Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters

Article abstract of DOI:10.1002/adsc.201800306

Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by-product are the main obstacles associated with the use of pyrophosphate in the phosphatase-catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl-compounds. Among them, acetyl phosphate delivered stable product levels and high phospho-transfer efficiency at the lower functional pH-limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative-scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L^?1 h^?1 space-time yield and 75% reduction of barium phosphate waste. (Figure presented.).

Full text of DOI:10.1002/adsc.201800306

Title: Evaluation of Natural and Synthetic Phosphate Donors for the
Improved Enzymatic Synthesis of Phosphate Monoesters
Authors: Gabor Tasnadi, Wolfgang Jud, Mélanie Hall, Kai Baldenius,
Klaus Ditrich, and Kurt Faber
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
UPDATE
Evaluation of Natural and Synthetic Phosphate Donors
for the Improved Enzymatic Synthesis of Phosphate Monoesters
Gábor Tasnádi,a,b Wolfgang Jud,b Mélanie Hall,b Kai Baldenius,c Klaus Ditrich,c Kurt Faberb,*
aAustrian Centre of Industrial Biotechnology, c/o
bDepartment of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse
28, 8010 Graz, Austria
Fax: +43-316-380-9840; phone: +43-316-380-5332; e-mail: Kurt.Faber@Uni-Graz.at
cWhite Biotechnology Research Biocatalysis, BASF SE, Carl-Bosch-Strasse 38, 67056
Ludwigshafen, Germany
Abstract: Undesired product hydrolysis along with large amounts of waste in form of inorganic
monophosphate by-product are the main obstacles associated with the use of pyrophosphate in the
phosphatase-catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both
limitations, we screened a broad range of natural and synthetic organic phosphate donors with
several enzymes on a broad variety of hydroxyl-compounds. Among them, acetyl phosphate
delivered stable product levels and high phospho-transfer efficiency at the lower functional pH-
limit, which translated into excellent productivity. The protocol is generally applicable to acid
phosphatases and compatible with a range of diverse substrates. Preparative-scale transformations
using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various
sugar phosphates with up to 433 g L-1 h-1 space-time yield and 75% reduction of barium phosphate
waste.
Keywords: Enzymatic phosphorylation, phosphatase, phosphate donor, acetyl phosphate.
Introduction
Synthesis of valuable phosphate (mono)esters, e.g. sugar phosphates,[1–3] nucleotides,[4,5]
metabolites[6,7] and prodrugs[8–10] can be achieved by laborious chemical[11–18] or mild enzymatic
routes.[19–21] The latter usually employ kinases and ATP in conjunction with a second enzyme for
the recycling of the phosphate donor. Although applied on preparative-scale,[22–25] kinases are rather
substrate-specific and efficient cofactor recycling systems are still in an emerging phase.[26]
The ability of phosphatases – naturally active in the hydrolysis mode – to catalyze the (reverse)
transphosphorylation reaction employing cheap high-energy phosphate donors (P-donors), e.g.
