Synthesis and Characterization of Conjugates Formed between

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Bioconjugate Chem. 1994, 5, 436-444

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Synthesis and Characterization of Conjugates Formed between Periodate-Oxidized Ribonucleotides and Amine-Containing Fluorophores Ronald E. Hileman, Kay Martin Parkhurst, Naba K. Gupta, a n d Lawrence J. Parkhurst* Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304. Received January 19, 1994@

The synthesis and purification of new fluorescently labeled derivatives of GDP and ATP are described. The fluorescent groups are coupled initially through amine-containing linker arms to periodate-oxidized nucleotides. Reduction of the initial product yields primarily a six-membered morpholine-like ring. Fluorescein-labeled GDP, rhodamine-labeled GDP, and fluorescein-labeled ATP were characterized by absorbance spectroscopy and TLC. NMR and FAB-MS studies were carried out on a single nucleotide derivative formed by reacting periodate-oxidized guanosine and benzylamine with subsequent reduction to establish the modification to the ribose moiety. The synthesis of the guanosine-benzylamine conjugate led to a mixture of products that were separated. The predominant product (70%) resulted in conversion of the ribose moiety to a six-membered morpholine-like ring having no hydroxyl group, and the minor product (30%) resulted in an open ring structure having one hydroxyl. When NaCNBH3 was used as the sole reductant, only the product with the morpholinelike ring was formed. These probes were prepared for use in solution studies of the interactions of eukaryotic initiation factor-2 with other components of mammalian protein synthesis initiation.

INTRODUCTION

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A

Fluorescent dyes have been used extensively as probes to study the microscopic environment of proteins ( 1 -3). In mammalian protein synthesis, the protein eIF-2I plays a n essential role in initiation. Its function has been characterized using radioactive filter assays ( 4 , 5 ) , and it is known to be required for both ternary (eIF-2GTP.MettRNApt) and 405 complex (40S.mRNA.Met-tRNApt) formation. Our interest in monitoring the behavior of eIF-2 in solution and in quantitating the interaction of eIf-2 with other components of the protein synthesis initiation process prompted our attempts to label eIF-2 directly with the 5(and 6)-N-hydroxysuccinimidylester of rhodamine. This labeling resulted in inactivation of the eIF2. Since eIF-2 is known to bind GDP and had been reported to bind ATP with high affinity (6),we opted to attach a dye to these nucleotides and label the eIF-2 indirectly. Previous investigators had reported that modification of the ribose has no apparent effect on the binding of these nucleotides to various proteins. For example, the 2’,3’-0-(2,4,6-trinitrocyclohexadienylidene)adenosine derivatives have been characterized to act as substrates for adenosine deaminase, alkaline phosphatase, adenylate kinase, myosin subfragment 1, and gastric H+,K+-ATPase(7-10). The natural product guanosine 3’,5’-bispyrophosphate has been implicated to act as a competitive inhibitor for enzymes involved in prokaryotic protein synthesis, in both the initiation and

* Author to whom correspondence should be addressed. Abstract published in Advance ACS Abstracts, August 1 , 1994. Abbreviations: GDP*F, fluorescein-labeled GDP; GDP*FH, fluorescein hydrazine-labeled GDP; ATP*F, fluorescein-labeled ATP; GDP*R, rhodamine-labeled GDP; oxG, periodate-oxidized guanosine; oxGDP, periodate-oxidized GDP; oxATP, periodateoxidized ATP; GBA, the adduct(s) formed from the synthesis using oxG and benzylamine; GDP-BA, the adduct formed between oxGDP and benzylamine; eIF-2, eukaryotic initiation factor-2; Tris, tris(hydroxymethy1)aminomethane. @

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Figure 1. Previously proposed structures formed by the reaction of a periodate-oxidized nucleotide with (A) an amine with subsequent reduction and (B) a hydrazine without subsequent reduction.

elongation steps (11, 12). Moreover, periodate-oxidized guanine nucleotides have been shown to bind to the prokaryotic elongation factor G (EF-G) and the 70s ribosome (13). GTP labeled by dansyl through the 2’ (and 3‘) ribose hydroxyl has been shown to bind to eIF-2 and histone H1 proteins ( 1 4 ) . Formation of Schiff base adducts by the reaction of periodate-oxidized ribonucleotides (or polyribonucleotides) with amines (or hydrazines) has been previously reported (15-17). No structural studies were carried out to clarify the modification a t the ribose ring. On the other hand, Khym (18) reported that the reaction of methylamine with periodate-oxidized adenosine and subsequent borohydride reduction yielded a 6-membered morpholine derivative with a hydroxyl group a t the 2’ position (Figure 1A). Girshovich et al. (19)proposed an alternative structure having a hydroxyl group at both the 2‘ and 3’ positions when periodate-oxidized GTP and 2-nitro-4-azidobenzoylhydrazineare reacted in the absence of reducing agents (Figure 1B). Hansske et al. (20) also proposed that a morpholine-like structure having 2’and 3’-hydroxyl groups resulted from the reaction of periodate-oxidized adenosine or adenosine monophosphate with carboxylic acid anhydrazides in the absence of reducing agents (Figure 1B). We report here the synthesis of GDP- and ATPfluorescein (GDP*F1and ATP*F, respectively) and GDPrhodamine (GDP*R)prepared by reducing the Schiff base 0 1994 American Chemical Society

Fluorescent Probes

formed in the reaction of the 2’,3’-dialdehyde of a periodate-oxidized nucleotide with amino derivatives of the dyes. In order to understand the structure of the labeled nucleotides, specifically, the nature of the ribose ring following the incorporation of the dye derivative, we synthesized a guanosine benzylamine derivative (GBA). The synthesis of this material was entirely analogous to that for the labeled nucleotides but gave NMR and mass spectra that were significantly simpler to interpret.

