Hydrolysis of phosphate esters in aqueous solution promoted by cobalt(III)-tetraamine metallohydrolases: Chromatographic and spectroscopic studies

Article abstract of DOI:10.1080/10426507.2018.1511557

A combination of chromatographic and spectroscopic (Q-ToF/MS; FTIR) studies were conducted to further understand hydrolytic processes in reactions of phosphate mono- and phosphodiesters with model Cobalt(III)-tetraamine metallohydrolases of the type [CoL(OH)(OH₂)]²⁺; L = (en)₂, (tn)₂; en = 1,2-diaminoethane, tn = 1,3-diaminopropane. High resolution mass spectroscopic (HRMS) analysis of the reaction fragments indicates that the monoester substrates undergo initial coordination to the metal center followed by an intramolecular hydroxyl attack on phosphorus, a process that is concerted with liberation of the bound alcoholic leaving group. The Co^III(tn)₂ complex displays twice as more reactivity towards both mono- and phosphodiesters compared to the (en)₂ analog under similar conditions, a behavior that is discussed in terms of the structural differences in the coordinated amine ligands. Plausible mechanisms for Co(III)-promoted hydrolysis of both the mono- and phosphodiesters are proposed.

Full text of DOI:10.1080/10426507.2018.1511557

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Hydrolysis of phosphate esters in aqueous
solution promoted by cobalt(III)-tetraamine
metallohydrolases: Chromatographic and
spectroscopic studies
Thokoza Bhengu, Tim Wood, Manelisi Shumane & Fikru Tafesse
To cite this article: Thokoza Bhengu, Tim Wood, Manelisi Shumane & Fikru Tafesse (2019)
Hydrolysis of phosphate esters in aqueous solution promoted by cobalt(III)-tetraamine
metallohydrolases: Chromatographic and spectroscopic studies, Phosphorus, Sulfur, and Silicon
and the Related Elements, 194:1-2, 87-101, DOI: 10.1080/10426507.2018.1511557
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
019, VOL. 194, NOS. 1-2, 87–101
2
https://doi.org/10.1080/10426507.2018.1511557
Hydrolysis of phosphate esters in aqueous solution promoted
by cobalt(III)-tetraamine metallohydrolases: Chromatographic
and spectroscopic studies
a
a
a
b
Thokoza Bhengu , Tim Wood , Manelisi Shumane , and Fikru Tafesse
a
b
Protechnik Laboratories, A Division of Armscor SOC Limited, Pretoria, South Africa; Department of Chemistry, College of Science,
Engineering and Technology, University of South Africa, Pretoria, South Africa
ABSTRACT
ARTICLE HISTORY
A combination of chromatographic and spectroscopic (Q-ToF/MS; FTIR) studies were conducted to
further understand hydrolytic processes in reactions of phosphate mono- and phosphodiesters
Received 28 March 2018
Accepted 9 August 2018
2þ
with model Cobalt(III)-tetraamine metallohydrolases of the type [CoL(OH)(OH
2
)] ; L ¼ (en)
2 2
, (tn) ;
KEYWORDS
en ¼ 1,2-diaminoethane, tn ¼ 1,3-diaminopropane.
Metallohydrolase; UHPLC;
Q-ToF/MS; phosphate
monoester;
High resolution mass spectroscopic (HRMS) analysis of the reaction fragments indicates that the
monoester substrates undergo initial coordination to the metal center followed by an intramolecu-
lar hydroxyl attack on phosphorus, a process that is concerted with liberation of the bound
phosphodiester; hydrolysis
III
alcoholic leaving group. The Co (tn)
2
complex displays twice as more reactivity towards both
analog under similar conditions, a behavior
mono- and phosphodiesters compared to the (en)
2
that is discussed in terms of the structural differences in the coordinated amine ligands. Plausible
mechanisms for Co(III)-promoted hydrolysis of both the mono- and phosphodiesters are proposed.
GRAPHICAL ABSTRACT
Introduction
The hydrolysis of phosphate esters is largely complicated
by the possible involvement of neutral or charged substrate
species including monoanionic and dianionic phosphate
Chemical reactions that involve the formation or cleavage of P–O
bonds in phosphate esters are of fundamental prominence in life
[1]
esters. Furthermore, these substrates can be attacked by H O
2
processes. The transfer of phosphoryl groups from one entity
to another is of’ crucial importance in nature. Indeed, the regula-
tion of virtually all cellular processes including the biosynthesis of
macromolecules such as proteins, RNA and DNA, energy pro-
duction, signal transduction and the control of protein activity is
dependent upon the phosphorylation and dephosphorylation of
ꢀ
[6–8]
and OH nucleophiles in solution.
Several plausible
mechanisms have been advanced in the literature for
[
9–11]
Phosphoryl Transfer Reactions (PTRs).
The potential
for both solvent and substrate-assisted pathways, which
involve proton transfer to the phosphoryl oxygens, further
complicates the interpretation of available experimental and
[
2,3]
various different biomolecules.
The reaction mechanisms that
[12]
computational tools.
These structural (X-ray structure
lead to the cleavage of the phosphoester bond are divided into
two major classes; those mechanisms that proceed via the
analysis), spectroscopic (UV-vis; 31P NMR) and computa-
tional (Linear Free Energy Relations, LFER’s; Kinetic Isotope
Effects, KIE’s; Entropic Effects - EF) tools have been
ꢀ
hydrated PO3 anion (metaphosphate) are called dissociative
(Equation 1), whereas mechanisms that require formation of an
collectively used to study the reaction pathways and the
intermediate or transition state with pentacovalent phosphorus
ꢀ
accumulated data has provided details of [PO ] transfer at
(
phosphorane) belong to the family of associative mechanisms
3
[1,4,5]
[9,12,13]
(Equation 2).
atomic resolution.
[14]
Recently, we reported
reactions of the phosphate monoesters, phenyl phosphate
PP) and 4-nitrophenyl phosphate (NPP), and the phospho-
diesters, diphenyl phosphate (DPP) and bis-4-nitrophenyl
on the uncatalyzed hydrolysis
2
ꢀ
ꢀ
3
ꢀ
HPO ꢀPO þ OH
(1)
(2)
4
2
4
ꢀ
ꢀ
3ꢀ
5
(
HPO þ OH ꢀH PO
2
CONTACT Fikru Tafesse
Tafesf@unisa.ac.za
Department of Chemistry, College of Science, Engineering and Technology, University of South Africa, Pretoria, USA.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
Supplemental data for this article can be accessed here.
ß 2018 Taylor & Francis Group, LLC

