Preparative aerobic oxidations with basidiomycetous enzymes: CH-functionalization of adamantane

Article abstract of DOI:10.1016/j.molcatb.2015.08.002

The potential of basidiomycetous enzymes for selective alkane CH-functionalizations in aqueous media has been disclosed utilizing surface and submerged cultures. A screening displayed the high catalytic activity of Dichomitus albidofuscus, Pholiota squarrosa, and Abortiporus biennis in aerobic oxidations of adamantane as a model hydrocarbon. With isopropanol as a co-solvent, the oxidation was accelerated significantly without changes of the fungal growth. 1-Adamantanol was obtained in 40% preparative yield from the oxidation of adamantane by D. albidofuscus. The CH-positional selectivity (3°/2°= 3.6) and the deuterium kinetic isotopic effect (k_H/k_D = 2.25) values provide evidence for the participation of fungal metalloenzymes in the CH-activation step.

Full text of DOI:10.1016/j.molcatb.2015.08.002

Journal of Molecular Catalysis B: Enzymatic 122 (2015) 87–92
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Preparative aerobic oxidations with basidiomycetous enzymes:
CH-functionalization of adamantane
Tatyana S. Zhuka,b,∗, Michael Goldmanna, Julia Hofmanna, Juliane C.S. Pohla,
Holger Zorna
a Institute of Food Chemistry and Food Biotechnology, Justus Liebig University, Heinrich-Buff-Ring, 58, 35392 Giessen, Germany
b Department of Organic Chemistry, Kiev Polytechnic Institute, pr. Pobedy, 37, 03056 Kiev, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2015
Received in revised form 5 July 2015
Accepted 4 August 2015
Available online 8 August 2015
The potential of basidiomycetous enzymes for selective alkane CH-functionalizations in aqueous media
has been disclosed utilizing surface and submerged cultures. A screening displayed the high catalytic
activity of Dichomitus albidofuscus, Pholiota squarrosa, and Abortiporus biennis in aerobic oxidations of
adamantane as a model hydrocarbon. With isopropanol as a co-solvent, the oxidation was accelerated
significantly without changes of the fungal growth. 1-Adamantanol was obtained in 40% preparative yield
from the oxidation of adamantane by D. albidofuscus. The CH-positional selectivity (3◦/2◦ = 3.6) and the
deuterium kinetic isotopic effect (kH/kD = 2.25) values provide evidence for the participation of fungal
metalloenzymes in the CH-activation step.
Keywords:
Alkane biotransformation
Aerobic oxidation
Adamantane
© 2015 Elsevier B.V. All rights reserved.
Basidiomycetes
Dichomitus albidofuscus
1. Introduction
and further to 1,3-adamantanediol [15] using Streptomyces sp. SA8,
have been reported. Additionally, many nickel, [16,17] ruthenium
The selective functionalization of saturated hydrocarbons (alka-
nes) still represents a major theoretical and practical challenge in
organic synthesis [1–6]. Laboratory scale and industrial approaches
are often based on hazardous chemicals (radicals, electrophiles
or oxidants) and, despite enormous efforts, are still unsatisfac-
tory. One of the challenges lays in overoxidation, as the primary
oxidation products are typically more reactive than the starting
alkanes. In Nature, however, C H-bond functionalizations of satu-
rated hydrocarbons often proceed highly selectively, namely the
oxidation of methane to methanol by methane monooxygenase
[18] and iron non-heme [19–21] complexes have been developed
as “artificial metalloenzymes” mimicking oxidative biocatalysts.
These catalytic systems, however, are much less effective than their
natural counterparts [22].