1
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
pyrophosphate (PPi), was recognized decades ago.[27,28] The avoidance of ATP recycling and the
relaxed substrate spectrum of phosphatases rendered these enzymes attractive alternatives for the
synthesis of a broad range of phosphate monoesters.[29] However, on large scale, this method suffers
from two major limitations: (i) the enzymatic hydrolysis of the formed phosphate ester leads to
product depletion and unpredictable (optimum) reaction times, and (ii) generates large quantities of
inorganic monophosphate (Pi) as by-product. The first problem could be overcome by applying high
substrate concentration,[30–32] by protein engineering[33–35] or by continuous flow technology.[31,36,37]
On the other hand, the formation of inorganic monophosphate as by-product is a result of the nature
of the P-donor. Though being cheap, PPi is associated with excessive release of Pi, owing to
liberation of a stoichiometric quantity of Pi during phosphate transfer and competing hydrolysis of
the P-donor. Application of triphosphate (PPPi) or other oligo- and polyphosphates would result in
higher product levels, however, the efficiency of the phosphate transfer does not correlate with the
number of transferable high-energy phosphate moieties.[30,31,37] The generation of high amounts of
inorganic monophosphate also leads to technical difficulties in downstream processing. A
straightforward and generally applicable way to separate Pi from the product phosphate monoester
consists in fractional crystallization, whereby Pi as well as the unreacted P-donor are sequentially
precipitated as barium salts, thereby creating vast amounts of barium waste.[31,35,37–41] Decreasing
the latter would render the process overall more benign. As alternative to crystallization, ion-
exchange chromatography might be applied, however, due to similar pKa values of Pi and product
phosphate monoesters, large amounts of eluent are required.[30,42,43]
Herein, we compare various organic P-donors as alternatives to inorganic oligophosphates for the
synthesis of phosphate monoesters by phosphatases. Organic P-donors have been used with
phosphatases in the hydrolysis- and transphosphorylation-mode, however, product isolation has
scarcely been reported.[27,44,45] Aryl phosphates (e.g. phenyl phosphate, p-nitrophenyl phosphate,
phenolphthalein phosphate) and various metabolites (e.g. nucleoside phosphates, sugar-phosphates)
have been commonly used in biochemical characterization of novel enzymes due to their
availability and features allowing facile spectrophotometric or NMR analysis. For reason of
comparison, literature reports on enzyme-catalyzed transphosphorylation reactions performed with
organic P-donors are collected in Table S1 (see the Electronic Supporting Information).
During the course of our study we identified a specific pH range in which the phosphotransferase
activity of the enzymes remained intact, while the phospho-hydrolase activity was reduced. This
proved to be a general feature among the tested acid phosphatases. The combination of pH control
with use of acetyl phosphate as donor enabled rapid product synthesis on preparative scale, and
hence formation of less barium phosphate waste.
2
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
Results and Discussion
Scheme 1 Substrates and phosphate donors tested in this study
PPi: disodium dihydrogenpyrophosphate; AcP: lithium potassium acetyl phosphate; PEP: potassium
phosphoenolpyruvate; CP: lithium carbamoylphosphate; PC: phosphocreatine disodium salt.
P-donor screening
Preliminary experiments were conducted at pH 4.2 with a range of acid phosphatases and an
alkaline phosphatase (Table S2) using 1,4-butanediol (1) as model substrate (500 mM) and natural
P-donors, such as acetyl phosphate (AcP), phosphoenol pyruvate (PEP), carbamoyl phosphate (CP)
or phosphocreatine (PC) (100 mM; Scheme 1). Importantly, at this pH value, AcP, CP and PC
hydrolyze spontaneously with a half-life of ~7 h, ~6 h and ~3 h, respectively, while PPi and PEP are
stable over at least 8 h (Fig. S1). The results were compared with those obtained with PPi. All
donors were accepted by the tested acid phosphatases, i.e. PhoN-Sf from Shigella flexneri, PhoN-Se
from Salmonella typhimurium LT2, PiACP from Prevotella intermedia, Lw from Leptotrichia
wadei, PhoC-Mm from Morganella morganii variant G92D/I171T, NSAP-Eb from Escherichia
blattae 11-variant and AphA-St from Salmonella typhimurium LT2 (Fig. S2). The latter is a Mg-
3
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
dependent enzyme which shows marginal transphosphorylation activity using inorganic
oligophosphates due to chelation of the metal causing inactivation. Magnesium supplementation is
insufficient to recover its activity.[31] However, using alternative donors, AphA-St showed good
activity comparable to that of other acid phosphatases, highlighting an important benefit of non-
chelating phosphate donors. PhoK from Sphingomonas sp. BSAR-1 at pH 9.0 and phytase from
Aspergillus niger at pH 2.5 and 4.2 furnished only traces of product and were not further
investigated. With less active variants, i.e. PhoC-Mm and NSAP-Eb,[35] spontaneous hydrolysis of
labile P-donors (AcP, CP and PC) competed with transphosphorylation leading to lower maximal
product levels.
