Development of new methodology for the synthesis of functionalized α- fluorophosphonates and its practical application to the preparation of phosphopeptide mimetics

Article abstract of DOI:10.1039/b002188l

New methodology for the synthesis of functionalized α- fluorophosphonates which utilizes organocopper-mediated reduction has been developed and applied to the preparation of a monofluoromethyl-substituted phosphoserine mimetic-containing peptide.

Full text of DOI:10.1039/b002188l

Development of new methodology for the synthesis of functionalized
a-fluorophosphonates and its practical application to the preparation of
phosphopeptide mimetics
Akira Otaka,* Etsuko Mitsuyama, Hideaki Watanabe, Hirokazu Tamamura and Nobutaka Fujii
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
E-mail: aotaka@pharm.kyoto-u.ac.jp
Received (in Cambridge, UK) 20th March 2000, Accepted 10th May 2000
Table 1 Reduction of 4 with several organo copper reagents
New methodology for the synthesis of functionalized a-
fluorophosphonates which utilizes organocopper-mediated
reduction has been developed and applied to the preparation
of a monofluoromethyl-substituted phosphoserine mimetic-
containing peptide.
Naturally occurring phosphate-containing molecules play im-
portant roles in various cellular processes, including signal
transduction.1 Therefore, nonhydrolyzable phosphate mimetics
have received considerable attention, with a,a-difluorophos-
phonates serving as potential phosphate mimics,2 extensive
synthetic and biological studies of which have been made.3 In
contrast, evaluation of a-monofluorophosphonates4 as bio-
logical phosphate mimics has been somewhat limited due to
lack of flexibility of practical synthetic methodology5 for this
kind of molecule. In our efforts to prepare difluoromethyl
(CF2)-substituted phosphothreonine mimetics,6 we attempted
conjugate addition of a methyl group to 3-(diethylphosphonodi-
fluoromethyl)but-2-enoate 1† using an organocopper reagent to
construct the secondary phosphonate unit. Unexpectedly, the
reaction predominantly afforded an organocopper-mediated
reduction product, a-fluorovinylphosphonate 3, and not the
corresponding conjugate addition product 2 (Scheme 1). This is
the first example of organocopper-mediated reduction of g-
difluoro-a,b-enoates yielding g-fluoro-b,g-enoates.
ing reduction product 5 with (E)-configuration§ in up to 80%
isolated yields. Similarly, using methyl copper reagents, the
formation of an alkylated product was also not observed. Use of
methyl copper reagents was critical for conversion of 4 to 5,
since exposure of 4 to a butyl-copper reagent (run 14) afforded,
besides 5, a Bu-substituted Michael adduct (24%). The reaction
presented here, different from other published protocols,7,10 is
conceptually a new methodology for the preparation of a-
vinylphosphonates. Hydrogenation of the resulting a-vinyl-
phosphonates affords the corresponding a-monofluorophos-
phonates, possessing a carboxy functionality which is amenable
to further derivatization. Furthermore, starting from a common
Scheme 1
The a-fluorovinylphosphonate7 represents a potential syn-
thetic intermediate for the preparation of a-fluorophosphonates.
Accordingly, we describe herein the feasibility studies of our
newly found reaction and its application to the synthesis of the
monofluoromethyl (CHF)-substituted phosphoserine (pSer)
mimetic 2-amino-4-fluoro-4-phosphonobutanoic acid (FPab) in
a form suitably protected for the preparation of pSer mimetic-
containing peptides.
Initially, we chose a difluoromethylphosphonate-bearing
conjugate (2S)-bornane-[10,2]-sultam (Xs-sultam)-imide8 4 as
a substrate for the copper-mediated reaction in order to allow
subsequent stereoselective introduction of amino functionality
under chiral auxiliary control. The sultam-imide 4 was treated
under various conditions9 and the results are shown in Table
1.‡
difluoromethylphosphonate intermediate, both the monofluoro-
and corresponding difluoro-methylphosphoryl counterparts can
be obtained.
Next, we applied this methodology to the synthesis of CHF-
substituted pSer mimetic (FPab) as shown in Scheme 2.
Hydrogenation of a-vinylphosphonate 5 over Pd–C in AcOEt
proceeded without diastereoselectivity to yield a-monofluor-
ophosphonate 6 in quantitative yield. Reaction of 1-chloro-
1-nitrosocyclohexane11 in THF (blue) with the Na-enolate,
resulting from treatment of 6 with NaHMDS in THF at 278 °C,
proceeded with high diastereoselectivity to instantaneously
afford a colorless solution of nitrone. Treatment of this solution
with aqueous 1 N HCl, followed by extractive work-up, gave
crude hydroxylamine 7, which was taken to the next step
without further purification. Reduction of 7 with Zn–AcOH in
THF, followed by introduction of Boc protection onto the
resulting NH2 group using (Boc)2O, gave Boc-protected 8. The
sultam moiety was then converted to the benzyl ester 9 utilizing
Reaction of 4 with either MeCu(CN)Li or Me2Cu(CN)Li2 in
the presence of LiCl and/or AlCl3 at 278 °C proceeded without
accompanying alkylation, but rather provided the correspond-
DOI: 10.1039/b002188l
Chem. Commun., 2000, 1081–1082
This journal is © The Royal Society of Chemistry 2000
1081

