A potent bicyclic inhibitor of a family 27 α-galactosidase

Article abstract of DOI:10.1039/b704509c

Two isomeric bicyclo4.1.0heptane analogues of the glycosidase inhibitor galacto-validamine, (1R*,2S,3S,4S,5S,6S*)-5-amino-1-(hydroxymethyl) bicyclo4.1.0heptane-2,3,4-triol, have been synthesized in 13 steps from 2,3,4,6-tetra-O-benzyl-d-galactose. The inh

Full text of DOI:10.1039/b704509c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A potent bicyclic inhibitor of a family 27 a-galactosidase†
Yi Wang and Andrew J. Bennet*
Received 26th March 2007, Accepted 16th April 2007
First published as an Advance Article on the web 30th April 2007
DOI: 10.1039/b704509c
Two isomeric bicyclo[4.1.0]heptane analogues of the glycosidase inhibitor galacto-validamine,
(1R*,2S,3S,4S,5S,6S*)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptane-2,3,4-triol, have been
synthesized in 13 steps from 2,3,4,6-tetra-O-benzyl-D-galactose. The inhibitory activities of the two
conformationally restricted amines, and their corresponding acetamides, were measured against
commercial a-galactosidase enzymes from coffee bean and E. coli. The activity of the glycosyl hydrolase
family GH27 enzyme (coffee bean) was competitively inhibited by the 1R,6S-amine (7), a binding
interaction that was characterized by a Ki value of 0.541 lM. The GH36 E. coli a-galactosidase
exhibited a much weaker binding interaction with the 1R,6S-amine (IC50 = 80 lM). The diastereomeric
1S,6R-amine (9) bound weakly to both galactosidases, (coffee bean, IC50 = 286 lM) and (E. coli,
IC50 = 2.46 mM).
certain facets of the galactopyranosylium ion (1), a high-energy
Introduction
intermediate formed during the acid-catalysed hydrolysis of galac-
tosides in aqueous solution.15–17 Regardless of whether the enzyme-
catalysed glycosylation and deglycosylation steps are dissociative
(DN * AN)18,19 or “exploded” associative (ANDN)19,20 reactions, it
is clear that there is a sizable degree of cationic character on the
sugar moiety at the critical enzymatic TSs,21,22 which therefore must
bear some resemblance to the galactopyranosylium ion. Thus, 1-
deoxy-galacto-nojirimycin (2),23,24 galacto-isofagomine (3)12,25 and
galacto-validamine (4)26 mimic charge development at the TS
by incorporating a basic nitrogen atom in place of O-5, C-1,
and O-1, respectively (Fig. 1). Whereas, galacto-valienamine (5)
mimics both the ring distortion and charge development present
in galactopyranosylium ions.27
An important field in glycobiology involves design, synthesis,
and biological evaluation of inhibitors for various glycosidase
enzymes.1 Tight-binding reversible inhibitors of glycosidases have
the potential to be therapeutic agents for the treatment of various
medical conditions, including diabetes,2 cancer,3 and influenza.4
Of particular importance with respect to this report, it is known
that Fabry disease is a lysosomal storage disorder caused by a
deficiency of a-galactosidase A (a-Gal A).5 This enzyme activity
deficiency results from impaired trafficking of misfolded a-Gal A
variants to the lysosome.5 Of note, the use of low concentrations
of galactosidase inhibitors, as chemical chaperones, ameliorates
the trafficking of mutant enzymes presumably by stabilizing the
correctly folded enzymatic structure.6,7
At the present time, a-galactosidase enzymes are classified into
four glycosidase families based on protein sequence alignment
methods.8 These families have been assigned glycosyl hydrolase
(GH) numbers 4, 27, 36, and 57. All of these categorized a-
galactosidases (EC 3.2.1.22) are retaining hydrolases, that is, the
first product formed during the catalysed hydrolysis of an a-
galactopyranoside is a-galactopyranose. Moreover, as family GH
4 members operate via an atypical mechanism involving oxidation
of the 3-OH group followed by an E1CB elimination reaction,9
compounds that are designed to inhibit glycopyranosylium ion-
like transitions states are not expected to inhibit enzymes from
glycosidase family GH 4. Consequently, discussion will be limited
to retaining glycosidases that hydrolyse sugar acetal linkages via
a glycosylated enzyme intermediate.10,11 Both enzyme-catalysed
glycosylation and deglycosylation steps occur via transition states
(TSs) that have oxacarbenium ion character and a distorted six-
membered ring.
Fig. 1 Structures of the galactopyranosylium ion (1) and selected
glycosidase inhibitors that contain a basic amino group (2–6).
While no single compound effectively inhibits all galacto-
sidases12–14 most inhibitors possess structural features that mimic
In 2001, Tanaka et al. reported that sub-micromolar inhibition
was obtained for yeast a-glucosidase using the carbocyclic bicy-
clo[4.1.0]heptylamine a-glucopyranose mimic 6.28 The current re-
port details the synthesis and inhibitory activity of the structurally
similar four cyclopropane containing bicyclic galactose analogues
7-10 (Fig. 2).
Department of Chemistry, Simon Fraser University, 8888 University Drive,
Burnaby, V5A 1S6, British Columbia, Canada. E-mail: bennet@sfu.ca
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of 7 and 9. See DOI: 10.1039/b704509c
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H-1 and H-6 in the 1H NMR spectrum (6.4 Hz) relative to
that of the 14-S diastereomer (4.3 Hz).31 In addition, an NOE
contact was observed for 14-R between H-1 and H-5. Following
separation of the R-diastereomer 14-R from the pseudo-axial
isomer 14-S, a process that was accomplished by the use of radial
chromatography,32 incorporation of an azide functionality was
accomplished with inversion of configuration to give the allylic
azide 15, along with a small amount of the SN2ꢀ product (16).
Reduction of the azide, followed by acetylation gave the acetamide
17.
Fig. 2 Structures of the designed bicyclo[4.1.0]heptane-based potential
galactosidase inhibitors (7–10).
Subsequent use of the Furukawa modification of the Simmons–
Smith reaction gave the two cyclopropyl isomers in a total yield
of 88%.33 Of note, the ratio of the D-galacto (18) and L-altro (19)
diastereomers formed in this reaction was unpredictable, that is, it
varied between 2.5 : 1.0 and 1.0 : 1.5. The absolute stereochemistry
for the two diastereomers was assigned based on observed NOE
contacts between one of the H-7 protons and either H-3 or H-4 in
the 1D NOE difference spectra (Fig. 3).
Results and discussion
The route chosen for the synthesis of these inhibitors utilized
2,3,4,6-tetra-O-benzyl-D-galactose (11), which can be made in two
steps from methyl a-D-galactopyranoside,29 as starting material
(Scheme 1).
