Microwave-Mediated Synthesis of Labeled Nucleotides with Utility in

Sep 2, 2010 - Mark Lefever, Jerome W. Kosmeder II, Michael Farrell, and Christopher Bieniarz. Ventana Medical Systems, Inc., a member of the Roche Gro...
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Bioconjugate Chem. 2010, 21, 1773–1778

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Microwave-Mediated Synthesis of Labeled Nucleotides with Utility in the Synthesis of DNA Probes Mark Lefever, Jerome W. Kosmeder II, Michael Farrell, and Christopher Bieniarz Ventana Medical Systems, Inc., a member of the Roche Group, 1920 East Innovation Park Drive, Tucson, Arizona 85755. Received January 7, 2010; Revised Manuscript Received July 22, 2010

A novel method of linking haptens to deoxycytidine 5′-triphosphate via microwave-mediated bisulfate-catalyzed transamination with hydrazine has been developed. This method enables the tethering of small molecule haptens to dCTP via a discrete polyethylene glycol (PEG) spacer, yielding N4-aminodeoxycytidine 5′-triphosphate-dPEGhaptens. This synthetic approach employs microwave-catalyzed hydrazinolysis that enables the attachment of spacers via hydrazine linkages. The microwave-mediated introduction of this hydrazine handle provides a significant improvement in yield over those of published thermal methods. The microwave reaction was shown to be scalable, and the final product was amenable to labeling with a wide variety of haptens. The resulting nucleotide triphosphates, N4-aminodeoxycytidine 5′-triphosphate-dPEG-haptens, can serve as unique substrates for the enzyme-mediated labeling of DNA probes. The efficacy of incorporation of one such novel nucleotide, N4-aminodeoxycytidine 5′-triphosphate-dPEG4-DNP, has been demonstrated in nick translation labeling of HER2 and HPV probes. The labeled probes have been shown to be effective in visualizing their target genes in tissue.

INTRODUCTION Chemically modified nucleic acids are used as probes in hybridization assays, e.g., Southern blots, Northern blots, dot blots, and in situ hybridization (ISH), to detect RNA transcripts, gene amplification, and translocations in cells, in microarray assays, and in surface attachment chemistries and to generate various kinds of bioconjugates (1-3). Recently, the use of ISH assays both by fluorescent in situ hybridization detection (FISH) and by enzymatic signal deposition for brightfield detection via silver in situ hybridization (SISH) and chromogenic in situ hybridization (CISH) has found important clinical applications (4, 5, 6, 7). The amplification of the human epidermal growth factor 2 (HER2) gene in breast and stomach cancers has been shown to identify patients who can benefit from Herceptin treatment (8, 9). The amplification status of other genes is being used increasingly to assist in diagnosis and to profile tumors as the practice of personalized medicine progresses (10). Detection of viral genomes in cells by ISH has many potential clinical applications [e.g., human papilloma virus (HPV) in cervical cancer] (11, 12). For the generation of chemically modified nucleic acids, uridine and cytidine are the most frequently used bases (13). Enzymatic methods are frequently used to make probes by incorporating modified nucleotide triphosphates to produce a nucleic acid containing a label or reactive residue to which a label, e.g., a hapten or fluorophore, can be subsequently coupled. The bisulfite-mediated displacement of the C4 amino group of cytosine, first reported in 1970 by Shapiro and Weisgras (21), has become a widely reported method for the introduction of bifunctional nucleophilic handles onto cytosine residues (14-22). These newly introduced nucleophilic handles can then serve as reaction centers for the introduction of reporter groups derivatized with appropriate electrophilic handles. Reporter groups that have been employed include immunoreactive groups, photochemical cross-linking agents, fluorescent tags, alkylating agents, and catalytically active metal chelates (14-22). The bisulfite-catalyzed transamination of cytosine involves a number of discrete steps: addition of bisulfite across the 5-6 double bond of cytosine, transamination via attack of the nitrogen

