Protection of the hydroxyphosphinyl function of phosphinic dipeptides by adamantyl. Application to the solid-phase synthesis of phosphinic peptides

Article abstract of DOI:10.1021/jo9603439

To develop solid-phase synthesis of phosphinic peptides, different FmocXaaΦ{PO(OAd)CH₂}XaaOH building blocks have been prepared, where Fmoc is (fluorenylmethoxy)carbonyl. In this respect, the protection of the hydroxyphosphinyl function in these phosphinic dipeptides by the adamantyl group turns out to be convenient. The phosphinic adamantyl esters are completely stable in basic conditions and can be removed under relatively mild acidic conditions. Using these building blocks, despite the bulkiness of the adamantyl group, no particular problem of coupling was observed during the solid-phase synthesis of phosphinic peptides by the Fmoc strategy. The developed methodology is of particular interest to facilitate the development of potent inhibitors of zinc-metalloproteases.

Full text of DOI:10.1021/jo9603439

J . Org. Chem. 1996, 61, 6601-6605
6601
P r otection of th e Hyd r oxyp h osp h in yl F u n ction of P h osp h in ic
Dip ep tid es by Ad a m a n tyl. Ap p lica tion to th e Solid -P h a se
Syn th esis of P h osp h in ic P ep tid es
Athanasios Yiotakis,†,* Stamatia Vassiliou,† J irˇ´ı J ira´cˇek,‡,§ and Vincent Dive‡
Department of Organic Chemistry, Laboratory of Organic Chemistry, University of Athens,
Panepistiomiopolis, Zografou, Athens, 15771, Greece, and De´partement d’Inge´nierie et d’Etudes
des Prote´ines, CEA-Direction des Sciences du Vivant, Centre d’Etudes de Saclay,
91191 Gif/ Yvette Cedex, France
Received February 20, 1996X
To develop solid-phase synthesis of phosphinic peptides, different FmocXaaΨ{PO(OAd)CH2}XaaOH
building blocks have been prepared, where Fmoc is (fluorenylmethoxy)carbonyl. In this respect,
the protection of the hydroxyphosphinyl function in these phosphinic dipeptides by the adamantyl
group turns out to be convenient. The phosphinic adamantyl esters are completely stable in basic
conditions and can be removed under relatively mild acidic conditions. Using these building blocks,
despite the bulkiness of the adamantyl group, no particular problem of coupling was observed during
the solid-phase synthesis of phosphinic peptides by the Fmoc strategy. The developed methodology
is of particular interest to facilitate the development of potent inhibitors of zinc-metalloproteases.
In tr od u ction
of phosphinic peptide inhibitors. To this end, we recently
elected to develop, by combinatorial chemistry, phos-
phinic peptide libraries which can be screened by various
zinc-metalloproteases. However, the use of such an
approach requires the development of a reliable and
convenient solid-phase synthesis of these phosphinic
peptides.
In this paper, we demonstrate that the adamantyl is
a suitable protecting group for the hydroxyphosphinyl
function of phosphinic dipeptides, which allows the
development of solid-phase synthesis of phosphinic pep-
tides by the Fmoc ((fluorenylmethoxy)carbonyl) strategy.
In the last decade, several studies have demonstrated
that the synthesis of phosphinic peptides is a very
effective approach to the development of highly potent
inhibitors of zinc-metalloproteases.1-6 Recently, the
resolution of the three-dimensional structure of a complex
between a zinc-metalloprotease and a phosphinic pep-
tide inhibitor has confirmed the view that these pseudo-
peptides are indeed good mimics of the substrate in the
transition state.7 Phosphinic peptides are more stable
than the phosphonamide peptides and are therefore more
suitable for the development of zinc-metalloprotease
inhibitors.5,8,9 However, in the view of pharmacological
studies such inhibitors should not only be potent but also
as selective as possible, in order to avoid their interac-
tions with multiple zinc-metalloproteases. This prob-
lem could be addressed by systematically investigating
the influence on selectivity of the different side-chains
Resu lts
As a protecting group for the hydroxyphosphinyl func-
tion of FmocXaaΨ{PO(OH)CH2}XaaOH phosphinic dipep-
tides, we looked for one which can be removed easily
under classical conditions. Among different possible
protecting groups, adamantyl appears promising. In fact,
1-adamantyl is a tertiary system which reacts by SN1
mechanism, because backside attack is hindered for steric
reasons. This means that 1-adamantyl ester must be
labile under acidic conditions due to tertiary cation
formation. On the other hand, due to its constrained
structure, the adamantyl is less stable than other tertiary
group cations.10 Consequently, 1-adamantyl esters are
expected to be less acid-sensitive than tert-butyl esters.11
Starting from the classical synthon 1, adamantyl and
Fmoc protecting groups can be introduced in three steps
(Scheme 1). To check the potential influence that the
residues framing the phosphinic bond may play on the
introduction of the adamantyl group, four different
phosphinic synthons were prepared (Table 1).
