Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates

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Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates Robert Puschmann, Robert K. Harmel, and Dorothea Fiedler Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00587 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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Biochemistry

Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates Robert Puschmann‡,1,2, Robert K. Harmel‡,1,2, Dorothea Fiedler*,1,2 1 Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Straße 10, 13125 Berlin, Germany; 2 Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin, Germany;

Supporting Information Placeholder scaffold. The enzymes responsible for synthesizing these hyperphosphorylated molecules fall into two classes of evolutionarily conserved small molecule kinases, the inositol hexakisphosphate kinases (IP6Ks) and the diphosphoinositol pentakisphosphate kinases (PPIP5Ks).5– 8 Genetic perturbation of these kinases have linked PPInsPs to tumor cell motility, insulin signaling, life span, and hearing loss.9–12 While the genetic studies have provided key insights into PP-InsP function, many questions regarding the underlying molecular mechanisms of action have remained unanswered.

ABSTRACT: The inositol pyrophosphates (PP-InsPs) are an

important group of cellular messengers which influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs (5PPInsP5, 1PP-InsP5 and 1,5(PP)2-InsP4) is reported, relying on the highly active inositol hexakisphosphate kinase A (IP6KA) from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2). A facile purification procedure using precipitation with Mg2+ ions and strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-32P, which is a valuable tool to study protein pyrophosphorylation. The chemoenzymatic method to obtain PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling functions of PP-InsP molecules.

To date, only a few proteins are known to selectively interact with PP-InsPs in eukaryotes.13–15 In addition, in vitro experiments have shown that PP-InsPs can transfer their β-phosphoryl group onto proteins in a process termed protein pyrophosphorylation, but so far the cellular relevance of this modification is unclear.16–20 It is evident that the genetic studies discussed above still need to be complemented by detailed biochemical investigations, so that the observed phenotypes can be explained by specific molecular mechanisms. To enable such studies, the small molecule messengers need to become commonly available to researchers across disciplines.21

Introduction Small molecule messengers are key components in cellular decision-making processes.1 Among cellular messengers, inositol-based signaling molecules occur ubiquitously in nature and include a unique, highly phosphorylated subgroup, termed the inositol pyrophosphates (PP-InsPs).2–4 In these molecules highenergy diphosphate groups are attached to the inositol

In the case of PP-InsPs, an efficient synthetic strategy to supply sufficient material of the structurally closely-related molecules for biochemical experiments and structural investigations is required. There are currently two main methodologies for the preparation of PP-InsPs: classical organic synthesis and an enzyme-mediated approach (Fig. 1). Enzymatic synthesis

Chemical synthesis OH OH HO

OH

OH OH

P

P P

P P

P

P

or

P

1,5(PP)2-InsP4

myo-inositol

+ high purity + mg quantities

P

7–9 steps

– labor intensive – 1–30 % yield

P

=

P

P P

P P

P

P

P P =

P

P

5PP-InsP5 O O P P O O O O O

P

1–2 step

P

P 1PP-InsP5

O P O O O

P

or

P

IP6K1 or/and Vip1

P

P

P

P

P InsP6

+ short synthesis – purification not scalable + high yield – µg quantities

Figure 1. Current synthetic strategies for the preparation of distinct PP-InsPs from commercially available starting materials. (left) Organic multistep synthesis produces PP-InsPs in high purity from myo-inositol with low overall yield due to several challenging

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Biochemistry

steps. (right) Enzymatic synthesis converts InsP6 in one or two steps into PP-InsPs. To chemoenzymatically obtain 5PP-InsP5 and 1PP-InsP5, IP6K1 and Vip1 have been applied, respectively.

