Efficient synthesis of protected l-phosphonophenylalanine (Ppa) derivatives suitable for solid phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2007.04.027

l-Phosphonophenylalanine (Ppa) derivatives were synthesized from l-4-iodophenylalanine and suitably protected phosphites using Michaelis-Arbuzov reaction conditions in the presence of Pd(0) and triethylamine at 70 ± 2 °C.

Full text of DOI:10.1016/j.tetlet.2007.04.027

Tetrahedron Letters 48 (2007) 4051–4054
Efficient synthesis of protected L-phosphonophenylalanine
(Ppa) derivatives suitable for solid phase peptide synthesis
Satendra S. Chauhan,* Arti Varshney, Bhavana Verma and Michael W. Pennington
BACHEM Bioscience Inc., 3700 Horizon Drive, King of Prussia, PA 19406, USA
Received 2 March 2007; revised 4 April 2007; accepted 4 April 2007
Available online 11 April 2007
Abstract—L-Phosphonophenylalanine (Ppa) derivatives were synthesized from L-4-iodophenylalanine and suitably protected phos-
phites using Michaelis–Arbuzov reaction conditions in the presence of Pd(0) and triethylamine at 70 2 ꢀC.
ꢁ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
OH
OH
X
P
O
Recently, we have designed a phosphotyrosine (pTyr)
containing analog of a potassium channel blocking pep-
tide derived from the sea anemone Stichodactyla helian-
thus (ShK), called ShK(L5) that selectively inhibits
Kv1.3 channels. ShK(L5) has been shown to suppress
the proliferation of human and rat effector memory T
cells (TEM) and inhibits interleukin-2 production at
picomolar concentrations. Thus, ShK(L5) is a very
interesting compound, which may be useful in treating
autoimmune disorders.1 The amino acid sequence of
ShK(L5) is H-pTyr-Aeea-Arg-Ser-Cys-Ile-Asp-Thr-Ile-
Pro-Lys-Ser-Arg-Cys-Thr-Ala-Phe-Gln-Cys-Lys-His-Ser-
Met-Lys-Tyr-Arg-Leu-Ser-Phe-Cys-Arg-Lys-Thr-Cys-
Gly-Thr-Cys-OH with disulfide bonds between Cys-5
and Cys-37, Cys-14 and Cys-30, and Cys-19 and Cys-
34. Aeea is a spacer amino acid in the sequence and is
introduced using commercially available Fmoc-Aeea-
OH (Fmoc-amino-ethoxy-ethoxy-acetic acid; Bachem
Product No. B-3635).
H2N
CO2H
1; X = O; Phosphotyrosine (pTyr)
2; X = CH2; Phosphonomethyl phenylalanine (Pmp)
3; X = CHOH; Hydroxyphosphonomethyl phenylalanine (HPmp)
4; X = CHF; Fluorophosphonomethyl phenylalanine (FPmp)
5; X = CF2; Difluorophosphonomethyl phenylalanine (F2Pmp)
HO OH
P
O
H2N
CO2H
6; Phosphonophenylalanine (Ppa)
Figure 1. Structures of phosphotyrosine mimetics.
In one of our first analogs for this study, pTyr of
ShK(L5) was substituted with Pmp (2), which is com-
mercially available in the protected form. However,
Pmp-substitution for pTyr led to significant reduction
in selectivity,1 possibly due to higher ionization constant
In our efforts to improve the stability of ShK(L5) and to
minimize the potential cleavage of the selectivity-pro-
moting phosphate moiety under physiological condi-
tions since the phosphoryl ester bond may be labile to
hydrolysis and may be cleaved by phosphatases
in vivo,2 we investigated several nonhydrolyzable
mimetics of pTyr. Figure 1 shows the structures of pTyr
mimicking moieties.3
of the phosphonate (pKa ¼ 7:1) relative to the phos-
2
phate (pKa ¼ 5:7) in ShK(L5).3 Thus, an analog incor-
2
porating 4-phosphonophenylalanine (6; Ppa) as a
nonhydrolyzable pTyr mimic was designed because the
ionization constant (pKa ) of Ppa is comparable to pTyr
2
and no reduction in selectivity was expected due to sim-
ilar interactions at the binding site.4 In this report, we
describe an efficient synthesis of suitably protected
*
Corresponding author. Tel.: +1 610 239 0300; fax: +1 610 239
0800; e-mail: schauhan@usbachem.com
0040-4039/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.04.027

4052
S. S. Chauhan et al. / Tetrahedron Letters 48 (2007) 4051–4054
4-phosphonophenylalanine (6; Fig. 1) derivatives start-
ing from L-4-iodophenylalanine.
