Bioconjugate Chem. 1990, 1, 138- 143
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Photoaffinity Heterobifunctional Cross-Linking Reagents Based on N-(Azidobenzoyl)tyrosines’ Nobuyuki Imai Tadashi Kometani,t Peter J. Crocker,t Jean B. Bowdan,+ Ayhan Demir,+ Lori D. Dwyer,i Dennis M. Mann,$ Thomas C. Vanaman,*Si and David S. Watt*yt Departments of Chemistry and Biochemistry, University of Kentucky, Lexington, Kentucky 40506. Received February 12, 1990
New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N - [N’-(4-azidobenzoyl)tyrosyl]-~-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct N-(N’-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N’-(3-azido-5-nitrobenzoyl)tyrosyl]-~-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calciumdependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2+-ATPaseand to a synthetic fragment (M13)containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency. The interaction of the ubiquitous calcium-binding protein calmodulin with a variety of other proteins including myosin light-chain kinase and membrane Ca2+pumping ATPases lies at the heart of certain regulatory events mediated by calcium ions and remains a topic of intense current interest (1-3). An effort to define these protein-protein interactions on a molecular level led to the construction of cross-linking reagents ( 4 , 5 )that possessed the following features: a radiolabel of high specific activity, an electrophilic succinimidyl ester terminus, and a photoactive aryl azide terminus. As an additional feature, an enzymatically cleavable linking arm connecting these termini was incorporated in the reagent with the expectation that a reagent with such a cleavable bond would permit the isolation of a fragment of the target protein in a cross-linking experiment as illustrated in the cartoon in Scheme I. In a study of two interacting proteins 2 and 4, reagent 1 would interact with a protein 2 to form a protein-reagent adduct 3. Subsequent binding of a second protein 4 and photolysis of complex 5 would lead to covalently cross-linked adduct
Scheme I. Cartoon Representation of a Cross-Linking Experiment
.
chemically cleavable
1 f enzymatically cleavable (chymotrypsin)
Legend: ArN, = aryl azide,
* Authors to whom correspondence should be addressed. Department of Chemistry. Department of Biochemistry. Part of this work was disclosed at the following meetings: Imai, N., Bowdan, J. B., Crocker, P. J., Watt, D. S., Vanaman, T. C., and Dwyer, L. D. Meeting of the American Chemical Society, Los Angeles, CA, September 1988 and Dwyer, L. D., Imai, N., Crocker, P. J., Mann, D. M., Watt, D. S., and Vanaman, T. C. FASEB Summer Research Conference on Calcium and Cell Function, Saxton’s River, VT, July 1989. The abbreviations used are as follows: BOC-ON, 2-[[(tertbutoxycarbonyl)oxy]imino]-2-phenylacetonitrile; Et,N, triethylamine; CaM, calmodulin; DCC, N,N’-dicyclohexylcarbodiimide; DMAP, 4-(dimethy1amino)pyridine;DMSO, dimethyl sulfoxide;DSO, disuccinimidyloxalate; EtOAc, ethyl acetate; EGTA, ethylene glycol bis(0-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HOAc, acetic acid; M13, a synthetic peptide containing the CaMbinding region of myosin light-chain kinase; TFA, trifluoroacetic acid; THF, tetrahydrofuran;TRIS, tris(hydroxymethy1)aminomethane.
.
