Bioconjugate Chem. 2005, 16, 465−470
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Optimized Synthesis of Functionalized Fluorescent Oligodeoxynucleotides for Protein Labeling Charles Goussu,†,‡ Jean-Jacques Vasseur,† Herve´ Bazin,‡ Eric Trinquet,‡ Fabrice Maurin,‡ and Franc¸ ois Morvan*,† Laboratoire de Chimie Organique Biomole´culaire de Synthe`se, UMR 5625 CNRS-UM II, Universite´ de Montpellier II, CC008, Place E. Bataillon, 34095 Montpellier Cedex 5, France, and CIS Biointernational, Marcoule, BP 84 175, 30204 Bagnols/Ce`ze Cedex, France. Received October 18, 2004; Revised Manuscript Received January 31, 2005
Phthalimido-alkanol solid supports were rapidly prepared from solid supported phthalic anhydride and amino alcohol condensation induced by microwaves. These supports were used to synthesize 5′fluorescent 3′-aminoalkyl oligodeoxynucleotides allowing a two-step deprotection necessary to avoid aminolink alkylation. After conversion into an NHS derivative using dissuccinimidyl suberate and an optimized isolation, they were conjugated with a protein.
INTRODUCTION
Although the labeling of proteins with fluorescent dyes is well documented and many prelabeled proteins are commercially available, it appears that the quality of the conjugates is not always satisfactory. The most common labeling method involves a selective acylation of an amino function (reaction of activated esters on -NH2 from lysines). Since several reactive sites are available on a given protein, the reaction leads to a “statistical labeling” (1) resulting in a population of multiple labeled molecules (2, 3). The labeling ratio (number of dyes per protein) usually measured from the UV-visible spectrum of the purified conjugate is a macroscopic value corresponding to the average ratio for the population. Organic dyes such as fluoresceins, cyanines, or Alexa are often preferred since they have a minimum impact on the biological properties (e.g. immunoreactivity for a antibody) and the labeling process is straightforward. The state of the art in immunochemistry is to develop, in a reproducible manner, a conjugate in which the unlabeled protein is largely in the minority and most of the molecules bear two or more labels. This has an important impact on the fluorescent properties of the conjugates, since most of fluorescent dyes undergo fluorescence quenching due to the proximity between two dye molecules (4, 5). Although some sulfonated Cy5 are claimed to yield conjugates presenting a low level of dye dimerization (6) characterized by a reduced hypochromic D-band, we observed a 50% decrease in the quantum yield even at a dye/protein ratio of two for the labeling of IgG with commercial Cy5 N-hydroxysuccinimidyl (NHS) ester, following the recommended protocol (Amersham Biosciences) as previously observed by others (7) who reported that for a ratio of 2 to 3 Cy5 labels per IgG, some moderate fluorescence was obtained and IgG bearing six Cy5 label was almost nonfluorescent. Furthermore, overlabeling should be avoided since it also has a negative impact on the biological activity (binding constants) of macromolecules. * Corresponding author. Telephone +33/ 467 144 961, fax +33/ 467 042 029, e-mail
[email protected]. † Universite ´ de Montpellier II. ‡ CIS Biointernational.
