1-(Acylamino)alkylphosphonic acids—alkaline deacylation

Article abstract of DOI:10.3390/molecules23040859

The alkaline deacylation of a representative series of 1-(acylamino)alkylphosphonic acids [(AC)-AA^P: (AC) = Ac, TFA, Bz; AA^P = Gly^P, Ala^P, Val^P, Pgl^P and Phe^P] in an aqueous solution of KOH (2M) was investigated. The results suggested a two-stage reaction mechanism with a quick interaction of the hydroxyl ion on the carbonyl function of the amide R-C(O)-N(H)- group in the first stage, which leads to instant formation of the intermediary acyl-hydroxyl adducts of R-C(O?)2-N(H)-, visible in the 31P NMR spectra. In the second stage, these intermediates decompose slowly by splitting of the RC(O?)2-N(H)- function with the subsequent formation of 1-aminoalkylphosphonate and carboxylate ions.

Full text of DOI:10.3390/molecules23040859

molecules
Article
1-(Acylamino)alkylphosphonic
Acids—Alkaline Deacylation
Marek Cypryk 1, Jozef Drabowicz 1,2,*, Bartlomiej Gostynski 1, Marcin H. Kudzin 3,
Zbigniew H. Kudzin 4,* and Pawel Urbaniak 4
ID
1
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 120a,
Lodz 90-363, Poland; mcypryk@cbmm.lodz.pl (M.C.); bgostyns@cbmm.lodz.pl (B.G.)
Department of Chemistry and Environment Protection, Jan Dlugosz University, Czestochowa 42-200, Poland
Textile Research Institute, Brzezinska 5/15, Lodz 92-103, Poland; kudzin@iw.lodz.pl
Faculty of Chemistry, University of Lodz, Tamka 12, Lodz 91-403, Poland; pawelurb@chemia.uni.lodz.pl
2
3
4
*
Correspondence: drabowicz@gmail.com (J.D.); zhkudzin@uni.lodz.pl (Z.H.K.)
Academic Editor: György Keglevich
Received: 5 March 2018; Accepted: 28 March 2018; Published: 9 April 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The alkaline deacylation of a representative series of 1-(acylamino)alkylphosphonic acids
[(AC)-AAP: (AC) = Ac, TFA, Bz; AAP = GlyP, AlaP, ValP, PglP and PheP] in an aqueous solution of
KOH (2M) was investigated. The results suggested a two-stage reaction mechanism with a quick
interaction of the hydroxyl ion on the carbonyl function of the amide R-C(O)-N(H)- group in the first
−
stage, which leads to instant formation of the intermediary acyl-hydroxyl adducts of R-C(O )2-N(H)-,
visible in the 31P NMR spectra. In the second stage, these intermediates decompose slowly by splitting
of the RC(O−)2-N(H)- function with the subsequent formation of 1-aminoalkylphosphonate and
carboxylate ions.
Keywords: amino acids; aminophosphonic acids; 1-aminoalkylphosphonic acids; 1-(acylamino)
alkylphosphonic acids; 31P NMR spectra of intermediates; hydrolytic deacylation
1. Introduction
Aminoalkylphosphonic acids (AAP) are structural analogues of amino acids (AAC) [
which are of natural origin [ ]. Due to the structural analogy, they present similar biological properties
1], some of
2
to the class and are important inhibitors of enzymes of the amino acids metabolism [1,3] (Table 1).
Table 1. Structures of aminophosphonic acids (AAP)—phosphonic analogs of amino acids (AAC) and
1-(acylamino)alkylphosphonic acids (AC)-AAP and phosphonopeptides AAC-AAP and AAP-AAC
[4–14].
AAC
ω-AAC
AAP
ω-AAP
AAP–R
Naturally occurring aminophosphonic acids.
Molecules 2018, 23, 859; doi:10.3390/molecules23040859
www.mdpi.com/journal/molecules

