Boranophosphate salts as an excellent mimic of phosphate salts: Preparation, characterization, and properties

Article abstract of DOI:10.1002/ejic.200400142

We report on the preparation of boranophosphate salts, BPi (2), and the exploration of their properties with a view to developing a new mimic of the parent phosphate. BPi salts were easily prepared in excellent yield in a one-pot two-step reaction from tris(trimethylsilyl) phosphite, and were characterized by X-ray crystallography and IR, ¹H and ³¹P NMR spectroscopy. We evaluated the acid/base character of BPi by determining its acidity constants. Likewise, we evaluated the stability of BPi at various pH values, and calculated the decomposition-rate constants at highly acidic pH. We also monitored the H-bonded clustering of BPi in organic solvents, including MeOH. Finally, we explored the chemical behavior of BPi with respect to various organic and inorganic reagents. BPi is stable under the following conditions: both basic and acidic pH (pH > 2), in the presence of amines, and in the presence of Mg²⁺ ions. However, a P-B bond cleavage is observed upon the reaction of BPi with carbodiimides or upon catalytic hydrogenation. The reducing nature of the BH₃ moiety is drastically decreased in BPi. Likewise, the nucleophilicity of BPi's oxygen atom is lower than in phosphate, Pi, salts. Based on its water solubility, acid-base character, and H-bonding properties, BPi appears as a perfect mimic of Pi and is an attractive alternative to the known phosphate isosters. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejic.200400142

FULL PAPER
Boranophosphate Salts as an Excellent Mimic of Phosphate Salts: Preparation,
Characterization, and Properties
Victoria Nahum[a] and Bilha Fischer*[a]
Keywords: Bioisosters / Boranophosphates / NMR spectroscopy / Phosphate mimics
We report on the preparation of boranophosphate salts, BPi
(2), and the exploration of their properties with a view to de-
veloping a new mimic of the parent phosphate. BPi salts were
BPi is stable under the following conditions: both basic and
acidic pH (pH Ͼ 2), in the presence of amines, and in the
presence of Mg2+ ions. However, a P−B bond cleavage is ob-
easily prepared in excellent yield in a one-pot two-step reac- served upon the reaction of BPi with carbodiimides or upon
tion from tris(trimethylsilyl) phosphite, and were characteri-
catalytic hydrogenation. The reducing nature of the BH3
moiety is drastically decreased in BPi. Likewise, the nucle-
1
zed by X-ray crystallography and IR, H and 31P NMR spec-
troscopy. We evaluated the acid/base character of BPi by de- ophilicity of BPi’s oxygen atom is lower than in phosphate,
termining its acidity constants. Likewise, we evaluated the
stability of BPi at various pH values, and calculated the de-
Pi, salts. Based on its water solubility, acid-base character,
and H-bonding properties, BPi appears as a perfect mimic of
composition-rate constants at highly acidic pH. We also mo- Pi and is an attractive alternative to the known phosphate
nitored the H-bonded clustering of BPi in organic solvents,
including MeOH. Finally, we explored the chemical behavior
isosters.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
of BPi with respect to various organic and inorganic reagents. Germany, 2004)
Introduction
This emerging field of novel nucleotide bioisosters has
Phosphates and phosphate-containing molecules play a
major role in numerous biological systems.[1] However, the
unwanted lability of the ester PϪO bond has promoted the
search for suitable bioisosters — phosphate analogues —
which retain the biological activity but possess diminished
lability. The search for bioisosters was initiated by the need
to produce phosphate probes for various studies, such as
probing the stereochemical requirements of enzymes.[2] In
addition, phosphate bioisosters have been developed for im-
proving the pharmacological effects of nucleotide-based
drugs, for example anti-sense agents.[3]
A widely used isoster of phosphate is phosphorothioate
and its analogues, proposed in the pioneering work of
Eckstein et al.[4] In these analogues, the nonbridging oxygen
atom is replaced by a sulfur atom. Other chemical modifi-
cations of the phosphate moiety include the substitution of
the labile phosphate ester oxygen atom by a carbon or ni-
trogen atom, to give phosphonates and phosphoramidate
analogues, respectively.[5]
expanded significantly and has provided many important
applications of the boranophosphate analogues. For in-
stance, nonterminal P-boronated nucleotides, existing as a
pair of diastereoisomers, have been used as stereochemical
probes to elucidate enzymatic catalysis.[8] Oligodeoxyribon-
ucleotides bearing boranophosphate linkages have been
used for polymerase chain reaction (PCR) sequencing and
DNA diagnostics,[9] and boranophosphate nucleotides have
been found to be highly potent and stable P2Y-receptor
agonists.[10] Oligonucleotides bearing boranophosphate
linkages have also been considered as potentially useful
anti-sense agents.[11] These analogues were also tested for
the treatment of cancer as carriers of 10B isotope in boron
neutron-capture therapy.[12]
As mentioned above, the field of boranophosphates co-
vers, in-depth, related nucleotide/oligonucleotide analogues.
However, to the best of our knowledge, no attention has
been given to the unique and chemically interesting inor-
ganic boranophosphate 2 (BPi). Although the related di-
methyl boranophosphate potassium salt 3 has been de-
scribed by Imamoto et al.[13] and by Wada and Saigo,[14]
the preparation of inorganic boranophosphate 2 has not
been reported.
During the last decade, pioneering studies by Spielvogel
and Ramsay-Shaw have proposed boranophosphate anaclo-
gues 1 as bioisosters of natural nucleotides[6] and as import-
ant tools for biochemists.[7]
[a]
We present here the preparation, characterization, and
unique chemical properties of inorganic boranophosphate.
In addition, we demonstrate that BPi is an excellent mimic
of phosphate.
