Cleavage of Phosphodiesters and of DNA by a Bis(guanidinium)naphthol Acting as a Metal-Free Anion Receptor

Article abstract of DOI:10.1002/cbic.201100022

Phosphoric acid diesters form anions at neutral pH. As a result of charge repulsion they are notoriously resistant to hydrolysis. Nucleophilic attack, however, can be promoted by different types of electrophilic catalysts that bind to the anions and reduce their negative charge density. Although in most cases phosphodiester-cleaving enzymes and synthetic catalysts rely on Lewis acidic metal ions, some exploit the guanidinium residues of arginine as metal-free electrophiles. Here we report that a combination of two guanidines and a hydroxy group yields highly reactive receptor molecules that can attack a broad range of phosphodiester substrates by nucleophilic displacement at phosphorus in a single-turnover mode. Some stable O-phosphates were isolated and characterized further by NMR spectroscopy. The bis(guanidinium)naphthols also cleave plasmid DNA, presumably by a transphosphorylation mechanism.

Full text of DOI:10.1002/cbic.201100022

DOI: 10.1002/cbic.201100022
Cleavage of Phosphodiesters and of DNA by a
Bis(guanidinium)naphthol Acting as a Metal-Free Anion
Receptor
Stefan Ullrich, Zarghun Nazir, Arne Bꢀsing, Ute Scheffer, Daniela Wirth, Jan W. Bats,
Gerd Dꢀrner, and Michael W. Gçbel*[a]
Phosphoric acid diesters form anions at neutral pH. As a result
of charge repulsion they are notoriously resistant to hydrolysis.
Nucleophilic attack, however, can be promoted by different
types of electrophilic catalysts that bind to the anions and
reduce their negative charge density. Although in most cases
phosphodiester-cleaving enzymes and synthetic catalysts rely
on Lewis acidic metal ions, some exploit the guanidinium resi-
dues of arginine as metal-free electrophiles. Here we report
that a combination of two guanidines and a hydroxy group
yields highly reactive receptor molecules that can attack a
broad range of phosphodiester substrates by nucleophilic dis-
placement at phosphorus in a single-turnover mode. Some
stable O-phosphates were isolated and characterized further
by NMR spectroscopy. The bis(guanidinium)naphthols also
cleave plasmid DNA, presumably by a transphosphorylation
mechanism.
Introduction
The remarkable kinetic stability of phosphoric acid diesters[1]
presents a major challenge for the design of biomimetic artifi-
cial nucleases.[2–6] Most enzymes involved in phosphoryl trans-
fer reactions[7] (and many synthetic analogues) rely on Lewis
acidic metal ions. These can simultaneously bind and polarize
substrates, together with the attacking nucleophiles, thus en-
hancing their intrinsic reactivity. Some phosphoryl transfer en-
zymes, on the other hand, do not require metal ions. Human
DNA topoisomerase I,[8] as an example, activates the phosphate
groups of DNA by forming ion pairs with two arginine resi-
dues. Reversible strand cleavage is then achieved by nucleo-
philic attack by a tyrosine hydroxy group. The bis(guanidinium)
motif, also known from staphylococcal nuclease,[9] has guided
chemists to the design of several synthetic anion receptors.[10]
Some of them induce large rate effects on phosphoryl transfer
reactions.[11] As an example, the bis(guanidinium) alcohol 1
forms ion pairs with catechol cyclic phosphate 2 in DMF (Ka =
2900mÀ1 at 308C; see Scheme 1). In the presence of 0.25m di-
isopropylethylamine, a fast and reversible reaction occurs, thus
leading to 3. Compared to simple uncharged alcohols, the
guanidinium ions of 1 accelerate the phosphorylation by six to
seven orders of magnitude.[11a] Compound 1, however, fails to
cleave less activated substrates such as phosphodiesters 5–6.
The reaction is sluggish even with substrate 4, and ring nitro-
gens (instead of oxygens) are the preferred phosphorylation
sites. It has been observed previously that the impressive rate
effects in “enzyme models” are often restricted to artificial sub-
strates that exceed, by far, the reactivity of their natural coun-
terparts.[12] To broaden the application of phosphodiester
cleavage by bis(guanidinium) alcohols, our strategy was to op-
timize the structure of the nucleophilic side chains.
Scheme 1. Top: Guanidinium alcohol 1 is phosphorylated in a supramolec-
ular process involving ion-pair complexes [1·2]. Bottom: Phosphodiester
substrates used in this study (TMG+: Tetramethylguanidinium; anion: pic-
rate).
[a] Dr. S. Ullrich, Dr. Z. Nazir, Dr. A. Bꢀsing, Dr. U. Scheffer, D. Wirth,
Dr. J. W. Bats, Dr. G. Dꢀrner, Prof. Dr. M. W. Gçbel
Institut fꢀr Organische Chemie und Chemische Biologie
Johann Wolfgang Goethe-Universitꢁt Frankfurt
Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany)
Fax: (+49)69-798-29464
E-mail: m.goebel@chemie.uni-frankfurt.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100022.
ChemBioChem 2011, 12, 1223 – 1229
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1223

M. W. Gçbel et al.
Results and Discussion
Among other analogues, guanidinium alcohols 7–10 were syn-
thesized and tested for their ability to cleave substrates 4 and
5 (BDNPP and BNPP, Scheme 2). To summarize a large number
Scheme 3. a) 1: CuCN, DMF, Py, D, 8 h, 73%; 2: HNO2, KI, 81%. b) 1:
Na2Cr2O7, H2O, H2SO4, 08C, 3 h, RT, 1 h, 70%; 2: MeOH, H2SO4, D, 8 h, 94%.
c) 1,8-dihydroxynaphthalene, NaH, DMF, 608C, 30 min, 70%. d) H2, Pd/C,
PtO2, Boc2O, MeOH, 45 bar, 558C, 24 h, 69%. e) 1: AcCl, MeOH; 2: 18, MeOH,
Et3N, RT, 1 h; 3: picric acid, 41%. f) 1,8-dihydroxynaphthalene, NaH, DMF,
908C, 6 h, 62%. g) H2, Pd/C, PtO2, Boc2O, MeOH, 40 bar, 558C, 42 h, 73%.
h) AcCl, MeOH. i) 1: 18, MeOH, H2O, Et3N, RT, 12 h; 2: picric acid, 70%.
Scheme 2. Structures of guanidinium alcohols 7–12 (counter ion: picrate).
of experiments, out of 7–10 the slowest reaction was observed
with glycol ether 9. Phenanthrene 10 can be regarded as a
rigid, cyclic version of 9. Both molecules have side chains of
identical lengths, but the rigid analogue was much more effec-
tive in terms of rates. However, reaction at oxygen atoms was
still accompanied by N-phosphorylation. Catechol 7 comes
close to 10 in its reactivity in DMF, but declines in DMF-water
mixtures, and also shows high levels of N-phosphorylation.