For all these reasons, it is highly desirable to find a way to avoid any detrimental interaction between individual fluorophores coupled to the same molecule. A few methods are known to ensure the controlled labeling of native proteins as antibodies, one possibility being the use of cyclodextrin-dye complex which allow multiple labeling with minimum fluorescence quenching (8). An alternative is the use of a fluorescent dye substituted by an oligonucleotide (ODN) moiety bearing a reactive linker enabling the conjugation of this probe to a target protein (9). For this unconventional application of ODNs, one needs is an efficient way to obtain doubly modified ODN bearing for example a Cy5 dye on its 5′-end and an aminoalkyl linker on its 3′-end. For routine labeling, these doubly modified ODNs should be readily available and it should be confirmed that the amino group is indeed fully unprotected and free of side-products. A linker bearing a primary amino group was chosen since it is the most versatile and can be converted into many reactive groups used in immunochemistry (e.g. NHS ester, maleimide, thiol, etc.) through the use of a panoply of homobifunctional reagents (e.g. dissuccinimidyl suberate, DSS) (10) or heterobifunctional reagents (e.g. succinimidyl trans-4-(N-maleimidylmethyl)cyclohexane1-carboxylate: SMCC) which are widely used in immunochemistry and are commercially available. ODNs with 3′-aminoalkyl arms are conveniently prepared through standard solid-phase synthesis on a 3′amino-protected support which releases the free 3′-amino during the final deprotection (11-15). The 3′-amino support based on an immobilized 3-nitro-6-succinyloxymethylbenzoyl group (15) or an immobilized phthalimido protective group (16) is particularly attractive since it releases an enantiomerically pure ODN (as compared to some amino links (12) which give rise to resolvable diastereoisomers). Unfortunately, the former was synthesized with a very low yield from 6-nitrophthalide and O-(4,4′-dimethoxytrityl)-1-hexanol-6-amine. A solid support from phthalimido groups exhibits two main advantages: it cannot react with acetic anhydride during the capping step of the elongation process, and furthermore, the cyanoethyl groups can be removed from the support before the phthamilido cleavage, avoiding the reaction
10.1021/bc049752z CCC: $30.25 © 2005 American Chemical Society Published on Web 02/24/2005
466 Bioconjugate Chem., Vol. 16, No. 2, 2005
Technical Notes
Figure 1. Crude HPLC profiles of (A) T6-(CH2)6-NH2 and (B) T15-(CH2)3-NH2 on column C18 nucleosil, gradient 5 to 36% acetonitrile in 50 mM TEAAc in 20 min. Table 1. Reagents and Conditions for the Preparation of Phthalimido Alkanols Solid Support 3a-c Using Microwaves entry
reagents
time (s)
temperature or watt
1 2 3 4
2+ NH2(CH2)6OH 2+ NH2(CH2)6OH 2+ NH2(CH2)2OH 2+ NH2(CH2)3OH
120 (6 × 20 s) 150 150 150
650 W 200 °C 150 °C 150 °C
of acrylonitrile with the free amine (17). Unfortunately, this support is expensive and its preparation requires three steps, with a crystallization and a chromatography step, to obtain the p-nitrophenyl active ester in 47% yield after which then reacts poorly with LCAA CPG (long chain aminoalkyl-controlled pore glass) to give a loading of only 20 µmol/g (16). We developed a very convenient, rapid, and efficient synthesis of phthalimido alkyl alcohol solid supports. All the reactions were performed on the solid support; hence, workups consisted of only filtrations and washes. Several free alkyl alcohols reacted on the immobilized phthalic anhydride to give the corresponding solid supports with high loading (∼60 µmol/g). Indeed, the hydroxyl function does not need to be protected since the reaction is chemoselective. From the solid support, 5′-fluorescent 3′alkylamino ODN was synthesized, and by an optimized procedure it was conjugated with an antibody. EXPERIMENTAL SECTION