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Table 1. Cont.
β-AlaP [4]
Aspβ–P [5]
IserP [6]
TyrP [7]
−γ–P(Me)
[8]
Glu
Representative, biologically active aminophosphonates.
(AC)-AAP[R=H,
(AC)-GlyP] [11]
AAC-AAP(R=R1=Me,
Ala-AlaP) [12]
Gluγ–P [10]
AAP-AAC [13,14]
PMG [9]
Abbreviation of AAP and (AC)-AAP follow the general rules elaborated by Kudzin et al. [15].
Several papers reflected the complexing abilities of this class of compounds [16
their pharmacological [19 22], agro-chemical [23] and industrial [11 24 25] applications. Therefore,
the studies on the synthesis [26 30] and physico-chemical properties [31
40] of AAP and their
–18] and
–
, ,
–
–
derivatives constitute an important topic in chemistry and biochemistry also in addition to material
science (e.g., Self-assembled monolayers agents, SAMs [41]).
The 1-(acylamino)alkylphosphonic acids (AC)-AAP belong to the interesting group of compounds
that is of potential pharmacological importance since the corresponding 1-N-aminoacylamino
derivatives of AAC-AAP (mixed P-terminal phosphono-dipeptides, e.g., Alaphosphaline) exhibit
antibacterial activity [1,23] (Table 1).
1-(Acylamino)alkylphosphonic acids [36,37] and esters [42] are easily formed from starting
aminoalkylphosphonic acids, the latter being substrates for selective deacylation to O,O-dialkyl
aminoalkylphosphonates [43]. Both of these groups are substrates for stereoselective enzymatic
hydrolysis [44–48] (Figure 1). 1-(Acylamino)alkylphosphonic acids are also formed in so-called
Engelman-Pikl-Oleksyszyn method synthesis of 1-aminoalkylphosphonic and 1-aminoalkylphosphonic
acids, which starts from appropriate amides and phosphorus chlorides [10,27,49,50] ( Figure 2). As some
secondary 1-aminoalkanephosphonates are unstable in the process of acidic degradation [40], alkaline
hydrolysis can present an alternative method in such procedures.
Figure 1.
R1-C(O)OH/[R1-C(O)]2O [36
(4) enzymatic hydrolysis of racemic (D,L)-(AC)-AAP and (D,L)-(AC)-AAP(OR)2 [44–48]].
Transformation of 1-aminoalkylphosphonic acids via N-acyl-derivatives [(1)
–
38]; (2) R1-C(O)OH/[R1-C(O)]2O/HC(OR2)3 [42]; (3) NaBH4/MeOH [43];

