On the reaction of carbonyl diphosphonic acid with hydroxylamine and O-alkylhydroxylamines: Unexpected degradation of P-C-P bridge

Article abstract of DOI:10.3390/molecules22071040

Derivatives of methylenediphosphonic acid possess wide spectra of biological activities and are used in enzymology as research tools as well as in practical medicine. Carbonyl diphosphonic acid is a promising starting building block for synthesis of functionally substituted methylenediphosphonates. Investigation of the interaction of carbonyl diphosphonic acid with hydroxylamine clearly demonstrates that it is impossible to isolate oxime within the pH range 2–12, while only cyanophosphonic and phosphoric acids are the products of the fast proceeding Beckmann-like fragmentation. In the case of O-alkylhydroxylamines, corresponding alcohols are found in the reaction mixtures in addition to cyanophosphonic and phosphoric acids. Therefore, two residues of phosphonic acid being attached to a carbonyl group provide new properties to this carbonyl group, making its oximes very unstable. This principally differs carbonyl diphosphonic acid from structurally related phosphonoglyoxalic acid and other α-ketophosphonates.

Full text of DOI:10.3390/molecules22071040

molecules
Article
On the Reaction of Carbonyl Diphosphonic Acid
with Hydroxylamine and O-alkylhydroxylamines:
Unexpected Degradation of P-C-P Bridge
Olga A. Khomich 1 , Dmitry V. Yanvarev , Roman A. Novikov , Alexey B. Kornev ,
,†
1,†
1
1
2
2
1
1,
Elina Puljulla , Jouko Vepsäläinen , Alex R. Khomutov and Sergey N. Kochetkov *
1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 11991 Moscow, Russia;
o.a.khomich@gmail.com (O.A.K.); JDmitry@hotmail.com (D.V.Y.); Novikovfff@bk.ru (R.A.N.);
abkornev@yandex.ru (A.B.K.); alexkhom@list.ru (A.R.K)
School of Pharmacy, Biocenter Kuopio, University of Eastern Finland, P.O. Box 1627,
2
FI-70211 Kuopio, Finland; elina.puljula@uef.fi (E.P.); jouko.vepsalainen@uef.fi (J.V.)
*
†
Correspondence: kochet@eimb.ru; Tel.: +7-499-135-0590
These authors contributed equally to this work.
Received: 21 May 2017; Accepted: 21 June 2017; Published: 23 June 2017
Abstract: Derivatives of methylenediphosphonic acid possess wide spectra of biological activities
and are used in enzymology as research tools as well as in practical medicine. Carbonyl
diphosphonic acid is a promising starting building block for synthesis of functionally substituted
methylenediphosphonates. Investigation of the interaction of carbonyl diphosphonic acid with
hydroxylamine clearly demonstrates that it is impossible to isolate oxime within the pH range
2–12, while only cyanophosphonic and phosphoric acids are the products of the fast proceeding
Beckmann-like fragmentation. In the case of O-alkylhydroxylamines, corresponding alcohols are
found in the reaction mixtures in addition to cyanophosphonic and phosphoric acids. Therefore,
two residues of phosphonic acid being attached to a carbonyl group provide new properties to this
carbonyl group, making its oximes very unstable. This principally differs carbonyl diphosphonic
acid from structurally related phosphonoglyoxalic acid and other α-ketophosphonates.
