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ˇ ınova´ Veronika Sol´ ´ Kaiser Martin Maxmilian Milosˇ Luka´ cˇ Zlatko Janeba ´ ˇ cka ˇ Vaclav Kasi Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic Received October 3, 2013 Revised November 8, 2013 Accepted November 10, 2013
Research Article
Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogs bearing [(3-hydroxypropan-2-yl)1H-1,2,3-triazol-4-yl]phosphonic acid, 2-[(diisopropoxyphosphonyl)methoxy]propanoic acid, or 2–(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6diaminopurine, uracil, and 5-bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5-bromouracil-derived acyclic nucleoside phosphonate with a 2-(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51–2.94, within a reasonable time, 13–28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85, and 10.30, respectively) containing 20 mg/mL -cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5-bromouracil-derived acyclic nucleoside phosphonate with 2-(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20:40 mM Tris/phosphate, pH 2.20) using 60 mg/mL quaternary ammonium -cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range. Keywords: Acyclic nucleoside phosphonates / CE / Chiral analysis / Cyclodextrins / Nucleotide analogs DOI 10.1002/jssc.201301092
1 Introduction Acyclic nucleoside phosphonates (ANPs) represent a distinguished class of antiviral agents and drugs [1–4]. Three ANPs have been approved worldwide for clinical use [1, 2]: (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidR ) for the treatment of cytomegalovirus reofovir, Vistide tinitis in AIDS (acquired immunodeficiency syndrome) patients, 9-[2-(phosphonomethoxy)ethyl]adenine (as its oral R ) for the treatment prodrug adefovir dipivoxil, Hepsera of chronic hepatitis B virus infections [5], and (R)-9-[2(phosphonomethoxy)propyl]adenine, PMPA, (as its oral pro´ ˇ cka, ˇ Correspondence: Dr. Vaclav Kasi Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic E-mail: [email protected] Fax: +420-220-183-592
Abbreviations: AIDS, acquired immunodeficiency syndrome; ANP, acyclic nucleoside phosphonate; CD, cyclodextrin; HIV, human immunodeficiency virus; PMPA, (R)-9-[2(phosphonomethoxy)propyl]adenine; QA--CD, quaternaryammonium-–cyclodextrin C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
R drug tenofovir disoproxil fumarate, Viread ) for the treatment of HIV (human immunodeficiency virus) infection (itself or in combination with other antivirals) and chronic hepatitis B virus infections [6]. Their mechanism of action is based on interfering with DNA polymerase or reverse transcriptase resulting in viral DNA chain termination. Many other ANPs were found to exhibit important antiviral properties, e.g. derivatives of 2,6-diaminopurine and “open ring” derivatives of 2,4-diaminopyrimidine. (S)-9[2-Hydroxy-3-(phosphonomethoxy)propyl]adenine possesses a broad spectrum activity against DNA viruses (including herpes-, adeno-, pox-, and iridoviruses), as well as against retroviruses [4]. Acyclic nucleoside analogs of adenosine, like (S)-9-(2,3-dihydroxypropyl)adenine produced in CzechosloR , and structurally related 3-(adeninvakia as Duvira gel 9-yl)-2-hydroxypropanoic acid constitute another class of broad-spectrum antivirals interfering with (S)-adenosyl-Lhomocysteine hydrolase, thus inhibiting viral RNA maturation. ANPs exhibit not only antiviral but also cytostatic [7], antiparasitic [8–10], and immunomodulatory activities [11].
ˇ Dedicated to Prof. Frantiˇsek Svec on the occasion of his 70th birthday.
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The registered as well as new potential ANPs-based antiviral drugs are frequently chiral compounds with asymmetric carbon atoms in their acyclic alkyl chain. The enantiomers and diastereomers of these compounds may significantly differ in their biological and pharmacological activities, antiviral potency, metabolism, and toxicity. For example, the R enantiomers of PMPA and (R)-9-[2(phosphonomethoxy)propyl]-2,6-diaminopurine were found to be 10–100 times more effective against HIV than their S enantiomers [6]. Consequently, the determination of the enantiomeric purity of these compounds is extremely important and necessary prior to their biological tests and medical applications. The most commonly used analytical method for these purposes is HPLC with chiral stationary phases based on cellulose, amylose or cyclodextrin (CD) [12,13] or using the ligand-exchange separation principle [14]. HPLC separations of diastereoisomers of nucleoside-based derivatives were also reported [15, 16]. Alternative HPCE methods, CZE, MEKC, and CEC with free or immobilized chiral discriminators (e.g. CDs and macrocyclic antibiotics) were shown to be also suitable tools for the analysis of nucleotides, nucleosides, and nucleic acid bases including their stereoisomers [17–21]. CE separation of ANPs stavudine and acyclovir was achieved with a sulfated--CD chiral selector in an acidic BGE composed of triethanolamine phosphate buffer, pH 2.5, in a fused-silica capillary dynamically coated with polyethylene oxide [17]. Under the same separation conditions, the enantioseparation of cis and trans diastereoisomeric thymine derivatives of isochroman aromatic analogs of stavudine was obtained [18]. The separation and analysis of pronucleotide diastereomers of 3 -azido-2 ,3 -dideoxythymidine in biological samples were performed by CE in sodium phosphate BGE, pH 6.2 or pH 2.5, with carboxymethyl--CD [19] and sulfated--CD and sulfated-␣-CD [20]. The R and S enantiomers of tenofovir, cidofovir, and related ANPs were successfully separated in 35–50 mM sodium tetraborate buffer, pH 10.0–10.5 with -CD as chiral selector [21, 22]. The enantiopurity of synthetic R and S isomers of acyclic nucleoside bisphosphonate (2-amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines) and 9-{3-hydroxy[1,4-bis(phosphonomethoxy)]butan-2-yl}derivatives of purines was determined in 40–50 mM sodium tetraborate/NaOH, pH 10.0–10.5, 20 mg/mL -CD [23, 24]. HPCE methods are used also for achiral separations and analyses of nucleotides, nucleosides, and nucleobases in biochemical, pharmaceutical, and clinical research [25]. A mixture of nucleosides (guanosine, thymidine, adenosine, cytidine, uridine) and thymine was separated by CEC in porous-layered open tubular capillaries in alkaline borate buffers, pH 8.5–10.5 [26]. The stability of entecavir, cyclopentyl guanosine analog, in pharmaceutical formulations was monitored by using a validated CE method using 20 mM sodium tetraborate buffer, pH 10.0, as BGE, and nimesulide as internal standard [27]. The CE separation of 6-mercaptopurine and 6-methylmercaptopurine in BGE consisting of 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid titrated by triethylamine to pH 11.2 with addition of 10% of methanol has become a methodic basis for screening C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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of thiopurine (S)-methyltransferase enzyme activity (methylation of thiopurine drugs used for treatment of leukemia and autoimmune disorders) in human erythrocytes [28]. Adenine nucleotides, AMP, ADP, ATP (adenosine mono-, di-, and triphosphates), coenzymes NAD+ (nicotinamide adenine dinucleotide), NADP+ (NAD phosphate), and their reduced forms, were determined in the bacterium Paracoccus denitrificans at various stages of the active respiratory chain by CZE in 100 mM sodium carbonate/hydrogencarbonate BGE, pH 9.6, containing 10 mM -CD, with submicromolar LODs within 20 min [29]. The aim of the present work was to develop HPCE methods for the chiral analysis of new types of six acyclic nucleoside phosphonates (ANPs), nucleotide analogs bearing either a [(3-hydroxypropan-2-yl)-1H-1,2,3-triazol-4-yl]phosphonic acid moiety as a side chain of guanine, 2,6-diaminopurine and uracil nucleobases, or 2-(phosphonomethoxy)propanoic acid or its diisopropyl derivative moieties as side chains of adenine, uracil, and 5-bromouracil nucleobases (Fig. 1). The developed methods should be used for the enantiopurity control of synthetic R and S enantiomers of these ANPs prior to their biological tests.