Compared to 7 and 9, benzylic alcohol 8 has a side chain of in-
termediate length, and is also much less acidic. In spite of the
distinct pKa difference, 8 did not react much slower than phen-
anthrene 10, and it showed clean O-phosphorylation. The
overall geometry of 8 therefore looked promising, and subse-
quent research was focused on the rigid naphthol analogues
11 and 12.
The synthesis of compound 12 is depicted in Scheme 3.
Starting from dibromotoluidine 13,[13] the cyano and iodo sub-
stituents of intermediate 14 were introduced in a Rosenmund-
von-Braun reaction,[14] followed by Sandmeyer replacement.
Oxidation and esterification then led to 15, a compound acti-
vated for nucleophilic displacement of iodide by the naph-
thol[15] moiety. Boc-protected diamine 17 was obtained by cat-
alytic hydrogenation of the nitriles. Removal of Boc then al-
lowed the diamine to be converted into the target com-
pound 12 by reagent 18.[11a] Analogous methods transformed
2-iodo-1,3-dicyanobenzene 19[16] via 20 into diamines 21a,
21b, and finally into target compound 11.
The crystal structure of the picrate salt of 21b (Figure 1)
shows how steric constraints limit the conformational space
accessible to this molecule, thus forcing the amino and hy-
droxyl residues into the same space region.[17] Hydrogen bonds
between the protonated nitrogens and the ether oxygen
might further stabilize this arrangement. Analogous factors are
expected to govern the conformation of guanidinium alcohols
11 and 12 as well.
The phosphorylation of 11 with the most reactive substrate,
4, was studied first by 31P NMR (250 mm diisopropylethylamine
in [D7]DMF, 308C). Apart from the substrate peak
(À13.42 ppm), a new signal at À11.40 ppm appeared; this was
assigned to the phosphorylated naphthol 22 (Scheme 4).
When the experiment was repeated with substrate
5
(À11.83 ppm),
a
single product peak was formed at
À11.29 ppm. 31P NMR shifts unfortunately do not allow a clear
distinction between O-phosphorylated and N-phosphorylated
products. We therefore isolated 24 by HPLC. The 1H NMR
signal of all eight imidazoline CÀH groups appeared as a broad
singlet at 3.36 ppm, thus ruling out N-phosphorylated asym-
metric structures.[18] Direct evidence for the structure of 24
comes from 2D NMR as shown in Figure 2.
1224
www.chembiochem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1223 – 1229

Cleavage of Phosphodiesters and DNA
Pseudo-first-order rates of phosphorylation were then deter-
mined by HPLC (10 mm 11 as picrate salt, 30 mm substrate,
250 mm diisopropylethylamine in DMF, 308C). At the chosen
concentrations, almost complete complexation of guanidinium
naphthols by phosphate substrates can be expected.[11a] For
substrate 4, the value of kobs (3.6ꢂ10À2 minÀ1) corresponds to a
half life of only 19 min. Bis-4-nitrophenyl phosphate 5 showed
an initial rate of kobs =2.2ꢂ10À4 minÀ1 (t = =53 h). The same
1
2
rates were observed for the reaction of substrate 5 with com-
pound 12a (chloride salt) to yield 26. Experimental errors of
rate determinations were Æ10%. At longer reaction times
some deviation from first order kinetics occur in all cases, pre-
sumably due to competitive binding of nitrophenolate ions.
Even the least reactive substrate, 6, slowly phosphorylated 11
to form 25 (kobs =1.4ꢂ10À5 minÀ1; t = =830 h).
1
2
The stability of ion pair complexes is distinctly reduced by
the addition of polar and protic solvents. Nevertheless, when
the reaction of 11 with substrate 4 was tested in DMF/aqueous
TRIS-HCl buffer (2:1; pH 10.7) an initial rate of kobs =2.8ꢂ
10À3 minÀ1 was observed. In the presence of water, 22 was
slowly hydrolyzed to form phosphomonoester 23 and dephos-
phorylated 11. Compared to the value found in DMF/diisopro-
pylethylamine, the reaction in the DMF/water mixture was re-
duced just 13-fold.
Figure 1. Crystal structure of the picrate salt of diamine 21b. One of two dis-
ordered conformations is shown (see also the Supporting Information).[17]
We therefore tested the ability of guanidinium alcohols 11
and 12 to cleave supercoiled pUC19 plasmid DNA. Each cyclic
strand consists of 2686 nucleotides. If at least one of the nucle-
otides gets disrupted, the supercoiled DNA (form I) uncoils,
and the nicked but still cyclic form II results. These forms can
be separated by electrophoresis on agarose gels. The assay,
therefore, produces an overlay of all processes that occurred at
the 5372 individual nucleotides. In addition to superhelical
strain, this statistical factor makes plasmid DNA sensitive
against cleavage. Good preparations of pUC19 contain less
than 5% of form II. After 20 h not much additional degradation
was observed in TRIS or HEPES buffers at pH 7.0 or above.
However, while stable at pH 6.0 or 6.5 in TRIS, almost complete
conversion into form II and even linear DNA (form III) was ob-
served in acidic HEPES buffers (a cationic alcohol!). When incu-
bated for 20 h at 378C, 10 mm of either 11 or 12 converted
about 37% of the plasmid into form II (HEPES, pH 7.0). Linear
DNA was not observed. Significant effects above background
occurred at cleaver concentrations down to 1 mm. DNA cleav-
age, however, might result from different mechanisms. To ex-
clude general acid catalyzed depurination—potentially exerted
by the naphthol or guanidinium moieties—the interdepend-
ence of pH and cleavage yields was tested (Figure 3 and
Table 1).
Scheme 4. Reaction products observed in phosphorylation studies with gua-
nidinium naphthols 11 and 12.
Although significant variation occurred in each individual ex-
periment, the data clearly shows an increase of cleavage yield
with increasing pH, thus arguing against acid-induced depuri-
nation. Up to 2 mm of EDTA did not significantly alter reaction
rates, whereas a slight reduction in cleavage yield was seen at
5 and 10 mm. In this concentration range, the polyanionic
EDTA is expected to interfere with DNA binding of the cationic
naphthols 11 and 12.[19] Only minor gains in plasmid cleavage
resulted from the addition of 1–10 mm Mg2+ ions. The reac-
Figure 2. 31P,1H HMBC spectrum of 24 ([D3]acetonitrile+CDCl3 +[D1]TFA,
1
250 MHz). Crosspeaks from 31P to the H signals of 4-nitrophenyl (8.28 ppm)
and H(7) of the naphthol (7.85 ppm) but not to the CH2 signal of the guani-
dines at 3.52 ppm demonstrate the attachment of the phosphate ester to
the naphthol moiety.
