IOOC route to substituted chiral pyrrolidines. Potential glycosidase inhibitors

Article abstract of DOI:10.1021/jo9815094

Branched-chain five-membered ring aza-sugar analogues, synthesized from amino acids by an intramolecular oxime-olefin cycloaddition reaction, the IOOC route, were found to be selective glycosidase inhibitors. The derivative 2,4(S)-di(hydroxymethyl)-3(S)-aminopyrrolidine, obtained from D-serine, was about 1 order of potency more active than its enantiomer obtained from L- serine.

Full text of DOI:10.1021/jo9815094

498
J . Org. Chem. 1999, 64, 498-506
IOOC Rou te to Su bstitu ted Ch ir a l P yr r olid in es. P oten tia l
Glycosid a se In h ibitor s
Eliezer Falb, Yosi Bechor, Abraham Nudelman,* Alfred Hassner,* Amnon Albeck,* and
Hugo E. Gottlieb
Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel
Received J uly 29, 1998
Branched-chain five-membered ring aza-sugar analogues, synthesized from amino acids by an
intramolecular oxime-olefin cycloaddition reaction, the IOOC route, were found to be selective
glycosidase inhibitors. The derivative 2,4(S)-di(hydroxymethyl)-3(S)-aminopyrrolidine, obtained from
D-serine, was about 1 order of potency more active than its enantiomer obtained from L-serine.
In tr od u ction
occurring amino acids and their enantiomers. These
reactions have been shown to be convenient stereoselec-
tive routes to isoxazolidine derivatives and hence to
amino alcohols. A preliminary communication describing
this system has appeared.8
Alkaloids1 and synthetic aza-sugars that structurally
resemble monosaccharides, where the ring O is replaced
by N, and display glycosidase inhibitory properties are
of significant importance not only for the potential
treatment of diabetes but also for chemotherapy of cancer
and AIDS.2 Recently reported syntheses of aza-sugars are
based primarily on manipulations of natural sugars and
their derivatives,2,3 non-sugar-based syntheses,4 enzyme-
based synthetic methodologies,5 and a few approaches
using amino acids as starting materials.6 Although
hydroxylated aza-sugars have been studied intensively,
amino-hydroxy aza-sugars, especially branched-chain
aza-sugars, remain virtually unexplored.
Resu lts a n d Discu ssion
Two general paths, A and B, were explored for the
synthesis of pyrrolidines I (Scheme 1). The planned
synthetic approach to compounds I involved reduction of
the N-O bond and deprotection of the respective bicyclic
(IIa ) or tricyclic (IIb) intermediates that contain a
pyrrolidine skeleton fused to an isoxazolidine ring. These
intermediates might be available via a key IOOC reaction
from the unsaturated oximes IIIa or IIIb, accessible from
the corresponding N,O-diprotected-R-amino esters, ob-
tained from IV. In this retrosynthetic analysis, the five-
membered ring aza-sugar derivatives I were selected,
because it has been observed9,5a-c that pyrrolidine-type
aza-sugars are frequently better glycosidase inhibitors
than the corresponding piperidine-type analogues. Fur-
thermore, this route enabled us to stereoselectively
introduce an amino function into the pyrrolidine ring and
to introduce either OH or SH groups into one of the side
chains. Although many polyhydroxylated pyrrolidines
have been studied and found to exhibit glyscosidase
inhibition, little is known about the effect of substituting
an NH2 group for an OH on the pyrrolidine ring.
We report here new approaches to substituted chiral
pyrrolidines based on an intramolecular oxime-olefin
cycloaddition (IOOC) route,7 starting from naturally
(1) (a) Elbein, A. D. Annu. Rev. Biochem. 1987, 56, 497. (b) Fellows,
L. E.; Kite, G. C.; Nash, R. J .; Simmonds, M. S. J .; Scofield, A. M. In
Plant Nitrogen Metabolism; Poulton, J . E., Romero, J . T., Conn, E. E.,
Eds.; Plenum: New York, 1989; p 395. (c) Legler, G. Adv. Carbohydr.
Chem. Biochem. 1990, 48, 319. (d) Fellows, L. E.; Nash, R. J .;
Simmonds, M. S. J .; Scotfield, A. M. In Nitrogen Metabolism of Plants;
Mengel, K., Ed.; 1992; pp 271-284.
(2) (a) Chen, Y.; Vogel, P. J . Org. Chem. 1994, 59, 2487. (b) Wong,
C.-H.; Provencher, L.; Porco, J . A., J r.; J ung, S.-H.; Wang, Y.-F.; Chen,
L.; Wang, R.; Steensma, D. H. J . Org. Chem. 1995, 60, 1492 and
references therein.
(3) (a) Baxter, E. W.; Reitz, A. B. J . Org. Chem. 1994, 59, 3175. (b)
J ohnson, C. R.; Miller, M. W.; Golebiowski, A.; Sundram, H.; Ksebati,
M. B. Tetrahedron Lett. 1994, 35, 8991. (c) Kilonda, A.; Compernolle,
F.; Toppet, S.; Hoornaert, G. J . Tetrahedron Lett. 1994, 35, 9047. (d)
Ryu, Y.; Kim, G. J . Org. Chem. 1995, 60, 103. (e) Ichikawa, M.;
Ichikawa, Y. Bioorg. Med. Chem. 1995, 3, 161. (f) Godskesen, N.; Lundt,
I.; Madsen, R.; Winchesters, B. Bioorg. Med. Chem. 1996, 4, 1857. (g)
Thomassigny, C.; Bennis, K.; Gelas, J . Synthesis 1997, 2, 191. (h)
J efferies, I.; Bowen, B. R. Bioorg. Med. Chem. Lett. 1997, 7, 1171.
(4) (a) Tchamber, T.; Backenstrss, F.; Neuburger, M.; Zehnder, M.;
Streith, J . Tetrahedron 1994, 50, 1135. (b) Paulvannan K.; Stille, J .
R. J . Org. Chem. 1994, 59, 1613. (c) Cook, G. R.; Beholz, L. G.; Stille,
J . R. J . Org. Chem. 1994, 59, 3575. (d) Barton, D. H. R.; Arau´jo Pereira,
M. M. M.; Taylor, D. K. Tetrahedron Lett. 1994, 35, 9157. (e) Nukui,
S.; Sodeoka, M.; Sasai, H.; Shibasaki, M. J . Org. Chem. 1995, 60, 398.
(f) McCort, I.; Dure´ault, A.; Depezay, J .-C. Tetrahedron Lett. 1996, 37,
7717. (g) Yu, L.; Li, J .; Ramirez, J .; Chen, D.; Wang, P. G. J . Org. Chem.
1997, 62, 903. (h) Defoin, A.; Sarazin, H.; Streith, J . Tetrahedron 1997,
53, 13783. (i) Degiorgis, F.; Lombardo, M.; Trombini, C. Synthesis 1997,
11, 1243. (j) Huwe, C. M.; Blechert, S. Synthesis 1997, 2, 61.
(5) (a) Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res. 1993,
26, 182. (b) Provencher, L.; Steensma, D. H.; Wong, C.-H. Bioorg. Med.
Chem. 1994, 2, 1179. (c) Wang, Y.-F.; Takaoka, Y.; Wong, C.-H. Angew.
Chem., Int. Ed. Engl. 1994, 33, 1242. (d) J ohnson, C. R.; Golebiowski,
A.; Schoffers, E.; Sundram, H.; Braun, M. P. Synlett 1995, 313. (e) Dax,
K.; Ebner, M.; Peinsipp, R.; Stu¨tz, A. E. Tetrahedron Lett. 1997, 38,
225.
According to the retrosynthetic analysis (Scheme 1),
derivatives I can be approached via noncyclic (path A)
or cyclic (path B) intermediates. Compounds 2 were
(6) (a) Koskinen, A. M. P.; Paul, J . M. Tetrahedron Lett. 1992, 33,
6853. (b) Dondoni, A.; Merino, P.; Perrone, D. Tetrahedron 1993, 49,
2939. (c) Burley, I.; Hewson, A. T. Tetrahedron Lett. 1994, 35, 7099.
(d) Blanco, M. J .; Sardina, F. J . Tetrahedron Lett. 1994, 35, 8493. (e)
Mart´ın-Lo´pez, M. J .; Bermejo-Gonza´lez, F. Tetrahedron Lett. 1994, 35,
8843. (f) Gurjar, M. K.; Ghosh, L.; Syamala, M.; J ayasree, V. Tetra-
hedron Lett. 1994, 35, 8871. (g) Oishi, T.; Iwakuma, T.; Hirama, M.;
Itoˆ, S. Synlett 1995, 404. (h) Huang, Y.; Dalton, D. R. J . Org. Chem.
1997, 62, 372.
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29, 5331. (b) Hassner, A.; Maurya, R. Tetrahedron Lett. 1989, 30, 2289.
(c) Hassner, A.; Maurya, R.; Padwa, A.; Bullock, W. H. J . Org. Chem.
1991, 56, 2775. (d) Chiacchio, U.; Corsaro, A.; Pistara`, V.; Rescifina,
A.; Romeo, G.; Romeo R. Tetrahedron 1996, 52, 7875.
(8) Hassner, A.; Falb, E.; Nudelman, A.; Albeck, A.; Gottlieb, H. E.
Tetrahedron Lett. 1994, 35, 2397.
(9) Takaoka, Y.; Kajimoto, T.; Wong, C.-H. J . Org. Chem. 1993, 58,
4809.
