α-Azido bisphosphonates: Synthesis and nucleotide analogues

Article abstract of DOI:10.1021/jo200045a

The first examples of α-azido bisphosphonates [(RO) ₂P(O)]₂CXN₃ (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4, R = H, X = H) and corresponding β,γ- CXN₃ dGTP (5-6) and α,β-CXN₃ dATP (7-8) analogues are described. The individual diastereomers of 7 (7a/b) were obtained by HPLC separation of the dADP synthetic precursor (14a/b).

Full text of DOI:10.1021/jo200045a

NOTE
pubs.acs.org/joc
r-Azido Bisphosphonates: Synthesis and Nucleotide Analogues
Brian T. Chamberlain, Thomas G. Upton, Boris A. Kashemirov, and Charles E. McKenna*
Department of Chemistry, University of Southern California, Los Angeles, California 90089-0744, United States
S Supporting Information
b
ABSTRACT: The first examples of R-azido bisphosphonates [(RO)2
P(O)]2CXN3 (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4,
R = H, X = H) and corresponding β,γ-CXN3 dGTP (5ꢀ6) and R,β-CXN3
dATP (7ꢀ8) analogues are described. The individual diastereomers of
7 (7a/b) were obtained by HPLC separation of the dADP synthetic precursor
(14a/b).
riphosphate analogues in which a bridging oxygen is re-
placed by a functionalized carbon atom have been of interest
T
as enzymatic probes since the early 1980s.1,2 Recently, we
described the preparation of a series of highly purified and
well-characterized β,γ-CXY3,4 and R,β-CXY5 dNTP derivatives
and applied them as probes of structure, function, and fidelity in
DNA polymerase β (pol β),3ꢀ5 a base excision repair (BER)
enzyme that is overexpressed in some cancers. By appropriate
substitution on the CXY carbon (X, Y= H, Me, F, Cl, Br) a range
of stereoelectronic properties is made available to probe binding
and catalytic interactions with the enzyme. Linear free-energy
relationship (LFER) plots of DNA pol β-catalyzed dGMP
incorporation rates into nascent DNA from β,γ-CXY dGTPs
have revealed a base match-dependent leaving group effect,4 and
X-ray crystallographic data have provided evidence for stereo-
specific binding of analogues containing a CXF group.3,6
Figure 1. Structures of R-azido bisphosphonate esters and acids.
Scheme 1. Synthesis of 1
To extend these studies, we wished to construct β,γ (5ꢀ6)
and R,β (7ꢀ8) dNTPs that incorporate a bridging CXN3
group. As a pseudohalide,7 the azido functionality could usefully
extend the tunability of the analogue series, providing more
information on molecular interactions at the active site arising
from the stereochemistry at the bridging carbon. Introduction of
the azido group might also facilitate separation of individual CXY
diastereomers, which has not been accomplished with the various
X,Y = H, methyl or halide derivatives prepared thus far.3,5 These
considerations, combined with the prospect of developing a new
and potentially useful bisphosphonate synthon, motivated an
investigation of R-azido bisphosphonic esters and acids, a pre-
viously unknown class of compounds (Figure 1).
Electrophilic azido transfer was explored as a methodology
offering a direct route to 1 and 2 from readily available starting
materials. In this approach, a sulfonyl azide reacts with a
carbanion species to generate a triazene intermediate that can
fragment into either diazo or azido products.8 Various factors
influence the fragmentation pathway, but the nature of the sulfonyl
azide is particularly important. Sulfonyl azides with sterically bulky
and electron-donating substituents, such as 2,4,6-triisopropylbenze-
nesulfonyl azide (trisyl azide; TrN3), favor azido transfer8,9 whereas
those containing strongly electron-withdrawing moieties, for exam-
ple trifluoromethylsulfonyl azide (triflyl azide; TfN3), favor diazo
transfer.9,10
Although the methylene group of a bisphosphonate ester such
as tetraisopropyl methylenebis(phosphonate) 9 is exposed in
principle to either pathway, replacement of one R-H by a methyl
group, as in tetraisopropyl ethylidenebis(phosphonate) 10, will
enforce azido transfer. In the synthesis of 1 from 10, methylation
of 9 with potassium tert-butoxide (t-BuOK) followed by methyl
iodide gave a mixture of 78% 10 and 20% R,R-dimethyl side
product. Fortunately, this crude 10 preparation could be directly
treated with t-BuOK and then p-toluenesulfonyl azide (tosyl
azide; TsN3) in DMSO at room temperature to give the azido
Received: January 7, 2011
Published: April 04, 2011
r
2011 American Chemical Society
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Scheme 2. Synthesis of 2
Scheme 4. Synthesis of Nucleotide Azidomethylene
Analogues
Scheme 3. Base-Induced Decomposition of 2
with a previously observed 3ꢀ4 ppm downfield effect of R-methyl-
ation in other bisphosphonate esters.20,21
ester 1 in 70% yield after purification by column chromatography
(Scheme 1).
