Synthesis of methionine- and norleucine-derived phosphinopeptides

Article abstract of DOI:10.1016/j.tetlet.2008.07.062

We present herein a straightforward synthesis of N-Fmoc-protected synthons derived from a phosphinic analogue of methionine. These precursors were used successfully for the solid-phase synthesis of methionine-mimic phosphinopeptides using BOP-catalyzed coupling without protection of the phosphoryl moiety. We also prepared a new type of pseudopeptide derived from a phosphinic analogue of norleucine with a -PO(OH)-CH₂-COOR moiety.

Full text of DOI:10.1016/j.tetlet.2008.07.062

Tetrahedron Letters 49 (2008) 5629–5631
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Tetrahedron Letters
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Synthesis of methionine- and norleucine-derived phosphinopeptides
*
ˇ
ˇ
´
ˇ
ˇ
Radek Liboska, Jan Pícha, Ivona Hanclová, Miloš Budešínsky, Miloslav Šanda, Jirí Jirácek
Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
We present herein a straightforward synthesis of N-Fmoc-protected synthons derived from a phosphinic
analogue of methionine. These precursors were used successfully for the solid-phase synthesis of methio-
nine-mimic phosphinopeptides using BOP-catalyzed coupling without protection of the phosphoryl
moiety. We also prepared a new type of pseudopeptide derived from a phosphinic analogue of norleucine
with a –PO(OH)–CH2–COOR moiety.
Received 2 June 2008
Revised 30 June 2008
Accepted 9 July 2008
Available online 12 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Phosphinopeptides1,2 (or phosphinic pseudopeptides) have
found important applications in biology and medicine, primarily
as inhibitors of metalloenzymes.3,4 The replacement of a hydrolyz-
able peptide bond by a non-hydrolyzable phosphinate moiety
mimics the hypothetical transition state of the native substrate
during hydrolysis and may result in a potent inhibition of the
respective peptidases.5
rather poor yields (about 30–35%). We were unable to prepare
synthon 4, which contains a phosphinyl moiety protected by an
adamantyl group (Ad). However, similar compounds were used
previously for the solid-phase synthesis of phosphinic pseudopep-
tides.9,10 The preparation of compound 4 failed due to low yields in
the adamantylation step and especially in the final catalytic hydro-
genation. This difficulty was probably caused by the hydrogena-
tion-poisoning effect of the sulfur atom present in our
compounds. Therefore, we returned to phosphinic acid 3 and in-
stead of further difficult purification attempts, we neutralized the
redundant phosphorus acid and its phosphoric acid oxidation
product with sodium carbonate, partially removed the methanol
in vacuo, and protected the amino group of crude compound 3
with di-tert-butyl dicarbonate in aqueous sodium carbonate/1,4-
dioxane mixture. The N-Boc-protected product was extracted from
the acidified (pH ꢀ 2) reaction mixture, and column chromatogra-
phy on silica gel gave a good yield of compound 5. P–C bond forma-
tion was performed according to Georgiadis et al.10 via silylation of
5 with chlorotrimethylsilane in dichloromethane/DIPEA followed
by addition of ethyl acrylate or ethyl methacrylate to give com-
pounds 6a (R = H) or 6b (R = CH3), respectively. Extraction of these
compounds into ethyl acetate after careful acidification and final
silica gel chromatography afforded pure compounds 6a and 6b in
good yields. Unprotected C-terminal amides 7a and 7b were pre-
pared from 6a and 6b, respectively, using a 20% (w/w) solution
of ammonia in anhydrous ethanol heated to 55 °C for 48 h in a
sealed container. Alternatively, the use of aqueous ammonia
yielded mainly the free acid. The solution was evaporated to dry-
ness in vacuo and the N-Boc-protecting group was cleaved with
TFA (100%, rt, 4 h). Finally, compounds 7a and 7b were purified
