Hydrolysis and desulfurization of the diastereomeric phosphoromonothioate analogs of uridine 2',3'-cyclic monophosphate

Article abstract of DOI:10.1021/jo960118r

Hydrolyses of the two diastereomeric phosphoromonothioate analogs of uridine 2',3'-cyclic monophosphate [(R(P))- and (S(P))-2',3'-cUMPS] at 363.2 K have been followed by HPLC over pH-range 0-12. In aqueous alkali (pH > 9) only base-catalyzed endocyclic phosphoester hydrolysis to a nearly equimolar mixture of uridine 2'- and 3'-phosphoromonothioates (2'- and 3'-UMPS) takes place, analogously to the hydrolysis of uridine 2',3'-cyclic monophosphate (2',3'-cUMP). The (R(P))- and (S(P))-2',3'-cUMPS are hydrolyzed 50 and 30%, respectively, more slowly than 2',3'-cUMP. Under neutral and acidic conditions, desulfurization of the cyclic thiophosphates to 2',3'-cUMP competes with the phosphoester hydrolysis, both reactions being acid-catalyzed at pH < 5. The desulfurization is most pronounced in strongly acidic solutions ([HCl] > 0.1 mol L^-1), where more than 90% of the starting material is degraded via this route. At pH < 2, the thioates are considerably, i.e., more than 1 order of magnitude, more stable than 2',3'-cUMP. While the hydrolysis of 2',3'-cUMP is second-order in hydronium-ion concentration, that of 2',3'-cUMPS exhibits a first-order dependence. The reactivities of the two diastereomers are comparable with each other over the entire pH-range studied, the most significant difference being that the pH-independent desulfurization at pH > 5 is with the R(P)-isomer 5-fold faster than with the S(P)-isomer. In contrast to 2',3'-cUMP, depyrimidination of the starting material (i.e., release of the uracil base) competes with the hydrolysis of the thiophosphate moiety under neutral conditions (pH 6-8).

Full text of DOI:10.1021/jo960118r

J . Org. Chem. 1996, 61, 3951-3955
3951
Hyd r olysis a n d Desu lfu r iza tion of th e Dia ster eom er ic
P h osp h or om on oth ioa te An a logs of Ur id in e 2′,3′-Cyclic
Mon op h osp h a te
Mikko Ora, Mikko Oivanen,* and Harri Lo¨nnberg
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
Received J anuary 18, 1996X
Hydrolyses of the two diastereomeric phosphoromonothioate analogs of uridine 2′,3′-cyclic mono-
phosphate [(RP)- and (SP)-2′,3′-cUMPS] at 363.2 K have been followed by HPLC over pH-range
0-12. In aqueous alkali (pH > 9) only base-catalyzed endocyclic phosphoester hydrolysis to a nearly
equimolar mixture of uridine 2′- and 3′-phosphoromonothioates (2′- and 3′-UMPS) takes place,
analogously to the hydrolysis of uridine 2′,3′-cyclic monophosphate (2′,3′-cUMP). The (RP)- and
(SP)-2′,3′-cUMPS are hydrolyzed 50 and 30%, respectively, more slowly than 2′,3′-cUMP. Under
neutral and acidic conditions, desulfurization of the cyclic thiophosphates to 2′,3′-cUMP competes
with the phophoester hydrolysis, both reactions being acid-catalyzed at pH < 5. The desulfurization
is most pronounced in strongly acidic solutions ([HCl] > 0.1 mol L-1), where more than 90% of the
starting material is degraded via this route. At pH < 2, the thioates are considerably, i.e., more
than 1 order of magnitude, more stable than 2′,3′-cUMP. While the hydrolysis of 2′,3′-cUMP is
second-order in hydronium-ion concentration, that of 2′,3′-cUMPS exhibits a first-order dependence.
The reactivities of the two diastereomers are comparable with each other over the entire pH-range
studied, the most significant difference being that the pH-independent desulfurization at pH > 5
is with the RP-isomer 5-fold faster than with the SP-isomer. In contrast to 2′,3′-cUMP, depyrim-
idination of the starting material (i.e., release of the uracil base) competes with the hydrolysis of
the thiophosphate moiety under neutral conditions (pH 6-8).
