A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach

Article abstract of DOI:10.1016/j.bioorg.2021.104810

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

Full text of DOI:10.1016/j.bioorg.2021.104810

Bioorganic Chemistry 110 (2021) 104810
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A dansyl-derivatized phytic acid analogue as a fluorescent substrate for
phytases: experimental and computational approach
a
b
c
b
a
´
Christophe Dussouy , Eric Dubreucq , Patrick Chemardin , Veronique Perrier ,
d
d
d
´
Josiane Abadie , Herve Quiquampoix , Claude Plassard , Jean-Bernard Behr
a
´
Universite de Reims Champagne Ardenne, CNRS, ICMR UMR 7312, 51097 Reims, France
b
´
IATE, Universite Montpellier, INRAE, Institut Agro, Montpellier, France
c
´
SPO, Universite Montpellier, INRAE, Institut Agro, Montpellier, France
d
´
Eco&Sols, Universite Montpellier, CIRAD, INRAE, Institut Agro, IRD, Montpellier, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The
fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the
catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the
new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases.
Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A
and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared
to their natural substrate.
Phosphate
Soil
Fertilizer
Inositol-phosphates
Phytases
Kinetics
Artificial substrate
1. Introduction
processed by plants, orthophosphate must be released from its organic
source by specific enzymes known as phytases. However, most of plant
Inositol-phosphates are phosphate esters of cyclohexanhexol, which
are involved in a wide range of biological processes [1]. Originally found
in plants [2], this family of inositol compounds was also identified later
in humans, where they play key roles in the regulation of ion uptake [3].
In particular, inositol 1,4,5-triphosphate promotes the release of Ca2+, a
biological mediator linked to cell proliferation, muscle contraction,
apoptosis or gene transcription [4]. Among the nine possible stereoiso-
mers of cyclohexanhexol, myo-inositol 1 is the most relevant, although
scyllo-, chiro- and neoinositol have also been isolated from animal tissues
and are present in soils [5] (Fig. 1). Myo-inositol hexakisphosphate, or
phytic acid 2 (abbreviated as InsP6 according to IUPAC recommenda-
tions) and its salts (named phytates) is sequentially biodegraded to
afford partially phosphorylated inositol penta- (InsP5), tetra- (InsP4), tri-
(InsP3), bis- (InsP2) or mono-phosphates (InsP1). Phytic acid is the major
storage form of phosphorus in plant seeds and makes inputs to soil
through two main ways [6a]. The first one is the fall of seeds to the
ground and the second one is the use of manures produced by mono-
gastric animals as they are not able to degrade phytate from seeds. Taken
as a whole, phytic acid might comprise up to 58% of soil organic
phosphorus [6b]. However, due to the lack of phytate membrane
transporters in roots [6a], plants cannot absorb directly phytate. To be
species are not able to secrete phytases in their root environment [6c]. In
contrast, microorganisms produce extracellular phytases responsible for
phytate hydrolysis, which can represent a crucial step in the P-cycle. So
far, four classes of phytases have been identified on phylogenetic kinship
[7]: purple acid phosphatase phytase (PAPhytase), histidine acid phy-
tase (HAP), cysteine phosphatase-like phytase (CP-phytase), and ß-pro-
peller phytase (BPP) [8]. Phytases are also characterized according to
their mechanism of action as 3-phytases (EC 3.1.3.8), which initiate
hydrolysis preferentially at the C-3 position, and 6-phytases (EC
3.1.3.26), which process at C-6. Although a great deal is known about
the biological relevance of microbial phytases in soil, gaps still remain in
the understanding of their accurate mode of action. This is due to the
difficulty to follow phytate hydrolysis in soil because the released
orthophosphate (i) will be trapped by adsorption on soil mineral sur-
faces such as clays or oxides and oxyhydroxides of Fe and Al and (ii) will
be difficult to distinguish from endogenous orthophosphate. Therefore,
it would be more efficient to extract and to identify by HPLC the various
phosphorylated inositols potentially generated by the microbial phy-
tases following phytate addition to the soil. Hence, to gain knowledge in
the localization, the role and the mode of action of phytases, more easily
detectable substrates are needed. A first artificial phytate analogue has
E-mail address: jb.behr@univ-reims.fr (J.-B. Behr).
https://doi.org/10.1016/j.bioorg.2021.104810
Received 10 December 2020; Received in revised form 3 February 2021; Accepted 5 March 2021
Available online 9 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

C. Dussouy et al.
Bioorganic Chemistry 110 (2021) 104810
detection of which is much more sensitive than UV-absorbance, would
address the sensitivity issues. The dansyl fluorescent label seemed
particularly attractive as it has been used with success for biological
purposes, in the synthesis of fluorescently labeled substrate analogues
for chitin synthase for instance [14]. The choice of the dansyl group
preferably to BODIPY dyes, cyanines, coumarins or other fluorogenic
labeling strategies for biological purposes was its ease of handling, its
stability in biological medium, its stability to various chemical trans-
formations, its commercial availability and low price and its compati-
bility with some enzymatic targets.
The compatibility of the dansyl derivative with the active site of
phytases was confirmed by molecular docking calculations performed
with structures of the Phy-A and Phy-C phytases crystallized in complex
with phytate analogue myo-inositol-1,2,3,4,5,6-hexakis sulfate and, for
Phy-C, Ca2+ [15,16] (PDB entries 3K4Q and 3AMR, respectively). As
shown on Fig. 2, the presence of the linked dansyl group did not seem to
interfere with the binding of the phytate moiety at the same location as
for the natural phytate and allowed a similar positioning of phosphate
groups in the catalytic cavity.
2.2. Chemistry
Since our final goal is to profile phytase activity in environments,
preparative amounts of artificial substrate are needed. For this reason
we wished to come up with a reliable synthetic route to 4a (Fig. 1),
which could be applicable on a large scale.
A three-stage method of myo-inositol protection/deprotection was
envisioned to keep hydroxyl at position C-5 free for further functional-
ization. For this, positions 1,3 and 5 were simultaneously protected by
reaction with triethyl orthoformate, according to a known reaction
(Scheme 1) [17]. Benzylation of the remaining free hydroxyls was then
performed under standard conditions to afford fully protected inositol 6.
