Synthesis of 13C-labeled 5-aminoimidazole-4-carboxamide-1-β-D-[13C5] ribofuranosyl 5′-monophosphate

Article abstract of DOI:10.1002/jlcr.3647

5-Aminoimidazole-4-carboxamide-1-β-D-[¹³C₅] ribofuranosyl 5′-monophosphate ([¹³C₅ ribose] AICAR-PO₃H₂) (6) has been synthesized from [¹³C₅]adenosine. Incorporation of the mass-label into [¹³C₅ ribose] AICAR-PO₃H₂ provides a useful standard to aid in metabolite identification and quantification in monitoring metabolic pathways. A synthetic route to the ¹³C-labeled compound has not been previously reported. Our method employs a hybrid enzymatic, and chemical synthesis approach that applies an enzymatic conversion from adenosine to inosine followed by a ring-cleavage of the protected inosine. A direct phosphorylation of the resulting 2′,3′-isopropylidine acadesine (5) was developed to yield the title compound in 99% purity following ion exchange chromatography.

Full text of DOI:10.1002/jlcr.3647

13
Synthesis of C-labeled 5-Aminoimidazole-
1
3
4
-carboxamide-1-β-D-[ C ]ribofuranosyl 5’-monophosphate
5
Allison K. Zarkin, Phyllis D. Elkins, Amanda Gilbert, Teresa L. Jester, Herbert H. Seltzman*
Corresponding Author
*
Author Affiliation: RTI International, Research Triangle Park, NC 27709
Abstract
1
3
5
-Aminoimidazole-4-carboxamide-1-β-D-[ C ]ribofuranosyl 5’-monophosphate
5
13
13
(
[ C ribose] AICAR-PO H ) (6) has been synthesized from [ C ]adenosine. Incorporation
5
3
2
5
1
3
of the mass-label into [ C ribose] AICAR-PO H provides a useful standard to aid in
5
3
2
metabolite identification and quantification in monitoring metabolic pathways. A synthetic
1
3
route to the C-labeled compound has not been previously reported. Our method employs a
hybrid enzymatic and chemical synthesis approach that applies an enzymatic conversion from
adenosine to inosine followed by a ring-cleavage of the protected inosine. A direct
phosphorylation of the resulting 2’,3’-isopropylidine acadesine (5) was developed to yield the
title compound in 99% purity following ion exchange chromatography.
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3647
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Introduction
AICAR-PO H (aka ZMP) is a biosynthetic precursor of purine nucleotides. It has
1
-2
3
2
been shown to be involved in cell growth through regulation of folate one-carbon metabolism
and is therefore of interest in, and a marker of, healthy (human) cell development as well as a
3
-4
potential target in the development of antibiotics. This compound also activates
monophosphate-activated protein kinase (AMPK), which is associated with numerous
5
cardiovascular benefits. AICAR-PO H is a potential biomarker of numerous metabolic
3
2
disease states, has antiproliferative effects by interaction with tumor promoting enzymes and
6
proteins, and improves muscle function. AICAR-PO H has potential medicinal uses, but it
3
2
7
has also been used as a performance enhancing drug. Therefore, a mass-labeled analog is of
interest to monitor metabolic pathways involving the AICAR-PO H intermediate or for
3
2
possible use as a standard in doping detection.
Preparations of AICAR-PO H have been disclosed in the literature, although the
3
2
synthesis of the carbon-13 labeled AICAR-PO H has not been reported previously. Notably,
3
2
Thomas et al. synthesized carbon-13 labeled AICAR (no phosphate) starting from carbon-13
7
ribose. In the first synthesis of AICAR-PO H , reported in 1961, the phosphorylation was
3
2
conducted on 2’,3’-isopropylidine inosine followed by subsequent ring cleavage to ultimately
8
yield AICAR-PO H . However, this paper cited low yields of 11-16% from the inosinic acid
3
2
derivative. The purity and isolation of the final compound was also unclear. Similarly,
Oliviero, et al. developed a solid-phase synthesis that conducts the phosphorylation on an
9
inosine derivative on a small scale. Shaw, et al. synthesized AICAR-PO H by
3
2
phosphorylating an acadesine derivative containing a methyl ester on the imidazole ring
1
0
which was later converted to the necessary amide. A direct phosphorylation of the
acadesine nucleoside core (AICAR) would be ideal. However, this reaction has been noted in
the literature as challenging and has only been done enzymatically with adenosine kinase on a
9
,11
2
.4 mg scale.