Keywords: Beckmann fragmentation; carbonyl diphosphonic acid; hydroxylamines
1
. Introduction
Non-hydrolyzable analogues of inorganic pyrophosphate, i.e., derivatives of methylenediphosphonic
acid, are used in biochemistry as a source of the inhibitors of the pyrophosphate- related enzymes
and for the investigation of their reaction mechanisms (for review see [
1
–
3
]). Medronic and etidronic
acids as well as their derivatives (Figure 1), having high affinity to Ca2 ions in bones, are used for
+
treatment of osteoporosis [
Complexes of medronic and etidronic acids, and their derivatives with
imaging of bone diseases [ ]; while, for example, 11-amino-1-hydroxyundecylidene-1,1-bisphosphonic
acid (Figure 1) has high practical potential as an effective chelator of heavy metals ions and rare-earth
elements [ ]. Besides, clodronic and pamidronic acids (Figure 1), as well as risedronic and zoledronic acids,
have antitumor activity [ ]. Lipophilic bisphosphonates (BPH-703 and BPH-629; Figure 1) are effective
and selective inhibitors of the growth of Plasmodium falciparum [ ] and Mycobacterium tuberculosis [ ],
3
]. Zoledronic and risedronic acids (Figure 1) are used to treat Paget disease [
4
].
99m
Tc, are used in radiometal-based
5
6
7
8
9
that cause malaria and tuberculosis, respectively. Predominant modification of the structure of
alkyl/aralkyl substituent (BPH-715, Figure 1) has resulted in powerful inhibitors of cholesterol
biosynthesis, effecting both farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase
in nanomolar concentrations [10]. Recently, the inhibitors of reverse transcriptase [10,11] and HIV
Molecules 2017, 22, 1040; doi:10.3390/molecules22071040
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1040
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integrase [12], as well as RNA-dependent RNA polymerase of hepatitis C virus [13] have been found
among the derivatives of methylenediphosphonic acids (Figure 1). It is assumed that the activity of these
inhibitors is determined by effective chelation of Mg2+ ions in the active center of the polymerases and
effective binding of aryl substituent in the hydrophobic pocket of the active enzyme center [11].
Thus, by varying the structure of the substituents at the carbon atom of methylenediphosphonic
acid and the structure of chelating group, it appeared possible to change the spectrum of biological
activity of bisphosphonates.
Figure 1. Methylenediphosphonic acid and some of its biologically active derivatives [3–7,9,11–16].
Methods of synthesis of functionally substituted methylene bisphosphonates can be divided into
two main groups. The first one is comprised of the reaction of phosphorous acids derivatives with
nitriles/acid chlorides/anhydrides, giving C-substituted methylenediphosphonic acid tetraesters with
amino or hydroxyl group at the carbon of the > P-C-P < backbone [10,17–19]. The second group of
methods proceeds from methylenediphosphonic acid tetraesters and consists in functionalization
of the methylene group that is a CH-acid. Target C-substituted methylenediphosphonic acids are
obtained from tetraesters by acidic hydrolysis [20], or using trimethylbromosilane [21]. In the case of
unstable alkoxyisoprenyl derivatives, trimethylbromosilane in collidine is used [16]. However, trialkyl
phosphonoglyoxalates, being structurally related to tetralkyl esters of carbonyl diphosphonic acid, are
too labile to be hydrolyzed by strong aqueous acids due to the very reactive ketone function, which,
unlike in simple acylphosphonates, can even undergo reaction with trimethylbromosilane [22].
Therefore, it seems reasonable to obtain a variety of functionally substituted methylenediphosphonates
directly from the corresponding acids containing reactive substituents at the central carbon atom, i.e.,
for example, carbonyl diphosphonic acid (1, Scheme 1) and vinylidene diphosphonic acid. The use
of the carbonyl diphosphonic acid as a starting compound seems promising for synthesis of different
O-substituted oximes in aqueous solutions using the click chemistry reaction. Surprisingly, unsubstituted
oxime and corresponding O-substituted oximes of carbonyl diphosphonic acid have not been a subject
of systematic study yet. Thus, the goal of the present paper is to investigate the reaction of carbonyl
diphosphonic acid with hydroxylamine and O-alkylhydroxylamines.

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2
. Results and Discussion
It is known that carbonyl diphosphonic acid
dihydroxymethylenediphosphonic acid (gem-diol)
1
2
in aqueous solutions exists in equilibrium with
, and the equilibrium (Scheme 1) depends on the
pH of the solution [23].
