Polyhydroxylated sapphyrins: Multisite non-metallic catalysts for activated phosphodiester hydrolysis

Article abstract of DOI:10.1021/ja0518474

Enhanced hydrolysis rates for the cleavage of bis(4-nitrophenyl)phosphate (BNPP), a model phosphodiester, may be achieved by using appropriately designed ditopic receptors containing the known phosphate-binding nucleus, sapphyrin, attached covalently to s

Full text of DOI:10.1021/ja0518474

Published on Web 12/21/2005
Polyhydroxylated Sapphyrins: Multisite Non-metallic
Catalysts for Activated Phosphodiester Hydrolysis
Vladim´ır Kra´l,*,† Kamil Lang,*,‡ Jarmila Kra´lova´, Michal Dvorˇa´k, Pavel Marta´sek,§
Aileen O. Chin,| Andrei Andrievsky,| Vincent Lynch,| and Jonathan L. Sessler*,|
Contribution from the Department of Analytical Chemistry, Institute of Chemical Technology,
Technicka´ 5, 166 28, Prague 6, Czech Republic, ZentiVa R & D, U KabeloVny 130,
10237 Prague 10, Czech Republic, Institute of Inorganic Chemistry, Academy of Sciences of the
Czech Republic, 250 68 Rˇezˇ, Czech Republic, Institute of Molecular Genetics, Academy of
Sciences of the Czech Republic, FlemingoVo na´m. 2, 166 37, Prague 6, Czech Republic, First
Medical Faculty, Charles UniVersity, Katerˇinska´ 1660/32, 120 00 Prague, Czech Republic, and
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology,
1 UniVersity Station-A5300, The UniVersity of Texas at Austin, Austin, Texas 78712-0165
Received March 23, 2005; E-mail: kral@vscht.cz; lang@iic.cas.cz; sessler@mail.utexas.edu
Abstract: Enhanced hydrolysis rates for the cleavage of bis(4-nitrophenyl)phosphate (BNPP), a model
phosphodiester, may be achieved by using appropriately designed ditopic receptors containing the known
phosphate-binding nucleus, sapphyrin, attached covalently to suitably oriented polyhydroxyl subunits.
Evidence for the interaction between sapphyrin and BNPP comes from solid-state X-ray diffraction analysis
of a diprotonated dihydroxylated sapphyrin-BNPP complex and from solution-phase 31P NMR spectroscopic
binding studies. The sapphyrins described in this paper may have a role to play as oligonucleotide cleavage
agents.
Introduction
with a general base provided from the side chains of His, Lys,
Arg, Asn, or Glu.2,5a,6 The activation of a water molecule,
Phosphate diester hydrolysis is a reaction of continuing
interest since such processes are of fundamental biological
importance. Under normal, uncatalyzed conditions, phosphate
diesters are extremely resistant to hydrolysis with DNA and
bis(4-nitrophenyl)phosphate (BNPP) displaying half-lives of 2
× 108 and 2 × 103 years, respectively, at pH 7.0 and 25 °C.1
On the other hand, several phosphodiesterases, including
staphylococcal nuclease,2 alkaline phosphatase,3 and DNase I,4
are known that are very efficient catalysts for the hydrolysis of
their phosphodiester substrates. This efficiency often derives
from the cooperation of various phosphate binding center(s),
such as metal ions5 (e.g., Zn2+, Mg2+, or Mn2+) and guanidinium
generating an efficient nucleophilic species, is also implicated
as playing a vital role in the proposed mechanism of staphy-
lococcal nuclease.2 Likewise, a serine OH group, together with
three metal cations and Arg, has been suggested as being
important in the functioning of alkaline phosphatase.3
Mimicking the myriad activities of nucleases presents a
formidable challenge to those who wish to design artificial
catalytic systems capable of promoting efficient hydrolysis of
phosphodiester bonds, including those of RNA and, perhaps,
even DNA. Yet meeting this challenge is considered important
in terms of advancing areas such as supramolecular chemistry
and antisense technology. Not surprisingly, therefore, consider-
able effort has been devoted to the problem, and currently a
number of metal-containing systems are known that are designed
to enhance catalytically phosphodiester hydrolysis. These
include systems wherein two metals are used in a bifunctional
catalytic sense,7 systems wherein a single metal center in
conjunction with some other group acts to augment hydrolysis
efficiency,8-12 and metal-free systems,13,14 including ones
† Institute of Chemical Technology and Zentiva R & D.