10.1021/jo9815094 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/31/1998

Intramolecular Oxime-Olefin Cycloaddition
J . Org. Chem., Vol. 64, No. 2, 1999 499
Sch em e 1. IOOC-Ba sed Retr osyn th etic An a lysis
cysteine was converted to thiazolidin-2-one L-15c by
treatment of 1 with triphosgene.15 Subsequent N-allyla-
tion, DIBAL-H reduction, and oximation of oxazolidino-
nes L-15a ,b and thiazolidinone L-15c afforded the
desired key intermediate ene-oximes L-17a -c. Reaction
of L-17a -c under IOOC conditions (heating at 165 °C)
gave the tricyclic L-18a -c as single diastereomers as
indicated by chromatographic behavior and1H and 13C
NMR (Scheme 4). The structural and stereochemical
assignments for the tricyclic L-18 and the bicyclic amino
alcohols L-19, as well as for the final pyrrolidines L-21,
1
are based on H and 13C NMR and NOE data shown for
L-18a and L-19b and NOESY of L-21a (Scheme 4). IOOC
ring closure of L-17a -c to L-18a -c produced solely an
anti-syn fused ring system consistent with the stereose-
lectivity of previous IOOC ring closures.7 Even the
threonine-derived product L-18b was formed stereospe-
cifically. The isoxazolidine ring of L-18a ,b underwent
N-O bond reduction with Raney Ni to produce L-19a ,b,
but thiazolidin-2-one L-18c also underwent desulfuriza-
tion to give pyrrolidine 20. However, the alternative
reduction of L-18c with Zn gave amino alcohol L-19c.
Oxazolidin-2-ones L-19a ,b and thiazolidin-2-one L-19c
were rather stable toward subsequent aqueous hydrolytic
conditions. O,N-deprotection to L-21a -c required over-
night reflux in the presence of catalytic amounts of Cs2-
prepared following path A by individual protection of the
O and N groups. Attempted N-allylation of 2 by the
method of Cheung and Benoiton10 was unsuccessful, as
even at 5 °C 2a underwent â-elimination concomitant
with N-allylation to give 3. A second L-serine derivative,
N-Boc-O-Bn-Ser11 2b (Scheme 2), expected to be less
susceptible to â-elimination when treated with allyl
iodide under the same conditions, led to the desired
intermediate 4, accompanied by minor amounts of the
corresponding â-elimination byproduct 3. Esterification
of 4 to give 5, followed by reduction, led to the diprotected
R-amino aldehyde 6. Although 6 contains an allylic group
and an aldehyde (as a precursor for an oxime), necessary
for the key IOOC step, the many steps involved and the
low yields obtained led us to set aside path A in favor of
path B (Scheme 1). First, L-serine was converted to
oxazolidine 7a ;12 however, even under mild conditions
(Me3SiCl/NaI13) Boc removal caused total deprotection of
7a to give 8. Next, N-Cbz-oxazolidine 7b was prepared,
but hydrogenolysis of the Cbz group proceeded with
concomitant N-deprotection and ring reduction to the
N-isopropyl derivative 13. The NMR spectra of 7a and
7b showed that these compounds were present as pairs
of amide rotamers. Different catalysts and reaction
conditions for Cbz removal were explored and Rh/Al2O3/
Et2O was found to be the best chemoselective catalyst
for Cbz reduction. With 9 in hand, it was expected that
N-allylation followed by reduction would provide the
desired R-amino aldehyde 11. Although reaction of 9 with
allyl bromide afforded 10, subsequent treatment with
DIBAL-H unfortunately again caused reductive ring
opening to give 14.14
CO3 for L-19a ,b and KOH17 for L-19c (Scheme 3). The
16
stereochemical 2H,3H-trans relationship in compounds
L-21 was unambiguously established on the basis of
NMR data (Scheme 4).18 Moreover, reaction of L-21a with
phenylisothiocyanate gave the corresponding thiourea
derivative L-22. The NMR spectrum of L-22 showed very
1
broad H and 13C NMR lines at room temperature due
to the dynamics of the thiourea group, which could be
sharpened by heating the sample to 320 K. Inspection of
the spectrum of L-22 by resolution enhancement revealed
a very small (1 Hz) coupling for J 2,3, indicating the trans
stereochemistry between these two H’s,19 which was
further confirmed by NOE (Scheme 4). By analogy with
L-21a , the stereochemistry of L-21b,c was assigned as
shown.
In a preliminary communication,8 we reported on the
presence of a stereoisomer of L-21a . Further examination
indicated that this species is a covalently bound adduct
of L-21a and CO2 (e.g., a carbamic acid derivative). The
presence of analogous adducts was detected in L-21b and
D-21a (see below) but was absent in the case of thiol
derivative L-21c. The relative amount of this material
could be reduced to virtually zero by heating the D2O
solution of L-21a used for NMR determination to 90 °C
for about 1 h. Conversely, the concentration of this adduct
could be increased considerably by exposing solid L-21a
to a CO2 atmosphere.
Because the substrates of glycosidases are the natural
sugars that possess the D configuration, it may be
expected that aza-sugar analogues that also possess this
In view of the difficulties encountered with protection
of L-serine as oxazolidines 7, the protection was switched
to the more stable oxazolidin-2-ones L-15a ,b derived from
L-serine and L-threonine, respectively. Analogously, L-
(15) Falb, E.; Nudelman, A.; Hassner, A. Synth. Commun. 1993, 23,
2839.
(16) Ishizuka, T.; Kunieda, T. Tetrahedron Lett. 1987, 28, 4185.
(17) Abd Elall, E. H. M.; Al Ashmawy, M. I.; Mellor, J . M. J . Chem.
Soc., Perkin Trans. 1 1987, 2729.
(10) Cheung, S. T.; Benoiton, N. L. Can. J . Chem. 1977, 55, 906.
(11) Sugano, H.; Miyoshi, M. J . Org. Chem. 1976, 41, 2352.
(12) Garner, P.; Park, J . M. In Organic Syntheses; Meyers, A. I.,
Ed.; J ohn Wiley and Sons: New York, 1991; p 18.
(13) Olah, G. A.; Narang, S. C. Tetrahedron 1982, 38, 2225.
(14) Hamdani, M.; Scholler, D.; Bouquant, J .; Feigenbaum, A.
Tetrahedron 1996, 52, 605.
(18) Before a NOESY experiment was carried out, L-21a had been
erroneously assigned in our preliminary communication8 as the 2,3-
cis isomer. The uncertainty in stereochemical assignment in five-
membered rings and the utility of NOE in such systems has been
pointed out.19
(19) Namboothiri, I. N. N.; Hassner, A.; Gottlieb, H. E. J . Org. Chem.
1997, 62, 485.

500 J . Org. Chem., Vol. 64, No. 2, 1999
Falb et al.
Sch em e 2
Sch em e 3
D configuration may be better inhibitors than their
enantiomers. The stereochemistry of the aza-sugar ana-
logues described above is L, because they were prepared
from the corresponding L-amino acids. To be able to
compare the biological activity of the enantiomers, we
chose to prepare also the D enantiomer of compound
L-21a ; the latter was found to be the most active of this
family of branch-chained aza-sugar analogues (vide in-
fra). Because the overall yield of L-21a prepared as de-
scribed in Scheme 3 was quite low, we considered
examining a slightly altered approach to the synthesis
of its D enantiomer. The synthetic steps up to D-18 were
as shown in Scheme 3. At this point, the sequence of
deprotection steps was switched so that initially the
carbamate group was hydrolyzed to give the bicyclic
isoxazolidine D-23, followed by subsequent reduction of
the N-O bond to give the desired enantiomer D-21a
(Scheme 5). Although a very clean reduction of D-23 to
D-21a was obtained, the hydrolysis of D-18 to D-23 was
found to be more difficult than that of L-19a to L-21a ,
so that the overall yield of D-21a was similar to that of
its enantiomer.
Biologica l Activity
In many recent examples, aza-sugars and their ana-
logues have shown glycosidase inhibition.1,2 It was pointed

Intramolecular Oxime-Olefin Cycloaddition
J . Org. Chem., Vol. 64, No. 2, 1999 501
Sch em e 4
Sch em e 5
position of the hydroxyl substituents is less important.
Pyrrolidine sugar analogues 21 contain a hydroxymeth-
ylene substituent at C-4 instead of a hydroxyl group and
thus could be considered “branched-chain aza-sugar”
analogues with potential inhibitory activity. In addition,
an amino group was introduced in place of one of the
hydroxy substituents, which is isosteric and is expected
to maintain hydrogen bonding. One of these sugar
analogues, L-21c, was further modified by replacing
another hydroxy group by a thiol. The three sugar
analogues L-21a -c were derived from the natural L-R-
amino acids, thus bearing R absolute configurations at
their C-2 and C-4 positions. The fourth analogue, D-21a ,
is the mirror image of L-21a , as it was synthesized from
(R)-serine. Thus, whereas the former three compounds
are analogous to L sugars, the latter resembles the
natural D sugar stereochemistry. In light of the suggested
limited importance of the hydroxy substitution pattern,
we hoped that these modifications would not significantly
affect binding. Thus, the four branched-chain sugar
analogues L-21a -c and D-21a were tested as potential
reversible inhibitors of four different glycosidases. Com-
pound L-21a was tested as an inhibitor for both R- and
out that, because pyrrolidine sugar analogues5a-c,9,20a and
piperidine sugar analogues20b,c are very good mimics of
the transition state for glycoside hydrolysis with respect
to both the flattened half-chair conformation and the
charge distribution, they should bind tightly to the
enzyme and thus make good inhibitors. Furthermore, it
was suggested20b that these factors provide the major
contribution to binding of some inhibitors, and the
(20) (a) Boutellier, M.; Ganem, B.; Horenstein, B. A.; Schramm, V.
L.; Synlett 1995, 510. (b) Tong, M. K.; Papandreou, G.; Ganem, B. J .
Am. Chem. Soc. 1990, 112, 6137. (c) Papandreou, G.; Tong, M. K.;
Ganem, B. J . Am. Chem. Soc. 1993, 115, 11682.

502 J . Org. Chem., Vol. 64, No. 2, 1999
Falb et al.