Regioselective reduction of the orthoformate with diisobutylaluminium
hydride (solution in hexanes) yielded compound 7, which featured an
unprotected OH group at C-5 as expected. This transformation was
highly efficient and afforded quantitatively 7 after 2 h at room tem-
perature in the presence of 2.5 equivalents of hydride. During this latter
stage, when a solution of DIBAL-H in THF was used instead of the so-
lution in hexanes, only poor conversion was reached under the same
experimental conditions. The choice of the source of hydride was thus
crucial to succeed in this transformation.
Fig. 1. Structures of key compounds.
been prepared by Berry et al in 2005 [9]. The benzoylated analogue 3
(Fig. 1) was used to measure phytase activity in soils through the
identification of hydrolysis products (InsP6-n) by HPLC. However, rela-
tively high concentrations of substrate are required in biological assays
due to the low absorbance of the benzoyl chromophore. Another arti-
ficial phytate from commercial sources was used more recently to the
same purpose, but no information was available on its synthetic prep-
aration [10a,b].
In this investigation we report the development of the new fluoro-
genic phytic acid analogue 4a (Fig. 1) and the validation of its value as
an efficient substrate for two recombinant phytases that are (i) the HAP
phytase (Phy-A) from Aspergillus niger (EC 3.1.3.8) [11] and (ii) the BPP
phytase (Phy-C) from Bacillus subtilis (EC 3.1.3.8) [12]. Although both
enzymes are 3-phytases, their end products are different with InsP1 for
PhyA and InsP3 for PhyC. In addition, PhyC requires calcium for its
activity. Our final goal is to use 4a as a chemical probe for assaying
phytase activity in soils. For this reason an efficient synthetic route
enabling the production of preparative amount of 4a is needed [10b].
The attachment of an ethyleneglycol derived linker to inositol 7 was
anticipated by simple nucleophilc substitution of an activated form
(mesylate) of the linker with C5-OH. The linker should also feature an
amino function at the opposite end to tether the chromophore. To this
aim we prepared mesylates 9 and 11 from commercial 2-(2-amino-
ethoxy)ethanol 8 and 2-(2-chloroethoxy)ethanol 9 respectively. Stan-
dard protection of amine 8 with di-tert-butyl dicarbonate followed by
mesylation with methanesulfonyl chloride afforded 9 in 90% overall
yield (Scheme 2). Chloride 10 was treated with NaN3 for 2 days in
refluxing water and the resulting hydroxyl-azide was mesylated as above
to give 11 (72%, two steps) [18]. In the next step, whereas azide 11
reacted efficiently with inositol 7 in the presence of NaH to afford 12
(90% yield), the same reaction with protected amine 9 proved unsuc-
cessful. Instead, intramolecular reaction was kinetically favored and
only morpholine 13 was isolated along with unreacted inositol.
In order to make the synthetic route versatile and applicable in the
future to a range of fluorogenic species we intended to attach the
chromophoric structure at the later steps of the synthesis, that is on the
fully deprotected inositol-amine 15 (Scheme 3). Thus, azide 12 was
treated with boron trichloride at 0 ◦C to remove both the acetal and the
benzyl groups in a very smooth manner affording azide-pentol 14 in
good yields. Reduction of 14 under Staudinger’s conditions afforded
amine 15 quantitatively. However, reaction of dansyl chloride with
unprotected 15 proved difficult and yields did not exceed 60%. A more
reliable sequence was obtained by switching the steps and by
2. Results and discussion
2.1. Substrate design
The preparation of a suitable substrate for 3- or 6- phytases raised
several issues. The choice of the tethered position was crucial to allow a
possible action of both classes of enzymes. Thus, we intended to install
the chromophoric substituent at the non-reactive position C-5.
Furthermore, synthetic modifications at this position would minimize
stereochemical issues, keeping the intermediates and the final com-
pound as meso forms. According to structure 3 (Fig. 1), moving the
chromophoric species away from the inositol by a spacer allows to retain
affinity for the active site of phytase. The introduction of a PEG linker
would also enhance hydrophilicity and biocompatibility of the artificial
substrate [13]. Finally, the introduction of a fluorogenic probe, the
2

C. Dussouy et al.
Bioorganic Chemistry 110 (2021) 104810
Fig. 2. Molecular modelling of the positioning of
the inositol phosphate moiety in the catalytic site
of Phy-A (A, B) or Phy-C (C, D, E) when myo-
inositol 6P (A, C) or myo-inositol 5P with a fluo-
rogenic group (dansyl group) (B, D, E) is sup-
plied. Figures show the poses with highest
docking score after energy minimization of the
structure in an explicit water box. Docking scores
by Autodock Vina were ꢀ 6.3 kcal/mol (A), ꢀ 7.3
(B), ꢀ 5.2 (C), ꢀ 6.0 (D) and ꢀ 6.0 (E). For both
enzymes, the red and blue colors indicate nega-
tive and positive electrostatic potential, respec-
tively. In Phy-C, calcium ions are in green. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.)
introducing the dansyl group before deprotection. Thus, reduction of
azide was effected first on compound 12 to afford quantitatively amine
17, which was reacted without further purification with DNS-Cl to give
the corresponding tethered inositol 18 in 81% yield. Final deprotection
with BCl3 proved as efficient as above affording 16 in 98% yield. In
parallel, amine 17 was functionalized likewise with a benzoyl group
affording inositol 19 and its deprotected analogue 20, showing the
versatility of the synthetic route.
The phosphorylation sequence of inositols is a standard procedure
requiring an excess (usually 2 equivalents per hydroxyl) of O-dibenzyl or
O-ortho-xylylene-phosphoramidite followed by oxidation to the ex-
pected phosphate with mCPBA [19]. The presence of an acid activator is
usually necessary to assume complete and rapid conversion. Tetrazole
and derivatives thereof are commonly used, but pyridinium tri-
fluoroacetate (PyTFA) showed also good results. Initial phosphorylation
attempts of dansyl-inositol 16 were conducted under various conditions,
using either dibenzyl N,N-diisopropylphosphoramidite or o-xylylene N,
N-diethylphosphoramidite as the phosphorylating agent and PyTFA or
5-phenyltetrazole as the activator (Scheme 4). Though complete con-
version of the starting material occurred, the expected pentaphosphate
21 was isolated in only poor yields. Conversely, a control reaction with
benzoylated inositol 20 proved successful, affording 22 in 90% yield.
