Binuclear copper(II) complexes discriminating epimeric glycosides and α- And β-glycosidic bonds in aqueous solution

Article abstract of DOI:10.1016/j.jcat.2015.12.026

Two chiral binuclear copper(II) complexes were synthesized and characterized for the first time as efficient chemoselective catalysts for the hydrolysis of aryl glycosides and disaccharides in aqueous solution at near neutral pH. Under these conditions, discrimination of epimeric aryl α-glycopyranosides was observed by both 29-fold different reaction rates and 3-fold different proficiency of the catalyst. Additionally, large differentiation of the nature of α- and β-glycosidic bond in aryl glycosides as model compounds is apparent, but also noted in selected disaccharides. The influence of the chirality of the complexes and the role of the configuration of the carbohydrate upon interaction with the catalyst is discussed in detail. Lastly, a putative mechanism for the metal complex-catalyzed hydrolysis is derived from the experimental evidence pointing at deprotonation of the hydroxyl group at C-2 as a pre-requisite for glycoside hydrolysis.

Full text of DOI:10.1016/j.jcat.2015.12.026

Journal of Catalysis 338 (2016) 349–364
Contents lists available at ScienceDirect
Journal of Catalysis
journal homepage: www.elsevier.com/locate/jcat
Binuclear copper(II) complexes discriminating epimeric glycosides
and a- and b-glycosidic bonds in aqueous solution
a
b
Susanne Striegler a, , Qiu-Hua Fan , Nigam P. Rath
⇑
a Department of Chemistry and Biochemistry, University of Arkansas, 345 N Campus Drive, Fayetteville, AR 72701, USA
b Department of Chemistry and Biochemistry and Center for Nanoscience, University of Missouri–St. Louis, St. Louis, MO 63121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two chiral binuclear copper(II) complexes were synthesized and characterized for the first time as effi-
cient chemoselective catalysts for the hydrolysis of aryl glycosides and disaccharides in aqueous solution
Received 31 October 2015
Revised 16 December 2015
Accepted 30 December 2015
at near neutral pH. Under these conditions, discrimination of epimeric aryl
observed by both 29-fold different reaction rates and 3-fold different proficiency of the catalyst.
Additionally, large differentiation of the nature of - and b-glycosidic bond in aryl glycosides as model
a-glycopyranosides was
a
compounds is apparent, but also noted in selected disaccharides. The influence of the chirality of the
complexes and the role of the configuration of the carbohydrate upon interaction with the catalyst is dis-
cussed in detail. Lastly, a putative mechanism for the metal complex-catalyzed hydrolysis is derived from
the experimental evidence pointing at deprotonation of the hydroxyl group at C-2 as a pre-requisite for
glycoside hydrolysis.
Keywords:
Catalysis
Glycosides
Selective hydrolysis
Binuclear copper(II) complexes
Chiral
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
synthesis, characterization and evaluation of chiral binuclear
copper(II) complexes that discriminate epimeric glycosides and
Chiral discrimination is displayed by many enzyme classes,
such as glycosidases, lipases, and esterases during bond formation
and cleavage [1]. While much progress has been made in the syn-
thesis and evaluation of selective catalysts mimicking such fea-
tures [2–6], most synthetic entities do not show sufficient
diastereoselective or even chiral discrimination ability in aqueous
solution. Man-made stereoselective catalysts for the discrimina-
tion of epimeric glycosides in aqueous solution or the selective
a- and b-glycosidic bonds upon hydrolysis in aqueous solution at
pH 7.5 for the first time (see Chart 1).
2. Results and discussion
2.1. Synthesis of the chiral binuclear copper(II) complexes
Symmetric complex Cu2bpdpo (1) was derived from
a
hydrolysis of a- and b-glycosidic bonds are not known yet.
reduced Schiff-base ligand bpdpo that was obtained from
1,3-diaminopropanol and pyridinecarbaldehyde as described [7].
The same strategy was employed to prepare chiral complexes
S-Cu2bpdbo (S-2) and R-Cu2bpdbo (R-2) by using enantiopure
S- and R-malic acid 3 as inexpensive starting material for the
synthesis of chiral 1,4-diaminobutanols (4) (see Scheme 1).
In short, the chiral acids 3 were converted into methyl malates
5 using methanol in the presence of acetyl bromide [13–15]. Treat-
ment of esters 5 with excess ammonia in methanol yielded mala-
mides 6 [16]. The hydrochlorides of S-4 and R-4 were obtained
after reduction of 6 with borane in THF and treatment of the reac-
tion products with hydrogen chloride in absolute ethanol [17]. In
analogy to the synthesis of bpdpo described above [18], condensa-
tion of the free diaminoalcohols with pyridinecarbaldehyde
afforded the chiral ligands S-bpdbo (S-7), and R-bpdbo (R-7),
We previously disclosed a symmetric binuclear copper(II) com-
plex, Cu2bpdpo (1), that interacts differently with glycopyrano-
sides upon binding in alkaline solution resulting in a 30-fold
stronger binding to mannose over glucose [7]. Similar observations
were made by others thereafter using related metal complexes
confirming our early results on carbohydrate-metal complex bind-
ing in alkaline aqueous solution [7–12].
These combined results imply that binuclear copper(II) com-
plexes may also be able to discriminate epimeric glycosides during
hydrolysis enabling chemoselective catalysis. We report here the
Abbreviations: HEPES, N-(2-hydroxyethyl)piperazine-N0-2-ethanesulfonic acid;
TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid.
⇑
Corresponding author. Fax: +1 479 575 4049.
E-mail address: Susanne.striegler@uark.edu (S. Striegler).
http://dx.doi.org/10.1016/j.jcat.2015.12.026
0021-9517/Ó 2016 Elsevier Inc. All rights reserved.

350
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
Chart 2. Structures of (A) S-2 and (B) R-2 in the solid state; atoms are shown as
ellipsoids with 60% probability; carbon (gray), oxygen (red), nitrogen (blue), copper
(magenta); H-atoms, disordered C-atoms, and perchlorate ions are omitted for
clarity.
coordinating methanol in S-2, the perchlorate ion coordinated with
Cu1, and the methylene C-chain C7–C10 are disordered in both
complexes, yet the anticipated absolute configurations are
confirmed by the solid-state data (Flack X = 0.021(5) and 0.020(3)
for S and R respectively). As S-2 and R-2 crystallize in the same
space group (P212121), the structures have opposite coordinates.
Absolute structure determination was carried out using Parson’s
method [19].
The Cuꢀ ꢀ ꢀCu distance was deduced from the structural data as
3.368(1) Å on average. The intermetallic Cu(II) distances in the chi-
ral complexes are shorter than in 1 (3.499(1) Å) confirming previ-
ous observations for a comparison between 2 and 1 [18]. The
average Cu1–O1–Cu2 angle (121.7°) is smaller in the chiral com-
plexes than in 1 (132.9°) due to distortion in the ligand backbone
caused by the additional methylene group. A full description and
all details of the crystal structure determination are given in the
supporting information.
Chart 1. Binuclear copper(II) complexes 1 and 2.
respectively, after reduction of the initially formed Schiff bases
with sodium borohydride in methanol. Enantiopure binuclear cop-
per(II) complexes S-2 and R-2 were prepared from the pentaden-
tate ligands 7 and copper(II) acetate in methanol following our
synthetic protocols for the synthesis of complex 2 [18]. Caution:
Perchlorate salts are potentially explosive. However, we did not expe-
rience any difficulty in handling or drying the complexes below 50 °C.
2.2. Characterization of the chiral complexes in the solid state
Blue needles suitable for X-ray crystallography were obtained
by diffusion of hexane into solutions of S-2, or R-2, in methanol.
X-ray diffraction studies of the single crystals revealed bond angles
and bond lengths for the central Cu1–O–Cu2 geometry similar to
those of previously studied racemic 2 [18](see Chart 2). The
2.3. Characterization of the binuclear complexes in solution
To evaluate the complexes for their ability to discriminate gly-
cosidic bonds, two pH values for kinetic evaluations were selected
reflecting different complex compositions. The composition of chi-
ral S-2 and R-2 in aqueous solution is identical to the speciation of
racemic complex 2 and deduced from data previously determined
using spectrophotometric titration methods [20,21]. At pH 10.5,
3+
the predominant species of S-2 and R-2 is still a [Cu2L-H
]
species
(57.5%), while only one other additional [Cu2L-H (OH)]2+ species
(42.5%) is formed (L = S- and R-bpdbo, respectively) (Scheme 2).
By contrast, complex 1 forms under these conditions [Cu2L-H
(OH)2]+ as the main species (88.5%) in equilibrium with a minor
[Cu2L-H(OH)]2+ species (11.5%) as previously discussed [7,22].
Mononuclear complexes formed from remaining ligand or free
metal ions can be neglected for catalysis under the applied condi-
tions or were demonstrated to be inactive [18]. At pH 7.5, com-
3+
plexes S-2 and R-2 exist predominantly as a binuclear [Cu2L-H
]
species (94%, L = S- and R-bpdbo, respectively), while complex 1
exists as [Cu2L-H(OH)]2+ species (98.6%, L = bpdpo) (Scheme 3).
To allow a comparison between all complexes and species
derived therefrom during the hydrolysis of glycosidic bonds, the
catalyst amounts used for the determination of kinetic parameters
are corrected to reflect the different amounts of the respective
catalytically active species.
Scheme 1. Synthesis of chiral copper(II) complexes S-2 and R-2; reagents and
conditions: (i) AcBr, MeOH, 0 °C ? r.t., 18 h, 59%; (ii) 7N NH3/MeOH, r.t., 24 h, 43–
48%; (iii) BH3/THF, HCl/EtOH, 77 °C, 1 h, 29–74%; (iv) NaOH, r.t., 5 h; C5H4NCHO, r.t.
22 h; NaBH4, r.t., 48 h, 59–83%; (v) Cu(OAc)2, MeOH, r.t., 12 h, 25–32%.

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
351
Scheme 2. Predominant species of complex 1 and S-2 at pH 10.5; R-2 likewise; X = H2O.
2.4. Differentiation of 4-nitrophenyl glycosides during catalytic
hydrolysis in alkaline solution
Based on previously established assays using UV/Vis spec-
troscopy to evaluate the catalytic activity of complexes 1 and 2
during glycoside hydrolysis [18], we employed chiral catalysts
S-2 and R-2, extended the substrate scope by employing six
commercially available 4-nitrophenylglycosides, and transferred
the previous assay from 1 mL standard cuvettes into 96-well plate
format. The adjusted procedure allows considerably faster catalyst
screening using smaller compound amounts and volumes, and
thereby circumvents previously observed limitations caused by
low substrate solubility.
Along these lines, the catalytic hydrolysis of 4-nitrophenyl-
mannopyranoside (8a), 4-nitrophenyl-b-D-mannopyranoside (8b),
4-nitrophenyl- -galactopyranoside (8c), 4-nitrophenyl-b-
galactopyranoside (8d), 4-nitrophenyl- -D-glucopyranoside (8e),
a-D-
a-
D
D-
Scheme 3. Predominant species of complex 1 and S-2 at pH 7.5; R-2 likewise;
X = H2O.
a
and 4-nitrophenyl-b-D-glucopyranoside (8f) (Chart 3) was studied
by UV/Vis spectroscopy recording the product formation at 405 nm
over time (Scheme 4).
(knon = 0.4–2.2 ꢂ 10ꢁ7 minꢁ1 Mꢁ1) [18]. For comparison of different
substrates, only the proficiency (kcat/(KM ꢂ knon)) of the catalysts is
discussed to account for the different strengths of glycosidic bonds
and the resulting different hydrolysis rates in the absence and
presence of catalysts.
The adjusted assay has a total volume of 200 lL and lowered
the catalyst concentration from 0.1 mM to 0.03 mM, while the sub-
strates were used between 6 and 10 mM. The substrate hydrolysis
depends linearly on the catalyst concentration under these condi-
tions [18]. The measured absorbance was converted into concen-
The catalytic proficiency of the symmetric complex 1 under the
employed conditions is very modest and shows only limited
tration using the apparent extinction coefficient eapp, corrected
differentiation of substrates with
a-glycosidic bond (8a, 8c and
for the catalyst concentration, its relative speciation amount, and
the uncatalyzed reaction, and then plotted versus the substrate
concentration. By applying a nonlinear fit to the resulting hyper-
bolic data, the catalytic rate constant kcat (minꢁ1) and the substrate
affinity KM (mM) were determined utilizing the Michaelis–Menten
model (Table 1).
8e) from substrates with b-glycosidic bond (8b, 8d and 8f) that
are typically hydrolyzed even less efficiently, if at all (Fig. 1A).
The substrates are not significantly discriminated by complex 1
for their epimeric sugar moiety reflecting previously stated
observations [18].
By contrast, both chiral complexes are more proficient for cleav-
ing the glycosides 8a–f than 1 (Fig. 1B). In addition, S-2 and R-2
The uncatalyzed reactions (knon
in the same order of magnitude as previously determined
) of all substrates remain
OH
OH
OH
O
HO
HO
OH
O
O
HO
HO
HO
HO
OH
OH
O
O
O
8e
OH
8a
8c
R
R
R
R =
OH
HO OH
OH
NO2
O
O
O
HO
HO
HO
HO
O
HO
O
O
R
R
R
OH
OH
8f
8b
8d
Chart 3. Structures of substrates 8a–f.

