Bioconjugate Chem. 1997, 8, 89−93
89
TECHNICAL NOTES Characterization of Oligodeoxyribonucleotide-Polyethylene Glycol Conjugates by Electrospray Mass Spectrometry Theodore M. Tarasow,* David Tinnermeier, and Carina Zyzniewski NeXstar Pharmaceuticals Inc., 2860 Wilderness Place, Boulder, Colorado 80301. Received September 26, 1996X
Electrospray mass spectrometry (ESMS) was used to characterize a number of differently functionalized polyethylene glycol-oligodeoxyribonucleotide conjugates. Sample preparation was found to be crucial to obtaining quality data. The resolution and precision of ESMS allowed for the identification of individual conjugates differing in MW by a single ethylene glycol unit with an accuracy of e1 (0.02% of the molecular weight). In addition, ESMS was shown to be valuable in identifying chromatographically unresolvable components of a derivatized polyethylene glycol-oligonucleotide conjugate mixture.
INTRODUCTION
Interest in oligonucleotides as research tools, diagnostic reagents, and therapeutics has increased dramatically. As scientific probes, oligonucleotides and oligonucleotide conjugates have been used in both macromolecular structural research and reaction mechanism investigations. From a commercial perspective, these molecules have shown promise as therapeutic and diagnostic agents (1). Potential oligonucleotide therapeutics include antisense molecules (2, 3), ribozymes (4), and oligonucleotides obtained from the systematic evolution of ligands by exponential enrichment (SELEX) (1, 5, 6). In particular, the SELEX-derived compounds are of intense pharmaceutical interest because of the high affinity and exquisite selectivity demonstrated for a broad range of targets. However, oligonucleotides as drugs suffer from two drawbacks, the first being their susceptibility to hydrolytic and enzymatic degradation and the second being their lack of bioavailability. A general strategy for overcoming these shortcomings has been chemical modification of oligonucleotides such that their inherent stability and bioavailability are increased (3, 7). In addition, oligonucleotides have been modified to facilitate their incorporation into stabilizers and delivery vehicles such as liposomes (8). Oligonucleotide modifications have included conjugation of cholesterol (9, 10), fatty acids (12, 13), polycations (8, 11), proteins (14), and polymers (15, 16) to name a few. Characterization of these molecules is crucial to gaining knowledge about their mode of action and to qualify them as drug candidates. Unfortunately, the process of chemically modifying oligonucleotides often results in multiple reaction products, especially when polymers are involved, making characterization difficult. Most product characterizations have included some change in physical property such as a band shift in gel electrophoresis or a peak shift in high-performance liquid chromatography (HPLC). Although these techniques suggest a modification has taken place, they do not directly allude to the identity of the reaction product or * Author to whom correspondence should be addressed. Telephone: (303) 546-7702. Fax: (303) 444-0672. X Abstract published in Advance ACS Abstracts, January 1, 1997.