* To whom correspondence should be addressed.
† University of Athens.
‡ Centre d’Etudes de Saclay.
§ Present address: Institute of Organic Chemistry and Biology,
Academy of Sciences of Czech Republic, Flemingovo nam. 2, 16610
Praha 6, Czech Republic.
X Abstract published in Advance ACS Abstracts, August 15, 1996.
(1) Petrillo, E. W.; Cushman, D. W.; Duggan, M. E.; Heikes, J . E.;
Karanewsky, D. S.; Ondetti, M. A.; O’Reilly, B.; Roonyak, G. C.;
Schwartz, J .; Spitzmiller, E. R.; Wang, N. Y. In Peptides: Structure
and Function, Proceedings of the Eighth American Peptide symposium;
Hruby, V. J ., Rich, D. H., Eds.; Pierce Chemical Co.: Rockford, IL,
1983; pp 541-550.
(2) Krapcho, J .; Turk, C.; Cushman, D. W.; Powell, J . R.; DeForrest,
J . M.; Spitzmiller, E. R.; Karanewsky, D. S.; Duggan, M.; Rovnak, G.;
Schwartz, J .; Natarajan, S.; Godfrey, J . D.; Ryono, D. E.; Neubeck, R.;
Atwa, K. S.; Petrillo, E. D., J r. J . Med. Chem. 1988, 31, 1148-1160.
(3) Karanewsky, D. S.; Badia, M. C.; Cushman, D. W.; DeForrest,
J . M.; Dejneka, T.; Loots, M. J .; Perri, M. G.; Petrillo, E. W.; Powell, J .
R. J . Med. Chem. 1988, 31, 204-212.
(4) Grobelny, D.; Goli, U. B.; Galardy, R. E. Biochemistry 1989, 28,
4948-4951.
(5) Yiotakis, A.; Lecoq, A.; Nicolaou, A.; Labadie, J .; Dive, V.
Biochem. J . 1994, 303, 323-327.
The first step involves the formation of a silver salt
which reacts with 1-adamantyl bromide to give the
corresponding ester (Scheme 2). However, it should be
stressed that our first attempts to prepare these ada-
mantyl esters resulted in poor yields. Closer examination
of this reaction led to the isolation of an unexpected silver
salt complex where silver and synthon 1 counterions
(6) Vincent, B.; Dive, V.; Yiotakis, A.; Smadja, G.; Maldonado, R.;
Vincent, J . P.; Checler, F. Br. J . Pharmacol. 1995, 115, 1053-1063.
(7) Grams, F.; Dive, V.; Yiotakis, A.; Yiallouros, I.; Vassiliou, S.;
Zwilling, D.; Bode, W.; Sto¨cker, W. Submitted for publication.
(8) Hanson, J . E.; Kaplan, A. P.; Bartlett, P. A. Biochemistry 1989,
28, 6294-6305.
(10) Fort, R. C.; Schleyer, P. R. Chem. Rev. 1964, 64, 277-300.
(11) Schleyer, P. R.; Nicolas, R. D. J . Am. Chem. Soc. 1961, 83,
2700-2705.
(9) Mookhtiar, K. A.; Marlowe, C. K.; Bartlett, P. A.; Van Wart, H.
E. Biochemistry 1987, 26, 1962-1965.
S0022-3263(96)00343-X CCC: $12.00 © 1996 American Chemical Society

6602 J . Org. Chem., Vol. 61, No. 19, 1996
Yiotakis et al.
Sch em e 1a
a
(a) Ag2O, (b) 1-Ad-Br, (c) NaOH, (d) H+, (e) HCOO-NH4+, 10% Pd/C, (f) Fmoc-Cl.
Ta ble 1. Ch a r a cter iza tion of th e Typ e 1 Syn th on s
synthons
FAB-MS (M + H)
TLC (Rf)
(1)a (2)a (3)a
analytical data (found/calcd)
yield
(%)
δ(ppm) 31
P
(CDCl3)
R1
R2
formula
calcd
found
C
H
N
P
C6H5CH2
C6H5CH2 CH3
C6H5CH2 (CH3)2CHCH2 C25H34NO6P
CH3
H
C21H26NO6P
C22H28NO6P
90
91
89
96
419.41
433.41
475.49
399.40
419.5
433.5
475.6
399.6
53.8, 53.8 0.52 0.80 0.64 60.35/60.14 6.15/6.25 3.26/3.23 6.98/7.39
53.1, 53.2 0.56 0.96 0.61 60.69/60.96 6.32/6.51 3.18/3.23 6.73/7.15
53.4, 53.9 0.70 0.91 0.65 62.89/63.15 7.22/7.20 2.81/2.94 6.12/6.52
54.2, 54.6 0.50 0.73 0.51 57.29/57.13 7.41/7.57 3.32/3.50 7.32/7.76
(CH3)2CHCH2 C19H30NO6P
a
See Experimental Section for the definition of the solvent systems.
