Phosphinate Analogues of Glutamyl-γ-glutamate
J . Org. Chem., Vol. 66, No. 15, 2001 5153
solution of n-octylphosphinic acid (6, R2 ) (CH2)6CH3, 0.10 g,
0.562 mmol) and dibenzyl 2-methylene glutarate (0.549 g, 1.69
mmol) in 5 mL of CH2Cl2 was added 0.686 g (3.37 mmol) of
N,O-(bis-trimethylsilyl)acetamide (BSA) at room temperature.
The reaction mixture was stirred overnight at room temper-
ature, after which time it was quenched with 1 N HCl and
extracted three times with CH2Cl2. The combined organic
extracts were dried over MgSO4 and then concentrated in
vacuo. The resulting crude phosphinic acid 8 (R1 ) H) was
used in the next reaction without further purification. Forma-
tion of the P-methyl ester was accomplished with ethereal
CH2N2 as described above to provide 0.185 g (62%, two steps)
of the desired phosphinic acid P-methyl ester 8: 1H NMR
(CDCl3) δ 0.90 (3 H, t, J ) 6.20 Hz), 1.20-2.40 (23 H, m), 2.70-
2.90 (1 H, m), 3.60 (3 H, d, J ) 11.8 Hz), 5.10 (2 H, s), 5.11 (2
H, s), 5.14 (2 H, s), 5.15 (2 H, s), 7.20-7.40 (5 H, m); 13C NMR
(CDCl3) δ 14.51, 21.96, 22.02, 22.07, 22.13, 22.98, 27.79, 28.20,
29.00, 29.10, 29.24, 29.30, 29.38, 29.41, 29.50, 30.40, 30.69,
30.80, 31.03, 31.24, 31.62, 31.70, 32.00, 32.13, 38.81, 38.86,
39.04, 39.08, 51.18, 51.27, 51.37, 66.68, 67.15, 67.21, 128.57,
128.70, 128.74, 128.79, 128.90, 135.92, 135.95, 136.16, 172.52,
174.25, 174.34; 31P NMR (CDCl3) δ 56.5, 56.2; MS (CI, NH3)
m/z 517.3 (MH+, 58% bp); HRMS calcd for C29H41O6P, 517.2719
(MH+), found 517.2720.
2-[(Eth oxy(vin yl)p h osp h in yl)m eth yl]p en ta n e-1,5-d io-
ic Acid Diben zyl Ester (12). 2-(Hydroxyphosphinoylmethyl)-
pentane-1,5-dioic acid dibenzyl ester (10) (2.7 g, 6.9 mmol) was
dissolved in dry 1,2-dibromoethane (2.4 mL, 27.6 mmol).
Hexamethyldisilazane (3 mL, 13.8 mmol) was added to the
solution, which was stirred at 120 °C for 20 h. The reaction
mixture was cooled to room temperature, after which time
EtOH (15 mL) was added, and the resulting solution was
stirred at reflux temperature for 0.5 h. Following removal of
the solvent in vacuo, the residue was dissolved into EtOAc (60
mL) and washed with water (3 × 15 mL). The organic layer
was dried over MgSO4 and evaporated to give 11 (2.4 g)
containing ca. 50% 12a resulting from dehydrohalogenation
of 11 in the presence of hexamethyldisilazane: 31P NMR
(CDCl3) δ 31.37, 38.97.
This residue was treated with HC(OEt)3 (4.0 mL, 19.2 mmol)
for 1.5 h at reflux temperature with simultaneous removal of
resulting EtOH, following which excess HC(OEt)3 was removed
in vacuo. The residue was subjected to silica gel chromatog-
raphy (EtOAc/acetone 3:1) to give 12 (1.71 g, 57% over two
steps) as a colorless oil: TLC Rf ) 0.48 (EtOAc/acetone 3:1);
1H NMR (CDCl3) 1.23-1.29 (m, 3H), 1.80-2.29 (dm, 2H, J )
120 Hz), 2.01-2.03 (m, 2H), 2.34 (t, 2H, J ) 8 Hz), 2.80-2.95
(m, 1H), 3.80-4.09 (dm, 2H, J ) 22 Hz), 5.09-5.14 (m, 4H),
5.96-6.35 (m, 3H), 7.29-7.38 (m, 10H); 13C NMR (CDCl3)
16.78, 16.86, 28.99, 30.67, 30.84, 31.72, 31.77, 32.00, 32.18,
38.96, 60.96, 61.05, 66.77, 67.18, 128.62, 128.66, 128.75,
128.79, 128.96, 129.89, 130.17, 136.20, 136.88, 172.64, 174.15,
174.19, 174.24, 174.28; 31P NMR (CDCl3) 39.0, 39.5; MS (CI,
NH3) m/z 445.2 (MH+, 100). HRMS (DCI with ammonia) calcd
for C24H29O6P 445.1780 [M + H]+, found 445.1780. Anal. Calcd
for C24H29O6P: C, 64.86; H, 6.58. Found: C, 64.89; H, 6.51.
