Bioconjugate Chem. 1003, 4, 425-433
425
Labeling of Proteins by Organometallic Complexes of Rhenium(1). Synthesis and Biological Activity of the Conjugates Michele Salmain, Michele Gunn, Abdellaziz Gorfti, Siden Top, and GBrard Jaouen* Laboratoire de Chimie OrganomBtallique, URA 403, Ecole Nationale SupBrieure de Chimie de Paris, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France. Received February 17, 1993"
We describe herein a totally new pathway for the introduction of rhenium in the form of low oxidation state organometallic complexescovalently attached to various proteins. The synthesis of several rhenium conjugates takes advantage of the specificity of N-succinimidyl esters for amino residues. Conjugation experiments were carried out under various conditions, and analysis of the conjugates was performed by FT-IR spectroscopy. Yields were optimized and reached 50%. Furthermore, the conjugate resulting from the coupling of N-succinimidyl4- [q5-cyclopentadienylrheniumtricarbonyl] 4-oxobutanoate to an anti-hTSH monoclonal antibody retained a satisfactory immunoreactivity. Finally, IR detection of conjugates adsorbed onto nitrocellulose membranes was achieved and response was found to be related to the coupling extent of the conjugate.
INTRODUCTION The main concern of our research is to provide new "cold" biological probes, mainly organometallic, suitable for quantitative and qualitative analysis of ligandlreceptor interactions. Thus our early purpose was the synthesis of steroid hormones modified by an organometallic moiety, and affinity measurements for the natural receptor protein (1). This approach was extended to the labeling of haptens for the purpose of designing a new immunological assay method that we called CMIAl for "carbonylmetalloimmunoassay" which associates metal-carbonyl probes and FT-IR spectroscopy as a detection technique ( 2 ) . More recently, we developed a method of introduction of cobaltcarbonyl fragments into proteins in order to extend the field of CMIA to antigen assays (3). The introduction of metals into biologicals has been the subject of numerous publications reflecting the various applications ( 4 ) . Among them, radiolabeling with the aim of radioimmunodetection of cancer tumors shows great promise at the moment. This technique requires the use of specificmonoclonal antibodies labeled with a y-emitting radioisotope. Among these, gbTc is particularly interesting because of its ideal properties (5). On the other hand, monoclonal antibodies labeled with an adequate radioisotope (p-emittor this time) could be used in radioimmunotherapy procedures. For this purpose, isotopes "Re and lWReare valuable (5). The synthesis of labeled antibodies proceeds either from direct introduction onto the protein (with prior reduction of generator-produced 99mT~04or '%Reo4- (6)to lower oxidation state forms) or indirect introduction by prior coupling of strong chelating agents followed by complexation of the conjugate by 9gmTcV (7).However, both of these labeling techniques suffer from possible nonspecific ~~~
* Author to whom correspondence should be addressed.
Abstract published in Advance ACS Abstracts, October 1,
1993. 1 Abbreviations used CMIA, carbonylmetalloimmunoassay; FT-IR,Fourier transform infrared;NHS, N-hydroxysuccinimide; DCC, dicyclohexylcarbodiimide;DTGS, deuteriotriglycinesulfate; BSA, bovine serum albumin; hTSH, human thyroid stimulating hormone; PBS, phosphate-buffered saline; mAb, monoclonal antibody; CE, coupling extent; CY, coupling yield; PR, protein recovery.
