An efficient synthesis of N-α-Fmoc-4-(phosphonodifluoromethyl)-L-phenylalanine

Full text of DOI:10.1021/jo9517508

J . Org. Chem. 1996, 61, 1537-1539
1537
An Efficien t Syn th esis of
N-r-F m oc-4-(P h osp h on od iflu or om eth yl)-L-
p h en yla la n in e
Dennis Solas,* Ron L. Hale, and Dinesh V. Patel
Affymax Research Institute, 3410 Central Expressway,
Santa Clara, California 95051
Received September 25, 1995 (Revised Manuscript Received
December 8, 1995)
Nonhydrolyzable O-phosphotyrosine analogs for use in
peptide synthesis have been of great interest over the
last several years. This interest stems from thier broad
utility in studying various cellular signal transduction
pathways. For example, generation of such mimetics and
incorporation into suitable peptides would afford systems
that could bind to a Src-homology 2 (SH2) domain and
potentially prevent interaction with receptor tyrosine
kinase molecules. Thus signaling events that can induce
changes in cell shape, mobility, or adhesiveness leading
to a variety of diseases such as cancer1 may be modu-
lated.
of Pmp. This methodology requires the chromatographic
separation of two amino acids resulting from the hy-
drolysis of the alkylated lactim to obtain the desired
phophonate.
We now report a new asymmetric convergent synthesis
of F2Pmp 1b using the commercially available imino
lactone 3 as the chiral auxiliary.
Much of the interest in nonhydrolyzable phospho-
tyrosine mimics has centered around phosphonic acids
such as (phosphonomethyl)phenylalanine (Pmp) 1a and
its variously substituted analogs such as 1a -1c. The
phosphotyrosine isostere 1b (F2Pmp), mainly in its phos-
phonic ester form, has been the focus of much synthetic
effort2-8 because of its lower pKa value relative to those
of 1a and 1c. The methods employed to date have
resulted in both racemic as well as enantioselective
syntheses of F2Pmp 1b. Most have involved the stepwise
modification of a commercially available starting mate-
rial. These methods, as mentioned earlier, have resulted
in phosphonate ester groups which must be deprotected
at a later stage of the peptide synthesis. It has been
shown recently8 that such protection of the phosphonic
acid group is unnecessary and the absence of such
protection prevents the resulting peptide from being
exposed to deprotection conditions which may degrade
the final compound. Therefore a convergent enantio-
selective synthesis, as well as one that would result in a
free phosphonic acid group, suitably protected at the
nitrogen, would be highly desirable.
Resu lts a n d Discu ssion
In this synthesis of F2Pmp 1b the necessary alkylating
reagent 5 (required to react with the imino lactone 39 )
was generated from the commercially available R-bro-
motoluic acid (2). Treatment of the acid 2 with phos-
phorus tribromide afforded the acid bromide. Following
low-temperature addition of triethyl phosphite to a
toluene solution of the acid bromide, the R-keto phos-
phonate 4 was obtained in 57% yield. Since the keto
phosphonate is rather unstable, rapidly decomposing on
silica, it was next treated with a large excess of (diethyl-
amido)sulfur trifluoride (DAST) to yield the desired
bromo difluorophosphonate 5 (Scheme 1).
Sch em e 1
One convergent synthesis that has been previously
described7 is one where a modified Fmoc-L-serine ester
is used to establish the correct stereochemistry of the
final compound. In this case the (phosphonodifluorom-
ethyl)phenyl section is attached via a palladium-cata-
lyzed cross-coupling reaction. This assembles the nec-
essary backbone quickly; however the coupling yield for
the desired Fmoc-protected material is not optimal.
Another such synthesis2 involves the use of the bis-
lactim ether of cyclo(D-Val-Gly) in the asymetric synthesis
It should be noted here that the reaction of triethyl
phosphite with the acid chloride of 2 was also investi-
gated, but it did not react cleanly, giving instead mainly
a polymeric tacky residue. This may be due to a
competitve reaction between the aromatic methyl bro-
mide and the acid chloride for the phosphite, even at low
temperature, whereas the more reactive acid bromide
adds rapidly to the phosphite.
With the desired reagent in hand, the next step
involved the alkylation of the imino lactone 39 with
bromide 5, in the presence of HMPA (10%), which
afforded a 78% yield of the alkylated material 6.