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Figure 2.
Scheme of Engelmann-Pikl-Oleksyszyn methods for synthesis of secondary
1-aminoalkylphosphonic (R2=OH) and 1-aminoalkylphosphinic (R2=H, alkyl) acids (1) aldehyde,
R-PCl2/AcOH; and (2) 5M HCl-H2O, ∆8–10 h.
The susceptibility of (AC)-AAP to hydrolysis/solvolysis constitutes an important factor influencing
their biological activity, especially during penetration through cell membranes. Therefore, the detailed
studies on deacylation of these compounds should be helpful in obtaining a deeper understanding of
this phenomenon. To tackle this problem, we have decided to carry out studies devoted to deacylation of
(AC)-AAP under different reaction conditions. Our preliminary results of experiments were carried
out in aqueous media in the pH range of 0–6.5, which occurs namely in an aqueous 2M HCl and
buffer solutions. These are briefly described in a recently published article [51]. As an obvious and
necessary continuation of this topic, this paper presents our results on the deacylation of representative
types of 1-(acylamino)alkylphosphonic acids (AC)-AAP in aqueous 2M KOH solution. These include
1-(acetylamino)alkylphosphonic Ac-AAP, 1-(trifluoroacetylamino)alkylphosphonic acids TFA-AAP and
1-(benzoylamino)alkylphosphonic Bz-AAP, derived from representative 1-aminoalkylphosphonic acids
AAP (GlyP, AlaP, ValP, PglP and PheP).
2. Results and Discussion
It is generally known that amides can be hydrolyzed with either acidic or basic catalysis, with
products being the free acid and the ammonium/substituted ammonium ions or the salts of the acid and
ammonia/amine, respectively. Both the acid- and base-catalyzed hydrolyses are essentially irreversible,
since salts are formed in both cases [52
The very low rate of amide hydrolysis by water has been measured by Kahne & Still [57].
kinetic study has been conducted on the alkaline hydrolyses of several types
of amides, including formanilides 58 59], N-methylformanilides and N-acetanilides 60],
–55]. Water alone is not sufficient to hydrolyze most of amides [56].
A
[
,
[
1,8-bis(trifluoroacetylamino)-naphthalene [61] also in addition to N-methylacetamide, N-methylbenzamide
and acetanilide [62]. A mild protocol for the alkaline hydrolysis of secondary and tertiary amides in
non-aqueous conditions at room temperature or under reflux has been recently described [63].
The structural analogy of 1-(acylamino)alkylphosphonic acids (AC)-AAP and 1-(acylamino)-alkanoic
acids (AC)-AAC or amides causes the mechanism of hydrolytic scission of the amide linkage R-C(O)-N in
common amides and in 1-(acylamino)alkylphosphonic acids. These should be ruled by similar if not the
same mechanisms.
2.1. Investigations on the Deacylation Course of 1-(acylamino)alkylphosphonic Acids
The (AC)-AAP contain amidic functions, which has a hydrolytic sensitivity that should be
dependent on the structure of their acyl moieties and on the type of applied hydrolytic medium [49].
For conducting an inquiry into the hydrolytic stability of (AC)-AAP, we undertook deacylation
investigations of these compounds in aqueous 2M KOH solutions.
The application of 31P NMR monitoring of the reaction course enabled identification and
quantification of phosphoric components of the reaction mixtures formed. Essentially, monitoring
of the deacylation course of Ac-GlyP in 2M KOH solution reveals the formation of GlyP (Figure 3).
The results of 31P NMR monitoring of the deacylation of the various types of (AC)-AAP (Ac-AAP,
Bz-AAP and TFA-AAP) at a temperature of 25 ◦C and exposition period up to 240 h are presented in
Figure 4 (Ac-AAP), Figure 5 (TFA-AAP) and Figure 6 (TFA-AAP), respectively.

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Figure 3. 31P NMR spectra of Ac-GlyP recorded in 2 M KOH. (
mixture, and (B) After 192 h of exposition of the reaction mixture. [Ac-GlyP: 13.4 ppm; GlyP: 19.0 ppm].
A) Immediately after mixing the reaction
Figure 4. A solution of 0.1 M 1-(acetylamino)alkylphosphonic acids Ac-AAP in 2 M KOH reacted at
◦
25 C, which was monitored by 31P NMR [DD (%) vs. time (h)].
Figure 5. A solution of 0.1 M 1-(trifluoroacetylamino)alkylphosphonic acids TFA-AAP in 2 M KOH
◦
reacted at 25 C, which was monitored by 31P NMR [DD (%) vs. time (h)].