We thank Dr Terrence R. Burke Jr., NCI, NIH, Bethesda, MD
20892, for proofing the manuscript. This work was supported in
part by The Japan Health Sciences Foundation and Grants-in-
Aid for Scientific Research from the Ministry of Education,
Science and Culture of Japan.
Notes and references
† (Z)-3-(diethyldifluoromethyl)but-2-enoate 1 was prepared by coupling of
ethyl (Z)-3-iodobut-2-enoate with BrZnCF2(O)(OEt)2 in the presence of
CuBr in DMF.14 The (E)-isomer was synthesized according to the literature
method.15 Sultam-imide 4 was synthesized via the following sequence of
reactions: (i) transesterification of ethyl (Z)-3-iodobut-2-enoate to the
corresponding p-methoxybenzyl (PMB) ester using Ti(OPri)4 in PMB-OH;
(ii) CuBr-mediated coupling, as mentioned above; (iii) removal of the PMB
group with 95% aqueous TFA; (iv) coupling of the sultam.
‡ To a solution of CuCN·2LiCl in THF (1 mol dm23, 4.2 cm3) was added
MeLi·LiBr in Et2O (1.5 mol dm23, 5.6 cm3) at 278 °C. The mixture was
allowed to warm to 0 °C and stirred at this temperature for 1–2 min. After
re-cooling to 278 °C, 4 (763 mg, 1.68 mmol) in THF (5 cm3) was added
with a syringe. After being stirred at 278 °C for 1.5 h, the reaction was
quenched by addition of sat. NH4Cl–28% NH4OH solution. Usual work-up
followed by flash chromatography gave 5 (639 mg, 87% yield).
§ Coupling constants of 5 (3JHF = 38.6, 3JHP = 7.3 Hz) are consistent with
those of a-fluorovinylphosphonate possessing (E)-configuration (3JHPtrans
= 39–40, 3JHPcis = 7.6–10 Hz).16
¶ Protected peptide resin (Boc–Gly–FPab(OEt)2–Val–Pro–Met–Leu–PAM
resin, 0.05 mmol) was treated with 1 mol dm23 TMSOTf–thioanisole
(molar ratio 1+1) in TFA (2.5 cm3) in the presence of m-cresol (125 mm3)
and EDT (125 mm3) at 4 °C. After being stirred at 4 °C for 60 min, DMS
(0.75 cm3) and TMSOTf (0.5 cm3) were successively added to the reaction
with additional stirring at room temperature for 2 h. The reaction was
quenched by addition of EtOH–H2O. The aqueous layer was subjected to
HPLC purification, yielding 22 mg of the desired peptide. Ion-spray MS m/z
calcd for C27H49N6O10SFP (MH+) 699.76; found 699.50. Purified peptides,
consisting of diastereomers derived from FPab, were eluted as two peaks
incompletely resolved on HPLC.
Scheme 2 Reagents: (i) H2/Pd–C, AcOEt; (ii) NaHMDS (1.1 eq.), 1-chloro-
1-nitrosocyclohexane (1.1 eq.). THF then 1 N HCl aq.; (iii) Zn (40 eq.).
AcOH (50 eq.) then (Boc)2O (2.0 eq.), CH3CN; (iv) Ti(CPri)4 (2.0 eq.),
BzOH (44 eq.), toluene.
Ti(OPri)4–benzyl alcohol in toluene at 120 °C. Hydrogenolytic
debenzylation (H2/10% Pd–C in AcOEt) of 9 gave the protected
L
-CHF-substituted pSer mimetic (Boc-FPab(OEt)2-OH 10).
Application of a similar sequence of reactions to 4 gave
enantiometically pure
-CF2-substituted pSer mimetic12
(F2Pab) 11. We speculate that FPab derivative 10 possesses the
2S configuration ( -amino acid), by analogy to F2Pab derivative
∑ Peptides possessing L-phosphotyrosine mimetics as an FPab replacement
were completely hydrolyzed by leucine amino peptidase, while
phosphotyrosine mimetic-containing peptides remained intact.17
D-
L
1 T. Hunter, Cell, 2000, 100, 113.
2 G. M. Blackburn, Chem. Ind., (London), 1981, 134.
L
11, which is obtained from a difluoromethylphosphonate-
containing Xs-sultam utilizing a similar reaction sequence and
has the 2S configuration. To our knowledge, this is the first
synthesis of a CHF-substituted pSer mimetic.
3 For a recent review see: M. J. Tozer and T. F. Herpin, Tetrahedron,
1996, 52, 8619; for an updated compilation of references see: J. M.
Percy, Top. Curr. Chem., 1997, 193, 131.