Overall, AcP and PEP delivered the highest product concentrations, similar to those obtained with
PPi (Fig. S2 and Table S3). In the case of PEP, pyruvic acid (pKa 2.5) released upon phosphate
transfer led to gradual decrease of the pH (to ~3.2) and concomitant enzyme deactivation, which
resulted in stable product concentrations but incomplete P-donor consumption. Upon pH re-
adjustment, however, enzyme deactivation was reversible (Fig. S3), indicating the existence of a pH
threshold below which the enzymes reversibly turned into an inactive 'stand-by' mode.
pH-controlled product hydrolysis
The dependence of catalytic activity on pH was tested with all enzymes. Since the use of AcP
barely influences the pH during the course of the reaction (data not shown), the wild-type enzymes
were screened at various pH values with this donor. We found that lowering the initial pH led to
dramatically reduced product hydrolysis. In contrast, the transphosphorylation activity was affected
to a much lesser extent, which ultimately allows control of one activity over the other. Below a
certain threshold the enzymes turned inactive in the synthesis mode as well (Fig. S4). This narrow
pH range (3.3-3.8), in which the transphosphorylation activity was conserved, while the hydrolytic
activity was strongly diminished, was identified for PhoN-Sf, PhoN-Se, PiACP, Lw and AphA-St
(Fig. 1). Overall, PiACP and Lw proved to be the most promising candidates for exploiting this
property. The product concentrations were in the same range as those obtained at pH 4.2 and could
be maintained over at least 1 d. Under these conditions, AcP was completely consumed when the
product plateau was reached and no bis-phosphorylated product was observed. Similar observations
were made with acid phosphatases and PPi as donor,[31,33,35,37] however, high product levels could
not generally be maintained, if any, at the cost of large Pi side-product formation. A difference
between the pH optimum of phosphotransferase and phosphohydrolase activities has been also
observed with calf intestine alkaline phosphatase.[30]
4
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
Fig. 1 Phosphorylation of 1a using AcP at optimum pH.
Reaction conditions: 1 U mL -1 (0.4-1.0 M) enzyme in 100 mM AcP at pH 3.8 (for PhoN-Sf), pH
3.5 (for PiACP and Lw), pH 3.3 (for PhoN-Se) or pH 2.9 (for AphA-St), 500 mM 1a.
With PPi as donor at the optimum pH (Fig. S5) the enzymes exhibited greater hydrolytic activity
compared to that observed with AcP, which can be associated with a pH-increase during 4 h (pH ~
+0.5 unit).
Next,
various
synthetic
P-donors,
i.e.
2,2,2-trifluoroethyl
hydrogenphosphate
monocyclohexylammonium salt (TFEP), 2,2,2-trichloroethyl dihydrogen phosphate (TClEP), 2,5-
dioxopyrrolidin-1-yl hydrogen phosphate monocyclohexylammonium salt (NPS) were screened
starting at the optimum pH (Fig. S6-S8; for synthesis of donors see ESI). Remarkably, most non-
natural P-donors delivered product concentrations and product/Pi ratios similar to those obtained
with AcP. However, as with PPi, hydrolytic activities were more pronounced due to increasing pH
over time. In summary, AcP proved to be the ideal alternative to PPi, associated with rapid product
formation, high phosphotransfer efficiency and low Pi waste.
P-Donor, enzyme and substrate concentration test
Since Lw and PiACP delivered generally highest product concentrations at optimum pH (3.3-3.5),
we investigated these enzymes in scale-up reactions with 1a. First, the concentration of AcP was
enhanced (up to 400 mM) at constant enzyme concentration (1 U mL-1) and pH 3.4. Maximal
product level and starting velocities decreased at above approx. 100 mM AcP (Fig. S9), which
indicates potential inhibition by AcP. Increasing the enzyme concentration (up to 10 U mL-1)
5
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
improved product levels drastically (up to ~270 mM; Fig. S10). Finally, the concentration of 1a was
raised to 1.0 M and 1.5 M, however, both enzymes seemed to be sensitive to higher substrate
concentrations (Fig. S11).