Measurement of Cy5 Dye Concentrations. For sulfonated Cy5 dye and conjugates, the concentration were measured from the absorbance values at λmax assuming a molar extinction coefficient of 250 000 M-1 cm-1 according to the product data sheet (Amersham Biosciences Cy5 monoreactive dye # PA25001) and literature (18). For nonsulfonated Cy5 dye linked to oligonucleotides, the concentrations was measured at λmax assuming a molar extinction coefficient of 250 000 M-1 cm-1 according to the product data sheet (Amersham Biosciences Cy5 phosphoramidite). Preparation of Phthalimido Alkyl Alcohol Solid Supports. One gram of LCAA-CPG (Sigma), pretreated for 2 h with 3% TCA in CH2Cl2 (19), was gently shaken with 63.2 mg (0.3 mmol) of 1,2-anhydrotrimellitic chloride and 105 µL (0.6 mmol) of diisopropylethylamine in 5 mL of CH2Cl2 for 3 h. The resin was filtered off, washed with CH2Cl2 and ether, and dried under vacuum. The resin was treated for 2 h with 10 mL of a mixture of Ac2O, pyridine, THF (1:1:8, v/v/v) (Cap A) and 10% N-methylimidazole in THF (Cap B). The resin was filtered off, washed with CH2Cl2 and ether, and dried under vacuum. The resulting resin was mixed with 0.3 mmol of amino alcohol in 5 mL of DMF and heated in a microwave synthesizer (Discover from CEM Corporation) set at 300 W and 200 °C for 150 s, or in a domestic MW oven at 650 W for 6 × 20 s with a cooling of the mixture between
solid support & loading 3a 3a 3b 3c
49 µmol/g 62 µmol/g 67 µmol/g 59 µmol/g
two runs. The resin was filtered off, washed with CH2Cl2 and ether, and dried. Approximately 20 mg of the solid support was poured into a column, and then a standard thymidine phosphoramidite was incorporated and detritylated. The loading was determined at 498 nm by the assay of the dimethoxytrityl cation released. (Table 1). Synthesis of 5′-Cy5,3′-Aminoalkyl Oligonucleotides. From the phthalimido alkyl alcohol solid support (1 µmol), the ODN was synthesized on a ABI 394 DNA synthesizer using standard reagents and 15 equiv of commercially available phosphoramidites. A standard elongation cycle was applied except that the first detritylation step was omitted since the solid support is hydroxyl free. Then, the Cy5 phosphoramidite (Amersham Biosciences) was incorporated using a coupling step of 120 s to ensure optimum coupling. Next, a treatment with a solution of 10% piperidine in CH3CN was applied for 10 min to remove the cyanoethyl groups. After washes with CH3CN, the supported ODN was treated overnight with aqueous concentrated ammonia in the dark. Ammonia was removed by evaporation under reduced pressure to yield a dark blue solution. Finally, the fluorescent aminoalkyl ODN was purified by HPLC on a Delta Pak column (Waters) 15 µm, C18, 100 Å, 7.8 × 300 mm, using a gradient of CH3CN in 50 mM TEAAc, pH 7, 2 mL/min (Figure 2). UV-visible spectrum for Cy5-T15-(CH2)6 NH2 (0.1 M phosphate buffer, pH 7) λmax ) 265 600 (sh), 646 nm, A265/A646 ) 0.639, A280/A650 ) 0.417, A600 /A650 ) 0.38. MALDI-TOF Mass Spectrometry. MALDI-TOF mass spectra were recorded on a Voyager DE mass spectrometer (Perseptive Biosystems, Framingham, MA) equipped with an N2 laser (337 nm). MALDI conditions are as follows: laser power 2400 (arbitrary units); accelerating voltage 24 000 V; guide wire 0.05% of accelerating voltage; grid voltage 94% of accelerating voltage; delay extraction time 550 ns and 100 scans averaged. All reported spectra here were obtained in negative ion mode and were not smoothed. THAP (trihydroxyacetophenone) as matrix; T6-(CH2)6 NH2 m/z for C66H92N13O43P6: Calcd 1941.38; Found 1941.82; T15-(CH2)3NH2 m/z for C153H203N31O106P15: Calcd 4637.08; Found 4633.78; Cy517mer-(CH2)6 NH2: m/z for C204H258N79O98P18: Calcd 5941.33; Found 5942.40. Labeling of Antibody with Reference Cy5 Dye. The antibody to be labeled was dialyzed against pH 9 0.1 M carbonate-bicarbonate buffer and the concentra-
Technical Notes
Bioconjugate Chem., Vol. 16, No. 2, 2005 467
Figure 2. HPLC profiles at 260 and 660 nm of purified Cy5-GCG AAA AAA AAA AGC TC-(CH2)6-NH2, UV/visible spectrum and MALDI-TOF mass spectrometry.