Ta ble 2. Ch a r a cter iza tion of th e Typ e 2 Syn th on s
synthons
FAB-MS (M + H)
TLC (Rf)
(4)a (5)a
analytical data (found/calcd)
yield
(%)
δ(ppm) 31
P
(CDCl3)
R1
R2
formula
calcd
found
C
H
N
P
C6H5CH2
C6H5CH2 CH3
H
C31H40NO6P
C32H42NO6P
84
79
553.64
567.64
554.1
567.8
48.8, 48.8
47.7, 47.7,
48.0, 48.5
47.4, 47.8,
48.0, 48.3
47.3, 47.3,
48.3, 49.0
0.52 0.63 67.39/67.25 7.02/7.28 2.41/2.53 5.18/5.59
0.57 0.70 68.02/67.71 7.41/7.45 2.32/2.46 5.21/5.46
C6H5CH2 (CH3)2CHCH2 C35H48NO6P
81
80
609.76
533.67
610
0.54 0.58 69.18/68.94 7.82/7.93 2.19/2.29 4.63/5.08
0.53 0.56 65.33/65.26 8.41/8.31 2.56/2.62 5.41/5.80
CH3
(CH3)2CHCH2 C29H44NO6P
534.5
a
See Experimental Section for the definition of the solvent systems.
Ta ble 3. Ch a r a cter iza tion of th e Typ e 3 Syn th on s
synthons
FAB-MS (M + H)
TLC (Rf)
(4)a (5)a
analytical data (found/calcd)
yield
(%)
δ(ppm) 31
P
(CDCl3)
R1
R2
formula
calcd
found
C
H
N
P
C6H5CH2
C6H5CH2 CH3
H
C29H36NO6P
C30H38NO6P
86
98
525.57
539.59
525.4
540.1
50.7-50.8
0.15 0.41 65.89/66.27 6.67/6.90 2.55/2.66 5.49/5.89
0.16 0.44 66.38/66.77 6.91/7.09 2.34/2.59 5.39/5.74
48.7, 50.1,
50.3, 51.0
47.9, 49.6,
50.2, 51.5
48.1, 50.0,
51.1, 51.8
C6H5CH2 (CH3)2CHCH2 C33H44NO6P
98
91
581.66
505.56
582.1
505.7
0.20 0.53 67.96/68.14 7.29/7.69 2.31/2.40 4.99/5.33
0.16 0.46 63.87/64.14 7.71/7.97 2.71/2.76 5.51/6.13
CH3
(CH3)2CHCH2 C27H40NO6P
a
See Experimental Section for the definition of the solvent systems.
Sch em e 2a
Sch em e 3a
a
(a) H2/Pd/C.
completely selective and allows for the preparation of
synthon 3 in good yields (Table 3).
a
(a) AgNO3, (b) 1-Ad-Br.
In the third step, in addition to the decarbobenzoxy-
lation reaction, a certain degree of deadamantylation was
observed during the catalytic hydrogenation (compound
7, Scheme 3). These observations are in contrast with
previous work showing that the adamantyl esters of
amino acids are completely stable to catalytic hydrogena-
tion.12 To avoid this problem, the fully protected synthon
2 was subjected to catalytic hydrogenation followed by
coprecipitate in a ratio of 1:2 (compound 6, Scheme 2).
To overcome this problem, a large excess of silver nitrate
was used to obtain a 1:1 molar ratio of the desired silver
and synthon 1 salt (see Experimental Section). Given
these precautions, the adamantylation reaction proceeds
in good yield (Table 2).
In the second step, the saponification of the ethyl ester
2, in spite of the presence of the adamantyl ester, is
(12) Froussios, C. Personal communication.

Solid-Phase Synthesis of Phosphinic Peptides
J . Org. Chem., Vol. 61, No. 19, 1996 6603
Ta ble 4. Ch a r a cter iza tion of th e Typ e 5 Syn th on s
synthons
FAB-MS (M + H)
TLC (Rf)
(4)a (5)a
analytical data (found/calcd)
yield
(%)
δ(ppm) 31
P
(CDCl3)
R1
R2
formula
calcd
found
C
H
N
P
C6H5CH2
C6H5CH2
H
CH3
C36H40NO6P
C37H42NO6P
61
65
613.68
627.74
614
627.7
50.7, 51.0
0.40 0.38 70.19/70.49 6.34/6.57 2.32/2.28 4.65/5.05
0.46 0.40 70.41/70.79 6.39/6.74 2.20/2.23 4.59/4.93
49.0, 49.0,
50.6, 51.3
48.2, 49.6,
50.2, 52.0
48.5, 50.4,
51.2, 52.2
C6H5-CH2 (CH3)2CHCH2 C40H48NO6P
67
62
669.77
593.67
669.6
593.4
0.57 0.49 71.58/71.73 7.13/7.22 2.12/2.09 4.22/4.62
0.54 0.50 68.37/68.78 7.51/7.47 2.21/2.35 4.89/5.22
CH3
(CH3)2CHCH2 C34H44NO6P
a
See Experimental Section for the definition of the solvent systems.
