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Published in final edited form as: Anal Bioanal Chem. 2021 Jan 23;413(7):1999–2006. doi: 10.1007/s00216-021-03168-z

Rapid, user-friendly and inexpensive detection of Azidothymidine

Ying Luo 1,#, Tianwei Jia 1,#, Jieqiong Fang 1, Dandan Liu 1, Varma Saikam 1, Xiaolin Sheng 1, Suri S Iyer 1,*
PMCID: PMC8276851  NIHMSID: NIHMS1671938  PMID: 33484329

Abstract

Strict adherence to Highly Active Anti-Retroviral Therapy (HAART) is very important to improve quality of life for HIV-positive patients to reduce new infections and determine treatment success. Azidothymidine (AZT) is an antiretroviral drug commonly used in HAART treatment. In this research, an “add, mix and measure” assay was developed to detect AZT within minutes. Three different probes designed to release fluorophores when samples containing AZT are added, were synthesized and characterized. The limit of detection to AZT in simulated urine samples was determined to be 4 μM in 5 minutes for one of the probes. This simple and rapid point-of-care test could potentially be used by clinicians and health care workers to monitor the presence of AZT in low resource settings.

Keywords: HIV, Azidothymidine, Therapeutic drug monitoring, Point-of-care, Fluorescence

Graphical Abstract

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Introduction

Human Immunodeficiency Virus (HIV) continues to be a deadly affliction, with over 38 million HIV-positive individuals and 1.7 million new infections in 2019 [1]. Approximately 25 million HIV positive individuals are receiving antiretroviral treatment [2]. HAART, the most successful treatment for HIV has been demonstrated to reduce viral resistance, decrease viral load, and improve quality of life [3,4]. HAART was first introduced in 1996 as a cocktail treatment involving a minimum of two antiretrovirals chosen from the class of protease inhibitors (PIs), entry/fusion inhibitors, nucleos(t)ide or non-nucleoside reverse transcriptase inhibitors (NRTIs or NNRTIs), and integrase strand transfer inhibitors (INSTIs) [3,5,6]. The strict adherence to HAART and regular testing to monitor viral load is critical to determine treatment success, reduce morbidity and mortality, and reduce drug resistance. Unfortunately, adherence is not optimal for several reasons, including side effects, drug-drug interactions, forgetfulness, and fatigue in taking medication daily. Pharmaceutical companies have been developing novel therapies by reducing the number of doses to overcome some of these issues and reduce patient burden. However, lack of adherence remains an issue [7]. If patients and healthcare professionals were equipped with rapid, user-friendly adherence monitoring diagnostics, patients would take the medication regularly. This strategy has found significant success with diagnostics that improve health and well-being in individuals with diabetes, obesity, and heart-related ailments. Indeed, data from wearable or stand-alone electronics have been demonstrated to change patient behavior [8–13]. Unfortunately, current methods to track adherence are unreliable (patient self-reporting, pill counting, electronic drug monitoring) or require expensive instrumentation (HPLC and/or mass spectroscopy) [5]. Lateral flow assays based on ELISA using antibodies developed against drugs are being designed [14], but antibody generation against small molecules is challenging and expensive. Briefly, small molecules have to be conjugated to a carrier protein and injected into animals to elicit a robust immune response. Extensive separation and purification to isolate the required antibody is required because a large number of distinct antibodies are produced against small molecules, carrier proteins, linkers, or a combination of different parts. Production of highly selective antibodies against low molecular weight compounds remains a major bottleneck [15–17]. After purification, antibodies are used in a competition lateral or flow through assay and these competition assays often have limited sensitivity because of the nature of the competition assays.

