Structure-activity relationship of highly potent galactonoamidine inhibitors toward β-galactosidase (Aspergillus oryzae)

Article abstract of DOI:10.1021/jm501111y

A small library of 22 N-substituted galactonoamidines was synthesized, and their structure-activity relationship for inhibition of the hydrolytic activity of β-galactosidase (Aspergillus oryzae) was evaluated. A fast screening assay in 96-well plate forma

Full text of DOI:10.1021/jm501111y

Article
pubs.acs.org/jmc
Structure−Activity Relationship of Highly Potent Galactonoamidine
Inhibitors toward β‑Galactosidase (Aspergillus oryzae)
Qiu-Hua Fan, Kailey A. Claunch, and Susanne Striegler*
Department of Chemistry and Biochemistry, University of Arkansas, 345 North Campus Drive, Fayetteville, Arkansas 72701, United
States
S
* Supporting Information
ABSTRACT: A small library of 22 N-substituted galactonoamidines was
synthesized, and their structure−activity relationship for inhibition of the
hydrolytic activity of β-galactosidase (Aspergillus oryzae) was evaluated. A fast
screening assay in 96-well plate format was used to follow the enzymatic hydrolysis
of 2-chloro-4-nitrophenyl-β-D-galactopyranoside using UV−vis spectroscopy. The
aglycon moiety of all compounds was found to have a profound effect on their
inhibitory ability. In general, galactonoamidines derived from cyclic aliphatic and
linear amines show higher inhibition activity than those derived from
benzylamines. Hydrophobic interactions of the methyl group rather than π−π stacking interactions of the aromatic ring in p-
methylbenzyl-D-galactonoamidine were identified to cause its transition-state-like character and the remarkably high inhibitory
ability (Ki = 8 nM). A flexible 3-carbon methylene spacer between the exo N atom of the sugar moiety and a phenyl group
furthermore increased the observed apparent inhibition drastically.
and gluconoamidines that did not give access to corresponding
N-substituted galactonoamidines. We thus developed a
synthetic strategy suitable for large-scale operation to fill this
gap and evaluated the inhibitory ability of N-benzylgalactono-
amidines and their derivatives toward β-galactosidase (Asper-
gillus oryzae) as a representative, conveniently accessible
member of GHF-35.17,18
Designed inhibitors were found to be very potent
competitive inhibitors with inhibition constants in the very
low nanomolar concentration range (12−48 nM).18 Encour-
aged by these results, we evaluated selected methyl- and fluoro-
N-arylgalactonoamidines as putative transition-state analogs
(TSA) following a kinetic procedure outlined by Barlett et
al.28,29 We thereby obtained experimental evidence suggesting
p-methylbenzyl-D-galactonoamidine as a suitable candidate
fulfilling the requirements as a TSA. The results furthermore
pointed at a strong influence of the aglycon moiety on the
overall inhibitory ability of the N-substituted galactonoami-
dines.29
These preliminary results prompted an in-depth investigation
on the contributions of the aglycon moiety toward the
interaction with the active site of the enzyme that may include
steric, electronic, hydrophobic, and spacer effects, π−π stacking
of the benzyl ring, and van der Waals interactions. We
hypothesized that the transition-state-like character of p-
methylbenzyl-D-galactonoamidine and its remarkably high
inhibitory ability are caused by π−π stacking interactions of
the aromatic ring of the aglycon moiety with aromatic side
chains of amino acids in the active site of the β-galactosidase, by
INTRODUCTION
■
Glycosidases are ubiquitous enzymes cleaving and transferring
glycosidic bonds.1 β-Galactosidases (EC 3.2.1.23), a subgroup
of glycosidases, can be classified based on the reaction
catalyzed, namely, hydrolysis of β-(1−3)- and β-(1−4)-
galactosyl bonds, or based on sequence homology among the
90 glycosyl hydrolase families (GHFs). The currently known β-
galactosidases belong to GHF-1, GHF-2, GHF-35, and GHF-
42.1
Frequently, β-galactosidases are used in industrial processes
requiring lactose hydrolysis of diary products.2−4 Additional use
of β-galactosidases is found as a reporter gene tool in molecular
biology. Therapeutic benefits for treatment of lysosomal
disorders have been associated with in vitro competitive
inhibitors of lysosomal hydrolases that might also act as
intracellular enhancers by stabilization of mutant proteins.5,6
Due to the limited number of structural information for
galactosidases across the corresponding families,2,7−13 design of
inhibitors to diminish or completely hamper glycosidase activity
serves a dual purpose, namely, to elucidate the mechanism
details and to interfere with the enzyme activity. Competitive
inhibitors of human lysosomal β-galactosidase, the most
prominent member of GHF-35, are envisioned as new agents
for pharmacological chaperone therapy to treat metabolic
disorders, such as GM1-gangliosidoses or Morquio B
syndrome.11,14−16
Following the outlined strategy to elucidate mechanistic
details of β-galactosidase activity, we recently focused on the
design and evaluation of N-benzylgalactonoamidines as
glycosidase inhibitors.17,18 Glyconoamidines are known as
potent inhibitors of glycosidases.19−27 However, the early
synthetic strategies developed by others focused on mannono-
Received: July 22, 2014
Published: October 8, 2014
© 2014 American Chemical Society
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hydrophobic interaction of the methyl group in the para
position of the N-benzyl ring with corresponding amino acids,
or by a combination of both.
To verify or reject this hypothesis, we synthesized a small
library of N-substituted galactonoamidines derived from
benzylic and aliphatic amines and determined the correlation
between galactonoamidine structure and resulting inhibitory
activity toward β-galactosidase (A. oryzae). The synthetic efforts
and determined bioactivity are summarized and discussed
below.
amine). The selection of amines results in different electronic,
steric, hydrophobic, and spacer effects, π−π stacking, van der
Waals interaction, and a variety of ring sizes of the aglycon of
the galactonoamidines used in the structure−activity study
described below.
The coupling reactions between the selected amines and 2
yielded N-4-tert-butylbenzyl-2,3,4,6-tetra-O-benzyl-D-galacto-
noamidine (3h), N-4-methoxybenzyl-2,3,4,6-tetra-O-benzyl-D-
galactonoamidine (3i), N-4-trifluoromethylbenzyl-2,3,4,6-tetra-
O-benzyl-D-galactonoamidine (3j), N-4-chlorobenzyl-2,3,4,6-
tetra-O-benzyl-D-galactonoamidine (3k), N-4-bromobenzyl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3l), N-propyl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3m), N-n-heptyl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3n), N-n-dodecyl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3o), N-2-ethylhex-
yl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3p), N-3-phenyl-
propyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3q), N-cy-
clopropyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3r), N-4-
cyclohexyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3s), N-
cyclopropylmethyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3t), N-cyclohexylmethyl-2,3,4,6-tetra-O-benzyl-D-galactonoa-
midine (3u), and N-cycloheptylmethyl-2,3,4,6-tetra-O-benzyl-
D-galactonoamidine (3v) in moderate to good yields after
chromatographic purification over silica gel.
RESULTS AND DISCUSSION
■
Chemistry. In order to verify or reject our hypothesis, we
extended our previously synthesized small library of galacto-
noamidines composed of N-benzyl-D-galactonoamidine (1a),17
2-methyl-N-benzyl-D-galactonoamidine (1b),17 3-methyl-N-
benzyl-D-galactonoamidine (1c),17 4-methyl-N-benzyl-D-galac-
tonoamidine (1d),17 2-fluoro-N-benzyl-D-galactonoamidine
(1e),17 3-fluoro-N-benzyl-D-galactonoamidine (1f),29 and 4-
fluoro-N-benzyl-D-galactonoamidine (1g)17 by additional gal-
actonoamidines derived from selected benzylic and aliphatic
amines following the same synthetic strategy (Scheme 1).17,20,30
a
Scheme 1. Synthesis of Galactonoamidines 1
Removal of the protecting benzyl ethers in 3 using Pd on
charcoal as catalyst in hydrogen atmosphere in the presence of
trifluoroacetic acid (TFA) afforded galactonoamidines 1,
namely, N-4-tert-butylbenzyl-D-galactonoamidine (1h), N-4-
methoxybenzyl-D-galactonoamidine (1i), N-4-trifluoromethyl-
benzyl-D-galactonoamidine (1j), N-propyl-D-galactonoamidine
(1m), N-n-heptyl-D-galactonoamidine (1n), N-n-dodecyl-D-
galactonoamidine (1o), N-2-ethylhexyl-D-galactonoamidine
(1p), N-3-phenylpropyl-D-galactonoamidine (1q), N-cyclo-
propyl-D-galactonoamidine (1r), N-cyclohexyl-D-galactonoami-
dine (1s), N-cyclopropylmethyl-D-galactonoamidine (1t), N-
cyclohexylmethyl-D-galactonoamidine (1u), and N-cycloheptyl-
methyl-D-galactonoamidine (1v) in very good to excellent
yields after filtration of the reaction mixture over a pad of
Celite, concentration of the filtrate to dryness, and freeze drying
of the remaining residue.
Removal of the protecting groups under the elaborated
conditions is effortless when the aglycon moiety of 3 is derived
from substituted benzylamines.17 However, applying these
conditions to 3k and 3l leads to complete or partial
dehalogenation of the N-benzyl group, yielding N-benzylga-
lactonoamidine 1a as reaction product depending on the
catalyst amount. Dehalogenation reactions of aryl halides under
similar hydrogenation conditions are known.31 As galactonoa-
midines derived from cyclic aliphatic and linear amines were
found to show higher inhibition activity than those derived
from benzylamines, a synthetic strategy to afford p-chloro- and
p-bromo-substituted benzylgalactonoamidines 1k and 1l was
not further explored. Deprotection of 2,3,4,6-tetra-O-benzylga-
lactonoamidines 3n and 3p−3u derived from aliphatic amines
under the conditions resulted in formation of TFA salts that
can be avoided by reducing the amount of TFA used during the
deprotection reaction. Remaining traces of TFA in the raw
products can then be removed by repetitive freeze-drying cycles
to afford 1n and 1p−1u as free bases. For control experiments,
D-galactonoamidine (1w)27 and D-galactono-δ-lactam30 (1x)
were synthesized as described by others earlier. All compounds
in the small library (1a−x) were subsequently employed to
probe the stabilizing and destabilizing effects of the aglycon
a
−
Reaction conditions: (i) Et3O+BF4 , CH2Cl2, 0 °C, 2 h; R−NH2,
0 °C → ambient temperature, 17 h to 8 days; (ii) H2, Pd/C, EtOH,
ambient temperature, 24 h.