Ta ble 1. In h ibitor y Activity of Aza -Su ga r An a logu es
inhibitors
enzyme
D-21a
L-21a
L-21b
L-21c
R-glucosidase
â-glucosidase
R-galactosidase
â-galactosidase
Ki ) 0.018 mM
Ki ) 0.15 mM
Ki ) 0.24 mM
nia
ndd
nd
nib
nic
nic
IC50 ) 1.22 mMe
IC50 ) 1.12 mMf
nd
nd
nia
nia
nd
ni; no inhibition observed: up to [I] ) 1.12 mM. Up to [I] ) 4.0 mM. c Up to [I] ) 1.4 mM. nd; not determined. e At [S] ) 1.1 mM.
a
b
d
f At [S] ) 0.54 mM.
was washed with brine, dried (MgSO4), and concentrated to
give 2a as a yellow oil (1.23 g, 62%): 1H NMR (CDCl3) δ 0.70
(s, 9H), 1.45 (s, 9H), 3.78 (s, 3H), 3.80 (dd, 1H, J ) 8, 3 Hz),
4.00 (dd, 1H, J ) 8, 3 Hz), 4.38 (m, 1H), 5.40 (bd, 1H); 13C
NMR (CDCl3) δ -0.7 (s), 28.4 (s), 52.2 (q), 55.6 (d), 63.1 (t),
80.3 (s), 155.7 (s), 171.2 (s); MS (CI) (NH3) m/z 292 (MH+, 5),
236, 192.
â-glucosidases. It did not inhibit â-glucosidase to any
extent with inhibitor concentrations of up to 4 mM. On
the other hand, L-21a inhibited R-glucosidase with an
apparent Ki ) 0.15 mM. Compound L-21b, derived from
L-threonine and bearing an additional methyl substituent
at the C-2 side chain, exhibited moderate inhibition of
both R- and â-glucosidases. Compound L-21c, the thio
analogue derived from L-cysteine, exhibited a different
selectivity by inhibiting â-glucosidase while having no
effect on R-glucosidase. Compounds L-21a and L-21c
failed to inhibit R- and â-galactosidases. Because com-
pounds L-21a -c are L sugar analogues, we compared the
inhibitory activity of D-21a , the analogue derived from
(R)-serine, toward R-glucosidase with that of its enanti-
omer L-21a . (It should be noted that as enantiomers all
three chiral centers are of opposite configuration, not only
the one corresponding to the sugar C-4 position). As
might be expected, D-21a inhibited R-glucosidase an
order of magnitude better than L-21a (Ki ) 0.018 mM).
These results are summarized in Table 1.
N-Boc-O-ben zyl-L-ser in e (2b). This compound was pre-
pared as described11 but was isolated as the free acid (22%):
1H NMR (CDCl3) δ 1.43 (s, 9H), 3.73 (dd, 1H, J ) 9, 3 Hz),
3.91 (dd, 1H, J ) 9, 2 Hz), 4.43 (m, 1H), 4.47 (s, 2H), 5.50 (d,
1H), 7.20-7.40 (m, 5H); 13C NMR (CDCl3) δ 28.3 (s), 54.2 (d),
70.2 (t), 73.3 (t), 79.8 (s), 127.6, 127.7, 128.3, 133.2 (Ph), 155.6
(s), 173.9 (s); MS (CI) (isobutane) m/z 296 (MH+, 95), 240.
N-Boc-N-a llyl-∆-a la n in e Meth yl Ester (3). To a stirred
solution of 2a (0.51 g, 1.75 mmol) in dry THF (5 mL) was added
NaH (60%, 0.13 g, 3.3 mmol) at 0 °C. After the evolution of H2
ceased, allyl iodide (1.2 mL, 13 mmol) in THF (5 mL) was
added. The reaction mixture was stirred at 25 °C for 24 h or
at 5 °C for 72 h and then filtered. The filtrate was diluted with
EtOAc (10 mL) and washed with aqueous Na2S2O3 (20 mL),
and the organic layer separated, dried (MgSO4), and evapo-
rated to give 3 as a yellow oil (0.2 g, 45%): 1H NMR (CDCl3)
δ 1.42 (bs, 9H), 3.78 (s, 3H), 4.10 (dt, 2H, J ) 6, 1 Hz), 5.15
(dq, 1H, J ) 10, 1 Hz), 5.18 (dq, 1H, J ) 18, 1 Hz), 5.40 (s,
1H), 5.83 (s, 1H), 5.85 (ddd, 1H, J ) 18, 10, 6 Hz); 13C NMR
(CDCl3) δ 28.2 (s), 46.0 (t), 52.3 (q), 81.2 (s), 116.6 (t), 116.8
(d), 133.9 (s), 135.4 (d), 153.6 (s), 169.8 (s); MS (CI) (NH3) m/z
242 (MH+, 63), 259.
N-Boc-N-a llyl-O-ben zyl-L-ser in e (4). To a stirred solution
of 2b (1 g, 3.39 mmol) in dry THF (11 mL) was added NaH
(60%, 0.41 g, 10 mmol) at 0 °C. After the evolution of H2 ceased,
allyl iodide (2.5 mL, 27 mmol) was added. The reaction mixture
was stirred at 5 °C for 72 h, and then the solvent was removed
to give a white residue which was dissolved in water and
acidified to pH 3 (3 N HCl). The yellow oil obtained was
separated from the solution and was extracted with EtOAc (2
× 10 mL); the organic phase washed with aqueous Na2S2O3
(10 mL), dried (MgSO4), and evaporated to give an oil (0.85 g,
75%): 1H NMR (CDCl3) δ 1.42 (s, 9H), 3.80-4.00 (m, 2H), 4.05
(dt, 1H, J ) 15, 0.5 Hz), 4.10 (dt, 1H, J ) 15, 1 Hz), 4.58 (s,
2H), 4.60 (bm, 1H), 5.10-5.20 (m, 2H), 5.80-6.00 (m, 1H),
7.20-7.40 (m, 5H); MS (CI) (isobutane) m/z 336 (MH+, 12).
N-Boc-N-a llyl-O-ben zyl-L-ser in e Meth yl Ester (5). To
a stirred solution of 4 (0.70 g, 2 mmol) in DMF (5 mL) was
added K2CO3 (0.31 g, 2.24 mmol) at 0 °C. After 10 min of
stirring, MeI (0.25 mL, 4 mmol) was added; stirring continued
for 30 min at 0 °C and for an additional hour at room
temperature. The reaction mixture was filtered, the filtrate
was partitioned between EtOAc (20 mL) and water (10 mL),
and the organic phase was washed with brine (10 mL),
separated, dried (MgSO4), and evaporated to give a yellow oil
(0.3 g, 43%): 1H NMR (CDCl3) δ 1.43 (s, 9H), 3.80 (s, 3H),
3.80-4.00 (m, 2H), 4.05 (dt, 1H, J ) 15, 0.5 Hz), 4.10 (dt, 1H,
J ) 15, 1 Hz), 4.56 (s, 2H), 4.60 (bs, 1H), 5.10-5.20 (m, 2H),
5.80-6.00 (m, 1H), 7.20-7.40 (m, 5H).
The results described above support the importance of
the flattened ring conformation and the positive charge
in the differential stabilization of the transition state of
the glycosidase catalytic activity. Although comparison
21
of Ki values of these inhibitors with Km
values of
glucosidase substrates suggests that the extensive modi-
fications in these compounds led to inferior interactions
with the enzyme, possibly due to a lack of similarity in
their substitution patterns or to steric hindrance, the
present results do indicate that the newly synthesized
branched-chain aza-sugar analogues 21 act as selective
glucosidase inhibitors.
Exp er im en ta l Section
1H and 13C NMR spectra were obtained at 200 or 300 MHz
and 50 or 75 MHz, respectively. NOE and NOESY experiments
were carried out at 600 MHz. For chloroform solutions,
chemical shifts are expressed in ppm downfield from Me4Si
as internal standard; for D2O solutions, the HOD peak was
taken as δ 4.80 (1H spectra), or the peak of a small amount of
added MeOH was taken as δ 49.50 (13C). Multiplicities in the
13C NMR spectra were determined by off-resonance decoupling.
Mass spectra were obtained in CI (chemical ionization), DCI
(desorption chemical ionization), EI (electron impact) or HRMS,
(high-resolution) modes. The progress of the reactions was
monitored by TLC on silica gel or alumina. Flash chromatog-
raphy was carried out on silica gel (32-63 µm).
2(S)-ter t-Bu toxyca r bon yla m in o-3-tr im eth ylsila n yloxy-
p r op ion ic Acid Meth yl Ester (2a ). N-Boc serine methyl
ester (1.5 g, 6.8 mmol), Et3N (1.14 mL, 8.2 mmol), DMAP (0.28
g, 2.25 mmol), and Me3SiCl (1 mL, 7.5 mmol) were stirred in
CH2Cl2 (10 mL) under N2 at room temperature. After 3 days,
the reaction mixture was poured into water (10 mL), the
organic phase was separated, and the aqueous phase was
extracted with Et2O (3 × 10 mL). The combined organic phase
N-Boc-N-a llyl-O-ben zyl-L-ser in a l (6). To a solution of 5
(0.3 g, 0.85 mmol) in dry toluene (5 mL), cooled to -78 °C
under N2, was added dropwise DIBAL-H in toluene (1.5 M, 2
mL, 3 mmol), while the temperature was maintained below
-75 °C. After 2 h, MeOH (1 mL) was added followed by Et2O
(10 mL) and saturated aqueous sodium potassium tartrate (10
mL). Vigorous stirring was continued for ca. 2 h until all the
(21) Fleet, G. W. J .; Nicholas, S. J .; Smith, P. W.; Evans, S. V.;
Fellows, L. E.; Nash, R. J . Tetrahedron Lett. 1985, 26, 3127.

