An Improved Synthesis of the Antibiotic Dehydrophos

Article abstract of DOI:10.1002/ejoc.201800689

Within this work an efficient procedure for the synthesis of the vinyl phosphonate tripeptide dehydrophos is described. This procedure constitutes a significant improvement over previously described strategies, since critical transformations in the synthesis of dehydrophos were carried out in only two synthetic steps, with higher yields and under smooth conditions. Thus, the dehydrophosphoalanine residue was generated by means of the Horner–Wadsworth–Emmons reaction of formaldehyde with a peptide bearing an aminomethylbis(phosphonate) moiety, whereas deprotection of the N-terminal position of the thus-obtained dehydrophosphonopeptide and partial hydrolysis of the dimethyl phosphonate residue took place simultaneously. In addition, we confirmed that the chiral integrity of the leucine residue was preserved throughout these transformations.

Full text of DOI:10.1002/ejoc.201800689

A Journal of
Accepted Article
Title: An Improved Synthesis of the Antibiotic Dehydrophos
Authors: María Mercedes Jiménez-Andreu, Francisco J. Sayago, and
Carlos Cativiela
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800689
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800689
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10.1002/ejoc.201800689
European Journal of Organic Chemistry
FULL PAPER
An Improved Synthesis of the Antibiotic Dehydrophos
M. Mercedes Jiménez-Andreu,[a] Francisco J. Sayago*[a] and Carlos Cativiela*[a]
Dedication ((optional))
Abstract: Within this work an efficient procedure for the synthesis of
the vinyl phosphonate tripeptide dehydrophos is described. This
procedure constitutes a significant improvement over previously
described strategies, since critical transformations in the synthesis of
dehydrophos were carried out in only two synthetic steps, with higher
where it is hydrolyzed to a toxic phosphopyruvate derivative after
cleavage of peptide bonds.[5] This mechanism differs from the
most common modes of action of -aminophosphonate
derivatives, which usually act as amino acid mimetics, analogues
of the transition state of the peptide bond cleavage or irreversible
inhibitors of serine proteases.[6] Furthermore, it is also different
from the mode of action of dehydropeptides, where the ,-
dehydroamino acid residues influence the bioactivity by means of
both their conformational properties and chemical reactivity.[7]
As a result of the unique features of the phosphonopeptide
dehydrophos, many efforts have been devoted to elucidate both
its biosynthetic pathway and gene cluster.[8–14]
Despite the great interest of dehydrophos, the synthetic
procedures leading to this antibiotic are very scarce in the
literature. Thus, Van der Donk and coworkers[4] first synthesized
dehydrophos for a reliable reassignment of its structure. The
critical steps in the synthesis of dehydrophos were the
introduction of the dehydroaminophosphonate moiety, which was
accomplished in low yields through the condensation of a suitable
amido peptide with dimethyl acetylphosphonate, and the
deprotection of the thus-obtained dehydrophosphonopeptide to
obtain dehydrophos, which was carried out under harsh basic
conditions and requires HPLC purification of the final compound
(Figure 1).
yields
and
under
smooth
conditions.
Thus,
the
dehydrophosphoalanine residue was generated by means of the
Horner-Wadsworth-Emmons reaction of formaldehyde with a peptide
bearing
an
aminomethylbisphosphonate
moiety,
whereas
deprotection of the N-terminal position of the thus-obtained
dehydrophosphonopeptide and partial hydrolysis of the dimethyl
phosphonate residue took place simultaneously. In addition, we
confirmed that the chiral integrity of the leucine residue was preserved
throughout these transformations.
Introduction
The peptide dehydrophos is a broad-spectrum antibiotic.[1–3] This
naturally
occurring
phosphonopeptide
possesses
a
dehydrophosphoalanine residue at the C-terminal position,[4]
which is responsible for its biological activity. Thus, dehydrophos
acts as a trojan horse and is taken by the target microorganism,
O
O
1. Et3SiH, TEA
PdCl2
O
O
P
O
O
O
O
H
H
OMe
MeO
CbzHN
CbzHN
NH2
CbzHN
N
P OMe
OMe
H2N
N
P OMe
OH
N
H
N
H
N
H
11%
2. NaOH(aq)
60%
O
O
O
Dehydrophos
3 steps
38%
O
H
N
P OMe
OMe
N
H
O
TBDMSO
Figure 1. Reported procedures for the synthesis of dehydrophos.
Alternatively, the dehydroaminophosphonate moiety was
generated by elimination of a phosphoserine residue.[15] However,
although the protected dehydrophos derivative was obtained in a
higher yield, the formation of the double bond requires a 3-step
procedure. Furthermore, the phosphoserine derivative must be
prepared from the commercially available N-
(benzyloxycarbonyl)serine by previously described protocols.[16–
18] In both cases, the two critical transformations in the synthesis
of dehydrophos occurred in low overall yields and/or required
many synthetic steps. Furthermore, the final unprotected
compound could be isolated only after HPLC purification.
[a]
Departamento de Química Orgánica, Instituto de Síntesis Química y
Catálisis Homogénea (ISQCH), CSIC-Universidad de Zaragoza,
Zaragoza 50009, Spain
Within this work we have developed a synthetic procedure which
permit
to
carry
out
the
generation
of
the
E-mail: jsayago@unizar.es, cativiela@unizar.es
dehydroaminophosphonate moiety[19,20] and the deprotection
leading to dehydrophos in only two steps (Figure 2). On one hand,
we have explored the Horner-Wadsworth-Emmons reaction as a
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/ejoc.201800689
European Journal of Organic Chemistry
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suitable strategy for generating the dehydroaminophosphonate
Firstly, we addressed the Horner-Wadsworth-Emmons reaction of
the corresponding bisphosphonate derivative with formaldehyde.
The reaction conditions described so far in the literature for the
synthesis of -amino vinylphosphonates through the Horner-
Wadsworth-Emmons reaction[19–22] involve the use of strong
bases, both ionic[21] and non-ionic.[22] However, while searching
for milder conditions, we found that the use of a 37%
formaldehyde aqueous solution in the presence of cesium
carbonate yielded the corresponding dehydrophosphoalanine
derivatives 4 and 5 in good yields (Scheme 2).
residue. On the other hand,
a high-yielding deprotection
methodology has been developed. Moreover, these
transformations involve the use of smooth bases and conditions,
which ensure the stereochemical integrity of the amino acid
residue and allow an easy purification of the final compound.
OMe
OMe
O P
H-W-E
OMe
OMe
OMe
OMe
Xaa
Xaa
Dehydrophos
N
H
P
O
N
H
P
O
OMe
OMe
Figure 2. Critical steps of the synthetic procedure described in this work.
O
P
HCHO(aq.), Cs2CO3
OMe
OMe
OMe
OMe
CbzHN
P
O
CbzHN
BocHN
P
O
THF/iPrOH 4:1
rt, 1h
Results and Discussion
2
63%
4
OMe
According to that exposed above, we first addressed the
synthesis of an appropriate aminomethylbisphosphonate
derivative and the preparation of a peptide containing it (Scheme
1). Thus, the aminomethylbisphosphonic acid 1 was prepared
from formamide using the conditions described by Schöllkopf et
al with slight modifications.[21] Benzyloxycarbonylation of
compound 1 followed by esterification of the phosphonic acid
groups using trimethyl orthoformate as alkylating agent, provided
the suitably protected aminophosphonate 2 in gram scale without
isolation of intermediates. Finally, hydrogenolysis of compound 2
followed by peptide coupling with Boc-Leu-OH yielded the
peptidyl-bisphosphonate 3.
OMe
O O P
O
HCHO(aq.), Cs2CO3
OMe
OMe
OMe
BocHN
N
H
P
O
N
P
THF/iPrOH 4:1
rt, 1h
OMe
H
O
3
5
79%
Scheme
2.