Department of Chemistry, Gonda-Goldschmied Medical
Research Center, Bar-Ilan University,
Ramat-Gan 52900, Israel
Fax: (internat.) ϩ 972-3-535-1250
E-mail: bfischer@mail.biu.ac.il
4124
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejic.200400142
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131

Boranophosphate Salts as an Excellent Mimic of Phosphate Salts
FULL PAPER
Eventually, we were able to obtain BPi in an excellent
overall yield in a two-step, one-pot reaction starting from
tris(trimethylsilyl) phosphite[16] (10; Scheme 2). Phosphite
10 was treated with BH3·SMe2 in dry acetonitrile under an
inert gas for 15 min. Subsequently, intermediate 11 was
treated with 2  methanolic ammonia for 1 h to give the
BPi 2a as a white solid in 93% yield. No further purification
was conducted, since volatile silyl derivatives and the unre-
acted BH3·SMe2 were removed by evaporation. Alterna-
tively, intermediate 11 was treated with NH4OH(aq) solution
(pH ϭ 10), Bu3N in MeOH, or 0.5  triethylammonium
hydrogencarbonate buffer (pH ϭ 7.5) and freeze-dried or
concentrated to provide 2a, 2b, or 2c, respectively.
Results and Discussion
Synthesis
Scheme 2
For the preparation of boranophosphate, we first at-
tempted the treatment of chlorobis(diisopropylamino)phos-
phane (4) with BH3·SMe2 complex,[15] followed by acidic
hydrolysis (pH ϭ 3 or 1; Scheme 1A). This attempt resulted
in a mixture of several phosphorus species. Alternatively,
dibenzyl H-phosphonate 6 was treated with bis(silyl)aceta-
mide, followed by boranation of intermediate 7 with
BH3·SMe2, and hydrolysis of 8 with concentrated am-
monium hydroxide. In this way, dibenzyl boranophosphate
Product 2a is highly water-soluble, whereas 2b dissolves
only in organic solvents such as MeOH, CH3CN, DMF,
and CHCl3. Product 2c is highly soluble both in water and
in organic solvents.
Characterization
9 was obtained in 71% overall yield (Scheme 1B). However, NMR Spectroscopy
attempts to remove the benzyl groups by either catalytic
Compound 2a in water was characterized by 31P NMR
hydrogenation or acidic hydrolysis (pH ϭ 1.3) resulted in
the cleavage of the PϪB bond, leading to phosphorus acid
instead of BPi.
spectroscopy. It shows a signal at δ ഠ 80 ppm (Figure 1).
The boranophosphate 31P NMR spectrum shows a typical
pattern including two overlapping signals: the larger signal
is due to coupling of P to the 11B isotope, and the smaller
signal is due to coupling with the 10B isotope. The relative
height of the smaller peak to the larger one is 0.14 (Fig-
ure 1A).[17] BPi’s hydrogen-coupled 31P NMR spectrum
shows further splitting of the lines into a quadruplet (Fig-
ure 1B). The 1H NMR spectrum shows a typical doublet of
quadruples pattern, at δ ഠ 0.2 ppm, due to coupling of H
to both 11B and 31P (Figure 1C). This pattern overlaps a
more complex pattern due to coupling of H to both 10B
and 31P.
The chemical shift of BPi is pH-dependent. For instance,
at pH ϭ 4.87 and 13.20, the phosphorus atom of BPi res-
onates at δ ϭ 84 and 63 ppm, respectively. Likewise, the
PϪB coupling constant is also pH-dependent, and is re-
duced as the pH decreases (e.g. 147 and 183 Hz for pH ϭ
4.87 and 13.2, respectively). The pH-dependent BPi spec-
trum indicates structural changes of BPi, which are due to
Scheme 1
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131
www.eurjic.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4125

V. Nahum, B. Fischer
FULL PAPER
Figure 2. X-ray crystal structure of BPi: A) unit cell includes eight
BPi molecules, eight H-phosphonate molecules, and 24 ammonium
ions; hydrogen atoms are omitted to clarify the BPi geometry; B)
ORTEP drawing of BPi; crystal data of 2a: monoclinic, P21/c; a ϭ
˚
˚
˚
23.616(5) A, b ϭ 6.3470(13) A, c ϭ 15.325(3) A; V ϭ 2172.9(8)
3
A ; Z ϭ 12; Dcalcd. ϭ 1.623 g/cm3; F(000) ϭ 1104; 3094 reflections
˚
collected, R ϭ 0.1015, Rw ϭ 0.2345, GOF ϭ 1.286; selected bond
lengths [A] and angles [°]: P(1)ϪO(1A) 1.524(7), P(1)ϪO(1B)
˚
1.617(7),
P(1)ϪO(1C)
1.583(7),
P(1)ϪB(1)
1.891(11);
O(1A)ϪP(1)ϪO(1C) 104.0(4), O(1A)ϪP(1)ϪO(1B) 105.3(4),
O(1C)ϪP(1)ϪO(1B) 104.4(4), O(1A)ϪP(1)ϪB(1) 118.2(5),
O(1C)ϪP(1)ϪB(1) 113.0(5), O(1B)ϪP(1)ϪB(1) 110.7(5)
1
Figure 1. NMR spectra of BPi: A) H-decoupled 31P NMR spec-
trum in D2O at 81 MHz; B) 1H-coupled 31P NMR spectrum in
1
D2O at 81 MHz; C) H NMR spectrum in D2O at 200 MHz
Comparison with X-ray crystal data obtained for the re-
the reduction of OϪPϪO angles upon protonation of the lated dimethyl boranophosphate salt (3)[13] indicated similar
˚
molecule.
values for the BϪP (1.895 A) and OϪP (1.490, 1.597 and
˚
1.612 A) bond lengths. For dimethyl boranophosphate, one
X-ray Crystallography
potassium ion was found near one of the oxygen atoms at
a distance of 2.66 A. Based on a comparison of the bond
˚
To obtain structural information on BPi, compound 2a
was crystallized from an aqueous solution (pH ϭ 7). In
addition to compound 2a, the crystal contained phos-
phorus acid (H-phosphonate) in a 1:1 ratio. This unexpec-
ted ratio does not reflect the molar ratio in the original BPi
solution, in which phosphorus acid was less than 5%.
