Hydrolysis of thymidine boranomonophosphate and stepwise deuterium substitution of the borane hydrogens. 31P and 11B NMR studies

Article abstract of DOI:10.1021/ja9540280

The α-P-boranophosphate nucleosides comprise a new class of modified nucleotides that may find use as therapeutic and DNA diagnostic agents. Hydrolysis of thymidine 5'-boranomonophosphate, d(p(B)T), has been studied in H₂O and D₂O using ¹H, ³¹P, and ¹¹B NMR spectroscopies. Although d(p(B)T) is quite stable at 25 °C, it hydrolyzes slowly at higher temperatures. At 50 or 60 °C, d(p(B)T) hydrolyzes first into thymidine (dT) and boranophosphate (O₃P-BH₃^3-), followed by subsequent hydrolysis of the O₃P-BH₃^3- to produce phosphonate and boric acid. A three-step deuterium substitution of the borane hydrogens in O₃P-BH₃^3- was detected in D₂O by the presence of a ³¹P isotope shift. The ³¹P resonances shifted downfield by 0.14 ppm upon substitution of each of three ¹H atoms by ²H. Exchange of the borane hydrogens with D₂O occurs as sequential processes superimposed upon hydrolysis of O₃P-BH₃^3-. The hydrolysis and deuteration steps were characterized in terms of pseudo-first-order rate constants. Hydrolysis of O₃P-BH₃^3- is an order of magnitude slower than deuterium substitution.

Full text of DOI:10.1021/ja9540280

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J. Am. Chem. Soc. 1996, 118, 6606-6614
Hydrolysis of Thymidine Boranomonophosphate and Stepwise
11
Deuterium Substitution of the Borane Hydrogens. 31P and B
NMR Studies†
Hong Li,‡ Charles Hardin,§ and Barbara Ramsay Shaw*,‡
Contribution from the Department of Chemistry, Duke UniVersity,
Durham, North Carolina 27708, and Department of Biochemistry,
North Carolina State UniVersity, Raleigh, North Carolina 27695
ReceiVed NoVember 30, 1995X
Abstract: The R-P-boranophosphate nucleosides comprise a new class of modified nucleotides that may find use as
therapeutic and DNA diagnostic agents. Hydrolysis of thymidine 5′-boranomonophosphate, d(pBT), has been studied
in H2O and D2O using 1H, 31P, and 11B NMR spectroscopies. Although d(pBT) is quite stable at 25 °C, it hydrolyzes
slowly at higher temperatures. At 50 or 60 °C, d(pBT) hydrolyzes first into thymidine (dT) and boranophosphate
3-
(O3P-BH33-), followed by subsequent hydrolysis of the O3P-BH3 to produce phosphonate and boric acid. A
three-step deuterium substitution of the borane hydrogens in O3P-BH33- was detected in D2O by the presence of a
31P isotope shift. The 31P resonances shifted downfield by 0.14 ppm upon substitution of each of three 1H atoms by
2H. Exchange of the borane hydrogens with D2O occurs as sequential processes superimposed upon hydrolysis of
O3P-BH33-. The hydrolysis and deuteration steps were characterized in terms of pseudo-first-order rate constants.
3-
Hydrolysis of O3P-BH3 is an order of magnitude slower than deuterium substitution.
Introduction
Scheme 1. Phosphate Ester Hydrolysis Reaction of the
d(pBT) Nucleotide in H2O
A series of R-P-boranomono- (1) or triphosphate (2) nucleo-
sides designed for use as potential therapeutic and DNA
diagnostic agents have been synthesized.1-3 These compounds
The hydrolytic stability of thymidine 5′-boranomonophos-
phate, d(pBT), has been studied in solution under various pH
and temperature conditions using reverse-phase HPLC.9 The
HPLC studies indicated that d(pBT) undergoes slow hydrolysis
in aqueous solution to produce thymidine (dT) (Scheme 1) with
a rate constant of 10-5 s-1 at pH 7.4, 37 °C, analogous to the
hydrolysis pathway of unmodified thymidine 5′-monophosphate,
d(pT)9. Notably, hydrolysis cleaves the P-O-sugar linkage
and not the P-B bond9 (see Scheme 1).
are distinctive in that one of the nonbridging oxygens in the
phosphate diester is replaced by a -BH3 borane moiety.
Nucleoside 5′-R-P-boranotriphosphates 2 can substitute for
normal dNTP and be successfully incorporated into DNA by
DNA polymerase,2,4,5 yet once in DNA, the boranophosphate
linkage is more resistant to exo and endo nucleases than normal
phosphate diesters.3,6-9a
Although HPLC methods were used to detect dT as one of
the products, conventional HPLC methods cannot detect the
other hydrolysis product, which is presumably boranophosphate
(O3P-BH33-),10 because this fragment does not absorb in the
accessible UV range. Questions arise as to (a) whether O3P-
* Author to whom correspondence should be addressed.
† Abbreviations: dT; thymidine; d(pT), thymidine 5′-monophosphate;
d(pBT), thymidine 5′-boranomonophosphate; d(TpBT), dithymidine bora-
nomonophosphate; EDTA, ethylenediaminetetraacetic acid; Et2OBF3, diethyl
ether-boron trifluoride; HPLC, high-performance liquid chromatography;
k1, observed pseudo-first-order rate constant; NMR, nuclear magnetic
resonance spectroscopy; ppm, parts per million; γ, magnetogyric ratio; TSP,
3-(trimethylsilyl)propionate-2,2,3,3-d4 sodium salt.
3-
BH3 indeed forms as a direct product of d(pBT) hydrolysis,
3-
(b) whether O3P-BH3 undergoes further hydrolysis, and if
‡ Duke University.
§ North Carolina State University.
X Abstract published in AdVance ACS Abstracts, June 15, 1996.
(1) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J. Am. Chem. Soc. 1990,
112, 9000-9001.
(6) (a) Porter, K.; Briley, J. D.; Tomasz, J.; Sood, A.; Spielvogel, B. F.;
Shaw, B. R. Genome Sequencing and Analysis Conference V, Hilton Head,
SC; Mary Ann Liebert, Inc.: New York, 1993; p 55. (b) Porter, K.; Briley,
J. D.; Shaw, B. R. Genome Sequencing and Analysis Conference VI, Hilton
Head, SC; Mary Ann Liebert, Inc.: New York, 1994; p 43.
(7) Ramaswamy, M.; Shaw, B. R. Manuscript in preparation.
(8) Huang, F.; Sood, A.; Spielvogel, B. F.; Shaw, B. R. J. Biomol. Struct.
Dyn. 1993, 10, a078.
(9) (a) Huang, F. Ph.D. Thesis; Duke University, Durham, NC, 1994.
(b) Huang, F.; Sood, A.; Spielvogel, B. F.; Shaw, B. R. Eighth International
Meeting on Boron Chemistry; The University of Tennessee: Knoxville,
TN, 1993; p 114. (c) Huang, F.; Shaw, B. R. Manuscript in preparation.
(2) Tomasz, J.; Shaw, B. R.; Porter, K.; Spielvogel, F.; Sood, A. Angew.
Chem., Int. Ed. Engl. 1992, 31, 1373-1375.
(3) Shaw, B. R.; Madison, J.; Sood, A.; Spielvogel, B. F. Methods Mol.
Biol. 1993, 20, 225-243.
(4) Porter, K.; Ealey, K.; Briley, J. D.; Huang, F.; Shaw, B. R. Genome
Sequencing and Analysis Conference VI, Hilton Head, SC; Mary Ann
Liebert, Inc.: New York, 1994; p 43.