1043-1802/93/2904-0425$04.00/0
binding that can induce leaching of the radioisotope and high background levels in vivo. On the other hand, the indirect introduction of 99mT~ (8)or "Re (9)as preformed coordination complexes may prevent this phenomenon. Organometallic complexes can also serve this purpose very well. Moreover, they have the advantage of being highly stable in biologicalmedia because of the very strong linkage between the metal and some well-chosen surrounding ligands (10). In the field of protein labeling, we have explored the organometallic chemistry of Re with a double aim in mind: first, the synthesis of new metal-carbonyl markers for CMIA assays and, second, the introduction of strongly bound Re into proteins as a model for radiolabeling with 99mTc, lWReor ls8Re. Our experience in the conjugation of organometallic entities of cobalt to proteins was based on the indirect introduction of the metal in its coordinated form (3). Moreover, theuse of N-succinimidyl esters which are specific for amino residues allows for the very strong attachment of the metal to the protein. This finding, and the absence of low-affinity binding of these markers, could make them very useful for in viuo studies. This was applied to rhenium and we describe in this article the synthesis of three rhenium carbonyl N-succinimidyl esters and their reactivity toward proteins like BSA and a monoclonal antibody anti-hTSH coded JOSS 2-2. Results of biological activity of the conjugate are also reported. EXPERIMENTAL PROCEDURES General. Rez(C0)lo was purchased from Strem Chemical Inc. (Newburyport, MO). Cyclopentadienylrhenium tricarbonyl (CpRe(C0)a)was synthesized in one step from Rez(CO)lo (yield = 90%) according to Tam et al. (11). Other chemicals were purchased from Aldrich (St. Louis, MO) or Janssen (Geel,Belgium). Solvents were purchased from Prolabo (Paris, France) or SDS (Peypin, France). Tetrahydrofuran was distilled under argon from a sodium/ benzophenone mixture before use. Bovine serum albumin (BSA) fraction V was obtained from Sigma (St. Quentin Fallavier, France) and the monoclonalantibody anti-hTSH coded JOSS 2-2 (3.2 mg/mL solution in PBS) was generously donated by Clonatec (Paris, France). The followingbuffers were prepared phosphate buffered saline (PBS, 0.01 M, 0.15 M NaC1, pH = 7.11, carbonate buffer (0.1 M, pH = 9.5), borate buffer A (0.1 M, pH = 8.01, 0 1993 American Chemical Society
426
Bioconjugate Chem., Vol. 4, No. 6, 1993
borate buffer B (0.1 M, pH = 8.6), and borate buffer C (0.1 M, pH = 9.0). Analytical and preparative thin-layer chromatography (TLC)were performed on silica gel glass plates (F60Merck, thickness 0.25 and 1 mm respectively), with UV visualization. Melting points were measured on a Kofler apparatus (Reichert, Model 184321). IR spectra were recorded on a Bomem (Quebec,Canada) Michelson 100 FT-spectrometer using a conventional DTGS detector for qualitative analysis and a liquidnitrogen-cooled InSb detector for quantitative analysis. lH NMR data were obtained with a Bruker AM-250 or -200 spectrometer (250 and 200 MHz, respectively). Mass spectra were recorded on a Nermag R 10-1OCspectrometer controlled by a digital