Reaction of 1 and 2 with bromotrimethylsilane (BTMS)22 in
acetonitrile at room temperature quantitatively gave the tetrakis-
(trimethylsilyl) esters, which display the expected upfield 31P
shifts, Δδ þ(16ꢀ17) ppm. Desilylation with aqueous ethanol
produced the acids 3 and 4 (>98% overall) as hygroscopic white
solids. These acids are stable in dilute aqueous solution pH 2ꢀ12
for at least several days at room temperature. They have IR bands
at 2126, 2085 (sh) cmꢀ1 (3) and 2113 cmꢀ1 (4) attributed to the
azido group. The 1H NMR spectra include resonances at δ 1.43
ppm (C(CH3)N3, t, JHP 13 Hz) for 3 and δ 3.26 ppm (CHN3, t,
Attempts to synthesize 2 from 9 using tosyl azide led to the
unwanted diazo transfer product. Reaction of a carbanion derived
from an activated methylene group with a sulfonyl azide is a
known route to tetraalkyl diazomethylenebis(phosphonates)
and trialkyl R-diazo phosphonoacetates.11 However, use of trisyl
azide with careful optimization of conditions (THF, ꢀ78 °C,
potassium counterion, 5-fold azide reagent excess, immediate
quenching by AcOH) generated 2 from the carbanion of 9 in
greater than 95% yield by 31P NMR (Scheme 2). Despite the
efficient conversion, purification of 2 proved to be problematic
initially due to difficulty in removing the large excess of trisyl
azide and a minor side product, tetraisopropyl diazomethylenebis
(phosphonate) (11), by preparative chromatography on silica
gel. Pure 2 (50% yield) was eventually obtained by crystalliza-
tion of remaining trisyl azide, filtration, and selective oxidation
of 11 with t-butyl hypochlorite12 in wet ethyl acetate13 prior to
chromatography.
Care must be taken during workup of 2, which retains a
relatively acidic R-H, because it decomposes slowly on silica gel,
more quickly in aqueous solution at pH 6, and rapidly at pH 8.
This contrasts with the reported stability of the malonate
derivative14 and presumably reflects the resonance stabilization
available in the enolate form of the latter compound.
Exposure of 2 to bases such as BuLi, NaH, KHMDS, and
t-BuOK results in the release of N2 with formation of diisopropyl
phosphorocyanidate (12)15 and diispropyl phosphite (13)
(Scheme 3). The base-induced elimination of nitrogen from
R-azido esters,16 malonates,14 and sulfones17 has been previously
reported and transition metal-catalyzed versions of this reaction
have found synthetic utility.18,19 In contrast to diethyl azidoma-
lonate, in which the carbanion was further derivatized at ꢀ15 °C
without the loss of N2,14 2 loses N2 rapidly upon exposure to a
variety of bases in organic solvents at ꢀ78 °C and does not give
appreciable amounts of adducts in the presence of a large excess
of N-chlorosuccinimide.
J
HP 17 Hz) for 4. The 13C NMR displays resonances at δ 23.5
(CH3) and δ 68.4 ppm (CR, t, JCP 129 Hz) for 3, and at δ 65.1
ppm (CR, t, JCP 124 Hz) for 4. The 31P chemical shifts are in the
expected regions, δ 18.3 ppm (3) and δ 13.5 ppm (4). The
HRMS m/z values for the [M ꢀ H]ꢀ anion of 3 and 4 match
calculated values within error, confirming the assigned structures.
