Modeling of the ribonucleotide reductases substrate reaction. Hydrogen atom abstraction by a thiyl free radical and detection of the ribosyl-based carbon radical by pulse radiolysis

Article abstract of DOI:10.1135/cccc2011085

The 1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol (1) was prepared from 1,2-O-isopropylidene-α-D-glucose in 10 steps. In a key step treatment of the 1,2-O-isopropylidenehexofuranose derivative with BF₃/Et ₃SiH effected deacetonization and reductive deoxygenation at carbon 1. Pulse radiolysis experiments with 6-thiohexofuranitol 1 and its disulfide derivative demonstrated formation of the ribosyl-based carbon-centered radical upon generation of 6-thiyl radical in basic medium. The proposed [1,5]-hydrogen shift abstraction with generation of the C3 radical mimics the initial substrate reaction of RNRs. The reversible H-atom transfer has been quantified and was correlated with the computed rate constants for the internal H atom abstraction from C1, C2, C3 and C4 by the thiyl radical. The energy barrier for the H3 and H4 abstractions were calculated to be most favorable with the corresponding barriers of 11.1 and 11.2 kcal/mol, respectively.

Full text of DOI:10.1135/cccc2011085

Modeling of the Ribonucleotide Reductases Substrate Reaction
1223
MODELING OF THE RIBONUCLEOTIDE REDUCTASES SUBSTRATE
REACTION. HYDROGEN ATOM ABSTRACTION BY A THIYL FREE
RADICAL AND DETECTION OF THE RIBOSYL-BASED CARBON
RADICAL BY PULSE RADIOLYSIS
a3
Stanislaw F. WNUKa1,*, Jaidev A. K. PENJARLAa2, Thao DANG
,
Alexander M. MEBELa4, Thomas NAUSER and Christian SCHÖNEICH
b
c
a
Department of Chemistry and Biochemistry, Florida International University,
Miami, Florida 33199, USA; e-mail: wnuk@fiu.edu, jaidev_org@yahoo.com,
1
2
3
4
dangtp@gmail.com, mebela@fiu.edu
b
c
Department of Chemistry and Applied Bioscience, ETH Zurich, CH-8093, Switzerland;
e-mail: nauser@inorg.chem.ethz.ch
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA;
e-mail: schoneic@ku.edu
Received April 21, 2011
Accepted May 24, 2011
Published online September 22, 2011
Dedicated to Professor Antonin Holý on the occasion of his 75th birthday in recognition of his
outstanding contributions to the area of nucleic acid chemistry.
The 1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol (1) was prepared from 1,2-O-isopropyl-
idene-α-D-glucose in 10 steps. In a key step treatment of the 1,2-O-isopropylidenehexo-
furanose derivative with BF3/Et3SiH effected deacetonization and reductive deoxygenation at
carbon 1. Pulse radiolysis experiments with 6-thiohexofuranitol 1 and its disulfide derivative
demonstrated formation of the ribosyl-based carbon-centered radical upon generation of
6-thiyl radical in basic medium. The proposed [1,5]-hydrogen shift abstraction with genera-
tion of the C3 radical mimics the initial substrate reaction of RNRs. The reversible H-atom
transfer has been quantified and was correlated with the computed rate constants for the in-
ternal H atom abstraction from C1, C2, C3 and C4 by the thiyl radical. The energy barrier
for the H3 and H4 abstractions were calculated to be most favorable with the corresponding
barriers of 11.1 and 11.2 kcal/mol, respectively.
Keywords: Radicals; Thiosugars; Pulse radiolysis; Ribonucleotide reductase; Carbohydrates;
Bioorganic chemistry.
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to
2′-deoxynucleotides providing the monomeric precursors required for DNA
replication and repair1. Inhibition of RNRs disrupts this primary source of
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011085

1224
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
DNA components and therefore is an appealing concept for rational drug
•
design2. In the first step of the mechanism of RNR a thiyl radical SCys439
is generated in proximity to the β face of the nucleotide-substrate is pro-
posed to abstract H3′ of the ribosyl, which promotes irreversible loss of
water (C2′-H2O) from the resulting C3′ radical1,3,4. Abstraction of H3′ by a
primary aliphatic thiyl radical (•SCys439) to generate C3′ radical has
aroused debate owing to the absence of appropriate chemical precedence.
Stubbe based her original hypothesis3,4 on the fact that thiyl radicals, gen-
erated via hydrogen atom abstraction by hydroxyl radical, were able to
isomerize cis-2,5-dimethyl-tetrahydrofuran to its trans isomer5. Moreover,
the rate constants for hydrogen atom abstraction from the activated C–H
bonds (α-Hs of alcohols and ethers) by thiyl radical are approximately 104
times slower than the reverse donation of hydrogen to alkyl radicals by
thiols6–7
.
4
–1 –1
1–4 × 10
M
s
R′H + RS•
R′• + RSH
(1)
7
–1 –1
1 × 10
M
s
Chemical modeling by Giese8 and Robins9,10 as well as enzymatic studies
with gemcitabine11 and McCarthy’s 2′-deoxy-2′-fluoromethylenecytidine12–14
preserve the fundamental concepts of the Stubbe mechanism but detection
of the ribosyl-based radical(s) during RNR-catalyzed deoxygenation of the
natural substrates remains elusive. Nevertheless, kinetic studies of cytidine
5′-diphosphate with the E441Q mutant of E. coli class I RNR identified two
radical species by EPR spectroscopy15. The first radical was established to be
a disulfide radical anion16 which gave rise to a second nucleotide-based rad-
ical of the semidione nature with a hyperfine coupling to two almost iden-
tical protons at C1′ and C4′ 17
.
Robins and Ewing18 demonstrated that thiyl free radical generated at C6
of hexofuranosyl model A abstracts H3 by a [1,5]-hydrogen shift (Scheme 1).