ChemBioChem 2011, 12, 1223 – 1229
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1225

M. W. Gçbel et al.
solution was then slowly added to an ice-cooled solution of KI
(21.12 g, 127.3 mmol) in water (300 mL). The ice bath was then re-
moved, and the foamy suspension was stirred for 2 h at ambient
temperature. CH2Cl2 (100 mL) was added, followed by a saturated
Na2S2O3 solution (50 mL). The organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (2ꢂ50 mL). The combined
CH2Cl2 layer was dried over MgSO4, the solvent was evaporated,
and the crude product was purified by column chromatography
(200 g silica gel, CH2Cl2/hexane/EtOAc 30:30:1) to yield 14 as color-
less crystals (2.75 g, 81%). m.p. 200–2018C; 1H NMR (250 MHz,
[D6]DMSO): d=7.98 (s, 2H; 2Ar-H), 2.32 (s, 3H; Ar-CH3); 13C NMR
(60 MHz, [D6]DMSO): d=140.2, 138.7, 121.1, 118.7, 102.2, 19.7; IR
(KBr): n=3056, 2236, 1815, 1560, 1448, 1418, 1409, 1380, 1286,
1231, 1030, 886, 706, 678, 626, 565 cmÀ1; elemental analysis: calcd
(%) for C9H5N2I: C 40.33, H 1.88, N 10.45; found: C 40.53, H 2.08, N
10.45.
Figure 3. Cleavage of pUC19 plasmid DNA by guanidinium naphthol 12 as a
function of pH. Bottom: superhelical DNA (form I); top: nicked circular DNA
(form II). Lane 1 (from left to right): control, pH 7.0; lane 2: 5 mm 12, pH 7.0;
lane 3: control, pH 7.5; lane 4: 5 mm 12, pH 7.5; lane 5: control, pH 8.0;
lane 6: 5 mm 12, pH 8.0; lane 7: control, pH 8.5; lane 8: 5 mm 12, pH 8.5;
lane 9: control, pH 9.0; lane 10: 5 mm 12, pH 9.0 (22 nm DNA, HEPES buffer,
378C, 20 h; electrophoresis on 1% agarose gel, ethidium staining; inverted
grayscale).
Methyl 3,5-dicyano-4-iodobenzoate (15): Water (2.5 mL) was carefully
added to a solution of 14 (2.00 g, 7.46 mmol) in conc. H2SO4
(30 mL), and the resulting mixture cooled to 08C. Na2Cr2O7·2H2O
(5.55 g, 18.65 mmol) was dissolved in water (20 mL) and added
dropwise over 3 h to the cooled solution of 14. After complete
addition the cooling was removed, water (10 mL) added, and the
solution was stirred at ambient temperature for another 60 min.
The deep green mixture was poured slowly into ice–water (50 mL).
EtOAc (50 mL) was added, and the mixture was stirred vigorously
for 30 min. The organic layer was separated, and the aqueous layer
was extracted with EtOAc (4ꢂ30 mL). The combined organic layer
was washed twice with 20% H2SO4, the solvent was evaporated,
and the residue was purified by column chromatography (200 g
silica gel, CH2Cl2/MeOH 4:1) to yield 3,5-dicyano-4-iodobenzoic acid
14a as a colorless solid (1.55 g, 70%). m.p. 254–2558C; 1H NMR
(250 MHz, [D6]DMSO): d=14.05 (br, 1H; COOH), 8.43 (s, 2H; 2Ar-
H); 13C NMR (60 MHz, [D6]DMSO): d=164.1, 137.7, 132.4, 122.3,
118.2, 112.7; IR (KBr): n=3238, 3076, 2246, 1734, 1624, 1586, 1412,
1393, 1377, 1265, 1224, 1182, 1138, 1126, 1027, 936, 910, 777, 744,
708, 687, 646 cmÀ1; ESIÀ MS: m/z: calcd for C9H3IN2O2-H+: 296.9;
found 296.8 [MÀH]À; elemental analysis calcd (%) for C9H3IN2O2: C
36.27, H 1.01, N 9.40; found: C 36.65, H 0.83, N 9.67.
Table 1. Yields of plasmid cleavage as a function of pH (conditions as
given in Figure 3).
pH
7.0
7.5
8.0
8.5
9.0
exp.1 [%]
exp.2 [%]
exp.3 [%]
Ø[a] [%]
32.1
31.0
35.3
49.8
50.4
59.3
67.5
33.2
32.0
41.9
51.6
39.8
65.4
55.8
30.0
31.1
42.1
45.4
48.8
66.1
65.1
31.8
31.4
39.8
48.9
46.3
63.6
62.8
9.4[b]
10.2[b]
[a] Mean value of the three independent experiments. For details of
quantification see the Supporting Information. [b] Separate experiments
not shown in Figure 3.
tions of 11 and 12 with DNA, therefore, do not depend on
metal ions (data not shown).
The carboxylic acid 14a (2.00 g, 6.71 mmol) was dissolved in
MeOH (75 mL), conc H2SO4 (10 mL) was added and the mixture
heated to reflux for 8 h. The solution was cooled to ambient tem-
perature and carefully diluted with water (50 mL), then the pH was
adjusted to ~3 with saturated NaHCO3. The aqueous layer was ex-
tracted with EtOAc (3ꢂ30 mL), the organic layers were combined,
and the solvent was evaporated. The crude product was purified
by column chromatography (200 g silica gel, hexane/EtOAc 2:1) to
yield 15 as a colorless solid (1.98 g, 94%). m.p. 1898C; 1H NMR
(250 MHz, [D6]DMSO): d=8.45 (s, 2H; 2Ar-H), 3.90 (s, 3H; OCH3);
13C NMR (60 MHz, [D6]DMSO): d=163.3, 140.1, 136.8, 131.8, 116.3,
113.5, 53.0; IR (KBr): n=3058, 2950, 2236, 1718, 1588, 1439, 1394,
1313, 1233, 1132, 1030, 992, 951, 891, 772, 762, 712, 639 cmÀ1; ele-
mental analysis calcd (%) for C10H5IN2O2: C 38.49, H 1.61, N 8.98;
found: C 38.76, H 1.73, N 9.18.
Conclusions
Compounds 11 and 12 resulted from a long-term attempt to
“synthesize” chemical reactivity by a proper assembly of simple
functional groups. Even though the bis(guanidinium) naph-
thols cannot compete at present with advanced metal-based
phosphate cleavers, they exhibit remarkable rates, and even
degrade DNA. What is the mechanism of plasmid cleavage? Ex-
perimental evidence is in accord with nucleophilic substitution
at phosphorus atoms. However, we cannot yet exclude path-
ways that involve naphthoxy radicals. Further studies on re-
action mechanisms and sequence specific DNA cleavers are
planned.