Hydrogenation of 6 using H2/THF:EtOH/PdCl2 gave
the amino acid 7 in quantitative yield. In most runs
(1) (a) Brugge, J . S. Science 1993, 260, 918. (b) Domchek, S. M.;
Auger, K. R.; Chatterjee, S.; Burke, T. R.; Shoelson, S. E. Biochemistry
1992, 31, 9865. (c) Cooper, J . A.; Howell, B. Cell 1993, 73, 1051.
(2) Cushman, M.; Lee, E.-S. Tetrahedron Lett. 1992, 33, 1193.
(3) Burke, T. R.; Smyth, M. S.; Nomizu, M.; Otaka, A.; Roller, P. P.
J . Org. Chem. 1993, 58, 1336.
(4) Otaka, A.; Burke, T. R.; Smyth, M. S.; Nomizu, M.; Roller, P. P.
Tetrahedron Lett. 1993, 34, 7039.
(5) Burke, T. R.; Smyth, M. S.; Otaka, A.; Roller, P. P. Tertrahedron
Lett. 1993, 35, 4125.
(6) Wrobel, J .; Dietrich, A. Tetrahedron Lett. 1993, 34, 3543.
(7) Smyth, M. S.; Burke, T. R. Tetrahedron Lett. 1994, 35, 551.
(8) Gordeev, M. F.; Patel, D. V.; Barker, P. L.; Gordon, E. M.
Tetrahedron Lett. 1994, 35, 7585.
(9) Williams, R. M.; Im, M.-N. J . Am. Chem. Soc. 1991, 113, 9276.
0022-3263/96/1961-1537$12.00/0 © 1996 American Chemical Society

1538 J . Org. Chem., Vol. 61, No. 4, 1996
Notes
4-morpholinecarboxylate were purchased from Aldrich Chemical
Co. and used without further purification. HPLC analysis was
performed using a 5 µm 4.6 × 150 mm reverse phase column
[eluent A, 0.1% TFA/water and eluent B, 0.1% TFA/CH3CN;
gradient from 30% B to 100% B over 35 min; flow rate 1 mL/
min; UV at 260 nm]. Optical purity of 1b was determined by
HPLC of its amide with (R)-(+)-1-(1-naphthyl)ethylamine using
the known procedure.10
Sch em e 2
Dieth yl (4-(r-Br om om eth yl)p h en yl)oxom eth yl)p h osp h o-
n a te (4). A mixture of R-bromotoluic acid (2) (2.15 g, 0.01 mol)
and PBr3 (6 mL) was refluxed together for 3 h whereupon a
yellow solution with some yellow semisolid was obtained. The
solution was decanted from the residue and rotary evaporated
at high vacuum to give a yellow semisolid. This residue was
taken up into toluene (20 mL), and the solution/suspension was
filtered under a blanket of argon. The filtrate was rotary
evaporated to dryness at high vacuum affording a white solid.
To a solution of the crude acyl bromide (3.1 g) in toluene (50
mL) at 0 °C was added triethyl phosphite (1.9 mL, 0.011 mol)
in a dropwise fashion. After addition was completed the mixture
was stirred for 40 min at 0 °C. The reaction was then rotary
evaporated to dryness at high vacuum, and the gummy residue
was dissolved in CH2Cl2 (40 mL) and then treated with acid-
washed silica (∼20 g). After 10 min the mixture was filtered
and the filtrate rotary evaporated to a yellow gum which
solidified under vacuum to give the keto phosphonate 4 (1.9 g,
57%). 1H NMR (CDCl3): δ 1.4 (t, 6H, J ) 7 Hz), 4.3 (q, 4H, J )
4 Hz), 4.5 (s, 2H), 7.52 (d, J ) 7 Hz), 8.27 (d, 2H, J ) 7 Hz). 13C
NMR (CDCl3): δ 16.48, 16.54, 45.27, 64.22, 64.29, 128.55, 129.03,
130.39, 131.10, 144.23.