2 Materials and methods 2.1 Chemicals and analyzed ANPs All chemicals used were of analytical reagent grade. Disodium tetraborate decahydrate (Na2 B4 O7 .10H2 O), sodium benzoate, naphthalene-1,6-disulfonic acid disodium salt, quaternary-ammonium--CD (QA--CD) (degree of substitution not specified, catalog no. 33805), 2-hydroxypropyl--CD (2-HP--CD, molar substitution 0.6), and 2-hydroxypropyl␥-CD (2-HP-␥-CD, molar substitution 0.5–0.7), heptakis(2,6dimethyl)--CD, -CD, and ␥-CD were obtained from Sigma– Aldrich (Prague, Czech Republic), ␣-CD was supplied by Fluka (Basel, Switzerland), acetic acid, formic acid, phosphoric acid, and dimethylsulfoxide (EOF marker) was from Lachema (Brno, Czech Republic), sodium hydroxide was obtained from Penta (Chrudim, Czech Republic) and Tris was supplied by Serva (Heidelberg, Germany). The analyzed ANPs were synthesized in our Institute following the procedure described by Kaiser et al. [30]; their molecular structures are shown in Fig. 1.
2.2 Capillary electrophoresis CE experiments were carried out in a P/ACE MDQ System (Beckman–Coulter, Fullerton, CA, USA), data acquisition and evaluation were performed using the software P/ACE System MDQ, version Karat supplied by Beckman, and Clarity station (DataApex, Prague, CR), respectively. The apparatus was equipped with the internally uncoated fused-silica capillary, total/effective length 40.0/30.3 cm, id/od 50/375 m (Polymicro Technologies, Phoenix, AR, USA). The analytes were www.jss-journal.com
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297
Figure 1. Molecular structures of the ANPs analyzed.
detected by a UV/Vis absorption spectrophotometric photodiode array detector (190–600 nm), set at two wavelengths, 206 and 254 nm. The new capillary was consecutively washed by 0.1 M NaOH, water and BGE at 2000 mbar for 10 min and conditioned at high voltage 12 kV for 20 min. Between runs, the capillary was washed by BGE at 2000 mbar for 2 min. The capillary was thermostatted by liquid coolant at 20 or 15⬚C. The ANPs were hydrodynamically injected at 6.9–13.8 mbar for 5–15 s. The ANPs were dissolved in deionized water. For identification of enantiomers, they were mixed in a 2:1 ratio, concentration of the R isomer was 0.2 mM and that of the S isomer 0.1 mM. The experimental conditions, namely BGE composition and pH, type and concentration of chiral selector, separation voltage, electric current, and temperature, of the successful enantioseparations of ANPs are listed in Table 1.
2.3 Calculation of effective electrophoretic mobility, resolution, selectivity factor, and separation efficiency The effective electrophoretic mobility, meff , of the analyte at the pH of the applied BGE was determined from the experimental data of its CE analysis using Equation (1):
meff =
L tot L eff Usep
1 tmig
−
1
(1)
teof
where Ltot is the total capillary length, Leff is the effective capillary length (from injection end to the detector position), Usep is the applied separation voltage, tmig is the migration time of the analyte (peak apex), and teof is the migration time of the electroneutral EOF marker (peak apex). The resolution, Rs , of CE-separated R and S enantiomers was calculated according to Equation (2): Rs =
2 × tmig,R − tmig,S (w R + w S )
(2)
where tmig,R and tmig,S are the migration times, and wR and wS are the peak widths of the R and S enantiomers, respectively. The selectivity factor, ␣, was calculated according to Equation (3): ␣=
meff ,S meff ,R
(3)
where meff,R and meff,S are the effective electrophoretic mobilities of the R and S enantiomers, respectively.
Table 1. CE separation conditions: composition and pH of the BGE; type and concentration, cCD , of chiral selector; separation voltage, Usep ; electric current, I; and temperature, T
BGE no.
Composition
pHa)
Chiral selector
cCD (mg/mL)
Usep (kV)
I (A)
T (⬚C)
I II III IV V VI
50 mM Na2 B4 O7 50 mM Na2 B4 O7 50 mM Na2 B4 O7 50 mM Na2 B4 O7 50 mM Na2 B4 O7 40 mM H3 PO4
10.31 9.85 9.85 9.60 9.85 2.20
-CD -CD ␣-CD QA--CD -CD QA--CD
20.0 20.0 40.0 10.0 20.0 60.0
20.0 20.0 15.0 −12.0 15.0 −14.0
108.2 94.3 68.7 82.4 98.1 181.2
15.0 15.0 20.0 20.0 20.0 15.0
a) Adjusted by NaOH in BGEs I–V, and by Tris in BGE VI.
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The separation efficiency was expressed by the number of theoretical plates per meter of capillary effective length, N, defined by Equation (4): tmig (4) N = 5.545 w1/2 L eff where tmig is the migration time of the analyte (peak apex), and w1/2 is the peak width of the analyte at half of the peak height.
3 Results and discussion 3.1 Development of the chiral CE method The development of experimental conditions for the enantioseparation of R and S isomers of ANPs derived from (i) purine nucleobases: guanine (1), 2,6-diaminopurine (2) and adenine (5), or (ii) from pyrimidine nucleobases: uracil (3 and 4) and 5-bromouracil (6) (see Fig. 1), was based on general screening strategy for the development of the chiral CE separation method [31] and on the solubility, acid– base and other physicochemical characteristics of the above compounds. According to the structure of the side chain of their nucleobases, the analyzed ANPs can be divided in two groups: (i) bearing [(3-hydroxypropan-2-yl)-1H-1,2,3-triazol4-yl]phosphonic acid moiety as a side chain of guanine, 2,6diaminopurine and uracil bases (1, 2, and 3), and (ii) containing 2-(phosphonomethoxy)propanoic acid or its diisopropyl derivative as a side chain of uracil, adenine, and 5-bromouracil bases (4, 5, and 6). Compounds 1–3 and 6 contain acidic divalent phosphonic acid group, compound 6 in addition to that also a carboxylic group of propanoic acid, therefore, they behave as divalent or trivalent anions in alkaline BGEs at pH > 7. ANPs with guanine, uracil, or bromouracil bases (1, 3, 4, and 6) contain enol group of these bases, which can be negatively charged at pH > 10, thus further enhancing anionic character of these ANPs in the alkaline BGEs. The ANPs 4 and 5 contain also carboxy group of propanoic acid but due to the presence of diisopropylesters of phosphonic acid group, they possess only a single negative charge at pH above ∼5. ANPs with guanine, 2,6-diaminopurine or adenine bases (1, 2, and 5) contain one or two amino groups on their purine moiety. Hence, these ANPs are amphoteric compounds but with respect to weak basicity of their amino groups (pKa ∼ 2–4), they can be positively charged only in the strongly acidic BGEs at pH < 2, where dissociation of phosphonic acid group of compounds 1 and 2 and carboxy group of compound 5 is suppressed and amino groups are protonated. Based on these acid–base properties, the first choice was to analyze all ANPs as anions in alkaline BGEs. From several potential chiral selectors (oligo- and polysaccharides, crown ethers, metal–amino acid or metal–peptide complexes, glycopeptide macrocyclic antibiotics, and proteins), cyclic oligosaccharides ␣-, -, and ␥-CDs and their derivatives were chosen as the most promising chiral discrim C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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inators. Because of their high