4 L. Schmitt, N. Cavusoglu, B. Spiess and G. Schlewer, Tetrahedron Lett.,
1998, 39, 4009; T. R. Burke Jr., M. S. Smyth, A. Otaka, M. Nomizu,
P. P. Roller, G. Wolf, R. Case and S. E. Shoelson, Biochemistry, 1994,
33, 6490.
5 B. Iorga, F. Eymery and P. Savignac, Tetrahedron, 1999, 55, 2671; X.
Zhang, W. Qiu and D. J. Burton, Tetrahedron Lett., 1999, 40, 2681.
6 A. Otaka, E. Mitsuyama, T. Kinoshita, H. Tamamura and N. Fujii, J.
Org. Chem., 2000, 65, in press.
7 G. M. Blackburn and M. J. Parratt, J. Chem. Soc., Perkin Trans 1, 1986,
1417.
8 W. Oppolzer, Pure Appl. Chem., 1990, 62, 1241.
9 Organocopper Reagents, A Practical Approach, ed. R. J. K. Taylor,
Oxford University Press, Oxford, 1994, pp. 85–28, 143–158, and
references cited therein.
10 A. J. Zapata, Y. Gu and G. B. Hammond, J. Org. Chem., 2000, 65, 227;
R. S. Gross, S. Mehdi and J. R. McCarthy, Tetrahedron Lett., 1993, 34,
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11 W. Oppolzer, O. Tamura and J. Deerberg, Helv. Chim. Acta, 1992, 75,
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12 A. Otaka, K. Miyoshi, T. R. Burke Jr., P. P. Roller, H. Kubota, H.
Tamamura and N. Fujii, Tetrahedron Lett., 1995, 36, 927; D. B.
Berkowitz, Q. Shen and J.-H. Maeng, Tetrahedron Lett., 1994, 35,
6445.
In order to examine the general applicability of protected
FPab 10 to peptide synthesis, 10 was incorporated into the
peptide sequence (H–Gly–FPab–Val–Pro–Met–Leu) using a
standard Boc-based solid-phase protocol. The resulting pro-
tected peptide resin was treated with a one-pot, two-step
deprotection methodology13 consisting of high-acidity [1 mol
dm23 TMSOTf–thioanisole in TFA, m-cresol, ethanedithiol
(EDT)] and low-acidity (1 mol dm23 TMSOTf–thioanisole in
TFA, m-cresol, EDT + DMS–TMSOTf), which was developed
for practical deprotection of protected phosphoamino acid-
containing peptide resins, to yield a crude deprotected peptide
without accompanying partially Et-deprotected peptides.¶ After
HPLC purification, an FPab-containing peptide was obtained in
63% yield. In order to confirm the 2S configuration of FPab,
purified peptide was subjected to enzyme digestion using
leucine amino peptidase (LAP).∑ Interestingly, it was found that
the parent peptides were converted to 5-residue peptides, H–
FPab–Val–Pro–Met–Leu–OH, with rates that varied between
the diastereomers derived from the fluorine substitution in FPab
and with only 10% of FPab being released from the resulting
5-residue peptide after 24 h of LAP treatment. On the other
13 A. Otaka, K. Miyoshi, M. Kaneko, H. Tamamura, N. Fujii, M. Nomizu,
T. R. Burke Jr. and P. P. Roller, J. Org. Chem., 1995, 60, 3967.
14 T. Yokomatsu, K. Suemune, T. Murano and S. Shibuya, J. Org. Chem.,
1996, 61, 7207.
15 K. Blades, A. H. Butt, G. S. Cockerill, H. J. Easterfield, T. P. Lequeux
and J. M. Percy, J. Chem. Soc., Perkin Trans 1, 1999, 3609.
16 R. Waschbu¨sch, J. Carran and P. Savignac, Tetrahedron, 1996, 52,
14199.
hand, -FPab-containing peptides remained intact after 24 h
D
digestion using LAP. The present methodology should allow
the facile preparation of functionalized a-fluorovinylphospho-
nates and a-fluorophosphonates. Furthermore, it is tempting to
speculate that FPab-containing peptides could serve as in-
hibitors against both proteases and phosphatases since peptides
having FPab residues at the N-terminal position are resistant to
the action of LAP.
17 T. R. Burke Jr., M. S. Smyth, M. Nomizu, A. Otaka and P. P. Roller,
J. Org. Chem.,1993, 58, 1336.
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Chem. Commun., 2000, 1081–1082