(Analogous H-3 abstractions were also observed by an aminyl and oxyl radi-
cals19.) Thus, treatment of A with Bu3SnH/AIBN/benzene/∆ generated thiyl
radical by a radical fragmentation of S-2-[(phenoxythiocarbonyl)oxy]ethyl
group, which initiated the abstraction of H3 via an intramolecular [1,5]-
hydrogen transfer reaction with accompanying elimination of phenyl-
sulfinyl radical from C2 (e.g. B) to generate C. The irreversible β-elimina-
tion was assumed to be a driving force for the equilibrium to shift towards
product C, in spite of the poor ability of thiyl radicals to abstract the hydro-
gen from the activated carbons. In case of RNRs abstraction of H3′ and a
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1225
[1,2]-electron shift coupled with hydrogen transfer from O3′ to O2′ and ir-
reversible loss of water from C2′ gives the stabilized oxyallyl radical1,10
.
SCHEME 1
Biomimetic modeling of the first step of substrate reaction at active site of RNR 18
We now report synthesis and pulse radiolysis experiments with a 1,4-
anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol 1 which allowed us to detect
the ribosyl-based carbon-centered radical(s) and to measure rate constant
for the H abstraction(s). The computed rate constants for the internal H
atom abstractions from C1, C2, C3 and C4 by the thiyl radical are corre-
lated with the experimental results. The conversion of thiyl free radical D
into carbon radical F through an intramolecular [1,5]-hydrogen shift (via an
obligated six-membered transition state E) mimics the initial substrate reac-
tion of RNRs (Scheme 2).
SCHEME 2
Proposed biomimetic modeling for the H3 abstraction by 6-thiyl radical
RESULTS AND DISCUSSION
Synthesis
Synthesis of the 6-thiohexofuranitol 1 started from readily available 1,2-O-
isopropylidene-α-D-glucose which was converted to 3-O-benzoyl-5-deoxy-1,2-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1226
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
O-isopropylidene-α-D-ribo-hexofuranose 2 in five steps (55%, Scheme 3)9,20
.
Attempted selective deoxygenation of 2 at the anomeric carbon with the
excess of boron trifluouride etherate and triethylsilane18,21–23 produced a
complex mixture. However, treatment of the 3,6-O-dibenzoyl hexofuranose
3 with BF3/Et3SiH (5 equiv.) effected deacetonization and reductive deoxy-
genation (at C1) to give a mixture of the 2,6- and 3,6-di-O-benzoyl hexo-
furanitols 4a and 4b due to the partial migration23 of the benzoyl group
from 3-OH to 2-OH group. Debenzoylation of 4a/4b mixture with NH3/
MeOH afforded a single 1,4-anhydroalditol 5. Selective tosylation of 5 gave
6-O-tosylate 6 and subsequent SN2 displacement with AcSK/DMF produced
6-S-acetyl hexofuranitol 7. Deacylation with NH3/MeOH and column chro-
matography yielded disulfide 8 instead of the expected free thiol 1.
SCHEME 3
(a) BzCl/pyridine. (b) Et3SiH/BF3·Et/CH2CH2. (c) NH3/MeOH. (d) TsCl/pyridine. (e) AcSK/DMF.
(f) PPh3/MeOH/H2O
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1227
Alternatively, tosylation of 2 produced 9, which upon treatment with
three equivalents each of BF3OEt2 and Et3SiH gave a product profile very
similar to the deoxygenation of 3,6-O-dibenzoyl sugar 3. Two closely mi-
grating on TLC 2-O-benzoyl and 3-O-benzoyl sugars 10a/10b were obtained
after column chromatography. Subsequent nucleophilic displacement with
AcSK/DMF afforded 6-S-thioacetate mixture 11a/11b. Deacylation with
NH3/MeOH (without exclusion of oxygen) resulted in the formation of di-
sulfide 8.
Reduction of disulfide 8 with PPh3/MeOH/H2O produced a more rapidly
migrating (TLC) thiol 1, in agreement with the migratory properties of
other carbohydrate alkanethiol-disulfide pairs19. The presence of the thiol
function was also qualitatively confirmed by the colorization upon treat-
1
ment with Ellman’s reagent. In the H NMR spectra, an upfield shift for the
H6,6′ signals of thiol 1 (δ 2.56–2.69) as compared to disulfide 8 (δ 2.78–
2.94) was observed.
Treatment of 1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol 1 with
2,2′-azobis-2-methylpropanimidamide dihydrochloride (AAPH) as a radical
initiator in D2O (pD = 5.5) at 35, 45 or 55 °C for up to 72 h under argon24
resulted in the recovery of unchanged 1 and/or formation of its disulfide 8
with no deuterium incorporation at any of the ribosyl carbons (1H NMR,
MS). Presumably very fast reverse reaction (Eq. (1)) and/or water elimi-
nation at the acidic pD might be responsible for the lack of deuterium in-
corporation. Also, treatment of 1 with Bu3SnD/AIBN in refluxed MeOH-d4
or benzene resulted in neither deuterium exchange nor epimerization on
C3 in agreement with the results reported for the analogouos thiosugar
models19.
Theoretical Calculations
Using density functional calculations at the B3LYP/6-311G** level of the-
ory25–27 we computed reaction energies and barrier heights for internal H
atom abstractions by the thiyl radical from C1, C2, C3 and C4 of 6-thio-
hexofuranitol 1 (Fig. 1 and Table I). The results show that all considered
H shift reactions are endothermic and the least endothermic of them are
H4 abstraction occurring by the 1,4-H shift (5.2 kcal/mol) and H1 abstrac-
tion taking place by the 1,6-H shift (5.4 kcal/mol). These are followed by
H3 abstraction (1,5-H shift, 7.7 kcal/mol) and H2 abstraction (1,6-H shift,
10.5 kcal/mol). In terms of the calculated barrier height, the H3 and H4
abstractions are most favorable, with the corresponding barriers of 11.1 and
11.2 kcal/mol, respectively. These calculations are in agreement with the
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1228
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
TABLE I
Reaction energies, barrier heights and rate constants for the internal H atom abstractions by
the thiyl radical from C1, C2, C3 and C4 of 6-thiohexofuranitol 1
Reaction channel H1 abstraction
H2 abstraction
H3 abstraction
H4 abstraction
∆E, kcal/mola
∆G, kcal/molb
∆E#, kcal/molc
∆G#, kcal/mold
kf, s–1 e
5.4
5.7
10.5
7.7
5.2
10.4
7.9
5.3
13.4
17.7
11.1
11.2
14.6
19.1
12.2
12.1
1.18 × 102
1.74 × 106
6.30 × 10–2
2.61 × 106
6.77 × 103
4.04 × 109
7.80 × 103
6.04 × 107
kr, s–1 f
a
b
c
d
Reaction energy. Reaction Gibbs free energy at T = 298.15 K. Barrier height. Gibbs free
e
energy barrier height at T = 298.15 K. Reaction rate constants in the forward direction.
