Determination of the Absolute Configuration of (?)-Hydroxynitrilaphos and Related Biosynthetic Questions

Article abstract of DOI:10.1002/chem.201702904

The ongoing search for bioactive natural products has led to the development of new genome-based screening approaches to identify possible phosphonate producing microorganisms. From the identified phosphonate producers, several until now unknown phosphonic acid natural products were isolated, including (hydroxy)nitrilaphos (4 and 5) and (hydroxy)phosphonocystoximate (7 and 6). We present the synthesis of phosphonocystoximate via an aldoxime intermediate. Chlorination and coupling with methyl N-acetylcysteinate furnished 6 after global deprotection. The obtained experimental data confirm the previously assigned structure of the natural product. We were also able to determine the absolute configuration of (?)-hydroxynitrilaphos. Chiral resolution of diethyl cyanohydroxymethylphosphonate (24) with Noe's lactol furnished both enantiomers of 4. Conversion of (+)-24 to (R)-2-amino-1-hydroxyethylphosphonic acid by reduction of the cyano-group showed (?)-hydroxynitrilaphos ultimately to be S-configured. Further, we present a ¹³C-isotope labeling strategy for 4 and 5 that will possibly solve the question of whether hydroxynitrilaphos is a biosynthetic intermediate or a downstream product of hydroxyphosphonocystoximate biosynthesis.

Full text of DOI:10.1002/chem.201702904

A Journal of
Accepted Article
Title: Determination of the absolute configuration of (-)-
hydroxynitrilaphos and related biosynthetic questions
Authors: Katharina Pallitsch, Barbara Happl, and Christian Stieger
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To be cited as: Chem. Eur. J. 10.1002/chem.201702904
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Determination of the absolute configuration of (–)-
hydroxynitrilaphos and related biosynthetic questions
Katharina Pallitsch,[a]* Barbara Happl[a] and Christian Stieger[a].
Abstract: The ongoing search for bioactive natural products led to the development of new genome-based screening approaches to identify
possible phosphonate producing microorganisms. From the identified phosphonate producers, several until now unknown phosphonic acid
natural products were isolated, including (hydroxy)nitrilaphos (4 and 5) and (hydroxy)phosphonocystoximate (7 and 6). We present the
synthesis of phosphonocystoximate via an aldoxime intermediate. Chlorination and coupling with methyl N-acetylcysteinate furnished 6 after
global deprotection. The obtained experimental data confirmed the previously assigned structure of the natural product. We were also able to
determine the absolute configuration of (–)-hydroxynitrilaphos. Chiral resolution of diethyl cyanohydroxymethylphosphonate (24) with Noe’s
lactol furnished both enantiomers of 4. Conversion of (+)-24 to (R)-2-amino-1-hydroxyethylphosphonic acid by reduction of the cyano-group
showed (–)-hydroxynitrilaphos ultimately to be (S)-configured. Further we present an 13C-isotope labeling strategy for 4 and 5 that will possibly
solve the question of whether hydroxynitrilaphos is a biosynthetic intermediate or a downstream product of hydroxyphosphonocystoximate
biosynthesis.
Introduction
Phosphonates are characterized by
a
direct, covalent
phosphorus to carbon bond (P-C bond), which is the key driving
force for their bioactivity against microbes.1,2 The substance
classes they can effectively mimic are ubiquitous in nature. Thus,
they are unrivalled in their range of possible targets.3 Consistent
therewith phosphonates with a large variety of biological
activities are known, ranging from antibacterial (1)4 to herbicidal
(2)5 or antiviral (3)6 drugs (Figure 1).
Figure 2. Some of the compounds that have been isolated from strains of
Actinobacteria identified as phosphonate producers by genome mining
techniques.
Figure 1. Some bioactive phosphonates of high industrial importance.
The commercialization rate of phosphonates is remarkably high
(15%),7 compared to the average of natural products (0.1%).8
High throughput genetic screening approaches, for which
phosphonates are ideal candidates, were designed to overcome
the general problems of natural product use in pharmaceutical
industry. All but two9,10 known phosphonate natural products
share a common first biosynthetic step – the rearrangement of
phosphoenol pyruvate (PEP) to phosphonopyruvate (PnPy),
catalyzed by PEP-mutase. The gene encoding this enzyme
(pepM) is hence ideal marker to identify phosphonate producing
microorganisms.11
Indeed, Metcalf et al. identified Streptomyces regensis WC-
3744 as phosphonate producer by genome screening and
isolated hydroxynitrilaphos (4) from its culture broth (Figure 2).11
This is remarkable as less than 0.1% of all isolated natural
products (approx. 150,000) contain a cyano moiety and even
less contain a phosphonic acid functionality.12 Subsequent
[a]
*Katharina Pallitsch
Institute of Organic Chemistry
University of Vienna
Währingerstraße 38, 1090 Vienna
E-mail: katharina.pallitsch@univie.ac.at
Telephone: + 43 1 / 4277-52105 or 52123
ORCID: K. Pallitsch: 0000-0003-2648-1044
Supporting information for this article (comprising 1H, 13C and 31
P
NMR spectra for all synthesized compounds) is available on the
WWW under http://dx.doi.org/10.1002/chem.2017xxxxx..
screenings of
a
library containing more than 10,000
actinomycetes, led to the identification of 244 pepM+ strains,
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among those 55 potential new producers of hydroxynitrilaphos.13
Another group of highly interesting, new phosphonic acid natural
products, called phosphonocystoximates, was isolated from
microorganisms of this pepM+ group. The genomes of the
identified organisms contained additional genes encoding a
flavin- and NAD+-dependent N-hydroxylase, suggesting
a
glucosinolate inspired biosynthesis.14 Mycothiol-dependent
transferase genes suggest mycothiol as source of N-
acetylcysteine in phosphonocystoximates, similar to a common
xenobiotic detoxification strategy in actinobacteria.15 An
additionally identified -ketoglutarate dependent/non-heme
Fe(II) dioxygenase showing sequence similarity to PhnY*,16,17
was suggested to be responsible for -hydroxylation next to the
phosphorus
atom
in
hydroxynitrilaphos
and
hydroxyphosphonocystoximate.13 Despite these first findings,
many questions concerning the structure and biosynthesis of
these fascinating new compounds remained elusive. In this work
we answer some of the pending questions.
Results and Discussion
Structure verification of phosphonocystoximate
The main purpose of establishing a genetic screening approach
was the search for new bioactive phosphonates. Unfortunately,
6 was not obtained in sufficient quantities to allow bioactivity
testing. Further the structure of phosphonocystoximate (6) was
assigned by a combination of various 2D-NMR experiments and
high-resolution mass spectrometry (ESI-MS).18 This information
allowed the assignment of the relative position of all but four
atoms, leaving the authors with three possible structures
meeting all necessary criteria. They found structure 6 to be the
most likely one due to the chemical shift of the -carbon atom
next to the phosphorus atom, which was supported by the strong
sequence homology to genes involved in nitrilaphos
biosynthesis. However, definite evidence for the proposed
structure was still missing. We developed a straight forward
synthetic strategy to access the proposed structure in six steps
(Scheme 1) in order to verify its structure. 6 was obtained in
sufficient quantitied to allow bioactivity testing.
Scheme 1. Synthesis of phosphonocystoximate (6) as its sodium salt.
using
a
mixture
of
TMSBr/allylTMS
(bromotrimethylsilane/allyltrimethylsilane). Finally, the methyl
ester was saponified in the presence of an excess of sodium
hydroxide (4 equiv). Neutralization and lyophilization furnished
phosphonocystoximate (6, 66%) as its sodium salt.
Comparison of the spectroscopic data of our synthetic sample
and phosphonocystoximate of natural origin showed both
compounds to be identical, thus verifying the structure
assignment by Metcalf et al [1H NMR of natural 6. H (CH2-P) 2.7
ppm; synthetic 6 H (CH2-P) 2.9 ppm.].13
Diethylphosphoryl acetaldehyde (10) was obtained from
commercially available bromoacetaldehyde diethyl acetal (8) in
two steps.19,20 It was then converted to a 3:2 mixture of (E)- and
(Z)-oximes 11 in the presence of hydroxylamine hydrochloride in
moderate yield (41%).21 The mixture of (E)- and (Z)-oximes was
chlorinated using N-chlorosuccinimide (NCS). The most
abundant by-product 13, could be removed by flash
chromatography. A significant temperature dependence of the
chlorination was observed with higher temperatures producing
more of the undesired -chlorination product 13. Thus, upscaling
to more than 2 mmol per reaction proved difficult due to local
overheating during NCS addition.21 We observed very poor
ratios (up to 60%13) on sunny days, even with careful
temperature control. Thus we recommend to perform the
reaction under protection from UV-light. The obtained chloro-
oxime 12 was coupled with the methyl ester of N-acetyl cysteine
(14) in the presence of Et3N as a base in 48% yield.22 The
obtained thio-oxime 15 was deprotected at the phosphorus atom
Determination of the absolute configuration of (−)-
hydroxynitrilaphos
Hydroxynitrilaphos was isolated from cultures of S. regensis
WC-3744, albeit in very low yield (1 mg).11 Nevertheless, the
authors succeeded in elucidating the structure of this fascinating
compound and to measure its optical rotation ([]D22= −180.0).