Results and Discussion Chemoenzymatic synthesis of 5PP-InsP5

P

P

P

P

IP6KA, ATP, pCr, CrK

P

P

buffer, 37 °C, 30 min

P

P

P

P

P

(80%)

InsP6

P

P

5PP-InsP5

b) 1) C18 plug 5PP-InsP5, Mg2+ salt

reaction mixture 2) MgCl2 2) precipitation

3) Mg2+ chelation 4) lyophilization 5PP-InsP5, NH4+ salt

c)

5

4

3

2

1

0

-1

-2

-3

-4

-5 -6 [ppm]

-7

-8

-9

1.13

Here, we report a strategy that merges the high efficiency of the enzymatic synthesis with the scalability of a classical synthetic approach. We developed high yielding procedures for the chemoenzymatic synthesis of 5PPInsP5, 1PP-InsP5 and 1,5(PP)2-InsP4 on a 100–350 mg scale, using the recently identified inositol hexakisphosphate kinase A (IP6KA) from E. histolytica and the kinase domain of human PPIP5K2 (PPIP5K2KD). Coupled to a purification method that relies on precipitation of the PP-InsP-Mg complexes and strong anion exchange chromatography on an FPLC system, the PP-InsPs could be obtained in 85–95% purity. The approach was also applied on a small scale to simplify the synthesis of radiolabeled 5PP-InsP5-32P, a compound used for the biochemical study of protein pyrophosphorylation. In the long term, the practical setup of the PP-InsP syntheses will enable researches across fields to apply these molecules in a variety of biological contexts and thereby enhance our molecular understanding of PP-InsP signaling in healthy and diseased states.

a)

1.00

Overall, the organic syntheses of PP-InsPs have been improved greatly in recent years, but they still require multistep procedures and remain in the hands of skilled organic chemistry labs. Organic chemistry, however, remains the only scalable solution to obtain mg quantities of PP-InsPs in high purity. On a small scale, PP-InsPs have been synthesized from InsP6 using two distinct classes of small molecule kinases: IP6Ks and PPIP5Ks. Specifically, 5PP-InsP5, as well as 1,5(PP)2-InsP4, have been prepared in one- and two-step reactions using human IP6K1 and the kinase domain of Vip1 from S. cerevisiae, respectively.16,27 While the number of synthetic steps is strongly reduced compared to chemical synthesis, the challenge of this enzyme mediated method lies in the separation of the PPInsPs from other reaction components such as salts, buffer, or other small molecules. High performance strong anion exchange chromatography and preparative high percentage acrylamide gel electrophoresis have been valuable tools to purify PP-InsP.16,27 Unfortunately, these techniques lack scalability and have thus far disqualified chemoenzymatic approaches as major sources for PPInsPs.

To synthesize 5PP-InsP5 chemoenzymatically, we recombinantly expressed IP6KA, an InsP6 kinase from the parasitic amoebozoan E. histolytica.28,29 The advantage of this enzyme, compared to the commonly used human IP6K1, is its fast reaction kinetics, good stability, and high expression yield (ca. 100 mg of protein per 1L of E. coli culture). The InsP6 substrate can either be prepared from myo-inositol using a straightforward two step literature procedure, or can be obtained commercially (Calbiochem).30,31 Next, the reaction conditions (pH, time, enzyme- and Mg2+-concentration) were optimized using 13C-labeled InsP ([13C ]InsP ) as a substrate to monitor PP6 6 6 InsP kinase activity in vitro by nuclear magnetic resonance (NMR) spectroscopy.32 Full conversion to the 5PP-InsP5 product was achieved within 30 min, applying 0.3 µM of IP6KA in combination with an ATP recycling system (pCr and CrK) (Fig. 2a, S1).32 Determining the right balance between phosphate containing species, Mg2+ ions, and pH proved to be an integral consideration to prevent precipitation of InsP6 during the reaction.

0.91

The chemical syntheses employed orthogonally protected myo-inositol to introduce the pyrophosphate groups at the desired positions.22 For example, 5PP-InsP5 could be obtained in 7 steps with 30% overall yield.13,23,24 For the unsymmetrical 1PP-InsP5 and 1,5(PP)2-InsP4 molecules, synthetic access was granted by resolution of a mixture of the two enantiomers with chiral protecting groups. Using this approach, 1PP-InsP5 and 1,5(PP)2-InsP4 were isolated in 9 steps with a total yield of 1–5%.23,25,26

