Synthesis of an enkephalin analogue containing an α,α- disubstituted heterocyclic aminophosphonic acid

Article abstract of DOI:10.1080/10426507.2010.535398

Synthesis of a phosphonopentapeptide enkephalin analogue was achieved from heterocyclic aminophosphonates via the coupling of Boc-Tyr-Gly-Gly-OH with phosphonodipeptide. The latter was formed by coupling Boc-Phe-OH with N-terminal α,α-disubstituted hetero

Full text of DOI:10.1080/10426507.2010.535398

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Phosphorus, Sulfur, and Silicon and the
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Synthesis of an Enkephalin Analogue
Containing an α,α-Disubstituted
Heterocyclic Aminophosphonic Acid
Nicolas Rabasso a & Antoine Fadel a
a Laboratoire de Synthèse Organique et Méthodologie, ICMMO,
Université Paris-Sud, Orsay, France
To cite this article: Nicolas Rabasso & Antoine Fadel (2011): Synthesis of an Enkephalin Analogue
Containing an α,α-Disubstituted Heterocyclic Aminophosphonic Acid, Phosphorus, Sulfur, and Silicon
and the Related Elements, 186:8, 1811-1819
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Phosphorus, Sulfur, and Silicon, 186:1811–1819, 2011
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2010.535398
SYNTHESIS OF AN ENKEPHALIN ANALOGUE
CONTAINING AN α,α-DISUBSTITUTED HETEROCYCLIC
AMINOPHOSPHONIC ACID
Nicolas Rabasso and Antoine Fadel
Laboratoire de Synthe`se Organique et Me´thodologie, ICMMO, Universite´
Paris-Sud, Orsay, France
GRAPHICAL ABSTRACT
Abstract Synthesis of a phosphonopentapeptide enkephalin analogue was achieved from het-
erocyclic aminophosphonates via the coupling of Boc-Tyr-Gly-Gly-OH with phosphonodipep-
tide. The latter was formed by coupling Boc-Phe-OH with N-terminal α,α-disubstituted hete-
rocyclic α-aminophosphonates.
Keywords Aminophosphonate; enkephalin analogue; pentapeptide; phosphonopeptide;
thiopyran
INTRODUCTION
Development of new drugs for treatment of pain represents one of the major areas
of research, since patients are more tolerant to and dependent on the existing medicines.
In this context, enkephalins and their analogues represent an under-investigated target.1
Enkephalin 1 is a naturally occurring pentapeptide isolated from the brain and spinal cord.
Few syntheses of enkephalin analogues have been reported in the literature, including some
derivatives of α-aminoalkylphosphonates.2–4
Previous studies on enkephalin pentapeptide analogues have shown that the acidic
group at the C-terminus is a key element for the δ opioid receptor selectivity.5 Increasing the
acidity at this position should improve the enkephalin-like activity of the peptide. Moreover,
Received 30 September 2010; accepted 15 October 2010.
The authors thank Mr. Ronny Wahlstro¨m and Ms. Alice Six for technical assistance.
Address correspondence to Nicolas Rabasso and Antoine Fadel, Laboratoire de Synthe`se Organique et
Me´thodologie, ICMMO, CNRS, UMR 8182, baˆt. 420, Universite´ Paris-Sud 91405, Orsay, France. E-mail:
antoine.fadel@u-psud.fr; nicolas.rabasso@u-psud.fr
1811

1812
N. RABASSO AND A. FADEL
Ph
O
O
O
H
N
H
H2N
N
N
H
N
H
OH
O
O
1
S
HO
Tyr-Gly-Gly-Phe-Met
enkephalin
Ph
O
H
O
O
P
H
OH
OH
N
H2N
N
N
H
N
H
O
O
S
2
HO
Tyr-Gly-Gly-Phe-cMetP
Figure 1 Met-enkephalin and its constrained phosphonic analogue.
conformational studies of the highly flexible enkephalin pentapeptides and their analogues
could be investigated by replacing the α-amino acids with α,α-disubstituted aminoacid.6
In continuation of our ongoing program on the synthesis of cyclic aminophosphonic
acids,7 we report our efforts for the synthesis of the new pentapeptide 2 bearing an S-
heterocyclic aminophosphonic fragment in the replacement of the methionine fragment in
natural met-enkephalin 1 (Figure 1).