After reduction of the protected hemiacetal, selective protection
of the primary alcohol as a trityl ether, followed by a Swern
oxidation and a Wittig reaction gave olefin 12 in a yield of 47% over
these 4 steps. Removal of the trityl ether using mild acid-catalysis
and subsequent Swern oxidation and addition of vinylmagnesium
bromide gave a 1 : 2 ratio of the R : S diastereomers of an acyclic
octadienol (13-R,S). Of note, the assignment of stereochemistry
is based on the ratio of 14-R to 14-S that is formed by a ring
closing metathesis reaction catalysed by a second generation
Grubbs’ catalyst.30 Specifically, the stereochemistry of 14-R was
assigned based on the larger coupling constant observed between
Fig. 3 Observed NOE contacts for the protected bicyclo[4.1.0]heptane-
based galactoside analogues (18 and 19).
Scheme 1 Reagents and conditions: i, NaBH4, EtOH, rt, 93%; ii, TrCl, pyridine, rt, 74%; iii, DMSO, Ac2O, rt, 86%; iv, Ph3PCH3I, BuLi, −78 to 0 ◦C,
79%; v, HCl·H2O, AcOH, rt, 61%; vi, (COCl)2, DMSO, −78 ◦C, then Et3N, vii, CH2CHMgBr, −78 ◦C, R : S 1 : 2, 60%; viii, Grubbs’ catalyst, CH2Cl2,
40 ◦C, R: 32%, S: 54%; ix, (PhO)2P(O)N3, PhCH3, DBU, 60 ◦C, 15: 76%, 16: 21%; x, H2S, Et3N, H2O, rt, then AcCl, pyridine, 80%; xi, ZnMe2, CH2I2,
10 ◦C, 18 : 19 1 : 1, 88%; xii, 10% Pd–C, H2, rt, xiii, LiOH, THF : H2O 1 : 1, 70 ◦C.
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Table 1 Inhibitory activity (IC50/lM) of 2, 3, 4, 5, 7, 8, 9, and 10 against two a-galactosidases
Enzyme
2
3
4
5
7
8
9
10
Coffee bean
E. coli
0.0016a
0.24a
200b
500c
890c
56d
(0.541 0.018)e
343 14
N.I.f
286 15
2460 130
N.I.f
N.I.h
N.R.g
N.R.g
80
6
a Reference 24. b Reference 25. c Reference 26. d Reference 27. e Ki value. f No inhibition at 1.0 mM. g Not reported. h No inhibition at 5.0 mM.
Removal of the benzyl protecting groups was accomplished
using conventional hydrogenation conditions to give the amides 8
and 10 in good yields. Subsequent hydrolysis of the amide groups,
using LiOH in THF–H2O gave the corresponding amines 7 and
9. It was not possible to obtain crystals of either 7 or 9, however,
their conformations in solution are likely similar to their respective
gluco-analogues28 because of the similarity of 1H–1H coupling
constants for the pseudo-anomeric hydrogen atom.
Amines 7 and 9, and their respective acetamido compounds 8
and 10 were tested as inhibitors against two commercially available
a-galactosidase enzymes. Measured inhibition parameters (Ki or
IC50 values) for these four bicyclo[4.1.0]heptane derivatives and
the reported values for 2, 3, 4 and 5 are listed in Table 1.
The plot (Fig. 4) for inhibition of 4-nitrophenyl a-D-galacto-
pyranoside hydrolysis catalysed by coffee bean a-galactosidase on
the addition of 7 shows that this compound binds competitively
to the active site of the enzyme. Specifically, fitting the kinetic
data displayed in Fig. 4 to three standard models of inhibition,
namely: competitive, non-competitive, and uncompetitive results
in the following “goodness of fit” (R2 values) of 0.999, 0.983, and
0.956, respectively.
(cf., 4, 5, and 7 in Table 1), an observation that matches the
inhibition of yeast a-glucosidase by compound 6, valienamine and
validamine.28
Given that all IC50 values were measured at low substrate
concentration (between 0.20 and 0.25 times Km) the reported
IC50 values are between 1.20 and 1.25 times larger than the
true inhibition constant Ki,34 assuming that all inhibitors bind
competitively to the enzyme. With respect to inhibition of the
GH27 enzyme (coffee bean), the comparison between the galacto-
bicyclic inhibitor 7 and both 5 and 9 is particularly interesting
since these compounds are structurally very similar, and 7 is ∼100
fold (11.9 kJ mol−1) tighter binder than either the alkene (5) or the
altro-isomer (9). This observation suggests that in compound 7
the hydroxymethyl group (CH2OH) has more optimal interactions
with the coffee bean enzyme, but that in both 5 and 9 no severe
steric interactions are introduced by changing the position of this
group relative to the carbasugar portion of the inhibitors.
Also, the contrast between the potency of amine 7 and
acetamide 8 supports the idea that a positive charge enhances
inhibitor binding to the enzymatic active site, in this case the
difference in the free energy of binding is 14.5 kJ mol−1.
Given that both glycosyl hydrolase families GH27 and GH36
share a commonality of mechanism,35 it is not surprising that
inhibitor 7 is also the tightest binder to the GH36 enzyme from
E. coli. (cf., 4, 7, and 9 in Table 1 bottom row). An extended
discussion on the differences in binding affinity of 7 between the
two GH family members is unwarranted until structural data is
available.
Also of note, the weakest binding inhibitor of the three com-
pounds that are present in 4C1 conformations (2, 3 and 4; Table 1)
is 4, the inhibitor where the basic nitrogen atom is incorporated in
place O-1. It remains to be seen whether analogues of 2 and 3 in
which the pyranosyl ring is held in non-chair conformations are
tighter binding inhibitors than the parent compounds themselves
as is the case for 7.
Conclusions
A
tight binding competitive inhibitor of coffee bean a-
galactosidase (7) was made in 13 steps from 2,3,4,6-tetra-O-
benzyl-D-galactose in an overall yield of 0.6%.
Fig. 4 Plot for inhibition of coffee bean a-galactosidase by 7. Drawn lines
are for the fit of the data to a competitive inhibition model. Concentrations
of inhibitor 7 are: ᭹ 0.0 lM, ꢀ0.3 lM, ᭺ 0.5 lM, ꢁ 1.5 lM, andꢁ3.0 lM.
Experimental
Thin-layer chromatography (TLC) was performed on aluminium-
Moreover, the measured Ki value (541 18 nM) for inhibition
of coffee bean a-galactosidase shows that 7 is the tightest binding
inhibitor of those in which the basic nitrogen atom of the inhibitor
is located in the position of a substrate’s glycosidic oxygen atom
backed TLC plates pre-coated with Merck silica gel 60 F254.
Compounds were visualized with UV light and/or staining with
phosphomolybdic acid (5% solution in EtOH). Flash chromatog-
raphy was performed using Avanco silica gel 60 (230–400 mesh).
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Radial chromatography was performed on a Harrison Research
Inc. model 8924 chromatotron. Solvents used for anhydrous
reactions were dried and distilled immediately prior to use.
Methanol was dried and distilled over magnesium methoxide.
Dichloromethane was dried and distilled over calcium hydride.