(5) Li, H.; Porter, K.; Huang, F.; Shaw, B. R. Nucleic Acids Res. 1995,
21, 4495-4501.
S0002-7863(95)04028-5 CCC: $12 00 © 1996 American Chemical Society

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J. Am. Chem. Soc., Vol. 118, No. 28, 1996 6607
signal-to-noise ratios. NMR spectra were processed and plotted using
a Sun SPARC 2 computer with Varian NMR software. NMR peak
intensities represent time-averaged values for a given acquisition period,
which was much less than the total reaction time. Since the line widths
did not change appreciably with time, NMR peak integrals could be
determined using signal heights.
so, what the identities of the hydrolysis products are, and (c)
whether stepwise hydrolysis and/or exchange of borane hydro-
gens with solvent occurs. These questions are important for
assessing the toxicity of boranophosphate-containing nucleotides
as therapeutic agents, as well as for enhancing our understanding
of the chemistry of boron-containing phosphorus compounds.
In order to address the fate of these B-P bond-containing
compounds, we have employed 1H, 11B, and 31P NMR to study
the d(pBT) hydrolysis. We describe new aspects of d(pBT)
hydrolysis, identify O3P-BH33- as the main hydrolysis product,
and document solvent exchange of the borane hydrogens.
31P NMR spectra were acquired at 202 MHz using 32k data points
and a sweep width of 33 113 Hz. 31P chemical shifts were referenced
externally to a solution of 85% H3PO4. The 31P spectra of d(pBT) and
3-
O3P-BH3 are complicated due to direct bonding of phosphorus to
the boron atom. Naturally abundant boron consists of 80.4% 11B (spin
I ) 3/2) and 19.6% 10B (spin I ) 3). When a 31P (I ) 1/2) atom is
3-
bonded to a single 11B, as in the -P-BH3 linkage, four equally-
spaced and equal-intensity lines (a quartet with a 1-1-1-1 pattern)
are expected in the 31P spectrum. If a 31P is coupled to a 10B atom,
seven equally-spaced and equal-intensity lines (a septet with a 1-1-
1-1-1-1-1 pattern) are expected. Thus, a P-B bond-containing
sample with naturally abundant boron could be expected to show a
complicated 31P spectrum. The 31P-10B coupling constant is about
one-third that of the 31P-11B coupling constant due to the difference
in magnetogyric ratios (γ(11B)/γ(10B) ) 2.99).13a The intensity of an
individual 31P peak coupled to a 10B is hence about 0.14 times the
intensity of a 31P peak coupled to a 11B in a naturally abundant boron
compound.13b Thus, underlying the quartet due to 31P coupling with
11B in the 31P spectrum is a septet due to 31P coupling with 10B at similar
chemical shifts. Experimentally, the 31P spectra usually appear as if
the boron effects derive only from 11B scalar coupling.14 However,
the intensities of the two middle peaks are often enhanced relative to
the intensities of the two outside peaks. In this study, we consider
only the 11B coupling and neglect the 10B coupling in the 31P spectra.
1H NMR spectra were collected at 499.843 MHz with a sweep width
of 5498.3 Hz and 16k data points. 1H chemical shifts were measured
relative to TSP (3-(trimethylsilyl)propionate-2,2,3,3-d4 sodium salt) as
internal reference.
Materials and Methods
Sample Preparation. Lyophilized d(pBT) powder in ammonium
form was graciously provided by Dr. J. Tomasz.2 NMR samples, which
typically contained about 85 mM d(pBT), were made by dissolving the
lyophilized d(pBT) powder directly into H2O or D2O. The concentration
of d(pBT) was estimated from the UV absorbance at 260 nm using the
molar extinction coefficient of unmodified thymidine monophosphate
d(pT).11 All samples were analyzed in 5 mm NMR tubes (Wilmad).
Since all samples were prepared in nonbuffered H2O or D2O solvents,
a gradual decrease in pH was observed as hydrolysis proceeded.
Initially, the pH was about 6; by the final stages of hydrolysis it had
decreased to about 5. This pH change may affect the rate constants12a
and chemical shifts12b to a limited extent.
Hydrolysis and Solvent Exchange Reactions. Hydrolysis of d(pBT)
was studied in H2O and D2O by examining 1H, 31P, and 11B NMR
spectra at different incubation times. Hydrolysis and deuterium
substitution rates were determined by measuring changes in the heights
of the respective NMR peaks with time. Intensity changes measured
as a function of time were fit to a pseudo-first-order reaction model.
In general, the correlation coefficients (R2) were better than 0.92. The
beginning of each time-dependent acquisition is referred to as t ) 0.
True zero times were not available in this kind of experiment due to
the elapsed time required for sample preparation and proper setup.
However, calculation of a pseudo-first-order rate constant from reactant
concentration changes does not require knowledge of the exact reaction
initial time.
11B NMR spectra were acquired at 160 MHz using 16k data points
and a sweep width of 22 447 Hz. 11B chemical shifts were referenced
externally to a solution of diethylether-boron trifluoride, Et2OBF3.
Although 11B is a quadrupolar nucleus, line-broadening is not severe
for the molecules studied. Boron in the glass NMR tube and the NMR
probe produce a broad background 11B signal, which did not obstruct
our observations.
NMR Experiments. All H, 31P, and 11B NMR experiments were
1
performed on a Varian Unity-500 NMR spectrometer at the Duke NMR
Center. Spectra were acquired in the X-nucleus channel of a 5 mm
reverse-detect probe which was tuned for either 31P or 11B. D2O was
used as the lock signal. For experiments performed in H2O, the D2O
was enclosed in a special NMR insert tube (Wilmad) to avoid unwanted
deuterium exchange between the sample and solvent. 31P and 11B NMR
spectra were recorded using enough transients to provide satisfactory
Results
1H, 31P, and 11B Spectra of d(pBT). The starting material
used for these studies was the mononucleotide thymidine 5′-
boranomonophosphate, d(pBT). The 1.5-7 ppm range of the
1H spectrum (Figure 1A) shows a peak pattern very similar to
that of normal thymidine 5′-monophosphate, d(pT),15 corre-
sponding to the protons on the sugar and base of d(pBT). An
eight-line pattern centered at about 0.33 ppm corresponds to a
1-1-1-1 quartet (Figure 1A,B) from borane hydrogens which
couple with 11B and are further split by 31P to produce a doublet
(10) The actual ionization states of thymidine boranomonophosphate,
boranophosphate, phosphonate (or phosphite), and boric acid in solution
are both pH- and ionic strength-dependent. For convenience, we show
thymidine boranomonophosphate, boranophosphate, and phosphonate in
their fully deprotonated forms, and boric acid in its fully protonated form.
This probably does not reflect the actual protonation states of these
molecules at pH 5-6 (Vide infra). The state of protonation is not the subject
of the present paper.
in each peak of the quartet (Figure 1B). Couplings to both 11
B
and 31P were verified by 11B- and 31P-decoupling experiments,
respectively (data not shown). The 11B-decoupled spectrum
shows only a doublet due to 31P coupling with borane hydrogens;
the 31P-decoupled spectrum shows a 1-1-1-1 pattern due to
11B coupling with borane hydrogens.
(11) Borer, P. N. Nucleic Acids. In Handbook of Biochemistry and
Molecular Biology, 3rd ed.; Fasman, G. D., Ed.; CRC Press: Cleveland,
OH, 1975; Vol. 1, p 589.
(12) (a) The rate-pH profile of the thymidine boranomonophosphate
hydrolysis in ref 9 indicated that the rate constants change by less than
25% over the pH range from 3 to 6.5. (b) Small but observable changes in
31P chemical shifts were observed during hydrolysis of d(pBT) mononucleo-
The 31P NMR spectrum of d(pBT) at 25 °C is a 1-1-1-1
quartet centered at about 85 ppm due to 11B coupling (Figure
1C). Further splitting of the 31P resonance by borane hydrogens
was not resolved under these conditions.