In tr od u ction
In spite of extensive usage of nucleoside phospho-
rothioates in biochemical studies, only few kinetically
relevant investigations on their chemical hydrolysis
have been reported. We have recently described the
kinetics for concurrent hydrolysis, desulfurization, and
intramolecular transesterification of the diastereomeric
phosphoromonothioate analogs (2a ,b) of uridylyl(3′,5′)-
uridine over a wide pH range.8 One of the parallel
reactions, viz. hydrolysis, proceeds via intermediary
formation a 2′,3′-cylic phosphorothioate (1a or 1b ),
although this initial product is usually not markedly
accumulated. A similar cyclic intermediate is also formed
by the action of RNases and hammerhead ribozymes
on internucleosidic phosphoromonothioate linkages.5,7
Accordingly, quantitative information on the hydroly-
tic behavior of 1a ,b appears relevant. It has been
shown previously1,2 that 1a is hydrolytically more
stable than its phosphate analog, uridine 2′,3′-cyclic
monophosphate (2′,3′-cUMP; 1c), the so-called “thio-
effect” being 6 in aqueous alkali and 200 in aqueous
acid (0.15 mol L-1 HClO4). The base-catalyzed reaction
was reported to yield a mixture of uridine 2′- and
3′-thiomonophosphates (3a ;4a ), whereas under acidic
conditions the major products (>70%) were desulfurized
nucleotides, 2′- and 3′-UMP (3b;4b). The present study
was undertaken to learn more about the kinetics of
the competing hydrolysis and desulfurization reactions
of 1a and 1b in comparison with the hydrolysis of the
natural nucleotide 1c. We feel that these results are
useful in analyzing the fate of phosphoromonothioate
linkages in ribozyme studies.
The usefulness of chiral nucleoside phosphorothioates
as stereochemical probes in mechanistic studies of enzyme-
catalyzed phosphate transfer was first demonstrated by
Eckstein et al., who synthesized the diastereomeric
uridine 2′,3′-cyclic phosphoromonothioates (2′,3′-cUMPS;
1a ,b)1 and showed that of these only the one having the
endo (RP) configuration at phosphorus (1a ) is a substrate
for RNase A.2,3 Subsequently, the diastereomers of 2′,3′-
cUMPS and their guanosine analog, 2′,3′-cGMPS, were
used to elucidate the stereochemistry of the second step
of RNA-hydrolysis catalyzed by RNase A and RNase T1,
respectively.4 Since these pioneering works, chiral thio-
phosphate esters have become one of the most widely
used tools for stereochemical and mechanistic studies of
biological reactions of nucleic acid constituents.5 More
recently, the stereochemical course of the ribozyme-
catalyzed phosphodiester hydrolysis and the potential
metal ion binding sites have been probed by inserting
an RP or SP phosphorothioate linkage at a given site of
the substrate chain.6,7
* To whom correspondence should be addressed. Fax: +35821-
3336700. E-mail: mikko.oivanen@utu.fi.
X Abstract published in Advance ACS Abstracts, May 15, 1996.
(1) Eckstein, F.; Gindl, H. Chem. Ber. 1968, 101, 1670.
(2) Eckstein, F. FEBS Lett. 1968, 2, 85.
(3) Saenger, W.; Eckstein, F. J . Am. Chem. Soc. 1970, 92, 4712.
(4) (a) Usher, D. A.; Richardson, D. I., J r.; Eckstein, F. Nature
(London) 1970, 228, 663. (b) Eckstein, F.; Schulz, H. H.; Ru¨terjans,
H.; Haar, W.; Maurer, W. Biochemistry 1972, 11, 3507.
(5) For reviews see: (a) Eckstein, F. Ann. Rev. Biochem. 1985, 54,
367. (b) Frey, P. Adv. Enzymol. Relat. Areas Mol. Biol. 1989, 62, 119.
(c) Hall, C. R.; Inch, T. D. Tetrahedron 1980, 36, 2059.
(6) Padgett, R. A.; Podar, M.; Boulanger, S. C.; Perlman, P. S. Science
1994, 266, 1685.
(7) Pyle, A. M. Science 1993, 261, 709.
(8) Oivanen, M.; Ora, M.; Almer, H.; Stro¨mberg, R.; Lo¨nnberg, H.
J . Org. Chem. 1995, 60, 5620.