Horner-Wadsworth-Emmons
reaction
of
aminomethylbisphosphonates 2 and 3.
This procedure has a great advantage over those described
before, since the use of a weak base avoid the racemization in
compounds possessing an amino acid moiety.
OH
OH
It should be noted that the introduction of a methylene group
under such smooth conditions is also very rare in the case of the
carboxylic acid counterparts. Indeed, the synthetic methodologies
described in the literature for the preparation of peptides
containing a dehydroalanine moiety comprise the elimination
reaction of serine,[23,24] cysteine[25] or selenocysteine[26] residues.
First attempts to deprotect the dimethyl phosphonate group in
derivative 4 was carried out using LiOH, which is preferred to
NaOH in compounds bearing a chiral amino acid residue.[27]
However, after stirring at room temperature overnight, the 31P
NMR spectrum of the crude reaction mixture showed many side
products in addition to remaining starting material.
P
O
O
a, b
OH
OH
+ H3PO3 + PCl3
H
NH2
H2N
P
O
53%
1
c, d, e
70%
OMe
OMe
OMe
O P
OMe
O O P
f, g
OMe
OMe
BocHN
N
H
P
O
CbzHN
P
74%
OMe
OMe
O
Alternatively, the use of a non-ionic nucleophile base, Dabco®,[28]
was tested. Thus, a solution of compound 4 in an acetone/toluene
1:1 mixture was refluxed in the presence of Dabco® for 5 hours.
2
3
Scheme 1. Reagent and conditions: (a) 70 ºC, 8 h., then H2O, room temp., 12
1
Both the H and 31P NMR spectra of the crude reaction mixture
h.; (b) 6
M HCl, reflux, 72 h.; (c) Cbz-OSu, Et3N, H2O/CH3CN 4:1, room temp.,
12 h.; (d) Dowex® 50WX8; (e) CH(OCH3)3, reflux, 72 h.; (f) H2, Pd/C, MeOH,
room temp., 1 h.; (g) Boc-Leu-OH, NMM, EDC, HOBt, DMF, 0 ºC, 18 h.
showed the total conversion of compound 4 into the 1-methyl-1,4-
diazabicyclo[2.2.2]octan-1-ium salt of the corresponding
phosphonic acid monoester, without significant presence of side
products (Scheme 3). It should be noted that these reaction
conditions compare favorably with the partial hydrolysis of
dimethyl -aminophosphonate derivatives using tert-butylamine
as the base,[29,30] which usually requires long reaction times.
The bisphosphonate derivatives 2 and 3 were used as suitable
models for exploring the introduction of a methylene group by
means of the Horner-Wadsworth-Emmons reaction, and the
cleavage of one methyl ester group to obtain the phosphonic acid
monoester.
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10.1002/ejoc.201800689
European Journal of Organic Chemistry
FULL PAPER
O
OMe
OH
a
b
OMe
OMe
OH
Cbz
4
+
CbzHN
P
O
N
P
O
P
O
B
O
6
O
O
O
a, b
OMe
OMe
OH
OMe
OH
BocHN
BocHN
5
+
+
N
H
P
O
N
P
O
P
OH
O
7
8
Scheme 3. Reagent and conditions: (a) Dabco®, acetone/toluene 1:1, reflux, 5 h.; (b) Dowex® 50WX8
Despite this promising result, treatment of the above-mentioned
salt with acidic resin Dowex® 50WX8 resulted in a mixture of Z/E
isomers of the iminophosphonic acid 6 instead of the desired -
amino vinylphosphonic acid monoester. Thus, the 1H NMR
spectrum of 6 showed two doublets for three protons at 1.50 and
1.57 ppm respectively. Both signals exhibit a 3JP,C of about 12 Hz,
which is typical in the methyl groups of compounds possessing a
OMe
OMe
O O P
a, b
64%
OMe
FmocHN
2
N
H
P
O
OMe
9
c
62%
iminoethylphosphonate structure.[31–35] However, compound 6
O
O
decomposed upon standing, and the 1H NMR spectrum also
showed the presence of the methyl acetylphosphonate[36] formed
from the hydrolysis of the imino group.
d, e
OMe
OMe
FmocHN
H2N
N
H
P
N
P
89%
OMe
OH
H
O
O
10
11
Interestingly, when compound 5 was subjected to the procedure
described above for 4, the main compound was the desired -
amino vinylphosphonic acid monoester 7, although both the H
and the 31P NMR spectra showed signals compatible with the
Scheme 4. Reagents and conditions: (a) H2, Pd/C, MeOH, room temp., 1 h.; (b)
Fmoc-Leu-OH, NMM, EDC, HOBt, DMF, 0 ºC, 18 h.; (c) 37% HCHO(aq),
Cs2CO3, THF/iPrOH 4:1, room temp., 1 h.; (d) Dabco®, acetone/toluene 1:1,
reflux, 5 h.; (e) Dowex® 50WX8.
1
presence of the iminophosphonate
8
and the methyl
acetylphosphonate. However, deprotection of the amine group in
compound 7 resulted in decomposition.
These results indicated that the -amino vinylphosphonic acid
monoesters were sensitive to acidic conditions, including their
own phosphonic acid group. Furthermore, we reasoned that the
presence of an amine group in the molecule would neutralize the
phosphonic acid group and thus would stabilize the final
compound. Accordingly, both deprotection of the amino group
and partial hydrolysis of the dimethyl phosphonate group should
take place at the same time, and procedures for the partial
hydrolysis of the phosphonate group based on acidic
conditions[37–41] or iodide salts[42–45] were discarded.
Interestingly, when compound 10 was treated with Dabco® partial
hydrolysis of the dimethyl phosphonate group and deprotection of
the amine occurred simultaneously. Thus, both the 31P and the 1H
NMR spectra of the reaction crude showed the total conversion of
10 into the 1-methyl-1,4-diazabicyclo[2.2.2]octan-1-ium salt of the
corresponding
deprotected
dehydrophosphonopeptide
monoester, in addition to the formation of 9-methylenefluorene
from the deprotection of the 9-fluorenylmethoxycarbonyl group.
To our delight, treatment of the crude reaction product with acidic
resin
Dowex®
50WX8
yielded
the
desired
free
With
this
aim
in
mind,
the
Fmoc
protected
dehydrophosphonopeptide 11 as the only product. This result
confirmed the above-mentioned hypothesis regarding the
stabilization of -amino vinylphosphonic acid monoesters.
The synthetic procedures developed both for the introduction of a
methylene group through the Horner-Wadsworth-Emmons
reaction and for the partial hydrolysis of the dimethyl phosphonate
group were then applied to the synthesis of dehydrophos
(Scheme 5).
dehydrophosphonopeptide 10 was prepared. Thus, the
hydrogenolysis of bisphosphonate 2 followed by the peptide
coupling
bisphosphonate 9, which was converted into 10 using the
procedure described above for the Horner-Wadsworth-Emmons
reaction (Scheme 4).
with
Fmoc-Leu-OH
afforded
the
peptidyl-
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10.1002/ejoc.201800689
European Journal of Organic Chemistry
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OMe
OMe
aqueous solution in the presence of cesium carbonate yielded the
dehydrophosphonopeptide 13, which afforded the antibiotic
dehydrophos in a 93% yield by the selective cleavage of a methyl
group of the dimethyl phosphonate moiety and simultaneous
deprotection of the amine group with Dabco®. It should be
mentioned that in this case the reaction crude was treated with
the weakly acidic resin Amberlite® CG50 instead of the strongly
acidic one Dowex® 50WX8, since the latter leaded to
decomposition of the desired compound.
