Preparation of phospho-L-azatyrosine suitably protected for the synthesis of signal transduction related peptides. A correction

Article abstract of DOI:10.1055/s-1998-3131

Synthesis of N^α-Fmoc 4-O,O-(di-tert-butyl)phospho-L-azatyrosine (6) and its use in Fmoc-based solid-phase peptide synthesis are reported. The di-tert-butyl phospho protecting groups were removed during TFA-mediated cleavage of the finished peptide (10) from the resin, providing the free phosphoazatyrosine-containing peptide directly. This is in contrast to diethyl phospho protection we previously reported [Ye, B.; Otaka, A.; Burke, T. R., Jr. Synlett. 1996, 459-460] in which the diethyl protection has proven impractical to remove. Due to incorrect assignment of starting material, the title compound of this latter report should be corrected to the isomeric L-β-(3-hydroxy-2-pyridyl)-alanine compound (5).

Full text of DOI:10.1055/s-1998-3131

428
LETTERS
SYNLETT
Preparation of Phospho-L-Azatyrosine Suitably Protected for the Synthesis of Signal
Transduction Related Peptides. A Correction
a
a
b
b
a*
Zhu-Jun Yao , Bin Ye , Kengo Miyoshi , Akira Otaka and Terrence R. Burke, Jr.
a
Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 5C06,
Bethesda, Maryland 20892, U.S.A.
FAX +301-402-2275; E-mail:tburke@helix.nih.gov
b
Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan
Received 15 August 1997
α
11,12
Abstract: Synthesis of N -Fmoc 4-O,O-(di-tert-butyl)phospho-
facile solid-phase synthesis. By analogy to pTyr itself,
such a
L-azatyrosine (6) and its use in Fmoc-based solid-phase peptide
synthesis are reported. The di-tert-butyl phospho protecting groups were
removed during TFA-mediated cleavage of the finished peptide (10)
from the resin, providing the free phosphoazatyrosine-containing
peptide directly. This is in contrast to diethyl phospho protection we
previously reported [Ye, B.; Otaka, A.; Burke, T. R., Jr. Synlett. 1996,
459-460] in which the diethyl protection has proven impractical to
remove. Due to incorrect assignment of starting material, the title
compound of this latter report should be corrected to the isomeric L-β-
(3-hydroxy-2-pyridyl)-alanine compound (5).
derivative of pAzaTyr was therefore designed bearing tert-butyl
phosphate and N Fmoc protection (6).
α
Protein-tyrosine kinase (PTK)-dependent pathways offer potentially
Utilizing unprotected AzaTyr (2) prepared by the enantioselective
enzymatic route of Stevens, the amino group was first derivatized in
interesting targets for therapeutic intervention in several diseases,
1,2
13
including cancers and immune disorders.
Since in proteins, the
phosphotyrosyl (pTyr) residue (1) provides a critical motif around
95% yield using Fmoc N-hydroxysuccinimide active ester (Fmoc-OSu).
The high water solubility induced by the zwitterionic pyridyl nitrogen
which PTK-dependent signalling centers, analogues of pTyr have
α
3
proven useful in the design of PTK inhibitors. Azatyrosine (L-β-
resulted in workup difficulties not encountered with N -Fmoc Tyr itself,
α
(5-hydroxy-2-pyridyl)-alanine) (2) is an antibiotic isolated from
and efficient extraction of the N Fmoc AzaTyr (7) was only achieved
4
using a H O:THF biphasic system. Next, protection of the carboxyl
Streptomyces chibanesis which has been shown to promote permanent
2
reversion of ras-dependent transformed cells to the normal phenotype in
group was required prior to phosphorylation of the phenolic hydroxyl.
Selective TBDMS esterification of the carboxyl group without silation
5
culture and to inhibit chemical induction of carcinogenesis in
α
6
of the 4’-hydroxyl is possible for N Fmoc Tyr when done in
transgenic mice bearing oncogenic human ras. Its phosphorylated
11,12
α
version, phosphoazatyrosine (pAzaTyr, 3), represents
a
pyridyl-
anhydrous THF.
However, the poor solubility of N Fmoc AzaTyr
containing variant of pTyr, which may be potentially interesting for the
study of signal transduction.
(7) in anhydrous THF or dioxane, necessitated the use of DMF, which
resulted in loss of selectivity and silation of both the carboxyl and
phenolic groups. Alternate esterification as the methyl ester was
therefore employed, since this could be accomplished under aqueous
conditions prior to phosphorylation, and subsequently removed post
phosphorylation without cleaving the tert-butyl or Fmoc groups.