S0022-3263(96)00118-1 CCC: $12.00 © 1996 American Chemical Society

3952 J . Org. Chem., Vol. 61, No. 12, 1996
Ora et al.
F igu r e 1. Time-dependent product distribution for the hy-
drolysis of (SP)-2′,3′-cUMPS (1b) in 0.1 mol L-1 aqueous
hydrogen chloride at 363.2 K. Notation: (O) (SP)-2′,3′-cUMPS;
(b) mixture of 2′- and 3′-UMPS; (9) mixture of 2′- and 3′-UMP;
(3) uridine.
Resu lts a n d Discu ssion
p H-Dep en d en t P r od u ct Distr ibu tion s. Decomposi-
tion of the diastereomeric monothioate analogs of 2′,3′-
cUMP (2′,3′-cUMPS; 1a ,b) was followed by HPLC over a
wide acidity range [from H0 ) 0 to -lg([H+]/mol L-1) )
12] at 363.2 K, and the products were identified by
spiking with authentic samples. Under strongly acidic
conditions ([H+] > 0.1 mol L-1), both diastereomers are
hydrolyzed to a mixture of 2′- and 3′-UMP ([3b]/[4b] )
2/3) and their thioate analogs 2′- and 3′-UMPS ([3a ]/[4a ]
) 2/3). During the early stages of the reaction the ratio
[2′/3′-UMP]/[2′/3′-UMPS] is 9:1 (the data for 1b at pH 1
given as an example in Figure 1). 2′- and 3′-UMPS (3a ,
4a ) are subsequently dethiophosphorylated to uridine (5)
and desulfurized to 2′/3′-UMP, as described previously.9
2′- and 3′-UMPS are neither interconverted nor cyclized
to 1a ,b under the experimental conditions.9 Accordingly,
1a and 1b undergo two parallel reactions: irreversible
ring opening to 2′/3′-UMPS and desulfurization to 2′,3′-
cUMP that is rapidly hydrolyzed10-12 to 2′/3′-UMP.
Under less acidic conditions (pH 2 to 5), appearance of
2′,3′-cUMP as an intermediate is experimentally observed
(Figure 2). Owing to relatively rapid dephosphorylation
of 2′- and 3′-UMP to uridine at pH > 2,11,13 these
compounds are not accumulated as markedly as in more
acidic solutions. In summary, the reactions taking place
under acidic conditions may be depicted by routes A and
B in Scheme 1.
F igu r e 2. Time-dependent product distribution for the hy-
drolysis of (SP)-2′,3′-cUMPS (1b) in a formic acid buffer at pH
4.0 ([HCOOH]/[HCOONa] ) 0.01/0.02 mol L-1; I ) 0.1 mol
L-1 with NaCl) at 363.2 K. Notation: (O) (SP)-2′,3′-cUMPS;
(0) 2′,3′-cUMP; (9) mixture of 2′- and 3′-UMP; (3) uridine.
Sch em e 1
Under neutral conditions (pH 6-8), hydrolysis of both
(RP)- and (SP)-2′,3′-cUMPS yields a considerable amount
of uracil (up to 40% of the products) in addition to 2′,3′-
cUMP, 2′/3′-UMP and uridine. In striking contrast,
neither 2′,3′-cUMP nor 2′/3′-UMP13 give uracil under the
same conditions, and only traces of uracil (<5%) were
detected to be formed from 2′- and 3′-UMPS. Accord-
ingly, the base moiety appears to be cleaved directly from
2′,3′-cUMPS. To verify the identity of the product
assumed to be uracil, this product was isolated by HPLC
and characterized spectroscopically. Both the mass
spectrum (EI) and H NMR spectrum were identical with
those of an authentic sample of uracil. It is worth noting
that the release of uracil is significant only at pH < pKa
of the uracil base (pH < 9). Evidently, deprotonation of
the base moiety of 2′,3′-cUMPS retards the depyrimidi-
nation. No attempts were made to clarify the mechanism
of this reaction.
1
(9) Ora, M.; Oivanen, M.; Lo¨nnberg, H. J . Chem. Soc., Perkin Trans.
2, in press.