In short, we reacted 2,3,4,6-tetra-O-benzyl-D-galactothiono-
lactam (2)17,20 with five p-substituted benzylamines (namely, 4-
tert-butylbenzylamine, 4-methoxybenzylamine, 4-trifluorome-
thylbenzylamine, 4-chlorobenzylamine, and 4-bromobenzyl-
amine), five aliphatic linear amines (namely, propylamine, n-
heptylamine, n-dodecylamine, 2-ethylhexylamine, and 3-phenyl-
propylamine), and five aliphatic amines with cyclic substituents
(namely, cyclopropylamine, cyclohexylamine, cyclopropylme-
thylamine, cyclohexylmethylamine, and cycloheptylmethyl-
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Table 1. Inhibition Constants of N-Substituted Galactonoamidines Obtained for Enzymatic Hydrolysis of 2-Chloro-4-
a
nitrophenyl-β-D-galactopyranoside (5) by β-Galactosidase (A. oryzae)
a
kcat = 2516 53 min−1; KM = 0.70 0.04 mM; knon = 1.04 0.31 × 10−5 min−1 M−1; Vt = 100 μL; 50 mM acetate buffer at pH 5.00 0.05; 30.0
0.1 °C; εapp,405 × d = 1532 M−1.
moiety upon interaction of the galactonoamidines with the
active site of β-galactosidase (A. oryzae). The determined
structure−activity relationship is described below.
chains of amino acids in the active site of the β-galactosidase, by
hydrophobic interaction of the methyl substituent with
corresponding amino acids, or by a combination of both.
In order to verify or reject this hypothesis, we developed a
fast screening tool based on a 96-well plate assay to evaluate
our small library of N-substituted galactonoamidines in a timely
manner. Along these lines, we replaced the previously used,
commercially available substrate 2-nitrophenyl-β-D-galactopyr-
anoside (4) by 2-chloro-4-nitrophenyl-β-D-galactopyranoside
(5) synthesized for a different project.29 The comparably low
value for the extinction coefficient of 4 in acetate buffer at pH 5
(ε400 ≈ 820 60 cm−1 M−1; Vt = 1000 μL)18 translates into an
even smaller apparent extinction coefficient in a kinetic assay
Biology. Substrate Choice and Kinetic Assay. We
previously established N-benzylgalactonoamidines 1a−g as
competitive inhibitors of β-galactosidase (A. oryzae)18 and p-
methyl-N-benzylgalactonoamidine 1d as putative transition-
state analog for hydrolysis of β-glycosidic bonds by β-
galactosidase (A. oryzae).29 We subsequently hypothesized a
significant influence of the aglycon moiety on the high
inhibition ability of 1d (Ki ≈ 8 nM) and its transition-state-
like character that might be due to π−π stacking interactions of
the aromatic ring of the aglycon moiety with aromatic side
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under these conditions in a 96-well plate (εapp,405 ≈ 125 cm−1
M−1; Vt = 100 μL), increases the error associated with the
kinetic data, and prompted this change. While mathematical
corrections of an apparent extinction coefficient can be done to
account for the reduced optical path length and the smaller
total volume in a 96-well plate assay, the thereby initially
obtained kinetic data showed a larger error than data obtained
from standard UV−vis cells and were consequently discarded.
To circumvent any uncertainty of kinetic data while relying on
a fast screening method, substrate 5 was used throughout the
kinetic structure−activity study described below.29
The hydrolytic activity of β-galactosidase (A. oryzae) was
determined in the presence and absence of the synthesized N-
substituted galactonoamidines 1 in 50 mM acetate buffer at pH
5 and 30 °C under steady state conditions. The absorbance
recorded for formation of 2-chloro-4-nitrophenolate was
transformed into the product concentration using the
uncorrected apparent extinction coefficient of 2-chloro-4-
nitrophenolate obtained separately from a calibration curve
under identical conditions (εapp,405 × d = 1532 M−1). Initial
rates of the hydrolysis were obtained from plots of product
formation over time. The apparent Michaelis−Menten
constants (KM) and rate constants (kcat) were determined by
plotting the initial rates corrected for the enzyme concentration
versus the substrate concentration (0−2 mM) and fitting the
hyperbolic data by nonlinear regression applying the
Michaelis−Menten model. All experiments were conducted in
triplicate, and the resulting data were averaged.
(Table 1, entries 8−10) with tert-butyl, methoxy, and
trifluoromethyl groups and compared their inhibition ability
to 1d. The increased size of the p-tert-butyl group decreases the
inhibition ability of 1h compared to the smaller p-methyl group
in 1d, indicating steric constraints for large substituents.
Likewise, the inhibition ability of 1i with a p-methoxy
substituent is smaller than that determined for 1d (Ki,1d
<
Ki,1i) but of the same order of magnitude as for 1h, indicating
that H-bonding interactions toward the oxygen atom in the
aglycon are absent (Ki,1h < Ki,1i). A similar lack of supporting H-
bonding interactions is apparent when comparing the effect of
the p-methyl substituent in 1d to the p-trifluoromethyl group in
1j, which is also considerably larger in size than the p-methyl
group in 1d. The study of para-substituted N-benzylgalacto-
amidines revealed size constraints and supportive hydrophobic
interactions between the inhibitors and the active site in β-
galactosidase (A. oryzae) but a lack of supporting H-bonding
interactions with the para substituent of the aglycon.
Galactonoamidines with p-chloro and p-bromo substituents
are not accessible with the current synthetic procedure
described above and resulted in partial or complete
dehalogenation of the corresponding precursor compounds
depending on the catalyst amount. However, given the size
constraints and lack of supporting H-bonding interactions
observed by comparing 1a, 1d, and 1h, we did not improve the
synthetic strategy toward these target compounds for
comparison to 1g and 1j and focused on evaluating
hydrophobic and π−π stacking interactions instead.
In the presence of three different concentrations of inhibitors
1a−w, the value of the apparent Michaelis−Menten constant
(K′M) increases while the apparent rate constant remains
unaffected. Competitive inhibition constants (Ki) were
estimated from the apparent Michaelis−Menten constants in
the presence and absence of 1a−w and are given as an average
of three independent experiments for each concentration
(Table 1).
Along these lines we synthesized a selection of aliphatic
galactonoamidines with linear aliphatic, branched aliphatic, and
aliphatic cyclic amines (Table 1, entries 11−20). The
distribution of the inhibition ability determined for galacto-
noamidines 1m−o with linear aliphatic side chains (Table 1,
entry 11−13) disclosed that a side chain consisting of 3 C
atoms has only a small contribution on the inhibition ability of
the compound, while a linear side chain of 7 C atoms promotes
almost the same interaction as the aromatic N-benzyl groups
(Ki,1d ≈ Ki,1n). A carbon side chain of 12 carbon atoms reveals
lower inhibition ability and points at steric constraints due to
the side chain length.
Galactonoamidines with aglycons consisting of cyclic amines
directly attached to the amidine group show insufficient
interactions with the active site for both a 3-membered ring
(1r) and a 6-membered ring (1s). Interestingly, galactonoami-
dines with a methylene spacer and aliphatic cyclic aglycon (1t−
v) show remarkable high inhibition ability toward the β-
galactosidase (Table 1, entries 18−20). This observation
indicates strong hydrophobic interactions of the active site
with the methylene spacer and also with the aliphatic cyclic
moiety of the aglycon in 1t−v. Most pronounced is a
comparison of the inhibition ability between 1r and 1t and
between 1s and 1u, respectively, revealing the importance of
the aliphatic methylene spacer in the aglyon, while a
comparison of 1a with N-benzyl aglycon to 1u with aliphatic
methylcyclohexyl aglycon clearly discloses a lack of favorable
π−π stacking interactions to the galactonoamidines in the
active site of the β-galactosidase (Ki,1u < Ki,1a).
Structure−Activity Relationship. Initially, we re-evaluated
already available inhibitors 1a−g containing methyl- or fluoro-
substituted benzyl aglycons with substrate 5 instead of
previously used substrate 4 in 96-well plate format under
otherwise identical conditions. We determined competitive
inhibition constants for those 7 inhibitors (Ki = 8−30 nM) that
reflect the same stereoelectronic effects as described
previously.18,29 In short, the fluoride substituent on the N-
benzyl aglycon mimics a hydrogen atom in size but has
electron-withdrawing properties that vary depending on the
position on the aromatic ring, while a methyl group substituent
is larger in size than the H atom and has electron-donating
properties. The unsubstituted N-benzylgalactonoamidine 1a
shows a smaller inhibition constant than the 2- or- 3-methyl-N-
benzylgalactonoamidines 1b−c and the 2- or 3-fluoro-N-
benzylgalactonoamidines 1e−f (Table 1, entries 1−3, 5−6).
In contrast, a methyl or fluoro substituent in the para position
of the benzyl substituent favor the interactions of inhibitors 1d
and 1g over those of 1a with the amino acids in the active site
of β-galactosidase (A. oryzae) (Table 1, entry 4 and 7). This
result indicates favorable stereoelectronic effects of substituents
in the para position of the benzyl aglycon upon interactions of
the inhibitors, like 1d or 1g, for inhibition of β-galactosidase (A.
oryzae) and possible hydrophobic interactions with the methyl
group in the active site (Ki,1d < Ki,1g).
These findings prompted the synthesis of galactonoamidines
1p with branched aliphatic and of galactonoamidines 1q with a
methylene spacer consisting of 3 C atoms attached to an
aromatic ring (Table 1, entries 14 and 15). Both compounds
were indeed found to inhibit the β-galactosidase in the same
order of magnitude as 1d and 1t−u (Ki ≈ 8 1 nM). A similar
To further explore the supporting effects of para substituents
on the N-benzyl aglycon, we synthesized inhibitors 1h−j
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phenomenon on the inhibition ability has been previously
observed by others after introduction of flexible and hydro-
phobic substituents.18,32 N-Ethylphenylgluconoamidine and
C(2)-phenylethyl-substituted glucoimidazole were identified
as very potent inhibitors with very low inhibition constants (Ki
≤ 1.2 nM) toward selected β-glucosidases (Almonds, A. niger,
and Caldocellum saccharolyticum.).19,32
Consequently, the previously observed high inhibition ability
of 1d is not based on π−π stacking interactions with the
aromatic N-benzyl aglycon but rather on stabilizing hydro-
phobic interactions with the p-methyl substituent on the
aromatic aglycon. For comparison and control experiments, we
resynthesized already described galactonoamidine 1w and
galactono-δ-lactam 1x (Table 1, entries 21 and 22).27,30 The
inhibition constant of 1w is about 1 order of magnitude above
the inhibition constant of galactonoamidines with an aglycon,
while the exchange of the amidine group in 1w against the
lactam group in 1x reveals the contribution of the exocyclic N
atom on the inhibition ability of the galactonoamidines 1a−v.