However, the absolute configuration of hydroxynitrilaphos
remained elusive. It was suggested to be produced by S.
regensis from 2-aminoethylphosphonic acid. The -hydroxyl
group was proposed to be inserted by an -ketoglutarate
dependent/non-heme Fe(II) dioxygenase (see Introduction). We
wanted to elucidate the stereo-selectivity of this enzymatic
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transformation by synthesizing both enantiomers of
hydroxynitrilaphos and comparing their optical rotations to that of
the natural product.
Unfortunately, the cyclic diazo compound 16 is very unstable
and could not be isolated in yields > 11%. Furthermore,
chromatographic separation of the diastereomers proved
extremely difficult. As an alternative,
a
phosphate/-
hydroxyphosphonate rearrangement based approach was
envisaged (method B), which did not give satisfactory results
either.30 Only traces of the desired product 19 could be observed
and the enantiomers were difficult to separate. In a third
Even though hydroxynitrilaphos is a very small molecule,
containing only two carbon atoms, its synthesis is not trivial.
Despite its size, it is a highly functionalized molecule containing
a phosphonic acid moiety, a cyano and a hydroxyl group. It is
not possible to introduce the cyano group by addition of cyanide
alternative
we
envisaged
diethyl
to
a
suitable
dialkyl
formylphosphonate.
Diethyl
benzyloxycyanomethylphosphonate (21) as
a
possible
formylphosphonate is not stable at room temperature and
eliminates CO at temperatures above −10°C.23 It is neither
possible to use a Pudovik reaction24 to form the intrinsic P-C
bond as formyl cyanide can only be obtained as a gas under
extremely harsh conditions (400°C, 0.02 Torr), in very low
yields.25 Furthermore, the structure of hydroxynitrilaphos
suggested the stereo center in -position next to the phosphorus
atom to be configurationally very labile under acidic conditions.
The hydroxyl-group could pose additional problems due to a
intermediate, which can be obtained from diethyl
cyanomethylphosphonate after diazotation and Rh-catalyzed
substitution with benzylic alcohol in 60% yield. The enantiomers
could be separated by chiral stationary phase HPLC, but the
benzyl group could not be removed hydrogenolytically without
partial reduction of the nitrile and catalyst poisoning by the
resulting amine (method C). Thus, we envisaged an alternative,
photocleavable
enantiomers
protecting
of
group.31 Unfortunately,
diethyl cyano-o-
the
possible
-hydroxyphosphonate/phosphate
rearrangement
under mild basic conditions at room temperature.26 Thus, very
mild reaction conditions are necessary for the synthetic
sequence. Further the applied synthetic strategy has to provide
an opportunity to determine the absolute configuration of the
product at an appropriate point. Therefore, we opted for a
crystalline key intermediate, to allow an X-ray structure analysis.
These considerations led to the design of compound 17 as key
intermediate. Former work from our group has shown that cyclic
-hydroxyphosphonates of type 17, are often easily separable
diastereomeric mixtures. The diastereomers are prone to
crystallize27 and can be deprotected under very mild conditions.
We wanted to obtain 17 by rhodium-catalyzed hydroxylation of
cyclic diazo-compound 16 (Scheme 2, method A).28,29
nitrobenzyloxymethylphosphonate (22) racemized rapidly
(complete racemization within weeks at −25°C) after
2
separation by chiral HPLC. Despite several attempts, we could
only obtain crystals from the racemate and further observed a
critical loss in ee (final ee 66%) during photolytic cleavage of the
o-nitrobenzyl ether.
Finally, the synthesis of hydroxynitrilaphos was accomplished in
a six-step sequence using Noe’s lactol32 for chiral resolution of
the enantiomers of the key intermediate 24 (Scheme 3). Diethyl
cyanomethylphosphonate was converted to the corresponding
diazo compound using NaN3/1,3-dimethyl-2-chloroimidazolinium
chloride as diazo-transfer reagent (Scheme 3).28
Scheme 3. Synthesis of racemic diethyl cyanohydroxymethylphosphonate
[(±)-24].
The yields could be raised significantly (up to 77%) compared
to the literature by using 2 equivalents of the transfer reagent
and very careful exclusion of water. The obtained diazo
compound was stable enough for chromatographic purification
Scheme 2. Initial strategies towards enantiomerically pure hydroxynitrilaphos;
method A: (a) separation of diastereomers, (b) H2/Pd/C, (c) pH 7 (NaOH);
method B: (a) separation of enantiomers with chiral HPLC, (b) H2/Pd/C, (c) pH
7 (NaOH); method C: (a) separation of enantiomers with chiral HPLC, (b)
H2/Pd/C for 21 and h for 22, (c) TMSBr/allylTMS, (d) pH 7 (NaOH).
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Scheme 4. Successful synthesis of ()-hydroxynitrilaphos of high ee and determination of its absolute configuration; structure of chiral solvating agent (+)-(R)-27.
at room temperature. The hydroxyl group was installed by
reaction with water in the presence of Rh2(OAc)4 as catalyst (2
mol%) in good yield (87%). The thus obtained racemate of
to have (S)-configuration.
Unfortunately, the chiral solvating agent described above
cannot be used in aqueous solution to determine the ee of the
obtained sample. (−)-4 was derivatized using a freshly prepared
ethereal solution of diazomethane to obtain (−)-(S)-dimethyl
cyanohydroxymethylphosphonate (28, Scheme 5). The ee of the
product could again be determined (ee > 96%) using the chiral
solvating agent (+)-(R)-27. This clearly shows the final
deprotection to proceed without any loss of chirality (Scheme 5).
diethyl
cyanohydroxymethylphosphonate
[(±)-24]
was
transformed into a pair of diastereomeric lactols using (+)-Noe’s
lactol dimer in the presence of a catalytic amount of p-toluene
sulfonic acid monohydrate (Scheme 4).32 These were separated
by a series of six sequential chromatographic separations (at a 2
mmol scale). Unfortunately, the diastereomeric acetals could not
be crystallized to determine the absolute configuration at the -
carbon atom next to the phosphorus atom by a single crystal X-
ray structure analysis.
The absolute configuration could be determined by different
means. The more polar acetal 25a was deprotected using
equimolar amounts of p-toluene sulfonic acid to give (+)-24 (ee >
96%). The ee of the sample was determined by addition of (+)-
(R)-tert-butylphenylphosphinothioic acid [(+)-(R)-27] as chiral
solvating agent to an NMR sample of (+)-24.33 Mosher
esterification could not be used to determine the ee of the
obtained sample due to racemization under the necessary
reaction conditions.34 Reduction of the nitrile using BH3∙THF,
followed by removal of the phosphorus protecting groups with 6
M HCl gave (−)-2-amino-1-hydroxyethylphosphonic acid [(−)-1-
OH-2-AEP, 26],16,35 another abundant phosphonic acid natural
product, of known configuration. (R)-1-OH-2-AEP (ee > 99%)
has an optical rotation of []D20= −36.8 (in H2O), while we
observed []D20= −25.7 (in H2O) for a sample obtained from (+)-
24. These results clearly showed (+)-24 to have (R)-
configuration, consequently (−)-24 is (S)-configured. We
attribute the decrease in optical rotation to partial racemization
during the borane reduction via imine/enamine tautomerization.
The less polar acetal 25b furnished (−)-(S)-24 upon
deprotection (ee > 94%). Final deprotection at the phosphorus
atom in the presence of TMSBr/allylTMS and neutralization gave
the sodium salt of (−)-hydroxynitrilaphos after lyophilization
([]D20= −16.7 for the disodium salt of 4 and []D20= −24.2 (for
the free acid). This proves (−)-hydroxynitrilaphos of natural origin
Scheme 5. Derivatization of synthetic ()-hydroxynitrilaphos with ethereal
diazomethane.
Labeling strategy for hydroxynitrilaphos
Even though nitriles are rare in nature, they have been isolated
from plants,36 bacteria12 and fungi.37 In plants the cyano
functionality is derived from amino acids, which are N-oxidized
to the corresponding aldoxim (29) and then dehydrated to give
the nitrile (31).12 While this has been studied extensively, the
biosynthesis of nitriles in bacteria is less studied. None of the
oxidoreductases that could be identified in the genome of
phosphonocystoximate producing microorganisms showed
sufficient sequence similarity to any of the known bacterial gene
clusters involved in nitrile biosynthesis, suggesting a completely
different mechanism.11 Due to the presence of 2-
aminoethylphosphonic acid (2-AEP, 32) in extracts from S.
regensis it was proposed to be converted to hydroxynitrilaphos
and hydroxyphosphonocystoximate after hydroxylation following
a similar pathway to the glucosinolate biosynthesis (Scheme
638).14
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diethyl cyanomethylphosphonate ([2-13C]23) by nucleophilic
subtitution. The very good overall yield for unlabeled (–)-
hydroxynitrilaphos from diethyl cyanomethylphosphonate made
the introduction of the 13C-label at such an early stage of the
reaction sequence acceptable.