This result calls into questions the compatibility of the dansyl group with
the reaction conditions. Particularly, oxidation with mCPBA, although it
is conducted at low temperature, could affect the sulfonamide group. To
control the stability of dansyl in the presence of mCPBA, a solution of
inositol 16 and the peracid was stirred for 1 h 30 in dichloromethane.
NMR analysis of the crude mixture revealed the conversion of 50% of the
starting material into degradation products, confirming the in-
compatibility of the DNS moiety with mCPBA. Iodine was also checked
Scheme 1. Synthesis of intermediate 7.
Scheme 2. Syntheses of linkers.
3

C. Dussouy et al.
Bioorganic Chemistry 110 (2021) 104810
Scheme 3. Synthesis of tethered myo-inositols, experimental conditions: i: BCl3, 0 ◦C, then MeOH; ii: PPh3, DMF/H2O, 50 ◦C, 24 h; iii: DNS-Cl, NEt3, DMF, rt.
reaction, method of purification of final compound) permitted to
improve the isolated yields up to 56%. A first preparative synthesis of
DNS-phytate 4a was conducted and permitted to isolate 0.8 g of final
compound by debenzylation of 3.55 g of protected inositol 21. Its use as
substrate for various phytases was examined further to evaluate its po-
tential as a chemical tool for biological purposes.
2.3. Use of fluorescent substrate 4a by phytases
◦
A sample of compound 4a might be stored at ꢀ 20 C without any
degradation. Only 5% autogenic release of phosphate was measured
with a sample that was stored at room temperature for several months.
Further control was performed by NMR analysis in water (D2O), which
proved its complete stability for at least 48 h in this solvent (pH 1),
without any degradation. The potential of fluorescent myo-inositol
phosphate 4a to act as a substrate for phytases was then verified using
two recombinant enzymes produced by gene overexpression in the yeast
Komagataella (Pichia) pastoris: one fungal histidine acid phytase from
Aspergillus niger (Phy-A) and one bacterial ß-propeller phytase from
Bacillus subtilis (Phy-C). Both enzymes were assayed with their natural
substrate myo-inositol hexakisphosphate and with our new fluorescent
5-(dansylaminoethoxy)ethyl-myo-inositol-pentaphosphate.
Reaction
rates were estimated by measurement of Pi release using the Malachite
green method (see experimental part).
In the reaction conditions tested, using myo-inositol hexaki-
sphosphate (sodium salt), Phy-A proved significantly more active than
Phy-C (0.65 µmol.sꢀ 1.mgꢀ 1 protein vs 0.10 µmol.sꢀ 1.mgꢀ 1 protein)
(Fig. 3). Despite belonging to two very different phytase families, both
enzymes released free Pi from our artificial substrate 4a at the same rate
as for the natural substrate (0.60 µmol.sꢀ 1.mgꢀ 1 protein and 0.13 µmol.
sꢀ 1.mgꢀ 1 protein, respectively) (Fig. 3). The calculated conversion after
5 min (end-point) are for PhyA, 37,47 ± 1,8% with Na-phytate and
37,41 ± 2,9% with 4a (n = 3), for PhyC, 21,6 ± 1,29% with Na-phytate
and 23,47 ± 3,7% with 4a (n = 3). Taken together, these results
demonstrated that 4a is a valuable phytase substrate and that the
addition of the fluorogenic group did not alter recognition of the phytate
moiety of the substrate by either enzyme.
Scheme 4. Phosphorylation/hydrogenolysis steps.
as the oxidant in a further assay, with no improvement in the yields.
Finally, the use of oxygen peroxide allowed selective oxidation of the
phosphoramidite function without degradation of the chromophore.
However, yields of the expected phosphate 21 did not exceed 35%.
Indeed, a side product rising from additional phosphorylation at nitro-
gen could also be isolated [20]. This side reaction could be minimized by
simply lowering the amount of phosphoramidite from 10 equivalents to
6.6 affording 21 in 72% yield. During the final debenzylation step, the
amount of Pd-C had to be carefuly checked. Indeed, uncoupling of DNS
occurred when performing the first assays of hydrogenation in the
presence of a 1.4-fold amount of Pd-C vs benzyl-protected substrate 21,
affording the expected pentaphosphate 4a in unsatisfactory 20% yield.
Optimization of the reaction conditions (amount of catalyst, time of
4

C. Dussouy et al.
Bioorganic Chemistry 110 (2021) 104810
(60% dispersion in mineral oil, 129.6 mg, 3.24 mmol, 3 eq). The mixture
was stirred for 20 min and a solution of 2-(2-Azidoethoxy)ethyl) meth-
anesulfonate 11 (452 mg, 2.16 mmol, 2 eq) in DMF (10 ml) was added
slowly. The mixture was stirred for 30 min at 0 ◦C and then heated to
80 ◦C. After 18 h the reaction mixture was cooled to RT, poured into ice
and extracted with 3x50 ml of Et2O. The organic layers were washed
with water (50 ml) and NaHCO3 (2x50 ml). The combined organic
phases were dried (MgSO4), filtered, and concentrated under reduced
pressure. The residue was purified by chromatography on silica gel in
Tol/EA 95/5 to 9/1 to give 12 (560 mg; 0.97 mmol; 90%) as a colorless
oil. Rf = 0.49 with Tol/EA 8/2. 1H NMR (500 MHz, CDCl3) δ 7.47–7.27
(m, 15H, CH (Bn)), 5.18 (d, JHaHb = 5.6 Hz, 1H, Ha), 4.85 (d, JHbHa = 5.6
Hz, 1H, Hb), 4.69 (d, J = 11.7 Hz, 2H, CH2 (Bn)), 4.67 (s, 2H, CH2 (Bn)),
4.64 (d, J = 11.7 Hz, 2H, CH2 (Bn)), 4.27 (d, JH1H2 = JH3H2 = 1.1 Hz, 2H,
H1, H3), 3.94 (d, JH4H5 = JH6H5 = 6.1 Hz, 2H, H4, H6), 3.84–3.80 (m, 3H,
H2, HA’), 3.65–3.59 (m, 4H, HB’ , HC’), 3.54 (t, JH5H4 = JH5H6 = 6.1 Hz,
1H, H5), 3.32 (t, JHD’HC’ = 5.1 Hz, 2H, HD’). 13C NMR (126 MHz, CDCl3) δ
137.88 (2C, Bn), 137.70 (C, Bn), 128.56, 128.51, 127.95, 127.93,
127.90, 127.86 (15CH, Bn), 85.61 (CH2, CHaHb), 82.29 (2CH, C4, C6),
81.80 (CH, C5), 72.24 (2CH, C1, C3), 71.90 (2xCH2, Bn), 71.31 (CH2,
CA’), 71.07 (CH2, Bn), 70.86 (CH2, CB’), 70.24 (CH, C2), 70.01 (CH2, CC’),
50.75 (CH2, CD’). HRMS m/z [M + Na]+ calculated for C32H37N3O7Na
598.2534, found 598.2529.