Recently we reported a straightforward synthesis of new heterocyclic α-
aminophosphonic acids 3 by nucleophilic addition of phosphite to ketimines (Scheme 1).8
The same strategy was successfully applied to the synthesis of the (3-amino-piperidin-
3-yl)phosphonic acids 4 on both enantiomerically pure forms.9 On the other hand, the
synthesis of ( )-aminophosphonic acids 5 has required the formation of hydrazone
intermediates to provide hydrazinophosphonates (Scheme 1).10 We also reported a
stereoselective synthesis of new α-amino-β-hydroxy-heterocyclic phosphonic acids 6 with
construction of two consecutive chiral centers.11
O
OH
OH
NH2
O
5
O
O
OH
P
OH
OH
OH
OH
P
P
P
OH
NH2
X
X
X
NH2
NH2
X
OH
4
6
3
X= O, S, NR, CH2
Scheme 1 Heterocyclic aminophosphonic acids.
With this library of new α,α-disubstituted aminophosphonic acids in hand, it is now
possible to explore their ability to be associated with other amino acids to prepare confor-
mationally constrained enkephalin analogues. This synthesis involved a peptide coupling
with an α,α-disubstituted aminophosphonate 9, which, to date, has been reported only once
in acyclic version in the late 1980s.12 Indeed, the coupling is strongly dependent on the
structure of the amino acid. The yields decrease rapidly by increasing steric hindrance on
both aminophosphonate and acylating agent.13

SYNTHESIS OF AN ENKEPHALIN ANALOGUE
1813
The retrosynthetic approach depicted in Scheme 2 includes the coupling of the
phosphono dipeptide 7 and tripeptide 8. The latter is prepared from Boc-Gly-Gly-OMe
11 and protected Boc-L-tyrosine 12, while the phosphonodipeptide is formed by coupling
between Boc-L-Phe-OH 10 and the aminophosphonate 9 (Scheme 2).
Ph
O
O
O
P
H
N
H
N
H
OEt
OEt
N
N
H
Boc
N
H
O
O
S
HO
O
2
Ph
O
O
P
H
H
N
H
N
OEt
OEt
N
H2N
OH
Boc
N
H
O
O
S
8
HO
7
O
P
OEt
OEt
O
H
N
Ph
H2N
O
H
N
Boc
OH
OMe
Boc
OH
Boc
N
H
N
H
O
S
O
RO
12
11
10
9
Boc-Gly-Gly-OMe
Boc-Tyr-OH
Boc-Phe-OH
Scheme 2 Retrosynthesis of phosphonic enkephalin analogue.
RESULTS AND DISCUSSION
In order to enlarge the applications of our targeted pentapeptides, we decided to
prepare both sulfur 9a and oxygen 9b heterocyclic aminophosphonates following our
previously described protocol.8
Next we turned our attention to the key coupling between the α,α-disubstituted
aminophosphonates 9a–b and Boc-L-phenylalanine 10. For this reaction, likely for the
synthesis of peptides, standard coupling conditions were used. Thus, the synthesis of
dipeptides 13a and 13b was achieved by the reaction of the Boc-L-phenylalanine 10 with
the heterocyclic aminophosphonate N-terminus 9a–b in the presence of dicyclohexylcar-
bodiimide (DCC) and hydroxybenzotriazole (HOBt) at room temperature for five days.
Due to the slow reaction, only 45% yield of dipeptide 13a was obtained (64% based on
recovered starting material, BRSM). This result could be explained by the intramolecular
hydrogen bonding between the phosphonate moiety and the free amino group, according
to Gilmore and McBride.14 The phosphonodipeptides 13a and 13b were obtained in good
yields after purification on silica gel (Scheme 3).