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Figure 6. A solution of 0.1 M 1-(benzoylamino)alkylphosphonic acids Bz-AAP in 2 M KOH reacted at
◦
25 C, which was monitored by 31P NMR [DD (%) vs. time (h)]. Thus, the presented graphs illustrate
much higher stability of benzoyl-derivatives Bz-AAP (Bz-GlyP: DD200h ~30%; for Bz-AlaP and Bz-PglP:
DD200h < 3%) in comparison with acetyl-derivatives Ac-AAP (Ac-GlyP: DD200h ~70%; for Ac-AlaP:
DD200h ~20%; and Ac-PglP: DD200h < 7%).
The comparison of hydrolytic stability of TFA-GlyP, Ac-GlyP and Bz-GlyP in 2M KOH is presented
in Figure 7.
Figure 7. A solution of 0.1 M 1-(acylamino)methylphosphonic acids (AC)-GlyP (AC: Ac, TFA, Bz) in
◦
2M KOH reacted at 25 C, which was monitored by 31P NMR [DD (%) vs. time (h)]. Points on the plots
corresponds to GlyP released from the parent (AC)-GlyP, which means GlyP from Ac-GlyP, GlyP from
Bz-GlyP and GlyP from TFA-GlyP.
Since no products of the scission of the C-P bond in all investigated cases were observed,
the deacylation mechanism of (AC)-AAP in alkaline solutions can be illustrated by an analogy to the
corresponding basic deacylation of 1-(acylamino)alkanoic acids (Scheme 1) [54,62,63].
Scheme 1. Hypothetical mechanism of deacylation of (AC)-AAP in 2 M KOH solution.

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This includes the prior interaction of hydroxyl ion on the carbonyl carbon of
with the formation of the tetrahedral intermediate state
1
, which occurs
2
(Scheme 1). These intermediates were
observed on 31P NMR spectra as signals down-shifted in comparison with the parent signals of
AC-AAP (e.g., Figure 2). The 31P NMR chemical shifts of (AC)-AAP (
1) and the corresponding adduct
compounds AC(OH)-AAP (2) formed are listed in Table 2.
Table 2. 31P NMR chemical shifts of (AC)-AAP and the corresponding adduct compounds
(AC)(OH)-AAP formed immediately after mixing the reaction mixture. ( 31P NMR chemical shifts of
31P NMR chemical shifts
A
)
Ac-AAP and the corresponding adduct compounds Ac(OH)-AAP formed. (
B)
of (AC)-GlyP and the corresponding adduct compounds [AC(OH)]-GlyP formed.
These compounds [AC(OH)]-AAP] slowly disappeared during the deacylation progress (Figure 8).
Figure 8. Ac-GlyP hydrolysis in 2M KOH [DD (%) vs. time (h)].
The rehybridization (sp3
→
sp2) of the carbonyl carbon in
2 enforces the splitting of the amide
bond R-C(O)-N and the subsequent formation of the aminophosphonate 3.
The comparison of (AC)-AAP stability in aqueous 2M KOH and 2M HCl solutions is given in Figure 9.

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Figure 9. (AC)-GlyP(Ac-GlyP, TFA-GlyP, TFA-GlyP) hydrolysis in aqueous solutions of 2M KOH and
2M HCl [DD (%) vs. time (h)].
2.2. Quantum Chemical Calculations for Deacylation of (AC)-AAP in Basic Conditions
The reaction illustrated in Scheme 1 was divided into two stages (reactions 1.1 and 1.2,
respectively), which were studied separately. Stage 1.1 concerns the addition of hydroxide to
amidophosphonate anion
product
for Ac-AlaP (R=R1=Me) is given in Figure 10). Stage 1.2 involves dissociation of the trianion
into aminophosphonate dianion and carboxylate anion . We assumed that the transfer of proton
from OH to the amide nitrogen in proceeds simultaneously with dissociation. The thermodynamics
1 with the formation of trianion 2 (the example structure of the addition
2
2
3
4
2
of this two-stage process was studied by density functional theory (DFT) methods in the gas phase and
in aqueous solution. Gas phase calculations were performed only for comparison with the aqueous
system, which was the real reaction system to illustrate the essential role of the solvation effect.
Figure 10. The structure of the addition product
2
(R=R1=Me, Ac(OH)-AlaP) with intramolecular
hydrogen bond between OH and phosphoryl group (orange—phosphorus, blue—nitrogen,
red—oxygen).
Stage 1: Hydroxide anion attack on the carbonyl moiety of (AC)-AAP dianion 1 (Scheme 1, r. 1.1).
This reaction stage presumably proceeds by the attack of a lone pair of negatively charged oxygen
atoms on sp2 carbon in the carbonyl group. This process is highly unfavorable since it involves the
reaction of two anions with the formation of the molecule with a total charge of
reaction in the gas phase is very high, so this process seems to be thermodynamically very unlikely
(Table 3).
−
3. The energy of this