stereoselectivity, commercial availability, and good solubility in aqueous buffers, UV transparency, and chemical stability, they belong to the prominent stereoselectors in chiral CE analysis of variable compounds as confirmed by several recent reviews and papers on chiral CE separations in general [32–34] and with CDs especially [35–41]. Chiral separations with CDs are usually based on the formation of host–guest complexes, i.e. on the inclusion of the whole or at least a part of the analyte molecule into the hydrophobic CD cavity and/or on the stereoselective interactions of the analyte with CD rim. When the derivatized charged CDs are employed, then also their electrostatic interactions with the ionogenic groups of analytes may contribute to enantioresolution of the analytes. In this study, taking into account the prevalence of acidic groups in the molecules of the ANPs analyzed and considering the results of our previous chiral and achiral analyses of ANPs [21, 42], first, several alkaline BGEs, such as Tris/Tricine, boric acid/NaOH, and sodium tetraborate/NaOH, in the pH range 8.1–10.5, were tested, using variable native and derivatized noncharged and charged CDs (␣-, -, and ␥-CDs, 2-HP--CD, 2-HP-␥-CD, heptakis(2,6dimethyl)--CD, and quaternary-ammonium-–cyclodextrin (QA--CD)) as chiral selectors. No or very low enantioseparations were observed when any of these CDs were added to the Tris/Tricine and boric acid/NaOH BGEs (data not shown). The first successful separations of enantiomers of ANPs 1–5 were achieved when sodium tetraborate was employed as the BGE and -CD as the chiral selector. Following this finding and after testing the sodium tetraborate concentration in the range 20–60 mM, pH in the range 9.3–10.5, and -CD at 5–20 mg/mL concentration range, the best separations of the R and S enantiomers of ANPs 1–5 with sufficient resolution of 1.51–2.94 were achieved in the BGEs composed of 50 mM sodium tetraborate adjusted with NaOH to pH 9.60 or 9.85 or 10.30, respectively, and containing 20 mg/mL -CD. The R and S enantiomers of ANPs bearing the relatively more hydrophobic moiety of diisopropyl derivative of 2-(phosphonomethoxy)propanoic acid as the alkyl side chain of the uracil and guanine bases, ANPs 4 and 5, were successfully separated not only with -CD but also with other CDs, particularly with 10 mM QA--CD in 50 mM sodium tetraborate BGE adjusted with NaOH, pH 9.6, with a resolution of 1.32 and 2.22, respectively, or with 40 mg/mL ␣-CD in 50 mM sodium tetraborate BGE adjusted with NaOH, pH 9.85, with a resolution of 1.80. A list of the compositions and pH values of the BGEs together with type and concentration, cCD , of chiral selector, the applied separation voltage, Usep , the electric current, I, and the capillary coolant temperature, T, are given in Table 1. The examples of successful chiral separation of R and S isomers of 4 with the addition of three types of cyclodextrins, ␣-CD, –CD and QA--CD, to alkaline sodium tetraborate BGEs are depicted in Fig. 2. The R and S enantiomers of 4 were fully separated with a resolution of 1.80–2.94. The analyses of all five ANPs 1–5 were relatively fast, migration time was in the range 12–30 min and no www.jss-journal.com
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Figure 2. CE separations of R and S enantiomers of 4 in alkaline sodium tetraborate BGEs, using different CDs as chiral selectors. (A) -CD contained in the BGE II, pH 9.85. (B) ␣-CD contained in the BGE III, pH 9.85; (C) QA--CD contained in the BGE IV, pH 9.60; For the BGE composition and other experimental conditions, see Table 1 and Section 2.2. A, absorbance at 206 nm.
Figure 3. CE separations of R and S enantiomers of analyzed ANPs under the optimized experimental conditions. (A) 1 in BGE I; (B) 2 in BGE I; (C) 3 in BGE II; (D) 4 in BGE II; (E) 5 in BGE V; (F) 6 in BGE VI. For the BGE composition and other experimental conditions, see Table 1 and Section 2.2. A, absorbance at 206 nm.
significant admixtures and impurities were observed. The CE separations of the R and S enantiomers of ANPs 1–5 under the optimized separation conditions are depicted in Fig. 3A–E. The separation of R and S enantiomers of 6 was not successful in these alkaline BGEs. Due to their high negative charge (3–4 negative charges originating from dissociation of divalent phosphonic acid and monovalent propanoic acid and enol group of bromouracil) and relatively weak interactions with CDs, these isomers migrated with anionic mobilities higher than the cationic mobility of the EOF and in this C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
way they escaped from the capillary into the anodic electrode vessel. Therefore, some other BGEs in the acidic pH range, such as acetic acid/NaOH, pH 4.5, formic acid/NaOH, pH 3.5, phosphoric acid/Tris, pH 2.75–1.8, with 20 mg/mL -CD or 10 mM QA--CD as chiral selectors were tested but no or very low resolutions were observed (data not shown). The best separation with a resolution of around 0.5 was achieved in 20: 40 mM Tris/phosphate BGE, pH 2.2, with 10 mM QA--CD chiral selector. The resolution has been improved up to 0.97 when the concentration of QA--CD was increased to 60 mM
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Table 2. Characteristics of CE separations of the enantiomers of 1–6. Rs , resolution; ␣, selectivity factor; NR and NS , numbers of theoretical plates/meter of R and S isomers; meff,R and meff,S , effective electrophoretic mobilities of the R and S isomers. For the composition of the BGEs and other experimental conditions, see Table 1 and Section 2
ANP
BGE
pH
Rs
␣
NR × 10−3 (m−1 )
NS × 10−3 (m−1 )
meff,R × 109 (m2 V−1 s−1 )
meff,S × 109 (m2 V−1 s−1 )
1 2 3 4
I I II I II III IV V IV VI
10.31 10.31 9.85 10.31 9.85 9.85 9.60 9.85 9.60 2.20
1.76 1.51 1.57 2.69 2.94 1.80 2.22 1.54 1.32 1.48
0.99 0.99 0.99 0.98 0.98 0.99 0.97 0.98 1.02 1.01
107.8 190.8 83.8 126.8 103.0 121.8 122.5 110.3 211.5 340.5
124.0 158.8 117.3 145.5 114.3 125.0 111.0 127.5 191.3 401.8
−20.6 −16.8 −21.7 −19.9 −20.0 −18.5 −16.8 −13.3 −12.0 −21.2
−20.5 −16.6 −21.6 −19.6 −19.7 −18.3 −16.3 −13.0 −11.8 −21.5
5 6
Table 3. Repeatability of migration times, tmig , corrected peak areas, Acor , and ratio of corrected peak areas, Acor,S /Acor,R , at detection wavelength 206 nm, for the R and S enantiomers of the analyzed ANPs in optimized BGEs. For the composition of the BGEs and other experimental conditions, see Table 1 and Section 2
ANP no.
Enantiomer
BGE no.
tmig [min] Mean ± SD
1 2 3 4 5 6
R S R S R S R S R S R S
I I II II V VI
28.44 27.54 14.48 12.79 22.19 21.52 21.62 20.25 12.25 11.98 7.67 7.52
± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.12 0.03 0.06 0.08 0.14 0.05 0.08 0.06 0.05 0.02 0.02
Acor [AU] RSD%
Mean ± SD
0.55 0.45 0.20 0.43 0.13 0.25 0.23 0.37 0.49 0.42 0.31 0.26
153.0 107.9 150.4 87.1 125.8 85.9 59.9 43.2 116.1 55.9 91.5 43.8
± ± ± ± ± ± ± ± ± ± ± ±
9.4 7.2 4.3 3.5 1.6 1.1 2.2 1.4 4.3 3.0 4.5 1.8
Acor,S /Acor,R RSD%
Mean ± SD
RSD%
6.1 6.6 2.8 2.8 1.3 1.3 3.7 3.2 3.7 5.4 5.0 4.1
1.418 ± 0.002
0.16
1.729 ± 0.088
5.12
1.462 ± 0.005
0.34
1.387 ± 0.010
0.75
2.080 ± 0.039
1.87
2.088 ± 0.022
1.07
n = 3.
