A phosphinate inhibitor of the meso-diaminopimelic acid-adding enzyme (MurE) of peptidoglycan biosynthesis

Full text of DOI:10.1021/jo981895p

J . Org. Chem. 1998, 63, 10081-10086
10081
A P h osp h in a te In h ibitor of th e
m eso-Dia m in op im elic Acid -Ad d in g En zym e
(Mu r E) of P ep tid oglyca n Biosyn th esis
Binqi Zeng,† Kenny K. Wong,‡ David L. Pompliano,‡
Sreelatha Reddy,‡ and Martin E. Tanner*,†
Department of Chemistry, University of British Columbia,
Vancouver, British Columbia, V6T 1Z1, Canada, and
Department of Biochemistry, Merck Research Laboratories,
P.O. Box 2000, Rahway New J ersey 07065-0900
Received September 17, 1998
The emergence of multi-drug-resistant bacteria has led
to an increased demand for new types of antibiotics.1 The
biosynthetic pathway for bacterial cell wall (peptidogly-
can) formation represents an attractive target for inhibi-
tor design since many clinically useful antibiotics (such
as penicillin and vancomycin) inhibit enzymes of the later
stages of this pathway.2 A set of ligases (MurC-F) is
responsible for the synthesis of the UDP-MurNAc-pen-
tapeptide (Figure 1) that is a common precursor to
peptidoglycan biosynthesis in Gram-negative bacteria.3
The meso-diaminopimelic acid-adding enzyme (MurE) is
an ATP-dependent amino acid ligase that is responsible
for the formation of UDP-MurNAc-L-Ala-D-Glu-m-Dap.4,5
Here we report the synthesis and initial evaluation of
the first effective inhibitor 1 of MurE.6
F igu r e 1. The reactions catalyzed by MurE and MurF and
The mechanism of the MurE reaction is assumed to
resemble that employed by many of the well-character-
ized ATP-dependent ligases.7-10 An initial phosphoryla-
the proposed intermediates formed during the MurE reaction.
tion of the glutamate γ-carboxylate is followed by attack
from the (S)-amino group of meso-diaminopimelic acid
to form a tetrahedral intermediate (Figure 1). A subse-
quent loss of phosphate yields the product amide. An
effective strategy for inhibiting such enzymes has been
to mimic the tetrahedral intermediate with a correspond-
ing phosphinic acid.10b-d,11-14 In certain cases, it has been
established that phosphorylation of the phosphinic acid
occurs and results in slow-tight binding inhibition. Previ-
ous work in this lab demonstrated that the D-glutamate-
adding enzyme (MurD) was strongly inhibited by a
phosphinic acid analogue in which the MurNAc moiety
was replaced by a hydrophobic hexanoate linker.13 With
this precedence in mind we designed the MurE inhibitor
1 (Scheme 1).
† University of British Columbia.
‡
Merck Research Laboratories.
(1) (a) Davies, J . Science 1994, 264, 375. (b) Cohen, M. L. Science
1992, 257, 1050. (c) Neu, H. C. Science 1992, 257, 1064.
(2) Gale, E. F.; Cundliffe, E.; Reynolds, P. E.; Richmond, M. H.;
Waring, M. J . The Molecular Basis of Antibiotic Action, 2nd ed.; Wiley
and Sons: London, 1981.
(3) van Heijenoort, J . In Escherichia coli and Salmonella; Neidhardt,
F. C., Curtis, R., III., Ingraham, J . L., Lin, E. C. C., Low, K. B.,
Magasanik, B., Resnikoff, W. S., Riley, M., Schaechter, M., Umbarger,
H. E., Eds.; American Society for Microbiology: Washington, DC, 1996;
pp 1025-1034.
(4) Abbreviations: UDP ) uridine diphosphate; UMP ) uridine
monophosphate; m-Dap ) meso-diaminopimelic acid; MurNAc )
N-acetylmuramic acid; HOBt ) 1-hydroxybenzotriazole; LSI-MS )
liquid secondary ion mass spectrometry.
(5) Michaud, C.; Mengin-Lecreulx, D.; van Heijenoort, J .; Blanot,
D. Eur. J . Biochem. 1990, 194, 853.
(6) Previous work has primarily focused on testing m-Dap analogues
as MurE inhibitors. See ref 5 and: (a) Auger, G.; van Heijenoort J .;
Vederas, J . C.; Blanot, D. FEBS Lett. 1996, 391, 171. (b) van Assche,
I.; Sokora, M.; Haemers, A.; Hooper, M.; Blanot, D.; van Heijenoort, J .
Eur. J . Med. Chem. 1991, 26, 505.
(7) Glutamine synthetase: (a) Meek, T. D.; J ohnson, K. A.; Vil-
lafranca, J . J . Biochemistry 1982, 21, 2158. (b) Meek, T. D.; Villafranca,
J . J . Biochemistry 1980, 19, 5513. (c) Midelfort, C. F.; Rose, I. A. J .
Biol. Chem. 1976, 251, 5881.
(8) D-Ala-D-Ala ligase: (a) Shi, Y.; Walsh, C. T. Biochemistry 1995,
34, 2768. (b) Fan, C.; Moews, P. C.; Walsh, C. T.; Knox, J . R. Science
1994, 266, 439. (c) Wright, G. D.; Walsh, C. T. Acc. Chem. Res. 1992,
25, 468. (d) Mullins, L. S.; Zawadzke, L. E.; Walsh, C. T.; Raushel, F.
M. J . Biol. Chem. 1990, 265, 8993.
(9) Other Mur ligases: (a) Emanuele, J . J ., J r.; J in, H.; Yanchunas,
J ., J r.; Villafranca, J . J . Biochemistry 1997, 36, 7264. (b) Bouhss, A.;
Mengin-Lecreulx, D.; Blanot, D.; van Heijenoort, J .; Parquet, C.