Glassware for anhydrous reactions was flame-dried and cooled
under a nitrogen atmosphere immediately prior to use. NMR
spectra were recorded on a Varian Unity 500 MHz spectrometer.
Chemical shifts (d) are listed in ppm downfield from TMS using
H-2), 4.00 (dt, 1 H, H-5), 4.18 (t, 1 H, H-3), 4.31 (d, 1 H, JA,B
=
11.7, CHAHBC6H5), 4.37 (d, 1 H, JC,D = 12.0, CHCHDC6H5), 4.42
(d, 1 H, CHCHDC6H5), 4.44 (d, 1 H, CHAHBC6H5), 4.51 (d, 1 H,
JE,F = 11.7, CHEHFC6H5), 4.62 (d, 1 H, JG,H = 11.0, CHGHHC6H5),
4.66 (d, 1 H, CHEHFC6H5), 4.73 (d, 1 H, CHGHHC6H5), 7.09–7.44
(m, 35 H, H-Ar), 13C NMR (CDCl3) d 63.3 (C-1), 70.2 (C-5),
71.5 (C-6), 72.8, 73.0, 73.4, 75.2 (4 × CH2C6H5), 77.1 (C-4), 78.9
(C-2), 79.8 (C-3), 87.1 (CPh3), 127.2, 127.7, 127.8, 127.9, 128.0,
128.2, 128.3, 128.4, 128.44, 128.8, 138.2, 138.2, 138.4, 138.5, 144.0.
Found, C, 80.5; H, 6.6. C53H52O6 requires C, 81.1; H, 6.7%.
1
the residual solvent peak as an internal reference. H and 13C
1
NMR peak assignments are made based on H–1H COSY and
1H–13C HMQC experiments. Coupling constants are reported in
Hz. IR spectra were recorded on a Bomem IR spectrometer and
samples were prepared as cast evaporative films on NaCl plates
from CH2Cl2. Optical rotations were measured using a Perkin-
Elmer 341 polarimeter and are reported in units of deg cm2 g−1
(concentrations reported in units of g per 100 cm3).
Green coffee bean (GH27) and E. coli (GH36) a-galactosidases
were purchased from Sigma-Aldrich and Calbiochem, respec-
tively.
(3R,4R,5S)-1,3,4,5-Tetrabenzyloxy-6-trityloxy-2-hexanone
Acetic anhydride (17.4 cm3) was added to a solution of 2,3,4,6-
tetra-O-benzyl-1-O-trityl-D-galactitol (6.7 g, 8.5 mmol) in dry
DMSO (27 cm3). The reaction mixture was stirred for 16 h at rt
under N2. Following the addition of water (40 cm3) to the reaction
mixture, aqueous NH3 was added. The resultant mixture was
extracted with Et2O (3 × 30 cm3) and the combined organic layers
were washed with brine (60 cm3), dried (MgSO4), and concentrated
under reduced pressure. The residue was purified by flash column
chromatograph (hexane : EtOAc, 9 : 1 v/v) to give a yellow syrup
2,3,4,6-Tetra-O-benzyl-D-galactitol
(5.72 g, 86%): [a]D20 +0.17 (c = 0.60, CH2Cl2), IR 1731 cm−1 (C O),
=
NaBH4 (2.8 g, 74 mmol) was added to a solution of 1129 (17.5 g,
32.4 mmol) in ethanol (180 cm3). After the reaction mixture had
been stirred for 15 h it was diluted with ether (300 cm3), and it was
then washed with water (2 × 100 cm3) and brine (100 cm3), dried
(MgSO4), and concentrated under reduced pressure. The residue
was purified by flash column chromatography (hexane : EtOAc,
2 : 1 v/v) to give a colourless syrup (16.3 g, 93%): [a]2D0 −5.5 (c =
1.85, CH2Cl2), IR 3443 cm−1 (br, O–H), 1H NMR (CDCl3) d 3.50
1H NMR (CDCl3) d 3.33 (dd, 1 H, J
= 10.4, J5,6 = 5.3, H-6),
ꢀ
6,6
= 3.7, H-6ꢀ), 3.84 (m, 1 H, H-5), 3.88 (d,
ꢀ
3.46 (dd, 1 H, J
5,6
1 H, J3,4 = 3.9, H-3), 4.22 (dd, 1 H, J4,5 = 5.7, H-4), 4.25–4.46
(m, 6 H, CH2C6H5), 4.51 (d, 1 H, JA,B = 11.6, CHAHBC6H5),
4.58 (d, 1 H, CHAHBC6H5), 4.60 (br s, 2 H, H-1, H-1ꢀ), 7.01–
7.44 (m, 35 H, H-Ar), 13C NMR (CDCl3) d 63.3 (C-6), 72.4, 73.0,
73.05, 74.4 (4 × CH2C6H5), 74.5 (C-1), 79.0 (C-5), 80.4 (C-4), 82.6
(C-3), 86.8 (CPh3), 127.0, 127.4, 127.5, 127.6, 127.7, 127.77, 127.8,
127.9, 128.1, 128.2, 128.26, 128.28, 128.3, 128.4, 128.6, 137.3, 137.6,
137.9, 138.3, 143.9, 207.4 (C-2). Found, C, 80.95; H, 6.7. C53H50O6
requires C, 81.3; H, 6.4%.
ꢀ
ꢀ
(dd, 1 H, J6,6 = 9.1, J5,6 = 6.7, H-6), 3.54 (dd, 1 H, J5,6 = 5.5,
H-6ꢀ), 3.68–3.73 (m, 2 H, H-3, H-1), 3.79 (m, 1 H, H-1ꢀ), 3.86–3.91
(m, 2 H, H-2, H-4), 4.03 (m, 1 H, H-5), 4.42 (d, 1 H, JA,B = 11.9,
CHAHBC6H5), 4.47 (d, 1 H, CHAHBC6H5), 4.49 (d, 1 H, JC,D
=
11.3, CHCHDC6H5), 4.60 (d, 1 H, JE,F = 11.6, CHEHFC6H5), 4.64
(d, 1 H, CHCHDC6H5), 4.67 (d, 1 H, CHEHFC6H5), 4.68 (d, 1 H,
JG,H = 11.3, CHGHHC6H5), 4.75 (d, 1 H, CHGHHC6H5), 7.22–7.36
(m, 20 H, H-Ar), 13C NMR (CDCl3) d 60.8, 69.7, 70.8, 72.2, 73.1,
73.6, 74.2, 77.3, 79.0, 79.9, 127.6, 127.7, 128.0, 128.2, 128.3, 137.7,
137.8, 137.9.