tion adjusted to 1 mg/mL. The NHS-activated Cy5 dye (‘to label 1 mg of protein” Amersham Biosciences kit) was dissolved in DMSO, and several labeling reactions were performed, varying the number of equivalents of Cy5 added. After 30 min, the reaction mixture was separated by gel filtration over SD 200 in an HR10/30 column (Amersham Biosciences), eluting at 1 mL/min in 0.1 M phosphate buffer, pH 7. The protein concentrations in the void volume were calculated from A280 according to 280 ) 210 000 M-1 cm-1 for the antibody, taking into account the contribution of the Cy5 dye at 280 nm (0.05 × A650) according to the manufacturer’s instruction and literature (18). The dye over protein ratio (D/P) and the A600/A650 ratio were calculated as previously described (7). Conjugation of Cy5-oligonucleotides with Antibody. Starting with the 5′-Cy5 labeled ODN bearing a 3′ aminolinker (10 nmol) in 12 µL of 50 mM N-(3sulfopropyl) morpholine (MOPS) pH 7.0, we added 4 µmol of DSS in 28 µL of DMF (Fluka), and the mixture was kept under vigorous mixing (Eppendorf vortexer) either at 20 °C for 5 h or at 4 °C for 16 h. The conversion into active ester was monitored by RP-HPLC for example the 5′-Cy5-(dT)15-3′-hexylamine (Rt ) 15 mn) gave the corresponding NHS-suberate derivative (Rt ) 16.1 mn). Column Lichrospher RP-18 E 5 µm 125 × 4 mm, elution 1 mL/min, gradient 9.5% to 50% of CH3CN in 25mM TEAAc in 30 min. The reaction mixture was then loaded on a NAP 5 column (Amersham Biosciences) equilibrated in 10 mM MOPS pH 6.0 (4 °C) to remove the excess DSS, and the exclusion fraction (1 mL) collected in a 2 mL eppendorf was then extracted by 1 mL of n-Butanol (vortexing, centrifuging and pipeting off the upper phase). The process was repeated three times and a fourth time after the addition of 1.5 mL n-Butanol and 2 min centrifuging (14 000 rpm) the Cy5-ODN-NHS ester was precipitated as a blue spot. The supernatant was removed and the precipitate was dried under vacuum (5 min, speed-Vac). This Cy5-ODN-NHS ester can be kept dried at -80 °C (or even -20 °C) for a few days with limited decomposition. The antibody (300 µg, 2 nmol) was added to the Cy5-ODN-NHS in 35 µL of 0.1 M sodium carbonate buffer pH 9, the reaction mixture was kept at room temperature for 30 min and then loaded on an HR10/30 Superdex 200 column (Amersham Biosciences). The
elution was carried out with 0.1 M phosphate buffer pH 7, the fastest blue band (tR ) 10 min) containing the conjugate was collected and was well resolved from the excess of Cy5-ODN (tR ) 17 min). An initial Cy5-ODNNHS/IgG ratio of 4 gave a conjugate (62% yield) for which the UV-visible spectrum (A650 ) 0.286, A600 ) 0.103, A280 ) 0.236) indicated the conjugate contained an average of two cyanines (Cy5) per antibody. An initial Cy5-ODNNHS/IgG ratio of 12 gave a conjugate (49% yield) for which the UV-visible spectrum (A650 ) 0.463, A604 ) 0.168, A280 ) 0.283) indicates that the conjugate contained an average of five cyanines (Cy5) per antibody. The contribution at 280 nm due to the Cy5-T15 moiety is 0.435 × A650. For long-term storage, 0.1% BSA (Sigma Bovine Serum Albumin “protease free”) was added as stabilizer, and aliquots were stored at -20 °C. The conjugates (ca. 150 pmol) were analyzed on a Superdex 200 column connected to a diode array detector, and a single peak was detected for the conjugates in both conditions (tR ) 12 min for 5(Cy5-T15)-Ab and tR ) 12.7 min for 5(Cy5-T15)-Ab), the native antibody being eluted at tR ) 13.6 min. The conjugates were well resolved from the excess of hydrolyzed Cy5-ODN labeling reagent, so the A650/A280 ratio could be used with confidence to assess the average number of Cy5 moieties covalently bound to the antibody. Furthermore, analysis of the conjugates by size exclusion chromatography showed a single species absorbing at 650 nm, and