The lack of an aglyon diminishes the inhibitory activity of the
investigated compounds, thereby revealing its significant
contribution.
inhibition efficacy for 1n (IC50 = 107 nM) is noted that is
attributed to insufficient hydrophobic interactions of the heptyl
aglycon in the active site. An further diminished inhibition
efficacy is noted for inhibitor 1t (IC50 = 0.86 μM) that contains
an even smaller methylenecyclohexyl aglycon.
Furthermore, the importance of the methylene spacer
relative to the size of the remaining algycon for the inhibitory
ability of the galactonoamidines becomes apparent by
comparing the inhibitory efficacy of 1t to that of 1s. Control
compound 1s lacks the methylene spacer that is contained in 1t
but contains a larger cyclohexyl aglycon instead of a
methylenecyclopropyl rest and shows a considerably lower
inhibitory efficacy (IC50 = 4.8 μM). Even more pronounced is
the difference in efficacy for galactonolactam 1x that was found
to require a 49 μM concentration to achieve 50% inhibition of
the reaction. Both control compounds, 1s and 1x, have
inhibition constants on the same order of magnitude (Table 1),
but compound 1x lacks an aglycon moiety and has a 10-fold
lower inhibitory efficacy than 1s. Overall, the efficacy studies
provide further evidence for the important contributions of the
aglycon to the performance of the galactonoamidines as
inhibitors and reveal high inhibitory efficacy for 1u, 1d, 1p−
q, and 1v (IC50 = 22−36 nM).
We then determined the efficacy of selected inhibitors to
characterize the concentrations needed to cause inhibition
effects for hydrolysis of 5 by β-galactosidase (A. oryzae).
Toward this goal, we varied the concentration of inhibitors 1d,
1n, 1p-q, 1s-v, and 1x between 5 × 10−4 and 50 μM at constant
substrate concentration (Figure 1).
CONCLUSION
■
A small library of 22 N-substituted galactonoamidines (1a−v)
was synthesized and evaluated for their ability to inhibit the
hydrolytic activity of β-galactosidase (A. oryzae). Along these
lines, a fast screening assay based on UV−vis spectroscopy was
developed suitable for small volumes in acidic conditions
employing a 96-well plate format to follow hydrolysis of 2-
chloro-4-nitrophenyl-β-D-galactopyranoside. The N-substituted
galactonoamidines were synthesized from a selection of
aliphatic and benzylic amines representing steric, electronic,
hydrophobic, and spacer effects, π−π stacking, and van der
Waals interactions. The aglycon moiety of all compounds was
found to have a profound effect on their inhibitory ability in
comparison to control compounds lacking aglycons, namely,
galactonolactam (1w) and galactonoamidines (1x). In general,
galactonoamidines derived from cyclic aliphatic and linear
amines were found to show higher inhibition activity than those
derived from benzylamines. Finally, hydrophobic interactions of
the methyl group rather than π−π stacking interactions of the
aromatic ring in p-methylbenzyl-D-galactonoamidine (1d) were
identified to cause its transition-state-like character and the
remarkably high inhibitory ability (Ki = 8 nM) and efficacy
(IC50 = 36 nM).
Figure 1. Efficacy of selected inhibitors during enzymatic hydrolysis of
5: black square, 1u; red circle, 1v; green up triangle, 1p; dark blue
down triangle, 1d; blue diamond, 1q; pink left side triangle, 1n; yellow
right side triangle, 1t; dark yellow hexagon, 1s; blue star, 1x.
Compounds 1d, 1n, 1p−q, and 1t−v were identified as very
potent inhibitors with inhibition constants equal to or less than
12 nM, while 1s and 1x have higher inhibition constants and
were selected for control experiments (Table 1). In general, the
apparent inhibition efficacy of 1d, 1n, 1p−q, and 1t−v is very
similar, showing high inhibition efficacy. All of the selected
inhibitors support hydrophobic interactions between the active
site of the enzyme and their aglycon as described above. A
significant difference in the overall inhibitor efficacy related to
the respective aglycon structure in 1d, 1p−q, and 1u−v is not
apparent. The maximal apparent inhibition effect for these
inhibitors is achieved at concentrations between 3 and 10 μM.
The concentration required to reach the half-maximal
inhibition (IC50) of the enzymatic hydrolysis of 5 was
determined to be as low as 22 (1u) and 36 nM (1d, 1p−q,
and 1v). Although the inhibition constant for compound 1n is
on the same order of magnitude, a somewhat diminished
EXPERIMENTAL SECTION
■
1
Instrumentation. H and 13C NMR spectra were recorded on a
400 MHz Bruker magnet with Z gradient and 5 mm broad-band head
using Topspin 2.1 software. High-resolution mass spectrometry data
were obtained in the state-wide mass spectrometry facility at the
University of Arkansas on a Bruker ultrOTOF-Q quadrupole time-of-
flight (qQ-TOF) mass spectrometer equipped with an electrospray
ionization source or the Mass Spectrometry Facility at Georgia State
University, Atlanta, GA. UV−vis data were recorded on a FilterMax F5
Multi-Mode Microplate Reader from Molecular Devices using 96-well,
medium-binding microlon Elisa plates from Greiner Bio-one.
Lyophilization was performed on a FreeZone 1 L benchtop freeze-
dry system from Labconco. Melting points were recorded on a Mel-
Temp melting point apparatus, and values are uncorrected. Nanopure
water at a resistance of 18.2 mΩ was obtained from a ThermoScientific
Barnstead E-pure water purification system. Purity assays were
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performed on an HPLC system from Shimadzu equipped with a SCL-
10Avp system controller, 2 LC-20AD analytical pumps, DGU-20A3R
three-channel online degassers, a SIL-20A UFLC autosampler with 96-
well capability, a CTO-20A/prominence column oven, and ELSD-
90LT light scattering and LC solution software, version 1.25 from
Shimadzu for data recording and analysis.
allowed to warm to ambient temperature. After 12 h to 8 days, all
volatile material was removed under reduced pressure. The resulting
residue was purified by column chromatography over silica gel,
yielding the title compounds 3h−v in fair to moderate yields.
N-4-tert-Butylbenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3h). Reaction: Meerwein’s reagent (0.54 g, 2.820 mmol, 1.5 equiv)
and 2 (1.05 g, 1.950 mmol) in 10 mL of dichloromethane; amine 4-
tert-butylbenzylamine (0.62 g, 3.800 mmol, 2.0 equiv); reaction time 8
days; eluent for chromatography cyclohexane/ethyl acetate = 6/1−3/
1, v/v; compound 3h (0.53 g, 0.776 mmol, 41%); colorless oil. Rf 0.27
(SiO2, cyclohexane/ethyl acetate = 3/1, v/v). δH (CD2Cl2) 7.21−7.49
(m, 20H), 7.14 (d, 8.3, 2H), 5.01 (br s, 1H), 4.94 (d, 11.0, 1H), 4.85
(t, 12.0, 2H), 4.63−4.69 (m, 2H), 4.62 (d, 3.8, 1H), 4.56 (s, 2H), 4.51
(d, 9.5, 1H), 4.32 (s, 1H), 4.21 (quind, 14.3, 4.3, 2H), 3.99 (dd, 9.7,
1.9, 1H), 3.67−3.76 (m, 3H), 3.59−3.66 (m, 2H), 1.31 (s, 9H). δC
(CD2Cl2) 156.2, 150.4, 139.7, 139.4, 138.9, 138.7, 137.3, 129.0, 129.0,
128.9, 128.7, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0,
127.9, 125.8, 82.5, 76.1, 75.2, 74.7, 74.2, 73.8, 72.0, 71.6, 60.1, 44.9,
34.9, 31.7. δC (CDCl3) 156.0, 149.8, 139.0, 138.4, 138.1, 137.7, 136.1,
128.5, 128.4, 128.3, 128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.6, 127.6, 127.4, 125.3, 81.8, 75.3, 74.5, 73.9, 73.7, 73.3, 71.1, 71.0,
59.5, 45.0, 34.4, 31.3. HRMS (ESI) calcd for C45H51N2O4 [M + H]+
683.3843; found, 683.3843.
N-4-Methoxybenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3i). Reaction: Meerwein’s reagent (0.62 g, 3.260 mmol, 2.0 equiv) and
2 (0.90 g, 1.630 mmol) in 6 mL of dichloromethane; amine 4-
methoxybenzylamine (0.45 g, 3.260 mmol, 2.0 equiv); reaction time
60 h; eluent for chromatography cyclohexane/ethyl acetate = 6/1−3/
1, v/v; compound 3i (0.64 g, 0.976 mmol, 63%); colorless oil. Rf 0.21
(SiO2, cyclohexane/ethyl acetate = 3/1, v/v). δH (CD2Cl2) 7.19−7.48
(m, 20H), 7.11 (d, 8.8, 2H), 6.80 (d, 8.8, 2H), 4.76−5.00 (m, 4H),
4.62 (dd, 11.4, 7.2, 4H), 4.55 (s, 2H), 4.50 (br s, 2H), 4.30 (br s, 1H),
4.17 (br s, 2H), 3.96 (d, 9.8, 1H), 3.77 (s, 3H), 3.54−3.74 (m, 3H). δC
(CD2Cl2) 159.3, 156.2, 139.7, 139.4, 138.9, 138.7, 132.3, 129.5, 129.0,
129.0, 128.9, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
114.2, 82.4, 76.0, 75.2, 74.7, 74.2, 73.8, 72.0, 71.6, 60.1, 55.7, 44.8. δC
(CDCl3) 158.6, 155.8, 139.0, 138.4, 138.0, 137.7, 131.3, 129.0, 128.5,
128.4, 128.3, 128.1, 128.1, 127.9, 127.8, 127.8, 127.6, 127.6, 127.5,
127.3, 113.7, 81.8, 75.2, 74.4, 73.9, 73.7, 73.3, 71.1, 70.9, 59.5, 55.2,
44.5. HRMS (ESI) calcd for C42H45N2O5 [M + H]+ 657.3323; found,
657.3321.