352
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
Table 1
Kinetic parameters for the hydrolysis of 4-nitrophenylglycosides 8a–f at pH 10.5 and 30 °C.
Entry
S
Cat
kcat ꢂ 10ꢁ3 (minꢁ1
)
KM (mM)
kcat/KM ꢂ 10ꢁ3 (minꢁ1 Mꢁ1
)
kcat/knon ꢂ 103 (M)
kcat/(KM ꢂ knon
)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
8a
1
3.09
6.32
4.42
0.30
0.76
0.64
0.65
1.07
1.15
0.12
0.28
0.26
0.27
0.83
0.81
0.15
0.81
1.17
107.5
107.7
57.7
21.0
26.6
23.9
70.5
42.1
18.7
12.1
10.2
16.8
7.5
28.7
58.7
76.6
14.3
28.6
26.7
9.2
25.4
61.7
9.9
27.5
15.5
35.8
91.5
129.0
2.8
20.7
42.4
29.7
1.5
3.9
3.3
18.2
29.9
32.1
0.9
2.1
1.9
2.5
7.8
7.6
0.3
1.4
20.0
193,000
394,000
514,000
74,000
147,000
138,000
258,000
710,000
1,720,000
74,000
206,000
116,000
335,000
857,000
1,210,000
13,000
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
S-2
R-2
8b
8c
8d
8e
8f
9.1
6.3
53.2
72.2
42.8
11.2
27.3
51,000
124,000
knon,8a = 1.5 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
,
.
knon,8b = 1.9 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
,
knon,8c = 0.4 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
,
knon,8d = 1.3 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
;
knon,8e = 1.1 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
,
knon,8f = 2.2 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
(A)
(B)
2.0x106
1.5x106
1.0x106
2.0x106
1.5x106
1.0x106
Cu2bpdpo
S-Cu2bpdbo
R-Cu2bpdbo
8a
8b
8c
8d
8e
8f
5.0x105
0.0
5.0x105
0.0
α
β
α
β
α
β
catalysts
4-nitrophenylglycoside
Fig. 1. Catalytic proficiency of binuclear complexes 1, S-2 and R-2 in aqueous solution at pH 10.5 during the hydrolysis of 4-nitrophenylglycosides; the data are grouped by
(A) catalysts promoting the hydrolysis of glycosides as 8a–f, and (B) glycosides 8a–f hydrolyzed by catalysts 1, S-2 and R-2.
show higher proficiency than 1 to hydrolyze
a
-over b-glycosidic
Unfortunately, the commercially available 4-nitrophenyl glycopy-
ranosides have a low molar extinction coefficient under these con-
ditions that hamper their use as model compounds at pH values
below 9 [18]. As a consequence, rapid catalyst screening in 96-
well plate format using such substrates is limited and results in
high uncertainty for the evaluation of the catalyst performance
due to the resulting small apparent absorbance changes, small
extinction coefficients, and large errors of the associated data. To
overcome this obstacle, two approaches come to mind: the use
of derivatized nitrophenyl glycosides with large extinction coeffi-
cients suitable to follow hydrolysis reactions at near physiological
pH by UV/Vis spectroscopy, and/or the use of ‘real’ saccharides
instead of phenylglycoside model compounds after further
modification of the current assay. Toward the development of
functional enzyme models, both approaches were followed and
the results are summarized below.
bonds. Complex R-2 hydrolyzes 8c with a 6.7-fold, and 8e with a
3.6-fold higher proficiency than 1, and shows a 1.3-fold, and
2.4-fold, respectively, higher proficiency for the hydrolysis of the
same substrates than S-2. Interestingly, while R-2 shows the
overall highest proficiency for hydrolyzing 4-nitrophenyl-
a
-
D
-galactopyranoside (8c) (kcat/KM ꢂ knon = 1,720,000), the same
complex shows an almost 10-fold higher proficiency for hydrolyzing
the b-glucosidic bond in 8f (R-2: kcat/KM ꢂ knon = 124,000) than 1
(1: kcat/KM ꢂ knon = 12,800).
While the results encourage further investigation of binuclear
Cu(II) complexes for their ability to discriminate glycosidic bonds
in natural systems, including disaccharides, initial attempts to
use these substrates under the above described assay conditions
were futile and led to catalyst destruction. Visibly to the naked
eye, the originally blue solutions will turn green and then orange
within 1–2 h indicating the formation of Cu(I) oxide without evi-
dence for a significant hydrolysis of the disaccharide at 30 or
40 °C. Decreasing the pH of the solution to pH 7 or 8 was found
to increase catalyst stability, but resulted in catalyst inactivation
below 40 °C (see below). Selective oxidation of the primary hydro-
xyl group at C-6 in methyl glycosides was previously observed for
complex 1 after activation with TEMPO in alkaline solution [23].
Following the hydrolysis of glycosides and natural saccharides
at near physiological pH appears more relevant for the synthesis
of functional enzyme models and modeling of enzyme activity.
2.5. Synthesis of 20-chloro-40-nitrophenyl glycosides
As noted above, the commercially available 4-nitrophenyl gly-
copyranosides 8 hydrolyze into saccharide and 4-nitrophenolate
(9) that both have fairly low molar extinction coefficients at pH
values below 9 [18]. However, a significantly higher apparent
extinction coefficient of 2-chloro-4-nitrophenolate (10) at near
neutral pH was noted in this and other laboratories prompting us

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
353
Chart 4. Structures of substrates 11a–i.
OH
OH
O
O
cat
HO
HO
HO
HO
−
R =
R
+
O
CAPS, pH 10.5
NO2
OH
OH
OH
OR
Scheme 4. Catalytic hydrolysis of 8e as representative example for the hydrolysis of substrates 8a–f.
to synthesize a small library of 20-chloro-40-nitrophenylglycosides
(11a–i) as substrates for this study (Chart 4) [24–30].
The aryl a-glycopyranosides 11a, 11c and 11e were obtained in
two steps after treatment of peracetylated glycosides with
2-chloro-4-nitrophenol in the presence of Lewis acids yielding per-
acetylated aryl a-glycopyranosides 12a, 12c, and 12e, and global
deacetylation [26,27,31,32]. The aryl b-D-glycopyranosides 11b,
11d and 11f were prepared via peracetylated glucosyl bromides
(13) as described [26–28].
glucoside (18). The reaction of 18 with trifluoromethanesulfonic
anhydride afforded triflyl b-glucoside (19) that was imme-
diately treated with tetrabutylammonium acetate to give
b-allopyranoside (20) as a result of the inversion of the configura-
tion at C-3 in the glycon [36]. Treatment of 20 with diluted acetic
acid afforded aryl b-allopyranoside (21), and the target compound
11i after global deprotection.
2.6. Discrimination of 20-chloro-40-nitrophenyl glycopyranosides
during catalytic hydrolysis at physiological pH
For mechanistic studies, we synthesized aryl 2-O-methyl-a-
galactopyranoside (11g), and its b-isomer 11h from galactoside
(14) (Scheme 5), which was obtained as an intermediate during
an unrelated study [33]. Here, galactoside 14 was peracetylated
with acetic anhydride in pyridine yielding galactoside 15 [34],
and treated with 2-chloro-4-nitrophenol in the presence of N-
With substrates 11a–i on hand, their hydrolysis was initially
studied under the same conditions as outlined above for the
hydrolysis of p-nitrophenyl glycopyranosides, i.e. at alkaline pH.
While the catalytic rate constants are of similar order of magni-
tude, the uncatalyzed hydrolyses of 20-chloro-40-nitrophenyl gly-
copyranosides (e.g. knon = 0.5–3.3 ꢂ 10ꢁ4 minꢁ1 Mꢁ1, 50 mM CAPS
buffer, pH 10.5, 30 °C) are considerably faster than the uncatalyzed
hydrolysis reactions of p-nitrophenyl glycopyranosides (Table 1)
rendering all complexes less efficient as catalysts and the differ-
ences in catalyst proficiency smaller. This observation accounts
for the decreased stability of the glycosidic bond after introduction
of the chloro-substituent in the ortho position of the aglycon. How-
ever, the decreased stability of the glycosidic bond in the 20-chloro-
40-nitrophenyl glycopyranosides does allow the investigation of
the catalyst performance at near neutral pH values as hypothesized
above.
iodosuccinimide and silver triflate to afford aryl
a-galactoside
12g. By contrast, the transformation of 15 with iodine monobro-
mide yielded bromide (16), which then allowed the coupling with
2-chloro-4-nitrophenol affording aryl b-galactoside 12h via phase
transfer catalysis as described for other b-glycosides [27]. The
target compounds 11h and 11g were finally obtained after global
deacetylation [35].
Lastly, allopyranoside 11i was obtained from substrate 11f as
readily accessible starting material (Scheme 6). Initially, the free
hydroxyl groups at C-4 and C-6 were protected as benzyl acetal
yielding glucoside (17). After treatment of 17 with dibutyltin oxide,
selective benzoylation of the hydroxyl group at C-2 yielded aryl
Scheme 5. Synthesis of aryl a- and b-galactoside 11g and 11h from 14; conditions and reagents: (i) Ac2O, C6H5N, r.t, 16 h, 95%; (ii) 2-chloro-4-nitrophenol, CH2Cl2, N-
iodosuccinimide, AgOTf, 0 °C, 30 min, 53%; (iii) IBr, CH2Cl2, 0 °C, 10 min, 81%; (iv) 2-chloro-4-nitrophenol, NaOH, CH2Cl2, Bu4NBr, 35 °C, 3 h, 80%; (v) 7 N NH3/MeOH, r.t., 24 h,
79–87%.