although the population of labeled antibodies was not resolved, the retention time (centroid of the population) had a tendency to become shorter when increasing the number of Cy5 moieties bound to antibodies. Fluorescence Measurements. All measurements were performed in 0.1 M phosphate buffer, pH 7, at 20 °C. The Cy5-T15 used as reference and the two conjugates 2(Cy5-T15)-Ab and 5(Cy5-T15)-Ab obtained above were diluted (triplicates) to 100 nM chromophore concentration (A650 ) 0.025) according to the A650 measured for the mother solutions. The fluorescence spectra were recorded on a LS50 fluorimeter, the excitation being set at 640 nm (10 nm slit), and scanning at 650-750 nm (5 nm slit). For each compound, the acquired spectra (triplicates) were averaged.
468 Bioconjugate Chem., Vol. 16, No. 2, 2005
Technical Notes
Scheme 1. Synthesis of Phthalimido Alkyl Solid Supporta
a Reagents and conditions: i: LCAA CPG, 1,2-anhydrotrimellitic chloride, CH Cl , iPr EtN 3 h; ii: CAP A: Ac O, pyridine, THF 2 2 2 2 (10/10/80) + CAP B: 10% N-methylimidazole, THF 2 h; iii: amino alcohol, DMF, MW 150 s; iv: classic ODN synthesis; v: 10% piperidine/ CH3CN 10 min; vi: aq NH3 16 h rt.
RESULTS AND DISCUSSION
Since solution-phase synthesis requires time-consuming workup and purification, we developed a strategy on solid support using only commercially available reagents. Thus, the preparation of the phthalimido solid supports was convenient, easy, inexpensive, fast, and also versatile since any aminoalkyl alcohol or amino-PEG-alcohol (e.g. 2-(2-aminoethoxy)ethanol)) could be anchored. This method was applied to the preparation of phthalimido-ethyl, -propyl, and -hexanyl alcohol solid supports. Finally, using the phthalimido-hexanol solid support, a 5′fluorescent 3′-amino ODN was synthesized, converted into an NHS derivative, and then conjugated with an antibody. The solid supports 3a-c were prepared in two steps (Scheme 1). The first step was the reaction of 1,2anhydrotrimellitic acid chloride 1 (0.3 mmol) on CPGLCAA (19) (1 g) to yield the phthalic anhydride CPG 2 within 3 h (14, 20). The resin was filtered off, washed, and dried. Unreacted amines were capped with commercial Cap A and Cap B solutions for 2 h. The resin was filtered off, washed, and dried. The key step was the formation of a phthalimido function which was performed under microwave irradiation (MW). It should be noted that a partial reaction yielding an amide linkage must be avoided since its hydrolysis under basic conditions is very long (t1/2 ) 17 500 h at 0.1 M NaOH at 30 °C) (21). Reactions of phthalic anhydride with amine occur with or without solvent by conventional heating (175-225 °C) (16) or by MW (22, 23). Note that the reaction is chemoselective since it occurs specifically with the amino moiety. In addition it was reported that reaction of phthalic anhydride with 6-amino-1-hexanol is faster when it occurred by MW than under conventional heating (23). Theoretically, since 6-amino-1-hexanol is a solid with a low melting point (56-58 °C), it could be used without solvent. In our case, to optimize the interaction between the solid supported phthalic anhydride and the 6-amino-1-hexanol, and later with the other alkylamino alcohol, DMF was selected as solvent. Reactions were performed with 0.3 mmol of alkylamino alcohol per gram of solid-supported phthalic anhydride 2 in 5 mL of dry DMF. The reaction was first performed using a domestic MW oven (650W full power) by 6 × 20 s with a cooling of the mixture between two runs (to avoid bumping and spillage), yielding the expected solid support 3a with a fairly high loading of 49 µmol per gram of resin (Table 1, entry 1). Initially, the loading was determined by dimethoxytritylation of a support sample followed by its detritylation and assay of the trityl cation at 498 nm. This