The present study and our previous interest in diazo transfer
chemistry23,24 prompted us to reinvestigate a reported azido
transfer to triethyl phosphonoacetate using triflyl azide in the
presence of triethyl amine.25 Triflyl azide is known to be a
powerful diazo transfer reagent9 and attempts by other authors
to extend the procedure to benzocyclic β-keto esters were
unsuccessful;8 nevertheless, the original reaction has been cited
as a viable azido transfer reaction in recent reviews.26,27 In our
hands, the procedure of Hakimelahi and Just25 produced triethyl
1
diazophosphonoacetate, characterized by H, 31P NMR and
MS (ESI and APCI) and generated no detectable amounts of
azido transfer product. In light of these results and analysis of the
literature,28 we propose that the reaction of triflyl azide with
triethylphosphonoacetate is consistent with known diazo trans-
fer chemistry and is not an anomalous case of azido transfer.
With acids 3 and 4 in hand, we then proceeded to the synthesis
of β,γ (5, 6) and R,β (7, 8) azido methylene nucleotide tripho-
sphate analogues. The β,γ-CXN3 dGTP analogues 5 and 6 were
prepared by coupling3,4 the Bu3NHþ salts of 3 and 4 with
dGMP-morpholidate.29 R,β-CXN3 analogues of dATP (7, 8)
were synthesized by reaction of the Bu4Nþ salt of 3 or 4 with dA-
50-tosylate followed by enzymatic phosphorylation of the result-
ing R,β-CXN3 dADP intermediates (14, 15) by ATP-phos-
phoenolpyruvate (PEP) catalyzed by pyruvate kinase (PK) and
nucleoside diphosphate kinase (NDPK) in HEPES buffer, pH
7.55 (Scheme 4). The final compounds were purified by dual-
pass (strong-anion exchange, SAX, followed by reverse phase,
RP) HPLC to g97% as determined by 31P NMR and analytical
HPLC and were characterized by NMR (1H, 31P) and HRMS.
The structures of 1ꢀ4 are supported by their suite of IR,
NMR, and MS data. Esters 1 and 2 are both colorless oils with
strong IR absorbances in the azido region (1: 2123, 2086 cmꢀ1; 2:
2104, sh. at 2134 cmꢀ1). The 1H NMR spectrum of 1 includes a
CH3 triplet at δ 1.60 ppm (JHP 15 Hz) and that of 2 has an R-H
triplet at δ 3.53 ppm (JHP 21 Hz). The 13C NMR spectrum of 1 has
resonances at δ 17.6 (CH3) and δ 59.8 ppm (CR, t, JCP 150 Hz).
The CR of 2 resonates at δ 55.0 ppm (t, JCP 145 Hz). The 31P
chemical shifts of 1 (δ 17.0 ppm) and 2 (δ 13.3 ppm) are consistent
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NOTE
diastereomers of the R,β-C(CH3)N3 dADP (14a, 14b) and
corresponding dATP analogues (7a, 7b) provides more refined
probes for stereochemical interactions of these compounds with
appropriate enzymes. A further use of these new bisphospho-
nates may be to prepare derivatives made more conveniently or
uniquely accessible by the presence of the synthetically versatile
azido function.
’ EXPERIMENTAL SECTION
General Experimental Methods. The nucleotide triphosphate
analogues were prepared by adapting our previously published methods
for β,γ-CXY3,4,6 and R,β-CXY5 dNTP analogues. Analytical HPLC
analysis was conducted on a Varian PureGel SAX 10 mm ꢁ 100 mm
7 μL column eluted with an A, H2O, B, 0.5 M (0ꢀ50%) LiCl gradient
over 30 min at a 4 mL/min flow rate. Products were detected at 259 nm
for adenosine derivatives and at 253 nm for guanosine derivatives. All
dNTP derivatives were prepared as triethylammonium salts.3ꢀ6 CD
spectra were obtained on a JASCO J-815 spectropolarimeter.
Figure 2. Separation of 14 diastereomers by RP preparative HPLC.
(Left) 31P NMR spectrum of 14a/b as synthesized displays two pairs of
doublets for both PR and Pβ. (Right) 31P NMR (14a and 14b) spectra of
separated diastereomers show single pairs of doublets for PR and Pβ.