(data not shown). However, at this high QA--CD concentration, the BGE electric conductivity and current increased and there was no potential for enhancing the separation efficiency and resolution by increasing the separation voltage. Hence, the temperature of capillary coolant was decreased from 20 to 15⬚C, which allowed increasing the separation voltage from –8 to –14 kV. Although the electric current and input power (Joule heat) were rather high (181 A, and 6.3 W per 1 m capillary length, respectively), a baseline separation of the R and S enantiomers of 6 was achieved under these conditions in rather short time of 7 min, as shown in Fig. 3F. The characteristics of the enantioseparations of all ANPs, such as resolution, selectivity factor, number of theoretical plates and effective mobilities are summarized in Table 2. As can be seen in Fig. 3, the migration order of enantiomers according to migration times was the same for all ANPs, i.e. the S isomers migrated first and the R isomers second. However, taking into account that the separation of the enantiomers of 1–5 was performed at positive separation voltage (anode at injection end, separation upstream strong C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cationic EOF) while the separation of enantiomers of 6 was run at a negative separation voltage (cathode at injection end, separation upstream very slow, almost zero cationic EOF), it is obvious that the electrophoretic migration order of the enantiomers of ANPs 1–5 is R first, S second, and it is opposite to the migration order S first, R second, of the enantiomers of ANP 6. Hence, from the observed migration order of ANPs enantiomers in the Fig. 3 and from their mobilities in the Table 2, it follows that the S isomers of ANPs 1–5 formed stronger complexes with -CD and migrated slower than the R isomers, whereas the S isomers of ANP 6 were less strongly complexed by cationic QA--CD and migrated faster than the R isomers.
3.2 Repeatability, linearity, LOD, and LOQ After optimization of the enantioseparations, the repeatability, and linearity of the developed methods were examined, and LODs and LOQs of the analyzed ANPs enantiomers www.jss-journal.com
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Table 4. LOD, LOQ, parameters of linear concentration dependence (slope, k; intercept value, q; coefficient of determination, R2 ), and linear range, for the R and S enantiomers of the analyzed ANPs in optimized BGEs. For the composition of the BGEs and other experimental conditions, see Table 1 and Section 2
ANP
1 2 3 4 5 6
Enantiomer
R S R S R S R S R S R S
BGE
I I II II V VI
LOD (M)
5.08 3.18 5.78 5.52 6.50 4.05 11.61 8.33 7.10 9.34 3.51 5.96
LOQ (M)
16.9 10.6 19.3 18.4 21.7 13.5 38.7 27.8 23.7 31.1 11.7 19.9
Linear regression:a) y = k*x+q k ± SD
q ± SD
2.053 ± 0.067 3.280 ± 0.097 2.650 ± 0.067 2.775 ± 0.070 1.679 ± 0.039 2.694 ± 0.063 1.268 ± 0.017 1.767 ± 0.023 2.690 ± 0.048 2.045 ± 0.037 3.529 ± 0.029 2.079 ± 0.017
0.023 0.036 −0.015 −0.015 0.014 0.023 0.001 0.001 0.020 0.015 −0.007 −0.004
Linear range (mM) R2
± ± ± ± ± ± ± ± ± ± ± ±
0.017 0.027 0.017 0.018 0.010 0.016 0.004 0.006 0.012 0.009 0.007 0.004
0.997 0.998 0.997 0.998 0.998 0.999 0.999 0.999 0.999 0.999 0.999 0.999
0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5
a) y = ratio of corrected peak areas of enantiomer and internal standard, x = concentration of the enantiomer [mM]; the measurements were performed at five concentrations, three times for each concentration; SD, standard deviation.
were determined. Repeatability of the migration times, tmig , and corrected peak areas (peak areas divided by migration times) at detection wavelength 206 nm, Acor , LODs and LOQs were estimated from three analyses of the mixtures of the R and S isomers of ANPs at the 0.1–0.2 mM level. The results presented in the Table 3 show very good repeatability of migration times, with RSD lower than 0.55%, RSD of corrected peak areas were about one order higher, ranging from 1.3–6.6%. The variability of the relative corrected peak area of enantiomer S (related to corrected peak area of enantiomer R, which was taken as internal standard), i.e. RSD of Acor,S /Acor,R ratio was usually few times lower than the RSD of the absolute corrected area of enantiomer S due to compensation of the variations of the injected volume. The LODs, calculated at S/N = 3, and LOQs, calculated at S/N = 10, were found to be in low micromolar level (3.2–11.6 M) and LOQs were about one order higher (10.6–38.7 M), see Table 4. The calibration curves were calculated by linear regression of a ratio of the corrected peak areas of the enantiomer and internal standard (0.5 mM sodium benzoate in alkaline
BGEs I, II, and V, and 0.1 mM napthalene-1,6-disulfonic acid disodium salt in acidic BGE VI) at a detection wavelength of 206 nm versus enantiomer concentration. The measurements were performed for five concentration values, three times for each concentration of all ANPs. The parameters of linear regression (slope and intercept of the calibration curve and the correlation coefficient) are presented in Table 4. The linearity of the response was confirmed in the range 0.025– 0.5 mM for all ANPs.
3.3 Enantiopurity control of synthetic ANPs The developed methods have been applied to determination of enantiomeric purity of synthetic preparations of enantiomers of all above ANPs. The examples of the determination of the enantiopurity of the R and S enantiomers of ANPs 1 and 6 under the optimized separation conditions are depicted in Fig. 4. As can be seen from these records and as follows from the CE analyses of enantiomers of the other ANPs (data
Figure 4. Determination of the enantiopurity of the R and S isomers of ANPs 1 and 6 and CE separations of mixtures of these enantiomers under the optimized experimental conditions. (A) 1 in the BGE I; (B) 6 in the BGE VI; a, mixture of 0.2 mM R and 0.1 M S isomers; b, 0.1 mM R isomer; c, 0.1 mM S isomer. For the BGE composition and other experimental conditions, see Table 1 and Section 2.2. A, absorbance at 206 nm.
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not shown), the preparations of all ANPs were found to be synthesized in pure enantiomeric forms.
ˇ cek, ˇ ´ D., Tichy, ´ T., Vrbkova, ´ [10] Keough, D. T., Spa P., Hockova, ˇ ınska, ´ L., Janeba, Z., Naesens, L., Edstein, M. S., Slavet´ D., Chavchich, M., Wang, T.-H., de Jersey, J., Guddat, L. W., J. Med. Chem. 2013, 56, 2513–2526.
4 Concluding remarks
ˇ P., Krecmerov ˇ ´ M., Kmon´ıckov ˇ ´ E., Holy, ´ A., [11] Potmeˇ sil, a, a, Z´ıdek, Z., Eur. J. Pharmacol. 2006, 540, 191–199.
CE with CD-based stereoselectors proved to be a highperformance method for the fast and sensitive chiral analysis of new types of six ANPs, nucleotide analogs with potential biological activities. From the CDs tested, -CD at 20 mg/ mL concentration was found to be the best chiral selector for the complete separation of the R and S enantiomers of five ANPs containing either [(3-hydroxypropan-2-yl)-1H1,2,3-triazol-4-yl]phosphonic acid moiety as a side chain of guanine, 2,6-diaminopurine and uracil nucleobases, or bearing [(diisopropoxyphosphonyl)methoxy]propanoic acid as a side chain of adenine and uracil nucleobases, by CE in alkaline 50 mM sodium tetraborate BGEs, pH 9.6–10.3. On the other hand, QA--CD at 60 mg/mL concentration served as the most effective chiral selector for baseline separation of the R and S enatiomers of the sixth ANP, containing a 2-(phosphonomethoxy)propanoic acid group linked to the N1 atom of 5-bromouracil, by CE in acidic 20/40 mM tris/phosphate BGE, pH 2.20. The analyses provided baseline resolution and a good repeatability. The developed methods are ready to be used for the enantiopurity analysis of R and S isomers of these new types of ANPs prior to testing their biological and pharmaceutical activity.
[12] Ward, T. J., Ward, K. D., Anal. Chem. 2012, 84, 626–635.
This work was supported by the Czech Science Foundation (grants nos. P206/12/0453 and 13-17224S), by the Academy of Sciences of the Czech Republic (Research Project RVO 61388963), by the Ministry of the Interior of the Czech Republic (VG20102015046) and by Gilead Sciences (Foster City, CA, USA). The authors have declared no conflict of interest.