Scheme 1. Interaction of carbonyl diphosphonic acid with hydroxylamine and O-alkylhydroxylamines.
Application of NMR is the most convenient method to monitor the reaction of
hydroxylamine and O-alkylhydroxylamines. However, 13C-NMR data for gem-diol
are not available.
We have determined that the carbon atom of gem-diol has the chemical shift of +92 ppm (D O, pH 3)
Figure 2). Having obtained crystalline acid we found chemical shift of its carbon atom to be
246 ppm (D O, pH 12) that is in agreement with earlier reported value of +245 ppm [24]. Such a
1 with
2
2
2
(
1
+
2
strong shift to a weak field is quite untypical to carbonyl compounds and confirms carbonyl group of
1
to be highly electrophilic. It should also be noted that in the IR spectrum of acid carbonyl stretching
1
−
1
frequency is also observed at the unusually low value of 1612 cm [23].
Figure 2. Fragments of 13C{ H}-NMR spectra of sodium carbonyl diphosphonate
1
1
at pH 12;
δ: 245.94
1
1
(t, J
118.4 Hz) and gem-diol 2 at pH 3; δ: 92.32 (t, J
181.1 Hz).
C-P
C-P
It is known that neutral and alkaline aqueous solutions of acid
and that also conveniently allows one to monitor transformations of
solution disappears immediately on the addition of 25% molar excess of hydroxylamine both at pH 12
and at pH 6–5. In the 31P-NMR spectrum of the reaction mixture at pH 12 no signal of the starting acid
is detected, while two singlet signals at +1.4 ppm, and 16.7 ppm at a ratio of 1:1 are observed (Figure 3a).
1
have yellow color (λmax 413 nm [23])
1. The yellow color of the acid
1
1
−
The first of them belongs to the sodium salt of phosphoric acid and introduction of sodium phosphate
into the NMR tube with the reaction mixture provides an increase of the intensity of +1.4 ppm signal.

Molecules 2017, 22, 1040
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To determine the origin of the compound with the chemical shift of
P-NMR-spectrum (Figure 3a), we re-set th reaction using hydroxylamine containing 10% NH OH
−
16.7 ppm in
31
15
2
and in this case the phosphorus atom signal in a strong field region is split into a doublet with
2
1
a constant of 5.8 Hz typical for J (Figure 3b). This all together with the satellites of J 145 Hz
PN
PC
13
(
Figure 3b), being equivalent to the P-C constant in the C-NMR spectrum (
cyanophosphonate nature of the compound having the value of
The chemical shift of the nitrogen atom ( 130.99 ppm) of this compound (Figure 3c) is typical for
the nitriles. The formation of cyanophosphonate was independently confirmed by HRMS. The signals
δ
= 126 ppm), points at the
16.7 ppm in 31P-NMR spectrum.
δ
−
δ
=
−
−
−
H] in the ratio of about
of the compounds with m/z = 105.9698 [M
−
H] and m/z = 106.9659 [M
−
15
9
:1, corresponding to 14N- and N-cyanophosphonate, respectively, were observed (data not shown).
Figure 3. Fragments of NMR spectra of the reaction on mixing the solutions of carbonyl diphosphonic
acid and 15NH OH
·
HCl (each solution was adjusted to pH 12 by alkali).
(
a)
31
P{ H}; (
1
b)
31
P
2
15
(not decoupled, high resolution); (c) N–inverse gated.
The same changes in 31P-NMR spectra of the reaction mixtures are observed when the reaction
of carbonyl diphosphonic acid with hydroxylamine is carried out at pH 2 and pH 5–6. Starting
acid is not detected even on mixing the reagents, just the equimolar amounts of phosphoric and
1
cyanophosphonic acids are formed.