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Figure 2. Biochemical synthesis and purification of 5PP-InsP5. (a) Optimized conditions for the conversion of InsP6 by IP6KA. Conditions: 250 µM InsP6, 2 mM ATP, 7 mM MgCl2, 5 mM creatine phosphate (pCr), 1 U/mL creatine kinase (CrK), 0.3 µM IP6KA, 50 mM NaCl, 20 mM MES pH 6.4. (b) Separation of the 5PP-InsP5 from the reaction components. Enzymes were removed by a C18 plug and the product precipitated as a PP-InsP-Mg complex by addition of excess MgCl2. Mg2+ions were subsequently exchanged by solid phase chelation in ammonium carbonate buffer and lyophilization

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Biochemistry

afforded the product as the ammonium salt. (c) spectrum of purified 5PP-InsP5.

31P

NMR

With the optimized conditions (0.3 µM IP6KA, 2 mM ATP, 7 mM MgCl2 and 250 µM InsP6), we scaled up the reaction to 350 mg of InsP6 starting material and a total reaction volume of 2 L. To accurately control the reaction time, it proved crucial to preincubate the reaction mixture at 37 °C before the addition of IP6KA, and to quench the solution by cooling to 4 °C within 3–5 min using a –78 °C dry ice bath. For large scale purification, the proteins were removed by passing the solution through a fritted filter carrying a short plug of C18 reversed phase silica gel (Fig. 2b). To separate 5PP-InsP5 from other components in the mixture, we took advantage of the magnesium-chelating properties of InsPs and PP-InsPs.33,34 At basic pH these highly negatively charged molecules form strong Mgcomplexes and precipitate almost quantitatively. Therefore, an excess of MgCl2 was added to the reaction mixture and the pH was raised to pH 8.8–9.0. The resulting precipitate could easily be isolated by centrifugation and separated from soluble impurities. To redissolve the 5PPInsP5-Mg complex, and to remove the Mg2+ ions, we used an immobilized chelator (Amberlite® IRC 748) in NH4HCO3 buffer (pH 7.5). The Mg2+ ions could be removed to release 5PP-InsP5 into solution without additional contaminants. Finally, 5PP-InsP5 was isolated by lyophilization as the ammonium salt in 80% yield (312 mg, > 95% purity, Fig. 2c). While 5PP-InsP5 was synthesized enzymatically before, the scale of those reactions was substantially smaller. The purification of the product was accomplished by preparative high percentage polyacrylamide gel electrophoresis (PAGE), followed by gel extraction (100–200 µg 5PP-InsP5 per gel).27 Therefore, the isolated 5PP-InsP5 was contaminated with buffer and gel components, as evidenced by 1H NMR analysis (Fig. S2). In summary, our biochemical synthesis of 5PP-InsP5 outperforms classical organic chemistry approaches in yield and accessibility. The precipitation of the 5PP-InsP5Mg complex offers a scalable purification procedure that can deliver large amounts of high purity 5PP-InsP5, compared to currently used protocols.

formation of 1PP-InsP5 using the labeled [13C6]InsP6 as substrate and found that an extended reaction time of 18 hours and increased enzyme concentration of 2 µM were necessary to guarantee full conversion. (Fig. 3a) Following precipitation and Mg2+-chelation, we noticed that 1PPInsP5 contained impurities, probably due to the formation of side products under the prolonged reaction times. To remove these impurities, 1PP-InsP5 was subjected to strong anion exchange (SAX) chromatography on an FPLC system. (Fig. 3b), allowing for the isolation of ca. 100 mg of 1PP-InsP5 in two purification runs. After lyophilization, 1PP-InsP5 was isolated as the ammonium salt in 68% yield (77 mg, > 95% purity). With robust procedures for enzymatic pyrophosphorylation at the 5- and the 1-position of the inositol ring in hand, we lastly sought to synthesize the most densely phosphorylated PP-InsP: 1,5(PP)2-InsP4. Full conversion from InsP6 to 1,5(PP)2-InsP4 using both enzymes in a one-pot reaction proved to be challenging, therefore, a one step protocol starting from purified 5PPInsP5 was pursued. Reaction optimization with [13C6]5PPInsP5 confirmed that 5PP-InsP5 is the preferred substrate for human PPIP5K2KD, compared to InsP6, and full conversion could be obtained with 1.5 µM enzyme in 5.5 hours. 1,5(PP)2-InsP4 was purified analogously to 5PP-InsP5 by precipitation with Mg2+ ions the product was isolated as the ammonium salt in 77% yield (111 mg, > 85% purity). If a purity higher than 85% is required, the SAX-based purification protocol described above can be adopted for 1,5(PP)2-InsP4 (Figure 3b). a) P P