On the other hand, the preparation of tripeptide 17a–b was achieved starting from
Boc-Gly 14. Thus, coupling 14 with Gly-OMe·HCl 15 was carried out as described15 using
DCC, Et3N, and a catalytic amount of dimethylaminopyridine (DMAP) in CH2Cl2. The
resulting Boc-Gly-Gly-OMe intermediate, after purification on silica gel, was deprotected
by use of trifluoroacetic acid (TFA) in excess in CH2Cl2 at room temperature. The dipeptide

1814
N. RABASSO AND A. FADEL
H
HN
O
P
Ph
OEt
OEt
Ph
OH
O
P
H
OEt
OEt
Boc
Boc
N
DCC, HOBt
CH2Cl2, rt
N
H
N
H
O
O
X
X
13a (45%, 64% BRSM)
10
9a X= S
9b X=O
13b (73%)
Scheme 3 Preparation of phosphonodipeptides.
TFA salt 16 was obtained in 80% overall yield and used without purification in the next
step.
Next, the synthesis of tripeptide 17a required the coupling between Boc-Gly-Gly-
OMe 16 and Boc-L-tyrosine 12a. Carried out with 3-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC·HCl), HOBt, and N-methylmorpholine (NMM) in
dimethylformamide (DMF) at room temperature, the coupling provided the tripeptide 17a
in an unexpected 19% yield. However, protection of the phenol moiety on tyrosine as tert-
butyldimethylsilyl ether 12b16 gave, after coupling under the same conditions, the desired
tripeptide 17b in 57% yield (Scheme 4).
Gly-OMe.HCl, 15
DCC, DMAP
O
O
H
N
H
OMe
N
Boc
N
H
Boc
OH
Et3N, CH2Cl2, 80%
O
14
16
O
H
N
1) TFA, xs
2) EDC.HCl
Boc
OH
HOBt, NMM
DMF, rt
12a X= TBDMS
12b X= H
XO
O
O
O
O
H
H
H
H
N
N
LiOH
then H+
N
N
N
OH
R
N
OMe
Boc
H
H
O
O
HO
XO
17a X= TBDMS, 57%
17b X= H, 19%
8ab R= H (10%)
8a R= Boc (51%)
Boc2O
K2CO3
quant.
TFA, xs
quant.
13a
7
EDC.HCl
HOBt, NMM
DMF, rt
71%
Ph
O
O
O
H
H
N
H
OEt
OEt
N
N
P
N
H
Boc
N
H
O
O
S
HO
2a
Scheme 4 Preparation of the phosphonopentapeptide enkephalin analogue.

SYNTHESIS OF AN ENKEPHALIN ANALOGUE
1815
Then the synthesis of pentapeptide 2a was achieved by coupling tripeptide 17b with
phosphonodipeptide 13a. First, saponification of the tripeptide 17b with 2N LiOH followed
by acidification with 1N H2SO4 furnished the acid 8a (R = Boc).17 This saponification
sequence is of particular interest, since both the carboxylic acid and the phenol are depro-
tected in one step. However, along with 8a, the fully deprotected tripeptide 8ab (R = H)
was observed in 10% yield, which was subjected to protection with Boc2O to quantitatively
produce tripeptide 8a. Secondly, the phosphonodipeptide 13a was deprotected with TFA to
quantitatively lead to the formation of 7 as TFA salt. This dipeptide 7 was used in the next
step without further purification (Scheme 4).
Then, the coupling reaction between 8a (R = Boc) and dipeptide 7 was carried out
with EDC·HCl, HOBt, and NMM in DMF at room temperature to provide the phospho-
nopentapeptide 2a in 71% yield, after purification on a silica gel column. It is noteworthy
that the presence of the unprotected tyrosine phenol function in 8a did not lower the cou-
pling yield. Indeed, the intramolecular hydrogen bond between the phenol moiety and the
carbonyl group in 8a might prevent the undesired side reaction with the coupling reagents.
EXPERIMENTAL
All reactions were carried out under argon with magnetic stirring. Triethyl phosphite
was distilled under reduced pressure and stored under argon. All other solvents and chemical
compounds were purified based on standard procedures. Reagent-grade solvents were used
without purification for all extractions and work-up procedures. Rf values refer to values
obtained by TLC on 0.25 mm silica gel plates (60-F254). Flash chromatography (FC) was
performed on silica gel 60 (0.035–0.070 mm). Yields refer to chromatographically and
spectroscopically pure compounds, unless otherwise mentioned. IR spectra were recorded
on a FT-IR and are reported in wavenumbers (cm−1) with polystyrene as a standard.