Biochemistry 1997, 36, 11556. (c) Falk, P. J .; Ervin, K. M.; Volk, K. S.;
Ho, H.-T. Biochemistry 1996, 35, 1417. (d) Vaganay, S.; Tanner, M.
E.; van Heijenoort, J .; Blanot, D. Microbial Drug Resistance 1996, 2,
51. (e) Eveland, S. S.; Pompliano, D. L.; Anderson, M. S. Biochemistry
1997, 36, 6223. (f) Anderson, M. S.; Eveland, S. S.; Onishi, H. R.;
Pompliano, D. L. Biochemistry 1996, 35, 16264.
(10) Glutathione-related synthetases: (a) Walsh, C. T.; Bradley, M.;
Nadeau, K. TIBS 1991, 16, 305. (b) Chen, S.; Lin, C.-H.; Walsh, C. T.;
Coward, J . K. Bioorg. Med. Chem. Lett. 1997, 7, 505. (c) Katoh, M.;
Hiratake, J .; Kato, H.; Oda, J . Bioorg. Med. Chem. Lett. 1996, 6, 1437.
(d) Hiratake, J .; Kato, H.; Oda, J . J . Am. Chem. Soc. 1994, 116, 12059.
(e) Kato, H.; Tanaka, T.; Yamaguchi, H.; Hara, T.; Nishioka, T.;
Katsube, Y.; Oda, J . Biochemistry 1994, 33, 4995.
(11) Phosphinothricin: (a) Abell, L. M.; Villafranca, J . J . Biochem-
istry 1991, 30, 6135 and references therein.
(12) D-Ala-D-Ala ligase/adding enzyme inhibition: (a) Miller, D. J .;
Hammond, S. M.; Anderluzzi, D.; Bugg, T. D. H. J . Chem. Soc., Perkin
Trans. 1 1998, 131-142. (b) Fan, C.; Park, I.-S.; Walsh, C. T.; Knox,
J . R. Biochemistry 1997, 36, 2531. (c) Ellsworth, B. A.; Tom, N. J .;
Bartlett, P. A. Chem. Biol. 1996, 3, 37. (d) McDermott, A. E.; Creuzet,
F.; Griffin, R. G.; Zawadzke, L. E.; Ye, Q. Z.; Walsh, C. T. Biochemistry
1990, 29, 5767. (e) Duncan, K.; Walsh, C. T. Biochemistry 1988, 27,
3709. (f) Parsons, W. H.; Patchett, A. A.; Bull, H. G.; Schoen, W. R.;
Taub, D.; Davidson, J .; Combs, P. L.; Springer, J . P.; Gadebusch, H.;
Weissberger, B.; Valiant, M. E.; Mellin, T. N.; Busch, R. D. J . Med.
Chem. 1988, 31, 1772. See also refs 8a and 8b.
(13) Tanner, M. E.; Vaganay, S.; van Heijenoort, J .; Blanot, D. J .
Org. Chem. 1996, 61, 1756.
10.1021/jo981895p CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/05/1998

10082 J . Org. Chem., Vol. 63, No. 26, 1998
Notes
conditions.19 Treatment of 3 with bis(trimethylsilyl)-
phosphonite under mild conditions (room temperature,
CH2Cl2) gave the D-Glu analogue 5 in 32% yield.17 The
Sch em e 1
1
main side product in this reaction displayed MS and H
NMR data that were consistent with an amino acid
containing a reduced propyl side chain. It may have been
possible to increase this yield by carrying out the
displacement at elevated temperatures in toluene, as has
recently been reported by Chen and Coward.20
Compound 2 was also protected as a tert-butoxycarbo-
nyl derivative to give 4. Compound 4 was converted to
the corresponding iodide and then directly coupled to the
methyl ester of 2-(bromomethyl)acrylate using zinc/
copper chemistry to give 6 in a 33% yield (both steps).
Excellent precedence was available for carrying out this
coupling reaction on amino acid side chains and it was
shown to proceed without racemization (albeit in rela-
tively low yields).21 Compound 5 was silylated with N,O-
(bistrimethylsilyl)acetamide and attached to compound
6 via a conjugate addition.15,22 The resulting phosphinic
acid was formed as an approximately 3:2 mixture of two
diastereomers, as judged by the intensity of the 31P NMR
signals (CD3OD, sodium salt; 37.79 and 37.63 ppm). The
phosphinic acid was then esterified with (trimethylsilyl)-
diazomethane to give 7 in an overall 50% yield (both
steps).23
With the dipeptide analogue in hand, the next task was
to attach the portion of the inhibitor designed to mimic
UDP-MurNAc-L-Ala. The linker compound 8 was pre-
pared from 5-carboxypentyl diphenyl phosphate13 via a
DCC-mediated addition of the tert-butyl ester of L-alanine
followed by treatment with 88% formic acid. The “D-
glutamate” amino group of 7 was deprotected by hydro-
genation and coupled to 8 by using DCC/1-hydroxyben-
zotriazole to give 9 in an 86% yield. In previous work on
MurD inhibitors, construction of the diphosphate linkage
was performed after the complete deprotection of the
phosphinic acid/peptide-linker moiety.13,14 The phospho-
ramidate coupling yields were quite low, however, and
in this work the coupling was performed prior to depro-
tection.24 Compound 9 was hydrogenated over PtO2 at
50 psi and the resulting phosphate was coupled to UMP-
morpholidate in the presence of 1H-tetrazole.25 Coupling
yields as high as 65% were obtained in this fashion.
Complete deprotection was achieved by treatment with
88% formic acid followed by 2.3 M lithium hydroxide to
The synthesis of 1 involves the preparation of a D-Glu-
m-Dap dipeptide analogue in which a methylenephos-
phinic acid replaces the amide bond (compound 7 in
Scheme 1). The preparation of â-carboxyphosphinic acids
can readily be achieved by using conjugate addition
chemistry15 and this strategy is appropriate for use in
the construction of the m-Dap-phosphinic acid linkage.