8
8
T. BHENGU ET AL.
phosphate (BNPP) probed by chromatographic and spectro-
The present study focusses on developing a more qualita-
scopic techniques. Here, we demonstrated, for the first time tive (HRMS) and quantitative (UV-Vis) picture in metal-
and using High Resolution Mass Spectrometry (HRMS; Q- ion-promoted phosphate ester hydrolysis that would hope-
ToF-MS), that under the experimental conditions of pH 7.0; fully produce a detailed experimental trend in a group of
ꢁ
H O 25 C, the monoesters react dissociatively to yield the selected phosphate esters with activated and inactivated leav-
2
;
ꢀ
free monomeric metaphosphate, [PO ] - m/z 78.9976, ing groups. Qualitative and quantitative data on metal ion-
3
(
E ¼ ꢀ0.0266 Da). It was further demonstrated that in the substrate reactivity profiles was collected using a combin-
r
absence of an unhindered nucleophile, this reactive species ation of chromatographic (ultra high performance liquid
is transferred to the solvent, water, with concerted proton chromatography; UHPLC) and spectroscopic techniques
transfer to the phosphate, producing predominantly othor- (Quadrupole-time-of-flight-mass spectrometry - Q-ToF-MS;
phosphoric acid, H PO – m/z 97.9954, (E ¼ ꢀ0.0014 Da). ultraviolet-visible spectrometry - UV-Vis, DAD and Fourier
3
4
r
These findings were found to be consistent with reported Transform Infrared Spectrometry – FTIR).
theoretical studies (KIE’s, LFER’s) that have been reported
[13,15]
for aqueous reactions of these compounds.
As a con-
Results and discussion
tinuation of our investigation on phosphate ester hydrolysis,
we report here the hydrolysis of some phosphate mono- and Characterisation of the metal complexes was conducted by
phosphodiesters (PP, NPP, DPP and BNPP) promoted by FTIR and significant vibrational modes are depicted in
Co(III) phosphatase models
of the type; cis- Table 1. As expected, the vibrational spectra of the com-
2þ
[
N Co(OH)(OH )]
,
N ¼ (en) - Compound 1; (tn)2- plexes display a striking similarity, particularly in those
4
2
4
2
Compound 2, (en ¼ 1,2-diaminoethane; tn ¼ 1,3-diamino- bands associated with direct bonding to the metal center
propane). Co(III) artificial metallonucleases stabilized by tet- such as v(M-N), v(M-O) and the coordinated water/
raamine ligands and that are analogous to compounds 1 and hydroxyl stretch, v(M-H O/OH). Vibrations that are affected
2
III
2
, such as Co -tren (tren ¼ tris- 2- aminoethyl amine), by the character of the amine ligand such as the v(CH ) and
2
III
III
Co -trpn (trpn ¼ tris- 3-aminopropyl amine) and Co - v(NH ) stretches show some slight differences. All these
2
cyclen (cyclen ¼ 1,4,7,10- tetraazacyclododecane), have been assignments were made by reference to available literature
[
15,26–29]
the subject of extensive research and they pass the test as data on analogous Co(III) complexes.
2
þ
the most suitable models for ATPase and phospha-
The neat Co(III) complexes, [N Co(OH)(H O)] ,
4 2
[
16–18]
tases.
They have been identified as some of the most N ¼ (en) ; (tn) were subjected to chromatography and
4
2
2,
7
active agents, providing up to 10 - fold rate enhancement spectroscopy at the conditions of the experiment in order
compared to the uncatalysed reactions. Most studies have to understand their dissociation profiles. It is expected
focused on their hydrolytic efficiency on phosphate mono- that in solution, the enzyme model complexes would lose
and phosphodiesters with good leaving groups such as p- the water ligand rather than the hydroxyl ligand and in
III
NPP and bis-dinitrophenyl phosphate, BDNPP, including the case of the Co -(en)
complex (MW ¼ 210.123 Da),
2
[
19–21]
phosphoanhydrides.
Model systems based on Co(III) are especially advanta- z ¼ 191.9952, which is associated with the loss of the water
geous in that metal coordination sites open for phosphate sub- ligand (E
, Da ¼ 0.1128). A similar behavior was observed
stitution can be limited thus permitting studies of a type not in the case of the Co -(tn)
readily available using labile cations such as Mg(II), Ca(II), where loss of the water ligand gave rise to a peak in the
this view was supported by a low intensity peak at m/
r
III
complex (MW ¼ 238.177 Da)
2
[
22,23]
Zn(II), Cu(II) and Mn(II).
Co(III) provides the oppor- mass spectrum at m/z ¼ 220.9595, giving an error of
tunity for a detailed mechanistic analysis in that isolated reac- ꢀ0.7975 Da in relation to the calculated exact mass of
tion species have defined metal ion-substrate interactions. The 220.162 Da. With reference to similar bidentate amine
relative substitutional inertness of Co(III) complexes allows ligand complexes of Co(III), the observed vibrational data,
unambiguous analysis of the reaction mixtures. Moreover, the coupled with their solution dissociation profile are in
Co(III) system has been chosen as a functional model in order agreement with the cis-arrangement of the ligands around
to take advantage of the high positive charge of Co(III) to acti- the metal center, leaving the water and hydroxyl ligands to
[
23,24]
[15,30–33]
vate substrates and/or nucleophiles.
occupy the axial positions.
Hydrolysis studies involving the selected phosphate
mono- and phosphodiester model compounds such as NPP
and BNPP, respectively, constitute significant reactions for
understanding the reactivity of Co(III) metalloenzymes and
The reaction profile of phosphate monoesters (PP, NPP)
with the enzyme models – qualitative analysis
metal complexes in biological systems. BNPP remains the The reaction profile of phosphate monoesters (PP, NPP)
most suitable DNA model since Co(III) complexes give with the cobalt metallohydrolases at various cobalt to sub-
comparable rate enhancements for the hydrolysis of phos- strate ratio (1:1, 2:1, 3:1 and 20:1) were conducted in aque-
phate esters with good or poor leaving groups. NPP is the ous medium at pH 7.0 and under ambient conditions
ꢁ
most researched model substrate for phosphate monoester (23 ± 2.0 C). Reactions were monitored at the predeter-
hydrolysis; it is known to have slower hydrolysis rates in mined time intervals of t ¼ 30 min (after adjusting the pH to
comparison to bisphosphates thus allowing more accurate 7.0), 60, 90, 120, 150 and 180 min. Qualitative data revealed
[24–26]
kinetic data to be accumulated.
that reactions at the 3:1 Co(III):substrate ratio contained the