354
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
Scheme 6. Synthesis of peracetylated 2-chloro-4-nitrophenyl b-D-allopyranoside (11i); conditions and reagents: (i) benzaldehyde dimethyl acetal, CSA, CH3CN, r.t, 2 h, 87%;
(ii) Bu2SnO, MeOH, reflux, 1.5 h; benzoyl chloride, C6H5CH3, 0 °C ? r.t., 16 h; (iii) Tf2O, CH2Cl2, C6H5N, 0 °C, 30 min, quantitative; (iv) TBAꢀOAc, DMF, 0 °C, 30 min, 91%; (v)
HOAc/H2O (4/1, v/v), 50 °C, 15 h, 97%; (vi) 7N NH3/MeOH, r.t., 16 h, 83%.
0.0006
Table 2
Kinetic parameters for the catalytic hydrolysis of 11a–f, i at pH 7.5 and 30 °C.
11a
Entry
S
Cat
kcat ꢂ 10ꢁ3 (minꢁ1
)
KM (mM)
kcat/(KM ꢂ knon
)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
11a
1
1.51
1.74
1.98
0.42
0.33
0.26
0.37
0.28
0.71
5.49
4.55
3.28
0.07
0.06
0.06
1.10
1.67
1.26
0.53
0.49
0.24
25.7
30.0
38.3
25.6
22.8
17.3
15.6
8.8
31.6
73.0
58.4
43.2
4.5
2.1
2.2
30.7
47.7
45.0
4.0
43,000
42,000
38,000
62,000
55,000
57,000
51,000
68,000
48,000
35,400
37,000
36,000
66,000
121,000
118,000
50,000
49,000
39,000
60,000
47,000
58,000
0.0004
0.0002
0.0000
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
S-2
R-2
1
11b
11c
11d
11e
11f
11i
11c
11e
0.000
0.005
0.010
S [M]
Fig. 2. Product formation over time, catalyst and substrate concentration for the
hydrolysis of 11a, 11c, and 11e by S-2 in 50 mM HEPES buffer at 7.50 0.05 and
30.0 0.1 °C.
the glycon of the substrates during metal complex-catalyzed
hydrolysis. Substrates with hydroxyl groups trans to each other
(11c, 11e) promote slower hydrolysis than a substrate with hydro-
xyl groups cis to each other (11c). This observation correlates with
known metal complex coordination abilities to trans-diols (weak)
and cis-diols (strong) in pyranosides [37]. To account for uncat-
alyzed background reactions and different substrate affinity
toward a complex, the catalyst proficiency (kcat/KM ꢂ knon) was cal-
culated (Fig. 3) [38].
S-2
R-2
4.8
1.9
knon,11a = 1.3 ꢂ 10ꢁ6 minꢁ1 Mꢁ1
;
knon,11b = 2.6 ꢂ 10ꢁ7 minꢁ1 Mꢁ1
;
knon,11c = 6.7
ꢂ 10ꢁ7 minꢁ1 Mꢁ1
;
knon,11d = 2.1 ꢂ 10ꢁ6 minꢁ1 Mꢁ1
;
knon,11e = 2.4 ꢂ 10ꢁ7 minꢁ1
Mꢁ1, knon,11f = 7.1 ꢂ 10ꢁ7 minꢁ1 Mꢁ1; knon,11i = 2.2 ꢂ 10ꢁ6 minꢁ1 Mꢁ. 1
Along these lines, the proficiency of the selected complexes
toward glycoside hydrolysis was evaluated in 50 mM HEPES buffer
at pH 7.50 and 30 °C. Typically, lag times around 200 min were
observed prior to the start of the catalytic hydrolyses that may
indicate substrate deprotonation, distortion or inversion of the gly-
con upon interaction with the catalyst. Data collection over 9–12 h
allowed the determination of all kinetic parameters after conver-
sion of the observed absorbance data into product concentrations
and application of the Michaelis–Menten model (Table 2).
The known lability of the
uncatalyzed hydrolysis (knon) to be about an order of magnitude
a-glycosidic bond in 11a causes its
The following discussion of the obtained data highlights the
most predominant discrimination trends by focusing on the perfor-
100,000
50,000
0
mance of catalyst S-2 for (i) the hydrolysis of epimeric
in comparison with symmetric 1, (ii) the hydrolysis of
a
-glycosides
- and b-
a
glycosides in comparison with R-2, and (iii) deducing a mechanism
for the observed glycoside hydrolysis.
2.6.1. Performance of S-2 during hydrolysis of epimeric a-
glycopyranosides
Catalyst S-2 discriminates epimeric a-glycosides 11a, 11c and
11a
11c
11e
11e by catalyzing their hydrolysis with significantly different rates
(Fig. 2). The rate for the catalyzed hydrolysis of 11a is more than
6-fold higher than for 11c and 29-fold higher than for 11e pointing
at a significant influence of the hydroxyl groups at C-2 and C-3 in
substrates
Fig. 3. Proficiency of S-2 for the catalytic hydrolysis of
11e in 50 mM HEPES buffer at pH 7.50 0.05 and 30.0 0.1 °C.
a
-glycosides 11a, 11c, and

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
355
faster than for 11c and 11e. Thus, the catalytic proficiency of S-2
decreases in the order of 11e > 11c > 11a showing nevertheless
for the first time a distinct discrimination of epimeric glycosides
by a metal complex at near neutral pH. For comparison, symmetric
complex 1 shows an overall lower proficiency to hydrolyze the epi-
meric substrates with negligible differences (1.5-fold or less).
2.6.4. Putative mechanism of the a-glycoside hydrolysis
The high catalytic proficiency of S-2 for the hydrolysis of
glucopyranosides 11e–f over those of 11a–d indicates again
different interactions of the catalyst with the glycon of the
substrates. As the epimeric substrates are overall only different
in their configuration at C-2 and C-4, respectively, we proposed
that the higher acidity of the hydroxyl group at C-2 over that of
the hydroxyl group at C-4 and its proximity to the anomeric center
promote substrate deprotonation and coordination with the metal
complex as pre-requisite for catalytic hydrolysis to occur. The coor-
dination is consequently stronger when a deprotonated cis-diol
structure (11a) is participating in metal complex chelation and
weaker when a trans-diol structure (11c or 11e) is present
accounting for above described observations confirming previous
observations by others [37].
For experimental evidence, the catalytic hydrolysis of sub-
strates 11g and 11h was evaluated (Chart 4). Both substrates are
methylated in the glycon at the hydroxyl group at C-2 preventing
deprotonation at this position upon interaction with the catalyst,
while weaker hydrogen-bonding interactions are still enabled.
The hydrolysis of 11g and 11h is unsuccessful with any studied
catalyst both at pH 7.5 and in alkaline solution at pH 10.5 indicat-
ing that all substrates indeed coordinate with the catalysts over a
deprotonated hydroxyl group at C-2. A putative mechanism for
2.6.2. Performance of S-2 during hydrolysis of
a- and b-
glycopyranosides
The rate of the S-2 catalyzed hydrolysis of
a
-mannoside 11a is
about 5-fold higher than for b-mannoside 11b, while the reverse
trend is observed for gluco- and galactosides where the
b-glycosides are hydrolyzed 16-fold (11d) and 28-fold (11f) faster
than their corresponding
11; 14 & 17). As the uncatalyzed hydrolyses of the manno- and
galactosides differ by an order of magnitude within each -/b-
pyranoside pair (Table 2, footnote), the catalytic proficiency of
the catalyst for the hydrolysis of the respective and
b-glycosides does not reflect the discrimination of its ability, but
reveals rather equivalent catalytic proficiency near 50,000
a-glycosides (Table 2, entries 2 & 5; 8 &
a
a
-
a
instead. Substrate 11i behaves likewise.
By contrast, the uncatalyzed reactions of the
a- and b-
glucosides 11e and 11f are of the same order of magnitude and
the S-2-catalyzed hydrolyses differ by almost 1.5 orders of magni-
tude or 28-fold (Fig. 4; Table 2, entries 14 & 17). This observation
translates into a 3-fold higher proficiency of S-2 to hydrolyze 11e
over 11f due to higher substrate affinity of 11f over 11e for S-2,
and demonstrates for the first time the ability of a metal complex
to discriminate a- and b-glycosidic bonds notably close to physio-
logical pH. This finding is of particular significance for catalyst
development, use of biomass or its transformation into fine chem-
the hydrolysis of
a-glycoside by S-2 at pH 7.5 is deduced from
the described experimental observations, and depicted for the
hydrolysis of 11a (Scheme 7).
Upon dissolving complexes S-2 and R-2 in solution, a binuclear
species I is formed that coordinates with the glycoside substrate
under release of water and protons resulting in half-acetal forma-
tion (Scheme 7, species II). Distortion of the configuration may
then yield a structure similar to the transition state proposed for
enzymatic glycoside hydrolyses encompassing substrate distortion
icals and fuel due to the abundance of
a- and b-glucopyranosyl
moieties as building blocks in natural products and oligosaccha-
rides including cellulose and starch.
to
a
half-chair conformation, sp2-character of the anomeric
C-atom, partially positively charged endocyclic O-atom and length-
ening of the glycosidic bond (Scheme 7, species III). Models suggest
a twisted boat-like structure for a similar species derived from
b-glycosides requiring further computational analyses in future
efforts. Hydration of the binuclear copper species and protonation
of the sugar by solvent molecules may release the coordinated
hydrolysis products to reform species I and close the catalytic
cycle.
2.6.3. Performance of R-2 during hydrolysis of glycopyranosides
As elaborated previously, the overall catalyst performance
correlates with the intramolecular Cuꢀ ꢀ ꢀCu distance in the metal
complex core [18]. As similar rates of the catalyzed substrate
hydrolyses are observed for S-2 and R-2 (Table 2), the chirality
of the complexes is, however, unlikely to have
a profound
contribution to the observed discrimination of the epimeric
or anomeric model compounds by S-2 and is echoed by R-2.
Instead, the configuration of the hydroxyl groups in the
glycon of the substrate presents itself as a rationale for the
observed glycoside discrimination. For simplicity, the following
discussion on the mechanistic insights is consequently limited to
complex S-2.
2.7. Discrimination of disaccharides during catalytic hydrolysis
In order to apply the catalysts to natural carbohydrates, all
complexes were explored in their ability to hydrolyze the
disaccharides maltose (22), cellobiose (23), and lactose (24)
(Fig. 5a). Disaccharides 22 and 23 differ in the nature of their gly-
cosidic bonds, and 23 and 24 are epimers in their non-reducing
sugar moiety. All disaccharides were hydrolyzed at 60 °C at pH 8
over 24 h in the presence and absence of 10 mol% of complex 1,
S-2, and R-2, respectively. The amount of remaining starting mate-
rial was then quantified by HPLC analysis (Fig. 5b) [39]. Evidence
for catalyst destruction or sugar oxidation was not apparent under
the elaborated conditions. The composition of the complexes is
similar to that at pH 7.5 with slightly different amounts of the
major catalytically active species.
3.0x10-4
11f
2.0x10-4
1.0x10-4
The data reveal indifference of 1 to hydrolyze the selected dis-
accharides in any significantly higher amount than buffer solution
11e
alone. By contrast, S-2 hydrolyzed twice as much of a-glycoside 22
0.0
0.000
as buffer, 1, or R-2. Likewise, R-2 hydrolyzed up to 2.5-fold more of
b-glycosides 23 and 24 than buffer solution or another catalyst.
The finding indicates stereoselective discrimination of the glyco-
sidic bonds in disaccharides by the chiral catalysts with a prefer-
0.005
0.010
S [M]
Fig. 4. Product formation during the hydrolysis of 11e and 11f by S-2 in 50 mM
HEPES buffer at pH 7.50 0.05 and 30.0 0.1 °C.
ence of S-2 for
a-glycosidic bonds and of R-2 for b-glycosidic

356
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
Scheme 7. Putative mechanism for the metal complex-catalyzed hydrolysis of a-glycopyranosides at pH 7.5 and 30 °C using S-2 and 11a as representative example.
bonds. This preliminary finding is promising for future applications
of the developed catalysts and prompted an in-depth study that is
topic of current investigation.
3. Conclusions
In conclusions, chiral binuclear copper(II) complexes S-2 and
R-2 were synthesized and fully characterized including analysis
by X-ray diffraction to confirm their stereochemistry. Subsequent
evaluations of the complexes as catalysts for the cleavage of
glycosidic bonds in aqueous alkaline solution showed moderate
proficiency during the hydrolysis of 4-nitrophenyl glycosides and
small discrimination ability among the selected epimeric
substrates.
However, at near physiological pH, complex S-2 shows distinct
discrimination of epimeric aryl
of a- and b-glycosidic bonds in manno- and galactopyranosides by
a-glycopyranosides. Discrimination
Fig. 5a. Disaccharide structures.
the same complex is apparent in their reaction rates, but masked in
the catalytic proficiency by the different rates of the uncatalyzed
reaction. By contrast, a 28-fold faster hydrolysis of aryl b- over
50
40
30
20
10
0
catalysts:
buffer
a-glucopyranoside is noted translating into 3-fold higher profi-
Cu2bpdpo, 1
ciency of S-2 for the hydrolysis of b-glucopyranoside, while the
uncatalyzed reactions are of the same order of magnitude for both
substrates. The discrimination is not related to the chirality of the
complexes, but rather due to the configuration of the glycosides
promoting cis- or trans-configured diol binding sites for catalyst
coordination. Mechanistic studies reveal deprotonation of the
hydroxyl group at C-2 as pre-requisite for catalysis.
S-Cu2bpdbo, S-2
R-Cu2bpdbo, R-2
An initial catalyst evaluation toward the hydrolysis of represen-
tative disaccharides revealed a preference of S-2 for the cleavage of
a-glycosidic bonds, and of R-2 for the hydrolysis of b-glycosidic
bonds. The study identifies for the first time catalysts that are able
to discriminate epimeric and anomeric model glycosides promot-
ing a stereoselective hydrolysis of glycosidic bonds in disaccha-
rides in aqueous solution at near neutral pH. The results point at
exciting possibilities for further catalyst development that may
mal
cel
lac
disaccharide
Fig. 5b. Catalyzed disaccharide hydrolysis.

