Synthesis of adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate (APBADP)
Adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate was synthesized by phosphorylation of 6chloropurine riboside and then reaction of the product with 2-amino-4-phosphonobutyric acid (Figure
2). This procedure was modified from that of Brandt and Lowenstein (1978) which yielded adenylophosphonobutyrate. However, the commercial 6-chloropurine riboside 5’-phosphate used by
Brandt and Lowenstein as their starting material is no longer commercially available. In our new method, POCl
3
(1.5 mmole) was added to 1 mmole 6-chloropurine riboside [Figure 2, structure (I)] in
5 ml trimethylphosphate, under N
2
at room temperature, and the reaction was monitored by HPLC
(Varian aerograph Model no. 502000-00). At the end of one hour, a second addition of 1.5 mmole
POCl
3
was made and the pH was adjusted to pH 5 using triethylamine. After 2 hours, 10 ml 0.4 M barium acetate was added, followed by the addition of 40 ml ethanol containing 2 mL triethylamine,
1

to precipitate the nucleotide. The supernatant was discarded after centrifugation in a table top centrifuge for 5 min; the precipitate was washed with 70% ethanol before dissolving it in 10 ml 1 M acetic acid. The reaction was monitored on an HPLC program of 0-30 minutes of solvent A, followed by a linear gradient to 20% solvent B at 50 minutes. The solvents used were MilliQ H
2
O with 0.1% TFA as solvent A and acetonitrile with 0.1% TFA as solvent B. The column used was a
Vydac C18 reverse- phase column (cat. # 218TP54C18). 6-Chloropurine riboside (I) has a peak at 40 min, in the gradient. The eluate was measured at 264 nm using a VARI-CHROM detector. The product (II) peak occurs at 10 minutes. The product was desalted on an AG-50 column (1.5 cm x 40 cm) equilibrated with 0.1 M HCl and washed with H
2
O. The fractions with absorbance at 264 nm were pooled and rotovaporated to dryness.
The phosphorylation product was dissolved in 8 ml (40%, dimethylformamide/H
2
O, v/v) and reacted with 2.37 mmole 2-amino-4-phosphonobutyric acid; the pH was adjusted to pH 8.5 with
NaHCO
3
(Figure 2, Step II). The reaction was allowed to proceed for 13 days at room temperature without stirring, as described by Brandt and Lowenstein (1978), and was monitored by HPLC using the program and same solvent system described earlier. The supernatant was decanted and applied to a DE52 column (1.5 cm x 35 cm) equilibrated with 20 mM NH
4
HCO
3
, pH 7.9, and the product was separated by a linear gradient from 20-300 mM NH
4
HCO
3
(1 L each) gradient. The product comes off as the last peak (around 300 ml) with absorbance at 267 nm. The peak fractions were evaluated by HPLC to check for purity before being pooled and rotovaporated to dryness. The product was then desalted on an AG-50 column (1.5 cm x 40 cm) equilibrated with 0.1 M HCl and washed with H
2
O.
Characterization of adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate (APBADP)
The proton and phosphorous NMR spectra of a 6 mM sample (III) in D
2
O were obtained using a
Bruker DRX400 spectrometer. The number of moles of organic phosphate/mole of compound was determined by the method described by Hess and Derr (1975) and Lanzetta et al. (1979), with slight
2