S1043-1802(96)00082-1 CCC: $14.00
products. Furthermore, when multiple products are produced, these techniques are often unable to resolve the resulting mixture and identify the individual components. Analytical hurdles such as these were encountered early on in our efforts to produce 5′ polyethylene glycol (PEG)-modified oligonucleotides using standard automated synthetic techniques. Somewhat similar conjugates had been prepared in the past (16); however, our oligonucleotide conjugate targets were designed with longer PEG units than previously described. As a result, new methods were required to prepare these compounds, and improved analytical analysis proved to be key in developing these procedures. Mass spectrometry (MS) offers a characterization technique superior to those discussed above by providing a direct measurement of the molecular weight of the molecule or molecules of interest (17). In addition, one of the many valuable uses of MS has been mixture analysis, making the technique potentially useful for analyzing reaction product mixtures produced by the chemical modification of oligonucleotides. Indeed, two accounts revealed matrix-assisted laser desorption/ ionization mass spectrometry (MALDI MS) to be a useful tool for the characterization of oligonucleotide-PEG conjugates (16, 18). However, the resolution and accuracy of MALDI are limited and decrease significantly with increasing molecular weight (MW). These limitations are especially evident in the MALDI analyses of the PEG-oligonucleotide conjugates where the polydispersity of the PEG polymer results in multiple species with similar molecular weights. These species became less resolved as the length of the oligonucleotide and/or the polymer was increased. Another MS technique which offers superior resolution and accuracy is electrospray mass spectrometry (ESMS). ESMS generates and detects multiple charge states of the sample, which upon reconstruction yields the molecular weights of the species present (19). Because of the mild ionization process and the increased measurable molecular weight range, ESMS has been particularly useful for characterizing large biomolecules (17), such as proteins (20), protein conjugates (21), protein-ligand complexes (22) and oligonucleotides (23, 24). Herein, we report the characterization of a variety of functionalized oligodeoxyribonucleotide-PEG conjugates by electrospray mass spec© 1997 American Chemical Society
90 Bioconjugate Chem., Vol. 8, No. 1, 1997
trometry. Further, we demonstrate the utility of ESMS in analyzing multiple reaction products as a result of the chemical modification of oligonucleotide-PEG conjugates. We also describe good yielding methods for the incorporation of PEG molecules with average molecular weights up to 2000 in oligonucleotide conjugates by automated synthesis using standard phosphoramidite chemistry. EXPERIMENTAL PROCEDURES
General. Monoamino-PEG-2000 (average Mn of ≈2000) and monomethoxy-PEG-2000 were purchased from Shearwater Polymers Inc. (Huntsville, AL) and used as received. All other chemicals were purchased from Aldrich Chemical Co. and used as received unless otherwise specified. HPLC was performed on a Rainin Dynamax HPLC instrument equipped with a Rainin UV D-II dualwavelength detector. Vydac C4 reverse phase HPLC columns were used with triethylammonium acetate (TEAA) at pH 7.0 and acetonitrile (MeCN) as eluents. NMR spectra were obtained on a Bruker AMX 300 instrument. Functionalized PEG. The functionalized PEG molecules used to produce compounds 2-4 were prepared by acylation of monoamino-PEG-2000 with either the N-hydroxysuccinimide (NHS)-activated ester or the pnitrophenyl-activated carbonate of the corresponding functional modification. The derivatized PEG compounds were purified by flash silica gel chromatography and precipitation from tetrahydrofuran (THF) with diethyl ether. The identities of the PEG molecules were confirmed by 1H NMR. The (N-acetylamino)cyclohexadienyl-PEG molecule used to synthesize compound 5 was prepared by addition of amino-PEG to the boron tetrafluoride salt of cyclohexadienyliumiron(0) tricarbonyl with subsequent oxidative elimination of the iron tricarbonyl using cerric ammonium nitrate (T. Dewey, C. Zyzniewski, and B. Eaton, unpublished results). The derivatized PEG molecule was purified and characterized in a manner similar to that described above. PEG Phosphoramidite Synthesis. All steps were carried out under an argon atmosphere using inert atmosphere techniques. THF was freshly distilled from potassium/benzophenone prior to use. The primary amine on the PEG used to prepare compound 1 was protected as the 9-fluorenylmethyl carbamate (fmoc) prior to phosphoramidite synthesis. The derivatized PEG (150 mg, 0.07 mmol) was dissolved in THF (1.5 mL) and added to a solution of 18.2 mg (0.077 mmol) of (2-cyanoethyl)N,N-diisopropylchlorophosphoramidite and 14.7 mg (0.114 mmol) of diisopropylethylamine in THF (0.5 mL). The reagents were allowed to react at room temperature for 30 min, after which 31P NMR indicated that the reaction was complete. Excess phosphoryl chloride as detected by 31P NMR was quenched by the addition of methanol (20 µL). The mixture was then filtered through glass wool and the solvent removed under reduced pressure. The PEG phosphoramidite was used immediately in the automated oligonucleotide conjugation described below. Oligonucleotide-PEG Conjugate Synthesis. All oligonucleotide-PEG conjugates were synthesized on an ABI 392 DNA synthesizer using standard cycles and conditions except for coupling of the PEG phosphoramidite. Reagents, including phosphoramidites, were obtained from Glen Research. All syntheses were performed at the 1 µmol scale using a Pharmacia polystyrene support. The PEG phosphoramidite was dissolved in anhydrous acetonitrile to a final concentration of 50 mM. Coupling of PEG phosphoramidite was achieved using a
Tarasow et al.
