K.; Withers, S. G. Carbohydr. Res. 1993,
250, 113e128.
4.6. Assay for the inhibition of the enzymatic 2-nitrophenyl-
b-D-galactopyranoside hydrolysis with substituted N-
10. Bols, M. Acc. Chem. Res. 1998, 31, 1e8.
benzylgalactonoamidines
11. Gloster, T. M.; Davies, G. J. Org. Biomol. Chem. 2010, 8, 305e320.
12. Tong, M. K.; Papandreou, G.; Ganem, B. J. Am. Chem. Soc. 1990, 112, 6137e6139.
13. Pan, Y. T.; Kaushal, G. P.; Papandreou, G.; Ganem, B.; Elbein, A. D. J. Biol. Chem.
1992, 267, 8313e8318.
4.6.1. Stock solutions. The enzyme and substrate stock solutions
were prepared as described for the non-inhibited hydrolysis (vide

52
R. Kanso et al. / Tetrahedron 68 (2012) 47e52
14. Andrews, C. W.; Fraser-Reid, B.; Bowen, J. P. J. Am. Chem. Soc.1991,113, 8293e8298.
15. Lindback, E.; Lopez, O.; Fernandez-Bolanos, J. G.; Sauer, S. P. A.; Bols, M. Org.
19. Field, R. A.; Haines, A. H.; Chrystal, E. J.; Luszniak, M. C.; Biochem, J. Biochem. J.
1991, 274, 885e889.
€
Lett. 2011, 13, 2908e2911.
20. Legler, G.; Finken, M.-T. Carbohydr. Res. 1996, 292, 103e115.
21. Kanso, R.; Striegler, S. Carbohydr. Res. 2011, 346, 897e904.
22. von Hoff, S.; Heightman, T. D.; Vasella, A. Helv. Chim. Acta 1998, 81, 1710e1725.
23. The detection limit for the analysis by microgel electrophoresis was 2.5 ng/ml
24. Haider, T.; Husain, Q. Int. Dairy J. 2009, 19, 172e177.
16. Bleriot, Y.; Genre-Grandpierre, A.; Tellier, C. Tetrahedron Lett.1994, 35,1867e1870.
17. Bleriot, Y.; Dintinger, T.; Guillo, N.; Tellier, C. Tetrahedron Lett.1995, 36, 5175e5178.
18. Bleriot, Y.; Dintinger, T.; Genre-Grandpierre, A.; Padrines, M.; Tellier, C. Bioorg.
Med. Chem. Lett. 1995, 5, 2655e2660.