by RP-HPLC (Phenomenex Luna C-18). If the N-terminal Boc group
was removed prior to the ammonolysis of the C-terminal ethyl
ester, the lactams 8a and 8b were formed. Similar reactivity was
observed previously by Yiotakis et al.9 The unprotected phosphinic
amino acids 9a and 9b were prepared in two steps; alkaline hydro-
lysis (4 M aq NaOH) of the ethyl esters 6a or 6b was followed, after
First, we present an efficient synthesis of a novel type of
pseudopeptides of formulas Met-
w
[PO(OH)CH2]-Ala(Gly), Met-
[PO(OH)CH2]-Ala(Gly)-Cys
w
[PO(OH)CH2]-Ala(Gly)-Val, and Met-
w
that mimic the sequences Met-Ala(Gly), Met-Ala(Gly)-Val, and
Met-Ala(Gly)-Cys, but contain the phosphinate analogue of methio-
nine at the N-terminus (Scheme 1). Second, we demonstrate the
preparation of a new type of pseudopeptides of formula Nle-
w
[PO(OH)]-Gly derived from the phosphinate analogue of norleu-
cine (Scheme 2). Both types of compounds represent potential
inhibitors of methionine or leucine aminopeptidases. The proposed
pseudopeptide sequences should fulfill the structural require-
ments of methionine aminopeptidases with Met or Nle at the P1
position and a small aliphatic residue, such as Ala or Gly, at the
P01 position. The C-terminal Val and Cys at the P02 position of the
methionine-derived phosphinates were chosen based on our re-
cent results with statin pseudopeptides as inhibitors of methionine
aminopeptidases.6
The synthesis of the methionine-derived phosphinates (Scheme
1) began with the addition of methanethiol to acrolein7 followed
by reaction of the aldehyde 1 with hydroxylamine hydrochloride
in pyridine to give oxime 2 in good yield. The phosphinate interme-
diate 3 was prepared according to Zhukov et al.8 by refluxing
oxime 2 with anhydrous hypophosphorus acid in dry methanol un-
der an argon atmosphere. The previously published8 procedures
for the isolation of phosphinic acid 3, such as crystallization from
2-propanol or chromatography (on cellulose or Dowex), afforded
* Corresponding author. Tel.: +420 220183441; fax: +420 220183571.
ˇ
E-mail address: jiracek@uochb.cas.cz (J. Jirácek).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.07.062

5630
R. Liboska et al. / Tetrahedron Letters 49 (2008) 5629–5631
OH
CH3
O
AdO
FmocHN
O
HO
H
O
N
O
H2N
P
P
CH3
H
OH
c
a
b
O
H2C
S
S
S
S
CH3
CH3
CH3
R
d
4
1
2
3
R
HO
O
HO
BocHN
HO
O
P
H2N
P
NH2
BocHN
P
OEt
f
e
O
O
O
S
S
S
CH3
CH3
CH3
7a, R = H
7b, R = CH3
g
f'
6a
5
, R = H
6b, R = CH3
R
O
R
R
HO
O
HO
O
OH
O
HN
P
FmocHN
P
OH
H2N
P
OH
h
O
O
S
S
S
CH3
8a
CH3
CH3
, R = H
8b, R = CH3
10a
, R = H
10b, R = CH3
9a, R = H
9b
i
i
, R = CH3
R
O
R
O
HO
O
HO
O
H
N
H
N
H2N
P
H2N
P
NH2
SH
NH2
O
O
H3C CH3
S
S
CH3
CH3
11a
11b, R = CH3
12a
, R = H
12b, R = CH3
, R = H
Scheme 1. Reagents, conditions, and yields: (a) MeSH, 0 °C (41%); (b) NH2OHꢁHCl, pyridine, 14 h rt (80%); (c) anhydrous H3PO2 (1 M) in methanol, reflux for 6 h; (d) Boc2O,
Na2CO3, water/1,4-dioxane, 0 °C for 1 h then 2 h rt (79%, calculated from 2); (e) ethyl acrylate, or ethyl methacrylate, TMSCl, DIPEA, rt overnight R = H (88%), R = CH3 (82%); (f)
(i) NH3/EtOH, 20% (w/w), 55 °C, 48 h; (ii) TFA, 100%, 4 h rt, R = H (46%, after HPLC), R = CH3 (42% after HPLC); (f0) (i) TFA (100%), 24 h rt; (ii) NH4OH (25%), 55 °C, 16 h, or aq
NaOH (4 M), 4 h rt. The yields of products 8a and 8b have not been determined; (g) (i) aq NaOH (4 M), rt overnight; (ii) TFA (100%), 4 h rt, R = H (78%), R = CH3 (74%); (h)
FmocOSu, Na2CO3, water/dioxane, 3 h rt R = H, CH3 (28–92%); (i) (i) Cys(Trt)-Rink Amide AM resin or Val-Rink Amide AM resin (1 equiv), 10a or 10b (1.5 equiv), BOP (3 equiv)/
DIPEA (5 equiv) in DMF, rt overnight and 6 h; (ii) piperidine/DMF; (iii) TFA (95%), ethanedithiol, water, triisopropylsilane, 2 h, rt (11a 21%, 11b 25%, 12a 23%, 12b 30%, yields
are given for compounds purified by RP-HPLC as mixtures of diastereoisomers).
