On the question of stepwise vs. concerted cleavage of RNA models promoted by a synthetic dinuclear Zn(ii) complex in methanol: Implementation of a noncleavable phosphonate probe

Article abstract of DOI:10.1039/b918310h

To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the 2-hydroxypropyl aryl and alkyl RNA models (1a-k) promoted by dinuclear Zn(ii) complex (4) at sspH 9.8 and 25 °C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by 4 are k_obs^6a = (1.47 ± 0.09) × 10^-4 s^-1 and k_obs^6b = (2.08 ± 0.09) × 10^-6 s^-1, respectively. The rates of methanolysis of a series of O-aryl phenylphosphonates (8a-f) in the presence of increasing [4] were analyzed to provide binding constants, K_b, and the catalytic rate constant, k_cat^max, for the unimolecular decomposition of the 8:4 Michaelis complex. A Bronsted plot of the log (k_cat^max) vs.sspK_a^phenol (acidity constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log k_cat^max = (-0.80 ± 0.07)sspK_a + (10.2 ± 1.0) which includes the datum for 6a. The datum for 6b, which reacts ～70-fold slower, falls significantly below the linear correlation. The data provide additional evidence consistent with a concerted cyclization of RNA models 1a-k promoted by 4. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b918310h

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
On the question of stepwise vs. concerted cleavage of RNA models promoted
by a synthetic dinuclear Zn(II) complex in methanol: implementation of a
noncleavable phosphonate probe†
David R. Edwards, Wing-Yin Tsang, Alexei A. Neverov and R. Stan Brown*
Received 4th September 2009, Accepted 6th November 2009
First published as an Advance Article on the web 23rd December 2009
DOI: 10.1039/b918310h
To address the question of concerted versus a stepwise reaction mechanisms for the cyclization of the
2-hydroxypropyl aryl and alkyl RNA models (1a–k) promoted by dinuclear Zn(II) complex (4) at
sspH 9.8 and 25 ◦C, the non-cleavable O-hydroxypropyl phenylphosphonate analogues 6a and 6b were
subjected to the catalytic reaction in methanol. These phosphonates did not undergo isomerization in
the study, the only observable methanolysis reaction being release of 1,2-propanediol and the formation
of O-methyl phenylphosphonate. The observed first order rate constants for methanolysis promoted by
4 are kobs = (1.47 0.09) ¥ 10-4 s-1 and kobs = (2.08 0.09) ¥ 10-6 s-1, respectively. The rates of
6a
6b
methanolysis of a series of O-aryl phenylphosphonates (8a–f) in the presence of increasing [4] were
analyzed to provide binding constants, Kb, and the catalytic rate constant, kcatmax, for the unimolecular
decomposition of the 8 : 4 Michaelis complex. A Brønsted plot of the log (kcatmax) vs. sspKa
(acidity
phenol
constant of the conjugate acid of the leaving group in methanol) was fitted to a linear regression of log
kcat = (-0.80 0.07)sspKa + (10.2 1.0) which includes the datum for 6a. The datum for 6b, which
max
reacts ~70–fold slower, falls significantly below the linear correlation. The data provide additional
evidence consistent with a concerted cyclization of RNA models 1a–k promoted by 4.