Diethyl hydroxymethylphosphonic acid (33) was prepared as a
precursor by a literature known procedure.39 The obtained
hydroxyphosphonate 33 was converted to either the
corresponding triflate 34 or tosylate 35 (Scheme 7). While the
yield of triflate 34 could not be raised above 56%, we succeeded
in preparing tosylate 35 in 71% yield in the presence of Et3N at
0°C. Unfortunately, the subsequent substitution with cyanide
was not possible with satisfying yields (39% maximum yield
starting from the triflate; 17% starting from the tosylate). The
unsatisfactory yield, combined with problems during work-up
and isolation of the desired product made this route very
inconvenient.
Compound 23 was finally prepared in satisfying yields by a
different method (Scheme 8).
Scheme 6. Comparison of proposed glucosinolate (A), nitrilaphos (B) and
hydroxynitrilaphos (C) biosynthesis.
However, it remains elusive whether hydroxynitrilaphos is a
biosynthetic intermediate or downstream product of
hydroxyphosphnocystoximate biosynthesis. The same question
remains unanswered for nitrilaphos and phosphonocystoximate.
In order to shed more light on these questions we designed an
easy strategy to introduce a 13C-label in -position to the
phosphorus atom of nitrilaphos and enantiomerically pure (–)-
hydroxynitrilaphos. We think that feeding experiments using
these
compounds
as
phosphonate-source
for
phosphonocystoximate producing microorganisms will show
whether the 13C label from these nitriles is incorporated in the
corresponding phosphonocystoximic acid derivatives. It will
possibly also allow the identification of other biosynthetically
related compounds or degradation products.
Sodium [13C]cyanide (99% 13C) is one of the cheapest starting
materials for 13C-isotope labeling. It is the most direct way to
label nitrilaphos and hydroxynitrilaphos, as both target
molecules [5 and (–)-4] bear a cyano functionality. We
Scheme 8.
13C]5.
-(S)-[2-13C]4 and [2-
[1-13C]chloroacetonitrile ([1-13C]36] was prepared by an
adapted literature procedure in two steps from sodium
[13C]cyanide, paraformaldehyde and SOCl2 in 65% yield.40
A following Arbusov reaction41 with triethyl phosphite gave
diethyl [2-13C]cyanomethylphosphonate ([2-13C]23) in sufficient
purity for all following transformations. It was treated similarly to
the unlabeled compound 23 to give the sodium salt of (–)-(S)-[2-
13C]hydroxynitrilaphos in six steps [as described in the section
on the determination of the absolute configuration of (–)-
hydroxynitrilaphos]. Furthermore, [2-13C]23 was converted to the
sodium salt of [2-13C]nitrilaphos ([2-13C]5) in two steps in
acceptable yield (36%).
Scheme 7. Initial strategies to incorporate
cyanomethylphosphonate (23).
a
13C-label in diethyl
envisaged introducing the 13C-label already at the stage of
Conclusions
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Diethyl 2-oxoethylphosphonate (10):20
In this work we showed that the proposed structure of
phosphonocystoximate is consistent with the experimental data.
Moreover, we proved (–)-hydroxynitrilaphos to have (S)-
configuration by independent chemical synthesis and
comparison of the experimental data of our synthetic sample
with that of the natural product. To further investigate how the
Diethyl 2,2-diethoxyethylphosphonate (9, 1.27 g, 5.00 mmol) was
dissolved in aqueous HCl (2 M, 15 mL) and the reaction mixture was
stirred at room temperature for 20 h. The aqueous mixture was extracted
with EtOAc (4 × 10 mL), dried (Na2SO4) and the solvent removed in
vacuo. The crude aldehyde could be purified by bulb-to-bulb distillation
(0.37 mbar / 65-90°C) to obtain 10 as colorless liquid (495 mg, 2.80
mmol, 55%). The product has to be stored at −25°C.
biosynthesis
of
(hydroxy)phosphonocystoximate
and
(hydroxy)nitrilaphos are linked, we designed a labeling strategy
to incorporate a 13C-label in -position to the phosphorus atom in
nitrilaphos and (–)-hydroxynitrilaphos. Feeding experiments with
these 13C-labeled compounds will hopefully help to further
investigate the biosynthesis of these fascinating compounds.
Further, we prepared sufficient quantities of all discussed
compounds to allow bioactivity testing.
H (CDCl3, 400.27 MHz) 9.64 (td, J 3.2 Hz, 0.9 Hz, 1H, CHO), 4.22-
4.09 (m, 4H, -OCH2), 3.05 (dd, J 21.9 Hz, 3.2 Hz, 2H, CH2-P), 1.32 (td, J
7.1 Hz, 0.2 Hz, 6H, CH3); P (CDCl3, 162.03 MHz) 33.65 (∫ 0.06, s,
unknown impurity), 18.90 (∫ 0.94, s, product).
Diethyl 2-hydroxyimino-ethylphosphonate (11):21
Diethyl 2-oxoethylphosphonate (10, 665 mg, 3.7 mmol) was dissolved
in dry EtOH (7 mL) and an aqueous solution of hydroxylamine
hydrochloride (514 mg, 7.4 mmol, dissolved in 3 mL H2O) was added
dropwise at room temperature. The resulting reaction mixture was stirred
for 20 h and the solvent removed in vacuo. The residual solid was
extracted with EtOAc (3 × 20 mL), dried (Na2SO4) and concentrated to
dryness. The crude product was purified by flash chromatography, Rf =
0.21 (EtOAc, iodine for staining) to give the desired oxime 11 as a 3 : 2
mixture of its E- and Z-isomer in sufficient purity for the next reaction
(299 mg, 1.5 mmol, 41%).
Experimental Section
General Experimental
1H, 13C and 31P NMR spectra were recorded in CDCl3 or D2O on a
Bruker Avance AV III 400 (1H: 400.27 MHz, 13C: 100.65 MHz, 31P: 162.03
MHz), AV III 600 (13C: 150.93 MHz) or AV III HD 700 (13C: 176.12 MHz)
spectrometer. Proton NMR chemical shifts were referenced to residual
CHCl3 (H 7.24 ppm) or HOD (H 4.80 ppm). 13C NMR spectra were
referenced to CDCl3 (C 77.23 ppm) and 31P NMR spectra to external
H3PO4 (85%) (P 0.0 ppm). Chemical shifts () are given in ppm and
coupling constants (J) in Hz. IR spectra were run on a Bruker VERTEX
70 IR spectrometer in ATR mode. Optical rotations were measured at
20°C on a Perkin-Elmer 341 polarimeter in a 1 dm cell. []D values are
given in 10-1 deg cm2 g-1. Melting points were determined on a Leica
Galen III Reichert Thermovar instrument and are uncorrected.
H (CDCl3, 400.27 MHz) 7.41 (q, J 6.3 Hz, 0.4H, =C-H), 6.82 (q, J 6.1
Hz,0.6H, =C-H), 4.20-4.02 (m, 4H, -OCH2 both isomers), 3.02 (dd, J 21.7
Hz, 6.1 Hz, 1.2H, P-CH2), 2.76 (dd, J 22.0 Hz, 6.3 Hz, 0.8H, P-CH2) 1.32
(t, J 7.0 Hz, 3.6H, CH3), 1.30 (t, J 7.0 Hz, 2.4H, CH3); P (CDCl3, 162.03
MHz) 33.74 (∫ 0.23, unknown impurity), 23.82 (∫ 0.39, s, Z-oxime), 23.43
(∫ 0.61, s, E-oxime).
TLC was carried out on 0.25 mm thick MilliporeSigma plates coated
with silica gel 60 F254. Spots were visualized by UV light and/or dipping
Diethyl 2-chloro-2-hydroxyimino-ethylphosphonate (12):
the plate into
a solution of (NH4)6Mo7O24·4H2O (25.0 g) and
The E-/Z-mixture of oxime 11 (299 mg, 1.5 mmol) was dissolved in dry
DMF (1 mL) at 0°C and N-chlorosuccinimide (200 mg, 1.5 mmol) was
added in small portions over a period of 30 min. Stirring was continued
for 3 h before addition of toluene (5 mL) and evaporation of the solvent.
The residue was purified by flash chromatography, Rf = 0.31(EtOAc) to
give chloro-oxime 12 (224 mg, 85 mol% purity).
Ce(SO4)2·4H2O (1.0 g) in aqueous 10% H2SO4 (500 mL), followed by
heating with a heat gun, if not stated differently. Flash (column)
chromatography was either performed with Macherey-Nagel silica gel 60
(230-400 mesh) or on a Biotage Isolera Prime flash purification system
using KP-Sil (50 g, 25 g or 10 g) silica cartridges depending on the total
amount of substance.