P

P

P

P InsP6

P

buffer, 37 °C, 18 h (68%)

P P

P

P P 1PP-InsP5

b) 1) C18 plug reaction mixture 2) MgCl2 2) precipitation

1PP-InsP5, NH4+ salt

Biochemical synthesis of 1PP-InsP5 and 1,5(PP)2-InsP4 The successful, large-scale chemoenzymatic synthesis of 5PP-InsP5 encouraged us to apply our strategy to the synthetically more challenging unsymmetrical PP-InsPs: 1PP-InsP5 and 1,5(PP)2-InsP4 (Fig. 3). The pyrophosphorylation at the 1-position of InsP6 and 5PPInsP5 is catalyzed by PPIP5Ks. We thus expressed the Histagged kinase domain of human PPIP5K2 (PPIP5K2KD) but its low solubility caused low overall protein yields. To increase the solubility, the His-tag of PPIP5K2KD was replaced with a solubility enhancing SUMO-tag, resulting in the isolation of the desired protein in acceptable yield (ca. 25 mg of protein per 1L of E. coli culture). Next, we optimized the biochemical reaction conditions for the

P

PPIP5K2KD, ATP, pCr, CrK

1PP-InsP5 and undesired side products, Mg2+ salt 3) Mg2+ chelation 4) lyophilization

5) SAX FPLC

1PP-InsP5 and undesired + 6) lyophilization side products, NH4 salt

c) P P

P

P

P

P 5PP-InsP5

P

P

PPIP5K2KD, ATP, pCr, CrK buffer, 37 °C, 5.5 h (77%)

P

P P

P

P

P

P

1,5(PP)2-InsP4

Figure 3. Biochemical synthesis and purification of 1PP-InsP5 and 1,5(PP)2-InsP4. (a) Optimized conditions for the synthesis of unsymmetrical 1PP-InsP5 using PPIP5K2KD: 250 µM InsP6, 2 mM ATP, 6 mM MgCl2, 6 mM creatine phosphate (pCr), 1 U/mL creatine kinase (CrK), 2 µM PPIP5K2KD, 250 mM NaCl, 20 mM MES pH 6.4. (b) Purification of 1PP-InsP5 by MgCl2 precipitation and chelation, followed by additional FPLC


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purification using a SAX column. Fractions were analyzed by metal dye detection assay and lyophilized to afford the product as ammonium salt. (c) Optimized conditions for the synthesis of 1,5(PP)2-InsP4 using PPIP5K2KD: 250 µM 5PPInsP5, 2 mM ATP, 5 mM MgCl2, 5 mM creatine phosphate (pCr), 1 U/mL creatine kinase (CrK), 1.5 µM PPIP5K2KD, 250 mM NaCl, 20 mM MES pH 6.4. Purification of the product was analogous to 5PP-InsP5 (see Fig. 2b).

4b). Since HPLC analysis of the product revealed appreciable amounts of ATPγ32P at this step, we included an apyrase treatment, followed by another Mg2+ precipitation, to remove the residual ATPγ32P (Fig. 4c). Subsequent HPLC analysis confirmed that the predominant product was 5PP-InsP5-β32P and amounts of ATPγ32P were insignificant. Attempts to conduct the apyrase treatment prior to the precipitation failed, due to co-precipitation of 32Pi.

To avoid hydrolysis of the synthesized compounds during storage, we recommend to store the solid at –20 °C. However, it is also possible to store stock solutions at –80 °C. In this case, the pH of the solution should be between pH 6 and pH 7 and repeated freeze-thaw-cycles should be minimized. PP-InsPs can also be stored in solution at 4 °C for several days, for an extended life span 100 µM EDTA can be added.