nucleophile and release of ammonia, and base-catalyzed elimination of bisulfite to regenerate the double bond (20). In initial attempts to obtain cytosine derivatives tagged with nucleophilic handles, the procedure described by Negishi was employed (23). Although the detailed procedure was strictly followed, the reaction produced more hydrolysis than product. The initial failure led to the exploration of alternative conditions employing microwave technology. This choice was based on a number of studies indicating that microwaves are capable of accelerating a wide range of thermally driven reactions, including additions, cycloadditions, condensations, substitutions, rearrangements, organometallic reactions, and a host of others (24-29). The application of microwave energy results in a very rapid transfer of energy to the reactant molecules enabling the activation energy to be reached quickly (30, 31). Consequently, thermodynamic products are produced more rapidly, often avoiding the generation of unwanted side products that occur with traditional thermal methods. The commercial microwave synthesizers that are available allow for precise control of energy and offer the option of simultaneous cooling. The ability to maintain strict control of reaction conditions has proven to be indispensible in achieving reproducibility. Failure to provide sufficient microwave energy led to incomplete reactions, whereas overheating led to product decomposition. Herein is described the microwave-mediated synthesis of N4amino-dCTP and its use in labeling DNA. The assembly of these reagents involved the tethering of various moieties to N4-aminodCTP via discrete water-soluble polyethylene glycol (PEG) linkers. The resulting haptenylated nucleotide triphosphates can serve as unique substrates for the enzyme-mediated labeling of DNA probes via nick translation and/or random priming (32-35). One such novel nucleotide, N4-aminodeoxycytidine 5′-triphosphate-(dPEG4-2,4-dinitrophenylamino)(N4-amino-dCTP-PEG4DNP) was used to label DNA for use as a probe.

EXPERIMENTAL PROCEDURES Materials. Deoxycytidine 5′-triphosphate, hydrazine monohydrate, sodium bisulfite, N-hydroxysuccinimide, 1-fluoro-2,4-

10.1021/bc100013b  2010 American Chemical Society Published on Web 09/02/2010

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dinitrobenzene, and all organic solvents were commercial products of Sigma Aldrich. Discrete dPEG amino acids were products of Quanta Biodesign Ltd. (Powell, OH). Concentrated HCl from VWR was utilized to make pH adjustments. Microwave and Thermal Hydrazinolysis. We prepared reaction solutions by combining sodium bisulfite (203 mg, 2.85 mmol) and hydrazine monohydrate (1.0 mL, 20.6 mmol), cooling the mixture on an ice bath, and adjusting the pH to 7.2 with the careful addition of concentrated HCl (1.48 mL). In this premade hydrazine/bisulfite solution was dissolved deoxycytidine 5′-triphosphate (300 mg, 0.587 mmol). The pH was adjusted to 7.2 with additional hydrazine hydrate, and the solution was transferred to a 10 mL glass microwave tube. The tubes were either heated in a CEM Discover microwave reactor at 55 °C (5 W max), with cooling, for times ranging from 1 to 60 min or placed in a 55 °C water bath for the appropriate length of time. Aliquots (20 µL) were withdrawn at intervals and diluted with 100 µL of methanol, and the extent of the reaction was determined by immediate HPLC analysis. Analysis was performed on a Waters BioAlliance HPLC system fitted with a 2996 photodiode array detector and a XBridge 5 µm, C18(2), 100A, 150 mm × 4.6 mm column (10 µL injection per run). The column was eluted with 125 mM triethylammonium carbonate and 1-4% ACN over 10 min at pH 8.5 and a flow rate of 1 mL/min. N4-Aminodeoxycytidine 5′-Triphosphate Purification. Reaction mixtures were fractionated by HPLC utilizing a Waters Delta 600 HPLC system fitted with a 2996 photodiode array detector and a Waters Sunfire Prep 10 µ, C18(2), 100A, 250 mm × 50 mm column (350 µL of reaction mixture per run). The column was eluted with 125 mM triethylammonium carbonate and 1-4% acetonitrile (ACN) over 20 min at pH 8.5 and a flow rate of 20 mL/min. The N4-amino-dCTP fraction eluted at 30-32 min with a λmax of 275 nm. The N4-aminodCTP fraction was lyophilized three times from deionized (DI) water to ensure removal of all residual triethylammonium carbonate and then stored at -80 °C until it was used. To obtain the sodium salt, cation exchange was performed on a 50 mL column packed with Sp Sephadex C-25 equilibrated with 0.2 N NaOH and washed with water until the pH of the eluant buffer was 7.0. The lyophilized triethylammonium salt was taken up in 1 mL (4 × 0.250 mL) of water, loaded on the cation exchange column, and eluted with water. The UV-vis spectra of the collected fractions were recorded on an Agilent 8453 UV-vis spectrophotometer, and the N4-amino-dCTP-containing fraction were pooled and lyophilized to give the sodium salt of N4-aminodCTP as a white crystalline solid (284 mg, 85%): 1H NMR (600 MHz, D2O) δ 2.22 (m, 1 H), 2.31 (m, 1 H), 3.99 (m, 1 H), 4.10 (m, 2 H), 4.53 (d, J ) 2.7 Hz, 1 H), 5.96 (br s, 1 H), 6.23 (m, 1 H), 7.74 (s, 1 H); 13C NMR (63 MHz, D2O) δ 42.42, 68.19 (d, J ) 5.5 Hz, 1 C), 73.53, 88.81 (d, J ) 9.1 Hz, 1 C), 89.32, 98.51, 146.42, 153.68, 163.84; 31P NMR (121 MHz, D2O) δ -8.01 (d, J ) 48 Hz, 1 P), 10.22 (d, J ) 50 Hz, 1 P), -21.56 (t, J ) 45 Hz, 1 P); ESI-MS m/z 481.0 (M - H); ESI-HRMS calcd for C9H17N4O13P3 (M - H) 480.9927, found 480.9943. Synthesis of 3-[2-(2-{2-[2-(2,4-Dinitrophenylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoic Acid (2). To a solution of 3-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]propanoic acid (0.5 g, 1.89 mmol) and Na2CO3 (0.317 g, 3.77 mmol) in 5 mL of water was added 1-fluoro-2,4-dinitrobenzene (227 mL, 1.89 mmol), and the solution was irradiated in a CEM Discovery microwave synthesizer set at 80 °C (30 W maximum), with cooling for 30 min. After cooling, the solution was acidified with 0.25 M HCl and extracted three times with 20 mL of ethyl acetate. Concentration of the combined organic fractions under vacuum provided a thick yellow oil that was purified utilizing an Isco CombiFlash automated chromatography