Compared to many biocatalytic systems, those based on basid-
iomycetous fungi are distinguished by an unusually high stability
of the enzymes, their nontoxicity and simplicity in use. Addi-
tionally, cultivation of the fungi as submerged cultures provides
almost unlimited amounts of the catalysts. The enormous syn-
thetic potential of basidiomycetes has been demonstrated recently
by the oxidation of cyclohexane to cyclohexanol and cyclohex-
anone with fungal unspecific peroxygenases (UPO) [23] and the
selective allylic oxidation of terpenes to the corresponding enones
with lyophilisates of Pleurotus sapidus [24]. The H2O2-dependent
UPOs from Agrocybe aegerita [25] and Coprinellus radians [4] are
heme thiolate proteins that are fairly stable in a large number of
organic solvents, such as acetone or dichloromethane, and catalyze
the hydroxylation of alkanes. The enzymes showed 100% selectiv-
ity for the conversion of propane to 1-propanol, while mixtures
of 2- and 3-alcohols were obtained from higher alkanes [4]. How-
ever, all of these biotransformations were studied for very low
substrate amounts (<0.25 mmol), and the reaction products were
analyzed by gas chromatography (GC–FID) and mass spectrome-
try (GC–MS) only, without isolation of the products. The suitability
sMMO [7,8] or the insertion of a sulfur atom into unactivated C
H
bonds by radical SAM enzymes in the final step of biotin biosyn-
thesis [9]. The cytochrome P450 enzymes catalyze a multitude
of oxidations in Nature and are intensively applied for new bio-
catalytic transformations [10] involving linear alkanes [11]. Many
other biocatalytic approaches for the functionalizations of C
H
bonds, such as the terminal hydroxylation of gaseous and liquid
n-alkanes (n = 2–9) by AlkB [12] and other bacterial enzymes, [13]
as well as the bioconversion of adamantane to 1-adamantanol [14]
∗
Corresponding author at: Department of Organic Chemistry, Kiev Polytechnic
Institute, pr. Pobedy, 37, 03056 Kiev, Ukraine. Fax: +380 44 236 97 74.
E-mail address: t.zhuk@xtf.kpi.ua (T.S. Zhuk).
http://dx.doi.org/10.1016/j.molcatb.2015.08.002
1381-1177/© 2015 Elsevier B.V. All rights reserved.

88
T.S. Zhuk et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 87–92
Table 1
The 3◦/2◦-selectivities for adamantane (1) oxidations by different oxygen-containing species.
#
1
Reactive system
3◦/2◦ selectivity
1.3
Ref.
[44]
H2O2/O2/[n-
Bu4N]+[VO3]−/pyrazine-2-
carboxylic
acid
2
3
4
5
6
7
8
H2O2/O2
1.4
6
33
62
2.2
3
[44]
[45]
[46]
[42]
[47]
[48]
[49]
t-BuOOH/Mn2+
phthalimido-N-oxyl
•
NO3
Fe2O-(HB(pz)3)2(OAc)2/Zn/O2
sMMO/O2
Fe2O(OAc)2Cl2-(2,2ꢀ-bipy)2 or
Fe2O(OAc(tmima)2(ClO4)3/H2O2
Fe(PA)2/H2O2
3.5
9
4.0
3–10
9.5
[50]
[51]
[52]
[53]
10
11
12
Mn-porphyrin/KHSO5
[FeCl2-(TPA)](ClO4)/t-BuOOH
Fe2O(2,2ꢀ -
10
bipy)4(H2O)2(ClO4)4/t-BuOOH
P-450
13
11–48
[54]
of basidiomycetes for the oxidation of alkanes on a preparative
scale is therefore still unknown. Possible drawbacks may comprise
large reaction volumes, long reaction times as well as losses of the
substrates and products by adsorption to the fungal mycelia. Fur-
thermore, alkanes are poorly soluble in water, which is usually used
as reaction medium for fungal oxidations [24,26].
In contrast to numerous mechanistic studies on the CH
functionalization with methanotrophic bacteria and cytochrome
oxygenases [27], only little is known about the oxidation of alkanes
by basidiomycetes. Comparatively few of their enzymes have been
isolated and intensely characterized [25,28–31] adding only little
to the understanding of the mechanisms of their action [32,33].
For this purpose we have analyzed the positional 3◦/2◦ substi-
tution selectivity, which is strongly reagent-dependent, and the
hydrogen/deuterium kinetic isotope effects (KIE) [34] resulting
from the differences in the reaction rates of C H and C D bonds.
Both experiments are useful in the analyses of CH-bond activation
mechanisms providing information on the nature of the transition
structures.
the solvent, is typical for Gif-type oxidations [43]. In addition, the
KIE values for the reaction of 1 with traditional electrophiles and
oxidants are very characteristic and also involve many examples
[36,40].