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Table 3. Gibbs free energies ∆G (298 K) for reaction 1.1 in gas phase (kcal/mol).
However, when carried out in aqueous solutions, this reaction is much less unfavorable due to
the stabilization of ionic structures by the polar solvent (Table 2). Similarly, this reaction should also
be facilitated by counterions, although these were omitted in order to keep the model system simple.
The values of the Gibbs free energy
phase are listed in Tables 3 and 4, respectively.
∆
G298 (kcal/mol) for reaction 1.1 in gas phase and in aqueous
Table 4. Gibbs free energies ∆G (298 K) for reaction 1.1 in aqueous phase (kcal/mol).
The free energy barriers, ∆
G‡ (298 K), calculated for reaction 1.1 in a water solution are shown
in Table 5. The lowest energy barriers for OH− addition (Scheme 1, r. 1.1) occur when R1 = CF3,
which results from stabilization of the negative charge by the electron-withdrawing CF3 group.
−
Table 5. Free energy barriers (∆G‡ (298 K), kcal/mol) for OH addition (reaction 1.1) in water.

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Stage 2: Dissociation of trianion
reaction 1.2).
2
into aminophosphonate dianion
3
and carboxylate anion
4
(Scheme 1,
This reaction stage presumably involves the proton transfer from the geminal hydroxyl group to
nitrogen with dissociation of the C-N bond. This hypothesis is supported by the transition structures
found for this reaction. The example of this structure for the proton transfer/dissociation of the C-N
bond in Ac-AlaP(3−) is shown in Figure 11. Vibrational analysis indicates that the imaginary vibration
is associated with proton transfer from OH to NH. In the structure of the transition state shown in
Figure 11, the geminal C-O bond distance is 1.284 Å, which corresponds well to C=O double bond.
Furthermore, the C-N distance is 1.802 Å compared to 1.503 Å in the starting amide, which indicates
that the bond breaking in the transition state is significantly advanced.
Figure 11. The transition state for proton transfer/dissociation of the C-N bond in Ac(OH)-AlaP(3−)
.
The Gibbs free energies for the dissociation reaction 1.2 in the gas phase and in water are presented
in Tables 6 and 7, respectively.
Table 6. Gibbs free energy ∆G (298 K) for the reaction 1.2 in gas phase (kcal/mol).
Table 7. Gibbs free energy ∆G (298 K) for the reaction 1.2 in an aqueous phase (kcal/mol).

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Comparing the thermodynamic quantities in Tables 3 and 6, it is evident that the total free energy
of the reaction 1 is negative in all cases by approximately 30 kcal/mol and thus, the entire process 1
−
is thermodynamically feasible. However, the very high energy barrier for the first step of reaction 1.1
(Table 3) prevents this reaction from proceeding in the gas phase.
Analogously, the reaction 1 in water is equally thermodynamically favorable (
30 kcal/mol, Tables 4 and 7), but the energy barrier associated with the first step 1.1 is much lower in this
case (Table 4). Table 8 presents the results of free energy barrier calculations for reaction 1.2 in water.
∆G =
−20 to
−
Table 8. Free energy barriers (
in water (reaction 1.2).
∆
G‡ (298 K), kcal/mol) for proton transfer/dissociation of the C-N bond
The total Gibbs free energy profiles of entire process 1 for two selected pairs of
1-(acylamino)alkylphosphonic acids [Ac-AlaP(2−), TFA-AlaP(2−) , Bz-PglP(2−) and TFA-PglP(2−)] in
water solution are shown in Figure 12.
Figure 12. Free energy profiles ∆
G298 (kcal/mol) for the reaction of Ac-AlaP(2−) (blue), TFA-AlaP(2−) (red),
Bz-PglP(2−) (green), and TFA-PglP(2−) (purple) with hydroxyl ions in water solution (the general reaction
scheme is given above).