Treatment of 7 with dimethoxypropane in aqueous HCl provided methyl
ester 8 in 97% yield. Phosphorylation to provide 9 was then achieved in
88% yield using di-tert-butyl diisopropylphosphoramidite and
1H-tetrazole followed by MCPBA-mediated oxidation of the
12
intermediate di-(tert-butyl)phosphite. Hydrolysis of the methyl ester
was accomplished using ice-cold 0.2 N LiOH without significant loss of
14
the Fmoc group, providing title compound 6 in 65% yield.
We have previously reported diethylphospho-L-azatyrosine bearing
α
N
Boc protection (4) as a reagent potentially useful for the synthesis of
7
signal transduction-related peptides. This latter synthesis was based on
our approach for the preparation of AzaTyr itself. However, as recently
8
pointed out, the putative starting material which we indicated as
5-hydroxy-2-iodopyridine,
was
actually
the
isomeric
9
8
3-hydroxy-2-iodopyridine. Accordingly, both in our original AzaTyr
7
and pAzaTyr papers, compounds derived from this starting material
have ring oxy-substituents at the 3-position rather than the 5-position as
we reported. When this isomeric pAzaTyr analogue (5) was
incorporated into peptides by solid-phase techniques, subsequent
removal of the phosphate O,O-diethyl protection proved to be
impractical, potentially indicating the unsuitability of the ethyl groups
In order to demonstrate the utility of 6 for the synthesis of pAzaTyr-
containing peptides, preparation of Ac-Asp-Ala-Asp-Glu-[pAzaTyr]-
10
15
for solid-phase synthesis. A need has therefore arisen for the synthesis
Leu-amide (10) was achieved using the acid-labile Rink amide resin
of pAzaTyr possessing phospho and amino protection amenable to
and standard Fmoc-based protocols. Cleavage of the peptide from the

April 1998
SYNLETT
429
resin with concomitant global deprotection was achieved using aqueous
TFA with triethylsilane (TES) scavenger. Finally, purification by HPLC
provided desired peptide 10 in 20% yield based on resin substitution.
4.51 (1H, m), 4.22 (3H, m), 3.61 (3H, s), 3.11 (2H, m), 1.42 (18H, s).
FABMS (m/z): 611 (MH , 79).
+
2(S)-2-((Fluorenyl-9-ylmethoxy)carbonylamino)-3-(5-(di-tert-butyl
α
phosphoryloxy)(2-pyridyl))propanoic acid [N Fmoc 4-O-(di-tert-
butyl)phospho-L-azatyrosine] (6): To a cooled solution of 9 (200 mg,
0.327 mg) in THF (7 mL) was added 0.2 N LiOH (3.5 mL, 0.70 mmol)
slowly at 0 °C. The reaction mixture was stirred at 0 °C (25 min) then
quenched by addition of 0.2 N HCl (3.5 mL, 0.700 mmol) and adjusted
to pH 5. The mixture was extracted with EtOAc (3 x 10 mL), washed
Experimental Section
Elemental analyses were obtained from Atlantic Microlab Inc.,
Norcross, GA and fast atom bombardment mass spectra (FABMS) were
acquired with a VG Analytical 7070E mass spectrometer under the
control of a VG 2035 data system. Amino acid analysis was obtained
with brine (10 mL), dried (Na SO ) and concentrated. Purification by
2
4
1
from Peptide Technologies, Corp., Gaithersburg, MD. H NMR data
silica gel column chromatography (MeOH : CHCl , from 1:10 to 1:3)
3
1
were obtained on a Bruker AC250 (250 MHz) instrument. Solvent was
removed by rotary evaporation under reduced pressure and silica gel
chromatography was performed using Merck silica gel 60 with a
particle size of 40 - 63 µ. Anhydrous solvents were obtained
commercially and used without further drying.
provided 6 as a white foam (126 mg, 65%),mp 172 °C (dec.). H NMR
(DMSO-d ) δ 8.30 (1H, s), 7.88 (2H, d, J =7.4 Hz), 7.63 (2H, d, J = 6.3
6
Hz), 7.40 (3H, m), 7.31 (3H, m), 7.03 (1H, m), 4.14 (4H, m), 2.99 (2H,
m), 1.40 (18H, s) ppm. HR FABMS calcd. For C
[(M-H) ]. Found: 595.2205.