It is worth noting that the synthetic procedure developed in this
work represents a significant improvement of the two key
transformations for the synthesis of dehydrophos. Thus, the yield
of both the generation of a dehydrophosphoalanine residue and
the deprotection of the amine and the dimethyl phosphonate
groups has increased notably (overall yield: 53%), whereas only
two synthetic steps were needed. Furthermore, the peptide
dehydrophos thus obtained was easily purified using a weakly
acidic resin instead of HPLC.
O O
P
H
a, b
3
OMe
OMe
N
FmocHN
88%
N
P
O
H
O
12
c
57%
O
H
d, e
OMe
OMe
N
Dehydrophos
FmocHN
N
H
P
O
93%
O
13
Scheme 5. (a) TFA, CH2Cl2, room temp., overnight; (b) Fmoc-Gly-OH, NMM,
EDC, HOBt, DMF, 0 ºC, 1 h., then 4 ºC, 14 h.; (c) 37% HCHO(aq), Cs2CO3,
THF/iPrOH 4:1, room temp., 1 h.; (d) Dabco®, acetone/toluene 1:1, reflux, 2 h.;
(e) Amberlite® CG50.
Finally, we confirmed that the chiral integrity of the leucine residue
was not affected throughout the synthetic route. Thus, peptides
14 and 17 bearing an (S)- and an (R)--methoxy--
(trifluoromethyl)phenylacetic acid residue respectively were
synthesized for this purpose (Scheme 6).
Accordingly, we synthesized an intermediate peptidyl-
bisphosphonate bearing a Fmoc protecting group at the N-
terminal position. Thus, compound 12 was prepared starting from
3 by the peptide coupling of a Fmoc-Gly-OH residue. Treatment
of the bisphosphonate derivative 12 with a 37% formaldehyde
OMe
OMe
OMe
O
O O P
OMe
O
O
OMe
O
O
Ph
Ph
Ph
H
N
H
N
H
N
a, b
c
e
OMe
OMe
OMe
OMe
OMe
3
F3C
N
H
P
O
F3C
N
H
P
O
F3C
N
H
P
O
62%
85%
O
B
H
H
H
14
15
16
OMe
OMe
Ph
O
O O P
Ph
O
O
Ph
O
O
MeO
F3C
MeO
F3C
MeO
F3C
H
N
H
N
H
N
a, d
c
e
OMe
OMe
OMe
OMe
OMe
3
N
P
O
N
H
P
O
N
H
P
O
73%
74%
O
B
H
H
H
H
17
18
19
Scheme 6. Reagents and conditions: (a) TFA, CH2Cl2, room temp., overnight; (b) (R)-MTPACl, CH2Cl2, DIPEA, room temp., 21 h.; (c) 37% HCHO(aq), Cs2CO3,
THF/iPrOH 4:1, room temp., 3 h.; (d) (S)-MTPACl, CH2Cl2, DIPEA, room temp., 21 h.; (e) Dabco®, acetone/toluene 1:1, reflux, 5 h.
Compounds 14 and 17 were submitted to the synthetic procedure
described above for the synthesis of dehydrophos. Thus, they
were firstly treated with a 37% formaldehyde aqueous solution in
the presence of cesium carbonate to afford the
vinylphosphonates 15 y 18, which were converted into the 1-
shielded for compounds bearing an (S)--methoxy--
(trifluoromethyl)phenylacetyl moiety. These data are in
agreement with the model proposed for Mosher derivatives[46] and
with that observed for leucine derivatives.[47] Furthermore, signals
corresponding to the methoxy group of the (S)--methoxy--
(trifluoromethyl)phenylacetyl amide (compounds 14, 15 and 16)
were less shielded than those observed for the (R)-isomer
(compounds 17, 18 and 19).
methyl-1,4-diazabicyclo[2.2.2]octan-1-ium
salts
of
the
corresponding phosphonic acid monoesters 16 and 19 by
reaction with Dabco®. It should be noted that 16 and 19 were not
treated with an acidic ion exchange resin to avoid decomposition,
and therefore NMR spectra of the reaction crude products were
recorded.
The analysis of 1H NMR spectra of compounds described in
scheme 6 showed that the stereochemistry of the -methoxy--
(trifluoromethyl)phenylacetyl amide did not substantially affect the
chemical shift of the -proton of the leucine residue. However,
signals corresponding to the leucine side chain were more
Considering that epimerization of the leucine residue in (S)- or
(R)--methoxy--(trifluoromethyl)phenylacetyl amide derivatives
would result in the enantiomer of the opposite derivative, it should
1
be easily detected in the H NMR spectra of the crude reaction
products. To our delight, given that signals corresponding to
epimerization products were not observed, we concluded that our
improved protocol for the synthesis of dehydrophos proceeded
without racemization.
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10.1002/ejoc.201800689
European Journal of Organic Chemistry
FULL PAPER
purified by column chromatography on silica gel (dichloromethane/diethyl
ether/methanol 55:40:5) to afford 2 as a yellow oil (1.35 g, 3.54 mmol, 70%
yield). 1H NMR (400 MHz, CDCl3, 23 °C) δ = 7.37–7.29 (m, 5H, Ph), 5.41
(d, J = 10.4 Hz, 1H, NH), 5.15 (s, 2H, CH2-Ph), 4.66 (td, J = 22.1, 10.4 Hz,
1H, CH-P2), 3.85–3.76 (m, 12H, CH3 OMe) ppm. 13C NMR (100 MHz, CDCl3,
25 °C) δ = 155.7 (t, J = 5.0 Hz, CO), 136.0 (Phipso), 128.6, 128.4, 128.1
(Ph), 67.8 (CH2-Ph), 54.2–54.0 (m, CH3 OMe), 45.3 (t, J = 148.5 Hz, CH-P2)
ppm. 31P NMR (162 MHz, CDCl3, 23 °C) δ = 18.85 ppm. IR (neat)  1708,
1270, 1240, 1149, 1053, 1024 cm–1. HRMS (ESI) C13H21NNaO8P2
[M+Na]+: 404.0635, found 404.0628.
Conclusions
We have developed an efficient procedure for the synthesis of
dehydrophos. The effectiveness of this procedure relies on the
Horner-Wadsworth-Emmons
dehydroaminophosphonate residue at the C-terminal position,
and in the simultaneous cleavage of a methyl ester of the
phosphonate moiety and the protecting group of the N-terminal
position amino acid. Furthermore, we have confirmed that the
chiral integrity of the leucine residue was not affected throughout
these synthetic transformations.
reaction
to
generate
a
Tetramethyl
[N-(tert-butoxycarbonyl)-L-
leucylamidomethyl]bisphosphonate (3): To a solution of 2 (2.50 g, 6.56
mmol) in methanol (75 mL) 10% wt. palladium on carbon (250 mg) was
added. The mixture was stirred under a hydrogen atmosphere at room
temperature for one hour. Then, the reaction mixture was filtered (Celite)
and evaporated to afford an oil which was used in the next step. A solution
Experimental Section
General: All reagents were used as received from commercial suppliers
without further purification. Anhydrous solvents were dried using a Solvent
Purification System (SPS). N,N-Dimethylformamide was purchased
(anhydrous grade) from Aldrich. Thin-layer chromatography (TLC) was
performed on Macherey–Nagel Polygram® SIL G/UV254 precoated silica
gel polyester plates. The products were visualized by exposure to UV light
or submersion in ninhydrin, phosphomolybdic acid or permanganate.