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ˇ ınova´ Veronika Sol´ ´ Kaiser Martin Maxmilian Milosˇ Luka´ cˇ Zlatko Janeba ´ ˇ cka ˇ Vaclav Kasi Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic Received October 3, 2013 Revised November 8, 2013 Accepted November 10, 2013
Research Article
Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogs bearing [(3-hydroxypropan-2-yl)1H-1,2,3-triazol-4-yl]phosphonic acid, 2-[(diisopropoxyphosphonyl)methoxy]propanoic acid, or 2–(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6diaminopurine, uracil, and 5-bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5-bromouracil-derived acyclic nucleoside phosphonate with a 2-(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51–2.94, within a reasonable time, 13–28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85, and 10.30, respectively) containing 20 mg/mL -cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5-bromouracil-derived acyclic nucleoside phosphonate with 2-(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20:40 mM Tris/phosphate, pH 2.20) using 60 mg/mL quaternary ammonium -cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range. Keywords: Acyclic nucleoside phosphonates / CE / Chiral analysis / Cyclodextrins / Nucleotide analogs DOI 10.1002/jssc.201301092
1 Introduction Acyclic nucleoside phosphonates (ANPs) represent a distinguished class of antiviral agents and drugs [1–4]. Three ANPs have been approved worldwide for clinical use [1, 2]: (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidR ) for the treatment of cytomegalovirus reofovir, Vistide tinitis in AIDS (acquired immunodeficiency syndrome) patients, 9-[2-(phosphonomethoxy)ethyl]adenine (as its oral R ) for the treatment prodrug adefovir dipivoxil, Hepsera of chronic hepatitis B virus infections [5], and (R)-9-[2(phosphonomethoxy)propyl]adenine, PMPA, (as its oral pro´ ˇ cka, ˇ Correspondence: Dr. Vaclav Kasi Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic E-mail: [email protected] Fax: +420-220-183-592
Abbreviations: AIDS, acquired immunodeficiency syndrome; ANP, acyclic nucleoside phosphonate; CD, cyclodextrin; HIV, human immunodeficiency virus; PMPA, (R)-9-[2(phosphonomethoxy)propyl]adenine; QA--CD, quaternaryammonium-–cyclodextrin C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
R drug tenofovir disoproxil fumarate, Viread ) for the treatment of HIV (human immunodeficiency virus) infection (itself or in combination with other antivirals) and chronic hepatitis B virus infections [6]. Their mechanism of action is based on interfering with DNA polymerase or reverse transcriptase resulting in viral DNA chain termination. Many other ANPs were found to exhibit important antiviral properties, e.g. derivatives of 2,6-diaminopurine and “open ring” derivatives of 2,4-diaminopyrimidine. (S)-9[2-Hydroxy-3-(phosphonomethoxy)propyl]adenine possesses a broad spectrum activity against DNA viruses (including herpes-, adeno-, pox-, and iridoviruses), as well as against retroviruses [4]. Acyclic nucleoside analogs of adenosine, like (S)-9-(2,3-dihydroxypropyl)adenine produced in CzechosloR , and structurally related 3-(adeninvakia as Duvira gel 9-yl)-2-hydroxypropanoic acid constitute another class of broad-spectrum antivirals interfering with (S)-adenosyl-Lhomocysteine hydrolase, thus inhibiting viral RNA maturation. ANPs exhibit not only antiviral but also cytostatic [7], antiparasitic [8–10], and immunomodulatory activities [11].
ˇ Dedicated to Prof. Frantiˇsek Svec on the occasion of his 70th birthday.
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The registered as well as new potential ANPs-based antiviral drugs are frequently chiral compounds with asymmetric carbon atoms in their acyclic alkyl chain. The enantiomers and diastereomers of these compounds may significantly differ in their biological and pharmacological activities, antiviral potency, metabolism, and toxicity. For example, the R enantiomers of PMPA and (R)-9-[2(phosphonomethoxy)propyl]-2,6-diaminopurine were found to be 10–100 times more effective against HIV than their S enantiomers [6]. Consequently, the determination of the enantiomeric purity of these compounds is extremely important and necessary prior to their biological tests and medical applications. The most commonly used analytical method for these purposes is HPLC with chiral stationary phases based on cellulose, amylose or cyclodextrin (CD) [12,13] or using the ligand-exchange separation principle [14]. HPLC separations of diastereoisomers of nucleoside-based derivatives were also reported [15, 16]. Alternative HPCE methods, CZE, MEKC, and CEC with free or immobilized chiral discriminators (e.g. CDs and macrocyclic antibiotics) were shown to be also suitable tools for the analysis of nucleotides, nucleosides, and nucleic acid bases including their stereoisomers [17–21]. CE separation of ANPs stavudine and acyclovir was achieved with a sulfated--CD chiral selector in an acidic BGE composed of triethanolamine phosphate buffer, pH 2.5, in a fused-silica capillary dynamically coated with polyethylene oxide [17]. Under the same separation conditions, the enantioseparation of cis and trans diastereoisomeric thymine derivatives of isochroman aromatic analogs of stavudine was obtained [18]. The separation and analysis of pronucleotide diastereomers of 3 -azido-2 ,3 -dideoxythymidine in biological samples were performed by CE in sodium phosphate BGE, pH 6.2 or pH 2.5, with carboxymethyl--CD [19] and sulfated--CD and sulfated-␣-CD [20]. The R and S enantiomers of tenofovir, cidofovir, and related ANPs were successfully separated in 35–50 mM sodium tetraborate buffer, pH 10.0–10.5 with -CD as chiral selector [21, 22]. The enantiopurity of synthetic R and S isomers of acyclic nucleoside bisphosphonate (2-amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines) and 9-{3-hydroxy[1,4-bis(phosphonomethoxy)]butan-2-yl}derivatives of purines was determined in 40–50 mM sodium tetraborate/NaOH, pH 10.0–10.5, 20 mg/mL -CD [23, 24]. HPCE methods are used also for achiral separations and analyses of nucleotides, nucleosides, and nucleobases in biochemical, pharmaceutical, and clinical research [25]. A mixture of nucleosides (guanosine, thymidine, adenosine, cytidine, uridine) and thymine was separated by CEC in porous-layered open tubular capillaries in alkaline borate buffers, pH 8.5–10.5 [26]. The stability of entecavir, cyclopentyl guanosine analog, in pharmaceutical formulations was monitored by using a validated CE method using 20 mM sodium tetraborate buffer, pH 10.0, as BGE, and nimesulide as internal standard [27]. The CE separation of 6-mercaptopurine and 6-methylmercaptopurine in BGE consisting of 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid titrated by triethylamine to pH 11.2 with addition of 10% of methanol has become a methodic basis for screening C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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of thiopurine (S)-methyltransferase enzyme activity (methylation of thiopurine drugs used for treatment of leukemia and autoimmune disorders) in human erythrocytes [28]. Adenine nucleotides, AMP, ADP, ATP (adenosine mono-, di-, and triphosphates), coenzymes NAD+ (nicotinamide adenine dinucleotide), NADP+ (NAD phosphate), and their reduced forms, were determined in the bacterium Paracoccus denitrificans at various stages of the active respiratory chain by CZE in 100 mM sodium carbonate/hydrogencarbonate BGE, pH 9.6, containing 10 mM -CD, with submicromolar LODs within 20 min [29]. The aim of the present work was to develop HPCE methods for the chiral analysis of new types of six acyclic nucleoside phosphonates (ANPs), nucleotide analogs bearing either a [(3-hydroxypropan-2-yl)-1H-1,2,3-triazol-4-yl]phosphonic acid moiety as a side chain of guanine, 2,6-diaminopurine and uracil nucleobases, or 2-(phosphonomethoxy)propanoic acid or its diisopropyl derivative moieties as side chains of adenine, uracil, and 5-bromouracil nucleobases (Fig. 1). The developed methods should be used for the enantiopurity control of synthetic R and S enantiomers of these ANPs prior to their biological tests.
