Application of in situ silylation for improved, convenient preparation of fluorenylmethoxycarbonyl (Fmoc)-protected phosphinate amino acids

Article abstract of DOI:10.1021/jo070266p

(Chemical Equation Presented) A convenient and efficient method has been developed for the preparation of 9-fluorenylmethoxycarbonyl (Fmoc)-protected 1-aminoalkylphosphinic acids. Reproducible procedures for the synthesis and purification of free α-amino H-phosphinates are provided. Protection of free amino phosphinates as the N-Fmoc derivative was achieved by in situ trimethylsilylation of aminoalkylphosphinic acids, which then reacted with Fmoc-Cl to provide corresponding products in excellent yields and in high purity after simple extractive isolation. Mechanistic aspects of the silylation are discussed, and the application of the procedure to another class of amino phosphorus acids is presented.

Full text of DOI:10.1021/jo070266p

Application of in Situ Silylation for Improved,
Convenient Preparation of
Fluorenylmethoxycarbonyl (Fmoc)-Protected
Phosphinate Amino Acids
hands, protocols for the synthesis of amino phosphinates and
their protected derivatives have not led to reproducible results
and often resulted in poor yields.
One difficulty we have invariably encountered is achieving
high purity of the free phosphinate amino acids. A number of
procedures in the literature report the precipitation of the free
amino acids by neutralization of the HCl or HBr salts by addition
†
†
Shunzi Li, John K. Whitehead, and Robert P. Hammer*
5,6
of propylene oxide, a non-ionic acid scavenger. This protocol
generally does not work in our hands, giving either no precipitate
or very impure products. Another difficulty that we have is
synthesizing the carbamate-protected phosphinate amino acids
under Schotten-Baumann conditions in the presence of Na-
Department of Chemistry, Louisiana State UniVersity,
232 Choppin Hall, Baton Rouge, Louisiana 70803
rphammer@lsu.edu
ReceiVed February 7, 2007
7,8
HCO3 or Na2CO3 in mixed aqueous organic solvents.
Particularly, the Fmoc amino acids are difficult to prepare as
both the phosphinate amino acid and Fmoc-OSu are not very
soluble under the previously reported solvent conditions, which
leads to long reaction times and low yields. Also, homogeneous
Fmoc amino acids can only be obtained after chromatography.
Herein, we report reproducible procedures for preparation of
H-phosphinate amino acids in analytically pure form by
modification of literature protocols and careful ion-exchange
chromatographic purification. Also, we have adapted a non-
aqueous amino acid Fmoc-protection strategy, which has been
utilized by our laboratory for preparation of natural, hydropho-
9
A convenient and efficient method has been developed for
the preparation of 9-fluorenylmethoxycarbonyl (Fmoc)-
protected 1-aminoalkylphosphinic acids. Reproducible pro-
cedures for the synthesis and purification of free R-amino
H-phosphinates are provided. Protection of free amino
phosphinates as the N-Fmoc derivative was achieved by in
situ trimethylsilylation of aminoalkylphosphinic acids, which
then reacted with Fmoc-Cl to provide corresponding products
in excellent yields and in high purity after simple extractive
isolation. Mechanistic aspects of the silylation are discussed,
and the application of the procedure to another class of amino
phosphorus acids is presented.
bic, and highly sterically hindered amino carboxylic acids that
involves trimethylsilylation for solubilization and protection of
the H-phosphinate amino acid as a trimethylsilyl ester. The
silylated amino phosphinate is then reacted in situ with Fmoc-
Cl to give the Fmoc-amino phosphinate in pure form after simple
extractive workup.
1
-Aminoalkylphosphinic acids are generally prepared
5
according to a procedure adapted from Baylis et al.,
which consists of the addition of hypophosphorous acid to
N-(diphenylmethyl) imines followed by hydrolysis to cleave the
benzhydryl group. The main drawback of this method is the
need for drastic acidic conditions such as 49% hydrobromic
acid to cleave the N-alkyl group. More recently, Jiao et al.
proposed an easy and fast general one-pot method to prepare
differently functionalized 1-aminoalkylphosphinic acids in high
yield, using an addition of bis(trimethylsilyl) phosphonite to
N-tritylalkanimines. Trityl protection was used for the amino
group and had the advantage of being rapidly removed by dilute
acid, such as 1 N hydrochloric acid in methanol. Herein, the
latter procedure was utilized to synthesize 1-aminoalkylphos-
A general approach to create inhibitors of proteolytic
enzymes, amidases, and esterases, as well as inhibitors of
transferases, ligases, and synthetases, involves replacing the
trigonal planar, scissile amide or ester bond of the substrate
with a tetrahedral analogue that mimics the intermediate formed
during enzyme-catalyzed nucleophilic attack on the carbonyl.