(3S,4S,5S)-3,4,5-Tribenzyloxy-2-((benzyloxy)methyl)-
6-trityloxy-1-hexene (12)
To a solution of methyltriphenylphosphonium iodide (1.13 g,
2.8 mmol) in THF (10 cm3), n-BuLi (2.5 M, 1.12 cm3, 2.8 mmol)
was added at −78 ◦C. After stirring the reaction at this temperature
for 1 h, the mixture was warmed up to 0 ◦C, and a solution
of the fully protected 2-hexanone (1.00 g, 1.28 mmol) in THF
(5 cm3) was added dropwise. The resulting mixture was allowed
to warm to rt over a period of 16 h. The reaction was quenched
by the addition of saturated NH4Cl, and it was then diluted with
water (20 cm3) and extracted with CH2Cl2 (3 × 20 cm3). The
combined organic layers were washed with aqueous H2SO4 (10%),
saturated NaHCO3, brine, dried (MgSO4), and concentrated at
reduced pressure. The resultant crude syrup was purified by flash
column chromatography (hexane : EtOAc, 9 : 1 v/v) to give the
2,3,4,6-Tetra-O-benzyl-1-O-trityl-D-galactitol
To a solution of 2,3,4,6-tetra-O-benzyl-D-galactitol (5.4 g,
10.0 mmol) in dry pyridine (17 cm3) chlorotriphenylmethane (3.1 g,
11.0 mmol) was added. The reaction mixture was stirred at rt for
72 h, and it was then poured onto ice (200 cm3). The resultant
slurry was stirred for 2 h at 0 ◦C, and the aqueous layer was
decanted from the syrup, which was then dissolved in chloroform
(120 cm3) and washed with aqueous acetic acid (10%, 30 cm3),
saturated NaHCO3, brine, dried (MgSO4), and concentrated under
reduced pressure. The resultant residue was purified by flash
column chromatography (hexane : EtOAc, 6 : 1 v/v) to give a
light yellow syrup (5.8 g, 74%): [a]2D0 +22.5 (c = 1.32, CH2Cl2), IR
product as a light yellow syrup (0.784 g, 79%): [a]2D0 +22.5 (c =
1
1.32, CH2Cl2), IR 1650 cm−1 (C C); H NMR (CDCl3) d 3.25 (dd,
=
1 H, J6,6 = 10.0, J6,5 = 5.6, H-6), 3.27 (dd, J6 ,5 = 5.3, H-6ꢀ), 3.81
ꢀ
ꢀ
ꢀ
(br q, 1 H, J6,5 + J6 ,5 + J5,4 = 14.7, H-5), 3.86 (dd, 1 H, J4,5 = 3.9,
1
3529 cm−1 (br, O–H), H NMR (CDCl3) d 3.23 (dd, 1 H, J1,1
=
J4,3 = 7.1, H-4), 4.04–4.16 (m, 4 H, H-3, H-7, H-7ꢀ, CHAHBC6H5),
ꢀ
ꢀ
10.3, J1,2 = 4.9, H-1), 3.41 (dd, 1 H, J6,6 = 9.4, J6,5 = 6.0, H-6), 3.45
4.43 (d, 1 H, JC,D = 11.0, CHCHDC6H5), 4.47 (d, 1 H, JB,A =
(dd, 1 H, J1 ,2 = 4.7, H-1ꢀ), 3.53 (dd, 1 H, J6 ,5 = 6.4, H-6ꢀ), 3.69 (dd,
11.6, CHAHBC6H5), 4.48 (d, 1 H, JE,F = 12.0, CHEHFC6H5), 4.49
(d, 1 H, JG,H = 11.6, CHGHHC6H5), 4.52 (d, 1 H, CHEHFC6H5),
ꢀ
ꢀ
ꢀ
1 H, J4,5 = 1.7, J4,3 = 5.2, H-4), 3.84 (q, 1H, J2,3 + J2,1 + J2,1 = 14.5,
1734 | Org. Biomol. Chem., 2007, 5, 1731–1738
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4.57 (d, 1 H, CHCHDC6H5), 4.64 (d, 1 H, CHGHHC6H5), 5.13
(br s, 1 H, H-1), 5.44 (m, 1 H, H-1ꢀ), 7.07–7.43 (m, 35 H, H-Ar),
13C NMR (CDCl3) d 63.7 (C-6), 70.4 (C-7), 71.0, 72.8, 73.7, 74.8
(4 × CH2C6H5), 78.6 (C-5), 79.8 (C-3), 81.2 (C-4), 87.0 (CPh3),
115.9 (C-1), 127.0, 127.4, 127.5, 127.6, 127.7, 127.8, 127.9, 128.1,
128.3, 128.38, 128.4, 128.8, 138.5, 138.53, 138.6, 139.0, 144.0 (C-2),
144.1. Found, C, 82.8; H, 6.8. C54H52O5 requires C, 83.05; H, 6.7%.
Compound 13-R. 1H NMR (CDCl3): d 2.66 (d, 1 H, JOH,3
5.6, OH), 3.66 (t, 1 H, J4,5 + J4,3 = 9.0, H-4), 3.80 (dd, 1 H, J5,4
=
=
4.8, J5,6 = 7.1, H-5), 4.05–4.18 (m, 3 H, H-6, H-9, H-9ꢀ), 4.24–4.31
(m, 2 H, H-3, CHAHBC6H5), 4.45–4.70 (m, 7 H, CH2C6H5), 5.15
(dt, 1 H, J1E,2 = 10.5, J1E,1Z + J1E,3 = 2.7, H-1E), 5.26 (dt, 1 H,
J1Z,2 = 17.2, J1Z,1E + J1Z,3 = 2.9, H-1Z), 5.44 (br s, 1 H, H-8), 5.56
(d, 1 H, J8,8 = 1.5, H-8ꢀ), 5.86 (m, 1 H, H-2), 7.36–7.21 (m, 20 H,
ꢀ
13
H-Ar), C NMR (CDCl3) d 70.5 (C-3), 81.1 (C-5), 82.1 (C-4),
116.3 (C-1), 116.4 (C-8), 138.2 (C-2), 143.8 (C-7).
(2S,3S,4S)-2,3,4-Tribenzyloxy-5-((benzyloxy)methyl)-
5-hexen-1-ol
Compound 13-S. 1H NMR (CDCl3): d 2.62 (d, 1 H, JOH,3 = 5.8,
Aqueous HCl (1 M, 1 cm3) was added to a solution of 12 (10.2 g,
13.1 mmol) in acetic acid (90%, 145 cm3). After stirring this
mixture for 40 min, the reaction was quenched by the addition
of saturated NaHCO3. The resultant solution was extracted
with ether (3 × 50 cm3) and the combined organic layers were
washed with brine, dried (MgSO4), and concentrated under
reduced pressure. The crude product was purified by flash column
chromatography (hexane : EtOAc, 3 : 1 v/v) to give a light yellow
syrup (4.2 g, 61%): [a]D20 + 22.8 (c = 0.71, CH2Cl2), IR 3470 cm−1
OH), 3.71 (dd, 1 H, J4,5 = 3.1, J4,3 = 5.5, H-4), 3.86 (dd, 1 H, J5,6
=
8.0, H-5), 4.05–4.19 (m, 4H, H-6, H-9, H-9ꢀ, CHAHBC6H5), 4.41
(m, 1 H, H-3), 4.45–4.70 (m, 7 H, CH2C6H5), 5.21 (dt, 1 H, J1E,2
=
10.6, J1E,1Z + J1E,3 = 3.2, H-1E), 5.37 (dt, 1 H, J1Z,2 = 17.2, J1Z,1E
+
ꢀ
J1Z,3 = 3.3, H-1Z), 5.47 (br s, 1 H, H-8), 5.58 (d, 1 H, J6,8 = 1.5,
H-8), 5.94 (m, 1 H, H-2), 7.36–7.21 (m, 20 H, H-Ar), 13C NMR
(CDCl3) d 72.2 (C-3), 80.4 (C-5), 80.6 (C-4), 116.2 (C-1), 116.5
(C-8), 138.1 (C-2), 143.8 (C-7).