3-
tide to dT and hydrolysis of O3P-BH3 in H2O or D2O, in addition to
intensity changes. The 31P chemical shifts of both the reactant d(pBT) and
product boranophosphate decreased with time (e.g., by -0.15 and -0.11
ppm, respectively, in H2O at 50 °C in 35 min). During subsequent hydrolysis
of boranophosphate, the 31P chemical shifts of non-deuterated and partially
and fully deuterated species I-IV increased with time in both D2O and
H2O (e.g., by +0.06 ppm for intermediates II-IV in 3 h at 60 °C), while
the 11B chemical shift decreased (e.g., by -0.06 ppm in 9 h at 60 °C). The
reason for the small observed 31P chemical shift changes is not apparent.
These changes could be due to a pH change as a result of hydrolysis,
chemical exchange among different species, or an equilibrium isotope effect.
(13) (a) Harris, R. K. Nuclear Magnetic Resonance Spectroscopy: A
Physicochemical View; Longman Scientific & Technical, Somerset, NJ,
1986. (b) Eaton, G. R.; Lipscomb, W. N. NMR Studies of Boron Hydrides
and Related Compounds; W. A. Benjamin., Inc.: New York, 1969.
(14) Schaeffer, R. Progress in Boron Chemistry; The MacMillan Co.:
New York, 1964; Vol. 1.
(15) Wood, D. J.; Hruska, F. E.; Ogilvie, K. K. Can. J. Chem. 1974, 52,
3353-3366.

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Figure 2. 1H-decoupled 31P spectra during d(pBT) hydrolysis in H2O
at 50 °C. The downfield quartet centered at ca. 87.1 ppm corresponds
to d(pBT) resonance; the upfield quartet centered at ca. 83.5 ppm is
3-
from O3P-BH3 resonance. A total of 52 scans were acquired for
each spectrum in about 1.5 min, and a line-broadening of 30 Hz was
applied. The exact initial time is not available but refers to the time of
the first measurement of the set of data (see the Materials and Methods).
but is similar to the 50 °C spectrum shown in Figure 4). The
11B pattern can be attributed to 11B coupling with a single 31P
and three equivalent hydrogens, indicating the presence of a
-P-BH3 linkage in the hydrolysis product.
1
Figure 1. 1H, 31P, and 11B NMR spectra of d(pBT) at 25 °C: (A) H
spectrum, 512 scans at 10 Hz line-broadening; (B) expanded region
from panel A for the -P-BH3 linkage; (C) 31P spectrum, 1024 scans
at 10 Hz line-broadening; peaks in the 81-83 ppm range are due to
the hydrolysis product of d(pBT); (D) 11B spectrum, 512 scans at 10
Hz line-broadening.
Formation of dT, as the other d(pBT) hydrolysis product, was
also detected by following the 1H NMR spectrum. As expected,
the integrals of the sugar and base 1H reasonances of dT
increased with time while those of d(pBT) decreased (data not
shown).
The intensity changes of the 31P peaks with time, demon-
strated in Figure 2, were fit to a pseudo-first-order exponential
decay rate equation with respect to d(pBT) concentration. The
calculated hydrolysis rate constant k1 determined at 50 °C with
21 data points acquired over 30 min is 4 × 10-4 s-1 (R2 )
0.98; data not shown).
In the 11B NMR spectrum of d(pBT) at 25 °C (Figure 1D),
two partially overlapped 1-3-3-1 patterns centered at about
-40.1 ppm are seen due to coupling with both the single 31P
and three equivalent hydrogens in the -P-BH3 linkage. Upon
1H decoupling of the 11B spectrum, a doublet was observed at
this chemical shift (data not shown), confirming the 11B
assignments in the -P-BH3 linkage.
Since phosphate ester hydrolysis of d(pBT) to produce dT
3-
Phosphate Ester Hydrolysis of d(pBT) in H2O. Hydrolysis
of d(pBT) in H2O was studied by 1H, 31P, and 11B NMR at higher
temperatures in order to accelerate the reaction for suitable NMR
observation. When d(pBT) was incubated at 50 °C and the 1H-
decoupled 31P spectrum was monitored with time, a new 1-1-
1-1 quartet (centered at ca. 83.5 ppm) was seen upfield from
the quartet of the starting d(pBT) material (centered at 87.1 ppm)
(Figure 2). The intensity of the new upfield quartet increased
with time as the intensity of the d(pBT) quartet decreased. This
is consistent with previous HPLC experiments, which indicated
that the d(pBT) mononucleotide undergoes slow phosphate ester
hydrolysis.9 The decomposition products are proposed to be
dT and O3P-BH33- as shown in Scheme 1. The upfield quartet
centered at 83.5 ppm indicates that the hydrolysis product retains
the P-B bond, since the chemical shift is nearly equal to that
of the d(pBT) quartet (only a 3.6 ppm difference) and both peaks
had the same splitting pattern.
and O3P-BH3 (Scheme 1) has been studied under various
conditions using quantitative HPLC methods, those results will
be reported elsewhere.9c Our emphasis in this work is to
3-
describe further reactions of O3P-BH3 in aqueous solution.
3-
O3P-BH3
Hydrolysis in H2O. The same solutions
described above, which had undergone nearly complete phos-
phate ester hydrolysis of d(pBT) mononucleotide (lacking the
87 ppm quartet in the 31P spectrum), were used to investigate
further hydrolysis of O3P-BH33-. Upon monitoring the H-
1
decoupled 31P spectrum in H2O at 50 °C, a singlet was seen at
ca. 3.6 ppm whose intensity increased with time while that of
3-
the O3P-BH3 quartet centered at about 83.5 ppm decreased
(Figure 3). For the same solution, the intensity of the O3P-
BH33- 11B spectrum (having two sets of quartets with a 1-3-
3-1 pattern centered at ca. -38.8 ppm, Figure 4) decreased
with time. Concomitantly, a singlet appeared at about 19.5 ppm
which increased in intensity. However, neither the 31P nor 11
B
Additional evidence that hydrolysis proceeds as shown in
Scheme 1 comes from an examination of the spectra of the
hydrolysis products. The 11B spectrum of O3P-BH33- in H2O
shows a doublet of quartets with 1-3-3-1 patterns (centered
at ca. 39.3 ppm at 25 °C). (The 25 °C spectrum is not shown,
spectral patterns changed over time in H2O. Such spectral
behaviors are consistent with the proposed hydrolysis reaction
of O3P-BH33- as depicted in Scheme 2, where the P-B bond
is broken accompanied by formation of phosphonate (corre-
sponding to a singlet at 3.6 ppm in the 1H-decoupled 31P

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Hydrolysis of Deoxynucleoside Boranophosphate
J. Am. Chem. Soc., Vol. 118, No. 28, 1996 6609
Figure 3. 1H-decoupled 31P spectra during O3P-BH33- hydrolysis in
H2O at 50 °C. The major quartet centered at ca. 83.5 ppm is the O3P-
3-
11BH3 resonance, the singlet at about 3.6 ppm is the phosphonate
resonance, and the three minor peaks centered at ca. 83.5 ppm are due
to the resonance of the 10B-form of O3P-BH33-. A total of 175 scans
were acquired in each spectrum in about 5 min, and a line-broadening
of 20 Hz was applied (see Figure 2 for t ) 0).