At high pH (pH > 9), the endocyclic phosphoester
hydrolysis of 2′,3′-cUMPS to a mixture of 2′- and 3′-UMPS
(route A, [3a ]/[4a ] ) 0.45/0.55) is the only reaction
detected. Accordingly, in addition to routes A and B,
(10) Oivanen, M; Lo¨nnberg, H. J . Org. Chem. 1989, 54, 2556.
(11) Eftink, M. R.; Biltonen, R. L. Biochemistry 1983, 22, 5134.
(12) Abrash, H. I.; Cheung, C.-C. S.; Davis, J . C. Biochemistry 1967,
6, 1298.
(13) Oivanen, M.; Lo¨nnberg, H. Acta Chem. Scand. 1990, 44, 239.

Analogs of Uridine 2′,3′-Cyclic Monophosphate
J . Org. Chem., Vol. 61, No. 12, 1996 3953
F igu r e 3. pH-rate profiles for the decomposition of (RP)-2′,3′-
cUMPS (1a , 3), (SP)-2′,3′-cUMPS (1b, ]), and 2′,3′-cUMP (1c,
O) at 363.2 K. The ionic strength of the solutions was adjusted
to 0.1 mol L-1 with sodium chloride.
F igu r e 4. pH-rate profiles for the hydrolytic reactions of (RP)-
2′,3′-cUMPS (1a ) at 363.2 K. The ionic strength of the
solutiuons adjusted to 0.1 mol L-1 with sodium chloride.
Notation: (]) phosphoester hydrolysis; ([) desulfurization; (3)
depyrimidination.
route C in Scheme 1 is also utilized under neutral and
slightly alkaline conditions.
To exclude the possibility of an oxidative desulfuriza-
tion at the pH-independent region (pH > 4), the reaction
solutions were, firstly, thoroughly purged with nitrogen
before initiating the reactions. Secondly, EDTA (1 mmol
L-1) was added to the reaction buffers (acetate and
triethanolamine buffers), and thirdly, the effect of added
dithiotreitol (1 mmol L-1) on reaction kinetics was also
determined. While purging with nitrogen and addition
of EDTA had no effect on the kinetics of desulfurization,
addition of dithiotreitol slightly accelerated both the
hydrolysis and desulfurization. Accordingly, no indica-
tion of possible oxidative nature of the desulfurization
could be obtained.
The time-dependent product distributions discussed
above were used to calculate the partial rate constants
for the phosphoester hydrolysis (k1), desulfurization (k2)
and depyrimidination (k5), of (RP)- and (SP)-2′,3′-cUMPS
(see Scheme 1), as described in detail in the Experimental
Section. The rate constants obtained at different hydro-
nium ion concentrations gave the pH-rate profiles
discussed below.
F igu r e 5. pH-rate profiles for the hydrolytic reactions of (SP)-
2′,3′-cUMPS (1b) at 363.2 K. The ionic strength of the
solutiuons adjusted to 0.1 mol L-1 with sodium chloride.
Notation: (]) phosphoester hydrolysis; ([) desulfurization; (3)
depyrimidination.
Sch em e 2
p H-Ra te P r ofiles a n d Mech a n ism s for th e P h os-
p h oester Hyd r olysis a n d Desu lfu r iza tion . Figure 3
shows the pH-rate profile for the decomposition of (RP)-
and (SP)-2′,3′-cUMPS at 363.2 K. As seen, the reactivities
of the two diastereomers are comparable, the SP-isomer
being 20-50% more reactive than the RP-diastereomer
over the entire pH range studied. Under basic and
neutral conditions, the diastereomeric 2′,3′-cUMPS react
approximately as rapidly as 2′,3′-cUMP, but under acidic
conditions they are considerably more stable. On com-
paring the decomposition rates of 2′,3′-cUMPS and 2′,3′-
cUMP, one has to bear in mind that with 2′,3′-cUMP the
only reaction over the entire pH range is the endocyclic
phosphodiester hydrolysis to 2′/3′-UMP, whereas with the
thioate analogs desulfurization to 2′,3′-cUMP competes
with the phosphodiester hydrolysis, in particular in acidic
solutions where this reaction predominates. Further-
more, depyrimidination markedly contributes to the total
decomposition rate at pH 6-8. The pH-rate profiles for
the individual partial reactions are depicted in Figures
4 and 5. Generally speaking, the competition between
hydrolysis, desulfurization, and depyrimidination is very
similar with both (RP)- and (SP)-2′,3′-cUMPS. In fact, the
only significant difference is that the desulfurization of
the RP-isomer is around neutral pH slightly more favored
over the phosphodiester hydrolysis than that of the SP-
isomer.