‡ Academy of Sciences of the Czech Republic, Rˇ ezˇ.
Academy of Sciences of the Czech Republic, Prague.
§ Charles University.
| The University of Texas at Austin.
(1) Chin, J.; Banaszczyk, M.; Jubian, V.; Zou, X. J. Am. Chem. Soc. 1989,
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Kady, I. O.; Tan, B.; Ho, Z. Y.; Scarborough, T. J. Chem. Soc., Chem.
Commun. 1995, 1137-1138.
(4) Lomonossoff, G. P.; Butler, P. J. G.; Klug, A. J. Mol. Biol. 1981, 149,
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Friedman, J. M.; Beese, L. S.; Sanderson, M. R.; Steitz, T. A. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 8924-8928. (c) Hough, E.; Hansen, L. K.;
Birknes, B.; Jynge, K.; Hansen, S.; Hordvik, A.; Little, C.; Dodson, E.;
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W. J. Mol. Biol. 1991, 218, 449-464.
9
432
J. AM. CHEM. SOC. 2006, 128, 432-437
10.1021/ja0518474 CCC: $33.50 © 2006 American Chemical Society

Polyhydroxylated Sapphyrins
A R T I C L E S
Scheme 1. Molecular Structures of BNPP and Macrocycles Tested as Hydrolysis Catalysts
wherein one or more guanidinium or guanidinium-like binding
sites15-18 are allowed to cooperate with other groups so as to
enhance the rate-limiting step of phosphodiester hydrolysis.
Systems based on the use of two rather different metal centers
(e.g., Zn(II) and Fe(III)) have been also reported.7f,19 A large
number of cyclodextrin-based inclusion complexes with func-
tionalized sidearms, including ones that do not rely on catalytic
metal centers, have also been shown to act as phosphodiester
hydrolysis catalysts.20 Nonetheless, the number of functioning,
non-metal-based catalyst systems remains rather limited.
In this paper we report an approach to achieving metal-free
catalytic phosphodiester hydrolysis that is based on the use of
sapphyrins, pentapyrrolic porphyrin-like macrocycles, as the key
phosphodiester binding entity. Earlier, we established that certain
expanded porphyrins, namely, sapphyrin and rubyrin, act as
efficient and selective phosphate binding receptors at neutral
pH.21 We have also described that appropriately designed
sapphyrin-based systems may be used as carriers for the through-
model-membrane transport of biologically important phospho-
rylated species, such as nucleotides.21 Therefore, we rationalized
that a combination of a sapphyrin phosphate binding unit and
a properly positioned “internal” nucleophile (i.e., appended to
the macrocyclic core) might provide a functioning metal-free
phosphodiester hydrolysis system. Here, we report that the
polyhydroxysapphyrin derivatives 1 and 2 (Scheme 1) act to
effect the catalytic hydrolysis of bis(4-nitrophenyl)phosphate
(BNPP) at near-neutral pH. We also report the results of
hydrolysis experiments carried out using the control porphyrins
8, 9, and 10, as well as the dimeric sapphyrin 6.
(9) For an example based on the use of a metal and hydroxamic acid group,
see: Hashimoto, S.; Yamamoto, K.; Yamada, T.; Nakamura, Y. Hetero-
cycles 1998, 48, 939-947.
(10) For an example based on the use of a metal and ammonium group, see:
Kovari, E.; Kramer, R. J. Am. Chem. Soc. 1996, 118, 12704-12709.
(11) For an example based on noncovalently bound amino acids, see: Roigk,
A.; Schneider, H. J. Eur. J. Org. Chem. 2001, 205-209.
(12) For an example based on the use of a metal and anthraquinone intercalator,
see: Boseggia, E.; Gatos, M.; Lucatello, L.; Mancin, F.; Moro, S.; Palumbo,
M.; Sissi, C.; Tecilla, P.; Tonellato, U.; Zagatto, G. J. Am. Chem. Soc.
