Bioconjugate Chem. 1996, 7, 380−384
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TECHNICAL NOTES S-[2-(4-Azidosalicylamido)ethylthio]-2-thiopyridine: Radioiodinatable, Cleavable, Photoactivatible Cross-Linking Agent Yon W. Ebright, Yan Chen, Younggyu Kim, and Richard H. Ebright* Department of Chemistry and Waksman Institute, Rutgers University, New Brunswick, New Jersey 08855. Received January 2, 1996X
S-[2-(4-Azidosalicylamido)ethylthio]-2-thiopyridine (AET) contains a 2-thiopyridyl moiety, which permits cysteine-specific incorporation into protein through a cleavable disulfide bond, and a 4-azidosalicylamido moiety, which permits radioiodination and photoactivatible cross-linking. In contrast to the related compound S-[2-[N-[4-(4-azidosalicylamido)butyl]carbamoyl]ethylthio]-2-thiopyridine [APDP; Zecherle, G., Oleinikov, A., and Traut, R. (1992) Biochemistry 31, 9526], AET contains a relatively short linker arm between the 2-thiopyridyl moiety and the 4-azidosalicylamido moiety. In a previous paper, it was shown that AET could be used in site-specific protein-protein photocross-linking to identify nearest-neighbor protein domains within a multiprotein complex [Chen, Y., Ebright, Y., and Ebright, R. (1994) Science 265, 90]. In this paper, the synthesis, radioiodination, and incorporation into protein of AET are described.
INTRODUCTION
Site-specific protein-protein photo-cross-linking followed by cross-link cleavage and radiolabel transfer is a powerful procedure for defining interactions within multiprotein complexes (1-4). The procedure permits identification of proteinssand amino acids within those proteinssthat are close to a site of interest within an intact, fully-assembled, functional multiprotein complex (3, 4). The procedure involves four steps: (i) incorporation of a radiolabeled, cleavable, photoactivatible crosslinking agent at a site of interest within one protein of a multiprotein complex; (ii) formation and photoirradiation of the derivatized multiprotein complex; (iii) cleavage of the resulting cross-link, with concomitant radiolabel transfer to the protein(s) and amino acid(s) at which cross-linking occurs; and (iv) identification of the protein(s) and amino acid(s) at which cross-linking occurs. 125I is a radioisotope of choice in site-specific proteinprotein photo-cross-linking followed by cross-link cleavage and radiolabel transfer, due to its ease of incorporation under mild conditions into phenol-containing agents and its availability at very high specific activities (up to 90 Bq/fmol) (3-5). In published work, two radioiodinatable agents have been used in site-specific proteinprotein photo-cross-linking followed by cross-link cleavage and radiolabel transfer: S-[2-[N-[4-(4-azidosalicylamido)butyl]carbamoyl]ethylthio]-2-thiopyridine (APDP; 3) and S-[2-(4-azidosalicylamido)ethylthio]-2-thiopyridine (AET or ACT; 4). The azidosalicylamido moiety in each agent permits radioiodination and photoactivatible crosslinking. The 2-thiopyridyl moiety present in each agent permits cysteine-specific incorporation into a protein through a disulfide linkagesa linkage cleavable under mild conditions. Each agent can be incorporated at a * Address correspondence to this author at Waksman Institute, Rutgers University, New Brunswick, NJ 08855 [telephone (908) 445-5179; fax (908) 445-5735]. X Abstract published in Advance ACS Abstracts, March 15, 1996.