removal of sodium ions using Dowex 50W (H+), by treatment with
TFA (100%, rt, 4 h). The products 9a and 9b were purified using sil-
ica gel chromatography. In order to prepare N-Fmoc-protected syn-
thons 10a and 10b, convenient precursors for the solid-phase
synthesis of phosphinic pseudopeptides, compounds 9a and 9b
were treated with 9-fluorenylmethyl N-succinimidyl carbonate
(FmocOSu) in aqueous sodium carbonate/1,4-dioxane. After com-
pletion of the reactions (monitored by TLC) and removal of the
dioxane, the reaction mixture was acidified with 1 M HCl, and
the product was taken up into ethyl acetate and purified by silica
gel chromatography. The irreproducible yields of compounds 10a
and 10b were due to the very low solubility of the products in
the available chromatography eluents. As an example, the NMR
spectral data for compounds 10 are given.11 We applied the tech-
niques of solid-phase synthesis for the preparation of pseudopep-
tides 11 and 12. The C-terminal valine and cysteine residues
were attached to Rink Amide AM resin using an Fmoc/HBTU/DIPEA
strategy.12 The subsequent coupling of the phosphinic synthons
10a or 10b was achieved with BOP/DIPEA reagents according to
Raguin et al.13 After the N-terminal deprotection, the pseudopep-
tides 11 and 12 were cleaved from the resin with a mixture of
TFA and scavengers, and purified by RP-HPLC in low yields.
In the second part of our study, we prepared a new type of
phosphinate with a –PO(OH)–CH2–COOR moiety. Similar com-
pounds were previously prepared by Allen et al.14 The above-
described difficulties with methionine-mimic phosphinic
pseudopeptides led us to abandon the methionine side chain and
to introduce the analogous norleucine-derived side chain.15 (1-
Benzhydrylamino)pentylphosphinic acid 13 was synthesized by
heating valeraldehyde and diphenylmethylamine hypophosphite
in THF according to the procedure of Baylis et al.16 The phosphinic
acid 13 was converted to (R,S)-1-aminopentylphosphonic acid by
refluxing with concentrated hydrobromic acid followed by treat-
ment with propylene oxide. Reaction of the free phosphinic acid

R. Liboska et al. / Tetrahedron Letters 49 (2008) 5629–5631
5631
HO
HO
NH2 H3PO2
H
O
H
O
H
N
H
N
P
BnO
P
a
b, c
O
CH3
CH3
d
14
13
O
O
O
O
HO
HO
O
HO
O
O
H
N
O
H2N
P
BnO
P
H2N
P
HN
P
OCH3
OCH3 f, g
+
e
OH
OH
O
CH3
CH3
CH3
18
CH3
16
17
15
Scheme 2. Reagents, conditions, and yields: (a) valeraldehyde, THF, reflux for 2 h (75%); (b) 48% HBr, reflux for 8 h, then propylene oxide, ethanol (69%); (c) benzyl
chloroformate, Na2CO3, water and dioxane, 0 °C 2 h then rt overnight (93%); (d) TMSCl, TEA, methyl bromoacetate, rt overnight (80%); (e) HCOOꢃNH4þ, 10% Pd/C, methanol, rt
overnight (20% for 16); (f) NaOH, methanol and water, rt overnight (79%); (g) HCOOꢃNH4þ, 10% Pd/C, methanol, rt overnight (48%).
4. Yiotakis, A.; Georgiadis, D.; Matziari, M.; Makaritis, A.; Dive, V. Curr. Org. Chem.
2004, 8, 1135.
which gave intermediate 15 upon Arbuzov reaction14 with methyl
5. Collinsova, M.; Jiracek, J. Curr. Med. Chem. 2000, 7, 629.
with benzyl chloroformate afforded the Z-protected derivative 14,
bromoacetate. For reference, the NMR spectral data for compound
15 are provided.11 The analogous Arbuzov reaction was also per-
formed with the methionine analogue of 14 but this attempt was
unsuccessful, probably due to the presence of a sulfur atom. Cata-
lytic transfer hydrogenation17 of 15 afforded the desired methyl
ester 16 along with a considerable amount of cyclic by-product
17. However, alkaline hydrolysis of 15 followed by catalytic trans-
fer hydrogenation afforded target compound 18.