indicates that the hydrolysis of 3¢5¢-UpU promoted by 2 generates
Introduction
cleavage product along with some isomerized 2¢5¢-UpU, providing
the first evidence that a dinuclear complex at near neutral pH can
stabilize a dianionic phosphorane intermediate enough to allow
pseudorotation and reversion to isomerized starting material.11
Conversely, complex 3 was recently shown, by heavy atom kinetic
isotope effect (kie) studies, to promote the cleavage of 1a by a
concerted process.12
The phosphate diester linkages present in the biological polymers
of RNA and DNA are highly resistant to cleavage as demonstrated
by the estimated t1/2 for hydrolysis at neutral pH and 25 ◦C of
1101 and 30 million years.2 Numerous metallo-enzymes including
paroxonase,3 alkaline phosphatase,4 phospholipase C5 and P1
Nuclease6 that catalyze the hydrolysis of neutral and anionic
phosphate esters have active sites containing a dinuclear Zn(II)
core. These are among Nature’s most proficient catalysts since
they accelerate the hydrolysis of their phosphate ester substrates
by as much as 1017.7
The cleavages of phosphate diesters of general structure 1 pro-
moted by di-Zn(II) complexes (2–4) have been actively studied as
model systems for metallo-RNase enzymatic cleavage of RNA.8,9,10
Of current interest is whether the cleavage reactions proceed in a
concerted fashion or through a 5-membered dianionic phospho-
rane intermediate.11,12 For simple RNA models like 1 and uridine-
5¢-phosphodiesters, LFE relationships13 demonstrated that the
base catalyzed cleavages of the latter, and in all probability the
former, proceed via stepwise processes. It is a current question
whether enzymes and/or small molecule catalysts that cleave
phosphate diesters select for transition states that are altered
relative to those of the uncatalyzed reaction.12 A recent study
We reported that the cleavage of a series of 2-hydroxypropyl
aryl and alkyl phosphates 1a–k was promoted by complex 4 in
methanol14 and ethanol.15 The Brønsted plot of the catalyzed
process in CH3OH was linear for the 1a–k series spanning a
sspKaphenol range from 11.3 to 18.5, with a fit of log(kcatmax) = (-0.85
0.024)sspKaphenol + (12.8 0.4), r2 = 0.9925.14b We also observed that
a synthetic mixture of 1j and 1j¢ (initial ratio 22 : 78) isomerizes in
Department of Chemistry, Queen’s University, 90 Bader Lane, Kingston,
Ontario, Canada K7L 3N6. E-mail: rsbrown@chem.queensu.ca
† Electronic supplementary information (ESI) available: Synthetic pro-
cedures for the preparation of 6a, 6b and 8a–f as well as analytical
data (1H, 31P NMR and HRMS). Partial 1H NMR spectra for the 4
promoted methanolysis of 6b. Description of kinetic procedures and tables
of observed rate constants. See DOI: 10.1039/b918310h
822 | Org. Biomol. Chem., 2010, 8, 822–827
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
s
s
the presence of 4 to generate a thermodynamic ratio of 72 : 28 and
that this process proceeds through cyclic intermediate 5 involving
cleavage of P-OCH3 that is faster than, or equal to, the rate of
isomerization (Scheme 1). Insofar as these studies failed to detect
the intermediacy of a 5-coordinate phosphorane, each is consistent
with the reaction proceeding via a concerted cyclization to form
the complexed cyclic phosphate 5.
ambient temperature, pH 9.8 0.1. Each of three reactions was
quenched after 5 min, 1 h and 24 h by the addition of 10 ¥
10-3 mol dm-3 HCl and 10 ¥ 10-3 mol dm-3 LiCl. The volatiles were
removed under reduced pressure and the reaction components
were reconstituted in CD3OD for H NMR analysis. To sharpen
further the signal intensities in the H NMR spectrum, an addi-
tional aliquot of LiCl was added to the NMR samples, bringing the
total concentration of chloride ion to 0.14 mol dm-3. An identical
procedure was used to study the reaction of isomerically pure 6b.
1
1
Kinetic studies (substrates 8a–f). The rates of methanolysis for
O-aryl phenylphosphonates 8a–f (5 ¥ 10-5 mol dm-3) promoted
by 4 were determined with an Applied Photophysics SX-17MV
stopped-flow reaction analyzer at sspH 9.8 0.1, 25.0 ◦C. Reactions
were monitored for the appearance of the product phenols at 320
(8a), 340 (8b), 284 (8c), 280 (8d), 292 (8e), 276 (8f) nm. Plots of
kobs versus [4]free were fit to eqn (2) from which the unimolecular
rate constants for decomposition of the 8:4 complex, kcatmax, and
the binding constants Kb could be determined. For the definition
of [4]free see footnote.‡
Scheme 1 Isomerization of 1j and 1j¢ promoted by catalyst 4 depicting
the cyclic intermediate 5.
Results
Since failure to detect does not disprove the existence of a
fleeting intermediate, we sought an additional test that adds infor-
mation as to whether the 4-promoted cyclizations are stepwise or
concerted. We disclose results of experiments where phosphonates
6a and 6b, that do not possess a suitable leaving group and
thus cannot form a cyclic ester, were subjected to reaction in the
presence of 4 in methanol. The observation that 6a and 6b undergo
interconversion would lend strong support for the existence of
phosphorane intermediate(s) of general structure 7 (Scheme 2)
and by implication a phosphorane intermediate in the 4-promoted
cyclization of substrates 1.