of pyrophosphate using BOP/HOBt (1:1)/NMM in
0.4 M LiCl/NMP,9 it was expected that Fmoc-Ppa-OH
would also afford the target peptide without pyrophos-
phate formation. However, high levels of pyrophosphate
formation were observed in all of the coupling protocols
employed (up to 70%) and were exceedingly difficult to
remove by RP-HPLC. Therefore, synthesis of the Ppa
analog of ShK(L5) had to be optimized in order to min-
imize the formation of this undesired pyrophosphate
impurity. It was determined that by protecting the phos-
phonate group, the formation of the pyrophosphate
could be prevented.
2. Results and discussion
Scheme 1 shows two plausible strategies for the synthe-
sis of phosphonophenylalanine (6; Ppa). Strategy 1 uti-
lizes L-tyrosine as the starting material and is reported
in the literature.5 However, Ppa (6) can also be syn-
thesized from L-4-iodophenylalanine by employing
Michaelis–Arbuzov reaction to form the aryl carbon–
phosphorus bond.6
The protecting groups for the phosphonate group had to
be chosen carefully. The simplest method was to keep
the phosphonate group protected as ethyl or methyl
ester. However, cleavage of the phosphonate ethyl or
methyl ester requires strong acidic conditions, such as
refluxing in 6 N aqueous HCl,10 45% HBr/AcOH,5
TMSBr,11 or HF.12 These cleavage conditions are not
suitable for synthesizing ShK(L5). The protecting
groups needed to be compatible with Fmoc chemistry
and should be cleavable with TFA. Thus, the choices
were limited to benzyl or t-butyl protecting groups.
R2O OR3
OTf
I
P
O
R1-HN
Scheme 1.
CO2R2
R1-HN
CO2H
R1-HN
CO2R2
As shown in Scheme 2, Boc-4-iodophenylalanine was
esterified with EtOH using BOP/DIEA as an activator
in good yield. The Boc-Phe(I)-OEt was treated with di-
ethylphosphite in the presence of (tetrakistriphenyl-
phosphine)Pd(0) and triethylamine in acetonitrile at
72 2 ꢀC for 20 h to afford Boc-Ppa(Et)2-OEt in 87%
yield.7 All the protecting groups were simultaneously
removed by refluxing Boc-Ppa(Et)2-OEt in 6 N aqueous
HCl at 110 ꢀC for 6 h. Concentration of the reaction
mixture followed by trituration with ether afforded the
product in quantitative yield. Ppa thus obtained was
reacted with Fmoc-OSu in the presence of sodium
carbonate to afford Fmoc-Ppa-OH in excellent yield.8
In order to synthesize Fmoc-Ppa(Bzl)-OH, we
attempted the same strategy as described in Scheme 2,
but starting with Fmoc-Phe(4-I)-OEt. Fmoc-Phe(4-I)-
OEt was prepared in quantitative yield from Fmoc-
Phe(4-I)-OH and EtOH/HCl(g). However, the reaction
of Fmoc-Phe(4-I)-OEt with dibenzylphosphite failed to
yield any Fmoc-Ppa(Bzl)2-OEt even after employing
long reaction times and with excesses of reagents and
catalyst. In the next attempt, we used di-t-butylphos-
phite to react with Fmoc-Phe(4-I)-OEt in the presence
of Pd(0).13 The reaction did proceed, albeit in low yield
(<10%). Efforts to improve the yield by prolonging the
reaction time and higher temperature as well as adding
excesses of reagents and catalyst were not successful. It
appears that Pd(0) reaction is not compatible with di-
benzylphosphite. However, with dialkylphosphite there
was some moderate reaction. As our efforts to produce
the benzyl derivative were not successful, we decided
to prepare Boc-Ppa(t-Bu)2-OH. This was facilitated by
the fact that Ppa is the N-terminal residue for the
ShK(L5) analog and the final Boc group would be
cleaved during standard cleavage of the peptide from
the resin.