Our approach (Scheme 1) started with C-13 labeled adenosine, which was converted
to inosine enzymatically, followed by ribose protection, inosine ring activation and cleavage
7
-9,11,12
to generate a 2’3’-protected acadesine derivative.
We then developed a direct and
scalable chemical route from 2’3’-protected acadesine to AICAR-PO H that will provide a
3
2
valuable standard for the metabolomics community.
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Results and Discussion
The synthetic route was first established on unlabeled adenosine and then applied to
13
the [ C -ribose] AICAR-PO H . In the carbon-13 labeled case, commercially available 1 was
5
3
2
1
3
12,13-
subjected to adenosine deaminase enzyme (ADA) to cleanly form [ C -ribose] inosine 2.
5
1
5
An acetonide protecting group was then installed as previous reports have indicated that
phosphorylation of a similar ribose 5’-hydroxyl in the presence of unblocked 2’- and 3’-
1
6
hydroxyls is not completely selective. The resulting inosine acetonide 2’ (not shown) was
1
7-
chromatographed to obtain 2’ free of buffer salts and enzyme in 69% yield over two steps.
1
8
Subsequent acetylation afforded compound 3 in 94% yield that was used without further
1
9
purification.
The protected inosine base 3 was arylated on N-1 of the hypoxanthene ring with 2,4-
dinitrochlorobenzene by the reported method to afford the derivative 4 in 67% yield after
19-20
chromatographic purification.
Compound 4 was then treated with ethylenediamine (EDA)
to ring-open the inosine nucleobase at C-2 with concomitant cleavage of the 5’-acetate group
2
0
in 61% yield following similar procedures as reported for a desoxy analog. This compound
showed instability on silica gel and was thus purified by reverse phase chromatography. This
step in the sequence was also particularly sensitive to water requiring strictly anhydrous
conditions and dried EDA.
As discussed above, direct phosphorylation of this riboside has been problematic.
Trimethyl phosphate, typically employed as the reaction solvent for phosphorylation, gave
minimal phosphorylated product 6 in our solution phase approach. However, a solvent
change from trimethyl phosphate to tri-n-butyl phosphate proved to be critical in affecting
this transformation. Treatment of compound 5 with 16 equivalents of phosphoryl chloride in
tri-n-butyl phosphate at -15 °C for 18 hours resulted in a successful phosphorylation. After
quenching with aqueous sodium hydroxide, the acetonide group cleaves during concentration
of the reaction mixture in a water bath at 30 °C to yield a residue containing the desired
=
compound (AICAR-PO ) 6.
3
Noteworthy factors impacting the phosphorylation were the amount of excess of
POCl , reaction time, temperature, and work-up pH, which influenced the formation of by-
3
products. Higher temperatures and longer reaction times yielded the cyclized
phosphoramidate ketal 7a and its deketalized analog 7b. The deketalized 5’-chlororibose
analog 8 was also observed under some reaction conditions. The identification of these by-
products was supported by mass spectrometry.
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=
The residue containing AICAR-PO 6 was neutralized and purified via cation
3
exchange chromatography. Initially, elution with dilute HCl yielded the HCl salt of the
desired compound in a 57% yield; however, decomposition occurred upon lyophilization.
The material was re-chromatographed slowly eluting with water to yield the target compound
1
3
13
5
-aminoimidazole-4-carboxamide-1-β-D-[ C ]ribofuranosyl 5’-monophosphate ([ C ]ribose
5
5
AICAR-PO H ) 6 in its neutral form with high purity (99%) in a 28% yield. This is in
3
2
contrast to commercial products obtained at >90% purity.