Materials and Methods. Chemical shifts (δ) in NMR data are
expressed in parts per million (ppm) and coupling constants (J) in
Hertz. Signal multiplicities are denoted as s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). Deuterated chloroform,
deuterated dichloromethane, DMSO-d6, acetone-d6, MeOH-d4, and
deuterium oxide were used as solvents. Chemical shift values are
reported relative to the residual signals of these solvents (CDCl3 δH
7.29, δC 77.0; CD2Cl2 δH 5.32, δC 54.0; DMSO-d6 δH 2.50, δC 39.5;
acetone-d6 δH 2.05, δC 29.8; MeOH-d4 δH 3.35, δC 49.0; D2O δH 4.80,
δC 49.0 after addition of a few drops of MeOH-d4). High-resolution
mass spectrometry data were obtained from samples that were mixed
with acetonitrile containing 0.1% formic acid and injected into the
source via a syringe pump operating at 3 μL/h. The dry gas
temperature was 180 °C, the dry gas flow was 5 L/min, and the
nebulizing gas pressure was 1 bar. The remaining instrument
parameters were optimized to obtain maximum signal for ions
between 100 and 1000 amu. Column chromatography was carried out
using silica gel 60 from Silicycle (40−63 μm, 230−240 mesh). Thin
layer chromatography (TLC) was performed using silica gel TLC
plates from SORBENT Technologies, 200 μm, 4 × 8 cm, aluminum
backed, with fluorescence indicator F254 and detection by UV light or
by charring with an ethanolic vanillin−sulfuric acid reagent and
subsequent heating of the TLC plate. Compound purity was
established on a Rezex RMN−Na+ column (Phenomenex) using
nanopure water as eluent with a flow rate of 0.4 mL/min at 80 °C and
electric light scattering detection (ELSD) at 50 °C (GAIN 5). In a
typical experiment, a 10 μL aliquot of a solution prepared from 5 mg
of compound in 1 mL of nanopure water was injected, and the
isocratic elution was followed over 30 min. All elution peaks were
integrated, and purity data were obtained as area percent values to
confirm a purity of all compounds above 95%. All pH values of the
buffer solutions were obtained using a Beckman Φ 250 pH meter
equipped with a refillable ROSS combination pH electrode from
Orion with epoxy body and an 8 mm semimicrotip. The pH meter was
calibrated before each set of readings (3-point calibration).
N-4-Trifluoromethylbenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoa-
midine (3j). Reaction: Meerwein’s reagent (0.25 g, 1.355 mmol, 1.5
equiv) and 2 (0.50 g, 0.906 mmol) in 6 mL of dichloromethane; amine
4-trifluoromethylbenzylamine (0.31 g, 1.812 mmol, 2.0 equiv);
reaction time 48 h; eluent for chromatography cyclohexane/ethyl
acetate = 6/1−3/1, v/v; compound 3j as a colorless oil (0.23 g, 0.331
mmol, 33%). Rf 0.20 (SiO2, cyclohexane/ethyl acetate = 2/1, v/v). δH
(CD2Cl2) 7.53 (d, 7.8, 2H), 7.41−7.46 (m, 2H), 7.23−7.40 (m, 20H),
5.09 (br s, 1H), 4.95 (d, 11.0, 1H), 4.92 (d, 11.5, 1H), 4.85 (d, 11.5,
1H), 4.62−4.71 (m, 3H), 4.55 (s, 3H), 4.52 (d, 1.3, 1H), 4.35 (s, 3H),
4.32 (t, 2.0, 1H), 3.99 (dd, 9.7, 1.9, 1H), 3.65−3.73 (m, 3H), 3.60 (dd,
Chemicals. All commercially obtained chemicals had reagent-grade
quality or better and were used as received, if not noted otherwise. 4-
Trifluoromethylbenzylamine, 4-methoxybenzylamine, 4-tert-butylben-
zylamine, 4-chlorobenzylamine, 4-bromobenzylamine, propylamine, n-
heptylamine, 2-ethylhexylamine, cyclopropylamine, cyclohexylamine,
cyclopropylmethylamine, cyclohexylmethylamine, benzylamine, cyclo-
hexane, and ethyl acetate were distilled prior to use; dichloromethane
was dried over molecular sieves 4 Å; β-galactosidase [3.2.1.23] from A.
oryzae was obtained from Sigma-Aldrich as lyophilized powder
stabilized on dextrin and stored at −18 °C. A BCA assay confirmed
that the supplied batch contains 10% of protein.18 The molar weight of
the tetrameric protein was determined as 347.2 kDa (86.8 kDa per
unit) by microchip electrophoresis.18
Synthetic Procedures. Synthesis of Galactonoamidines. 2,3,4,6-
Tetra-O-benzyl-D-galactothionolactam (2),17,20,30 N-benzyl-D-galacto-
noamidine (1a),17 N-2-methylbenzyl-D-galactonoamidine (1b),17 N-3-
methylbenzyl-D-galactonoamidine (1c),17 N-4-methylbenzyl-D-galacto-
noamidine (1d),17 N-2-fluorobenzyl-D-galactonoamidine (1e),17 N-3-
fluorobenzyl-D-galactonoamidine (1f),29 N-4-fluorobenzyl-D-galacto-
noamidine (1g),17 D-galactonoamidine (1w),27 and D-galactono-δ-
lactam (1x)30 were prepared as described. Quantitative HPLC analysis
for all galactonoamidines used in structure−activity relationship
studies was performed to verify a compound purity above 95% (see
Supporting Information).
11.3, 9.8, 1H). δC (CD2Cl2) 156.1, 144.9, 139.5 (d, J2 =40.3 Hz),
C,F
138.8 (d, J3 =22.7 Hz), 129.1, 129.1, 129.0, 128.9, 128.8, 128.7,
C,F
128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.1, 125.7 (q, J4
=
C,F
3.7 Hz), 125.0 (q, J1 =270.7 Hz), 82.6, 76.1, 75.3, 74.6, 74.5, 73.8,
C,F
71.9, 71.6, 60.1, 44.7. HRMS (ESI) calcd for C42H43F3N2O4 [M + H]+
695.3097; found 695.3092.
N-4-Chlorobenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3k). Reaction: Meerwein’s reagent (0.51 g, 2.72 mmol, 2.0 equiv) and
2 (0.75 g, 1.36 mmol) in 10 mL of dichloromethane; amine 4-
chlorobenzylamine (0.38 g, 2.72 mmol, 2.0 equiv); reaction time 5
days; eluent for chromatography cyclohexane/ethyl acetate = 8/1−4/
1, v/v; compound 3k (0.40 g, 0.608 mmol, 45%); colorless oil. Rf 0.25
(SiO2, cyclohexane/ethyl acetate = 2/1, v/v). δH (CD2Cl2) 7.18−7.45
(m, 22H), 7.14 (d, 8.5, 2H), 5.00 (br s, 1H), 4.94 (d, 11.0, 1H), 4.88
(d, 11.5, 1H), 4.84 (d, 11.8, 1H), 4.59−4.68 (m, 3H), 4.55 (s, 2H),
4.51 (d, 9.8, 1H), 4.31 (t, 2.0, 1H), 4.24 (br s, 2H), 3.97 (dd, 9.5, 1.8,
1H), 3.64−3.74 (m, 2H), 3.56−3.64 (m, 1H). δC (CD2Cl2) 156.1,
139.7, 139.3, 138.9, 138.6, 132.9, 129.6, 129.1, 129.0, 128.9, 128.9,
General Procedure for Synthesis of N-Aryl- and N-Alkyl-2,3,4,6-
tetra-O-benzyl-D-galactonoamidines (3h−v). Meerwein’s salt was
added to a solution of 2,3,4,6-tetra-O-benzyl-D-galactothionolactam
(2)17,20,30 in dry dichloromethane under nitrogen atmosphere at 0 °C.
After 2 h, freshly distilled amine was added and the solution was
9004
dx.doi.org/10.1021/jm501111y | J. Med. Chem. 2014, 57, 8999−9009

Journal of Medicinal Chemistry
Article
128.8, 128.7, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 82.5, 76.0,
75.2, 74.6, 74.4, 73.8, 71.9, 71.6, 60.0, 44.5. δC (CDCl3) 155.7, 139.0,
138.3, 138.0, 137.8, 137.7, 132.5, 129.0, 128.6, 128.4, 128.4, 128.3,
128.1, 128.0, 127.9, 127.8, 127.8, 127.7, 127.6, 127.6, 127.4, 81.9, 77.2,
75.3, 74.5, 74.1, 73.6, 73.3, 71.0, 71.0, 59.5, 44.2. HRMS (ESI) calcd
for C41H42ClN2O4 [M + H]+, 661.2828 and 662.2861; found 661.2825
and 662.2845.
N-4-Bromobenzyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3l). Reaction: Meerwein’s reagent (0.54 g, 2.820 mmol, 1.5 equiv) and
2 (1.05 g, 1.900 mmol) in 10 mL of dichloromethane; amine 4-
bromobenzylamine (0.71 g, 3.817 mmol, 2.0 equiv); reaction time 8
days; eluent for chromatography cyclohexane/ethyl acetate = 6/1−3/
1, v/v; compound 3l (0.73 g, 1.035 mmol, 55%); colorless oil. Rf 0.20
(SiO2, cyclohexane/ethyl acetate = 3/1, v/v). δH (CD2Cl2) 7.19−7.45
(m, 22H), 7.08 (d, 8.5, 2H), 4.99 (br s, 1H), 4.92 (d, 10.8, 1H), 4.87
(d, 11.3, 1H), 4.83 (d, 11.8z, 1H), 4.58−4.67 (m, 3H), 4.54 (s, 2H),
4.49 (d, 9.5, 1H), 4.30 (t, 1.8, 1H), 4.22 (br s, 2H), 3.96 (dd, 9.7, 1.9,
1H), 3.63−3.71 (m, 2H), 3.54−3.62 (m, 1H). δC (CD2Cl2) 156.1,
139.7, 139.3, 138.9, 138.6, 131.8, 129.9, 129.2, 129.1, 129.0, 128.9,
128.8, 128.6, 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
120.9, 82.5, 76.0, 75.2, 74.6, 74.4, 73.8, 71.8, 71.6, 60.0, 44.6. HRMS
(ESI) calcd for C41H42BrN2O4 [M + H]+, 705.2322 and 707.2302;
found 705.2322 and 707.2309.
N-Propyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3m). Reac-
tion: Meerwein’s reagent (0.25 g, 1.355 mmol, 1.5 equiv) and 2 (0.50
g, 0.906 mmol) in 6 mL of dry dichloromethane; amine propylamine
(0.11 g, 1.812 mmol, 2.0 equiv); reaction time 36 h; eluent for
chromatography cyclohexane/ethyl acetate = 3/1−1/2, v/v; com-
pound 3m (0.30 g, 0.520 mmol, 58%); colorless oil. Rf 0.23 (SiO2,
cyclohexane/ethyl acetate = 1/2, v/v). δH (CD2Cl2) 7.25−7.44 (m,
20H), 6.56−6.94 (m, 1H), 5.04 (d, 11.8, 1H), 4.88 (d, 10.8, 1H),
4.67−4.84 (m, 4H), 4.50−4.65 (m, 3H), 4.33 (t, 2.3, 1H), 4.04 (dd,
9.9, 1.9, 1H), 3.90−3.98 (m, 1H), 3.84 (dd, 8.9, 6.4, 1H), 3.70 (dd,
9.0, 8.0, 1H), 3.16 (dquin, 45.7, 7.0, 2H), 1.50 (qd, 7.3, 1.3, 2H), 0.85
(t, 7.4, 3H). δC (CD2Cl2) 162.9, 138.2, 138.1, 137.7, 136.9, 129.4,
129.3, 129.2, 129.1, 128.9, 128.9, 128.9, 128.7, 128.6, 128.6, 128.5,
128.5, 128.4, 79.9, 75.9, 75.7, 73.9, 72.7, 72.4, 71.9, 67.9, 56.7, 54.5,
54.3, 53.7, 53.5, 44.1, 21.3, 11.2. HRMS (ESI) calcd for C37H43N2O4
[M + H]+ 579.3223; found, 579.3200.