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The total Gibbs free energies and reaction barriers for the entire process 1 are shown in Table 9.
Moreover, Figure 12 reveals that the energy profiles for both TFA-substituted phosphonic acids are
essentially identical and all stationary points are significantly lower in energy than Ac-substituted
phosphonic acids.
Table 9. Total Gibbs free energies
aqueous phase and total Gibbs free energy barriers (
∆
G298(1.1) +
∆
G298(1.2) for the overall deacylation process in the
G298(1.1) + G‡(1.2), in parentheses) (kcal/mol).
∆
∆
Therefore, the presence of a TFA group is the only factor determining the free energy barrier of the
reaction. From the energy profile, it is obvious that the overall energy barrier in water
∆
G‡ is G298[TS
−
(1.2)]
−
(G298
(
1
) + G298(OH )), while the net Gibbs energy effect for the reaction in water solution can
be calculated as ∆Gr298 = G298(3) + G298(4) −[(G298(1) + G298(OH−)] = ∆G298(1.1) + ∆G‡(1.2).
The total energetic effect of the process 1 in water is negative, which means that the overall
reaction is thermodynamically feasible (Table 8). The free energy of the process when R1=CF3
is also approximately 8 kcal/mol more favorable than for R1=Me. The comparison of values in
Table 9 also reveals that the transformation 1 of substrates with R1=Ph are thermodynamically more
favored by approximately 3 kcal/mol than R1=Me. Thus, the thermodynamic effect of reaction 1
depends on the R1 substituents in the order of: CH3 < Ph < CF3. Table 9 also leads to the conclusion
that the substrates with a trifluoromethyl group in the R1 position systematically show free energy
barriers that are approximately 10–15 kcal/mol lower than those for other substituents R1, due to the
additional stabilization of the trianion 2 by the inductive effect of the strongly electron-withdrawing
CF3 group. This suggests that trifluoromethyl-substituted anions should be more reactive than the
other derivatives.
3. Materials and Methods
3.1. General Information
The 31P NMR spectra were recorded on a Bruker AV 200 spectrometer (Rheinstetten, Germany)
operating at 81.01 MHz and on a Bruker Avance III 600 spectrometer (Bruker BioSpin, Rheinstetten,
Germany) operating at 242.9 MHz. The1H NMR spectra were recorded on a Bruker Avance III
600 spectrometer operating at 600 MHz. The positive chemical shift values of 31P were reported for
compounds that were absorbing at lower fields than H3PO4. The purity of the 1-aminoalkylphosphonic
acids and corresponding 1-(acylamino)alkylphosphonic acids were determined by pH-metric titration
using a computer aided automatic titrator connected to the EMU-meter (Wroclaw University of Science
and Technology, Wrocław, Poland), which was fitted with a combined glass microelectrode Crison 5028.