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unit fitted with a 40 g silica gel column (MeOH/DCM, 0 to 20% MeOH over 25 min), affording the aniline as a yellow oil (748 mg, 92%): 1H NMR (250 MHz, CDCl3) δ 2.58 (t, J ) 6.0 Hz, 2 H), 3.57-3.84 (m, 18 H), 6.94 (d, J ) 9.5 Hz, 1 H), 8.25 (dd, J ) 9.5, 2.6 Hz, 1 H), 8.81 (br s, 1 H), 9.12 (d, J ) 2.7 Hz, 1 H); 13C NMR (126 MHz, CD3OD) δ 36.0, 44.2, 67.9, 70.0, 71.5, 71.6, 71.71, 71.72, 71.74, 71.77, 116.2, 124.8, 131.1, 131.6, 137.1, 150.0, 175.4; ESI-MS m/z 430 (M - H); TOFHRMS calcd for C17H24N3O10 (M - H) 430.1462, found 430.1441. Synthesis of 3-[2-(2-{2-[2-(2,4-Dinitrophenylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanate-N-hydoxysuccinimide Ester (3). The acid 1 (748 mg, 1.73 mmol) was dissolved in 1.9 mL of anhydrous dichloromethane (DCM); 1.0 M dicyclohexylcarbodiimide (DCC) in DCM (1.92 mL, 1.92 mmol) and Nhydroxysuccinimide (NHS) (221 mg, 1.92 mmol) were added. The solution was blanketed with dry argon and shaken at ambient temperature for 16 h. After the solvent had been removed in vacuo, the residue was taken up in dry DCM and the urea byproduct was removed by filtration. Purification was performed by silica gel chromatography utilizing an Isco CombiFlash automated chromatography unit fitted with a 40 g silica gel column (2-propanol/DCM, 0 to 20% 2-propanol over 25 min) and afforded the active ester as a thick yellow oil (561 mg, 61%): 1H NMR (600 MHz, CDCl3) δ 9.05 (d, J ) 2.6, 1 H), 8.75 (s, 1 H), 8.20 (dd, J ) 9.5, 2.6 Hz, 1 H), 6.94 (d, J ) 9.5 Hz, 1 H), 3.87-3.75 (m, 5 H), 3.71-3.55 (m, 16 H), 2.84 (t, J ) 6.4 Hz, 3 H), 2.80 (dd, J ) 19.3, 7.8 Hz, 4 H), 1.99 (s, 1 H); 13C NMR (63 MHz, CDCl3) δ 25.27, 31.79, 42.96, 65.36, 68.27, 70.10, 70.27, 70.32, 70.38, 114.09, 123.85, 129.89, 130.04, 135.58, 148.19, 166.48, 168.90; ESI-MS m/z 529 (M + H); ESI-HRMS calcd for C21H28N4O12 (M + NH4) 546.20475, found 546.20655. Synthesis of N4-Aminodeoxycytidine 5′-TriphosphatedPEG4-DNP (4). Active ester 3 (124 mg, 0.234 mmol) in 2 mL of dry dimethyl sulfoxide (DMSO) was added to a solution of N4-aminodeoxycytidine 5′-triphosphate triethylammonium salt (173 mg, 0.195 mmol) in 1.5 mL of anhydrous DMSO. The solution was blanketed with dry argon and vortexed until all solids had dissolved. After being stirred for 16 h at room temperature, the reaction mixture was fractionated by HPLC utilizing a Waters Delta 600 HPLC system fitted with a model 2996 photodiode array detector and a Sunfire Prep 10 µ, C18(2), 100A, 250 mm × 50 mm column. Elution with 125 mM triethylammonium carbonate and 5-50% ACN over 60 min at pH 8.5 and a flow rate of 20 mL/min provided the triethylammonium salt of N4-amino-dCTP-dPEG4-DNP. Cation exchange was performed on a 50 mL column packed with Sp Sephadex C-25 equilibrated with 0.2 N NaOH and washed with water until the pH of the eluent buffer was 7.0. The lyophilized triethylammonium salt was taken up in 1 mL (4 × 0.250 mL) of DI water loaded on top of the cation exchange column and eluted with water. The UV-vis spectra of the collected fractions were recorded on an Agilent 8453 UV-vis spectrophotometer, and the N4-amino-dCTP-dPEG4-DNP fractions were pooled and lyophilized to give 145 mg of the tetrasodium salt of N4amino-dCTP-dPEG4-DNP as a yellow crystalline solid: 1H NMR (250 MHz, D2O) δ 2.27 (m, 1 H), 2.36 (br m, 1 H), 2.63 (t, J ) 4.8 Hz, 2 H), 3.85-3.63 (m, 18 H), 4.18 (d, J ) 4 Hz, 3 H), 4.58 (br s, 1 H), 6.19 (br s, 2 H), 7.15 (d, J ) 8 Hz), 7.94 (br s, 1 H), 8.27 (d, J ) 8.3 Hz, 1 H), 9.04 (s, 1 H); 13C NMR (126 MHz, D2O) δ 34.4, 39.8, 42.8, 65.47, 65.51, 66.62, 68.82, 69.94, 70.02, 70.06, 70.10, 70.72, 71.94, 85.98, 86.37, 92.7, 115.4, 124.9, 130.3, 130.8, 135.7, 141.7, 143.6, 149.3, 173.2, 173.6; 31P NMR (121 MHz, D2O) δ -8.39 (d, J ) 41 Hz, 1 P), 10.58 (d, J ) 48 Hz, 1 P), -22.00 (t, J ) 48 Hz, 1 P); ESI-MS