Melting points were determined on a Bu¨chi B-545 capillary melting point apparatus and
1
are uncorrected. H NMR spectra were recorded on a Bruker DRX250 (250 MHz) or
Bruker AC360 (360 MHz) spectrometer. Chemical shifts are given in ppm from the solvent
resonance (CDCl3 at 7.27 and D2O at 4.8 ppm). 13C NMR spectra were recorded on a
Bruker DRX250 (62.9 MHz) or Bruker AC360 (90.56 MHz) spectrometer. Chemical shifts
are reported in ppm from the solvent resonance (CDCl3 at 77.16 ppm). 31P NMR spectra
were recorded on a Bruker DRX250 (101.25 MHz), and chemical shifts are quoted relative
to internal 85% H3PO4 (δ = 0 ppm). High-resolution mass spectra were recorded on a
Bruker MicrOTOFq using the following ionization techniques: chemical ionization (CI),
electron impact (EI), and electrospray (ESI). All new compounds were determined to be
>95% pure by 1H NMR spectroscopy.
Tert -Butyl (S)-1-[4-(Diethoxyphosphoryl)-tetrahydro-2H-thiopyran-4-
ylamino]-1-oxo-3-phenylpropan-2-carbamate (13a)
To a solution of aminophosphonate 9a (1.265 g, 5 mmol) in CH2Cl2 (60 mL), Boc-
N-Phe 10 (1.59 g, 6 mmol), DCC (1.55 g, 7.5 mmol), and HOBt (1.35 g, 10 mmol) were
added. The reaction was slightly exothermic. The mixture was stirred at r.t. for 5 d and
then filtered over a pad of Celite. The precipitate was washed with CH2Cl2 (2 × 50 mL).
The combined filtrate was concentrated under vacuum, the residue was dissolved in 30 mL
of a citric acid solution (10%) and extracted with EtOAc (3 × 100 mL). The organic
layer was washed with NaHCO3 solution, dried over MgSO4, filtered, and concentrated.

1816
N. RABASSO AND A. FADEL
The residue was purified on a silica gel column (eluent EtOAc/CH2Cl2, 50:50→80:20) to
provide 1.125 g (45%) of pure phosphonodipeptide 13a.
[α]D = −10.8 (c = 1, CHCl3); Rf = 0.27 (MeOH/CH2Cl2, 10:90); IR (film): νmax
=
3430 (OH), 3285 (NH), 1701 (NCOO), 1686 (COamide), 1231 (P O), 1024 (P O), 909;
1H NMR (360 MHz, CDCl3) δ = 1.29 (t, J = 7.2 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H), 1.41
(s, 9H), 1.92–2.12 (m, 2H, 1H-3 and 1H-5), 2.12–2.40 (m, 2H, 1H-3 and 1H-5), 2.40–2.76
(m, 3H, 1H-2 and 2H-6), 2.76–2.95 (m, 1H, H-2), 3.05 (dd, J = 7.2, J = 13.7 Hz, 1H
benzyl), 3.11 (dd, J = 7.2, J = 13.7 Hz, 1H benzyl), 3.94–4.22 (m, 4H, CH2OP), 4.36 (ddd,
J = 7.2, 7.2, 7.0 Hz, 1H, CH-Bn), 5.13 (d, J = 7.0 Hz, 1H, NH), 6.17 (br s, 1H, NH),
7.10–7.40 (m, 5H); 13C NMR (62.9 MHz, CDCl3) δ = 16.6 (d, J = 4.8 Hz, CH3), 22.0 (d,
J = 14.0 Hz, C-5), 22.2 (d, J = 14.7 Hz, C-3), 28.4 (s, C(CH3)3), 30.3 (C-6), 31.2 (C-2),
1
2
38.2 (CH2 benzyl), 56.0 (d, JPC = 161.2 Hz, C-4), 56.9 (CH-Bn), 62.9 (d, JPC = 7.5 Hz,
CH2OP), 63.1 (d, 2JPC = 7.3 Hz, CH2OP), 80.5 (C(CH3)3), [6 arom C: 127.1 (CH), 128.8
(2 CH), 129.3 (2 CH), 136.7 (Cq)], 155.5 (OCON), 171.2 (CON); 31P NMR (145.78 MHz,
CDCl3) δ = 23.82; HRMS (ES) m/z [M + Na]+ calcd. for C23H37N2O6PS + Na: 523.2002;
found: 523.2003.