Concerning practical aspects the biotransformation of 1 is an
appropriate model reaction due its availability from Nature [55]
or synthetically [56] and wide use of adamantane derivatives as
pharmaceuticals [57–60], polymeric [61], and electronic [62–64]
materials. We, thus, selected 1 as a model compound for oxidation
by surface and submerged cultures of basidiomycetes.
2. Experimental
2.1. Organisms
The basidiomycetes used in this work were supplied by the
Centraalbureau voor Schimmelcultures (CBS, Baarn, Netherlands),
the Deutsche Sammlung für Mikroorganismen und Zellkulturen
(DSMZ, Braunschweig, Germany), the Friedrich-Schiller-University
Jena (FSU), and the culture collection of the Justus Liebig University
Giessen (LCB).
The stock cultures of the fungi were maintained on a solid
medium containing 15 g/L malt extract (Fluka, Neu-Ulm, Germany)
and 15 g/L Agar–Agar (Roth, Karlsruhe, Germany).
The tricyclic saturated hydrocarbon adamantane (1) containing
two types of carbon atoms (CH and CH2) was used as a model for
many mechanistic studies on radical, electrophilic, and oxidative
alkane activations [1,3,35–41]. Due to large amount of accumulated
data concerning the 3◦/2◦-selectivities of the functionalization as
well as KIE values, 1 is a perfect substrate for disclosing the mecha-
nistic details of alkane oxidation by basidiomycetes, namely for the
identification of the nature of the H-abstracting species. Depend-
ing on the oxidizing reagent the ratios of 3◦-(1-adamantanol,
(2))–2◦- (2-adamantanol (3) and adamantanone (4)) vary signifi-
cantly (Table 1). The existing examples involve different oxidative
systems, e.g., H2O2/O2, H2O2/O2/[n-Bu4N]+[VO3]−/pyrazine-2-
carboxylic acid, t-BuOOH/Mn2+ and many others, where, HO•,
MeOO•, and t-BuO•, respectively, are involved in the H-abstraction
step. (Table 1, entries 1–3) The selectivities increase signifi-
cantly for bulky t-BuO [42], I3C [40] and highly electrophilic
2.2. Submerged cultures
The culture medium was prepared by dissolving malt extract
(30 g) and soy peptone (3 g) in 1 L of deionised water. The pH was
adjusted to 5.6 with 1.0 M NaOH prior to autoclaving. For prepara-
tion of the precultures, a 1 cm2 agar plug from the leading mycelial
edge was transferred into 100 mL malt extract medium and then
homogenized with a T 25 digital Ultra-Turrax homogenizer (IKA,
Staufen, Germany; 30 s, 10.000 r min−1). The precultures were
grown on an incubation shaker (Orbitron, Infors HAT, Bottmin-
gen, Switzerland; 150 r min−1, deflection 25 mm) under exclusion
of light at 24 ◦C for 7 days. Afterwards, the precultures were homog-
enized, and 10% (v/v) of the homogenate were inoculated for
•
phthalimido-N-oxyl, NO3 radicals. (Table 1, entries 4, 5) Concern-
ing biotransformations of 1 the ratio of 3◦/2◦ varies from 2.2 up to
11–48 depending on the use of bio- or biomimetic systems. (Table 1,
entries 6–13) An average value of 2.6–4.2, slightly dependent on

T.S. Zhuk et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 87–92
89
Table 2
was dried over anhydrous MgSO4 and evaporated to dryness. Purifi-
cation of the crude product by column chromatography on silica
gel (eluent pentane, then pentane/diethyl ether, 1/1) gave 71 mg
of adamantane (1) and 65.4 mg of 1-adamantanol (2) as a color-
less solid with 1H and 13C NMR spectra identical to the authentic
reference standard.
Oxidation of adamantane (1) to 1-adamantanol (2) and 2-adamantanone (3) by A.
biennis, D. albidofuscus, and P. squarrosa in the presence of alcohols (according to
GC–MS analyses).