Sch em e 4a
a
(a) H2, 10% Pd/C, (b) NaOH.
saponification (Scheme 4). With this protocol, no cleav-
age of the adamantyl group was observed during the
hydrogenation. However, in this case, depending on the
substituents R1 and R2 in the synthon 2, the saponifica-
tion leads to the formation of two products, the expected
pseudodipeptide 4 and the pseudodiketopiperazine 8
(Scheme 4). For instance, when the R1 and R2 substit-
uents were benzyl and isobutyl groups, respectively, the
pseudodiketopiperazine turned out to be the main prod-
uct of the reaction. Finally, satisfactory results for the
decarbobenzyloxylation of synthon 3 were obtained using
the method described by Anwer and Spatola,13 which
utilizes ammonium formate as the hydrogen donor and
palladium/carbon as the catalyst in methanol (Scheme
1). The N-deprotected synthon 4 was easily converted
to the final Fmoc product, synthon 5, in good yield
(Table 4).
Using the synthons prepared in this study and the
acid-sensitive 2-chlorotrityl resin14 as a solid support, the
different phosphinic pseudopeptides 9-12 were prepared
by classical Fmoc solid-phase peptide synthesis (see
Experimental Section). Satisfactory couplings were
achieved using only 1.5 equiv of these synthons with
HBTU, where HBTU is 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate, as an activat-
ing agent and a 60 min reaction time. In the same way,
coupling of Fmoc amino acids to these synthons was
found to proceed in good yield using standard conditions.
As shown by the HPLC profiles of crude materials (Figure
1), this method yields relatively pure phosphinic peptides,
the main peaks corresponding to the different diastere-
oisomeric forms of these compounds.
F igu r e 1. HPLC traces of Pro-(L,D)-PheΨ(PO2CH2)Gly-Phe
(9), Cbz-Pro-Lys-(L,D)-PheΨ(PO2CH2)-(L,D)-Ala-Pro-Leu-Val
(10), Cbz-Pro-Gln-(L,D)-PheΨ(PO2CH2)-(L,D)-Leu-Trp-Ala (11),
and Cbz-Pro-Gln-(L,D)-AlaΨ(PO2CH2)-(L,D)-Leu-Trp-Ala (12).
The profiles correspond to the crude material obtained after
the cleavage of the protecting groups and the peptide from the
resin (Vydac C18 column, 4.66 × 25 cm).
peptides by solution methods and the synthesis of phos-
phonate peptides by a solid-phase approach have been
described using the methyl ester as a protecting group
of the hydroxyphosphinyl function.4,20-22 However, the
final deprotection of methyl ester of phosphinic or phos-
phonate peptides has usually been achieved under drastic
conditions (e.g., lithium propanethiolate, trimethylsilyl
bromide, concentrated sodium, or lithium hydroxide
solutions, or a mixture of thiophenol, triethylamine and
dioxane).4,20-22
The need to protect the hydroxyphosphinyl function
with a protecting group which can be removed easily
under classical conditions led us to investigate the
adamantyl group for this purpose.23,12 Interestingly, we
observed that phosphinic adamantyl esters can be re-
moved under relatively mild acidic conditions (50% TFA/
DCM (dichloromethane), 30 min). However, these esters
were also found to be quite resistant to acidic conditions,
since they remain stable during acidification which
follows saponification. It should be noted that the benzyl
esters, especially the diphenylmethyl phosphinic esters,
Discu ssion
Although a number of phosphinic peptides have been
synthesized by the solution-phase method without pro-
tection of the hydroxyphosphinyl group, the coupling of
a R-XaaΨ(PO(OH)CH2)Xaa′ unit to an amino peptidyl
moiety is problematic.15-19 The synthesis of phosphinic
(17) Kafarski, P.; Lejczac, B.; Mastalerz, P. In Bietraege zur Wirk-
stofforschung; Oehme, P., Loewe, H., Gores, E., Axt, J ., Eds.; Institut
fu¨r Wirkstofforshung: Berlin, Germany, 1984; Vol. 25.
(18) Bartlett, P. A.; Acher, F. Bull. Soc. Chim. Fr. 1986, 5, 771-
775.
(19) Campagne, J . M.; Coste, J .; Guillou, L.; J ouin, P. Tetrahedron
Lett. 1993, 34, 4181-4184.
(20) Bartlett, P. A.; J ohnson, W. S. Tetrahedron Lett. 1970, 46,
4459-4462.
(13) Anwer, M. K.; Spatola, A. E. Synth. Commun. 1980, 929-932.
(14) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J . Pept.