Microwave-Mediated Synthesis of Labeled Nucleotides

m/z 984.1 (M + H); FAB-HRMS calcd for C26H37N7Na4O22P3 (M + H) 984.0795, found 984.0790. Labeling DNA with DNP-PEG4-dCTP. Double-stranded DNA was labeled with DNP-dPEG4-dCTP by nick translation (34). No unmodified dCTP was added to the nick translation reaction mixture. The reaction was stopped when the size of the DNA was reduced to ∼300 bp as judged by agarose gel electrophoresis. The resulting DNA was purified by column chromatography (Qiagen) and precipitation by 2-propanol. The extent of labeling was estimated from optical absorption measurements at 260 and 365 nm. Typically, from ∼1 to 5% of the nucleotides in the resulting DNA was labeled with DNP. Tissue Staining. The BenchMark XT automated slide processing system (Ventana Medical Systems, Inc., Tucson, AZ) was used for to assess performance of the in situ hybridization assay for HER2 and HPV DNA targets. Pretreatments. Automated deparaffinization, pretreatment, hybridization, stringency wash, signal detection, and counterstaining were performed on the BenchMark XT system. Formalin-fixed paraffin-embedded tissue sections on glass slides were baked at 65 °C for 20 min prior to the deparaffinization step with EZ Prep (Ventana) at 75 °C for 16 min. Deparaffinized tissue sections were exposed to a combination of heat treatment with reaction buffer [Tris-based solution (pH 7.6), Ventana] and ISH Protease 2 or ISH Protease 3 (Ventana) to unmask DNA targets. Hybridization and Signal Development. For HER2 gene detection, the INFORM HER2 DNA Probe (Ventana) was applied to the glass slide and the probe and sample DNA were codenatured by incubation at 95 °C for 10 min. Then, the hybridization step was conducted at 52 °C for 2 h. After three stringency wash steps were performed at 72 °C with 2× SCC (Ventana), tissue sections were incubated with monoclonal rabbit anti-DNP antibody (Ventana) for 20 min and then with HRPconjugated anti-rabbit antibody for 16 min at 37 °C. The peroxidase catalyzed silver deposition was developed using the ultraView SISH Detection Kit (Ventana) (36). HER2 probe slides were counterstained with Hematoxylin II (Ventana) for 4 or 8 min and Bluing Reagent (Ventana) for 4 min. For the HPV assays, the DNP-labeled HPV probe was detected using the Ventana ISH iView Blue Plus detection kit on the BenchMark XT system.