5ioconjugate Chem., Vol. 5, No. 5, 1994 437

Fluorescence anisotropy measurements were made using the method of Meuser and Parkhurst (24), a modification of the Desa and Wampler method (22)with modulation of the exciting light. The same fluorimeter described above was used with a Lexel Model 75 argon ion laser (Palo Alto, CA) with 488-nm light as the excitation light source. A photoelastic modulator (Model 01 01 PEM 80, Hinds International, Inc., Portland, OR) was positioned between the laser and the cuvette in order to modulate the excitation light (24). The laser power EXPERIMENTAL PROCEDURES supply was set between 5.5 and 6.5 A. For each emission anisotropy measurement 100 data Materials. The reactive fluorophores purchased from points were collected in 1 s, first for unmodulated Molecular Probes (Eugene, OR) were 5-((2-aminoethyl)excitation, followed immediately by modulated excitation, thioureidyl)fluorescein2 (catalog no. A-458), 5-((2-((carwith the emission monochromator set a t 518 nm for bohydrazinomethy1)thio)acetyl)amino)fluorescein ((2-3561, fluorescein or 575 nm for rhodamine. The PEM 80 dial and Xand 6)-((N-(5-aminopentyl)amino)carbonyl)tetsetting was 595.4 nm (retardation 1.2227 radians, since ramethylrh~damine~ “rhodamine cadaverine” (A-1318). the instrument setting corresponds to , Iin nm for n Periodate-oxidized GDP, ATP, and guanosine, as well as retardation). Each set of points was fit by linear regresNaCNBH3, trichloroacetic acid, NjV-dimethylformamide, sion analysis; the last point of the fitted line was recorded sephadex G-25 superfine, DEAE-sephacel, and the coloas the emission intensity for the unmodulated reading, rimetric inorganic phosphorus determination kit were and the first point of the fitted line was used for the from Sigma (St. Louis, MO). Silica gel 60 f254 TLC plates second, modulated, case. The very slight effect of phowere from EM Science (Darmstadt, Germany), extra fine tobleaching of the fluorescein by the exciting light was Biogel P-2 was from BioRad (Richmond, CA), and NaBH4 thus essentially eliminated. The cuvette holder was and benzylamine were from Aldrich Chemical Co. (Milthermostated a t 15 “C (Lauda K-2R constant temperawaukee, WI). All other materials used were reagent ture circulating bath). Typically, 250 pL of the fluoresceingrade or better. labeled material was thermally equilibrated in the Buffers used: buffer A, 10 mM TrisCl, pH 8.0; buffer cuvette, and three to five anisotropy measurements were B, 20 mM TrisC1, pH 7.8, 100 mM KCl, 5 mM p-mertaken, recorded, and averaged. The corrections to the captoethanol, and 10% (v/v) glycerol; buffer C , 50 mM anisotropy due to scattering were calculated as described TrisC1, pH 7.8, 100 mM KC1, 1 mM MgC12, 5 mM earlier (14)and were found to be negligible (less than P-mercaptoethanol, and 10% (v/v) glycerol. 0.001). Instrumentation and Data Analysis. All purifica( 2 ) Synthesis of Fluorescein-Labeled GDP. (A) A modition was done using a n FPLC system that consisted of fication of the method of Ingham and Brew (22),which an LCC-500 Plus gradient programmer, two P-500 pumps, describes a method for labeling the periodate-oxidized P-1 peristaltic pump, UV-1 monitor, FRAC-100 fraction sialic acid moiety of glycoproteins with dansylethylenecollector and REC 481 chart recorder (Pharmacia, Uppdiamine, fluoresceinamine, or dansylhydrazine, was used sala, Sweden) unless otherwise noted. The Pharmacia for the synthesis of GDP*F. Our conditions reflect those columns used were Mono-Q HR 5/5, PepRPC 5/5 o r 10/ that were found to be optimal in that work (22). Pre10, and G-25 SF HR10/10 fast desalting columns. Abliminary PepRPC 10/10 chromatography (conditions desorbance measurements were made using a Hewlettscribed below) of oxGDP was done to ascertain purity; Packard photodiode array spectrophotometer HP 8452A four peaks were obtained (relative amounts given in (Palo Alto, CAI. parentheses) based on the absorbance a t 280 nm: oxGDP All NMR and FAB mass spectra were obtained from (83%), oxGMP (9%), oxG (8%), and unknown (‘0.4%). the instrument facilities a t the University of NeThe third chromatographic peak of oxGDP had the same braska-Lincoln Chemistry Department. The NMR specretention time as oxG (Sigma) under the same conditions, tra were acquired using a General Electric Omega-500 and the second peak was thus assumed to be oxGMP. NMR system operating a t 500.1 MHz for proton observaAll reactions were carried out a t room temperature and tion. The mass spectra were obtained using an Analytiin the dark to minimize photobleaching. Eighteen pmol cal Instruments VG ZAB-T BlElBzEz configured mass of 5-((2-aminoethyl)thioureidyl)fluorescein(8.7 mg) disspectrometer with a Cs-Ion gun for FAB desorption or a solved in 0.4 mL of DMF was added a t once to 6 pmol Kratos MS50 ElBEz configured mass spectrometer with (3.0 mg) of periodate-oxidized GDP dissolved in 0.8 mL an Ar-Ion Saddle-Field gun for FAB desorption. The of 100 mM sodium phosphate buffer, pH 7.1, while matrix used was either 5050 (v/v) 3-nitrobenzyl alcohol: stirring. The reaction mixture was stirred for 30 min a t glycerol or 1%(v/v) trifluoroacetic acid in glycerol. room temperature in a fume hood. After the addition of For steady-state measurements, the fluorimeter (Al120 pmol (7.5 mg) of solid NaCNBH3, the solution was phaScan, Photon Technology, Inc.) with single excitation stirred another 30 min followed by the addition of 120 and emission f4.4 ruled monochromators was fitted with pmol of NaBH4 (120 pL of a freshly prepared 1M NaBH4 a 150-W Xenon compact arc lamp and a Lexel Model 7 5 solution in 10 mM NaOH) for a final NaBH4 concentraArgon ion laser as light sources with a reference cell tion of 90 mM. The solution was stirred for 1h a t room quantum counter. Emission scans were collected through temperature and then acidified to pH 5.0 to remove any PTI’s Alphascan software (version 2.060). The backexcess NaCNBH3 and NaBH4 by the addition 150 pL of ground fluorescence from the buffer alone was subtracted 1.0 M acetic acid. The pH was then adjusted to 7.8 with for all data reported. Each scan was corrected for 1M Tris base, and the solution was left overnight on ice dilution when necessary. in order to fully oxidize any remaining trace amounts of NaCNBH3 and NaBH4. In our modification, the reaction mixture was loaded Fluorescein,2-(3,6-dihydroxy-9H-xanthen-9-yl)benzoic acid. Tetramethylrhodamine,N-[9-(2-~arboxypheny1)-6-(dimethy- onto a PepRPC 10/10 column (0.5 mL for each sample lamino)-3H-xanthen-3-ylidene]-N-methylaminium chloride. injection) previously equilibrated with 1%(w/v) potas-