(S)-4-[2-N-tert-Butoxycarbonylamino-3-phenylpropanamido]-
tetrahydro-2H-pyran-4-yl Phosphonate (13b)
Following the procedure applied for 13a: Aminophosphonate 9b (495 mg, 2.08
mmol), Boc-N-Phe 10 (665 mg, 2.5 mmol), DCC (650 mg, 3.15 mmol), and HOBt (570 mg,
4.205 mmol) in CH2Cl2 (30 mL) after 3 d of stirring at r.t. and flash chromatography on silica
gel (eluent MeOH/CH2Cl2, 2.5:97.5) yielded 740 mg (73%) of pure phosphonodipeptide
13b.
[α]D = −14.1 (c = 1.0, CHCl3); mp 171.7◦C white solid; Rf = 0.60
(MeOH/CH2Cl2, 10:90); IR (film): νmax = 3424, 3290 (NH2), 2980, 1708 (CON),
1
1685 (OCON), 1530, 1247 (P O), 1025 (P O), 968; H NMR (250 MHz, CDCl3)
δ = 1.31 (t, J = 7.0 Hz, 3H, CH3), 1.32 (t, J = 7.0 Hz, 3H, CH3), 1.43 (s, 9H),
1.95–2.10 (m, 3H, 2H-3, 1H-5), 2.0–2.60 (m,1H-5), 3.01 (dd, JAB = 13.7, J =
7.2 Hz, 1H benzyl), 3.14 (dd, JAB = 13.7, J = 7.2 Hz, 1H benzyl), 3.20–3.50
(m, 2H, H-2, H-6), 3.52–3.84 (m, 2H, H-2 H-6), 4.00–4.21 (m, 4H, 2CH2OP),
4.39 (dd, J = 7.2, 7.2 Hz, JNH-H, = 7.0 Hz, N-CH), 5.15 (d, J = 7.0 Hz, 1H,
BocNH), 6.26 (br s, NH), 7.10–7.40 (m, 5H); 13C NMR (62.90 MHz, CDCl3) δ =
1
16.5 (2CH3), 28.3 (C(CH3)3), 29.4.0 (C-3), 30.5 (C-5), 38.0 (CH2 benzyl), 54.3 (d, JPC
=
158.7 Hz, C-4), 56.8 (CH-N), 62.0 (d, 2JPC = 11.0 Hz, CH2OP), 62.2 (d, 2JPC = 11.1 Hz,
2
2
CH2OP), 62.8 (d, JPC = 7.2 Hz, C-2), 63.0 (d, JPC = 7.0 Hz, C-6), 80.4 (C(CH3)3),
[6 arom C: 127.0 (CH), 128.7 (2 CH), 129.3 (2 CH), 136.7 (Cq)], 155.5 (OCON), 171.4
(CON); 31P NMR (101.25 MHz, CDCl3) δ = 23.28; HRMS (ES) m/z [M+Na]+ calcd. for
C23H37N2O7P + Na: 507.2231; found: 507.2240.
Methyl N-(tert-Butoxycarbonyl)glycylglycinate (16)
Prepared by the following reported procedure:15 Rf = 0.59 (acetone/petroleum ether,
50:50); IR (film): νmax = 3330 (NH), 1750 (COOMe), 1700 (CON), 1677 (OCON), 1529,
1
1171; H NMR (360 MHz, CDCl3) δ = 1.46 (s, 9H), 3.76 (s, 3H, H ester), 3.86 (d, J =
7.2 Hz, 2H, CH2-N), 4.07 (d, J = 7.2 Hz, 2H, CH2-N), 5.24 (m,1H, NH Boc), 6.73 (m,
1H, NH amide); 13C NMR (90.56 MHz, CDCl3) δ = 28.3 (C(CH3)3), 41.0 (CH2N), 44.0

SYNTHESIS OF AN ENKEPHALIN ANALOGUE
1817
(CH2N), 52.3 (CH3 ester), 80.1 (C(CH3)3), 156.2 (OCON), 170.2 (CON), 170.3 (COOMe).