RESULTS Synthesis. A novel methodology involving the bisulfitemediated, microwave-promoted hydrazinolysis of cytosine was utilized to efficiently produce nucleotide conjugates suitable for the labeling of DNA via nick translation. The synthesis of one such conjugate, N4-amino-dCTP-dPEG4-DNP, is outlined in Scheme 1. A number of microwave-mediated transamination reactions of deoxycytosine triphosphate (dCTP) with bidentate nitrogen nucleophiles were explored. Of these, the reaction with hydrazine to produce N4-aminodeoxycytidine 5′-triphosphate (N4-amino-dCTP) proved to be the highest yielding and most reproducible. To obtain optimal results, strict control of reaction parameters, including applied microwave power, pH, and temperature, had to be maintained. On a 300 mg scale, under optimal microwave conditions, the hydrazinolysis of cytosine proceeded nearly quantitatively within 30 min. The corresponding thermal reaction produced a 90% yield after irradiation for only 30 min. The corresponding reaction in organic solvent in the absence of microwave irradiation never afforded a >60% yield and required several hours to complete. The synthesis of DNP-dPEG4-NHS was accomplished by conversion of the acid intermediate to the NHS ester via treatment with NHS and DCC in DCM overnight. The crude NHS ester obtained following the removal of the urea byproduct was sufficiently stable to allow for purification on the silica gel. Overnight reaction of the nucleotide (1) and the NHS ester (3) in anhydrous DMSO proceeded smoothly to give the final nucleotide conjugate that was efficiently purified by ion pair chromatography. Ion exchange provided the sodium salt of N4amino-dCTP-dPEG4-DNP that was used in the labeling of DNA. DNA Labeling with DNP-dPEG4-dCTP and Tissue Staining. Double-stranded HER2 and HPV DNA were labeled with DNP-dPEG4-dCTP by nick translation using Escherichia coli DNA polymerase I. The resulting probes were typically labeled at 1-5% of the nucleotide residues. The probes were used to visualize gene amplification of HER2 DNA in HER+ breast cancer and to detect human papilloma virus DNA in cervical tumors. In situ hybridization assays for HER2 and HPV DNA signals utilizing the BenchMark XT automated slide processing system (Ventana) were effective in visualizing the HER2 and HPV DNA targets in tissue. HPV was visualized utilizing a chromogenic ISH protocol, whereas HER2 was visualized via SISH (Figure 1). The observed signals were strong and easily discernible with little or no background (Figure 2).

DISCUSSION Efforts to efficiently produce nucleotide conjugates suitable for the labeling of double-stranded DNA via nick translation have led to a novel methodology involving the bisulfitemediated, microwave-promoted hydrazinolysis of cytosine at the C4 position. By utilizing microwave acceleration, the hydrazine moiety was effectively installed while side reactions were prevented and phosphate hydrolysis was minimized. HPLC analysis of thermally promoted and microwave-promoted reactions indicated that microwave energy accelerates the hydrazinolysis reaction with no effect on the rate of phosphate hydrolysis. This observation allowed the optimization of 4-amino-dCTP formation by employing the minimum effective reaction time. The reaction was shown to be scalable within the capacity limits of the microwave synthesizer. As the scale was increased, slightly longer microwave reaction times were required to affect complete hydrazinolysis. As such, larger scale reactions produced slightly lower yields. There are several examples of nucleotides that have been synthetically modified to contain nucleophilic handles (37, 38). To the best of our knowledge, the methodology that we reported here represents a particularly efficient route for producing such nucleotides. The utility of 4-amino-dCTP as a reagent for the assembly of hapten-labeled nucleotides that can be incorporated into DNA via nick translation has been demonstrated via the synthesis of DNP-dPEG4-dCTP. Nick translation of double-stranded HER2 and HPV DNA in the prescence of DNP-dPEG4-4-aminodCTP consistently produced probes labeled at 3-4% of the nucleotide residues. Visualization of HER2 and HPV DNA targets in tissue via in situ hybridization utilizing the BenchMark