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
357
include biomass transformation into valuable chemical synthons
and fuels, applications in pharmaceutical industry, or the develop-
ment of functional enzyme mimics.
resistance of 18.2 m
X
was obtained from a ThermoScientific Barn-
stead E-pureTM water purification system.
4.2. Chemicals
4. Experimental section
Pyridincarboxaldehyde was distilled in vacuum prior to use;
p-tolyl 2-O-methyl-1-thio-b-
2-chloro-4-nitrophenyl b-D-mannopyranoside (11b) [28], 2-
chloro-4-nitrophenyl b-
-galactopyranoside (11d) [26], N,N0-
[1,4-bis[(pyridin-2-ylmethyl)amino]propan-2-ol]ato dicopper(II)
-acetato) diperchlorate, Cu2bpdpo (1) [7], and (S)-methyl malate,
D-galactopyranoside (14) [33],
4.1. Instrumentation
1H and 13C NMR spectra were recorded on a 400 MHz Bruker
spectrometer (Bruker Biospin) with Z gradient and broadband
probe (400.2 MHz for 1H, and 100.6 MHz for 13C). Chemical shifts
(d) in 1H NMR are expressed in parts per million (ppm) and cou-
pling constants (J) in Hz. Signal multiplicities are denoted as s (sin-
glet), d (doublet), t (triplet), q (quartet) and m (multiplet). CDCl3,
CD2Cl2, MeOH-d4, DMSO-d6 and D2O were used as solvents, and
chemical shift values are reported relative to the residual signals
of these solvents (CDCl3: dH = 7.29, dC = 77.0; (CD3)2SO: dH = 2.50,
dC = 39.5; D2O: dH = 4.80, dC = 49.0 for 13C after addition of a few
drops of CD3OD or dC = 39.5 for 13C after addition of a few drops
of DMSO-d6). IR spectra were obtained on a Perkin-Elmer spectrum
100 series FT-IR spectrophotometer with a resolution 0.5 cmꢁ1 as
D
(l
(S-5) [40–42] were prepared as described; all other chemicals were
used as received from commercial suppliers.
4.3. Metal complex syntheses
The intermediates and copper(II) complexes were prepared fol-
lowing protocols by this laboratory for the synthesis of racemic
complex Cu2bpdbo (2) [18].
thin films on KBr plates or as KBr pellets (m
in cmꢁ1). The software
4.3.1. (R)-methyl malate (R-5) [43,44]
used to record the spectrum is PerkinElmer Spectrum Express ver-
sion 1.01.00. High resolution mass spectrometry data were
obtained in the state-wide mass spectrometry facility at Arkansas
University 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. Elemental analyses were obtained
from Atlantic Microlab, Atlanta, GA. X-ray diffraction data were
collected in the X-ray diffraction laboratory of the University of
Missouri at St. Louis on a Bruker X8 diffractometer.
The hydrolysis of saccharides was monitored on an HPLC sys-
tem from Shimadzu equipped with SCL-10Avp system controller,
2 LC-20AD analytical pumps, DGU-20A3R three channel online
degassers, SIL-20A UFLC autosampler with 96 well capability,
CTO-20A/prominence column oven and ELSD-90LT light scattering
and LC solution software, version 1.25 from Shimadzu for data
recording and analysis. Lyophilization was performed on a Free-
Zone 1 liter benchtop freeze dry system from Labconco. Melting
points (uncorrected) were recorded on a Mel-Temp melting point
apparatus. Apparent optical rotations were measured at 589 nm
on an Autopol III polarimeter from Rudolph Research Analytical
at ambient temperature in a 1 mL cell glass center fill stainless
steel jacketed cell with an optical path length l of 100 mm. The
Acetyl bromide (2.2 mL, 0.031 mol) was added dropwise to
20 mL cold methanol. The solution was stirred for 30 min in ice.
Then R-malic acid, R-3, (12.5 g, 93.28 mmol) was added. The acid
dissolved in about 5 min, and the resulting solution was stirred
at ambient temperature. After 18 h, sodium bicarbonate (4.00 g,
0.048 mol) was added to the pale yellow solution. After 15 min
of stirring, the mixture was filtered, the filtrate collected, and all
volatile compounds evaporated in vacuum, yielding a sticky, oily
raw material containing a white precipitate. The desired ester
was distilled from this mixture in vacuum yielding 8.88 g of R-5
(54.77 mmol, 59%) of a colorless liquid; bp 65–80 °C (0.9 torr);
½
a 2D3:5
ꢃ
¼ þ9:7 (c 0.288, EtOH) (lit. [44] ½a D25
¼ þ9:6 (c 2.3, EtOH);
ꢃ
dH (CDCl3) 4.51 (dd, 6.0, 4.3, 1H), 3.81 (s, 3H), 3.71 (s, 3H), 3.28
(br. s, 1H), 2.87 (dd, 16.6, 4.5, 1H), 2.79 (dd, 16.3, 6.0, 1H); dC
(CDCl3) 173.6, 170.9, 67.1, 52.6, 51.9, 38.3; m
max cmꢁ1 (thin film
on KBr) 3470 (br), 2958, 1737 (s), 1442, 1220. The NMR, IR and
optical rotation data agree well with literature data [43,44].
4.3.2. (S)-malamide (S-6) [16,45,46]
(S)-methyl malate S-5 (6.20 g, 38.27 mmol) was dissolved in
45 mL of 7 N ammonia in methanol under inert atmosphere and
stirred at ambient temperature. After 24 h, the formed precipitate
was separated by filtration and washed with methanol. The raw
material was recrystallized from methanol and dried in vacuum
yielding 2.20 g of S-6 (16.54 mmol, 43%) as a colorless solid;
154–155 °C (lit. [16] 157–158 °C; lit. [46] 156–158 °C);
specific optical rotation was calculated as ½a DT
ꢃ
¼
a
ꢂ 100=c ꢂ l,
where the value for the concentration c is expressed in g/100 mL
solvent and l is 1 dm; the values are not temperature-corrected.
UV/Vis absorbances in 96 well, non-binding microlon Elisa-plates
½
a 2D2
ꢃ
¼ ꢁ36:6 (c 0.333, H2O) (lit. [16] ½a D23
¼ ꢁ34:8 (c 1.5, H2O);
ꢃ
from Greiner Bio-one were recorded on
a FilterMax F5
lit. [46] ½a 1D8
ꢃ
=-36.7 (c 3.1, H2O)); dH (D2O) 4.45 (dd, 8.5, 4.0, 1H),
Multi-Mode Microplate Reader from Molecular Devices at
405 nm and 30 0.1 °C.
2.72 (dd, 15.3, 4.0, 1H), 2.57 (dd, 15.3, 8.8, 1H); dC (D2O + DMSO-
d6) 179.9, 176.7, 69.7, 41.0;
m
max/cmꢁ1 (KBr) 3388, 1677, 1440.
Column chromatography was carried out using silica gel 60
from SilicycleÒ (40–63
lm, 230–240 mesh) or basic aluminum
The NMR, IR and optical rotation data agree well with literature
data [16,46].
oxide (pH range 9.7 0.4, activity I), 32–63 lm, surface area
150 m2 gꢁ1 from Sorbent Technologies as stationary phase. Thin
layer chromatography (TLC) was performed using silica gel TLC
4.3.3. (R)-malamide (R-6) [47–49]
plates from SORBENT Technologies, 200
l
m, 4 ꢂ 8 cm, aluminum
The title compound was prepared from 6.84 g (42.22 mmol) of
R-methyl malate, R-5, as described for S-malamide from S-5, yield-
ing 2.68 g (20.15 mmol, 48%) of R-6 as a colorless solid; 154–
backed, with fluorescence indicator F254 and detection by UV light
or by charring with an aqueous vanillin-sulfuric acid reagent and
subsequent heating of the TLC plate. The pH values were measured
156 °C; ½a 2D2
¼ þ33:7 (c 0.309, H2O); dH (D2O) 4.44 (dd, 8.7, 3.9,
ꢃ
using a Beckman
U 250 pH meter equipped with refillable ROSS
Orion combination pH electrode with a 165 mm long epoxy body,
a 95 mm long semi-micro tip and an 8 mm diameter. The pH meter
was calibrated before each set of readings. Nanopure water at a
1H), 2.72 (dd, 15.3, 4.0, 1H), 2.56 (dd, 15.3, 8.8, 1H); dC (D2O
+ DMSO-d6) 180.0, 176.7, 69.7, 41.1;
1440.
m
max cmꢁ1 (KBr) 3388, 1677,