Tetraisopropyl (1-Azidoethane-1,1-diyl)bis(phosphonate),
1. Tetraisopropyl methylenebis(phosphonate) (1.75 g, 5 mmol) was
treated with 1.2 equiv of t-BuOK and 1.2 equiv of MeI in anhydrous
acetonitrile overnight at rt. Volatiles were removed at reduced pressure and
the residue was dissolved in water, extracted into DCM, and dried over
MgSO4. Evaporation of the solvent left an oil (1.84 g) that contained ∼20%
dimethylated product, ∼2% unmethylated material, and 78% monomethy-
lated product. This mixture (∼4 mmol monomethylated product) was
dissolved in 20 mL of DMSO and stirred with 0.689 g (6 mmol) of t-BuOK.
TsN3 (1.05 g, 5 mmol) was added dropwise. The reaction mixture was
stirred for 30 s, quenched with 1.5 mL of AcOH, and then allowed to stir for
several hours at rt. The reaction mixture was extracted with hexanes and the
organic phase was dried over MgSO4. Following concentration by evapora-
tion, the product was purified on silica gel using ACE/DCM (1:3), Rf =
0.69, giving 1as a colorless oil. Yield: 1.45 g (70% overall). IR: v (film) 2123,
2086 cmꢀ1 (N3). δH (399.8 MHz; CDCl3): 1.33ꢀ1.37 (24 H, m,
4 ꢁ OCH(CH3)2), 1.60 (3H, t, JHP 15.2 Hz, PCCH3N3P), 4.83 (4 H,
m, 4 ꢁ OCH(CH3)2); δC (100.5 MHz; CDCl3): 17.6 (t, JCP 3.9 Hz,
PCCH3N3P), 23.7ꢀ23.8, 24.3ꢀ24.4 (OCH(CH3)3, 59.8 (t, JCP 150.3 Hz,
PCP), 72.6ꢀ72.8 ((OCH(CH3)3; δP (161.9 MHz; CDCl3; ext. 85%
H3PO4): 17.0 ({1H} s; coupled JPH 15.2 Hz (qm), JPC 150 Hz (satellites)).
Tetraisopropyl (Azidomethanediyl)bis(phosphonate), 2.
In a 500 mL RB flask under N2, ∼6 g (19 mmol) of TrN3 were dissolved
in 30 mL of anhydrous THF and stirred vigorously at ꢀ78 °C. A solution
of 1.517 g (4.41 mmol) of tetraisopropyl methylenebis(phosphonate)
and 4.41 mL of 1 M KHMDS in 10 mL THF was mixed at rt, cooled to
ꢀ78 °C and added at once to the TrN3 solution by syringe. The solution
was stirred for 10 s and then quenched with 2.5 mL AcOH. The flask was
moved to a ꢀ20 °C freezer and allowed to stand for 48 h, or until the
triazene had decomposed as determined by 31P NMR. The precipitate
was filtered and volatiles removed at reduced pressure. The reaction
mixture was then seeded with solid TrN3 and allowed to sit overnight at
0 °C. The resulting precipitate was washed with H2O and filtered off.
After removing water by rotovap, the residue was dissolved in 20 mL
wet EtOAc. Ten drops of t-BuOCl were added and stirred at rt until
evolution of N2 ceased. EtOAc was washed with a saturated aqueous NH4Cl
solution, dried, and concentrated. The residue was purified on silica gel
eluted with ACE/DCM (1:9), Rf = 0.36. Contaminating phosphite was
removed by rotary evaporation (oil pump) with gentle warming to afford
0.83 g of 2 as a colorless oil (yield: 50% overall). IR: v (film) 2134 (m),
2104 cmꢀ1 (N3). NMR: δH (399.8 MHz; CDCl3): 1.31ꢀ1.34 (24 H, m, 4
ꢁOCH(CH3)2), 3.53 (1H, t, JHP 20.8 Hz, PCHN3P), 4.79 (4 H, m, 4 ꢁ
OCH(CH3)2); δC (100.5 MHz; CDCl3): 23.7ꢀ23.8, 24.1ꢀ24.2
(OCH(CH3)3, 55.0 (t, JCP 144.8 Hz, PCP), 72.7, 72.9 (2t, JCP 3.5 Hz
Nucleotide triphosphate R,β-CXY or β,γ-bisphosphonate
analogues with an asymmetrically substituted bridging carbon
(X ¼ Y) are obtained as mixtures of diastereomers by existing
synthetic procedures starting from prochiral bisphosphonate
precursors, and thus far none have been separated chromato-
graphically.3,5,6 Diastereomeric β,γ-fluoromethylene dNTPs can
be individually observed in the synthetic mixture by 19F NMR.3,6
In addition to the expected nucleoside resonances for dGuo,
1
the H NMR of 5a/b shows a pair of doublets centered at δ
1.58ꢀ1.59 ppm (C(CH3)N3) and that of 6a/b an apparent
triplet at δ 3.40 ppm (CHN3). However, although the proton-
decoupled PR and Pγ 31P NMR signals of 5a/b are doublets at δ
ꢀ9.45 and 14.5 ppm respectively, two resolvable (Δδ 0.03 ppm)
diastereomeric pairs of doublets (J = 34, 19 Hz) are observed for
Pβ. The δ vs J assignments were confirmed by comparing 31P
spectral data obtained at 162 and 202 MHz.