#
Alcohol
Abortiporus
biennis
Dichomitus
albidofuscus
Pholiota
squarrosa
1
i-PrOH
1, 80%
2, 20%
1, 20%
2, 79%
1, 100%
2.6. Determination of kinetic isotope effects
traces of 3
1, 100%
1, ca. 100%
traces of 2
1, 100%
2
3
i-BuOH
t-BuOH
1, 100%
1, 100%
1, 100%
1, ca. 100%
traces of 2
1, 100%
All kinetic experiments were performed competitively
for
adamantane
(1)
(68 mg,
0.5 mmol)
vs
1,3,5,7-
4
5
n-BuOH
s-BuOH
1, 100%
1, 100%
tetradeuterioadamantane (70 mg, 0.5 mmol) using the same
conditions as for preparative oxidation of 1 by D. albidofuscus. The
relative concentrations of deuterated and nondeuterated products
were determined by GC/MS analysis (HP5890 Series II GC, col-
umn HP Ultral (50 m × 0.2 mm, cross-linked methyl silicon) with
HP5971A mass selective detector by mass selective integration.
1, 100%
1, 100%
submerged cultivation into a 1000 mL Erlenmeyer flask containing
400 mL medium.
2.3. Biotransformation of adamantane (1)
3. Results and discussion
(a)1 (125 mg, 0.92 mmol per plate) was placed as a solution in
diethyl ether into the agar of petri dishes. The plates were inocu-
lated with a 1 cm2 agar plug of the respective fungus and incubated
at 24 ◦C for 14 days under aerobic conditions. The cultures were
mixed with 12 mL of saturated NaClO4 solution, and the mycelium
was removed by filtration. The aqueous solution was extracted
with diethyl ether (3 × 30 mL) and EtOAc (1 × 30 mL). The com-
bined organic phases were dried over Na2SO4 and concentrated
to a defined volume of 8 mL at a pressure of 240 mbar. 1 ␮L was
measured by GC–MS.
3.1. Screening of basidiomycetous oxidative activity towards
saturated C H-bonds
In a broad screening of various basidiomycetes, 30 different
species from the orders Agaricales, Gloeophyllales, Polyporales,
Corticiales, Hymenochaetales, Russulales, and Auriculariales (see
SI for details) were tested for the oxidation of 1. Either malt
extract (MEA) or a minimal medium with or without glucose were
employed for fungal growth.
(b) 1 (50 mg, 0.37 mmol) was added to 110 mL submerged
cultures of ABI, DAL, or PSQU after 168 h of growth in malt
extract medium in 250 mL Erlenmeyer flasks. For each fungus
30 flasks were prepared. During 15 days, 2 flasks were ana-
lyzed every day. The cultures were mixed with EtOAc/n-hexane
(1/1, 30 mL) and centrifuged (4.500 g /10 min /4 ◦C). The super-
natant was separated and extracted with EtOAc/n-hexane (3/1,
3 × 30 mL). The combined organic phases were dried over Na2SO4
and concentrated to a defined volume of 10 mL at a pressure of
240 mbar. Quantitative analyses were performed by GC–FID using
1,3-dimethyladamantane as internal standard (IST). 300 ␮L from a
stock solution of 0,36 mg mL−1 1,3-dimethyladamantane in EtOAc
were mixed with 1 mL of the sample, from which 1 ␮L was analyzed
by GC–FID.
This first screening round indicated high oxidative activities of
Abortiporus biennis, Pholiota squarrosa, and Dichomitus albidofus-
cus where 1-adamantanol (2) was identified as the main product
together with trace amounts of 2-adamantanone (3). This set of
products is characteristic for the oxidation of adamantane (1) with
oxygen-centered radicals that are involved in the CH activation step
(Scheme 1).
3.2. Time course for adamantane (1) biotransformation
In the next step, the selected fungi were grown submerged
in malt extract medium containing 50 mg of 1. According to the
GC–MS data, the adamantane derivatives 2 and 3 were formed. Dur-
ing the culture period of 14 days the formation of fungal biomass
and the pH of the media were monitored. In parallel, the produc-
tion of adamantane derivatives was analyzed quantitatively using
1,3-dimethyladamantane as an internal standard in GC–FID anal-
ysis (Fig. 1). After 3 days, about 1.4 g of dry biomass was obtained
with A. biennis per 110 mL of culture medium (Fig. 1a). This amount
remained constant for 3 days and then slightly decreased together
with an increase of the pH from 5 to 7.5, indicating a beginning
lysis of the fungal mycelium. Only traces of 2 were detected in
the reaction mixture after 24 h, and the maximum formation of
2 (0.64%) was achieved after 4 days (Fig. 1b). In contrast, D. albido-
fuscus grew well until the 8th culture day and formed up to 0.54%
of 2 (Figs. 1c,d). P. squarrosa formed only traces of 2 together with
further oxidation products of 1 (data not shown).