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3.2. Investigated Compounds
Phosphonoglycine (GlyP) was obtained according to
a
previous study
[64].
The 1-Aminoalkylphosphonic acids of phosphonoalanine (AlaP); phosphonovaline (ValP);
phosphonophenylglycine (PglP) and phosphonophenylalanine (PheP) have been prepared
according to a previous study [65]. 1-(Acetylamino)alkylphosphonic acids (Ac-AAP: Ac-GlyP;
Ac-AlaP; Ac-ValP; Ac-PglP and Ac-PheP) and 1-(benzoylamino)alkylphosphonic acids (Bz-AAP:
Bz-GlyP; Bz-AlaP; Bz-ValP; Bz-PglP and Bz-PheP) were synthesized according to a previous study [36].
1-(Trifluoroacetylamino)alkylphosphonic acids (TFA-AAP: TFA-GlyP; TFA-AlaP; TFA-ValP; TFA-PglP
and TFA-PheP) were synthesized according to a previous study [37]. (Structures, names and
abbreviations of aminophosphonic acids and corresponding 1-(acylamino)phosphonic acids discussed
in this work are given in Table 10). Purity of all (AC)-AAP synthesized was determined by 31P NMR
and 1H NMR in addition to potentiometric titration. Analytical standards (fixanals) and other reagents
were purchased from Aldrich–Sigma (Poznan´, Poland).
Table 10. Structures, names and abbreviations of aminophosphonic acids and corresponding
1-(acylamino)phosphonic acids discussed in this work a.
Name
Trivial Name
Abbr./(No)
Ref.
Phosphono-Amino
Acid
AAP (3)
Aminoalkyl-phosphonic acid
R
Aminomethyl-phosphonic
acid
GlyP (3a)
AlaP (3b)
ValP (3c)
PglP (3d)
PheP (3e)
H
Phosphono-glycine
Phosphono-alanine
Phosphono-valine
[64]
[65]
1-Aminoethyl-phosphonic
acid
Me
iPr
1-Amino-1-methylethyl-
phosphonic acid
1-Amino-1-phenylmethyl-
phosphonic acid
Phosphono-phenyl-
glycine
Ph
1-Amino-1-phenylethyl-
phosphonic acid
Phosphono-phenyl-
alanine
PhCH2
1-(Acylamino)alkyl-
phosphonic acid
Acylamino-
Phosphono Acid
(AC)-AAP (2)
R1
R
H, Me, iPr, Ph,
PhCH2
1-(Acetylamino)alkyl
-phosphonic acid
Acetylamino-
Phosphono Acid
Ac-AAP (2A)
Ac-GlyP (2Aa)
CH3
[36]
1-(Acetylamino)methyl-
phosphonic acid
Phosphono-
Acetylamino Glycine
CH3
CH3
H
Phosphono-
Acetyl-amino-
PhenylGlycine
1-(Acetylamino)-1-phenyl-
methyl-phosphonic acid
Ac-PglP (2Ad)
TFA-AAP (2B)
Ph
Phosphono-
Trifluoroacetyl-amino
Acid
H, Me, iPr, Ph,
PhCH2
1-(Trifluoroacetylamino)-alkyl-
phosphonic acid
CF3
[37]
[36]
1-(Trifluoroacetylamino)-2-
methylethyl-phosphonic acid Trifluoroacetyl-Valine
Phosphono-
TFA-ValP (2Bc)
Bz-AAP (2C)
CF3
Ph
iPr
H, Me, iPr, Ph,
PhCH2
1-(Benzoylamino)alkyl-
phosphonic acid
Phosphono-
Benzoylamino Acid
Phosphono-
Benzoyl-Phenyl-
alanine
1-(Benzoylamino)-2-phenyl-
ethyl-phosphonic acid
Bz-PheP (2Ce)
Ph
PhCH2
a
Applied names were in accordance with the IUPAC rules, and the abbreviations were in agreement with the
general rules elaborated by Kudzin et al. [15].