PDP 11 system using electronic impact E1 (70 eV) or desorption chemical ionization (DCI) with NH3 as the reactant gas as indicated below. UVvisible spectra were recorded on a Kontron Uvikon 860 (Zurick, Switzerland). Elemental analyses (C, H, N) were performed at the SIAR of Jussieu (Paris, France). Synthesis of Rhenium Carbonyl Markers. All the experiments were performed under dry argon atmosphere, using standard Schlenk techniques. @-AlanineChloroacetamide Ethyl Ester 2. Chloroacetic acid (0.01 mol, 1 g) in THF was treated with equimolar quantities of NHS and DCC. After a night, the white precipitate of dicyclohexylurea was removed by filtration and the solvent was evaporated to dryness. The residue was redissolved in dry CHzClz and dry pentane was added to precipitate N-succinimidyl chloroacetate 1 (1.3 g, 68%): mp 120 "C; lH NMR (CDCl3) 6 4.4 (s,2H, CHZ), 2.89 (s,4H, NHS); IR (CHzClz) YCO 1837w, 1790w, 1746m. To @-alanineethyl ester hydrochloride (1g, 6.5 mmol) dissolved in THF was added triethylamine (0.66 g, 6.6 mmol). A white precipitate was formed. N-Succinimidyl chloroacetate (1.5 g, 6.3 mmol) was added and the mixture was stirred at room temperature for 4 h. After filtration and evaporation of the solvent, an oilyresidue was obtained which was washed with 0.1 M HC1 and water. The organic fraction was evaporated and @-alaninechloroacetamide ethyl ester 2 (1.2 g, 72 % ) was obtained as a yellow oil: IR (CH2C12) uco 1729 m, 1677 m, 1530 m; MS (EI) mlz 194 [MI*+,158 [M - Cl]*+,148 [M - OEt]*+,116 [H~NCHZCHzCOOEtl'+, 77 [ClCHzCO]*+;'H NMR (CDCl3) 6 7.25 (9, l H , NH), 4.18 (q,2H, J = 7 Hz, COOCHZCH~), 4.05 (s, 2H, ClCHZ), 3.58 (9, 2H, J = 6 Hz, NCHz), 2.59 (t, 2H, J = 6 Hz, CHzCO), 1.29 (t, 2H, J = 7 Hz, COOCH2CH3). Anal. Calcd for C7H1203NCl: C, 43.31; H, 6.20; N, 7.20. Found: C, 43.77; H, 6.20; N, 7.52. @-Alanine[Rhenium pentacarbonyllacetamideEthyl Ester 3. Rez(C0)lo (0.50 g, 0.76 mmol) was allowed to react with a 1% Na/Hg amalgam (0.05 g of Na, 5 g of Hg) in THF. The colorless solution turned yellow and then red. The reaction was followed by IR upon the disappearance of the 2069-cm-1uco band of Rez(CO)loand was completed in 3 h. NaRe(C0)E formed in situ was then allowed to react with @-alaninechloroacetamide ethyl ester 2 (0.3 g, 1.5 mmol). The mixture returned immediately to colorless. After 30 min, the mixture was filtered and the solvent evaporated. The residue was redissolved in ethyl ether and purified by TLC (eluent ethyl ether/THF 2ll). A 0.58-g (80%)portion of 3 was obtained as a white solid after recrystallization from an ethyl etherlpentane mixture: mp 90 OC; lH NMR (CDCl3) 6 5.84 (s, lH, NH), 4.13 (4, 2H, J = 7 Hz, COOCHZCH~), 3.49 (4, 2H, J = 6 Hz, NCHz), 2.52 (t, 2H, J = 6 Hz, CH2COO); 1.68 (s, 2H, ReCHd, 1.26 (t, 3H, J = 7 Hz, COOCH2CH3);IR (CH2C12)
Gunn et al.