As an alternative, we are developing assays that could be eventually used in Point-Of-Care (POC) diagnostics to monitor drug adherence. The assays are designed to be an “add, mix and measure” assay, where synthetic probes can react with specific functionalities in drug molecules to produce color or fluorescence and no antibodies are required. AZT (AZidoThymidine or ZiDoVudine ZDV) was chosen as a model therapeutic since it is an antiretroviral drug commonly used in HAART treatment in developing countries and has a distinct azide functionality that is not present in naturally occurring compounds or in the human body. AZT is converted to 5’-glucuronylzidovudine (GZDV) metabolically and is excreted through the kidney. Approximately 14% of AZT and 74% of GZDV are excreted in urine. Hence, the existence of any azide moieties in urine directly corresponds to AZT consumption and can be correlated to adherence. [18,19]. The azide group can react with suitable substrates via a Staudinger ligation or an azide-alkyne Huisgen cycloaddition to release fluorescent products [20,21]. The reactions can be carried out in aqueous or biological media. Since serum, urine, hair, or stool samples are the preferred source of samples for adherence testing [22,23], if the reaction can be performed directly under aqueous conditions, sample pretreatment can be minimized or eliminated. This is an important consideration when developing a POC diagnostic for a resource-poor environment.

Two different strategies were investigated for the rapid detection of AZT (Figure 1). First, we used Staudinger ligation, where the azide functionality can react with a phosphine to generate an aza-ylide [24,25]. In the presence of water, the intermediate hydrolyzes to yield a primary amine and the corresponding phosphine oxide [20]. As Bertozzi and coworkers have previously demonstrated, an appropriately designed molecule with an electrophilic trap, such as methyl ester, can capture the aza-ylide intermediate by intramolecular cyclization and release methanol [26]. Fluorogenic 4-methyl-2-oxo-2H-chromen-7-yl-2-(diphenylphosphaneyl) benzoate derivatives (Probe-1 and Probe-2) were used for AZT detection. In an alternate method, azide-alkyne Huisgen cycloaddition, which occurs between an alkyne and an azide, was also used to detect AZT. Fluorogenic Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reactions have emerged as a powerful tool for bioconjugation, materials science, organic synthesis, and drug discovery [27]. A water-soluble fluorogenic 4-acetylene-1,8-naphthalimide derivative (Probe-3) was used in this study. The triazole unit generated during the reaction modulates the fluorescence via electron-donating properties [28]. In total, three probes were synthesized as fluorescent probes in the presence of AZT.

Figure 1.

Figure 1.

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a When AZT is added to the Probe-1 or Probe-2, fluorescence is generated in 20 min and recorded at 360/460 nm. b When AZT is added to the Probe-3, fluorescent is generated in 5 min and recorded at 357/462 nm.

Materials and methods

Materials

Azidothymidine (AZT) and 4-Methylumbelliferone (4-MU) were purchased from Sigma-Aldrich. Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) was purchased from TCI America (Portland, OR). 96 and 384 microwell black plates were purchased from Thermo Scientific (Waltham, MA). Simulated urine without glucose was purchased from Carolina Biologicals (Burlington, NC). Fluorescence intensity was detected by Enspire Plate Reader (PerkinElmer, Inc., Waltham, MA).

Detection of AZT using Probe-1 or Probe-2 in 80% v/v ethanol/H2O:

Different concentrations of Probe-1 (150 μL, 250 μM-63 μM) or Probe-2 (150 μL, 400 μM-50 μM) were mixed with different concentrations of AZT (150 μL, 1 mM-63 μM). The fluorescence intensity of this solution was recorded in 5 min intervals at 360/460 nm for 2h at 37 °C.

Detection of AZT using Probe-2 in 80% v/v ethanol/simulated urine:

Different concentrations of Probe-2 (150 μL, 100 μM) were mixed with different concentrations of AZT (150 μL, 500 μM-63 μM). The fluorescence intensity of this solution was recorded in 5 min intervals at 360/460 nm for 2h at 37 °C.

Detection of AZT using Probe-3 in 10% v/v DMSO /H2O:

Different concentrations of Probe-3 (70 μL, 100 μM-1 μM) were mixed with L-ascorbic acid (200 μM), CuSO4 (50 μM), and TBTA (50 μM) first. Different concentrations of AZT (70 μL, 100 μM-0.1 μM) were added in the final step to mimic realworld situations [29]. The fluorescence intensity of this solution was recorded in 5 min intervals at 360/460 nm for 2h at 37 °C.