Intramolecular Oxime-Olefin Cycloaddition
J . Org. Chem., Vol. 64, No. 2, 1999 503
solids dissolved. The organic layer was separated, dried
(MgSO4), and evaporated to give 6a as a yellow oil (0.1 g,
37%): 1H NMR (CDCl3) δ 1.40 (s, 9H), 3.80-4.00 (m, 2H), 4.10
(dt, 2H, J ) 15, 1 Hz), 4.40 (m, 1H), 4.50 (s, 2H), 5.00-5.20
(m, 2H), 5.80-6.00 (m, 1H), 7.15-7.40 (m, 5H), 9.52 (bd, 1H).
Hz), 3.75 (s, 3H), 3.76 (dd, 1H, J ) 10.5, 4 Hz);13C NMR
(CDCl3) δ 22.0 (q), 23.2 (q), 47.4 (d), 51.9 (q), 60.2 (d), 62.8 (t),
173.8 (s); MS (EI) m/z 162 (M+, 1), 130, 102.
N-Allyl-N-isop r op yl-L-ser in e Meth yl Ester (14). Com-
pound 10 was reduced as described for 6 to give 14 as a yellow
oil (60%): bp 62 °C/0.02 Torr; 1H NMR (CDCl3) δ 1.00 (d, 3H,
J ) 6 Hz), 1.11 (d, 3H, J ) 6 Hz), 3.20 (septet, 1H, J ) 6 Hz),
3.00-3.20 (m, 1H), 3.35-3.45 (m, 1H), 3.38 (dd, 1H, J ) 6.5,
4 Hz), 3.60 (dd, 1H, J ) 10.5, 6.5), 3.65 (dd, 1H, J ) 10.5, 4
Hz), 3.70 (s, 3H), 5.07 (dq, 1H, J ) 10, 1 Hz), 5.20 (dq, 1H, J
) 17, 1 Hz), 5.78 (dddd, 1H, J ) 17, 10, 8, 7 Hz); MS (CI)
(isobutane) m/z 202 (MH+, 100).
N-Boc-4(S)-car bom eth oxy-2,2-dim eth yloxazolidin e (7a)
a n d N-Cbz-4(S)-ca r bom eth oxy-2,2-d im eth yloxa zolid in e
(7b). Boc-serine, Cbz-serine, and their methyl esters were
prepared by standard procedures.12,22 Esterification of Cbz-L-
serine with MeI was carried out in 75% yield with K2CO3
instead of NaHCO3, without the need of chromatography. 7a
was obtained as a pale yellow oil (50%): bp 70 °C/0.5 Torr; 1H
NMR (CDCl3) for the major rotamer δ 1.42 (s, 9H), 1.54 (s,
3H), 1.67 (s, 3H), 3.76 (s, 3H), 4.04 (dd, 1H, J ) 9, 3 Hz), 4.16
(dd, 1H, J ) 9, 7 Hz), 4.39 (dd, 1H, J ) 7, 3 Hz); for the minor
rotamer 1.50 (s, 9H), 1.50 (s, 3H), 1.64 (s, 3H), 3.76 (s, 3H),
4.06 (dd, 1H, J ) 9, 2 Hz), 4.14 (dd, 1H, J ) 9, 7 Hz), 4.49 (dd,
1H, J ) 7, 2 Hz); 13C NMR (CDCl3) for major rotamer δ 24.4
(q), 25.0 (q), 28.3 (s), 52.2 (q), 59.3 (d), 66.3 (t), 80.3 (s), 95.1
(s), 151.2 (s), 171.6 (s, 3H); for minor rotamer δ 25.24 (q), 26.04
(q), 28.3 (s), 52.4 (q), 59.3 (d), 66.0 (t), 80.9 (s), 94.4 (s), 152.1
(s), 171.2 (s); MS (CI) (NH3) m/z 277 (MNH4+, 48), 260; The
N-Cbz derivative 7b was obtained as a yellow oil in 86% yield
Gen er a l P r oced u r e for th e Syn th esis of Oxa zolid in -
2-on es L-15 fr om L-1 Am in o Acid s or th eir Resp ective
Ester s. P r oced u r e A: Compounds L-15 were obtained from
the amino acids 1 as described.15 P r oced u r e B: To a solution
of a methyl or ethyl ester of 1 (64 mmol) and K2CO3 (13.8 g,
0.1 mol) in water (65 mL) at room temperature was added
triphosgene (7.6 g, 25.6 mmol) in toluene (65 mL), and the
mixture was stirred for 3 h. The solvents were concentrated
to dryness, and the solid residue was triturated with warm
EtOAc (2 × 40 mL). The solution was filtered and evaporated,
and the residue was distilled to give 15.
4(S)-Ca r bom eth oxy-oxa zolid in e-2-on es (L-15a ).15 This
compound was obtained in 47% by Procedure A and in 62%
from L-serine methyl ester by Procedure B.
after distillation: bp 128-130 °C/0.02 Torr; [R]20 -40.86° (c
D
) 0.046, CHCl3); 1H NMR (CDCl3) for the major rotamer δ
1.56 (s, 3H), 1.71 (s, 3H), 3.64 (s, 3H), 4.09 (dd, 1H, J ) 9, 3
Hz), 4.17 (dd, 1H, J ) 9, 7 Hz), 4.48 (dd, 1H, J ) 7, 3 Hz),
5.04 (d, 1H, J ) 12 Hz), 5.17 (d, 1H, J ) 12 Hz), 7.30-7.40
(m, 5H); for the minor rotamer δ 1.49 (s, 3H), 1.65 (s, 3H),
3.77 (s, 3H), 4.17 (dd, 1H, J ) 9, 6.5), 4.19 (dd, 1H, J ) 9, 2.5
Hz), 4.56 (dd, 1H, J ) 6.5, 2.5 Hz), 5.17 (d, 1H, J ) 12 Hz),
5.21 (d, 1H, J ) 12 Hz), 7.30-7.40 (m, 5H); 13C NMR (CDCl3)
δ for the major rotamer 24.1 (q), 24.9 (q), 52.3 (q), 58.8 (d),
66.5 (t), 66.7 (t), 95.4 (s), 127.7, 127.9, 128.3, 136.3 (Ph), 151.7
(s), 171.1 (s); for the minor rotamer δ 25.2 (q), 26.0 (q), 52.4
(q), 59.5 (d), 66.1 (t), 67.5 (t), 94.8 (s), 127.9, 128.1, 128.6, 136.0
(Ph), 152.8 (s), 170.9 (s); MS (CI) (NH3) m/z 311 (MNH4+, 95),
294. HRMS (DCI) calcd for C15H19NO5 + H 294.1341, found
294.1331.
4(R)-Ca r bom eth oxy-oxa zolid in e-2-on es (D-15a ). An ice-
cold solution of D-serine methyl ester (6.66 g, 42.8 mmol), Et3N
(18 mL, 3 equiv) and triphosgene (6.35 g, 0.5 equiv) in dry
CH2Cl2 (260 mL) was stirred for 2 h, followed by additional
stirring at room temperature for 48 h. The mixture was then
filtered through a silica gel column, and the filtrate was
evaporated to give D-15a in ca. quantitative yield. [R]20
D
+14.18° (c ) 0.013 CH2Cl2). The 1H and 13C NMR spectra were
identical to those of its L-enantiomer.
4(S)-Ca r bom eth oxy-5(R)-m eth yl-oxa zolid in e-2-on e (L-
15b).15 Obtained (56%) by Procedure A.
4(S)-Ca r b om et h oxyt h ia zolid in e-2-on e (L-15c).15 Ob-
tained (66%) by Procedure A.
4(S)-Ca r boeth oxyth ia zolid in e-2-on e (L-15c′). This com-
pound was obtained as a clear yellow oil in 68% by Procedure
4(S)-Ca r bom eth oxy-2,2-d im eth yloxa zolid in e (9). A so-
lution of 7b (2 g, 6.8 mmol) in dry Et2O (100 mL) was
hydrogenated at 1 atm over 5% Rh/Al2O3 for 18 h. The catalyst
was filtered, and the filtrate was evaporated under reduced
pressure to give 9 as a colorless liquid (0.91 g, 84%): bp 40
B from L-cysteine ethyl ester hydrochloride: bp 150-152 °C/
1
0.3 Torr; [R]20 -16.48° (c ) 0.018, CHCl3); H NMR (CDCl3)
D
δ 1.32 (t, 3H, J ) 7 Hz), 3.62 (dd, 1H, J ) 11, 5 Hz), 3.71 (dd,
1H, J ) 11, 8 Hz), 4.28 (q, 2H, J ) 7 Hz), 4.44 (ddd, 1H, J )
8, 5, 1 Hz), 6.61(bs, 1H, NH); 13C NMR (CDCl3) δ 14.1 (q), 31.8
(t), 56.1 (d), 62.4 (t), 170.0 (s), 174.6 (s); MS (CI) (NH3) m/z
193 (MNH4+, 100), 176; HRMS (CI CH4) calcd for C6H9NO3S
+ H 176.0381, found 176.0284.
1
°C/1 Torr; H NMR (CDCl3) δ 1.31 (s, 3H), 1.48 (s, 3H), 2.50
(bs, 1H, NH), 3.77 (t, 1H, J ) 8 Hz), 3.78 (s, 3H), 4.05 (dd, 1H,
J ) 14, 8 Hz), 4.11 (dd, 1H, J ) 14, 8 Hz); 13C NMR (CDCl3)
δ 26.0 (q), 26.7 (q), 52.4 (q), 59.7 (d), 67.7 (t), 96.2 (s), 172.9
(s); MS (CI) (NH3) m/z 160 (MH+, 100), 144.