Column chromatography was performed using 60 Å (0.04–0.063 mm)
silica gel from Macherey–Nagel. Melting points were determined on a
Gallenkamp apparatus. Optical rotations were measured in a JASCO P-
1020 polarimeter. IR spectra were registered on a Nicolet Avatar 360 FTIR
spectrophotometer; max is given for the main absorption bands. 1H, 13C
and 31P NMR spectra were recorded on a Bruker AV-400 instrument at
room temperature using the residual solvent signal, the solvent signal, or
the chemical shift of the lock solvent as the internal standard; chemical
shifts (δ) are expressed in parts per million and coupling constants (J) in
Hertz. High-resolution mass spectra were recorded on a Bruker Microtof-
Q spectrometer.
of previous oil and Boc-L-Leucine (1.79 g, 7.18 mmol) in dry N,N-
dimethylformamide (30 mL) was cooled to 0 °C. N-Methylmorpholine (0.93
mL, 8.46 mmol) and 1-hydroxybenzotriazole hydrate (12% H2O) (1.20 g,
7.82 mmol) were added and the mixture was stirred for 15 minutes at 0 °C.
Then N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (1.50
g, 7.82 mmol) was added and the reaction mixture was stirred for 18 hours
at 0 °C. Solvent was removed under reduced pressure, the residue was
dissolved in ethyl acetate (30 mL) and washed with an aqueous 5%
solution of sodium bicarbonate. The aqueous phase was extracted with
ethyl acetate (3 x 20 mL) until no final product was detected by TLC in the
aqueous phase. The combined organic layers were dried over anhydrous
magnesium sulphate, filtered and solvent removed under reduced
pressure. The resulting oil was purified by column chromatography on
silica gel (dichloromethane/2-propanol 9:1) to afford 3 as a colorless oil
(2.22 g, 4.82 mmol, yield 74%). []23D = –26.7 (c = 0.36, CHCl3). 1H NMR
(400 MHz, CDCl3, 25 °C) δ = 7.18 (br s, 1H, NH), 5.14–4.97 (m, 2H, CH-
P2+NH), 4.23–4.12 (m, 1H, CHα Leu), 3.84–3.75 (m, 12H, CH3 OMe), 1.74–
1.41 (m, 3H, CH2 Leu+CHLeu) overlapped with 1.41 (s, 9H, CH3 Boc), 0.95–
0.88 (m, 6H, CH3 Leu) ppm. 13C NMR (100 MHz, CDCl3, 25 °C) δ = 172.4
(CO), 155.5 (CO), 80.3 (CBoc), 54.3–54.0 (m, CH3 OMe), 53.4 (CHα Leu), 42.7
(t, J = 147.7 Hz, CH-P2), 41.0 (CH2 Leu), 28.3 (CH3 Boc), 24.8 (CHLeu), 23.0
(CH3 OMe), 22.0 (CH3 OMe) ppm. 31P NMR (162 MHz, CDCl3,24 °C) δ = 18.55
ppm. IR (KBr)  3280, 2959, 2241, 1716, 1266, 1030 cm–1. HRMS (ESI)
C16H34N2NaO9P2 [M+Na]+: 483.1632, found 483.1624.
Aminomethylbisphosphonic acid (1): A mixture of phosphorous acid
(4.55 g, 55.49 mmol), phosphorus trichloride (20.20 mL, 166.51 mmol) and
formamide (8.80 mL, 222.04 mmol) was stirred under argon for 8 hours at
70 °C. It was then cooled to room temperature, water was added dropwise
(30 mL) and then stirred 12 hours at room temperature. Solvent was
removed and the crude mixture was suspended in a hydrogen chloride 6N
aqueous solution and stirred under reflux for 3 days. Solvent was removed
under reduced pressure and then a solvent mixture of methanol/diethyl
ether 2:1 (70 mL) was added in order to form a precipitate. This precipitate
was filtered and washed with a methanol/water 2:1 mixture (25 mL) to give
the product as a white solid (11.15 g, 58.37 mmol, yield: 53%). M.p. 298–
300 °C. 1H NMR (400 MHz, D2O, 24 °C) δ 3.13 (t, J = 16.4 Hz, 1H, CH-P2)
ppm. 13C NMR (100 MHz, D2O, 24 °C) δ 50.83 (t, J = 119.6 Hz, CH-P2)
ppm. 31P NMR (162 MHz, D2O, 30 °C) δ 9.53 ppm. IR (KBr)  3162, 1200,
1162 cm–1. HRMS (ESI) CH6NO6P2 [M-H]−: 189.9676, found 189.9673.
Dimethyl [1-(benzyloxycarbonylamino)ethen-1-yl]phosphonate (4):
To a solution of 2 (395 mg, 1.04 mmol) in a mixture of tetrahydrofuran/2-
propanol 4:1 (8 mL) was added cesium carbonate (422 mg, 1.29 mmol)
and 37% aqueous formaldehyde solution (78 µL, 1.04 mmol). After stirring
for one hour at room temperature, solvent was removed under reduced
pressure, the crude suspended in dichloromethane and the solid removed
by filtration. Then, solvent was removed under reduced pressure and the
resulting oil was purified by column chromatography on silica gel (ethyl
acetate/hexane 3:1) to afford 4 as a yellow oil (188 mg, 0.66 mmol, yield:
63%). 1H NMR (400 MHz, CDCl3, 24 °C) δ = 7.39–7.31 (m, 5H, Ph), 6.62
(d, J = 6.6 Hz, 1H, NH), 6.39 (d, J = 41.6 Hz, 1H, E-CH2=C), 5.47 (d, J =
19.1 Hz, 1H, Z-CH2=C), 5.15 (s, 2H, CH2-Ph), 3.76 (d, J = 11.2 Hz, 6H,
CH3 OMe) ppm. 13C NMR (100 MHz, CDCl3, 25 °C) δ = 153.4 (d, J = 16.7
Hz, CO), 135.7 (Phipso), 130.2 (d, J = 202.1 Hz, CH2=C-P), 128.8 (Ph),
128.6 (Ph), 128.5 (Ph), 111.5 (d, J = 10.0 Hz, CH2=C), 67.4 (CH2-Ph), 53.4
(d, J = 5.5 Hz, CH3 OMe) ppm. 31P NMR (162 MHz, CDCl3, 25 °C) δ = 15.18
ppm. IR (neat)  3238, 3034, 2955, 1732, 1237, 1030 cm–1. HRMS (ESI)
C12H16NNaO5P [M+Na]+: 308.0658, found 308.0661.
Tetramethyl (benzyloxycarbonylaminomethyl)bisphosphonate (2):
To a solution of 1 (969 mg, 5.07 mmol) in water (5 mL) and triethylamine
(3.0 mL, 21.50 mmol) was added
a
solution of N-
(benzyloxycarbonyloxy)succinimide (1.28 g, 5.14 mmol) in acetonitrile (1.3
mL). The reaction mixture was stirred 12 hours at room temperature, and
solvent was removed under reduced pressure. The residue was dissolved
in water, washed with diethyl ether, treated with an ion-exchange resin
(Dowex® 50WX8, hydrogen form) and evaporated to afford a colourless
oil which was used in the next step without further purification. Previous
residue was dissolved in trimethyl orthoformate (15.32 mL, 0.14 mol) and
stirred at reflux under argon for 72 hours. Subsequently, the orthoformate
in excess was removed under reduced pressure. The resulting oil was
Dimethyl
{1-[N-(tert-butoxycarbonyl)-L-leucylamido]ethen-1-
yl}phosphonate (5): To a solution of 3 (518 mg, 1.12 mmol) in a mixture
of tetrahydrofuran/2-propanol 4:1 (10 mL) was added cesium carbonate
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800689
European Journal of Organic Chemistry
FULL PAPER
(459 mg, 1.41 mmol) and 37% aqueous formaldehyde solution (84 µL, 1.12
mmol). After stirring two hours at room temperature, solvent was removed
under reduced pressure, the crude suspended in dichloromethane and the
solid removed by filtration. Then, the solvent was removed under reduced
pressure and the resulting oil was purified by column chromatography on
silica gel (ethyl acetate/hexane 3:1) to afford 5 as a yellow oil (322 mg,
0.88 mmol, yield: 79%). []21D = –54.83 (c 0.50. CHCl3). 1H NMR (400 MHz,
CDCl3, 27 °C) δ = 7.92 (br s, 1H, NH), 6.69 (d, J = 42.0 Hz, 1H, E-CH2=C),
5.62 (dd, J = 19.3, 1.0 Hz, 1H, Z-CH2=C), 4.87 (br s, 1H, NH), 4.19–4.05
(m, 1H, CHα Leu), 3.76 (d, 3H, J = 11.2 Hz, CH3 OMe), 3.75 (d, 3H, J = 11.2
Hz, CH3 OMe), 1.77–1.61 (m, 2H, CH2 Leu+CHLeu), 1.53–1.45 (m, 1H, CH2
Leu) overlapped with 1.44 (s, 9H, CH3 Boc), 0.98–0.90 (m, 6H, CH3 Leu) ppm.
