Identification of lower-order inositol phosphates (IP<sub>5</sub> and IP<sub>4</sub>) in soil extracts as determined by hypobromite oxidation and solution <sup>31</sup>P NMR spectroscopy

Abstract

<p><strong>Abstract.</strong> Inositol phosphates (IP) are a major pool of identifiable organic phosphorus (P) in soil. However, insight on their distribution and cycling in soil remains limited, particularly of lower-order IP (IP<sub>5</sub> and IP<sub>4</sub>). This is because their quantification typically requires a series of chemical extractions, including hypobromite oxidation to isolate IP, followed by chromatographic separation. Here, for the first time, we identify the chemical nature of organic P in four soil extracts following hypobromite oxidation using solution <sup>31</sup>P NMR spectroscopy and transverse relaxation (T<Sub>2</sub>) experiments. Soil samples analysed include the A horizon of a Ferralsol from Colombia, of a Cambisol from Switzerland, of a Gleysol from Switzerland and of a Cambisol from Germany. Solution <sup>31</sup>P NMR spectra of the phosphomonoester region on soil extracts following hypobromite oxidation revealed an increase in the number of sharp signals (up to 70), and an on average 2-fold decrease in the concentration of the broad signal compared to the untreated soil extracts. We identified the presence of four stereoisomers of IP<sub>6</sub>, four stereoisomers of IP<sub>5</sub>, and <i>scyllo</i>-IP<sub>4</sub> (using solution <sup>31</sup>P NMR spectroscopy). We also identified for the first time two isomers of <i>myo</i>-IP<sub>5</sub> in soil extracts: <i>myo</i>-(1,2,4,5,6)-IP<sub>5</sub> and <i>myo</i>-(1,3,4,5,6)-IP<sub>5</sub>. Concentrations of total IP ranged from 1.4 to 159.3 mg P/kg<sub>soil</sub> across all soils, of which between 9 % and 50 % were comprised of lower-order IP. Furthermore, we found that the T<sub>2</sub> times, which are considered to be inversely related to the tumbling of a molecule in solution and hence its molecular size, were significantly shorter for the underlying broad signal compared to the sharp signals (IP<sub>6</sub>) in soil extracts following hypobromite oxidation. In summary, we demonstrate the presence of a plethora of organic P compounds in soil extracts, largely attributed to IP of various order, and provide new insight on the chemical stability of complex forms of organic P associated with soil organic matter.</p>

Phosphorus retention in constructed wetlands enhanced by zeolite‐ and clinopyroxene‐dominated lava sand

“…Pools of unknown sharp peaks in the phosphomonoester region (phosphomonoester ‘other’ in Table 1) ranged from 22 to 82 mg kg −1 across all samples, which comprised on average 17% of total phosphomonoesters (Table 1). The identity of these Po species is unknown, but is likely due to a myriad of lower order (IP 5 to IP 1 ) inositol phosphates (Reusser et al, 2019). This is because a variety of enzymes are known to dephosphorylate IP 6 along a continuum of lower order inositol phosphates (Cosgrove, 1970; Sun et al, 2017), which could then become stabilized by metal surfaces.…”

Soil phosphomonoesters in large molecular weight material comprise multiple components

“…These results also reveal a greater number of Po components than that revealed by chromatography, which generally show one (or two) large pools of Po in large MW fractions (>5 kDa) (Jarosch et al, 2015). Nevertheless, further separation of this Po using high-performance liquid chromatography or specific chemical extractants (e.g., bromine) may reveal an even greater diversity of P components (Reusser, Verel, Zindel, et al, 2020). Whereas solution 31 P NMR spectra on 10-kDa retentates as reported in were of low spectral quality, the authors described the presence of some distinct spectral features.…”

“…The phosphomonoester region exhibited two main spectral features based on a visual assessment: (a) a multitude of sharp signals (between 22 and 31 sharp signals), and (b) an underlying broad signal. On average, 37% of the sharp signals in this region could be identified, which were largely arising from IP 6 (Reusser, Verel, Zindel, et al, 2020;Turner et al, 2012).…”

“…It is likely that the quantification of scyllo-IP 6 in previous NMR studies may be overestimated because of signal overlap with these two signals. Reusser, Verel, Zindel, et al (2020) has recently identified these peaks as belonging to that of myo-and scyllo-IP 5 , respectively.…”

“…This approach has revealed that phosphomonoesters (ROPO 3 2-, where R is an organic moiety; Condron et al, 2005) are the predominant (average 80%) class of Po in most soils (McLaren, Smernik, McLaughlin, Doolette, et al, 2020). A plethora of sharp peaks have been identified within this region, particularly those arising from IP 6 (Newman & Tate, 1980;Turner et al, 2012), lower-order inositol phosphates (IP 5 and IP 4 ) (Neal et al, 2021;Reusser, Verel, Zindel, et al, 2020), RNA mononucleotides (Vestergren et al, 2012), and α-glycerophosphate and β-glycerophosphate (Doolette et al, 2009). However, compounds arising from these sharp signals typically comprise less than half of the total pool of Po in most soils (McLaren, Smernik, McLaughlin, Doolette, et al, 2020).…”