S1043-1802(96)00016-X CCC: $12.00
single, defined site of interest within a protein by a twostep procedure consisting of (i) site-directed mutagenesis to introduce a unique surface cysteine at the site of interest, followed by (ii) cysteine-specific chemical modification. APDP results in a long linker arm between CR of the amino acid of interest and the photoreactive atom (21 Å if fully extended). The length of this linker arm is comparable to the diameter of an average protein domain (25 Å; 6) and is greater than the diameters of the smallest protein domains (19-20 Å; see 7). As such, the length of this linker arm precludes reliable identification of nearest-neighbor protein domainssmuch less nearestneighbor amino acidssin multiprotein complexes. AET results in a shorter linker arm between CR of the amino acid of interest and the photoreactive atom (14 Å if fully extended) and, as such, permits identification of nearest-neighbor protein domainssand, possibly, nearest-neighbor amino acidsswithin multiprotein complexes. In this report, we describe the synthesis, radioiodination, and conjugation to protein of AET. MATERIALS AND METHODS
S-(2-Aminoethylthio)-2-thiopyridine Hydrochloride. S-(2-Aminoethylthio)-2-thiopyridine hydrochloride was synthesized according to a modification of the procedure in ref 8. Thiopyridyl disulfide [Aldrich (Aldrithiol-2); 4.41 g, 20.0 mmol] was dissolved in 20 mL of methanol and 0.8 mL of acetic acid. Into this solution was added dropwise over a period of 0.5 h 2-aminoethylthiol hydrochloride (Aldrich; 1.14 g, 10.0 mmol) in 10 mL of methanol. The reaction mixture was stirred for an additional 48 h and then evaporated under high vacuum to a yellow oil. The product was washed with 50 mL of diethyl ether and was dissolved in 10 mL of methanol. The product was precipitated by addition of 200 mL of diethyl ether, chilled for 12 h at -20 °C, and collected by vacuum filtration (twice). Yield: 1.7 g, 77%; TLC (silica gel; ammonium hydroxide/95% ethanol, 1:99 © 1996 American Chemical Society
Technical Notes
Bioconjugate Chem., Vol. 7, No. 3, 1996 381
Figure 1. (A) Synthesis of AET. (B) Radioiodination of AET, yielding [125I]IAET. (C) Reaction of [125I]IAET with a protein having a unique surface cysteine residue, yielding a conjugate of the form [S-[2-([125I]iodo-4-azidosalicylamido)ethylthio]-Cys]protein. In the conjugate, the length of the linker arm between the R carbon of the cysteine residue and the photoreactive atom is 14 Å (1.9 times the length of an arginine side chain). The length of the linker arm is suitable for analysis of interactions occurring at, or just beyond, side-chain contact distance. (D) UV irradiation of a multiprotein complex containing the conjugate and nearest-neighbor protein X. (E) Cleavage of the cross-link, with concomitant radiolabel transfer to nearest-neighbor protein X.
v/v) Rf ) 0.8; NMR (CD3OD; tetramethylsilane as reference) δ 3.15 (2H, t, J ) 5.8 Hz), 3.30 (2H, t, J ) 6.0 Hz), 7.32 (1H, t, J ) 5.0 Hz), 7.64 (1H, d, J ) 8.3 Hz), 7.80 (1H, t, J ) 7.4 Hz), 8.54 (1H, d, J ) 4.4 Hz). S-[2-(4-Azidosalicylamido)ethylthio]-2-thiopyridine (AET). S-(2-Aminoethylthio)-2-thiopyridine hydrochloride (90 mg, 0.40 mmol) and sodium bicarbonate (70 mg, 0.80 mmol) were dissolved in 1.5 mL of water. Into this solution was added in three aliquots over a period of 1 h N-hydroxysuccinimidyl-4-azidosalicylic acid (9; Pierce; 100 mg, 0.36 mmol) in 4.5 mL of tetrahydrofuran. The reaction mixture was stirred for an additional 0.5 h and then extracted three times with 5 mL of diethyl ether. The ether extracts were pooled, washed twice with 10 mL of 1% HCl and once with 10 mL of brine, dried over anhydrous sodium sulfate, and evaporated. The resulting clear oil was dissolved in 1 mL of methanol. White crystals formed upon standing for 3 h at 4 °C and were collected by vacuum filtration. Yield: 39 mg, 31%; TLC (silica gel; ethyl acetate/chloroform, 1:3 v/v) Rf ) 0.8;
NMR (CD3OD; tetramethylsilane as reference) δ 3.18 (2H, t, J ) 5.3 Hz), 3.75 (2H, t, J ) 5.3 Hz), 6.53-6.62 (2H, m, J ) 2.0 Hz), 7.31 (1H, t, J ) 6.0 Hz), 7.58 (1H, d, J ) 8.3 Hz), 7.77-7.87 (2H, m, J ) 4.0 Hz), 8.65 (1H, d, J ) 4.9 Hz), 8.95 (1H, br s); FAB-MS m/e 348 (M + H)+. All operations in this and subsequent steps were performed in darkness or under safelamp illumination. S-[2-(Iodo-4-azidosalicylamido)ethylthio]-2-thiopyridine (IAET). In standard-scale reactions, 1,3,4,6tetrachloro-3R,6R-diphenylglycoluril [10, 11; Pierce (IODOGEN); 52 µg, 120 nmol] was plated on the walls of a 1.5 mL flat-bottom vial by evaporation under nitrogen of a 100 µL chloroform solution. AET (5.2 µg, 15 nmol) in 100 µL of 100 mM sodium borate (pH 8.4) and 0.5% dimethyl sulfoxide was mixed with KI (Aldrich; 2.5 µg, 15 nmol) in 20 µL of 30 mM sodium borate (pH 8.4), and the mixture immediately was added to the vial containing 1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril. After 30 s at 22 °C with vigorous stirring, reactions were terminated by transfer to a vial containing 15 µL of 8 mM methionine
382 Bioconjugate Chem., Vol. 7, No. 3, 1996
Figure 2. Radioiodination of AET. AET was allowed to react with 125I- and the solid-phase oxidant 1,3,4,6-tetrachloro-3R,6Rdiphenylglycoluril (10, 11). Reaction products were analyzed by TLC, with detection by UV shadowing (A) or autoradiography (B).