All spectral data are identical with those reported.15
(S)-2-[2-N-tert-Butoxycarbonylamino)-3-[4-(tert-butyldimethylsilyloxy)
phenyl]-propanoic Acid (N-Boc-O-(tert-Butyldimethyl)-L-tyrosine) (12b)
Prepared following the reported procedure,16 [α]D = +45.3 (c = 1, CHCl3); Rf =
0.25 (MeOH/CH2Cl2/AcOH, 10:90:1); IR (film): νmax = 3431 (OH), 1714 (NCOO), 1657
(COOH), 1620, 1265, 1170; 1H NMR (250 MHz, CDCl3) δ = 0.19 (s, 6H, CH3-Si), 0.98
(s, 9H), 1.42 (s, 9H Boc), 3.07 (m, 2H, H-3), 4.57 (m,1H, H-2), 4.94 (d, J = 8.0 Hz, 1H,
NH), 6.77 (d, J = 8.5, Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 10.70 (COOH); 13C NMR
(62.90 MHz, CDCl3) δ = −4.3 (CH3-Si), 18.3 (s, Si-C(CH3)3, 25.8 (s, Si-C(CH3)3), 28.4
(s, Si-C(CH3)3 Boc), 37.2 (s, C–3), 54.5 (s, C-2), 80.3 (s, C(CH3)3), [6 arom C: 120.3 (CH),
128.5 (2 CH), 130.5 (2 CH), 155.5 (Cq)], 154.9 (OCON), 177.1 (C-1). The 1H NMR data
are only reported.16
Methyl (S)-[N-(tert-Butoxycarbonyl)-O-tert-butyldimethylsilyl]
tyrosylglycylglycinate (17b)
A solution of protected N-Boctyrosine 12b (330 mg, 1.1 mmol) and Gly-Gly-
OMe·TFA 16 (435 mg, 1.1 mmol) in DMF (5 mL) was stirred at 0◦C for 10 min. Then
EDC·HCl (211 mg, 1.1 mmol), HOBt (150 mg, 1.1 mmol), and NMM (330 µL, 3.0 mmol)
were successively added. The reaction mixture was stirred at 0◦C for 1 h, then at r.t. for
4 d. The mixture was extracted with AcOEt (100 mL). The organic layer was washed
successively with (4 × 2 mL) of citric acid solution (5%) and brine (10 mL), and then
dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified on
a silica gel column (eluent, MeOH/CH2Cl2, 3:97 → 15:85) to provide 230 mg (57%) of
pure tripeptide 17b as a pale yellow oil.
[α]D = +12.5 (c = 0.65, CHCl3); Rf = 0.38 (MeOH/CH2Cl2, 10:90); IR (film): νmax
=
3663, 3330, 2932, 1750 (COO), 1671 (CON), 1510, 1255 (P O), 1020 (P O); 1H NMR
(250 MHz, CDCl3) δ = 0.16 (s, 6H), 0.95 (s, 9H), 1.36 (s, 9H, Boc), 2.80–2.98 (m, 1H
benzyl), 3.00 (dd, JAB = 14.0, J = 6.5 Hz, 1H benzyl), 3.70 (s, 3H, H ester), 3.80–4.05 (m,
4H, 2 CH2-N), 4.29 (ddd, J = 7.0, J = 7.0, J = 6.5 Hz, 1H, CH-N), 5.29 (s, 1H, NH Boc),
6.74 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.16 (m, 2H, NH); 13C NMR (62.9
MHz, CDCl3) δ = −4.4 (CH3-Si), 18.2 (Si-C(CH3)3, 25.7 (Si-C(CH3)3), 28.3 (C(CH3)3),
37.4 (CH2 benzyl), 41.1 (CH2N), 52.4 (CH3 ester), 56.5 (CH-N), 80.5 (C(CH3)3), [6 arom
C: 120.3 (2CH), 129.1 (Cq), 130.3 (2 CH), 156.0 (Cq)], 154.7 (OCON), 169.5 (CON),
170.2 (CON), 172.5 (COOMe); HRMS (ES) m/z [M + Na]+ calcd. for C28H39N4O4SiNa:
546.2633; found: 546.2619.
