(Received for publication, August 28, 1995)
From the
2-Deoxy-2,2-difluoroglycosides are a new class of
mechanism-based inhibitors of -glycosidases, which function via
the accumulation of a stable difluoroglycosyl-enzyme intermediate. Two
members of this new class of inhibitor have been synthesized and
kinetic studies performed with their target glycosidases. Thus
2,4,6-trinitrophenyl 2-deoxy-2,2-difluoro-
-glucoside is shown to
inactivate yeast
-glucosidase with a second order rate constant of k
/K
= 0.25 min
mM
. The equivalent difluoromaltoside
inactivates human pancreatic
-amylase with k
/K
= 0.0073 min
mM
. Competitive inhibitors protect the
enzyme against inactivation in each case, showing reaction to occur at
the active site. A burst of release of one equivalent of
trinitrophenolate observed upon inactivation of human pancreatic
-amylase proves the required 1:1 stoichiometry. These are the
first mechanism-based inhibitors of this class to be described, and the
first mechanism-based inhibitors of any sort for the medically
important
-amylase. In addition to having potential as
therapeutics, compounds of this class should prove useful in subsequent
structural and mechanistic studies of these enzymes.
Specific inhibitors of glycosidases have proved valuable in a
number of applications ranging from mechanistic studies (Legler, 1990;
Sinnott, 1990) through their use to study protein glycosylation (Elbein et al., 1984), to possible therapeutic uses such as the
control of blood glucose levels via control of the degradation of
dietary disaccharides and starch (Truscheit et al., 1981) or
control of viral infectivity through interference with normal
glycosylation of viral coat proteins (Elbein, 1984; Prasad et
al., 1987). A number of naturally occurring reversible glycosidase
inhibitors are known such as nojirimycin, castanospermine, swainsonine,
and acarbose (Legler, 1990), and these have been subjected to intensive
study including the synthesis and testing of a number of analogues.
Another class of inhibitors that has been less well studied is that of
the covalent, irreversible type, typically affinity labels. These are
generally synthetic analogues of sugars containing reactive groups such
as epoxides, isothiocyanates and -halocarbonyls as reviewed
recently (Legler, 1990; Withers and Aebersold, 1995). Less common are
the more selective mechanism-based inhibitors whose efficacy depends
upon binding and subsequent enzymatic action to generate a reactive
species. These include the conduritol epoxides (Legler, 1968, 1970),
the quinone methide-generating glycosides (Halazy et al.,
1990; Briggs et al., 1992), and the glycosylmethyl triazenes
(Marshall et al., 1980; Sinnott and Smith, 1976).
Interestingly, two naturally occurring inhibitors of this class have
now been described: the hydroxymethylconduritol epoxide, cyclophellitol
(Atsumi et al., 1990; Withers and Umezawa, 1991), isolated
from Phellinus sp.; and the putative quinone
methide-generating glycoside salicortin, isolated from Salix (Clausen et al., 1990).
An additional, relatively
recently described class of mechanism-based inhibitor that has proved
successful is that of the 2-deoxy-2-fluoro (Withers and Aebersold,
1995; Withers et al., 1987, 1988, 1990). These function as
excellent inactivators of retaining glycosidases (glycosidases that
hydrolyze the glycosidic linkage with net retention of anomeric
configuration) by formation of a stable glycosyl-enzyme intermediate
which turns over to product only very slowly. As shown in Fig. S1for an -glucosidase, the normal mechanism of action
of this class of enzyme involves the formation and hydrolysis of a
glycosyl-enzyme intermediate with general acid/base catalytic
assistance via transition states with substantial oxocarbenium ion
character (Sinnott, 1987, 1990). A combination of inductive
destabilization of these positively charged transition states by the
electronegative fluorine at C-2 and loss of crucial hydrogen bonding
interactions with the 2-hydroxyl serves to substantially destabilize
these two transition states, dramatically slowing both steps.
Incorporation of a relatively reactive leaving group such as fluoride
or 2,4-dinitrophenolate as aglycone accelerates the first
(glycosylation) step sufficiently that the glycosyl-enzyme intermediate
is formed, but then hydrolyzes only very slowly, thereby resulting in
inactivation.
Figure S1: Scheme 1
This strategy has proved very successful with a wide
range of retaining -glycosidases, and has allowed the
characterization of this intermediate and the identification of the
catalytic nucleophiles in a number of enzymes (Withers and Aebersold,
1995). These compounds have also proved effective in vivo,
selectively inactivating the expected glycosidases in all organs tested
in rats, including the brain (McCarter et al., 1994). However,
as noted early on (Withers et al., 1988), and as has been
confirmed in subsequent studies (McCarter et al., 1993), this
approach has not been successful with
-glycosidases, despite the
fact that ample evidence exists that equivalent mechanisms are followed
in the two cases.