modified ABI 1 µmol CE coupling cycle. The coupling time was increased to 45 min, and an additional coupling cycle was inserted prior to capping. Oligonucleotide cleavage from the polystyrene support and deprotection were carried out in concentrated NH4OH at 55 °C for 1216 h. The oligonucleotide-PEG conjugates were purified by HPLC using a 15 to 40% acetonitrile gradient over 20 min. Retention times of the oligonucleotide conjugates varied depending on what functionality was present at the end of the PEG. Due to the degree of polymerization and polydisperse nature of the PEG, conjugates typically eluted as an envelope of moderately resolved peaks. Oligonucleotide-PEG conjugates were collected, concentrated to dryness, dissolved in water, and quantitated by UV spectroscopy (260 ≈ 91 600). Yields were typically 40-60%. These samples were used directly in the MS experiments described below. The alternative route to functionalized oligonucleotide-PEG conjugates 2 and 4 involved reaction of the amino-PEG-oligonucleotide (1) with the NHS ester of either 4-acetobenzoic acid or biotin, respectively. The resulting reaction mixture was HPLC purified as above and subjected to ESMS analysis. In both cases, HPLC was ineffective at separating the desired compounds, 2 and 4, from undesired side products. MS Analysis of Oligonucleotide-PEG Conjugates. Electrospray mass spectrometry was performed on a Fisons Quattro II (Beverly, MA) triple-quadrapole mass spectrometer using the negative ion mode. The first detector was used for mass detection. Nitrogen was used as the nebulizer gas at 20 L/h and the drying gas at 300 L/h. The cone voltage was -30 V, the capillary 2.65 kV, and the HV lens 0.33 kV. The source temperature was maintained at 80 °C. Samples (10 µL) were injected via flow injection using a Rheodyne 7125 (Cotati, CA) manual injector with a 20 µL stainless steel loop. A Harvard apparatus model 22 (South Natick, MA) syringe pump was used to deliver 1:1 methanol/H2O (v/v) containing 0.1% triethylamine at 10 µL/min to the mass spectrometer. All solvents used were HPLC grade and were obtained from Fisher Scientific (Fair Lawn, NJ). Data were acquired at 5 ms dwell, 0.1 step size between m/z 350 and 2000. The spectra were averaged across the entire sample plug, and background was subtracted. The instrument was calibrated using a 2 µg/µL NaI solution. At least three charge states were used to determine the reported weights. The deviation reported is the error associated with the molecular weight calculation using several charge states. Transformation of the raw data was accomplished using MaxEnt, included in the MassLynx software from Fisons. RESULTS AND DISCUSSION
Synthesis of Oligonucleotide Conjugates. A variety of oligonucleotide-PEG conjugates have been prepared in good yields using automated synthesis. The more efficient approach involved preparing the desired monohydroxy-PEG compound, converting it to the phosphoramidite, and coupling it to the oligonucleotide using a modified automated synthesis reaction cycle. This strategy resulted in good yields of HPLC-purified PEG2000 (average Mn of ≈2000)-oligonucleotide conjugates as determined by UV quantitation. Crucial to obtaining reasonable yields were the support, coupling times, and repetitive coupling procedure. Yields were found to be lower with CPG supports than with polystyrene resins. In addition, CPG yields were highly dependent on pore size, with 2000 Å supports giving the best yields. Increasing the coupling time to 45 min and including an