A similar pattern is seen with 6a/6b except that resolvable
diastereomeric multiplets are observed for both PR (δ ꢀ9.78,
ꢀ9.81 ppm) and Pβ (δ 9.04, 9.06 ppm), but not for Pγ
(10.4 ppm). This difference between 5a,b and 6a,b with respect
to the sensitivity of the PR chemical shift to the Pβγ CXN3 chiral
center invites further investigation.
In the preparation of the R,β-C(CH3)N3 dATP analogues 7a
and 7b, we found that the corresponding dADP bisphosphonate
intermediates (14a/b) could be isolated individually by isocratic
RP HPLC (Figure 2). Subsequent enzymatic phosphorylation as
described above then generated the individual diastereomers 7a
and 7b. CD spectra measured for 7a and 7b showed prominent
features of opposite polarity centered near 260 nm (7a, (ꢀ); 7b
(þ)). Curiously, the R,β-CHN3 dADP intermediate (15a/b) did
not separate under similar conditions and its phosphorylation
yielded the triphosphates 8a/b as a mixture of diastereomers
which could not be distinguished by 31P NMR.
In conclusion, we have described the first examples of R-azido
bisphosphonate esters and acids. The latter compounds can be
used to synthesize novel nucleotide analogues containing a
CHN3 or C(CH3)N3 at either the R,β or β,γ bridging position.
The ability to obtain for the first time the individual
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NOTE
OCH(CH3)3); δP (161.9 MHz; CDCl3; ext. 85% H3PO4): 13.3 ({1H} s;
coupled JPH 20.5 Hz (dm), JPC 145 Hz (satellites)).
yield a white solid, 31.1 mg (23.5%) 6a/b after dual pass (SAX then RP)
HPLC purification.4 Analytical HPLC: retention time = 10.3 min,
purity = 98%. NMR: δH (499.8 MHz; D2O; pH 10.88): 2.48ꢀ2.53,
2.71ꢀ2.76, 3.53 (ddd, PCHN3P), 4.10ꢀ4.20, 4.25, 6.31 (t, J 6.0 Hz)
8.03, 8.03; δP (161.9 MHz; D2O; ext. 85% H3PO4; pH 10.88): ꢀ9.78 (d,
JRβ 27.8 Hz, PR), ꢀ9.81 (d, J 27.8 Hz PR), 9.04 (dd, JRβ 27.8 Hz, Jβγ 12.9
Hz, Pβ), 9.06 (dd, JRβ 27.8 Hz, Jβγ 12.9 Hz, Pβ) 10.41 (d, Jβγ 12.9 Hz,
Pγ). HRMS (ESI/APCI) [M ꢀ H]ꢀ: calcd for C11H16N8O12P3,
545.0106. Found: 545.0121.
Base-Induced Decomposition of 2. Five mg (13 μmol) of 2 was
dissolved in 1.5 mL of anhydrous THF and cooled to ꢀ78 °C.
t-BuOK (1.5 equiv) was added at once, causing rapid evolution of gas.