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sium acetate in water, pH 7.8 a t 6 "C. A 10-mL linear 45% methanol in 1%(w/v) potassium gradient of 0 acetate in water was run, followed by a 40-mL linear gradient of 45 90% methanol in 1%(w/v) potassium acetate in water, a t a flow rate of 0.5 mumin. Unlabeled GDP was eluted a t 0% methanol in potassium acetate, free fluorescein a t approximately 45% methanol and fluorescein-labeled GDP at approximately 75% methanol. One-mL fractions were collected, and an absorbance spectrum from 230-650 nm was obtained for each fraction. The concentrations of GDP and fluorescein were calculated using the following extinction coefficients: GDP, pH 7.8,13.7 mM-l cm-I a t 252 nm (231,fluorescein, pH 7.8, 72.6 mM-' cm-l at 492 nm, and 32.3 mM-l cm-' a t 252 nm. The extinction coefficient for fluorescein a t 252 and 492 nm were calculated for pH 7.8 buffer using the extinction coefficients of 75 mM-l cm-I a t 492 nm in 20 mM TrisCl, pH 9.0 (24). All fractions with a mole ratio of GDP to fluorescein between 0.9 and 1.5 were dried by vacuum centrifugation, weighed (approximately 1 mg product was obtained), and redissolved in a minimum volume of HzO (total of 0.2 mL) and stored a t -80 "C. A 20-21% yield was achieved based on the initial moles of oxGDP and calculated from the ratio of the product absorbance to that of the starting material a t 252 nm (for oxGDP) correcting for the contribution of *F (for GDP*F) to the absorbance. For the molar absorptivity of oxidized nucleotides in the 252-nm region, we used the values for the intact nucleotides. The spectra of oxGDP and GDP could be scaled to overlay exactly throughout this region. The labeled GDP was stored frozen a t -80 "C and remained stable for a t least 4 months through repeated freeze-thaw cycles. (B) A second procedure for preparing fluoresceinlabeled GDP was developed for the 5-((2-((carbohydrazinomethy1)thio)acetyl)amino)fluorescein (*FH, hydrazine derivative of fluorescein) that followed closely the procedure of Wells and Cantor (25) who describe the preparation of dansyl-labeled tRNA but only used dialysis to purify the labeled material. The motivation for this alternative method was to improve the product yield since the hydrazine moiety forms the relatively stable hydrazone intermediate (compared to the relatively unstable Schiff base formed from amines) (24). Purification was performed using silica gel TLC and sizeexclusion chromatography instead of reversed-phase chromatography as in the GDP*F synthesis described in procedure 1A. All reactions were carried out a t room temperature in a fume hood and in the dark. Twelve pmol of fluorescein hydrazine (6 mg, weighed on an analytical balance) were added to 8 pmol (4 mg) of periodate-oxidized GDP dissolved in 2 mL of 100 mM sodium phosphate buffer, pH 7.1. The reaction was monitored by TLC using silica gel 60 f254 plastic-backed plates and 80% acetonitrile in water as the solvent. A new fluorescent spot appeared a t Rf = 0.15 as the reaction progressed. The optimal reaction time (room temperature) for maximum product formation was 4.5 h. A 10-fold molar excess (NaCNBH3:fluorescein)of solid NaCNBH3 (7 mg) was added a t once, and the reaction mixture was stirred for 1 h and then stored at 4 "C overnight before preparative TLC purification was carried out. Using 25- x 25-cm Kieselgel-60 0.2-mm coated plastic TLC plates, 0.4-0.5 mL of the above reaction mixture, which was stored overnight, was applied by repeated applications followed by drying under a stream of nitrogen. The plates were developed using a 20% water/80% acetonitrile mixture. The newly appearing band (Rf= 0.15, visualized by room light o r a 365-nm lamp) was cut

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Hileman et al.

out from each plate with a razor blade and scraped into a 30-mL siliconized Corex tube. UV-vis absorption measurements later showed that this band corresponded to 1 : l guanosine nuc1eotide:fluorescein. Fluorescent bands (and the relative amounts estimated visually given in parentheses) were observed a t R f = 0.15 (40%), 0.3 (20%), 0.5 (20%),0.68 (lo%), and 0.75 (10%). Elution of the GDP*FH was performed using four successive extractions using 5 mL of 20% water in acetonitrile followed by centrifugation at 2000 rpm for 5 min in an SA-600 rotor (DuPont). No visible fluorescein remained in the gel after the fourth extraction. The pooled material was dried to completion by vacuum centrifugation, resuspended in a minimum volume of water (0.75 mL), and loaded onto a 1- x 60-cm Biogel P-2 column preequilibrated with sterile water. A P-1 pump (Pharmacia)was used to maintain a flow rate of 0.02 mL/h while collecting 1-mL fractions. The major fluorescein-containing peak eluted in the void volume of 21 mL and was dried by vacuum centrifugation and weighed. The material was dissolved in 1mL of water, and an absorbance spectrum was obtained from 230 to 550 nm. The concentration of GDP was calculated from the absorbance a t 252 nm, correcting for the contribution of fluorescein at that wavelength and using the extinction coefficients given in procedure 1A. Based on the absorbance a t 252 nm (for GDP), 38% of the initial oxGDP was recovered as GDP*FH. The material was stored a t -80 "C. ( 2 )Synthesis of Fluorescein-Labeled ATP. The quality of the oxATP starting material was ascertained by PepRPC 10/10 chromatography; two peaks were obtained (relative amounts given in parentheses) based on the absorbance at 280 nm: oxATP (98%),unknown (2%).This synthesis was identical to that used for GDP*F, procedure A, through the step calling for the solution to be stored overnight on ice to ensure complete oxidation of any remaining borohydride. A different chromatographic method was used, however, since ATP*F and free fluorescein nearly coelute on the PepRPC 5/5 column under the conditions for the fluorescein-GDP preparation. First, nearly all the free fluorescein was removed as follows. The solution (after sitting on ice overnight) was diluted to 100 times the reaction volume by the addition of water to 200 mL and loaded onto a 1.0- x 7.0-cm gravity flow DEAE-Sephacel anion exchange column preequilibrated with buffer A (10 mM TrisC1, pH 8.0). The column was washed at a flow rate of 0.5 mumin with buffer A until no absorbance a t 492 nm could be detected (approximately 50 mL of buffer A was required). Both the ATP*F and unlabeled ATP were then eluted with 0.4 M NaCl in buffer A by batch elution. The eluant (3 mL total) was loaded in separate runs (0.5 mL for each sample injection) directly onto a PepRPC 5/5 column preequilibrated with 1% (w/v)potassium acetate in water and washed with 5 mL of the same solution. A 70-mL linear gradient from 0 to 80% methanol in 1%(wlv) potassium acetate solution a t a flow rate of 0.2 mL/min was used and resulted in four major peaks a t the following methanol concentrations: 0% (reduced ATP), 60% (free fluorescein), 70% (ATP*F), and 85% (unknown, fluorescein:ATP ratio approximately 4:1 based on absorbances a t 492 and 258 nm). One-mL fractions were collected and an absorption spectrum obtained for each from 230 to 650 nm. The ratio of ATP to fluorescein was calculated using the following extinction coefficients.The concentration of ATP was determined from the absorbance a t 258 nm, using €258 nm (pH 7.8) of 15.4 mM-l cm-' (23),and the concentration of fluorescein from the absorbance at 492 nm, using €492 nm (pH 7.8) of 72.6 mM-l cm-' and 6258 nm of 28.3 mM-' cm-l. The extinction