uco 2133w, 2063sh, 2016vs, 1978m, 1778m, 1735w, 1642w, 1518w;MS (E11mlz (Re = 187)485 [Ml'+,457 [M-COl*+, 429 [M - 2CO]'+. @-AZanine[rheniumpentacarbonyllacetamide 4. (C0)sReCHzCONHCHzCH2COOEt3 (0.5g, 1mmol) was saponified by 0.03 M NaOH in ethanollwater 2/1. After 4 h, the solution was acidified by HCl and the solvent evaporated under reduced pressure. The residue was washed with ethyl ether to eliminate the unreacted ester. A 0.34g (73% ) portion of a white powder was obtained after recrystallization from an acetonelpentane mixture: mp 150 "C; 'H NMR (DMSO-de)6 12.06 (8, l H , COOH), 7.50 (t,l H , NH), 3.20 (9, 2H, NCHz), 2.30 (t, 2H, CHzCO), 1.60 (9, 2H, ReCH2); 13C NMR ( D M s 0 - d ~6) 184.24 (4 CO, Re(CO)), 181.67, 181.31, 173.27 (3 CO, Re(CO), CONH, COOH), 34.82, 34.75 (CHZ-CHZ),-4.23 (ReCHz); IR (THF) uco 2133w, 2063w, 2016vs, 1977m, 1733w, 1642w, 1517w; MS (EI) mlz (Re = 187) 457 [MI*+,429 [M - COl*+,401 [M - 2COI'+. 4-[Rhenium tricarbonyl(q5-cyclopentadieny1)l4-Oxobutanoic Acid 6. To a suspension of AlC13 (0.347 g, 1.3 mmol) in dichloromethane was added dropwise a solution containing equimolar amounts of succinic anhydride and CpRe(C0)s in CHzC12. The mixture was then heated to reflux for 24 h. It was cooled to room temperature and hydrolyzed with crushed ice and 0.3 mL of concentrated HC1. The mixture was treated with 20% sodium carbonate (w/v). The aqueous phase was acidified to pH 2 and extracted with ethyl ether. Then evaporation of the organic phase gave 0.29 g (42 % of a white solid which was recrystallized in an ethyl acetatelhexane mixture; 0.2 g of unreacted CpRe(C0)a was also recovered: mp 134 OC; lH NMR (CDCl3) 6 6.03 (t,2H, J = 2.4 Hz, Cp), 5.41 (t, 2H, J = 2.4 Hz,Cp),2.91 (m,2H,COCH2),2.75 (m,2H,CHzCOOH); IR (CH2C12) uco 2032~,1939~, 1 7 1 4 ~1, 6 8 9 ~MS ; (EI) mlz (Re = 187) 436 [MI*+,408 [M - COl'+, 322 [M - 2COl'+. Anal. Calcd for Cl2H906Re: C, 33.10; H, 2.06. Found: C, 33.14; H, 2.18. [q5-Cyclopentadienyl carboxylic acid] Rhenium Tricarbonyl 8. To CpRe(C0)s (0.5 g, 1.5 mmol) in THF at -78 OC was added sec-BuLi (1.7 mL, 2.1 mmol, 1.4 M solution in hexane). After 45 min, solid COZ (2 g) was added to the mixture, which was kept at -78 "C for 10 min. The cooling bath was removed, and after 5 min the mixture was diluted with CH2C12 and aqueous HC1 was added. The organic compounds were extracted with CH2Cl2. After evaporation of the organic phase, the crude product was washed with pentane and 0.46 g of a white powder was obtained. After crystallization from a CHzCldpentane mixture, 0.38 g of 8 was collected as white crystals (67%): mp 210 OC (lit. (12)mp 206-208 "C); 'H NMR (acetone-d6) 6 6.21 (t, 2H, J = 2.2 Hz, Cp), 5.71 (t, 2H, J = 2.2 Hz, Cp); IR (CH2Clz)YCO 2033~,1 9 3 9 ~ , 1 7 3 8 ~ , 1653w;MS (EI) mlz (Re = 187)380 [MI *+, 352 [M - COI *+, 324 [M - 2COl*+,296 [M - 3CO]*+. Synthesis of the N-Succinimidyl Esters of 4 6 , and 8. Actiuation of 4. To 4 (0.2 g, 0.56 mmol) in 15 mL of THF were added NHS (0.07 g, 0.61 mmol) and DCC (0.126 g, 0.61 mmol). After one night at room temperature, dicyclohexylurea was filtered out and the solvent evaporated under reduced pressure. The resulting white powder was identified to compound 5 by IR (see Table I) and used in labeling trials with no further purification. Actiuation of 6. 6 (0.1 g, 0.23 mmol) was activated into its N-succinimidyl ester in a similar way in 10 mL of THF by addition of NHS (0.031 g, 0.26 mmol) and DCC (0.054 g, 0.26 mmol). The resulting white powder was identified to 7 by IR analysis (see Table I).