It has been shown previously10,11 that the observed
pseudo-first-order rate constant, k1, of the hydrolysis of
nucleoside 2′,3′-cyclic monophosphates may be expressed
by eq 1 over the pH range where the predominant ionic
k1 ) ka[H+]2 + kb[H+] + kc + kd[OH-]
(1)
form is the phosphodiester monoanion (pH > 0.5). The
partial rate constants ka, kb, kc, and kd are those indicated
in Scheme 2. Table 1 records the values obtained for the
decomposition of (RP)- and (SP)-2′,3′-cUMPS and 2′,3′-

3954 J . Org. Chem., Vol. 61, No. 12, 1996
Ora et al.
Ta ble 1. Ra te Con sta n ts for th e Va r iou s P a r tia l
Rea ction s of th e Hyd r olysis a n d Desu lfu r iza tion of (RP )-
a n d (SP )-2′,3′-cUMP S (1a ,b) a n d of Hyd r olysis of
2′,3′-cUMP (1c) a t 363.2 Ka
Sch em e 3
(RP)-cUMPS
(SP)-cUMPS
cUMP
ka/L2 mol-2 s-1
kb/10-3 L mol-1 s-1
kc/10-6 s-1
b
3.1
0.29
0.085
0.045
6.0
b
4.5
16.1
51
0.98
0.18
0.57
0.13
0.030
10.1
0.24
kd/L mol-1 s-1
ke/L2 mol-2 s-1
kf/10-3 L mol-1 s-1
kg/10-6 s-1
1.5
a
The ionic strength adjusted to 0.1 mol L-1 with sodium
b
chloride. For the rate constants see the schemes. Could not be
determined.
cUMP by fitting the observed rate constants of hydrolysis
(k1) to eq 1 and those of desulfurization to eq 2.
k2 ) ke[H+]2 + kf[H+] + kg
(2)
These data, and the shape of the pH-rate profiles
(Figures 4 and 5), reveal one basic difference between
the hydrolysis of 2′,3′-cUMP and 2′,3′-cUMPS. The
dominant kinetic term at pH < 4 is kb[H+] with the
hydrolysis and desulfurization of 2′,3′-cUMPS and ka[H+]2
with the hydrolysis of 2′,3′-cUMP. In other words, 2′,3′-
cUMPS exhibits first-order and 2′,3′-cUMP second-order
dependence on hydronium ion concentration. Only the
desulfurization to 2′,3′-cUMP shows some indication of
second-order dependence on hydronium ion concentration
at pH < 2. Mechanistically, this means that the hydro-
lytic reactions of 2′,3′-cUMPS proceed via neutral phos-
phodiester, while 2′,3′-cUMP reacts via the phosphodi-
ester monocation. A similar difference has previously
been observed in the behavior of 3′,5′-UpU (2c) and its
phosphoromonothioate analogs (2a ,b).8 Replacement of
one of the nonbridging phosphate oxygens with sulfur
appears to retard protonation of neutral phosphodiester
to such an extent that hydrolysis and desulfurization via
the monocationic starting material cannot compete with
the reactions occuring via the neutral species. Conse-
quently, the monothioates are in very acidic solutions
hydrolytically much more stable than their oxygen
counterparts, while the hydrolysis rates under mildly
acidic conditions are comparable.
Sch em e 4
migration at pH > 1 but becomes slower than these
reactions under more acidic conditions. Why the thio-
phosphorane intermediates derived from 2′,3′-cUMPS
and 3′,5′-Up(s)U do behave so differently remains to be
answered.