Fig. 3. Rates of inorganic Pi release, expressed in µmol.sꢀ 1.mgꢀ 1 protein, by
Phy-A and Phy-C incubated with myo-inositol-hexaphosphate (Phytate) or 5-
(dansylaminoethoxy)ethyl-myo-inositol-pentaphosphate (Dansyl-phytate) sup-
plied as their sodium salts. Note that calcium, as a required co-factor, was
added to the substrate for incubating Phy-C. Values are means with standard
deviation between brackets (n = 3). For each enzyme, no significant differences
were found between means measured with either substrate (NS, Student’s t-test).
3. Conclusions
4.1.2.2. 2,3,4-tri-O-Benzyl-5-O-[2-(2-dansylaminoethoxy)ethyl)-1,3-O-
methylidene-myo-inositol (18). To a solution of inositol 12 (1.2 g, 2.18
mmol) in THF (40 ml) under an atmosphere of argon, was added PPh3
(821 mg, 3.12 mmol, 1.5 eq). The mixture was heated at 50 ◦C. After 8 h
the reaction mixture was cooled to RT and H2O was added (6 ml) while
keeping stirring overnight. The mixture was concentrated under reduced
pressure and directly used in the next step. The crude extract was dis-
solved in dry DMF (30 ml) under an atmosphere of argon at 0 ◦C, then
In conclusion, we prepared a chromogenic analogue of myo-inositol
hexaphosphate, which features a dansyl group at position C-5. Com-
pound 4a might be used as a non-natural substrate for phytases in order
to follow enzyme kinetics more conveniently. Preliminary docking ex-
periments conducted with structures of Phy-A and Phy-C (PDB entries
3K4Q and 3AMR) confirmed the compatibility of the dansyl derivative
with the active site of phytases. The presence of the tethered dansyl
group might not impede the binding of the phytate moiety and allows a
similar positioning of phosphate groups into the catalytic cavity. Two
enzymes, (Phy-A from Aspergillus niger and Phy-C from Bacillus subtilis)
were shown to operate experimentally with the chromogenic substrate,
at rates which were comparable to those observed for the natural sub-
strate (sodium phytate). Hence, the new substrate 4a will be useful to
assess the actual phytate hydrolysis in soil by microbial extracellular
phytases.
triethylamine (507 μL, 3.64 mmol, 2 eq) and dansyl chloride (834 mg,
3.1 mmol, 1.7 eq) were added. The mixture was stirred overnight at RT,
after which EA and water were added. The aqueous layer was extracted
twice with EA and the combined organic phases were dried (MgSO4),
filtered, and concentrated under reduced pressure. The residue was
purified by chromatography on silica gel in PE/EA 7/3 to 5/5 to give the
product (1.15 g; 1.76 mmol; 81%) as a yellow oil. Rf = 0.30 with PE/EA
5/5. 1H NMR (500 MHz, CDCl3) δ 8.51 (d, J = 8.5 Hz, 1H, CH (DNS)),
8.30 (d, J = 8.6 Hz, 1H, CH (DNS)), 8.18 (dd, J = 7.3, 0.9 Hz, 1H, CH
(DNS)), 7.53–7.43 (m, 2H, CH (DNS)), 7.39–7.24 (m, 15H, CH (Bn)),
7.13 (d, J = 7.5 Hz, 1H, CH (DNS)), 5.63 (t, JNHHD’ = 5.8 Hz, 1H, NH),
5.20 (d, JHaHb = 5.5 Hz, 1H, Ha), 4.81 (d, JHbHa = 5.5 Hz, 1H, Hb), 4.67
(d, J = 11.8 Hz, 2H, CH2 (Bn)), 4.63 (s, 2H, CH2 (Bn)), 4.58 (d, J = 11.7
Hz, 2H, CH2 (Bn)), 4.28 (s, 2H, H1, H3), 3.92 (d, JH4H5 = JH6H5 = 6.0 Hz,
4. Experimental section
4.1. Chemistry
4.1.1. Reagents and methods
2H, H4, H6), 3.86 (s, 1H, H2), 3.69–3.59 (m, 2H, HA’), 3.53 (t, JH5H4
=
Reactants and reagents were purchased from Aldrich and Sigma and
were used without further purification. Silica gel F254 (0.2 mm) was
used for TLC plates, detection being carried out by spraying with an
alcoholic solution of phosphomolybdic acid, followed by heating. Flash
column chromatography was performed over silica gel M 9385 (40–63
JH5H6 = 6.0 Hz, 1H, H5), 3.38–3.35 (m, 2H, HB’), 3.34 (t, JHC’HD’ = 5.0
Hz, 2H), 3.02 (q, JHD’HC’ = 5.4 Hz, 2H, HD’), 2.86 (s, 6H, CH3 (DNS)). 13
C
NMR (126 MHz, CDCl3) δ 151.99 (C, DNS), 137.85 (2C, Bn), 137.81 (C,
Bn), 135.42 (C, DNS), 130.35 (CH, DNS), 130.03 (C, DNS), 129.81 (C,
DNS), 129.22 (CH, DNS), 128.56, 128.54 (CH, Bn), 128.34 (CH, DNS),
127.99, 127.96, 127.94, 127.91 (CH, Bn), 123.28 (CH, DNS), 119.15
(CH, DNS), 115.28 (CH, DNS), 85.61 (CH2, CHaHb), 82.07 (2CH, C4,
C6), 81.31 (CH, C5), 72.12 (2CH, C1, C3), 71.96 (2xCH2, Bn), 71.07 (CH2,
Bn), 70.92 (CH2, CA’), 70.73 (CH2, CB’), 70.29 (CH, C2), 69.29 (CH2, CC’),
45.52 (2CH3, DNS), 43.18 (CH2, CD’). HRMS m/z [M + H]+ calculated
for C44H51N2O9S 783.3315, found 783.3312.