Bioconjugate Chem., Vol. 5, No. 5, 1994 439

Fluorescent Probes

coefficients for fluorescein a t 258 and 492 nm were determined as before in the GDP*F preparation using 75 mM-l cm-l a t 492 nm in pH 9.0 (24). Fractions that eluted a t 70 f 1%methanol in 1%(w/v) potassium acetate solution and that had a molar ratio of ATP: fluorescein from 0.9 to 1.5 were pooled, dried by vacuum centrifugation, weighed (approximately 1mg of product), and redissolved in a minimum volume of sterile water (0.2 mL). A 24% yield was calculated based on the initial amount of oxATP, with e o d p = EATP a t 258 nm. The material was stored a t -80 "C and was stable for up to 4 months through repeated freeze-thaw cycles. (3)Synthesis of Rhodamine-Labeled GDP. This synthesis followed the same protocol used to prepare ATP*F, with the following exceptions: A 2-fold molar excess of tetramethylrhodamine cadaverine to oxGDP (12 and 6 pmol, respectively) was used. A 1.0- x 5.0-cm gravity flow DEAE-Sephacel column was equilibrated with buffer A, the sample was applied, and the absorbance of the eluant was monitored a t 556 nm to determine when the unreacted rhodamine had been nearly all removed (approximately 25 mL was required) before elution of the product, as in procedure 2 above. Purified rhodamineGDP was obtained by PepRPC 5/5 chromatography. The column was preequilibrated with 5% methanol in 1%(w/ v) potassium acetate in water, and the total sample (2 mL) was loaded. A 40-mL linear gradient of 5 95% methanol in 1%(w/v)potassium acetate solution a t a flow rate of 0.4 m u m i n was used. Under these conditions, two peaks were obtained from the column. Unlabeled GDP does not bind and elutes in the void volume, and GDP*R elutes a t approximately 40% methanol. The fractions containing GDP*R were determined by absorption spectroscopy, using values for €556 nm of 70 mM-' cm-l (24)and 6252 nm of 25.9 (pH 7.8) for rhodamine and a value for 6252 nm of 13.7 mM-' cm-l for GDP (23). These fractions were pooled, dried by vacuum centrifugation, and redissolved in a minimum volume (0.4 mL) of 10% ethanol in HzO. The purified material was stored frozen a t -80 "C. (4)Synthesis of the Guanosine-Benzylamine Derivative. (A) This synthesis was similar to that used for GDP*F, procedure 1A. Ten mg of oxG (33 pmol) was dissolved in 1 mL of 100 mM sodium phosphate buffer, pH 7.1, and added to 36 pL (330 pmol) of benzylamine mixed into 2 mL of the same buffer. This resulted in a solution that was 110 mM in benzylamine and 11 mM in oxG (lO:l, benzy1amine:oxG). While the solution was stirring, the progress of the reaction was monitored by spotting 2-5 pL a t 20-min intervals on silica gel 60 f254 plastic-backed TLC plates, using an 80%(v/v) acetonitrile in water as the mobile phase. A new spot was observed using a 254-nm lamp a t Rf = 0.78 (Rfvalues for oxG and benzylamine were 0.52 and 0.42, respectively). After the solution was stirred for 1 h, 10 mg of NaCNBH3 was added and the solution stirred for 1h. Ten mg of NaBH4 was added and the solution stirred for another 1h. The solution was acidified to pH 6.0 (measured by the color change after spotting 5 pL on pH paper) by the addition of 50 pL of 4 M acetic acid and stirred for 10 min and the pH adjusted to pH 9.5 (also measured by pH paper) with 75 pL of 1 M NaOH. Reversed-phase chromatography using the PepRPC 101 10 column was performed using a 0 100% methanol gradient in water over 40 mL while 1-mL fractions were collected a t a flow rate of 1 mumin. Six runs (0.5 mL each) were required to apply the total sample of 3 mL. As shown in Figure 2, three major peaks were obtained by monitoring absorbance a t 280 nm, corresponding to reduced oxG a t 20% methanol, benzylamine a t 50%

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I

20

40 80 Time (minutes)

100

80

Figure 2. Reversed-phase chromatography of the crude guanosine-benzylamine mixture using a PepRPC loll0 column as described in the Experimental Procedures. The relative absorbance is shown as a solid line and the percent methanol as a dotted line. 2

c

1

+PEAK

I (73%)

f 15 I n

s W

PEAK II (27%)

Y ' 28 0 5

-c

9 0 15

25

35

45

55

VOLUME (mL)