H (CDCl3, 400.27 MHz) 4.20-4.11 (m, 4H, -OCH2), 3.09 (d, J 20.9 Hz,
THF was refluxed over potassium and distilled prior to use. All other
chemicals were used as purchased from Sigma Aldrich, Acros, Alfa
Aesar or Cambridge Isotope Laboratories.
2H, P-CH2), 1.33 (br t, J 7.1 Hz, 6H, CH3); P (CDCl3, 162.03 MHz) 21.13
(∫ 0.85, s, desired product), 20.72 (∫ 0.08, d, unknown impurity), 15.98 (∫
0.07, s, 13).
Structure verification of phosphonocystoximate
Diethyl 2,2-diethoxyethylphosphonate (9):19
(R)-Methyl N-acetyl cysteinate [(R)-14]:
(R)-N-acetyl cysteine (2.00 g, 12.3 mmol) was dissolved in dry MeOH
(15 mL) under argon at 0°C. Chlorotrimethylsilane (2.00 g, 18.4 mmol,
2.34 mL) was added dropwise and stirring was continued for 45 min at
0°C, followed by 1 h at room temperature. All volatiles were removed in
vacuo and the residue was purified by flash chromatography, Rf = 0.48
(EtOAc), to give the desired methyl ester 14 (1.22 g, 6.9 mmol, 56%) as
colorless solid; all recorded spectra were identical to those reported in
the literature.42
Bromoacetaldehyde diethylacetal (8, 19.71 g, 100 mmol, 15.04 mL)
was added to triethyl phosphite (33.23 g, 200 mmol, 34.26 mL). A Dean-
Stark head was fitted to the reaction flask and the mixture was heated
under argon at 155°C for 4 h. The reaction mixture was cooled to room
temperature and afterwards fractionated under reduced pressure (0.19
mbar/80-86°C) to obtain phosphonoacetal 9 (14.80 g, 58 mmol, 58%) as
colorless oil.
Methyl N-acetyl-S-(2-diethoxyphosphoryl-1-hydroxyiminoethyl)
H (CDCl3, 400.27 MHz) 4.87 (q, J 5.7 Hz, 1H, CH), 4.12-4.03 (m, 4H,
cysteinate (15):22
P-OCH2), 3.58 (ABX-system, 2H, OCH2, A-part: qd, J 7.0 Hz, J 9.3 Hz, B-
part: qd, J 7.0 Hz, J 9.3 Hz), 2.17 (dd, J 5.7 Hz, 18.6 Hz, 2H, CH2-P),
1.29 (td, J 7.0 Hz, 0.3 Hz, 6H, 2 × CH3CH2-O-P), 1.18 (t, J 7.0 Hz, 6H, 2
× CH3), P (CDCl3, 162.03 MHz) 33.65 (∫ 0.04, s, unknown impurity),
26.36 (∫ 0.96, s, product).
Chloro-oxime 12 (525 mg, 2.3 mmol) was dissolved in dry CH2Cl2 (10
mL) at 0°C before addition of (R)-methyl N-acetyl cysteinate [(R)-14, 405
mg, 2.3 mmol] in CH2Cl2 (5 mL). Et3N (233 mg, 2.3 mmol, 0.32 mL) was
added dropwise and stirring was continued for 90 min at 0°C, followed by
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10.1002/chem.201702904
Chemistry - A European Journal
FULL PAPER
2 h at room temperature. The reaction mixture was concentrated and
dried in vacuo. The thus obtained residue was purified by flash
chromatography [EtOAc, Rf = 0.05 (EtOAc), then CH2Cl2/EtOH 19:1, Rf =
0.09 (CH2Cl2/EtOH)] to give the desired product 15 as colorless, highly
viscous oil (409 mg, 1.1 mmol, 48%).
whereupon the mixture turned pale yellow. Stirring was continued for 2 h
during which the reaction mixture became turbid and red. It was diluted
with water (10 mL) and CH2Cl2 (10 mL) and the phases were separated.
The aqueous layer was washed with CH2Cl2 (2 × 10 mL). The combined
organic portions were washed with water (1 × 10 mL), dried (Na2SO4)
and the solvent was removed in vacuo.
[]D20= +6.0 (c = 0.77 in CH2Cl2); H (400.27 MHz, CDCl3) 9.10 (br s,
1H, OH), 7.38 (br d, J 7.5 Hz, 1H, NH), 4.80 (ddd, J 7.5 Hz, 5.8 Hz, 4.4
Hz, 1H, CH), 4.20-4.07 (m, 4H, 2 × OCH2), 3.75 (s, OCH3), 3.60 (ABX-
system, 2H, CH2-S, A-part: dd, J 13.9 Hz, 4.4 Hz; B-part: dd, J 13.9 Hz,
5.8 Hz), 2.99 (ABX-system, 2H, CH2-P, A-part: dd, J 15.7 Hz, 20.7 Hz; B-
part: dd, J 15.7 Hz, 20.8 Hz), 2.03 (s, CH3C(O)-N), 1.32 (t, J 7.1 Hz, 3H,
CH3), 1.31 (t, J 6.9 Hz, 3H, CH3); C (176.12 MHz, CDCl3) 171.0 (s,
C(O)CH3), 170.7 (s, COOMe), 144.2 (d, J 9.6 Hz, C=N), 63.1 (d, J 6.6
Hz, OCH2), 63.0 (d, J 6.7 Hz, OCH2), 53.0 (s, COOCH3), 52.8 (s, CH),
33.0 (s, CH2-S), 32.4 (d, J 141.5 Hz, CH2-P), 23.0 (s, CH3CO-), 16.58 (s,
CH3CH2O-), 16.55 (s, CH3CH2O-); P (162.03 MHz, CDCl3) 23.02 (s);
The residue was purified by column chromatography, Rf = 0.55
(EtOAc), to give diazo compound 20 as yellow oil (426 mg, 2.1 mmol,
77%).
H (400.27 MHz, CDCl3) 4.29-4.15 (m, 4H, 2 × OCH2), 1.39 (td, J 7.1
Hz, 0.8 Hz, 6H, 2 × CH3); C (100.65 MHz, CDCl3) 108.7 (d, J 10.6 Hz,
CN), 64.8 (d, J 5.9 Hz, 2C, 2 × OCH2), 16.3 (d, J 6.7 Hz, 2C, 2 × CH3),
C=N2 not detected; P (162.03 MHz, CDCl3) 8.58 (s).
(±)-Diethyl cyanohydroxymethylphosphonate [(±)-24]:29

max/cm-1 3251, 2986, 1742, 1656, 1544, 1437, 1373, 1220, 1020;
elemental analysis calcd. (%) for C12H23N2O7PS (370.36 g/mol): C 38.92;
H 6.26; N 7.56; O 30.24, S 8.66; found: C 38.90; H 6.38; N 7.36; O
30.61; S 8.53.
Diethyl cyanodiazomethylphosphonate (20, 426 mg, 2.1 mmol), water
(119 mg, 6.6 mmol) and Rh2(OAc)4 (9 mg, 20 mol) were combined in
dry toluene (10 mL). The mixture was slowly heated to 85°C and stirred
for 1 h. The greenish suspension was cooled to room temperature,
filtered over Celite and concentrated in vacuo. The residue was purified
by flash chromatography, Rf = 0.44 (EtOAc), to yield the desired racemic
hydroxyphosphonate (±)-24 as colorless solid (370 mg, 1.9 mmol, 87%).
Phosphonocystoximate as its sodium salt (6):
15 (118 mg, 0.32 mmol) was dissolved in dry 1,2-dichloroethane (5
mL) under argon at room temperature. Allyltrimethylsilane (219 mg, 1.92
mmol, 0.30 mL) and bromotrimethylsilane (811 mg, 3.8 mmol, 0.51 mL)
were added and the solution was stirred at room temperature for 22 h. All
volatiles were removed in vacuo and another portion of 1,2-
dichloroethane (5 mL) was added. The solvent was again evaporated
and this procedure was repeated one more time. The residue was
dissolved in water (3 mL) and an aqueous solution of sodium hydroxide
was added (0.1 M, 1.28 mL). After 45 min, the reaction mixture was
applied to Dowex 50W × 8, H+ and eluted with water. All acidic fractions
were pooled and titrated to pH 7.0 with aqueous NaOH (0.5 M). The
sodium salt of phosphonocystoximate was obtained as colorless powder
(84 mg) after lyophilization.
m.p. 66-69°C; H (400.27 MHz, CDCl3) 4.67 (br d, J 17.6 Hz, 2H, CH-P
+ OH), 4.39-4.21 (m, 4H, 2 × OCH2), 1.40 (td, J 7.0 Hz, 0.6 Hz, 3H, CH3),
1.38 (td, J 7.1 Hz, 0.5 Hz, 3H, CH3); C (100.65 MHz, CDCl3) 115.4 (s,
CN), 65.9 (d, J 7.4 Hz, OCH2), 65.1 (d, J 7.1 Hz, OCH2), 57.7 (d, J 169.4
Hz , CH-P), 16.60 (d, J 5.3 Hz, CH3), 16.58 (d, J 5.3 Hz, CH3); P (162.03
MHz, CDCl3) 12.36 (s);

max/cm-1 3233, 2988, 2916, 1248, 1020;
elemental analysis calcd. (%) for C6H12NO3P (193.14 g/mol): C 37.31; H
6.26; N 7.25; O 33.13; found: C 37.43; H 6.21; N 6.75; O 32.93.