μ
m) Kieselgel 60. NMR spectra were recorded on Bruker AC 250 (250
MHz for 1H, 62.5 MHz for 13C) or 500 (500 MHz for 1H, 125 MHz for
13C) spectrometers. Chemical shifts are expressed in parts per million
(ppm) and were calibrated to the residual solvent peak. Coupling con-
stants are in Hz and splitting pattern abbreviations are: br, broad; s,
singlet; d, doublet; t, triplet; m, multiplet. High Resolution Mass Spectra
(HRMS) were performed on Q-TOF Micro micromass positive ESI (CV =
30 V).
4.1.2.3. 5-O-[2-(2-Dansylaminoethoxy)ethyl-myo-inositol (16). A solu-
tion of boron trichloride 1 M in DCM (5.3 ml, 5.3 mmol, 10 eq) was
added to a solution of inositol (18) (415 mg, 0.53 mmol) at 0 ◦C. The
reaction mixture was stirred for 5 h and MeOH was added to quench the
reaction. After evaporation the crude extract was taken into MeOH/Et2O
to form a precipitate which was filtered to give 16 (260 mg, 0.52 mmol;
4.1.2. Syntheses
4.1.2.1. 5-O-[2-(2-Azidoethoxy)ethyl)-2,3,4-tri-O-benzyl-1,3-O-methyl-
idene-myo-inositol (12). To a solution of inositol 7 (500 mg, 1.08 mmol)
in DMF (10 ml) at 0 ◦C under an atmosphere of argon, was added NaH
5

C. Dussouy et al.
Bioorganic Chemistry 110 (2021) 104810
98%) as a light brown solid. Mp = 213 ◦C. 1H NMR (500 MHz, MeOD) δ
8.57 (d, J = 8.5 Hz, 1H, CH (DNS)), 8.36 (d, J = 8.7 Hz, 1H, CH (DNS)),
8.22 (dd, J = 7.3, 1.2 Hz, 1H, CH (DNS)), 7.61 (dd, J = 8.6, 7.6 Hz, 1H,
CH (DNS)), 7.59 (dd, J = 8.5, 7.3 Hz, 1H, CH (DNS)), 7.28 (d, J = 7.5 Hz,
1H, CH (DNS)), 3.95 (t, JH2H1 = JH2H3 = 2.8 Hz, 1H, H2), 3.81–3.76 (m,
2H, HA’), 3.66 (d, JH4H5 = JH6H5 = 9.5 Hz, 2H, H4, H6), 3.41–3.37 (m,
4H, HB’, HC’), 3.36 (d, JH1H2 = JH3H2 = 3.0 Hz, 2H, H1, H3), 3.07 (t,
JHD’HC’ = 5.5 Hz, 2H, HD’), 2.98 (t, JH5H4 = JH5H6 = 9.3 Hz, 1H, H5), 2.88
(s, 6H, CH3 (DNS)). 13C NMR (126 MHz, MeOD) δ 153.17 (C, DNS),
137.21 (C, DNS), 131.20 (C, DNS), 131.16 (CH, DNS), 130.94 (C, DNS),
130.03 (CH, DNS), 129.19 (CH, DNS), 124.34 (CH, DNS), 120.62 (CH,
DNS), 116.48 (CH, DNS), 85.95 (CH, C5), 73.89 (3CH, C2, C4, C6), 73.28
(2CH, C1, C3), 72.67 (CH2, CA’), 71.77 (CH2, CB’), 70.59 (CH2, CC’),
45.81 (2CH3, DNS), 43.63 (CH2, CD’). HRMS m/z [M + Na]+ calculated
for C22H32N2O9NaS 523.1726, found 523.1718.
CH3 (DNS)), 3.39–3.33 (m, 3H, HA’, H5), 3.28 (t, JHC’HD’ = 4.7 Hz, 2H,
HC’), 3.23–3.18 (m, 2H, HD’), 3.12–3.07 (m, 2H, HB’). 13C NMR (126
MHz, D2O) δ 138.32 (C, DNS), 135.78 (C, DNS), 130.48 (CH, DNS),
128.63 (C, DNS), 128.13 (CH, DNS), 127.07 (CH, DNS), 126.75 (CH,
DNS), 125.69 (CH, DNS), 125.51 (C, DNS), 119.51 (CH, DNS), 79.68
(CH, C5), 76.54 (t, J = 5.6 Hz, 2CH, C4, C6), 75.94 (d, J = 5.6 Hz, C2),
73.39–72.71 (m, 2CH, C1, C3), 71.15 (CH2, CA’), 69.42 (CH2, CB’), 68.51
(CH2, CC’), 46.84 (2CH3, DNS), 41.82 (CH2, CD’). 31P NMR (202 MHz,
D2O) δ ꢀ 0.11 (2P), ꢀ 0.62 (2P), ꢀ 1.19 (1P). HRMS m/z [M + H]+
calculated for C22H38N2O24P5S 901.0223, found 901.0216.
4.2. Enzymatic assays
4.2.1. Recombinant enzymes
We worked with recombinant proteins produced by gene over-
expression in the yeast Pichia pastoris, with methanol as a promotor,
following reported procedures [21,22]. Both enzyme extracts (Phy-A
and Phy-C) were produced at the ‘‘Halle de Biotechnologie’’ (UMR IATE,
Montpellier, France). The Phy-A enzyme extract was of the same batch
as the one described in the literature [11]. It contained 45 mg/mL of
total protein mass assayed by the BCA Protein Assay (Pierce), of which
99% is the Phy-A enzyme. The yeast over-expressing the Phy-C enzyme
was obtained following a procedure from the literature [12]. The
maximal production of recombinant enzyme (17 mg of total protein,
pure at 99%) was obtained after five days of methanol induction. The
two enzyme extracts were kept at –20 ◦C before use.