modifications. Samples containing organic phosphorous (up to 10 nmoles) were digested prior to phosphorous analysis. The sample was mixed with 20 µl 10 N H
2
SO
4
and the solutions were incubated in an aluminum foil closed tube at 190 ˚ C for 2 hours. The samples, when cooled to room temperature, were analyzed for inorganic phosphate. Samples (150 µl) containing inorganic phosphate were mixed with 20 µl 10 N H
2
SO
4
and 800 µl of fresh mixture of Malachite Green and ammonium molybdate (3:1) and the absorbance at 660 nm was read after 5 minutes. The standard curve was generated using 1 to 10 nmoles of SAMP. IMP, ADP and SAMP (5 nmole) were also tested as controls. The adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate was measured from
0.5 – 3 nmole.
Characterization of adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate (APBADP)
APBADP (Figure 2, structure III) exhibits one peak at ~ 3 minutes on HPLC (in the solvent system and program described in Materials and methods), as compared to ~ 40 minutes for 6-chloropurine riboside (Figure 2, structure I) and ~10 minutes for 6-chloropurine riboside, 2’(3’),5’-diphosphate
(Figure 2, structure II). The UV-Vis spectrum also exhibits one peak with a
λ max
at 267 nm.
The proton NMR spectrum shows the peaks summarized in Table S1. The NMR spectrum of
APBADP appears to be a composite of that of adenylosuccinate (SAMP) and 2-amino-4phosphonobutyric acid (APBA). The peaks at δ 1.9 and 2.3 ppm of the APBADP correspond to δ 1.7 and 2.1 ppm of the -CH
2
and -CH
2
PO
3
2-
of APBA. The δ 4.1 ppm triplet of APBADP corresponds to the δ 4.1 and 4.0 ppm of the -CH of SAMP and APBA, respectively. The δ 6 ppm doublet corresponds to the SAMP δ 6.3 (1H, ribosyl H1’). The APBADP δ 8.45 ppm singlet corresponds to the SAMP δ 8.1 (1H, purine 2-H), and APBADP δ 8.59 ppm corresponds to the SAMP δ 8.32 ppm
(broadened singlet, 1H, purine 8-H).
3

Table S1. Proton NMR spectra of adenylosuccinate (SAMP), adenosine phosphonobutyric acid,
2’(3’), 5’-diphosphate (APBADP), and 2-amino-4-phosphonobutyric acid (APBA) a
SAMP APBADP APBA
CH
2
CH
H5’
H4’
H3’
H2’
H1’
H2
H8
2.8(d,2,CH
2
)
4.1(t,1,CH)
3.8
4.3
4.4
5.0
6.0(d,1,H1)
8.1(s,1,H2)
8.3(2,1,H8)
2.3 (t,2,CH
2
)
1.9(m,2,CH
2
)
4.1(t,1,CH)
4.2
4.4
4.5
5.1
6.3(d,1,H1)
8.5(s,1,H2)
8.6(s,1,H8)
2.1(t,2,CH
2
)
1.7(m,2,CH
2
)
4.0(t,1,CH) a
The NMR samples of the substrate analogue, APBADP, and APBA were dissolved in D
2
O and run on a DRX 400 NMR spectrometer.
The phosphorous NMR spectrum of APBADP, in Figure S1, exhibits 2 peaks: around 27 and
0 ppm. The 27 ppm peak corresponds to the phosphate group observed in the spectrum of 2-amino-
4-phosphonobutyric acid, and the 0 ppm peak to that of ribose phosphate groups, respectively.
Integration of the 2 peaks indicates a total of 3 phosphorous groups, with the peak of chemical shift 0 ppm having twice the area of the peak of 27 ppm. Of the two phosphate groups on the ribose ring, one is on the 5’-OH and the other on either the 2’-OH or 3’-OH, since these both have similar reactivity, but 5’-OH is the most reactive. Chemical analysis of the organic phosphate content of
APBADP shows the presence of 2.92 ± 0.21 moles of phosphorous per mole of compound, with the concentration determined from the A
267nm
(ε = 19900 M
-1
cm
-1
). The control samples, IMP, ADP and
4

SAMP, yield 0.81 ± 0.06, 1.86 ± 0.10, and 1.05 ± 0.26 moles of organic phosphorous per mole of compound, respectively. These results are all consistent with the structure of adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate in Figure 2 (Structure III).
Figure S1
Figure S1. 31 P NMR spectrum of Adenosine phosphonobutyric acid, 2’(3’), 5’-diphosphate
(APBADP) in D
2
O, pH ~ 3.
5