These observations are completely unexpected, since it is known that both E- and Z-isomers of
structurally related α-(hydroxyimino)phosphonoacetic acid (“troika acid”) (Figure 4) are stable at
alkali pH [25]. The ionization of oxime HO-group, having pKa > 10, restricted the fragmentation of
troika acid since the ionized oxime oxygen can hardly function as a leaving group. Moreover, E- and
Z-isomers of troika acid are obtained by alkaline hydrolysis of the corresponding C-monomethyl esters
(
Z-isomer is hydrolyzed more than 50 times faster than E-isomer [25]). However, E-troika acid is a mild
phosphorylating agent at neutral and slightly acidic pH. The cleavage of the P-C bond (τ1/2< 10 min)
is likely to result in the formation of the meta-phosphoric acid, which has been frequently postulated
to exist as a highly reactive intermediate in dissociative phosphorylation mechanisms. When the
reaction is carried out in alcohols, mono-alkyl phosphates are formed in high yields [22
,25]. On the
contrary, at neutral or slightly acidic pH, Z-troika acid undergoes the Beckmann-like fragmentation

Molecules 2017, 22, 1040
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(
reported τ1/2~15 min) with the formation of cyanophosphonate [25]. Hence, these transformations
proceed stereospecifically and the position of the oxime hydroxyl group in troika acid mediates
phosphorylation vs. nitrile formation [25].
OH
HO
N
N
(
HO) (O)P
COOH
(HO) (O)P
COOH
2
2
E- -(Hydroxyimino)-
phosphonoacetic acid
ʺtroika acidʺ
Z- -(Hydroxyimino)-
phosphonoacetic acid
ʺtroika acidʺ
Figure 4. E- and Z-isomers of troika acid.
It should be specially pointed out that, unlike the structurally-related troika acid, the oxime of
carbonyl diphosphonic acid is unstable not only at acidic or slightly acidic pH, but also at alkaline
pH due to the fast (τ1/2< 1 min) Beckmann-like fragmentation leading to cyanophosphonic acid, or
its salts. We assume that oxime and O-substituted oximes of carbonyl diphosphonic acid undergo
the Beckmann-like fragmentation via six-membered intermediate (Figure 5). In acidic and neutral
aqueous solutions in addition to cyanophosphonic acid, a reactive metaphosphate intermediate may
be formed (Figure 5A), while at alkali pH the Beckmann-like fragmentation directly leads to the salts
of cyanophosphonic and phosphoric acids (Figure 5B).
Figure 5. Presumed mechanism of the Beckmann-like fragmentation leading to the degradation of
P-C-P bridge under acidic (A) and basic conditions (B).
Therefore, the substitution of the carboxyl group of troika acid to the phosphonic one dramatically
changes its chemical properties and the oxime of carbonyl diphosphonic acid immediately rearranges
to cyanophosphonic acid even at alkali pH.
It is known that stable O-substituted oximes of acyl phosphonic acids are smoothly obtained by
the reaction of
α-ketophosphonates with O-alkylhydroxylamines and possess chemical properties
typical for oximes. O-Methyl oxime of benzoylphosphonic acid can be considered an interesting
◦
◦
exception. It is stable at acidic and neutral pH at 20 C, while in aqueous alkali at 20 C it very slowly
days) decomposes to phosphoric acid and benzonitrile, in contrast to the non-substituted oxime,
which is stable under these conditions [26].