2004, 126, 4543-4549.
(13) For an example of diamine-catalyzed hydrolysis, see: Komiyama, M.;
Yoshinary, K. J. Org. Chem. 1997, 62, 2155-2160.
(14) (a) For an example of imidazole-based hydrolysis, see: Beloglazova, N.
G.; Fabani, M. M.; Zenkova, M. A.; Bichenkova, E. V.; Polushin, N. N.;
Silnikov, V. V.; Douglas, K. T.; Vlassov, V. V. Nucleic Acids Res. 2004,
32, 3887-3897. (b) Scheffer, U.; Strick, A.; Ludwig, V.; Peter, S.; Kalden,
E.; Gobel, M. W. J. Am. Chem. Soc., in press.
Experimental Section
Materials and Methods. BNPP hydrolysis rates were monitored
by following the increase in absorbance at 400 nm, reflecting the
(15) Jubian, V.; Veronese, A.; Dixon, R. P.; Hamilton, A. D. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 1237-1239.
(16) Perreault, D. M.; Cabell, L. A.; Anslyn, E. V. Bioorg. Med. Chem. 1997,
5, 1209-1220.
(21) (a) Sessler, J. L.; Furuta, H.; Kra´l, V. Supramol. Chem. 1993, 1, 209-220.
(b) Kra´l, V.; Sessler, J. L. Tetrahedron 1995, 51, 539-554. (c) Kra´l, V.;
Furuta, H.; Shreder, K.; Lynch, V.; Sessler, J. L. J. Am. Chem. Soc. 1996,
118, 1595-1607. (d) Iverson, B. L.; Shreder, K.; Kra´l, V.; Sansom, P.;
Lynch, V.; Sessler, J. L. J. Am. Chem. Soc. 1996, 118, 1608-1616. (e)
Sessler, J. L.; Kra´l, V.; Genge, J. W.; Thomas, R. E.; Iverson, B. L. Anal.
Chem. 1998, 70, 2516-2522. (f) Sessler, J. L.; Camiolo, S.; Gale, P. A.
Coord. Chem. ReV. 2003, 240, 17-55. (g) Sessler, J. L.; Davis, J. M.;
Kra´l, V.; Kimbrough, T.; Lynch, V. Org. Biomol. Chem. 2003, 1, 4113-
4123.
(17) Ait-Haddou, H.; Sumaoka, J.; Wiskur, S. L.; Folmer-Andersen, J. F.; Anslyn,
E. V. Angew. Chem., Int. Ed. 2002, 41, 4014-4016.
(18) Piatek, A. M.; Gray, M.; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, 9878-
9879.
(19) Irisawa, M.; Takeda, N.; Komiyama, M. J. Chem. Soc., Chem. Commun.
1995, 1221-1222.
(20) (a) Breslow, R. Acc. Chem. Res. 1995, 28, 146-153. (b) Hengge, A. C.;
Tobin, A. E.; Cleland, W. W. J. Am. Chem. Soc. 1995, 117, 5919-5926.
9
J. AM. CHEM. SOC. VOL. 128, NO. 2, 2006 433

A R T I C L E S
Kra´l et al.
Table 1. Rate Constants for the Hydrolysis of BNPP by
1:1 mixture of dichloromethane and DMF for 1,2,3,4-tetra-O-acetyl-
2-amino-2-deoxy-D-glucopyranose. As the coupling reagent, either 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) or
diisopropylcarbodiimide was used. Final purification of the various
water-soluble macrocyclic compounds was achieved using reverse-phase
column chromatography.
Sapphyrins and Porphyrinsa
1
1
catalyst
rate constant (10-5 h-
)
catalyst
rate constant (10-5 h-
)
1
2
3
4
5
38
37
23
17
11
6
8
9
9.5
4.3
6.7
7.0
10
X-ray Diffraction Analysis. Experimental procedure for (C42H55N5O2)-
[((C6H4NO2)O)2PO2]2: Crystals were grown as dark plates from 1:1
chloroform-methanol of sapphyrin 7 and BNPP (1:2 molar ratio) by
allowing diethyl ether to diffuse into it over the course of 3 weeks.