Bioconjugate Chem., Vol. 8, No. 1, 1997 91
Technical Notes Table 1. Functionalized Polyethylene Glycol-Oligodeoxyribonucleotide Conjugates Studied, Their Calculated Molecular Weights, and Their Measured Molecular Weights as Determined by ESMS
n
calculated MW
measured MW
1
50
5323.4
5324.2 ( 0.3
2
50
5469.4
5468.3 ( 0.3
3
43
5139.2
5140.2 ( 0.9
4
43
5241.2
5240.5 ( 0.5
5
43
5135.2
5135.7 ( 0.2
6
42
4986.1
4986.4 ( 0.9
compound
R
additional coupling step were also found to dramatically increase the yield of the conjugate. An alternative, less efficient approach to differently functionalized PEG-oligonucleotide molecules was chemical modification of the PEG after conjugation to the 10mer oligonucleotide. For example, the amino-PEG-200010-mer 1 was modified with various acylating reagents and the resulting functionalized oligonucleotide-PEG conjugate HPLC purified. Modification of the amino group generally resulted in a significant shift in the retention time, thus allowing for facile purification. However, some modifications resulted in unresolvable mixtures, in which case the MS proved invaluable to analysis of the product mixture (vide infra). In all cases, this approach resulted in significantly lower yields compared to yields obtained by modifying the PEG prior to automated oligonucleotide conjugation. ESMS Analysis of Oligonucleotide Conjugates. Representative ESMS spectra for the conjugates listed in Table 1 are shown in Figure 1. The peaks fall into charge state envelopes in which each envelope consists of molecules of like charge and is spread over a range of m/z ratios due to the polydisperse nature of the PEG. The resolution of ESMS is demonstrated by the near baseline separation of peaks within an envelope, where the difference between two peaks represents one ethylene glycol unit. The smaller peaks observed within an envelope correspond to salt adducts of the parent molecule. Sample preparation is crucial to obtaining quality ESMS data. Conjugates should be purified using volatile buffers such as triethylammonium acetate or triethylammonium bicarbonate and should always be dissolved in deionized water. Contaminating cations, particularly sodium and potassium ions, can render a spectrum impossible to assign if present in high enough amounts. Undesired counterions can be exchanged using triethyl-
Figure 1. Representative ESMS spectra of selected oligo conjugates listed in Table 1. All data were obtained under conditions described in Experimental Procedures. Mass envelope charge states have been labeled: (a) compound 1, (b) compound 3, and (c) compound 5.
ammonium buffers in either ion exchange HPLC, reverse phase HPLC, size exclusion chromatography, or dialysis. Reconstruction of Figure 1a yielded the spectrum shown in Figure 2. Like the primary spectrum, the reconstructed spectrum shows the polydisperse nature of the PEG-oligonucleotide conjugate, but it also illustrates the accuracy of the ESMS technique. The molecular weight values listed in Table 1 agree well with the calculated molecular weights. Typically, a difference of less than 0.02% was obtained. Accuracy does not
92 Bioconjugate Chem., Vol. 8, No. 1, 1997
Figure 2. Reconstructed spectrum of Figure 1a. The value of n is indicated for the maximum and minimum number of polyethylene glycol units, as well as for the most abundant species. The peak corresponding to the measured molecular weight reported in Table 1 is also labeled.
Tarasow et al.