Synthesis of acetyl phosphate (AcP)
So far, commercially available (expensive) acetyl phosphate was employed, setting serious
limitations to large-scale applications. However, this disadvantage along with the unstable character
of AcP can be compensated by facile synthesis from cheap starting materials. AcP was synthesized
by a modified protocol of Crans and Whitesides starting from phosphoric acid and acetic anhydride,
followed by aqueous extraction and pH adjustment, to afford a mixture of 1.01 M AcP, containing
only 47 mM Ac2P (diacetyl phosphate), 86 mM Pi and 12 mM PPi (Scheme S1, Fig. S17).[46] When
stored at pH 7 at -20 °C, this crude solution exhibited only ~5% degradation within 3 months
without opening of the container and ~10% degradation by regular usage.
Substrate scope
The crude AcP preparation was diluted to ~400 mM concentration and used for the phosphorylation
of a range of substrates (1a-9a) with PiACP and Lw at previously identified optimum pH (~3.4). In
order to compensate for slight differences between batches of synthesized AcP, increased enzyme
concentration was employed (15 U mL-1 corresponding to 6 M PiACP and 9 M Lw). In general,
both enzymes could maintain stable product levels over at least 24 h (Fig. 2 and Fig. S12). PiACP
yielded ~260 mM 4-hydroxybutyl phosphate (1b), which is approx. twice as much as that formed
by Lw (~130 mM). The ratio of mono- versus bis-phosphorylated product was ~80:20 (PiACP) and
~90:10 (Lw). In all other cases, however, PiACP delivered product concentrations lower than those
obtained by Lw (Fig. S12). Because of the sensitivity of this enzyme towards high substrate
loadings, other substrates 3a-5a, 7a and 8a were used at 300 mM. In contrast, Lw had remarkable
activity on 500 mM methyl -D-glucopyranoside (2a), D-glucosamine (3a) and maltotriose (5a)
resulting in ~420 mM 2b, ~360 mM 3b and ~350 mM 5b (Fig. 2). These concentration levels
correspond to >99%, 86% and 83% conversion (with respect to AcP), respectively. Furthermore,
approx. 170 mM N-acetyl-D-glucosamine-6-phosphate (4b) was obtained (conv. ~40%). Inosine
(6a) and 6-amino-1-hexanol (7a) proved to be poor substrates (product concentration <30 mM),
while allyl alcohol (8a) and glycerol (9a) gave moderate results (~50 mM and ~100 mM, resp.).
The amount of 4b increased from 170 mM to 200 mM, while that of 8b from 40 mM to 120 mM by
employing elevated Lw loadings (from 15 to 20 and 25 U mL-1) (Fig. S13-S14). The formation of
7b was boosted by employing wild-type PhoC-Mm from Morganella morganii[35] finally reaching
6
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
140 mM with 25 U mL-1 enzyme (Fig. S15). The phosphorylation took place on the alcohol moiety
as observed previously.[35] 40 mM 6b could be also obtained by PhoC-Mm (Fig. S16).
Fig. 2 Substrate screening of Lw using crude AcP
Reaction conditions: 15 U mL -1 (9 M) Lw in ~400 mM AcP at pH 3.4, 500 mM 1a-5a, 7a-9a, 80
mM 6a.
Preparative-scale synthesis
Finally, scale-up of conversions of 1a-5a (500 mM) was performed in 20-50 mL volume using
~400 mM crude AcP preparation (Table 1, Fig. 3) to avoid potential AcP inhibition. In the cases of
2a, 3a and 5a, conversions (based on consumption of the limiting component AcP) reached >90%,
highlighting a nearly perfect P-transfer efficiency accompanied by remarkable space-time yields
(433, 197 and 425 g L-1 h-1, respectively). Turnover numbers (TONs) up to ~50.000 highlight the
robustness of the enzymes. The product phosphate esters were isolated as Ba salts.
In order to compare the AcP protocol with the one employing PPi as donor, the phosphorylation of
1a was performed using 400 mM PPi (Fig. 3, dashed line). Similar conversion, isolated yield and
somewhat reduced reaction rate were obtained, however, the amount of BaHPO4 waste was
significantly more with PPi (8.0 g vs. 2.1 g). Phosphorylation of 2a-5a took place on the C6-OH
position as proven by NMR (ESI).