Methyl 3,5-dicyano-4-(8-hydroxynaphthalen-1-yloxy)benzoate (16): A
solution of 1,8-dihydroxynaphthalene (0.77 g, 4.81 mmol) in anhy-
drous DMF (8 mL) was added dropwise to a suspension of NaH
(0.18 g of a 60% dispersion in mineral oil, 4.49 mmol) in anhydrous
DMF (10 mL) under argon. When the addition was complete, the
resulting yellow solution was stirred at ambient temperature for
30 min. Subsequently, a solution of 15 (1.00 g, 3.20 mmol) in anhy-
drous DMF (10 mL) was added, and the mixture was stirred for
30 min at 608C. The red solution was allowed to cool to room tem-
Experimental Section
Syntheses
2-Iodo-5-methylisophthalonitrile (14): 2-Amino-5-methylisophthaloni-
trile[14] (2.00 g, 12.72 mmol) was suspended in H2SO4 (40 mL of a
50% aqueous solution), cooled to À58C, and stirred for 60 min. A
solution of NaNO2 (1.31 g, 19.09 mmol) in water (5 mL) was added
slowly, and the mixture stirred for 30 min at 08C. The pale yellow
1226
www.chembiochem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1223 – 1229

Cleavage of Phosphodiesters and DNA
1
perature, poured into acidic water (50 mL of 2n HCl) and extracted
with EtOAc (2ꢂ20 mL). The combined organic layer was dried over
MgSO4 and concentrated in vacuo, and the crude product was
purified by column chromatography (65 g silica gel, hexane/EtOAc
3:1) to yield 16 as a slightly yellow solid (0.77 g, 70%). m.p. 2078C;
1H NMR (250 MHz, [D6]DMSO): d=10.06 (br, 1H; Ar-OH), 8.56 (s,
2H; phenyl-H), 7.86 (dd, J=0.8, 8.3 Hz, 1H; naphthyl-H), 7.48–7.33
(m, 4H; naphthyl-H), 6.84 (dd, J=0.9, 7.6 Hz, 1H; naphthyl-H), 3.88
(s, 3H; OCH3); 13C NMR (60 MHz, [D6]DMSO): d=163.5, 163.0,
152.2, 149.1, 140.0, 136.7, 127.4, 125.6, 124.3, 119.0, 118.0, 117.5,
113.5, 110.7, 103.3, 52.8; IR (KBr): n=3510, 3084, 2957, 2238, 1733,
1712, 1629, 1610, 1597, 1578, 1458, 1433, 1395, 1352, 1315, 1285,
1234, 1213, 1195, 1107, 1021, 987, 933, 903, 821, 760, 724,
614 cmÀ1; ESIÀ MS: m/z: calcd for C20H12N2O4-H+: 343.1; found
343.1 [MÀH]À; elemental analysis calcd (%) for C20H12N2O4: C 69.76,
H 3.51, N 8.14; found: C 69.53, H 3.62, N 7.95.
(21 mg, 41%). M.p. 189–1918C; H NMR (250 MHz, [D6]DMSO): d=
9.91 (s, 1H; OH), 8.75–8.67 (m, 2H; 2benzyl-NH), 8.50–8.00 (brs,
6H; 4dihydroimidazole-NH, 2phenyl-H), 7.54 (d, J=7.9 Hz, 1H;
naphthyl-H), 7.41 (m, 2H; naphthyl-H), 7.22 (t, J=8.0 Hz, 1H; naph-
thyl-H), 7.10–7.02 (m, 1H; naphthyl-H), 6.22 (dd, J=1.4, 7.6 Hz, 1H;
naphthyl-H), 4.44–4.25 (m, 4H; 2benzene-CH2), 3.92 (s, 3H;
COOCH3), 3.43 (s, 8H; 4dihydroimidazole-CH2); 13C NMR (60 MHz,
[D6]DMSO): d=165.3, 159.4, 153.7, 153.3, 137.0, 131.5, 130.0, 127.5,
127.1, 125.9, 122.6, 118.7, 115.0, 111.4, 107.3, 52.4, 42.3, 40.8; IR
(KBr): n=3411, 3158, 3053, 1723, 1670, 1603, 1578, 1490, 1456,
1436, 1386, 1355, 1317, 1289, 1222, 1197, 1070, 1025, 1004, 932,
902, 819, 758, 706, 632 cmÀ1; ESI+ MS: m/z: calcd for C26H28N6O4 +
H+: 489.2; found 489.3 [M+H]+, 245.0 [M+2H]2+
.
2-(8-Hydroxynaphthalen-1-yloxy)isophthalonitrile (20): NaH (0.25 g of
a 60% dispersion in mineral oil, 6.2 mmol) was suspended in dry
DMF (3 mL) under argon. A solution of 1,8-dihydroxynaphthalene
(1 g, 6.2 mmol) in dry DMF (8 mL) was added slowly, and the result-
ing mixture was stirred for an additional 30 min. A solution of 19[15]
(1.6 g, 6.2 mmol) in dry DMF (8 mL) was added dropwise, and the
mixture was heated (908C, 6 h). The solution was cooled to room
temperature and quenched by pouring into ice-cooled aqueous
HCl (30 mL, 1m). EtOAc (20 mL) was added, then the organic layer
was separated and dried over MgSO4. The solvent was removed in
vacuo, the crude product was purified by flash chromatography
(EtOAc/hexane 1:1) and recrystallized from EtOAc/hexane to yield
20 as a crystalline solid (1.1 g, 62%). m.p. 149–1518C; 1H NMR
(250 MHz, [D6]DMSO): d=9.96 (s, 1H; -OH), 8.16 (d, J=7.8 Hz, 2H;
2Ar-H), 7.78 (d, J=8.0 Hz, 1H; naphthyl-H), 7.44–7.34 (m, 4H;
4naphthyl-H), 7.11 (d, J=7.2 Hz, 1H; naphthyl-H), 6.88 (d, J=
7.2 Hz, 1H; Ar-H); 13C NMR (60 MHz, [D6]DMSO): d=160.2, 152.8,
150.6, 139.4, 136.9, 127.4, 126.3, 125.6, 123.7, 118.8, 117.3, 115.9,
114.3, 110.6, 104.0; IR (KBr): n=3854, 3839, 3676, 3650, 3506, 3494,
3069, 2490, 2240, 1978, 1930, 1858, 1755, 1734, 1629, 1610, 1573,
1514, 1451, 1394, 1301, 1247, 1217, 1194, 1157, 1111, 1081, 1022,
968, 932, 866, 854, 819, 800, 780, 754, 669, 628, 612, 570 cmÀ1; ESIÀ
MS: m/z: calcd for C18H10O2N2-H+: 285.1; found: 284,9 [MÀH]À; ele-
mental analysis calcd (%) for C18H10N2O2: C 75.52, H 3.52, N 9.79;
found: C 75.54, H 3.56, N 9.80.