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
89
most reactive species in solution and therefore all accumu-
lated qualitative chromatographic and spectroscopic data
concentrated on the 3:1 E:S reaction medium. The 20:1 E:S
reaction medium was used to confirm the identification of
MS fragment ions and it was found that no significant dif-
ference in qualitative data existed in reactions conducted at
the 3:1 versus those at the 20:1 Co(III):substrate ratios.
Figure 1 shows the negative electrospray total ion chro-
III
matogram (TIC) of the aqueous reaction between Co -(en)
2
and the monoester substrate, NPP. The corresponding chro-
III
matographic and spectroscopic data for the Co -(en) -PP
2
reaction is included in the supplemental material.
A common feature in the chromatographic eluent ana-
III
lysis of the Co -(en) -monoester reactions is that below the
2
retention time (t ) of 0.50 min, the identifiable fragments
R
are the conjugate alcohols and the four phosphorus oxy-
acids, hypophosphoric acid (A ; MW ¼ 161.974 Da), pyro-
1
phosphoric acid (A ; MW ¼ 177.973 Da), orthophosphoric
2
acid (A ; MW ¼ 97.994 Da) and the dibasic phosphoric/
3
phosphorus acid (A ; MW ¼ 81.995 Da).
4
The extracted mass spectra (in negative ionization mode)
depicting the identification of these molecular species in the
III
reactions of Co -(en) with NPP are shown in Figures 2
2
and 3. A similar reaction profile was observed in the reac-
III
tions of the Co -(tn) system with the monoester substrates
2
and therefore the corresponding chromatographic and mass
spectral data are not included in this manuscript but are
III
available as supplemental material (Co -(tn) /NPP reac-
2
tion mixtures).
ꢀ
The nitrophenolate conjugate (O N–PhO ) appears at m/
2
z 138.0732 (MW ¼ 138.102 Da; E ¼ 0.0288); peaks at m/z
r
1
60.9801 and 177.9051 are attributed to the two phosphorus
oxy-acids,
hypophosphoric
acid
(MW ¼ 161.974 Da;
E ¼ 0.9939) and pyrophosphoric acid (MW ¼ 177.973 Da;
r
E ¼ 0.0679), respectively. A similar pattern is observed in
r
the extracted mass spectra in reactions of the PP substrate
with CoN . In both substrates, the most abundant of the
4
oxy-acids is othorphosphoric acid, H PO (t ¼ 0.465 min)
3
4
R
and appears as
a
base peak at m/z 98.9970
(
MW ¼ 97.994 Da; E ¼ ꢀ1.0030). The peak at m/z 82.9952
r
III
in the extracted mass spectra of the Co (en) - NPP reac-
2
tion mixtures is associated with the dibasic, phosphorus/
phosphonic acid, H PO (MW¼ 81.995 Da; E ¼ ꢀ1.0002).
3
3
r
All these assignments are common for the monoesters and
III
III
in their reactions with the Co -(en) and Co -(tn) metal-
2
2
lohydrolases. They have been confidently assigned to the
respective fragments at an average error of ꢂ0.7575 Da.
The actual hydrolysis reaction can be considered to occur
in the retention time region of t ¼ 1.0 to 4.5 min for the
R
III
monoesters. Focussing on the reaction between the Co -
(
en) system and NPP, significant fragment ions identified
2
in reaction mixtures in this region are shown in Figures 4
and 5.
The reactivity of Co(III) complexes is largely influenced
by isomeric configurations and acid–base equilibria as
depicted in Equations 3 and 4. Therefore in solution, each
of the Co(III) complexes exists in three different oxidation
states with the Co(III)-aquo-hydroxo form being the active

9
0
T. BHENGU ET AL.
III
ꢁ
Figure 1. Negative Electrospray TIC of Co -(en) - NPP, pH 7.0; H O, 25 C.
2 2
III
Figure 2. Extracted MS Spectrum of Co (en)
2
- NPP at 0.270 min.
2
þ
species under conditions of the present experiment (pH
In solution, inactive trans-[Co(L) (OH)(OH )] (L ¼ en,
2
2
[
23,33–36]
ꢁ
6
.8–7.2).
tn) isomerizes to the cis-isomer rapidly (t1/2 ¼1s, 25 C) and
III
þ
completely so that under the present experimental condi-
N4CoðOH2Þ ! N4CoðOHÞðOH2Þ þ H
2
(
3) tions (pH 6.8 – 7.2), the chemistry of Co (en) and
2
pH < 6:0
pH ꢂ 6:0 – 8:0
III
Co (tn) can be attributed exclusively to the hydrolytically
2
[23,36,37]
active cis-isomers.
þ
N CoðOHÞðOH Þ ! N CoðOHÞ þ H
4
2
4
2
(4)
Mass spectral data for the Substrate Fragments in the
Reactions of PP/NPP with CoN , N ¼ (en) ; (tn) show
pH > 8:0
4
4
2
2