The unit cell contains eight BPi ions, eight H-phosphon-
ate ions, and 24 ammonium ions (Figure 2A). Apparently,
for each BPi anion, one ammonium counterion is observed,
whereas two ammonium counterions are observed around
each H-phosphonate group.
lengths of dimethyl boranophosphate salt with BPi, we as-
sume that the BPi bears two H atoms, which were not found
in the crystallographic data.
˚
The shortest PϪO bond (1.524 A) indicates a partial
double-bond character, and is in accordance with values
˚
found in the structures of phosphate diesters (1.47Ϫ1.51 A)
˚
and monoesters (1.49Ϫ1.53 A). This PϪO bond is signifi-
cantly longer than the bond observed in phosphate triesters
[18]
˚
(1.38Ϫ1.44 A).
˚
IR Spectroscopy
For BPi, the average PϪB bond length is 1.892 A,
whereas for the three PϪO bonds, the average lengths are
The IR spectra of 2a or 2b in KBr pellet indicated
˚
˚
1.585, 1.605 A, and 1.524 A, respectively (Figure 2B). A characteristic bands for PϪB and BϪH in addition to
deviation from tetrahedral angles was observed with values bands associated with PϪOH and PϭO. Specifically, three
of 111Ϫ118° for BϪPϪO, and 104Ϫ105° for the OϪPϪO absorptions at 2350, 2381, 2407 cmϪ1 (s) correspond to
angles.
BϪH stretches, and the absorption at 654 cmϪ1 (m) is the
4126
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131

Boranophosphate Salts as an Excellent Mimic of Phosphate Salts
FULL PAPER
PϪB stretch.[19] Typical absorptions were observed for
PϪOH and PϭO stretches at 900Ϫ1080 cmϪ1 and at
1140Ϫ1250 cmϪ1, respectively.
For an evaluation of the effects of the solvents on the H-
bonds between BPi ions, IR spectra of BPi 2a in aqueous
and methanolic solutions (see below ‘‘H-bonding of BPi’’)
were measured in a germanium cell and compared to the
corresponding spectrum of a neat sample of BPi (Fig-
ure 3B). Comparison of those spectra indicated only minor
differences. For instance, a shift of about 10 cmϪ1 to lower
frequencies was observed for the PϭO stretch of BPi, either
in the neat sample or in MeOH, relative to BPi in aqueous
solution. This shift is probably due to H-bonding-based
clustering in the neat sample and MeOH. The typical fine-
structure for the PϭO stretch in a neat sample of BPi, in
the range of 1144Ϫ1178 cmϪ1, which is possibly also due
to H-bonded clusters, is lost in water. The corresponding
spectrum in MeOH appears as an average of the neat
sample and aqueous solution spectra, probably indicating
the presence of both BPi clusters and solvent H-bonded
species.
Figure 4. Determination of pKa values of BPi; plot of BPi’s 31P
NMR chemical shift in H2O as a function of the pH; two inflection
points are observed in the pH range 4.87Ϫ13.20
Stability of BPi
BPi is stable in neutral and basic solutions. For instance,
after 48 h at room temperature at pH ϭ 13.7, no degra-
dation of BPi was observed by 31P NMR spectroscopy. BPi
is also relatively stable in acidic solution at pH values Ͼ 2.
At pH ϭ 2, BPi slowly degrades to phosphorus acid at a
rate of 7 ϫ 10Ϫ7 sϪ1, R2 ϭ 1.00 (t1/2 ϭ 275 h), as monitored
by 31P NMR spectroscopy.
Under highly acidic conditions (pH Ͻ 2), the evolution
of H2 is clearly observed, the PϪB bond is cleaved, and
boric acid is formed together with phosphorus acid
(Scheme 3).[20] Phosphorus acid was observed in the 31P
NMR spectrum as a doublet at δ ϭ 3.5 ppm (J ϭ 633 Hz).
The borane reacts with water to liberate hydrogen gas and
boric acid.
Figure 3. IR spectra of BPi 2c (germanium cell; 1300Ϫ1100 cmϪ1
;
cutoff: 680 cmϪ1): A) methanolic solution; B) aqueous solution; C)
neat sample
Scheme 3
The stability of inorganic boranophosphate, resulting
from neutral hydrolysis of thymidine 5Ј-boranomono-
phosphate at 50 °C, has been reported earlier.[20]
Chemical Properties
H-Bonding of BPi
Solutions of 2b or 2c in organic solvents (CHCl3,
Acid/Base Properties
CH3CN, DMF, and even MeOH), show unexpected 31P
The acid/base character of BPi was studied by 31P NMR- NMR spectra. Product 2b in MeOH apparently consists of
monitored pH-titration. The chemical shift of compound three different but pattern-related signals. The signals with
2a was plotted against the pH (Figure 4). For the pH range chemical shifts of δ ϭ 80.0 (A), 86.2 (B), and 90.8 (C) ppm,
4.8Ϫ13.2, two inflection points were observed. The second each have an identical BPi-typical pattern (Figure 5).