We used side-chain unprotected Fmoc-Ppa-OH (10) to
synthesize Ppa analog of ShK(L5) on the solid support
using standard protocols. Since side-chain unprotected
Fmoc-pTyr can be used with only minimal formation
I
I
a
b
Boc-HN
CO2Et
Boc-HN
CO2H
As shown in Scheme 3, Boc-Phe(4-I)-OEt (8) was
reacted with di-t-butylphosphite, Pd(0), and triethylamine
8
7
HO
EtO
OH
OEt
tBuO
tBuO
P
P
O
OtBu
OtBu
O
c, d
P
P
O
O
a
b
8
Fmoc-HN
CO2H
Boc-HN
CO2Et
Boc-HN
CO2Et
Boc-HN
CO2H
10
9
11
12
Scheme 2. Reagents and conditions: (a) BOP/DIPEA, EtOH, 72%; (b)
(Ph3P)4 Pd(0), HPO3Et2, TEA, MeCN, 76%; (c) 6 N aq HCl, reflux,
6 h, 92%; (d) Fmoc-OSu, Na2CO3, dioxane/H2O, 90%.
t
Scheme 3. Reagents and conditions: (a) (Ph3P)4Pd(0), HPO3 Bu2/
TEA, MeCN, 62%; (b) 1 N NaOH, 4 h, 67%.

S. S. Chauhan et al. / Tetrahedron Letters 48 (2007) 4051–4054
4053
in anhydrous acetonitrile at 70 2 ꢀC for a total of 40 h.
The reaction was monitored periodically and one addi-
tional equivalent each of the catalyst, di-t-butylphosph-
ite and triethylamine, were added after 24 h of reaction.
Boc-Ppa(t-Bu)2-OEt (11) was obtained in 62% yield
after silica gel column purification. The ethyl ester was
hydrolyzed with 1 N aqueous sodium hydroxide in
MeOH/water mixture at ambient temperature for 4 h.
No racemization was observed during hydrolysis. Purifi-
cation of the crude product using a silica gel column
chromatography gave pure 12 in 67% yield as a white
solid.14
selectively hydrolyzed the ethyl ester without affecting
the benzyl phosphonate ester. However, contrary to
the reports, there was 25–30% cleavage of the Fmoc
group over the course of reaction and no hydrolysis
was observed with sodium bicarbonate. It should also
be mentioned here that we did not observe any racemi-
zation during the base-mediated hydrolysis of the ethyl
ester. The optimized hydrolysis conditions utilized
2 equiv of 1 N aqueous lithium hydroxide in THF at
0 ꢀC followed by addition of 1 equiv of Fmoc-OSu to
the reaction mixture after hydrolysis was complete and
allowing it to stir at the same temperature overnight.
The basic solution was neutralized with 1 N aqueous
HCl and the product was extracted with dichloro-
methane. After washing the organic layer with brine,
Fmoc-Ppa(Bzl)-OH (14) was precipitated from dichloro-
methane/hexane as white solid in 80% yield.18
In continuation of our efforts to prepare Fmoc-
Ppa(Bzl)-OH and having failed to force the Michaelis–
Arbzov reaction between Fmoc-Phe(4-I)-OEt and
dibenzylphosphite, we decided to carry forward Fmoc-
Ppa-OH (10) to selectively protect phosphonic acid
group as benzyl ester.
It should be mentioned that both Boc-Ppa(t-Bu)2-OH
and Fmoc-Ppa(Bzl)-OH were successfully incorporated
in the sequence of ShK(L5) using DIC/HOBt as the
activator. Apparently, no pyrophosphate was formed
during the solid phase peptide synthesis using the side-
chain protected amino acid derivatives of Ppa.