In all likelihood, the acid-catalyzed hydrolysis and
desulfurization of 2′,3′-cUMPS proceed via a common
pentacoordinated intermediate14 obtained by the attack
of a water molecule on the phosphorus atom of the
neutral (and possibly monocationic) cyclic phosphorothio-
ate (Scheme 3). Cleavage of the mercapto ligand as
dihydrogen sulfide gives 2′,3′-cUMP (desulfurization),
while departure of one of the sugar hydroxy groups
produces either 2′- or 3′-UMPS (hydrolysis). At pH 2-3,
breakdown of the intermediate to desulfurization prod-
ucts is moderately favored; desulfurization is twice as fast
as hydrolysis. Under more acidic conditions, the product
distribution is even more strongly biased in favor of
desulfurization. Evidently, this change in product dis-
tribution results from the fact that the reaction via
monocationic phosphorothioate becomes more important,
and this partial reaction appears to favor desulfurization
products over hydrolysis products more strongly than the
reaction via neutral phosphorothioate. In this respect
the situation is opposite to that observed for 3′,5′-Up-
(s)U.8 With the latter compound, desulfurization pre-
dominates over phosphodiester hydrolysis and phosphate
The base-catalyzed hydrolysis of the diastereomeric
2′,3′-cUMPS to an almost equimolar mixture of 2′- and
3′-UMPS exhibits first-order dependence on hydroxide-
ion concentration at pH > 9, analogously to the hydrolysis
of 2′,3′-cUMP. The rate constants kd in Table 1 indicate
that (RP)- and (SP)-2′,3′-cUMPS are hydrolyzed 50% and
30% less rapidly than 2′,3′-cUMP, respectively. The
hydrolysis under these conditions most likely involves an
“in-line” displacement of the leaving 2′- or 3′-hydroxy
group by hydroxide ion.14 In other words, the reaction
takes place via a pentacoordinated transition state rather
than via a pentacoordinated intermediate (Scheme 4).
According to quantum chemical calculations,15 the dian-
ionic phosphorane species can hardly be stable enough
to undergo ligand reorganization by a pseudorotation
process. Consistent with the assumed instability of the
(14) (a) Westheimer, F. Acc. Chem. Res. 1968, 1, 70. (b) Thatcher,
G. R. J .; Kluger, R. Adv. Phys. Org. Chem. 1989, 25, 99.
(15) (a) Lim, C.; Karplus, M. J . Am. Chem. Soc. 1990, 112, 5872.
(b) Kluger, R.; Taylor, S. D. J . Am. Chem. Soc. 1991, 113, 5714. (c)
Taira, K.; Uchinaru, T.; Storer, J . W.; Yliniemela¨, A.; Uebayasi, M.;
Kazutoshi, T. J . Org. Chem. 1993, 58, 3009.

Analogs of Uridine 2′,3′-Cyclic Monophosphate
J . Org. Chem., Vol. 61, No. 12, 1996 3955
Sch em e 5
less ready protonation of phosphorothioate diesters to a
reactive monocationic form. Unlike with dinucleoside
phosphoromonothioates, the pentacoordinated intermedi-
ate derived from the monocationic 2′,3′-cUMPS is decom-
posed to desulfurization products to a larger extent than
the one obtained from neutral starting material. In other
words, the predominance of desulfurization to 2′,3′-cUMP
over hydrolysis to 2′/3′-UMPS is increased with decreas-
ing pH. Near neutral pH, release of uracil base from the
starting material (depyrimidination) competes with the
reactions of the phosphate moiety.
dianionic phosphorane, desulfurization of 2′,3′-cUMPS
does not compete with the phosphoester hydrolysis in
alkaline solutions. According to Westheimer’s concept
on pseudorotating phosphorane intermediates,14 the at-
tacking hydroxide ion adopts an apical position on
formation of a pentacoordinated transition state. The
remaining apical position is occupied by either the 2′- or
3′-sugar oxygen, since one of these must be apical and
the other equatorial. If the pentacoordinated species
obtained is too unstable to pseudorotate, the sulfur ligand
cannot adopt an apical position, and hence it is unable
to leave. The small “thio-effect” of the alkaline hydrolysis
is comparable to that reported for the base-catalyzed
hydrolysis of 3′,5′-Up(s)U,8,16 which proceeds by an “in-
line” displacement of the 5′-linked nucleoside by the
neighboring 2′-oxyanion.
Exp er im en ta l Section
Ma ter ia ls. The preparation of the monothioates (RP)- and
(SP)-2′,3′-cUMPS (1a ,b) and 2′- and 3′-UMPS (3a , 4a ) has been
described previously.8 Uridine, uridine monophosphates, and
uracil were products of Sigma. They were used as received
after the purity was checked by HPLC.