One of the most popular and effective classes of such “transition
1
0
1
state” mimetics are phosphorus acid derivatives in which the
(
4) Bartlett, P. A.; Kezer, W. B. J. Am. Chem. Soc. 1984, 106, 4282-
amide or ester bond is replaced by either a phosphonate ester
4283. Buchardt, J.; Ferreras, M.; Krog-Jensen, C.; Delaisse, J. M.; Foged,
N. T.; Meldal, M. Chem.-Eur. J. 1999, 5, 2877-2884. Gall, A. L.; Ruff,
M.; Kannan, R.; Cuniasse, P.; Yiotakis, A.; Dive, V.; Rio, M. C.; Basset,
P.; Moras, D. J. Mol. Biol. 2001, 307, 577-586.
2
3
(
-PO2-O-), a phosphonamide (-PO2-NH-), or a phosphinate
4
linkage (-PO2-CH2-). The synthesis of each type of these
phosphonopeptide mimetics depends on the ready availability
of a protected form of a reduced phosphorus amino acid,
typically the Boc or Fmoc amino H-phosphinic acid. In our
(5) Baylis, E. K.; Campbell, C. D.; Dingwall, J. G. J. Chem. Soc., Perkin
Trans. 1 1984, 2845-2853.
(
(
6) Grobelny, D. Synthesis 1987, 942-943.
7) Dumy, P.; Escale, R.; Girard, J. P.; Parello, J.; Vidal, J. P. Synthesis
1
992, 1226-1228.
†
Contributed equally to this work.
(8) Georgiadis, D.; Matziari, M.; Yiotakis, A. Tetrahedron 2001, 57,
(
1) Collinsova, M.; Jiracek, J. Curr. Med. Chem. 2000, 7, 629-647.
3471-3478.
(2) Bartlett, P. A.; Hanson, J. E.; Giannousis, P. P. J. Org. Chem. 1990,
(9) Fu, Y. W.; Hammarstrom, L. G. J.; Miller, T. J.; Fronczek, F. R.;
McLaughlin, M. L.; Hammer, R. P. J. Org. Chem. 2001, 66, 7118-7124.
Fu, Y. W.; Etienne, M. A.; Hammer, R. P. J. Org. Chem. 2003, 68, 9854-
9857.
5
8
3
5, 6268-6274. Kaplan, A. P.; Bartlett, P. A. Biochemistry 1991, 30, 8165-
170. Bartlett, P. A.; Giangiordano, M. A. J. Org. Chem. 1996, 61, 3433-
438.
(
3) Jacobsen, N. E.; Bartlett, P. A. J. Am. Chem. Soc. 1981, 103, 654-
(10) Jiao, X. Y.; Verbruggen, C.; Borloo, M.; Bollaert, W.; Degroot,
A.; Dommisse, R.; Haemers, A. Synthesis 1994, 23-24.