HZN
-
H,N
-
6
41
= radio label; = cleavable = covalent bond between
bond, 0 = F , Y , K ; reagent 1 and p r o t e i n
6. Whereas chemical cleavage of a specific bond in the linking arm of 6 and SDS-PAGE separation would permit the identification of the entire protein 4 by autoradiography, the enzymatic cleavage, depicted in Scheme I for the case of chymotrypsin, would provide only a fragment 7 of target protein 4. Clearly, an enzymatically cleavable reagent of this type would be best suited for the delination of binding sites between two purified proteins 3 and 4 in which the covalent attachment site in 3 was defined and in which enzymatic cleavage of the linking arm would provide a fragment that presumably could be separated and sequenced in order to define the binding site on protein 4. Before surmounting the separation problems necessary to reduce this notion to practice, it was necessary to synthesize heterobifunctional reagents of this type, test the feasibility of using these enzymatically cleavable reagents in model systems, and
1043-1802/90/2901-0138$02.50/0 0 1990 American Chemical Society
Tyrosine-Based Cross-Linking Reagents
perform preliminary experiments to verify that these reagents would effect a photochemical cross-link. EXPERIMENTAL PROCEDURES General Chemical Procedures. Infrared spectra were recorded on a Perkin-Elmer Model 1310 spectrometer. Nuclear magnetic resonance spectra were determined on a Varian EM390 or Gemini 200 MHz NMR spectrometer. Chemical shifts are reported in parts per million relative to tetramethylsilane as an internal standard. Mass spectra were determined on a VG ZAB spectrometer. Elemental analyses were performed by Atlantic Microlabs, Norcross, GA. Column chromatography using Macherey Nagel silica gel 60 is referred to as "chromatography on silica gel", preparative-layer chromatography on Macherey Nagel silica gel F254 is referred to as "chromatography on a silica gel plate", and the drying of an organic solution over anhydrous magnesium sulfate is simply indicated by the phrase "dried". The following experimental section contains detailed procedures only for the reagent that was successfully employed in biochemical ,cross-linking experiments (16C). Experimental data for other compounds appear in the supplementary material. 3-Azido-5-nitrobenzoic Acid (12C). To a solution of 2.00 g (9.46 mmol) of methyl 3,5-dinitrobenzoate (6) in 135 mL of acetone, 25 mL of acetic acid, and 25 mL of water a t reflux was added 2.17 g (38.9 mmol) of iron in three portions at intervals of 20 min. After stirring at the reflux temperature for an additional 35 min, the mixture was cooled and filtered. The filtrate was adjusted to pH 8 with sodium carbonate, extracted with EtOAc,' washed with brine, and dried. The crude product was chromatographed on silica gel using 2:3 EtOAc-hexane to afford 0.71 g (38%) of methyl 3-amino-5-nitrobenzoate: mp 159-160 "C [lit (7,8) mp 159-160 "C]. To a solution of 1.45 g (7.39 mmol) of methyl 3-amino-5-nitrobenzoate in 170 mL of water was added 17 mL of concentrated HC1 at 0-5 "C. To this solution at 0 "C was slowly added 663 mg (9.61 mmol) of NaNO,. The solution was stirred for 45 min a t 0 "C, and 577 mg (8.87 mmol) of NaN, was slowly added. The mixture was stirred for 45 min at 0 "C and was extracted with EtOAc. The organic layer was washed with brine and dried. The product was chromatographed on silica gel using 2:3 EtOAchexane to afford 1.37 g (84%) of methyl 3-azido-5nitrobenzoate: mp 93-94 "C; IR (KBr) 2114 (N,), 1721 (CO) cm-'; 'H NMR (CDCl,) 6 4.01 (s, 3, OCH,), 8.01 (dd, J = 1.2, 2.2 Hz, 1, ArH), 8.04 (t,J = 2.2 Hz, 1, ArH), 8.60 (dd, J = 1.2, 2.2 Hz, 