f
Reaction rate constants in the reverse direction.
FIG. 1
Optimized geometries of the 6-thiyl radical and products and transition states of its internal H
abstraction (H shift) reactions from C3 (path a), C4 (path b), C1 (path c) and C2 (path d). Cal-
culated reaction energies ∆E, reaction Gibbs free energies ∆G at 298.15 K, barrier heights ∆E#,
and Gibbs free energy barrier heights ∆G# at 298.15 K (all in kcal/mol), as well reaction rate
constants in forward (kf) and reverse (kr) directions are also shown
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1229
theoretical values for reaction energy (5.9 kcal/mol) and energy barrier
(9.2 kcal/mol)28 for H3′ abstraction by thiyl radicals in the enzymatic
model systems for natural substrates28–30. The barriers for the H1 and H2
abstractions are higher by 2.2 and 6.5 kcal/mol.
Rate constant calculations at room temperature using transition state the-
ory (TST) confirm the kinetic preference of the H3 and H4 abstraction
channels given similar values of about 7–8 × 103 s–1. The computed rate
constant for the H1 abstraction is about 60 times slower and that for the H2
abstraction is negligibly small. It should be also noted that coupled clus-
ters31 CCSD(T)/6-311G** calculations confirmed the B3LYP results for the
barrier heights giving very close barriers for the H3 and H4 abstractions,
with the barriers for the H1 and H2 abstractions being 2.5 and 6.6 kcal/mol
higher, respectively. In summary, according to the theoretical calculations,
the H3 and H4 abstractions are expected to give comparable product yields,
whereas the H1 and H2 abstractions would not contribute significantly.
Pulse Radiolysis
The radiolysis of disulfide 8 in an argon saturated aqueous solution con-
taining tert-butanol under alkaline conditions (pH 12) generates the absorp-
–1
tion of disulfide radical anion (ε420 ≈ 2 × 103
M
cm–1; Fig. 2, panel a). This
absorption bleaches quickly (kdecay = 2 × 105 s–1) and the equilibrium is irre-
versibly shifted to a carbon centered radical (ε260 ≈ 4.2 × 103
M
cm–1;
–1
Fig. 2, panel a) with kbuild-up of 2 × 105 s–1. The absorption of the carbon
radical(s) at 260 nm turned out to be astonishingly stable at pH 12. At this
pH the HSR• radical (R = ribose fragment) is deprotonated to the thiolate-
containing radical SR•. Because the reverse reaction relies on protonated
–
thiol groups, the equilibrium is shifted fully to the carbon centered radi-
cal(s). Experiments with methyl viologen (MV2+) proved the reducing na-
ture of our radical product(s) (Fig. 2, panel b and Fig. 3). At 600 nm, the
absorption-time profile is dependent on the concentration of methyl violo-
gen radicals (MV•+) only. There are three processes governing this profile
RSSR•– + MV2+ → RSSR + MV•+
(2)
(3)
(4)
RR′C•OH + MV2+ → RR′CO + H+ + MV•+
•
MV•+ + (CH2 )(CH3)2COH + H+ → MV2+ + (CH3)3COH
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1230
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
Reactions (2) and (3) contribute to the build up of the absorptivity, reac-
tion (4) causes its decay. Because these reactions are concurrent, a direct de-
termination of reaction (3) and its yield is not possible. Quantification of
a
b
FIG. 2
Pulse radiolysis of 0.9 mM of thiosugar disulfide 8 in 1 M tert-butanol solution saturated with
Ar in basic medium (pH 12): Panel a shows both the decay of the disulfide radical ion moni-
tored at 420 nm and the build-up of a carbon-centered radical monitored at 260 nm after a
pulse of 30 Gy. Panel b demonstrates the reducing nature of the radical product(s) in experi-
ments with methyl viologen (MV2+, 25 µM) detected at 600 nm. The build up is caused by re-
duction of MV2+ to MV•+ by both the disulfide radical anion and an additional reducing
species, most probably a ribosyl based radical (Dose 16 Gy). The decay visible is caused by re-
–1
combination of •CH2(CH3)2COH radicals with MV•+ (k4 = 2.5 × 106
M
s–1
)
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1231
the yield of reaction (3) however is necessary for the carbon-centered radi-
cal to be identified as reducing (located at an alcoholic carbon). We there-
fore analyzed the kinetics traces in more detail. Because of the low concen-
tration of MV2+ (25 µM), the kinetics of reaction (2) is governed by the com-
peting intramolecular hydrogen abstraction from the ribose by thiyl radi-
cals and therefore by k1 (Eq. (1)). The rate constant of reaction (3) will be
comparable with the known rate constants of 1,2-dihydroxycyclohexanyl
–1
radicals with MV2+ 32. The rate constant k4 ≈ 2.5 × 106
M
s–1 can easily be
derived in the time range 30–500 µs after the electron pulse (not shown),
when reactions (2) and (3) are completed. We then corrected our experi-
mental trace for reaction (4) and analyzed if the corrected trace could be ex-
plained by reaction (2) only or if reaction (3) must be invoked to explain
experimental results. Figure 3 shows residuals for least squares fits with the
known rate constants k2 and k2 plus k3, respectively. Without invoking re-
action (3) with a contribution of at least 15% of the total reducing equiva-
lents, there is a systematic and significant deviation between experiment
and fit. The total yield of MV•+ with respect to initially formed sulfur radi-
cals is >95%. Thus, we confirmed intramolecular abstraction of a H from a
carbon adjacent to either an ether (C1 or C4) or an hydroxyl group (C2 or
FIG. 3
Residuals of least square fits to the build-up of methyl viologen radicals. The rate constants k2
(reduction of MV2+ by RSSR•–) and k3 (reduction by ribosyl radicals) are taken from independ-
ent measurements. The single exponential fit is based on reaction (2) only. Clearly part of the
reduction is caused by ribosyl radicals
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1232
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
C3) by a thiyl radical which produces carbon radical species that are sub-
stantially reducing. Steenken et al.32 showed, that such radicals indeed un-
dergo rapid dehydration reactions.