Acetals of (±)-diethyl cyanohydroxymethylphosphonate [(±)-24]
derived from (+)-Noe´s lactol (25a and 25b):32
NMR spectroscopic analysis using 2-aminoethylphosphonic acid as
internal standard showed the obtained powder to contain 2.5 mol
phosphonocystoximate per mg. Thus, the obtained powder equals a total
amount of 0.21 mmol of the free phosphonic acid (66% yield).
The crude -hydroxyphosphonate [(±)-24, 370 mg, 1.9 mmol], (+)-
Noe´s lactol dimer [(+)-MBF-OH dimer, 431 mg, 1.1 mmol] and p-toluene
sulfonic acid monohydrate (14 mg, 74 mol) were dissolved in dry CH2Cl2
(10 mL). Molecular sieves (4 Å) were added and the resulting mixture
was stirred for 1 h at room temperature. The solution was filtered, and
the solvent was removed under reduced pressure.
H (400.27 MHz, D2O) 4.45 (dd, J 7.9 Hz, 4.1 Hz, 1H, CH-N), 3.55
(ABX-system, 2H, CH2-S, A-part: dd, J 13.4 Hz, 4.1 Hz; B-part: dd, J 13.4
Hz, 7.9 Hz), 2.91 (ABP-system, 2H, CH2-P, A-part: dd, J1=J2 17.9 Hz; B-
part: dd, J1=J2 17.9 Hz), 2.10 (s, CH3C(O)-N); C (150.93 MHz, D2O)
176.2 (s, C=O), 173.8 (s, C=O), 153.7 (d, J 10.1 Hz, C=N), 54.7 (s, CH),
33.0 (d, J 124.4 Hz, CH2-P), 32.2 (s, CH2-S), 21.8 (s, CH3-); P (162.03,
D2O) 13.76 (s); max/cm-1 3250, 2850, 2452, 1598, 1392, 1067.
The diastereomeric lactols 25a and 25b were separated by six
subsequent flash chromatography runs under the same conditions
[heptanes/EtOAc, gradient: 12% EtOAc  100% EtOAc, Rf (25a) = 0.52
(heptanes/EtOAc 1:1) and Rf (25b) = 0.39 (heptanes/EtOAc 1:1)].
Fractions containing only diastereomer A or B were pooled separately
and set aside. Mixed fractions were combined and subjected to the next
separation run. This yielded the diastereomeric acetals 25a (226 mg, 0.6
mmol, 32%) and 25b (233 mg, 0.6 mmol, 32%) as colorless oils.
Determination
of
the
absolute
configuration
of
(−)-
hydroxynitrilaphos:
Diethyl cyanodiazomethylphosphonate (20):28
Diastereomer 25a: []D20= +162.3 (c = 1.09 in CH2Cl2); H (400.27
MHz, CDCl3) 5.50 (d, J 4.9 Hz, 1H, O-CH-O), 4.79 (d, J 19.8 Hz, 1H, CH-
P), 4.31-4.21 (m, 5H, 2 × OCH2 + 1 × CH-O), 2.96-2.85 (m, 1H, CH),
1.94 (ABX-system, 2H, CH2, A-part: ddd, J 14.1 Hz, 6.9 Hz, 5.0 Hz; B-
part: dd, J 14.1 Hz, 10.0 Hz), 1.63-1.58 (m, 1H, CH), 1.61 (ABX-system,
2H, CH2, A-part: m; B-part: m), 1.42-1.31 (m, 7H, 2 × CH3CH2O + 1H
from ABX-system), 1.25-1.12 (m, 1H, 1H from ABX-system), 0.94 (3 H, s,
CH3), 0.91 (3 H, s, CH3), 0.85 (3 H, s, CH3); C (150.93 MHz, CDCl3)
114.5 (d, J 1.8 Hz, CN), 107.6 (d, J 10.9 Hz, O-CH-O), 91.2 (s, CH-O),
65.0 (d, J 6.6 Hz, OCH2), 64.9 (d, J 6.7 Hz, OCH2), 58.9 (d, J 177.9 Hz,
CH-P), 53.1 (s, Cq), 48.9 (s, Cq), 47.4 (s, CH), 39.8 (s, CH), 32.2 (s, CH2),
26.5 (s, CH2), 21.1 (s, CH3), 20.6 (s, CH2), 18.9 (s, CH3), 16.61 (d, J 5.8
Hz, CH3CH2O), 16.59 (d, J 5.8 Hz, CH3CH2O), 14.9 (s, CH3); P (162.03
1,3-Dimethyl-2-chloroimidazolinium chloride (980 mg, 5.8 mmol) was
weighed into a flame dried reaction flask, which was immediately purged
with argon. Sodium azide (234 mg, 3.6 mmol) was added to the solution
of the imidazolinium chloride in dry CH3CN (15 mL) under argon at 0°C
and the resulting mixture was stirred at 0°C for 30 min, whereupon it
turned beige and turbid.
Meanwhile Et3N (587 mg, 5.8 mmol, 0.80 mL) was added to a stirred
solution of diethyl cyanomethylphosphonate (23, 495 mg, 2.8 mmol) in
dry THF (15 mL) under argon at room temperature. The phosphonate
solution was added dropwise to the diazo transfer reagent solution at 0°C
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10.1002/chem.201702904
Chemistry - A European Journal
FULL PAPER
MHz, CDCl3) 10.94 (s); max/cm-1 2986, 2954, 1273, 1022, 980; HRMS
NMR spectroscopic analysis using 2-aminoethylphosphonic acid as
internal standard showed the obtained powder to contain 5.3 mol (−)-
hydroxynitrilaphos per mg. Thus, the obtained powder equals a total
amount of 0.47 mmol of the free phosphonic acid (89% yield) and its
composition is very close to the disodium salt of (−)-hydroxynitrilaphos.
calcd. for C18H30NO5P: [M-H]- 3370.1789; found: [M-H]- 370.1824.
Diastereomer 25b: []D20= +81.6 (c = 1.04 in CH2Cl2); H (400.27
MHz, CDCl3) 5.45 (d, J 4.6 Hz, 1H, O-CH-O), 4.54 (d, J 18.3 Hz, 1H, CH-
P), 4.41 (dd, J 9.5 Hz, J 1.4 Hz, 1H, CH-O), 4.37-4.21 (m, 4H, 2 × OCH2),
3.03-2.92 (m, 1H, CH), 1.91 (ABX-system, 2H, CH2, A-part: ddd, J 14.0
Hz, 7.1 Hz, 4.8 Hz; B-part: dd, J 14.0 Hz, 4.0 Hz), 1.63-1.58 (m, 1H, CH),
1.59 (ABX-system, 2H, CH2, A-part: ddd, J 17.5 Hz, 9.4 Hz, 4.9 Hz; B-
part: m), 1.41-1.29 (m, 1H, 1H from AB system), 1.38 (td, J 7.1 Hz, 0.4
Hz, 3H, CH3CH2O-), 1.37 (td, J 7.1 Hz, 0.4 Hz, 3H, CH3CH2O-), 1.23-
1.13 (m, 1H, 1H from AB system), 0.97 (3 H, s, CH3), 0.90 (3 H, s, CH3),
0.88 (3 H, s, CH3); C (150.93 MHz, CDCl3) 115.4 (s, CN), 111.3 (d, J 8.0
Hz, O-CH-O), 91.3 (s, CH-O), 65.2 (d, J 6.7 Hz, OCH2), 64.7 (d, J 7.0 Hz,
OCH2), 60.5 (d, J 174.8 Hz, CH-P), 53.1 (s, Cq), 48.9 (s, Cq), 47.6 (s,
CH), 40.0 (s, CH), 32.8 (s, CH2), 26.6 (s, CH2), 21.1 (s, CH3), 20.6 (s,
CH2), 19.0 (s, CH3), 16.64 (d, J 5.3 Hz, CH3CH2O), 16.63 (d, J 5.6 Hz,
CH3CH2O), 14.8 (s, CH3); P (162.03 MHz, CDCl3) 10.90 (s, ∫ 0.97, 25b),
12.42 (s, ∫ 0.03, 24); max/cm-1 3235, 2984, 2951, 1447, 1392, 1372,
1267, 1103, 1031, 979; HRMS calcd. for C18H30NO5P: [M-H]- 370.1789;
found: [M-H]- 370.1827.
[]D20= −16.7 (c = 0.69 in D2O),43 H (400.27 MHz, D2O) 4.48 (d, J 16.0
Hz, 1H, CH-P); C (150.93 MHz, D2O) 120.72 (s, CN), 59.10 (d, J 136.3
Hz, CH-P); P (162.03 MHz, D2O) 10.23 (s).