1, ArH). Anal. Calcd for C,H,N,O,: C, 43.25; H, 2.72. Found: C, 43.37; H, 2.87. To a solution of 1.34 g (6.06 mmol) of methyl 3-azido5-nitrobenzoate in 225 mL of MeOH was added 1.25 mL of water and 0.59 g (13.9 mmol) of lithium hydroxide monohydrate. The mixture was stirred for 7 h at 50 "C. The solution was acidified with 2 N aqueous HCl solution and extracted with EtOAc. The organic layer was washed with brine, dried, and concentrated to afford 1.41 g (100%) of 12C (9): mp 186-188 "C dec. Succinimidyl3-Azido-5-nitrobenzoate (13C). To a solution of 1.38 g (6.62 mmol) of 12C and 0.84 g (7.3 mmol) of N-hydroxysuccinimide in 50 mL of anhydrous T H F was added 1.50 g (7.28 mmol) of DCC. The mixture was stirred for 16 h at 25 "C and filtered. The filtrate was concentrated, dissolved in EtOAc, washed with aqueous NaHCO, solution and brine, and dried. The solvent was removed to afford 1.98 g (98%) of 13C that was sufficiently pure to be used in the next reaction: mp 163-164 "C dec; IR (KBr) 2128 (N,), 1809, 1788 (CON), 1743 (CO,) cm-l; 'H NMR (CDC1,) 6 2.96 (s, 4, CH,CH,), 8.07
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(dd, J = 2.3 and 1.4 Hz, 1, ArH), 8.17 (t, J = 2.1 Hz, 1, ArH), 8.72 (dd, J = 2.0 and 1.4 Hz, 1, ArH); exact mass spectrum calcd for C,,H,N,O, 305.0396, found 305.0396. Methyl N-[N'-(tert-Butoxycarbonyl)tyrosyl]-8alanate (15). The procedure described for the preparation of 13C was repeated with 5.62 g (20 mmol) of N-(tertbutoxycarbony1)tyrosine (14), 2.76 g (24 mmol) of N-hydroxysuccinimide, and 4.53 g (22 mmol) of DCC in 60 mL of anhydrous T H F to afford, after stirring for 16 h a t 25 "C, 5.98 g (79%) of succinimidyl N-(tert-butoxycarbony1)tyrosine as white prisms: mp 183-184 "C (from EtOAc-hexane); IR (KBr) 3426 (OH), 3372 (NH), 1820, 1788 (CON of NHS), 1741 (CO,NHS), 1683 (CO of BOC) cm-';'H NMR (DMSO-$) 6 1.32 (s, 9, C(CH,),), 2.79 (s, 4, (CH,), of NHS), 2.72-3.12 (m, 2, CHCH,), 4.25-4.62 (m, 1, CHCH,), 6.68 and 7.13 (2 d, J = 8.1 Hz, 4, ArH), 7.53 (d, J = 8.1 Hz, 1,NH), 9.20 (s, 1,OH). Anal. Calcd for C,,H,,N,O,: C, 57.14; H, 5.86; N, 7.40. Found: C, 57.02; H, 5.94; N, 7.37. To a solution of 5.98 g (15.8 mmol) of succinimidyl N-(tert-butoxycarbony1)tyrosinateand 2.65 g (19.0 mmol) of methyl /3-alanate hydrochloride in 60 mL of DMSO was added 3.19 g (31.6 mmol) of Et,N. The mixture was stirred for 19 h a t 50 "C, diluted with EtOAc, washed successively with dilute HC1 solution and brine, and dried. The crude product was chromatographed on silica gel using 1:l EtOAc-CHC1, to afford 3.83 g (66%) of 15: mp 104106 "C; IR (KBr) 3348 (OH), 1732 (CO,Me), 1698 (CON of BOC), 1664 (CON) cm-'; 'H NMR (CDC1,) 6 1.37 (s, 9, C(CH,),), 2.41 (t, J = 5.4 Hz, 2, MeO,CCH,), 2.93 (d, J = 6.3 Hz, 2 , CHCH,), 3.42 (t, J = 5.4 Hz, 2, MeO,CCH,CH,), 3.66 (s, 3, OCH,), 5.27 (d, J = 7.2 Hz, 1,CHCH,), 6.58 (t, J = 6.3 Hz, 1, NH), 6.75 and 7.01 (2 d, J = 9 Hz, 4, ArH), 7.68 (br s, 1,OH). Anal. Calcd for C18H,,N,06: c, 59.00; H, 7.15. Found: c , 59.02; H, 7.18. S uccinimidyl N-[N '-(3-Azido-5-nitrobenzoy1)tyrosyl]-/3-alanate (16C). To a solution of 1.1g (3 mmol) of 15 in 20 mL of anhydrous MeOH was bubbled HC1 gas for 5 min at 0 "C. The mixture was stirred for 2 h a t 25 "C and concentrated to afford 856 mg (94%) of crude methyl N-tyrosyl-/3-alanate hydrochloride: 'H NMR (CD,OD) 6 2.40 (t, J = 6.3 Hz, 2, MeO,CCH,), 2.96 (d, J = 7.2 Hz, 2, CHCH,), 3.33 (t, J = 6.3 Hz, 2, Me0,CCH,CH,), 3.62 (s, 3, OCH,), 3.95 (t, J = 7.2 Hz, 1, CHCH,), 6.75 and 7.05 (2 d, J = 9 Hz, 4, ArH). To a solution of 484 mg (1.60 mmol) of methyl N-tyrosyl-/3-alanate hydrochloride and 406 mg (1.33 mmol) of 13C in 8 mL of DMSO was added 263 mg (2.66 mmol) of Et,N. The mixture was stirred for 20 h at 25 "C, diluted with EtOAc, washed successively with dilute HCl solution and brine, and dried. The crude product was chromatographed on silica gel using 1:l EtOAc-CHCl, to afford 368 mg (69%) of methyl N-[N'-(3-azido-5-nitrobenzoyl)tyrosyl]-/3-alanate: mp 137-139 "C dec (EtOAc-hexane); IR (KBr) 3498 (OH), 3370,3300 (NH), 2128 (N3), 1734 (CO,), 1643, 1628 (CON) cm-'; 'H NMR (DMSOde) 6 2.46 (t, J = 7.0 Hz, 2, MeO,CCH,), 2.79-3.08 (m, 2, CHCH,), 3.24-3.55 (m, 2, MeO,CCH,CH,), 3.60 (s, 3, OCH,), 4.55-4.74 (m, 1, CHCH,), 6.63 and 7.11 (2 d, J = 8.3 Hz, 4, ArH of Tyr), 7.95 (d, J = 1.4 Hz, 1, ArH of ArN,), 8.00 (t, J = 1.4 Hz, 1,ArH of ArN,), 8.31 (t, J = 5.8 Hz, 1, CH,NHCO), 8.43 (d, J = 1.4 Hz, 1, ArH of ArN,), 9.12 (d, J = 6.8 Hz, 1, CHNHCO), 9.18 (s, 1, OH). Anal. Calcd for C,,H,,N,O,: C, 52.63; H, 4.42. Found: C, 52.73; H, 4.43. To a solution of 137 mg (0.3 mmol) of methyl N-[N'(3-azido-5-nitrobenzoyl)tyrosyl] - @ - a h a t ein 3.6 mL of 1:5 water-MeOH was added 76 mg (1.8 mmol) of lithium
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hydroxide monohydrate. The mixture was stirred for 18 h at 25 "C and concentrated. The residue was acidified with 2 N HCl solution. The solution was extracted with EtOAc, washed with brine, and dried. Concentration afforded 142 mg of crude N- [N'-(3-azido-5-nitrobenzoyl)tyrosyl]-P-alanine: mp 94-96 OC dec; IR (KBr) 3316 (OH), 2128 (NJ, 1719 (CO,), 1647,1620 (CON) cm-l; 'H NMR (CD,OD) 6 2.52 (t, J = 6.8 Hz, 2, HO,CCH,), 3.05 (d, J = 9.0 Hz, 2, CHCH,), 3.23-3.73 (m, 2, HO,CCH,CH,), 6.72 and 7.16 (2 d, J = 8.1 Hz, 4, ArH of Tyr), 7.77, 7.94, and 8.33 (3 s, 3, C,H,(NO,)N,). The procedure described in the preparation of 13C was repeated with 118 mg (0.27 mmol) of N-[Nt-(3-azido-5nitrobenzoyl)tyrosyl]-@-alanine,37 mg (0.32 mmol) of N hydroxysuccinimide, and 55 mg (0.27 mmol) of DCC in 10 mL of anhydrous T H F to afford, after stirring for 16.5 h at 25 "C, 117 mg (81%) of 16C: mp 142-145 "C dec (from EtOAc-hexane); IR (KBr) 3320 (NH), 2122 (N3), 1813,1780 (CON of NHS), 1731 (CO,), 1640 (CONH). Preparation of Calmodulin. CaM was isolated from bovine testes according to the procedure of Jamieson and Vanaman (10). Preparation of Human Erythrocyte Plasma Membrane Ca2+,Mg2+-ATPase,Ca2+,Mg2+-ATPasewas isolated from outdated human blood according to the procedure of Niggli et al. (11). Activity assays were performed by the method described by Niggli et al. (11) by using a colorimetric test for free phosphate. Synthetic Fragment (M13) of Myosin LightChain Kinase Containing the CaM Binding Region. The peptide (12) called M13 was synthesized a t the Macromolecular Structure Analysis Facility at the University of Kentucky, Lexington, KY with Biosearch 9600 peptide synthesizer. Functionalization of CaM with Succinimidyl N'-[N"-(3-Azido-5-nitrobenzoyl)tyrosyl]-j3-alanate (16C). A mixture containing 30 mM HEPES (pH 7.4), 10 pM CaM, 20 pM 16C dissolved in HPLC-grade acetonitrile (1mg/mL) immediately prior to use, and either 2 mM CaCl, or 5 mM EGTA was incubated for 2 h at 25 "C, and the reaction was quenched by the addition of lysine to a final concentration of 10 mM. The modification of CaM was monitored by HPLC on a C-3 RPSC column (Altex). The calcium dependence of cross-linking was determined by substituting 2 mM EGTA for CaC1, in separate experiments. Separation of native and modified CaM was achieved with a gradient increasing from 25% to 45% acetonitrile in 10 mM sodium dihydrogen phosphate (pH 6.0) and 2 mM EGTA as described by Mann and Vanaman (13) and shown in Figure 1. The purified monoadduct of CaM and 16C was digested with Staphococcus