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
91
III
Figure 3. Extracted MS Spectrum of Co (en)
2
- NPP at 0.463 min.
III
ꢁ
Figure 4. Major Ionic Fragments in the Reaction of Co (en)
2
with NPP, pH 7.0, 25 C.
liberation of the conjugate alcohols as depicted in Figure 6. substitution of the water ligand for phosphate through coord-
The underlying mechanism for the reaction is believed to ination of one of the non-bridging oxygen atoms of the ester
undergo similar patterns of reactivity observed for aqueous to the metal center to form complexes C , C , C and C .
1
4
6
10
dissociation
profile
of
complexes, which dissociate via the dissociation of the enzyme models coupled with reported
loss of the water ligand to yield the coordinatively unsatur- work on similar Co(III) complexes, that in solution, formation
the
neat
hydroxoaqua, This observation is in agreement with data from the aqueous
2
þ
[
N Co(OH)(OH )]
4
2
III
ated N Co (OH) species.
of the monodentate CoN (OH)–phosphato complexes, C , C ,
4 1 4
4
Under the experimental conditions, the monoesters exist in C6 and C10 proceeds via the slow, rate limiting substitution of
[
36–38]
aqueous solution in their dianionic form,
the detected the monoester substrates for H O in the reactive aquohydroxo
2
[37–39]
monodentate Co(III)-phosphato complexes result from the complex.

9
2
T. BHENGU ET AL.
III
ꢁ
Figure 5. Major Ionic Fragments in the Reaction of Co (en) with PP, pH 7.0, 25 C.
2
III
Figure 6. Significant Co – Substrate Fragments in the Reactions of PP/NPP with CoN
4
, N
4
¼ (en)
2 2
; (tn) .
III
Liberation of the nitrophenolate/phenolate conjugates from chelate, C (m/z 268.8761; E ¼ 1.1949 Da) for the Co -(en)
2
R
2
the hydroxo-(p-nitrophenyl/phenylphosphato) (tatraamine) – NPP reaction (Figure 4). These reactions proceed via intra-
cobalt(III) complexes is believed to be fast and yields the molecular attack of the metal-coordinated hydroxyl (HO )
ꢀ
bidentate (phosphato)(tetraamine)Co(III) 4-membered ring group on the phosphorus center, a route that is favored by

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
93
entropic effects over the alternative intermolecular attack by involves attack of cis-coordinated OH/NH on phosphorus
2
[
23,38,39]
the solvent, H O.
Moreover, intramolecular attack by with a concerted release of the nitrophenolate moiety.
the Co -coordinated hydroxyl nucleophile is favored since it
Table 2 presents the calculated isotope effects from these
is 10 – 10 more nucleophilic than uncoordinated hydrox- studies, focusing on the reactions of Co(III)-coordinated p-NPP:
2
III
5
6
[36]
ide.
From mass spectral analysis, it is detected that the 4-
With reference to Table 2, evidence of intramolecular
membered ring chelate, C is highly susceptible to H-bonding hydroxide attack/nitrophenolate release is indicated by the
2
III
18
at the bridging oxygen atoms to yield the N Co –m(OH) –P large primary 18O isotope effect ( O-bridge) of 2.13%, indi-
4
2
i
complex, C (Figure 4). Such a structural arrangement con- cating that the P–O bond is largely cleaved in the transition
3
state (TS). The 0.21% 15N isotope effect demonstrates con-
siderable electron delocalization at the leaving group sub-
stituent. In addition, it was concluded that the relatively
small secondary isotope effect ( O non-bridge) of 0.06%
suggests a slightly associative TS. By contrast, the smaller
forms to that of natural enzymes which are normally bridged
in the enzyme active sites by either the hydroxyl groups from
amino acid residues or by –OH nucleophiles. Hydrogen bond-
18
III
ing at the Co –O–Pi bridge assists the metal ion by providing
additional electrostatic stabilization including activation and
[39–41]
primary 18O isotope effect of 0.98% in the [Co(NH ) ] com-
orientation of the substrate.
3 5
plex suggests a more associative mechanism with no intra-
molecular activity, reflecting the different properties of the
Reactions involving intramolecular attack by coordinated
hydroxyl ion have previously been reported for a number of
–
ꢀ
[
37–39]
OH and NH moieties as nucleophiles . In the case of the
analogous Co(III) complexes.
orkers
of
Sargeson and cow-
conducted 18O-labelling studies in the hydrolysis
coordinated p-NPP in the complex, cis-
Co(en) (OH)O POC H NO ], Complex A, which is an
2
[
37]
cyclen complex, hydrolysis isotope effects are similar to
those of the Co(en) system which can be attributed to the
2
[
40]
cis-OH attack on the phosphorus center
.
[
2
3
6
4
2
On the basis of the observed qualitative, spectroscopic
data coupled with supporting evidence from related studies
involving analogous Co(III)-tetraamine complexes, the pro-
posed mechanism for the reaction of the selected monoest-
equivalent of Compounds C , C C6 and C10 shown in
1
4,
Figure 6. Complex A was synthesized and fully character-
ized afterwhich hydrolysis reaction with NPP was carried
out in the pH range of 7–14 and monitored using 31P NMR
and UV-vis spectroscopy. These workers detected that intra-
III
ers with the N Co complexes of (en) and (tn) may be
4
2
2
–
proposed to proceed via the following sequential pathway:
molecular 18O-labeled metal-coordinated OH initially
2þ
In the proposed mechanism for [N Co(OH)(OH )]
-
4
2
yielded
a
five-coordinate phosphorane which rapidly
promoted hydrolysis of PP and NPP, the metal ion plays a
dual role; as a Lewis acid and also as a template for the
reaction. From spectroscopic data, it is believed that the
intramolecular hydroxyl attack on phosphorus (Step I) and
the phenolate leaving group departure is a concerted process
that leads to the 4-membered, bidentate phosphorane inter-
mediate, (3). In the case of the bidentate, 4-membered inter-
mediate, (3), chelate-ligand geometry and basicity control
metal ion reactivity, phosphate ligand substitution and the
decayed to the [Co(en) PO ] chelate. In addition, the labeled
2
4
1
8O was detected bonded between Co and the P atom, sug-
–
gesting direct transfer from the attacking OH nucleophile.
When the same experiment was conducted using unlabeled
complex but in 18O-labeled water, it was demonstrated that
only 16% of the 18O label was incorporated into inorganic
phosphate. These findings clearly demonstrated that the
hydrolysis proceeds through an intramolecular Co(III)-
[37]
hydroxide attack on the phosphorus atom.
[
39,41,42]
initial hydrolytic step.
Relief of ring strain on the
Further supporting evidence for intramolecular attack by
coordinated hydroxide was demonstrated using known
experimental markers and mathematical models such as
phosphorus atom in the 4-membered ring complex, (3), may
be thought to provide a driving force for the subsequent
[
40] solvent/H
2
O attack and generation of the hydrolysis prod-
Kinetic Isotope Effects (KIE’s). Cleland and coworkers
used the analogous Co(III) complexes,
Co(en) (OH)(OH )] and [Co(NH ) (OH)(NH )] to
3þ
ucts and the N Co
enzyme residues. On attack by the
4
water solvent, the cyclic phosphorane intermediate decom-
2
þ
[
2
2
3 5
2
poses exclusively by exocyclic Co–O- cleavage to yield the
determine KIE’s on the hydrolysis of Co(III)-coordinated p-
NPP. The experimental approach was through the measure-
ment of mass ratios in liberated p-NP and also in dissoci-
ated p-NPP. In this way, these workers were able to
calculate isotope effects on both the hydrolysis and dissoci-
ation processes. It was observed that the hydrolysis process
[42–44]
hydrolysis products.
Observations from the present work are that solvent
attack at the phosphorus of the strained 4-memberered ring
system does not lead to the formation of the starting inter-
mediate, monodentate Co(III)-tetraamine phosphato com-
plexes, C , C C6 and C10 (Figure 6) . Such a pathway
1
4,
would result in the possible regeneration of the starting
Table 2. Isotope Effects (%) on Reactions of Cobalt(III)-Coordinated p-
[40]
aquohydroxo(tetraamine)cobalt(III)
complex,
Nitrophenyl Phosphate.
Reaction, Complex
Hydrolysis:
2þ
15
18
18
[N Co(OH)(OH )]
and production of phosphoric acid.
4
2
K
Bridge
K
Non-bridge
K
Whilst phosphoric acid is the major hydrolysis product
Co(en)
Co(NH
Co(cyclen)]
Dissociation:
Co(en)
Co(NH
2
0.21
0.12
0.16
2.13
0.98
2.07
0.06
0.45
0.57
from the present study, the starting CoN complexes have
4
3 5
)
not been detected and they are not regenerated under condi-
tions of the present experiments.
[
2
[
44]
2
ꢀ0.02
ꢀ0.02
0.63
0.11
1.35
1.67
Previous studies by Horn and coworkers
reveal
3 5
)
that the unique attribute of the metal complex in these