The optical rotation of the free phosphonic acid is more difficult to
determine than for the disodium salt as it is very hygroscopic. Therefore,
the sample of the disodium salt, which was taken to determine the optical
rotation, was passed through Amberlyst 15, H+ (3 mL) and the resin was
washed with 10 mL MeOH. MeOH was then removed in vacuo and the
residue diluted to
a
total volume of
2
mL with D2O. 2-
Aminoethylphosphonic acid (2.4 mg) was added to 0.7 mL of the solution
as internal standard. 31P NMR of this mixture showed the D2O solution to
contain 3.3 mg (−)-hydroxynitrilaphos per mL (this equals a concentration
of 0.33). The remaining 1.3 mL were used to measure the optical rotation
of the free phosphonic acid.
[]D20= −24.2 (c = 0.33 in D2O)
Deprotection of acetals 25a and 25b:
p-Toluene sulfonic acid monohydrate (109 mg, 0.57 mmol) was added
to a stirred solution of diastereomer 25b (225 g, 0.60 mmol) in a mixture
of dry CH2Cl2 (3 mL) and dry methanol (1 mL) at room temperature. The
resulting solution was stirred for 1 h and then concentrated under
reduced pressure. The residue was purified by flash chromatography
[heptanes/EtOAc, gradient: 12% EtOAc  100% EtOAc, Rf = 0.10
(EtOAc)] to give (−)-24 (98 mg, 0.51 mmol, 89%) as a colorless solid;
[]D20= −30.9 (c = 0.47 in CH2Cl2).
The enantiomeric purity of the sodium salt of (−)-hydroxynitrilaphos
could not be determined using the same chiral solvating agent as for the
precursor substances (due to solubility problems). Therefore the ee of
the sample had to be determined after derivatization with diazomethane
(CH2N2):
(−)-Dimethyl cyanohydroxymethylphosphonate [(−)-28]:
The sodium salt of (−)-hydroxynitrilaphos (4, 13 mg, equivalent to 69
mol, see above) was dissolved in water (0.25 mL) and charged on
Amberlyst 15, H+ (4 mL). It was eluted with water (0.75 mL) and MeOH
(20 mL). The eluate was cooled to −45°C and a distilled ethereal CH2N2
solution was added until a yellow color persisted. All volatiles were
immediately removed under reduced pressure at room temperature.
CH2Cl2 (5 mL) was added and the solvent was again evaporated. Then,
CDCl3 (1 mL) was added, the solution was filtered over cotton and
evaporated similarly. Finally, the obtained sample was dried in vacuo to
constant weight (1 h). The desired product 28 was obtained as glassy
solid (8 mg, 48 mol, 70%).
(+)-24 Was prepared similarly starting from diastereomer 25a (212 mg,
0.57 mmol); colorless oil (88 mg, 0.46 mmol, 80%); []D20= +31.5 (c =
0.39 in CH2Cl2).
The measured spectroscopic data of both enantiomers were identical
to those of the racemic compound. The ee of each samples was
determined by addition of (+)-(R)-tert-butylphenylphosphinothioic acid {ee
≥ 93%, []D20= +28.7 (c = 1.01 in MeOH)} to an NMR probe of the
compound. Relevant NMR signals are denoted below:
NMR of (−)-24 with chiral solvating agent: P (162.03 MHz, CDCl3)
98.03 (∫ 1.79, chiral solvating agent), 12.28 [∫ 0.97, complex of chiral
solvating agent with (−)-24], 12.07 [∫ 0.03, complex of chiral solvating
agent with (+)-24]  ee ≥ 94%.
[]D20= −35.2 (c = 0.25 in CDCl3); H (400.27 MHz, CDCl3) 4.73 (d, J
17.4 Hz, 1H, CH-P), 3.97 (d, J 10.8 Hz, 3H, OCH3), 3.91 (d, J 10.9 Hz,
3H, OCH3); C (150.93 MHz, CDCl3) 115.2 (s, CN), 57.3 (d, J 171.9 Hz,
63.1, CH-P), 55.9 (d, J 7.0 Hz, OCH3), 55.3 (d, J 7.2 Hz, OCH3); P
(162.03 MHz, CDCl3) 14.56 (s).
NMR of (+)-24 with chiral solvating agent: P (162.03 MHz, CDCl3)
98.34 (∫ 1.68, chiral solvating agent), 12.30 [∫ 0.02, complex of chiral
solvating agent with (−)-24], 12.14 [∫ 0.98, complex of chiral solvating
agent with (+)-24]  ee ≥ 96%.
The ee of the sample was determined by addition of (+)-(R)-tert-
butylphenylphosphinothioic acid (ee ≥ 93%) to an NMR probe of the
compound; relevant signals are denoted below:
(−)-Hydroxynitrilaphos as its sodium salt [(−)-4]:
NMR of (−)-28 with chiral solvating agent: P (162.03 MHz, CDCl3)
98.05 (∫ 3.17, chiral solvating agent), 14.49 [∫ 0.98, complex of chiral
solvating agent with (−)-28], 14.24 [∫ 0.02, complex of chiral solvating
agent with (+)-28]  ee ≥ 96%.
(−)-24 (102 mg, 0.53 mmol) was dissolved in dry 1,2-dichloroethane (5
mL) under argon at room temperature. Allyltrimethylsilane (300 mg, 2.6
mmol, 0.42 mL) and bromotrimethylsilane (811 mg, 5.3 mmol, 0.70 mL)
were added and the solution was stirred at room temperature for 17 h. All
volatiles were removed in vacuo and another portion of 1,2-
dichloroethane (5 mL) was added. The solvent was again evaporated
and this procedure was repeated again. An aqueous solution of sodium
hydroxide was added (0.1 M, 6.3 mL at once, followed by dropwise
addition) until the pH of the product solution was 7.0. The sodium salt of
(−)-hydroxynitrilaphos was obtained as colorless powder (89 mg) after
lyophilization.
(−)-(R)-2-Amino-1-hxdroxyethylphosphonic acid [(–)-26]:
BH3∙THF (3.72 mL, 1 M solution in THF) was added dropwise to a
stirred solution of (+)-diethyl cyanohydroxymethylphosphonate [(+)-24, 91
mg, 0.47 mmol) in dry THF (5 mL) under argon at 0°C. The reaction
mixture was stirred for 30 min before careful addition of MeOH (5 mL).
The solvent was evaporated and the residue was dissolved in HCl (10
mL, 6 M). The solution was refluxed for 8 h, cooled to room temperature
This article is protected by copyright. All rights reserved.

10.1002/chem.201702904
Chemistry - A European Journal
FULL PAPER
and concentrated. The residual liquid was dried in vacuo at 100°C for 2 h
before applying it to Dowex 50W × 8, H+ (29 mL, water as eluent). The
product containing fractions {Rf = 0.18 [iPrOH/H2O/NH3(25%)] 6:3:2; 1%
ninhydrin in EtOH for staining} were pooled and concentrated under
reduced pressure. The residue was dried in vacuo at 60°C for 2 h to give
(−)-(R)-2-amino-1-hydroxyethylphosphonic acid (–)-26 as colorless solid
(47 mg, 0.33 mmol, 71%).
(EtOAc), to give diazo compound [2-13C]20 as yellow oil (594 mg, 2.9
mmol, 77%).
H (400.27 MHz, CDCl3) 4.29-4.15 (m, 4H, 2 × OCH2), 1.39 (td, J 7.1
Hz, 0.8 Hz, 6H, 2 × CH3); C (100.65 MHz, CDCl3, not j-modulated) 108.7
(d, J 10.8 Hz, 13CN), 64.8 (d, J 6.0 Hz, 2C, 2 × OCH2), 16.3 (d, J 6.7 Hz,
2C, 2 × CH3), C=N2 not detected; P (162.03 MHz, CDCl3) 8.58 (d, J 11.2
Hz).
[]D20= −25.7 (c = 0.75 in D2O); H (400.27 MHz, D2O) 4.09-3.95 (m,
1H, CH-P), 3.31 (AB-system, 2H, CH2, A-part: m, B-part: m); P (162.03
MHz, D2O) 17.66 (s).16
(±)-Diethyl [2-13C]cyanohydroxymethylphosphonate {(±)-[2-13C]24}:
(±)-[2-13C]24 was prepared by the same procedure as (±)-24, using
Synthesis of the 13C-labeled substances:
[1-13C]Chloroacetonitrile ([1-13C]36):40
the
following
substance
amounts:
Diethyl
[2-
13C]cyanodiazomethylphosphonate ([2-13C]20, 594 mg, 2.9 mmol), water
(156 mg, 8.7 mmol) and Rh2(OAc)4 (25 mg, 57 mol) in dry toluene (15
mL).