Figure 5. 1H-decoupled 31P spectra during O3P-BH33- hydrolysis in
D2O at 60 °C. The multiple quartets centered at about 81.7 ppm are
from boranophosphate resonances, and the triplet centered at about 3.4
ppm is from deuterated phosphonate. Subquartets I-IV correspond to
the 31P resonances of non-deuterated, partially deuterated, and fully
deuterated boranophosphate populations O3P-BH33-, O3P-BH2D3-
,
O3P-BHD23-, and O3P-BD33-, respectively, in Scheme 3. A total of
344 scans were collected in each spectrum in about 10 min, and a line-
broadening of 10 Hz was applied (see Figure 2 for t ) 0).
the hydrolysis mechanism, we determined whether the borane
hydrogens exchanged with solvent. When the time course of
3-
O3P-BH3 incubation in D2O at 50 or 60 °C was followed
1
3-
by H-decoupled 31P NMR, the peak intensity of O3P-BH3
(centered at ca. 81.7 ppm; Figure 5) decreased, accompanied
by an increase in peak intensity of phosphonate (at ca. 3.4 ppm;
Figure 5). This behavior was similar to that in H2O solvent
(Figures 3 and 4); however, the peak patterns differed. Specif-
Figure 4. 11B spectra during O3P-BH33- hydrolysis in H2O at 50 °C.
The peaks centered at -38.8 ppm are from the O3P-BH33- resonance,
and the singlet at about 19.5 ppm is from the resonance of boric acid.
A total of 136 scans were collected in each spectrum in about 5 min,
and a line-broadening of 5 Hz was applied (see Figure 2 for t ) 0).
3-
ically, the O3P-BH3 quartets (centered at about 81.7 ppm)
measured in D2O at 60 °C (Figure 5) consisted of two or more
subquartets (labeled I, II, III, and IV) each having a 1-1-1-1
pattern. The total intensity of the quartets decreased with time;
simultaneously the relative intensities within each of the
subquartets changed. The results in Figure 5 show that the
intensity of subquartet II increases and the intensity of subquartet
I decreases with time in the early phase of the reaction. Then,
subquartet III starts to appear, and its intensity increases with
time. At the same time, subquartet I starts to disappear and
the intensity of subquartet II decreases. As the reaction
proceeds, subquartet IV starts to appear while the intensity of
subquartet III decreases and subquartet II disappears. Finally,
only quartet IV exists, and its intensity decreases further with
time (data not shown). Intensity changes of subquartets I-IV
are plotted in Figure 6; the relative 31P NMR intensity changes
of subquartets I, II, III, and IV increase through a maximum,
and then decay. This behavior is characteristic of a reaction
consisting of four sequential pseudo-first-order steps.
3
Scheme 2. Hydrolysis of O3P-BH3 - in H2O
spectrum, Figure 3) and boric acid (a singlet at ca. 19.5 ppm in
the 11B spectrum, Figure 4, close to the literature value of 18.8
((1.0) ppm at 25 °C14,16). When the 31P spectrum was collected
without 1H decoupling, a doublet was observed at ca. 3.6 ppm
with a coupling constant of 618 Hz instead of a singlet (data
not shown), confirming that a P-H bond exists in the phos-
3-
phonate, as expected for the O3P-BH3 hydrolysis product.
After most of the d(pBT) had hydrolyzed (as in Scheme 1),
3-
changes in O3P-BH3 peak intensities observed by 1H-
The 31P subquartet II is 0.14 ppm downfield from subquartet
I. Downfield shifts of this magnitude were also observed for
subquartet III relative to subquartet II, and for subquartet IV
relative to subquartet III. The shift values are independent of
reaction time and insensitive to temperature changes in the 25-
60 °C range (data not shown). Since subquartets I-IV are
1-1-1-1 quartets and have chemical shifts very similar to
those of O3P-BH33- (quartet I), subquartets II, III, and IV must
correspond to species which all retain P-B bonds (as proposed
in Scheme 3).
decoupled 31P and 11B NMR experiments gave reasonable
pseudo-first-order kinetics fits. Equal hydrolysis rate constants
of 2 × 10-5 s-1 (R2 ) 1.00) were calculated from 31P and 11
B
NMR data at 50 °C (16 and 31 points, respectively; data not
shown). There is a 20-fold difference in the rates between
3-
d(pBT) and O3P-BH3 hydrolysis (Schemes 1 and 2).
O3P-BH33- Hydrolysis and Solvent Exchange of Borane
Hydrogens in D2O. To further understand the chemical
3-
reactions of O3P-BH3 in aqueous solution, and to elucidate
1
In the same H-decoupled 31P spectra (Figure 5), a triplet
(16) Onak, T. P.; Labdesman, H.; Williams, R. E.; Shapiro, I. J. Phys.
Chem. 1959, 63, 1533-1535.
with a 1-1-1 pattern (peak V) centered at about 3.4 ppm in

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6610 J. Am. Chem. Soc., Vol. 118, No. 28, 1996
Li et al.
Figure 7. 11B spectra of O3P-BD33- at 25 °C. A total of 1024 scans
were acquired, and a line-broadening of 2 Hz was applied.
doublet can be attributed to further coupling between 1H/D and
11B during stepwise deuterium substitution of the borane
hydrogens. After extensive incubation in D2O, when the 31P
spectrum only contains quartet IV, the corresponding 11B
spectrum (Figure 7) contains a doublet and each peak of the
doublet has a 1-3-5-6-5-3-1 pattern. This 11B spectrum
Figure 6. Time dependence of 31P NMR intensities for species I-IV
3-
during O3P-BH3 hydrolysis in D2O at 60 °C obtained from
experiments illustrated in Figure 5.
1
is unaffected by H decoupling (data not shown). The 11B
3
Scheme 3. Hydrolysis of O3P-BH3 - in D2O Superimposed
pattern shown in Figure 7 is due to the presence of three
equivalent deuterium atoms bonded to boron, as in the -P-
BD3 linkage (species IV in Scheme 3). The 11B NMR results
hence corroborate the conclusion obtained from the 1H-
decoupled 31P NMR studies that the borane hydrogens undergo
stepwise deuterium substitution.
upon Stepwise Deuterium Substitution in D2Oa
Discussion
Hydrolysis of Boranophosphate Mononucleotide Involves
P-O-R Bond Cleavage in H2O. 1H, 31P (Figure 2), and 11
B
NMR studies indicate that d(pBT) is hydrolyzed to produce
3-
thymidine dT and boranophosphate O3P-BH3 in aqueous
solution at 50 °C as shown in Scheme 1. This result is in
agreement with previous HPLC experiments which showed that
dT, not thymidine monophosphate d(pT), was the observed
product at 260 nm.9 From these results, it can be concluded
that the P-BH3 bond in deoxythymidine borano-monophos-
phates is hydrolyzed less readily than the P-O-R bond.
It is interesting to compare these experimental results with
conclusions from ab initio SCF calculations by Thatcher and
Campbell,20 who assumed that phosphate hydrolysis proceeds
Via a trigonal bipyramidal pentacoordinate intermediate in which
incoming and leaving groups occupy the apical position.18,19
These calculations predicted that the P-O bond would be more
labile to hydrolysis than the P-B bond.20 This is consistent with
our experimental results. It remains to be determined whether
hydrolysis of the P-O bond in boranophosphate nucleotides
proceeds Via a similar intermediate.
a Stepwise deuteration produces boranophosphate intermediates II-
IV. Hydrolysis of intermediates I-IV produced deuterated phosphonate
(V). The -BH3 group carries one negative charge.