aureus V8 protease (Sigma) a t a ratio of 1:20 V8/CaM in the presence of EGTA. Glutamatespecific cleavage was achieved by conducting the digest in ammonium bicarbonate buffer at pH 8.0. The peptides were separated with a Phenyl pBondpak HPLC (Waters) column using a 1% /min gradient of 0.05% TFA and 88% acetonitrile in 1% TFA. The single peptide exhibiting 310-nm absorbance characteristic of the nitroaryl azide was sequenced with an Applied Biosystems 477A peptide sequencer. Preparation of lZ5I-LabeledMonoadduct of CaM and Succinimidyl N'-[Nt'-(3-Azido-5-nitrobenzoyl)tyrosyl]-j3-alanate (16C). Radioiodination of 100 pg of the monoadduct of CaM and reagent 16C was achieved with 100 pCi of sodium ['251]iodide and Enzymobeads (Bio-Rad) according to the procedure supplied by BioRad.
0
IO TIME (min)
3
Figure 1. Reverse-phase HPLC profiles (C-3column) of adducts of CaM and cross-linking reagent 16C: Upper trace in the presence of Ca2+ and lower trace in the absence of Ca2+ as monitored a t 230 nm. The arrow denotes the Lys-75 monoadduct of CaM and 16C.
Photolabeling of Ca2+,Mg2+-ATPase with lZ5I-Labeled Monoadduct of CaM and 16C. A solution of 0.7 pM Ca2+,Mg2+-ATPasewas incubated with 2 pM of 1251labeled monoadduct of CaM and 16C (0.5 mCi/pmol) in 30 mM HEPES (pH 7.4), 130 mM NaCl, 2 mM MgCl,, 100 pM CaCl,, 0.05% (v/v) Triton X-100,5% (v/v) glycerol, and 0.5 mg/mL phosphatidylcholine. The calcium dependence of cross-linking was determined by substituting 2 mM EGTA for CaC1, in separate experiments. Solutions were photolyzed with a hand-held ultraviolet light with the glass face removed (4600 pW/cm2, Model IJVS-11, Ultra-violet Products, Inc.) at a distance of 4 cm for 1 min, suspended in a protein-solubilizing mixture of 65 mM Tris, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, and 0.001% (w/v) bromophenol blue, and boiled for 5 min. The resulting solution was resolved on 7.5% SDS-PAGE, and the gel was silver-stained and autoradiographed for 48 h. Photolabeling of the M13 Peptide. A solution of 4 pM monoadduct of CaM and 16C, 12 pM M13 peptide, and 2 mM CaC1, in 30 mM HEPES (pH 7.4) was photolyzed with a hand-held ultraviolet light as described above. The calcium dependence of cross-linking was again determined by substituting 2 mM EGTA for CaC1, in separate experiments. The photolyzed mixture was suspended in the protein-solubilizing mixture described above and boiled for 5 min. The product was resolved on 15% SDS-PAGE, and the gel was silver-stained. RESULTS
Chemical Synthesis. After selecting tyrosine as the central structural element for these cross-linking reagents, we explored the addition of the photoactive aryl azide group a t either the C-terminus or the N-terminus of tyrosine. As shown in Scheme 11, the straightforward acylation of 4-azidoaniline (8) (14) with N-(tert-butoxycarbonyl)tyrosine, deblocking, and subsequent acylation with disuccinimidyl adipate furnished the potential crosslinking reagent 11 in which the C-terminus of the tyrosine residue held the photoactive portion of the reagent. As shown in Scheme 11, incorporating the photoactive group at the N-terminus in the cross-linking reagents 16A, 16B, or 16C proceeded through a route that involved the acylation of @-alaninemethyl ester with N-(tert-butoxy-
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Scheme 11." Synthesis of Tyrosine-Based Reagents 0
N3
OH 11
14
OH
OH 16
15 N1
A series: Ar = O
N
B series: Ar =
l
4 N1
C series: Ar =
NO2
Legend for reagents: (a) DCC, 4-azidoaniline (8); (b) HC1, HOAc; (c) NHSOCO(CH,),CO,NHS, E t N; (d) NHS-OH, MeO,CCH,CH,NH,-HCl, Et,N; (e) HCl, MeOH; (f) ArC0,NHS (13); (g) LiOH; (h) N-hydroxysuccinimide, DC& Scheme 111." Model Studies for t h e Iodination and Enzymatic Cleavage NHCOAr