358
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
4.3.4. (S)-1,4-diamino-2-butanol hydrochloride S-4 [48,49]
(m, 1H), 7.18–7.08 (m, 1H), 3.95–3.78 (m, 5H), 3.50–3.26 (m, 3H),
2.96–2.87 (m, 1H), 2.84–2.74 (m, 1H), 2.65 (dd, 5.0, 11.8, 1H), 2.57
(dd, 7.8, 11.8, 1H), 1.66–1.55 (m, 2H); dC (CD2Cl2) 160.7, 159.9,
149.7, 149.6, 136.9, 136.8, 122.7, 122.5, 122.4, 122.3, 71.3, 56.1,
Under an inert atmosphere, 100 mL of 1 M borane in THF was
added to ice-cooled S-6 (1.50 g, 11.28 mmol), and the resulting
solution was heated to 77 °C. After 11 h, the solution was cooled
in an ice bath, and 40 mL of methanol was added in small portions
to control the gas development. The solution was then heated to
reflux for 1 h. After cooling, all volatile components were removed
by rotary evaporation leaving the crude material as yellowish oil.
The oil was dried in vacuum yielding a gummy-like off-white
solid that was triturated with 200 mL water-free ethanol and fil-
tered. The filtrate was subjected to gaseous HCl in the cold yielding
a precipitate. The precipitate was isolated, washed once with 2 mL
ice-cold ethanol and dried in vacuum over drierite yielding 0.575 g
of S-4 (3.248 mmol, 29%); mp 233–240 °C (lit. [49] 249–254 °C); dH
(D2O) 3.94 (tdd, 9.5, 6.5, 3.3, 1H), 3.02–3.20 (m, 3H), 2.92 (dd, 13.1,
9.8, 1H), 1.82–1.93 (m, 1H), 1.70–1.82 (m, 1H); dC (D2O + MeOH-d4)
66.0, 44.6, 36.8, 31.5.
55.5, 55.2, 48.0, 34.3;
1597, 1476, 1117; HR ESI-MS calcd for
m
max cmꢁ1 (KBr) 3304, 2927, 2843, 1593,
C
16H23N4O (M+H)+
287.1866; found 287.1860.
4.3.5.3. 2S, N,N0-[1,4-bis[(pyridin-2-ylmethyl)amino]butan-2-ol]ato
dicopper(II) ( -acetato) diperchlorate, (S)-Cu2bpdbo, S-2. Copper(II)
l
acetate monohydrate (2.00 g, 10.00 mmol) was dissolved in
20 mL water and 400 mL methanol at ambient temperature. To
the greenish-blue solution, 4.5 mL of the 1 M stock solution of S-
7
in ethanol was added followed by a solution of 4.00 g
(32.68 mmol) of sodium perchlorate in 10 mL water and 40 mL
ethanol. The resulting dark blue solution was stirred for 12 h, fil-
tered and concentrated below 50 °C to about 40 mL. Caution: Per-
chlorate salts are potentially explosive. However, we did not
experience any difficulty in handling or drying the complex below
45 °C. Upon standing at ambient temperature, a precipitate formed
that was isolated by filtration and dried at ambient temperature in
air yielding 2.85 g (4.155 mmol) of a blue raw material. The raw
material was recrystallized from aqueous methanol yielding
4.3.5. (R)-1,4-diamino-2-butanol hydrochloride, R-4 [49]
The title compound was obtained as colorless solid in 74% yield
(5.96 g, 33.67 mmol) from 6.0 g (44.8 mmol) (R)-malamide R-6 as
described above for the synthesis of S-4 from S-6; 244–246 °C
(lit. [49] 232–236 °C); dH (D2O) 3.96 (tt, 3.3, 9.5, 1H), 3.21–3.03
(m, 3H), 2.92 (dd, 9.5, 13.1, 1H), 1.88–1.88 (m, 1H), 1.98–1.84 (m,
1H), 1.83–1.68 (m, 1H); dC (D2O + MeOH-d4) 66.6, 45.4, 37.6, 32.3.
ꢁ1
~
2.19 g (3.193 mmol, 32%) of S-2 as a blue solid;
m cm 1611,
1569, 1085; calcd for C18H26Cl2Cu2N4O12 [Cu2L-H(OAc)(ClO4)2 + H2-
O, L = S-7] C 31.40, H 3.81, N 8.14; found: C 31.66, H 3.66, N 8.07;
4.3.5.1. 2S, N,N0-bis(2-pyridylmethyl)-1,4-diaminobutan-2-ol, S-
bpdbo, S-7. Sodium hydroxide (1.53 g, 38.25 mmol) was added to
a solution of S-4 (1.50 g, 8.475 mmol) in 80 mL methanol at
ambient temperature. The initially turbid solution became clear
and after 5 min turbid again. After 5 h, 2.27 g (21.19 mmol) of dis-
tilled 2-pyridinecarbaldehyde was added. After additional 22 h, the
solution was diluted with 80 mL methanol prior to the addition of
3.04 g (0.080 mol) of sodium borohydride. After further 48 h, all
volatile material was removed in vacuum to yield a residue that
was taken up in 50 mL chloroform and 15 mL ice water. The
organic layer was separated and extracted two times with 15 mL
of ice water each. The combined organic layer was dried over
sodium sulfate, filtered and concentrated to dryness yielding the
raw product as yellowish oil (1.42 g, 4.958 mmol, 59%). Typically,
the ligand obtained by this procedure was diluted in an appropri-
ate amount of ethanol to yield a 1 M stock solution, which was
then used without further purification or characterization for the
synthesis of copper(II) complexes.
[a]
22.4 + 23.6 (c 0.1116, MeOH); n-hexane was diffused into a solu-
D
tion prepared from 20 mg of S-2 in 1 mL of methanol to give dark
blue needles suitable for X-ray diffraction within 24 h at ambient
temperature.
4.3.5.4. 2R, N,N0-[1,4-bis[(pyridin-2-ylmethyl)amino]butan-2-ol]ato
dicopper(II) (l-acetato) diperchlorate, R-Cu2bpdbo, R-2. The title
compound was prepared from 4.5 mL of a 1 M stock solution of
R-7 as described for the preparation of S-Cu2bpdbo from S-7 yield-
ꢁ1
~
ing 1.70 g (2.478 mmol, 25%) of R-2 as a blue solid;
m cm 1611,
1569, 1085; calcd for C18H26Cl2Cu2N4O12 [Cu2L-H(OAc)(ClO4)2ꢀH2O,
L = R-7] C 31.40, H 3.81, N 8.14; found: C 31.77, H 3.70, N 8.07;
22.4
[a]
ꢁ23.5 (c 0.0895, MeOH); n-hexane was diffused into a solution
D
prepared from 20 mg of S-2 in 1 mL methanol to give dark blue nee-
dles suitable for X-ray diffraction within 48 h at ambient
temperature.
To obtain analytical data, 1.0 g (3.491 mmol) of the raw mate-
rial was dissolved in dichloromethane and purified by column
chromatography over silica gel (dichloromethane/methanol,
20/1–1/1, v/v) yielding 0.52 g (1.815 mmol, 52%) of S-7 as a pale
4.4. X-ray crystallography
Crystals of S-2 and R-2 were mounted on MiTeGen cryoloops in
random orientations. Preliminary examination and data collection
were performed using a Bruker X8 Kappa Apex II Charge Coupled
Device (CCD) Detector system single crystal X-ray diffractometer
equipped with an Oxford Cryostream LT device. All data were
collected using graphite monochromated Mo
(k = 0.71073 Å) from a fine focus sealed tube X-ray source. Prelim-
inary unit cell constants were determined with a set of 36 narrow
frame scans. Typical data sets consist of combinations of x and /
yellowish oil; [a]
23.4 + 3.7 (c 0.782, EtOH); dH (CD2Cl2) 8.57–8.46
D
(m, 2H), 7.62 (tt, 1.6, 7.7, 2H), 7.33–7.24 (m, 2H), 7.17–7.10 (m,
2H), 3.96–3.78 (m, 5H), 3.34 (br. s, 3H), 2.94–2.86 (m, 1H),
2.83–2.72 (m, 1H), 2.64 (dd, 3.8, 11.5, 1H), 2.57 (dd, 7.8, 12.0, 1H),
1.68–1.51 (m, 2H); dC (CD2Cl2) 160.7, 159.9, 149.6, 149.6, 136.8,
136.8, 122.6, 122.5, 122.4, 122.2, 71.2, 56.1, 55.5, 55.3, 48.0, 34.3;
Ka radiation
m
max/cmꢁ1 (KBr) 3304, 2927, 2843, 1593, 1597, 1476, 1117; HR
ESI-MS calcd for (C16H22N4O + H)+ 287.1866; found 287.1858.
scan frames with a scan width of 0.5° and counting time of 15
s/frame at a crystal to detector distance of 4.0 cm. The collected
frames were integrated using an orientation matrix determined
from the narrow frame scans. Apex II and SAINT software packages
were used for data collection and data integration [50]. Analysis of
the integrated data did not show any decay. Final cell constants
were determined by global refinement of reflections harvested
from the complete data set. Collected data were corrected for sys-
tematic errors using SADABS [50] based on the Laue symmetry
using equivalent reflections. The disorder was modeled with
4.3.5.2. 2R, N,N0-bis(2-pyridylmethyl)-1,4-diaminobutan-2-ol, R-
bpdbo, R-7. The title compound was prepared from R-6 using the
same procedure as described for the synthesis of S-7 from S-6
above yielding 2.42 g (8.450, quantitative) of R-7 as raw material.
Purification of the raw material by column chromatography over
silica gel (dichloromethane/methanol, 20/1–1/1, v/v) yielded
2.01 g (7.0189 mmol, 83%) of R-7 as a pale yellowish oil; Rf 0.12
23.4
(SiO2, CH2Cl2/MeOH, 4/1, v/v); [
a]
ꢁ3.8 (c 0.603, EtOH); dH
D
(CD2Cl2) 8.50 (qd, 1.4, 4.7, 1H), 7.63 (tt, 1.8, 7.7, 1H), 7.35–7.23

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
359
partial occupancy atoms and geometrical restraints for both
structures.
70.3, 68.9, 68.4, 65.4, 61.8, 20.8, 20.6, 20.6; Calcd for C20H22ClNO12
C, 47.68; H, 4.40; N, 2.78; found: C, 47.65, H, 4.50; N, 2.68.
Structure solution and refinement were carried out using the
SHELXTL-PLUS software package [51]. The structures were solved
by direct methods and refined successfully in the space group
P212121. Full matrix least-squares refinements were carried out
4.5.2. General description for the synthesis of aryl 2,3,4,6-tetra-O-
acetyl-a-D-gluco- and galactopyranosides [31]
A 75 mL aliquot of 1 M anhydrous tin(IV) chloride in dichloro-
methane was added to 15.0 g (38.46 mmol) peracetylated
monosaccharide and 9.76 g (56.24 mmol) 2-chloro-4-nitrophenol
in 50 mL dichloromethane. The resulting reaction mixture was
refluxed for 5 days and then poured into 200 mL of a saturated
aqueous sodium bicarbonate solution. The solution was neutral-
ized by further addition of sodium bicarbonate. The suspension
was filtered on Celite, and the recovered solid was extracted with
three 150-mL portions of ethyl acetate. The combined organic lay-
ers were extracted with 450 mL of 1 M aqueous sodium hydroxide
solution, dried over sodium sulfate, filtered and concentrated to
dryness. The residue was purified by column chromatography on
silica gel using hexane/ethyl acetate = 3/1 (v/v) as eluent, yielding
the target compound.
by minimizing
R
w(F2o–Fc2)2. The non-hydrogen atoms were refined
anisotropically to convergence. All hydrogen atoms were treated
using appropriate riding models.
X-ray data for S-2 (C19H28Cl2Cu2N4O12), blue needles
(0.567 ꢂ 0.149 ꢂ 0.102 mm3, V 2640.4(3) Å3), were collected at
100 K. The crystals are orthorhombic, space group P212121 with
a = 7.2680(5) Å, b = 14.5809(10) Å and c = 24.9155(17) Å, and
Z = 4. The h-range for data collection was 1.618–30.743°. The num-
ber of reflections collected was 61,147, with 8157 unique reflec-
tions (Rint = 0.0402). Refinement by full-matrix least-squares on
F2, 392 parameters, gave final R indices (I > 2
rI) R1 = 0.0290,
weighted R2 = 0.0706; R indices on all data were R1 = 0.0335 and
weighted R2 = 0.0721. The absolute structure parameter x was
ꢁ0.020(3); CCDC 1431768.
X-ray data for R-2 (C19H28Cl2Cu2N4O12), blue needles
(0.516 ꢂ 0.104 ꢂ 0.066 mm3, V 2650.10(13) Å3), were collected at
100 K. The crystals are orthorhombic, space group P212121 with
a = 7.2854(2) Å, b = 14.5841(4) Å and c = 24.9419(7) Å, and Z = 4.
The h-range for data collection was 1.633–34.970°. The number
of reflections collected was 88769, with 11,457 unique reflections
(Rint = 0.0662). Refinement by full-matrix least-squares on F2, 385
4.5.3. 2-Chloro-4-nitrophenyl 2,3,4,6-tetra-O-acetyl-a-D-
galactopyranoside (12c)
Off-white solid; 6.25 g (12.40 mmol, 32%); mp 175–176 °C; Rf
0.15 (SiO2, hexane/ethyl acetate = 3/1 (v/v); dH (CDCl3) 8.33 (d,
2.4, 1H), 8.15 (dd, 9.0, 2.7, 1H), 7.32 (d, 9.3, 1H), 5.95 (d, 3.4, 1H),
5.55–5.62 (m, 2H), 5.30 (ddd, 11.7, 3.7, 1.2, 1H), 4.33 (t, 6.8, 1H),
4.11 (ddd, 17.1, 11.7, 6.8, 2H), 2.19 (s, 3H), 2.11 (s, 3H), 2.05 (s,
3H), 1.98 (s, 3H); dC (CDCl3) 170.5, 170.1, 169.9, 169.8, 156.9,
142.9, 126.2, 125.0, 123.6, 115.8, 96.0, 68.1, 67.4, 67.4, 67.2, 61.3,
parameters, gave final R indices (I > 2rI) R1 = 0.0398, weighted
R2 = 0.0832; R indices on all data were R1 = 0.0570 and weighted
R2 = 0.0891. The absolute structure parameter x was ꢁ0.021(5);
CCDC 1431767.
Crystal data and intensity data collection parameters, the final
residual values and structure refinement parameters, complete
listings of positional and isotropic displacement coefficients for
hydrogen atoms, anisotropic displacement coefficients for the
non-hydrogen atoms and tables of calculated and observed struc-
ture factors are available in Supplementary Information.
20.7, 20.6, 20.5, 20.5; Calcd for
4.40; found: C, 47.90, H, 4.47; HRMS, ESI-TOF+ m/z calcd. for
20H22ClNO12Na [M+Na]+ 526.0722; found 526.0712.
C20H22ClNO12 C, 47.68; H,
C
4.5.4. 2-Chloro-4-nitrophenyl 2,3,4,6-tetra-O-acetyl-a-D-
glucopyranoside (12e)
Off-white solid; 2.75 g (5.458 mmol, 14%); an analytical sample
(0.5 g, 1.0 mmol) was purified by precipitation of the target com-
pound from a 30 mL solution in cyclohexane/ethyl acetate (4/1,
v/v) by cyclohexane addition. The recovered material was sub-
jected to the precipitation procedure two more times, and the
recovered solid was dried in vacuum; mp 117–118 °C; Rf 0.19
(SiO2, hexane/ethyl acetate 3/1, v/v); dH (CDCl3) 8.34 (d, 2.5, 1H),
8.15 (dd, 9.0, 2.8, 1H),7.32 (d, 9.0, 1H), 5.91 (d, 3.8, 1H), 5.74 (t,
9.8, 1H), 5.20 (t, 9.9, 1H), 5.06 (dd, 10.2, 3.6, 1H), 4.29–4.24 (m,
1H), 4.17–4.07 (m, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 2.07 (s, 6H); dC
(CDCl3) 170.3, 170.2, 169.9, 169.5, 156.7, 143.0, 126.2, 125.0,
123.6, 115.7, 95.4, 70.2, 69.5, 69.0, 67.8, 61.3, 20.6, 20.6, 20.5,
20.5; HRMS, ESI-TOF+ m/z calcd. for C20H22ClNO12Na (M+Na)+:
526.0722; found 526.0718.
4.5. Substrate syntheses
4.5.1. 2-Chloro-4-nitrophenyl 2,3,4,6-tetra-O-acetyl-a-D-
mannopyranoside (12a)
The title compound was obtained using a general protocol for
the synthesis of related aryl -D-mannopyranosides as described
a
by Han et al. [32] Boron trifluoride diethyl etherate (2.28 mL,
18.14 mmol, 3.0 equiv.) was added in nitrogen atmosphere to a
solution of peracetylated mannose (2.34 g, 6.0 mmol) and 2-
chloro-4-nitrophenol (2.08 g, 12.0 mmol, 2.0 equiv.) in 40 mL of
dry dichloromethane. The solution was then brought to reflux.
After 96 h, the solution was cooled to ambient temperature and
diluted with 100 mL dichloromethane and 50 mL of water. The
separated aqueous layer was extracted twice with 60 mL of
dichloromethane each. The combined organic layers were washed
with 40 mL of saturated bicarbonate solution, 40 mL water, and
40 mL brine, and dried over anhydrous sodium sulfate. After filtra-
tion, the filtrate was concentrated under reduced pressure to dry-
ness. The obtained residue was purified on silica gel by column
chromatography (hexane/ethyl acetate = 6/1–4/1, v/v) to give 12a
as a colorless solid (1.45 g, 2.883 mmol, 48%); mp: 144–145 °C; Rf
0.36 (SiO2, cyclohexane/ethyl acetate = 3/2, v/v); dH(CDCl3) 8.35
(d, J = 2.8 Hz, 1H), 8.15 (dd, J = 2.8, 9.3 Hz, 1H), 7.32 (d, J = 9.0 Hz,
1H), 5.70 (d, J = 1.8 Hz, 1H), 5.59 (dd, J = 3.5, 10.3 Hz, 1H), 5.41 (t,
J = 10.0 Hz, 1H), 4.33–4.23 (m, 1H), 4.12–4.00 (m, 2H), 2.23 (s,
3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H); dC(CDCl3) 170.3,
169.9, 169.7, 169.6, 155.9, 142.9, 126.3, 124.8, 123.6, 115.3, 96.3,
4.5.5. 2-Chloro-4-nitrophenyl 3,4,6-tri-O-acetyl-2-O-methyl-a-D-
galactopyranoside (12g)
The synthesis was performed according to a general protocol
described by Olsson et al. for the preparation of related compounds
with
a-glycosidic bonds [52]. A solution of p-tolyl 3,4,6-tri-
O-acetyl-2-O-methyl-1-thio-b-
D
-galactopyranoside 15 (2.70 g,
6.308 mmol) and 2-chloro-4-nitrophenol (1.64 g, 9.474 mmol,
1.50 equiv.) in 50 mL of dry dichloromethane was stirred at 0 °C.
After
5 min,
2.84 g
(12.622 mmol,
2.0 equiv.)
of
N-
iodosuccinimide and 0.65 g (2.519 mmol, 0.40 equiv.) of silver tri-
fluoromethanesulfonate were added and stirring continued at 0 °C.
After 30 min, 3 mL of triethylamine was added, and the resulting
mixture was filtered through a pad of Celite. The filtrate was
diluted with 200 mL dichloromethane and washed with 100 mL
of 10% aqueous sodium disulfite solution, 50 mL water, 50 mL
brine and dried over anhydrous sodium sulfate. After filtration