Bioconjugate Chem., Vol. 4, No. 6, 1993 427
Labeling of Proteins by Complexes of Rhenium(1)
Activation of 8. 8 (0.1 g, 0.26 mmol) was activated into ita N-succinimidyl ester in a similar way in 8 mL of THF by addition of NHS (0.031 g, 0.26 mmol) and DCC (0.054 g, 0.26 mmol). The remaining white powder was washed with dry pentane and dried under vacuum. It was identified to compound 9 by IR analysis (see Table I). Labeling Studies of Amino Acids with 7'. In THF. Complete studies were carried out with the manganese equivalent of 7 called 7', which was synthesized in the same manner according to the procedure described by Le Plouzennec and Dabard (13). In a typical experiment, equimolar amounts of 7', the C-protected amino acid hydrochloride, and triethylamine in THF were allowed to react at room temperature for 3 h. The main compounds were purified by TLC and characterized by classical spectroscopic methods. The results are reported in Table 11. In a WaterlTHF Mixture. Equimolar quantities of 8-alanine and NaHC03 were dissolvedin water and allowed to react with 7' in THF (final mixture, water/THF 1/31. The mixture was stirred for 3 h at room temperature. It was then acidified to pH = 2 and extracted with ethyl ether. After evaporation of the organic phase, 0.08g (32%) of a yellow solid was obtained: mp 156 OC; IR (KBr pellet) vco 2023s,1938s,1679m,1626m,1560m;lH NMR (acetonede) 6 10.70 (9, l H , COOH) 7.22 (9, lH, NH), 5.72, 5.13 (9, 9, 2H, 2H, 2Cp) 3.42 (9,2H, NHCHz), 2.96 (9,2H, COCHz), 2.51 (s, 4H, CHzCOOH and CHzCO). Anal. Calcd for CldI1407NMn: C,48.00,H,3.73;N,3.73. Found C,48.09; H, 3.88; N, 3.63. Labeling Studies of BSA with 5 and 7. All experiments were performed on 21 nmol(l.5 mg; final protein concentration 0.75 mg/mL) of BSA following the same general protocol and using 60 molar equiv of the N-succinimidyl markers. Solutions (1.5 X lk3M) of the markers in THF were prepared just before use. A 1.5 mg/mL solution of protein was prepared in carbonate buffer (0.1 M, pH = 9.5). To 1mL of protein solution was added 1 mL of 5 or 7. Mixtures were incubated for 15 h at 4 OC. Labeling Studies of BSA with 9. All labeling experiments were performed from a 1 mg/mL stock solution of BSA in the incubation buffer (final protein concentration, 0.5 mg/mL) and from a 1 X le2M stock solution of 9 in ethanol or DMF (60 molar equiv; final concentration 5.1 mM). Effect of Reaction Time on the Labeling Ratio. To 6 mL of BSA in carbonate buffer, pH = 9.5, were added 600 pL of the ethanolic solution of 9 and 5.4 mL of ethanol (ethanol/water 1/11 or 600 pL of the DMF solution of 9, 600 pL of DMF and 4.8 mL of buffer (DMF/water 1/91. The mixtures were incubated at 4 OC and periodically assayed for (C0)3Re binding. Effect of pH on the Labeling Ratio. To 1mL of BSA in PBS (pH = 7.1) or borate buffer A (pH = 8.0) borate buffer B (pH = 8.6), or borate buffer C (pH = 9.0) or carbonate buffer (pH = 9.5) were added 100 pL of 9 in DMF, 100 pL of DMF, and 800 pL of buffer (DMF/water 1/9). The mixtures were incubated for 24 h at 4 OC. Effect of Percent Solvent on the Labeling Ratio. Twomilliliter solutions containing BSA and 9 were incubated in ethanol/water or DMF/water (pH = 9.5) mixtures for 24 h at 4 "C. Stability of BSA-Re(C0)3 Conjugates. A PBS solution of conjugate was incubated for 24 h at 37 "C. Protein and marker concentrations were assayed as described below and the results were compared to nontreated conjugate. Labeling of JOSS 2-2 with 7. A monoclonal antibody
anti-hTSH called JOSS 2-2 (IgG form, 99% purity) available as a PBS stock solution (3.2 mg/mL) constituted the source of protein. To 312.5 pL of stock solution (1mg, 6.7 nmol) was added 100 pL of a 2 X 10-3 M solution of 7 in acetonitrile (Le. 30-fold molar excess, 201 nmol, final concentration 100 pM). The volume was completed to 2 mL with carbonate buffer (0.1 M, pH = 9.5). The mixture was stirred a t 4 OC for 2 h. Purification of the Rhenium Carbonyl Conjugates. All conjugates (BSA and JOSS 2-2) were purified on a gel filtration column (5 cm X 2 cm; Sephadex G-25, ready to use PDlO column, Pharmacia). The incubation mixtures were completed to 2.5 mL, filtered on Millex-GV (0.22 pm), and poured onto the top of the column previously equilibrated with PBS. The elution was performed with PBS. Ten 1-mL fractions were collected. Each fraction was assayed for a metal-carbonyl fragment by IR and protein by the Coomassie Blue method as previously described (3). Fractions containing protein were combined and coupling extent (CE),coupling yield (CY),and protein recovery (PR) were calculated from the followingformulas: CE =