The pulse radiolysis experiments in acidic medium (pH 3) increased the
decay rate of disulfide radical, kobs ≥ 5 × 105 s–1, and decreased formation of
absorbing species at 260 nm (25% compared to pH 12). If we take this in-
formation to be indicative of equilibrium (1), we derive an equilibrium con-
stant of K ≈ 0.3, which implies ∆G ≈ 1 kcal/mol. This is comparable, but
lower, than the known value for the equilibrium between thiyl radicals and
tetrahydrofuran, ∆G = 4 kcal/mol 6.
CONCLUSION
We have synthesized 1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol
disulfide (8) and demonstrated efficient formation of the ribosyl-based
carbon-centered radical upon generation of 6-thiyl radical using pulse
radiolysis experiments. One electron reduction of 8 by hydrogen atoms and
solvated electrons under alkaline conditions (pH 12) generated the disulfide
radical anion which bleached quickly and equilibrium was irreversibly
shifted to the more stable carbon centered radical (k ≈ 2 × 105 s–1). Experi-
ments with methyl viologen (MV2+) proved the reducing nature of our radi-
cal product(s). The reversible H-atom transfer has been quantified and was
correlated with the computed rate constants for the internal H atom ab-
straction from the C1, C2, C3 and C4 of the sugar ring by the thiyl radical.
The energy barrier for the H3 abstraction by 6-thiyl radical was calculated
to be about 11.1 kcal/mol.
EXPERIMENTAL
1H (400 MHz), 1H (600 MHz) and 13C (100.6 MHz) NMR spectra (δ, ppm; J, Hz) were re-
corded on a Bruker NMR spectrometer with solutions in CDCl3, unless otherwise specified.
Mass spectra were obtained by atmospheric pressure chemical ionization (APCI) technique.
Merck Kieselgel 60-F254 sheets were used for TLC, and products were detected by visualiza-
tion with Ce(SO4)2/(NH4)6Mo7O24·4H2O/H2SO4/H2O reagent or under 254 nm light. Merck
Kieselgel 60 (230–400 mesh) was used for column chromatography. Reagent grade chemicals
were used, and solvents were dried by reflux over distillation from calcium hydride under
argon atmosphere or by passing through the activated cartridges using solvent purifying sys-
tem.
Pulse radiolysis experiments were conducted using the facility at ETH Zürich as described
before33. Curve fitting was carried out with Excel software and its Solver plugin which al-
lowed for determination of yields and rate constants.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1233
3,6-O-Dibenzoyl-5-deoxy-1,2-O-isopropylidene-α-D-ribo-hexofuranose (3)
BzCl (508 µl, 2 mmol) was added to a solution of 2 20 (290 mg, 0.94 mmol) in pyridine
(5 ml) at ambient temperature and stirring was continued for 3 h. The volatiles were evapo-
rated and the residue was partitioned (CHCl3/H2O). The organic phase was washed (1
M
HCl/H2O, NaHCO3/H2O, brine) and dried (Na2SO4). Volatiles were evaporated and the resi-
due was column chromatographed (25% EtOAc/hexanes) to give 3 as a white solid (342 mg,
88%). 1H NMR: 1.32, 1.54 (2 × s, 2 × Me), 2.10–2.25 (m, 2, H5), 4.44–4.53 (m, 3, H4,6,6′),
4.76 (dd, J = 9.2, 4.8, 1, H3), 4.97 (t, J = 4.5, 1, H2), 5.89 (d, J = 3.9, 1, H1), 7.38–7.43 (m, 4,
Ar) 7.52–7.58 (m, 2, Ar), 7.98–8.02 (m, 4, Ar). 13C NMR: 21.77, 26.66, 26.76, 31.98, 67.04,
73.70, 76.50, 77.37, 104.27, 113.26, 128.08, 128.65, 129.33, 130.02, 130.10, 133.45, 133.65,
144.91, 165.98. MS m/z: 413 (100, MH+).
1,4-Anhydro-2,6-O-dibenzoyl-5-deoxy-D-ribo-hexofuranitol (4a) and
1,4-Anhydro-3,6-O-dibenzoyl-5-deoxy-D-ribo-hexofuranitol (4b)
Et3SiH (659 µl, 4.13 mmol) and BF3·Et2O (314 µl, 2.48 mmol) were added to a solution of 3
(340 mg, 0.59 mmol) in CH2Cl2 (5 ml) at 0 °C. The reaction mixture was stirred for 3 h at
ambient temperature, and then carefully partitioned between saturated NaHCO3/H2O
(15 ml) and CH2Cl2 (25 ml). The aqueous layer was washed with CH2Cl2 (3 × 10 ml), and
the combined organic phase was washed (brine), dried (Na2SO4) and column chromatog-
raphed (30% EtOAc/hexanes) to give partially separated mixtures of 4a/4b (~2:3; 148 mg,
52% total).