Identical results were obtained with BuLi and KHMDS. NMR: δP (202.5
MHz; CDCl3; ext. 85% H3PO4): 22.3 (diisopropyl phosphorocyanidate),15
4.8 (diispropyl phosphite) with integration 1:1.
(1-Azidoethane-1,1-diyl)bis(phosphonic acid),
3 and
20-Deoxyadenosine 50-Diphosphate, r,β-C(CH3)N3, 14a/b.
The tris(tetrabutylammonium) salt from 60 mg (0.260 mmol) of 3 was
allowed to react with 115 mg (∼1.1 equiv) of dA-50-Ts in anhydrous
acetonitrile5 to yield 65.1 mg of the nucleoside diphosphonate diaster-
omers in a ratio of 2:3 (54% crude conversion), which could be separated
by RP HPLC. 14a: NMR: δH (399.8 MHz; D2O; pH 10.88): 1.50 (dd,
JHP 12.4, JHP 15.2 Hz, PCCH3N3P), 2.57ꢀ2.63, 2.82ꢀ2.91, 4.10ꢀ4.16,
4.22ꢀ4.27, 6.48 (t, 6.4 Hz), 8.25, 8.52; δP (161.9 MHz; D2O; 85%
H3PO4; pH 10.88) 14.5 (d, J 15 Hz, Pβ); 21.7 (d, J 15 Hz, PR). 14b:
NMR: δH (399.8 MHz; D2O; pH 10.88): 1.50 (dd, JHP 12.8, 15.2 Hz,
PCCH3N3P), 2.57ꢀ2.63, 2.82ꢀ2.88, 4.10ꢀ4.16, 4.22ꢀ4.27, 6.47 (t,
6.4 Hz), 8.22, 8.50; δP (161.9 MHz; D2O; ext. 85% H3PO4; pH 10.88)
14.4 (d, J 15 Hz, Pβ); 21.6 (d, J 15 Hz, PR).
20-Deoxyadenosine 50-Triphosphate r,β-C(CH3)N3, 7a/b.
Enzymatic phosphorylation5 of 14a and 14b followed by HPLC
purification of each product gave 17.3 mg (yield: 12.2% overall from
starting acid) of 7a and 14.9 mg (10.5%) of 7b. 7a: Analytical HPLC:
retention time = 9.5 min; purity = 98%. NMR: δH (499.8 MHz; D2O;
pH 10.88): 1.58 (t, JHP 15.0 Hz, PCCH3N3P), 2.58ꢀ2.63, 2.83ꢀ2.89,
4.21ꢀ4.29, 6.50 (t, 6.5 Hz), 8.25, 8.54; δP (202.5 MHz; D2O; 85%
H3PO4; pH 10.88) ꢀ4.5 (d, Jβγ 31.9 Hz, Pγ); 7.0 (dd, Pβ); 18.51 (d, JRβ
19.0 Hz, PR). HRMS (ESI/APCi) [Mꢀ H]ꢀ: calcd for C12H18N8O11P3,
543.0313. Found: 543.0333. 7b: Analytical HPLC: retention time = 9.5
min; purity = 96%. NMR: δH (499.8 MHz; D2O; pH 10.88): 1.59 (t, JHP
15 Hz, PCCH3N3P), 2.58ꢀ2.63, 2.85ꢀ2.90, 4.17ꢀ4.22, 6.51 (t, 6.5
Hz), 8.26, 8.56; δP (202.3 MHz; D2O; ext. 85% H3PO4; pH 10.88) ꢀ4.4
(d, Jβγ 30.1 Hz, Pγ); 8.0 (dd, Pβ); 18.47 (d, JRβ 18.2 Hz, PR). HRMS
(ESI/APCI) [M ꢀ H]ꢀ: calcd for C12H18N8O11P3, 543.0315. Found:
543.0315.
20-Deoxyadenosine 50-Diphosphate, r,β-CHN3, 15a/b. The
tris(tetrabutylammonium) salt was prepared from 60 mg (0.28 mmol)
of 4 and allowed to react with 120 mg (∼1.1 equiv) of dAdo-50-Ts in
anhydrous acetonitrile5 to yield a white solid, 32 mg (26%). NMR: δp
(242.8; D2O; pH 10.88): 9.9 (Pβ); 17.2 (PR).