derivatives of the fitted function provided two pKa values —
Several minutes after the dissolution of 2b in MeOH, sig-
7.12 and 12.54 — with R2 values of 0.999 and 0.997, respec- nals A, B, and C are observed in the 31P NMR spectrum,
tively. These values are similar to the corresponding values with A and B as the major peaks (C constitutes ca. 5% of
of the second and third protonation equilibria of phos- the mixture). The composition of the initial mixture is time-
phoric acid (7.21 and 12.67), and they are higher than those dependent due to the interconversion of the species. When
for phosphorus acid (1.8 and 6.2).
monitoring this process with 0.14  2b in CD3OD (ε ϭ 33)
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131
www.eurjic.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4127

V. Nahum, B. Fischer
FULL PAPER
cur.[28] Indeed, three signals were clearly observed at δ ϭ
3.63, 3.23, and 2.93 ppm, demonstrating that different H-
bonded phosphate clusters can be detected by 31P NMR
spectroscopy. These three phosphate signals, seen in ben-
zene, converged into one in acetonitrile and MeOH, indicat-
ing the collapse of the Pi clusters in polar/protic solvents.
Based on our observations of the pH-dependent chemical
shift of BPi, and on the determination of BPi’s acidity con-
stants (Figure 4), we propose the following assignment of
signals A, B, and C. Signal A corresponds to the mono-
meric BPi, whose chemical shift at δ ഠ 80 ppm indicates
that half the BPi monomer population bears two protons,
and the other half bears one proton (Figure 4). Signal B, at
δ ϭ 86 ppm, corresponds to a BPi moiety that has one BPi
H-bonded neighbor. Namely, signal B could result both
from BPi dimers and higher clusters (Scheme 4). In these
Figure 5. 31P NMR spectrum of BPi 2b in methanolic solution
at room temperature for 160 h, we noted the conversion of cases, each BPi is associated with an additional proton
A and B to C, with a final C/B ratio of 4.4:1 (A disappeared (Bu3NHϩ ions neutralize the negative charges). Therefore,
1
completely). The H-coupled 31P NMR spectrum indicated the chemical shift of the BPi dimer shifts downfield (δ ϭ
H-split quadruplet signals for B and C — no D/H ex- 86 ppm, as at pH ϭ 4.7). As indicated by signal C, BPi also
change occurred.
forms clusters, corresponding to a BPi moiety that has two
A spectrum similar to the one shown in Figure 5, and H-bonded BPi neighbors (Scheme 4). A BPi moiety in the
time-dependent interconversion of the species, was also ob- middle of a cluster is associated with three protons, re-
served for 2b in DMF, CH3CN, and CHCl3.
sulting in an additional downfield shift to δ ϭ 91 ppm, cor-
The possibility that the additional BPi-like species are the responding to that of BPi at pH ϭ 2.
corresponding mono- or dimethyl esters, due to a reaction
1
of 2b with MeOH, was ruled out because their 13C and H
NMR spectra in CDCl3 are devoid of a methyl ester signal.
The three signals seen in the 31P NMR spectrum of 2c
in organic solvents converged into one, probably A (δ ϭ
79.8 ppm) after solvent evaporation and dissolution in D2O.
The possibility that signals B and C are due to borano-
phosphate anhydrides, resulting from 2c in the NMR
sample, is unlikely as the formation of the related phos-
phoric acid anhydrides requires drastic conditions (up to
250 °C at very low pH, or the presence of a condensation
Scheme 4
agent).[21] Obviously, these conditions do not exist in the
NMR samples of BPi.
The possibility of B and C being anhydrides formed dur-
Small H-bonded clusters (i.e. dimers and trimers) are
ing the preparation of BPi, was also ruled out as the 31P
NMR spectrum of 2c, which exhibits several signals in or-
ganic solvents, shows only one signal when measured in
water. In contrast, the hydrolysis of condensed phosphates
is known to be quite slow (i.e. several weeks at 60 °C,
pH ϭ 5).[22]
formed almost instantaneously. This is probably the stage
of nucleation. Once a critical nucleus is formed, a slow pro-
cess of high-order clustering occurs. At this stage the con-
centration of A in solution is drastically reduced. This H-
bonding-based clustering mechanism is also supported by
the observation that, upon evaporation of the organic sol-
vent from the species mixture and dissolution in water, only
A is detected.
The fact that BPi forms clusters even in MeOH, whereas
Pi forms clusters only in benzene, implies that BH3 may
play a role in the pre-organization of the BPi clusters. The
lipophilic BH3 moieties possibly form the core of the cluster
due to hydrophobic interactions (in MeOH). This core is
then further stabilized by PϪOϪ···HOϪP hydrogen bonds.
Phosphoric acid and its derivatives form strongly H-
bonded clusters including cyclic dimers, trimers, and
chains.[23,24] Dimerization occurs in the solid state,[25] in
highly concentrated phosphoric acid,[24,26] in freons,[23b] or
in aprotic organic solvents.[27,28] Therefore, it is likely that
the new BPi-like signals are indicative of clustering of BPi
in organic solvents, and even in a highly polar and H-bond-
ing solvent such as MeOH. These clusters are not large
˚
(Ͻ 30 A) based on light-scattering measurements.