The data crystal was a plate of approximate dimensions; 0.13 × 0.56
× 0.56 mm. The data were collected at 188 K on a Siemens P3
diffractometer, equipped with a Nicolet LT-2 low-temperature device
and using a graphite monochromator with Mo KR radiation (λ )
0.710 73 Å). Four reflections (-1,2,4; 2,3,0; 2,-1,-5; 2,0,2) were
remeasured every 96 reflections to monitor instrument and crystal
stability. A smoothed curve of the intensities of these check reflections
was used to scale the data. The scaling factor ranged from 0.970 to
1.01. The data were corrected for Lp effects but not absorption. Data
reduction and decay correction were performed using the SHELXTL/
PC software package.25 The structure was solved by direct methods
and refined by full-matrix least- squares on F2 with anisotropic thermal
parameters for the non-H atoms.25 The hydrogen atoms were calculated
in idealized positions (C-H, 0.96 Å; N-H, 0.90 Å) with isotropic
temperature factors riding at 1.2 or 1.5 × Ueq of the attached atom.
The higher factor is used for all methyl hydrogens. The function,
a Conditions: pH 7.5, 1 × 10-5 M catalyst, 1 × 10-4 M BNPP, 37 °C.
For further details, see: Experimental Section.
production of 4-nitrophenolate anion, as a function of time. All reactions
were carried out in doubly distilled water, which was boiled prior to
use. Appropriate amounts of NaNO3 and HEPES buffer were introduced
in order to make up test solutions with total concentrations of 0.1 and
0.01 M in these two entities, respectively. These solutions were then
adjusted to pH 7.5 and, in both cases, filtered through a Nylon 66 (0.45
µm) Millipore filter. Typically, the kinetic experiments were carried
out at 37 °C using solutions that were 0.1 M in NaNO3, 0.01 M in
HEPES buffer, 1 × 10-5 M in the studied sapphyrin or porphyrin
derivative, and 1 × 10-4 M in BNPP. In the sapphyrin- and porphyrin-
free control experiments, the concentrations of glucamine, glucosamine,
and imidazole were 2 × 10-5 M, with the concentration of BNPP being
1 × 10-4 M at pH 7.5. The rate constants shown in Table 1 were
estimated from kinetic runs monitored over a 9 or 10 day period and
were determined from the method of initial rates. The concentration
of the 4-nitrophenolate anion produced was obtained from absorption
at 400 nm (ꢀ400 ) 18 500 M-1 cm-1) in aliquots whose pH was adjusted
to pH 10. The concentration of BNPP at each time period was calculated
by subtracting the observed 4-nitrophenolate anion concentration from
that of the BNPP initially present. The listed rate constants were then
determined by dividing the slopes of the linear traces, obtained by
plotting this derived BNPP concentration versus time, by the initial
BNPP concentration. All rate constants reported in Table 1 were then
corrected for the rate of hydrolysis observed in a “blank” run consisting
of a mixture of BNPP (1 × 10-4 M) and NaNO3 (0.1 M) in HEPES
buffer (0.01 M) at pH 7.5. All kinetic experiments were run at least
three times and were found to give rate constant values reproducible
to within 10%, a margin of error considered acceptable for the purposes
of the present study.
Synthesis of Catalysts. The sapphyrin compounds were prepared
in accord with literature procedures reported earlier.22 The sapphyrins
and control porphyrins were prepared by introducing the desired
functionality onto the macrocycle periphery via the formation of amide
bonds. This was done by reacting the activated bis-acid form of the
appropriate macrocycle with a suitable, functionality-bearing amino
component. As the amino components, we have used histamine for
the preparation of 3, 1-amino-1-deoxy-D-glucitol (D-glucamide) for the
preparation of 1, 6, 8, and 10, and 1,2,3,4-tetra-O-acetyl-2-amino-2-
deoxy-D-glucopyranose for the preparation of the precursors to 2 and
9. In the case of the latter species, deprotection of the acetylated forms
was achieved using standard procedures,23 giving 2 and 9 in good yields.