2 Materials and methods 2.1 Chemicals and analyzed ANPs All chemicals used were of analytical reagent grade. Disodium tetraborate decahydrate (Na2 B4 O7 .10H2 O), sodium benzoate, naphthalene-1,6-disulfonic acid disodium salt, quaternary-ammonium--CD (QA--CD) (degree of substitution not specified, catalog no. 33805), 2-hydroxypropyl--CD (2-HP--CD, molar substitution 0.6), and 2-hydroxypropyl␥-CD (2-HP-␥-CD, molar substitution 0.5–0.7), heptakis(2,6dimethyl)--CD, -CD, and ␥-CD were obtained from Sigma– Aldrich (Prague, Czech Republic), ␣-CD was supplied by Fluka (Basel, Switzerland), acetic acid, formic acid, phosphoric acid, and dimethylsulfoxide (EOF marker) was from Lachema (Brno, Czech Republic), sodium hydroxide was obtained from Penta (Chrudim, Czech Republic) and Tris was supplied by Serva (Heidelberg, Germany). The analyzed ANPs were synthesized in our Institute following the procedure described by Kaiser et al. [30]; their molecular structures are shown in Fig. 1.
2.2 Capillary electrophoresis CE experiments were carried out in a P/ACE MDQ System (Beckman–Coulter, Fullerton, CA, USA), data acquisition and evaluation were performed using the software P/ACE System MDQ, version Karat supplied by Beckman, and Clarity station (DataApex, Prague, CR), respectively. The apparatus was equipped with the internally uncoated fused-silica capillary, total/effective length 40.0/30.3 cm, id/od 50/375 m (Polymicro Technologies, Phoenix, AR, USA). The analytes were www.jss-journal.com
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Figure 1. Molecular structures of the ANPs analyzed.
detected by a UV/Vis absorption spectrophotometric photodiode array detector (190–600 nm), set at two wavelengths, 206 and 254 nm. The new capillary was consecutively washed by 0.1 M NaOH, water and BGE at 2000 mbar for 10 min and conditioned at high voltage 12 kV for 20 min. Between runs, the capillary was washed by BGE at 2000 mbar for 2 min. The capillary was thermostatted by liquid coolant at 20 or 15⬚C. The ANPs were hydrodynamically injected at 6.9–13.8 mbar for 5–15 s. The ANPs were dissolved in deionized water. For identification of enantiomers, they were mixed in a 2:1 ratio, concentration of the R isomer was 0.2 mM and that of the S isomer 0.1 mM. The experimental conditions, namely BGE composition and pH, type and concentration of chiral selector, separation voltage, electric current, and temperature, of the successful enantioseparations of ANPs are listed in Table 1.
2.3 Calculation of effective electrophoretic mobility, resolution, selectivity factor, and separation efficiency The effective electrophoretic mobility, meff , of the analyte at the pH of the applied BGE was determined from the experimental data of its CE analysis using Equation (1):
meff =
L tot L eff Usep
1 tmig
−
1
(1)
teof
where Ltot is the total capillary length, Leff is the effective capillary length (from injection end to the detector position), Usep is the applied separation voltage, tmig is the migration time of the analyte (peak apex), and teof is the migration time of the electroneutral EOF marker (peak apex). The resolution, Rs , of CE-separated R and S enantiomers was calculated according to Equation (2): Rs =
2 × tmig,R − tmig,S (w R + w S )
(2)
where tmig,R and tmig,S are the migration times, and wR and wS are the peak widths of the R and S enantiomers, respectively. The selectivity factor, ␣, was calculated according to Equation (3): ␣=
meff ,S meff ,R
(3)
where meff,R and meff,S are the effective electrophoretic mobilities of the R and S enantiomers, respectively.
Table 1. CE separation conditions: composition and pH of the BGE; type and concentration, cCD , of chiral selector; separation voltage, Usep ; electric current, I; and temperature, T
BGE no.
Composition
pHa)
Chiral selector
cCD (mg/mL)
Usep (kV)
I (A)
T (⬚C)
I II III IV V VI
50 mM Na2 B4 O7 50 mM Na2 B4 O7 50 mM Na2 B4 O7 50 mM Na2 B4 O7 50 mM Na2 B4 O7 40 mM H3 PO4
10.31 9.85 9.85 9.60 9.85 2.20
-CD -CD ␣-CD QA--CD -CD QA--CD
20.0 20.0 40.0 10.0 20.0 60.0
20.0 20.0 15.0 −12.0 15.0 −14.0
108.2 94.3 68.7 82.4 98.1 181.2
15.0 15.0 20.0 20.0 20.0 15.0
a) Adjusted by NaOH in BGEs I–V, and by Tris in BGE VI.
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The separation efficiency was expressed by the number of theoretical plates per meter of capillary effective length, N, defined by Equation (4): tmig (4) N = 5.545 w1/2 L eff where tmig is the migration time of the analyte (peak apex), and w1/2 is the peak width of the analyte at half of the peak height.
3 Results and discussion 3.1 Development of the chiral CE method The development of experimental conditions for the enantioseparation of R and S isomers of ANPs derived from (i) purine nucleobases: guanine (1), 2,6-diaminopurine (2) and adenine (5), or (ii) from pyrimidine nucleobases: uracil (3 and 4) and 5-bromouracil (6) (see Fig. 1), was based on general screening strategy for the development of the chiral CE separation method [31] and on the solubility, acid– base and other physicochemical characteristics of the above compounds. According to the structure of the side chain of their nucleobases, the analyzed ANPs can be divided in two groups: (i) bearing [(3-hydroxypropan-2-yl)-1H-1,2,3-triazol4-yl]phosphonic acid moiety as a side chain of guanine, 2,6diaminopurine and uracil bases (1, 2, and 3), and (ii) containing 2-(phosphonomethoxy)propanoic acid or its diisopropyl derivative as a side chain of uracil, adenine, and 5-bromouracil bases (4, 5, and 6). Compounds 1–3 and 6 contain acidic divalent phosphonic acid group, compound 6 in addition to that also a carboxylic group of propanoic acid, therefore, they behave as divalent or trivalent anions in alkaline BGEs at pH > 7. ANPs with guanine, uracil, or bromouracil bases (1, 3, 4, and 6) contain enol group of these bases, which can be negatively charged at pH > 10, thus further enhancing anionic character of these ANPs in the alkaline BGEs. The ANPs 4 and 5 contain also carboxy group of propanoic acid but due to the presence of diisopropylesters of phosphonic acid group, they possess only a single negative charge at pH above ∼5. ANPs with guanine, 2,6-diaminopurine or adenine bases (1, 2, and 5) contain one or two amino groups on their purine moiety. Hence, these ANPs are amphoteric compounds but with respect to weak basicity of their amino groups (pKa ∼ 2–4), they can be positively charged only in the strongly acidic BGEs at pH < 2, where dissociation of phosphonic acid group of compounds 1 and 2 and carboxy group of compound 5 is suppressed and amino groups are protonated. Based on these acid–base properties, the first choice was to analyze all ANPs as anions in alkaline BGEs. From several potential chiral selectors (oligo- and polysaccharides, crown ethers, metal–amino acid or metal–peptide complexes, glycopeptide macrocyclic antibiotics, and proteins), cyclic oligosaccharides ␣-, -, and ␥-CDs and their derivatives were chosen as the most promising chiral discrim C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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inators. Because of their high