360
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
and concentration to dryness, the residue obtained was purified by
column chromatography over silica gel (cyclohexane/ethyl acetate,
5/1–3/1, v/v) yielding compound 12g as a colorless solid (1.60 g,
3.361 mmol, 53%); Rf 0.25 (SiO2, cyclohexane/ethyl acetate, 2/1,
v/v); mp: 87–89 °C; dH(CDCl3) 8.34 (d, 2.8, 1H), 8.15 (dd, 2.8, 9.3,
1H), 7.31 (d, 9.3, 1H), 5.87 (d, 3.5, 1H), 5.56 (dd, 1.4, 3.4, 1H),
5.49 (dd, 3.3, 10.5, 1H), 4.26 (dt, 1.5, 6.8, 1H), 4.13–4.02 (m, 2H),
3.90 (dd, 3.5, 10.5, 1H), 3.54 (s, 3H), 2.19 (s, 3H), 2.08 (s, 3H),
1.96 (s, 3H); dC(CDCl3) 170.1, 169.9, 157.0, 142.6, 126.2, 125.0,
123.5, 115.4, 96.5, 74.8, 69.4, 68.3, 67.7, 61.3, 59.4, 20.8, 20.6,
20.5; calcd for C19H22ClNO11 C, 47.96; H, 4.66; found: C, 48.12, H,
4.64.
4.5.10. 2-Chloro-4-nitrophenyl a-D-mannopyranoside (11a) [24,25]
Colorless solid; 0.64 g (0.192 mmol, 71%) after column chro-
matography on silica gel (ethyl acetate/methanol, 30/1–20/1, v/
v); mp 156–157 °C; Rf 0.35 (SiO2, ethyl acetate/methanol, 10/1, v/
v); dH (DMSO-d6) 8.34 (dd, 2.8, 0.8, 1H), 8.20 (ddd, 9.2, 2.8, 0.9,
1H), 7.56 (d, 8.8, 1H), 5.74 (d, 1.3, 1H), 5.24 (dd, 4.5, 0.8, 1H),
4.96 (dd, 5.6, 0.6, 1H), 4.92 (dd, 5.9, 0.6, 1H), 4.46 (dd, 6.3, 5.8,
1H), 3.90 (t, 4.5, 1H), 3.70–3.77 (m, 1H), 3.48–3.62 (m, 2H), 3.39–
3.47 (m, 1H), 3.33 (s, 3H), 3.22–3.30 (m, 1H); dC (DMSO-d6)
156.6, 141.5, 125.4, 124.2, 122.6, 116.2, 99.2, 75.9, 70.5, 69.6,
66.3, 60.8; HRMS, FTMS+ m/z calcd. for C12H14ClNNaO8 [M+Na]+:
358.0306; found 358.0304.
4.5.11. 2-Chloro-4-nitrophenyl a-D-galactopyranoside (11c)
4.5.6. General procedure for the synthesis of aryl 2,3,4,6-tetra-O-
acetyl-b-D-glycopyranosides [26]
Off-white solid, 1.50 g (4.468 mol, 31%); mp 90–115 °C; Rf 0.89
(SiO2, MeOH); dH (DMSO-d6) 8.33 (d, 2.8, 1H), 8.20 (dd, 9.2, 2.9,
1H), 7.53 (d, 9.3, 1H), 5.86 (d, 3.3, 1H), 5.16 (d, 5.5, 1H), 4.97 (d,
5.3, 1H), 4.68 (d, 3.8, 1H), 4.51 (dd, 6.1, 5.4, 1H), 3.88 (dd, 5.1,
3.4, 1H), 3.76–3.86 (m, 2H), 3.56–3.65 (m, 1H), 3.46–3.55 (m,
1H), 3.35–3.42 (m, 1H); dC (DMSO-d6) 157.6, 141.2, 125.4, 124.2,
The syntheses were achieved by phase-transfer-catalyzed gly-
cosylation of phenols with peracetylated glycopyranosyl bromide
as described by Kroger et al. and us earlier [53,54]. Typically,
15 mL of a 1 N aqueous sodium hydroxide solution was added to
a solution of the peracetylated glycosyl bromide (3.5 mmol), tetra-
122.8, 115.8, 98.5, 73.5, 69.1, 68.3, 67.4, 60.1; Calcd for C12H14
-
butylammonium
bromide
(1.45 equiv.)
and
2-chloro-4-
ClNO8: C, 42.93; H, 4.20; found: C, 42.89, H, 4.29; HRMS, ESI-
TOF+ m/z calcd. for C12H14ClNO8Na [M+Na]+: 358.0300; found
358.0291.
nitrophenol (2 equiv.) in 25 mL dichloromethane. The resulting
two phase system was vigorously stirred at 35 °C for 3 h. After
cooling to ambient temperature, the mixture was diluted with
100 mL ethyl acetate. After separation of the organic layer, the
aqueous layer was extracted two times with 50 mL ethyl acetate
each. The combined organic layers were washed with 1 N aqueous
sodium hydroxide solution, 20 mL water, 20 mL brine and dried
over anhydrous sodium sulfate. After filtration, the filtrate was
concentrated to dryness yielding a residue that was purified by
column chromatography over silica gel affording the target
compounds.
4.5.12. 2-Chloro-4-nitrophenyl b-
D
-galactopyranoside (11d)
The compound was prepared as described by us previously [26].
4.5.13. 2-Chloro-4-nitrophenyl a-D-glucopyranoside (11e)
Off-white solid; 1.20 g (3.575 mmol, 25%); mp 157–159 °C; Rf
0.66 (SiO2, MeOH); dH (DMSO-d6) 8.33 (d, 2.8, 1H), 8.21 (dd,
9.3, 2.8, 1H), 7.54 (d, 9.5, 1H), 5.83 (d, 3.5, 1H), 5.33 (d, 5.3,
1H), 5.13 (t, 5.4, 2H), 4.48 (t, 5.9, 1H), 3.70 (td, 9.2, 5.0, 1H),
3.53 (ddd, 12.0, 5.8, 2.5, 1H), 3.41–3.50 (m, 2H), 3.35–3.40 (m,
1H), 3.22 (ddd, 10.0, 8.8, 5.3, 1H); dC (DMSO-d6) 157.5, 141.3,
125.5, 124.2, 122.8, 115.8, 98.2, 74.9, 72.6, 71.2, 69.4, 60.5; Calcd
for C12H14ClNO8: C, 42.93; H, 4.20; found: C, 43.05, H, 4.23;
ESI-TOF+ m/z calcd. for C12H14ClNO8Na [M+Na]+: 358.0300; found
358.0302.
4.5.7. 2-Chloro-4-nitrophenyl 2,3,4,6-tetra-O-acetyl-b-D-
glucopyranoside (12f)
Off-white solid, 4.40 g (8.733 mmol, 23%); mp 152–153 °C; Rf
0.29 (SiO2, hexane/ethyl acetate = 2/1 (v/v); dH (CDCl3) 8.32 (d,
2.6, 1H), 8.14 (dd, 9.0, 2.6, 1H), 7.24 (s, 1H), 5.12–5.46 (m, 4H),
4.30 (dd, 12.4, 5.3, 2H), 4.22 (dd, 12.4, 2.6, 2H), 3.90–3.98 (m,
2H), 2.10 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H); dC (CDCl3)
170.3, 170.0, 169.2, 168.9, 157.1, 143.0, 126.0, 124.8, 123.5, 116.3,
99.1, 72.4, 71.9, 70.3, 67.8, 61.6, 20.6, 20.5, 20.5, 20.5; calcd for
4.5.14. 2-Chloro-4-nitrophenyl b-D-glucopyranoside (11f)
Off-white solid; 1.94 (5.779 mmol, 40%); mp 181–183 °C; Rf
0.66 (SiO2, MeOH); dH (DMSO-d6) 8.15 (t, 9.0, 1H), 7.21 (dd, 13.3,
2.5, 1H), 7.04 (dd, 9.3, 2.3, 1H), 5.48 (d, 4.5, 1H), 5.18 (d, 4.5, 1H),
5.06–5.14 (m, 2H), 4.61 (t, 5.6, 1H), 3.69 (dd, 9.8, 5.3, 1H), 3.40–
3.52 (m, 2H), 3.10–3.33 (m, 3H); dC (DMSO-d6) 157.8, 141.5,
125.5, 124.3, 122.2, 115.7, 99.9, 77.3, 76.6, 73.0, 69.3, 60.5; calcd
for C12H14ClNO8, C 42.93, H 4.20; found: C 42.96, H 4.19.
C20H22ClNO12 C, 47.68; H, 4.40; found: C, 47.83, H, 4.43.
4.5.8. 2-Chloro-4-nitrophenyl 3,4,6-tri-O-acetyl-2-O-methyl-b-
D-
galactopyranoside (12h)
Purification by column chromatography over silica gel (hexane/
ethyl acetate, 3/1–1/1, v/v) as colorless solid (1.30 g, 2.731 mmol,
80%); mp 148–149 °C; Rf 0.40 (SiO2, hexane/ethyl acetate, 1/1,
v/v); dH (CDCl3) 8.34 (d, 2.5, 1H), 8.15 (dd, 2.8, 9.0, 1H), 7.20 (d,
9.3, 1H), 5.46 (dd, 0.9, 3.4, 1H), 5.10 (d, 7.5, 1H), 5.02 (dd, 3.5,
10.3, 1H), 4.26–4.02 (m, 3H), 3.76 (dd, 7.5, 10.3, 1H), 3.69 (s, 3H),
2.20 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H); dC (CDCl3); 170.2, 170.0,
157.4, 142.7, 126.2, 124.1, 123.6, 115.0, 101.1, 77.5, 72.0, 71.4,
66.9, 61.5, 61.5, 20.7, 20.7, 20.6; calcd for C19H22ClNO11 C, 47.96;
H, 4.66; found: C, 47.96, H, 4.64.
4.5.15. 2-Chloro-4-nitrophenyl 2-O-methyl-a-D-galactopyranoside
(11g)
Chromatographic purification over silica gel (hexane/ethyl acet-
ate 2/1–0/1, v/v), colorless solid; 0.81 g (2.314 mmol, 79%); mp
130–132 °C; dH (DMSO-d6) 8.33 (d, 2.8, 1H), 8.22 (dd, 2.8, 9.3,
1H), 7.55 (d, 9.3, 1H), 6.15 (d, 3.5, 1H), 5.15 (d, 6.0, 1H), 4.81 (d,
4.3, 1H), 4.52 (t, 5.8, 1H), 3.91 (ddd, 3.1, 6.1, 9.8, 1H), 3.81 (t, 3.5,
1H), 3.64–3.45 (m, 3H), 3.42 (s, 3H), 3.39–3.30 (m, 2H); dC
(DMSO-d6) 157.3, 141.3, 125.5, 124.2, 122.8, 115.8, 95.7, 77.3,
73.3, 68.5, 68.5, 60.0, 58.4; HRMS, ESI-TOF+ m/z calcd. for C13H16
-
4.5.9. General procedure for deacetylation reactions
ClNNaO8 [M+Na]+: 372.0462; found 372.0456.
Typically, the peracetylated aryl glycopyranosides 12 were sus-
pended in 12–15 mL 7 M ammonia in methanol and stirred at
ambient temperature. After 5–24 h, all volatile material was evap-
orated. The resulting residue was recrystallized from ethyl acetate
or purified by column chromatography on silica gel as noted yield-
ing the target compounds [26].
4.5.16. 2-Chloro-4-nitrophenyl 2-O-methyl-b-
(11h)
Chromatographic purification over silica gel (hexane/ethyl acet-
ate 4/1–0/1, v/v), colorless solid; 0.77 g (2.200 mmol, 87%); Rf 0.17
(SiO2, ethyl acetate); mp 193–195 °C; dH (DMSO-d6) 8.36 (d, 2.8,
D-galactopyranoside