N-n-Heptyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3n). Re-
action: Meerwein’s reagent (0.51 g, 2.717 mmol, 1.5 equiv) and 2
(1.00 g, 1.82 mmol) in 10 mL of dichloromethane; amine n-
heptylamine (0.42 g, 3.665 mmol, 2.0 equiv); reaction time 41 h;
eluent for chromatography cyclohexane/ethyl acetate = 4/1−2/1, v/v;
compound 3n (0.67 g, 1.055 mmol, 58%); colorless oil. Rf 0.25 (SiO2,
cyclohexane/ethyl acetate = 3/2, v/v). δH 7.18−7.49 (m, 20H), 6.65
(br s, 1H), 5.04 (d, 11.5, 1H), 4.88 (d, 10.8, 1H), 4.78−4.83 (m, 1H),
4.75 (d, 12.0, 1H), 4.68−4.72 (m, 1H), 4.53 (d, 11.3, 1H), 4.33 (t, 2.0,
1H), 4.03 (dd, 9.9, 1.9, 1H), 3.94 (d, 5.5, 1H), 3.80−3.90 (m, 1H),
3.69 (t, 8.5, 1H), 3.04−3.33 (m, 2H), 1.38−1.54 (m, 2H), 1.11−1.33
(m, 9H), 0.88 (t, 7.0, 3H). δC 162.5, 138.3, 138.2, 137.8, 137.0, 129.6,
129.5, 129.3, 129.2, 129.0, 129.0, 128.8, 128.7, 128.6, 128.6, 128.5,
128.5, 79.9, 76.1, 75.8, 74.1, 72.8, 72.1, 68.1, 57.1, 54.4, 42.7, 32.1,
29.2, 28.1, 27.0, 23.1, 14.4. HRMS (ESI) calcd for C41H51N2O4 [M +
H]+ 635.3849; found 635.3848.
HRMS (ESI) calcd for C46H61N2O4 [M + H]+ 705.4626; found
705.4632.
N-2-Ethylhexyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3p).
Reaction: Meerwein’s reagent (0.51 g, 2.717 mmol, 1.5 equiv) and 2
(1.00 g, 1.82 mmol) in 10 mL of dichloromethane; amine 2-
ethylhexylamine (0.47 g, 3.623 mmol, 2.0 equiv); reaction time 41 h;
eluent for chromatography cyclohexane/ethyl acetate = 4/1−2/1, v/v;
compound 3p (0.47 g, 0.724 mmol, 40%); colorless oil. Rf 0.21 (SiO2,
cyclohexane/ethyl acetate = 3/2, v/v). δH (CD2Cl2) 7.25−7.45 (m,
20H), 6.30 (br s, 1H), 5.01 (d, 11.5, 1H), 4.90 (d, 10.5, 1H), 4.82 (d,
11.5, 1H), 4.75 (d, 12.5, 1H), 4.67−4.72 (m, 2H), 4.63 (d, 10.8, 1H),
4.59 (d, 11.8, 1H), 4.54 (d, 11.8, 1H), 4.33 (d, 2.0, 1H), 4.03 (dd, 9.8,
1.8, 1H), 3.88−3.95 (m, 1H), 3.83 (dd, 8.8, 6.3, 1H), 3.69 (t, 8.5, 1H),
3.15 (dd, 14.1, 6.5, 1H), 3.05 (dd, 13.3, 6.3, 1H), 1.35−1.47 (m, 1H),
1.07−1.31 (m, 8H), 0.84 (dt, 19.0, 7.2, 6H). δC (CD2Cl2) 161.8, 138.5,
138.4, 138.0, 137.2, 129.5, 129.4, 129.3, 129.2, 129.2, 129.1, 129.0,
128.9, 128.9, 128.8, 128.8, 128.7, 128.6, 128.6, 128.5, 128.5, 128.4,
128.3, 128.3, 128.2, 80.4, 75.7, 75.6, 73.9, 73.2, 72.5, 68.8, 57.5, 54.4,
45.4, 38.7, 38.6, 31.2, 31.1, 29.1, 29.0, 24.5, 23.4, 14.3, 11.0, 11.0.
HRMS (ESI) calcd for C42H53N2O4 [M + H]+ 649.4005; found
649.4003.
N-3-Phenylpropyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3q). Reaction: Meerwein’s reagent (0.54 g, 2.820 mmol, 1.5 equiv)
and 2 (1.05 g, 1.950 mmol) in 10 mL of dichloromethane; amine 3-
phenylpropylamine (0.51 g, 3.800 mmol, 2.0 equiv); reaction time 8
days; eluent for chromatography cyclohexane/ethyl acetate = 4/1−1/
1, v/v; compound 3q (0.64 g, 0.977 mmol, 51%); colorless oil. Rf 0.20
(SiO2, cyclohexane/ethyl acetate = 1/1, v/v). δH (CD2Cl2) 7.46−7.07
(m, 25H), 4.99−4.78 (m, 3H), 4.59−4.69 (m, 4H), 4.56 (s, 2H), 4.47
(d, 9.5, 1H), 4.30 (br s, 1H), 3.95 (d, 9.5, 1H), 3.77−3.52 (m, 3H),
3.09 (br s, 2H), 2.56 (t, 7.3, 2H), 1.73 (quin, 7.0, 2H); δC (CD2Cl2)
156.4, 142.6, 139.7, 139.3, 138.9, 138.8, 129.1, 128.9, 128.9, 128.8,
128.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
126.2, 82.4, 76.1, 75.2, 74.6, 74.3, 73.8, 71.9, 71.6, 60.0, 40.8, 33.8,
31.4. HRMS (ESI) calcd for C43H47N2O4 [M + H]+ 655.3530; found
655.3535.
N-Cyclopropyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3r).
Reaction: Meerwein’s reagent (0.51 g, 2.72 mmol, 1.5 equiv) and 2
(1.00 g, 1.82 mmol) in 10 mL of dichloromethane; amine
cyclopropylamine (0.22 g, 3.62 mmol, 2.0 equiv) was added, and the
solution was allowed to warm to ambient temperature. After 3 days, all
volatiles were removed; reaction time 3 days; eluent for chromatog-
raphy cyclohexane/ethyl acetate = 2/1−1/2, v/v; compound 3r (0.54
g, 0.938 mmol, 52%); colorless solid; mp 128−130 °C. Rf 0.14 (SiO2,
cyclohexane/ethyl acetate = 1/2, v/v). δH (CDCl3) 7.72 (br s, 1H),
7.28−7.44 (m, 17H), 7.19−7.26 (m, 3H), 4.98 (d, 11.5, 1H), 4.73−
4.83 (m, 2H), 4.65−4.73 (m, 3H), 4.53−4.60 (m, 3H), 4.26 (t, 2.3,
1H), 4.10 (dd, 7.0, 3.5, 1H), 4.06 (dd, 9.3, 2.0, 1H), 3.89 (dd, 9.2, 7.4,
1H), 3.69 (dd, 9.3, 7.3, 1H), 2.66 (dd, 6.5, 3.5, 1H), 1.60 (s, 1H),
0.88−1.00 (m, 2H), 0.53−0.64 (m, 1H), 0.42−0.50 (m, 1H). δC
(CDCl3) 164.8, 137.3, 137.2, 136.9, 136.3, 128.8, 128.7, 128.5, 128.5,
128.4, 128.4, 128.1, 128.0, 128.0, 127.9, 127.8, 78.8, 75.4, 74.8, 73.4,
72.3, 71.7, 70.9, 67.4, 55.8, 22.9, 6.6. HRMS (ESI) calcd for
C40H41N2O4 [M + H]+ 577.3061; found 577.3067.
N-4-Cyclohexyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3s).
Reaction: Meerwein’s reagent (0.52 g, 2.720 mmol, 1.5 equiv) and 2
(1.00 g, 1.820 mmol) in 10 mL of dichloromethane; amine
cyclohexylamine (0.36 g, 3.620 mmol, 2.0 equiv); reaction time 4
days; eluent for chromatography cyclohexane/ethyl acetate = 8/1−4/
1, v/v; compound 3s (0.30 g, 0.485 mmol, 27%); colorless oil. Rf 0.20
(SiO2, cyclohexane/ethyl acetate = 1/1, v/v). δH (CD2Cl2) 7.04−7.66
(m, 20H), 6.36 (br s, 1H), 5.02 (d, 11.8, 1H), 4.89 (d, 10.8, 1H), 4.81
(d, 11.5, 1H), 4.62−4.75 (m, 4H), 4.57 (dd, 19.1, 11.8, 2H), 4.34 (t,
2.1, 1H), 4.02 (dd, 9.9, 1.9, 1H), 3.80−3.96 (m, 2H), 3.70 (t, 8.5, 1H),
3.52 (br s, 1H), 1.89 (d, 9.5, 1H), 1.49−1.73 (m, 4H), 1.24−1.48 (m,
2H), 1.05−1.22 (m, 2H), 0.95 (q, 11.0, 1H). δC (CD2Cl2) 160.9,
138.3, 138.3, 137.9, 137.1, 129.6, 129.3, 129.3, 129.1, 128.9, 128.9,
128.7, 128.6, 128.5, 128.5, 128.4, 79.8, 75.8, 75.7, 73.9, 72.9, 72.6, 72.2,
68.2, 57.0, 51.3, 32.1, 31.6, 25.4, 24.4. HRMS (ESI) calcd for
C40H47N2O4 [M + H]+ 619.3536; found 619.3531.
N-n-Dodecyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3o).