20-Deoxyadenosine 50-Triphosphate r,β-CHN3, 8a/b. En-
zymatic phosphorylation5 of 15a/b above followed by HPLC purifica-
tion gave 33.2 mg (22.7%) 8a/b. Analytical HPLC: retention time = 10.4
min; purity = 98%. NMR: δH (400.2 MHz; D2O; pH 10.88): 2.57ꢀ2.63,
2.82ꢀ2.90, 3.86 (dt, PCHN3P), 4.11ꢀ4.25, 4.29, 6.50 (t, J 6.5 Hz), 8.24,
8.528, 8.532; δP (202.3 MHz; D2O; ext. 85% H3PO4; pH 10.88) ꢀ5.1
(d, Jβγ 25.1 Hz, Pγ); 2.8 (dd, Pβ); 14.3 (d, JRβ 13.8 Hz, PR). HRMS
(ESI/APCI) [M ꢀ H]ꢀ: calcd for C11H16N8O11P3, 529.0157. Found:
529.0147.
(Azidomethanediyl)bis(phosphonic acid), 4. The appropriate
bisphosphonate ester was dissolved in 2 mL anhydrous acetonitrile and
6 equiv. of BTMS were added by syringe. Stirring overnight gave the
tetrakis(trimethylsilyl) esters (100% by 31P NMR). From 1: δP (202.5
MHz; CH3CN, ext. 85% H3PO4) 0.9. From 2: δP ꢀ3.2 (s). Hydrolysis
with ∼1 mL of EtOH/H2O (1:1) and removal of volatiles by prolonged
evaporation at low pressure provided the acids as hygroscopic white
solids (>98% overall yield). The acids were further dried under vacuum
in a desiccator containing P2O5.
3: IR: v (KBr): 2085 (m), 2126 cmꢀ1 (N3). NMR: δH (399.8 MHz;
D2O; pH 10.88): 1.43 (t, JHP 13.2 Hz, PCCH3N3P); δC (100.5 MHz;
D2O; ext. pH 10.88): 23.5 (s, PCCH3P), 68.4 (t, JCP 129 Hz, PCP); δP
(161.9 MHz; D2O; 85% ext. H3PO4; pH 10.88): 18.3 ({1H} s; coupled,
JPH 13.2 Hz (q); JPC 129 Hz (satellites)). HRMS (ESI/APCI) [M ꢀ
H]ꢀ: calcd for C2H6N3O6P2: 229.9737. Found: 229.9734.
4: IR: v (KBr): 2113 cmꢀ1 (N3). δH (399.8 MHz; D2O; pH 10.88):
3.26 (t, JHP 16.8 Hz, PCHN3P); δC (100.5 MHz; D2O; pH 10.88): 65.1
(t, JCP 124 Hz, PCP); δP (161.9 MHz; D2O; 85% ext. H3PO4; pH
10.88): 13.5 ({1H} s; coupled, JPH 16.5 Hz (d); JPC 122 Hz (satellites));
HRMS (ESI/APCI) [M ꢀ H]ꢀ: calcd for CH4N3O6P2, 215.9581.
Found: 215.9584.
Ethyl diazo(diethoxyphosphoryl)acetate from trifyl azide
and ethyl(diethylphosphoryl)acetate. Following the procedure
of Hakimelahi and Just,25 72 mg of NaN3 (1.1 mmol) was suspended in
20 mL of anhydrous DMF and 106 μL (1.0 mmol) of CF3SO2Cl was
added under N2. After several min, a solution of 222 mg (1.0 mmol) of
ethyl (diethylphosphoryl)acetate and 101 mg (1.0 mmol) of Et3N in
5 mL anhydrous DMF was added dropwise. After 40 min, a mixture of
compounds were detected by 31P NMR with δ (DMF) 20.8, 18.3, 10.4
in a ratio of 5:4:1. Gentle heating for an additional 30 min gave a binary
mixture of the compounds at δP 10.4 (40%) and 20.8 (60%). Ether (30
mL) was added and the reaction mixture was washed 5ꢁ with 5 mL
water. The organic layer was dried over MgSO4 and evaporated. A
portion of the residue was purified by preparative TLC on silica gel
eluted with ACE/DCM (1:9) giving a single mobile band (UV), Rf =
0.65, which was identified as ethyl diazo(diethoxyphosphoryl)acetate.