9
4
T. BHENGU ET AL.
III
ꢁ
Figure 7. Major Ionic Fragments in the Reaction of Co (en)
2
with BNPP, pH 7.0, 25 C.
III
ꢁ
Figure 8. Major Ionic Fragments in the Reaction of Co (en)
2
with BNPP, pH 7.0, 25 C.
hydrolysis reactions is its ability to generate a higher The reaction profile of phosphate diesters (DPP, BNPP)
concentration of the reactive nucleophile at a much lower with the enzyme models – qualitative analysis
pH than would be possible. It is further noted that the pH
for which the rate of the intramolecular process reaches a As observed in the reactions of monoesters with the CoN
4
maximum is shifted into the physiological range (pH systems, the corresponding diester reactions for DPP and
.8–7.2), which is a result of potential significance to the BNPP follow the same reactivity profile in that hydrolysis is
mechanism of action of the enzyme E. coli Alkaline detected in the retention time region of 1.0–4.5 min.
6
[
44,45]
Phosphatase (AP)
.
Significant ionic fragments detected in the reactions of

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
95
III
ꢁ
Figure 9. Major Ionic Fragments in the Reaction of Co (en)
2
with BNPP, pH 7.0, 25 C.
III
ꢁ
Figure 10. Major Ionic Fragments in the Reaction of Co (en)
2
with BNPP, pH 7.0, 25 C.
III
BNPP with the Co -(en) system are depicted in Figures (A , hypophosphoric acid; A , pyrophosphoric acid) are
2
1
2
7
–10. A similar reaction profile was obtained for the reac- detected. The chromatographic peak in the region
III
tions of the Co -(tn) system with the diesters and for pur- 0.45–0.48 min is the area where othorphosphoric acid pre-
2
poses of focusing on the discussion of the observed diester dominates, always detected as a base peak at m/z 98.9970
reaction results, the corresponding chromatographic and (MW ¼ 97.994). The dibasic phosphonic acid, A , m/z
4
III
spectroscopic data for the Co -(tn) – BNPP reaction are 82.8963 (M/W ¼ 81.995) is also detectable in this region.
2
included in the supplemental material.
The hydrolysis reactions of the diesters may best be
In the total ion chromatograms (TIC’s) of the diester described in terms of the observed initial dissociation prod-
reactions with the CoN enzyme models, retention times in ucts, monophosphate and the alcoholic conjugate fragments.
4
III
the region t
0.10–0.47 min represent the area where the In the Co -(en) – BNPP reaction mixtures, the dissociated
2
R ¼
conjugate alcohols and the polymeric phosphorus oxyacids monoester and the alcoholic leaving group fragments can be

9
6
T. BHENGU ET AL.
2
þ
Scheme 1. Proposed Mechanism for the Reaction of PP/NPP With [N
4
Co(OH)(OH
2
)] ; N
4
¼ (en)
2
, (tn)
2
; Ar ¼ Phenolate/Nitrophenolate.
detected in the solution mixtures of the BNPP substrate and pNPP in the reactions of BNPP, it is evident that in the two
the enzyme system, signifying that aqueous dissociation of possible reactions in solution, which are, dissociation and
the substrate occurs prior to the hydrolysis process. With hydrolysis, the dissociation of the diester to the monoester
reference to the previously reported uncatalyzed aqueous and the alcoholic conjugate occurs first. Therefore it is safe
[
14]
reactions of the diesters , the dissociated NPP monoester to say that it is this dissociated monoester product that sub-
fragment could be detected in reaction solutions of BNPP sequently coordinates to the Co(III) center to complete the
(protonated or deprotonated form), an indication that it is hydrolysis process which facilitates the release of the second
one of the solution dissociation products following the equivalent of the conjugate alcohol as depicted in scheme 1.
release of the first equivalent of the conjugate alcohol.
These observations are in direct contrast to earlier views
[17,43]
Extracted mass spectral data indicated that the monoanion by Chin and coworkers
who studied the hydrolysis of
of the deprotonated molecular ion in both DPP and BNPP BNPP promoted by Co(III) complexes of the type; cis-
2
þ
is detectable as a base peak in the reaction mixtures of these [(L)Co(OH)(OH )] , L ¼ cyclen, tren, trpn. These workers
2
substrates: DPP ¼ 249.0294 (E ¼ 0.1576); BNPP ¼ 339.0928 proposed a mechanism involving coordination of the diester
r
(
E ¼ 0.0812). Since the deprotonated molecular anion is monoanion to the Co(III) center via the non-bridging oxy-
r
detected together with the dissociation products, pNP and gen atom, followed by ring closure through intramolecular