Methyl 3,5-bis((tert-butoxycarbonylamino)methyl)-4-(8-hydroxynaph-
thalen-1-yloxy)benzoate (17): A solution of 16 (200 mg, 0.581 mmol)
and Boc2O (500 mg, 2.29 mmol) in anhydrous MeOH (10 mL) was
added to a suspension of Pd/C (73 mg, 10% on active charcoal)
and PtO2 (34 mg) in anhydrous MeOH (10 mL). The mixture was
stirred in a steel autoclave under H2 (45 bar) for 24 h at 558C. The
catalyst was removed by filtration through Celite, the solvent was
evaporated, and the crude product was purified by column chro-
matography (60 g silica gel, hexane/EtOAc 4:1) to yield 17 as color-
1
less solid (220 mg, 69%). m.p. 808C; H NMR (250 MHz, [D6]DMSO):
d=9.50 (s, 1H; OH), 7.89 (s, 2H; 2Ar-H), 7.51 (dd, J=0.8, 8.3 Hz,
1H; naphthyl-H), 7.42–7.35 (m, 4H; 2naphthyl-H, 2NH), 7.21 (t, J=
7.9 Hz, 1H; naphthyl-H), 6.94 (m, 1H; naphthyl-H), 6.22 (d, J=
7.5 Hz, 1H; naphthyl-H), 4.21 (dd, J=5.4/16.6 Hz, 2H; Ar-CH2), 3.93–
3.84 (m, 5H; COOCH3, Ar-CH2), 1.36 (s, 18H; 2OC(CH3)3); 13C NMR
(60 MHz, [D6]DMSO): d=165.6, 155.6, 153.8, 153.4, 152.3, 137.0,
133.4, 127.5, 126.5, 125.8, 122.3, 118.6, 114.7, 110.8, 106.8, 78.0,
52.1, 38.2, 28.0; IR (KBr): n=3467, 3373, 3058, 2977, 2931, 1721,
1634, 1606, 1581, 1514, 1456, 1437, 1394, 1367, 1314, 1298, 1249,
1221, 1169, 1117, 1078, 1049, 1026, 1001, 968, 941, 901, 862, 834,
818, 773, 756 cmÀ1; ESI+ MS: m/z: calcd for C30H36N2O8+H+: 553.3;
found 553.4 [M+H]+; elemental analysis calcd (%) for C30H36N2O8: C
65.20, H 6.57, N 5.07; found: C 65.28, H 6.78, N 4.87.
tert-Butyl (2-(8-hydroxynaphthalen-1-yloxy)-1,3-phenylene)bis(methyl-
ene)dicarbamate (21a): Pd/C (0.28 g, 10% on active charcoal) and
PtO2 (0.09 g) were suspended in dry MeOH (10 mL). A solution of
di-tert-butyl dicarbonate (0.64 g, 2.9 mmol) and 20 (0.4 g,
1.4 mmol) in dry MeOH (20 mL) was added, and the mixture was
stirred under hydrogen (40 bar) at 558C in a steel autoclave for
42 h. H2 was replaced with argon, the catalyst was filtered off
(Celite), and the solvent was evaporated. The crude product was
purified by flash chromatography (EtOAc/hexane 1:1), and the
solid was recrystallized from EtOAc/hexane (0.5 g, 73%). m.p. 908C;
1H NMR: (250 MHz, [D6]DMSO): d=9.39 (s, 1H; OH), 7.48 (d, J=
8.1 Hz, 1H; naphthyl-H), 7.39–7.21 (m, 8H; 3Ar-H, 2NH, 3naphthyl-
H), 6.93–6.90 (m, 1H; naphthyl-H), 6.21 (d, J=7.6 Hz, 1H; naphthyl-
H), 4.16 (dd, J=5.3, 16.2 Hz, 2H; CH2), 3.88 (dd, J=6.4, 16.1 Hz, 2H;
CH2), 1.34 (s, 18H; 2C(CH3)3); 13C NMR (60 MHz, [D6]DMSO): d=
155.5, 153.9, 153.8, 148.1, 136.9, 132.6, 127.4, 126.6, 125.8, 125.6,
121.9, 118.6, 114.5, 110.7, 106.3, 77.8, 38.3, 28.0; IR (KBr): n=3854,
3822, 3802, 3752, 3736, 3712, 3690, 3676, 3650, 3630, 3461, 3366,
3056, 2979, 2932, 2345, 1924, 1683, 1630, 1609, 1580, 1528, 1457,
1392, 1366, 1299, 1277, 1251, 1223, 1166, 1088, 1047, 1028, 976,
895, 859, 851, 819, 775, 757, 636, 570 cmÀ1; ESI+ MS: m/z: calcd for
C28H34O6N2 +H+: 495.2; found: 495.5 [M+H]+; elemental analysis
calcd (%) for C28H34N2O6: C 68.00, H 6.93, N 5.66; found: C 67.96, H
7.01, N 5.36.
Dihydrochloride of methyl 3,5-bis((4,5-dihydro-1H-imidazol-2-ylami-
no)methyl)-4-(8-hydroxynaphthalen-1-yloxy)benzoate
(12a):
Dry
MeOH (2 mL) was cooled to 08C, and acetyl chloride (1 mL) was
slowly added dropwise, followed by stirring for 30 min. Com-
pound 17 (50 mg, 90.5 mmol) was dissolved in anhydrous MeOH
(0.5 mL), added dropwise to the acidic MeOH solution, which was
stirred for 30 min at 08C, then 30 min at ambient temperature.
When the reaction was complete, the solvent was evaporated, and
the residue was dried in vacuo. The deprotected diamine was dis-
solved in MeOH/NEt3 (2:1, 3 mL), 18 (41 mg, 0.27 mmol) was
added, and the solution was stirred for 1 h at ambient tempera-
ture. The solvent was evaporated, and the residue was purified by
column chromatography (20 g silica gel, EtOAc/EtOH/H2O/HOAc
11:4:4:1). After the solvent had been removed, the residue was
redissolved in MeOH, picric acid (138 mg, water content 40%;
0.36 mmol, 4 equiv) was added, followed by addition of water until
the solution became turbid. This mixture was heated to reflux,
slowly cooled to ambient temperature, and then stored at 48C
overnight. The orange-brown solid was filtered and dried in vacuo.
To replace the counterion of 12 with chloride, the residue was dis-
solved in a little MeOH, and filtered through a small column filled
with Dowex 1ꢂ8 (ClÀ form). Further elution of the column with
MeOH and removal of the solvent yielded 12a as a yellow solid
ChemBioChem 2011, 12, 1223 – 1229
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1227