method was deemed inefficient, because it was timeconsuming and poorly reproducible. Thus, a coupling with commercial thymidine cyanoethyl diisopropylphosphoramidite was preferred, and the loading was determined from the cation released during a detritylation step. This method is fast and gives reproducible values. The reaction was also performed in a sealed tube using a microwave synthesizer with the temperature set at 200 °C, 300 W for 150 s (Table 1, entry 2). Next, the reaction was extended to ethanolamine and 3-amino-1-propanol, setting the microwave synthesizer at 150 °C, 300 W for 150 s (Table 1, entries 3 and 4). For all the solid supports, a similar loading was obtained around 60 µmol/g, demonstrating that reaction is fast and efficient. The use of a microwave synthesizer for the preparation of the phthalimido-alkanol solid support is convenient and gives reproducible results and high loading. However, this solid support could also be prepared using a common domestic MW oven with a slightly lower but still high loading (49 µmol/g). Using this procedure, all the steps were performed on solid supports that allow rapid and easy workups. The use of MW speeds up the reaction, and several new solid supports were prepared in less than 1 day using inexpensive, commercially available reagents. To evaluate the properties of the resins, a few short ODN T6-NH2 were synthesized on these various supports (Figure 1A), and for each the same trend was observed in the HPLC profiles. Longer ODNs were synthesized as T15-(CH2)3-NH2 (Figure 1B) and also a hetero ODN [5′Cy5-d(GCG AAA AAA AAA AGC TC3′-(CH2)6-NH2)], labeled with the cyanine Cy5 on its 5′ position (Figure 2). The syntheses were carried out on a 1 µmol scale using 15 equiv of phosphoramidite and regular reagents on an ABI 394 DNA synthesizer. The first detritylation step was omitted. The average yield was better than 98.5%. ODNs were deprotected by a two-step procedure. First, cyanoethyl groups were removed by a piperidine treatment (24) (10% piperidine in acetonitrile for 10 min at room temperature and washing) to remove the resulting acrylonitrile that could further react with the amino function (17). Then, on the partially deprotected ODN still attached to the CPG, a treatment with concentrated ammonia at 55 °C for 17 h for unlabeled ODN or 16 h at room temperature in the dark for the Cy5 ODN yielded the fully deprotected 3′-amino ODNs in solution (Figures 1 and 2), which were purified by HPLC. ODNs were characterized by MALDI-TOF MS (see Experimental Section). The activation of amine-tethered ODN by homobifunctional reagents such as DSS (dissuccinimidyl suberate)
Bioconjugate Chem., Vol. 16, No. 2, 2005 469
Technical Notes Scheme 2. Conjugation of Fluorescent Alkylamino ODN to Antibodya
a Reagents and conditions: i: 50 mM MOPS buffer, pH 7, DMF; ii: antibody, NaHCO3 pH 9.
Table 2. A600/A650 Ratios of Cy5 Dye-Antibody Conjugates Cy5/Ab ratio A600/A650
a “free” Cy5-ODN molecule in which the cyanine dye was in a monomeric form. For a conjugate containing an average of five Cy5-ODN moieties per antibody, we observed that the A600/A650 ratio was 0.37; again, this value was close to the one observed for the monomeric Cy5. The measured fluorescence intensity was I665 ) 140.9 ( 4.3 a.f.u. (arbitrary fluorescence units) for the 2(Cy5-T15)-Ab conjugate, I665 ) 141.1 ( 3.8 a.f.u. for 5(Cy5-T15)-Ab, and I665 ) 163.1 ( 3.8 a.f.u. for the reference Cy5-T15 oligodeoxynucleotide. These measurements performed at a constant chromophore concentration (100 nM) showed that the relative quantum yields of the Cy5 moieties were almost the same for both conjugates and only about 15% lower than compound Cy5-T15 taken as reference. This shows that using the new labeling strategy results in no loss of fluorescence efficiency, by increasing the number of bound Cy5-T15 units.