Protein. Res. 1991, 37, 513-520.
(21) McKenne, C. E.; Higa, M. T.; Cheung, N. H.; McKenne, M. E.
Tetrahedron Lett. 1977, 2, 155-158.
(15) Allen, M. C.; Fuhrer, W.; Tuck, B.; Wade, R.; Wood, J . M. J .
Med. Chem. 1989, 32, 1652-1661.
(22) Campbell, D. A.; Bermak, J . C. J . Am. Chem. Soc. 1994, 116,
6039-6041.
(16) Bartlett, P. A.; Kezer, W. B. J . Am. Chem. Soc. 1984, 106, 4282-
4283.
(23) Okada, Y.; Iguchi, S.; Kawasaki, K. J . Chem. Soc., Chem.
Commun. 1987, 20, 1532-1534.

6604 J . Org. Chem., Vol. 61, No. 19, 1996
Yiotakis et al.
ously described,4,5 with some modifications. A suspension of
the corresponding N-benzyloxycarbonyl phosphinic acid [ Cbz-
HN-(R,S)-CH(R1)PO(OH)H]25 (1 mmol) and hexamethyldisi-
lazane (5 mmol) was heated at ca. 110 °C for 1 h under an
argon atmosphere. After cooling to 90 °C, the appropriate
ethyl alkylacrylate [CH2dC(R2)COOEt]26 (1.3 mmol) was added
dropwise over 30 min, and the reaction mixture was stirred
for an additional 3.5 h at 90 °C. The resulting mixture was
allowed to cool to ca. 70 °C, and absolute ethanol (3 mL) was
added dropwise. After cooling to rt, the volatile products were
removed under vacuo and the residue was dissolved in 10%
NaHCO3. The aqueous phase was rinsed with diethyl ether,
acidified to pH 1.5 with 1N HCl, and the precipitated product
was extracted with ethyl acetate. The organic layer was rinsed
with water, dried over Na2SO4 and evaporated to dryness to
give the synthons 1 in very good yields.
(B) Gen er a l P r oced u r e for th e Syn th esis of Syn th on s
2, Cbz-HN-(R,S)-CH(R1)P O(OAd)-CH2-(R,S)-CH(R2)COOEt.
Meth od 1. The type 1 synthon (1 mmol) was dissolved in 95%
ethanol (25 mL). This solution was added dropwise over 10
min to a stirred 0.5 M aqueous solution of silver nitrate (10
mL) (precautions should be taken to avoid exposure of silver
synthons to the light). After 10 min, water (15 mL) was added
to this reaction mixture and the ethanol was removed under
vacuum. The remaining aqueous phase, containing the pre-
cipitated silver salt of the synthon, was cooled in an ice-water
bath for 1 h. The product was filtered, washed with cold water,
are much more sensitive under similar acidic conditions.
This study also demonstrates the stability of the ada-
mantyl esters under basic conditions. These esters
remain intact even after 24 h under strong alkaline
conditions. In comparison, the saponification of ethyl or
methyl esters is usually completed in 2 h under the same
conditions. Despite the bulkiness of the adamantyl
group, its presence does not create particular problems
of coupling during the solid-phase synthesis of phosphinic
peptides, even with the synthon containing the benzyl
and isobutyl side chains. This solid-phase strategy, the
first reported to date for phosphinic peptides, now enables
the development of libraries of these pseudopeptides
without restrictions on peptide size. The efficiency of this
approach was recently demonstrated by the discovery of
the most potent and selective inhibitor of the mammalian
zinc-endopeptidase 24-15 by screening a library of phos-
phinic peptides.24
Exp er im en ta l Section
Gen er a l. All of the compounds, for which analytical and
spectroscopic data are quoted, were homogeneous by TLC. TLC
analyses were performed using silica gel plates (E. Merck silica
gel 60 F-254), and components were visualized by the following
methods: ultraviolet light absorbance, iodine vapor, and
charring after spraying with a solution of (NH4) HSO4 and
ninhydrin spray. The solvent systems used for TLC develop-
ments were (1) 1-butanol-acetic acid-water (4:1:1), (2) chlo-
roform-methanol-acetic acid (7:2:1), (3) chloroform-methanol-
acetic acid (7:0.5:0.5), (4) chloroform-methanol (9.5:0.5), (5)
hexane-ethyl acetate-acetic acid (3:3:0.2), and (6) chloroform-
methanol (9:1). In most solvent systems close, but different,
Rf values have been observed for the various stereoisomers of
these compounds, due to the presence of asymmetric centers
in these compounds. Thus, the Rf values quoted in Tables 1,
2, 3 and 4 correspond to an average value. The presence of
these asymmetric centers complicates the use of 1H-NMR
spectroscopy to characterize these compounds, especially when
the hydroxyphosphinyl function is protected by the adamantyl
group. For these reasons, the compounds were characterized
only by 31P NMR spectroscopy, elemental analyses, and mass
spectrometry. (31P, 1H)-decoupled spectra of the compounds
dissolved in CDCl3 were recorded on a WM 250 Bruker
spectrometer (31P, 101 MHz). 31P chemical shifts are reported
on δ scale (in ppm) downfield from 85% H3PO4. Before
microanalyses, samples were dried under high vacuum at 35
°C for 12 h in a dry pistol. These analyses were obtained from
Service de Microanalyses, ICSN CNRS, 91198 Gif/Yvette,
France. Fast atom bombardment mass spectrometry (FAB-
MS) was performed on a Nermay R3010 3-fold quadrupole
instrument by Dr. Virelizier (DCC/DPE/SPEA/SAIS/CEA, CE-
Saclay, 91191 Gif/Yvette, France).