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
97
Scheme 2. BNPP Hydrolysis Promoted by cis -[(L)Co(OH)(OH2)]2þ, L ¼ cyclen, tren, trpn[17,43]
.
hydroxide attack at phosphorus. Hydroxide attack at phos- complexes reveal that the preferred angle at P in the 4-
ꢁ
phorus is reported to be concerted with the cleavage of one membered ring chelate is 108–109 and provided the chelate
of the P–O–R bonds, as shown in Scheme 2.
spans an axial-equatorial position, the strain remains consid-
Under the present experimental conditions, such a mech- erably less.[
39,45]
anism does not account for the observed monodentate
The influence of hydrogen bonding at the bridging oxy-
Co(III)-phosphato fragments detected in the reactions of gen atoms deserves some comment. In a recent study
III
III
3þ
2
DPP and BNPP with the Co -(en) and Co -(tn) model involving the Co(III) complexes, cis-[Co(cyclen)(H O) ]
2
2
2
3þ
[41]
systems (C - Figure 8). Therefore this mechanism cannot and [Co(tren)(H O) ] , Basallote and coworkers
demon-
4
2
2
be applicable under conditions of the present study since no
strated that the formation of mono- and bis-hydroxo-
bridged species in solution is relevant for understanding
their speciation and reactivity. It was further shown that in
the reactivity of biologically relevant polyphosphates and
coordinated diester monoanion complexes in the form of
DPP/BNPP–Co(III) fragments could be detected in any of
the reaction mixtures using accurate mass measurements.
Therefore from the foregoing spectroscopic evidence, the
diester monoanions are not involved in the hydrolysis reac-
tion but undergo initial dissociation to yield the correspond-
ing monoester and one equivalent of the conjugate alcohol.
The monoester fragments may be protonated but it is
believed that under conditions of the present experiment,
the dianionic form is predominant and readily coordinates
to the Co(III) center to yield the monodentate Co(III)-phos-
inorganic phosphate, P , outersphere hydrogen bonding
i
interactions remain a dominant factor for the hydrolysis
process. Thus in view of the obtained experimental evidence,
the present work demonstrates that coordination of a phos-
phate substrate to a Co(III) center that is positioned cis- to
a hydroxo ligand results in an enhanced rate of cleavage of
the ester. The study further demonstrates the efficacy of
metal-coordinated –OH ion as a powerful nucleophile in the
intramolecular pathway for cleaving phosphate esters under
neutral pH conditions.
Therefore, on the basis of the accumulated spectroscopic
data that is supported by relevant analogous studies involv-
ing Co(III) complexes, the mechanism for the Co(III)-pro-
moted aqueous hydrolysis of the phosphodiesters, DPP and
BNPP can be demonstrated as shown in Scheme 3.
phato complex, C , similar to monodentate phosphato spe-
4
cies detected in the case of monoester reactions with the
enzyme models (refer to Figure 6).
The detection of compounds C and C (BNPP reactions
4
10
with CoN ) in the mass spectra provides sufficient evidence
4
that the dissociated monoester products, NPP and PP are
responsible for coordination to the Co(III) center before any
second equivalent of the conjugate alcohol can be released.
Thus as is the case with the monoester reactions reported
earlier, attack of the metal-coordinated hydroxyl nucleophile Conclusion and future considerations
at the phosphorus center results in the formation of the
III
It has been demonstrated in the present study that the prep-
Co –m(O) –phosphato, four-membered ring chelates
2
aration of the Co(III) model compounds comes with min-
imal challenges yet with good yields under favorable
laboratory conditions. The complexes are kinetically robust
thereby allowing the characterization of all the species pre-
sent in solution. The kinetics and mechanism of the
observed cobalt(III) complex substitution reactions is easily
understood, a knowledge that has facilitated the identifica-
tion of mechanisms involved in complex-promoted phos-
phate ester hydrolysis.
(
Compound C Figure 9).
As mentioned earlier, compounds C and C are detected
2,
2
7
to be highly susceptible to hydrogen bonding at the bridging
oxygen atoms. This, coupled with the ring strain at the
Co –m(OH) –P center presents an ideal situation for the
hydrolysis process to progress. From spectroscopic data and
reports from similar work involving analogous Co(III) tetra-
amine complexes,
released through exocyclic Co–O-ring opening leading to
the hydrolysis products and some enzyme residues.
III
2
[
36,37]
it is believed that ring strain is
In the practical context, the present study has permitted
the identification of experimental techniques that may facili-
tate future investigations of similar reactions. It has been
In the present study, the 4-membered Co(III)-phosphato
[
37]
chelates postulated by other workers
are essentially repre-
sented by compound C (Figure 9). Based on data from the demonstrated that the UHPLC-QToF method of analysis
2
present studies, and with reference to studies involving other offers numerous advantages over other researched methods
analogous Co(III) complexes, it is believed that Co(III)- such as the UV-Vis and NMR spectroscopic techniques. The
bound hydroxyl attack and the P–O–L bond cleavage are challenge with other techniques is that they offer limited
g
concerted processes and determine steps towards the forma- knowledge from direct experimental investigations, particu-
tion of the Co(III)-m(O )-phosphato, 4-membered ring che- larly on the mechanism of the cleavage of the phosphate
2
[
38,39]
late.
X-ray studies of similar Co(III)-tetraamine esters promoted by metal complexes. This is due to lack of