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
361
1H), 8.22 (dd, 2.8, 9.3, 2H), 7.44 (d, 9.3, 2H), 5.24 (d, 7.5, 2H), 5.14
(d, 6.0, 2H), 4.76 (d, 4.8, 2H), 4.71 (t, 5.5, 1H), 3.77–3.66 (m, 2H),
3.63–3.45 (m, 6H), 3.41 (dd, 7.5, 9.5, 1H); dC (DMSO-d6) 157.5,
141.6, 125.6, 124.4, 122.0, 115.5, 100.3, 80.5, 75.8, 72.3, 68.1,
60.4, 60.1; HRMS, ESI-TOF+ m/z calcd. for C13H16ClNNaO8 [M
+Na]+: 372.0462; found 358.0451.
4.5.20. 20-Chloro-40-nitrophenyl 4,6-benzylidene-b-D-glucopyranoside
(17)
The synthesis was performed according to a general protocol for
related compounds [56]. Benzaldehyde dimethyl acetal (0.43 g,
2.799 mmol, 1.25 equiv.) and camphorsulfonic acid (0.10 g,
0.431 mmol, 0.14 equiv.) were added to the solution of 11f
(0.75 g, 2.239 mmol) in 15 mL acetonitrile at ambient temperature.
After 2 h, the solution was neutralized with 0.3 mL trimethy-
lamine, concentrated to yield a residue that was purified by
column chromatography over silica gel (hexane/ethyl acetate =
4/1–1/1, v/v) to give 17 (0.82 g, 1.953 mmol, 87%) as a colorless
solid; Rf 0.17 (SiO2, cyclohexane/ethyl acetate = 2/1, v/v); mp
222–224 °C; dH (CD3OD) 8.33 (d, 2.8, 1H), 8.20 (dd, 9.0, 2.8, 1H),
7.49–7.56 (m, 2H), 7.46 (d, 9.3, 1H), 7.31–7.40 (m, 2H), 5.62 (s,
1H), 5.34 (d, 7.8, 1H), 4.33 (dd, 9.8, 4.5, 1H), 3.66–3.84 (m, 4H),
3.61 (t, 9.2, 1H); dC (CD3OD) 159.1, 143.8, 139.0, 130.0, 129.1,
127.5, 126.8, 124.9, 124.9, 116.8, 103.0, 102.1, 81.7, 75.4, 74.7,
69.4, 67.9; HRMS, ESI-TOF+ m/z calcd. for C19H18ClNNaO8 [M
+Na]+: 446.0619; found 446.0622.
4.5.17. 20-Chloro-40-nitrophenyl b-D-allopyranoside (11i)
Chromatographic purification over silica gel (ethyl acetate/
methanol, 1/0–10/1, v/v); colorless solid; 0.42 g, 1.254 mmol,
83%; Rf 0.27 (SiO2, ethyl acetate/methanol = 10/1, v/v); mp 153–
154 °C; dH (CD3OD) 8.30 (d, 2.5, 1H), 8.19 (dd, 9.3, 2.8, 1H), 7.43
(d, 9.3, 1H), 5.48 (d, 7.8, 1H), 4.16 (t, 2.9, 1H), 3.91–3.97 (m, 1H),
3.88 (dd, 12.0, 2.3, 1H), 3.66–3.77 (m, 2H), 3.63 (dd, 9.8, 3.0, 1H);
dC (CD3OD) 159.6, 143.4, 126.6, 124.9, 124.6, 116.7, 100.1, 76.1,
73.1, 71.7, 68.4, 62.7. The spectral data match those reported by
others [28].
4.5.18. p-Tolyl 3,4,6-tri-O-acetyl-2-O-methyl-1-thio-b-
galactopyranoside (15)
D-
4.5.21. 20-Chloro-40-nitrophenyl 2-benzoyl-4,6-benzylidene-b-D-
glucopyranoside (18)
The synthesis was performed using a general protocol by Li
et al. for related compounds [34]. A solution of 14 (0.86 g,
2.85 mmol) in 8 mL pyridine and 5 mL acetic anhydride was stirred
at ambient temperature for 3 h. The solution was then diluted with
100 mL ethyl acetate and neutralized with saturated sodium bicar-
bonate solution. The separated organic layer was extracted with
20 mL water, 20 mL brine, and dried over anhydrous sodium sul-
fate. After filtration and concentration, a residue was obtained
and purified by column chromatography over silica gel (hexane/
ethyl acetate = 6/1–4/1, v/v) to give compound 15 (1.20 g, 94%) as
a colorless foam; Rf 0.27 (SiO2, cyclohexane/ethyl acetate = 3/1,
v/v); dH (CDCl3) 7.48–7.52 (m, 2H), 7.15 (dd, 8.5, 0.5, 2H), 5.40
(dd, 3.5, 1.0, 1H), 4.96 (dd, 9.7, 3.4, 1H), 4.58 (d, 9.8, 1H), 4.18
(dd, 11.5, 7.0, 1H), 4.11 (dd, 11.3, 6.3, 1H), 3.87 (td, 6.5, 1.0, 1H),
3.55 (s, 3H), 3.44 (t, 9.7, 1H), 2.37 (s, 3H), 2.15 (s, 3H), 2.05 (s,
6H); dC (CDCl3) 170.3, 170.1, 169.9, 138.0, 132.8, 129.6, 129.1,
87.8, 77.2, 76.5, 74.2, 74.0, 67.6, 61.7, 61.0, 21.1, 20.7, 20.6; HRMS
(ESI-TOF+) m/z calcd. for C20H26NaO8S [M+Na]+ 449.1246; found
449.1238.
The synthesis was performed according to the general protocol
for related compounds [36].
A suspension of 17 (1.50 g,
3.538 mmol) and dibutyltin oxide (0.97 g, 3.891 mmol, 1.1 equiv.)
in 50 mL absolute methanol was kept under reflux. After 1.5 h,
the homogenous solution was concentrated and co-evaporated
with toluene twice. The residue was suspended in 30 mL toluene
and cooled in ice.
A solution of benzoyl chloride (0.55 g,
3.891 mmol) in 2 mL toluene was then added dropwise at 0 °C.
Lastly, the solution was allowed to warm gradually to ambient
temperature. After 16 h, the reaction mixture was cooled to 0 °C,
diluted with 150 mL ethyl acetate and filtered through a short
pad of Celite. The pad was washed with cold ethyl acetate. The
organic layers were combined, all volatile material was evaporated
in vacuum, and the resulting residue was purified by column chro-
matography over silica gel (hexane/ethyl acetate = 8/1–3/1, v/v) to
give compound 18 (1.30 g, 2.675 mmol, 76%) as an off-white solid;
Rf 0.14 (SiO2, cyclohexane/ethyl acetate = 4/1, v/v); mp 190–
192 °C; dH (CD2Cl2) 8.21 (d, 2.8, 1H), 8.13 (dd, 9.0, 2.8, 1H), 8.02–
8.09 (m, 2H), 7.57–7.65 (m, 1H), 7.36–7.55 (m, 7H), 7.31 (d, 9.3,
1H), 5.64 (s, 1H), 5.57 (dd, 9.0, 7.8, 1H), 5.38 (d, 7.8, 1H), 4.46
(dd, 10.3, 4.8, 1H), 4.19 (t, 9.2, 1H), 3.90 (t, 9.8, 1H), 3.84 (t, 9.2,
1H), 3.72–3.80 (m, 1H), 2.76 (br. s., 1H); dC (CD2Cl2) 165.9, 157.9,
143.6, 137.5, 134.1, 130.3, 129.9, 129.0, 128.8, 126.8, 126.6,
125.2, 124.2, 116.9, 102.5, 100.5, 80.8, 74.2, 72.6, 68.9, 67.4; HRMS
(ESI-TOF+) m/z calcd. for C26H23ClNO9 [M+H]+ 528.1061; found
528.1050.
4.5.19. 3,4,6-Tri-O-acetyl-2-O-methyl-a-D-galactopyranosyl bromide
(16)
The synthesis was performed according to a protocol described
for related compounds [55]. Iodine monobromide (0.12 g,
0.580 mmol, 1.13 equiv.) was added to a solution of 15 (0.22 g,
0.514 mmol) in 5 mL dichloromethane at 0 °C. After 10 min, the
solution was diluted with 100 mL dichloromethane and
neutralized with 5 mL saturated aqueous sodium bicarbonate solu-
tion. The organic layer was separated and washed with 5 mL satu-
rated aqueous sodium bisulfite solution, 20 mL water, 20 mL brine
and dried over anhydrous sodium sulfate. After filtration and con-
centration of the filtrate to dryness, a residue was obtained that
was purified by column chromatography over silica gel (hexane/
ethyl acetate, 4:1–3:1, v/v) to give compound 16 (0.16 g,
0.419 mmol, 81%) as a colorless foam; Rf 0.20 (SiO2, cyclohexane/
ethyl acetate = 2/1, v/v); dH (CDCl3) 6.65 (d, 3.8, 1H), 5.49 (dd,
3.3, 1.3, 1H), 5.27 (dd, 10.3, 3.3, 1H), 4.46–4.48 (m, 1H), 4.17 (dd,
12.0, 4.0, 1H), 4.11 (dd, 12.0, 4.0, 1H), 3.63 (dd, 10.2, 3.9, 1H),
3.48 (s, 3H), 2.16 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H); dC (CDCl3)
170.3, 169.8, 169.8, 90.1, 75.1, 71.1, 70.0, 67.1, 60.8, 58.3, 20.7,
20.6, 20.6; HRMS (ESI-TOF+) m/z calcd. for C13H19BrNaO8 [M+Na]+
405.0161 and 407.0141; found 405.0157 and 407.0137.
4.5.22. 20-Chloro-40-nitrophenyl 2-O-benzoyl-4,6-benzylidene-3-O-
triflyl-b-D-glucopyranoside (19)
The synthesis was performed according to the general protocol
[36]. A solution of 18 (1.20 g, 2.469 mmol) in 20 mL dry dichloro-
methane and dry pyridine (0.78 g, 0.988 mmol, 4.0 equiv.) was
cooled to ꢁ5 °C. Then, a solution of trifluoromethanesulfonic anhy-
dride (1.39 g, 4.938 mmol. 2.0 equiv.) in 2 mL dry dichloromethane
was added dropwise. The yellow solution was stirred at 0 °C for
0.5 h and then poured into 50 mL of iced water. The resulting mix-
ture was extracted with 80 mL dichloromethane three times each.
The combined organic layer was washed with 50 mL water, 40 mL
brine, separated, and dried over anhydrous sodium sulfate. After
filtration and concentration, 19 was obtained as a yellowish foam
(1.63 g, 2.469 mmol, quantitative) and used immediately in the