NMR: δH (400.2 MHz; CDCl3): 1.32 (3H, t, JHH 7 Hz), 1.38 (6H, dt,
JHH 7 Hz, JHP 0.8 Hz), 4.22 (4H, m), 4.29 (2H, q, JHH 7 Hz); δP (202.5
MHz, CDCl3; 85% H3PO4) 10.3 (s). MS (APCI, m/z), 251 (M þ 1)þ;
an MS/MS analysis of the m/z 251 species gave a major fragment at
m/z 223.11
20-Deoxyguanosine 50-triphosphate β,γ-C(CH3)N3, 5a/b.
Compound 3 (2 equiv of the 1.5 tributylammonium salt) was coupled to
100 mg (0.242 mmol) of dGuo-morpholidate in anhydrous DMSO to
yield a white solid, 32.5 mg (23.8%) after dual pass (SAX then RP)
HPLC purification.4 Analytical HPLC: retention time = 9.6 min; purity
= 98%. NMR: δH (399.8 MHz; D2O; pH 10.88): 1.58 (dd, PCCH3N3P)
2.48ꢀ2.54, 2.72ꢀ2.79, 4.11ꢀ4.21, 4.12, 6.32 8.05. δP (161.9 MHz;
D2O; 85% H3PO4; pH 10.88): ꢀ9.5 (d, JRβ 33.7 Hz, PR), 13.28 (dd, JRβ
33.7 Hz, Jβγ 19.4 Hz, Pβ), 13.31 (dd, JRβ 33.7 Hz, Jβγ 19.4 Hz, Pβ) 14.5
(d, Jβγ 19.4 Hz, Pγ). HRMS (ESI/APCI) [MꢀH]ꢀ: calcd for C12H18-
N8O12P3, 559.0263. Found: 559.0266.
’ ASSOCIATED CONTENT
Supporting Information. IR (1ꢀ4), 1H NMR (1ꢀ8, 14),
S
b
gCOSY (1, 2), 13C NMR (1ꢀ4), 31P NMR (1ꢀ8, 14, 15), HRMS
(3ꢀ8), UV/vis and CD spectra (7a, 7b), HPLC data for 5ꢀ8, 31P
NMR spectrum for conversion of 2 to 12 and 13, 31P NMR of 5a/b
at two field strengths, and 1H and 31P NMR and MS data for the
product from reaction of phosphonoacetate carbanion with trifyl
azide. This material is available free of charge via the Internet at
http://pubs.acs.org.
20-Deoxyguanosine 50-Triphosphate β,γ-CHN3, 6a/b. Com-
pound 4 (2 equiv of the 1.5 tributylammonium salt) was coupled to
100 mg (0.242 mmol) of dGuo-morpholidate in anhydrous DMSO to
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dx.doi.org/10.1021/jo200045a |J. Org. Chem. 2011, 76, 5132–5136

The Journal of Organic Chemistry
NOTE
’ AUTHOR INFORMATION
identify a PC(HX)C hydrogen. The R-proton is distinguishable in
analogous HꢀF,33 HꢀCl, and HꢀBr34 triethyl phosphonoacetates
and should be apparent in authentic trialkyl azidophosphonoacetate.
The C.I. MS data in the original report identifies a m/z 223 peak which
was assigned to a [M ꢀ N3]þ species. However, this peak also corre-
sponds to a prominent fragment which we observe in the MS/MS of the
triethyl diazophosphonoacetate parent peak [M þ 1]þ at m/z 251.
Finally, the authors supported their structure by reducing their product
to the aminophosphonoacetate with H2 (Pd/C) in EtOH. t-Butyl
diazo(diethoxyphosphoryl)acetate is converted to a cognate PCH-
(NH2)C product under these conditions,35 demonstrating that the
reduction would not necessarily distinguish the azido compound from
triethyl diazophosphonoacetate.
Corresponding Author
*E-mail: mckenna@usc.edu.
’ ACKNOWLEDGMENT
This research was supported by NIH grant CA105010. We
thank Ms. Inah Kang for assistance in preparing the manuscript
and Dr. Ron New (UC Riverside High Resolution Mass Spectro-
metry Facility) for obtaining the HRMS data.
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