and 1 mM tyrosine. TLC (silica gel; chloroform/ethyl acetate, 95:5 v/v) Rf ) 0.75 (monoiodinated AET) and 0.88 (diiodinated AET). In preparative-scale reactions, 1,3,4,6-tetrachloro3R,6R-diphenylglycoluril [10, 11; Pierce (IODO-GEN); 200 mg, 460 µmol] was plated on the walls of a 500 mL Erlenmeyer flask by evaporation under house vacuum of a 25 mL chloroform solution. AET (20 mg, 60 µmol) in 2 mL of dimethyl sulfoxide and KI (10 mg, 60 µmol) in 26 mL of 100 mM sodium borate (pH 8.4) were mixed and then added to the prepared flask containing 1,3,4,6tetrachloro-3R,6R-diphenylglycoluril. After 15 min at 22 °C with vigorous stirring, reaction mixtures were filtered and extracted three times with 20 mL of chloroform. The chloroform extracts were pooled, washed with 20 mL of brine, dried over anhydrous sodium sulfate, and evaporated to an oil. Products were purified by preparative TLC (silica gel, 1000 µm; ethyl acetate/chloroform, 95:5 v/v) Rf ) 0.7 (monoiodinated AET) and 0.8 (diiodinated AET); FAB-MS m/e 474 [monoiodinated AET, (M + H)+] and 599 [diiodinated AET, (M + H)+]. S-[2-([125I]Iodo-4-azidosalicylamido)ethylthio]-2thiopyridine ([125I]IAET). Standard-scale reactions
Ebright et al.
were performed as described for IAET using KI (Aldrich; 2.2 µg, 13 nmol) and Na125I (New England Nuclear; 0.30 µg, 2.0 nmol, 190 MBq). Vials were equipped with rubber septa vented through 10 g activated-charcoal filters, and transfers were made using syringes (see 5). Photolysis of AET and IAET. Samples contained 6 µM AET in 100 mM sodium borate (pH 8.4), 0.5% dimethyl sulfoxide or 4 µM IAET in 80 mM sodium borate (pH 8.4), 0.4% dimethyl sulfoxide, 0.9 mM methionine, and 0.1 mM tyrosine. Photolyses were carried out for 0-640 s at 22 °C (350 nm; 1 × 105 erg mm-2 s-1) in a Rayonet RPR100 photochemical reactor (Southern New England Ultraviolet). Reaction vessels were 1 mL polystyrene microcentrifuge tubes (Fisher) held inside 13 × 100 mm borosilicate glass tubes; these reaction vessels exclude wavelengths less than 290 nm. Absorbance spectra were recorded using a Lambda 3A UV-vis spectrophotometer (Perkin-Elmer). [S-[2-([125I]Iodo-4-azidosalicylamido)ethylthio]Cys152;Ser178]CAP ([125I]IAE]CAP). Reaction mixtures contained (in 100 µL) the following: 70 µM [125I]IAET (12 Bq/fmol; radioiodinated immediately before use); 4 µM [Cys152;Ser178]CAP or [Ser178] (prepared as described for [Ser178] in ref 12); 50 mM sodium borate (pH 8.3); 8 mM Tris-HCl (pH 8.3); 80 mM KCl; 0.4 mM EDTA; 5 mM methionine; 0.6 mM tyrosine; 2% glycerol; and 0.2% dimethyl sulfoxide. Reactions were carried out for 20 min at 22 °C. [[125I]IAE]CAP was purified by chromatography on Bio-Gel P6DG (Bio-Rad) and was stored at -70°C in 40 mM Tris-HCl (pH 8.0), 100 mM KCl, 10 mM MgCl2, and 5% glycerol. 125I was quantified using a Gamma 5500 gamma counter (Beckmann), 2-thiopyridone was quantified using a Lambda 3A UV-vis spectrophotometer (Perkin-Elmer) [343 ) 8080 M-1 cm-1 (1)], and protein was quantified using Protein Gold reagent (Integrated Separation Systems). RESULTS AND DISCUSSION