13
Purified [ C ribose] AICAR-PO H was characterized to establish the identity and
5
3
2
relative purity of the compound for its use as a qualitative metabolite reference identification
1
13
31
standard. Identification was confirmed using NMR spectroscopy ( H-NMR, C-NMR, P-
NMR) and high-resolution mass spectrometry (HRMS). Chromatographic purity was
established by UPLC using an RP-UPLC/UV impurity profile method developed and
qualified for use in measuring related organic impurities in test samples. Analysis of the
purified standard using the qualified UPLC/UV method revealed a chromatographic purity of
9
9.0%. The labeled composition of the compound (isotopic purity) was determined at 95.5%
1
3
13
13
C 4.0% C , 0.5% C by mass spectrometry, calculated using the peak areas from
5,
4
3
extracted ion chromatograms.
Conclusion
1
3
We have successfully synthesized and purified [ C ribose] AICAR-PO H from
5
3
2
13
[
C ribose] adenosine. Development of effective phosphorylation conditions has allowed an
5
efficient synthesis of this C-13 labeled metabolite in combination with modification of
literature methods to afford the high purity mass labeled standard needed for metabolomics
studies.
Acknowledgement
The authors state that they have no financial conflict of interest in this work. The
research was supported through Contract No. HHSN268201300021C to HHS from the
National Heart, Lung, and Blood Institute under the NIH Common Fund Metabolomics
program.
Experimental
1
13
H and C NMR spectra were obtained on a Bruker Avance 300 MHz NMR
spectrometer or a Varian Unity Inova 500 MHz NMR spectrometer. Mass spectra were
obtained with a Perkin-Elmer Sciex API 150 EX mass spectrometer. High Resolution Mass
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Spectra of the final compound were obtained with an Agilent 1290 Infinity UHPLC/6230
TOF mass spectrometer. Column chromatography was conducted using a Teledyne Isco
Combiflask Rf system with RediSep Rf silica and C18 cartridges. Reverse phase high
pressure liquid chromatography (HPLC) for in-process monitoring was conducted with a
Waters 1525 pump unit, a Waters 717plus autosampler, and a Waters 2487 detector driven by
Waters Empower2 software. Chromatographic purity of the final compound was confirmed
by ultrahigh pressure liquid chromatography (UPLC) using a Waters Acquity System with a
Waters Acquity UPLC PDA Detector driven by Waters Empower3 software. An Acquity
HSS T3 column was used under gradient, reversed phase conditions with detection at 254
1
3
nm. [ C ribose] adenosine was purchased from Cambridge Isotopes. The isotopic
5
1
3
13
distribution of the [ C ribose] adenosine starting material was determined to be 96.8% C ,
5
5
1
3
13
2
.9% C and 0.3% C by HRMS. Unlabeled AICAR-PO H was purchased from
4 0 3 2
CalBioChem. Neither human nor animal subjects were employed in this research.
1
3
13
Inosine ([ C ]-ribose) (2). Adenosine ([ C ]-ribose) 1 (787 mg, 2.89 mmol) was suspended
5
5
in 28 mL (0.1 M) of 0.1 M buffer solution (1:1 NaH PO :Na HPO ). Adenosine deaminase
2
4
2
4
(239 mg) was added and the mixture was stirred under nitrogen at room temperature for 1.5
h. The solution became clear upon completion. The reaction was filtered through a 0.45 m
membrane and then lyophilized to yield a white powder (1.16 g) containing both product 2
1
3
and salt. It was carried forward without further purification. C NMR (75.5 MHz, D O) 
2
8
8.28 (d, J=42 Hz), 85.52 (t, H=40 Hz), 73.95 (t, J=40 Hz), 70.27 (t, J=38 Hz), 61.25 (d, J=41
Hz). HPLC: 99.69% (XBridge C18 5 m, 4.6x100 mm, 95:5 H O:MeOH, 254 nm, retention
2
time 1.94 min.). After labeled material had been carried forward past this step, additional
unlabeled material was synthesized, and it was found that this reaction can be carried out in
water without a buffer solution. Unlabeled adenosine (500 mg, 1.87 mmol) was suspended in
H O (30 mL, 0.06M) and adenosine deaminase (4 mg) was added. The reaction was complete
2
in 16 h and was concentrated to yield pure inosine in quantitative yield.