Three separate experiments were run in which a mixture of
phosphonates 6a and 6b (2.43 ¥ 10-3 mol dm-3, isomeric ratio
of 96 : 4) were reacted in the presence of 2.44 ¥ 10-3 mol dm-3 4 in
CH3OH at room temperature, sspH 9.8 0.1. The three reactions
were terminated after 5 min, 1 h and 24 h using a quench and
solvent removal/reconstitution protocol identical to that previ-
ously employed by us14 in studies of the 4-catalyzed cyclization
of a series of 2-hydroxypropyl alkyl phosphate diesters. In Fig. 1
1
are the partial H NMR spectra (600 MHz) of the reconstituted
reaction contents that indicate the CH3CH< doublet of 6a at d
1.078 ppm (3H, d, J = 6.60 Hz) is converted into one centered at d
1.119 ppm (3H, d, J = 6.60 Hz). This was independently identified
as belonging to the CH3CH< group of 1,2-propanediol resulting
from cleavage of 6a according to the process shown in eqn (1).
6a
The observed rate constant for cleavage of 6a is kobs = (1.47
Scheme 2 Putative isomerization of 6a and 6b promoted by catalyst 4
depicting the hypothetical intermediate 7.
0.09) ¥ 10-4 s-1 which is the average of the two rate constants 1.38 ¥
10-4 s-1 and 1.56 ¥ 10-4 s-1, determined from the signal intensities
of starting material and product for the spectra obtained after
5 min and 1 h. Substrate 6b was prepared in isomerically pure
form (Supporting Information†) and subjected to 4-catalyzed
methanolysis. As before, the reactions were quenched at 5 min,
1 h and 24 h. Integration of the signal intensities corresponding
to 1,2-propanediol and the methyl substituent of 6b (d 1.085 ppm
(3H, d, J = 6.6 Hz)) at 60 min and 24 h leads to a calculated
Experimental
Materials
Methanol (99.8% anhydrous), sodium methoxide (0.5 mol dm-3
solution in methanol) and Zn(CF3SO3)2 were purchased from
Aldrich and used as supplied. 1,3-Bis-N1-(1,5,9-triazacyclo-
dodecyl)propane was synthesized according to the published
procedure.16 The dinuclear (CH3O-):Zn(II)2 complex 4 was pre-
pared as a 2.5 mmol dm-3 solution in methanol by sequential
addition of aliquots of stock solutions of sodium methoxide, 1,3-
bis-N1-(1,5,9-triazacyclododecyl)propane and Zn(CF3SO3)2 such
that the relative amounts were 1 : 1 : 2. This order of addition is
essential: complete formation of 4 requires ~ 40 min (as monitored
by optimization of catalytic activity).14 Preparation of O-aryl
phenylphosphonates 8a–f and 6a, 6b is described in the Supporting
Information.†
kobs = (2.08 0.09) ¥ 10-6 s-1. Shown in Fig. 2 are the partial 1H
6b
NMR spectra obtained following reaction of isomer 6b.
(1)
‡ Previously we have established that triflate ion is an inhibitor of the
catalysis afforded by the dinuclear Zn(II) complex with an inhibition
constant of 14.9 mmol dm-3 from which one can determine the free catalyst
concentration or [4]free at any [triflate]. See ref. 14.
Kinetics studies (substrates 6a and 6b). A 2.43 ¥ 10-3 mol dm-3
mixture of phosphonate 6a (containing 4% 6b) was reacted with
2.44 ¥ 10-3 mol dm-3 of catalyst 4 in anhydrous methanol at
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 822–827 | 823
©

View Article Online
max
Table 1 Catalytic rate constants kcat
and Kb determined for the 4-
catalyzed cleavage of phosphonates 8a–f and 6a,b
phenol
Substrate
sspKa
kcatmax/s-1a
Kb/dm3 mol-1a
6a
6b
8a
8b
8c
8d
8e
8f
17.17b
17.70b
11.30
12.41
13.59
14.33
14.77
15.04
(1.75 ¥10-4)c
(2.48 ¥10-6)c
5.29 0.1
—
—
(2.8 0.2) ¥103
(5.1 0.6) ¥104
(1.1 0.1) ¥104
(4.6 1.2) ¥104
(2.3 0.8) ¥104
(2.0 1.5) ¥104
3.09 0.09
0.48 0.01
(9.2 0.2) ¥10-2
(4.5 0.2) ¥10-2
(1.1 0.1) ¥10-2
a Determined from fit of kobs versus [4]free data to eqn (2). b For the calcu-
Fig. 1 Partial 1H NMR (600 MHz, CD3OD, 25 ◦C) spectra obtained
following the 4-catalyzed methanolysis of a mixture of 6a and 6b (2.43 ¥
10-3 mol dm-3, isomeric ratio of 96 : 4) showing the conversion of starting
material into 1,2-propanediol after a) 24 h; b) 60 min and c) 5 min. The
CH3CH< doublets of 6a and 1,2-propanediol appear at d 1.078 ppm (3H,
d, J = 6.60 Hz) and d 1.119 ppm (3H, d, J = 6.60 Hz), respectively.