Reaction: Meerwein’s reagent (0.54 g, 2.82 mmol, 1.5 equiv) and 2
(1.05 g, 1.90 mmol) in 10 mL of dichloromethane; amine n-
dodecylamine (0.70 g, 3.80 mmol, 2.0 equiv); reaction time 72 h;
eluent for chromatography; cyclohexane/ethyl acetate = 8/1−4/1, v/v,
compound 3o (0.70 g, 0.993 mmol, 52%); colorless oil. Rf 0.27 (SiO2,
cyclohexane/ethyl acetate = 2/1, v/v). δH (CD2Cl2) 7.12−7.59 (m,
20H), 4.86 (td, 19.7, 11.3, 3H), 4.57−4.67 (m, 4H), 4.54 (s, 2H),
4.42−4.42 (m, 1H), 4.43 (d, 9.8, 1H), 4.22−4.31 (m, 1H), 3.92 (dd,
9.8, 1.8, 1H), 3.48−3.73 (m, 3H), 3.01 (dd, 12.5, 6.8, 2H), 1.13−1.48
(m, 20H), 0.84−0.91 (m, 3H). δC (CD2Cl2) 156.3, 139.8, 139.4, 139.0,
138.9, 129.1, 129.0, 128.9, 128.8, 128.7, 128.5, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 82.4, 76.2, 75.3, 74.7, 74.2, 73.8, 72.1, 71.6, 60.1,
41.3, 32.6, 30.3, 30.3, 30.2, 30.2, 30.1, 30.0, 29.8, 27.8, 23.3, 14.6.
9005
dx.doi.org/10.1021/jm501111y | J. Med. Chem. 2014, 57, 8999−9009

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N-Cyclopropylmethyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3t). Reaction: Meerwein’s reagent (0.51 g, 2.717 mmol, 1.5 equiv)
and 2 (1.00 g, 1.82 mmol) in 10 mL of dichloromethane; amine
cyclopropylmethylamine (0.22 g, 3.62 mmol, 2.0 equiv); reaction time
17 h; eluent for chromatography cyclohexane/ethyl acetate = 4/1−1/
1, v/v; compound 3t (0.86 g, 1.692 mmol, 80%); colorless oil. Rf 0.17
(SiO2, cyclohexane/ethyl acetate = 3/2, v/v). δH (CD2Cl2) 7.12−7.55
(m, 20H), 6.76 (br s, 1H), 5.05 (d, 11.5, 2H), 4.90 (d, 10.8, 3H), 4.82
(d, 11.8, 1H), 4.77 (d, 11.5, 1H), 4.72 (dd, 11.5, 9.8, 2H), 4.61 (t, 11.8,
2H), 4.53 (d, 11.5, 1H), 4.32 (t, 2.3, 1H), 4.05 (dd, 10.2, 1.6, 1H),
3.88−3.98 (m, 1H), 3.82 (dd, 8.9, 6.7, 1H), 3.68 (t, 8.5, 1H), 3.13 (dd,
14.1, 7.8, 1H), 2.99 (dd, 13.7, 7.4, 1H), 0.87 (td, 8.0, 4.1, 1H), 0.51−
0.62 (m, 2H), 0.14−0.31 (m, 2H). δC (CD2Cl2) 162.5, 138.2, 138.1,
137.8, 136.9, 129.5, 129.4, 129.3, 129.1, 128.9, 128.9, 128.7, 128.6,
128.5, 128.5, 128.4, 79.8, 75.8, 75.7, 73.9, 72.7, 72.5, 72.1, 68.2, 56.9,
47.4, 9.3, 4.2, 4.0. HRMS (ESI) calcd for C38H43N2O4 [M + H]+
591.3223; found 591.3222.
N-Cyclohexylmethyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3u). Reaction: Meerwein’s reagent (0.25 g, 1.355 mmol, 1.5 equiv)
and 2 (0.50 g, 0.906 mmol, 1 equiv) in 6 mL of dichloromethane;
amine cyclohexylmethylamine (0.21 g, 1.812 mmol, 2.0 equiv);
reaction time 72 h; eluent for chromatography cyclohexane/ethyl
acetate = 4/1−1/1, v/v; compound 3u (0.34 g, 0.538 mmol, 59%);
colorless oil. Rf 0.27 (SiO2, cyclohexane/ethyl acetate = 1/2, v/v). δH
(CD2Cl2) 7.10−7.55 (m, 20H), 6.50 (br s, 1H), 5.04 (d, 11.8, 1H),
4.88 (d, 10.5, 1H), 4.81 (d, 11.5, 1H), 4.67−4.77 (m, 3H), 4.47−4.66
(m, 4H), 4.35 (br s, 1H), 4.03 (dd, 9.9, 1.9, 1H), 3.83−3.98 (m, 2H),
3.72 (t, 8.8, 1H), 3.53 (br s, 1H), 1.89 (d, 10.0, 1H), 1.51−1.73 (m,
4H), 1.38 (sxt, 12.8, 2H), 1.05−1.24 (m, 2H), 0.95 (q, 11.5, 1H); δH
(CD2Cl2) 161.4, 138.3, 138.2, 137.8, 136.9, 129.7, 129.5, 129.4, 129.1,
129.0, 128.9, 128.8, 128.8, 128.6, 128.6, 128.6, 128.5, 128.4, 128.2,
79.6, 76.0, 75.8, 74.0, 72.8, 72.7, 72.0, 67.8, 56.8, 51.4, 32.0, 31.6, 25.3,
24.3. HRMS (ESI) calcd for C41H49N2O4 [M + H]+ 633.3692; found,
633.3674.
N-Cycloheptylmethyl-2,3,4,6-tetra-O-benzyl-D-galactonoamidine
(3v). Reaction: Meerwein’s reagent (0.46 g, 2.446 mmol, 1.5 equiv)
and 2 (0.90 g, 1.630 mmol) in 10 mL of dichloromethane; amine
cycloheptylmethylamine (0.41 g, 3.260 mmol, 2.0 equiv); reaction
time 17 h; eluent for chromatography cyclohexane/ethyl acetate = 4/
1−2/1, v/v; compound 3v (0.63 g, 0.974 mmol, 60%); colorless oil. Rf
0.20 (SiO2, cyclohexane/ethyl acetate = 3/2, v/v). δH 7.10−7.68 (m,
20H), 6.33 (br s, 1H), 5.00 (d, 11.8, 1H), 4.90 (d, 10.8, 1H), 4.79−
4.85 (m, 1H), 4.74 (d, 11.5, 1H), 4.66−4.71 (m, 2H), 4.63 (d, 10.8,
1H), 4.59 (d, 11.8, 1H), 4.54 (d, 11.5, 1H), 4.33 (t, 1.8, 1H), 4.02 (dd,
9.8, 1.8, 2H), 3.86−3.94 (m, 1H), 3.82 (dd, 8.8, 6.3, 1H), 3.68 (t, 8.5,
1H), 3.06 (dd, 12.8, 6.8, 1H), 2.97 (dd, 13.1, 6.3, 1H), 1.28−1.71 (m,
13H), 1.01−1.19 (m, 2H). δC 161.6, 138.5, 138.4, 138.0, 137.3, 129.4,
129.3, 129.2, 129.1, 129.0, 129.0, 128.9, 128.9, 128.6, 128.5, 128.4,
128.3, 80.4, 75.6, 73.9, 73.2, 72.6, 72.5, 68.9, 57.5, 48.6, 38.7, 32.3,
32.2, 28.7, 26.6, 26.5. HRMS (ESI) calcd for C42H51N2O4 [M + H]+
647.3849; found 647.3846
General Description for Synthesis of N-Aryl- and N-Alkyl-D-
galactonoamidines (1h−v) from N-Aryl- and N-Alkyl-2,3,4,6-tetra-
O-benzyl-D-galactonoamidine (3h−v). Compounds 3h−v were
suspended with Pd/C (10% Pd and 50% wet with water) and 0.1−6
mL of trifluoroacetic acid in 5−30 mL of ethanol and stirred under
hydrogen atmosphere at room temperature. After 8−24 h, the mixture
was filtered through a pad of Celite. The pad was rinsed with 2−3 mL
of ethanol three times. The combined filtrates were concentrated
under reduced pressure, and the resulting residues were lyophilized,
yielding compounds 1h−v in excellent yields.
HRMS (ESI) calcd for C17H27N2O4 [M + H]+ 323.1965; found
323.1971.
N-4-Methoxybenzyl-D-galactonoamidine (1i). Suspension: com-
pound 3i (0.32 g, 0.488 mmol), Pd/C (0.64 g, 0.301 mmol, 0.617
equiv) in 1.5 mL of trifluoroacetic acid and 7.5 mL of ethanol; reaction
time 24 h; ethanol amount for rinsing 3 mL each; compound 1i (0.14
g, 0.473 mmol, 92%), pale yellow foam. Rf 0.12 (SiO2, ethyl acetate/
methanol = 3/1, v/v). δH (D2O) 7.21 (d, 8.8, 2H), 6.88 (d, 8.8, 2H),
4.54 (d, 10.3, 1H), 4.46 (d, 3.5, 2H), 4.20 (t, 2.3, 1H), 3.88 (dd, 10.0,
2.3, 1H). δC (D2O/MeOH-d4) 164.9, 159.8, 130.0, 127.1, 115.4, 71.5,
67.9, 67.4, 61.0, 58.3, 56.3, 45.7. HRMS (ESI) calcd for C14H21N2O5
[M + H]+ 297.1445; found 297.1441.
N-4-Trifluoromethylbenzyl-D-galactonoamidine (1j). Suspension:
compound 3j (0.24 g, 0.346 mmol), Pd/C (0.48 g, 0.226 mmol, 0.653
equiv) in 2 mL of trifluoroacetic acid and 10 mL of ethanol reaction
time 20 h; ethanol amount for rinsing 2 mL each; compound 1j (0.10
g, 0.299 mmol, 87%); colorless oil. Rf 0.27 (SiO2, ethyl acetate/
methanol = 3/1, v/v). δH (D2O) 7.66 (d, 8.0, 2H), 7.42 (d, 8.0, 2H),
4.67 (d, 2.5, 2H), 4.62 (d, 10.0, 1H), 4.25 (t, 2.3, 1H), 3.94 (dd, 10.2,
2.4, 1H), 3.69−3.77 (m, 2H), 3.65 (dd, 3.3, 9.5, 1H). δC (D2O/
MeOH-d4) 165.6, 139.0 (q, 1.5), 130.7 (q, 32.3), 128.5, 126.9 (q, 3.7),
125.0 (q, 271.0), 71.5, 68.0, 67.5, 60.9, 58.4, 45.6. HRMS (ESI) calcd
for C14H17F3N2O4 [M + H]+ 335.1213; found 335.1211.
N-Benzyl-D-galactonoamidine (1a) from N-4-Chlorobenzyl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3k). Suspension: com-
pound 3k (0.16 g, 0.242 mmol), Pd/C (0.16 g, 0.0751 mmol, 0.310
equiv) in 1 mL of trifluoroacetic acid and 5 mL of ethanol; reaction
time 9 h; ethanol amount for rinsing 2 mL each; compound 1a (0.06 g,
0.223 mmol, 92%); colorless oil. Rf 0.21 (SiO2, ethyl acetate/methanol
= 3/1, v/v). δH (D2O) 7.44 (d, 8.3, 7H), 7.25 (d, 8.0, 2H), 4.49−4.63
(m, 3H), 4.24 (t, 1.8, 1H), 3.92 (dd, 10.0, 2.3z, 1H), 3.60−3.78 (m,
3H). δC (D2O + MeOH-d4) 165.2, 134.7, 130.1, 129.4, 128.3, 71.5,
67.9, 67.4, 60.9, 58.3, 46.2.