1
3
13
2
’,3’-O-(1-methylethylidene)-inosine ([ C ]-ribose) (2’). To the mixture of inosine ([ C ]-
5 5
ribose) 2 and inorganic salts (1.135 g) was added acetone (37 mL), p-toluenesulfonic acid
0.934 g, 4.91 mmol), and triethyl orthoformate (2.18 g, 14.7 mmol). The mixture was stirred
(
under nitrogen at room temperature overnight. After observation of starting material by TLC,
additional triethyl orthoformate (250 mg, 1.69 mmol) and p-toluenesulfonic acid (45 mg, 0.24
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mmol) were added. Solids were filtered off and the filtrate was concentrated to a white solid.
The crude material was purified by column chromatography (30-100% CMA-80 (80%
’
CHCl , 18% MeOH, 2% ammonium hydroxide):DCM (CH Cl ), SiO cartridge) to yield 2
3
2
2
2
1
as a white solid (630 mg, 2.01 mmol, 69% yield over 2 steps). H NMR (300 MHz, DMSO-
d6)  8.30 (s, 1H, H-2), 8.09 (s, 1H, H-8), 6.10 (d, 1H, J=165 Hz, H-1’), 5.26 (d, 1H, J=159
Hz, H-2’), 4.93 (d, 1H, J=162 Hz, H-3’), 4.22 (d, 1H, J=150 Hz, H-4’), 3.77 (s, 1H, H-5’a),
13
H-5’b under H O peak, 1.53 (s, 3H, CH ), 1.32 (s, 3H, CH ). C NMR (75.5 MHz, DMSO-
2
3
3
d6)  89.55 (d, J=40 Hz), 86.58 (t, J=40 Hz), 83.82 (t, J=32 Hz), 81.12 (t, J=38 Hz), 61.40 (d,
J=41 Hz).
1
3
2
’,3’-O-(1-methylethylidene)-5’-acetate-inosine ([ C ]-ribose) (3). In a round bottom
5
1
3
flask, 2’,3’-O-(1-methylethylidene)-inosine ([ C ]-ribose) 2’ (536 mg, 1.71 mmol) was
5
dissolved in pyridine (30 mL, 0.06M). Acetic anhydride (9.37 g, 9.17 mmol) was then added
over 1 min. The solution was stirred overnight at room temperature under nitrogen. The
reaction was complete by TLC (CMA-80:CHCl 1:1). The solution was placed in an ice bath
3
and quenched with methanol (18 mL). The solution was concentrated to ~30 mL volume,
diluted with H O (30 mL), and then extracted with ethyl acetate (3x80 mL). The combined
2
organic layers were washed with brine, dried with sodium sulfate and concentrated to yield
the product 3 as a white solid (570 mg, 1.60 mmol, 94% yield). The solid was carried forward
1
without further purification. H NMR (300 MHz, acetone-d )  8.15 (s, 2H, H-2, 8), 6.23 (d,
6
1
1
H, J=168 Hz, H-1’), 5.45 (d, 1H, J=159 Hz, H-2’), 5.11 (d, 1H, J=153 Hz, H-3’), 4.44 (d,
H, J=150 Hz, H-4’), 4.57-4.47 (m, 1H, H-5’a), 4.06-3.96 (m, 1H, H-5’b), 2.06 (s, 3H,
1
3
COCH ), 1.59 (s, 3H, CH ), 1.38 (s, 3H, CH ). C NMR (75.5 MHz, acetone-d )  90.99 (d,
3
3
3
6
J=41 Hz), 85.97-84.82 (m), 82.86-81.92 (m), 64.67 (d, J=44 Hz).
1
3
1
-(2,4-dinitrophenyl)- 2’,3’-O-(1-methylethylidene)-5’-acetate-inosine ([ C ]-ribose) (4).
5
1
3
2
’,3’-O-(1-methylethylidene)-5’-acetate-inosine ([ C ]-ribose) 3 (556 mg, 1.56 mmol) was
5
dissolved in DMF (7 mL, 0.22M). To the solution, K CO (538 mg, 3.9 mmol) and 1-chloro-
2
3
2
,4-dinitrobenzene (790 mg, 3.9 mmol) were added. The mixture was heated to 80 C for 4 h.