This approach to preparing 2 and 9 is based on the use of an O-protected
starting compound and thus represents a different strategy than that
used earlier to prepare analogous protoporphyrin derivatives.24 The
reactions were performed in water-DMF (1:1; DMF ) dimethylfor-
mamide) for histamine and D-glucamide, and in dichloromethane or a
2
2
∑w(|Fo| - |Fc| )2, was minimized, where w ) 1/[(σ(Fo))2 + (0.04P)2]
2
2
and P ) (|Fo| + 2|Fc| )/3. The absolute configuration was determined
by internal comparison. Neutral atom scattering factors and values used
to calculate the linear absorption coefficient are from the International
Tables for X-ray Crystallography (1990).26 Other computer programs
used in this work are listed elsewhere.27 All figures were generated
using SHELXTL/PC.25 Tables of positional and thermal parameters,
bond lengths, angles and torsion angles, figures, and lists of observed
and calculated structure factors are located in the Supporting Informa-
tion.
Results and Discussion
Design of the Catalysts. The functionalized sapphyrins were
designed in consideration of the mechanism of phosphodiester
hydrolysis; this is believed to be of an ANDN type wherein the
transition state resembles a pentavalent phosphorane or is close
in energy to a stable pentavalent phosphorane intermediate.20b
In our design we also took into consideration previous studies
showing that nucleophilic displacement plays an important role
in mediating phosphodiester hydrolysis.28 Therefore, we com-
bined the known phosphate ester binding species, sapphyrin,
with additional groups that could possibly function as “internal”
or “tethered” nucleophiles. Here, our thoughts were that if the
latter entities could position themselves near the phosphodiester
bond of the putative substrate, good phosphodiester cleavage
rates might be obtained. We also appreciated that the fast
hydrolysis rates seen for RNA relative to DNA are generally
ascribed to the 2′ hydroxyl group that is present in RNA but
absent from DNA. In any event, the use and utility of pendant
hydroxyl groups in phosphate ester hydrolysis has been dem-
onstrated previously in the case of zinc(II) complexes bearing
(22) (a) Gorden, A. E. V.; Davis, J.; Sessler, J. L.; Kra´l, V.; Keogh, D. W.;
Schroeder, N. L. Supramol. Chem. 2004, 16, 91-100. (b) Sessler, J. L.;
Kra´l, V. U.S. Patent 5,543,514, Aug. 6, 1996. (c) Sessler, J. L.; Iverson,
B. L.; Kra´l, V.; Shreder, K.; Furuta, H. U.S. Patent 5,457,195, Oct. 10,
1995. (d) Kra´l, V.; Val´ık, M.; Shishkanova, T. V.; Sessler, J. L. Dekker
Encyclopedia of Nanoscience and Nanotechnology; Dekker: New York,
2004; pp 2721-2738.
(25) Sheldrick, G. M. SHELXTL/PC (Version 5.03); Siemens Analytical X-ray
Instruments, Inc.: Madison, WI, 1994.
(26) International Tables for X-ray Crystallography; Wilson, A. J. C., Ed.;
Kluwer Academic Press.: Boston, 1992; Vol. C, Tables 4.2.6.8 and 6.1.1.4.
(27) Gadol, S. M.; Davis, R. E. Organometallics 1982, 1, 1607-1613.
(28) (a) Ba-Saif, S. A.; Waring, M. A.; Williams, A. J. Chem. Soc., Perkin
Trans. 2 1991, 1653-1659. (b) Hengge, A. C.; Cleland, W. W. J. Am.
Chem. Soc. 1991, 113, 5835-5841.
(23) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups in Organic Synthesis,
2nd ed.; Wiley: New York, 1991; p 90.
(24) Fuhrhop, J. H.; Demoulin, C.; Boettcher, C.; Ko¨ning, J.; Siggel, U. J. Am.
Chem. Soc. 1992, 114, 4159-4165.
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434 J. AM. CHEM. SOC. VOL. 128, NO. 2, 2006

Polyhydroxylated Sapphyrins
A R T I C L E S
to two of the sapphyrin NH sites (the oxygen-to-H hydrogen
bonding distances are 1.83 and 2.18 Å, respectively). In contrast,
the second type of binding mode is new in our experience and
involves not only “normal” oxyanion-to-NH hydrogen bonding
interactions but also a set of hydrogen bonding interactions
between the bipyrrole subunit and another oxygen of the BNPP
substrate (oxygen-to-NH distance, 2.20 Å). This observed
interaction provides a possible “hint” of how the suggested
sapphyrin-derived phosphodiester activation process could take
place, namely, that the macrocycle-derived binding of these two
oxygen atoms renders the central P atom more susceptible to
nucleophilic attack.