2-[((3-Am in o-3-ca r boxyp r op yl)(h yd r oxy)p h osp h in yl)-
m et h yl]p en t a n e-1,5-d ioic Acid (2). A solution of R,â-
unsaturated phosphinic acid ester 12 (0.79 g, 1.8 mmol) in dry
EtOH (1 mL) was added to a solution of diethyl acetoami-
domalonate (0.35 g, 1.8 mmol) in dry EtOH (2 mL) containing
metallic Na (0.04 g, 1.8 mmol). The reaction mixture was
stirred at 80 °C overnight. Ethanol was evaporated. The
residue was dissolved in CHCl3 (30 mL) and washed with 3%
HCl (5 mL) and water (2 × 5 mL). CHCl3 was evaporated.
Unreacted diethyl acetoamidomalonate was removed by silica
gel plug filtration (CHCl3). The resulting 2-(ethoxycarbonyl)-
2-(a cet a m ido)-4-[(2,4-diben zoxyca r bon ylbu t yl)(et h oxy)-
phosphinyl]butanoic acid ethyl ester (0.78 g) was dissolved in
EtOH (5 mL). Pd/C (0.021 g, 0.02 mmol) was added to the
solution, which was shaken overnight under an atmosphere
of H2. Pd/C was removed by filtration, after which EtOH was
evaporated.
solution was effected. The resulting solution was cooled to room
temperature. Light organic byproducts were removed by
extraction with ether. The aqueous layer was evaporated to
give 2 as a white solid (0.39 g, 88%); mp 121-125 °C dec; TLC
1
on cellulose Rf ) 0.5 (BuOH/AcOH/H2O 4:1:1) H NMR (D2O,
pH 0.5-1) δ 1.71-1.85 (m, 5H), 2.00-2.16 (m, 3H), 2.30 (t,
2H, J ) 7 Hz), 2.57-2.72 (m, 1H), 4.07 (t, 1H, J ) 6 Hz); 13C
NMR (D2O, pH 0.5-1) δ 22.5, 24.0, 25.2, 28.3, 28.5, 29.5, 30.7,
31.2, 38.6, 52.8, 53.1, 171.1, 177.4, 178.2, 178.3; 31P NMR (D2O,
pH 0.5-1) δ 52.7; MS (FAB) m/z 312.2 (MH+, 41.4); HRMS
(FAB) calcd for
312.0840. Anal. Calcd for C10H18NO8P‚HCl: C, 34.54; H, 5.51;
N, 4.03. Found: C, 34.84; H, 5.77; N, 3.90.
C
10H18NO8P 312.0800 [M + H]+, found
P ter oyl Azid e, 13a .40 Ice-cold TFA (32 mL) was added to
a 100 mL round-bottom flask containing pteroyl hydrazide (4.1
g, 12.5 mmol) and cat. KSCN (0.061 g, 0.63 mmol), and the
mixture was stirred until all solids dissolved. The reaction
mixture was cooled to -10 °C, and tBuONO (1.49 mL, 12.5
mmol) was added dropwise. The reaction was monitored by
analytical HPLC. The reaction was stirred at -10 °C for 6.5
h, at which time HPLC indicated that none of the starting
material was still present in the mixture. The reaction mixture
was allowed to warm to room temperature, and 60 mL of
2-propanol was slowly added to afford an orange precipitate
that was collected by centrifugation (30 min × 10 000 rpm).