N-Allyla tion of Oxa zolid in e-2-on es 15 to 16. Gen er a l
P r oced u r e. To a stirred solution of 15 (40 mmol) in dry DMF
(60 mL) at 0 °C was added NaH (60%, 1.86 g, 46.5 mmol). After
the evolution of H2 ceased, allyl iodide (40 mmol) was added.
The reaction mixture was stirred at room temperature for 24
h and was then partitioned between EtOAc (200 mL) and
aqueous Na2S2O3 (150 mL). The organic layer was separated,
and the aqueous phase was extracted again with EtOAc (200
mL). The combined organic layers were dried (MgSO4) and
concentrated, and the residue was chromatographed (hex-
anes-EtOAc) to give 16.
N-Allyl-4(S)-ca r b om et h oxy-2,2-d im et h yloxa zolid in e
(10). A heterogeneous mixture of 9 (0.98 g, 6.2 mmol), allyl
bromide (0.6 mL, 6.9 mmol), diisopropylethylamine (1 mL, 5.7
mmol), K2CO3 (0.95 g, 6.9 mmol), and NaI (7 mg) in dry MeCN
was refluxed for 4 h. The mixture was filtered, and the filtrate
was evaporated to give a dark oil that was washed with Et2O
(15 mL). The organic layer was decanted, the solvent was
evaporated, and the residue was distilled to give 10 as a
colorless liquid (0.69 g, 56%): bp 85 °C/20 Torr; 1H NMR
(CDCl3) δ 1.25 (s, 3H), 1.38 (s, 3H), 3.19 (ddt, J ) 14, 7, 1 Hz,
1H), 3.45 (ddt, J ) 14, 8, 1 Hz, 1H), 3.65 (dd, 1H, J ) 8, 5 Hz),
3.70 (s, 3H), 3.94 (dd, 1H, J ) 8, 5 Hz), 4.11 (t, 1H, J ) 8 Hz),
5.07 (dq, 1H, J ) 10, 1 Hz), 5.18 (dq, 1H, J ) 17, 1 Hz), 5.85
(dddd, 1H, J ) 17, 10, 8, 7 Hz); MS (CI) (NH3) m/z 200 (MH+,
65), 160.
N-Isop r op yl-L-ser in e Meth yl Ester (13). A solution of 7b
(0.5 g, 1.7 mmol) in MeOH (45 mL) was hydrogenated at 1
atm over 5% Pd/C for 4 h. The catalyst was filtered, and the
filtrate was evaporated under reduced pressure to give 3 as a
yellow oil (0.174 g, 63%): 1H NMR (CDCl3) δ 1.05 (d, 3H, J )
6 Hz), 1.10 (d, 3H, J ) 6 Hz), 2.40 (bs), 2.83 (septet, 1H, J )
6 Hz), 3.48 (dd, 1H, J ) 6.5, 4 Hz), 3.54 (dd, 1H, J ) 10.5, 6.5
N-Allyl-4-ca r bom eth oxy-oxa zolid in e-2-on es L-16a a n d
D-16a . The respective enantiomers 16a were obtained from
15a (chromatography, hexane-EtOAc 1:1) as a pale yellow oil
(50%): bp 110-112 °C/0.2 Torr; L-16a [R]20 -14.38° (c )
D
0.014, CHCl3) and D-16a [R]20 +10.51° (c ) 0.075, CHCl3);
D
1H NMR (CDCl3) δ 3.74 (ddt, 1H, J ) 15, 8, 0.5 Hz), 3.81 (s,
3H), 4.25 (ddt, 1H, J ) 15, 5, 1 Hz), 4.34 (dd, 1H, J ) 11, 4
Hz), 4.36 (dd, 1H, J ) 10, 4 Hz), 4.49 (dd, 1H, J ) 11, 10 Hz),
5.24 (dq, 1H, J ) 17, 1 Hz), 5.26 (dq, 1H, J ) 8, 1 Hz), 5.77
(dtd, 1H, J ) 17, 8, 5 Hz); 13C NMR (CDCl3) δ 46.1 (t), 52.7
(q), 56.3 (d), 64.3 (t), 119.4 (t), 131.4 (d), 157.5 (s), 170.0 (s);
MS (CI) (NH3) m/z 203 (MNH4+, 100), 186 (MH+, 50); HRMS
(CI CH4) calcd for C8H11NO4 + H 186.0766, found 186.0810.
N-Allyl-4(S)-ca r bom eth oxy-5(R)-m eth yloxa zolid in e-2-
on e (L-16b). Obtained from L-15b (chromatography, hexane-
(22) Hwang, D.-R.; Helquist, P.; Shekhani, M. S. J . Org. Chem. 1985,
50, 1264.

504 J . Org. Chem., Vol. 64, No. 2, 1999
Falb et al.
EtOAc 2:1, then hexane-EtOAc 1:1) as a yellow oil (75%): bp
45.8 (t), 56.6 (d), 74.4 (d), 119.5 (t), 131.3 (d), 149.5 (d), 157.3
(s); MS (CI) (NH3) m/z 202 (MNH4+, 100), 185; HRMS (CI CH4)
calcd for C8H12N2O3 + H 185.0926, found 185.0970.
100-102 °C/0.1 Torr; [R]20 +31.57° (c ) 0.019, CHCl3); 1H
D
NMR (CDCl3) δ 1.51 (d, 3H, J ) 6 Hz), 3.72 (ddt, 1H, J ) 15,
8, 0.5 Hz), 3.80 (s, 3H), 3.90 (d, 1H, J ) 5 Hz), 4.26 (ddt, 1H,
J ) 15, 5, 2 Hz), 4.57 (dq, 1H, J ) 6, 5 Hz), 5.23 (dq, 1H, J )
18, 1 Hz), 5.25 (dq, 1H, 10, 1 Hz), 5.76 (dddd, 1H, J ) 17, 10,
8, 5 Hz); 13C NMR (CDCl3) δ 21.0 (q), 45.9 (t), 52.8 (bq), 63.0
(d), 73.0 (v), 119.5 (q), 131.5 (d), 156.8 (s), 169.8 (q); MS (CI)
(NH3) m/z 217 (MNH4+, 100), 200; HRMS (CI CH4) calcd for
C9H13NO4 + H 200.0922, found 200.0908.
N-Allyl-th ia zolid in e-2-on e-4(S)-ca r boxa ld eh yd e Oxim e
(L-17c). Obtained from L-16c′ as a white solid when chro-
matographed (hexane-EtOAc 1:1) (50%): mp 95-97 °C; [R]20
D
+18.18° (c ) 0.011, CHCl3); 1H NMR (CDCl3) for the major
syn-isomer δ 3.17 (dd, 1H, J ) 11, 4.5 Hz), 3.56 (dd, 1H, J )
11, 8 Hz), 3.57 (ddt, 1H, J ) 15, 8, 0.5 Hz), 4.28 (ddt, 1H, J )
15, 5, 0.5 Hz), 4.45 (dt, 1H, J ) 8, 4.5 Hz), 5.18-5.30 (m, 2H),
5.73 (dddd, 1H, J ) 17, 10, 7, 5 Hz), 7.45 (d, 1H, J ) 8 Hz),
7.95 (bs, 1H). 1H NMR (CDCl3) for the minor anti-isomer δ
5.14 (ddd, 1H, J ) 8, 6, 4 Hz), 6.93 (d, 1H, J ) 6 Hz), 8.34 (bs,
1H); 13C NMR (CDCl3) for the major isomer δ 29.5 (t), 45.9 (t),
57.8 (d), 119.0 (t), 131.6 (d), 147.9 (d); for the minor isomer δ
29.3 (t), 46.5 (t), 52.8 (d), 119.1 (t), 131.5 (d), 149.9 (d); MS
(CI) (NH3) m/z 204 (MNH4+, 100), 187; HRMS (DCI CH4) calcd
for C7H10N2O2S + H 187.0541, found 187.0567.
N-Allyl-4(S)-car beth oxy-th iazolidin e-2-on e (L-16c′). Ob-
tained from L-15c′ (chromatography, hexane-EtOAc 1:1) as
a yellow oil (68%): [R]20D -47.00° (c ) 0.023, CHCl3); 1H NMR
(CDCl3) δ 1.32 (t, 3H, J ) 7 Hz), 3.39 (dd, 1H, J ) 11, 2.5 Hz),
3.58 (ddt, 1H, J ) 16, 7.5, 1 Hz), 3.61 (dd, 1H, J ) 11, 8 Hz),
4.28 (q, 2H, J ) 7 Hz), 4.36 (dd, 1H, J ) 8, 2.5 Hz), 4.49 (ddt,
1H, J ) 16, 4.5, 1.5 Hz), 5.20 (dq, 1H, J ) 17, 1 Hz), 5.24 (dq,
1H, J ) 10, 1 Hz), 5.77 (dddd, 1H, J ) 17, 10, 7.5, 4.5 Hz); 13
C
NMR (CDCl3) δ 14.2 (q), 29.1 (t), 46.7 (t), 59.6 (d), 62.2 (t),
119.0 (t), 132.0 (d), 169.9 (s), 171.3 (s); MS (CI) (NH3) m/z 233
(MNH4+, 100), 216; HRMS (CI CH4) calcd for C9H13NO3S + H
216.0694, found 216.0670.
Gen er a l P r oced u r e for IOOC of 17 to Isoxa zolid in es
18. A mixture of 17 (0.59 mmol) in dry toluene (30 mL) was
heated at 165-170 °C for 16 h in a sealed tube, under Ar. The
tube was opened (TLC indicated complete consumption of
starting material), and the solvent was evaporated to give 18.