This evidence includes the
C
NMR detection of a glycosyl-enzyme intermediate on an
-amylase
(Tao et al., 1989), and the denaturation trapping of such an
intermediate on a glycosyl transferase, a mechanistically analogous
enzyme (Mooser et al., 1991; Mooser, 1992). Instead, the
2-deoxy-2-fluoro-
-glycosides function as substrates, albeit poor,
for the enzymes studied. Thus the lack of inactivation must be a
consequence of the fluorine substituent not sufficiently slowing the
deglycosylation step relative to glycosylation, and a possible
stereoelectronic rationale for this observation has been proposed
(Withers, 1995; Kempton and Withers, 1992). The generation of an
effective inhibitor of this class for an
-glycosidase therefore
requires further slowing of the deglycosylation step. This could be
achieved via the incorporation of a second fluorine at C-2 to further
inductively destabilize the transition state, in conjunction with the
incorporation of a more reactive leaving group to ensure that
glycosylation is not rate-limiting. Since the second fluorine is only
slightly larger than the hydrogen it replaces, it would likely not
result in any significant steric repulsive interactions upon binding.
In this paper we describe such an approach which has led to the
development of novel mechanism-based inactivators of both yeast
-glucosidase and human pancreatic
-amylase. This is the first
mechanism-based inhibitor described for human pancreatic
-amylase,
which should prove valuable as a probe of the structure and mechanism
of this medically important enzyme. Compounds of this general class
also have considerable potential as therapeutics agents, particularly
in the control of post-prandial blood glucose levels by inhibition of
digestive glycosidases.
2,4,6-Trinitrophenyl
2-deoxy-2,2-difluoro--D-arabino-hexopyranoside
(TNPDFG)
data were as follows:
H NMR
(D
O):
9.10 (s, 2 H, aryl Hs), 5.89 (d, 1 H, J
4.9 Hz, H-1), 4.16 (dt, 1 H, J
19.6, J
7.5, J
7.5 Hz, H-3), 3.8-3.6 (m, 4 H, H-4,
H-5, H-6a, H-6b);
F NMR (D
O):
-120.6 (dd, J
261, J
6.9 Hz, Fe-2), -122.7 (ddd, J
261, J
19.7, J
6 Hz,
Fa-2).
2,4,6-Trinitrophenyl
2-deoxy-2,2-difluoro-4-O-(-(1,
4)-D-glucosyl)-
-D-arabino-hexopyranoside
(TNPDFM) data were as follows:
H NMR (D
O):
9.14 (s, 2 H, aryl H's), 5.91 (d, 1 H, J
3.6 Hz, H-1), 5.46 (d, 1 H, J
3.9 Hz,
H-1`);
F NMR (D
O):
-120.6 (dd, J
261, J
6.9 Hz,
Fe-2), -122.7 (ddd, J
261, J
19.7, J
6 Hz,
Fa-2).
Synthesis of the required glycone portion posed no great synthetic problem since a method for the synthesis of a 2-deoxy-2,2-difluoroglucose derivative based upon the addition of fluorine to 2-fluoro-D-glucal had been published (McCarter et al., 1993). However, the incorporation of an aglycone of greater leaving group ability than fluoride or dinitrophenolate necessitated more careful synthetic considerations. The most attractive candidate as a leaving group was chloride. However, repeated attempts to synthesize this derivative via displacement chemistry were unsuccessful, with no reaction occurring. This is likely the consequence of the very effect sought, the resistance of 2,2-difluoroglycosides toward nucleophilic displacements. An alternative strategy for the installation of a good leaving group was therefore followed, which did not require the displacement reaction. This involved reaction of the protected hemiacetal of the 2-deoxy-2,2-difluoro sugar with fluoro-2,4,6-trinitrobenzene, to yield the trinitrophenyl glycoside. Using this approach TNPDFG (1) and TNPDFM (2) were synthesized as shown in Fig. Z1.
Figure Z1: Structure 1
Treatment of 3,4,6-tri-O-acetyl
2-deoxy-2-fluoro--D-glucopyranosyl bromide (McCarter et al., 1993) with triethylamine in acetonitrile at reflux for
20 h yielded 2-fluoro-D-glucal, which was fluorinated with
acetyl hypofluorite to give 1,3,4,6-tetra-O-acetyl
2-deoxy-2,2-difluoro-D-glucopyranose. Selective anomeric
deprotection with hydrazine acetate in dimethyl formamide at 50 °C
yielded the free hemiacetal after 3 days. This was reacted with
fluoro-2,4,6-trinitrobenzene in dichloromethane containing a hindered
base (2, 6-di-tert-butylpyridine) for 10 days at room
temperature in the dark to give the protected glycoside.
De-O-acetylation with hydrogen chloride in methanol yielded
the desired product 1 after chromatographic work-up. An
equivalent approach was used for the synthesis of the maltose
derivative 2, starting with maltal hexa-O-acetate. Both
compounds provided the expected elemental and spectroscopic analyses.
Incubation of both yeast -glucosidase with TNPDFG and human
pancreatic
-amylase (
-amylase) with TNPDFM resulted in
time-dependent inactivation, as shown for
-amylase in Fig. 1a. Inactivation followed pseudo-first order
kinetic behavior for at least two half-lives. However some deviation
was observed at longer incubation times, which was shown to be due to
the spontaneous decomposition of the inhibitor in the incubation
mixtures (see below).