D2O is observed, instead of a singlet as previously seen in H2O
(Figure 3); the peak intensity increases with time (Figures 5
1
and 6). Removal of H decoupling did not change the nature
of the triplet (data not shown), suggesting that a D nucleus (spin
I ) 1) is coupled to a 31P. Therefore, peak V was assigned to
a deuterated phosphonate which contains a P-D bond (see
Scheme 3). The measured coupling constants J(31P-1H) and
J(31P-D) for the 1H- and D-forms of phosphonate species were
584.5 and 89.9 Hz, respectively, giving a J(31P-1H)/J(31P-D)
ratio of 6.50. Agreement with the theoretical value of γH/γD
Hydrolysis of Boranophosphate, O3P-BH33-, in H2O.
Hydrolysis of O3P-BH33-, as followed by 31P and 11B NMR
3-
with time, indicated that O3P-BH3 decomposes into phos-
phonate and boric acid in H2O at 50 °C as shown in Scheme 2.
1
) 6.51417 for the H- and D-forms of phosphonate species
3-
Does O3P-BH3 hydrolyze in a stepwise manner? Several
confirms the assignment. Thus, the data in D2O solvent are
consistent with O3P-BH33- undergoing hydrolysis to produce
the deuterated phosphonate species V (as proposed in Scheme
3).
researchers have proposed that stepwise hydrolysis of tetrahy-
droborate (BH4-) is possible Via the hydrated intermediates
- 21-26
BH4-n(OH)n
.
We attempted to find evidence for analo-
gous O3P-B3-n(OH)n3- intermediates; however, the 31P or 11
B
We also observed evidence for deuterium substitution of the
borane hydrogens in the 11B spectra. Upon 1H decoupling, the
11B resonance of O3P-BH33- in H2O at 50 °C became a doublet
(18) Thatcher, G. R. J.; Kluger, R. H. AdV. Phys. Org. Chem. 1989, 25,
99-265.
(19) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70-78.
(data not shown), due to coupling between bonded 11B and 31
P
(20) Thatcher, G. R. J.; Campbell, S. J. Org. Chem. 1993, 58, 2272-
atoms. However, each peak of the doublet developed a
complicated splitting pattern and changed with time upon
incubation in D2O (data not shown). The 11B doublet is the
result of coupling between the 11B and 31P atoms due to the
existence of a P-B bond. The changing peak pattern in the
2278.
(21) Davis, R. E.; Swain, C. G. J. Am. Chem Soc. 1960, 82, 5949-
5950.
(22) Mesmer, R. E.; Jolly, W. L. Inorg. Chem. 1962, 1, 608-612.
(23) Davis, R. E.; Bromels, E. B.; Kibby, C. L. J. Am. Chem. Soc. 1962,
84, 885-892.
(24) Gardiner, J. A.; Collat, J. W. J. Am. Chem. Soc. 1965, 87; 1692-
1700.
(17) Stec, W. J.; Goddard, N.; van Wazer, J. R. J. Phys. Chem. 1971,
75, 3547-3549.
(25) Gardiner, J. A.; Collat, J. W. Inorg. Chem 1965, 4, 1208-1212.

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Hydrolysis of Deoxynucleoside Boranophosphate
J. Am. Chem. Soc., Vol. 118, No. 28, 1996 6611
3-
3-
peak patterns and J coupling constants of O3P-BH3 did not
change with time during O3P-BH33- hydrolysis in H2O (shown
in Figures 3 and 4). This suggests that either the hydrated
intermediates O3P-BH2(OH)3-, O3P-BH(OH)23-, and O3P-
O3P-BH3 31P spectrum in D2O consists of two or more
subquartets (Figure 5), while only one quartet was observed in
H2O (Figure 3) throughout the reaction period. We propose
that deuterium substitution of the borane hydrogens in O3P-
BH33- in D2O occurs in a three-step manner with three different
rate constants. The observed multiple subquartets noted in 1H-
decoupled 31P spectra at different stages in D2O can be explained
as “isotope shifts” (see below) due to individual stepwise D
substitution of 1H atoms in the -P-BH3 linkage. Boranophos-
phate hydrolysis and deuterium substitution reactions in D2O
are summarized in Scheme 3, in which I, II, III, and IV
correspond to stepwise deuterated boranophosphate intermedi-
3-
B(OH)3 do not form or they are too unstable to be detected
by NMR.
Natural boron consists of about 80% 11B and 20% 10B. This
can be detected by the presence of major and minor peaks in
the 31P spectrum of O3P-BH33-. The major peak patterns
(Figure 3) can be explained by the proposed hydrolysis reaction
of O3P-BH33- shown in Scheme 2. However, three additional
minor 31P peaks of equal intensity are present throughout the
hydrolysis time course, superimposed upon the major O3P-
BH33- quartet (Figure 3). While the pattern of these peaks did
not change with time, their intensities did, similar to the observed
intensity decrease of the major quartet corresponding to O3P-
BH33-. The three minor peaks are adequately explained as
corresponding to the 10B-form of O3P-BH33-, while the major
quartet is due to the 11B-form of O3P-11BH33-. Since 10B has
a nuclear spin of 3, it is expected that (1) the 31P spectrum of
ates O
3P-BH33-, O3P-BH2D3-, O3P-BHD23-, and O3P-
BD33-, respectively. From these data we propose that hydrolysis
of O3P-BH33- occurs superimposed upon deuterium exchange.
Borane hydrogens exchange with deuteriums in D2O to produce
deuterated borane; at the same time, hydrolytic P-B bond
cleavage occurs, producing phosphonate and boric acid.
To further confirm deuterium substitution of the borane
hydrogens in O3P-BH3 in D2O, a reverse exchange experi-
ment was conducted by utilizing the reaction mixture which
3-
3-
the 10B-form of O3P-BH3 should be a septet with equal
intensities (a 1-1-1-1-1-1-1 pattern), (2) the individual
had been hydrolyzed in D2O to give fully deuterated O3P-
3-
31P peak intensity of the 10B-form in O3P-BH3 should be
3-
BD3 (determined by 11B and 31P NMR), followed by lyo-
about 0.14 times that of the 31P peak in the 11B-form in the
same solution (see Materials and Methods), and (3) the coupling
constant ratio J(31P-11B)/J(31P-10B) should be about 3.13b,27
Experimentally, only three minor peaks with equal intensities
are resolved in Figure 3. Upon closer inspection, one can see
that the two middle peaks in the major quartet have broader
base regions than those of the two outside peaks. This suggests
that the four hidden resonances of the septet from the 10B-form
are probably buried under the two middle peaks of the major
quartet of the 11B-species. Under this assumption, parameters
for the resolved minor peaks in Figure 3 can be estimated: (1)
The 31P chemical shifts of the 10B- and 11B-forms of O3P-
philizing and transferring the molecule into H2O. Phosphorus-
31 and 11B spectra both showed the expected peaks (data not
shown) due to stepwise hydrogen substitution intermediates as
depicted in Scheme 3.
We think it likely that the 31P multiplets at ca 81.7 ppm in
Figure 5 arise as a result of stepwise hydrolysis or deuterium
exchange. If stepwise hydrolysis were to occur instead of the
three-step deuterium substitution reaction in D2O as proposed
in Scheme 3, formation of partially or fully hydrated intermedi-
ates (e.g., O3P-B(OD)33-) would result. The deuterium in the
-B-OD linkage is expected to be in fast exchange with D2O,
so coupling of 11B to D may not be observable in the 11B NMR
spectrum with the 1-3-5-6-5-3-1 pattern shown in Figure
7. In this case, the 1-3-5-6-5-3-1 pattern obtained in the
11B spectrum provides further evidence that the species corre-
3-
BH3 are both ca. 83.5 ppm. (2) The J coupling constants
J(31P-B) are 149.8 and 50.0 Hz for the 11B- and 10B-forms,
respectively, giving an experimental J(31P-11B)/J(31P-10B) ratio
of 3.0. (3) The estimated individual 31P peak intensity ratio
for the 10B-form relative to the 11B-form is 0.16. Thus, the
1
sponding to subquartets I, II, III, and IV in H-decoupled 31P
spectra (Figure 5) are due to intermediates in stepwise deuterium
substitution of the borane hydrogens in D2O, and are not
produced by intermediates in stepwise hydrolysis of O3P-
BH33-. We concluded above that stepwise hydrolysis of borane
hydrogens in O3P-BD33- was not observable in H2O. The D2O
results are consistent with this conclusion.