H N O P: 595.2209
31 36 2 9
-
Solid Phase Synthesis of Ac-Asp-Ala-Asp-Glu-[pAzaTyr]-Leu-
amide (10): Solid-phase peptide synthesis of 10 was accomplished on
Fmoc-protected Rink amide resin using 20% piperidine deblock with
1-hydroxybenzotriazole (HOBt) active ester coupling of amino acids in
2(S)-2-((Fluorenyl-9-ylmethoxy)carbonylamino)-3-(5-hydroxy(2-
pyridyl))propanoic acid (7): To a suspension of L-azatyrosine (2) (420
15
mg, 2.30 mmol) and NaHCO (966 mg, 11.5 mmol) in dioxane (8 mL)
3
α
α
the order: N -Fmoc-L-Leu-OH, N -Fmoc-L-Azatyr(PO (tert-butyl) )-
3
2
with water (8 mL) was added Fmoc-OSu (775 mg, 2.30 mmol) at rt. The
reaction was stirred at rt (6 hr), then 1 N HCl was added dropwise at 0
°C until the pH was adjusted to 4.5~5.0. The solution was then extracted
with THF (3 x 40 mL), the combined organic solutions washed with
α
α
OH (6), N -Fmoc-L-Glu(O-tert-butyl)-OH, N -Fmoc-L-Asp(O-tert-
butyl)-OH, Fmoc-L-Ala-OH and Fmoc-L-Asp(O-tert-butyl)-OH
followed by 1-actylimidazole mediated N-terminal acetylation. The
finished resin was cleaved using a mixture of TFA-water-TES (92.5 : 5 :
2.5, 1.5 mL) (2 h). The crude peptide was subjected to HPLC
purification (peak retention time of 14.3 min) using a Vydac Protein &
brine (50 mL), dried over Na SO and concentrated. The residue was
2
4
dried in vacuo to provide 7 as a white foam (868 mg, 95%), which was
1
pure enough for the next step. H NMR (DMSO-d ) δ 9.72 (1H, brs),
6
Peptide C column (218TP1022) using an elution of B = 0% (from 0 -
18
8.04 (1H, s), 7.87 (2H, d, J = 7.5 Hz), 7.62 (3H, m), 7.40 (2H, t, J = 7.3
Hz), 7.29 (2H, dd, J = 7.2 Hz, 11.6 Hz), 7.08 (2H, s), 4.34 (1H, m), 4.18
(3H, m), 3.07 (1H, dd, J = 4.6 Hz, 14.0 Hz), 2.91 (1H, dd, J = 9.9 Hz,
5 min) with a linear gradient of from B = 0% to 50% over 20 min.
Solvent A = 0.1% TFA in H O and B = 0.1% TFA in acetonitrile.
2
Product 10 was obtained as a white solid (8.3 mg, 20% overall based on
+
13.9 Hz). FABMS (m/z): 405 (MH , 25).
1
resin substitution). H NMR (D O) δ 8.55 (1H, s), 8.18 (1H, m), 7.81
2
2(S)-2-((Fluorenyl-9-ylmethoxy)carbonylamino)-3-(5-hydroxy(2-
pyridyl))propanoic acid methyl ester (8): To a suspension of 8 (261
mg, 0.646 mmol) in 2,2-dimethoxypropane (10 mL) was added 36%
HCl (0.6 mL) and the resulting homogeneous solution was stirred at rt
overnight. The reaction mixture was quenched by addition of saturated
(1H, d, J = 8.8 Hz), 4.69 (3H, m), 4.31 (3H, m), 3.54 (1H, m), 3.43 (1H,
m), 2.88 (4H, m), 2.49 (2H, m), 2.19 (1H, m), 2.04 (4H, s), 1.63 (3H,
m), 1.41 (3H, d, J = 7.2 Hz), 0.94 (3H, d, J = 5.0 Hz), 0.87 (3H, d, J =
5.0 Hz). FABMS (m/z) [(M-H) , 14]. Amino acid analysis: Asp 2.04 (2),
Glu 1.00 (1), Ala 0.92 (1), Leu 1.05 (1).