362
S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
next step; HRMS (ESI-TOF+) m/z calcd. for C27H21ClF3NNaO11S [M
5.1.2. Catalyst stock solution
+Na]+ 682.0374; found 682.0378.
Typically, 3–4 mg of the binuclear metal complexes was dis-
solved in 25 mL of buffer solution yielding 0.27 mM stock solu-
tions. The catalyst stock solutions were then used in constant
4.5.23. 20-Chloro-40-nitrophenyl 3-O-acetyl-2-O-benzoyl-4,6-
25 lL aliquots for all experiments resulting in 0.03 mM catalyst
benzylidene-b-D-allopyranoside (20)
concentrations.
The synthesis was performed according to a general protocol for
related compounds [36]. Tetrabutylammonium acetate (1.92 g,
6.383 mmol, 3.0 equiv.) was added to a solution of 19 (1.40 g,
2.128 mmol) in 15 mL dry DMF at 0 °C. After 15 min, the solution
was poured into 50 mL of ice-water. The resulting mixture was
extracted with 100 mL ethyl acetate three times each. The com-
bined organic layer was washed with 50 mL water, 40 mL brine,
separated, and dried over anhydrous sodium sulfate. After filtra-
tion and concentration, a residue was obtained that was purified
by column chromatography over silica gel column (hexane/ethyl
acetate = 8/1–6/1, v/v) to give compound 20 (1.10 g, 1.941 mmol,
91%) as a colorless foam; Rf 0.33 (SiO2, cyclohexane/ethyl acet-
ate = 4/1, v/v); dH (CD2Cl2) 8.24 (d, 2.8, 1H), 8.17 (dd, 9.0, 2.8,
1H), 7.92–7.98 (m, 2H), 7.56–7.64 (m, 1H), 7.41–7.48 (m, 4H),
7.34–7.40 (m, 4H), 6.00 (t, 2.9, 1H), 5.68 (d, 8.3, 1H), 5.63 (s, 1H),
5.55 (dd, 8.2, 3.1, 1H), 4.49 (dd, 10.5, 5.0, 1H), 4.27 (td, 9.9, 5.1,
1H), 4.00 (dd, 9.5, 2.5, 1H), 3.89 (t, 10.3, 1H), 2.16 (s, 3H); dC (CD2-
Cl2) 170.0, 165.2, 158.1, 143.6, 137.4, 134.1, 130.2, 129.7, 129.7,
129.1, 128.8, 126.7, 126.6, 125.2, 124.2, 116.8, 102.3, 98.9, 76.5,
5.1.3. Substrate hydrolysis assay
All experiments were performed in triplicate in 96-well plate
format with a 200
tributed in the plate in aliquots with regular increments between 0
and 175 L. The overall volume of the solutions in each well was
then adjusted to 175 L by addition of appropriate aliquots of buf-
fer solution followed by addition of 25 L of the respective metal
lL volume. The substrate stock solution was dis-
l
l
l
complex stock solutions. The covered plate was shaken and equili-
brated at 30 °C for 30 min. The hydrolysis of the substrates was fol-
lowed by monitoring the formation of 4-nitrophenolate and 2-
chloro-4-nitrophenolate, respectively, using UV/Vis spectroscopy
at 405 nm over 1 and 12 h, respectively. Control experiments were
conducted in similar fashion by substituting the aliquots of the
metal complex stock solution with buffer solution.
5.1.4. Data analysis
The absorbance recorded was plotted versus time in minutes.
The rate of the reaction was determined at each substrate concen-
tration from the slope of the linear fit of the data after conversion
of the absorbance into product concentration using the apparent
extinction coefficients as described below. The rate of the reaction
was corrected for the catalyst concentration and the uncatalyzed
reaction, and then plotted versus the substrate concentration. By
applying a nonlinear fit of the resulting hyperbolic data, the cat-
alytic rate constant kcat (minꢁ1) and the substrate affinity KM
(mM) were determined utilizing the Michaelis–Menten model.
All experiments were conducted in triplicate and the data were
averaged.
70.3, 69.3, 68.7, 65.6, 21.1; HRMS (ESI-TOF+) m/z calcd. for C28H25
-
ClNO10 [M+H]+ 570.1167; found 570.1152.
4.5.24. 20-Chloro-40-nitrophenyl 3-O-acetyl-2-O-benzoyl-b-D-
allopyranoside (21)
The synthesis was performed according to protocol for related
compounds [57]. A suspension of 20 (0.90 g, 1.579 mmol) in
25 mL aqueous acetic acid/water (4/1, v/v) was stirred at 50 °C.
After 15 h, the suspension was cooled to ambient temperature
and concentrated to dryness. The obtained residue was purified
by column chromatography over silica gel (hexane/ethyl acet-
ate = 1/2–2/1, v/v) to give 21 as
a colorless foam (0.74 g,
1.535 mmol, 97%); Rf 0.12 (SiO2, cyclohexane/ethyl acetate = 1/1,
v/v); dH (CDCl3) 8.25 (d, 2.8, 1H), 8.16 (dd, 9.0, 2.8, 1H),
7.93–8.00 (m, 2H), 7.56–7.63 (m, 1H), 7.41–7.48 (m, 2H), 7.32 (d,
9.0, 1H), 5.89 (t, 3.0, 3H), 5.62 (d, 8.0, 1H), 5.48 (dd, 8.0, 3.0, 1H),
4.18 (dd, 9.7, 2.9, 1H), 3.99–4.11 (m, 2H), 3.92 (dd, 12.5, 5.0, 1H),
2.20 (s, 3H), 1.32–2.02 (br s, 2H); dC (CDCl3) 170.7, 165.0, 157.6,
142.9, 133.6, 129.7, 129.0, 128.5, 126.1, 124.8, 123.7, 116.0, 97.8,
77.2, 75.0, 71.2, 69.4, 66.1, 62.0, 20.8; HRMS (ESI-TOF+) m/z calcd.
for C21H20ClNNaO10 [M+Na]+ 504.0673; found 504.0664.
5.1.5. Determination of the apparent extinction coefficient
Typically, 5 mg of the respective nitrophenol was dissolved in
250
100
l
l
L DMSO and diluted into 5 mL with buffer solution. Then,
L of this solution was further diluted into 5 mL with buffer
solution yielding the phenol stock solution. Subsequently, 25
aliquot increments of this stock solution were diluted into 200
with buffer solution in a 96-well plate. The solutions were thor-
oughly mixed and then equilibrated at 30.0 0.1 °C for 30 min
prior to determination of their absorbance at 405 nm.
l
l
L
L
The obtained absorbance values were plotted versus the phenol
concentrations. The linear fit of the data equals the product of
extinction coefficient e405 times the unknown path length d for
5. Kinetic assays
product absorbance in 96-well plates containing 200 lL solutions
All experiments were conducted in triplicate in 50 mM HEPES
buffer at pH 7.50 0.05 and 30.0 0.1 °C, 100 mM TES buffer at
pH 8.00 0.05 and 60 0.1 °C, or 50 mM CAPS buffer at pH
10.50 0.05 and 30 0.1 °C.
in the respective buffer, i.e. for 4-nitrophenolate in 50 mM
CAPS buffer at pH 10.5 and 30 °C, eapp ꢂ d = 8120 Mꢁ1
, for
2-chloro-4-nitrophenolate in 50 mM HEPES buffer at pH 7.5 and
30 °C, eapp ꢂ d = 7580 Mꢁ1. The values were obtained from three
independent experiments by averaging the data.
5.1. Substrate hydrolysis followed by UV/Vis spectroscopy
5.1.1. Substrate stock solution
5.2. Substrate hydrolysis followed by HPLC
Typically,
25–30 mg
(75–90 mmol)
of
the
aryl-D-
All experiments were conducted in 100 mM TES buffer at pH 8.0
and 60 °C in triplicate, and the obtained data were averaged. The
substrate stock solutions were prepared from maltose (22), cel-
lobiose (23) or lactose (24). The metal complex stock solutions
were prepared from Cu2bpdpo (1), S-Cu2bpdbo (S-2) or
R-Cu2bpdbo (R-2).
glycopyranoside substrates was dissolved in 500
diluted in a total volume of 10 mL with buffer solution yielding
7.5–10.0 mM substrate stock solutions. The substrate stock solu-
lL DMSO and
tions were then used in 25
175 L for all experiments.
lL aliquot increments between 0 and
l

S. Striegler et al. / Journal of Catalysis 338 (2016) 349–364
363
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5.2.1. Substrate stock solution
Typically, 25–30 mg (73–83 mmol) of the disaccharides was
dissolved in 1 mL nanopure water yielding 70–83 mM substrate
stock solutions. A constant 50 lL aliquot of each stock solution
was then used for all experiments.
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Anal. Chim. Acta 539 (2005) 91–95.
5.2.2. Catalyst stock solution
Typically, 5 mg of the binuclear metal complexes was dissolved
in 10 mL of buffer solution yielding 0.73 mM stock solutions. The
catalyst stock solutions were then used in constant 450
for all experiments.
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copper(II)-carbohydrate complexes, Eur. J. Inorg. Chem. (2002) 487–495.
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5.2.3. Substrate hydrolysis assay
The 50
lL aliquot of the substrate stock solutions was diluted
with 450
lL nanopure water, buffer or catalyst stock solutions
and heated to 60 °C. After 24 h, the solutions were cooled in ice,
and 100 L aliquot was taken and 30 mM sodium sulfide solution
l
SAR of thrombin inhibitors incorporating
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5.2.4. HPLC assay and data analysis
All experiments were conducted on a Shimadzu HPLC with a
Rezex-Carbohydrate Na+ (8%) column 300 ꢂ 7.8 mm and
50 ꢂ 7.8 mm guard column (Phenomenex) using nanopure water
as eluent isocratic with a flow rate of 0.4 mL/min at 80 °C and
ELS detection. The filtered samples were diluted with an equal vol-
ume of 50 mM acetic acid buffer at pH 5.0. The resulting solution
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951.
monitored for 30 min. Disaccharides elute under these conditions
between 14 and 18 min, monosaccharides elute between 20 and
24 min and the TES buffer elutes at 22.5 min. The area of the peaks
in the chromatograms was integrated using the software supplied
by Shimadzu. The percentage of hydrolysis for each sugar aliquot
was determined by correlation of the area in the respective sample
to a reference samples in water.
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properties and hydrolytic activity of an alkoxo-bridged dinuclear copper(II)
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(2006) 9109–9114.
a dinuclear copper(II) complex, Tetrahedron 62
Acknowledgments
[24] O. Sperling, A. Fuchs, T.K. Lindhorst, Evaluation of the carbohydrate
recognition domain of the bacterial adhesin FimH: design, synthesis and
binding properties of mannoside ligands, Org. Biomol. Chem. 4 (2006) 3913–
3922.
Support by the National Science Foundation (CHE-1305543)
and the Arkansas Biosciences Institute to S.S. is gratefully acknowl-
edged. 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 number P30
GM103450.
[25] A. Vervoort, C.K. De Bruyne, Synthesis of substituted phenyl
mannopyranosides, Carbohyd. Res. 12 (1970) 277–280.
a-D-
[26] Q.-H. Fan, S. Striegler, R.G. Langston, J.D. Barnett, Evaluating N-
benzylgalactonoamidines as putative transition state analogs for b-
galactoside hydrolysis, Org. Biomol. Chem. 12 (2014) 2792–2800.
[27] L. Kröger, J. Thiem, Synthesis and evaluation of glycosyl donors with novel
leaving groups for transglycosylations employing b-galactosidase from bovine
testes, Carbohydr. Res. 342 (2007) 467–481.
Appendix A. Supplementary material
[28] R.W. Gantt, P. Peltier-Pain, W.J. Cournoyer, J.S. Thorson, Using simple donors to
drive the equilibria of glycosyltransferase-catalyzed reactions, Nat. Chem. Biol.
7 (2011) 685–691.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jcat.2015.12.026.
[29] eapp,9 ꢂ d = 1532 Mꢁ1
;
e
mol,9 ~ 6000 cmꢁ1 Mꢁ1
;
eapp,10 ꢂ d = 61 Mꢁ1
, emol,10 ~
223 cmꢁ1 Mꢁ1, 50 mM acetate buffer, pH 5.00 0.05, 30.0 0.1 °C.
[30] D.R. Hwang, M.E. Scott, Synthesis, characterization, kinetic parameters, and
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