N-Benzyl-D-galactonoamidine (1a) from N-4-Bromobenzyl-
2,3,4,6-tetra-O-benzyl-D-galactonoamidine (3l). Suspension: com-
pound 3l (0.10 g, 0.142 mmol), Pd/C (0.10 g, 0.0469 mmol, 0.330
equiv) in 0.5 mL of trifluoroacetic acid and 5 mL of ethanol; reaction
time 8 h; ethanol amount for rinsing 2 mL each; compound 1a (37.70
mg, 0.142 mmol, quantitative); colorless foam. Rf 0.21 (SiO2, ethyl
acetate/methanol = 3/1, v/v). δH (D2O) 7.44 (d, 8.3, 7H), 7.25 (d,
8.0, 2H), 4.49−4.63 (m, 3H), 4.24 (t, 1.8, 1H), 3.92 (dd, 10.0, 2.3z,
1H), 3.60−3.78 (m, 3H). δC (D2O + MeOH-d4) 165.2, 134.7, 130.1,
129.4, 128.3, 71.5, 67.9, 67.4, 60.9, 58.3, 46.2.
N-Propyl-D-galactonoamidine (1m). Suspension: compound 3m
(0.32 g, 0.554 mmol), Pd/C (0.64 g, 0.301 mmol, 0.543 equiv) in 4
mL of trifluoroacetic acid and 20 mL of ethanol; reaction time 24 h;
ethanol amount for rinsing 3 mL each; compound 1m (0.119 g, 0.505
mmol, 92%) as a pale white foam. Rf 0.25 (SiO2, ethyl acetate/
methanol = 2/1, v/v). δH (D2O) 4.48 (d, 10.0, 1H), 4.23 (t, 2.5, 1H),
3.87 (dd, 10.2, 2.4, 1H), 3.74−3.79 (m, 2H), 3.67−3.73 (m, 1H), 3.24
(t, 7.0, 2H), 1.55 (sxt, 7.3, 2H), 0.84 (t, 7.5, 3H). δC (D2O/MeOH-d4)
164.8, 71.6, 68.1, 67.3, 61.0, 58.1, 44.4, 21.4, 11.3. HRMS (ESI) calcd
for C9H19N2O4 [M + H]+ 219.1339; found 219.1338.
N-n-Heptyl-D-galactonoamidine (1n). Suspension: compound 3n
(0.45 g, 0.709 mmol), Pd/C (0.45 g, 0.211 mmol, 0.298 equiv) in 0.2
mL of trifluoroacetic acid and 10 mL of ethanol; reaction time 19 h,
ethanol amount for rinsing 2 mL each; compound 1n (0.19 g, 0.693
mmol, 97%); pale white foam. Rf 0.25 (SiO2, ethyl acetate/methanol =
4/1, v/v). δH (D2O) 4.53 (d, 10.0, 1H), 4.28 (t, 2.3, 1H), 3.92 (dd,
10.2, 2.1, 1H), 3.69−3.86 (m, 3H), 3.33 (t, 6.9, 2H), 1.60 (quin, 6.8,
2H), 1.14−1.37 (m, 8H), 0.75−0.87 (m, 3H). δC (D2O + MeOH-d4)
164.8, 71.5, 68.0, 67.2, 61.0, 58.1, 42.8, 32.1, 29.2, 27.9, 27.0, 23.0,
14.4. HRMS (ESI) calcd for C13H27N2O4 [M + H]+ 275.1971; found
275.1968.
N-4-tert-Butylbenzyl-D-galactonoamidine (1h). Suspension: com-
pound 3h (0.27 g, 0.395 mmol), Pd/C (0.27 g, 0.127 mmol, 0.322
equiv) in 0.75 mL of trifluoroacetic acid and 7.5 mL of ethanol;
reaction time 24 h; ethanol amount for rinsing 3 mL each; compound
1h (0.12 g, 0.373 mmol, 95%), pale gray foam. Rf 0.19 (SiO2, ethyl
acetate/methanol = 3/1, v/v). δH (D2O) 7.47 (d, 8.5, 2H), 7.26 (d,
8.3, 2H), 4.62−4.50 (m, 3H), 4.24 (t, 2.3, 1H), 3.92 (dd, 10.2, 2.4,
1H), 3.77−3.62 (m, 3H), 1.23 (s, 9H). δC (D2O + MeOH-d4) 165.1,
153.0, 131.9, 128.3, 127.1, 71.5, 67.9, 67.4, 61.0, 58.3, 45.7, 35.0, 31.4.
N-n-Dodecyl-D-galactonoamidine (1o). Suspension: compound
3o (0.36 g, 0.511 mmol), Pd/C (0.36 g, 0.169 mmol, 0.330 equiv) in
0.8 mL of trifluoroacetic acid and 8 mL of ethanol; reaction time 21 h;
ethanol for rinsing 2 mL each; compound 1o (0.17 g, 0.494 mmol,
97%); pale white solid; mp 69−70 °C. Rf 0.14 (SiO2, ethyl acetate/
methanol = 3/1, v/v). δH (acetone-d6) 9.36 (br s, 1H), 8.32 (br s, 1H),
9006
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5.05 (br s, 1H), 4.63 (d, 9.0, 1H), 4.31 (br s, 1H), 3.76−4.01 (m, 4H),
3.49 (dd, 11.8, 6.3, 2H), 1.73 (quin, 7.3, 2H), 1.15−1.49 (m, 20H),
0.87 (t, 6.8, 3H). δC (acetone-d6) 166.8, 72.9, 69.6, 68.7, 62.2, 59.5,
43.3, 33.1, 30.8, 30.8, 30.8, 30.5, 30.4, 28.9, 27.9, 23.8, 14.8. HRMS
(ESI) calcd for C18H37N2O4 [M + H]+ 345.2748; found 345.2752.
N-2-Ethylhexyl-D-galactonoamidine (1p). A suspension of com-
pound 3p (0.17 g, 0.262 mmol), Pd/C (0.17 g, 0.0798 mol, 0.305
equiv) in 0.2 mL of trifluoroacetic acid and 6 mL of ethanol; reaction
time 24 h; ethanol for rinsing 2 mL each; compound 1p (0.07 g, 0.244
mmol, 93%); colorless foam. Rf 0.21 (SiO2, ethyl acetate/methanol =
4/1, v/v). δH (D2O) 4.54 (d, 10.0, 1H), 4.27 (s, 1H), 3.93 (dd, 10.0,
1.8, 1H), 3.72−3.87 (m, 3H), 3.25 (d, 4.3, 2H), 1.68 (quin, 5.5, 1H),
1.12−1.42 (m, 9H), 0.82 (t, 7.0, 6H). δC (D2O + MeOH-d4) 165.0,
71.6, 68.1, 67.4, 61.2, 58.2, 46.3, 38.3, 38.3, 30.8, 30.7, 28.8, 24.3, 23.2,
14.3, 10.7, 10.7. HRMS (ESI) calcd for C14H29N2O4 [M + H]+
289.2127; found 289.2125.
N-3-Phenylpropyl-D-galactonoamidine (1q). Suspension: com-
pound 3q (0.21 g, 0.320 mmol), Pd/C (0.21 g, 0.0986 mmol, 0.308
equiv) in 0.1 mL of trifluoroacetic acid and 5 mL of ethanol; reaction
time 24 h; ethanol for rinsing 2 mL each; compound 1q (0.09g, 0.320
mmol, quantitative); colorless foam. Rf 0.17 (SiO2, ethyl acetate/
methanol = 3/1, v/v). δH (D2O) 7.15−7.38 (m, 5H), 4.42 (d, 10.3,
1H), 4.24 (t, 2.5, 1H), 3.86 (dd, 10.3, 2.5, 1H), 3.72−3.81 (m, 2H),
3.65−3.71 (m, 1H), 3.32 (t, 7.0, 2H), 2.67 (t, 7.5, 2H), 1.95 (quin, 7.3,
2H). δC (D2O + MeOH-d4) 164.7, 142.0, 129.6, 129.4, 127.2, 71.5,
68.0, 67.2, 61.0, 58.1, 42.1, 32.9, 29.1. HRMS (ESI) calcd for
C15H23N2O4 [M + H]+ 295.1652; found 295.1650.
N-Cyclopropyl-D-galactonoamidine (1r). Suspension: compound
3r (0.28 g, 0.486 mmol), Pd/C (0.12 g, 0.132 mmol, 0.272 equiv) in
0.5 mL of trifluoroacetic acid in 5 mL of ethanol; reaction time 24 h;
ethanol for rinsing 2 mL each; compound 1r (0.11 g, 0.486 mmol,
quantitative); pale gray foam. Rf 0.12 (SiO2, ethyl acetate/methanol =
3/1, v/v). δH (D2O) 4.50 (d, 10.0, 1H), 4.27 (s, 1H), 3.92 (dd, 10.2,
2.1, 1H), 3.77−3.86 (m, 3H), 2.53−2.66 (m, 1H), 0.85−1.02 (m, 2H),
0.63−0.82 (m, 2H). δH (D2O + MeOH-d4) 167.0, 71.5, 68.0, 67.0,
61.0, 58.1, 23.3, 7.2, 6.7. HRMS (ESI) calcd for C9H17N2O4 [M + H]+
217.1188; found 217.1179.
N-Cyclohexyl-D-galactonoamidine (1s). Suspension: compound 3s
(0.25 g, 0.404 mmol), Pd/C (0.25 g, 0.117 mmol, 0.290 equiv) in 0.5
mL of trifluoroacetic acid and 5 mL of ethanol; reaction time 17 h;
ethanol for rinsing 2 mL; compound 1s (0.10 g, 0.387 mmol, 96%);
pale gray foam. Rf 0.14 (SiO2, ethyl acetate/methanol = 3/1, v/v). δH
(D2O) 4.49 (d, 10.0, 1H), 4.26 (br s, 1H), 3.90 (dd, 10.2, 1.9, 1H),
3.69−3.85 (m, 3H), 3.50−3.68 (m, 1H), 1.80−1.93 (m, 2H), 1.72 (d,
4.8, 2H), 1.58 (d, 12.5, 1H), 1.21−1.46 (m, 4H), 1.03−1.20 (m, 1H).