This strategy cannot be used with simple primary phos-
phinic acids such as the γ-phosphinic acid analogue of
glutamate, 5 (note that the linkage of the MurE product
is via the γ-carboxylate of D-Glu). While routes to the
racemic phosphinic acid analogue of glutamate are
known,16 we wished to prepare optically pure 5, and since
2 could be used as a common precursor to both amino
acid analogues, we chose to prepare 5 using Arbuzov-
like chemistry.17
(18) (a) Baldwin, J . E.; North, M.; Flinn, A. Tetrahedron Lett. 1987,
28, 3167. (b) Dubois, G. E.; Stephenson, R. A. G.; Crosby, G. A. U.S.
Patent 4 226 804, 1980.
(19) Procha´zka, Z.; Smol´ıkova´, J .; Malon, P.; J ost, K. Collect. Czech.
Chem. Commun. 1981, 46, 2935.
(20) Chen, S.; Coward, J . K. J . Org. Chem. 1998, 63, 502.
(21) Dunn, M. J .; J ackson, R. F. W.; Pietruszka, J .; Turner, D. J .
Org. Chem. 1995, 60, 2210.
Compound 2 was prepared from D-aspartate in six
steps,18 protected as a benzyloxycarbonyl derivative and
then converted to iodide 3 under previously reported
(22) (a) Caldwell, C. G.; Sahoo, S. P.; Polo, S. A.; Eversole, R. R.;
Lanza, T. J .; Mills, S. G.; Niedzwiecki, L. M.; Izquierdo-Martin, M.;
Chang, B. C.; Harrison, R. K.; Kuo, D. W.; Lin, T.-Y.; Stein, R. L.;
Durette, P. L.; Hagmann, W. K. Bioorg. Med. Chem. Lett. 1996, 6, 323.
(b) Morgan, B. P.; Scholtz, J . M.; Ballinger, M. D.; Zipkin, I. D.; Bartlett,
P. A. J . Am. Chem. Soc. 1991, 113, 297.
(23) Reiter, L. A.; J ones, B. P. J . Org. Chem. 1997, 62, 2808.
(24) In a very recent report on the synthesis of the UDP-MurNAc-
pentapeptide (L-Lys containing), a similar strategy was used: Hitch-
cock, S. A.; Eid, C. N.; Aikins, J . A.; Zia-Ebrahimi, M.; Blaszczak, L.
C. J . Am. Chem. Soc. 1998, 120, 1916.
(14) Gegnas, L. D.; Waddell, S. T.; Chabin, R. M.; Sreelatha, R.;
Wong, K. K. Bioorg. Med. Chem. Lett. 1998, 8, 1643.
(15) Thottathil, J . K.; Ryono, D. E.; Przybyla, C. A.; Moniot, J . L.;
Neubeck, R. Tetrahedron Lett. 1984, 25, 4741.
(16) (a) Maier, L.; Rist, G. Phosphorus Sulfur 1983, 17, 21. (b) Baylis,
E. K.; Campbell, C. D.; Dingwall, J . D. J . Chem. Soc., Perkin Trans I
1984, 2845.
(17) (a) Boyd, E. A.; Regan, A. C. Tetrahedron Lett. 1994, 35, 4223.
(b) Boyd, E. A.; Boyd, M. E. K.; Kerrigan, F. Tetrahedron Lett. 1996,
37, 5425.
(25) Wittmann, V.; Wong, C.-H. J . Org. Chem. 1997, 62, 2144.

Notes
J . Org. Chem., Vol. 63, No. 26, 1998 10083
under argon at room temperature. The reaction was quenched
with 3% HCl and the separated organic layer washed with 3%
HCl (2 × 20 mL) and water (20 mL) and then dried over Na2SO4.
Filtration and removal of the solvent in vacuo yielded an oil.
The crude product was purified by chromatography on silica gel
(4% CH3OH/CH2Cl2) to give compound 5 as an N,N-diisopropyl-
ethylamine salt (0.3 g, 32%) with Rf ) 0.4 in 2% CH3OH/CH2-
Cl2: 1H NMR (200 MHz, CDCl3) δ 7.26 (m, 5 H), 7.02 (d, 1 H, J
) 540 Hz), 6.58 (br d, 1 H, J ) 8.0 Hz), 5.02 (s, 2 H), 4.40-4.20
(m, 1 H), 3.60 (s, 3 H), 3.75-3.40 (m, 2 H), 3.02-2.80 (m, 2 H),
2.20-1.40 (m, 4 H), 1.42-1.10 (m, 15 H); 31P NMR (81 MHz,
give the target inhibitor 1 as a mixture of two diastere-
omers (overall 35% yield from 9).26
Compound 1 was found to be a potent inhibitor of the
E. coli MurE. Using an HPLC based assay, the concen-
tration of the inhibitor necessary to reduce the rate of
reaction by one-half (IC50 value) was determined to be
1.1 ( 0.1 µM. This result supports the notion that the
MurE reaction follows a mechanism similar to that of
other ATP-dependent amino acid ligases and that the
enzyme will bind tightly to compounds which mimic the
structure of the putative tetrahedral intermediate (Figure
1). When compound 9 was fully deprotected and tested
as an inhibitor, the IC50 was only 700 ( 50 µM, indicating
that the complete UDP moiety is necessary for good
inhibition and a simple phosphate will not suffice.
This work provides a starting point for the rational
design of even more potent inhibitors of MurE. In studies
on analogous phosphinate inhibitors of MurD it was
found that the incorporation of a MurNac residue in place
of the simple hydrophobic linker increased the potency
of the inhibitor by almost 2 orders of magnitude.14 The
preparation of a MurNAc-containing MurE inhibitor is
currently under way in our laboratories.