13C NMR (100 MHz, CDCl3, 27 °C) δ = 172.1 (d, J = 9.9 Hz, CO), 155.9
(CO), 130.1 (d, J = 200.5 Hz, CH2=C), 115.3 (d, J = 9.8 Hz, CH2=C),
80.7(CBoc), 54.0 (CHα Leu), 53.4–53.2 (m, CH3 OMe), 40.6 (CH2 Leu), 28.4
(CH3 Boc), 24.9 (CHLeu), 23.1 (CH3 Leu), 21.9 (CH3 Leu) ppm. 31P NMR (162
MHz, CDCl3, 27 °C) δ = 15.07 ppm. IR (neat)  3297, 2957, 1700, 1525,
1257, 1029 cm–1. HRMS (ESI) C15H29N2NaO6P [M+Na]+:387.1655, found
387.1668.
(303 mg, 0.62 mmol, yield: 62%). M.p. 151.1–151.8 °C. []29 = –40.7 (c
D
0.41, CHCl3). 1H NMR (400 MHz, CDCl3, 23 °C) δ = 7.90 (d, JH = 4.8 Hz,
1H, NH), 7.75 (d, J = 7.5 Hz, 2H, ArFmoc), 7.57 (d, J = 7.4 Hz, 2H, ArFmoc),
7.39 (t, J = 7.5 Hz, 2H, ArFmoc), 7.30 (t, J = 7.4 Hz, 2H, ArFmoc), 6.70 (d, J =
41.8 Hz, 1H, E-CH2=C), 5.63 (dd, J = 19.2, 0.6 Hz, 1H, Z-CH2=C), 5.39 (d,
J = 8.1 Hz, 1H, NHLeu), 4.47–4.35 (m, 2H, CH2 Fmoc), 4.33–4.18 (m, 2H,
CHFmoc+CHα Leu), 3.76–3.67 (m, 6H, CH3 OMe), 1.75–1.51 (m, 3H, CH2
Leu+CHLeu), 1.00–0.88 (m, 6H, CH3 Leu) ppm. 13C NMR (100 MHz, CDCl3,
23 °C) δ = 171.9 (d, J = 10.1 Hz, CO), 156.4 (CO), 143.9, 143.7, 141.4
(Aripso), 130.0 (d, J = 200.9 Hz, CH2=C), 127.9, 127.2, 125.1, 120.1 (ArFmoc),
115.7 (d, J = 9.9 Hz, CH2=C), 67.3 (CH2 Fmoc), 54.3 (CHα Leu), 53.5–53.2 (m,
CH3 OMe), 47.2 (CHFmoc), 41.2 (CH2 Leu), 24.9 (CHLeu), 23.1, 22.0 (CH3 Leu
)
ppm. 31P NMR (162 MHz, CDCl3, 25 °C) δ = 14.77 ppm. IR (KBr)  3306,
2954, 1713, 1686, 1536, 1257, 1035 cm–1. HRMS (ESI) C25H31N2NaO6P
[M+Na]+: 509.1812, found 509.1814.
[1-(L-leucylamido)ethen-1-yl]phosphonic acid monomethyl ester
(11): To a solution of 10 (129 mg, 0.27 mmol) in a mixture of
acetone/toluene 1:1 (5 mL) 1,4-diazabicyclo[2.2.2]octane (74 mg, 0.55
mmol) was added. The reaction mixture was stirred at reflux for 5 hours.
Solvent was removed, and the resulting residue was purified using an
acidic ion-exchange resin (Dowex® 50WX8, Hydrogen form) to afford 11
Tetramethyl
[N-(9-fluorenylmethoxycarbonyl)-L-
leucylamidomethyl]bisphosphonate (9): To a solution of 2 (512 mg,
1.34 mmol) in methanol (30 mL) 10% wt. palladium on carbon (75 mg) was
added. The mixture was stirred under a hydrogen atmosphere at room
temperature for one hour. Then, the reaction mixture was filtered (Celite)
and evaporated to afford an oil which was used in the next step. A solution
as a white solid (61 mg, 0.24 mmol, 89% yield). []24 = +33.3 (c 0.41,
D
MeOH) M.p. 148.2–149.9 °C. 1H NMR (400 MHz, MeOD, 23 °C) δ = 6.37
(d, J = 35.2 Hz, 1H, E-CH2=C), 5.57 (d, J = 16.4 Hz, 1H, Z-CH2=C), 4.04–
3.97 (m, 1H, CHα Leu), 3.50 (d, J = 11.0 Hz, 3H, CH3 OMe), 1.83–1.65 (m,
3H, CH2 Leu+CHLeu), 1.04–0.98 (m, 6H, CH3 Leu) ppm. 13C NMR (100 MHz,
MeOD, 23 °C) δ = 169.7 (d, J = 9.7 Hz, CO), 137.8 (d, J = 187.1 Hz,
CH2=C), 113.5 (d, J = 9.5, CH2=C), 53.7 (CHα Leu), 52.3 (d, J = 5.3 Hz, CH3
OMe), 41.5 (CH2 Leu), 25.5 (CHLeu), 23.1 (CH3 Leu), 22.3 (CH3 Leu) ppm. 31P
NMR (162 MHz, MeOD, 23 °C) δ 7.99 ppm. IR (KBr)  2959, 2360, 1684,
1540, 1207, 1049 cm–1. HRMS (ESI) C9H19N2NaO4P [M+Na]+: 273.0975,
found 273.0976.
of previous oil and Fmoc-L-Leucine (475 mg, 1.34 mmol) in dry N,N-
dimethylformamide (6.7 mL) was cooled to 0 °C. N-Methylmorpholine (190
µL, 1.73 mmol) and 1-hydroxybenzotriazole hydrate (12% H2O) (248 mg,
1.61 mmol) were added and the mixture was stirred for 15 minutes at 0 °C.