Figure 3. Separation of the two major products formed during the guanosine-benzylamine synthesis using a Biogel P-2 column. methanol, and guanosine-benzylamine at 70% methanol, as identified by the Rf values on silica TLC of an aliquot from each fraction. Five fractions from each of the six runs that eluted between 65-75% methanol (total volume 30 mL) were pooled and concentrated to approximately 2 mL by vacuum centrifugation. This solution (1 muinjection) was rechromatographed on the same column using a linear gradient from 0 to 40% methanol 90% linear in water over 5 mL followed by a 40 gradient over 70 mL a t a flow rate of 2.0 mumin. Elution was monitored by absorbance a t 280 nm, and 1-mL fractions were collected. The predominant absorbance peak occurred at 70 f 1%methanol, and these fractions were spotted and checked for purity on silica TLC. The four fractions that yielded a spot a t Rf = 0.78 were pooled, dried by vacuum centrifugation, and redissolved in 0.5 mL water, and the absorption spectrum was obtained. A 38.3%yield was achieved based on the initial oxG, calculated from the absorbance a t 254 nm for guanosine (€254 nm = 13.7 mM-' cm-l) and benzylamine ( E nm~ = ~1.2~ mM-' cm-l, (26)). The results of the initial lH NMR analysis suggested that two different products were present. Two peaks (which when integrated) with a ratio of 7:3 were seen for the characteristic guanylyl H-8 downfield chemical shift (approximately 8 ppm) as well as for the anomeric ribosyl H-1' (approximately 5.7 ppm). Further purification was obtained by Biogel P-2 size-exclusion chromatography. The total sample (0.5 mL) was applied to a 1.5- x 25-cm column previously equilibrated with water. Fractions of 1 mL each were collected a t a flow rate of 0.08 m u m i n and were analyzed by absorbance scans from 230 to 350 nm. Two major peaks were observed in the chromatogram when monitored a t 254 nm: the first (I) eluted a t 24 mL and the second (11) a t 46 mL water (see Figure 3). The fractions eluting a t 20-23 mL were pooled, and fractions eluting a t 45-49 mL were pooled and labeled as GBA (I) and GBA (II), respectively. The

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440 Bioconjugate Cbem., Vol. 5, No. 5, 1994 160

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Hileman et al. Table 1. Summary of TLC Resultsa

Rf value(s)

detected by 365-nm excitation

compd

oxGDP oxATP oxG 210 230 250 270 290 310 330

220 280 340 400 460 520 580

WAVELENGTH (nm)

WAVELENGTH (nm)

Figure 4. (A) Absorption spectra of the Biogel P-2-separated components of the guanosine benzylamine (GBA) synthesis. GBA I (solid line) and GBA I1 (dotted line) correspond to the major product (peak I) and the minor product (peak 111, respectively. (B) Absorption spectra of ATP*F. The y-axes units are mM-l cm-l. two small peaks eluting in fractions 34 to 44 mL were discarded. The U V absorption spectra are shown in Figure 4. Both GBA (I) and (11)were submitted for NMR and FAB-MS analysis. (B) In a second procedure, the synthesis of the guanosine-benzylamine derivative was carried out with the following changes to procedure 4A. The reaction was carried out using 100 mM sodium phosphate buffer, pH 6.5, and NaBH4 was omitted. Following PepRPC 10/10 chromatography, the predominant peak material occurring a t approximately 70% methanol was submitted for FAB-MS analysis. (C) In a third procedure, to determine whether phosphates were lost during the reaction, oxGDP was reacted with benzylamine and the resulting product used for phosphate analysis. The same procedure described in section 1A for the synthesis of GDP*F was used with the following changes. In order to obtain sufficient product for analysis, both the volume and amount of reactants were scaled up. Benzylamine (300 mmol) dissolved in 4 mL of DMF was added a t once to 60 mmol of oxGDP dissolved in 8 mL of 100 mM sodium phosphate buffer, pH 7.1, while stirring. Similarly, the amounts of reductants added were scaled up, using 1.2 mmol of solid NaCNBH3 and 1.2 mmol of NaBH4 (1.2 mL of a 1 M NaBH4 solution in 10 mM NaOH). The GDP-BA derivative was isolated as a single peak a t approximately 60% methanol from the PepRPC 10/10 chromatography. The product was dried by vacuum centrifugation and redissolved in 4 mL of HzO. Silica gel TLC of the nucleotide derivatives was extremely useful during the synthesis to check the progress of reaction and after each chromatographic step t o check the purity. Table 1summarizes the R,value(s) for each material and the method of detection used to visualize the plate. Inorganic Phosphorus Determination. The GDPBA derivative prepared in section 4C above was subjected to acid and enzymatic hydrolysis in order to release inorganic phosphorus as follows. To 124.8 nmol of GDPBA (determined from the absorbance a t 254 nm) was added HzS04 to a final concentration of 0.5 M in a total volume of 1.30 mL and the mixture incubated a t 100 "C for 1h. Acid hydrolysis of 1mol of GDP results in 1mol of guanine, 1 mol of ribose 5-phosphate, and 1 mol of inorganic phosphorus (23). After treatment, the acidhydrolyzed sample was analyzed using the colorimetric inorganic phosphorus kit by comparison with a KHzP04 standard curve. Measured a t 660 nm, for each inorganic phosphate standard (0, 65.1, 130.2, 195.3, 259.9, and 325.5 nmol, each in 2.625 mL) the absorbance values were 0, 0.0841, 0.1762, 0.2671, 0.3556, and 0.4465, respectively. The acid-hydrolyzed GDP-BA sample re-

*F *FH

*R

0.18 0.30 0.55 0.68 0.72 0.75 0.18 0.30 0.55 0.68 0.72 0.75 0.19 0.54 0.63

benzylamine

GDP*F GDP*FH GDP*R ATP*F GBA(1)

GBA(I1)

detected by 254-nm excitation 0 0 0.52

0.42 0.15 0.15 0.15 0.10 0.78 0.13

a All values were observed using acetonitrile/water 8:2 as the mobile phase.

sulted in a n A660 nm value of 0.1729. The sample was compared to the standard curve (correlation coefficient, r , of 0,9999). Purification of eIF-2. The preparation of both four(eIF-2 containing the 67-kDa subunit in addition to the three subunits, a, P, and y ) and three-subunit eIF-2 was described earlier (5, 27).