CDCl3) δ 33.69; -LSI-MS (thioglycerol/CHCl3 matrix) 300 (M
+
- CH3 100%), 314 (M - H+, 53%). -LSI-HRMS calcd for
,
C
13H17NO6P: 314.0794. Found: 314.0799.
Dim eth yl (2R)-[(ter t-Bu tyloxyca r bon yl)a m in o]-6-m eth -
ylen eh ep ta n ed ioa te (6). A solution of methyl (2R)-[(tert-
butyloxycarbonyl)amino]-4-bromobutyrate, (4) (0.89 g, 3 mmol),
and NaI (0.67 g, 4.5 mmol) in acetone (10 mL) was stirred at
room temperature for 20 h. Precipitated NaBr was removed by
filtration and the solution was evaporated to dryness. The
residue was partitioned between Et2O (50 mL) and H2O (30 mL).
The ethereal solution was washed with a 5% sodium thiosulfate
solution (30 mL) and water (2 × 30 mL), dried over Na2SO4,
and evaporated to give methyl (2R)-[(tert-butoxycarbonyl)amino]-
4-iodobutyrate as a pale brown oil (0.96, 93%): 1H NMR (200
MHz, CDCl3) δ 5.20 (d, 1 H, J ) 7.8 Hz), 4.45-4.18 (m, 1 H),
3.72 (s, 3 H), 3.12 (t, 2 H, J ) 7.5 Hz), 2.50-2.00 (m, 2 H), 1.39
(s, 9 H). This compound was used directly in the next reaction.
A suspension of zinc (1.1 g, 16.8 mmol) in dry THF (1.2 mL)
and 1,2-dibromoethane (0.16 g, 0.8 mmol) was heated under
argon to 60 °C for 3 min in a 50-mL two-neck flame-dried flask
fitted with a septum and a condenser. After allowing the mixture
to cool to room temperature, trimethylsilyl chloride (0.02 g, 0.18
mmol) was added and the mixture was vigorously stirred for 30
min. The solution was warmed to 35 °C, methyl (2R)-[(tert-
butoxycarbonyl)amino]-4-iodobutyrate (0.98 g, 3.0 mmol) in dry
THF (5.6 mL) was slowly added, and the mixture was stirred
for 1 h until no starting material remained (as judged by TLC,
10:1 toluene/ethyl acetate). The solution was then cooled to -10
°C, and a solution prepared from CuCN (0.27 g, 3.0 mmol) and
LiCl (0.25 g, 6.0 mmol) in THF (5.6 mL) was added. The solution
was stirred at 0 °C for 10 min. Methyl 2-(bromomethyl)acrylate
(0.55 g, 3.0 mmol) was added at -25 °C, and the flask contents
were stirred at 0 °C for 3 h. The cooling bath was removed, and
once the solution reached room temperature, the mixture was
diluted with ethyl acetate (50 mL), washed with aqueous sodium
hydrogen carbonate (25 mL, saturated) and water (2 × 25 mL),
dried, and concentrated under reduced pressure to yield crude
oil. Flash chromatography over silica gel (8% hexanes/ethyl
ether, v/v) afforded the product 6 as an oil (0.31 g, 35%) with Rf
) 0.3 in 1:1 hexanes/ethyl ether: 1H NMR (200 MHz, CDCl3) δ
6.10 (d, 1 H, J ) 1.5 Hz), 5.48 (d, 1 H, J ) 1.4 Hz), 5.02 (br d,
1 H, J ) 8.3 Hz), 4.24 (t, 1 H, J ) 6.9 Hz), 3.70 (s, 6 H), 2.27 (t,
2 H, J ) 7.5 Hz), 2.00-1.20 (m, 4 H), 1.20 (s, 9 H); +LSI-MS
(thioglycerol matrix) 216 (M + H+ - C4H8 - CO2, 100%), 260
(M + H+ - C4H8, 25%), 316 (M + H+, 11%). +LSI-HRMS calcd
for C15H26NO6: 316.1760. Found: 316.1770.
Exp er im en ta l Section
Gen er a l P r oced u r es. Pyridine was refluxed with KOH for
24 h and then distilled from BaO under dry argon. CH2Cl2 was
distilled under N2 from P2O5. THF, Et3N, and DMF were dried
and distilled from CaH2 under nitrogen. Hydrophilic size-
exclusion chromatography was performed on Bio-Gel P-2 gel,
fine (Bio-Rad Laboratories). Lipophilic size-exclusion chroma-
tography was performed on Sephadex LH-20 (Sigma). Methyl
(2R)-amino-4-bromobutyrate hydrochloride (2) and methyl (2R)-
[(benzyloxycarbonyl)amino]-4-iodobutyrate (3) were prepared
according to previously published procedures.18,19
Meth yl (2R)-[(ter t-Bu tyloxycar bon yl)am in o]-4-br om obu -
tyr a te (4). Methyl (2R)-amino-4-bromobutyrate hydrochloride
(2) (3.0 g, 11 mmol) was suspended in THF (25 mL) and Et3N
(2.4 g, 24 mmol). The suspension was cooled to 0 °C and a
solution of di-tert-butyl dicarbonate (2.2 g, 10 mmol) in THF (5
mL) was added dropwise over 10 min. The mixture was allowed
to warm to room temperature and stirred for 6 h and then
warmed to 50 °C for a further 2 h. The solvent was removed in
vacuo and the residue partitioned between Et2O (30 mL) and
H2O (30 mL). The aqueous phase was extracted with Et2O (2 ×
30 mL), and the combined organic phases were washed with 3%
HCl (50 mL), NaHCO3 (50 mL, saturated), and NaCl (50 mL,
saturated). Drying (Na2SO4) and evaporation of the solvent
afforded the title compound as an oil (2.9 g, 89%) with Rf ) 0.3
in 3% CH3OH/CHCl3: 1H NMR (200 MHz, CDCl3) δ 5.22 (d, 1
H, J ) 8.1 Hz), 4.44-4.20 (m, 1 H), 3.75 (s, 3 H), 3.39 (t, 2 H, J
) 7.1 Hz), 2.45-2.05 (m, 2 H), 1.40 (s, 9 H); +LSI-MS (thioglyc-
erol) 240 (M(79Br) + H+ - C4H8, 100%), 242 (M(81Br) + H+
-
Com p ou n d 7. Compound 5 (0.07 g, 0.16 mmol) was dissolved
in dry CH2Cl2 (0.8 mL), and compound 6 (0.07 g, 0.23 mmol)
and N,O-bis(trimethylsilyl)acetamide (0.1 g, 0.5 mmol) were
added. The solution was stirred at room temperature for 20 h.