To assess the possibility of observing different H-bond-
clustered species on the NMR timescale, we measured the
31P NMR spectrum of the parent phosphate bis(tributylam-
Reactions of BPi with Selected Reagents
The reactivity of BPi towards various organic and inor-
monium) salt in benzene, where clustering is known to oc- ganic reagents was explored as part of the characterization
4128
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org Eur. J. Inorg. Chem. 2004, 4124Ϫ4131

Boranophosphate Salts as an Excellent Mimic of Phosphate Salts
FULL PAPER
of the chemical nature of BPi. These reagents include aque- Conclusions
ous acid solution, nitrile, amide, carbodiimide, pyridine and
The quest for phosphate bioisosters over the last several
decades has included the synthesis of phosphonates, α-halo
phosphonates,[32,33] phosphorothioates,[4] and borano-
phosphate analogues.[6,34,35] Despite the extensive study of
boranophosphate nucleoside analogues, the exploration of
the parent inorganic boranophosphate has not been re-
ported. The various potential applications of a phosphate
isoster, together with the limitations of the currently avail-
able isosters, justify the continued search for the perfect in-
organic phosphate mimic. Therefore, the unique and chemi-
cally interesting inorganic boranophosphate 2 has been in-
vestigated here as a mimic of phosphate with respect to
properties such as water solubility, geometry, acid/base
character, H-bonding, and chemical reactivity. The great
similarity of BPi to the inorganic phosphate, Pi, is demon-
strated here by the BPi’s high water solubility, and geometry
that is in accordance with that of the parent compound,
imidazole, tosyl chloride, phosphorus oxychloride, H2, and
Zn2ϩ and Mg2ϩ ions.
Although BH3, in complexes with a variety of sulfur/am-
ine/oxygen compounds, is an efficient reducing agent, its
reducing nature is drastically altered in BPi. For instance,
while hydride transfer from ‘‘BH3’’ to water occurs readily,
the BH3 moiety in BPi transfers hydride only in a highly
acidic solution (pH Ͻ 2). Likewise, while BH3·THF com-
plex readily reduces nitriles and amides to the correspond-
ing amines,[29] the borane moiety in BPi does not reduce
acetonitrile and dimethyl formamide, as evidenced by the
complete stability of BPi in these solutions.
A carbodiimide reagent is used for the condensation of
phosphate derivatives to provide the corresponding phos-
phoric anhydride. The reaction of 2a with an excess of 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was
explored in water (pH ϭ 6.5) at 37 °C for 4 h. The addition
of EDC to BPi resulted in excessive loss of this compound
due to complete PϪB bond cleavage of 2a to yield phos-
phorus acid (72% of 2a was degraded after 4 h, based on
the 31P NMR spectrum). This finding is in contrast to di-
ethyl phosphite (cyano- or methoxycarbonyl)borane ana-
logues, which are stable to dicyclohexylcarbodiimide
(DCC).[30]
˚
except for the long PϪB bond (1.892 A) and BϪPϪO
angles that are slightly larger than tetrahedral angles. Fur-
thermore, the acid/base character of BPi is essentially not
altered in comparison to Pi. This finding is in contrast to
the corresponding phosphorothioate isoster, where there is
a reduction of about two log units in the acidity relative to
Pi.[36] Likewise, pKa2 values of α-mono- and -difluorophos-
phonate isosters are one and two log units, respectively,
lower than the pKa2 value of phosphoric acid.[33]
The PϪB bond was also found to be sensitive to catalytic
hydrogenation. Thus, when compound 9 was subjected to
hydrogenation (in the presence of Pd/C), the PϪB bond was
also reduced, yielding phosphorus acid.
Indications for H-bond-based clustering of BPi in or-
ganic solvents, including polar and protic solvents, were ob-
tained from the IR and 31P NMR spectra.
The reactivity of BPi with imidazole and pyridine was
also studied. Specifically, a solution of BPi with 2 or 10
equiv. of imidazole in CD3OD remained unchanged for
96 h, based on the 31P NMR spectra. Likewise, only a negli-
gible cleavage of the PϪB bond was observed after 113 h
for a solution of BPi in pyridine. BPi is apparently more
stable to imidazole and pyridine than the related analogue
tetramethyl boranopyrophosphate.[31] The reaction of 5Ј-
DMT-2Ј-deoxythymidine with tetramethyl boranopyro-
phosphate in the presence of N-methylimidazole was re-
ported to proceed with the partial removal of the borane
group. Likewise, when pyridine was used as a solvent, par-
tial removal of the borane group was observed.[31]
BPi is stable under both highly basic and acidic con-
ditions (at pH Ͼ 2). In addition, BPi is stable in the pres-
ence of imidazole, pyridine, and Mg2ϩ ions. However, the
PϪB bond cleavage is observed upon the reaction of BPi
with carbodiimides. A loss of BPi’s borane moiety also oc-
curs at pH values Ͻ 2.
A drastic alteration in the chemical nature of BPi as com-
pared to Pi and BH3 complexes is observed. While Pi is
a nucleophile,[37] BPi is a poor nucleophile. Likewise, the
reducing nature of the BH3 group in BPi is drastically lower
than in other BH3 complexes.
Based on the geometry, water solubility, acid/base
character, and H-bonding properties, BPi appears to be a
perfect mimic of Pi, and an attractive alternative to the thi-
ophosphate and α-halophosphonate isosters.
The presence of divalent metal ions such as Zn2ϩ and
Mg2ϩ in DMF and water for 48 h and 4 h, respectively, left
BPi unchanged.
Whereas dimethyl boranophosphate monopotassium salt
(3) plays the role of an efficient nucleophile,[13] the related
BPi is a poor nucleophile. Thus, when BPi was treated with
tosyl chloride or mesyl chloride (with or without amine) in
acetonitrile for 24 h, even at 60 °C, no reaction occurred.
Likewise, the reaction of BPi with phosphorus oxychloride
and its derivatives [P(O)Cl2R] yielded no product. The rea-
son for BPi’s reduced nucleophilicity, as compared to 3,
might be due to the ‘‘carboxylate-like’’ nature of BPi, as
compared to the ‘‘alkoxide-like’’ nature of dimethyl borano-
phosphate.