6
57. Bartlett, P. A.; Marlowe, C. K. Biochemistry 1983, 22, 4618-4624.
10.1021/jo070266p CCC: $37.00 © 2007 American Chemical Society
3
116
J. Org. Chem. 2007, 72, 3116-3118
Published on Web 03/22/2007

SCHEME 1. Preparation of 1-Aminoalkylphosphinic Acids
SCHEME 2. Preparation of Fmoc-Protected Amino
Phosphinic Acids
the formation of dipeptides and tripeptides, which are difficult
to remove by crystallization, and yielded products with purity
superior to that achievable by traditional methods. This reaction
also occurs in organic solvents such as methylene chloride or
acetonitrile as the amino acids become soluble in organic
solvents once they are silylated. Our lab has successfully
employed this procedure to protect the amino group of very
sterically hindered R,R-dialkylated amino carboxylic acids. 9
Herein, we present the first preparation of Fmoc-protected
phinic acids 3a-c with different R substitution groups that
correspond to phosphorus analogues of natural amino acids Gly,
Ala, and Val, respectively (Scheme 1). Jiao and co-workers
reported that during the workup 3a-c were precipitated from
an ethanolic solution with excess of propylene oxide. The
precipitated products were isolated by suction, washed with
ethanol, and dried in a vacuum to obtain the desired pure
compound. In contrast with their work, we generally do not
observe a precipitate after treatment with propylene oxide. In
general, we have found the only reliable and reproducible
method for purifying amino acids is by ion exchange chroma-
tography. In addition, we found that using equimolar
N-tritylalkanimine 1 and bis(trimethylsilyl) phosphonite 2 as
described by Jiao and co-workers produces a significant (20-
1
-aminoalkylphosphinic acids using an in situ silylation pro-
cedure (Scheme 2). In this method, 3a-c were refluxed in an
aprotic solvent with TMS-Cl, and then diisopropylethylamine
was added to give a clear solution. NMR-scale silylation
3
1
reactions with 3a (R ) H) were analyzed by P NMR, which
revealed a mixture of major peaks at 11.8 and 150.4 ppm in a
3
3
:1 ratio, along with a number of smaller peaks at 49.0, 45.6,
2.5, and 28.3 ppm. We assign the major peak at 11 ppm to
the O-silyl H-phosphinate ester A and the smaller peak at 150
5
0%) amount of undesired dialkylated phosphinic acids 4. To
1
3 1
ppm to the O,O-bistrimethylsilyl phosphonite B. H NMR
analysis still shows the distinctive doublet of the P-H bond
overcome this problem, a 2-fold excess of phosphonite 2 over
imine 1 was used in our procedures, which then produced pure
(6.85 ppm, JPH ) 525 Hz), which is presumably O-silyl
amino acids 3 with only minor contamination with disubstituted
phosphinic acid 4 (<5% as estimated by 31P NMR of the crude
H-phosphinate ester A. Also, trimethylsilyl peaks at 0.25 ppm
indicate some N-silylation, suggesting that some of the inter-
mediates A and B may have been N-silylated (Scheme 2, x )
product). The diamino phosphinate 4 side-product could be
eliminated almost entirely by use of larger (5-fold) excesses of
bis(trimethylsilyl) phosphonite (2), but workup of these reactions
was impractical because of the large amount of hypophos-
phorous acid and its disproportionation products generated (data
not shown). Crude amino acids were purified by cation exchange
chromatography by elution with ammonium hydroxide to obtain
1
, y ) 1). The mixture of silylated intermediates A and B was
treated in situ with a slightly less than 1 equiv (based on starting
1
4
amino acid) of Fmoc-Cl. After consumption of the Fmoc-Cl,
the reaction mixture was subjected to a simple aqueous workup
where the phosphinates are extracted into aqueous NaHCO3,
washed with Et2O, and then precipitated from the aqueous phase
by addition of HCl to pH ∼2, to afford the corresponding
products 5a-c as white powders in high yields (85-88%),
which were of high purity as judged by NMR and elemental
analysis. While we are unsure of what all of the components of
the silylation reaction are, the high yields and purity of resultant
Fmoc-protected amino phosphinates 5 suggest that the majority
of the silylated products are converted to the desired material.
3
4
a-c in high purities as the small amount of diaminophospinate
present was more strongly retained on the cation exchange
resin.
The synthesis of Fmoc-protected 1-aminoalkylphosphinic
7
acids has been previously reported by Dumy et al. and
8
Georgiadis et al., who carried out the reaction using Schotten-
Baumann-type methods as described (e.g., water-dioxane
mixtures, pH 9-10). Our difficulties with these procedures led
us to think about conditions that would not require the use of
aqueous organic mixtures. For the synthesis of carbamate-
protected amino carboxylic acids, Bolin et al. developed an
efficient procedure involving in situ intermediate N,O-bis-
(
13) We assign the peak at 11 ppm as A in analogy to literature
31
compounds Me3SiO-P(O)(H)CH2NR2, P NMR δ ∼17 ppm (Prishchenko,
A. A.; Livantsov, M. V.; Lorents, K. L.; Grigor’ev, E. V.; Luzikov, Yu, N.
Zh. Obsh. Khim. 1994, 64, 1577-1578) and the peak at 150 ppm as B in
trimethylsilyl) amino acids1,1 that subsequently reacted with
acylating agents such as Fmoc-Cl. This strategy minimized
analogy to (Me SiO) PCH NR , 31P NMR δ ∼152 ppm (Prishchenko, A.