(1R*,4S,5S,6S)-4,5,6-Tribenzyloxy-3-((benzyloxy)methyl)-
cyclohex-2-enol (14-R and 14-S)
1
ꢀ
(br, O–H), H NMR (CDCl3) d 3.59 (dd, 1 H, J1,2 = 4.8, J1,1
=
11.7, H-1), 3.66 (dd, 1 H, J1 ,2 = 4.5, H-1ꢀ), 3.70–3.76 (m, 2 H,
ꢀ
H-2, H-3), 4.06–4.18 (m, 3 H, H-4, H-7, H-7ꢀ), 4.22 (d, 1 H, JA,B
=
To a solution of the diastereomeric mixture of 13-R and 13-S (2.3 g,
4.1 mmol) in dry CH2Cl2 (1.1 L) was added second generation
Grubbs’ catalyst (174.3 mg, 0.21 mmol). This reaction mixture
was heated to reflux for 3 h under N2, and then the volatiles
were removed under reduced pressure. The resultant residue was
purified by flash column chromatography (hexane : EtOAc, 2 :
1 v/v) to give a colourless syrup. The two diastereomers were
separated by radial chromatography (CH2Cl2 : EtOAc, 25 : 1 v/v)
to give 14-R (0.7 g, 32%) and 14-S (1.2 g 54%) as colourless syrups.
11.4, CHAHBC6H5), 4.48–4.62 (m, 7 H, CH2C6H5), 5.45 (br s,
1 H, H-6), 5.55 (br s, 1 H, H-6ꢀ), 7.20–7.34 (m, 20 H, H-Ar), 13
C
NMR (CDCl3) d 62.0 (C-1), 70.6 (C-7), 70.5, 72.8, 73.2, 74.4 (4 ×
CH2C6H5), 79.7, 81.0 (C-3, C-2), 80.1 (C-4), 116.2 (C-6), 127.6,
127.7, 127.7, 127.9, 128.0, 128.3, 128.4, 138.0, 138.2, 138.3, 138.6,
143.7 (C-5). Found, C, 77.78; H, 6.87. C35H38O5 requires C, 78.0;
H, 7.1%.
(2S,3S,4S)-2,3,4-Tribenzyloxy-5-((benzyloxy)methyl)-5-hexenal
Compound 14-R. [a]D20 +15.8 (c = 1.61, CH2Cl2), IR 3420 cm−1
To a solution of oxalyl chloride (0.230 cm3, 2 M in CH2Cl2,
(br, O–H), 1H NMR (CDCl3): d 3.64 (dd, 1 H, J5,4 = 3.5, J5,6 = 9.2,
◦
0.46 mmol) in CH2Cl2 (2.6 cm3) at −78 C, DMSO (0.11 cm3)
ꢀ
H-5), 3.88 (d, 1 H, J7,7 = 12.1, H-7), 3.96 (dd, 1 H, J6,1 = 6.4, H-6),
4.08 (d, 1 H, H-7ꢀ), 4.14 (m, 1 H, H-1), 4.27 (d, 1 H, H-4), 4.38
was added slowly. This mixture was stirred for 10 min, and then a
solution of the hexenol (206 mg, 0.38 mmol) in CH2Cl2 (1.5 cm3)
was added dropwise. After 10 min, triethylamine (0.26 cm3) was
added to the reaction mixture, which was kept at −78 ◦C for
40 min. The solvent was then evaporated under reduced pressure
to give a light yellow syrup of aldehyde (195.6 mg), which was used
in the next step without being purified further.
(d, 1 H, JA,B = 11.8, CHAHBC6H5), 4.44 (d, 1 H, CHAHBC6H5),
4.58 (d, 1 H, JC,D = 11.3, CHCHDC6H5), 4.75 (d, 1 H, JE,F
=
11.6, CHEHFC6H5), 4.78–4.81 (m, 2 H, CH2C6H5), 4.89 (d, 1 H,
CHCHDC6H5), 4.91 (d, 1 H, CHEHF C6H5), 5.76 (d, 1 H, J2,1 = 2.7,
H-2), 7.25–7.40 (m, 20 H, H-Ar), 13C NMR (CDCl3) d 70.6 (C-7),
71.1 (C-1), 72.4 (CHAHBC6H5), 73.3 (CHGHHC6H5), 73.5 (C-4),
74.3 (CHEHFC6H5), 74.4 (CHCHDC6H5), 79.5 (C-5), 80.6 (C-6),
127.6, 127.64, 127.66, 127.7, 127.8, 128.0, 128.01 (C-2), 128.1, 128.3,
128.4, 128.42, 128.48, 135.4, 138.1, 138.4, 138.6, 138.7. Found, C,
78.0; H, 6.85. C35H36O5 requires C, 78.3; H, 6.8%.
(3R*,4S,5S,6S)-4,5,6-Tribenzyloxy-7-((benzyloxy)methyl)octa-
1,7-dien-3-ol (13-R and 13-S)
To a solution of the freshly prepared aldehyde in dry THF
(50 cm3) at −78 ◦C under N2 was slowly added a solution of
vinylmagnesium bromide in THF (0.30 cm3, 1.0 M, 0.30 mmol).
The reaction mixture was then stirred for 1 h at rt, after which it
was quenched by the addition of a saturated aqueous NH4Cl. The
resultant solution was extracted with EtO2 (3 × 60 cm3) and the
combined organic layers were washed with brine, dried (MgSO4),
and concentrated under reduced pressure to give crude product
as an orange coloured syrup. This material was purified by flash
column chromatography (hexane : EtOAc, 4 : 1 v/v) to give a
pale yellow syrup (129 mg, ratio 13-R : 13-S = 1 : 2, 60% over 2
steps): IR 3463 cm−1 (br, O–H). Found, C, 78.7; H, 7.05. C37H40O5
requires C, 78.7; H, 7.1%. The NMR spectral assignments given
below were made on the mixture of diastereomers.