[3a -(3a r,8a â,8br)]-Hexa h yd r o-3H,6H-oxa zolo[3′4′:1,2]-
p yr r olo[3,4-c]isoxa zol-6-on e (L-18a a n d D-18a ). Obtained
from 17a as white solids upon chromatography (EtOH-EtOAc
Gen er a l P r oced u r e for Red u ction a n d Oxim a tion of
16 to Ald oxim es 17. To a solution of 16 (0.43 mmol) in dry
CH2Cl2 (13 mL) under N2 was added DIBALH in hexane (1
M, 9.1 mL, 9.1 mmol) dropwise over 30-60 min, while the
temperature was maintained below -75 °C. After 2 h, MeOH
(1 mL) was added. The reaction mixture was then partitioned
between EtOAc (25 mL) and saturated aqueous sodium
potassium tartrate (15 mL), NH2OH‚HCl (0.64 g, 9.2 mmol),
and NaOH (0.4 g, 10 mmol) at pH ) 11-12. Vigorous stirring
was continued at room temperature for 12 h until all the solids
dissolved. The organic layer was separated, and the aqueous
phase was again extracted with EtOAc (2 × 20 mL). The
combined organic layers were dried (MgSO4) and concentrated.
The residue was chromatographed on silica gel to give 17.
N-Allyl-4-oxa zolid in e-2-on e-4-ca r boxa ld eh yd e Oxim es
L-17a a n d D-17a . The respective oximes 17a were obtained
from 16a as colorless needles when chromatographed (hex-
ane-EtOAc 1:4) followed by crystallization from toluene
(60%): mp 113-115 °C; L-17a [R]20D +30.0° (c ) 0.01, CHCl3);
1:4) (80%): mp 114-115 °C; L-18a [R]20 -68.86° (c ) 0.033,
D
CHCl3); D-18a [R]20 +38.20° (c ) 0.009, CHCl3); 1H NMR
D
(CDCl3) δ 2.95 (dd, 1H, J ) 12.5, 7 Hz), 3.30 (ddddd, 1H, J )
9, 9, 7, 6, 0.5 Hz), 3.52 (dd, 1H, J ) 9.5, 6 Hz), 3.76 (ddd, 1H,
J ) 7.5, 6.5, 2.5 Hz), 3.96 (dd, 1H, J ) 9, 6.5 Hz), 4.02 (d, 1H,
J ) 9.5 Hz), 4.20 (dd, 1H, J ) 12.5, 9 Hz), 4.35 (dd, 1H, J )
9.5, 2.5 Hz), 4.59 (dd, 1H, J ) 9.5, 7.5 Hz), 5.21 (bs, 1H); 13C
NMR (CDCl3) δ 49.3 (d), 51.7 (t), 64.63 (d), 67.5 (t), 71.7 (d),
76.8 (t), 160.7 (s); MS (DCI) (NH3) m/z 188 (MNH4+, 100), 171;
HRMS (DCI) calcd for C7H10N2O3 + H 171.0769, found
171.0747.
[3a R-(3a r,8r,8a â,8br)]-Hexa h yd r o-3H,6H-8-m eth yl-ox-
a zolo[3′4′:1,2]p yr r olo[3,4-c]isoxa zol-6-on e (L-18b ). Ob-
tained from L-17b as a white solid when chromatographed
(EtOH-EtOAc 1:4) (55%): [R]20 -34.00° (c ) 0.010, CHCl3);
D
D-17a [R]20 -20.63° (c ) 0.016, CHCl3); 1H NMR (CDCl3) for
1H NMR (CDCl3) δ 1.53 (d, 3H, J ) 6 Hz), 2.92 (dd, 1H, J )
12.5, 7 Hz), 3.31 (ddddd, 1H, J ) 9, 9, 7, 6, 0.5 Hz), 3.38 (dd,
1H, J ) 6, 2.5 Hz), 3.50 (dd, 1H, J ) 9.5, 6 Hz), 3.95 (dd, 1H,
J ) 9, 6 Hz), 4.00 (d, 1H, J ) 9.5 Hz), 4.18 (dd, 1H, J ) 12.5,
9 Hz), 4.60 (dq, 1H, J ) 6, 2.5, Hz), 5.20 (bs, 1H); 13C NMR
(CDCl3) δ 21.4 (q), 49.2 (d), 51.2 (t), 70.9 (d), 71.4 (d), 76.4 (d),
76.6 (t), 156.0 (s); MS (CI) (NH3) m/z 202 (MNH4+, 100), 185;
HRMS (CI CH4) calcd for C8H12N2O3 + H 185.0926, found
185.0930.
D
the major syn-isomer δ 3.64 (ddt, 1H, J ) 15.5, 7, 1 Hz), 4.10
(ddt, 1H, J ) 15.5, 5, 1.5 Hz), 4.19 (dd, 1H, J ) 8, 5 Hz), 4.45
(ddd, 1H, J ) 9, 7, 5 Hz), 4.50 (t, 1H, J ) 8.5 Hz), 5.24 (ddt,
1H, J ) 17, 1.5, 1 Hz), 5.26 (ddt, 1H, J ) 10, 1.5, 1 Hz), 5.76
(dddd, 1H, J ) 17, 10, 7, 5 Hz), 7.35 (d, 1H, J ) 7 Hz), 7.85
(bs, 1H). 1H NMR (CDCl3) for the minor anti-isomer δ 3.69
(ddt, 1H, J ) 15.5, 7, 1 Hz), 4.11 (dd, 1H, J ) 9, 6 Hz), 4.12
(ddt, 1H, J ) 15.5, 5, 1.5 Hz), 4.60 (t, 1H, J ) 9 Hz), 5.01
(ddd, 1H, J ) 9, 6, 5.5 Hz), 5.27 (ddt, 1H, J ) 17, 1.5, 1 Hz),
5.28 (ddt, 1H, J ) 10, 1.5, 1 Hz), 5.80 (dddd, 1H, J ) 17, 10,
7, 5 Hz), 6.90 (d, 1H, J ) 5.5 Hz), 8.12 (bs, 1H); 13C NMR
(CDCl3) for the major isomer δ 45.2 (t), 54.8 (d), 65.0 (t), 119.5
(t), 131.2 (d), 147.5 (d), 157.8 (s); for the minor isomer δ 45.9
(t), 50.6 (d), 65.8 (t), 119.6 (t), 131.2 (d), 149.0 (d), 158.2 (s);
MS (CI) (isobutane) m/z 171 (MH+, 100); HRMS (CI, CH4) calcd
for C7H10N2O3 + H 171.0769, found 171.0746.
[3aR-(3ar,8aâ,8br)]-Hexah ydr o-3H,6H-th iazolo[3′4′:1,2]-
p yr r olo[3,4-c]isoxa zol-6-on e (L-18c). Obtained from L-17c
as a white solid when chromatographed (EtOH-EtOAc 1:4)
(75%): mp > 170 °C (dec); [R]20 -81.81° (c ) 0.022, CHCl3);
D
1H NMR (CDCl3) δ 2.87 (dd, 1H, J ) 12.5, 7 Hz), 3.30 (dd, 1H,
J ) 11.5, 8 Hz), 3.36 (ddddd, 1H, J ) 9.5, 8.5, 7, 6.5, 1 Hz),
3.53 (dd, 1H, J ) 9.5, 6.5 Hz), 3.62 (dd, 1H, J ) 11.5, 4.5 Hz),
3.90 (ddd, 1H, J ) 8, 7, 4.5 Hz), 3.96 (dd, 1H, J ) 8.5, 7 Hz),
4.01 (dd, 1H, J ) 9.5, 1 Hz), 4.24 (dd, 1H, J ) 12.5, 9.5 Hz),
5.25 (bs, 1H); 13C NMR (CDCl3) δ 30.0 (t), 49.3 (d), 49.7 (t),
66.4 (d), 70.1 (d), 76.9 (t), 172.5 (s); MS (CI) (NH3) m/z 204
(MNH4+, 100), 187; HRMS (DCI CH4) calcd for C7H10N2O2S +
H 187.0541, found 187.0470.
N-Allyl-5(R)-m eth yloxa zolid in e-2-on e-4(S)-ca r boxa ld e-
h yd e Oxim e (L-17b). Obtained from L-16b as colorless
needles when chromatographed (hexane-EtOAc 1:4) followed
by crystallization (60%): mp 140-142 °C; [R]20 +58.33° (c )
D
0.012, CHCl3); 1H NMR (CDCl3) for the major syn-isomer δ
1.47 (d, 3H, J ) 6 Hz), 3.63 (ddt, 1H, J ) 15, 6, 1 Hz), 3.97
(dd, 1H, J ) 6, 8 Hz), 4.07 (ddt, 1H, J ) 15, 5, 1.5 Hz), 4.44
(apentet, 1H, J ) 6 Hz), 5.23 (dq, 1H, J ) 16, 1 Hz), 5.27 (dq,
1H, J ) 10, 1 Hz), 5.75 (dddd, 1H, J ) 16, 10, 6, 5 Hz), 7.31
(d, 1H, J ) 8 Hz), 7.85 (bs, 1H); 1H NMR (CDCl3) for the minor
anti-isomer δ 1.52 (d, 3H, J ) 6 Hz), 3.65 (ddt, 1H, J ) 15, 6,
1 Hz), 4.12 (ddt, 1H, J ) 15, 5, 1.5 Hz), 4.42 (apentet, 1H, J )
6 Hz), 4.64 (t, 1H, J ) 6 Hz), 5.26 (dq, 1H, J ) 16, 1 Hz), 5.27
(dq, 1H, J ) 10, 1 Hz), 5.77 (dddd, 1H, J ) 16, 10, 6, 5 Hz),
6.82 (d, 1H, J ) 6 Hz), 8.11 (bs, 1H); 13C NMR (CDCl3) for the
major isomer δ 19.3 (q), 45.3 (t), 61.8 (d), 73.4 (d), 119.4 (t),
131.3 (d), 147.7 (d), 157.1 (s); for the minor isomer δ 20.4 (q),
Gen er a l P r oced u r e for Ra n ey Ni Red u ction of 18 to
Am in o Alcoh ols 19. A solution of 18 (0.43 mmol) in MeOH-
water (5:1, 9 mL) was hydrogenated (H2, 1 atm) over Raney
Ni for 24 h. The catalyst was filtered, and the filtrate was
evaporated under reduced pressure to give 19.