Dieth yl ((4-r-Br om om eth yl)p h en yldiflu or om eth yl)p h os-
p h on a te (5). To ice cold keto phosphonate 4 (1.8 g, 0.0054 mol)
was added DAST (10 mL, 0.08 mol). After stirring overnight at
room temperature the mixture was diluted with CH2Cl2 (40 mL)
and then added dropwise to an ice cold solution of Na2CO3 (100
mL). The CH2Cl2 layer was separated and dried (Na2SO4), and
the solvent was removed via rotary evaporation yielding the
desired difluoro phosphonate 5 (1 g, 52%). 1H NMR (CDCl3): δ
1.32 (t, 6H, J ) 7 Hz), 4.2 (qq, 4H, J ) 7 Hz), 4.5(s, 2H), 7.46 (d,
2H, J ) 7 Hz), 7.6 (d, 2H, J ) 7 Hz). 13C NMR (CDCl3): δ 16.5,
16.6, 32.4, 65.0, 65.1, 119,1, 126.88, 126.9, 129.28, 140.7. ESI
MS (M + H) 358.7. Anal. Calcd for C12H16BrF2O3P: C, 40.36;
H, 4.52. Found: C, 40.31; H, 4.66.
Ben zyl (2R,3S)-(-)-6-Oxo-2,3-d ip h en yl-5-[(4-((d iet h yl-
p h osp h on o)d iflu or om eth yl)ben zyl)]-4-m or p h olin eca r box-
yla te (6). To a solution of the bromide 5 (1.3 g, 0.0033 mol),
the lactone 3 (1.28 g, 0.0036 mol), and HMPA (7 mL) in THF
(70 mL) at -78 °C was added in a dropwise fashion 1 M Li-
(TMS)2 in THF (3.6 mL, 0.0036 mol). After stirring for 45 min
at -78 °C, EtOAc (50 mL) was added and the mixture was
washed with water and saturated NaCl and dried (Na2SO4) and
the solvent removed via rotary evaporation. Column chroma-
tography with 5% EtOAc/CH2Cl2 of the crude afforded the
desired alkylated lactone 6 (1.7 g, 78%). TLC: Rf ) 0.5 (10%
EtOAc/CH2Cl2). tR ) 28.09 (100%). 1H NMR (CDCl3): δ 1.15
(t, 3H, J ) 7 Hz), 1.3 (t, 3H, J ) 7 Hz), 3.4 (m, 2H), 3.7 (dd, 1H,
J ) 4 Hz), 4.15 (m, 5H), 4.4 (s, 1H), 5.1 (m, 1H), 5.35 (m, 1H),
6.49 (d, 2H, J ) 7 Hz), 6.6 (m, 3H), 6.8 (d, 2H, J ) 7 Hz), 7.0-
7.65 (m, 12H). 13C NMR (CDCl3): δ 16.38, 16.43, 16.5, 16.55,
38.90, 40.40, 59.21, 60.38, 60.48, 64.92, 64.98, 65.08, 65.14, 68.03,
68.62, 78.85, 79.19, 126.6, 126.69, 127.09, 127.69, 128.03, 128,
13, 128.50, 128.72, 128.96, 129.02, 130.09, 130.28, 133.88, 133.93,
135.16, 135.70, 135.80, 139.36, 154.65, 169.14. ESI MS (M +
H): 664.1 Anal. Calcd for C36H36F2NO7P: C, 65.15; H, 5.47; N,
2.11. Found: C, 65.20; H, 5.65; N, 1.87.
4-[(Die t h ylp h osp h on o)d iflu or om e t h yl]-L -p h e n yla la -
n in e (7). To a solution of PdCl2 (112 mg) in EtOH (8 mL) and
THF (4 mL) was added the alkylated lactone 6 (1.4 g, 0.0021
mol) in a small volume of MeOH. After aspiration the mixture
was stirred for 20 h under 50 psi of H2. The reaction was then
filtered through Celite and the filtrate rotary evaporated to
dryness. The residue was then triturated three times with ether
and dried under vacuum affording the desired amino acid 7 (800
mg, quant.). 1H NMR (DMSO-d6): δ 1.33 (t, 6H, J ) 7 Hz), 3.3
(d, 2H, J ) 4 Hz), 4.2 (m, 4H), 4.48 (t, 1H, J ) 6 Hz), 7.5 (d, 2H,
J ) 7 Hz), 7.61 (d, 2H, J ) 7 Hz). 13C NMR (CD3OD): δ 16.63,
16.70, 37.10, 54.84, 66.57, 66.67, 127.86, 127.97, 128.05, 130.84,
139.25, 170.96.
contamination by palladium salts tended to give slightly
higher than quantitative yields, but subsequent reaction
steps removed the impurities.
The last operation in the synthesis involved the protec-
tion of the amine group using Fmoc-NHS ester followed
by deprotection of the phosphonate with a mixture of
iodotrimethylsilane (TMSI) and bis(trimethylsilyl)tri-
fluoroacetamide (BSTFA).8 The overall yield of this two-
step reaction was quantitative affording the desired title
compound 1b (Scheme 2).