(3S,4S,5S,6S)-3-Azido-4,5,6-tribenzyloxy-1-((benzyloxy)methyl)-
cyclohexene (15) and (3S,4S,5R,6S)-3-azido-4,5,6-tribenzyloxy-3-
((benzyloxy)methyl)cyclohexene (16)
To a solution of 14-R (754.2 mg, 1.4 mmol) in dry toluene (62 cm3)
was added diphenyl phosphoryl azide (1.8 cm3, 8.4 mmol) followed
◦
by the slow addition of DBU (1.2 cm3, 8.0 mmol) at 0 C under
a N2 atmosphere. The reaction mixture was stirred for 1.5 h at
0
◦C, after which sodium azide (374 mg, 5.8 mmol) and 15-
crown-5 (0.6 cm3, 3 mmol) were added. The reaction mixture was
subsequently heated to 60 ◦C for 14 h. The resultant mixture
was then diluted with EtOAc (120 cm3), and washed with aqueous
H2SO4 (10%), saturated aqueous NaHCO3, brine, dried (Na2SO4),
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and concentrated under reduced pressure. The syrupy residue was
purified by flash column chromatography (hexane : EtOAc, 8 :
1 v/v) to give colourless syrups of 15 (600 mg, 76%), and the SN2ꢀ
tertiary azide product 16 (160 mg, 21%).
(1R*,2S,3S,4S,5S,6S*)-5-Acetamido-2,3,4-tribenzyloxy-1-
((benzyloxy)methyl)bicyclo[4.1.0]heptane (18: 1R,6S) and
(19: 1S,6R)
Under N2, a solution of compound 17 (523 mg, 0.91 mmol) in
dry toluene (125 cm3) was cooled down to −10 ◦C. Dimethylzinc
(2 M in toluene, 8.2 cm3, 1◦6.3 mmol) was added dropwise. The
reaction was stirred at −10 C for 15 min, then CH2I2 (1.68 cm3,
2.083 mmol) was added slowly. The reaction mi◦xture was stirred
for 16 h while being allowed to warm up to 20 C. The reaction
was quenched by the addition of aqueous H2SO4 (10%: 4 cm3),
and subsequently EtOAc (200 cm3) was added. The resulting
organic layer was washed with saturated aqueous NaHCO3, brine,
dried (MgSO4), and concentrated under reduced pressure. The
two cyclopropyl isomers were separated and purified by radial
chromatography (CH2Cl2 : MeOH 200 : 1 v/v) to give colourless
syrups of 18 and 19 (total weight 470 mg, 88%).
Compound 15. [a]D20 +76.0 (c = 1.23, CH2Cl2), IR 2030 cm−1
(N3), 1H NMR (CDCl3) d 3.86 (dd, 1 H, J5,6 = 3.5, J5,4 = 8.1, H-5),
3.89 (d, 1 H, J7,7 = 12.9, H-7), 4.07–4.15 (m, 3 H, H-7ꢀ, H-3, H-4),
ꢀ
4.24 (d, 1 H, H-6), 4.39 (d, 1 H, JA,B = 11.9, CHAHBC6H5), 4.46 (d,
1 H, CHAHBC6H5), 4.52 (d, 1 H, JC,D = 11.3, CHCHDC6H5), 4.68–
4.79 (m, 4 H, CH2C6H5), 4.81 (d, 1 H, CHCHDC6H5), 5.72 (d, 1 H,
J2,3 = 3.7, H-2), 7.19–7.42 (m, 20 H, H-Ar), 13C NMR (CDCl3)
d 58.5 (C-3), 70.1 (C-7), 72.0 (CHAHBC6H5), 73.4 (CH2C6H5),
73.8 (C-6), 73.83 (CH2C6H5), 74.3 (CHCHDC6H5), 76.5 (C-4), 76.6
(C-5), 120.1, 120.14, 122.2 (C-2), 125.6, 127.63, 127.65, 127.71,
127.73, 127.78, 127.8, 127.9, 128.2, 128.31, 128.38, 128.4, 129.8,
138.0, 138.1, 138.4, 138.5, 139.0. Found, C, 74.5; H, 6.2; N, 7.3.
C35H35N3O4 requires C, 74.8; H, 6.3; N, 7.5%.
Compound 18. [a]D20 +67.2 (c = 0.661, CH2Cl2), 1H NMR
(CDCl3) d 0.63–0.71 (m, 2 H, H-7, H-7ꢀ), 1.33 (m, 1 H, H-6),
1
Compound 16. IR 2099 cm−1, H NMR (CDCl3) d 3.65 (d,
ꢀ
1.92 (s, 3 H, CH3), 2.72 (d, 1 H, J8,8 = 9.5, H-8), 3.54 (dd, 1 H,
1 H, J7,7 = 9.7, H-7), 3.69 (d, 1 H, H-7ꢀ), 3.91 (m, 1 H, H-4), 3.98
J3,2 = 2.6, J3,4 = 6.8, H-3), 3.95 (t, 1 H, J4,5 + J4,3 = 13.7, H-4), 4.09
ꢀ
(d, 1 H, H-8ꢀ), 4.29 (d, 1 H, H-2), 4.38–4.42 (d, 2 H, CH2C6H5),
(dd, 1H, J5,6 + J5,4 = 9.6, H-5), 4.40 (m, 1 H, H-6), 4.52 (d, 1 H,
JA,B = 12.0, CHAHBC6H5), 4.56 (d, 1 H, CHAHBC6H5), 4.59 (d,
1 H, JC,D = 11.2, CHCHDC6H5), 4.64–4.76 (m, 4 H, CH2C6H5),
4.90 (d, 1 H, CHCHDC6H5), 5.61–5.67 (m, 1 H, H-2), 6.01 (dd,
1 H, J1,2 = 10.0, J1,6 = 2.2, H-1), 7.19–7.40 (m, 20 H, H-Ar).
4.45 (d, 1 H, JA,B = 12.0, CHAHBC6H5), 4.49 (d, 1 H, JC,D
=
11.5, CHCHDC6H5), 4.60 (d, 1 H, JE,F = 11.3, CHEHFC6H5), 4.65
(d, 1 H, JG,H = 11.9, CHGHHC6H5), 4.74 (m, 1 H, H-5), 4.76 (d,
1 H, CHGHHC6H5), 4.83 (d, 1 H, CHEHFC6H5), 6.00 (d, 1 H,
J5,NH = 7.1, NH), 7.18–7.37 (m, 20 H, H-Ar), 13C NMR (CDCl3)
d 13.8 (C-7), 20.2 (C-6), 23.6 (CH3), 26.1 (C-1), 45.4 (C-5), 72.6,
73.1, 73.3 (3 × CH2C6H5), 74.3 (C-8), 74.7 (CH2C6H5), 75.0 (C-2),
77.1 (C-4), 79.3 (C-3), 127.5, 127.62, 127.67, 127.7, 127.95, 127.97,
(3S,4S,5S,6S)-3-Acetamido-4,5,6-tribenzyloxy-1-
((benzyloxy)methyl)cyclohexene (17)
=
128.0, 128.4, 128.5, 128.6, 138.0, 138.4, 138.6, 139.2, 169.8 (C O).