Labeling eIF-2 with Fluorescein-GDP or Fluorescein-ATP. The eIF-2 preparations were labeled with a dye-labeled nucleotide by adding a 30-fold molar excess of fluorescein-labeled GDP (GDP*F) or ATP*F to eIF-2 as follows. Ten or 20 pL of 1.5 mM ATP*F in water was added to 0.5 or 1.0 nmol (72 or 144 pg) of eIF-2 in buffer B (20 mM TrisC1, pH 7.8, 100 mM KC1, 5 mM P-mercaptoethanol and 10% (v/v) glycerol), respectively, in a total volume of 0.2 mL, and incubated for 15 min a t 37 "C in the dark. All subsequent steps were carried out a t 6 "C. The incubation mixture was centrifuged a t 15 000 rpm for 10 min and loaded onto a Sephadex G-25 10/10 superfine fast desalting column equilibrated with buffer C (50 mM TrisCl, pH 7.8, 100 mM KC1, 1 mM MgC12,5 mM P-mercaptoethanol, and 10% (dv) glycerol) a t a flow rate of 0.2 mumin. The eluant was collected in 0.5-mL fractions and monitored for absorbance a t 280 nm. Fluorescently labeled ATP*F.eIF-2 or eIF-2.GDP*F eluted in the void volume of 4.5 mL, well separated from the peak of excess ATP*F or GDP*F which eluted a t 1213 mL. Although near-base-line resolution was obtained based on the chromatogram (monitored a t 280 nm), the fluorescence intensity was commonly 10% above background in the interpeak fractions. The labeled eIF-2 was freshly prepared a t the beginning of each set of fluorescence experiments. The portion of material not used immediately was kept on ice and used within 2-3 h or discarded. Attempts were made to optimize the labeling procedure by varying the incubation conditions: time, temperature, and nucleotidelprotein concentrations. A n increase in steady-state anisotropy ((r))of the chromatographic peak from the gel filtration separation was used as the criterion to determine the optimal conditions. Incubation with greater than a 30-fold molar excess of GDP*F or ATP*F over eIF-2 reduced the resolution of the chromatography. Typical ( r )values ranged from 0.1 to 0.15 for ATP*F labeling when a 60-fold molar excess was used, approximately 40% less than the optimal 0.205. Using less than a 30-fold excess only reduced the yield of labeled eIF-2. When a 10-fold molar excess of ATP*F was used over eIF-2, the measured IF value was approximately 50% less than that obtained for a 30-fold excess, but the

Bioconjugate Chem., Vol. 5, No. 5, 1994 441

Fluorescent Probes

anisotropy was normally greater than 0.160. Millipore filtration assays were performed to measure eIF-2 ternary complex activity. When eIF-2 was incubated a t 37 "C for 1h, then used to form ternary complex by addition of GTP and L3%1Met-tRNAfas described elsewhere (51,a 30% loss of ternary complex activity was observed (compared to a control where the same eIF-2 was stored on ice for the same time period). Thus, to maintain eIF-2 activity, incubation times greater than 15 min a t 37 "C were not tried for labeling. When the temperature was lowered to 0 "C, long incubation times could be tried without harm to eIF-2 activity. However, incubation of a 30-fold molar excess of ATP*F over eIF-2 for 12 h on ice resulted in essentially no binding, with ( r ) = 0.060.

I

6.0

RESULTS Synthesis of Nucleotide Conjugates. Reaction of amine-containing dyes with periodate-oxidized nucleotides with subsequent reduction produced nucleotide labeled through the ribose (rather than the base). The absorption spectra of the benzylamine derivatives of guanosine and a representative dye-labeled nucleotide (ATP*F) synthesized a t pH 7.1 using both NaCNBH3 and NaBH4 reductants are shown in Figure 4. The absorption spectra of both Biogel P-2-purified GBA (I) and (11) are essentially identical. The GDP*F, GDP*FH, and GDP*R spectra are very similar to that of ATP*F and are therefore not shown. A 1:l molar ratio was calculated for fluorescein:ATP based on the absorbance values a t 492 and 258 nm. When the reductive amination was carried out with only NaCNBH3 a t pH 6.5 (see Experimental Procedures section 4B), only one product was obtained corresponding to GBA (I). Inorganic Phosphorus Determination. To address the possibility of base-catalyzed p-elimination of the phosphate moiety, a GDP-BA derivative was isolated and analyzed using a quantitative inorganic phosphorus colorimetric method. Analysis of the acid hydrolysis of 125 nmol of GDP-BA yielded a value of 127 nmol of inorganic P from the standard curve. FAB-MSAnalysis. For GBA (I), positive ion lowresolution FAB in 5050 (vlv) 3-nitrobenzyl alcohol: glycerol showed mlz = 357. Identification of the molecular ion species was accomplished after doping the matrix with Na2C03 for which mlz = 379 for (M Na)+. For GBA (11), low-resolution FAB in both 5050 (vlv) 3-nitrobenzyl alcoho1:glyceroland 1%(vlv)trifluoroacetic acid in glycerol showed mlz = 375. Similarly, identification of the molecular ion species was accomplished after doping (separately) the matrix with Na2C03 and KI for which mlz = 397 (M Na)+ and mlz = 413 (M K)+, respectively, Two separate high-resolution FAB-MS analyses for each GBA (I) and (11) were performed using CsI as the standard. A search for C, H, 0, and Ncontaining compounds within 10 ppm of the observed mass was also done to obtain the following: GBA (I), (M H)+ = 357.168 30 and 357.166 90, predicted formula C17H21N603(calculated mass = 357.167 513 745, deviation = 2.2 ppm and -1.7 ppm, respectively) and C19H23N304 (calculated mass = 357.168 856 435, deviation = -1.5 ppm and -5.4 ppm, respectively); GBA (111, (M HI+ = 375.176 60 and 375.178 30, predicted formula C17H23N6O4 (calculated mass = 375.178 078 459, deviation = -3.9 ppm and 0.5 ppm, respectively) and C I ~ H ~ ~(calculated N ~ O ~ mass = 375.179 421 149, deviation = -7.5 ppm and -2.9 ppm, respectively). The low-resolution FAB-MS analysis of the GBA derivative synthesized a t pH 6.5 using only NaCNBH3 resulted in a (M + H)+ of 357.2, corresponding to GBA (I) from the synthesis a t pH 7.1 with both reductants.