(
3
2
2
2
12
A.; Livantsov, M. V.; Pisarnitskii, D. A.; Shagi-Mukhametova, N. M.;
Petrosyan, V. S. Zh. Obsh. Khim. 1990, 60, 699-701). Smaller peaks in
the range of 28-50 ppm are consistent with the phosphinate anhydride
species that may have formed under the silylation conditions. For examples
of mixed phosphinate anhydride species, see: Plack, V.; Schmutzler, R. Z.
Anorg. Allg. Chem. 1999, 625, 497-502.
(
11) Birkofer, L.; Ritter, A. Angew. Chem., Int. Ed. Engl. 1965, 4, 417-
29. Kricheldorf, H. R. Liebigs Ann. Chem. 1972, 763, 17-38.
12) Bolin, D. R.; Sytwu, I. I.; Humiec, F.; Meienhofer, J. Int. J. Pept.
Protein Res. 1989, 33, 353-359.
4
(
(14) The purity of the Fmoc-Cl should be rigorously checked.
J. Org. Chem, Vol. 72, No. 8, 2007 3117

To test the potential generality of this method for other types
of amino phosphorus acids, we utilized the phosphate ester of
ethanolamine as a substrate under identical conditions as for
the phosphinate amino acids (eq 1). While it is not clear what
that are present in the matrix. This resin was loaded as a slurry
into a large chromatography column (3 cm diameter). This column
is suitable for purification of up to 50 mmol of crude amino
phosphinate, but we use it routinely for the 15 mmol preparations
as described here.
The neutral solution containing the crude amino phosphinate was
then loaded onto the column packed with activated Dowex 50WX8-
4
00. Eluent fractions were monitored by 31P NMR. The column
was then washed with double distilled water (300 mL), absolute
ethanol (300 mL), and then again with water (300 mL). This wash
31
removes unreacted hypophosphorus acid ( P ∼5 ppm; elutes in
the structure of the silylated intermediate is in this case, an N,O-
bis(trimethylsilyl) or an O,O-bis(trimethylsilyl) phosphate de-
rivative, as before the reaction became homogeneous after
addition of the DIEA and Fmoc reaction proceeded readily to
give a high yield of the Fmoc amino phosphate 6. Presumably
this silylation strategy would work for amino-protection of other
amino acids including amino sulfonates, amino sulfates, and
amino phosphonates. It is a practical advance for the synthesis
of protected H-phosphinate amino acids both in terms of yields,
ease of workup, and purity of final products. Also, since
silylation can be carried out under very mild (nonacidic)
conditions (e.g., bistrimethylsilylacetamide; see ref 12), this
method may be applicable to the synthesis of protected
trifunctional phosphinate amino acids such as side-chain-
protected glutamic acid or lysine phosphinate analogues.
These detailed procedures for the synthesis of phosphinate
amino acids and the use of the in situ silylation procedure for
carbamate protection should provide ready access to Fmoc-
protected amino phosphinates that are valuable in a variety of
biological applications such as the synthesis of protease inhibi-
the aqueous phase) and any residual organics not removed by the
extraction (ethanol fraction). Next, 2 N NH
4
OH was added to the
column, and 100 mL fractions were collected, which eluted first
31
the desired phosphinate amino acid ( P ∼15-22 ppm) and
31
subsequently the double addition product ( P ∼30 ppm). The
aqueous eluate containing 3a was evaporated and dried over P
to provide a white powder. Yield: 43%. Mp: 274-275 °C (lit.
2
O
5
10
5
1
2
mp 179-185 °C, 254-256 °C ). H NMR (250 MHz, D O): δ
31
7
.1 (d, JP-H ) 540.0 Hz, 1H), 3.0 (d, J ) 9.2 Hz, 2H). P NMR
(
101 MHz, D O): δ 15.4. ESMS m/z: 96.0 (M + H, calcd 96.0),
2
118.1 (M + Na, calcd, 118.0). Anal. Calcd for CH NO P: C, 12.64;
6
2
H, 6.36; N, 14.74. Found: C, 13.03; H, 6.14; N, 14.50.
1-(N-(9-Fluorenylmethoxycarbonyl)amino)-methylphosphin-
ic Acid (5a). Finely ground amino phosphinate 3a (47.5 mg, 5
mmol) was placed in a 100-mL round-bottom flask fitted with a
heating mantle and condenser. The solid was suspended in
2 2
anhydrous CH Cl (20 mL) and stirred vigorously. TMS-Cl (1.27
mL, 10 mmol) was added in one portion and then refluxed for 2 h.