Found, C, 76.9; H, 7.1; N, 2.65. C38H41NO5 requires C, 77.1; H,
7.0; N, 2.4%.
Through a solution containing 15 (77 mg, 0.14 mmol) in pyridine,
Et3N, and H2O (4 : 1 : 1 v/v/v, 2 cm3) H2S gas was bubbled for
3 h at rt. Then N2 gas was used to purge the aqueous reaction
mixture, and this resulting mixture was concentrated under
reduced pressure. The syrupy residue was dissolved in dry toluene
(2.6 cm3) that contained pyridine (0.260 cm3). Acetyl chloride
(0.052 cm3) was then added slowly. The reaction mixture was
stirred for 1 h at rt, after which it was diluted with EtOAc (10 cm3),
and washed with H2SO4 (10%), saturated aqueous NaHCO3, brine,
dried (Na2SO4), and concentrated under reduced pressure. The
residue was purified by flash column chromatography (hexane :
EtOAc, 2 : 1 v/v) to give 17 as a colourless syrup (63 mg, 80%):
Compound 19. [a]D20 +18.7 (c = 1.20, CH2Cl2), 1H NMR
ꢀ
(CDCl3) d 0.63 (dd, 1 H, J6,7 = 9.4, J7,7 = 5.1 H-7), 0.91–0.96
(m, 1 H, H-6), 1.28 (t, 1 H, J7 ,6 + J7 ,7 = 10.8, H-7ꢀ), 1.93 (s, 3 H,
ꢀ
ꢀ
ꢀ
CH3), 2.81 (d, 1 H, J8,8 = 10.2, H-8), 3.66 (m, 1 H, H-4), 3.75–3.80
(m, 2 H, H-3, H-8ꢀ), 4.32 (d, 1 H, J2,3 = 4.3, H-2), 4.37–4.43 (m, 3 H,
H-5, 2 × CH2C6H5), 4.45–4.60 (m, 6 H, CH2C6H5), 6.10 (d, 1 H,
JNH,5 = 8.5, NH), 7.22–7.36 (m, 20 H, H-Ar), 13C NMR (CDCl3)
d 13.1 (C-7), 22.7 (C-6), 23.6 (CH3), 24.2 (C-1), 45.4 (C-5), 72.6
(CH2C6H5), 71.9 (C-2), 72.5, 72.6, 72.7 (3 × CH2C6H5), 74.8 (C-
3), 75.9 (C-8), 76.8 (C-4), 127.6, 127.7, 127.8, 128.0, 128.1, 128.4,
[a]D20 +69.7 (c = 3.76, CH2Cl2), IR 3291 cm−1 (N–H), 1650 cm−1
1
=
(C O); H NMR (CDCl3) d 1.92 (s, 3 H, CH3), 3.81 (dd, 1 H,
=
128.5, 128.7, 138.0, 138.3, 138.6, 138.7, 169.6 (C O). Found, C,
77.2; H, 7.0; N, 2.1. C38H41NO5 requires C, 77.1; H, 7.0; N, 2.4%.
J5,4 + J5,6 = 9.8, H-5), 3.88–3.93 (m, 2 H, H-7, H-4), 4.25 (d, 1 H,
J7 ,7 = 12.1, H-7ꢀ), 4.30 (br s, 1 H, H-6), 4.42 (d, 1 H, JA,B = 11.8,
ꢀ
CHAHBC6H5), 4.43 (d, 1 H, JC,D = 11.8, CHC HDC6H5), 4.45 (d,
1 H, CHAHBC6H5), 4.48 (d, 1 H, JE,F = 11.5, CHEHFC6H5), 4.49
(d, 1 H, CHCHDC6H5), 4.60 (d, 1 H, CHEHF C6H5), 4.67 (d, 1 H,
JG,H = 12.3, CHGHHC6H5), 4.71 (d, 1 H, CHGHHC6H5), 4.91–4.97
(m, 1 H, H-3), 5.63 (br s, 1 H, H-2), 6.73 (d, 1 H, JNH,3 = 8.9, NH),
7.16–7.37 (m, 20 H, H-Ar), 13C NMR (CDCl3) d: 23.4 (CH3), 45.8
(C-3), 70.1 (C-7), 72.4 (CHEHFC6H5), 72.8 (CHCHDC6H5), 72.9
(CHGHHC6H5), 73.0 (CHAHBC6H5), 73.2 (C-6), 73.7 (C-5), 75.8
(C-4), 125.4 (C-2), 127.5, 127.6, 127.7, 127.8, 128.0, 128.02, 128.05,
128.08, 128.3, 128.4, 128.5, 135.9 (C-1), 137.8, 138.3, 138.32, 138.4,
(1R,2S,3S,4S,5S,6S)-5-Acetamido-1-(hydroxymethyl)bicyclo-
[4.1.0]heptane-2,3,4-triol (8)
A mixture of 18 (467 mg, 0.79 mmol) and 10% Pd–C (217 mg)
in MeOH (90 cm3) was stirred at room temperature under an
atmosphere of H2 for 14 h. The mixture was filtered through a
Celite pad, which was washed thoroughly with MeOH (50 cm3).
The filtrate and washings were combined and concentrated under
pressure to give a colourless oil. This material was purified by
flash column chromatography (CH2Cl2–MeOH, 4 : 1 v/v) to give
compound 8 (151 mg, 83%) as a colourless syrup: [a]2D0 +92.7 (c =
=
169.4 (C O). Found, C, 76.7; H, 7.0; N, 2.5. C35H39NO5 requires
1
C, 76.9; H, 6.8; N, 2.4%.
0.52, MeOH), H NMR (D2O) d 0.58–0.70 (m, 2 H, H-7, H-7ꢀ),
1736 | Org. Biomol. Chem., 2007, 5, 1731–1738
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to give 9 as a colourless syrup (20.7 mg, 84%): [a]2D0 +72.7 (c =
0.85, MeOH : H2O 1 : 1 v/v), 1H NMR (CD3OD) d 0.54 (dd, 1 H,
ꢀ
1.35 (m, 1 H, H-6), 2.01 (s, 3 H, CH3), 2.89 (d, 1 H, J8,8 = 11.5,
H-8), 3.48 (dd, 1 H, J3,4 = 10.0, J3,2 = 3.0, H-3), 3.91 (dd, 1 H,
J4,5 = 7.3, H-4), 4.0 (d, 1 H, H-8ꢀ), 4.34 (d, 1 H, H-2), 4.60 (t, 1 H,
H-5), 13C NMR (D2O) d 10.9 (C-7), 19.9 (C-6), 22.0 (CH3), 28.0
(C-1), 46.5 (C-5), 65.9 (C-8), 66.6 (C-4), 68.6 (C-2), 68.8 (C-3),
J7,7 = 5.2, J7,6 = 9.7, H-7), 0.92 (t, 1 H, J6,7 + J7,7 = 11.3, H-7ꢀ),
ꢀ
ꢀ
ꢀ
ꢀ
1.05 (m, 1 H, H-6), 2.99 (d, 1 H, J8,8 = 11.2, H-8), 3.49 (m, 1 H,
H-5), 3.61 (dd, 1 H, J4,5 = 4.2, J4,3 = 9.2, H-4), 3.66 (dd, 1 H,
J3,2 = 4.7, H-3), 3.79 (d, 1 H, H-8ꢀ), 4.37 (d, 1 H, H-2), 13C NMR
(CD3OD) d 9.2 (C-7), 22.3 (C-6), 28.0 (C-1), 49.5 (C-5), 66.1 (C-4),
66.5 (C-2), 68.0 (C-8), 68.9 (C-3). Found 190.1074 (ESI-HRMS)
C8H16NO4 (M + H+) requires 190.1079.