6a
6b
max
lation of sspKa and spKa see Supporting Information. c kcat computed
assuming that the Ksb for these two substrates can be approximated by the
average Kb of 2.6 ¥ 104 dm3 mol-1 determined for 8a–f as discussed in the
main text.
Fig. 3 Plot of kobs versus [4]free for the catalyzed methanolysis of 8a (5 ¥
10-5 mol dm-3) determined from the rate of appearance of product phenol
at 320 nm, sspH 9.8 0.1 and 25 ◦C. Fitting of the data to the expression
Fig. 2 Partial 1H NMR (600 MHz, CD3OD, 25 ◦C) spectra obtained
following the 4-catalyzed methanolysis of6b (2.43 ¥ 10-3 mol dm-3) showing
the conversion of starting material into 1,2-propanediol after a) 24 h; b)
60 min and c) 5 min. The CH3CH< doublet of 6b appears at d 1.085 ppm
(3H, d, J = 6.6 Hz).
max
given in eqn (2) gives kcat
= 5.29 0.1 s-1 and Kb = (2.80 0.17) ¥
103 dm3 mol-1, and an intercept (A) = (0.186 0.006) ¥10-3 mol dm-3.
Phosphonates 8a–f were prepared by standard means (Support-
ing Information†) and subjected to 4-catalyzed cleavage at 25 ◦C,
and sspH 9.8 0.1. Shown in Fig. 3 is a representative plot of kobs
versus [4]free for the catalyzed methanolysis of 8a. All saturation
plots showed strong substrate binding and a slight x-intercept.14
These data were analyzed by fitting to a strong binding eqn (2)14,15
to give the binding constants (Kb) and kcatmax values for the cleavage
of the P-OAr group from the 8 : 4 complex listed in Table 1. In
Fig. 4 is the Brønsted plot of log kcat vs. sspKa
which gives a
+ (10.2 1.0). The
max
phenol
best fit of log kcat = (-0.80 0.07)sspKa
max
phenol
fit includes the 6a data; 6b reacts 70-fold slower and its cleavage
rate constant falls below the line.
Fig. 4 Brønsted plot of the log (kcatmax) vs. sspKa (phenol) for the 4 catalyzed
cleavage of phosphonates 8a–f and 6a. Fit of the data to a standard linear
= (-0.80 0.07)sspKa
+ (10.2 1.0).
max
phenol
regression provides log kcat
Data for cleavage of diol from 6a and 6b are indicated by arrows, and their
sspKavalues were assessed as given in the Supporting Information.†
Discussion
kobs = kcatmax(1 + Kbind[S] + [4]freeKb - X)/(2Kb)/[S]
(2)
(a) General considerations
where:
X = (1 + 2Kb[S] + 2[Cat]Kb + Kb [S]2 - 2Kb [Cat][S]
2
2
The methanolysis of 6a (containing 4% of isomer 6b) promoted
by catalyst 4, monitored over a 24 h time period, revealed that
the 2-hydroxypropyl substituent exclusively cleaves to produce
+ [Cat]2Kb )0.5
2
824 | Org. Biomol. Chem., 2010, 8, 822–827
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
1,2-propanediol and O-methyl phenylphosphonate, eqn (1). The
analogous cleavage of the 1-hydroxypropan-2-yl group from
isomerically pure 6b is also effected by 4, albeit about 70-times
The hydrolysis of O-aryl methylphosphonates is subject to base
LG
catalysis with a reported b of -0.69,20 and their hydrolysis,
when doubly coordinated to an exchange inert dinuclear Co(III)
slower than for 6a, kobs = (1.47 0.09) ¥ 10-4 s-1 and kobs = (2.08
0.09) ¥ 10-6 s-1, respectively. No interconversion of 6a and 6b was
detected in either reaction mixture. It is essential to investigate
the reactions of both isomers, since a finding of no isomeric
interconversion during investigation of only one starting material
could be due to a fortuitous choice of the thermodynamically most
stable one.