δH (D2O + MeOH-d4) 163.4, 71.6, 68.1, 67.3, 61.2, 58.2, 52.3, 32.0,
31.5, 25.4, 25.0, 25.0. HRMS (ESI) calcd for C12H23N2O4 [M + H]+
259.1652; found 259.1650.
N-Cycloheptylmethyl-D-galactonoamidine (1v). A suspension of
compound 3v (0.47 g, 0.726 mmol), Pd/C (0.47 g, 0.221 mmol, 0.304
equiv) in 0.2 mL of trifluoroacetic acid and 10 mL of ethanol; reaction
time 24 h; ethanol for rinsing 2 mL each; compound 1v (0.21 g, 0.726
mol, quantitative); pale white foam. Rf 0.20 (SiO2, ethyl acetate/
methanol = 4/1, v/v). δH (D2O) 4.53 (d, 10.3, 1H), 4.26 (br s, 1H),
3.91 (dd, 10.2, 2.4, 1H), 3.67−3.85 (m, 3H), 3.17 (dd, 7.2, 2.9, 2H),
1.74−1.93 (m, 1H), 1.54−1.71 (m, 4H), 1.31−1.53 (m, 6H), 1.15 (qd,
10.3, 2.0, 2H). δC (D2O + acetone-d6) 164.4, 71.1, 67.5, 66.8, 60.6,
57.6, 48.2, 37.8, 31.7, 31.7, 28.3, 25.9, 25.9. HRMS (ESI) calcd for
C14H27N2O4 [M + H]+ 287.1971; found 287.1963.
D-Galactonoamidine (1w).27 A suspension of perbenzylated
galactonoamidine (0.18 g, 0.336 mmol), Pd/C (0.18 g, 0.0845
mmol, 0.251 equiv), 0.12 mL of trifluoroacetic acid, and 6 mL of
ethanol; reaction time 12 h; ethanol for rinsing 2 mL; compound 1w
(0.06 g, 0.336 mol, quantitative); pale yellow foam. Rf 0.27 (SiO2, ethyl
acetate/methanol = 4/1, v/v). δH (D2O) 4.55 (d, 10.3, 1H), 4.24 (t,
2.3, 1H), 3.96 (dd, 10.2, 2.4, 1H), 3.79 (dd, 9.8, 4.3, 1H), 3.64−3.76
(m, 2H). δC (D2O + MeOH-d4) 176.9, 168.4, 80.9, 74.6, 73.8, 71.6,
69.8, 68.2, 67.0, 62.9, 61.3, 57.9; the compound matches the
characterization data described by Ganem et al. and consists of two
resonance isomers.27
D-Galactono-δ-lactam (1x).30 The title compound was prepared
from perbenzylated galactonolactam (2.50 g, 4.655 mmol) and
Pd(OH)2/C (0.48 g, 0.343 mmol, 0.137 equiv) in a solvent mix of
methanol, ethanol, and 1 M aqueous HCl (67.5 mL, 27.0 mL, 45 μL)
as described by Overkleeft et al.,30 yielding compound 1x (0.16 g,
0.090 mmol, 20%) as a pale gray solid; mp 190−192 (lit.30 mp 192−
195 °C). δH (D2O) 4.11−4.19 (m, 2H), 3.88 (dd, 10.0, 2.3, 1H),
3.67−3.76 (m, 1H), 3.55−3.65 (m, 2H). δC (D2O + MeOH-d4) 174.5,
73.2, 70.2, 68.8, 61.8, 55.8; the compound matches the character-
ization data described.30
Kinetic Assays. General Remarks. All kinetic experiments were
performed in 50 mM acetate buffer at pH 5.00 0.05 at 30 °C with β-
galactosidase (A. oryzae) as catalyst and 2-chloro-4-nitrophenyl-β-D-
galactopyranoside (5) as substrate in a 96-well plate format and a total
volume of 100 μL. All experiments were performed at least in
triplicate, and obtained data were averaged.
Enzyme Stock Solution. In a typical experiment, 4−5 mg of
commercially available β-galactosidase (A. oryzae) was dissolved in 5
mL of acetate buffer in a volumetric flask. Then 250.0 μL of this
solution was diluted into 5.0 mL of acetate buffer in a similar fashion
to obtain the enzyme stock solution. For each experiment, 20 μL of
this stock solution was used to initiate hydrolysis immediately prior to
data recording. Using a BCA assay, the protein concentration of the
corresponding protein batch was determined as 10%.18 The molar
weight of β-galactosidase in this enzyme batch was previously
determined as 86 800 g mol−1.18
N-Cyclopropylmethyl-D-galactonoamidine (1t). Suspension: com-
pound 3t (0.45 g, 0.763 mmol), Pd/C (0.45 g, 0.211 mmol, 0.277
equiv) in 0.2 mL of trifluoroacetic acid and 10 mL of ethanol; reaction
time 24 h; ethanol for rinsing 2 mL each; compound 1t (0.17 g, 0.760
mmol, quantitative); pale gray foam. Rf 0.21 (SiO2, ethyl acetate/
methanol = 3/1, v/v). δH (D2O) 4.55 (d, 10.3, 1H), 4.28 (s, 1H), 3.93
(dd, 10.0, 2.3, 1H), 3.71−3.86 (m, 3H), 3.20 (d, 5.8, 2H), 0.99−1.13
(m, 1H), 0.53−0.60 (m, 2H), 0.25 (ddd, 9.5, 4.8, 1.5, 2H). δC (D2O +
MeOH-d4) 164.7, 71.6, 68.1, 67.3, 61.1, 58.1, 47.5, 9.2, 4.0, 3.8. HRMS
(ESI) calcd for C10H19N2O4 [M + H]+ 231.1345; found 231.1343.
N-Cyclohexylmethyl-D-galactonoamidine (1u). Suspension: com-
pound 3u (0.47 g, 0.741 mmol), Pd/C (0.94 g, 0.442 mmol, 0.596
equiv) in 6 mL of trifluoroacetic acid and 30 mL of ethanol; reaction
time 24 h; ethanol for rinsing 2 mL each; compound 1u (0.26 g, 0.698
mol, 93%); white foam. Rf 0.30 (SiO2, ethyl acetate/methanol = 3/1,
v/v). δH (D2O) 4.44 (d, 10.0, 1H), 4.18 (t, 2.1, 1H), 3.83 (dd, 10.0,
2.3, 1H), 3.69−3.75 (m, 2H), 3.62−3.68 (m, 1H), 3.01−3.16 (m, 2H),
1.42−1.65 (m, 6H), 0.93−1.18 (m, 3H), 0.72−0.90 (m, 2H). δC
(D2O/MeOH-d4) 164.9, 71.5, 68.0, 67.3, 61.1, 58.1, 48.6, 37.0, 30.9,
30.9, 26.6, 26.1, 26.1. HRMS (ESI) calcd for C13H25N2O4 [M + H]+
273.1814; found 273.1804.
Substrate Stock Solution. In a typical experiment, 5.0 mg (0.015
mmol) of 2-chloro-4-nitrophenyl-β-D-galactopyranoside was dissolved
in 5.0 mL of acetate buffer in a volumetric flask immediately prior to
use. For determination of the substrate decomposition in the absence
of catalyst, 5.0 mg of substrate was dissolved in 10 mL of acetate buffer
under otherwise identical conditions.
Inhibitor Stock Solution. In a typical experiment, 50 μM stock
solutions of the inhibitors 1a−v in nanopure water were diluted into
0.25−10.0 μM stock solutions using volumetric flasks. For each
experiment with inhibitors, 10 μL of the respective stock solution was
used.
Assay To Determine Uncatalyzed Substrate Decomposition. The
substrate stock solution was pipetted in 0−70 μL aliquots and adjusted
to a total volume of 100 μL with buffer solution, thoroughly mixed and
thermostated at 30 °C for 60 min prior to data recording. Formation
of 2-chloro-4-nitrophenolate was followed at 405 nm over 2 h, and the
change in absorbances was recorded every 6 min. Product
concentration was derived from these values by correction for the
apparent molar extinction coefficient (εapp × d = 1532 M−1) that was
previously determined from purified 2-chloro-4-nitrophenol by a
calibration curve.29 The product concentration was then plotted over
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time and the resulting data fitted linearly to derive the rate constant of
the uncatalyzed reaction from the slope.
ASSOCIATED CONTENT
■
S
* Supporting Information
Assay To Determine Uninhibited Enzyme Activity. The substrate
stock solution was pipetted in 0−70 μL aliquots and adjusted to a total
volume of 80 μL with buffer solution, thermostated at 30 °C for 30
min prior to addition of a 20 μL aliquot of enzyme stock solution,
thorough mixing, and data recording. Formation of 2-chloro-4-
nitrophenolate was followed at 405 nm for 5−15 min, and the change
in absorbances was recorded every 27 s. Product formation in
dependence of time was derived from these values by correction for
the apparent molar extinction coefficient (εapp × d =1532 M−1).
A plot of the initial rates corrected for the enzyme concentration
over substrate concentration resulted in hyperbolic data that were
analyzed by the Michaelis−Menten model as described previously to
derive the rate constant (kcat) and the Michaelis−Menten constant
(KM) for noninhibited enzyme activity.18,29
Assay To Determine Inhibition of Enzyme Activity. Substrate stock
solution was pipetted in 0−70 μL aliquots, 10 μL of inhibitor stock
solution was added for each inhibitor stock solution, and the total
volume of the solutions was then adjusted to 80 μL with buffer
solution. Resulting solutions were thermostated at 30 °C for 30 min
prior to addition of 20 μL of enzyme stock solution, thorough mixing,
and data recording. Formation of 2-chloro-4-nitrophenolate was
followed at 405 nm for 5−15 min, and the change in absorbances
was recorded every 27 s as described for the assay in the absence of
inhibitor.
Analytical data for intermediates 3h−v and final galactonoami-
dines 1h−x; HPLC traces of 1a−x. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 479-575-5079. Fax: 479-575-4049. E-mail: Susanne.
striegler@uark.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
An undergraduate research fellowship of the University of
Arkansas Honors College to K.A.C. and support by the
National Science Foundation (CHE-1305543) to S.S. are
gratefully acknowledged. The facilities used in this study were
supported by the National Institute of General Medical
Sciences (NIGMS) of the National Institutes of Health
(NIH) by grant no. P30 GM103450.
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Inhibitor Efficacy. General Remarks. All experiments were
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0.05 at 30 °C
with β-galactosidase (A. oryzae) as catalyst and 2-chloro-4-nitrophenyl-
β-D-galactopyranoside (5) as substrate in a 96-well plate using a total
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after 60 min. All experiments were conducted in duplicate, and
obtained data were averaged.
Data Analysis. Recorded absorbances were transformed into
percentage using the blank experiments of each plate as reference
and then plotted against inhibitor concentrations in a logarithmic scale.
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