Experimental Section
General: All air- and moisture-sensitive reactions were performed
in flame-dried, nitrogen-flushed flasks sealed with rubber septa; the
reagents were introduced with a syringe. The progress of the reac-
tions was monitored by TLC on precoated Merck silica-gel plates
(60 K-254). Column chromatography was performed with Merck
silica gel 60 (230Ϫ400 mesh). Compounds were characterized by
NMR spectroscopy using Bruker DPX-300, DMX-600, or AC-200
spectrometers. NMR spectra were recorded with a Bruker AC-200
spectrometer with a 31P NMR probe (isotope frequency of
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131
www.eurjic.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4129

V. Nahum, B. Fischer
FULL PAPER
81 MHz) using 85% H3PO4 as an external reference. IR spectra
of BPi in KBr pellets were recorded with a Nicolet Impact 400D
temperature. Solutions of 2a (0.15Ϫ0.18 ) at different pH values
were prepared by adding dilute sodium hydroxide or hydrochloric
spectrometer using the OMNIC program. IR spectra of BPi in acid solutions. The 31P NMR chemical shift was monitored as a
solution were measured using a Bruker Vector 22 equipped with a
liquid-nitrogen-cooled MCT detector. For the ATR measurements,
a Harrick variable-angle ATR accessory was used. For one spec-
trum, 100 scans were co-added at a resolution of 4 cmϪ1. The clean
ATR germanium crystal (Harrick Scientific Corporation) was
measured for the background spectra (cutoff 680 cmϪ1). Crystallo-
graphic data were collected with a Nonius KappaCCD dif-
fractometer at 120 K with scans of 1° collected at a speed of 1°/20 s;
the merging R-factor on the data was 0.046 with 36867 reflections
collected and 2979 unique. BPi crystals were obtained as colorless
needles. Further details of the crystal structure investigation may
be obtained from the Fachinformationzentrum Karlsruhe, 76344
Eggenstein-Leopoldshafen, Germany, on quoting the depository
number CSD-413735. Melting points were measured using a
Fisher-Johns melting-point apparatus. Apparent pH values were
function of the pH. A five-parameter sigmoid function was fitted
to the data using Sigma Plot 2000 (SPSS, Inc.): δ ϭ δ0 ϩ a/[1 ϩ
e
Ϫ{(pHϪpH°)/b}]c. The inflection point, which is determined by the
second derivative of the fitted sigmoid function, is the pKa value.
Determination of the Decomposition Rate of BPi 2a at pH ؍ 2:
The stability of 2a in acidic solution was evaluated by 31P NMR
spectroscopy at room temperature, monitoring the formation of the
deboranation product (phosphorus acid). A 0.16  solution of 2a
at pH ϭ 2 was prepared by adding dilute hydrochloric acid to a
ϩ
solution of inorganic boranophosphate (NH4 salt) in H2O and
10% D2O. The percentage of decomposition of 2a is based on inte-
grations of PBi and phosphorus-acid signals (δ ϭ 90.93 and
3.3 ppm, respectively). The decomposition rate was determined by
measuring changes in the integration of the respective NMR signals
within 96 h.
measured with
a Hanna Instruments pH-meter (HI 8521),
equipped with an Orion micro-combination pH electrode (9802).
Compounds 2 are all inorganic boranophosphate salts having dif-
ferent ammonium counterions (ammonium in 2a, tributylammon-
ium in 2b, triethylammonium in 2c, and tetrabutylammonium in
2d, see Experimental for 2a below). The preparation of salts 2aϪ2c
for elemental analysis involved a freeze-drying process. Unfortu-
nately, it was impossible to obtain reliable elemental analyses for
salts 2aϪ2c due to loss of some of the amine during this freeze-
Acknowledgments
The authors would like to thank the Marcus Center for Medicinal
Chemistry, and the German Bundesministerium fuer Bildung und
Forschung (BMBF) and the Israeli Ministry of Science, Culture
and Sport (MOS) for financial support (grant number 1812). In
addition, the authors gratefully acknowledge Dr. D. T. Major for
the calculation of the IR spectrum of BPi, Prof. S. Specher and I.
Mastai for a helpful discussion, and Prof. C. Sukenik and Dr. T.
Bayer for their help with IR measurements in a Ge cell.
1
drying process (as observed in their H NMR spectra). Therefore,
we chose the tetrabutylammonium salt 2d for performing a re-
presentative elemental analysis, since in this case there is no partial
loss of the amine during freeze-drying.
[1]
[1a] F. H. Westheimer, Science 1987, 1173Ϫ1178. [1b] F. H. West-
Boranophosphate 2a: BH3·SMe2 complex in THF (2 , 1.35 mL,
2.7 mmol) was added to a solution of tris(trimethylsilyl) phosphite
(600 µL, 1.795 mmol) in dry CH3CN (5 mL) under N2 at 0 °C. The
resulting solution was kept at room temperature for 15 min. Dry
MeOH (15 mL) and 2  NH3 in EtOH (1.8 mL, 3.6 mmol) were
added and the mixture was stirred at room temperature for 1 h.
The solvent was then removed under reduced pressure, and the
product was obtained as a white solid in 93% yield (202 mg,
heimer, Phosphorus Chemistry Ϫ Developments in American
Science (Eds.: E. N. Walsh, E. J. Griffith, R. W. Parry, L. D.
Quin), ACS Symposium Series 486ACS, Washington DC,
1992.
[2] [2a]
P. J. Roumaniuk, F. Eckstein, J. Biol. Chem. 1981, 256,
[2b]
7322Ϫ7328.
B. A. Conolly, F. Eckstein, Biochemistry 1982,
21, 6158Ϫ6167.