complex, is proposed to be concerted on the basis of kinetic isotope
6a
6b
18
LG
effect data (18knuc and klg).20 In that instance a b of -1.12 was
reported which is larger than that determined here for the 4-
LG
promoted methanolysis of 8a–f and 6a (b = -0.80). It is difficult
to determine if the respective Brønsted slopes determined here
and in ref. 20 indicate a different degree of P-OAr bond cleavage
in consideration of the fact that neither is normalized by a known
EQ
The methanolytic cleavage of a series of O-aryl phenylphos-
phonates 8a–f promoted by dinuclear catalyst 4 was studied at
sspH 9.8 and 25 ◦C. The Kb values (Table 1) determined for the
series vary from 0.3 to 5.1 ¥ 104 dm3 mol-1 in no particular order
with an average binding constant of (2.7 1.9) ¥ 104 dm3 mol-1.
The binding constants determined for the 4-catalyzed cleavage of
O-aryl phenylphosphonates are similar to, and perhaps slightly
larger than those previously determined by us for the reaction of
methyl aryl phosphate diesters,17 suggesting that replacement of
the P-OMe substituent of a phosphate diester with a C6H5 group to
form the phosphonate has a minor effect on the relative stability of
the 4:substrate complex. This observation is in line with a proposed
mode of binding illustrated in 9 where the actual ligand is ear-
muff like when bound to the two Zn(II) ions and accommodates
b
value in water or methanol nor are the effects of substitution
of a P-CH3 for P-Ph group known.
(b) Mechanistic implications for the cleavage of 2-hydroxypropyl
alkyl and aryl phosphate diesters
The 4-catalyzed cyclization of 2-hydroxypropyl alkyl and aryl
phosphate diesters 1a–k in methanol is a highly efficient reaction
which is accelerated by at least 108 relative to the corresponding
CH3O- promoted reaction. The catalytic reactions were shown
to exhibit transition state stabilization in excess of 20 kcal mol-1
relative to the base-promoted reaction.14 A combination of the
large rate acceleration of the catalytic reaction and the fact that
substrates of general structure 1 are widely used in mechanistic
studies as models for RNA cleavage8–15,18 has prompted detailed
study of the reaction mechanism for this transformation. Two
classical ways to probe the existence of transient intermediates
are to determine if a break exists in a LFE relationship such as
a Brønsted or Hammett plot, or to observe some change in a
recovered starting material such as isomerization or positional
isotopic exchange.21 The absence of a break in the Brønsted plot
determined for the 4-catalyzed cyclization for the entire series of
1a–k spanning sspKaphenol values from 11.3 to 18.5, was considered to
support a concerted cyclization reaction.14b However, as pointed
out in the original report, this assertion rests on the lack of an
anticipated observation of a break in the Brønsted plot at the
-
the bridging substrate >PO2 group. The unbound RO-P, ArO-P
and/or R-P groups are perpendicular to the plane defined by the
two Zn(II) ions and thus point away from the catalytic pocket being
relatively free from steric interactions with the azacyclic ligand.18
quasi-symmetrical point (where sspKa = spKa = 17.7) which is
Nu
s
LG
close to its high pKa end.
max
The kcat
values for the unimolecular decomposition of the
Experiments also show that a synthetic mixture of 1j/1j¢ (see
Scheme 1, initial ratio of 22/78) undergoes fast equilibration in
the presence of equimolar 4 to generate a 72/28 thermodynamic
mixture. The pseudo-first order rate constant for the approach to
equilibrium is (7.0 1.0) ¥ 10-3 s-1 (t1/2 = 99 s). Positional isotope
exchange studies in CD3OH indicate that both 1j and 1j¢ lose
OCL3 on the same timescale as the isomerization occurs. Taken
together, these results indicate that isomerization of 1j/1j¢ occurs
through a cyclic phosphate intermediate (5) with concomitant
loss of OCL3 as in Scheme 1. The data are consistent with
concerted cleavage of the P-OLG bond although not uniquely so.