Compound 4a. 1H NMR: 2.10–2.00 (m, 1, H5), 2.28–2.20 (m, 1, H5′), 3.97 (td, J = 8.0, 4.3,
1, H4), 3.99 (dd, J = 10.8, 3.2, 1, H1), 4.14 (dd, J = 7.7, 5.6, 1, H3), 4.34 (dd, J = 10.8, 5.4, 1,
H1), 4.58–4.45 (m, 2, H6,6′), 5.46 (td, J = 5.4, 3.1, 1, H2), 7.47–7.42 (m, 4, Ar), 7.61–7.54 (m,
2, Ar), 8.07–8.04 (m, 4, Ar). 13C NMR: 32.57, 61.90, 70.79, 74.30, 75.91, 79.13, 128.51,
128.68, 129.53, 129.76, 129.92, 130.43, 133.07, 133.67, 166.54, 166.72. MS m/z: 357 (100,
MH+).
Compound 4b. 1H NMR: 2.14–2.00 (m, 1, H5), 2.24–2.16 (m, 1, H5′), 3.81 (dd, J = 9.8, 5.0,
1, H1), 4.24 (dd, J = 9.8, 5.7, 1, H1′), 4.28 (ddd, J = 8.1, 6.4, 4.5, 1, H4), 4.56–4.44 (m, 2,
H6,6′), 4.62 (“q”, J = 5.4, 1, H2), 5.03 (t, J = 5.9, 1, H3), 7.39–7.47 (m, 4, Ar), 7.61–7.52 (m,
2, Ar), 8.07–7.99 (m, 4, Ar). 13C NMR: 32.71, 61.71, 70.43, 72.73, 77.75, 78.11, 128.47,
128.67, 129.31, 129.71, 129.91, 130.28, 133.05, 133.70, 166.33, 166.59. MS m/z: 357 (100,
MH+).
1,4-Anhydro-5-deoxy-D-ribo-hexofuranitol (5)
A solution of 4a/4b (142 mg, 0.4 mmol) in NH3/MeOH was stirred overnight at ambient
temperature in a sealed flask. Volatiles were evaporated, and the residue was partitioned be-
tween H2O/Et2O. The aqueous layer was evaporated to give 5 (53 mg, 86%). 1H NMR (D2O):
1.71–1.80 (m, 1, H5), 1.86–1.94 (m, 1, H5′), 3.65–3.76 (m, 2, H6,6′), 3.73 (dd, J = 10.5, 2.8,
1, H1), 3.84 (td, J = 8.2, 4.1, 1, H4), 3.91 (dd, J = 7.4, 4.9, 1, H3), 4.08 (dd, J = 10.2, 4.7, 1,
H1′), 4.26 (td, J = 4.7, 2.9, 1, H2). 13C NMR: 35.04, 58.66, 70.66, 71.93, 75.50, 78.45. MS
m/z: 149 (100, MH+).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1234
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
1,4-Anhydro-5-deoxy-6-O-tosyl-D-ribo-hexofuranitol (6)
TsCl (58 mg, 0.3 mmol) was added to a solution of 5 (30 mg, 0.2 mmol) in pyridine (3 ml)
at ambient temperature and stirring was continued for 12 h. The volatiles were evaporated
and the residue was partitioned (CHCl3/H2O). The organic phase was washed (1 M HCl/H2O,
NaHCO3/H2O, brine) and dried (Na2SO4). Volatiles were evaporated and the residue was col-
umn chromatographed (80% EtOAc/hexanes) to give 6 as a syrup (37 mg, 61%). 1H NMR:
1.81–1.90 (m, 1, H5), 2.04–2.12 (m, 1, H5′), 2.46 (s, 3, Me), 3.69 (dd, J = 10.2, 3.3, 1, H1′),
3.73 (dd, J = 8.0, 4.2, 1, H3), 3.79 (t, J = 7.1, 1, H4), 4.05 (dd, J = 10.2, 5.2, 1, H1), 4.13–4.24
(m, 3, H2,6,6′), 7.36 (d, J = 8.3, 2, Ar), 7.80 (d, J = 8.1, 2, Ar). 13C NMR: 21.79, 32.74, 67.61,
70.97, 73.16, 75.85, 78.59, 128.10, 130.03, 133.14, 133.70, 145.01. MS m/z: 303 (100, MH+).
6-S-Acetyl-1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol (7)
AcSK (21 mg, 0.18 mmol) was added to a stirred solution of 6 (35 mg, 0.12 mmol) in a dried
DMF (4 ml) at ambient temperature. After 2 h, volatiles were evaporated and the residue was
column chromatographed (5% MeOH/CHCl3) to give 7 (19 mg, 72%). 1H NMR: 1.79 (dddd,
J = 13.0, 8.3, 8.1, 6.8, 1, H5), 1.91 (dddd, J = 13.0, 8.5, 6.5, 5.5 1, H5′), 2.32 (s, 3, Me), 2.92
(ddd, J = 13.7, 8.3, 6.8, 1, H6), 3.01 (ddd, J = 13.7, 8.5, 6.1, 1, H6′), 3.21 (br, 1, OH), 3.32
(br, 1, OH), 3.67–3.73 (m, 2, H1,4), 3.80 (t, J = 6.0, 1, H3), 4.08 (dd, J = 10.6, 5.2, 1, H1),
4.22 (dd, J = 9.0, 5.1, 1, H2). 13C NMR: 25.67, 30.74, 33.44, 71.11, 72.92, 75.78, 81.07,
196.69. MS m/z: 207 (100, MH+).
Bis(1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol) Disulfide (8)
Method A. Solution of 7 (18 mg, 0.08 mmol) in NH3/MeOH was stirred overnight at ambi-
ent temperature in a sealed flask. Volatiles were evaporated and the residue was partitioned
between H2O/Et2O. The aqueous layer was evaporated to give 8 (11 mg, 67%). 1H NMR
(D2O): 1.84–1.98 (m, 1, H5), 2.07–2.16 (m, 1, H5′), 2.78–2.94 (m, 2, H6,6′), 3.75 (dd, J =
10.2, 2.9, 1, H1), 3.90 (td, J = 7.9, 4.0, 1, H4), 3.97 (dd, J = 7.3, 4.8, 1, H3), 4.10 (dd, J =
10.2, 4.7, 1, H1′), 4.29 (td, J = 4.7, 3.0, 1, H2). 13C NMR: 32.13 (C5), 34.16 (C6), 70.79 (C2),
71.92 (C1), 75.25 (C3), 79.74 (C4). MS m/z: 327 (100, MH+). HRMS (CI) m/z: calculated for
C
12H23O6S2 [M + H]+ 327.0936, found 327.0941.