nmol of bound metal-carbonyl fragments (1) nmol of protein CY =
nmol of bound marker initial nmol of marker
(2)
PR = nmol of recovered protein (3) initial nmol of protein Immunoreactivity Assay. Dilutions (1/2 to 1/20 000) of (CO)sRe-labeled or native JOSS 2-2 (initial concentration 0.2 mg/mL in PBS) were incubated for 3 h at room temperature with a constant amount of lZSI-hTSH(10 000 cpm; NEN). After equilibration, bound antibody fractions were separated by immunoprecipitation by addition of a 1/50 dilution of a goat anti-mouse IgG antibody (Calbiochem) in PBS-Tween 20 0.1% containing 5% of PEG (incubation for 30 min at room temperature). Suspensions were centrifuged at 3000 rpm and precipitates counted for radioactivity. Detection of BSA-Re(C0)3 Conjugates by FT-IR Spectroscopy. Various dilutions of two conjugates with CE of 17 and 21 in a PBS solution were prepared. A 9-mm nitrocellulose membrane filter (0.45-pm porosity, Alltech, Templeuve, France) was dipped into each tube and incubated for 1or 1.5 h at room temperature. The filters were then removed, washed with PBS, and dried under vacuum for 30 min. The FT-IR spectrum was then recorded with a measurement time of 1 min (20 scans) taking a filter incubated in PBS as the reference. The absorbance of the 2030 cm-' carbonyl band was serially measured and plotted versus the protein concentration. RESULTS
Synthesis of the Rhenium-Carbonyl Carboxylic Acids. Two families of low oxidation state organometallic complexes of rhenium have been synthesized from commercial available Rez(C0)lo during this work. The first family has the general structure R-Re(CO)S, where Re is attached to a carbon via a Q bond and R bears a carboxylic function at the end of a linear bridge arm. This first type of marker has been chosen because it is readily accessible by condensation of sodium pentacarbonylrhenate which is strongly nucleophilic, with alkyl halides RC1, in which R possesses an ester group. This ester can be then hydrolyzed to the carboxylic acid which is condensed with a protein. This family of complexes presents a typical
428
Bioconjugate Chem., Vol. 4, No. 6, 1993
Gunn et ai.
Scheme I. Synthesis of Rhenium Pentacarbonyl Acid 4
Scheme 11. Synthesis of Rhenium Tricarbonyl Acids 6 and 8
CiCH@Ofi
0 1
1
H2NCH&&COOE1. NE13 in THF
1
CICH2CONHCH2CH2COOEt
I
NaNg 1% in M F
NaRe(COh
1.6- BuLi 2. co,
]
1
t @-COOH
2 R~(CO),
ie(~o), 6
t (CO)~R&H~CONHCH~CHZCCOE~
1::
6
Scheme 111. N-Succinimidyl Re, Mn, and Co Carbonyl Esters
3
3
(CO)5ReCHzCONHCHzCH2COON
Plethand
0
5
(CO)5R&H&ONHCH2CH@OH
0
4
infrared vco spectrum consisting of a sharp and intense band at about 2020 cm-l and a weaker band at about 1980 cm-l. The parent compound of this series, (CO)sReCH2COOH, was first prepared in this manner (14). However its activation into an N-succinimidyl ester failed. This nonactivation is the consequence of the Re(CO)5 effect, which causes a dramatic decrease of the acidity of the carboxylic function. In fact a pK, of 8.5 has been found for this acid, proving its weakness (14). One might think about using the acid (CO)sReCH2CH2COOH,where the effect of the metal-carbonyl entity on the acid function is diminished by addition of another CH2 radical, but we also failed to activate this acid. This lack of success may be attributed to the &elimination process which is commonly observed in organometallic systems (15). As a consequence, we oriented our research toward derivatives possessing a bridge with a long arm and no hydrogen in the 0-position of the Re(C0)S group. The carboxylic acid 4 was synthesized as shown in Scheme I in four steps, the last one being the saponification of the ester to form the acid. The bridge was obtained separately by condensation of chloroacetic acid with /3-alanine ethyl ester. The manganese analog of 4 was obtained in the same manner (data not shown). The second family can be represented by (RC5H4)Re(CO)3,where R bears a carboxylic function and is attached to the cyclopentadienyl ligand (Cp),which is very strongly wcoordinated to the metal. Like its homolog cymantrene, cyclopentadienylrhenium tricarbonyl reacts with succinic anhydride in the presence of AlC13 accordingto the FriedelCrafts reaction (Scheme11). Compound 