The mixture was cooled in an ice bath, and DIEA (1.59 mL, 9.1
mmol) and Fmoc-Cl (1.20 g, 4.6 mmol) were added sequentially.
The solution was stirred with cooling for 20 min and at room
temperature for 6 h. The mixture was concentrated and then
1
5
tors.
distributed between Et
The phases were separated, and the aqueous layer was extracted
with Et
O (5 × 30 mL). The aqueous layers were acidified to pH
with 6 N HCl. After standing for 24 h at 0 °C, the solid precipitate
2 3
O (30 mL) and 2.5% NaHCO (35 mL).
2
Experimental Section
-Aminomethylphosphinic Acid (3a). N-Tritylmethanimine 1a
2
1
was filtered, washed with cold water (3 × 20 mL) and Et O (3 ×
2
10
was prepared as described previously. Ammonium phosphinate
2.6 g, 31.4 mmol) and hexamethyldisilazane (6.6 mL, 31.4 mmol)
20 mL), and dried over P O to provide 5a as a white powder.
2
5
Yield: 87%. Mp: 178-180 °C; 1H NMR (250 MHz, CD3-
(
were heated together at 120 °C under argon for 2 h in a 100-mL
round-bottom flask fitted with a heating mantle and condenser to
prepare bis(trimethylsilyl) phosphonite 2 (31.4 mmol). After cooling
to room temperature, the reaction was cooled in an ice bath, and
SOCD ): δ 7.9 (d, J ) 7.5 Hz), 7.7 (d, J ) 7.5 Hz), 7.4 (t, J ) 7.4
3
Hz), 7.3 (t, J ) 7.4 Hz), 6.9 (d, J
P-H
) 539 Hz, 1H), 4.3 (m, 3H),
3.3 (m, J ) 7.3 Hz, 2H). 13C NMR (63 MHz, CD SOCD ): δ
3
3
156.4, 143.8, 140.8, 127.7, 127.1, 125.2, 120.1, 65.9, 46.6, 40.7
(d, JP-C ) 103.3 Hz). 31P NMR (101 MHz, CD SOCD ): δ 25.1.
CH
additional 5 min, a solution of 1a (4.3 g, 15.7 mmol) in CH
25 mL) was then gradually injected at 0 °C, and stirring was
2
Cl
2
(10 mL) was added to the in situ generated 2. After an
3
3
2
Cl
2
ESMS m/z: 318.1 (M + H, calcd 318.1), 340.1 (M + Na, calcd
(
340.1). Anal. Calcd for C H NO P: C, 60.57; H, 5.08; N, 4.41.
16
16
4
continued at room temperature for 12 h. The solvent was removed
under reduced pressure, and the residue was dissolved in 1 N HCl
in MeOH (30 mL) and refluxed for 15 min. The solvent was
Found: C, 60.34; H, 5.13; N, 4.56.
Acknowledgment. We thank Dr. Dale Treleaven, Mr.
Guangyu Li, Dr. Tracy McCarley, and Dr. Michelle Beeson
for expert technical assistance. We are indebted to Prof. Gregg
B. Fields of Florida Atlantic University for inspiring our work
on phosphinate amino acids. This work was supported by a grant
from the National Cancer Institute of the National Institutes of
Health (CA98799) and an NSF IGERT (CHE-9987603) Fel-
lowship (to JWK).
evaporated under reduced pressure, H
the mixture was extracted with Et
layer was retained for subsequent ion-exchange chromatography.
Dowex 50WX8-400 resin (∼250 g) was placed in a Buchner
funnel and activated by washing it with 2 N HCl until acidic (pH
2
O (30 mL) was added, and
O (3 × 30 mL). The aqueous
2
∼
1). The resin was then washed with double distilled water until
neutral (pH ∼7) followed by 2 N NH OH until basic (pH ∼14).
4
The resin was then washed again with double distilled water until
neutral (pH ∼7). The whole process was repeated again. This
washing procedure removes colored impurities and soluble polymer
Supporting Information Available: General procedures and
1H, C, and P NMR spectra for the compounds 3a-c, 5a-c,
and 6. This material is available free of charge via the Internet at
http://pubs.acs.org.
13
31
(15) Yiotakis, A.; Georgiadis, D.; Matziari, M.; Makaritis, A.; Dive, V.
Curr. Org. Chem. 2004, 8, 1135-1158.
JO070266P
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118 J. Org. Chem., Vol. 72, No. 8, 2007