The reaction was quenched with 1 M HCl, and the separated
organic layer was washed with 1 M HCl (2 × 10 mL) and water
(10 mL) and then dried over Na2SO4. Filtration and concentra-
tion in vacuo gave an oil. The crude product was purified by
chromatography on silica gel (7% CH3OH/CH2Cl2) to give the
disubstituted phosphinic acid as a white foam (0.07 g, 69%): 1H
NMR (200 MHz, CDCl3) δ 7.65 (br s, 1 H), 7.20 (s, 5 H), 6.02 (br
s, 1 H), 5.60 (br s, 1 H), 5.18 (br s, 1 H), 4.95 (s, 2 H), 4.40-4.00
(m, 2 H), 3.70-3.30 (m, 9 H), 2.60 (br s, 1 H), 2.20-1.00 (m, 21
C4H8, 98%), 196 (M(79Br) + H+ - C4H8 - CO2, 88%), 198 (M(81Br)
+ H+ - C4H8 - CO2, 80%), 296 (M(79Br) + H+, 40%), 298
(M(81Br) + H+, 38%). +LSI-HRMS calcd for C10H19NO479Br:
296.0497. Found: 296.0495.
[(3R)-[3-(Ben zyloxycar bon yl)am in o]-3-car bom eth oxypr o-
p yl]p h osp h in a t e (5), As a n N,N-Diisop r op ylet h yla m in e
Sa lt. To a solution of ammonium phosphinate (0.20 g, 2.4 mmol)
in dry CH2Cl2 (6 mL) was added N,N-diisopropylethylamine
(0.68 g, 5.3 mmol) and trimethylsilyl chloride (0.58 g, 5.3 mmol)
at 0 °C under argon. The solution was stirred at room temper-
ature for 3 h and then cooled to 0 °C. Methyl (2R)-[(benzyloxy-
carbonyl)amino]-4-iodobutyrate (3) (0.67 g, 1.8 mmol) in dry
CH2Cl2 (2 mL) was added and the reaction was stirred for 20 h
H); 31P NMR (81 MHz, CD3OD) δ 37.79 and 37.63; -LSI-MS
+
(thioglycerol/CHCl3 matrix) 629 (M - H+, 100%), 615 (M - CH3
14%). This compound was used directly in the next reaction.
,
(26) To check for possible epimerization during the final deprotection
step, a sample was deprotected by using 2.3 M LiOD/D2O. Mass
spectral analysis (-LSI-MS) of the resulting compound 1 indicated that
not more than 10% of the material contained deuterium and that
significant epimerization had not occurred.
The disubstituted phosphinic acid from above (0.07 g, 0.11
mmol) was taken up in toluene/methanol (4:1 v/v, 0.5 mL) and
treated with (trimethylsilyl)diazomethane (TMSCHN2) until
evolution of nitrogen ceased and the yellow color of diazomethane

10084 J . Org. Chem., Vol. 63, No. 26, 1998
Notes
remained permanent. The solution was stirred for 45 min at
room temperature and the excess TMSCHN2 was quenched with
acetic acid. The reaction mixture was evaporated to dryness and
was chromatographed (ethyl acetate) to give 7 as a colorless oil
(0.05 g, 71%) with Rf ) 0.2 in ethyl acetate: 1H NMR (400 MHz,
CDCl3) δ 7.30 (s, 5 H), 5.80-5.60 (m, 1 H), 5.10 (s, 2 H), 5.10-
5.00 (br, 1 H) 4.45-4.20 (m, 2 H), 3.80-3.50 (m, 12 H), 2.68 (br
s, 1 H), 2.20-2.05 (m, 2 H) 2.00-1.50 (m, 8 H), 1.40 (s, 9 H)
1.35-1.25 (m, 2 H); 31P NMR (81 MHz, CDCl3) δ 53.44, 52.84;
mL). The organic layer was washed with water (2 × 3 mL) and
dried over Na2SO4. The solvent was removed in vacuo to yield
an oil. The crude product was purified on a Sephadex LH-20
column (1.5 × 100 cm in diameter/length), eluted with CH3OH/
CHCl3 (4:3 v/v). The fastest moving fractions gave 9 as colorless
oil (83 mg, 86%) with Rf ) 0.4 in 20% methanol/ethyl acetate:
1H NMR (400 MHz, CDCl3) δ 7.50-7.10 (m, 10 H), 6.12 (br, 1
H), 5.07 (br, 1 H), 4.60-4.45 (m, 2 H), 4.23 (m, 3 H), 3.80-3.60
(m, 13 H), 2.72 (br s, 1 H), 2.40-1.30 (m, 20 H), 1.42 (s, 9 H),
1.36 (d, J ) 7.0 Hz, 3 H); 31P NMR (81 MHz, CDCl3) δ 55.94,
55.73, 55.49, 55.11, -12.04; +LSI-MS (thioglycerol/CH3OH
matrix) 828 (M + H+ - C4H8 - CO2, 100%), 928 (M + H+, 22%).