The stereoselectivity of the akylation was determined
by measurement of the optical rotation of the FMOC-
protected intermediate of compound 7. In this synthesis
a value of [R]D ) 44° (c ) 1.1 in chloroform) was obtained
which compares well with the literature value7 of [R]D )
42° (c ) 1.1 in chloroform). As well as this information
a HPLC of the compound 6 was run and found to be a
single peak showing a diastereomeric mix was not
present at this stage. Also a HPLC of the final compound
1b derivatized as an amide with the chiral amine (R)-
(+)-1-(1-naphthyl)-ethylamine10 afforded a single peak
again demonstrating that a diastereomeric mix was not
present.
Con clu sion
Utilizing both commercially available R-bromotoluic
acid (2), as a ready source for the synthesis of the
alkylating agent 5, and the commercially available imino
lactone 3, as the chiral glycine enolate, a new and
efficient synthesis of the N-R-Fmoc-4-(phosphonodifluo-
romethyl)-L-phenylalanine (1b) has been accomplished.
This method allows a quick entry into the preparation
of other potential phosphotyrosine isoteres.
Exp er im en ta l Section
Gen er a l. 1H and 13C NMR were recorded on a 400 MHz
spectrometer in the indicated solvents. Low-resolution mass
spectra were obtained via the ESI technique, while high-
resolution mass spectra were obtained via the FAB technique.
R-Bromotoluic acid and benzyl (2R,3S)-(-)-6-oxo-2,3-diphenyl-
(10) (a) Fujisawa, T.; Sato, T.; Kawara, T.; Ohashi, K. Tetrahedron
Lett. 1981, 22, 4823. (b) Castro, B.; Dormoy, J . R.; Evin, G.; Selve, E.
Tetrahedron Lett. 1975, 16 , 1219.

Notes
N-r-F m oc-4-(P h osp h on od iflu or om et h yl)-L-p h en yla la -
J . Org. Chem., Vol. 61, No. 4, 1996 1539
brown oil. The oil was treated with CH3CN (4 mL), TFA (0.5
mL), and water (1 mL) and stirred for 2.5 h at room temperature.
The reaction was then rotary evaporated to an oil which was
redissolved in EtOAc (50 mL) and washed with 5% Na2S2O4
(acidified) followed by a wash with saturated NaCl. The EtOAc
layer was then dried (Na2SO4) and the solvent removed via
rotary evaporation affording the phosphonic acid 1b (1.19 g,
quant. from 6). tR ) 11.43 (100%) as free acid. tR ) 19.39 (100%
ee) as amide. 1H NMR (DMSO-d6): δ 2.91 (dd, 1H, J ) 10 Hz),
3.12 (dd, 1H, J ) 10 Hz), 4.2 (m, 4H), 7.2-7.9 (m, 12H). 13C
NMR (CD3OD): δ 38.13, 57.63, 67.93, 120.85, 126.85, 126.20,
127.52, 128.15, 128.74, 129.97, 140.61, 142.51, 145.13, 145.19,
158.39, 174.92. ESI MS (M + H): 518. HRMS calcd for (M +
H)+: C25H22F2NO7P 518.1180, found 518.1168.
n in e (1b). To a solution of the amino acid 7 (800 mg, 2.1 mmol)
in water (5 mL) with NaHCO3 (235 mg, 2.8 mmol) was added
dioxane (5 mL). The mixture was then cooled in an ice bath
and then treated with FMOC-NHS (944 mg, 2.8 mmol) in a small
amount of dioxane. After stirring for 3 h at room temperature,
the reaction was diluted with saturated NaHCO3 (30 mL) and
then extracted with ether. The aqueous layer was then acidified
to pH 2 with 6 N HCl and then extracted with EtOAc. The
extracts were then dried (Na2SO4), and the solvents were
removed yielding the FMOC amino acid (1.29 g) as a white solid.
Without further purification the phosphonate in CH2Cl2 (20 mL)
was then treated with BSTFA (6.4 mL, 0.0242 mol). After
stirring for 1 h at room temperature the mix was cooled to -20
°C and TMSI (2.5 mL, 0.0176 mol) was added. After stirring
for 1 h at -20 to 0 °C the reaction was stirred for an additional
1 h at room temperature and then rotary evaporated to a viscous
J O9517508