0.73
1.32
2.64
3.56
5.40
CONCLUSION
0.353
0.394
0.490
0.592
0.613
We developed a convenient, easy, inexpensive, fast, and versatile protocol for the preparation of phthalimido alkanol solid supports used for the synthesis of 3′ aminoalkyl-ODNs which can be labeled on their 5′ position with classic fluorescent dyes or other tags. This was exemplified on the 5′-Cy5-labeled ODN. After release from the solid support, the Cy5-labeled ODN was converted into an activated NHS ester and was conjugated with an antibody. By adjusting the molar excess of the reactive Cy5-ODN, conjugates exhibiting a dye-to-protein ratio up to five were isolated. Using this labeling strategy and in contrast with classical Cy5 antibody labeling, no decrease of the fluorescence efficiency was observed by increasing the number of dyes per antibody. This should ensure a higher brightness using the conjugates, for example as acceptors in fluorescent resonance energy transfer (FRET)based immunoassays.
a
For free sulfonated Cy5 the A600/A650 ratio is 0.315 (0.1 M phosphate buffer pH 7).
(10) or EGS (ethylene glycol-bis-succinic acid NHS ester) (25) is reported for the preparation of ODN-enzyme conjugates. However, the protocols described were not found to be satisfactory since the diafiltration process to remove reagent excesses was too slow, and extensive breakdown of the NHS ester was observed by HPLC monitoring. DSS was preferred (Scheme 2) since it is less reactive than EGS. It therefore leads to a more stable and storable ODN-NHS ester, and, as an intermediary, allows conjugation to proteins in a short time at pH 9. Thus, the Cy5-ODN-NH2 in DMF and MOPS buffer, pH 7, was reacted with DSS. The isolation protocol combines the use of a fast size exclusion column and simple n-butanol extractions to yield an Cy5-ODN-NHS ester in a dry state allowing its storage and later use. The overall process can be easily scaled up and is highly reproducible. Finally, the Cy5-ODN-NHS ester was conjugated with antibody (Ab) in a pH 9 sodium carbonate buffer, and the Cy5-ODN-Ab conjugates were isolated by size exclusion chromatography. As reference compounds we prepared several conjugates using commercially available sulfonated Cy5 dye, for the free Cy5 dye in phosphate buffer we observed a A600/A650 ratio of 0.315 which is in agreement with previous data (7), and as shown in Table 2 this ratio increased with the number of Cy5 dyes bound per antibody as previously reported (7). In contrast with the appearance of a higher 600 nm absorbance, increasing with the number of bound Cy5 dyes observed for antibodies labeled with sulfonated Cy5 and the concomitant loss of fluorescence efficiency (7), in the labeling strategy presented here we do not observe such a phenomenom. Ideally, the spectrophotometric properties of the dye in Cy5-ODN-antibodies should be compared with the nonsulfonated dye; unfortunately, this one is only available as a phosphoramidite and is not soluble in aqueous buffers, therefore the Cy5-ODN which is the closest related compound available is used as reference. The UV-visible spectrum of the commercial Cy5-phosphoramidite in acetonitrile showed a λmax ) 644 nm which is close to λmax ) 645 nm observed for the Cy5ODNs in water or buffer; for the Cy5-phosphoramidite, the A600/A650 ratio was 0.35 in acetonitrile solution. The UV-visible spectra of a conjugate containing two Cy5ODN moieties per antibody displayed a A600/A650 ratio of 0.36 which is very close to the value of 0.38 measured on
ACKNOWLEDGMENT
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