Then N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (309
g, 1.61 mmol) was added, and the reaction mixture was stirred for 18 hours
at 0 °C. Solvent was removed under reduced pressure, the residue was
dissolved in ethyl acetate (30 mL) and washed with an aqueous 5%
solution of sodium bicarbonate (2 x 20 mL). The aqueous phase was
extracted with ethyl acetate (3 x 15 mL) until no final product was detected
by TLC in the aqueous phase. The combined organic layers were dried
over anhydrous magnesium sulphate, filtered and solvent removed under
reduced pressure. The resulting oil was purified by column
chromatography on silica gel (dichloromethane/2-propanol 95:5) to afford
Tetramethyl
[N-(9-fluorenylmethoxycarbonyl)glycyl-L-
leucylamidomethyl]bisphosphonate (12): A solution of 3 (1.44 g, 3.13
mmol) in dichloromethane (60 mL) was cooled to 0 °C, trifluoroacetic acid
(4.84 mL, 62.68 mmol) was added and then the solution stirred overnight
at room temperature. Subsequently, solvent was removed under reduced
pressure and the residue lyophilized to afford a colourless oil which was
9 as a colourless oil (502 mg, 0.86 mmol, 64% yield). []21 = –19.86 (c
D
used without further purification. A solution of previous oil and Fmoc-L-
1
0.51, CHCl3). H NMR (400 MHz, CDCl3, 25 °C) δ = 7.74 (d, J = 7.6 Hz,
Leucine (1.02 g, 3.43 mmol) in dry N,N-dimethylformamide (16 mL) was
cooled to 0 °C. N-Methylmorpholine (0.86 mL, 7.82 mmol) and 1-
hydroxybenzotriazole hydrate (12% H2O) (577 mg, 3.76 mmol) were
added and the mixture was stirred for 15 minutes at 0 °C. Then N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (720 mg, 3.76
mmol) was added, the reaction mixture was stirred for 1 hour at 0 °C and
then for 14 hours at 4 °C. Solvent was removed under reduced pressure,
the residue was dissolved in ethyl acetate (30 mL) and washed with an
aqueous 5% solution of sodium bicarbonate. The aqueous phase was
extracted with ethyl acetate (3 x 15 mL) until no final product was detected
by TLC in the aqueous phase. The combined organic layers were dried
over anhydrous magnesium sulphate, filtered and solvent removed under
reduced pressure. The resulting oil was purified by column
chromatography on silica gel (dichloromethane/diethyl ether/methanol
85:10:5) to afford 12 as a colorless oil (1.76 g, 2.75 mmol, yield 88%).
[]25D = -22.7 (c 0.41, CHCl3). 1H NMR (400 MHz, CDCl3, 24 °C) δ = 7.75
(d, J = 7.5 Hz, 2H, ArFmoc), 7.65–7.56 (m, 3H, ArFmoc+NH), 7.39 (t, J = 7.4
Hz, 2H, ArFmoc), 7.30 (tt, J = 7.5, 1.0 Hz, 2H, ArFmoc), 6.77 (d, J = 8.0 Hz,
1H, NHLeu), 6.09–6.02 (m, 1H, NHGly), 5.07 (td, J = 22.0, 10.0 Hz, 1H, CH-
P2), 4.75–4.65 (m, 1H, CHα Leu), 4.45–4.35 (m, 2H, CH2 Fmoc), 4.21 (t, 1H,
J = 7.0 Hz, CHFmoc), 4.00–3.84 (m, 2H, CH2 Gly), 3.84–3.72 (m, 12H, CH3
OMe), 1.73–1.50 (m, 3H, CH2 Leu+CHLeu), 0.96–0.83 (m, 6H, CH3 Leu) ppm.
13C NMR (100 MHz, CDCl3, 25 °C) δ = 172.0 (COLeu), 169.7 (COGly), 156.9
2H, ArFmoc), 7.56 (d, J = 7.5 Hz, 2H, ArFmoc), 7.38 (t, J = 7.5Hz, 2H, ArFmoc),
7.29 (td, J = 7.5, 1.1 Hz, 2H, ArFmoc), 5.42 (d, J = 8.3 Hz, 1H, NH), 5.08 (td,
J = 21.9, 10.0 Hz, 1H, CH-P2), 4.46–4.29 (m, 3H, CH2 FMoc+CHα Leu), 4.24–
4.15 (m, 1H, CHFmoc), 3.86–3.69 (m, 12H, CH3 OMe), 1.71–1.50 (m, 3H, CH2
Leu+CHLeu), 0.97–0.90 (m, 6H, CH3 Leu) ppm. 13C NMR (100 MHz, CDCl3,
26 °C) δ = 172.2 (CO), 156.1 (CO), 143.9, 143.8, 141.4, 141.3 (Aripso),
127.9, 127.2, 125.2, 125.1, 120.2, 120.1 (ArFmoc), 67.1 (CH2 Fmoc), 54.4–
54.0 (m, CH3 OMe), 53.6 (CHα Leu), 47.2 (CHFmoc), 42.8 (t, J = 148.2 Hz, CH-
P2), 41.5 (CH2 Leu), 24.8 (CHLeu), 23.0, 22.0 (CH3 Leu) ppm. 31P NMR (162
MHz, CDCl3, 25 °C) δ 18.59, 18.37 (AB spin system, 2P, J = 31.5 Hz) ppm.
IR (KBr)  3270, 2960, 1720, 1670, 1270, 1050 cm–1. HRMS (ESI)
C26H36N2NaO9P2 [M+Na]+: 605.1788, found 605.1805.
Dimethyl {1-[N-(9-fluorenylmethoxycarbonyl)-L-leucylamido]ethen-1-
yl}phosphonate (10): To a solution of 9 (582 mg, 1.00 mmol) in a mixture
of tetrahydrofuran/2-propanol 4:1 (12 mL) was added cesium carbonate
(407 mg, 1.25 mmol) and 37% aqueous formaldehyde solution (75 µL, 1.00
mmol). After stirring for 1 hour at room temperature, solvent was removed
under reduced pressure, the crude suspended in dichloromethane and the
solid removed by filtration. Then, the solvent was removed under reduced
pressure and the resulting oil was purified by column chromatography on
silica gel (dichloromethane/2-propanol 97:3) to afford 10 as a white solid
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800689
European Journal of Organic Chemistry
FULL PAPER
(COFmoc), 143.9, 141.4 (Aripso), 127.9, 127.2, 125.2, 120.1 (ArFmoc), 67.4
(CH2 Fmoc), 54.4–54.1 (m, CH3 OMe), 51.8 (CHα Leu), 47.2 (CHFmoc), 44.7 (CH2
Gly), 42.8 (t, J = 148.3 Hz, CH-P2), 41.2 (CH2 Leu), 24.9 (CHLeu), 22.9, 22.3
(CH3 Leu) ppm. 31P NMR (162 MHz, CDCl3, 24 °C) δ = 18.60 ppm. IR (KBr)
temperature under an argon atmosphere for 21 hours. Solvent was
removed under reduced pressure and the resulting oil was purified by
column chromatography on silica gel (dichloromethane/2-propanol 93:7)
to afford 14 as a colorless oil (225 mg, 0.39 mmol, 62% yield). []25D = –
37.2 (c 0.41, CHCl3). 1H NMR (400 MHz, CDCl3, 24 °C) δ = 7.52–7.46 (m,
2H, Ar), 7.42–7.36 (m, 4H, Ar+NH), 7.20 (d, J =8.5 Hz, 1H, NHLeu), 5.06
(td, J = 22.0, 9.9 Hz, 1H, CH-P2), 4.63 (td, J = 8.7, 6.0 Hz, 1H, CHα Leu),
3.86–3.76 (m, 12H, CH3 POMe), 3.43 (d, J = 1.3 Hz, 3H, CH3 OMe), 1.70–1.44
(m, 3H, CH2 Leu+CHLeu), 0.89 (d, J = 6.5 Hz, 3H, CH3 Leu) overlapped with
0.88 (d, J = 6.4 Hz, 3H, CH3 Leu) ppm. 13C NMR (100 MHz, CDCl3, 24 °C)
δ = 171.2 (t, J = 4.1 Hz, CO), 166.5 (CO), 132.5 (Aripso), 129.7, 128.7, 127.5
(Ar), 123.8 (q, J = 290.0 Hz, CF3), 84.1 (q, J = 26.3 Hz, CS), 55.2 (d, J =
1.8 Hz, CH3 OMe), 54.4–54.0 (m, CH3 POMe), 51.6 (CHα Leu), 42.8 (t, J = 148.0
Hz, CH-P2), 41.1 (CH2 Leu), 24.8 (CHLeu), 22.9, 22.0 (CH3 Leu) ppm. 31P NMR
(162 MHz, CDCl3, 24 °C) δ = 18.42, 18.18 (AB spin system, 2P, J = 30.6
Hz) ppm. IR (KBr)  3245, 2959, 1693, 1678, 1666, 1513, 1263, 1165,
1035 cm–1. HRMS (ESI) C21H33F3N2NaO9P2 [M+Na]+: 599.1506, found
599.1504.