Gen er a l P r oced u r es. (R,S)-(1-((Benzyloxycarbonyl)ami-
no)ethyl)phosphinic acid and (R,S)-(1-((benzyloxycarbonyl)-
amino)-2-phenylethyl)phosphinic acid were synthesized as
described by Baylis et al.25 2-Isobutylpropenoic acid ethyl ester
was prepared according to the method reported by Stetter and
Kuhma.26 The propenoic and 2-methylpropenoic acid ethyl
esters were purchased from Aldrich.
In order to increase the yield of the Michael addition to
obtain the synthons 1, the method described in the literature4,5
was modified as follows: hexamethyldisilazane was used in
large excess (5 equiv excess, instead of 1 equiv) and the
reaction time of the Michael addition was increased from 25
min to 4 h.
and dried over P2
O5 to give the silver synthon salt in about
90% yield. This silver synthon salt (1 mmol) was added to a
solution of 1-adamantyl bromide (1.2 mmol) in chloroform (10
mL). This reaction mixture was refluxed for 30 min. The
silver bromide, which precipitated, was removed by filtration
or centrifugation, and the filtrate was concentrated to dryness.
This residue was purified by silica gel column chromatography
(eluent: chloroform/2-propanol, 97:3) to give the pure products
in about 80% yields as oils.
Meth od 2. The type 1 synthon (1 mmol) was dissolved in
0.1 M NaOH (10 mL) (solution 1). Silver nitrate (1 mmol) was
dissolved in water (10 mL) (solution 2). Solutions 1 and 2 were
added simultaneously to a reaction flask containing water (5
mL). This mixture was cooled in an ice-water bath, and after
1 h, the precipitated silver salt was filtered, washed with cold
water, and dried in a desiccator over P2O5 to give the silver
synthon salt in about 90% yield. The adamantylation and its
yield were similar to those of the procedure described in
method 1.
Meth od 3. The type 1 synthon (1 mmol) and 1-adamantyl
bromide (1 mmol) were dissolved in chloroform (10 mL), and
the reaction mixture was refluxed. To this refluxing mixture,
silver oxide (2 mmol) was added in five equal portions over 50
min. After the solution was refluxed for an additional 30 min,
the solvents were removed in vacuo and the residue was
treated with diethyl ether. The silver bromide and the excess
silver oxide, as well as the traces of unreacted synthon, were
removed by filtration through a mixture of celite and alumina.
The filtrates were concentrated to dryness to give the pure
products in about 85% yield after column chromatography.
(C) Gen er a l P r oced u r e for th e Syn th esis of Syn th on s
3, Cbz-HN-(R,S)-CH(R1)P O(OAd )CH2-(R,S)-CH(R2)COOH.
The type 2 synthon (1 mmol) was dissolved in ethanol (10 mL),
and 4 N NaOH (1 mL) was added dropwise to the reaction
mixture. After 2 h (for the synthon containing the isobutyl
group, the completed saponification was only achieved after
12 h), the ethanol was removed in vacuo and the residue was
diluted with water (20 mL). This reaction mixture was then
cooled in an ice-water bath and acidified to pH 2 with 0.5 N
HCl. The precipitated product was taken up in diethyl ether
and the organic phase was rinsed with water, dried over Na2-
SO4, and concentrated to dryness to give the pure saponified
solid products (90-98% yields).
(A) Gen er a l P r oced u r e for th e Syn th esis of Syn th on s
1, Cb z-H N-(R,S)-CH (R 1)P O2H CH 2-(R,S)-CH (R 2)COOE t .
These compounds were synthesized using a procedure previ-
(D) Gen er a l P r oced u r e for th e Syn th esis of Syn th on s
5, Fm oc-HN-(R,S)-CH(R1)P O(OAd)CH2-(R,S)-CH(R2)COOH.
To a solution of methanol (5 mL), containing type 3 synthon
(1 mmol) and ammonium formate (4 mmol), 0.25 g of 10% Pd/C
was added. After 12 min at rt, the catalyst was removed by
filtration through celite, and the filtrate was evaporated to
(24) J iracek, J .; Yiotakis, A.; Vincent, B.; Lecoq, A.; Nicolaou, A.;
Checler, F.; Dive, V. J . Biol. Chem. 1995, 270, 21701-21706.