Figure 1:
Inactivation of human pancreatic
-amylase by TNPDFM. a, semilogarithmic plot of residual
activity versus time at the indicated inactivator
concentrations:
, 7.5 mM;
, 5.6 mM;
, 3.7 mM;
, 2.5 mM;
, 1.9
mM. b, replot of first-order rate constants from a. c, inactivation with 5.6 mM TNPDFM in the
absence (
) and presence (
) of 65 µM acarbose.
Inactivation data were analyzed according to the kinetic scheme shown below ().
Species E, TNP, and DFM represent, respectively,
enzyme, 2,4,6-trinitrophenol and
2-deoxy-2,2-difluoro-D-maltose or
2-deoxy-2,2-difluoro-D-glucose. A reciprocal replot of the
pseudo-first order rate constants (k) at each
inhibitor concentration, taken from slopes of the lines in Fig. 1a versus inhibitor concentration, is shown in Fig. 1b. The slope of this plot yielded a value of k
/K
= 0.0073
min
mM
for reaction of
human pancreatic
-amylase. Individual values of k
and K
could not be reliably determined
because the solubility of the inhibitor (maximum concentration achieved
= 11 mM) precluded measurements at concentrations
anywhere near the estimated K
value (based upon Fig. 1b) of 90 mM.
The active site-directed
nature of the inhibition was tested by measuring the rate of
inactivation at a fixed concentration of TNPDFM (5.6 mM) both
in the absence and presence of the known competitive inhibitor
acarbose. As is shown in Fig. 1c, the rate of
inactivation was decreased from 0.044 min to 0.008
min
by the addition of acarbose, indicating that
inactivation is a consequence of modification of the active site. In a
second control (not shown) TNP (picric acid) was shown not to result in
inactivation of the enzyme, thereby removing any concerns that the true
inactivator was the released product.
In order to demonstrate the
stoichiometry of inactivation, the reaction of -amylase, with the
inactivator was studied by monitoring the release of TNP.
-Amylase
(10 µl, 52 µg) in buffer was added to a solution of TNPDFM (10
µl, 0.98 mM) in the same buffer, placed quickly into a
microcell in a spectrophotometer, and the absorbance at 400 nm
monitored. The biphasic time course shown in Fig. 2was
obtained. The first, fast phase corresponds to the release of TNP upon
inactivation of the enzyme, and the second, slower phase to the
breakdown of the substrate. TNP was released at an equivalent, slow
rate from a second mixture to which was added buffer but no enzyme.
Back extrapolation of the essentially linear steady state phase and of
the initial burst phase to time zero revealed that 26 µM TNP was released in the initial burst phase. This corresponds well
to the concentration of
-amylase in the cell (23 µM),
indicating a stoichiometry of one TNP released per enzyme molecule. A
repeat of this experiment with twice the amount of enzyme resulted, as
required, in a burst of twice the magnitude (data not shown). These
data therefore show that inactivation of
-amylase is a consequence
of the formation of a stable difluoroglycosyl-enzyme intermediate at
the active site of the enzyme.
Figure 2:
Release of TNP from the reaction mixture
containing 0.49 mM TNPDFM and 0.023 mM -amylase.
Similar data were also acquired with
yeast -glucosidase inactivated by TNPDFG, and kinetic parameters
for this process along with other data are shown in Table 1. In
this case again, a competitive inhibitor, 1-deoxynojirimycin (45
µM, K
= 12.6 µM),
was shown to protect the enzyme against inactivation, reducing the rate
of inactivation in the presence of 1.6 mM TNPDFG from 0.13
min
to 0.073 min
.
It is of
interest to compare the rate reductions consequent upon introduction of
the fluorine substituents at C-2 in the two cases. As can be seen in Table 1, introduction of the first fluorine at C-2 reduces the
glycosylation rate (from k/K
values)
some 2500-fold for
-amylase but only 80-fold for the
-glucosidase. Comparison of parameters for the trinitrophenyl
difluoroglycosides with those for the 2-fluoroglycosyl fluorides
reveals a 330-fold reduction for
-amylase and again an 80-fold
reduction for the
-glucosidase. This reveals a much greater
sensitivity of the
-amylase-catalyzed reaction to the introduction
of fluorine substituents than that for
-glucosidase, indicating
either a greater degree of oxocarbenium ion character at the transition
state for
-amylase, or more important interactions with the
2-hydroxyl. These massive reductions in glycosylation rate constants
consequent upon the introduction of two fluorines (almost
10
-fold for
-amylase) were also reflected in reduced
deglycosylation rate constants. No reactivation of either inactivated
enzyme was seen when a sample of the inactivated enzyme was dialyzed to
remove excess inactivator, then incubated for up to 30 days and
aliquots removed for assay.
In summary, a new class of
mechanism-based inactivator of human pancreatic -amylase and of
yeast
-glucosidase has been synthesized and shown to function via
the stoichiometric trapping of a covalent glycosyl-enzyme intermediate.
In addition to providing powerful evidence of the commonality of
mechanisms of
- and
-glycosidases, such inhibitors should
prove useful in identifying the active site nucleophiles of these and
other glycosidases, and compounds of this class may well prove valuable
as therapeutic agents.