5382.2 ( 0.4, Mr-calc ) 5381.4, n ) 48) and bis (Mr-meas ) 5527.4 ( 0.2, Mr-calc ) 5527.4, n ) 48) acetophenone adducts. The synthetic modification of oligonucleotides quite often results in multiple products such as these, and while ESMS does not identify the regiochemistry of modification, it does provide data to more accurately characterize the reaction products. Conjugation of PEG to oligonucleotides has been used to increase hydrolytic stability (16), to provide a flexible linker between oligonucleotides and other molecules (14, 25), and to purify oligonucleotides (26). While the utility of oligonucleotide-PEG conjugation is obvious, the characterization of the reaction products has been incomplete. The polymeric nature of PEG introduces additional analytical hurdles. Mass spectrometry techniques such as MALDI are less than ideal for the analysis of oligonucleotide-PEG conjugates with high degrees of polymerization (n > 30) due to poor resolution between MS peaks (16, 18). We have shown ES-quadrapole MS to be a valuable tool for the accurate and resolute analysis of a variety of highly polymerized, functionalized oligonucleotide-PEG conjugates. We anticipate that this technique will be widely applicable to the analysis of other polymer-modified oligonucleotides as well. In the future, the concept may even be extended to collisioninduced dissociation ESMS/MS experiments (23) in which more detailed structural data of synthetically modified oligonucleotides may be obtained. ACKNOWLEDGMENT
The authors thank Pamela Crain, James McCloskey, Bruce Eaton, and Russ Lehrman for thoughtful discussions and suggestions. LITERATURE CITED
Figure 3. Reconstruction of mixture analysis data showing the mono (M) and bis (B) adduct mass envelopes. The two differently modified conjugates were the result of the derivatization of compound 1 with the NHS ester of 4-acetylbenzoic acid. The measured molecular weights for the n ) 48, mono, and bis adducts have been labeled.
appear to be sequence dependent because ESMS analyses of oligonucleotide-PEG conjugates similar to those reported here, differing only in sequence, have generated equally accurate results (T. M. Tarasow, D. Tinnermeier, and C. Zyzniewski, unpublished results). Furthermore, the resolution of ESMS provides data for the determination of oligonucleotide-PEG physical characteristics such as the range and distribution of PEG chain lengths. For example, using the reconstructed spectrum shown in Figure 2, the number of polyethylene glycol units ranges from 45 to 60 with a maximum around 49 units. These values can be easily determined for each batch of oligonucleotide conjugate to assure consistency in product and to help correlate physical differences between lots. Figure 3 demonstrates the ability of ESMS to deconvolute chromatographically unresolvable mixtures. Modification of the amino-PEG-10-mer (1) with the NHS ester of 4-acetylbenzoic acid generated mono and bis addition products which were not resolvable by HPLC. Although the mass envelopes for the two products overlap, the ESMS-reconstructed spectrum was baseline resolved (Figure 3). The identity of the two components in the mixture could be assigned as the mono (Mr-meas )
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Mass Spectrometry: Electrospray Ionization. Anal. Chem. 62, 882-899. (21) Bennet, K. L.; Smith, S. V., Lambrecht, R. M., Truscott, R. J. W., and Sheil, M. M. (1996) Rapid Characterization of Chemically-Modified Proteins by Electrospray Mass Spectrometry. Bioconjugate Chem. 7, 16-22. (22) Przybylski, M., and Glocker, M. O. (1996) Electrospray mass spectrometry of biomacromolecular complexes with noncovalent interactions-new analytica perspectives for supramolecular chemistry and molecular recognition processes. Angew. Chem., Int. Ed. Engl. 35, 806-826. (23) Ni, J., Pomerantz, S. C., Rozenski, J., Zhang, Y., and McCloskey, J. A. (1996) Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and Tandem Mass Spectrometry. Anal. Chem. 68, 1989-1999. (24) Little, D. P., and McLafferty, F. W. (1995) Sequencing 50mer DNAs Using Electrospray Tandem Mass Spectroscopy and Complementary Fragmentation Methods. J. Am. Chem. Soc. 117, 6783-6784. (25) Durand, M., Chevrie, K., Chassignol, M., Thuong, N. T., and Maurizot, J. C. (1990) Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability. Nucleic Acids Res. 18, 6353-6359. (26) Jaschke, A., Furste, J. P., Erdmann, V. A., and Cech, D. (1994) Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides. Nucleic Acids Res. 22, 1880-1884.
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