[6R-(6r,7r,7a r)-]-7-Am in otetr a h yd r o-6-(h yd r oxym eth -
yl)-1H ,3H -p yr r olo[1,2-c]oxa zol-3-on e (L-19a ). Obtained
from L-18a as a colorless oil when chromatographed (MeOH-
NH4OH) (76%). [R]20 -35.00° (c ) 0.020, H2O); 1H NMR
D
(CDCl3) δ 2.55 (ddddd, 1H, J ) 8.5, 8.3, 6.5, 5, 4.5 Hz), 3.22
(dd, 1H, J ) 12, 4.5 Hz), 3.33 (t, 1H, J ) 8.3 Hz), 3.72 (dd, 1H,
J ) 11, 6.5 Hz), 3.76 (dd, 1H, J ) 12, 8.5 Hz), 3.77 (dd, 1H, J

Intramolecular Oxime-Olefin Cycloaddition
J . Org. Chem., Vol. 64, No. 2, 1999 505
) 11, 5 Hz), 3.90 (td, 1H, J ) 8.3, 4.5 Hz), 4.37 (dd, 1H, J )
9, 4.5 Hz), 4.64 (dd, 1H, J ) 9, 8 Hz); 13C NMR (D2O) δ 44.1
(d), 48.4 (t), 57.3 (d), 60.5 (t), 64.9 (d), 69.1 (t), 163.9 (s); MS
(CI) (NH3) m/z 190 (MNH4+, 100), 173, 160; HRMS (CI CH4)
calcd for C7H12N2O3 + H 173.0926, found 173.0950.
CH4) calcd for C6H14N2O2 + H 147.1133, found 147.1090. A
small amount of a minor L-21a ‚CO2 adduct was also de-
tected: 1H NMR (D2O) δ 2.60 (m, 1H), 3.24 (dd, 1H, J ) 10.5,
9.5 Hz), 3.47 (dd, 1H, J ) 10.5, 8 Hz), 3.49 (dd, 1H, J ) 5.5,
1.5 Hz), 3.65 (dd, 1H, J ) 7.5, 6, 1.5 Hz), 3.72 (dd, 1H, J ) 11,
8 Hz), 3.78 (dd, 1H, J ) 11.5, 7.5 Hz), 3.80 (dd, 1H, J ) 11, 5
Hz), 3.88 (dd, 1H, J ) 11.5, 6 Hz); 13C NMR (D2O) δ 42.7 (d),
47.9 (t), 54.1 (d), 60.1 (t), 63.3 (t), 67.4 (d), 163.8 (s).
2(S),4(S)-Di(h ydr oxym eth yl)-3(S)-am in opyr r olidin e (D-
21a ) a n d D-21a ‚CO2 Ad d u ct. To a solution of D-23 (27.6 mg,
0.2 mmol) dissolved in 1:5 MeOH-H2O (4.5 mL) was added
an aqueous slurry of Ra-Ni (2 mL), and the mixture was
hydrogenated for 28 h. The mixture was filtered, and the
filtrate was evaporated. The crude residue was chromato-
graphed (MeOH-NH4OH 9:1) to give D-21a , 16.5 mg (66.5%
yield): [R]20D +12.1° (c ) 0.016, H2O); 1H and 13C NMR spectra
were identical to those of L-21a . The relative amount of the
minor D-21a ‚CO2 adduct detected in this case was consider-
ably lower than that found in the case of L-21a .
3(R)-Am in o-2(R)-[1′(R)-h yd r oxyeth yl]-4(R)-h yd r oxym -
eth ylp yr r olid in e (L-21b). Obtained from L-19b as an oil (22
mg, 70%): 1H NMR (D2O) δ 1.24 (d, 3H, J ) 6.5 Hz), 2.37
(sextet, 1H, J ) 7 Hz), 2.93 (dd, 1H, J ) 11.5, 7 Hz), 2.94 (dd,
1H, J ) 6, 5.5 Hz), 3.24 (dd, 1H, J ) 11.5, 7 Hz), 3.39 (dd, 1H,
J ) 7, 6 Hz), 3.64 (dd, 1H, J ) 11, 6.5 Hz), 3.76 (dd, 1H, J )
11, 6.5 Hz), 3.93 (qd, 1H, J ) 6.5, 5.5 Hz); 13C NMR (D2O) δ
20.7 (q), 44.5 (d), 47.5 (t), 54.3 (d), 60.0 (t), 67.4 (d), 72.0 (d);
MS (CI) (NH3) m/z 161 (MH+, 100).
[6R-(6r,7r,7a r)-1′R]-7-Am in o-tetr a h ydr o-6-(1′-h ydr oxy-
eth yl)-1H,3H-p yr r olo[1,2-c]oxa zol-3-on e (L-19b). Obtained
from L-18b as an oil when chromatographed (MeOH-NH4-
OH) (82%): [R]20 -16.47° (c ) 0.017, H2O); 1H NMR (D2O) δ
D
1.52 (d, 3H, J ) 6.5 Hz), 2.59 (ddddd, 1H, J ) 8.2, 8, 6.3, 5.1,
4.7 Hz), 3.24 (dd, 1H, J ) 11.8, 4.7 Hz), 3.38 (dd, 1H, J ) 8.2,
8 Hz), 3.55 (dd, 1H, J ) 8.2, 4.7 Hz), 3.74 (dd, 1H, J ) 11.4,
6.3 Hz), 3.77 (dd, 1H, J ) 11.8, 8.2 Hz), 3.80 (dd, 1H, J ) 11.4,
5.1 Hz), 4.73 (qd, 1H, J ) 6.5, 4.7 Hz); 13C NMR (D2O) δ 21.0
(q), 44.5 (d), 48.0 (t), 57.0 (d), 60.4 (t), 71.6 (d), 79.2 (d), 163.2
(s); MS (CI) (NH3) m/z 204 (MNH4+, 100), 187; HRMS (CI CH4)
calcd for C8H14N2O3 + H 187.1082, found 187.1064.
[6R-(6r,7r,7a r)]-7-Am in o-tetr a h yd r o-6-(h yd r oxym eth -
yl)-1H,3H-p yr r olo[1,2-c]th ia zol-3-on e (L-19c). To a solution
of isoxazolidine L-18c (51 mg, 0.28 mmol) in MeOH-water
(1:1, 4 mL) was added Zn (80 mg) and HCl (32%, 5 drops).
The reaction mixture was stirred at room temperature for 12
h, and the solvent was evaporated to give an oil which was
chromatographed (MeOH-NH4OH, 30 mL:7 drops) to give
L-19c as a colorless oil (22 mg, 42%): [R]20D -12.40° (c ) 0.025,
H2O); 1H NMR (D2O) δ 2.71 (tddd, 1H, J ) 8.1, 6.7, 5, 4.3 Hz),
3.19 (dd, 1H, J ) 12.1, 4.3 Hz), 3.40 (t, 1H, J ) 8.1 Hz), 3.40
(dd, 1H, J ) 11.0, 9.3 Hz), 3.50 (dd, 1H, J ) 11.0, 7.7 Hz),
3.72 (dd, 1H, J ) 11.5, 6.7 Hz), 3.73 (ddd, 1H, J ) 12.1, 8.1,
0.7 Hz), 3.80 (dd, 1H, J ) 11.5, 5.0 Hz), 4.20 (dddd, 1H, J )
9.3, 8.1, 7.7, 0.7 Hz); 13C NMR (D2O) δ 32.9 (t), 45.6 (d), 46.1
(t), 56.9 (d), 60.5 (t), 69.0 (d), 174.7 (s); MS (CI) (NH3) m/z 206
(MNH4+, 45), 189; HRMS (DCI CH4) calcd for C7H11N2O2S (M
- H)+ 187.0541, found 187.0540.
3(S)-Am in o-4(R)-h yd r oxym eth yl-2(S)-m eth yl-p yr r oli-
d in e 1-Ca r ba ld eh yd e (L-20). Ra-Ni reduction of L-18c (43
mg, 0.23 mmol) and preparative TLC (MeOH-NH4OH, 10:
0.1) gave desulfurization product L-20 as a colorless oily solid
(39%): 1H NMR (D2O) δ 1.21 (d, 3H, J ) 6.5 Hz, minor rotamer
Me), 1.28 (d, 3H, J ) 6.5 Hz, major rotamer Me), 2.60-2.75
(m, 4H), 3.20-3.35 (m, 1.5H), 3.40-3.85 (m, 4.5H), 8.11 (s,
1H, minor rotamer CHO), 8.14 (s, 1H, major rotamer CHO);
13C NMR (D2O) for the major rotamer δ 20.3 (q), 41.2 (t), 44.8
(d), 57.2 (d), 60.0 (t), 62.8 (d), 164.1 (d); for the minor rotamer
δ 17.5 (q), 41.8 (t), 47.5 (d), 57.6 (d), 59.6 (t), 60.6 (d), 164.4
(d); MS (CI) (NH3) m/z 159 (MH+, 100).