To complement the above analyses, we have also studied the
interactions between BNPP and sapphyrins 1 and 2 in methanol-
d4 using 31P NMR spectroscopy. For equimolar 1:1 mixtures
of these sapphyrins and BNPP we observed that the chemical
shifts of the phosphorus atom were displaced to higher field by
ca. 8 ppm compared to what is observed for BNPP alone (for
which δ ) -12.62 ppm); this, presumably, is the result of the
induced ring current associated with the aromatic sapphyrin
nucleus. Similar upfield shifts were seen when analogous
experiments were performed using sapphyrin 7 in D2O-
methanol-d4 but not when the water-soluble porphyrin 10 was
used instead under the same experimental conditions. These
findings, in conjunction with the solid-state results presented
immediately above, thus provide support for our previously
postulated contention,21 namely, that the protonated forms of
sapphyrin can act as specific and efficient receptors for different
phosphates. In the context of the present study, these results
are also important in that they are consistent with the suggestion
that appropriately functionalized sapphyrins can act to enhance
BNPP hydrolysis as the result of, in part, phosphate binding.
This postulate is discussed further below.
While 31P NMR spectroscopic measurements in pure water
were complicated by precipitation, UV-vis titrations of sap-
phyrin 2 and porphyrin 9 with BNPP could be carried out at
pH 7.5 (0.01 M HEPES and 0.1 M NaNO3). From these, Ka
values of ca. 400 and e100 M-1 could be calculated for the
formation of the 1:1 sapphyrin-BNPP and porphyrin-BNPP
complexes, respectively, through use of a standard curve fitting
program.32 The sapphyrin 2-BNPP binding constant, Ka, for
the formation of complex 2-BNPP is at least four times higher
than that for the formation of the corresponding porphyrin
complex (i.e. 9-BNPP). This factor of 4 (or more) thus
correlates well with the differences in BNPP hydrolysis rates
observed for these two putative catalysts (factor of 5; see Table
1). This observation, together with the finding that the rate of
hydrolysis increases with decreasing pH (Figure 1), conditions
under which the sapphyrin-to-phosphate binding affinity also
increases,21 further supports our contention that phosphate
binding plays an important role in inducing phosphodiester
hydrolysis. Consistent with this conclusion is the finding that
various putative nucleophiles (hydroxyls, imidazole, D-gluco-
soamine, D-glucamine) fail to effect much in the way of BNPP
hydrolysis rate enhancement when tested on their own (data
not shown).
Figure 1. Absorbance changes monitored after 2 days for the production
of p-nitrophenolate anion from BNPP in the presence (a) and absence (b)
of 1 at differing pH values. For further details pertaining to these
measurements, see Experimental Section.
an alcohol group8 and in the case of guanidinium derivatives
bearing attached OH groups.16 In our case, the use of such
groups was found to lead not only to apparent catalytic
efficiency as described below but also to acceptable water
solubility.
Hydrolysis of BNPP. For the present study, we have chosen
to use bis(4-nitrophenyl)phosphate (BNPP) as a model phos-
phodiester substrate and to study the efficacy of all 10 systems
given in Scheme 1 at pH 7.5.29 From Table 1, it is evident that
the polyhydroxylated sapphyrins, 1 and 2, are the most efficient
BNPP hydrolysis catalysts within the present sapphyrin- and
porphyrin-based set. Not surprisingly, compounds 1 and 2 are
far less effective than most of the metal-containing systems
reported in the literature. However, in contrast to at least some
of these latter systems, 1 and 2 appear to be true catalysts, with
a turnover of 1.2 being obtained for both sapphyrins over the
course of 10 days. Further, at 37 °C and pH 7.5, Km, Vmax, and
k
cat values of 2.0 × 10-3 M (500 M-1), 6.4 × 10-7 M‚h-1, and
3.2 × 10-4 h-1, respectively, could be derived for sapphyrin 1
on the basis of a Michaelis-Menten plot. Explanations for these
findings now follow. In the case of 1, we also studied the effect
of pH on the rate of hydrolysis. The rate increases considerably
at pH values below 7.5 (Figure 1).