Detection of AZT using Probe-3 in 10% v/v DMSO/simulated urine:

Probe-3 (70 μL, 10 μM) was mixed with L-ascorbic acid (200 μM), CuSO4 (50 μM), and TBTA (50 μM). Different concentrations of AZT (70 μL, 100 μM-0.1 μM) were added in the final step to mimic real-world situations [29]. The fluorescence intensity of this solution was recorded in 5 min intervals at 360/460 nm for 2h at 37 °C.

Determination of the limit of detection and limit of quantification

To calculate the Limit of Detection (LOD) and Limit of Quantification (LOQ) based on the calibration curves of probes, % RFU was plotted as a concentration of AZT. The LOD of probes was calculated as the blank signal plus 3 times its standard deviation. The standard deviation of blank measurement was achieved by measuring the fluorescence of probes three times. The LOQ of probes was calculated as 10 times the standard deviation of the blank signal divided by the slope of the calibration curve.

Additional experimental details, including synthesis of probes, fluorescence spectra of AZT probes, determination of LOD and LOQ of AZT using the probes, and pertinent NMR spectra, are provided in the Supplementary Information (ESM, Figs. S1–S6, Tables S1–S2).

Results and Discussion

Two different methods, Staudinger ligations and azide-alkyne Huisgen cycloadditions, were used to detect and quantify AZT. The application of Staudinger ligations was carried out with an electrophilic trap, coumarin ester, to capture the aza-ylide intermediate by intramolecular cyclization. Briefly, the phosphorus atom of Probe-1 or Probe-2 nucleophilic reacts with the azide functionality of AZT to form the aza-ylide intermediate and release nitrogen. In the subsequent step, the electrophilic trap, coumarin ester captures the nucleophilic attack of aza-ylide by intramolecular cyclization. In the presence of water, the intermediate hydrolyzes to form a stable phosphine oxide product. The process of intramolecular cyclization is depicted in Scheme 1. The synthesis of Probe-1 and Probe-2 is depicted in Schemes 2, 3. Compound 1 was reacted with 4-Methylumbelliferone (4-MU) to yield Probe-1. The methyl protons resonated at 2.44 ppm in the 1H NMR corresponding carbon of methyl resonates at 18.74 ppm in the 13C NMR and phosphine signal at −3.78 ppm in 31P NMR spectroscopy. To improve the solubility of Probe-1, an ethylene glycol spacer was introduced using compound 3 [30,31]. These spacers are expected to improve solubility in aqueous media like urine and/or serum [32–38]. Saponification to realize the free acid, subsequent esterification with 4-MU and final tert-butyloxycarbonyl group deprotection produced Probe-2 in 70% overall yield. The methyl protons resonated at 2.43 ppm in the 1H NMR corresponding carbon of methyl resonates at 18.76 ppm in the 13C NMR and phosphine signal at −3.23 ppm [39,30].

Scheme 1.

Scheme 1.

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Postulated mechanism of the fluorescence-releasing probe via Staudinger ligation.

Scheme 2.

Scheme 2.

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Reagents and conditions: (a) DCC, DMAP, DCM, rt, 12 h, 85%.

Scheme 3.

Scheme 3.

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Reagents and conditions: (a) 95% EtOH, NaOH, rt, 1.5 h, 60%; (b)HBTU, DIPEA, 4-MU, DCM/THF, rt, 12 h, 80%; (c) TFA, DCM, 0°C, 1 h, 70%.