[3] [3a]
[3b]
S. Agrawal, Biochim. Biophys. Acta 1999, 1489, 53Ϫ67.
1
C. A. Stein, Chem. Biol. 1996, 3, 319Ϫ323.
1.556 mmol), m.p. Ͼ 240 °C. H NMR (D2O, 200 MHz): δ ϭ 0.27
[4] [4a]
S. R. Nahorski, V. B. L. Potter, Trends Pharamacol. Sci.
(d of 1:1:1:1 quadruplet, JP,H ϭ 22, JB,H ϭ 87 Hz, 3 H) ppm. 31P
NMR (D2O, 81 MHz): δ ϭ 80.38 (1:1:1:1 quadruplet, J ϭ 156 Hz;
1:1:1:1:1:1:1 septuplet, J ϭ 52 Hz) ppm. IR (KBr): ν˜ ϭ 2412, 2378,
2352, 1181, 1149, 1077Ϫ903, 654 cmϪ1. Compound 2a was con-
verted into the corresponding tetraethylammonium salt as follows:
2a was passed through a Sephadex-CM C-25 Ϫ tetraethylam-
monium-form column (prepared from the corresponding sodium-
form resin upon loading with excess Et4NCl) and the column was
washed with about 20 volumes of deionized water. The solution
was freeze-dried to yield tetraethylammonium BPi (2d) as a white
solid. Based on the pH value of the 2d solution, the 31P NMR
spectrum, and correlation with the plot of BPi 31P NMR shifts vs.
pH (Figure 4), the expected empirical formula is BH3O3-
PH1.5(Et4N)1.5 (289.3): calcd. H 11.9, P 10.7; found H 11.3, P 9.5.
[4b]
1989, 10, 139Ϫ144.
F. Eckstein, Ann. Rev. Biochem. 1985,
54, 367Ϫ402. [4c] F. Eckstein, Angew. Chem. Int. Ed. Engl. 1983,
22, 423Ϫ439. [4d] F. Eckstein, Antisense Nucleic Acid Drug Dev.
2000, 10, 117Ϫ121.
[5]
R. Engel, Chem. Rev. 1977, 77, 349Ϫ367.
[6] [6a]
A. Sood, B. R. Shaw, B. F. Spielvogel, J. Am. Chem. Soc.
[6b]
1990, 112, 9000Ϫ9001.
B. R. Shaw, D. Sergueev, K. He, K.
Porter, J. S. Summers, Z. Sergueeva, V. Rait, Methods Enzymol.
2000, 313, 226Ϫ257.
[7] [7a]
V. Rait, D. S. Sergueev, J. S. Summers, K. He, F. Huang, B.
Krzyzanowska, B. R. Shaw, Nucleosides Nucleotides 1999, 18,
1379Ϫ1380. [7b] J. Zhang, T. Terhorst, M. D. Matteucci, Tetra-
hedron Lett. 1997, 38, 4957Ϫ4960. [7c] K. W. Porter, D. J. Briley,
B. R. Shaw, Nucleic Acids Res. 1997, 25, 1611Ϫ1617.
Z. A. Sergueeva, D. S. Sergueev, A. A. Ribeiro, J. S. Summers,
B. R. Shaw, Helv. Chim. Acta 2000, 83, 1377Ϫ1391, and refer-
ences cited therein.
[8]
Boranophosphate 2b: The tributylammonium salt of borano-
phosphate was prepared as described above for 2a. However, Bu3N
(0.85 mL, 3.57 mmol) was added instead of NH3/EtOH. The prod-
[9] [9a]
K. He, K. W. Porter, A. Hasan, J. D. Briley, B. R. Shaw,
Nucleic Acids Res. 1999, 27, 1788Ϫ1794. [9b] K. W. Porter, J. D.
Briley, B. R. Shaw, Nucleic Acids Res. 1997, 25, 1611Ϫ1617.
V. Nahum, G. Zündorf, S. A. Levesque, R. Beaudoin, G. Re-
iser, B. Fischer, J. Med. Chem. 2002, 45, 5384Ϫ5396.
J. S. Summers, B. R. Shaw, Curr. Med. Chem. 2001, 8,
1147Ϫ1155.
uct was obtained as
1.385 mmol), m.p. 83Ϫ84 °C. IR (KBr): ν˜: 2407, 2381, 2350, 1184,
1150, 1100Ϫ850, 655 cmϪ1
a white solid in 93% yield (645 mg,
[10]
[11]
[12]
.
Determination of the pKa Values of Boranophosphate 2a: The pKa
values of 2a were evaluated by 31P NMR spectroscopy at room
B. F. Spielvogel, A. Sood, B. R. Shaw, I. H. Hall, R. G. Fairch-
4130
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131

Boranophosphate Salts as an Excellent Mimic of Phosphate Salts
FULL PAPER
[23b]
ild, B. H. Laster, C. Gordon, Prog. Neutron Capture Ther. Can-
cer 1992, 211Ϫ213.
T. Imamoto, E. Nagato, Y. Wada, H. Masuda, A. Yamaguchi,
Cryst. Liq. Cryst. 2002, 379, 401Ϫ406.
C. Detering, P. M.
Tolstoi, N. S. Golubev, G. S. Denisov, H. H. Limbach, Dok.
Akad. Nauk 2001, 379, 353Ϫ356; Chem. Abstr. 2002, 136,
20119.
[13]
T. Uchimaru, J. Am. Chem. Soc. 1997, 119, 9925Ϫ9926.
T. Wada, M. Shimizu, N. Oka, K. Saigo, Tetrahedron Lett.
[14]
[24]
[25]
Y. Kameda, K. Sugawara, T. Hosaka, T. Usuki, O. Uemura,
Bull. Chem. Soc. Jpn. 2000, 73, 1105Ϫ1112.