stereoselectivity, commercial availability, and good solubility in aqueous buffers, UV transparency, and chemical stability, they belong to the prominent stereoselectors in chiral CE analysis of variable compounds as confirmed by several recent reviews and papers on chiral CE separations in general [32–34] and with CDs especially [35–41]. Chiral separations with CDs are usually based on the formation of host–guest complexes, i.e. on the inclusion of the whole or at least a part of the analyte molecule into the hydrophobic CD cavity and/or on the stereoselective interactions of the analyte with CD rim. When the derivatized charged CDs are employed, then also their electrostatic interactions with the ionogenic groups of analytes may contribute to enantioresolution of the analytes. In this study, taking into account the prevalence of acidic groups in the molecules of the ANPs analyzed and considering the results of our previous chiral and achiral analyses of ANPs [21, 42], first, several alkaline BGEs, such as Tris/Tricine, boric acid/NaOH, and sodium tetraborate/NaOH, in the pH range 8.1–10.5, were tested, using variable native and derivatized noncharged and charged CDs (␣-, -, and ␥-CDs, 2-HP--CD, 2-HP-␥-CD, heptakis(2,6dimethyl)--CD, and quaternary-ammonium-–cyclodextrin (QA--CD)) as chiral selectors. No or very low enantioseparations were observed when any of these CDs were added to the Tris/Tricine and boric acid/NaOH BGEs (data not shown). The first successful separations of enantiomers of ANPs 1–5 were achieved when sodium tetraborate was employed as the BGE and -CD as the chiral selector. Following this finding and after testing the sodium tetraborate concentration in the range 20–60 mM, pH in the range 9.3–10.5, and -CD at 5–20 mg/mL concentration range, the best separations of the R and S enantiomers of ANPs 1–5 with sufficient resolution of 1.51–2.94 were achieved in the BGEs composed of 50 mM sodium tetraborate adjusted with NaOH to pH 9.60 or 9.85 or 10.30, respectively, and containing 20 mg/mL -CD. The R and S enantiomers of ANPs bearing the relatively more hydrophobic moiety of diisopropyl derivative of 2-(phosphonomethoxy)propanoic acid as the alkyl side chain of the uracil and guanine bases, ANPs 4 and 5, were successfully separated not only with -CD but also with other CDs, particularly with 10 mM QA--CD in 50 mM sodium tetraborate BGE adjusted with NaOH, pH 9.6, with a resolution of 1.32 and 2.22, respectively, or with 40 mg/mL ␣-CD in 50 mM sodium tetraborate BGE adjusted with NaOH, pH 9.85, with a resolution of 1.80. A list of the compositions and pH values of the BGEs together with type and concentration, cCD , of chiral selector, the applied separation voltage, Usep , the electric current, I, and the capillary coolant temperature, T, are given in Table 1. The examples of successful chiral separation of R and S isomers of 4 with the addition of three types of cyclodextrins, ␣-CD, –CD and QA--CD, to alkaline sodium tetraborate BGEs are depicted in Fig. 2. The R and S enantiomers of 4 were fully separated with a resolution of 1.80–2.94. The analyses of all five ANPs 1–5 were relatively fast, migration time was in the range 12–30 min and no www.jss-journal.com
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Figure 2. CE separations of R and S enantiomers of 4 in alkaline sodium tetraborate BGEs, using different CDs as chiral selectors. (A) -CD contained in the BGE II, pH 9.85. (B) ␣-CD contained in the BGE III, pH 9.85; (C) QA--CD contained in the BGE IV, pH 9.60; For the BGE composition and other experimental conditions, see Table 1 and Section 2.2. A, absorbance at 206 nm.
Figure 3. CE separations of R and S enantiomers of analyzed ANPs under the optimized experimental conditions. (A) 1 in BGE I; (B) 2 in BGE I; (C) 3 in BGE II; (D) 4 in BGE II; (E) 5 in BGE V; (F) 6 in BGE VI. For the BGE composition and other experimental conditions, see Table 1 and Section 2.2. A, absorbance at 206 nm.
significant admixtures and impurities were observed. The CE separations of the R and S enantiomers of ANPs 1–5 under the optimized separation conditions are depicted in Fig. 3A–E. The separation of R and S enantiomers of 6 was not successful in these alkaline BGEs. Due to their high negative charge (3–4 negative charges originating from dissociation of divalent phosphonic acid and monovalent propanoic acid and enol group of bromouracil) and relatively weak interactions with CDs, these isomers migrated with anionic mobilities higher than the cationic mobility of the EOF and in this C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
way they escaped from the capillary into the anodic electrode vessel. Therefore, some other BGEs in the acidic pH range, such as acetic acid/NaOH, pH 4.5, formic acid/NaOH, pH 3.5, phosphoric acid/Tris, pH 2.75–1.8, with 20 mg/mL -CD or 10 mM QA--CD as chiral selectors were tested but no or very low resolutions were observed (data not shown). The best separation with a resolution of around 0.5 was achieved in 20: 40 mM Tris/phosphate BGE, pH 2.2, with 10 mM QA--CD chiral selector. The resolution has been improved up to 0.97 when the concentration of QA--CD was increased to 60 mM
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Table 2. Characteristics of CE separations of the enantiomers of 1–6. Rs , resolution; ␣, selectivity factor; NR and NS , numbers of theoretical plates/meter of R and S isomers; meff,R and meff,S , effective electrophoretic mobilities of the R and S isomers. For the composition of the BGEs and other experimental conditions, see Table 1 and Section 2
ANP
BGE
pH
Rs
␣
NR × 10−3 (m−1 )
NS × 10−3 (m−1 )
meff,R × 109 (m2 V−1 s−1 )
meff,S × 109 (m2 V−1 s−1 )
1 2 3 4
I I II I II III IV V IV VI
10.31 10.31 9.85 10.31 9.85 9.85 9.60 9.85 9.60 2.20
1.76 1.51 1.57 2.69 2.94 1.80 2.22 1.54 1.32 1.48
0.99 0.99 0.99 0.98 0.98 0.99 0.97 0.98 1.02 1.01
107.8 190.8 83.8 126.8 103.0 121.8 122.5 110.3 211.5 340.5
124.0 158.8 117.3 145.5 114.3 125.0 111.0 127.5 191.3 401.8
−20.6 −16.8 −21.7 −19.9 −20.0 −18.5 −16.8 −13.3 −12.0 −21.2
−20.5 −16.6 −21.6 −19.6 −19.7 −18.3 −16.3 −13.0 −11.8 −21.5
5 6
Table 3. Repeatability of migration times, tmig , corrected peak areas, Acor , and ratio of corrected peak areas, Acor,S /Acor,R , at detection wavelength 206 nm, for the R and S enantiomers of the analyzed ANPs in optimized BGEs. For the composition of the BGEs and other experimental conditions, see Table 1 and Section 2
ANP no.
Enantiomer
BGE no.
tmig [min] Mean ± SD
1 2 3 4 5 6
R S R S R S R S R S R S
I I II II V VI
28.44 27.54 14.48 12.79 22.19 21.52 21.62 20.25 12.25 11.98 7.67 7.52
± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.12 0.03 0.06 0.08 0.14 0.05 0.08 0.06 0.05 0.02 0.02
Acor [AU] RSD%
Mean ± SD
0.55 0.45 0.20 0.43 0.13 0.25 0.23 0.37 0.49 0.42 0.31 0.26
153.0 107.9 150.4 87.1 125.8 85.9 59.9 43.2 116.1 55.9 91.5 43.8
± ± ± ± ± ± ± ± ± ± ± ±
9.4 7.2 4.3 3.5 1.6 1.1 2.2 1.4 4.3 3.0 4.5 1.8
Acor,S /Acor,R RSD%
Mean ± SD
RSD%
6.1 6.6 2.8 2.8 1.3 1.3 3.7 3.2 3.7 5.4 5.0 4.1
1.418 ± 0.002
0.16
1.729 ± 0.088
5.12
1.462 ± 0.005
0.34
1.387 ± 0.010
0.75
2.080 ± 0.039
1.87
2.088 ± 0.022
1.07
n = 3.