The pellet was washed with 60 mL of H2O, 60 mL of
acetonitrile, and 60 mL of diethyl ether, centrifuging after each
wash (30 min × 10 000 rpm). The pellet was dried under high
vacuum (0.25 mmHg) for 24 h to give 3.52 g of 13a as an
orange solid (83%): mp 185-190 °C dec (lit.40 mp ∼180 °C);
IR (KBr) 2134 cm-1 (N3); 1H NMR (300 MHz, DMSO-d6) δ 8.69
(s, 1H), 7.69 (d, 2H, J ) 8.7 Hz), 7.42 (broad s, D2O exchange-
able, 1H), 6.70 (d, 2H, J ) 8.7 Hz), 4.56 (s, 2H). 13C NMR (75
MHz, DMSO-d6) δ 170.3, 160.4, 154.0, 153.7, 153.4, 148.8,
148.3, 131.4, 128.1, 116.9, 111.7, 45.5; UV/vis (HPLC diode
array, phosphate buffer, pH 7.0/acetonitrile) λmax 235 nm, 275
nm, 338 nm; (0.01 M NaOH) 256, 280 sh, 365 nm; (0.01 M
HCl) 333 nm; MS (FAB) m/z 338 (MH+); analytical HPLC tR
) 24.3 min (flow rate: 0.7 mL/min; eluent A: water and
phosphate buffer, pH 7.0; eluent B: acetonitrile; gradient: 0
min, 2% B, 25 min, 50% B; column: Chrompack Kromasil 100
C18, 250 mm × 4.6 mm).
2-[[[3-Ca r boxy-3-[[4-[[(2-a m in o-3,4-d ih yd r o-4-oxop ter i-
d i n -6 -y l ) m e t h y l ] a m i n o ] b e n z o y l ] a m i n o ] p r o p y l ] -
h yd r oxyp h osp h in yl]m eth yl]p en ta n e-1,5-d ioic Acid Tr is-
(tr ieth yla m in e) Sa lt (1a ). To a stirring suspension of pteroyl
azide (13a , 0.21 g, 0.63 mmol) and 2 (0.33 g, 0.95 mmol) in
anhydrous DMSO (10 mL) was added neat tetramethylguani-
dine (0.48 mL, 3.8 mmol). The reaction was stirred at room
temperature for 18 h. Acetonitrile (30 mL) was slowly added
to the reaction with vigorous stirring. The orange-yellow
precipitate was collected by centrifugation (10 000 rpm × 30
min). The pellet was washed with aqueous 1% HCl (30 mL),
acetonitrile (30 mL), and diethyl ether (30 mL × 2), centrifug-
ing after each wash (30 min × 10 000 rpm). The pellet was
dried under high vacuum (0.25 mmHg) for 24 h to give 0.200
g of crude 1a as an orange solid (52%; analytical HPLC
indicates 85% 1a , 15% pteroic acid). A portion of the crude
product (100 mg) was purified by ion-exchange chromatogra-
phy on a DEAE-cellulose (DE52) column (15 mL of wet resin
volume poured into a 22 × 2 cm glass Bio-Rad column)
equilibrated with 0.01 M triethylammomium bicarbonate, pH
8.0. The column was eluted with a linear gradient ranging from
0.01 to 1.0 M triethylammonium bicarbonate, pH 8.0, with a
flow rate of 1 mL/min and a total volume of 120 mL. Fractions
containing the product were lyophilized to give 130 mg of 1a
as a fluffy, yellow solid (45%): mp 200 °C dec; 1H NMR (D2O)
δ 8.68 (s, 1H), 7.63 (d, 2H), 6.63 (d, 3H), 4.47 (s, 2H), 4.31 (d,
1H), 3.15 (q, 6H) 2.54 (m, 1H), 2.23 (m, 2H), 2.05 (m, 1H), 2.0-
1.65 (m, 4H), 1.62 (m, 3H), 1.24 (t, 9H); 13C NMR (D2O) δ
181.94, 181.89, 180.13, 180.00, 179.01, 178.92, 169.88, 169.81,
165.11, 154.60, 153.39, 150.89, 150.84, 149.51, 149.42, 149.32,
129.41, 127.44, 127.40, 121.54, 112.44, 112.35, 59.02, 56.49,
47.01, 46.86, 45.96, 45.79, 45.77, 45.74, 45.56, 42.62, 41.62,
HCl (6 N, 5 mL) was added to the resulting residue (0.49
g). The mixture was heated for 20 h at reflux temperature as