7
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
Fig. 3 Preparative-scale transformations of 1a-5a
Table 1 Preparative-scale synthesis of 1b-5b
Yieldh
(%)
STYa
Enzyme
Conv.
P-donor
TON
53434
51970
41126
39465
9534
(%)
(g L-1 h-1)
(U, g mL-1)
PiACP
(750, 159)
PiACP
38g
1bb,c
AcP
PPi
73f
99
64
(2.3 g)
46g
(2.8 g)
68
(5.6 g)
78
(5.1 g)
33
71f
99
95
51
91
(750, 159)
Lw
2bb
3bb
4bd
5be
AcP
AcP
AcP
AcP
433
197
61
(750, 263)
Lw
(750, 263)
Lw
(600, 350)
Lw
(1.4 g)
62
(3.56 g)
425
37803
(300, 263)
a
Space-time yield with respect to conversion (measured as depletion of substrate),
monobasic form of phosphate product and reaction time needed to reach maximal
b
c
product level; 50 mL reaction volume; ratio of mono-/bis-phosphorylated products
~80:20; d 30 mL reaction volume; e 20 mL reaction volume; f mono-/bis-phosphorylated
products ~80:20; g mono-phosphorylated product; h products isolated as Ba salt.
-1
Reaction conditions: 15-20 U mL (5.4-12.8 M) enzyme in ~400 mM AcP or PPi at
pH 3.4, 500 mM 1a-5a, stirring at 30 °C.
Conclusion
A range of natural and synthetic high-energy phosphate donors were evaluated to replace inorganic
oligophosphates in phosphatase-catalyzed transphosphorylation to reduce formation of inorganic
8
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
monophosphate by-product. Acetyl phosphate was identified as the most suitable alternative.
Furthermore, a defined pH at which the hydrolytic activity of the enzymes could be disentangled
from that of the phosphotransferase was identified, which turned highly practical to prevent product
depletion. This, combined with the use of an easily obtained crude acetyl phosphate mixture
allowed gram-scale synthesis of valuable phosphorylated sugars with high space-time yields and
drastically reduced barium phosphate waste. The method is generally applicable to acid
phosphatases and their substrates.
Experimental Section
General remarks
All chemicals were purchased from commercial suppliers and used as received. Disodium
pyrophosphate (PPi; purity ≥99%), lithium potassium acetyl phosphate (AcP), dilithium
(carbamoyloxy)phosphonate
(phosphonocarbamimidoyl)glycinate hydrate (phosphocreatine disodium salt hydrate, PC) were
purchased from Sigma, potassium 2-(phosphonooxy)acrylate (phosphoenolpyruvate
hydrate
(CP)
and
disodium
N-methyl-N-
monopotassium salt, PEP), 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP) and
dimethyl methylphosphonate were from Alfa Aesar. Ni-NTA column for His-tag purification was
from GE Healthcare Life Sciences. NMR spectra were recorded on a Bruker Avance III 300 MHz
NMR spectrometer. Chemical shifts (δ) are g3
,iven in parts per million (ppm) relative to TMS or H3PO4 as a reference. HPLC analysis was
carried out on a Dionex Ultimate 3000 system equipped with Shodex RI-101 refractory index
detector (HPLC-RI; Alltech IOA-2000 Organic Acids column, eluent: 8 mM H2SO4, flow rate: 0.4
mL min-1, 50 °C, injection volume: 20 L).
Products were identified on an Agilent 1260 Infinity system equipped with Agilent Q6120
quadrupole mass spectrometer using electrospray ionization (HPLC-MS; Zorbax 300-SCX cation
exchanger column, eluent: 0.1% (v/v) formic acid, flow rate: 1 mL min-1, 40 °C, injection volume:
10 L) and via co-injection with reference materials. The ratio of mono- versus bis-phosphorylated
products in the phosphorylation of 1a was determined as reported.[31]
PiACP from Prevotella intermedia,[31] AphA-St[31] and PhoN-Se[35] from Salmonella typhimurium
LT2, PhoN-Sf from Shigella flexneri,[35] PhoK from Sphingomonas sp. BSAR-1,[47] PhoC-Mm
wild-type and G92D/I171T from Morganella morganii[35] and NSAP-Eb 11-variant from
Escherichia blattae[35] were expressed reported. Phytase from Aspergillus niger was from BASF.