Synthesis of AET. AET was prepared in two steps: (i) reaction of 2-aminoethylthiol hydrochloride with thiopyridyl disulfide, yielding S-(2-aminoethylthio)-2-thiopyridine hydrochloride; and (ii) reaction of S-(2-amino-
Figure 3. Photolysis of AET (A) and IAET (B). Photolyses were carried out for 0-40 s at 22 °C (350 nm; 1 × 105 erg mm-2 s-1).
Technical Notes
Bioconjugate Chem., Vol. 7, No. 3, 1996 383
A
Figure 5. Cleavage of linkage to protein. [S-[2-([125I]Iodo-4azidosalicylamido)ethylthio]-Cys152;Ser178]CAP was allowed to react with 0.25 mM β-mercaptoethanol for 5 min at 22 °C. Reaction products were analyzed by SDS-PAGE followed by autoradiography.
B
Figure 4. Incorporation into protein. In parallel experiments, [125I]IAET in large molar excess was allowed to react with [Cys152;Ser178]CAP (a CAP derivative having a single surface cysteine residue; solid circles) and [Ser178] [a CAP derivative having no surface cysteine residues (12); open circles]. Reaction products were isolated by gel filtration chromatography, and efficiencies of reaction were determined by quantitation of 125I incorporated (A) and 2-thiopyridone released (B).
ethylthio)-2-thiopyridine hydrochloride with N-hydroxysuccinimidyl-4-azidosalicylic acid (Figure 1A). The overall yield was 24%. Radioiodination of AET. AET was radioiodinated using 125I- and the solid-phase oxidant 1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril (10, 11) (Figure 1B). TLC indicated that >90% of the AET was consumed and that two main products were generated, in the ratio 1.5:1 (Figure 2A) (cf. 2, 11). TLC followed by autoradiography indicated that the two products contained iodine (Figure 2B). After a parallel reaction using nonradioactive I-, the two products were isolated by preparative TLC, analyzed by FAB-MS, and found to have masses corresponding to monoiodinated AET (3-iodo-AET) and diiodinated AET (3,5-diiodo-AET) (see Materials and Methods). All further analysessphotolysis, attachment to protein, and site-specific protein-protein photo-cross-linking (see below and ref 3)swere carried out using unfractionated reaction products (i.e., monoiodinated AET and diiodinated AET). The specific activity of the unfractionated reaction products was 70% of the specific activity of the input 125I-. Photolysis of AET and IAET. AET has absorbance maxima at 273 ( ) 26 000 M-1) and 330 nm ( ) 9500 M-1 cm-1) (Figure 3A). UV irradiation at 350 nm and 1 × 105 erg mm-2 s-1 for 10 s results in loss of absorption
at 273 and 330 nm, indicative of destruction of the azido group (Figure 3A). Further photolysis results in no further loss of absorption. IAET has absorbance maxima at 273 ( ) 23 000 M-1) and 343 nm ( ) 8000 M-1 cm-1) (Figure 3B). UV irradiation at 350 nm and 1 × 105 erg mm-2 s-1 for 2-4 s results in loss of absorption at 273 and 343 nm, and further photolysis results in no further loss of absorption (Figure 3B and data not shown). The ability to carry out photolysis with relatively long wavelengths (350 nm) and relatively short times (