O-substituted oximes of acid are not described in literature. However, carbonyl diphosphonic
esters have been reported to react smoothly with H NOCH forming O-methyl oxime of tetraisopropyl
(
1
2
3
carbonyl diphosphonate at a 60% yield [22]. At the same time, chemical properties of this O-methyl

Molecules 2017, 22, 1040
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oxime, as well as possibilities of its transformation into O-methyl oxime of acid 1, are have not been
studied at all. Therefore, O-methyl hydroxylamine, branched-chain O-isopropyl hydroxylamine,
O-benzyl hydroxylamine, aminooxyacetic acid, and 1-aminooxy-2-aminoethane were studied in the
reaction with acid
1 (Scheme 1). 1-Aminooxy-2-aminoethane was expected to have the accelerated
rate of oxime formation, since Schiff bases are formed faster than oximes and are much more
reactive towards hydroxylamines, hydrazines, and semicarbazones, if compared with free carbonyl
group [27,
28]. The signal of starting carbonyl diphosphonic acid could not be detected in 31P-NMR
spectra, even immediately, on mixing of carbonyl diphosphonic acid with any of tested O-substituted
◦
hydroxylamines at 20 C and pH 2–12. The reaction products are sodium salts of phosphoric acid
(
s, +1.4 ppm) and cyanophosphonic acid (s,
−
16.7 ppm)—the same as in the case of hydroxylamine
1
itself. The formation of cyanophosphonic acid was independently confirmed by HRMS. H-NMR
spectra of the reaction mixtures contain signal(s) of the alcohols derived from the corresponding
O-alkylhydroxylamines as well as the signal(s) of O-substituted hydroxylamines that are used in
these reactions in a 25% molar excess. Respectively, short-living O-substituted oximes undergo the
Beckmann-like fragmentation at a wide range of pH, while alkoxy groups serve as leaving groups.
Therefore, two residues of phosphonic acid attached to carbonyl group provide a unique set of
properties, making it impossible to synthesize oximes of carbonyl diphosphonic acid via treatment
◦
with hydroxylamine itself and even with O-substituted hydroxylamines. In all cases, at 20 C and
within pH 2–12, oximes undergo the Beckmann-like fragmentation leading to cyanophosphonic acid
and inorganic phosphate. This common feature of oxime and O-substituted oximes of acid
1
principally
differs them from the structurally relative troika acid and oximes of α-ketophosphonates.
3
. Materials and Methods
1
5
N-Enriched hydroxylamine hydrochloride was prepared by the reduction of 15N sodium nitrite
15
(
10 atom % 15N) with sodium metabisulfite. Crude NH OH
to acetone oxime, and the subsequent acidic hydrolysis provided NH OH HCl with the overall
yield of 65%. 1-Aminooxy-2-aminoethane dihydrochloride was synthesized as described in [29], while
hydrochloride of O-isopropyl hydroxylamine was synthesized as described in [30]. All other reagents
were purchased from Sigma-Aldrich (Steinheim, Germany) or Acros Organics (Geel, Belgium).
NMR spectra were recorded on 500.1 MHz ( H), 202.5 MHz ( P), 125.8 MHz ( C), and 50.8 MHz
N) Bruker Avance 500 DRX (Bruker, Karlsruhe, Germany) spectrometer. Chemical shifts are reported
in parts per million (ppm) using tetramethylsilane ( H), tert-butanol-d ( C) as internal standards,
P(OMe) in CDCl ( P, δ = 139.8 ppm) [31], and CH NO + 10% CDCl ( N) as external standards.
3 2 3
C- and P-NMR spectra were proton-decoupled unless otherwise specified. High-resolution
·
HCl was purified after its conversion
2
15
·
2
1
31
13
(15
1
13
1
31
15
3
3
1
3
31
electrospray mass spectra were obtained using Applied Biosystems/MDS Sciex QSTAR XL (USA).
The measurements were acquired in a negative ion mode with the following parameters: interface
capillary voltage 4700 V; mass range from m/z 50 to 3000; nebulizer pressure 0.4 Bar; flow rate
◦
3
µ
L/min; nitrogen was applied as a dry gas (4 L/min); interface temperature was set at 190 C. VWR
Scientific pH-meter (model 2000 with EW-5991-61-electrode, “Cole Parmer” (Vernon Hills, IL, USA)
was used to perform pH measurements.