Method B. A solution of 11a/11b (98 mg, 0.31 mmol) in NH3/MeOH (4 ml) was stirred
overnight at ambient temperature in a sealed flask. Volatiles were evaporated and the resi-
due was partitioned (H2O/Et2O). The aqueous layer was evaporated to give 8 (48 mg, 96%)
with spectroscopic data as in method A.
3-O-Benzoyl-5-deoxy-1,2-O-isopropylidene-6-O-tosyl-α-D-ribo-hexofuranose (9)
TsCl (441 mg, 2.31 mmol) was added to a solution of 2 20 (283 mg, 0.96 mmol) in pyridine
(3 ml) at ambient temperature and stirring was continued for 2 h. The volatiles were evapo-
rated and the residue was partitioned (CHCl3/H2O). The organic phase was washed (1
M
HCl/H2O, NaHCO3/H2O, brine) and dried (Na2SO4). Volatiles were evaporated and the resi-
due was column chromatographed (40% EtOAc/hexanes) to give 9 as a syrup (306 mg, 72%).
1H NMR: 1.30, 1.52 (2 × s, 2 × Me), 1.88–1.97 (m, 1, H5), 2.09–2.31 (m, 1, H5′), 2.42 (s, 3,
Me), 4.09–4.26 (m, 2, H6,6′), 4.31 (td, J = 8.9, 3.6, 1, H4), 4.63 (dd, J = 9.2, 4.8, 1, H3), 4.89
(t, J = 4.4, 1, H2), 5.79 (d, J = 3.8, 1, H1), 7.30 (d, J = 8.0, 2, Ar), 7.48 (t, J = 7.5, 2, Ar), 7.61
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1235
(tt, J = 7.4, 1.3, 1, Ar), 7.78 (d, J = 8.2, 2, Ar), 8.04–8.06 (m, 2, Ar). 13C NMR: 21.77, 26.66,
26.76, 31.98, 67.04, 73.70, 76.50, 77.37, 104.27, 113.26, 128.08, 128.65, 129.33, 130.02,
130.10, 133.45, 133.65, 144.91, 165.98. MS m/z: 463 (100, MH+).
1,4-Anhydro-2-O-benzoyl-5-deoxy-6-O-tosyl-D-ribo-hexofuranitol (10a) and
1,4-Anhydro-3-O-benzoyl-5-deoxy-6-O-tosyl-D-ribo-hexofuranitol (10b)
Et3SiH (477 µl, 2.90 mmol) and BF3·Et2O (227 µl, 1.79 mmol) were added to a solution of 47
(275 mg, 0.595 mmol) in CH2Cl2 (5 ml) at 0 °C. The solution was stirred for 3 h at ambient
temperature, and then carefully partitioned between saturated NaHCO3/H2O (15 ml) and
CH2Cl2 (30 ml). The aqueous layer was washed with CH2Cl2 (3 × 10 ml), and the combined
organic phase was washed (brine), dried (Na2SO4) and column chromatographed (50%
EtOAc/hexanes) to give partially separated mixtures of 10a/10b (~2:3; 68 mg, 28% total).
Compound 10a. 1H NMR: 1.83–1.91 (m, 1, H5), 2.07–2.15 (m, 1, H5′), 3.79 (td, J = 8.3, 4.0,
1, H4), 3.87 (dd, J = 10.9, 2.9, 1, H1), 3.97–4.02 (m,1, H3), 4.15–4.25 (m, 3, H1′,6,6′), 5.37
(td, J = 5.4, 2.9, 1, H2), 7.30–7.32 (m, 2, Ar), 7.43–7.47 (m, 2, Ar) 7.57–7.60 (m, 1, Ar),
7.78–7.80 (m, 2, Ar), 8.02–8.04 (m, 2, Ar). 13C NMR: 21.74, 32.63, 67.16, 70.32, 72.67,
75.75, 77.78, 128.10, 128.74, 129.24, 129.97, 133.13, 133.81, 144.92, 166.23. MS m/z: 407
(100, MH+).
Compound 10b. 1H NMR: 1.84–1.93 (m, 1, H5), 2.03–2.15 (m, 1, H5′), 3.71 (dd, J = 9.8,
4.9, 1, H1), 4.09–4.24 (m, 4, H1,2,6,6′), 4.53 (dt, J = 10.1, 4.9, 1, H4), 4.89 (t, J = 6.0, 1, H3),
7.30–7.32 (m, 2, Ar), 7.45–7.49 (m, 2, Ar), 7.58–7.63 (m, 1, Ar) 7.75–7.81 (m, 2, Ar),
8.03–8.05 (m, 2, Ar). 13C NMR: 21.74, 32.88, 67.43, 70.92, 74.09, 76.70, 77.92, 128.07,
128.71, 129.90, 129.46, 133.18, 133.73, 144.92, 166.44. MS m/z: 407 (100, MH+).
6-S-Acetyl-1,4-anhydro-2-O-benzoyl-5-deoxy-6-thio-D-ribo-hexofuranitol (11a) and
6-S-Acetyl-1,4-anhydro-3-O-benzoyl-5-deoxy-6-thio-D-ribo-hexofuranitol (11b)
AcSK (20.9 mg, 0.18 mmol) was added to a stirred solution of compound 10a/10b (68 mg,
0.17 mmol) in a dried DMF (6 ml) at ambient temperature. After 2 h, volatiles were evapo-
rated and the residue was partitioned between H2O/CH2Cl2. The organic phase was washed
(NaHCO3/H2O and brine), dried (Na2SO4) and column chromatographed (1% MeOH/CHCl3)
to give partially separated mixtures of 11a/11b (2:3; 37 mg, 72% total).