4.1.2.4. 5-O-[2-(2-Dansylaminoethoxy)ethyl-1,2,3,4,6-pentakis-O-[bis
(benzyloxy)-phosphoryl-myo-inositol (21). To a solution of 16 (80 mg,
0.16 mmol) and 5-phenyltetrazole (257 mg, 1.76 mmol, 11 eq) in dry
dichloromethane (8 ml) under argon was added bis(benzyloxy)(N,N-
diisopropylamino)phosphine (290 μL, 0.88 mmol, 5.5 eq). Stirring was
continued for 3 h30 at room temperature. The reaction mixture was
cooled to 0 ◦C and H2O2 30% in water (228
μ
L, 2.23 mmol, 14 eq) was
´
added while stirring. The mixture was allowed to reach room temper-
ature and stirring was continued for 1 h at RT. The reaction mixture was
diluted with dichloromethane and washed with water and NaHCO3. The
aqueous layers were extracted with DCM (3 times) and the organic
layers were combined, dried over MgSO4, and concentrated under
reduced pressure. The crude extract was washed twice with Et2O/PE (1/
2; 50 ml) and the residue was taken in Et2O, filtrated and concentrated
under reduced pressure to give the product (208 mg; 1.27 mmol; 72%)
4.2.2. Phytase measurements
We quantified the rate of release of orthophosphate ions (Pi) either
from ultrapure sodium phytate (Sigma-Aldrich, Ref P0109) or from
dansyl-phytate 4a to measure the phytase activities with the two sub-
strates. The enzyme assay was carried out as described [11] with some
modifications. Briefly, the reaction was carried out in 1.5 ml Eppendorf
tubes containing 0.3 ml of 0.025 M acetate buffer pH 5.5, 0.1 ml of 1 mM
substrate and 0.1 ml of each diluted enzyme solution (containing 10 and
20 µg of protein.mlꢀ 1 for PhyA and Phy-C, respectively) or deionized
water for control. Calcium was added to the substrate solution (1/3,
phytate/calcium) to measure the activity of Phy-C. Two sets of reaction
mixtures were prepared to measure the Pi concentrations at time 0 and
after 5 min at 25 ◦C. For each time, the reaction was terminated by
adding 0.5 ml of tri-chloro-acetic acid (TCA) 10% (w/v). Reaction
mixtures were then diluted 21 times with deionized water before
measuring Pi concentrations in microplates using the Malachite green
method [23] carried out on 200 µL of the diluted solution and 40 µL of
each of the two reactants. After 30 min of incubation, absorbance of
solutions was measured at 630 nm on a microplate reader. Phytase ac-
tivity was expressed in µmol.sꢀ 1.mgꢀ 1 of protein.
1
as a yellow oil. Rf = 0.38 with PE/EA 5/5. MW: 1800.48 g.molꢀ 1. H
NMR (500 MHz, CDCl3) δ 8.51 (d, J = 8.7 Hz, 1H, CH (DNS)), 8.45 (d, J
= 8.5 Hz, 1H, CH (DNS)), 8.22 (d, J = 7.1 Hz, 1H, CH (DNS)), 7.44–7.34
(m, 2H, CH (DNS)), 7.30–7.11 (m, 50H, CH (Bn)), 7.05 (d, J = 7.5 Hz,
1H, CH (DNS)), 5.62 (d, JH2H1 = JH2H3 = 8.9 Hz, 1H, H2), 5.17–4.86 (m,
22H, CH (Bn), H4, H6), 4.39 (t, JH1H2 = JH3H2 = JH1H6 = JH3H4 = 9.5 Hz,
2H, H1, H3), 3.83–3.72 (m, 2H, HA’), 3.41 (t, JH5H4 = JH5H6 = 9.3 Hz, 1H,
H5), 3.31–3.20 (m, 4H, HB’, HC’), 3.06 (q, JHD’HC’ = 5.2 Hz, 2H, HD’),
2.81 (s, 6H, CH3 (DNS)). 13C NMR (126 MHz, CDCl3) δ 151.60 (C, DNS),
136.55 (C, DNS), 135.97, 135.92, 135.86, 135.82, 135.78, 135.75,
135.73, 135.68 (10C, Bn), 129.98 (C, DNS), 129.95 (C, DNS), 129.76
(CH, DNS), 128.67, 128.59, 128.56, 128.53, 128.52, 128.47, 128.45,
128.36, 128.31, 128.28, 128.23, 128.19, 128.13, 128.03, 128.00,
127.96 (50CH, Bn, 2CH, DNS), 123.23 (CH, DNS), 120.01 (CH, DNS),
115.14 (CH, DNS), 79.49 (CH, C5), 77.01 (t, J = 6.12 Hz, 2CH, C4, C6),
76.24 (d, J = 4.98 Hz, CH, C2), 73.83 (m, 2CH, C1, C3), 72.76 (CH2, CA’),
70.17, 70.13, 70.10, 70.06, 70.01, 69.96, 69.92, 69.86, 69.81, 69.61,
69.57 (12CH2, CB’, CC’, Bn), 45.50 (2CH3, DNS), 43.23 (CH2, CD’). 31
P
4.3. Computational details
NMR (202 MHz, CDCl3) δ ꢀ 1.30 (2P), ꢀ 1.44 (2P), ꢀ 2.69 (1P). HRMS m/
z [M + Na]+ calculated for C92H97N2O24NaP5S 1823.4738, found
1823.4751.
The 3D structures of Phy-A and Phy-C in complex with the phytate
analogue myo-inositol hexakis sulfate were obtained from the Protein
Databank (https://www.rcsb.org) under entries 3K4Q and 3AMR,
respectively. The structure of the subtrates was drawn and turned into
3D models using Marvin Sketch v20.16 (ChemAxon, http://www.che
maxon.com). Molecular docking was performed with Autodock Vina
v1.1.2 [24] using the UCSF Chimera v1.14 [25] interface. The structures
corresponding to the poses with the highest docking score were pro-
tonated for pH 5.5 using PROPKA 3.0 [26], placed in a TIP3P water shell
(8 nm thick) and their energy was minimized within UCSF Chimera with
default parameters using the AMBER ff14SB and the AM1-SCC force
fields for atomic partial charge calculations in the protein and the
ligand, respectively, with the protein backbone constrained to fixed
positions. The surface electrostatic potential was calculated with APBS
[27]. Molecular graphics were obtained using UCSF ChimeraX v1.1
[28].