+

+

+ +

+

5.0

4.0

3.0

2.0

PPM Figure 5. 'H-COSY spectrum of Biogel P-2 purified GBA (I) in DzO. Table 2. Measured Steady-State Anisotropy Values of the Free Labeled Nucleotides and after Binding to 3-Subunit eIF-2a material GDP*F GDP*R ATP*F eIF-2-GDP*F eIF-2-GDP*R eIF-2-ATP*F

."

measured anisotrotw 0.050 i 0.001 0.055 i 0.002 0.045 i 0.001 0.103 i 0.005 0.110 i 0.004 0.172 i 0.007

a Excitation was 488 nm for both *F and *R, emission was detected at 518 nm for *F and 575 nm for *R. The sample volume was 250 p L for all measurements.

No GBA (11) product was detected from the modified synthesis procedure. NMR Analysis. The proton assignments for the onedimensional lH NMR spectra of GBA (I) and (11) in D2O are as follows. GBA (I): 6 (ppm) 7.94 (s, 1 H, guanylyl H-8), 7.48-7.38 (m, 5 H, benzyl H), 5.73 (d, J = 10.5 Hz, 1H, ribosyl H-1'),4.04-4.01 (m, 1H, ribosyl H-4'),3.893.55 (bm, 4 H, ribosyl H-5' CH2 and benzylamine CHZ), 3.15 (d, J = 12.1 Hz, 1 H, ribosyl H-2', equatorial), 2.96 (d, J = 12.5 Hz, 1 H, ribosyl H-3', equatorial), 2.73 (t, J = 10.9 Hz, 1 H, ribosyl H-2', axial), 2.30 (t,J = 11.7 Hz, 1H, ribosyl H-3', axial). GBA (11): 6 (ppm) 7.96 (s, 1H, guanylyl H-81, 7.48-7.38 (m, 5 H, benzyl H), 5.38 (d, J = 10.5 Hz, 1H, ribosyl H-1'1, 4.28-4.26 (m, 1H, ribosyl H-4'1, 3.92-3.46 (bm, 6 H, ribosyl H-5' CH2, H-2' CH2, and benzylamine CHz), 3.02-2.99 (dd, J = 2.8 Hz, 1 H, ribosyl H-3'),1.93 (s, 1H, ribosyl H-3'). The assignments for the ribosyl protons were made possible from the lHCOSY NMR spectrum (Figure 5) of GBA (I) which was used to further characterize the material.

Labeling eIF-2 with GDP*F,GDP*R,and ATP*F. The fluorescent nucleotide derivatives synthesized have been demonstrated to bind to 3-subunit eIF-2. Both GDP*F and ATP*F have also been shown to bind 4-subunit eIF-2 (28). Incubation using a 30-fold molar excess of labeled nucleotide over eIF-2 and subsequent size-exclusion chromatography to remove unbound labeled nucleotide resulted in a significant increase in anisotropy above that for the labeled nucleotide (see Table 2). DISCUSSION

Initially, PepRPC 10l10-purified GDP*F and Biogel P-%purified GDP*FH were submitted for analysis by NMR. The spectra obtained were complex and ambiguous with regard to the nature of the ribose ring and the position of the dye. The three main factors contributing

Hileman et al.

442 Bioconjugate Chem., Vol. 5, No. 5, 1994

to the complexity were as follows: (1) the peaks were too crowded, overlapped, and not sufficiently resolved, (2) the 'H NMR spectrum of *F alone (not shown) integrated to a total of 28 protons, when only 13 would be expected for *F in DzO (this indicated that *F was impure), TLC analysis of *F (Table 1) showed several fluorescent species of different mobilities (a spectrum of pure *F was needed to facilitate analysis of GDP*F), and (3) the GDP*F (and *F) NMR spectra showed a large number of aromatic and heteroaromatic proton peaks between 8.3 and 6.4 ppm as well as a-monosubstituted aliphatic proton peaks between 4.4 and 2.4 ppm, making it difficult to identify the guanylyl H-8 peak a t -8 ppm and any ribosyl protons in the anticipated morpholine-like structure (expected between 5 and 2 ppm). A similar synthesis using periodate-oxidized guanosine and benzylamine was chosen for the following reasons: (1)the chemistry of the synthesis was expected to be very similar since the pKa of benzylamine and the fluorescein amine derivative are approximately equal (pKa x 91, (2) both benzylamine and the fluorescein amine derivative have similar solubility properties, (3) the expected product would have a relatively small molecular weight, facilitating FAB-MS studies, (4) benzylamine can be obtained in much greater purity than the fluorescein amine derivative, and (5)p-elimination of the phosphates need not be considered. Periodate-oxidized nucleotides can undergo p-elimination, a reaction catalyzed by base (29-31). Near pH 7, however, p-elimination can be prevented (32). Lowe and Beechey (33)have reported no loss of inorganic P over a period of 1 h when a t pH 8-9 during the NaBH4 reduction of oxATP. Hansske et at!. (20)measured the half-lives of oxAMP decomposition (due to p-elimination) a t 4 , 2 0 , and 37 "C (pH 7) to be 17 days and 45 and 15 h, respectively. In our procedure, reaction materials are a t pH 7.1 a t room temperature for no more than 90 min, through the final reduction. Inorganic phosphorus analysis of the purified GDP-BA product synthesized a t pH 7.1 yielded 2 mol of Pi per mole of GDP-BA. Thus, p-elimination appears t o be insignificant under our conditions. The guanosine-benzylamine derivative gave significantly simpler NMR and mass spectra from which we have drawn several conclusions. (1) Following the described synthesis and purification procedure to the PepRPC 10/10stage leads to a mixture of products (Experimental Procedures section 4A). The mixture was separated using a Biogel P-2 size-exclusion column. By comparing the total absorbance (at 254 nm) found in peak I (GBA I) and peak I1 (GBA IIj, the mixture was calculated to be 73%I and 27%11. The two relatively insignificant peaks eluting before GBA (11) were not included in GBA (11)or in the above percentages. In a modified synthesis (Experimental Procedures section 4B) where only NaCNBH3 was used in the reaction a t pH 6.5, only one product was synthesized, corresponding to GBA (I). Fluorescent dyes have been coupled as the amine (15) or as the thiosemicarbazide ( I 7) to oxidized polyribonucleotides without subsequent reduction. Although there are several reports that describe the use of only NaBH4 during the reduction step (18,30,31,34,35), the synthesis method we chose to follow was based on the use of NaCNBH3 and NaBH4 (22). When Ingham and Brew (22) used only NaBH4 during the reduction step, they obtained low yields of a relatively unstable product, but when both NaCNBH3 and NaBH4 were used, a greater yield of a thermally stable product was obtained. Other authors have also reported the use of both NaBH4 and NaCNBH3 in similar reductive amination procedures

0

GBA ( I )

0

0

CH,

I I

&,OH

HOCH,

k~, I

NH

NH

y 2

FHZ

C6H5

C6H5

I

GBA ( II )

Figure 6. Proposed structures for GBA (I) and GBA (11) from the synthesis starting from periodate-oxidized guanosine and benzylamine.