The reaction mixture was then cooled and diluted with CHCl (50 mL). The solids were
3
1
3
filtered off (243 mg with no C label). The filtrate was concentrated to a brown residue.
The residue was taken up in MeOH, however some solid did not go into solution. The filtered
1
3
solid was indeed the C labeled desired product (377 mg). The remaining filtrate was
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concentrated to a residue and chromatographed (0-50% gradient 7% MeOH in CHCl :CHCl ,
3
3
1
SiO ) to yield the desired product 4 (169 mg, 546 mg total, 1.04 mmol, 67% yield). H NMR
2
of filtered solid (300 MHz, DMSO-d )  8.94 (s, 1H, H-3 ), 8.81 (d, 1H, J=9 Hz, H-4 ),
6
Ar
Ar
8
1
4
.67 (s, 1H, H-2), 8.43 (s, 1H, H-8), 8.16 (d, 1H, J=9 Hz, H-5 ), 6.23 (d, 1H, J=168 Hz, H-
Ar
’), 5.44 (d, 1H, J=162 Hz, H-2’), 5.04 (d, 1H, J=159 Hz, H-3’), 4.45 (d, 1H, J=147 Hz, H-
1
3
’), 4.45 (m, 1H, H-5’a), 3.98 (m, 1H, H-5’b). C NMR (75.5 MHz, DMSO-d )  89.40 (d,
6
J=44 H), 84.35-83.15 (m), 80.80 (t, J=33 Hz), 63.63 (d, J=41 Hz). HPLC: filtered solid
7.95%, chromatographed material 98.25% (XBridge C18 5 m, 4.6x100 mm, 40-60%
MeCN:H O, 254 nm, retention time 4.95 min.).
9
2
5
-amino-1-[2’,3’-O-(1-methylethylidene)-β-D-ribofuranosyl]-1H-Imidazole-4-
1
3
carboxamide ([ C ]-ribose) (5). An oven dried 50 mL round bottom flask was charged with
5
1
3
1
-(2,4-dinitrophenyl)- 2’,3’-O-(1-methylethylidene)-5’-acetate-inosine ([ C ]-ribose) 4 (445
5
mg, 0.85 mmol) under nitrogen. To the solid was added freshly opened anhydrous
ethylenediamine (4 mL) and the reaction was stirred at room temperature for 2 h. Upon
completion, 2.5 g of celite was added and the volatiles were removed on under high vacuum.
The material was solid-loaded onto a C18 column and purified by reverse phase
chromatography (0-20% MeOH:H O, 9 mL/min) to yield the material as a white solid (158
2
1
mg, 0.52 mmol, 61% yield). H NMR (300 MHz, CD OD)  7.36 (s, 1H, H-2), 5.74 (dt, 1H,
3
J=3, 165 Hz, H-1’), 5.28 (d, 1H, J=45 Hz, H-2’), 4.65 (d, 1H, J=99 Hz, H-3’), 4.34 (d, 1H,
13
J=213 Hz, H-4’), 3.75 (dd, 2H, J=3, 138, H-5’), 1.58 (s, 3H, CH ), 1.37 (s, 3H, CH ). C
3
3
NMR (125 MHz, CD OD)  168.12, 144.07, 129.73, 114.64, 112.59, 91.25 (d, J=43 Hz),
3
8
5.52 (t, J=39 Hz), 82.56 (t, J=33 Hz), 81.07-80.73 (m), 61.22 (d, J=42 Hz), 26.35, 24.34.
-
ESI MS: Calculated for C H N O : 303.13. Found: 302.3 [M-H ]. HPLC: 95.46% (Atlantis
12
18
4
5
dC18 5 m, 4.6x100 mm, 5-15% MeCN:0.1% Formic Acid in H O, 254 nm, retention time
2
1
2.24 min.).