Additional intermediates formed along the reaction coordinate,
such as 5-coordinate phosphoranes, could be formed, but these
must exclusively break down to 5 and thus do not play any
extensive role in the direct 1j to 1j¢ isomerization process.14b
In this work we have used a third test, employing poorly
reactive phosphonates 6a and 6b, to attempt to distinguish
between concerted and step-wise mechanisms for the 4-catalyzed
cyclization of 1a–k.22 Such a test has been used by Lo¨nneberg and
co-workers23 and Mikkola and Williams11 to study isomerization
of phosphoryl groups between 2¢- and 3¢- positions of ribose
4 : 8a–f Michaelis complexes span a factor of ~500. Direct compar-
ison can be made between the kcatmax terms determined for 8a–e and
a series of methoxy aryl phosphate diesters17 containing identical
leaving groups which indicates that the phosphonates react some
130–200 times faster than the diester containing the same leaving
max
group. The kcat
values for 6a and 6b can be calculated based
on the reasonable assumption that their binding constants with
catalyst 4 are the same as the average binding constant determined
ave
for substrates 8a–f (Kb = 2.7 ¥ 104 dm3 mol-1). Accordingly one
computes that substrates 6a and 6b were each 84% bound19 to
max
4 under the experimental conditions and so kcat
= kobs6/0.84
(Table 1).
The Brønsted plot of log kcat
regression of log kcat
max
s
s
phenol
vs. pKa
(Fig. 4) fits a
max
s
= (-0.80 0.07) pKa + (10.2 1.0).
s
The fit includes the datum for 6a but not that for 6b, since the
latter reacts 70 times slower and its datum falls below the line. A
6a
phenol
factor of 2.5 between the kobs and kobs6b, is due to the DsspKa
(17.17 for 6a and 17.70 for 6b see Supporting Information†),
while the remaining ~30-fold is suggested to result from the
secondary/primary alcohol substitution pattern.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 822–827 | 825
©

View Article Online
rings. In these and the present cases, a concerted process which
requires an in-line attack through a trigonal bipyramidal TS
with leaving and entering groups at the apical position cannot
occur, leaving the only possible pathway to be a two step one
which would be clearly evident if isomerization of 6a/6b could
be demonstrated. Phosphonates are generally at least 10 times
more susceptible to nucleophilic attack than their correspond-
ing phosphates24,25 (130–300 fold faster in this work with aryl
phenylphosphonates vs. methoxy aryl phosphates vide supra) and
4-catalyzed intramolecular attack of the 2-hydroxypropyl oxygen
on the central P of phosphate diesters is 1000 to 5000 times faster
than direct nucleophilic attack of coordinated methoxide.14–17,18
Thus, it is difficult to envision that a similar attack cannot occur in
phosphonates 6a/6b to give a 5-coordinate intermediate, although
isomerization of these species via a cyclic ester as was seen with
the diesters 1j/1j¢ is precluded. A proposed mechanism by which
phosphonates 6a and 6b might undergo isomerization is via a suite
of pseudo-rotating phosphoranes 10a,b and 11a,b as in Scheme 3.
the intramolecular cyclization, and thus should occur independent
of whether the 2-hydroxypropyl group is present. The fact that the
kcat for cleavage of the leaving group from 6a lies on the line of
max
the Brønsted plot constructed for the O-aryl phenylphosphonates
(8) suggests that all these phenylphosphonate esters react by a
common mechanism that probably involves a concerted attack of
Zn(II) coordinated methoxide on the P. This is similar to one of
the mechanisms that was proposed for cleavage of aryl methyl
phosphate diester DNA models by 4.17 That the rate constant for
the cleavage of 6b lies considerably below this line likely results
from a steric effect in the displacement of a secondary alcoholate
relative to a primary one.14b
Conclusions
An observation that 6a ↔ 6b isomerization occurred during
the slow cleavage of these phosphonates would provide pow-
erful evidence for the existence of 5-coordinate phosphorane
intermediates, and thus lend credence to the possibility that
such intermediates could occur during the cleavage of phosphate
diesters 1a–k. However, no observed isomerization does not
provide as convincing evidence for the absence of phosphorane
intermediates. Such intermediates could be formed but their
lifetime is too short, or they are too conformationally restricted
when bound to the catalyst, to allow the pseudorotations required
for isomerization. It was noted,11 in a recent report of a man-
made catalyst promoting the isomerization of 3¢5¢-UpU to 2¢5¢-
UpU concurrent with cleavage of the former, that “enzymes or
ribozymes that catalyze RNA cleavage do not appear to catalyze
isomerization as well, but presumably in these cases, the confines of
the active site even more firmly direct the reaction toward cleavage
if similar phosphoranes are involved”.
Scheme 3 Postulated process by which isomerization of 6a and 6b might
occur. Equatorial substituents on phosphoranes identified by dashed
triangle.
On the other hand, the lack of isomerization of 6a/6b and the
previous data14b are in full accord with the simplest explanation of
a concerted process for the catalytic cleavage of 1a–k.