3280, 2957, 1679, 1538, 1264, 1086 cm–1
C28H39N3NaO10P2 [M+Na]+: 662.2003, found 662.2016.
.
HRMS (ESI)
Dimethyl
{1-[N-(9-fluorenylmethoxycarbonyl)glycyl-L-
leucylamido]ethen-1-yl}phosphonate (13): To a solution of 12 (344 mg,
0.54 mmol) in a mixture of tetrahydrofuran/2-propanol 4:1 (7 mL) was
added cesium carbonate (219 mg, 0.67 mmol) and 37% aqueous
formaldehyde solution (43 µL, 0.57 mmol). After stirring at room
temperature for 1 hour, the solvent was removed under reduced pressure,
the crude suspended in dichloromethane and the solid removed by
filtration. Solvent was removed under reduced pressure and the resulting
oil was purified by column chromatography on silica gel (ethyl
acetate/hexane/2-propanol 7:2:1) to afford 13 as a yellow oil (168 mg, 0.31
1
mmol, yield: 57%). []27 = –36.2 (c 0.40, CHCl3). M.p. 63.7–65.1 °C. H
D
NMR (400 MHz, CDCl3, 25 °C) δ = 8.03 (d, J = 7.2 Hz, 1H, NH), 7.76 (d, J
= 7.5 Hz, 2H, ArFmoc), 7.59 (d, J = 7.4 Hz, 2H, ArFmoc), 7.39 (t, J = 7.5 Hz,
2H, ArFmoc), 7.30 (tt, J = 7.5, 1.0 Hz, 2H, ArFmoc), 6.66 (d, J = 7.9 Hz, 1H,
NHLeu) overlapped with 6.65 (d, J = 41.7 Hz, 1H, E-CH2=C), 5.77 (t, J = 5.6
Hz, 1H, NHGly), 5.61 (d, J = 19.0 Hz, 1H, Z-CH2=C), 4.62–4.54 (m, 1H, CHα
Leu), 4.41 (d, J = 6.9 Hz, 2H, CH2 Fmoc), 4.21 (t, J = 7.0 Hz, 1H, CHFmoc),
3.95–3.90 (m, 2H, CH2 Gly), 3.77–3.70 (m, 6H, CH3 OMe), 1.74–1.51 (m, 3H,
CH2 Leu+CHLeu), 0.94–0.87 (m, 6H, CH3 Leu) ppm. 13C NMR (100 MHz,
CDCl3, 25 °C) δ = 171.8 (d, J = 10.2 Hz, CO), 169.8, 156.9 (CO), 143.8,
141.4 (Aripso), 130.2 (d, J = 201.3 Hz, CH2=C), 127.9, 127.2, 125.2, 120.1
(ArFmoc), 115.9 (d, J = 9.9 Hz, CH2=C), 67.4 (CH2 Fmoc), 53.6–53.3 (m, CH3
OMe), 52.6 (CHα Leu), 47.2 (CHFmoc), 44.5 (CH2 Gly), 40.9 (CH2 Leu), 24.9
(CHLeu), 23.0, 22.0 (CH3 Leu) ppm. 31P NMR (162 MHz, CDCl3, 24 °C) δ =
14.65 ppm. IR (KBr)  3281, 3064, 2955, 1700, 1669, 1539, 1258, 1032
cm–1. HRMS (ESI) C27H34N3NaO7P [M+Na]+: 566.2027, found 566.2025.
Dimethyl
{1-{N-[(S)-α-methoxy-α-(trifluoromethyl)phenylacetyl]-L-
leucylamido}ethen-1-yl}phosphonate (15): To a solution of 14 (152 mg,
0.26 mmol) in a mixture of tetrahydrofuran/2-propanol 4:1 (5 mL) cesium
carbonate (108 mg, 0.33 mmol) and 37% aqueous formaldehyde solution
(20 µL, 0.26 mmol) were added. After stirring for 3 hours at room
temperature, the solvent was removed under reduced pressure, the crude
suspended in dichloromethane and the solid removed by filtration. Then,
the solvent was removed under reduced pressure and the resulting oil was
purified by column chromatography on silica gel (dichloromethane/2-
propanol 95:5) to afford 15 as a white solid (104 mg, 0.22 mmol, 85% yield).
[]29D = –66.5 (c 0.42, CHCl3). 1H NMR (400 MHz, CDCl3, 24 °C) δ = 7.87
(d, J = 6.7 Hz, 1H, NH), 7.54–7.47 (m, 2H, Ar), 7.43–7.37 (m, 3H, Ar), 7.15
(d, J = 8.2 Hz, 1H, NHLeu), 6.67 (d, J = 41.8 Hz, 1H, E-CH2=C), 5.68 (d, J
= 19.3 Hz, 1H, Z-CH2=C), 4.54 (td, J = 9.0, 5.5 Hz, 1H, CHα Leu), 3.80–3.71
(m, 6H, CH3 POMe), 3.45 (d, J = 1.3 Hz, 3H, CH3 OMe), 1.76–1.56 (m, 2H,
CH2 Leu), 1.55–1.42 (m, 1H, CHLeu), 0.93–0.84 (m, 6H, CH3 Leu) ppm. 13C
NMR (100 MHz, CDCl3, 24 °C) δ = 170.7 (d, J = 10.3 Hz, CO), 167.1 (CO),
132.4 (Aripso), 130.2 (d, J = 201.6 Hz, CH2=C), 129.8, 128.7, 127.5 (Ar),
123.7 (q, J = 290.1 Hz, CF3), 116.0 (d, J = 10.1 Hz, CH2=C), 84.1 (q, J =
26.5 Hz, CS), 55.3 (d, J = 1.9 Hz, CH3 OMe), 53.4–53.2 (m, CH3 POMe), 52.4
Dehydrophos: To a solution of 13 (146 mg, 0.27 mmol) in a mixture of
acetone/toluene 1:1 (5 mL) 1,4-diazabicyclo[2.2.2]octane (76 mg, 0.68
mmol) was added. The reaction mixture was stirred at reflux for 2 hours.
Solvent was removed, the crude dissolved in water (10 mL) and filtered
through 0.22 µm HPLC filter. Then, the aqueous solution was washed with
dichloromethane (2 x 5 mL) and ethyl acetate (2 x 5 mL) and concentrated
in vacuo. The resulting residue was purified using a weakly acidic ion-
exchange resin (Amberlite® CG50, hydrogen form) to afford dehydrophos
as a pale yellow solid (76 mg, 0.25 mmol, 93% yield). []23D = –46.5 (c 0.39,
H2O). M.p. 137.2–138.4 °C (dec). 1H NMR (400 MHz, D2O, 25 °C) δ = 6.20
(d, J = 36.3 Hz, 1H, E-CH2=C), 5.71 (d, J = 15.9 Hz, 1H, Z-CH2=C), 4.50–
4.42 (m, 1H, CHα Leu), 3.90 (s, 2H, CH2 Gly), 3.52 (d, J = 11.0 Hz, 3H, CH3
OMe), 1.74–1.63 (m, 3H, CH2 Leu+CHLeu), 0.97 (d, J = 6.1 Hz, 3H, CH3 Leu),
0.92 (d, J = 6.1 Hz, 3H, CH3 Leu) ppm. 13C NMR (100 MHz, D2O, 25 °C) δ
= 173.6 (d, J = 7.6 Hz, CO), 167.1 (CO), 134.5 (d, J = 189.9 Hz, CH2=C),
117.1 (d, J = 11.7 Hz, CH2=C), 53.3 (CHα Leu), 52.0 (d, J = 5.2 Hz, CH3 OMe),
(d, J = 1.6 Hz, CHα Leu), 40.4 (CH2 Leu), 24.8 (CHLeu), 23.0, 21.8 (CH3 Leu
)
ppm. 31P NMR (162 MHz, CDCl3, 24 °C) δ = 14.37 ppm. IR (KBr)  1682,
1537, 1248, 1169, 1045 cm–1. HRMS (ESI) C20H28F3N2NaO6P [M+Na]+:
503.1529, found 503.1550.