Kin etic Mea su r em en ts. Reactions were followed by the
HPLC method described previously,8,19 employing analytical
methods and reaction solutions similar to those of the earlier
study.8
The rate constants reported refer to buffer concentration
zero. Two runs at different buffer concentrations were carried
out at each pH studied. The effect of the buffer concentration
([buffer] < 0.05 mol L-1) on the rate of phosphoester hydrolysis
and desulfurization was relatively small (<30%).
Ca lcu la tion of th e Ra te Con sta n ts. The pseudo-first-
order rate constants for the decomposition of 2′,3′-cUMPS (kdec
)
The pH-rate profiles of the phosphoester hydrolysis
of neither 2′,3′-cUMP nor the diastereomeric 2′,3′-cUMPS
consist of a clearcut pH-independent part. In contrast,
the desulfurization of 2′,3′-cUMPS is pH-independent at
pH > 5. One may tentatively assume that this reaction
proceeds either by attack of a molecule of water on the
tetracoordinated phosphorus of a phosphate monoanion
or by attack of a hydroxide ion on a neutral phosphate.
The mercapto ligand may depart as hydrogen sulfide ion
after protolytic rearrangement and pseudorotation of the
monoanionic pentacoordinated intermediate obtained
(Scheme 5). Similarly, the desulfurization of 3′,5′-Up-
(s)U has been shown to be pH-independent at pH > 3
and suggested to proceed by departure of hydrogen
sulfide ion from the monoanionic pentacoordinated in-
termediate.8
Con clu sion s. The (RP)- and (SP)-diastereomers of
2′,3′-cUMPS are hydrolyzed in aqueous alkali to an
almost equimolar mixture of 2′- and 3′-UMPS 2.1 and
1.5 times less readily than 2′,3′-cUMP to 2′- and 3′-UMP.
Accordingly, the thiosubstitution only moderately retards
the nucleophilic attack on tetracoordinated phosphorus,
consistent with the earlier observations on the alkaline
hydrolysis of methyl 2,4-dinitrophenyl phosphate17 and
dinucleoside monophosphates.8,16-18 However, (RP)- and
(SP)-2′,3′-cUMPS are in aqueous acid from 1 to 2 orders
of magnitude more stable than 2′,3′-cUMP, the hydrolytic
desulfurization to 2′,3′-cUMP predominating over the
phosphoester hydrolysis. A similar stability difference
has previously8 been observed between dinucleoside
monophosphates and their thioate analogs under acidic
conditions. These large thio effects largely result from
were obtained by applying the integrated first-order rate
equation to the time-dependent concentration of the starting
material. The first-order rate constants (k1) for the phosphodi-
ester hydrolysis of the diastereomers of 2′,3′-cUMPS were
calculated by eq 3, where c0(cUMPS) stands for the initial
ct(UMPS)
k1
)
[exp(-kdect) - exp(-k3t)] (3)
k3 - kdec
c0(cUMPS)
concentration of 2′,3′-cUMPS, ct(UMPS) denotes the sum
concentration of 2′- and 3′-UMPS at moment t, and k3 is the
first-order rate constant of the disappearance of 2′/3′-UMPS.
The values of k3 have been reported previously.9
The first-order rate constants, k2, for the desulfurization of
2′,3′-cUMPS under acidic conditions were calculated by eq 4,
ct(cUMP)
k2
)
[exp(-kdect) - exp(-k4t)] (4)
k4 - kdec
c0(cUMPS)
where c0(cUMPS) stands for the initial concentration of the
starting material, ct(cUMP) denotes the concentration of 2′,3′-
cUMP at moment t, and k4 is the first-order rate constant of
the hydrolysis of 2′,3′-cUMP.
The rate constant for depyrimidination (k5) was calculated
by eq 5, where ct(Ura) and ct(cUMPS) stand for the concentra-
k5 ) [ct(Ura)/[c0(cUMPS) - ct(cUMPS)]]kdec
(5)
tions of uracil and 2′,3′-cUMPS at moment t, respectively.
Usually, only either k1 or k2 could be calculated by means
of the equation of two consecutive first-order reactions (eqs 3
and 4). Equation 6 was then applied to obtain the other rate
constant.
kdec ) k1 + k2 + k5
(6)
J O960118R
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