2(R)-Mer ca p t om et h yl-4(R)-h yd r oxym et h yl-3(R)-a m i-
n op yr r olid in e (L-21c). An aqueous (7 mL) solution of L-19c
(40 mg, 0.21 mmol) and KOH (0.188 g, 3.35 mmol) under N2
was refluxed for 24 h. The solvent was evaporated, and the
residue was chromatographed on silica gel (MeOH-NH4OH
4:1) to give L-21c as a dark yellow oil (21 mg, 61%): [R]20
D
-65.93° (c ) 0.018, H2O); 1H NMR (D2O) δ 2.56 (asextet, 1H,
J ) 7 Hz), 2.88 (dd, 1H, J ) 14.5, 9 Hz), 3.07 (dd, 1H, J ) 12,
7.5 Hz), 3.24 (dd, 1H, J ) 14.5, 4 Hz), 3.35 (dd, 1H, J ) 12,
7.5 Hz), 3.48 (dd, 1H, J ) 7, 6.5 Hz), 3.53 (ddd, 1H, J ) 9, 6.5,
4 Hz), 3.76 (dd, 1H, J ) 11.5, 6.5 Hz), 3.83 (dd, 1H, J ) 11.5,
6.5 Hz); 13C NMR (D2O) δ 41.4 (t), 44.1 (d), 47.4 (t), 52.2 (d),
60.4 (t), 65.1 (d); MS (CI) (isobutane) m/z 163 (MH+, 100);
HRMS (DCI CH4) calcd for C6H14N2OS + H 163.0905, found
163.0820.
2(R),4(R)-Bish ydr oxym eth yl-3(R)-(3-ph en yl-th iou r eido)-
p yr r olid in e-1-ca r both ioic Acid P h en yla m id e (L-22). Pyr-
rolidine L-21a (12.7 mg, 0.087 mmol) and phenylisothiocyanate
(0.1 mL, 0.83 mmol) in EtOAc (2 mL) were mixed at room
temperature for 72 h. The solvent was then decanted, and the
solution was evaporated to give a solid. The residue was
washed with acetone, and the mixture was filtered and
evaporated to give additional solid. The solids were combined
(22 mg) and chromatographed (EtOAc) to give 22 as a yellow
3a(S),6(R),6a(S)-(Hexahydro-pyrrolo[3,4-c]isoxazol-6-yl)-meth-
a n ol (D-23). A mixture of D-18a (23.4 mg, 0.12 mmol) and
Cs2CO3 (17 mg, 0.12 mmol, 0.6 equiv) in MeOH-H2O (1:3, 1.0
mL) was stirred at 83 °C for 14 h and at 103 °C for another 5
h. Chromatography (MeOH-NH4OH 25%, 9:1) gave 13b as a
light yellow oil (15.1 mg, 0.11 mmol, 75%): [R]20 +5.26° (c )
solid (10 mg, 27%): mp 118-120 °C (dec); [R]20 +19.41° (c )
D
D
1
0.0038, H2O); H NMR 320 K (D2O) δ 3.32 (dd, 1H, J ) 11.5,
0.017, acetone); 1H NMR 320 K (acetone-d6) δ 2.88 (ddddd, 1H,
J ) 10.5, 8.5, 6, 5.5, 4.5 Hz), 3.76 (dd, 1H, J ) 11, 4.5 Hz),
3.83 (dd, 1H, J ) 11, 6 Hz), 3.84 (dd, 1H, J ) 12, 8.5 Hz), 3.91
(dd, 1H, J ) 10.5, 7.5 Hz), 3.99 (dd, 1H, J ) 12, 10.5 Hz), 4.16
(dd, 1H, J ) 10.5, 4 Hz), 4.69 (ddd, 1H, J ) 7.5, 4, 1 Hz), 4.92
7 Hz), 3.05 (dd, 1H, J ) 6.5, 4.5 Hz), 3.32 (dd, 1H, J ) 12, 7.5
Hz), 3.48 (dd, 1H, J ) 11.5, 8.5 Hz), 3.64 (bdd, 1H), 3.72 (dd,
1H, J ) 12, 6.5 Hz), 3.82 (dd, 1H, J ) 12, 4.5 Hz), 3.93 (dd,
1H, J ) 8.5, 6.5 Hz), 4.00 (d, 1H, J ) 7.5 Hz); 13C NMR (D2O)
δ 48.3 (d), 51.3 (t), 61.3 (t), 66.0 (d), 66.7 (d), 76.4 (t); HRMS
(CI CH4) calcd for C6H13N2O2 + H 145.0977, found 145.0975.
1
(dd, 1H, J ) 5.5, 1 Hz), 7.00-7.60 (m, 10 H); H NMR 25 °C
(acetone-d6) for a mixture of rotamers δ 2.90 (bs, 1H), 3.75-
4.00 (m, 6H), 4.18 (bd, 1H), 4.29 (bs, 1H), 4.72 (bdd, 1H), 4.89
(bt, 1H), 7.00-7.55 (m, 10 H), 7.98 (d, 1H, J ) 6 Hz), 9.06 (s,
1H), 9.50 (bs, 1H); 13C NMR 25 °C (acetone-d6) for a mixture
of rotamers δ 42.2 (d), 52.4 (t), 59.7 (d), 60.1 (t), 64.4 (t), 71.4
(d), 124.6, 124.7, 124.9, 125.8, 128.8, 129.7, 141.7 (Ph), 181.6
(s), 182.3 (s).
Gen er a l P r oced u r e for Hyd r olysis of P yr r olo-Oxa zo-
lid in on es 19 to P yr r olid in es 21. To an aqueous (5 mL)
solution of 19 (0.33 mmol) was added Cs2CO3 (20 mg, <0.2
equiv). The reaction mixture was refluxed for 17 h. Evapora-
tion of the solvent followed by chromatography on silica gel
(MeOH-NH4OH) gave 21.
2(R),4(R)-Di(h ydr oxym eth yl)-3(R)-am in opyr r olidin e (L-
En zym e In h ibition Assa ys. R-Glucosidase (EC 3.2.1.20)
from baker’s yeast, â-glucosidase (EC 3.2.1.21) from almonds,
R-galactosidase (EC 3.2.1.22) from green coffee beans, â-ga-
lactosidase (EC 3.2.1.23) from Escherichia coli, and the p-
nitrophenyl R-D-gluco-, â-D-gluco-, R-D-galacto-, and â-D-galacto-
pyranosides were purchased from Sigma Co.
Enzymatic assays were carried out by following the hydroly-
sis of the appropriate p-nitrophenyl glycoside substrates
spectrophotometrically at 400 nm (ꢀ 11 150 at pH 7.2) at 25
21a ) a n d L-21a ‚CO2 Ad d u ct. Obtained from L-19a as an oil
1
(53%): [R]20 -14.92° (c ) 0.02, H2O); H NMR (D2O) δ 2.44
D
(sextet, 1H, J ) 7 Hz), 2.96 (dd, 1H, J ) 11.5, 7 Hz), 3.15 (td,
1H, J ) 6, 5 Hz), 3.25 (dd, 1H, J ) 11.5, 7 Hz), 3.39 (dd, 1H,
J ) 7, 6 Hz), 3.68 (dd, 1H, J ) 12, 6 Hz), 3.73 (dd, 1H, J ) 11,
7 Hz), 3.79 (dd, 1H, J ) 12, 5 Hz), 3.84 (dd, 1H, J ) 11, 6.5
Hz); 13C NMR (D2O) δ 43.7 (d), 47.3 (t), 54.0 (d), 60.0 (t), 61.9
(t), 67.2 (d); MS (CI) (NH3) m/z 147 (MH+, 100); HRMS (CI

506 J . Org. Chem., Vol. 64, No. 2, 1999
Falb et al.
°C. Enzyme concentration was set to obtain a hydrolysis rate
of about 10-3 OD/sec under substrate saturation. R-Glu cosi-
d a se (at 2 mg/mL) was assayed in 45 mM sodium acetate
buffer, pH 7.2. Substrate concentration was 0.25-4.0 mM (Km
0.2-0.3 mM under our experimental conditions, similar to a
published value of 0.3 mM).21 â-Glu cosid a se (at 2.3 mg/mL)
was assayed in 45 mM sodium acetate buffer, pH 7.2.
Substrate concentration was 0.54 and 1.08 mM (Km 2.84 mM
under our experimental conditions, similar to a published
value of 2.1-3.5 mM).21 R-Ga la ctosid a se (at 0.045 units/mL)
was assayed in 90 mM potassium phosphate buffer, pH 6.5.
Substrate concentration was 0.1-1.1 mM (Km 0.35 mM under
our experimental conditions). â-Ga la ctosid a se (at 0.4 units/
mL) was assayed in 45 mM potassium phosphate buffer, pH
7.0, containing 130 mM NaCl and 0.9 mM MgCl2. Substrate
concentration was 0.027-0.2 mM (Km 0.03 mM under our
experimental conditions).
Inhibition studies were carried out by measuring the initial
velocity of hydrolysis of an appropriate substrate (in the above-
mentioned concentrations) in the presence of varying inhibitor
concentrations. Lineweaver-Burk plots (1/v vs 1/[S]) for the
different inhibitor concentrations yielded the Km app values
from the X-axis intercepts (on the basis of the competitive
inhibition kinetics equation v ) vmax[S]/{[S] + Km(1 + [I]/Ki)},
where Km app ) Km(1 + [I]/Ki). Replotting of the Km app values
against inhibitor concentration yielded the kinetic parameter
Ki (as Km/slope). In some of the cases, a single inhibitor
concentration was used.
Ack n ow led gm en t. Generous support for this work
by the Marcus Center for Pharmaceutical and Medicinal
Chemistry at Bar Ilan University, Minerva Foundation
Otto Mayerhoff Center for the Study of Drug-Receptor
Interactions at Bar Ilan University, the Leah Dubovsky
Fund for Basic Research, the TAMI Institute of Re-
search and Development and the U.S.-Israel Binational
Science Foundation is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H and 13C
NMR spectra for compounds 2a ,b, 3-7a ,b, 8-11, 13, 14, 15a -
c, 16a -c, 17a -c, 18a -c, 19a -c, 20, 21a -c, 21a ‚CO2 adduct,
22, and 23 and the spectrum of L-21a after the sample was
heated to 90 °C (70 pages). This material is contained in
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, and can be ordered from the
ACS; see any current masthead page for ordering information.
J O9815094