3
.1. Synthesis of Tetraethyl 1,1-Dichloromethylene Bisphosphonate
To an ice-cold 13% water solution of NaClO (20 mL) 37% HCl (0.5 mL) was added and the
pH of the resulting mixture was adjusted to 9–10 with Na CO following with the addition of 4 g
2
3
(
13.9 mmol) of tetraethyl methylenebisphosphonate. Heterogeneous reaction mixture was stirred
◦
for 2 h at 20 C, then DCM (20 mL) was added, organic phase was separated, washed with brine,
and dried over Na SO to afford 2.78 g (56%) of tetraethyl 1,1-dichloromethylene bisphosphonate
2
4
◦ ◦
b.p. 160–166 C/0.1 mm Hg; lit.: 119–120 C/0.05 mm Hg [32]). C{ H}-NMR (CDCl ): δ 72.84
3
13
1
(
(
1
3
31
1
t, J
151.2 Hz), 66.46 (m), 16.76 (t, J
3.1 Hz). P{ H}-NMR (CDCl ): δ 8.95 (s).
C-P 3
C-P

Molecules 2017, 22, 1040
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3
.2. Synthesis of Carbonyl Diphosphonic Acid (1)
A mixture of 0.7 g (2 mmol) of 1,1-dichloromethylene bisphosphonate in 20% HCl (10 mL) was
refluxed for 3 h and then concentrated in vacuo. The residue was co-evaporated with water (3 10 mL)
×
and then refluxed in 2 M NaOH (6 mL) for 4 h. Yellow solution was concentrated in vacuo to 3 ml
◦
and left at 4 C for 24 h. The precipitate was recrystallized from water to give tetrasodium salt of
1
378 mg, 68%). 13C{ H}-NMR (D O): δ 245.94 (t, J
1
1
118.4 Hz). P{ H}-NMR (D O): δ 0.3 (s).
C-P 2
31
1
(
2
3
.3. Reaction of Carbonyl Diphosphonic Acid with Hydroxylamine and Its Esters, General Procedure
Reaction at pH 12: To a yellow solution of 10 mg (0.036 mmol) of tetrasodium salt of
1
in
D O (0.3 mL) a solution of hydroxylamine hydrochloride or hydrochloride of O-alkylhydroxylamine
2
(
1.25 eq) in water (0.3 mL, pH was adjusted to 12) was added. Reaction mixtures became colorless on
mixing the reagents and NMR spectra were registered. All transformations proceed according to NMR
with quantitative yields.
With 15NH OH: C{ H}-NMR (D O):
13
1
δ
126.0 (d, 1JP-C 145.0 Hz). 31P{ H}-NMR (D O):
1
δ
1.4 (s),
−
16.7
2
2
2
2
5.8 Hz). N{ H} inverse gated NMR (D O): δ −131 (d, 2JN-P 5.8 Hz). HRMS (ESI): calcd.
15
1
(d, J
P-N
2
14
−
15
−
H] : 106.9664;
for CH2 NO P [M
−
H] : 105.9694; found: 105.9698. Calcd. for CH NO P [M
−
3
2
3
found: 106.9659.
1
13
1
With NH OMe: H-NMR (D O):
δ
3.60 (s, 3H, NH OCH ), 3.31 (s, 3H, HOCH ). C{ H}-NMR (D O):
2
2
2
3
3
2
1
31
1
δ 126.0 (d, J
145.0 Hz), 61.3, 47.8. P{ H}-NMR (D O): δ 1.4 (s), −16.7 (s).
With NH O-i-Pr: H-NMR (D O):
6.2 Hz), 1.35 (d, 6H, NH OCH(C
C{ H}-NMR (D O):
P-C
2
1
3
δ
4.35 (sept., 1H, NH OC
H
, J
6.1 Hz), 4.08 (sept., 1H, HOC
H
,
2
2
2
H-H
3
3
3
J
H
) , J
3 2
6.1 Hz), 1.24 (d, 6H, HOCH(C
H
) , J
3 2
6.2 Hz).
H-H
H-H
1
2
H-H
13
1
31
1
δ
126.0 (d, J
145 Hz), 81.3, 67.1, 26.6, 22.5. P{ H}-NMR (D O): δ 1.4 (s),
P-C 2
2
−
16.7 (s).