M. W. Gçbel et al.
Picrate of 1-(2,6-bis((4,5-dihydro-1H-imidazol-2-ylamino)methyl)phe-
noxy)naphthalen-8-ol (11): Acetyl chloride (2 mL) was added to a
solution of 21a (0.27 g, 0.55 mmol) in MeOH (3 mL) and stirred at
ambient temperature for 2 h. The solvent was evaporated, and the
residue (21b) was dissolved in NEt3/H2O/MeOH (1:1:1, 6 mL). Com-
pound 18 (0.18 g, 1.2 mmol) was added, and the reaction mixture
was stirred at room temperature for another 12 h. H2O (10 mL) and
2n NaOH were added to a final pH of 13, and the mixture was ex-
tracted twice with CH2Cl2 to remove NEt3. After evaporation of the
aqueous phase to about 50%, it was acidified with 2n HCl, evapo-
rated completely, and dried in vacuo. Separation of the hydrochlo-
ride of 11 from NaCl was achieved by extracting the solid residue
three times with hot, dry MeOH, and the solution was reduced to
10 mL. Picric acid (0.44 g; water content 40%, 2 equiv) in MeOH
(5 mL) was added, then water was added until the mixture became
turbid. The precipitate was redissolved by heating, and yellow crys-
tals of 11 formed upon slow cooling (0.35 g, 70%). Rf =0.16
(EtOAc/EtOH/H2O/HOAc 15:5:4:1); m.p. 215–2168C; 1H NMR:
(250 MHz, [D6]DMSO); d=9.35 (s, 1H; OH), 8.80–6.80 (brs, 4H;
4ethylene-NH), 8.59 (s, 4H; 4picrate-H), 8.52–8.38 (m, 2H; Ar-H),
7.54–7.41 (m, 6H; Ar-H, 3naphthyl-H, 2NH), 7.32 (t, J=7.8 Hz, 1H;
naphthyl-H), 7.00 (t, J=3.8, 1H; naphthyl-H), 6.18 (d, J=7.6 Hz, 1H;
naphthyl-H), 4.34 (dd, J=4.6, 15.3 Hz, 2H; Ar-CH2), 4.21 (dd, J=6.2/
15.5, 2H; Ar-CH2), 3.39 (s, 8H; 4CH2); 13C NMR (60 MHz, [D6]DMSO):
d=160.7, 159.0, 158.9, 153.9, 153.6, 148.8, 141.7, 136.9, 130.4,
129.3, 127.6, 125.9, 125.1, 124.1, 122.3, 118.8, 114.5, 110.7, 42.5,
42.3, 41.1, 40.9; IR (KBr): n=3903, 3890, 3870, 3854, 3838, 3821,
3806, 3785, 3770, 3758, 3750, 3744, 3735, 3724, 2711, 2701, 3689,
3676, 3656, 3649, 3628, 2618, 3608, 3587, 3567, 3366, 3331, 3243,
3081, 2928, 2373, 2344, 2278, 1910, 1870, 1845, 1751, 1740, 1734,
1718, 1698, 1675, 1654, 1636, 1600, 1578, 1560, 1534, 1522, 1508,
1499, 1490, 1475, 1466, 1458, 1450, 1431, 1394, 1384, 1364, 1334,
1314, 1296, 1270, 1188, 1162, 1134, 1078, 1025, 942, 930, 910, 848,
818, 806, 788, 778, 746, 711, 682, 670, 662, 638, 620, 609, 584, 576,
559 cmÀ1; elemental analysis calcd (%) for C36H32N12O16·1H2O: C
47.68, H 3.75, N 18.53; found: C 47.74, H 3.88, N 18.57.
Phosphorylation kinetics: All reactants and substances were
weighed as precisely as possible, and dissolved in the required
amounts of solvent. An NMR tube (6 cm long) was loaded with the
solution, and sealed with a standard cap. 1-Nitronaphthalene (re-
crystallized from n-hexane) was used as the internal standard. All
kinetic experiments were carried out at 308C (Æ0.18C) in a Lauda
RM6 thermostat (Lauda, Kçnigshofen, Gernamy) . Samples (3 mL)
were taken at defined times, the reaction was quenched with
57 mL of a 1:1 mixture of water and MeOH (containing 3% TFA),
and samples stored in liquid nitrogen. Aliquotes (20 mL) were then
analyzed by HPLC with UV detection at 275 nm on a Merck Li-
Chrospher 100 RP-18e (5 mm) column. For details see the Support-
ing Information.
DNA cleavage assay: The pUC19 plasmid DNA, prepared from
DH5a cells, was purified by using a HiSpeed Plasmid Purification
Kit (Qiagen, Hilden, Germany). The assay mixture (10 mL) contained
11 or 12 (0.1–10 mm, as hydrochlorides) in a 50 mm HEPES-NaOH
(pH 7.0, if not otherwise indicated) and pUC19 DNA (22.5 nm). In-
cubation was performed at 378C for 20 h. Prior to electrophoresis,
gel loading buffer (2 mL, 0.2% crocein orange G, 40% sucrose) and
20% SDS (1 mL) were added to each sample. The addition of SDS,
and replacement of the usual tracking dye (bromophenol blue)
with crocein orange G is recommended to prevent aggregation of
compound and DNA during sample loading. Aliquots (10 mL) pre-
pared in TBE buffer (90 mm Tris, 90 mm boric acid, 2 mm EDTA)
were loaded on a 1% agarose gel that contained 0.5 mgmLÀ1
ethidium bromide (EtBr). The same buffer was used as the running
buffer. Subsequent to electrophoresis (120 V, 150–180 min), the gel
was placed on a transilluminator (302 nm) and photographed
through a yellow-orange filter (Etbr filter 540/640, P575; Biostep,
Jahnsdorf, Germany). The digital photos were analyzed by using
Phoretix 1D Quantifier software (Nonlinear dynamics, UK). The
peak areas of the supercoiled form and the open circle form were
determined, and the percentage open circle form was calculated.
To normalize the decreased ability of EtBr to intercalate into form I
DNA (relative to forms II and III) a factor of 1.22 was used.[20]
Isolation of phosphorylation product 24: Compound 11 (40 mg,
0.044 mmol) was dissolved in anhydrous DMF (2 mL) and di-isopro-
pylethylamine (0.07 mL) under argon. Bis-4-nitrophenylphosphate
5 (0.05 g, 0.11 mmol) was added, and the resulting mixture was
stirred at ambient temperature for one week. The solution was
diluted with water (10 mL), NaOH (2 mL, 2 n) was added, and the
mixture was extracted with CH2Cl2 (2ꢂ15 mL). The organic layers
were combined, the solvent was evaporated, and the solid was
dried in vacuo. To replace the counter ions with chloride, the resi-
due was dissolved in MeOH and filtered through a small column
filled with Dowex 1ꢂ8 (ClÀ form). The solvent was evaporated
again, and the residue was purified by flash chromatography
(EtOAc/EtOH/H2O/HOAc 14:4:4:1) to yield 24 (15 mg, 50%). The
analytical sample was purified further by HPLC (Maisch Reprosil-
Pur C18-AQ, 10 m, 250ꢂ20 mm; water/acetonitrile (10:5), +0.1%
trifluoroacetic acid; 9 mLminÀ1). Rf =0.20 (EtOAc/EtOH/H2O/HOAc
Acknowledgements
Financial support by the European Community is gratefully ac-
knowledged (European Network on the Development of Artificial
Nucleases, HPRN-CT-1999-00008).
Keywords: BDNPP · BNPP · enzyme models · guanidine ·
nucleic acids
[1] G. K. Schroeder, C. Lad, P. Wyman, N. H. Williams, R. Wolfenden, Proc.
Natl. Acad. Sci. USA 2006, 103, 4052–4055.
[2] Metal ion based artificial DNA cleavers as tools for gene manipulation:
a) H. Katada, M. Komiyama, ChemBioChem 2009, 10, 1279–1288; b) Y.
Yamamoto, A. Uehara, T. Tomita, M. Komiyama, Nucleic Acids Res. 2004,
32, e153; c) S. Durai, M. Mani, K. Kandavelou, J. Wu, M. H. Porteus, S.
Chandrasegaran, Nucleic Acids Res. 2005, 33, 5978–5990.