Compound 11a. 1H NMR: 1.82–1.92 (m, 1, H5), 1.98–2.06 (m, 1, H5′), 2.34 (s, 3, Me),
2.96–3.12 (m, 2, H6,6′), 3.83 (td, J = 7.9, 4.4, 1, H4), 3.97 (dd, J = 10.8, 3.2, 1, H1′), 4.07 (dd,
J = 7.5, 6.3, 1, H3), 4.31 (dd, J = 10.8, 5.3, 1, H1), 5.43 (td, J = 5.5, 3.2, 1, H2), 7.45–7.49 (m,
1, Ar), 7.58–7.62 (m, 2, Ar), 8.05–8.08 (m, 2, Ar). 13C NMR: 25.66, 30.76, 33.39, 70.76,
74.36, 75.75, 80.78, 128.71, 129.57, 129.93, 133.70, 166.51, 195.93. MS m/z: 311 (100,
MH+).
Compound 11b. 1H NMR: 1.82–1.92 (m, 1, H5), 1.98–2.06 (m, 1, H5′), 2.34 (s, 3, Me),
2.94–3.06 (m, 2, H6,6′), 3.78 (dd, J = 14.8, 5.1, 1, H1), 4.14 (dt, J = 8.4, 5.1, 1, H4), 4.21 (dd,
J = 9.7, 5.7, 1, H1′), 4.58 (td, J = 5.3, 10.6, 1, H2), 4.97 (t, J = 5.8, 1, H3), 7.46–7.50 (m, 1,
Ar), 7.59–7.63 (m, 2, Ar), 8.06–8.08 (m, 2, Ar). 13C NMR: 25.57, 30.73, 33.55, 70.61, 72.60,
77.36, 77.88, 79.56, 128.74, 129.38, 129.97, 133.77, 166.30, 195.64. MS m/z: 311 (100,
MH+).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1236
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
1,4-Anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol (1)
Ph3P (16 mg, 0.06 mmol) and water (40 µl) were added to a stirred solution of disulfide 8
(9.6 mg, 0.03 mmol) in MeOH (3 ml). After 72 h, the volatiles were evaporated, and the resi-
due was dissolved in 3 ml of deoxygenated water and quickly extracted with dichloro-
methane. The aqueous layer was evaporated to give 1 (6.6 mg, 69%). 1H NMR (D2O):
1.82–1.97 (m, 2, H5,5′), 2.69–2.56 (m, 2, H6,6′), 3.73 (dd, J = 10.2, 2.8, 1, H1), 3.86–3.93
(m, 2, H3,4), 4.07 (dd, J = 10.2, 4.6, 1, H1′), 4.26 (dd, J = 4.5, 3.0, 1, H2). 13C NMR: 20.21,
36.71, 70.77, 71.88, 75.21, 79.60. HRMS (CI) m/z: calculated for C6H13O3S [M + H]+
165.0585, found 165.0592.
Chemical Biomimetic Studies with 6-Thiohexafuranitol 1
The 2,2′-azobis-2-methylpropanimidamide dihydrochloride (AAPH; 49 mg, 0.18 mmol) dis-
solved in deoxygenated D2O (0.5 ml) was added under argon atmosphere to a solution of 1
(5 mg, 0.03 mmol) in deoxygenated D2O (0.5 ml) in an NMR tube at ambient temperature.
The solution was flushed with argon for 15 min, and then the NMR tube was tightly sealed
and heated at 55 °C, and the reaction progress was followed by 1H NMR. After 17 h, about
80% of AAPH decomposed as judged by the disappearance of a singlet at 1.53 ppm in
1H NMR. After 72 h, the reaction mixture was concentrated to the 50% of the volume and
was stirred in the open air for 48 h. Volatiles were evaporated and the residue was column
chromatographed (3 → 8% MeOH/CHCl3) to give a product with spectroscopic properties
identical to disulfide 8.
Bu3SnD (32 µl, 0.12 mmol) and AIBN (~1 mg) were added to a suspension of 1 (6 mg,
0.03 mmol) in deoxygenated benzene (0.5 ml) and N,N-dimethylacetamide (0.3 ml). The re-
action mixture was deoxygenated (Ar, 20 min) and refluxed for 5 h. Volatiles were evapo-
rated and the residue was dissolved in H2O. The aqueous solution was washed (CH2Cl2) and
evaporated. The residue was column chromatographed (1 → 8% MeOH/CHCl3), and the ap-
propriate fractions were analyzed by 1H NMR and MS but no incorporation of deuterium
was detected for the isolated thiol 1 and disulfide 8.
Pulse Radiolysis Experiments with 8
Aqueous solutions of 900 µM of thiosugar disulfide 8 were freshly prepared in 1
M
tert-butanol solution at pH 12 (10 mM KOH) and pH 3 (0.5 mM H2SO4). The solutions were
degassed in Schlenk tubes by evacuation to boiling and subsequently filled with Ar twice.
The solutions were then anaerobically transferred to a gas-tight syringe (Hamilton, Bonaduz,
Switzerland) and then with a syringe pump transferred batchwise through standard HPLC
tubing to a quartz flow cell with 1 cm optical pathlength. Irradiations were always carried
out on fresh samples.
This investigation was supported by grants from the NIGMS/NCI (SC1CA138176). T. D. is grateful
to FIU University Graduate School for her Doctoral Evidence Acquisition Fellowship.
REFERENCES
1. Stubbe J., van der Donk W. A.: Chem. Biol. 1995, 2, 793.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Modeling of the Ribonucleotide Reductases Substrate Reaction
1237
2. Cerqueira N. M. F. S. A., Fernandes P. A., Ramos M. J.: Recent Pat. Anti-Cancer Drug
Discov. 2007, 2, 11.