(S)-2-[2-(tert-Butoxycarbonyl)-3-(4-hydroxyphenyl)propanamido]
acetamidoacetic Acid (S)-N-(tert-Butoxycarbonyl)tyrosylglycylglycine) (8a)
Prepared following reported procedure.17 A solution of tripeptide TBDMS-O-Boc-
N-Tyr-Gly-Gly-OMe 17b (90 g, 0.172 mmol) in 1.4 mL of MeOH/H2O mixture (1:1)
was treated with LiOH (4.5 mg, 0.26 mmol) and stirred at r.t. for 17 h. The mixture was
acidified with 1N H2SO4 to pH 2 and concentrated to dryness. The residue was purified on
a silica gel column (eluent MeOH/EtOAc/AcOH, 5:95:1 → 20:80:1) to give 35 mg (51%)

1818
N. RABASSO AND A. FADEL
of Boc-Tyr-Gly-Gly-OH 8a, 5.2 mg (10%) of fully deprotected tripeptide 8ab, and 10 mg
(12%) of TBDMS-O-Boc-N-Tyr-Gly-Gly-OH.
8a: Rf = 0.17 (MeOH/CH2Cl2/AcOH, 10:90:1); 1H NMR (360 MHz, D2O) δ = 1.42
(s, 9H, Boc), 2.82–2.98 (m, 1H benzyl), 2.98–3.15 (m, 1H benzyl), 3.81 (s, 2H, CH2-N), 3.97
(s, 2H, CH2-N), 4.32 (m, 1H, CH-N), 6.77 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H).
All spectral data are identical with those reported.17
Diethyl (S)-4-[2-Amino-3-phenylpropanamido]tetrahydro-2H-
thiopyran-4-yl-phosphonate—Trifluoroacetic Salt (7)
To a solution of Boc-dipeptide 13a (76 mg, 0.150 mmol) in CH2Cl2 (3 mL),
CF3COOH (345 µL, 4.60 mmol) was added at r.t. and stirred for 22 h. The solvent and the
excess of reagent were removed under vacuum to give 7 (80 mg), which was used in the
next step without purification.
[α]D = +31.5 (c = 1.05, CHCl3); Rf = 0.32 (MeOH/CH2Cl2, 15:85); IR (film): νmax
=
1
3405 (NH2), 3240, 1777, 1673 (CON), 1266 (P O), 1203, 1024 (P O); H NMR (300
MHz, CDCl3) δ = 1.29 (m, 6H), 1.57 (m, 2H, H-3 and H-5), 2.00–3.00 (m, 4H, 2H-6 and
2H-2), 3.00–3.50 (m, 2H, H benzyl), 3.80–4.30 (m, 4H, 2CH2OP), 4.50–5.00 (m, 1H, N-
CH), 7.10–7.50 (m, 5H), 8.22 (br s, 2H, NH2), 10.40 (br s, H acid); 13C NMR (90.56 MHz,
CDCl3) δ = 16.35 (d, 3JPC = 2.6 Hz, CH3), 16.4 (d, 3JPC = 2.9 Hz, CH3), 21.5 (d, 2JPC
=
12.4 Hz, C–3), 21.9 (d, 2JPC = 13.2 Hz, C–5), 29.8 (C-2), 31.3 (C-6), 37.9 (CH2 benzyl) 55.1
(CH-N), 56.5 (d, 1JPC = 162.5 Hz, C–4), 63.4 (d, 2JPC = 7.8 Hz, CH2OP), 63.7 (d, 2JPC
=
7.6 Hz, CH2OP), 116.3 (d, 1JCF = 290.1 Hz, CF3), [6 arom C: 128.0 (CH), 129.3 (2CH),
129.5 (2 CH), 134.3 (Cq)], 161.5 (d, 2JCF = 37.0 Hz, CF3COO), 168.7 (CON). 31P NMR
(101.25 MHz, CDCl3) δ = 23.38 major, 22.97 minor (rotamer). HRMS (ES) m/z [M+Na]+
calcd. for C18H29N2O4PS + Na: 423.1473; found: 423.1469.