-
aqueous NaHCO until the pH was adjusted to 5, then it was extracted
3
with EtOAc (40 mL). The organic layer was washed with saturated
Acknowledgement. Appreciation is expressed to Dr. James Kelly and
Ms. Pamela Russ of the LMC for providing mass spectral analysis.
aqueous NH Cl (2 x 20 mL), dried (Na SO ) and concentrated. Residue
4
2
4
was purified by silica gel chromatography (MeOH : CHCl , 1:40) to
3
1
provide 8 as a white solid (262 mg, 97%), Mp 109.5-111.0 °C. H NMR
(DMSO-d ) δ 9.73 (1H, s), 8.04 (1H, s), 7.89 (2H, d, J = 7.4 Hz), 7.81
References and Notes
6
(1H, d, J = 8.1 Hz), 7.63 (2H, dd, J = 3.6 Hz, 7.3 Hz), 7.42 (2H, t, J =
7.3 Hz), 7.32 (2H, dd, J = 3.8 Hz, 7.2 Hz), 7.08 (2H, s), 4.44 (1H, m),
4.22 (3H, m), 3.60 (3H, s), 3.00 (2H, m). FABMS (m/z): 419 (MH , 74).
(1) Levitzki, A. Curr. Opin. Cell. Biol. 1996, 8, 239-244.
(2) Patrick, D. R.; Heimbrook, D. C. Drug Discov. Today 1996, 1,
+
325-330.
Analysis (C
H N O ): C, 68.89, H, 5.30, N, 6.69; Found: C 68.84, H,
24 22 2 5
(3) Burke, T. R., Jr.; Yao, Z.-J.; Smyth, M. S.; Ye, B. Curr. Pharm.
Design 1997, 3, 291-304.
5.38, N, 6.65.
2(S)-2-((Fluorenyl-9-ylmethoxy)carbonylamino)-3-(5-(di-tert-butyl
phosphoryloxy)(2-pyridyl))propanoic acid methyl ester (9) To a
solution of 8 (132 mg, 0.316 mmol) in anhydrous DMF (1.5 mL) was
added 1H-tetrazole (72 mg, 0.950 mmol) followed by di-tert-butyl
diisopropylphosphoramidite (90 mg, 0.395 mmol) under argon. The
reaction mixture was stirred at rt (2 hr), then cooled to -40 °C and a
solution of 80% MCPBA (81 mg, 0.411 mmol) in anhydrous
dichloromethane (2 mL) was added. The reaction mixture was stirred on
ice (30 min) then diluted with EtOAc (15 mL), washed with saturated
(4) Inouye, S.; Shomura, T.; Tsuruoka, T.; Ogawa, Y.; Watanabe,H.;
Yoshida, J.; Niida, T. Chem. Pharm. Bull. 1975, 23, 2669-2677.
(5) Shindo-Okada, N.; Makabe, O.; Nagahara, H.; Nishimura, S. Mol.
Carcinog. 1989, 2, 159-167.
(6) Izawa, M.; Takayama, S.; Shindo-Okada, N.; Doi, S.; Kimura, M.;
Katsuki, M.; Nishimura, S. Cancer Res. 1992, 52, 1628-1630.
(7) Ye, B.; Otaka, A.; Burke, T. R., Jr. Synlett 1996, 459-460.
(8) Ye, B.; Burke, T. R., Jr. J. Org. Chem. 1995, 60, 2640-2641.
aqueous NaHCO (5 mL x 3) and dried (Na SO ). Concentration and
3
2
4
(9) Sheldrake, P. W.; Powling, L. C.; Slaich, P. K. J. Org. Chem.
1997, 62, 3008-3009.
purification by silica gel column chromatography (MeOH : CHCl ,
3
1
from 1:100 to 1:40) provided 9 as a clear oil (170 mg, 88%). H NMR
(10) Unpublished results.
(DMSO-d ) δ 8.35 (1H, s), 7.88 (3H, m), 7.63 (2H, d, J =7.4 Hz), 7.53
6
(1H, dd, J =8.5 Hz), 7.41 (2H, t, J =7.3 Hz), 7.32 (3H, d, J =8.6 Hz),
(11) Perich, J. W.; Reynolds, E. C. Synlett 1991, 577-578.

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LETTERS
SYNLETT
(12) Kitas, E. A.; Knorr, R.; Trzeciak, A.; Bannwarth, W. Helv. Chim.
Acta 1991, 74, 1314-1328.
(14) Burke, T. R.; Smyth, M. S.; Otaka, A.; Roller, P. P. Tetrahedron
Lett. 1993, 34, 4125-4128.
(15) Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790.
(13) Cooper, M. S.; Seton, A. W.; Stevens, M. F. G.; Westwell, A. D.
Bioorg. Med. Chem. Lett. 1996, 21, 2613-2616.