1
With NH OBn: H-NMR (D O):
δ
7.57 (s, 5H, NH O-CH C6H5), 7.52–7.50 (m, 5H, HOCH C6H5), 5.07
2
2
2
2
2
13
1
(
1
s, 2H, NH OCH2), 4.72 (s, 2H, HOCH2). C{ H}-NMR (D O):
δ
143.2, 136.7, 132.5, 132.3, 132.0, 131.7,
2
2
1
31
1
30.8, 130.5, 126.0 (d, J
145 Hz), 80.2, 66.9. P{ H}-NMR (D O): δ 1.4 (s), −16.7 (s).
With NH O(CH ) NH : H-NMR (D O):
P-C
1
2
3
δ
4.18 (t, 2H, NH OCH2, J
4.7 Hz), 3.90 (t, 2H, HOCH2
,
2
2 2
2
2
2
H-H
3
3
3
JH-H 5.1 Hz), 3.38 (t, 2H, NH OCH CH2NH , J
4.7 Hz), 3.22 (t, 2H, HOCH CH2NH , J
145.0 Hz), 73.8, 60.6, 44.3, 41.0. P{ H}-NMR (D O):
5.06
H-H
2
2
2
H-H
2
2
Hz). 13C{ H}-NMR (D O):
1
δ
126.0 (d, J
1
31
1
δ
1.4
2
P-C
2
(
s), −16.7 (s).
With NH OCH COOH: H-NMR (D O):
1
4.48 (s, 2H, NH OCH2), 4.20 (s, 2H, HOCH2). 13C{ H}-NMR
1
δ
2
2
2
2
1
31
1
(
D O): δ 180.9, 178.0, 126.0 (d, J
145 Hz), 75.2, 63.4. P{ H}-NMR (D O): δ 1.4 (s), −16.7 (s).
2
P-C
2
Reaction at pH 2: To a solution of 12 mg (0.043 mmol) of tetrasodium salt of
1 in 10% citric acid
(
(
(
0.5 mL), a solution of hydroxylamine hydrochloride or O-substituted hydroxylamine hydrochlorides
31
1.25 eq) in D O (0.1 mL) was added and P NMR spectra were registered on mixing the reagents
2
pH of the reaction mixtures was ~2.0) demonstrating the formation of inorganic phosphate and
cyanophosphonic acid with quantitative yields similar to that described above for the reaction
performed at pH 12.
Reaction at pH 5–6: To a solution of 10 mg (0.036 mmol) of tetrasodium salt of
1 in water (0.5 mL),
a solution of hydroxylamine hydrochloride or hydrochloride of O-alkylhydroxylamine (1.25 eq) in
31
D O (0.1 mL) was added and P-NMR spectra were registered on mixing the reagents (pH of the
2
reaction mixtures were ~5–6, depending on O-substituted hydroxylamine structure) demonstrating
the formation of inorganic phosphate and cyanophosphonic acid with quantitative yields similar to
that described above for the reaction performed at pH 12.

Molecules 2017, 22, 1040
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Acknowledgments: The authors thank T. Bazhenova for her kind supply of 15N-labelled sodium nitrite and
P. Solyev for his thoughtful comments on the manuscript. NMR studies were supported by the Academy of Finland
(decision No. 292574), while the rest of the research by the Russian Science Foundation (grant No. 14-50-00060).
Author Contributions: O.A.K., D.V.Y. and A.B.K. performed chemical synthesis, R.A.N. and E.P. carried out
the physical–chemical experiments, A.R.K., S.N.K. and J.V. wrote the paper, and all co-authors commented on
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the
decision to publish the results.
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Sample Availability: Samples of the compounds are available from the authors.
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