Isotope Shifts in the O3P-BH33- NMR Spectra. Changes
in chemical shifts and J coupling constants can occur upon
isotopic substitution. Factors that influence the magnitude of
the isotope shift include the number of intervening bonds
between sites of isotopic substitution and the nucleus of interest,
the fractional change in mass upon isotopic substitution, and
the number of atoms in the molecule that are substituted by
isotopes. Larger shifts occur when the substitution is linked
directly to the nucleus, when a larger mass change occurs, and
with a larger number of substitutions. Although the isotope
shift leads to complications in interpreting NMR results, it can
provide useful information regarding molecular structure and
reaction mechanism, as shown here.
experimental J coupling ratio (J(31P-11B)/J(31P-10B)) and 31
P
intensity ratio (10B-form/11B-form) are in good agreement with
theoretical values of 2.99 and 0.14, respectively.13b,27 This
confirms our assumptions that the three minor 31P peaks in
3-
Figure 3 arise from the 10B-form of O3P-BH3 and that the
other four peaks of the 10B-form septet overlap with the two
middle peaks of the major quartet of the 11B-form. By following
the decrease in 31P intensities of the minor peaks illustrated in
Figure 3, we calculated a pseudo-first-order rate constant of 2
× 10-5 s-1 at 50 °C (with 21 data points acquired over 1.8 h;
data not shown), which is the same as that observed for the
11B-form in the same experiments.
3-
The three minor 31P peaks from the 10B-form of O3P-BH3
seen in H2O (Figure 3) were not observed in D2O (Figure 4).
This is presumably because the four subquartets of stepwise
3-
D-substituted 11B-forms of O3P-BH3 broaden the 31P reso-
nances and thereby obscure observation of the minor 31P septet
3-
from the 10B-form of O3P-BH3
.
3-
Hydrolysis and Deuterium Exchange of O3P-BH3 in
31P isotope shifts provide additional, quantitative evidence
3-
D2O. Incubation of O3P-BH3 in D2O gave 31P (Figure 5)
for the premise of Scheme 3 that the subquartet patterns in 1H-
and 11B spectral patterns different from those found when the
same reaction was followed in H2O (Figures 3 and 4). The
3-
decoupled 31P spectra of O3P-BH3 in D2O are the result of
a three-step deuterium substitution of the borane hydrogens. The
(26) Levine, L. A.; Kreevoy, M. M. J. Am. Chem. Soc. 1972, 94, 3346-
31P isotope shift is 0.14 ppm for each deuterium substitution of
3394.
the borane hydrogens, and the shifts are additive; subquartets
(27) Kidd, R. G. NMR of Newly Accessible Nuclei; Academic Press, Inc.,-
3-
New York, 1983; Vol. 2.
II (O3P-BH2D3-), III (O3P-BHD23-), and IV (O3P-BD2
)

+
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6612 J. Am. Chem. Soc., Vol. 118, No. 28, 1996
Li et al.
shift downfield relative to subquartet I (O3P-BH33-) by 0.14,
0.28, and 0.42 ppm, respectively. Even though the phosphorus
atom is separated by two bonds from the substituted position
in the -P-B(H/D)3 linkage, the observed 31P isotope shift is
quite large, possibly due to the large relative mass difference
Table 1. Hydrolysis and Deuterium Substitution Rate Constants
3-
for d(pBT)a and O3P-BH3
k1 (s-1
in H2O
)
k1 (s-1
in D2O
)
temp (°C)
Phosphate Ester Hydrolysis of d(pBT)
d(pBT) f dT +O3P-BH3
4 × 10-4
50
3-
1
2
between H and H (a 100% change).
3-
1
One would expect to see 11B isotope shifts due to H/D
substitution. These were in fact observed, but the 11B isotope
shift values from each deuterium substitution were obscured
by broadening and severe overlap of the 11B resonances from
the stepwise deuterated intermediates (data not shown). Yet
these results showed clearly that deuterium substitution shifts
the 11B resonance upfield, as expected on the basis of zero-
point vibrational energy arguments.28 In contrast, the 31P
resonances shifted downfield upon deuterium substitution. It
is possible that factors other than mass changes dominate in
determining the 31P isotope shift. One such possibility is that
pH changes occur as a result of O3P-BH33- hydrolysis to form
phosphonate and boric acids under unbuffered conditions (see
Materials and Methods).
Hydrolysis of O3P-BH3
O3P-BH33- f O3P-H2- + B(OH)3 2 × 10-5
7 × 10-5
50
60
60
O3P-BD33- f O3P-D2- + B(OD)3
8 × 10-6
3-
Deuterium Substitution of O3P-BH3
-P-BH3- f -P-BH2D-
-P-BH2D- f -P-BHD2
1 × 10-4 b
50
60
50
60
50
60
2 × 10-4 b
4 × 10-5 b
9 × 10-5 b
2 × 10-5 b
4 × 10-5 b
-
-P-BHD2- f -P-BD3
-
a d(pBT) is as shown in Scheme 1. b Upper limit value (see the
Discussion).
centration of species I decreases monotonically with time due
to the two concurrent processes at this stage. Assuming that
hydrolysis occurs more slowly than the rate of deuterium
substitution, and that the reverse exchange reaction is negligible
at early stages in the reaction, the rate of transformation from
species I into species II can be calculated from pseudo-first-
order plots of the changes in species I intensity with time. Rate
constants were 1 × 10-4 and 2 × 10-4 s-1 at 50 and 60 °C,
respectively (data not shown). Since the hydrolysis and
deuterium substitution reactions occur concurrently, the calcu-
lated substitution rate constants are only upper limit values.
We assume that hydrolysis is slower than deuterium substitu-
tion of boranophosphates. The fact that the hydrolysis rate for
the same species (e.g., I in Scheme 3) is 10-fold smaller than
the calculated upper limit deuterium substitution rate constant
(see Table 1) validates this assumption. When the boranophos-
phate is completely deuterated, the decrease in species IV
intensity can be attributed exclusively to hydrolysis, and the
hydrolysis rate constant is estimated to be 8 × 10-6 s-1 at 60
°C. Estimated rate constants for hydrolysis and deuterium
substitution are given in Table 1. Hydrolysis rate constants for
intermediates O3P-BH33-, O3P-BDH23-, and O3P-BD2H3-
in D2O could not be assessed at this time. Although 31P spectral
data offered only upper limit rates for stepwise deuterium
substitution of O3P-BH33-, Figures 5 and 6 clearly show that
the borane hydrogens in boranophosphate can exchange with
D2O in a stepwise manner.
A
31P isotope shift due to 10B/11B substitution is expected
theoretically; however, the observed 31P chemical shifts of the
3-
10B-forms of O3P-BH3 have almost the same values as the
11B-forms in H2O at 50 °C (in Figure 3). Thus, the secondary
31P isotope shift due to 11B/11B substitution is very small, even
though the boron is only one bond removed from the phosphorus
atom. This is not surprising since the mass change due to 10B/
11B substitution is only ca. 10%.