[3] Recent reviews on metal ion promoted cleavage of phosphodiesters
and of DNA: a) A. Sreedhara, J. A. Cowan, J. Biol. Inorg. Chem. 2001, 6,
337–347; b) F. Mancin, P. Scrimin, P. Tecilla, U. Tonellato, Chem.
Commun. 2005, 2540–2548; c) T. Niittymꢃki, H. Lçnnberg, Org. Biomol.
Chem. 2006, 4, 15–25; d) T. A. Shell, D. L. Mohler, Curr. Org. Chem. 2007,
11, 1525–1542; e) F. Mancin, P. Tecilla, New J. Chem. 2007, 31, 800–817.
[4] Cleavage of phosphodiesters and of DNA by metal ions: a) A. Berkessel,
D. A. Hꢄrault, Angew. Chem. 1999, 111, 99–102; Angew. Chem. Int. Ed.
1999, 38, 102–105; b) M. E. Branum, A. K. Tipton, S. Zhu, L. Que, Jr., J.
Am. Chem. Soc. 2001, 123, 1898–1904; c) R. Hettich, H.-J. Schneider, J.
1
14:4:4:1); H NMR (400 MHz, [D6]DMSO): d=8.80 (t, J=5.3 Hz, 3H;
exchangeable with D2O, NH), 8.25 (d, J=9.1 Hz, 2H), 7.75 (d, J=
8.3 Hz, 1H), 7.61 (d, J=8.2 Hz, 1H), 7.55–7.49 (m, 5H), 7.42–7.37
(m, 1H), 7.32–7.27 (m, 2H; 1 proton exchangeable with D2O), 7.17
(d, J=7.6 Hz, 1H), 7.10 (s, 1H; exchangeable with D2O, NH), 6.93 (s,
1H; exchangeable with D2O, NH), 6.30 (d, J=7.7 Hz, 1H), 4.35–4.17
(m, 4H; 2Ar-CH2), 3.36 (s, 8H; 2CH2CH2); 31P NMR (121.5 MHz,
[D6]DMSO): d=À11.91; ESI+ MS: m/z: calcd for C30H31N7O7P: 632.2;
found 632.7 [M]+.
1228
www.chembiochem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1223 – 1229

Cleavage of Phosphodiesters and DNA
Am. Chem. Soc. 1997, 119, 5638–5647; d) R. Hettich, H.-J. Schneider, J.
Chem. Soc. Perkin Trans. 2 1997, 2069–2072; e) A. Roigk, H.-J. Schneider,
Eur. J. Org. Chem. 2001, 205–209; f) A. Bencini, E. Berni, A. Bianchi, C.
Giorgi, B. Valtancoli, D. K. Chand, H.-J. -Schneider, Dalton Trans. 2003,
793–800; g) F. H. Zelder, A. A. Mokhir, R. Krꢃmer, Inorg. Chem. 2003, 42,
8618–8620; h) A. Schiller, R. Scopelliti, K. Severin, Dalton Trans. 2006,
3858–3867; i) R. A. Mathews, C. S. Rossiter, J. R. Morrow, J. P. Richard,
Dalton Trans. 2007, 3804–3811; j) I. Szilꢅgyi, S. Mikkola, H. Lçnnberg, I.
Libꢅdi, I. Pꢅlinkꢆ, J. Inorg. Biochem. 2007, 101, 1400–1403; k) C. Li, R.-Z.
Qiao, Y.-Q. Wang, Y.-F. Zhao, R. Zeng, Bioorg. Med. Chem. Lett. 2008, 18,
5766–5770; l) M. S. Deshpande, A. A. Kumbhar, A. S. Kumbhar, M.
Kumbharkar, H. Pal, U. B. Sonowane, R. R. Joshi, Bioconjugate Chem.
2009, 20, 447–459; m) R. P. Megens, T. A. van den Berg, A. D. de Bruin,
B. L. Feringa, G. Roelfes, Chem. Eur. J. 2009, 15, 1723–1733; n) Y. Kou, J.
Tian, D. Li, W. Gu, X. Liu, S. Yan, D. Liao, P. Cheng, Dalton Trans. 2009,
2374–2382; o) D. Li, J. Tian, Y. Kou, F. Huang, G. Chen, W. Gu, X. Liu, D.
Liao, P. Cheng, S. Yan, Dalton Trans. 2009, 3574–3583; p) M. C. Bof Oli-
veira, D. Mazera, M. Scarpellini, P. C. Severino, A. Neves, H. Terenzi, Inorg.
Chem. 2009, 48, 2711–2713; q) Y. Huang, S.-Y. Chen, J. Zang, X.-Y. Tan,
N. Jiang, J.-J. Zhang, Y. Zhang, H.-H. Lin, X.-Q. Yu, Chem. Biodiversity
2009, 6, 475–486; r) S. Anbu, M. Kandaswamy, P. Suthakaran, V. Muru-
gan, B. Varghese, J. Inorg. Biochem. 2009, 103, 401–410; s) R. Bonomi, P.
Scrimin, F. Mancin, Org. Biomol. Chem. 2010, 8, 2622–2626; t) C. Li, C.
Du, H. Tian, C. Jiang, M. Du, Y. Liu, R.-Z. Qiao, Y.-X. Jia, Y.-F. Zhao, Chem.
Eur. J. 2010, 16, 12935–12940; u) J.-T. Wang, X.-H. Zheng, L.-N. Ji, Z.-W.
Mao, J. Coord. Chem. 2010, 63, 2440–2449; v) W. Xu, X. Yang, L. Yang,
Z.-L. Jia, F. Liu, G.-Y. Lu, New J. Chem. 2010, 34, 2654–2661.
J. P. Priebe, T. A. S. Brand¼o, F. Nome, ARKIVOC 2009, (iii), 28–38;
h) A. M. Pia˛tek, M. Gray, E. V. Anslyn, J. Am. Chem. Soc. 2004, 126, 9878–
9879; i) J. Li, J. Zhang, Q.-S. Lu, Y. Yue, Y. Huang, D.-W. Zhang, H.-H. Lin,
S.-Y. Chen, X.-Q. Yu, Eur. J. Med. Chem. 2009, 44, 5090–5093.
[7] a) N. Strꢃter, W. N. Lipscomb, T. Klabunde, B. Krebs, Angew. Chem. 1996,
108, 2158–2191; Angew. Chem. Int. Ed. Engl. 1996, 35, 2024–2055;
b) E. A. Galburt, B. L. Stoddard, Biochemistry 2002, 41, 13851–13860;
c) A. Pingoud, M. Fuxreiter, V. Pingoud, W. Wende, Cell. Mol. Life Sci.
2005, 62, 685–707.
[8] M. R. Redinbo, L. Stewart, P. Kuhn, J. J. Champoux, W. G. J. Hol, Science
1998, 279, 1504–1513.
[9] F. A. Cotton, E. E. Hazen, Jr., M. J. Legg, Proc. Natl. Acad. Sci. USA 1979,
76, 2551–2555.