With the two probes in hand, assays to detect AZT were performed. The workflow is fairly straightforward; Probe-1 or 2 were mixed with different concentrations of AZT and the fluorescence was measured at 360/460 nm for 2 h at 37 °C. First, 125 μM of Probe-1 was mixed with 500 μM of AZT and incubated at 37°C for 2 h. The color change of the mixture can be visualized by human eyes under UV light (ESM Fig. S1a). When detected with a fluorescent plate reader, the signal for the mixture of Probe-1 and AZT increased significantly compared to the solution without AZT (Figure 2a). This result indicates that Probe-1 could be used to detect AZT. Next, the solubility of Probe-2 was evaluated as Probe-1 was sparingly soluble in water. As expected, the solubility of Probe-2 was determined twice than that of Probe-1. When 100 μM of Probe-2 was mixed with 500 μM of AZT (Figure 2a), fluorescent intensity was not as high as Probe-1, presumably because the activity of Probe-2 for the reaction is not as high as Probe-1. To determine the analytical limit of detection, different concentrations of AZT were added to probes (ESM Figs. S2 and S3). As shown in ESM Table S3, the detection range of AZT was determined to be 33–500 μM and 0.19–1.0 mM in 120 min by Probe-1 and Probe-2, respectively. To verify that there were no matrix problems in urine, a similar assay for Probe-2 was performed in 80% ethanol/simulated urine solution (ESM Fig. S4). As seen in ESM Table S3, the detection range of AZT was 99–500 μM in 120 min by Probe-2. As the concentration of AZT in the urine of HIV patients with AZT treatment has been determined previously to be 940 μM to 3.1 mM [5], these experiments demonstrated that AZT can be reliably detected using these synthetic probes. If concentrations in urine are in the high mM range in real world samples, samples could be diluted. Additionally, high frequency testing of urine samples collected over multiple time points in a day could provide additional actionable information for the physician to modify the dose.

Figure 2.

Figure 2.

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a Fluorescence intensity generated using Probe-1 (125 μM) and Probe-2 (100 μM) to detect AZT (500 μM) in 80% v/v ethanol/H2O. Mixtures were detected at 37 °C for 120 min at 360/460 nm. The y-axis, % RFU, represents the RFU of the sample divided by the RFU of 4-MU (63 μM) at the same time. 4-MU (63 μM) is using as a control. b Fluorescence intensity generated using Probe-3 (10 μM) to detect AZT (100 μM) in 10% v/v DMSO/H2O. The mixtures were detected at 37°C for 30 min at 357/462 nm. The y-axis, % RFU, represents the RFU of the sample divided by the RFU of Probe-3 + AZT (100 μM) at 30 min. Results are reported as the mean of three trials with the error reported as the standard deviation of the trials.

Next, Probe-3 was synthesized with the expectation of improving the fluorescence intensity signal compared to the Staudinger ligation assay [21] (Scheme 4). As demonstrated previously, 1,8 naphthalimide derivatives fluorescence properties can be modulated by the formation of the triazole unit via electron-donating properties. In brief, the alkyne was introduced to the 4-position by palladiummediated coupling reaction of trimethylsilylacetylene to afford compound 8. The deprotection of the silyl protecting group gave the desired Probe-3. Protons of trimethylsilyl that resonated at 0.39 ppm in 1H NMR disappeared with a concomitant increase of the peak at 3.77 ppm, corresponding to the alkyne functionality. The terminal carbon of alkyne moiety resonates at 80.2 ppm in the 13C NMR [40]. The characterization of Probe-3 was done after azide-alkyne cycloaddition reaction by fluorescence spectroscopy. The excitation and emission are 357 nm and 462 nm. NMR spectra of all intermediates and final compounds are provided in the ESM.

Scheme 4.

Scheme 4.

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Reagents and conditions: (a) Ethanol, R2-NH2, reflux, 12 h, 70%; (b) Trimethylsilylacetylene, CuI, DIPEA, Pd(PPh3)4, THF, rt, 12 h, 75%; (c) TBAF, MeOH, 0 °C, 2 h, 80%.

When Probe-3 was mixed with AZT, there is a significant increase in fluorescence intensity in 20 min (Figure 2b). To determine the analytical limit of detection, 10 μM of Probe-3 was introduced to different concentrations of AZT (ESM Fig. S5), and the detection range, as shown in ESM Tables S1 and S2 was 13–100 μM in 5 min. A similar assay was performed for Probe-3 in 10% DMSO/simulated urine solution (Figure 3), and as seen in ESM Table S3, the range of detection was 4–100 μM in 5 min, indicating that Probe-3 could be used for the rapid detection of AZT at micromolar concentrations.

Figure 3.

Figure 3.