(32,36). Wells and Cantor (25)described a procedure to label periodate-oxidized tRNA with dansyl hydrazine using only a single reductant, NaCNBH3. Stirchak et aZ. (37)used (NH4jB407, also with only NaCNBH3, to form a morpholine nucleoside for incorporation into nucleoside oligomers. Our observations show that the use of both NaCNBH3 and NaBH4 results in formation of a second product, GBA (11). (2) On the basis of the high-resolution FAB mass spectra, GBA (I) had a molecular ion mass consistent with two possible (M HI+ formulas for GBA (I), C17H2103N6 and CIgHz304N3, the latter of which is not possible from the synthesis because of the two additional carbon atoms. Similarly, GBA (11) had a molecular ion mass consistent with two possible (M H)+ formulas, C17Hz304N6 and ClgHZ503N5, the latter of which is again not possible because of the two additional carbon atoms. These results suggest one structure for the major product GBA (I) in which the ribose moiety has been converted to a morpholine ring structure (Figure 6). The two possible isomers for the minor product, GBA (II), that are consistent with the predicted formula are also shown in Figure 6. (3) The predominant product, GBA (I), as well a s the minor product, GBA (111, contained one guanosine per benzylamine based on lH NMR analyses. No free aldehyde group remained on the ribose moiety, indicated by the absence of the characteristic downfield chemical shift for aldehydic protons a t 9-10 ppm. (4) Further analysis performed on GBA (I) using COSY lH NMR (Figure 5) was used to assign chemical shift values to each proton and was completely consistent with the structure proposed from the high-resolution FABmass spectra analysis of GBA (I). The diagonal peaks (from the lower left to upper right corners) represent the one-dimensional spectrum. The guanylyl H-8 (-8 ppm, region not shown) is expected to appear downfield since it is on a carbon adjacent to two electronegative nitrogen atoms which would deshield the proton. The ribosyl H-1' peak (5.7 ppmj is correlated to two cross-peaks at 3.1 and 2.7 ppm which must therefore correspond to the two nonequivalent ribosyl H-2' protons. Furthermore, since the cross-peak at 2.7 ppm is larger, we may assign it as

+

+

Fluorescent Probes

Bioconjugate Chem., Vol. 5, No. 5, 1994 443

whether only one or both forms are recognized by the eIF-2 nucleotide binding site. The form in which the ribose ring remains open, however, is expected to have added flexibility compared to the morpholine-like closed ring derivative, and could facilitate binding. These fluorescent nucleotide derivatives have been very useful probes for studies involving eIF-2 interactions with several of the components necessary for the initiation of eukaryotic protein synthesis using steady-state fluorescence anisotropy (28).

N- 2 N H

&

b

&--

ACKNOWLEDGMENT

The authors are grateful to Dr. Patrick A. Dussault for discussions concerning the syntheses, Dr. Richard Shoemaker and Dr. Charles A. Kingsbury for discussions and technical assistance concerning NMR, and Dr. Michael L. Gross and Dr. Ronald Cerny for discussions and technical assistance concerning FAB-MS. This work was supported by grants from the Center for Biotechnology, University of Nebraska-Lincoln, and by NIH Grant GM 22079 (N.K.G.) and NIH Grant DK 36288 (L.J.P.). LITERATURE CITED

AP”F

0

Figure 7. Proposed structures of GDP*R and ATP*F corresponding to GBA (I).

the axial H-2’ proton and the 3.1 ppm cross-peak as the equatorial H-2’ proton. The peak at 4.0 ppm was assigned a s the H-4’ proton since it would be deshielded by the adjacent oxygen (thus shifted downfield) and would be expected to show complex splitting patterns due to the adjacent H-3’ and H-5’ protons. Indeed, the peak a t 4.0 ppm (x-axis) in the one-dimensional spectrum has cross-peaks that connect peaks in the one-dimensional spectrum (y-axis) both in the 3.9-3.6 ppm range (assigned as the two ribosyl H-5’ protons) as well as the 2.3 ppm peak (assigned as the axial H-3’ proton because of the large cross-peak size). The smaller cross-peak to the equatorial H-3’ is not seen due to suppression of axial peaks. However, the H-3’ peak a t 2.3 ppm has a crosspeak to 3.0 ppm, which was thus assigned as the equatorial ribosyl H-3’ proton. The assignments given for the H-2’ and H-3’ protons are reinforced by the fact that they show the same splitting patterns: H-2’)doublet and triplet (3.1 and 2.7 ppm, respectively); H-3’, doublet and triplet (3.0 and 2.3 ppm, respectively). In contrast to previously reported work involving periodate-oxidized nucleotides and their reactions with amines (18) or carboxylic hydrazides (19,20)the ribose moiety of the major product, following reduction, does not contain a hydroxyl on either the 2’ or 3’ carbon. The minor product, however, does contain one hydroxyl group. By inference, we propose that the structure(s) of the fluorescent nucleotide conjugates are the same as for that of the guanosine benzylamine conjugate(s1 in regard to the modification at the ribose ring. In Figure 7, we show putative structures for GDP*R and ATP*F corresponding to GBA (I). Since a mixture of products was obtained from the guanosine benzylamine synthesis with two reductants, we also infer that a similar mixture is obtained for the fluorescent nucleotide conjugates. Similarly, we presume that only the ring structures shown in Figure 7 should be formed when only the single reductant NaCNBH3 is used. It is unknown a t this point

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