Influence of Electronic Environment on Spectral Line-
3-
Broadening. The 1H, 31P, and 11B peaks of O3P-BH3
1
(Figures 2 and 4; H spectrum not shown) are considerably
narrower than those of d(pBT) mononucleotide (Figures 1 and
2). The broadened resonance of d(pBT) might be ascribed to a
less symmetric electronic environment around the 11B atom. The
line width of a quadrupolar nucleus is largely determined by
its electric field gradient, which is related to the local electronic
symmetry about the nucleus.13a,29 Tetrahedral, octahedral, cubic,
or spherical binding sites, in principle, are electronically
symmetric and thus have zero field gradients; under these
circumstances, sharp lines are observed for symmetric simple
ions in aqueous solutions (e.g., BH4- and BF4-). Lower local
asymmetry can cause profound line-broadening. The 11B
nucleus in the d(pBT) nucleotide is in a less symmetric geometric
environment than in the simpler O3P-BH33-, and thus broader
peaks would be expected for d(pBT), as was observed here.
Rates of O3P-BH33- Hydrolysis and Deuterium Substitu-
tion. The 31P NMR experiments provided evidence that
stepwise deuterium substitutions involving four intermediates
occur and that each intermediate undergoes hydrolysis concur-
rently in D2O as in Scheme 3. Intermediate concentrations of
O3P-BH2D3-, O3P-BHD23-, and O3P-BD33- first increased
The rates of boranophosphate hydrolysis and deuteration are
temperature-dependent. For O3P-BH33- hydrolysis in H2O, k1
values are 2 × 10-5 and 7 × 10-5 s-1 at 50 and 60 °C,
respectively (Table 1).
The rates of boranophosphate hydrolysis and deuteration are
solvent- and isotope-dependent. Each stage of deuterium
1
and then decreased with time during H-decoupled 31P NMR
3-
substitution of borane hydrogens in O3P-BH3 in D2O takes
experiments (Figure 6). From these data, rate constants for
hydrolysis and deuterium substitution steps can be estimated
from the changing concentrations of reactants in situations in
which the previous reaction is finished and the concentration
of the species of interest decreases monotonically with observa-
tion time. For example, after the d(pBT) phosphate ester is
completely hydrolyzed to form O3P-BH33- (species I, Scheme
3) in D2O, the latter species undergoes hydrolysis to produce
phosphonate (species V) and boric acids; deuterium substitution
also occurs to produce species II (O3P-BH2D3-). The con-
place at a progressively slower rate. Thus, the presence of more
deuterium substituents in the -BH3 group slows further
substitution. For example, the upper limit deuterium substitution
rate constant k1 for the transformation of -P-BH3 into -P-
BH2D is ca. 2 × 10-4 s-1, the rate of -P-BH2D deuteration
is ca. 9 × 10-5 s-1, and the rate of -P-BHD2 deuteration is
ca. 4 × 10-5 s-1 at 60 °C (Table 1). In addition, hydrolysis
rates for O3P-BH33- in H2O and O3P-BD33- in D2O are 7 ×
10-5 and 8 × 10-6 s-1 at 60 °C, respectively. Thus, O3P-
3-
3-
BH3 hydrolyzes ca. 10-fold faster in H2O than O3P-BD3
in D2O. These observed kinetic isotope effects are probably
(28) Hansen, P. E. Prog. NMR Spectrosc. 1988, 20, 207-255.
(29) Rankothge, H. M.; Hook, J.; van Gorkom, L.; Moran, G. Magn.
Reson. Chem. 1994, 32, 446-451.
the result of a combination of isotope solvent (H2O Vs D2O)
3-
and composition (O3P-BH3 Vs O3P-BD33-) effects. Since

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Hydrolysis of Deoxynucleoside Boranophosphate
J. Am. Chem. Soc., Vol. 118, No. 28, 1996 6613
Scheme 4. One Possible Mechanism for Hydrolysis and
3
Solvent Exchange Reactions of O3P-BH3 - in D2O
Figure 8. 1H-decoupled 31P spectrum of d(pBT) in D2O buffer at 25
°C. The sample was incubated at ca. 25 °C for at least 30 h before
collecting the spectrum. The downfield multiple quartets centered at
ca. 80.9 ppm correspond to d(pBT) resonances, and the upfield multiple
quartets centered at ca. 78.2 ppm are from boranophosphate resonances
(the right-most peaks of the downfield quartets overlap with the left-
most peaks of the upfield quartets). In the downfield multiple quartets,
subquartet B is due to the resonance of partially deuterated borane (P-
BH2D) in d(pBT), and subquartet A is the resonance of the non-
deuterated d(pBT). In the upfield multiple quartets, subquartets I-IV
are the same as assigned in Figure 5. A total of 1024 scans were
collected, and a line-broadening of 5 Hz was applied.
and 1 mM EDTA at 30 °C for at least 30 h showed no
1
experimental evidence for isotope shifts in the H, 31P, or 11B
spectra (data not shown). There are two reasonable explanations
for why isotope shifts have been detected in the NMR spectra
of the mononucleotide, but not those of the dinucleotide. First,
the NMR studies utilized 40-85-fold higher concentrations of
mononucleotide than the dinucleotide (85 mM Vs 1-2 mM),
so a small fraction of dinucleotide showing the isotope shift
may be difficult to observe. Second, the 11B atom in the
3-
we were not able to measure the hydrolysis rates of O3P-BH3
1
dinucleotide gives broadened H, 31P, and 11B peaks, reducing
in D2O or fully deuterated boranophosphate, O3P-BD33-, in
H2O without interfering with the solvent exchange process, these
isotope effect contributions could not be analyzed.
the “effective” signal/noise ratio. Thus, observation of deuter-
ium substitution by an isotope shift on the 31P or 11B spectra of
d(TpBT) dinucleotides may be obscured.
Tentative Evidence for Solvent Exchange of Borane
Hydrogens in the d(pBT) Mononucleotide. Whereas we
observed stepwise deuterium substitution of borane hydrogens
in O3P-BH33- in D2O, we did not observe deuterium-induced
Possible Mechanisms for the Hydrolysis and Deuteration
of O3P-BH33-. The overall hydrolysis and deuteration reac-
tions of boranophosphate are shown in Scheme 3, and one
possible mechanism involving a five-coordinate activated
complex is shown in Scheme 4. Pentavalent intermediates, e.g.,
BH5 and BH4CN, were previously proposed for the hydrolyses
of hydroborate (BH4-) and cyanoborate (BH3CN-), respec-
tively.22,30,31 If both hydrolysis and deuterium exchange reac-
tions share the same intermediate, an analogous intermediate
containing a -P-BH4 linkage may operate here for the
1
isotope shifts in H-decoupled 31P spectra of d(pBT) during
hydrolysis to form dT and O3P-BH33- in D2O at 50 °C (Figure
2). However, we observed deuterium substitution of the borane
hydrogens in one d(pBT) sample in D2O buffer which contained
100 mM NaCl, 1 mM EDTA, and 10 mM phosphate, pH 7.4,
1
upon incubation at or below 25 °C for over 30 h. The H-
decoupled 31P spectrum (Figure 8) showed that there are at least
two resolved subquartets (labeled A and B) from the original
d(pBT), whose chemical shifts differed by 0.14 ppm, the same
value as observed for each of the deuterium-substituted bora-
nophosphate forms I-IV (Figure 5). We believe that subquartet
A in Figure 8 corresponds to non-deuterated d(pBT) and that
subquartet B corresponds to a population of partially deuterated
d(pBT) (containing the -P-BH2D linkage). Thus, under these
conditions d(pBT) is observed to undergo stepwise solvent
exchange, but deuterium substitution is slow relative to hy-
drolysis of the -P-O-sugar linkage and is thus overtaken by
it.