(data not shown). However, at this high QA--CD concentration, the BGE electric conductivity and current increased and there was no potential for enhancing the separation efficiency and resolution by increasing the separation voltage. Hence, the temperature of capillary coolant was decreased from 20 to 15⬚C, which allowed increasing the separation voltage from –8 to –14 kV. Although the electric current and input power (Joule heat) were rather high (181 A, and 6.3 W per 1 m capillary length, respectively), a baseline separation of the R and S enantiomers of 6 was achieved under these conditions in rather short time of 7 min, as shown in Fig. 3F. The characteristics of the enantioseparations of all ANPs, such as resolution, selectivity factor, number of theoretical plates and effective mobilities are summarized in Table 2. As can be seen in Fig. 3, the migration order of enantiomers according to migration times was the same for all ANPs, i.e. the S isomers migrated first and the R isomers second. However, taking into account that the separation of the enantiomers of 1–5 was performed at positive separation voltage (anode at injection end, separation upstream strong C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cationic EOF) while the separation of enantiomers of 6 was run at a negative separation voltage (cathode at injection end, separation upstream very slow, almost zero cationic EOF), it is obvious that the electrophoretic migration order of the enantiomers of ANPs 1–5 is R first, S second, and it is opposite to the migration order S first, R second, of the enantiomers of ANP 6. Hence, from the observed migration order of ANPs enantiomers in the Fig. 3 and from their mobilities in the Table 2, it follows that the S isomers of ANPs 1–5 formed stronger complexes with -CD and migrated slower than the R isomers, whereas the S isomers of ANP 6 were less strongly complexed by cationic QA--CD and migrated faster than the R isomers.
3.2 Repeatability, linearity, LOD, and LOQ After optimization of the enantioseparations, the repeatability, and linearity of the developed methods were examined, and LODs and LOQs of the analyzed ANPs enantiomers www.jss-journal.com
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Table 4. LOD, LOQ, parameters of linear concentration dependence (slope, k; intercept value, q; coefficient of determination, R2 ), and linear range, for the R and S enantiomers of the analyzed ANPs in optimized BGEs. For the composition of the BGEs and other experimental conditions, see Table 1 and Section 2
ANP
1 2 3 4 5 6
Enantiomer
R S R S R S R S R S R S
BGE
I I II II V VI
LOD (M)
5.08 3.18 5.78 5.52 6.50 4.05 11.61 8.33 7.10 9.34 3.51 5.96
LOQ (M)
16.9 10.6 19.3 18.4 21.7 13.5 38.7 27.8 23.7 31.1 11.7 19.9
Linear regression:a) y = k*x+q k ± SD
q ± SD
2.053 ± 0.067 3.280 ± 0.097 2.650 ± 0.067 2.775 ± 0.070 1.679 ± 0.039 2.694 ± 0.063 1.268 ± 0.017 1.767 ± 0.023 2.690 ± 0.048 2.045 ± 0.037 3.529 ± 0.029 2.079 ± 0.017
0.023 0.036 −0.015 −0.015 0.014 0.023 0.001 0.001 0.020 0.015 −0.007 −0.004
Linear range (mM) R2
± ± ± ± ± ± ± ± ± ± ± ±
0.017 0.027 0.017 0.018 0.010 0.016 0.004 0.006 0.012 0.009 0.007 0.004
0.997 0.998 0.997 0.998 0.998 0.999 0.999 0.999 0.999 0.999 0.999 0.999
0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5 0.025–0.5
a) y = ratio of corrected peak areas of enantiomer and internal standard, x = concentration of the enantiomer [mM]; the measurements were performed at five concentrations, three times for each concentration; SD, standard deviation.
were determined. Repeatability of the migration times, tmig , and corrected peak areas (peak areas divided by migration times) at detection wavelength 206 nm, Acor , LODs and LOQs were estimated from three analyses of the mixtures of the R and S isomers of ANPs at the 0.1–0.2 mM level. The results presented in the Table 3 show very good repeatability of migration times, with RSD lower than 0.55%, RSD of corrected peak areas were about one order higher, ranging from 1.3–6.6%. The variability of the relative corrected peak area of enantiomer S (related to corrected peak area of enantiomer R, which was taken as internal standard), i.e. RSD of Acor,S /Acor,R ratio was usually few times lower than the RSD of the absolute corrected area of enantiomer S due to compensation of the variations of the injected volume. The LODs, calculated at S/N = 3, and LOQs, calculated at S/N = 10, were found to be in low micromolar level (3.2–11.6 M) and LOQs were about one order higher (10.6–38.7 M), see Table 4. The calibration curves were calculated by linear regression of a ratio of the corrected peak areas of the enantiomer and internal standard (0.5 mM sodium benzoate in alkaline
BGEs I, II, and V, and 0.1 mM napthalene-1,6-disulfonic acid disodium salt in acidic BGE VI) at a detection wavelength of 206 nm versus enantiomer concentration. The measurements were performed for five concentration values, three times for each concentration of all ANPs. The parameters of linear regression (slope and intercept of the calibration curve and the correlation coefficient) are presented in Table 4. The linearity of the response was confirmed in the range 0.025– 0.5 mM for all ANPs.
3.3 Enantiopurity control of synthetic ANPs The developed methods have been applied to determination of enantiomeric purity of synthetic preparations of enantiomers of all above ANPs. The examples of the determination of the enantiopurity of the R and S enantiomers of ANPs 1 and 6 under the optimized separation conditions are depicted in Fig. 4. As can be seen from these records and as follows from the CE analyses of enantiomers of the other ANPs (data
Figure 4. Determination of the enantiopurity of the R and S isomers of ANPs 1 and 6 and CE separations of mixtures of these enantiomers under the optimized experimental conditions. (A) 1 in the BGE I; (B) 6 in the BGE VI; a, mixture of 0.2 mM R and 0.1 M S isomers; b, 0.1 mM R isomer; c, 0.1 mM S isomer. For the BGE composition and other experimental conditions, see Table 1 and Section 2.2. A, absorbance at 206 nm.
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not shown), the preparations of all ANPs were found to be synthesized in pure enantiomeric forms.
ˇ cek, ˇ ´ D., Tichy, ´ T., Vrbkova, ´ [10] Keough, D. T., Spa P., Hockova, ˇ ınska, ´ L., Janeba, Z., Naesens, L., Edstein, M. S., Slavet´ D., Chavchich, M., Wang, T.-H., de Jersey, J., Guddat, L. W., J. Med. Chem. 2013, 56, 2513–2526.
4 Concluding remarks
ˇ P., Krecmerov ˇ ´ M., Kmon´ıckov ˇ ´ E., Holy, ´ A., [11] Potmeˇ sil, a, a, Z´ıdek, Z., Eur. J. Pharmacol. 2006, 540, 191–199.
CE with CD-based stereoselectors proved to be a highperformance method for the fast and sensitive chiral analysis of new types of six ANPs, nucleotide analogs with potential biological activities. From the CDs tested, -CD at 20 mg/ mL concentration was found to be the best chiral selector for the complete separation of the R and S enantiomers of five ANPs containing either [(3-hydroxypropan-2-yl)-1H1,2,3-triazol-4-yl]phosphonic acid moiety as a side chain of guanine, 2,6-diaminopurine and uracil nucleobases, or bearing [(diisopropoxyphosphonyl)methoxy]propanoic acid as a side chain of adenine and uracil nucleobases, by CE in alkaline 50 mM sodium tetraborate BGEs, pH 9.6–10.3. On the other hand, QA--CD at 60 mg/mL concentration served as the most effective chiral selector for baseline separation of the R and S enatiomers of the sixth ANP, containing a 2-(phosphonomethoxy)propanoic acid group linked to the N1 atom of 5-bromouracil, by CE in acidic 20/40 mM tris/phosphate BGE, pH 2.20. The analyses provided baseline resolution and a good repeatability. The developed methods are ready to be used for the enantiopurity analysis of R and S isomers of these new types of ANPs prior to testing their biological and pharmaceutical activity.
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This work was supported by the Czech Science Foundation (grants nos. P206/12/0453 and 13-17224S), by the Academy of Sciences of the Czech Republic (Research Project RVO 61388963), by the Ministry of the Interior of the Czech Republic (VG20102015046) and by Gilead Sciences (Foster City, CA, USA). The authors have declared no conflict of interest.
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