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3.3. Reaction of Deacylation of 1-(acylamino)alkylphosphonic Acids
A sample of (AC)-AAP (0.5 mmol) was placed into 5-mL V-vials, before being dissolved in 5 mL
of appropriate solution fortified with D2O (2 M KOH). The vials were maintained at 25 ◦C 0.2 ◦C.
±
At various time intervals, the vials were removed from the baths and the samples of 0.5 mL were taken
for 3lP NMR analysis. The concentration of the substrate and formed products were determined from
the integration of its NMR signal.
3.4. Computational Methods
All quantum mechanical calculations were performed using the Gaussian 09 suite of programs [66].
Geometries of model compounds in the gas phase and in water solution were optimized using the
B3LYP hybrid functional containing three-parameter Becke (B3) exchange and Lee-Yang-Parr (LYP)
correlation functional and the 6–31+G(d) basis set. All stationary points were identified as stable
minima by frequency calculations. The vibrational analysis also provided the thermal enthalpy and
entropy corrections at 298 K within the rigid rotor/harmonic oscillator/ideal gas approximation.
Computed frequencies were scaled by a factor of 0.98. Transition states were verified by the intrinsic
reaction path (IRC) procedure to confirm if they did link the substrates and products along the
reaction path.
Free energies of the reaction in water were calculated within a continuum (implicit) solvent
approximation using the Conductor-like Polarizable Continuum Model (CPCM) with UFF (Universal
Force Field) cavities (SCRF = CPCM option as defined in Gaussian 09 program) [67]. This approximation
assumes that molecule of the solute is placed in a cavity within the solvent, which is treated as a dielectric
continuum (self-consistent reaction field). Thermodynamic functions in solution were calculated based on
vibrational analysis, which was described above.
4. Conclusions
The analysis of 31P NMR results presented above leads to the following conclusions:
•
•
•
The alkaline deacylation of (AC)-AAP occurs through the hydroxyl adduct intermediates,
which was observed on 31P NMR spectra of the reaction mixtures;
The deacylation ability of investigated (AC)-AAP derivatives exhibited strong dependence of
electron-acceptor character of the acyl group, with CF3-C(O)- > CH3-C(O)-;
The 1-(acylamino)alkylphosphonic acids are present in aqueous solutions with substantial stability
at ambient temperatures, which decreases with temperature elevation;
•
•
•
The deacylation of (AC)-AAP increases substantially in basic (2 M KOH) solutions;
The lowest deacylation ability (highest stability) was found for the 1-(benzoylamino acids Bz-AAP;
For the same type of 1-(acylamino)-derivatives (AC)-AAP, the highest stability was found
in(AC)-ValP and (AC)-PheP; lower stability in (AC)-AlaP; and the lowest in (AC)-GlyP and
(AC)-PglP;
•
All examined 31P NMR spectra of deacylation mixtures did not reveal any trace of
H3PO3 and/orH3PO4, which represent the products of dephosphonylation or oxidative
dephosphonylation of (AC)-AAP.
The theoretical calculations lead to the following conclusions:
The substrates with the trifluoromethyl group in R1 position are predicted to be significantly more
reactive than the other phosphonic amino acids studied, which is consistent with the experimental
results. The intermediates and products when R1=CF3 are also more thermodynamically favored
than the other derivatives. This difference in reactivity is due to additional stabilization of the
trianion 2 by the inductive effect of the strongly electron-withdrawing CF3 group. The calculations
did not provide unequivocal information about relative reactivities of other AA derivatives as the
calculated reaction energy barriers are similar (within a computational error), which is shown in

Molecules 2018, 23, 859
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Table 9 and Figure 12. The calculation results are consistent with those reported for acidic deacylation
of 1-(acylamino)alkylphosphonic acids [51]. As CF3 is strongly electron-withdrawing, this group
plays an activating role in basic media, increasing the acidity of carbonyl moiety. In contrast, it has a
deactivating effect in acidic media, decreasing the basicity of carbonyl oxygen.
Acknowledgments: The studies at the Textile Research Institute, Łódz´, in 2017 were partly financed by the Polish
Ministry of Science and Higher Education within statutory research founds (for M.H.K.). DFT calculations were
supported by the PL-Grid infrastructure. We would like to thank the National Science Center (Krakow, Poland)
for the support in the frame of the grant UMO 2014/15/B/ST5/05329 (for J.D.). Dedicated to the memory of
Professor Andrzej Okruszek.
Author Contributions: J.D. and Z.H.K. designed the research study and contributed to the data interpretation
and to the manuscript drafting and revisions. M.C. and B.G. carried out the Quantum Chemical Calculations
and contributed to writing the manuscript. M.H.K. performed the synthesis and purification of investigated
1-(acylamino)alkylphosphonic acids and was involved in the conception of the research study, analyzed the data,
and contributed to writing the manuscript. P.U. recorded NMR spectra and analyzed the experimental data.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all 1-(acylamino)phosphonic acids discussed in this paper are available from the
authors. Calculated geometries of all modeled species are available upon request.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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