+LSI-HRMS calcd for C42H64N3O16P2: 928.3762.Found: 928.3771.
Com p ou n d 1. A solution of 9 (76 mg, 82 µmol) in methanol
(3 mL) containing PtO2 monohydrate (120 mg) was stirred under
50 psi of hydrogen at room temperature for 48 h. The mixture
was filtered, and the solvent was removed in vacuo to give the
phosphoric acid as colorless oil (62 mg). A mixture of the acid
from above (36 mg, 46 µmol) and 4-morpholine-N,N′-dicyclo-
hexylcarboxamidinium uridine 5′-monophosphomorpholidate (70
mg, 0.1 mmol) was dried by the addition of dry pyridine (3 × 1
mL) and evaporation under reduced pressure. 1H-Tetrazole (15
mg, 0.2 mmol) and dry pyridine (0.8 mL) were added, and the
solution was stirred under argon at room temperature for 48 h.
Additional uridine 5′-monophosphomorpholidate (1 equiv) was
dried as above and then added to the reaction in dry pyridine
(0.5 mL). The reaction was stirred for additional 24 h, and the
solvent was removed in vacuo to yield an oil. The oil was
dissolved in water (10 mL) and washed with CH2Cl2 (2 × 10
mL). Removal of the water in vacuo yielded an oil that was
purified on a Bio-Gel P-2 column (1.5 × 100 cm) eluted with 0.1
M NH4HCO3. The fastest moving UV active fractions were
collected and concentrated under reduce pressure to give the
protected 1 as a white foam (32 mg, 65%) with Rf ) 0.5 in
2-propanol/0.1 M NH4HCO3 (3:1 v/v): 1H NMR (400 MHz, D2O)
δ 7.91 (d, 1 H, J ) 8.1 Hz), 5.91-5.85 (m, 2 H), 4.50-4.05 (m, 8
H), 3.89 (dt, 2 H, J ) 7.5 Hz, J ) 6.6 Hz), 3.80-3.60 (m, 12 H),
2.74 (br s, 1 H), 2.40-1.20 (m, 20 H), 1.38 (s, 9 H), 1.35 (d, J )
7.2 Hz, 3 H); 31P NMR (121.5 MHz, D2O) δ 63.02, 62.86, 62.33,
62.17, -10.50 (d, J ) 20.7 Hz), -11.19 (d, J ) 21.0 Hz); -LSI-
MS (thioglycerol matrix) 1080 (M - H+, 100%), 1066 (M - CH3,
+LSI-MS (3-nitrobenzyl alcohol/CHCl3 matrix) 545 (M + H+
-
C4H8 - CO2, 100%), 645 (M + H+, 13%). +LSI-HRMS calcd for
C29H46N2O12P: 645.2788. Found: 645.2779.
N-(2-S-P r op a n oic a cid )-6-[(d ip h en oxyp h osp h in yl)oxy]-
h exa n a m id e (8). The precursor to 8, 5-carboxypentyl diphenyl
phosphate, was prepared by a slight modification of the previ-
ously reported synthesis.13 A solution of ꢀ-caprolactone (8.0 g,
70 mmol) and NaOH (3.0 g, 75 mmol) in water (2 mL) was heated
to reflux for 2 h. The solvent was evaporated to yield a white
solid. To a solution of the sodium 6-hydroxyhexanoate from
above (0.8 g, 5 mmol) in dry pyridine (25 mL) was slowly added
diphenyl phosphorochloridate (2.3 g, 8.5 mmol). The mixture was
stirred at room temperature for 12 h, and the solvent was
removed in vacuo. The residue was dissolved in CH2Cl2 and
washed with 1 M HCl (2 × 30 mL) and water (30 mL) and dried
over Na2SO4. Filtration and concentration in vacuo gave an oil.
The crude product was purified by flash chromatography on
silica gel (1:1 hexanes/ethyl ether) to give 5-carboxypentyl
diphenyl phosphate as a colorless oil (1.5 g, 83%). The spectral
characteristics were identical to those reported earlier.13
A solution of 5-carboxypentyl diphenyl phosphate (1.1 g, 2.9
mmol) in CH2Cl2/DMF (4:1 v/v, 5 mL) was prepared. To this
solution were added 1-hydroxybenzotriazole monohydrate (0.44
g, 3.2 mmol) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (1.3 g, 2.9 mmol). The mixture was
stirred at room temperature for 1 h during which time a white
precipitate formed. A solution of tert-butyl L-alanine hydrochlo-
ride (0.55 g, 2.9 mmol) and dry pyridine (0.4 g, 5 mmol) in DMF
(2 mL) was then added, and the mixture was allowed to stir at
room temperature for 24 h. The solution was filtered and the
filtrate was partitioned between water (60 mL) and CH2Cl2 (60
mL). The organic layer was washed with water (2 × 60 mL) and
dried over Na2SO4. The solvent was removed in vacuo to yield
an oil. The crude product was purified by flash chromatography
on silica gel (3:1 v/v, ethyl ether/hexanes) to give tert-butyl ester
of 8 as a pale yellow oil (0.87 g) with Rf ) 0.8 in ethyl ether: 1H
NMR (400 MHz, CDCl3) δ 7.40-7.10 (m, 10 H), 6.03 (br d, 1 H,
J ) 6.8 Hz), 4.55-4.35 (m, 1 H), 4.21 (dt, 2 H, J ) 7.6, 6.6 Hz),
2.12 (t, 2 H, J ) 7.6 Hz), 1.75-1.55 (m, 4 H), 1.45 (s, 9 H), 1.34
(m, 2 H), 1.32 (d, J ) 7.1 Hz, 3 H); 31P NMR (81 MHz, CDCl3)
δ -12.00; -LSI-MS (glycerol/H2O matrix) 414 (M - C6H5, 100%),
490 (M - H+, 5%). -LSI-HRMS calcd for C25H33NO7P: 490.1995.