-Aminoimidazole-4-carboxamide-1-β-D-[ C ]ribofuranosyl 5’-monophosphate
1
3
5
5
[
AICAR-PO H ] (6). At 0 C, 5-amino-1-[2’,3’-O-(1-methylethylidene)-β-D-ribofuranosyl]-
3
2
13
1
H-Imidazole-4-carboxamide ([ C ]-ribose) 5 (158 mg, 0.52 mmol) was dissolved in tri-n-
5
butyl phosphate (1.5 mL). POCl (0.8 mL, 8.49 mmol) was then added as a solution in tri-n-
3
butyl phosphate (3.7 mL). The reaction was placed in a freezer at -15 C overnight. The
reaction was quenched with NaOH (2.7 g in 17 mL H O). The aqueous solution was then
2
extracted with diethyl ether (2x20 mL) and ethyl acetate (2x20 mL). The aqueous layer was
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concentrated at 30 C. During concentration, the acetonide protecting group is cleaved. The
salt mixture is then purified by cation exchange chromatography. Initially, dilute HCl (slow
gradient up to 0.05 M) was used to elute the product as the HCl salt (113 mg obtained, 57%
yield, Dowex 50Wx8 resin, H+ form, ~30 grams of resin to form column, up to 2.5 g crude
salt mixture [dissolved in H O and neutralized by adding 1 M HCl dropwise] loaded onto
2
column). However, although the fractions were initially pure by HPLC, upon lyophilization
some decomposition occurred, and the material turned light brown. The material was then re-
purified eluting with only water (Dowex 50Wx8 resin, H+ form, loaded in H O, eluted with
2
~
200 mL H O). The fractions were monitored by HPLC and lyophilized to yield a white solid
2
(50 mg, 0.15 mmol, 28% yield). The material is stored under nitrogen, protected from light
1
and moisture at -20 C. H NMR (300 MHz, D O+DCl (3 drops))  7.93 (s, 1H, H-2), 5.25
2
(d, 1H, J=171 Hz, H-1’), 3.98 (d, 1H, J=150 Hz, H-4’), 3.80 (d, 2H, J=150 Hz, H-2’ and H-
1
3
1
3
9
’), 3.91-3.79 (m, 1H, H-5’a), 3.41-3.30 (m, 1H, H-5’b). H NMR (300 MHz, H O) 8.25 (s,
2
H), 5.79 (dd, 1H), 4.63 (m, 1H), 4.35 (m, 1.5H [other 0.5 under H O peak]), 4.13 (m, 1H),
2
1
3
.86 (m, 1H). C NMR (125 MHz, D O+DCl(3 drops))  162.57, 142.72, 128.10, 103.85,
2
3
1
0.45-88.89 (m), 84.58-83.67 (m), 74.21-73.40 (m), 69.89-69.11 (m), 65.18-64.42 (m). P
+
(
300 MHz, D O)  -0.01. HRMS (ESI+): Calculated for C H N O P [M+H] : 344.0868 m/z.
2
9
15
4
8
1
3
13
13
Found: 344.0890 m/z. Isotopic distribution: 95.5% C 4.0% C , 0.5% C . In-Process
5
,
4
3
HPLC: 99% (Atlantis dC18 5 m, 4.6 x 100 mm, 1:99 MeCN:0.1% Formic acid in H O, 254
2
nm, retention time 2.8 min.). GLP UPLC: 99.0% (Acquity HSS T3, 2.1 x 100 mm, 1.8 μm
particle size analytical column; 254 nm detection; 0.5 mL/min flow rate for gradient of 0.1%
formic acid in water (MPA) and 0.1% formic acid in acetonitrile (MPB); 5 μL 0.5 mg/mL
sample in MPA; gradient curve 6: % MPA/at min: 100/5, 80/10, 10/13, 10/14, 100/14.01-17;
retention time 1.312 min, largest impurity 1.673 min, 0.54%). Both HPLC methods were
developed using commercial unlabeled AICAR-PO H which supports identification of the
3
2
final product (see supplemental).
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13
Scheme 1. Chemical Synthesis of [ C -ribose] AICAR-PO H a. ADA b. (EtO) CH,
5
3
2.
3
acetone, TsOH c. Ac O, pyridine d. Dinitrochlorobenzene, K CO , DMF, 80 C e. EDA f.
2
2
3
POCl , (BuO) PO g. concentration, cation exchange chromatography
3
3
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