Sodium
[
13C]cyanide (99% 13C, 1.12 g, 22.4 mmol) and
paraformaldehyde (691 mg, 23.0 mmol) were dissolved in water (8 mL)
and the pH of the solution was adjusted to 2.0 with H2SO4 (conc.). The
mixture was stirred at 0°C for 2 h before addition of CH2Cl2 (100 mL). [1-
13C]Hydroxyacetonitrile was extracted continuously into CH2Cl2 over 20
h. The solvent was removed in vacuo. The crude [1-13C]hydroxynitrile
was again dissolved in dry CH2Cl2 (50 mL), pyridine (5.84 g, 73.9 mmol,
5.96 mL) and SOCl2 (6.13 g, 51.5 mmol, 3.70 mL) were added slowly at
0°C and the solution was allowed to come to room temperature over
night (19 h). Brine (5 mL) was added slowly (gas evolution!) and the
phases were separated. The organic phase was dried (Na2SO4) and
CH2Cl2 was removed by distillation (ambient pressure). The red, oily
residue was purified by bulb-to-bulb distillation (97 Torr/65-90°C) to give
[1-13C]chloroacetonitrile ([1-13C]36, 1.12 g, 14.6 mmol, 65%) as colorless
liquid.
The residue was purified by flash chromatography [heptanes/EtOAc,
gradient: 24% EtOAc  100% EtOAc, Rf = 0.44 (EtOAc)] to yield the
desired hydroxyphosphonate (±)-[2-13C]24 as colorless solid (429 mg, 2.2
mmol, 76%).
mp 66-69°C; H (400.27 MHz, CDCl3) 5.23 (br s, 1H, OH), 4.68 (dd, J
17.1 Hz, 9.4 Hz, 1H, CH-P), 4.41-4.21 (m, 4H, 2 × OCH2), 1.40 (td, J 7.1
Hz, 0.4 Hz, 3H, CH3), 1.37 (td, J 7.1 Hz, 0.4 Hz, 3H, CH3); C (150.93
MHz, CDCl3, not j-modulated) 115.4 (s, 13CN), 66.0 (d, J 7.2 Hz, OCH2),
65.2 (d, J 7.1 Hz, OCH2), 57.6 (dd, J 170.5 Hz, 63.7, CH-P), 16.59 (d, J
5.2 Hz, CH3), 16.56 (d, J 5.1 Hz, CH3); P (162.03 MHz, CDCl3) 12.36 (s);

max/cm-1 3233, 2988, 2916, 1248, 1020; HRMS calcd. for [2-
13C]C6H12NO3P: [M+Na]+ 217.0430; found: [M+Na]+ 217.0423.
Acetals of (±)-diethyl [2-13C]cyanohdroxymethylphospho-nate (±)-24
with (+)-Noe´s lactol ([2-13C]25a and [2-13C]25b):32
H (400.27 MHz, CDCl3) 4.08 (d, J 7.9 Hz, 2H, CH2).
[2-13C]Diethyl cyanomethylphosphonate ([2-13C]23):
[2-13C]25a and [2-13C]25b can be prepared by the same procedure as
25a and 25b, using the following substance amounts: -
hydroxyphosphonate {(±)-[2-13C]24, 429 mg, 2.2 mmol}, (+)-Noe´s lactol
dimer[(+)-MBF-OH dimer, 494 g, 1.3 mmol] and p-toluene sulfonic acid
monohydrate (17 mg, 89 mol) in dry CH2Cl2 (10 mL).
[1-13C]Chloroacetonitrile ([1-13C]36, 583 mg, 7.6 mmol) and triethyl
phosphite (1263 mg, 7.6 mmol, 1.06 mL) were combined and heated to
180°C under reflux. After 1 h an additional amount of triethyl phosphite
(249 mg, 1.5 mmol, 0.21 mL) was added and heating was continued for 2
h. The product [2-13C]23 was obtained from the reaction mixture by bulb-
to-bulb distillation (0.35 mbar/100-150°C) in 77 mol% purity (1.21 g,
equals about 6.1 mmol, 80%). It was used for the following reaction
without further purification.
The diastereomeric lactols [2-13C]25a and [2-13C]25b were separated
by the same method as the unlabeled compounds 25a and 25b: [2-
13C]25a (276 mg, 0.74 mmol, 34%) and [2-13C]25b (343 mg, 0,92 mmol,
42%); colorless oils.
Diastereomer [2-13C]25a: []D20= +163.13 (c = 1.06 in CH2Cl2); H
(400.27 MHz, CDCl3) 5.50 (d, J 4.8 Hz, 1H, O-CH-O), 4.78 (dd, J 19.8
Hz, 7.2 Hz, 1H, CH-P), 4.33-4.19 (m, 5H, 2 × OCH2 + 1 × CH-O), 2.91
(m, 1H, CH), 1.94 (ABX-system, 2H, CH2, A-part: ddd, J 14.0 Hz, 6.9 Hz,
5.0 Hz; B-part: dd, J 14.0 Hz, 10.0 Hz), 1.63 (ABX-system, 2H, CH2, A-
part: ddd, J 13.0 Hz, 9.4 Hz, 4.9 Hz; B-part: m), 1.28 (ABX-system, 2H,
CH2, A-part: m; B-part: m), 1.37 (m, 6H, 2 × CH3CH2O), 0.94 (3 H, s,
CH3), 0.91 (3 H, s, CH3), 0.85 (3 H, s, CH3); C (150.93 MHz, CDCl3, not
j-modulated) 114.5 (d, J 1.5 Hz, 13CN), 107.6 (d, J 10.9 Hz, O-CH-O),
91.1 (s, CH-O), 64.9 (d, J 6.6 Hz, OCH2), 64.8 (d, J 6.6 Hz, OCH2), 58.8
(dd, J 177.8 Hz, 60.6 Hz, CH-P), 53.0 (s, Cq), 48.9 (s, Cq), 47.4 (s, CH),
39.8 (s, CH), 32.2 (s, CH2), 26.5 (s, CH2), 21.1 (s, CH3), 20.6 (s, CH2),
18.9 (s, CH3), 16.60 (d, J 5.5 Hz, CH3CH2O), 16.59 (d, J 5.5 Hz,
CH3CH2O), 14.9 (s, CH3); P (162.03 MHz, CDCl3) 10.93 (s); max/cm-1
2985, 2952, 2933, 1447, 1392, 1372, 1272, 1040, 1021, 979; HRMS
calcd. for [2-13C]C18H30NO5P: [M+Na]+ 395.1787, [M2+Na]+ 767.3682;
found: [M+Na]+ 395.1779, [M2+Na]+ 767.3667.
A small sample (400 mg) was distilled two more times (0.31-0.68
mbar/98-115°C) for analytical purpose.
H (400.27 MHz, CDCl3) 4.30-4.16 (m, 4H, 2 × OCH2), 2.84 (dd, J 20.9
Hz, 10.6 Hz, 2H, CH2-P), 1.38 (td, J 7.1 Hz, 0.3 Hz, 6H, 2 × CH3); C
(150.93 MHz, CDCl3, not j-modulated) 112.8 (d, J 11.1 Hz, 13CN), 64.1
(d, J 6.6 Hz, 2C, 2 × OCH2), 16.7 (dd, J 144.0 Hz, 60.2 Hz, CH2-P), 16.5
(d, J 5.8 Hz, 2C, 2 × CH3); P (162.03 MHz, CDCl3) 14.22 (d, J 11.7 Hz);

max/cm-1 3055, 2985, 2916, 1266, 1023, 976; HRMS calcd. for [2-
13C]C6H12NO3P: [M+H]+ 179.0661, [M+Na]+ 201.0481; found: [M+H]+
179.0621, [M+Na]+ 201.0437.
Diethyl [2-13C]cyanodiazomethylphosphonate ([2-13C]20):
[2-13C]20 was prepared by the same procedure as 20, using the
following substance amounts: 1,3-Dimethyl-2-chloroimidazolinium
chloride (1.82 g, 10.7 mmol) and sodium azide (357 mg, 5.5 mmol) in dry
CH3CN (20 mL); Et3N (850 mg, 8.4 mmol, 1.16 mL) and [2-13C]diethyl
cyanomethylphosphonate ([2-13C]23, 753 mg, 77 mol% purity, about 3.7
mmol) in dry THF (20 mL).