Found: 490.1985.
63%). +LSI-HRMS calcd for
C39H67N5O24P3: 1082.3389.
Found: 1082.3406.
A solution of 88% HCO2H (0.5 mL) containing the protected
1 (32 mg, 30 µmol) was stirred at room temperature for 3 h.
The formic acid was removed in vacuo to yield the amine formate
salt as a colorless oil. This was dissolved in 2.3 M LiOH (0.5
mL) and stirred at room temperature for 24 h. The solution was
carefully brought to pH 9 by the addition of Dowex resin (H+
form) and filtered. The solution was concentrated to 0.5 mL
under reduced pressure and applied to a Bio-Gel P-2 column
(1.5 × 19 cm) that was eluted with 0.1 M NH4HCO3. The fastest
moving UV active fractions were collected, concentrated, and
pumped under high vacuum to give 1 as a white solid (15 mg,
54% for 2-steps): 1H NMR (400 MHz, D2O) δ 7.94 (d, 1 H, J )
8.2 Hz), 6.00-5.92 (m, 2 H), 4.40-4.10 (m, 7 H), 3.91 (dt, 2 H,
J ) 6.8, 6.4 Hz), 3.75-3.66 (m, 1 H), 2.52 (br s, 1 H), 2.27 (t, 2
H, J ) 7.5 Hz), 2.08-1.26 (m, 21 H); 31P NMR (121.5 MHz, D2O)
δ 43.12, -10.45 (d, J ) 20.0 Hz), -11.14 (d, J ) 20.2 Hz); +LSI-
MS (thioglycerol/CH3OH, matrix) 926 (M + H+, 100%). +LSI-
HRMS calcd for C30H51N5O22P3: 926.2239. Found: 926.2214.
En zym e Assa ys. The activity of MurE was assayed at pH
8.1 in 100 mM Bis-Tris propane, containing 5 mM ATP, 75 µM
UDP-MurNAc-L-Ala-D-Glu (KM ) 76 µM),5 and 10 µM meso-DAP
(KM ) 36 µM).5 Commercially available [3H]meso-DAP was used
as a tracer at 10 µCi/mL final concentration. The reactions were
initiated by the addition of MurE to a final concentration of 0.8
µg/mL. After 30 min, the reactions were quenched with 300 mM
potassium phosphate, pH 3.5. The reactions were analyzed by
HPLC cation exchange chromatography (Shimadzu) with radio-
flow detection (INUS) using an isocratic system of 150 mM
potassium phosphate, pH 3.5. Extent of reaction as a function
of inhibitor concentration was calculated from the ratio peak
area of product/(peak area of substrate + peak area of product).
To determine the IC50 value, the data were first graphically
analyzed by plotting the inhibitor concentration versus the
activity by using a Sigma Plot (J andel Scientific) and fitted to
A solution of 88% HCO2H (5 mL) containing the tert-butyl
ester of 8 (0.87 g, 1.8 mmol) was stirred at room temperature
for 48 h. The formic acid was completely removed in vacuo and
the oil was dissolved in CH2Cl2 (20 mL). The organic phase was
washed with water (3 × 10 mL) and dried over MgSO4. Filtration
and concentration in vacuo gave the acid 8 as a pale yellow oil
1
(0.70 g, 57% in two steps). H NMR (200 MHz, CDCl3) δ 7.35-
7.00 (m, 10 H), 6.55 (d, 1 H, J ) 7.2 Hz), 4.60-4.35 (m, 1 H),
4.18 (dt, 2 H, J ) 7.2, 7.0 Hz), 2.13 (t, 2 H, J ) 7.2 Hz), 1.75-
1.45 (m, 4 H), 1.40-1.15 (m, 5 H); 31P NMR (81 MHz, CDCl3) δ
-12.27; +LSI-MS (thioglycerol matrix) 436 (M + H+, 100%).
+LSI-HRMS calcd for C21H27NO7P: 436.1525. Found: 436.1514.
Com p ou n d 9. A solution of compound 7 (67 mg, 0.10 mmol)
in methanol (3 mL) containing 10% Pd/C (46 mg) was stirred
under 1 atm of hydrogen at room temperature for 10 h. The
mixture was filtered, and the solvent was removed in vacuo to
give the amine as colorless oil (52 mg). To a solution of the acid
8 (53 mg, 0.12 mmol) in CH2Cl2/DMF (4:1 v/v, 1 mL) were added
1-hydroxybenzotriazole monohydrate (19 mg, 0.14 mmol) and
dicyclohexylcarbodiimide (29 mg, 0.13 mmol). The mixture was
stirred at room temperature for 1 h, during which time a white
precipitate formed. A solution of the amine from above (46 mg)
in DMF (1 mL) was added, and the mixture was allowed to stir
at room temperature for 20 h. The solution was filtered and the
filtrate was partitioned between water (3 mL) and CH2Cl2 (3

Additions and Corrections
J . Org. Chem., Vol. 63, No. 26, 1998 10085
the equation y ) 1/(1 + x/b), where x is the inhibitor concentra-
tion, y is the relative activity, and b is the IC50 value. The
concentrations of stock solutions of 1 were calculated from A262
for support in the form of a Young Investigator Award
for Organic Chemistry.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
compounds 4-9 and 1 (7 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.
measurements using ꢀ ) 9890 M-1 cm-1
.
Ack n ow led gm en t. We thank the British Columbia
Health Research Foundation, the Natural Sciences and
Engineering Research Council of Canada (NSERC), and
Merck-Frosst Canada Inc. for financial support. M.E.T.
thanks Bio-Me´ga/Boehringer Ingelheim Research Inc.