As shown in Scheme 4, a suspension of Fmoc-Ppa-OH
(10) was stirred in EtOH saturated with HCl(g) over-
night. Fmoc-Ppa-OEt precipitated out from the reaction
mixture as a white solid. Further concentration and pre-
cipitation with isopropyl ether afforded the product in
quantitative yield. Fmoc-Ppa-OEt was then reacted with
benzyl alcohol (3 equiv) using BOP/DIPEA (2 equiv) as
an activator. However, there was no reaction possibly
due to intramolecular quenching of the HOBt-active
ester with the second hydroxyl function of the phosphonic
acid moiety. It should be noted that phosphonate mono-
esters are known to successfully react with alcohols to
afford mixed phosphonate diesters using BOP or PyBOP
as activating agent.15 Side-chain phosphonic acid
reacted with benzyl alcohol (3 equiv) in quantitative
yield (13) when activated with either DCC/DMAP
(2 equiv) or pivaloyl chloride (2 equiv)16 in aceto-
nitrile/pyridine (1:1) solvent mixture. It should also be
noted that only mono benzyl phosphonate ester formed
under these conditions and no diester was detected.
In conclusion, syntheses of Fmoc-Ppa-OH, Boc-Ppa(t-
Bu)2-OH, and Fmoc-Ppa(Bzl)-OH were accomplished
in good yields using Michaelis–Arbuzov reaction condi-
tions. These amino acids can be used in solid phase pep-
tide synthesis using an Fmoc strategy. Fmoc-Ppa-OH
was obtained in excellent yield from Boc-Phe(4-I)-OEt
and diethylphosphite. Boc-Ppa(t-Bu)2-OH was prepared
from Boc-Phe(4-I)-OEt and di-t-butylphosphite in mod-
erate yield. Fmoc-Ppa(Bzl)-OH could not be prepared
directly from Fmoc-Phe(4-I)-OEt and dibenzylphosph-
ite. Nonetheless, it was synthesized from Fmoc-Ppa-
OH by selectively protecting and deprotecting the
carboxylic and phosphonic acid moieties.
It is also important to report that that during solid phase
coupling of Fmoc-Ppa-OH, a significant amount of
pyrophosphate forms. The Fmoc group is also suscepti-
ble to cleavage during hydrolysis with either LiOH or
Na2CO3. The problem can be circumvented by adding
Fmoc-OSu to the reaction mixture once hydrolysis is
complete.
In the final step, selective hydrolysis of the carboxylic
acid ethyl ester was tried. Due to the presence of both
acid and base labile functional groups present in
Fmoc-Ppa(Bzl)-OEt (13), the choice was limited to
milder hydrolysis conditions. Mild bases, such as lithium
hydroxide in THF/dioxane at 0 ꢀC,14 or sodium carbon-
ate/sodium bicarbonate in MeOH/H2O17 are reported
to selectively hydrolyze carboxylic alkyl esters without
affecting the Fmoc group. Both lithium hydroxide and
sodium carbonate under the above reaction conditions
References and notes
1. Beeton, C.; Pennington, M. W.; Wulff, H.; Singh, S.;
Nugent, D.; Crossley, G.; Khaytin, I.; Calabresi, P. A.;
Chen, C.-Y.; Gutman, G. A.; Chandy, K. G. Mol.
Pharmacol. 2005, 67, 1369–1381.
2. Burke, T. R., Jr.; Smyth, M. S.; Nomizu, M.; Otaka, A.;
Roller, P. J. Org. Chem. 1993, 58, 1336–1340.
3. Burke, T. R., Jr.; Yao, Z.-J.; Liu, D.-G.; Voigt, J.; Gao, Y.
Biopolymers (Petide Sci.) 2001, 60, 32–44.
BzlO
BzlO
OH
OH
P
P
O
O
a,b
c
10
Fmoc-HN
CO2H
Fmoc-HN
CO2Et
4. Liu, W.-Q.; Olszowy, C.; Bischoff, L.; Garbay, C. Tetra-
hedron Lett. 2002, 43, 1417–1419.
14
13
Scheme 4. Reagents and conditions: (a) EtOH/HCl(g), 95%; (b) DCC/
DMAP or (CH3)3COCl, MeCN/pyridine (1:1), benzyl alcohol, 95%;
(c) (i) 1 N LiOH/THF, 4 h; (ii) Fmoc-OSu, overnight, 80%.
5. Thurieau, C.; Simonet, S.; Paladino, J.; Prost, J.-F.;
Verbeuren, T.; Fauchere, J.-L. J. Med. Chem. 1994, 37,
625–629.

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S. S. Chauhan et al. / Tetrahedron Letters 48 (2007) 4051–4054
6. Hirao, T.; Masunaga, T.; Ohshiro, Y.; Agawa, T.
9. Garcia-Echeverria, C. Lett. Peptide Sci. 1995, 2, 369–373.
10. Hammerschmidt, F.; Hanbauer, M. J. Org. Chem. 2000,
65, 6121–6131.