9
8
T. BHENGU ET AL.
2
þ
Scheme 3. Proposed Mechanism for the Reaction of DPP/BNPP With [N
4
Co(OH)(OH
2
)] ; N
4
2 2
¼ (en) , (tn) ; Ar ¼ Phenolate/Nitrophenolate.
direct observations and first hand evidence during the reac-
It was further demonstrated that on their own, the model
2
þ
tion which results in uncertainties of the binding mode of compounds, [N Co(OH)(OH )] , N ¼ (en) ; (tn) undergo
4
2
4
2
2,
the substrate-metal complexes and interactions between the spontaneous dissociation in aqueous medium and at pH 7.0
phosphate esters and the metal complexes. The UHPLC- via the release of the water ligand to yield the coordinatively
QToF procedure adopted in the present study permits separ- unsaturated N Co–OH species. Therefore, under the current
4
2þ
ation and detection of charged species in solution (negative experimental conditions and in the [N Co(OH)(OH )]
-
4
2
and positive); it is therefore convenient and should have promoted hydrolysis of the monoesters, PP and NPP, the
general applications to metal-ion complex-promoted catalytic reaction is initiated by the substitution of the water ligand
reactions, in general. Using this technique, we have been able for the monoester to the cobalt(III) center via one of the
to demonstrate that hydroxoaquotetraaminecobalt(III) com- non-bridging oxygen atoms in a monodentate fashion. From
plexes are capable of hydrolyzing organophosphorus com- spectroscopic data, it was demonstrated that this is followed
pounds with a clear identification of critical reaction by the intramolecular attack of the Co(III)-cis-coordinated
intermediates. The catalytic nature of the reaction is derived hydroxyl ligand on phosphorus which is believed to be a
from the relatively rapid hydrolysis of the intermediate phos- concerted process with the departure of the alcoholic pheno-
phate- cobalt(III) complexes to yield inorganic phosphates as late leaving group. This results in the formation of a
the predominant products.
strained, 4-membered, bidentate phosphorane intermediate,

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
99
where chelate-ligand geometry and basicity control metal Puripac PP8 cartridges using
ion reactivity, phosphate ligand substitution and the initial Purification System (Lasec).
a Purite Select Water
hydrolytic step. Relief of ring strain on the phosphorus atom
Hydrolysis studies for qualitative analysis were conducted
in the 4-membered ring chelate provides a driving force for on a STEM RS 600 Electrothermal Reaction Station
equipped with a magnetic stirring devise and a temperature
probe (Lasec).
the subsequent solvent/H O attack. In the process, the cyclic
phosphorane intermediate decomposes exclusively by Co–O-
2
In the preparation of samples for FTIR analysis, reac-
tion aliquots (6–8 mL) were separately transferred into
glass tubes and evaporated to dryness using a centrifugal
evaporator. Equipment parameters: Centrifuge: RVC 2–33
exocyclic cleavage to yield the hydrolysis products and some
3þ
N Co enzyme residues.
4
One of the key reaction elements that has been demon-
strated in the present study is that in aqueous medium,
phosphate diesters first undergo solution dissociation to
yield the monophosphate and one equivalent of the conju-
gate alcohol before they can embark on any hydrolysis pro-
ꢁ
ꢁ
IR, T C set at 30 ± 3 C, 900 rpm; Freeze-drier: Martin
Christ Freeze Drier Alpha 2–4 LSC Plus, Vacuum set at
ꢁ
ꢁ
2
0 ± 1 mbar, T C set at ꢀ80 ± 5 C. Solid samples were
2
þ
then reconstituted as mulls in liquid paraffin between KBr
discs and the instrument resolution was maintained at
cm . Characteristic vibrational bands were assigned by
cess involving the [N Co(OH)(OH )] model systems. This
4
2
signifies that under the experimental conditions, the energy
barrier towards the dissociation pathway is favored to that
of the hydrolysis pathway via the non-bridging oxygen atom
of the diester. Once the dissociation step has been achieved,
the hydrolysis reaction simply follows that observed in the
case of monoesters and can be summarized as follows:
ꢀ
1
4
comparison with spectral data of similar Co (III) coordin-
[15,27,43]
ation compounds.
Preliminary kinetic studies of the reaction mixtures were
conducted using the UHPLC-QToF instrument, monitoring
the depletion of the substrate against formation of the prod-
uct at the respective m/z values. Confirmatory kinetic studies
were conducted in-situ inside the cuvette using the UV-Vis
method by monitoring the release of the conjugate alcohols
1
.
.
Rate-limiting substitution of the phosphate ester for the
water ligand in the hydroxoaquo Co(III) complexes;
Fast, intramolecular attack of Co(III)-coordinated hydrox-
ide on phosphorus leading to the formation of a strained,
2
(
(
pNP at 405 nm in the reactions of NPP and BNPP; phenol
Ph) at 290 nm in the reactions of PP and DPP).
4-membered ring chelate, a process that is concerted with
the liberation of the conjugate leaving group alcohol;
Intermolecular attack by the solvent water on the phos-
phorus of the 4-membered ring chelate to yield the
hydrolysis products and some enzyme residues;
21
Synthesis of the enzyme models: [co(tn) (OH)(H O)]
2
2
3
4
.
.
21
and [co(en) (OH)(H O)]
2
2
The synthetic route leading to the preparation of the
complexes followed
the phosphorus center depends, amongst other factors, the initial synthesis of the intermediate, sodium tris-
on the number and nature of electron withdrawing sub- carbonatocobaltate (III) trihydrate, Na ]Co(CO ].3H O,
The intermediate
The rate at which the potent M–OH nucleophile attacks Co(III)-hydroxoaquotetraamine
)
3
3
3
2
[
46]
developed by Bauer and Drinkard.
stituents attached to the bridging O-atom of the sub-
has certain advantages as a precursor for the preparation
of a representative group of Cobalt (III) coordination
compounds in that it is very reactive under mild condi-
tions and it is fairly stable on storage, if kept dry and
away from UV rays.
strate, and, on the nature of the N ligand coordinated
to the Co(III) ion;
The hydrolysis pathway does not lead to the generation of
the starting hydroxoaquo (tetraamine)cobalt(III) complex.
4
5.
The intermediate was converted to the respective perchlo-
[
23,28,29]
Experimental
rate salts using established literature procedures.
Conversion of the perchlorate salts to the hydroxoaquo
complexes was achieved by the addition of 6 M perchloric
acid (2.5 mmol) to 1 mmol of finely divided carbonato spe-
Materials and methods
Synthetic reagents including the organophosphorus sub-
strates (PP, DPP, NPP and BNPP; Sigma-Aldrich) and all
other reagents used were either analytical reagent grade or
the purest available commercially and were used without
further purification. Standard solutions of both the metal
complexes and the substrates were freshly prepared before
any reactions were attempted, otherwise solutions were gen-
erally kept refrigerated.
pH measurements were performed on a Jenway 3520 pH
meter equipped with a combination electrode and connected
to a temperature probe (Lasec). Instrument calibration was
performed using standard buffer solutions (pH 4.0 and pH
cies. The mixture was then stirred in an aspirator vacuum
ꢁ
for 30 min at 50 ± 0.1 C to liberate CO . The resulting
2
purple solution was then diluted with deionized water
(10–15 m‘) and the pH adjusted to 7.0 ± 2.0 using varying
concentrations of NaOH or HClO4.
Liquid samples of the hydroxoaquo complexes were
evaporated to dryness under vacuum and kept in a dessica-
tor, in the dark until ready for use.
Protocol of the hydrolysis reactions –
qualitative analysis
ꢀ
3
7
.0). Deionised water was purified by continuously passing The substrate solution (6 mL; 5 x 10 M) was transferred
through a dual filtration system; Purite Pre-treat 8/Purite into
a
thermostated reaction vessel equilibrated at