(25) Baylis, E. K.; Campbell, C. D.; Dingwall, J . G. J . Chem. Soc.
Perkin. Trans. 1 1984, 2845-2853.
(26) Stetter, H.; Khman, H. Synthesis 1979, 29-30.

Solid-Phase Synthesis of Phosphinic Peptides
J . Org. Chem., Vol. 61, No. 19, 1996 6605
dryness. Methylene chloride was added to the residue, and
the solution was evaporated to dryness. This procedure was
repeated twice. The residue was treated with chloroform (50
mL), and excess insoluble unreacted ammonium formate was
removed by filtration. The filtrate was evaporated to dryness,
and the residue, synthon type 4, was dissolved in 10% Na2-
CO3 (3 mL). The reaction mixture was concentrated in vacuo
until half of the volume was removed, and then water (1.5 mL)
and dioxane (2 mL) were added. A solution of Fmoc-Cl (1.2
mmol) in dioxane (2 mL) was added dropwise to this cold
mixture (ice-water bath). After the solution was stirred for 2
h at 4 °C and 4 h at rt, the reaction mixture was diluted with
water (20 mL), cooled in an ice-water bath, and acidified to
pH 2.5 with 2 N HCl. The solid product, which precipitated,
was quickly taken up in diethyl ether, and the organic layer
was rinsed with water, dried over Na2SO4, and evaporated to
dryness to give the crude product type 5 synthon, which was
purified on a silica gel column (eluent: chloroform/methanol,
9.5:0.5) to give the synthons 5 (65% overall yields).
dried over Na2SO4 and concentrated to dryness to give the
unprotected pseudodiketopiperazine (0.035 g, 60% yield): Rf-
(2), 0.47; 31P NMR (CDCl3) 49.38, 43.74; FAB-MS (M + H) calcd
for C15H22NO3P 296.33, found 296.3. Anal. Calcd: C, 61; H,
7.50; N, 4.74; P, 10.49. Found: C, 60.76; H, 5.58; N, 4.81; P,
9.97.
Solid -P h a se Syn th esis of P h osp h in ic P ep tid es. These
syntheses were realized using the 2-chlorotrityl resin. The
first amino acids were attached to the 2-chlorotrityl resin
according to the procedure of Barlos et al.14 The degree of
substitution of each resin sample was determined according
to the procedure of Meienhofer et al.,27 using ꢀ 7040 M-1 cm-1
at 300 nm.28 The Fmoc groups were removed with 30% piperi-
dine in N-methylpyrrolidone. Coupling of the next residue was
achieved using the 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate/diisopropylethylamine in
situ strategy. Typically, 3 equiv of Fmoc amino acid, 3 equiv
of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate, and 4 equiv of diisopropylethylamine as solu-
tions in N-methylpyrrolidone were added to the resin, and the
reaction was allowed to proceed for 30 min. The coupling of
the phosphinic dipeptide synthon 5 was performed using the
coupling conditions described above, except that only 1.5 equiv
of the synthon was used and that the reaction was allowed to
proceed for 60 min. Under these conditions for coupling, a
negative Kaiser test was observed for the different phosphinic
building blocks. Fully protected peptides were cleaved from
the 2-chlorotrityl chloride resin with a mixture of glacial acetic
acid, trifluoroethanol, and dichloromethane (2:2:6) over 2 h.
Solutions of protected peptides were dried in vacuo. Protective
groups were removed by the action of trifluoroacetic acid
containing 5% H20, 5% thioanisole, 5% phenol, 2.5% ethanedithi-
ol, 2.5% triisopropylsilane, and 20% dichloromethane. Solu-
tions of deprotected peptides were concentrated in vacuo, and
peptides were treated with 2.5 equiv of NaHCO3 in water.
Aqueous solutions were repeatedly extracted with cold diethyl
ether and lyophilized. Purification and separation of the
diastereoisomeric forms of these phosphinic peptides were
performed on Gilson gradient system equipped with a variable-
wavelength detector. Compounds were detected at 254 and
230 nm. The following conditions were used: Vydac C18 (4.66
× 25 cm) column; mobile phase A ) 0.1% TFA and 10%
acetonitrile in water, B ) 0.1% TFA and 10% water in
acetonitrile; flow rate of 8 mL/min. The gradients used for
the peptide 9 (Pro-(L,D)-PheΨ(PO2CH2)Gly-Phe) was t ) 0 min
(0% B), t ) 40 min (50% B), t ) 60 min (100% B), and those
used for the peptides 10 (Cbz-Pro-Lys-(L,D)-PheΨ(PO2CH2)Ala-
Pro-Leu-Val), 11 (Cbz-Pro-Gln-(L,D)-PheΨ(PO2CH2)-(L,D)-
Leu-Trp-Ala), and 12 (Cbz-Pro-Gln-(L,D)-AlaΨ(PO2CH2)-(L,D)-
Leu-Trp-Ala) were t ) 0 min (0% B), t ) 10 min (25% B), t )
45 min (42% B), t ) 60 min (100% B), respectively. Mass
spectroscopy analysis of the main HPLC peaks observed for
the different phosphinic peptides shows that these correspond
to the different diastereoisomeric forms of these peptides:
FAB-MS (M + H) calcd for peptide 9 501.46, found 501.5; calcd
for peptide 10 930.23, found 930; calcd for peptide 11 929.3,
found 929; calcd for peptide 12 853.14, found 853.