6 was thus directly obtained by this method with its carboxylic function removed to the y-position from the cyclopentadienyl ring. From Rez(CO)lo,which is the starting material, an overall yield of 38 % was obtained. The cyclopentadienylrhenium tricarbonyl carboxylic acid 8 was prepared in one step by reaction of dry ice with the salt (C0)3Re(CsH4Li)(Scheme 11)(overallyield fromRe2(CO)lo,60%1. All thecompounds were characterized by classical spectroscopic methods and the results were in accordance with the expected structures. Activation of the Acids by N-Hydroxysuccinimide (NHS). The acids 4, 6, and 8 were activated into the corresponding esters 5, 7, and 9 (Scheme 111). The
0
7
9
0 7'
0 10
formation of the N-succinimidyl ester function was confirmed by its typical IR features in the vco region. Yields were quantitative in all cases. Three bands are typically observed for the three carbonyls of N-succinimidyl esters (see Table I). All the activated esters are stable under a dry atmosphere. Reactivity of Activated Ester 7' with a Series of Amino Acids. The reactivity of 7' was evaluated on a series of amino acids in THF (Table 11) and in a water/ THF mixture. In THF, expected condensation products were obtained with good yields (60% for @-alanineand cysteine, 40% for lysine). One can notice that in the case of lysine, the product of dicondensation was the only one separated, which explains the lower yield. In a water/ T H F mixture, the expected condensation product of 7' with unprotected &alanine was obtained with a yield of 32%. Side compounds observed were different, depending on the reaction medium. In THF, unreacted 7' was the only compound observed. In the THF/water mixture, a mixture of 7' and its corresponding carboxylic acid were observed but were not quantified. We have checked that 5,7, and 9 had a behavior very similar to that of 7' in the reaction with j3-alanine ethyl ester. Yields in rhenium carbonyl amides were on the same order of magnitude (50430%). Labeling Studies of BSA with 5,7, and 9. Labeling
Bioconjugate Chem., Vol. 4,
Labeling of Proteins by Complexes of Rhenlum(1)
Table I. IR Spectroscopic Data of Rhenium Carbonyl N-Sucoinimidyl Esters marker vco (cm-l) assignment 1816w
0
1741m 1785w
COON>
0
5
1745m 1787w
0 COON> 0
2028s 1936s
(COhRe
in THF 1818w (lit. 1825) 1784w (lit. 1784) 1739m (lit. 1739) c"$ (13b) 7'
2027s (lit. 2021) 1945s (lit. 1937) in CHzClz 1805w 1773w 1742m
0
(C0)3Mn
0
0
9
8
2s
-
20
-
i
15-
2134w 2063w 2019s 1988s in CHzClz 1816w
7
30 7
No. 6, 1993 429
2030s 1959s 1934s
(COhRe
in KBr
studies of BSA with rhenium carbonyl markers 5 and 7 were performed within the conditions previously described for the dicobalt hexacarbonyl marker 10 (3), except that ethanol was replaced by THF as the organic cosolvent (i.e. 15-h incubation time, % solvent = 50, pH = 9.5). An initial 60-fold molar excess of marker was chosen because it corresponds to the number of potentially reactive amino functions of BSA (59 lysine residues + 1 N-terminal residue). Purification of the conjugates was performed by gel-filtration chromatography. Calculation of the coupling extent, coupling yield, and protein recovery was carried out from IR measurements on KBr micropellets (for the quantitation of bound and free metal-carbonyl markers) and spectrophotometric measurements (for protein quantitation, Coomassie Blue method). The Coomassie Blue reagent is known to react mostly with arginine residues and can then be used to quantify the conjugates as well as the native protein (16). On the other hand, the intensity of the infrared signals of the bound marker is proportional to the number of metal-carbonyl entities. As a consequence, the sensitivity of a CMIA assay including a metal-carbonyl conjugate should increase with its CE. The results are reported in Table 111. Coupling extents of 43 and 27 were measured for reagents 5 and 7, respectively. Protein recovery was found equal to 80 % . As observed during our previous work with marker 10 (3), not all the amino functions are labeled and it seems that only approximately40 lysines are accessiblein the presence of an excess of reagent. Several parameters were studied in the course of labeling of BSA with 9, including the reaction time, the percent cosolvent (and the type of solvent), and the pH of the
1
06
7
8
4 10
PH
Figure 1. Effect of pH on the extent of labeling of BSA with 9 (mean of two experiments).