We next applied 5PP-InsP5-β32P in a pyrophosphorylation assay and chose a fragment of yeast nucleolin (Nsr1) as a well-established model substrate.16,17 As expected, prephosphorylated Nsr1 was pyrophosphorylated by 5PP-InsP5-β32P but not the unphoshorylated Nsr1. (Fig. S3) Incubation of Nsr1 with casein kinase 2 (CK2) and either 5PP-InsP5-β32P or ATPγ32P showed only phosphoryl-transfer in the case of ATPγ32P, indicating that the 5PP-InsP5-β32P contained negligible amounts of ATPγ32P.

Overall, the synthesis of unsymmetrical 1,5(PP)2-InsP4 was achieved in a fashion analogous to 5PP-InsP5 with only minor changes in the purification protocol. The purification of 1PP-InsP5, by contrast, required an additional step, limiting access to this compound to 100 mg batches. Preparation of similar amounts of 1PP-InsP5 by chemical means, however, also remains challenging and demands specialized chemistry equipment that is typically not present in biochemistry and molecular biology laboratories. Enzymatic synthesis of 5PP-InsP5-β32P More than ten years ago, it was proposed that PP-InsPs can modify proteins non-enzymatically by transferring a βphosphoryl group onto phosphoserine residues in a process termed pyrophosphorylation.16–20 In vitro detection of pyrophosphosphorylation was enabled by treating cell lysates, or isolated proteins, with radiolabeled 5PP-InsP532P, an inositol pyrophosphate carrying a radiolabel at the β-position. In previous studies, 5PP-InsP5-32P was prepared enzymatically using human IP6K1, InsP6, and radiolabeled ATP (ATPγ32P).16 Separation of 5PP-InsP5-32P from ATPγ32P was accomplished via strong anion exchange chromatography on an HPLC system. While this approach works efficiently on a small scale, a major limitation is the reliance on HPLC instrumentation for the purification of radioactive material, thus restricting the access to these compounds to only a few research groups worldwide. We envisioned that our newly developed procedure for the preparation and purification of 5PP-InsP5 could surmount this limitation, making 5PP-InsP5-32P more generally available. Importantly, to be a reliable source for in vitro protein pyrophosphorylation, 32P in 5PP-InsP5-32P cannot contain any residual ATPγ32P, to avoid background labeling by protein and small molecule kinases. The incorporation of the 32P-radiolabel into 5PP-InsP5was optimized by reducing the concentration of unlabeled ATP and prolonging the reaction time (Fig. 4a). 5PP-InsP5-β32P was then precipitated as Mg2+ salt, filtered, and resolubilized by treating with Chelex® 100 resin (Fig. β32P

Overall, the newly developed workflow for the synthesis of 5PP-InsP5-32P is a user-friendly alternative to previously established procedures. By omitting HPLC purification, which requires instrumentation that is not considered standard equipment in a radioactive isotope lab, the investigation of protein pyrophosphorylation, an understudied PTM, will become accessible to a broader research community. Conclusions Over the last decade we have witnessed significant progress in the synthesis of PP-InsPs. Still, the general availability of these compounds in good quantities, especially of the unsymmetrical regioisomers, has been limited. Here, we have established a robust workflow for PP-InsP synthesis and purification. With the required enzymes and relevant substrates in hand, all major mammalian PP-InsPs were synthesized on a milligram scale in one or two steps, without the need for specialized chemistry equipment. The facile precipitation procedure of PP-InsPs proved to be equally useful on a large and a small scale, as demonstrated for the synthesis of 5PP-InsP532P. In the latter case, HPLC purification of the radioactive compound was no longer necessary to obtain high purity 5PP-InsP5-32P. While our method is efficient in synthesizing the reported PP-InsPs, some limitations remain to be addressed in the future. The current protocol is restricted by the availability and activity of the small molecule kinases that selectively install the pyrophosphate groups. For example, Dictyostelium discoideum, a slime mold containing the highest concentration of PP-InsPs measured to date, produces PP-InsP regioisomers that appear to be different from other eukaryotes.35