Synthesis 1981, 56–57.
7. A typical procedure was as follows: Boc-Phe(4-I)-OEt
(2.4 g, 5.72 mM) and Pd(PPh3)4 (198.2 mg, 0.172 mM)
were dissolved in 100 mL of dry MeCN. An orange
colored solution was formed. To this solution, diet-
hylphosphite (1.2 g, 8.7 mM) and TEA (1.2 g, 11.8 mM)
were added. The reddish orange color faded to yellowish
and became almost colorless. The reaction mixture was
heated at 72 2 ꢀC for 20 h. The reaction mixture
was evaporated and the resultant syrupy residue (ꢀ6 g)
was taken in EtOAc (100 mL) and washed with 10%
aqueous citric acid (25 mL · 3), saturated NaHCO3
(25 mL · 3), H2O (25 mL · 2), and brine (25 mL · 2).
After drying over MgSO4, the organic layer was evapo-
rated to an oil. It was triturated with hexane to give a
white solid (2.6 g), which was recrystallized from MTBE/
hexane to afford 2.1 g pure solid (87%). 1H NMR (CDCl3):
d 7.75–7.70 (m, 2H), 7.70–7.65 (m, 1H), 7.50–7.45 (m, 1H),
4.95–5.1 (d, br, 1H), 4.65–4.55 (m, br, 1H), 4.20–4.10
(m, 4H), 4.10– 4.04 (m, 2H), 3.25–3.15 (dd, 1H), 3.15–3.05
(dd, 1H), 1.40 (s, 9H), 1.30 (t, 6H), 1.25 (t, 3H).
11. Hannour, S.; Roumestant, M.-L.; Viallefont, P.; Riche,
C.; Martinez, J.; El Hallaoui, A.; Ouazzani, F. Tetrahe-
dron Asymmetry 1998, 9, 2329–2332.
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Lett. 2002, 43, 4103–4106.
13. Smyth, M. S.; Burke, T. R., Jr. Tetrahedron Lett. 1994, 35,
551–554.
14. Compound 12. ESI-MS: expected m/e 457 (C22H36-
24
N1O7P), observed m/e 479.8 (M+Na)+, ½aꢁD ꢂ15.89 (c
0.5, DMF). 1H NMR (CDCl3): d 7.76–7.54 (m, 2H), 7.37–
7.24 (m, 2H), 5.32–5.15 (d, 1H), 4.69–4.55 (m, 1H), 3.34–
3.17 (m, 2H), 1.43 (s, 9H), 1.41 (s, 9H), 1.40 (s, 9H).
15. Campagne, J.-M.; Coste, J.; Jouin, P. Tetrahedron Lett.
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17. Kaestle, K. L.; Anwer, M. K.; Audhya, T. K.; Goldstein,
G. Tetrahedron Lett. 1991, 32, 327–330.
18. Compound 14. ESI-MS: expected m/e 557 (C31H28-
24
8. Compound 10. ESI-MS: Expected m/e 467 (C24H22-
N1O7P), observed m/e 579.8 (M+Na)+, ½aꢁD +17.2 (c
24
N1O7P), observed m/e 490 (M+Na)+, ½aꢁD ꢂ32.3 (c 0.25,
0.25, DMF). H NMR (DMSO-d6): d 7.87–7.81 (d, 2H),
1
1
DMF). H NMR (DMSO-d6): d 7.95–7.85 (d, 2H), 7.80–
7.70–7.55 (br, m, 4H), 7.55–7.35 (m, 2H), 7.35– 7.25 (m,
2H), 7.25–7.10 (br, m, 7H), 4.65 (s, 2H), 4.30–4.12
(overlapping, m, 4H), 3.12–3.05 (br, m, 1H), 2.95–2.88
(br, m, 1H).
7.75 (d, 1H), 7.75–7.65 (m, 2H), 7.65– 7.55 (m, 2H), 7.45–
7.35 (m, 2H), 7.35–7.25 (m, 2H), 4.28–4.10 (overlapping,
m, 4H), 3.25–3.15 (m, 1H), 2.95–2.85 (m, 1H).