From X-ray crystallographic structure considerations of the
14a
18b
Zn(II)2
and Cu(II)2
complexes of the 1,3-bis-N1-(1,5,9-
triazacyclododecyl)propane ligand, intramolecular attack of the
hydroxypropyl anion on a doubly Zn(II)-coordinated 6a or 6b
can occur most easily perpendicular to the plane defined by the
Zn(II)-O--P–O--Zn(II) moiety and in line with the P-Ph bond. This
gives phosphoranes 10a or 10b in which the attacking group and
the phenyl substituent occupy apical positions while the two O--
Zn(II) units occupy equatorial positions. Interconversion of 10a
to 10b requires two pseudorotations to form 11a and then 11b
in which each of the oxygens of the 5-membered cycle is in turn
apical to an apical O-Zn(II) group, violating one of the Westheimer
rules.26 The final formation of the isomerized product (6b from 6a
or vice versa) from the process proposed in Scheme 3 requires a
third pseudorotation placing the Ph and departing oxyanion in
apical positions.
An alternative explanation, less likely in our opinion for this
system due to the restrictive geometry of the complex, deems that
the initial attack of the hydroxypropyl oxyanion occurs anti to
one of the O--Zn(II) units, meaning that 6a would directly give 11b
and 6b would give 11a. These would have to interconvert to effect
isomerization again violating a Westheimer rule.
Acknowledgements
The authors acknowledge the financial assistance of NSERC and
the Canada Foundation for Innovation. This project also received
support from the Defense Threat Reduction Agency-Joint Science
and Technology Office, Basic and Supporting Science Division,
Grant # HDTRA-08-1-0046.
Notes and references
1 N. H. Williams, B. Takasaki, M. Wall and J. Chin, Acc. Chem. Res.,
1999, 32, 485.
2 G. K. Schroeder, C. Lad, P. Wyman, N. H. Williams and R. Wolfenden,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 4052.
3 H. Yang, P. D. Carr, S. Y. McLoughlin, J. W. Liu, I. Horne, X. Qiu,
C. M. J. Jeffries, R. J. Russell, J. G. Oakeshott and D. L. Ollis, Protein
Eng., 2003, 16, 135.
4 (a) E. E. Kim and H. W. Wyckoff, J. Mol. Biol., 1991, 218, 449; (b) B.
Stec, K. M. Holtz and E. R. Kantrowitz, J. Mol. Biol., 2000, 299, 1303.
5 (a) S. Hansen, L. K. Hansen and E. Hough, J. Mol. Biol., 1993, 231,
870; (b) S. Hansen, L. Kristian, H. Hough and E. Hough, J. Mol. Biol.,
1992, 225, 543; (c) E. Hough, L. K. Hansen, B. Birknes, K. Jynge, S.
Hansen, A. Hordvik, C. Little, E. Dodson and Z. Derewenda, Nature,
1989, 338, 357.
Despite the above considerations, 6a/6b isomerization is not
observed and the only reaction products detected were those of the
cleavage of the O-alkyl substituents, namely 1,2-propanediol and
O-methyl phenylphosphonate. This latter reaction does not involve
6 C. Romier, R. Dominguez, A. Lahm, O. Dahl and D. Suck, Proteins,
1998, 32, 414; A. Volbeda, A. Lahm, F. Sakiyama and D. Suck,
EMBO J., 1991, 10, 1607.
7 P. J. O’Brien and D. Herschlag, Biochemistry, 2001, 40, 5691.
826 | Org. Biomol. Chem., 2010, 8, 822–827
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
AVE
8 (a) F. Mancin and P. Tecillia, New J. Chem., 2007, 31, 800; (b) J. Weston,
Chem. Rev., 2005, 105, 2151; (c) F. Mancin, P. Scrimin, P. Tecilla and
U. Tonellato, Chem. Commun., 2005, 2540; (d) J. R. Morrow and O.
Iranzo, Curr. Opin. Chem. Biol., 2004, 8, 192.
9 (a) G. Feng, J. C. Mareque-Rivas and N. H. Williams, Chem. Commun.,
2006, 1845; (b) G. Feng, D. Natale, R. Prabaharan, J. C. Mareque-Rivas
and N. H. Williams, Angew. Chem., Int. Ed., 2006, 45, 7056.
10 (a) M.-Y. Yang, O. Iranzo, J. P. Richard and J. R. Morrow, J. Am.