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Biochemistry

a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

P P

P

P P

P

IP6KA, ATP32P, pCr, CrK P buffer, 37 °C, 1 h

InsP6 32

P =

O 32 P O O O

P

P

P

P

32

P

P

5PP-InsP5-32P

b) 1) C18 plug reaction mixture 2) MgCl2 2) precipitation

5PP-InsP5-32P, NH4+ salt

6) Mg2+ chelation

5PP-InsP5-32P and trace amounts of ATP32P 3) Mg2+ chelation 4) apyrase 5) MgCl2 precipitation 5PP-InsP5-32P and 32Pi

Overall, the scalable biochemical syntheses of the major mammalian PP-InsPs make these compounds readily available to a broader research community. Just as access to other small molecule messengers has greatly aided the structural and biochemical elucidation of their signaling functions, the method presented here will facilitate a thorough and granular analysis of the varied properties of PP-InsPs. Such detailed investigations are much needed, to determine the molecular mechanisms underlying the pleiotropic phenotypes associated with PP-InsP signaling, to ultimately guide the development of novel therapeutic strategies.

ASSOCIATED CONTENT Supporting Information

7) speed vac

The Supporting Information is available free of charge on the ACS Publications website. Material and Methods, and Supplementary Figures (PDF)

c)

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions ‡ R.P. and R.K.H. contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT Figure 4. Biochemical synthesis and purification of 5PP-InsP5β32P. (a) Optimized conditions for the conversion of InsP6 by IP6KA. Conditions: 0.2 mM InsP6, 2000 µCi ATPγ32P (6000 Ci/mmol, 10 mCi/mL), 0.2 mM ATP, 2 mM MgCl2, 5 mM creatine phosphate (pCr), 1 U/mL creatine kinase (CrK), 0.3 µM IP6KA, 1 mM DTT, 50 mM NaCl, 20 mM MES pH 6.4 (buffer). (b) Separation of the 5PP-InsP5-β32P from the reaction components by MgCl2 precipitation as described for 5PP-InsP5. Apyrase treatment removed traces impurities of ATPγ32P. Subsequent MgCl2 precipitation and removal of Mg2+ by solid phase chelation followed by speed vac afforded the product as ammonium salt. (c) HPLC analysis of purified 5PPInsP5-β32P. (black) Purification by MgCl2 precipitation shows residual ATPγ32P. (green) Purification by apyrase treatment followed by precipitation shows co-precipitation of 32P-Pi that is liberated from the ATPγ32P hydrolysis. (red) Purification by precipitation followed by apyrase treatment and a second precipitation afforded highly pure 5PP-InsP5-β32P.

Since the kinase(s) responsible for these unusual regioisomers have not been recombinantly expressed so far, the biochemical generation of the corresponding PPInsPs remains elusive as well. In addition, our method is not suited to synthesize analogs of PP-InsPs, such as nonhydrolyzable bisphosphonate analogs or photo-caged derivatives.36–38 Especially in the case of large structural perturbations of the PP-InsPs we envision those syntheses remain in the realm of classical organic chemistry.

R.K.H. and R.P. gratefully acknowledge funding from the Leibniz-Gemeinschaft (SAW-2017-FMP-1).

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Table of content P P P

P

P

1 or 2 steps P

P InsP6

P

IP6KA or/and PPIP5K2

+ short synthesis + high yield (68–80%) + scalable purification + 100–350 mg quantities

P

P

P

P 5PP-InsP5 P

P

P P

P P

P

P 1PP-InsP5

P

P

32

P

P

5PP-InsP5-32P P

P

P

P

P

P P

P

P

P

P

1,5(PP)2-InsP4

Proteins IP6KA: N9UNA8 IP6K1: Q92551


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

126x53mm (300 x 300 DPI)


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Biochemistry

251x57mm (300 x 300 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

111x145mm (300 x 300 DPI)


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Biochemistry

116x122mm (300 x 300 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

127x196mm (300 x 300 DPI)


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Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates

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