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Limit of Detection of AZT by Probe-3 in 10% v/v DMSO/simulated urine. Reaction solutions contained Probe-3 (10 μM), L-ascorbic acid (200 μM), CuSO4 (50 μM) and TBTA (50 μM). Different concentrations of AZT (100 μM, 10 μM, 1 μM, 0.1 μM) were added in the final step to mimic real-world situations. The fluorescence intensity (λex = 357 nm, λem = 462 nm) was detected at 5-min intervals for 30 min at 37 °C. The y-axis, % RFU, represents the RFU of the sample divided by the RFU of Probe-3+AZT (100 μM) at 30 min. Results are reported as the mean of three trials with the error reported as the standard deviation of the trials.

These results compare very well with other techniques that have been used to detect AZT. For example, the colorimetric detection of AZT using an alkyne-modified dextran substrate has been previously reported by Pratt and coworkers [5]. The authors reported a LOD of 750 μM of AZT in urine. Another report by Tiwari and coworkers, where the authors detected AZT electrochemically using chitosan stabilized silver nanoparticles in the range of 1–718 μM in PBS buffer and 10–533 μM in biological samples using voltametric techniques [41]. In contrast, the assay reported here has a lower LOD determined by Probe-1 (33–500 μM), Probe-2 (0.19–1.0 mM) in 120 min and Probe-3 (4 −100 μM) in 5 min. Other assays use HPLC. Even though HPLC method has been developed and validated over a lower range 0.05 to 50 μg/mL (0.187–187 μM) than our approach, HPLC is an expensive equipment, requires trained personnel and not conducive to low resource settings. [42]

Conclusion

In summary, we have developed three different probes to detect AZT rapidly and confirmed that these novel synthetic probes are highly efficient for the “add, mix and measure” assay in simulated urine samples. The results described here are highly significant because it uses a unique concept to detect small molecules unlike lateral flow or ELISA based assays. Detection of small molecules using lateral flow strips or ELISA assays require the development of highly specific antibodies. Generating antibodies against small molecules can be challenging and expensive. The typical process involves attaching the small molecule to a carrier protein in a mouse or rabbit, generating an immune response, bleeding the animal and purifying the antibodies. This is a labor and cost intensive process, because the animal generates antibodies against the protein, the linker, the small molecule and combinations (eg. small molecule and the linker). An extensive purification protocol is required, which increases cost and can fail, because the animal often doesn’t generate sufficient antibodies only against the small molecule. Even if a suitable antibody is generated, the ELISA assay is usually a competitive assay as the target is a small molecule and there aren’t sufficient epitopes for the capture and reporter antibody for a standard sandwich assay. False negative results for small molecules detection are common in ELISAs due to the insufficient blocking and washing steps. This approach, compared with lateral flow strips and ELISA assays, does not require technical expertise leading to be readily used in resource-limited settings. The probes can be synthesized in large quantities and the total cost of the synthesis and purification is minimal compared to antibody generation. Short oligoethylene spacers can be introduced in probes to improve the solubility in aqueous media. This method requires calibration and a fluorometer compared to a lateral flow assay; however, with the increased use of smartphone-based fluorescence microscopy, we envision the following work format. A patient or health care professional can introduce the sample into the vial containing the probe, take an image with a smartphone and transmit the image electronically to a central laboratory. This test can be performed in the privacy of a home or other low resource settings. The image can be processed, compared to a database, and the concentration of the drug can be accurately determined. [43] Finally, while the method described here focuses on well-known biorthogonal reactions related to azides, it is anticipated that similar strategies could be used to monitor other drugs [44–46] because there is an urgent need to monitor real-time concentrations in biological fluids for personalized medicine.

Supplementary Material

1671938_Sup_Info

Acknowledgments and Funding information

We are grateful to the National Institute of Allergy and Infectious Diseases (Grant number 5R61AI140475) for funding.

Footnotes

Declarations

Conflict of interest The authors declare that they have no conflict of interest.

Availability of data Synthesis of probes, data of fluorescence assays, determination of detection limits of AZT by probes, and characterization of all compounds and intermediates are provided in the ESM.

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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Associated Data

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Supplementary Materials

1671938_Sup_Info
NIHMS1671938-supplement-1671938_Sup_Info.pdf (3.2MB, pdf)

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