reaction mixture. Concentrations of protein (0.5 mg/mL) and marker (5.1 mM) and temperature (4 "C) were kept constant. The kinetics of labeling of BSA at pH 9.5 was performed in ethanol/water 50/50 and DMF/water 10/90 media. In the first case, the coupling extent is found nearly constant (average value, 18 f 2; n = 9). In the second case, the average CE is slightly higher (24 f 2; n = 4). The influence of percent cosolvent on the final coupling extent was studied for ethanol and DMF mixtures. A percentage of cosolvent ranging from 5 to 50% (v/v) was added to the mixture, which was incubated for 24 h at pH 9.5. When the cosolvent was ethanol, the maximum labeling was obtained for 25 % and a slight decrease was observed with a higher percentage (18 f 2; n = 9). When the solvent was DMF, the coupling extent was nearly constant and led to overall better results than ethanol (average value 27 f 2; n = 7). We finally studied the influence of pH of the incubation mixture on the coupling extent of marker 9 to BSA. The results are presented in Figure 1. This figure clearly indicates that the coupling extent of 9 to BSA increases with the pH in the range 7.1-9.5. Protein recovery remained constant and was almost quantitative. Surprisingly, the labeling of BSA occurs at neutral pH but to a lesser extent. In conclusion, the best conditions are pH = 9.5 and use of DMF as the cosolvent (10 % 1. As for the reaction time, 1 h seems sufficient. The average coupling extent with 60 equiv of 9 is then 24. Labeling of JOSS 2-2 with 7. The mAb JOSS 2-2 labeled with horseradish peroxidase is included as the second antibody in the ELISA kit for the assay of hTSH commercialized by Clonatec. Its immunological features have been reported (17). The labeling of native JOSS 2-2 with 7 was performed using conditions precedently described for the cobalt carbonyl marker 10 (3) and using a 30-fold initial excess of marker. This initial ratio of 30 to 1 was chosen because we had previously demonstrated that maximum CY was obtained within these conditions and that the resulting conjugate kept an unchanged immunoreactivity toward its antigen. The percentage of acetonitrile was decreased to 5% in order to limit denaturation processes which had been noticed. The results are reported in Table 111. The coupling yield is comparable to that reported for BSA (CY = 50%). Immunoreactivity Study. The immunoreactivity of Re(C0)s-labeled JOSS 2-2 (CE = 15) was evaluated by a classical dilution assay using a 1251-radiotracer. The binding of 125I-hTSHto Re(C0)3-JOSS 2-2 was measured and compared to that of native JOSS 2-2. For an antibody
430
Gunn et al.
Bioconjugate Chem., Vol. 4, No. 6, 1993
Table 11. Condensation of Some C-Protected Amino Acids with 7' in THF amino acid HzNCHZCHzCOOEt
IR vco yield (cm-l) 60% 2030s 1953sh 1944s 1730w 1681m 1522w
main compound
HSCH2CHCOOEt
NH2 HSCH~CH