2.4. Biotransformation of adamantane (1) with co-solvents
Hydrcarbon 1 (50 mg, 0.37 mmol) was dissolved in the respec-
tive alcohol (Table 2) (5 mL) and added to cultures of ABI, DAL,
or PSQU grown for 168 h in malt extract medium. After incuba-
tion for further 8 days, the culture media (115 mL) were extracted
with CH2Cl2 (3 × 10 mL) and washed with brine (1 × 10 mL) and
water (2 × 10 mL). The organic extract was dried over anhydrous
MgSO4 and evaporated to dryness. The resulting reaction mixtures
were analyzed by GC/MS (HP5890 Series II GC, column HP Ultral,
50 m × 0.2 mm with HP5971A detector), where the ratios of the
oxidation products 2–3 were determined by the integration and
statistically corrected to give the 3◦/2◦ functionalization sectivities.
2.5. Preparative oxidation of adamantane (1) by D. albidofuscus
3.3. Biotransformation of adamantane (1) with co-solvents
150 mg (1.1 mmol) of 1 were dissolved in 5 mL of iso-propanol
and added to a 168 h old culture of D. albidofuscus (125 DAL) (40 mL
of pre-culture) grown in malt extract medium (400 mL). After 8
days of incubation, the culture medium (440 mL) was extracted
with pentane (3 × 30 mL), CH2Cl2 (5 × 30 mL), washed with brine
(1 × 30 mL) and water (2 × 30 mL). The combined organic extract
In these preliminary experiments, A. biennis and D. albidofuscus
displayed similar efficiencies and selectivities in the oxidation of 1.
A. biennis grew faster and produced higher amounts of biomass
which may complicate the preparative isolation of the reaction
products. In all experiments, the conversion rates of 1 were lower
than 1%, most probably due to its poor solubility in water. Higher

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T.S. Zhuk et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 87–92
Scheme 1. Aerobic oxidation of adamantane (1) by surface cultures of D. albidofuscus (DAL).
conversion rates were achieved by addition of various C3 C4 alco-
hols that increased the actual concentration of 1 in the reaction
media. The addition of up to 5% of these organic solvents to the cul-
ture media did not retard the growth of the fungi, but dramatically
increased the efficiency of the oxidation (Table 2). According to
the GC–MS data, the presence of i-PrOH increased the conversion
rates of 1 to 20% with A. biennis and up to 80% with D. albidofus-
cus. Only traces of products were detected with t-BuOH and no
reaction occurred with other C4-alcohols. No oxidation products
of 1 were identified with P. squarrosa in the presence of alcohols,
as the alcohols strongly impeded the growth of this fungus. These
results clearly indicated the ability of the selected basidiomycetes
to activate the aliphatic CH-bonds.
3.4. Preparative biotransformation of adamantane (1)
To evaluate the potential of this bioconversion for preparative
applications, the oxidation of 1 by D. albidofuscus in the presence
of i-PrOH as a co-solvent was scaled-up. Using 1.1 mmol of 1 in
400 mL medium and 1% i-PrOH, a nearly quantitative conversion of
1 was achieved. The extraction of the reaction mixture (including
culture supernatant and mycelium) with diethyl ether resulted in
Fig. 1. (a) pH and production of dry mass in submerged cultures of A. biennis; (b) formation of oxidation products of 2 in submerged cultures of A. biennis; (c) pH and
production of dry mass in submerged cultures of D. albidofuscus; (d) formation of oxidation products of 2 in submerged cultures of D. albidofuscus.