The synthesis of methionine-derived phosphinic pseudopep-
tides is difficult due to the presence of a side-chain sulfur atom.
However, phosphinopeptides and phosphonopeptides can be pre-
pared by BOP-catalyzed coupling using N-protected synthons
without protection of the phosphoryl moiety. We successfully used
this strategy for the synthesis of several methionine-derived phos-
phinic pseudopeptides as potential inhibitors of aminopeptidases.
We also prepared a new type of pseudopeptide derived from the
phosphinic analogue of norleucine with a –PO(OH)–CH2–COOR
moiety. The target compounds 7, 9, 11, 12, 16, and 18 will be tested
for their inhibitory activities toward leucine and methionine
aminopeptidases.
6. Mitra, S.; Dygas-Holz, A. M.; Jiracek, J.; Zertova, M.; Zakova, L.; Holz, R. C. Anal.
Biochem. 2006, 357, 43.
7. Catch, J. R.; Cook, A. M.; Graham, A. R.; Heilbron, I. J. Chem. Soc. 1947, 1609.
8. Zhukov, Y. N.; Khomutov, A. R.; Osipova, T. I.; Khomutov, R. M. Russ. Chem. Bull.
1999, 48, 1348.
9. Yiotakis, A.; Vassiliou, S.; Jiracek, J.; Dive, V. J. Org. Chem. 1996, 61, 6601.
10. Georgiadis, D.; Matziari, M.; Yiotakis, A. Tetrahedron 2001, 57, 3471.
11. Compound 10a: 1H NMR (600 MHz, DMSO): 1.79 (2H, m, P–CH2); 1.79 and 1.97
(2ꢂ 1H, 2ꢂ m, C–CH2–C); 2.04 (3H, s, S–CH3); 2.40 and 2.46 (2ꢂ 1H, 2ꢂ m,
CH2–CO); 2.38 and 2.55 (2ꢂ 1H, 2ꢂ m, S–CH2); 3.81 (1H, m, CH–P); 4.23 (1H,
dd, J = 7.2 and 7.0, CH(Fmoc)); 4.31 (1H, dd, J = 10.6 and 7.2, O–CHa); 4.38 (1H,
dd, J = 10.6 and 7.0, O–CHb); 7.32 (1H, m, arom.H); 7.34 (1H, m, arom.H); 7.43
(2H, m, arom.H); 7.64 (1H, d, J = 9.4, NH); 7.73 (1H, m, arom.H); 7.74 (1H, m,
arom.H); 7.90 (2H, m, arom.H). 13C NMR (150.9 MHz, DMSO): 14.83 (S–CH3);
21.99 (d, J(C,P) = 90.3, P–CH2); 26.63 (d, J(C,P) = 2.8, CH2–CO); 27.15 (d,
J(C,P) = 2.3, C–CH2–C); 30.25 (d, J(C,P) = 13.1, S–CH2); 47.02 (˜CH– (Fmoc));
49.13 (d, J(C,P) = 106.0, P–CH); 65.83 (O–CH2); 120.40(2C), 125.48, 125.55,
127.31, 127.35, 127.92, 127.94, 141.00, 141.01, 143.94 and 144.15 (12 arom.C);
156.59 (d, J(C,P) = 3.8, O–CO–N); 173.90 (d, J(C,P) = 15.9, COOH). Compound
10b: 1H NMR (600 MHz, DMSO): 1.17 (3H, d, J = 7.0, CH3); 1.56 and 2.04 (2ꢂ
1H, 2ꢂ m, P–CH2); 1.78 and 1.96 (2ꢂ 1H, 2ꢂ m, C–CH2–C); 2.02 (3H, s, S–CH3);
2.38 and 2.53 (2ꢂ 1H, 2ꢂ m, S–CH2); 2.67 (1H, m, CH–CO); 3.78 (1H, m, CH–P);
4.22 (1H, t, J = 7.1, CH(Fmoc)); 4.30 (1H, dd, J = 10.6 and 7.1, O–CHa); 4.33 (1H,
dd, J = 10.6 and 7.1, O–CHb); 7.31 (1H, m, arom.H); 7.32 (1H, m, arom.H); 7.41
(2H, m, arom.H); 7.63 (1H, d, J = 9.4, NH); 7.71 (2H, m, arom.H); 7.89 (2H, m,
arom.H). 13C NMR (150.9 MHz, DMSO): 14.80 (S–CH3); 18.73 (d, J(C,P) = 5.8, C–