[10] K. A. Schug, W. Lindner, Chem. Rev. 2005, 105, 67–114.
[11] a) M.-S. Muche, M. W. Gçbel, Angew. Chem. 1996, 108, 2263–2265;
Angew. Chem. Int. Ed. Engl. 1996, 35, 2126–2129; b) M.-S. Muche, P. Ka-
malaprija, M. W. Gçbel, Tetrahedron Lett. 1997, 38, 2923–2926; c) V.
Jubian, R. P. Dixon, A. D. Hamilton, J. Am. Chem. Soc. 1992, 114, 1120–
1121; d) V. Jubian, A. Veronese, R. P. Dixon, A. D. Hamilton, Angew. Chem.
1995, 107, 1343–1345; Angew. Chem. Int. Ed. Engl. 1995, 34, 1237–
1239; e) J. Smith, K. Ariga, E. V. Anslyn, J. Am. Chem. Soc. 1993, 115,
362–364; f) D. M. Perreault, L. A. Cabell, E. V. Anslyn, Bioorg. Med. Chem.
1997, 5, 1209–1220; g) T. Oost, M. Kalesse, Tetrahedron 1997, 53, 8421–
8438.
[12] F. M. Menger, M. Ladika, J. Am. Chem. Soc. 1987, 109, 3145–3146.
[13] P. V. Vyas, A. K. Bhatt, G. Ramachandraiah, A. V. Bedekar, Tetrahedron
Lett. 2003, 44, 4085–4088.
[5] Phosphodiester cleaving metal complexes with pendant amino or gua-
nidino groups: a) E. Kçvꢅri, R. Krꢃmer, J. Am. Chem. Soc. 1996, 118,
12704–12709; b) H. Aꢇt-Haddou, J. Sumaoka, S. L. Wiskur, J. F. Folmer-
Andersen, E. V. Anslyn, Angew. Chem. 2002, 114, 4185–4188; Angew.
Chem. Int. Ed. 2002, 41, 4014–4018; c) M. Livieri, F. Mancin, U. Tonellato,
J. Chin, Chem. Commun. 2004, 2862–2863; d) M. Livieri, F. Mancin, G.
Saielli, J. Chin, U. Tonellato, Chem. Eur. J. 2007, 13, 2246–2256; e) G.
Feng, J. C. Mareque-Rivas, R. T. M. de Rosales, N. H. Williams, J. Am.
Chem. Soc. 2005, 127, 13470–13471; f) G. Feng, J. C. Mareque-Rivas,
N. H. Williams, Chem. Commun. 2006, 1845–1847; g) X. Sheng, X.-M. Lu,
Y.-T. Chen, G.-Y. Lu, J.-J. Zhang, Y. Shao, F. Liu, Q. Xu, Chem. Eur. J. 2007,
13, 9703–9712; h) Y. Shao, X. Sheng, Y. Li, Z.-L. Jia, J.-J. Zhang, F. Liu, G.-
Y. Lu, Bioconjugate Chem. 2008, 19, 1840–1848; i) R. Bonomi, F. Selves-
trel, V. Lombardo, C. Sissi, S. Polizzi, F. Mancin, U. Tonellato, P. Scrimin, J.
Am. Chem. Soc. 2008, 130, 15744–15745; j) J. He, J. Sun, Z.-W. Mao,
L.-N. Ji, H. Sun, J. Inorg. Biochem. 2009, 103, 851–858; k) M. J. Belousoff,
L. Tjioe, L. Spiccia, Inorg. Chem. 2008, 47, 8641–8651; l) L. Tjioe, T. Joshi,
J. Brugger, B. Graham, L. Spiccia, Inorg. Chem. 2011, 50, 621–635.
[14] N. Detzer, O. Burkhard, H. Schaffrin, W. Liptay, Z. Naturforsch. B 1987, 42,
1129–1141.
[15] A. G. M. Barrett, F. Blaney, A. D. Campbell, D. Hamprecht, T. Meyer,
A. J. P. White, D. Witty, D. J. Williams, J. Org. Chem. 2002, 67, 2735–
2750.
[16] T. D. Krizan, J. C. Martin, J. Org. Chem. 1982, 47, 2681–2682.
[17] Salt of diamine 21b with picric acid (C18H20N2O2·2C6H2N3O7·H2O·
0.5CH3OH). Mr =786.6. Crystal size: 0.04ꢂ0.45ꢂ0.60 mm. Crystal
¯
system: rriclinic. Space group P1. Cell dimensions: a=10.344 (3) ꢈ, b=
11.479 (5) ꢈ, c=14.960 (9) ꢈ, a=79.08 (5)8, b=70.99 (4)8, g=88.60 (7)8,
V=1647.8 (13) ꢈ3, Z=2, 1ber =1.585 mgmÀ3. m=0.133 mmÀ1. MoKa radi-
ation, l=0.71073 ꢈ. T=141 K. q=1.47–27.948. 14284 reflections col-
lected, 6915 independent (Rint =0.1739). R1=0.1226, wR2=0.2164 (I>
2s(I)). CCDC 743278 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif
1
[18] O-phosphorylation product of compound 7: H NMR signals of the dihy-
[6] Cleavage of phosphodiesters and of DNA by metal free reagents: a) F.
Avenier, J. B. Domingos, L. D. Van Vliet, F. Hollfelder, J. Am. Chem. Soc.
2007, 129, 7611–7619; b) C. Schmuck, J. Dudaczek, Org. Lett. 2007, 9,
5389–5392; c) J. Razkin, J. Lindgren, H. Nilsson, L. Baltzer, ChemBioChem
2008, 9, 1975–1984; d) X. Sheng, X.-M. Lu, J.-J. Zhang, Y.-T. Chen, G.-Y.
Lu, Y. Shao, F. Liu, Q. Xu, J. Org. Chem. 2007, 72, 1799–1802; e) Y. Shao,
Y. Ding, Z.-L. Jia, X.-M. Lu, Z.-H. Ke, W.-H. Xu, G.-Y. Lu, Bioorg. Med. Chem.
2009, 17, 4274–4279; f) L. Fernandes, F. L. Fischer, C. W. Ribeiro, G. P. Sil-
veira, M. M. Sꢅ, F. Nome, H. Terenzi, Bioorg. Med. Chem. Lett. 2008, 18,
4499–4502; g) A. J. Kirby, A. M. Manfredi, B. S. de Souza, M. Medeiros,
droimidazolium ions ([D6]DMSO, 250 MHz): 3.43 ppm, s, 8H. N-phos-
phorylation product of compound 7: 3.74 (m, 2H), 3.53 (m, 6H).
[19] B. Dietrich, D. L. Fyles, T. M. Fyles, J.-M. Lehn, Helv. Chim. Acta 1979, 62,
2763–2787.
[20] M. E. Branum, A. K. Tipton, S. Zhu, L. Que, J. Am. Chem. Soc. 2001, 123,
1889–1904.
Received: January 12, 2011
Published online on April 15, 2011
ChemBioChem 2011, 12, 1223 – 1229
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1229