3. Mao S. S., Holler T. P., Yu G. X., Bollinger J. M., Jr., Booker S., Johnston M. I., Stubbe J.:
Biochemistry 1992, 31, 9733.
4. Mao S. S., Yu G. X., Chalfoun D., Stubbe J.: Biochemistry 1992, 31, 9752.
5. Akhlaq M. S., Schuchmann H. P., Von Sonntag C.: Int. J. Radiat. Biol. 1987, 51, 91.
6. Schöneich C., Asmus K.-D., Bonifacic M.: J. Chem. Soc., Faraday Trans. 1995, 91, 1923.
7. Pogocki D., Schoneich C.: Free Radical Biol. Med. 2001, 31, 98.
8. Lenz R., Giese B.: J. Am. Chem. Soc. 1997, 119, 2784.
9. Robins M. J., Guo Z., Wnuk S. F.: J. Am. Chem. Soc. 1997, 119, 3637.
10. Robins M. J., Guo Z., Samano M. C., Wnuk S. F.: J. Am. Chem. Soc. 1999, 121, 1425.
11. van der Donk W. A., Yu G., Perez L., Sanchez R. J., Stubbe J., Samano V., Robins M. J.:
Biochemistry 1998, 37, 6419.
12. van der Donk W. A., Yu G., Silva D. J., Stubbe J., McCarthy J. R., Jarvi E. T., Matthews
D. P., Resvick R. J., Wagner E.: Biochemistry 1996, 35, 8381.
13. Gerfen G. J., van der Donk W. A., Yu G., McCarthy J. R., Jarvi E. T., Matthews D. P.,
Farrar C., Griffin R. G., Stubbe J.: J. Am. Chem. Soc. 1998, 120, 3823.
14. van der Donk W. A., Gerfen G. J., Stubbe J.: J. Am. Chem. Soc. 1998, 120, 4252.
15. Persson A. L., Sahlin M., Sjoberg B.-M.: J. Biol. Chem. 1998, 273, 31016.
16. Lawrence C. C., Bennati M., Obias H. V., Bar G., Griffin R. G., Stubbe J.: Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 8979.
17. Zipse H., Artin E., Wnuk S., Lohman G. J. S., Martino D., Griffin R. G., Kacprzak S.,
Kaupp M., Hoffman B., Bennati M., Stubbe J., Lees N.: J. Am. Chem. Soc. 2009, 131, 200.
18. Robins M. J., Ewing G. J.: J. Am. Chem. Soc. 1999, 121, 5823.
19. Guo Z., Samano M. C., Krzykawski J. W., Wnuk S. F., Ewing G. J., Robins M. J.:
Tetrahedron 1999, 55, 5705.
20. Wnuk S. F., Valdez C. A., Khan J., Moutinho P., Robins M. J., Yang X., Borchardt R. T.,
Balzarini J., De Clercq E.: J. Med. Chem. 2000, 43, 1180.
21. Lewis M. D., Cha J. K., Kishi Y.: J. Am. Chem. Soc. 1982, 104, 4976.
22. Bennek J. A., Gray G. R.: J. Org. Chem. 1987, 52, 892.
23. Ewing G. J., Robins M.: J. Org. Lett. 1999, 1, 635.
24. Nauser T., Schoneich C.: J. Am. Chem. Soc. 2003, 125, 2042.
25. Becke A. D.: J. Chem. Phys. 1993, 98, 5648.
26. Lee C., Yang W., Parr R. G.: Phys. Rev. 1988, B37, 785.
27. Frisch M. J., Trucks G. W., Schlegel H. B., Scuseria G. E., Robb M. A., Cheeseman J. R.,
Zakrzewski V. G., Montgomery J. A., Stratmann R. E., Burant J. C., Dapprich S., Millam J.
M., Daniels R. E., Kudin K. N., Strain M. C., Farkas O., Tomasi J., Barone V., Cossi M.,
Cammi R., Mennucci B., Pomelli C., Adamo C., Clifford S., Ochterski J., Petersson G. A.,
Ayala P. Y., Cui Q., Morokuma K., Salvador P., Dannenberg J. J., Malick D. K., Rabuck A.
D., Raghavachari K., Foresman J. B., Cioslowski J., Ortiz J. V., Baboul A. G., Stefanov B.
B., Liu G., Liashenko A., Piskorz P., Komaromi I., Gomperts R., Martin R. L., Fox D. J.,
Keith T., Al-Laham M. A., Peng C. Y., Nanayakkara A., Challacombe M., Gill P. M. W.,
Johnson B., Chen W., Wong M. W., Andres J. L., Gonzalez C., M. Head-Gordon M.,
Replogle E. S., Pople J. A.: Gaussian 98, Revision A.11. Gaussian, Inc., Pittsburgh (PA)
2001.
28. Siegbahn P. E. M.: J. Am. Chem. Soc. 1998, 120, 8417.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

1238
Wnuk, Penjarla, Dang, Mebel, Nauser, Schöneich:
29. Cerqueira N. M. F. S. A., Fernandes P. A., Eriksson L. A., Ramos M. J.: J. Comput. Chem.
2004, 25, 2031.
30. Bennati M., Lendzian F., Schmittel M., Zipse H.: Biol. Chem. 2005, 386, 1007.
31. Purvis G. D., Bartlett R. J.: J. Chem. Phys. 1982, 76, 1910.
32. Steenken S., Davies M. J., Gilbert B. C.: J. Chem. Soc., Perkin Trans. 2 1986, 1003.
33. Nauser T., Casi G., Koppenol W. H., Schöneich C.: J. Phys. Chem. B 2008, 112, 15034.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 10, pp. 1223–1238

Copyright of Collection of Czechoslovak Chemical Communications is the property of Institute of Organic
Chemistry & Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. and its content may not be
copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.