First we felt it appropriate to confirm that BNPP can bind to
sapphyrin and can thus serve as a potential substrate for a
putative sapphyrin-based catalysis process. The interaction
between sapphyrin and this model phosphodiester was estab-
lished in the solid state on the basis of a single-crystal X-ray
diffraction analysis of the 1:2 complex formed between dipro-
tonated dihydroxylated sapphyrin 7 and BNPP. This complex,30
which was obtained following extraction experiments carried
out at pH 3.4, is characterized by two different kinds of
phosphate-to-protonated sapphyrin interactions (Figure 2). The
first of these is analogous to what was seen in earlier sapphyrin
phenyl phosphate and diphenyl phosphate structures,21c,e,31 in
that it involves a single phosphate oxygen atom hydrogen bound
(29) Kinetics studies were not performed with 7 as it is insoluble in water.
(30) High-resolution FAB MS and combustion analyses are consistent with the
formation of a 1:1 monoprotonated sapphyrin-BNPP complex following
extraction experiments carried out at pH 6.0. HRMS. Calcd for C54H63N7O10P
(31) Sessler, J. L.; Andrievsky, A.; Genge, J. W. Anion Binding by Sapphyrins.
In AdVances in Supramolecular Chemistry, Vol. 4; Gokel, G. W., Ed.; JAI
Press: Greenwich, CT, 1997; pp 97-142.
(32) (a) Sheridan, R. E.; Whitlock, H. W. J. Am. Chem. Soc. 1986, 108, 7120-
7121. (b) Connors, K. A. Binding Constants; Wiley: New York, 1987; p
141.
[M + H]+: 1000.437. Found: 1000.440. Anal. Calcd for C54
H
N7O10
P
62
(1000.11): 64.86% C; 6.25% H; 9.80% N. Found: 64.61% C; 6.31% H;
9.65% N.
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J. AM. CHEM. SOC. VOL. 128, NO. 2, 2006 435

A R T I C L E S
Kra´l et al.
Figure 2. Two views of the H-bound complex, 7-(BNPP)2, showing partial atom labeling scheme. Also shown is the sapphyrin macrocycle with the BNNP
anions removed for clarity. Thermal ellipsoids are scaled to the 30% probability level. The mode of coordination is different for the two anions. One of the
bis(p-nitrophenyl)phosphate (BNPP) anions is coordinated by a single oxygen to four imine NH groups to one side of the macrocycle. The second BNPP
anion is coordinated by two oxygen atoms. For both anions, one of the nitrophenyl groups lies approximately parallel to macrocycle mean plane. The
relevant hydrogen bonding interactions are as follows: (N2-H2‚‚‚O1a) N‚‚‚O, 3.02(1) Å; N-H‚‚‚O, 147.3(8)°. (N3-H3‚‚‚O1a) N‚‚‚O, 2.73(1) Å; N-H‚
‚‚O, 150.4(8)°. (N4-H4‚‚‚O1a) N‚‚‚O, 3.09(1) Å; N-H‚‚‚O, 138.5(8)°. (N5-H5‚‚‚O1a) N‚‚‚O, 2.97(1) Å; N-H‚‚‚O, 148.9(8)°. (N1-H1‚‚‚O1b) N‚‚‚O,
2.76(1) Å; (N-H‚‚‚O) 119.1(8)°. (N4-H4‚‚‚O2b) N‚‚‚O, 2.76(1) Å; N-H‚‚‚O, 134.4(8)°. The phosphorus to oxygen distances are as follows: O1A‚‚‚P1A,
1.506(6) Å; O2A‚‚‚P1A, 1.480(6) Å; O3A‚‚‚P1A, 1.626(7) Å; O4A‚‚‚P1A, 1.568(8) Å; O1B‚‚‚P1B, 1.431(7) Å; O2B‚‚‚P1B, 1.483(7) Å; O3B‚‚‚P1B,
1.569(7) Å; O4B‚‚‚P1B, 1.630(8) Å.