Tetramethyl
{N-[(R)-α-methoxy-α-(trifluoromethyl)phenylacetyl]-L-
leucylamidomethyl}bisphosphonate (17): A solution of 3 (295 mg, 0.64
mmol) in dichloromethane (13 mL) was cooled to 0 ºC and trifluoroacetic
acid (0.98 mL, 12.70 mmol) was added. The mixture was stirred overnight
at room temperature. Then, solvent was removed under reduced pressure
and the resulting residue lyophilized. The resulting oil was dissolved in dry
dichloromethane (10 mL), N,N-diisopropylethylamine (0.28 mL, 1.61
mmol) and (S)-(+)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride
(0.14 mL, 0.75 mmol) were added. The mixture was stirred at room
temperature under an argon atmosphere for 22 hours. Solvent was
removed under reduced pressure and the resulting oil was purified by
column chromatography on silica gel (dichloromethane/2-propanol 9:1) to
afford 17 as a colorless oil (272 mg, 0.47 mmol, 73% yield). []25D = –23.6
(c 0.42, CHCl3). 1H NMR (400 MHz, CDCl3, 24 °C) δ = 7.50–7.45 (m, 2H,
Ar), 7.40–7.32 (m, 5H, Ar+NH+NH), 5.05 (td, J = 22.1, 9.9 Hz, 1H, CH-P2),
4.64–4.57 (m, 1H, CHα Leu), 3.84–3.72 (m, 9H, CH3 POMe), 3.61 (d, J = 11.0
Hz, 3H, CH3 POMe), 3.33 (d, J = 1.1 Hz, 3H, CH3 OMe), 1.74–1.61 (m, 3H,
CH2 Leu+CHLeu), 1.00–0.89 (m, 6H, CH3 Leu) ppm. 13C NMR (100 MHz,
CDCl3, 24 °C) δ = 171.5 (t, J = 4.1 Hz, CO), 166.4 (CO), 132.0 (Aripso),
129.6, 128.7, 128.1 (Ar), 123.9 (q, J = 290.2 Hz, CF3), 84.1 (q, J = 26.4 Hz,
40.3 (CH2 Gly), 39.6 (CH2 Leu), 24.3 (CHLeu), 22.1 (CH3 Leu), 20.7 (CH3 Leu
)
ppm. 31P NMR (162 MHz, D2O, 22 °C) δ = 9.56 ppm. IR (KBr)  3290, 3046,
2949, 1688, 1665, 1531, 1246, 1072 cm–1. HRMS (ESI) C11H22N3NaO5P
[M+Na]+: 330.1189, found 330.1179.
Tetramethyl
{N-[(S)-α-methoxy-α-(trifluoromethyl)phenylacetyl]-L-
leucylamidomethyl}bisphosphonate (14): A solution of 3 (290 mg, 0.63
mmol) in dichloromethane (12 mL) was cooled to 0 ºC and trifluoroacetic
acid (0.96 mL, 12.6 mmol) was added. The mixture was stirred overnight
at room temperature. Then, solvent was removed under reduced pressure
and the resulting residue lyophilized. The resulting oil was dissolved in dry
dichloromethane (10 mL), N,N-diisopropylethylamine (0.27 mL, 1.55
mmol) and (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride
(0.14 mL, 0.75 mmol) were added. The mixture was stirred at room
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800689
European Journal of Organic Chemistry
FULL PAPER
CR), 54.9 (d, J = 1.6 Hz, CH3 OMe), 54.3–54.0 (m, CH3 POMe), 51.8 (CHα Leu),
42.8 (t, J = 147.9 Hz, CH-P2), 41.2 (CH2 Leu), 24.8 (CHLeu), 22.9, 21.9 (CH3
Leu) ppm. 31P NMR (162 MHz, CDCl3, 25 °C) δ = 18.48, 18.15 (AB spin
system, 2P, J = 30.5 Hz) ppm. IR (KBr)  3412, 3245, 2959, 2360, 1684,
1507, 1266, 1163, 1037 cm–1. HRMS (ESI) C21H33F3N2NaO9P2 [M+Na]+:
599.1506, found 599.1492.
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Dimethyl
{1-{N-[(R)-α-methoxy-α-(trifluoromethyl)phenylacetyl]-L-
leucylamido}ethen-1-yl}phosphonate (18): To a solution of 17 (272 mg,
0.47 mmol) in a mixture of tetrahydrofuran/2-propanol 4:1 (6 mL) cesium
carbonate (191 mg, 0.59 mmol) and 37% aqueous formaldehyde solution
(35 µL, 0.47 mmol) were added. After stirring at room temperature for 7
hours, the solvent was removed under reduced pressure, the crude
suspended in dichloromethane and the solid removed by filtration. Solvent
was removed under reduced pressure and the resulting oil was purified by
column chromatography on silica gel (dichloromethane/2-propanol 95:5)
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to afford 18 as a white solid (168 mg, 0.35 mmol, 74% yield). []28 = –
D
69.82 (c 0.33, CHCl3). 1H NMR (400 MHz, CDCl3, 25 °C) δ = 7.83 (d, J =
6.9 Hz, 1H, NH), 7.51–7.44 (m, 2H, Ar), 7.42–7.37 (m, 3H, Ar), 7.34 (d, J
= 8.0 Hz, 1H, NHLeu), 6.65 (d, J = 41.8 Hz, 1H, E-CH2=C), 5.65 (d, J = 19.3
Hz, 1H, Z-CH2=C), 4.52 (td, J = 8.5, 5.5 Hz, 1H, CHα Leu), 3.70 (d, J = 11.2
Hz, 3H, CH3 POMe), 3.61 (d, J = 11.1 Hz, 3H, CH3 POMe), 3.34 (d, J = 1.3 Hz,
3H, CH3 OMe), 1.80–1.62 (m, 3H, CH2 Leu+CHLeu), 0.98 (d, J = 6.1 Hz, 3H,
CH3 Leu), 0.94 (d, J = 6.2 Hz, 3H, CH3 Leu) ppm. 13C NMR (100 MHz, CDCl3,
26 °C) δ = 170.7 (d, J = 10.4 Hz, CO), 167.1 (CO), 131.8 (Aripso), 130.3 (d,
J = 201.5 Hz, CH2=C), 129.7, 128.8 (Ar), 128.1 (d, J = 1.4 Hz, Ar), 123.9
(q, J = 290.2 Hz, CF3), 115.8 (d, J = 10.1 Hz, CH2=C), 84.2 (q, J = 26.5 Hz,
CR), 55.0 (d, J = 1.7 Hz, CH3 OMe), 53.4–53.2 (m, CH3 POMe), 52.7 (d, J =
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Acknowledgements
This work was supported by the Ministerio de Economía y
Competitividad (grant CTQ2013-40855-R) and the Gobierno de
Aragón-FEDER (Research group E19_17R) M.M.J.-A. thanks the
Gobierno de Aragón-FSE for predoctoral grant.
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