4.1.2.5. 5-O-[2-(2-Dansylaminoethoxy)ethyl-1,2,3,4,6-pentakis-O-[bis
(benzyloxy)-phosphoryl]-myo-inositol (4a). To a solution of Inositol (21)
(86 mg, 0.047 mmol) in MeOH (8.6 ml), 10% Pd/C (41 mg) was added
and the mixture was stirred under H2 atmosphere at RT for 2 h. The
reaction mixture was filtered over Celite (washed with MeOH and H2O)
and the filtrate was evaporated under reduced pressure. The residue was
purified by reverse phase chromatography on C-18 (H2O as eluent) to
give (4a) (24 mg, 56%) as a white powder after lyophilization. 1H NMR
(500 MHz, D2O) δ 8.74 (d, J = 8.8 Hz, 1H, CH (DNS)), 8.44 (d, J = 8.6
Hz, 1H, CH (DNS)), 8.36 (d, J = 7.3 Hz, 1H, CH (DNS)), 8.11 (d, J = 7.8
Hz, 1H, CH (DNS)), 7.90 (q, J = 8.4 Hz, 2H, CH (DNS)), 4.90 (d, JH2H1
=
JH2H3 = 9.8 Hz, 1H, H2), 4.37–4.23 (m, 4H, H1, H3, H4, H6), 3.51 (s, 6H,
6

C. Dussouy et al.
Bioorganic Chemistry 110 (2021) 104810
[10] (a) D.F. Berry, K. Harich, Tetrachlorofluorescein TInsP(5) as a substrate analog
probe for measuring phytase activity in surface water: proof of concept, J. Environ.
Qual. 42 (2013) 56–64. https://doi.org/10.2134/jeq2012.0204. (b) Another
synthetic phytate has been prepared on a 16 mg scale, to visualize internalization
of inositol phosphates by cancer cells, see A.M. Riley, S. Windhorst, H.-Y. Lin, B.V.
L. Potter, Cellular Internalisation of an Inositol Phosphate Visualised by Using
Fluorescent InsP5, ChemBioChem. 15 (2014) 57–67. https://doi.org/https://doi.
org/10.1002/cbic.201300583.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
[11] C.M. Trouillefou, E. Le Cadre, T. Cacciaguerra, F. Cunin, C. Plassard, E. Belamie,
Protected activity of a phytase immobilized in mesoporous silica with benefits to
plant phosphorus nutrition, J. Sol-Gel Sci. Technol. 74 (2015) 55–65, https://doi.
org/10.1007/s10971-014-3577-0.
This work was supported by the ANR project “UNLOCKP” (ANR 11
BSV7 015 01; France) awarded to C. P.. C. D. was also supported by the
UNLOCKP project through a 12-month post-doctoral fellowship. The
authors would like to thank François-Xavier Sauvage (SPO) for his help
and advices to produce the recombinant enzymes and Jean-Marc Sou-
quet (Eco&Sols) for his help for phytase activities measurement.
[12] M. Guerrero-Olazaran, L. Rodriguez-Blanco, J. Gerardo Carreon-Trevino, J.
A. Gallegos-Lopez, J.M. Viader-Salvado, Expression of a Bacillus Phytase C gene in
pichia pastoris and properties of the recombinant enzyme, Appl. Environ.
Microbiol. 76 (2010) 5601–5608, https://doi.org/10.1128/AEM.00762-10.
[13] N.A. Alcantar, E.S. Aydil, J.N. Israelachvili, Polyethylene glycol–coated
biocompatible surfaces, J. Biomed. Mater. Res. 51 (2000) 343–351, https://doi.
org/10.1002/1097-4636(20000905)51:3<343::AID-JBM7>3.0.CO;2-D.
[14] A.R. Yeager, N.S. Finney, Synthesis of fluorescently labeled UDP-GlcNAc analogues
and their evaluation as chitin synthase substrates, J. Org. Chem. 70 (2005)
1269–1275, https://doi.org/10.1021/jo0483670.
Appendix A. Supplementary material
[15] A.J. Oakley, The structure of Aspergillus niger phytase PhyA in complex with a
phytate mimetic, Biochem. Biophys. Res. Commun. 397 (2010) 745–749, https://
doi.org/10.1016/j.bbrc.2010.06.024.
Supplementary data to this article (experimental procedures for
compounds 5-7,11,14-16 and NMR data for all compounds) can be
found online at https://doi.org/10.1016/j.bioorg.2021.104810.
[16] Y.-F. Zeng, T.-P. Ko, H.-L. Lai, Y.-S. Cheng, T.-H. Wu, Y. Ma, C.-C. Chen, C.-S. Yang,
K.-J. Cheng, C.-H. Huang, R.-T. Guo, J.-R. Liu, Crystal structures of Bacillus alkaline
phytase in complex with divalent metal ions and inositol hexasulfate, J. Mol. Biol.
409 (2011) 214–224, https://doi.org/10.1016/j.jmb.2011.03.063.
[17] F. Song, J. Zhang, Y. Zhao, W. Chen, L. Li, Z. Xi, Synthesis and antitumor activity of
inositol phosphotriester analogues, Org. Biomol. Chem. 10 (2012) 3642–3654,
https://doi.org/10.1039/C2OB00031H.
References
[1] (a) M.P. Thomas, S.J. Mills, B.V.L. Potter, The “other” inositols and their
phosphates: synthesis, biology, and medicine (with recent advances in myo-inositol
chemistry), Angew. Chem. Int. Ed. 55 (2016) 1614–1650, https://doi.org/
10.1002/anie.201502227;
[18] X. Yu, S. Eymur, V. Singh, B. Yang, M. Tonga, A. Bheemaraju, G. Cooke,
C. Subramani, D. Venkataraman, R.J. Stanley, V.M. Rotello, Flavin as a photo-
active acceptor for efficient energy and charge transfer in a model donor–acceptor
system, Phys. Chem. Chem. Phys. 14 (2012) 6749–6754, https://doi.org/10.1039/
C2CP40073A.
(b) B.V.L. Potter, D. Lampe, Chemistry of inositol lipid mediated cellular signaling,
Angew. Chem. Int. Ed. English. 34 (1995) 1933–1972, https://doi.org/10.1002/
anie.199519331.
[2] (a) F.A. Loewus, P.P.N. Murthy, myo-Inositol metabolism in plants, Plant Sci. 150
(2000) 1–19, https://doi.org/10.1016/S0168-9452(99)00150-8;
(b) C. Cridland, G. Gillaspy, Inositol pyrophosphate pathways and mechanisms:
what can we learn from plants? Mol. 25 (2020) https://doi.org/10.3390/
molecules25122789.
[19] (a) N.T. Rudolf, T. Kaiser, A.H. Guse, G.W. Mayr, C. Schultz, Synthesis and
metabolism of the myo-Inositol pentakisphosphates, Liebigs Ann./Recueil, (1987),
1861-1869, https://doi.org/10.1002/jlac.199719970909; (b) N.W. Brown Jr, A.M.