3-
hydrolysis and deuteration of O3P-BH3 (Scheme 4).
Addition of a D+ from the D2O to O3P-BH33- would form
the -P-BH3D linkage. This intermediate could lead down
either of two paths: proton-deuterium exchange or hydrolysis.
Thus, the intermediate may transfer either an existing H or the
incoming D to phosphorus and P-B bond breakage can occur
to produce a -P-H or -P-D bond in the phosphonate,
resulting in hydrolysis. Alternatively, loss of H+ occurs,
resulting in deuterium substitution. A third possibility is loss
of D+, resulting in no observed solvent exchange.
In this mechanism, we expect to see both -P-H and -P-D
bond formation in the phosphonates if P-H and P-D bonds
are formed at similar rates under a given condition. Under these
circumstances, the 1H-decoupled 31P spectrum should then
contain a singlet and a triplet, and the 31P spectrum should
Exchange between borane hydrogens in the -P-BH3 linkage
and solvent protons is significant because it demonstrates that
the hydrogens in the borane group (-BH3) in O3P-BH33- and
d(pBT) mononucleotide are in dynamic equilibrium with the
solvent. This ready ability of borane hydrogens to exchange
1
contain a doublet and a triplet, corresponding to the H- and
3-
with D2O distinguishes the -BH3 functionality in O3P-BH3
D-forms of phosphonate, respectively. Qualitatively, at the
beginning of boranophosphate hydrolysis, more H-form phos-
phonate should be present, while at later stages most of the
and d(pBT) mononucleotide from the -CH3 in methylphosphon-
ate-modified nucleotides.
3-
In contrast to the mononucleotide, the dithymidine borano-
monophosphate dinucleotide d(TpBT) is very stable to hydroly-
sis; hydrolysis rates for the latter (diester) are about 5 orders of
magnitude slower than for d(pBT) (monoester), i.e., on the order
of 10-9 s-1 at 50 °C in 0.2 M phosphate (pH 7) buffer.8,9a If
we assume that the deuterium substitution rate in D2O obtained
boranophosphate should be in the O3P-BD3 form and only
the deuterated phosphonate should be produced as a hydrolysis
product. Results from our O3P-BH33- hydrolysis experiments
in D2O demonstrate the presence of a triplet in 31P spectra both
1
with (Figure 5) and without H decoupling (data not shown),
indicating that D-form phosphonate forms throughout the course
of the reactions.
3-
for O3P-BH3 (ca. 10-4-10-5 s-1 at 60 °C) is applicable to
the dinucleotide, it is possible that deuterium replacement of
the borane hydrogens in the d(TpBT) dinucleotide occurs more
easily than its hydrolysis in D2O. However, incubating a
d(TpBT) sample in 100 mM NaCl, 10 mM phosphate (pH 7.4),
(30) Kreevoy, M. M.; Hutchins, J. E. C. J. Am. Chem. Soc. 1969, 91,
4329-4330.
(31) Kreevoy, M. M.; Hutchins, J. E. C. J. Am. Chem. Soc. 1972, 94,
6371-6376.

+
+
6614 J. Am. Chem. Soc., Vol. 118, No. 28, 1996
Li et al.
Scheme 5. Tautomeric Equilibrium of Phosphonic Acid and
Hypothetical Deuterium Exchange Mechanism
consistent information. Of special utility were 31P and 11B
isotope shifts due to H/D substitution.
1
In aqueous solution, d(pBT) slowly decomposes, first into
thymidine dT and boranophosphate O3P-BH33- (k1 ) 4 × 10-4
s-1 at 50 °C in H2O), followed by an even slower hydrolysis of
O3P-BH33- to produce phosphonate and boric acid (k1 ) 2 ×
10-5 s-1 at 50 °C in H2O). Therefore, the P-B bond in the
d(pBT) mononucleotide is less susceptible to hydrolysis in
aqueous solution than the P-O-sugar linkage. In addition to
hydrolysis, the hydrogens in the -P-BH3 linkage of both
d(pBT) and O3P-BH33- exchange with solvent. The exchange
process was inferred from the presence of 31P and 11B isotope
shifts and changes in coupling patterns to involve three-step
deuterium substitution of borane hydrogens in D2O. Each
deuterium substitution causes a 0.14 ppm downfield shift of
One possible reason for not observing the 1H-form of
phosphonate by 31P NMR is that, in D2O, the 1H-form of
phosphonate is readily converted into the D-form. It is known
that phosphonic acid is in tautomeric equilibrium with its
trivalent phosphorous acid isomers32,33 (Scheme 5), which can
then be converted into deuterated phosphonate (product V in
Scheme 3). If the conversion reaction from the 1H-form to the
D-form of phosphonate in Scheme 5 were to occur faster (e.g.,
>500-fold) than the rate of P-B bond hydrolysis, the D-form
of phosphonate would be the major observed product in this
mechanism. Another possible reason for not observing the 1H-
form phosphonate by 31P NMR is that existing H and incoming
D in the intermediate (Scheme 4) are not in equivalent positions
so that the incoming deuterium is transferred more efficiently
to phosphorus during the hydrolysis process than the existing
H. In this inequivalent transfer rate case, the D-form of
3-
the 31P resonance of the -P-BH3 linkage. Deuterium
3-
substitution of borane hydrogens in O3P-BH3 occurs in a
three-step manner to form intermediates O3P-BDH23-, O3P-
BD2H3-, and O3P-BD33- and is superimposed upon hydrolysis
of partially and fully deuterated boranophosphate intermediates,
producing deuterated phosphonate and boric acid in D2O. At
3-
50 °C deuterium substitution of O3P-BH3 occurs an order
3-
of magnitude faster than its hydrolysis.
phosphonate would be the dominant product of O3P-BH3
hydrolysis. The present data cannot discriminate between the
two possibilities or other possible mechanisms for hydrolysis
and solvent exchange of boranophosphate.
Acknowledgment. NMR spectra were recorded at the Duke
University NMR Center established with fundings from the NIH,
NSF, NC Biotechnology Center, and Duke University. We
thank Dr. Jeno Tomasz for synthesis of the thymidine 5′-
boranomonophosphate and Dr. Faqing Huang for providing
HPLC hydrolysis data and helpful discussions. We thank Drs.
Tony Ribeiro, Louis D. Quin, and Edward M. Arnett for critical
reading of the paper and Drs. Edward Budowsky and Mark Burk
for suggestions in preparing the paper. C.H. was supported by
NIH Grant GM 47431 and the North Carolina Agricultural
Research Service. This work was supported by an R. J.
Reynolds-Leon Golberg Memorial Fellowship (Duke University
Toxicology Program) to H.L. and by Grant NP-741 from the
American Cancer Society and Grant 5U01-CA60139 from the
NIH to B.R.S.
Conclusion
Reactions of thymidine 5′-boranomonophosphate, d(pBT), in
aqueous solution were studied with the hope that the results
would offer important insights regarding the behavior of these
compounds in potential pharmaceutical applications. Unlike
many borane-containing compounds, phosphate borane is quite
stable in aqueous solution due to its unique structure. Thus,
1H, 31P, and 11B NMR could be used to follow hydrolysis of
the P-B bond and deuterium substitution of the borane
hydrogens by providing a set of interdependent and internally
(32) Crutchfield, M. M.; Dungan, C. H; van Wazer, J. R. Top. Phosphorus
Chem. 1967, 5, 1-74.
(33) Stawinski, J. Handbook of Organophosphorus Chemistry; Marcel
Dekker, Inc.: New York, 1992; pp 377-434.
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