Chem. Soc., 2005, 127, 1064; (b) O. Iranzo, T. Elmer, J. P. Richard and
J. R. Morrow, Inorg. Chem., 2003, 42, 7737; (c) O. Iranzo, J. P. Richard
and J. R. Morrow, Inorg. Chem., 2004, 43, 1743.
11 H. Linjalahti, G. Feng, J. C. Mareque-Rivas, S. Mikkola and N. H.
Williams, J. Am. Chem. Soc., 2008, 130, 4232.
12 T. Humphrey, S. Iyer, O. Iranzo, J. R. Morrow, J. P. Richard, P. Paneth
and A. C. Hengge, J. Am. Chem. Soc., 2008, 130, 17858.
13 H. Lo¨nnberg, R. Stro¨mberg and A. Williams, Org. Biomol. Chem.,
2004, 2, 2165.
14 (a) S. E. Bunn, C. T. Liu, Z.-L. Lu, A. A. Neverov and R. S. Brown,
J. Am. Chem. Soc., 2007, 129, 16238; (b) W.-Y. Tsang, D. R. Edwards,
S. A. Melnychuk, C. T. Liu, A. A. Neverov, N. H. Williams and R. S.
Brown, J. Am. Chem. Soc., 2009, 131, 4159.
15 C. T. Liu, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc., 2008,
130, 16711.
16 J. Kim and H. Lim, Bull. Korean Chem. Soc, 1999, 20, 491.
17 A. A. Neverov, C. T. Liu, S. E. Bunn, D. Edwards, C. J. White, S. A.
Melnychuk and R. S. Brown, J. Am. Chem. Soc., 2008, 130, 6639.
18 (a) R. S. Brown, Z.-L. Lu, C. T. Liu, W. Y. Tsang, D. R. Edwards and
A. A. Neverov, J. Phys. Org. Chem., 2009, DOI: 10.1002/poc.1584;
(b) Z.-L. Lu, C. T. Liu, A. A. Neverov and R. S. Brown, J. Am. Chem.
Soc., 2007, 129, 11642.
19 Even considering the relatively large error limit on the Kb
((2.7
1.9) ¥ 104 dm3 mol-1) the binding of substrates 6/6a only varies from
76% to 87%.
20 G. Feng, E. A. Tanifum, H. Adams, A. C. Hengge and N. H. Williams,
J. Am. Chem. Soc., 2009, 131, 12771.
21 A. Williams, Concerted Organic and Bio-organic Mechanisms, CRC
Press, Boca Raton, 2000.
22 The use of a non-cleavable phosphonate probe was instrumental in
the detection of a pentacoordinate phosphorane intermediate in the 2
catalyzed cleavage of 3¢5¢-UpU (ref. 11).
23 (a) T. Lo¨nnberg, S. Kralikova, I. Rosenberg, E. Maki and H. Lo¨nnberg,
Collect. Czech. Chem. Commun., 2006, 71, 859; (b) E. Maki, M.
Oivanen, P. Poijarvi and H. Lo¨nnberg, J. Chem. Soc., Perkin Trans.
2, 1999, 2493.
24 N. H. Williams, W. Cheung and J. Chin, J. Am. Chem. Soc., 1998,
120, 8079, provide data that the dicobalt(III) complex of methyl p-
nitrophenyl phosphate reacts with -OH with a second order rate
constant of 4.6 ¥ 104 dm3 mol-1 s-1while uncoordinated phosphate has
-OH
a k2
of ~1.3 ¥ 10-6dm3 mol-1 s-1. By contrast, the k2-OHrate constant
with the Co(III) complex of p-nitrophenyl methylphosphonate is 4.1 ¥
105 dm3 mol-1 s-1 while that with p-nitrophenyl methylphosphonate
itself is 1.2 ¥ 10-5dm3 mol-1 s-1(ref. 20).
25 Methoxide- and La3+-promoted methanolysis diethyl aryl phosphate
triesters is generally about 60–300 times slower than the corresponding
reactions on ethyl aryl methylphosphonate esters: see: T. Liu, A. A.
Neverov, J. S. W. Tsang and R. S. Brown, Org. Biomol. Chem., 2005,
3, 1525; R. E. Lewis, A. A. Neverov and R. S. Brown, Org. Biomol.
Chem., 2005, 3, 4082.
26 (a) F. H. Westheimer, Acc. Chem. Res., 1968, 1, 70; (b) G. R. Thatcher
and R. Kluger, Adv. Phys. Org. Chem., 1989, 25, 99.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 822–827 | 827
©