Marmelstein, D. Fiedler, Chemical tools for interrogating inositol pyrophosphate
structure and function, Chem. Soc. Rev., 45 (2016) 6311, DOI: 10.1039/
c6cs00193a ; W. Bannwarth, A. Trzeciak, A simple and effective chemical
phosphorylation procedure for biomolecules, Helv. Chim. Acta 70 (1987) 175-186,
https://doi.org/10.1002/hlca.19870700122.
[3] (a) J.B. Parys, H. De Smedt, Inositol 1,4,5-trisphosphate and its receptors, Adv.
Exp. Med. Biol. 2012, 740 (2012) 255–279, doi: 10.1007/978-94-007-2888-2_11.
(b) S.B. Shears, S.B. Ganapathi, N.A. Okhale, T.M.H. Schenk, H. Wang, J.D.
Weaver, A. Zaremba, Y. Zhou, Defining signal transduction by inositol phosphates,
Subcell. Biochem. 59 (2012) 389–412, doi: 10.1007/978-94-007-3015-1_13.
[4] H. Sterb, R.F. Irvine, M.J. Berridge, I. Schulz, Release of Ca2+ from
nonmitochondrial store in pancreatic acinar cells by inositol-1, 4, 5-triphosphate,
Nature 306 (1983) 67–69.
[20] B.T. Burlingham, T.S. Widlanski, Synthesis and biological activity of N-
sulfonylphosphoramidates: probing the electrostatic preferences of alkaline
phosphatase, J. Org. Chem. 66 (2001) 7561–7567, https://doi.org/10.1021/
jo010495q.
[21] C. Laborde, P. Chemardin, F. Bigey, Y. Combarnous, G. Moulin, H. Boze,
Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to
leptin production and characterization of the recombinant hormone, Yeast 21
(2004) 249–263, https://doi.org/10.1002/yea.1073.
´
[5] B.L. Turner, M.J. Paphazy, P.M. Haygarth, I.D. Mckelvie, Inositol phosphates in the
environment, Philos. Trans. R. Soc. London. Ser. B Biol. Sci. 357 (2002) 449–469,
https://doi.org/10.1098/rstb.2001.0837.
[6] (a) C.K. Madsen, H. Brinch-Pedersen, Globoids and phytase: the mineral storage
and release system in seeds, Int. J. Mol. Sci. 21 (2020) 7519, https://doi.org/
10.3390/ijms21207519;
[22] L. Brunel, V. Neugnot, L. Landucci, H. Boze, G. Moulin, F. Bigey, E. Dubreucq,
High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia
pastoris, J. Biotechnol. 111 (2004) 41–50, https://doi.org/10.1016/j.
jbiotec.2004.03.007.
˙
´
(b) J. Wierzbowska, S. Sienkiewicz, M. Zalewska, P. Zarczynski, S. Krzebietke,
Phosphorus fractions in soil fertilised with organic waste, Environ. Monit. Assess.
192 (2020) 315, https://doi.org/10.1007/s10661-020-8190-9;
[23] T. Ohno, L.M. Zibilske, Determination of low concentrations of phosphorus in soil
extracts using malachite green, Soil Sci. Soc. Am. J. 55 (1991) 892–895, https://
doi.org/10.2136/sssaj1991.03615995005500030046x.
(c) A.E. Richardson, T.S. George, I. Jakobsen, R.J. Simpson, Plant utilization of
inositol phosphates, inositol phosphates link, Agric. Environ. (2007) 242–260.
[7] (a) E.J. Mullaney, A.H.J. Ullah, The term phytase comprises several different
classes of enzymes, Biochem. Biophys. Res. Commun. 312 (2003) 179–184,
https://doi.org/10.1016/j.bbrc.2003.09.176;
[24] O. Trott, A.J. Olson, AutoDock Vina: Improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J. Comput.
Chem. 31 (2010) 455–461, https://doi.org/10.1002/jcc.21334.
[25] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera—A visualization system for exploratory research and
analysis, J. Comput. Chem. 25 (2004) 1605–1612, https://doi.org/10.1002/
jcc.20084.
(b) E.J. Mullaney, A.H.J. Ullah, B. Turner, A. Richardson, E. Mullaney, Phytases:
attributes, catalytic mechanisms and applications, Inositol Phosphates Link, Agric.
Environ. (2007) 97–110.
[8] B. Singh, I. Boukhris, Pragya, V. Kumar, A.N. Yadav, A. Farhat-Khemakhem, A.
Kumar, D. Singh, M. Blibech, H. Chouayekh, O.A. Alghamadi, Contribution of
microbial phytases to the improvement of plant growth and nutrition: A review,
Pedosphere. 30 (2020) 295–313. https://doi.org/https://doi.org/10.1016/S1002-
0160(20)60010-8.
[26] C.R. Søndergaard, M.H.M. Olsson, M. Rostkowski, J.H. Jensen, Improved treatment
of ligands and coupling effects in empirical calculation and rationalization of pKa
values, J. Chem. Theory Comput. 7 (2011) 2284–2295, https://doi.org/10.1021/
ct200133y.
[27] N.A. Baker, D. Sept, S. Joseph, M.J. Holst, J.A. McCammon, Electrostatics of
nanosystems: Application to microtubules and the ribosome, Proc. Natl. Acad. Sci.
98 (2001) 10037–10041, https://doi.org/10.1073/pnas.181342398.
[28] E.F. Pettersen, T.D. Goddard, C.C. Huang, E.C. Meng, G.S. Couch, T.I. Croll, J.
H. Morris, T.E. Ferrin, U.C.S.F. ChimeraX, Structure visualization for researchers,
educators, and developers, Protein Sci. 30 (2021) 70–82, https://doi.org/10.1002/
pro.3943.
[9] (a) D.F. Berry, D.A. Berry, Tethered phytic acid as a probe for measuring phytase
activity, Bioorg. Med. Chem. Lett. 15 (2005) 3157–3161, https://doi.org/10.1016/
j.bmcl.2005.04.009;
(b) D.F. Berry, C. Shang, L.W. Zelazny, Measurement of phytase activity in soil
using a chromophoric tethered phytic acid probe, Soil Biol. Biochem. 41 (2009)
192–200, https://doi.org/10.1016/j.soilbio.2008.09.011.
7