J O981895P
Additions and Corrections
Vol. 62, 1997
Ta ble 1A. Com p lex Sta bility Con sta n t (Ks) a n d Gibbs
F r ee En er gy Ch a n ge (-∆G) for th e Su p r a m olecu la r
System F or m ed by
Yu Liu ,* Yi-Min Zh a n g, Ai-Di Qi, Ron g-Ti Ch en , Keik o
Ya m a m oto, Ta k eh ik o Wa d a , a n d Yosh ih isa In ou e*. Mo-
lecular Recognition Study on a Supramolecular System. 10.
Inclusion Complexation of Modified â-Cyclodextrins with Amino
Acids: Enhanced Enantioselectivity for L/D-Leucine.
6-(m -Tolu id in yl)-6-d eoxy-â-cyclod extr in (1) a n d Som e
Alip h a tic Am in o Acid sa
-∆G
-∆∆G
host
guest
L-Ala 1197 3.08
Ks log Ks (kJ /mol) (kJ /mol)
6-(m-toluidinyl)-6-
17.3
17.7
16.4
17.7
14.7
15.6
17.7
16.8
0.4
1.3
Page 1827. After publishing the paper, we found that
mono[6-(m-toluidinyl)-6-deoxy]-â-cyclodextrin (1) was con-
taminated by the starting material, mono[6-O-(p-tolu-
enesulfonyl)]-â-cyclodextrin, probably due to the insuf-
ficient reaction period and the subsequent incomplete
purification. As for the 1H NMR data reported, the
signals observed at δ 7.41-7.44 and 7.73-7.75, which
had been erroneously assigned to the aromatic protons
of 1, should be assigned to those of the starting material,
mono[6-O-(p-toluenesulfonyl)]-â-cyclodextrin. From the
integrated area of the original spectrum, we found that
the “toluidinyl-CD” prepared previously contained ca.
40-50% of the starting material.
We therefore prepared a pure sample of compound 1
again and determined the binding constants for the
enantiomeric pairs of the same amino acids. The revised
synthetic procedure described below is similar to that
reported earlier, except for the extended reaction period
and the isolation procedure. The enantioselectivities cal-
culated from the corrected binding constants have turned
out to be considerably smaller than the previous values.
Mon o[6-(m-tolu idin yl)-6-deoxy]-â-cyclodextr in was
prepared by the reaction of mono[6-O-(p-toluenesulfo-
nyl)]-â-cyclodextrin with m-toluidine (10 mL) in N,N-
dimethylformamide (20 mL) at 85 °C with stirring for 3
days under N2. The reaction mixture was evaporated in
vacuo at 40 °C to dryness. The residue was dissolved in
water, and actone was added to the resulting solution to
give a gray precipiate. After drying, the product was
purified by chromatography over Sephadex to give the
pure sample of 1 in 50% yield. MS: m/e 1246 (calcd for
(M + Na - H) 1246). IR (KBr)/cm-1: 3364, 2909, 1716,
1625, 1542, 1412, 1361, 1338, 1307, 1233, 1147, 1071,
1018, 933, 841, 749, 695, 573, 518. 1H NMR (DMSO-d6,
TMS, ppm): δ 2.1 (s, 3H, CH3), 3.1-3.8 (m, 42H), 4.3-
4.6 (m, 6H), 4.6-4.9 (m, 7H), 5.0-5.2 (m, 1 H, NH), 5.6-
5.9 (m, 14H), 6.2-6.4 (m, 3H, Ar-H), 6.8-6.9 (t, 1H, Ar-
H). Anal. Calcd for C49O34H77N‚5H2O: C, 44.78; H, 6.67;
N, 1.07. Found: C, 44.63; H, 6.75; N, 1.01.
deoxy-â-cyclodextrin D-Ala 1430 3.16
L-Ser 843 2.93
D-Ser 1406 3.15
L-Val
D-Val
416 2.62
609 2.78
0.9
L-Leu 1439 3.16
D-Leu 1002 3.00
-0.9
a
Measured in a phosphate buffer (pH 7.20) at room temperature
(20-23 °C).
ferent from the previous data. One reasonable explana-
tion is that the compound reported previously contained
a considerable amount of mono[6-O-(p-toluenesulfonyl)]-
â-cyclodextrin, which is not fluorescent at all but yet
influences the inclusion complexation. Another possible
reason would be that there are some technical problems
inherently in the Benesi-Hildebrand method, so the
constants obtained in that way should be taken with
some reservations (Szejtli, J . Cyclodextrins and Their
Inclusion Complexes; Akademiai Kiado: Budapest (Hun-
gary), 1982; p 199).
Finally, we thank Prof. J erald S. Bradshaw and Dr.
Guoliang Yi for their helpful advice.
J O984014N
10.1021/jo984014n
Published on Web 11/01/1998
Lin -Hu a Zh a n g,* Goss S. Ka u ffm a n , J a a n A. P esti, a n d
J ia n gu o Yin . Rearrangement of NR-Protected L-Asparagine
with Iodosobenzene Diacetate. A Practical Route to â-Amino-L-
alanine Derivatives.
Page 6919, Table 1. Compounds 3 and 4 mentioned in
this table have been previously prepared by Dr. R. Pascal
et al. using 1,1-diacetoxyiodobenzene in DMF (Mendre,
C.; Pascal, R.; Calas, B. Tetrahedron Lett. 1994, 35,
5429-5432. Sola, R.; Saguer, P.; David, M.-L.; Pascal,
R. Chem. Commun. 1993, 1786-1788). We thank Dr.
Pascal for bringing this to our attention.
We further repeated the complexation experiment with
the pure host 1 prepared above to determine the complex
stability constants (Ks), according to the reported proce-
dure. The data obtained are listed in Table 1A.
J O984023W
As can be seen from Table 1A, the stability constants,
especially the enantioselectivities, are significantly dif-
10.1021/jo984023w
Published on Web 12/03/1998