The gene of acid phosphatase Lw from Leptotrichia wadei F0279 (signal peptide removed; UniProt
9
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
Accession Number: U2QAK5) was purchased from IDT and subcloned into pET28a vector using
standard molecular biology protocols. The enzyme was overexpressed in E. coli (ESI).
General screening conditions
A standard reaction mixture contained substrate and P-donor in H2O at a concentration and pH
indicated in the footnotes of tables and captions of figures and 1% (v/v) DMSO as internal standard
in 1 mL final volume. No additional buffering agent was added. The reaction was initiated by
adding the indicated amount enzyme. The mixture was shaken in 1mL glass vials at 30 °C and 600
rpm in an Eppendorf thermoshaker. Samples of 25 L volume were taken at intervals, diluted with
475 L of 8 mM aq. H2SO4 and analyzed on HPLC-RI. Experiments were performed in duplicate.
Product levels were determined by consumption of substrate.
In the case of D-glucosamine (3a), maltotriose (5a), inosine (6a) and 6-amino-1-hexanol (7a), a 100
L sample was taken at intervals and added to a mixture of 490 L H2O, 100 L 350 mM dimethyl
methylphosphonate (internal standard, final concentration: 50 mM) in D2O and 10 L 1 M HCl (for
31
quenching). Then, a P-NMR spectrum was recorded using inverse gated decoupling (ns 32, d1 =
30 s, pw = 11 s).
Preparative-scale synthesis of 1b-5b
Preparative-scale transformations were performed in 50 mL (1a-3a), 30 mL (4a) or 20 mL (5a)
reaction volume in a round-bottom flask containing 500 mM 1a-5a and 400 mM crude AcP or PPi.
The mixture was adjusted to pH 3.4, the reactions were initiated by addition of enzyme [1a: 15 U
mL-1 (159 g mL-1) PiACP; 2a, 3a, 5a: 15 U mL-1 (263 g mL-1) Lw; 4a: 20 U mL-1 (350 g mL-1)
Lw] and were stirred at 30 °C. The progress of the reaction was monitored via HPLC-RI (1a, 2a
31
and 4a) or P-NMR (3a and 5a). When the product level reached a maximum, the mixture was
ultrafiltered for enzyme removal. 450 mM Ba(OH)2×8H2O (for AcP reactions) or 800 mM
Ba(OAc)2 (for PPi reaction) was added, the pH was set to 9-10 and the mixture was stirred for 15
min (AcP reactions) or 1 h (PPi reaction) at room temperature followed by filtration. To the filtrate
was added EtOH (80 vol% final concentration) and the mixture was allowed to stand at 4 °C
overnight to precipitate the barium salt of the product. In the case of 3a and 4a, the pH of the filtrate
was set to 4 before the addition of EtOH. The solids were filtered and the products were dried at
room temperature. The products were characterized without further purification (purity 1b: ~90%;
2b: ~93%; 3b: ~95%; 4b: ~88%; 5b: ~90%) by NMR (ESI).
10
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10.1002/adsc.201800306
Advanced Synthesis & Catalysis
Acknowledgements: Funding by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol,
Government of Lower Austria and ZIT through the Austrian FFG-COMET-Funding Program is
gratefully acknowledged.
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Advanced Synthesis & Catalysis
UPDATE
Evaluation of Natural and Synthetic Phosphate Donors
for the Improved Enzymatic Synthesis of Phosphate Monoesters
Graphical Abstract
pH ~ 3.4
X
Features:
- mild conditions, easy reaction setup
- stable product levels
- substrates: monoalcohols and diols, mono-
and oligosaccharides, nucleosides
- high productivity (TONs up to 53.000)
- preparative-scale (400 mM)
- high isolated amounts (up to 5.6 g)
- up to 75% less waste
acid
O
P
O
O
P
phosphatase
R
H
R
+
O
O
O
O
O
OH
OH
HOAc
pH > 4
Pi
H2O
13
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