Diastereomer [2-13C]25b: []D20= +80.8 (c = 1.15 in CH2Cl2); H
(400.27 MHz, CDCl3) 5.45 (d, J 4.6 Hz, 1H, O-CH-O), 4.53 (dd, J 18.3
Hz, 8.9 Hz, 1H, CH-P), 4.40 (dd, J 9.5 Hz, J 1.2 Hz, 1H, CH-O), 4.35-4.20
(m, 4H, 2 × OCH2), 2.97 (m, 1H, CH), 2.00-1.83 (m, 2H, CH2), 1.72-1.58
The product was purified by column chromatography, Rf = 0.55
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10.1002/chem.201702904
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(m, 2H, CH2), 1.410-1.28 (m, 7H, 2 × CH3CH2O-+ 1H from an A
system), 1.23 (s, 1H, CH), 1.22-1.12 (m, 1H, 2H from AB system), 0.97
(3 H, s, CH3), 0.91 (3 H, s, CH3), 0.88 (3 H, s, CH3); C (150.93 MHz,
CDCl3, not j-modulated) 115.4 (s, 13CN), 111.3 (d, J 7.8 Hz, O-CH-O),
91.3 (s, CH-O), 65.2 (d, J 6.7 Hz, OCH2), 64.7 (d, J 7.4 Hz, OCH2), 60.5
(dd, J 174.4 Hz, 65.2 Hz, CH-P), 53.1 (s, Cq), 48.9 (s, Cq), 47.5 (s, CH),
40.0 (s, CH), 32.8 (s, CH2), 26.6 (s, CH2), 21.1 (s, CH3), 20.6 (s, CH2),
19.0 (s, CH3), 16.62 (d, J 5.4 Hz, CH3CH2O), 16.61 (d, J 5.9 Hz,
CH3CH2O), 14.8 (s, CH3); P (162.03 MHz, CDCl3) 10.90 (s); max/cm-1
2984, 2951, 2926, 2872, 1447, 1392, 1372, 1269, 1103, 1035, 979;
HRMS calcd. for [2-13C]C18H30NO5P: [M+Na]+ 395.1787, [M2+Na]+
767.3682; found: [M+Na]+ 395.1779, [M2+Na]+ 767.3667.
(−)-(S)-Dimethyl [2-13C]cyanohydroxymethylphosphonate {(−)-[2-
13C]28}:
The sodium salt of (−)-[2-13C]hydroxynitrilaphos {(−)-[2-13C]4, 7.8 mg,
equivalent to 41 mol} was dissolved in water (0.2 mL) and charged on
Amberlyst 15, H+ (3 mL). It was eluted with water (0.2 mL) and MeOH (10
mL). The obtained solution was cooled to −45°C and distilled ethereal
CH2N2 solution was added until a yellow color persisted. All volatiles
were immediately removed under reduced pressure at room temperature.
CH2Cl2 (2 mL) was added and the solvent was again evaporated. Then
CDCl3 (2 mL) was added and evaporated similarly. Finally, the obtained
sample was dried in vacuo to a constant weight (1 h). The desired
product (−)-(S)-[2-13C]28 was obtained as glassy solid (4 mg, 24 mol,
59%).
(−)-(S)-Diethyl [2-13C]cyanohydroxymethylphosphonate {(−)-[2-
13C]24}:
H (400.27 MHz, CDCl3) 4.72 (dd, J 17.3 Hz, 9.5 Hz, 1H, CH-P), 3.97
(d, J 10.8 Hz, 3H, OCH3), 3.91 (d, J 10.9 Hz, 3H, OCH3); C (150.93
MHz, CDCl3, not j-modulated) 115.2 (s, 13CN), 57.3 (dd, J 170.9 Hz, 63.1,
CH-P), 55.9 (d, J 6.8 Hz, OCH3), 55.2 (d, J 7.6 Hz, OCH3); P (162.03
MHz, CDCl3) 14.58 (s).
(−)-[2-13C]24 can be prepared by the same procedure as (−)-24, using
the following substance amounts: p-toluene sulfonic acid monohydrate
(143 mg, 0.75 mmol) and diastereomer [2-13C]25b (295 mg, 0.79 mmol)
in a mixture of dry CH2Cl2 (3 mL) and dry methanol (1 mL).
The residue was purified by flash chromatography [heptanes/EtOAc,
The ee of the sample was determined by addition of (+)-(R)-tert-
butylphenylphosphinothioic acid (ee ≥ 93%) to an NMR probe of the
compound; relevant signals as denoted below:
gradient: 12% EtOAc  100% EtOAc, Rf = 0.11 (heptanes/EtOAc 1:1)] to
20
give (−)-[2-13C]24 (117 mg, 0.60 mmol, 76%) as a colorless solid); []D
=
−29.2 (c = 0.47 in CH2Cl2).
NMR of (−)-[2-13C]28 with chiral solvating agent: P (162.03 MHz,
CDCl3) 98. (∫ 2.56, chiral solvating agent), 14.49 [∫ 0.96, complex of chiral
solvating agent with (−)-28], 14.24 [∫ 0.04, complex of chiral solvating
agent with (+)-28]  ee ≥ 92%.
The spectroscopic data was identical to that of the racemic compound
(±)-[2-13C]24. The ee of the sample was determined by addition of (+)-
(R)-tert-butylphenylphosphinothioic acid (ee ≥ 93%) to an NMR probe of
the compound; relevant signals are denoted below:
[2-13C]Nitrilaphos as its sodium salt ([2-13C]5):
NMR of (−)-[2-13C]24 with chiral solvating agent: P (162.03 MHz,
CDCl3) 98.45 (∫ 3.27, chiral solvating agent), 12.26 [∫ 0.97, complex of
chiral solvating agent with (−)-24], 12.01 [∫ 0.03, complex of chiral
solvating agent with (+)-24]  ee ≥ 94%.
Diethyl [2-13C]cyanomethylphosphonate ([2-13C]23, 186 mg, 1.0 mmol)
was dissolved in dry 1,2-dichloroethane (5 mL) under argon at room
temperature. Allyltrimethylsilane (570 mg, 5.0 mmol, 0.79 mL) and
bromotrimethylsilane (1.60 g, 10.4 mmol, 1.38 mL) were added and the
resulting reaction mixture was stirred at room temperature for 20 h. All
volatiles were removed in vacuo and another portion of 1,2-
dichloroethane (5 mL) was added. The solvent was again evaporated
and this procedure was repeated one more time. An aqueous solution of
sodium hydroxide (0.52 M) was added dropwise until the pH of solution
reached 7.1. The sodium salt of [2-13C]nitrilaphos was obtained as
colorless powder (65 mg) after lyophilization.
(−)-(S)-[2-13C]Hydroxynitrilaphos as its sodium salt {(−)-[2-13C]4}:
(−)-[2-13C]24 (102 mg, 0.53 mmol) was dissolved in dry 1,2-
dichloroethane (5 mL) under argon at room temperature.
Allyltrimethylsilane
(300
mg,
2.6
mmol,
0.38
mL)
and
bromotrimethylsilane (928 mg, 6.1 mmol, 0.80 mL) were added and the
solution was stirred at room temperature for 22 h. All volatiles were
removed in vacuo and another portion of 1,2-dichloroethane (5 mL) was
added. The solvent was again evaporated and this procedure was
repeated one more time. An aqueous solution of sodium hydroxide was
added (0.52 M, 1.9 mL at once followed by dropwise addition) until the
pH of the product solution was 6.9. CH2Cl2 (2 mL) was added and the
biphasic mixture was stirred for 2 min. The aqueous layer was separated
and residual CH2Cl2 was removed under reduced pressure. The sodium
salt of (−)-[2-13C]hydroxynitrilaphos was obtained as colorless powder
(89 mg) after lyophilization.
NMR spectroscopic analysis using 2-aminoethylphosphonic acid as
internal standard showed the obtained powder to contain 5.5 mol (−)-[2-
13C]nitrilaphos per mg. Thus, the obtained powder equals a total amount
of 0.36 mmol of the free phosphonic acid (36% yield) in 95 mol% purity.
H (400.27 MHz, D2O) 2.61 (dd, J 10.2 Hz, 17.5 Hz, 2H, CH-P); C
(150.93 MHz, D2O, not j-modulated) 119.6 (d, J 9.1 Hz, 13CN), 17.5 (dd, J
113.7 Hz, 55.5 Hz, CH-P); P (162.03 MHz, D2O) 13.81 (∫ 0.05, s,
unknown impurity), 7.20 (∫ 0.95, product, d, J 9.1 Hz); max/cm-1 3239,
2901, 2185, 1162, 1132, 1099, 984.
NMR spectroscopic analysis using 2-aminoethylphosphonic acid as
internal standard showed the obtained powder to contain 5.3 mol (−)-[2-
13C]hydroxynitrilaphos per mg. Thus, the obtained powder equals a total
amount of 0.47 mmol of the free phosphonic acid (89% yield).
Acknowledgements
H (400.27 MHz, D2O) 4.48 (dd, J 16.0 Hz, 7.8 Hz, 1H, CH-P); C
(150.93 MHz, D2O, not j-modulated) 120.72 (s, 13CN), 59.09 (dd, J 136.3
Hz, 57.4 Hz, CH-P); P (162.03 MHz, D2O) 7.66 (s); max/cm-1 3121,
2814, 2719, 1090, 972.
We greatly acknowledge the financial support by the Austrian
Science Fund (FWF): P27987-N28. We thank S. Felsinger for
recording NMR spectra and J. Theiner for performing
combustion analysis. Further we thank J. Schörghuber for
sharing her methodology on introducing 13C-labels.
The ee of the free phosphonic acid was determined to be ≥92% after
derivatization with diazomethane (CH2N2):
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Keywords: phosphonate • nitrile • natural product • biosynthesis
• hydroxynitrilaphos • hydroxyphosphonocystoximate • absolute
configuration
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