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Scheme 1. Synthesis of N4-Amino-dCTP-PEG4-DNP

XT automated slide processing system (Ventana) consistently provided strong and easily discernible signals with little or no background.

As the percent of labeled nucleotide incorporation increases for a given probe, effects on the DNA melting temperature and hybridization specificity became evident. These phenomena are

Figure 1. Alkaline phosphatase (AP)-mediated chromogenic detection (SISH) and horseradish peroxidase (HRP)-mediated silver deposition detection (SISH) (5).

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an individual patient sample. This last objective is of critical importance in the fast developing area of individualized medicine and diagnostics, which seeks to do away with the cancer paradigms of “trial and error” and “one drug fits all”, replacing them with targeted therapies tailored for the specific genomic profiles of an individual patient’s disease. This has become possible because of the recent advent of multiplexed light microscopy-based in situ hybridization methods, which allow for the visualization of individual copies of genes and amplification states. The use of HER2 as a prognostic and predictive biomarker for the determination of breast cancer patient eligibility for Herceptin (Genentech) treatment exemplifies this emerging approach to personalized cancer diagnosis and therapy.

ACKNOWLEDGMENT We thank Dr. Lidija Pestic-Dragovich for biological tissue staining data and many discussions and Mr. Philip Miller for continuing support of this work. Ventana, BenchMark, INFORM, and UltraView are trademarks of Roche. All other trademarks are property of their respective owners. Figure 2. (a) Detection of HPV DNA in cervical cancer tissues by in situ hybridization with a DNP-labeled HPV DNA probe cocktail followed by immunohistochemical detection of the probe with a rabbit anti-DNP monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-rabbit antibody and Ventana’s IViewBlue Plus Detection kit. The blue nuclear signal indicates the presence of HPV DNA. (b) Detection of HER2 genes in breast cancer cell lines. In situ hybridization with a DNP-labeled HER2 DNA probe was followed by reaction with a rabbit anti-DNP monoclonal antibody that was detected by a peroxidase-conjugated goat antirabbit antibody. Peroxidase-catalyzed silver deposition generated the black spots representing the HER2 genes (Ventana’s INFORM HER2 SISH assay). MCF-7 cells contain the normal two copies of the gene. In BT474 cells, the HER2 gene is amplified as indicated by the many spots in each nucleus.

likely a result of the weakened ability of labeled nucleotides to form stable hydrogen bonds with complementary bases and may also be a function of hydrophobic hapten-hapten interactions, particularly at higher hapten loadings. At ∼7% incorporation of labeled nucleotides, the probe performance suffers as shown by the increased background. If fluorescent derivatives are used, then quenching also becomes significant at high labeling levels. We have found the optimal level of labeled nucleotide incorporation as it relates to the quality of ISH or SISH signals to be in the range of 3-5% of the total nucleotides. The labeling level is typically controlled by adding a proportion of the natural nonlabeled nucleotide to the labeling reaction. The conditions for producing desirable labeling levels are diverse and depend on the particular nucleotide derivative (spacer composition and length, size, and chemical composition of the label being introduced) and the enzyme used for incorporation of the derivative. Many natural enzymes and mutated derivatives of natural enzymes are now in use, especially for PCR-related methods and in DNA sequencing reactions. We used the described derivative to label DNA by nick translation to show that a compound made by this method behaves as expected in DNA labeling and that the resulting probe functions in hybridization assays. We intend to utilize this methodology to assemble a wide variety of hapten-dPEG-amino-dCTP derivatives to be used in the construction of a diverse array of DNA probes. The construction of such a probe library has the potential to facilitate the simultaneous visualization of multiple DNA targets within

Supporting Information Available: Characterization of synthesized small molecules, including 1H NMR, 13C NMR, 31P NMR, and HRMS data. This material is available free of charge via the Internet at http://pubs.acs.org.

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