Syn t h esis of [Cb z-(R,S)-P h e (P O2--CH2)-(R,S)-Leu -
OEt]2Ag Com p lex. The synthon Cbz-(R,S)-PheΨ(PO(OH)-
CH2)-(R,S)-LeuOEt (0.951 g, 2 mmol) was dissolved in ethanol
(25 mL) and water (5 mL), and 1 M AgNO3 (2.5 mL, 2.5 mmol)
was added dropwise with stirring. After 15 min, most of the
ethanol was removed in vacuo and water (30 mL) was added
to the reaction mixture. After 1 h of cooling in an ice-water
bath, the solid was filtered, washed with cold water, and dried
to give 0.95 g of the title complex (90% yield): FAB-MS (M +
H) calcd for C50H67O12N2P2Ag 1057.87, found 1057.4. Anal.
Calcd: C, 56.82; H, 6.29; N, 2.64; P, 5.86; Ag, 10.20. Found:
C, 56.41; H, 5.94; N, 2.52; P, 4.92; Ag, 9.69.
Deter m in a tion of th e Silver Syn th on Com p lex Mola r
Ra tio. The above silver synthon complex [Cbz-(R,S)-PheΨ-
(PO2--CH2)-(R,S)-LeuOEt]2Ag (1.055 g, 1 mmol) was sus-
pended in chloroform (50 mL), and 4 N HCl in dioxane was
added (4 mL). The resulting solid precipitate was filtered,
washed with chloroform, and dried to give 0.131 g of AgCl (0.91
mmol, 91% yield). The filtrate was evaporated in vacuo, and
the residue was dried over P2O5 and NaOH to give 0.913 g
(1.92 mmol, 96% yield) of the Cbz-(R,S)-PheΨ(PO(OH)CH2)-
(R,S)-LeuOEt compound.
F or m a tion a n d Isola tion of th e P seu d od ik etop ip er a -
zin e. (A) Cyclo[(R,S)-P h e (P O(OAd)CH2)-(R,S)-Leu ]. The
synthon Cbz-(R,S)-PheΨ(PO(OAd)CH2)-(R,S)-LeuOEt (0.61 g,
1 mmol) was dissolved in 95% ethanol (10 mL) and was
hydrogenated for 5 h in the presence of 10% Pd/C catalyst (0.25
g). The catalyst was removed by filtration, and the filtrate
was concentrated to dryness to give the synthon H2N-(R,S)-
PheΨ(PO(OAd)CH2)-(R,S)-LeuOEt in 96% yield (Rf(4), 0.43).
This product was dissolved in 95% ethanol (5 mL), and 4 N
NaOH (1 mL) was added to the reaction mixture. After 5 h of
stirring, the solvents were removed in vacuo and the residue
was treated with diethyl ether. The organic layer was rinsed
successively with 0.1 N HCl and 10% NaHCO3, dried over Na2-
SO4, and concentrated to dryness to give the pseudodiketopip-
erazine (40% yield): Rf(5), 0.54; Rf(4), 0.48; 31P NMR (CDCl3):
45.59, 44.08, 41.45, 37.31; FAB-MS (M + H) calcd for C25H37
-
NO3P 430.56, found 430.5. Anal. Calcd: C, 69.90; H, 8.44;
N, 3.25; P, 7.21. Found: C, 69.64; H, 8.31; N, 3.42; P, 6.87.
(B) Cyclo[(R,S)-P h e (P O(OH )CH 2)-(R,S)-Leu ]. The
above compound (0.086 g, 0.2 mmol) was dissolved in a mixture
of CH2Cl2-TFA 1:1 (5 mL), and after 1 h of stirring, the
solvents were removed in vacuo. The residue was dissolved
in 10% Na2CO3 (10 mL). The aqueous phase was rinsed with
diethyl ether and acidified to pH 1.5 with 4 N HCl, and the
product was taken up in ethyl acetate. The organic layer was
J O9603439
(27) Meienhofer, J .; Waki, M.; Heimer, E. P.; Lambros, T. J .;
Makofske, R. C.; Chang, C. Int. J . Pept. Prot. Res. 1979, 13, 35-42.
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J ewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe,
C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen,
F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367-
9371.