Diethyl [(S,S)-N-(tert-Butoxycarbonyl)tyrosylglycylglycylphenylalanyl]-
4-amino-tetrahydro-2H-thiopyran-4-yl-phosphonate (2a)
To a solution of dipeptide 7·TFA (30 mg, 0.058 mmol) in DMF (2.5 mL), tripeptide
8a (20.5 mg, 0.052 mmol) was added, and then EDC·HCl (11 mg, 0.057 mmol), HOBt (8
mg, 0.059 mmol), and NMM (12 µL, 0.110 mmol, 2 eq) were successively added. After
3 d of stirring at r.t., a white precipitate was formed. The mixture was concentrated under
vacuum, and the residue was purified on a silica gel column (eluent, MeOH/CH2Cl2, 0:100
→ 100:0) to provide 32 mg (71%) of phosphonopentapeptide 2a as a viscous oil.
[α]D = −6.0 (c = 0.65, MeOH); Rf = 0.31 (MeOH/CH2Cl2/NH4OH, 10:90:1); IR
(KBr): νmax = 3422, 3330, 1677 (CON), 1294, 1248 (P O), 1153, 1043, 1025 (P O),
1
810; H NMR (250 MHz, DMSO-d6) δ = 1.12 (t, J = 4.3 Hz, 3H), 1.22 (t, J = 4.3 Hz,
3H), 1.30 (s, 9H, Boc), 1.64–1.93 (m, 2H, H-3 and H-5), 2.20–2.43 (m, 2H, H-3 and H-5),
2.43–3.20 (m, 7H, 2H-2, 2H-6 and 3H benzyl), 3.10–3.30 (m, 1H benzyl), 3.67 (d, J = 5.0 Hz,
2H, CH2-N), 3.73 (d, J = 4.8 Hz, 2H, CH2-N), 3.88–4.23 (m, 5H, 4H and CH-N), 4.67
(m, 1H, CH-N), 6.64 (d, J = 8.0, Hz, 2H), 6.93 (d, J = 7.7 Hz, NH Boc), 7.04 (d, J = 8.0,
Hz, 2H), 7.10–7.42 (m, 6H, 5H
and NH), 7.75 (br s, NH), 8.02 (br s, NH), 8.20 (br
arom
s, NH), 9.20 (br s, OH); 13C NMR (62.9 MHz, DMSO-d6) δ = 19.4 (2CH3), 29.3 (C-3),
31.1 (C(CH3)3), 32.8 (C-2), 33.2 (C-6), 39.5 (CH2 benzyl), 40.5/40.5 (CH2 benzyl), 44.8/45.1
(CH2N), 48.0/48.0 (CH2N), 57.1 (d, 1JPC = 161.5 Hz, C-4), 57.5/57.6 (CH-N), 59.0 (CH-
N), 65.3 (d, 2JPC = 7.0 Hz, CH2OP), 65.6 (d, 2JPC = 6.8 Hz, CH2OP), 81.0 (C(CH3)3), [12
arom C: 117.8 (2CH), 129.4 (CH), 130.6 (Cq), 131.1 (2 CH), 132.1 (2CH), 133.0 (2CH),

SYNTHESIS OF AN ENKEPHALIN ANALOGUE
1819
140.6 (Cq), 155.6 (Cq)], 158.7 (OCON), 171.5 (CON), 172.1 (CON), 174.2 (CON), 175.2
(CON); 31P NMR (101.25 MHz, CDCl3) δ = 24.30. HRMS (ES) m/z [M+Na]+ calcd. for
C36H52N5O10PSNa: 800.3076; found: 800.3070.
CONCLUSION
In summary, we have achieved the synthesis of the new pentapeptide 2, an enkephalin
analogue, that contains an α,α-disubstituted heterocyclic aminophosphonic ester. The
coupling reaction at the N-terminal of hindered tetrasubstituted α-aminophosphonate and
the C-terminal of aminocarboxylic acid was achieved in good yield. It was also shown that
the oxygen analogue can be accessible by the same methodology. Biological tests on the
activity of the phosphono enkephalin and intermediates are underway.
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