E. Gebert, A. H. Reis Jr., S. W. Peterson, L. I. Katzin, G. W.
Mason, D. F. Peppard, J. Inorg. Nucl. Chem. 1981, 43,
1451Ϫ1464.
D. L. Wertz, G. A. Cook, J. Solution Chem. 1985, 14, 41Ϫ8.
M. Hojo, H. Hasegawa, Z. Chen, Bull. Chem. Soc. Jpn. 1996,
69, 2215Ϫ2220.
2002, 43, 4137Ϫ4140.
[15]
A. Longeau, P. Knochel, Tetrahedron Lett. 1996, 37,
6099Ϫ6102.
[16]
A. Sood, C. K. Sood, I. H. Hall, B. F. Spielvogel, Tetrahedron
[26]
[27]
1991, 47, 6915Ϫ6930.
R. K. Harris, Nuclear Magnetic Resonace Spectroscopy: A
[17]
Physicochemical View, Longman Scientific and Technical, So-
merset, NJ, 1986.
D. E. C. Corbridge, The Structural Chemistry of Phosphorous,
[28] [28a]
D. F. Peppard, J. R. Ferraro, G. W. Mason, J. Inorg. Nucl.
[18]
[28b]
Chem. 1958, 7, 231Ϫ244.
D. F. Peppard, J. R. Ferraro, G.
[28c]
Elsevier, Amsterdam, 1974.
W. Mason, J. Inorg. Nucl. Chem. 1957, 4, 371Ϫ372.
[19] [19a] D. E. C. Corbridge, Phosphorous: An Outline of its Chemis-
Ref.[18a], pp. 1049Ϫ1064.
[29]
[30]
try, Biochemistry and Technology; Studies in Inorganic Chemis-
H. C. Brown, Borane in Organic Chemistry, Cornell University
Press, Ithaca, 1972, pp. 230Ϫ235.
K. Vyakaranam, G. Rana, B. F. Spielvogel, J. A. Maguire, N.
S. Hosmane, Nucleosides, Nucleotides Nucleic Acids 2002, 21,
581Ϫ598.
T. Wada, M. Shimizu, N. Oka, K. Saigo, Tetrahedron Lett.
2002, 43, 4137Ϫ4140.
G. M. Blackburn, Chem. Ind. 1981, 134. G. M. Blackburn, D.
A. England, F. Kolkman, J. Chem. Soc., Chem. Commun.
1981, 930Ϫ932.
G. M. Blackburn, D. Brown, S. J. Martin, M. J. Parratt, J.
Chem. Soc., Perkin Trans. 1 1987, 181Ϫ186.
J. S. Summers, D. Roe, P. D. Boyle, M. Colvin, B. R. Shaw,
Inorg. Chem. 1998, 37, 4158Ϫ4159.
[19b]
try, Elsevier, Amsterdam, 1995, vol. 20, pp 1141Ϫ2.
We
also based our IR assignment on quantum mechanical calcu-
lations as follows. The boranophosphate anion was optimized
using the B3LYP functional in conjunction with the 6-31ϩ6(d)
basis set. Frequency calculation was performed to obtain the
IR spectrum and the harmonic vibrational frequencies were
scaled by a factor of 0.9614. The calculations employed the
Gaussian 98 program: M. J. Frisch, G. W. Trucks, H. B. Schle-
gel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G.
Zakrzewski, J. A. Montgomery, R. E. Stratmann, J. C. Burant,
S. Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C.
Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi,
B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski,
G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma, D. K.
Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J.
Cioslowski, J. V. Ortiz, B. B. Stefanov, G. Liu, A. Liashenko,
P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin, D. J. Fox,
T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C.
Gonzalez, M. Challacombe, P. M. W. Gill, B. G. Johnson, W.
Chen, M. W. Wong, J. L. Andres, M. Head-Gordon, E. S. Re-
plogle, J. A. Pople, Gaussian 98 (Revision A.7), Gaussian, Inc.,
Pittsburgh, PA, 1998.
[31]
[32]
[33]
[34]
[35]
B. R. Shaw, J. Madison, A. Sood, B. F. Spielvogel, Methods
Mol. Biol. (Totowa, NJ, U. S.), 1993, 20, 225Ϫ243.
[36] [36a] E. K. Jaffe, M. Cohn, Biochemistry 1978, 17, 652Ϫ657. [36b]
J. A. Gerlt, M. A. Reynolds, P. C. Demou, G. L. Kenyon, J.
Am. Chem. Soc. 1983, 105, 6469Ϫ6474.
[37] [37a]
S. Saxena, Ind. J. Chem. Sect. A 2002, 41A, 718Ϫ722;
[37b]
Chem. Abstr. 2002, 137, 140554.
O. A. El Seoud, M.-F.
Ruasse, W. A. Rodrigues, J. Chem. Soc., Perkin Trans. 2 2002,
[37c]
1053Ϫ1058.
P. M. Cullis, G. A. Fawcett, M. J. Harger, M.
[20]
[37d]
H. Li, C. Hardin, B. R. Shaw, J. Am. Chem. Soc. 1996, 118,
6606Ϫ6614.
Lee, J. Am. Chem. Soc. 2001, 123, 4147Ϫ54.
H.
Bundgaard, J. Hansen, Int. J. Pharm. 1981, 9, 273Ϫ283.
Ref.[18], p. 180, 214, 261.
Received February 19, 2004
Early View Article
Published Online August 12, 2004
[21]
[22]
Ref.[18], p. 213.
[23] [23a]
A. Onoda, T.-A. Okamura, Y. Hitoshi, N. Ueyama, Mol.
Eur. J. Inorg. Chem. 2004, 4124Ϫ4131
www.eurjic.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4131