1
00
T. BHENGU ET AL.
ꢁ
ꢀ2
2
5 ± 0.1 C. The enzyme solution (6 mL: 5 ꢃ 10 M) was confidence and in complex mixtures. QToF-MS combines
transferred, drop-wise followed by the addition of the solv- high full spectral sensitivity with high mass resolution,
ent, deionized water (18 m‘). The mixture was then stirred allowing the mass of any ionizable component in the sample
continuously for about 30 min after which the pH of the to be accurately measured. The system can provide a notable
solution was adjusted to 7.0 ± 0.2 using varying concentra- amount of chemical information in a single experiment; this
tions of NaOH or HClO (0.05 M, 0.10 M, 0.25 M, 0.5 M, being facilitated by the interface with UV or Diode Array
4
0
.75 and 1.0 M). After pH adjustment, the first aliquot was Detection (DAD) which provides additional information
drawn for analysis (t ¼ 0 min). Subsequent aliquots were that is useful if molecules are not readily ionizable or missed
then drawn at the predetermined time intervals of 30 min,
0 min, 90 min, 120 min and 180 min. Aliquots were then
prepared for analysis using Q-ToF-MS, DAD-UV-Vis and
FTIR techniques.
by the QToF method. Besides providing accurate mass
measurements of the deprotonated molecular anions, the
instrument in full scan mode provides accurate masses of
their collision induced dissociation (CID) products.
6
Protocol of the hydrolysis reactions –
quantitative analysis
UV-Vis Spectroscopy (Thermo- Scientific Multiscan Go
Version 100.40/SkanIt Software Version 3.2)
Initial kinetic studies were conducted using the UHPLC-
QToF method by tracking substrate depletion against prod-
uct formation. This method had to be abandoned due to
clogging of the HPLC pumping system presumably from
insoluble Co(III)- substrate residues from the reac-
tion mixtures.
The UV-Vis method was then followed using the
Thermo-Scientific Multiscan Go Version 100.4 instrument
equipped with a SkanIt Software (Version 3.2). In this
method, basis for selecting the right software parameters
were set by initially conducting spectral scanning of all the
substrates and expected reaction products in the range 200
to 600 nm. This was done to confirm kmax values of the sub-
strates and reaction products. Using the instrument soft-
ware, at least three (3) kinetic loops were programed with 1
x loop consisting of 1000 readings at a kinetic interval of
UV-vis studies were designed to examine the effects of long
reaction times between the Co(III) complex and the sub-
strate and also to assess the effects of the reaction medium
on substrate depletion versus alcoholic leaving group forma-
tion. In conducting in-situ kinetic studies, substrate and
enzyme solutions were separately transferred into a 5.0 m‘
cuvette and the reaction monitored over at least three (3)
kinetic runs. Substrate-enzyme reactions were carried out
under pseudo-first order conditions with a large excess
(
ꢂx10) of the Co(III) complex over the phosphate ester.
Observed first norder rate constants were obtained by fitting
the first 3 half-lives of the reaction according to the first
order kinetics equation, ln[A] ¼ ln[A] – kobsd. t. The half-
t
0
lives of the reactions were determined using the expression
t1/2 ¼ ln2/k.
Data from these studies is available as supplementary
material.
1
0 s. Photometric settings were put at two (2)/dual wave-
lengths; one corresponding to the reactant substrate and the
other corresponding to the expected phenolate product
(300 nm and 405 nm for NPP/BNPP reactions; 270 nm and Acknowledgements
2
90 nm for PP/DPP reactions, respectively).
The hydrolysis reaction was initiated inside the cuvette
The authors would like to extend their sincere appreciation to the
South African Defense Research and Development Board (DRDB) for
financial assistance in this project.
ꢀ
3
by first transferring the substrate solution (ꢂ 10 M;
ꢀ
2
2
.0 mL) followed by the enzyme solution (ꢂ10 M; 2.0 mL)
in a 1:10, S:E stoichiometric ratio, assuming the hydroxoa-
ORCID
qua complex is the only reactive species under the experi-
ꢁ
mental conditions; pH 7.0, 25 C, aqueous medium. Each Fikru Tafesse
kinetic loop accumulated data related to changes in absorb-
ance over time whilst photometrically monitoring substrate
http://orcid.org/0000-0002-9321-5697
depletion against product formation.
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