It is also important to appreciate that the presence of a
sapphyrin phosphate binding element alone (i.e., without the
cooperation of a suitable nucleophilic group) does not, in and
of itself, suffice to obtain an efficient BNPP hydrolysis catalyst.
Support for this conclusion comes from a consideration of
sapphyrins 5 and 6. The amino groups in 5 are protonated at
neutral pH and are thus not expected to be able to function as
nucleophiles. Compound 6, on the other hand, is a bis-sapphyrin
conjugate bearing a polyhydroxyl substituent on only one side
of the compound. The size of this group and the obvious bulk
of the second sapphyrin subunit are not expected (on the basis
Figure 3. Proposed structure of the 1-BNPP complex (R ) p-nitrophenyl).
of Corey-Pauling-Koltun (CPK) models) to allow the side-
chain hydroxyls to orient themselves in such a way as to interact
favorably with the phosphodiester (i.e., in a manner conducive
to bond cleavage).
“arms” does not allow these hydroxyl groups to come within
reasonable reach of the targeted phosphate ester. The same kind
of argument can be invoked in the case of 4. There is not
sufficient sidearm length and flexibility in 4 to allow for
We found that sapphyrin 3, containing somewhat more tightly
bound imidazole substituents, gave lower (but still appreciable)
hydrolysis rates than 1 and 2. This, we believe, further
underscores the importance of establishing appropriate geometric
orientations. Consistent with this supposition, sapphyrin 4,
containing four hydroxyl substituents, was also found to be less
effective than 1 or 2 in terms of promoting BNPP hydrolysis.
Again, this finding is rationalized in terms of steric effects. The
crystal structure of 7 (Figure 2), a sapphyrin bearing two
n-hydroxylpropyl substituents, revealed no hydrogen bonding
between the hydroxyl groups and the phosphate group of the
phosphodiester. In this particular case, the length of the tethering
effective OH-to-P orbital overlap. In the case of 1 and 2, on
the other hand, the longer arms should allow precisely these
kinds of hydrolysis-promoting interactions, as suggested sche-
matically in Figure 3.
Conclusions
In summary, we have shown that appropriately functionalized
sapphyrins can act as efficient catalysts for activated phos-
phodiester bond hydrolysis. From the mechanistic point of view,
our results are consistent with phosphate binding to a sapphyrin
macrocycle serving to enhance the efficacy of appropriately
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Polyhydroxylated Sapphyrins
A R T I C L E S
bound nucleophilic (e.g., OH) groups. While the relative
importance of these two limiting factors is still being assessed
in semiquantitative terms, it is nonetheless clear that phosphate
binding plays an important role, as evidenced by the faster
hydrolysis rates seen for the sapphyrin-based systems as
compared to those for the porphyrin controls. These findings
lead us to suggest that the present ditopic approach could prove
useful in the construction of catalysts capable of enhancing the
hydrolysis of other less-active phosphodiester substrates includ-
ing oligonucleotides. In fact, in preliminary work, it was found
that incubation of a dA-dT mixed 10-mer with sapphyrins 1-3
at pH 7.5 for 2-48 h yielded fragments that were, at least in
part, phosphorylated (cf. Supporting Information), as would be
expected for a cleavage mechanism where hydrolysis plays an
important role. Current work is thus designed to explore further
this intriguing possibility.
Acknowledgment. Financial support from the Ministry of
Education of the Czech Republic, Research Project Nos. MSM
6046137307 and LC512 and the Grant Agency of the Czech
Republic (Grant Nos. 301/04/1315 and 303/05/0336) is grate-
fully acknowledged. This work was supported in part by the
National Institutes of Health (Grant CA 68682 to J.L.S.), as
well as by Project No. AV0Z50520514 awarded by the
Academy of Sciences of the Czech Republic (to J.K. and M.D.).
Supporting Information Available: Description of X-ray data
analysis of the 1:2 complex formed between diprotonated
dihydroxylated sapphyrin 7 and BNPP. Mass spectrometric
analysis of fragments produced upon incubation of dODNs. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA0518474
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