CH3); 27.16 (d, J(C,P) = 2.7, C–CH2–C); 29.73 (d, J(C,P) = 89.2, P–CH2); 30.22 (d,
J(C,P) = 2.9, S–CH2); 33.31 (d, J(C,P) = 3.3, CH–CO); 46.94 (˜CH– (Fmoc)); 49.86
(d, J(C,P) = 105.6, P–CH); 65.83 (O–CH2); 120.36(2C), 125.47, 125.51, 127.27,
127.30, 127.88(2C), 140.96(2C), 143.94 and 144.11 (12 arom.C); 156.43 (d,
J(C,P) = 3.8, O–CO–N); 176.89 (d, J(C,P) = 12.0, COOH). Compound 15: 1H NMR
(600 MHz, CDCl3): 0.87 (3H, t, J = 7.0, CH3); 1.28 and 1.34 (2ꢂ 1H, 2ꢂ m, CH2);
1.34 and 1.44 (2ꢂ 1H, 2ꢂ m, CH2); 1.54 and 1.84 (2ꢂ 1H, 2ꢂ m, CH2); 4.13 (1H,
m, CH–P); 2.98 (1H, dd, J = 14.8 and 16.8, P–CHa); 3.01 (1H, dd, J = 14.8 and
15.8, P–CHb); 3.67 (3H, s, COOCH3); 5.09 and 5.14 (2ꢂ 1H, 2ꢂ d, J = 12.3, O–
CH2); 5.39 (1H, d, J = 10.2, NH); 7.30–7.35 (5H, m, C6H5); 10.33 (1H,br s, P–OH).
13C NMR (150.9 MHz, CDCl3): 13.84 (CH3); 22.15 (CH2); 27.68 (CH2); 27.84 (d,
J(C,P) = 11.9, CH2); 34.94 (d, J(C,P) = 82.1, P–CH2); 50.08 (d, J(C,P) = 111.7, P–
CH); 52.63 (OCH3); 67.19 (O–CH2); 127.97(2C), 128.16, 128.48(2C) and 136.14
(5 arom.C); 156.35 (d, J(C,P) = 5.9, O–CO–N); 166.56 (d, J(C,P) = 4.4, COOCH3).
12. Fields, G. B.; Noble, R. L. Int. J. Pept. Prot. Res. 1990, 35, 161.
Acknowledgments
This project was supported by Grant 203/06/1405 (to J.J.) from
the Grant Agency of the Czech Republic, by the Chemical Genetics
Consortium No. LC060777 of the Ministry of Education, Youth and
Sports of the Czech Republic (to J.J.) and by Research Project Z4 055
0506 of the Academy of Sciences of the Czech Republic.
References and notes
1. Kafarski, P.; Lejczak, B. In Aminophosphinic and Aminophosphonic Acids.
Chemistry and Biological Activity; Kukhar, V. P., Hudson, H. R., Eds.; Synthesis
of Phosphono- and Phosphinopeptides; John Wiley & Sons Ltd: Chichester,
2000; pp 173–203.
13. Raguin, O.; Fournie-Zaluski, M. C.; Romieu, A.; Pelegrin, A.; Chatelet, F.;
Pelaprat, D.; Barbet, J.; Roques, B. P.; Gruaz-Guyon, A. Angew. Chem., Int. Ed.
2005, 44, 4058.
14. Allen, M. C.; Fuhrer, W.; Tuck, B.; Wade, R.; Wood, J. M. J. Med. Chem. 1989, 32,
1652.
15. Picha, J.; Budesinsky, M.; Sanda, M.; Jiracek, J. Tetrahedron Lett. 2008, 49, 4366.
16. Baylis, E. K.; Campbell, C. D.; Dingwall, J. G. J. Chem. Soc., Perkin Trans. 1 1984,
2845.
2. Kafarski, P.; Lejczak, B. In Aminophosphinic and Aminophosphonic Acids.
Chemistry and Biological Activity; Kukhar, V. P., Hudson, H. R., Eds.; The
Biological Activity of Phosphono- and Phosphinopeptides; John Wiley & Sons
Ltd: Chichester, 2000; pp 407–442.
3. Dive, V.; Georgiadis, D.; Matziari, M.; Makaritis, A.; Beau, F.; Cuniasse, P.;
Yiotakis, A. Cell. Mol. Life Sci. 2004, 61, 2010.
17. Anwer, M. K.; Spatola, A. F. Synthesis 1980, 929.