=
174.3 (C O). Found, C, 51.6; H, 7.6; N, 6.1. C10H17NO5 requires
C, 51.9; H, 7.4; N, 6.1%.
(1S,2S,3S,4S,5S,6R)-5-Acetamido-1-(hydroxymethyl)bicyclo-
[4.1.0]heptane-2,3,4-triol (10)
Enzyme kinetics
A mixture of 19 (533 mg, 0.90 mmol) and 10% Pd–C (247 mg) in
MeOH (100 cm3) was stirred at rt under an atmosphere of H2 for
14 h. The reaction mixture was filtered through a Celite pad, which
was then washed thoroughly with MeOH (50 cm3). The filtrate and
washings were combined and concentrated under pressure to give
a colourless syrup. The resultant crude product was purified by
flash column chromatography (CH2Cl2–MeOH, 4 : 1 v/v) to yield
10 (185 mg, 88%) as a colourless syrup: [a]2D0 +64.5 (c = 0.53, H2O),
The activity of both the coffee bean and E. coli enzymes were
assayed by monitoring the rate of hydrolysis of p-nitrophenyl a-
D-galactopyranoside (PNPG). For the measurement of Ki values
4 different concentrations of substrate and 4 of inhibitor were
used. Whereas, for IC50 values a single concentration of PNPG
was utilized and this was 0.14 and 0.01 mM for the coffee bean
and E. coli enzymes, respectively. The corresponding Michaelis
constants (Km) for the PNPG substrate are 0.7 and 0.04 mM for
the coffee bean and E. coli enzymes, respectively.
The coffee bean a-galactosidase activity assay solutions con-
tained 0.62 mU cm−3 enzyme in 50 mM sodium phosphate buffer
(pH 6.52) with 0.1% bovine serum albumin in total volume of
0.400 cm3. Each experiment was initiated by the addition of
enzyme to an equilibrated assay solution held at 37 ◦C.
The activity of the E. coli enzyme was assayed using a solution
that comprised of 5 mU cm−3 enzyme containing 0.1% bovine
serum albumin in 50 mM sodium phosphate buffer (pH 7.26),
the total volume of the assay solution was 0.400 cm3. Each
analysis began by the addition of a stock solution of enzyme to an
equilibrated assay solution held at 25 ◦C.
For both enzymes, the absorbance at 400 nM was monitored for
10 min using a Cary 3E spectrophotometer equipped with a Peltier
temperature controller. The measured initial rate versus inhibitor
concentration data were fit to standard enzyme kinetic equations
using a nonlinear least squares program (Prism).
1H NMR (D2O) d 0.60 (dd, 1 H, J7,7 = 5.7, J7,6 = 9.8, H-7), 0.83 (t,
ꢀ
1 H, J7 ,6 + J7,7 = 11.7, H-7ꢀ), 1.10 (ddd, 1 H, J6,5 = 1.4, H-6), 2.03
ꢀ
ꢀ
ꢀ
(s, 3 H, CH3), 3.06 (d, 1 H, J8,8 = 11.7, H-8), 3.65–3.67 (m, 2 H,
H-3, H-4), 3.71 (d, 1H, H-8ꢀ), 4.42 (m, 1 H, H-2), 4.48 (br s, 1 H,
H-5), 13C NMR (D2O) d 9.7 (C-7), 22.2 (CH3), 22.6 (C-6), 27.6
(C-1), 48.7 (C-5), 66.0 (C-4), 68.1 (C-2), 68.2 (C-8), 69.2 (C-3),
=
174.4 (C O). Found, C, 52.2; H, 7.5; N, 5.9. C10H17NO5 requires
C, 51.9; H, 7.4; N, 6.1%.
(1R,2S,3S,4S,5S,6S)-5-Amino-1-(hydroxymethyl)bicyclo-
[4.1.0]heptane-2,3,4-triol (7)
LiOH (32 mg, 0.76 mmol) was added to a solution of amide 8
(82 mg, 0.35 mmol) in THF–H2O (1 : 1, 12 cm3). This mixture was
heated at 70 ◦C for 32 h. The solution was then neutralized by the
addition of Amberlite IR resin (H+ form), and filtered. The resin
was washed thoroughly with water and the product was eluted
with NH4OH (5%, 100 cm3). The eluted solution was concentrated
under pressure to give a light yellow oil. This substance was further
purified by flash chromatography (MeOH–CHCl3–28% NH3 in
H2O 5 : 5 : 1 v/v/v) to give 7 as a colourless syrup (35 mg, 52%):
[a]20 +76.5 (c = 0.51, MeOH), 1H NMR (CD3OD) d 0.67 (dd, 1 H,
Acknowledgements
J7,7D = 5.5, J7,6 = 9.3, H-7), 0.75 (m, 1 H, H-7ꢀ), 1.33 (m, 1 H, H-6),
The authors gratefully acknowledge the Natural Sciences and
Engineering Research Council of Canada for financial support
of this work.
ꢀ
ꢀ
3.41 (d, 1 H, J8,8 = 11.3, H-8), 3.54 (dd, 1 H, J3,4 = 8.9, J3,2 = 3.1,
H-3), 3.74 (d, 1 H, H-8ꢀ), 3.82 (t, 1 H, J5,4 + J5,6 = 13.9, H-5), 3.93
(dd, 1 H, J4,5 = 6.6, H-4), 4.30 (d, 1 H, H-2), 13C NMR (CD3OD) d
11.6 (C-7), 20.8 (C-6), 29.1 (C-1), 48.7 (C-5), 67.6 (C-8), 68.5 (C-4),
70.5 (C-3), 70.52 (C-2). Found 190.1074 (ESI-HRMS) C8H16NO4
(M + H+) requires 190.1079.
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◦
was heated at 70 C for 32 h, when it was neutralized by adding
Amberlite IR resin (H+ form). After filtration the resin was
washed thorough by water. The product was eluted using NH4OH
(5%, 20 cm3) and the eluent was concentrated under pressure
to give a light yellow oil. This material was purified by flash
chromatography (MeOH–CHCl3–28% NH3 in H2O 5 : 5 : 1 v/v/v)
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