T.S. Zhuk et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 87–92
91
exclude the participation of the free HO-radicals. Both, the 3◦/2◦
selectivity and the KIE value for the oxidation of 1 strongly indicate
the participation of fungal metalloenzymes in the oxygen trans-
fer. Metalloenzymes like catalases [70] and multi-copper laccases
[71–74] play a key role in the proteome of basidiomycetes. Cata-
lases [70,75] contain an iron ion in their active sites and transform
H2O2 into oxygen and water [76]. They are involved in the nat-
ural degradation of lignocelluloses and also catalyze the oxidation
of various organic compounds including phenols, methoxyphenols,
aminophenols, and diamines [77]. Fungal UPOs, which have been
shown to oxidize aromatic as well as aliphatic compounds contain
iron and magnesium ions [32,78]. The extracellular enzymes of A.
biennis, D. albidofuscus, and P. squarrosa responsible for the oxida-
tion of adamantane remain to be identified on a molecular level
in ongoing research. However, based on the data presented here,
the contribution of peroxidase type metalloenzymes in the aerobic
oxidation of adamantane is to be expected.
Fig. 2. Ratio of non-deuterated and deuterated 2 formed by oxidation of 1 by D.
Albidofuscus.
a combined yield of 44% of crude 2 with small amounts of 1 and 3.
We supposed that substrates and products were tightly adsorbed
to the mycelia due to the high hydrophobicity of the adamantane
cage. Extraction with pentane added 10% of 1–2 mixture to the bal-
ance. Therefore, CH2Cl2 was chosen as a solvent, and additional 20%
of 1 was extracted. Lyophilization and disruption of the mycelia by
grinding under liquid nitrogen with subsequent extraction did not
further improve the reaction balance. After combining all organic
extracts and purification by column chromatography on silica gel,
39% of pure 2 were obtained. The physico-chemical properties of
2 were identical to those of an authentic reference standard [65].
To the best of our knowledge, this is the first example of a prepara-
tive aerobic functionalization of an alkane by basidiomycetes which
opens up new perspectives for the mild aerobic oxidation of alkanes
in aqueous media. Futhermore, based on the observed 3◦/2◦ ratio
of ca. 3.6, the participation of free OH-radicals may be excluded
for the aerobic oxidation of 1 by D. albidofuscus suggesting that
fungal metalloenzymes are most likely accountable for the oxygen
transfer.
4. Conclusions
An efficient aerobic oxidation of adamantane by submerged
cultures of basidiomycetes in aqueous media was confirmed by
analytical and preparative experiments. The 3◦/2◦ selectivity of the
introduction of the hydroxyl group as well as the KIE values agree-
able suggested the participation of fungal metalloenzymes as key
players in the activation of the C H bond. The suggested hydrocar-
bon oxidations do not require hazardous chemicals, occur under
room temperature and pressure and is almost waste-free. Further
extension of this approach to wider range of mono-, poly- and
non-cyclic aliphatic compounds that challenge the CH-activations
methods are currently underway in our labs.
Acknowledgements
We are grateful to Prof. Peter R. Schreiner (Giessen University)
and Prof. Andrey A. Fokin (Kiev Polytechnic Institute) for fruitful
discussions. The study was financially supported by the excellence
initiative of the Hessian Ministry of Science and Art which encom-
passes a generous grant for the LOEWE focus “Insect Biotechnology”
and the Center for Insect Biotechnology & Bioresources.
3.4. Kinetic isotope effects of adamantane (1) biotransformation
Additional proof for the participation of metal-oxo complexes
in the CH activation step arises from the kH/kD KIE values. The
competitive method, where two substrates are present in the same
vessel, was chosen for the determination of KIEs [34]. This approach
avoids unintended variations of the experimental conditions and
allows to estimate the ratio of reactants with high precision. A
KIE value of approximately 1 indicates that the cleavage of the
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molcatb.2015.08.
002.
C
H-bond is not the rate limiting step and is characteristic for
processes where HO-radicals participate in C H-activation [66]
as found for Fenton-type reactions (≤1.18) [43]. The KIE values
increase for high-valent metal-oxo-catalyzed hydroxylations of 1,
namely, for RuO4-mediated oxidations (4.8–7.8 depending on the
solvent) [67,68]. For the metalloporphyrine-catalyzed oxidation of
1, the KIEs are often higher for iron (2.8–8.7) [69] than for man-
ganese based cores (3.1–4.9) and are also sensitive to the oxidant
[69].
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Accordingly, the KIE was calculated for 1 by oxidizing a mixture
of its protio- (AdH4) and 1,3,5,7-tetradeuterio- (AdD4) [38] forms
from the ratio of non-deuterated and deuterated forms of 2 with
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