NHSOCO
NHCOAr
OH
16
NHCOAr
nBuNHCO+
OH
19XE Y =I
Aseriis: Ar= e
OH
NHCOAr
OH
22
dN3 4 N3
N
3 Bseries: A r =
Cseriis: Ar=
No2
Legend for reagents: (a) n-BuNH,; (b) NaI, chloramine T; (c) LiOH; (d) N-hydroxysuccinimide, DCC; (e) chymotrypsin, H,NCOCH(NH,)CH,C,H,OH. a
carbonyl)tyrosine, deblocking, and subsequent acylation with succimidyl esters 13 of 4-azidobenzoic acid (12A), 3-azidobenzoic acid (12B),or 3-azido-5-nitrobenzoic acid (12C), respectively. An alternate route to succinimidyl esters 16 in which the acylation of tyrosine methyl ester with succinimidyl esters 13 preceded acylation with 6alanine methyl ester furnished the same products (16) but in poorer overall yield than the route shown in Scheme 11. In comparison, cross-linking reagents 16 derived from azide-substituted benzoates were stable and readily purified whereas cross-linking reagent 11 derived from 4azidoaniline (8) was produced in poor yield and was not readily generalized to incorporate other substituents such as a nitro group in the azidophenyl ring. The electrophilic succinimidyl ester terminus in these reagents was expected to react with various nucleophilic groups in the target protein calmodulin. Since mammalian calmodulin lacked cysteine residues, the reactivity of succinimidyl esters with amino and hydroxyl groups was evaluated in a model study. Although succinimidyl esters are used routinely in reactions with amines (1517), there was little precedent for the reactions of suc-
cinimidyl esters with alcohols. The alcoholysis of various succinimidyl esters afforded esters in good yields from primary alcohols, secondary alcohols, and phenols only if the alcohol component was used in excess (18). Under conditions useful for protein labeling, the cross-linking reagents were expected to exhibit selectivity for lysine residues rather than serine or threonine residues, a fact which we subsequently confirmed in studies using calmodulin. As a model study for the radioiodination (19) and acylation processes needed for biochemical studies, the iodination (20,21) of succinimidyl ester 16A followed by treatment with n-butylamine, a surrogate for a lysine residue, led, as shown in Scheme 111, to a mixture of monoand diiodinated products (18A and 19A) that could not be separated. For characterization purposes, the iodination of ester 20A, separation of the mono- and diiodinated esters, saponification, and coupling with n-butylamine furnished either pure 18A or 19A. Although iodinated amide 18A was not identical with the cross-linked adduct 6 in Scheme I in the sense that the azide group in 18A was still intact, we tested whether chymotrypsin
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200-
31 22
-
11697
42
hv
8
1AI
hu
-
.
+
-
+
Figure 3. Silver-stained SDS-PAGE gel of cross-linking exper-
+
+
-
-
+ +
Figure 2. SDS-PAGE and autoradiograms of cross-linking experiments with CaM. Lanes 1, 3, and 5 correspond to the silverstained gels, and lanes 2,4, and 6 correspond to the autoradiograms of lanes 1, 3, and 5, res ectively. Lanes 1 and 2 correspond to the photolysis of the l2 I-labeled CaM-16C monoadduct and Ca2+,Mg2+-ATPasein the presence of Ca2+. Lanes 3 and 4 correspond to an experiment conducted in the absence of light, and lanes 5 and 6 correspond to an experiment conducted in the absence of Ca2+. Less protein was used in lane 5, and this accounted for the reduced intensity of bands in this lane. Molecular weight markers are indicated a t the left of lane 1.
t
would accept such a substrate. As shown in Scheme 111, exposure of iodinated amide 18A to chymotrypsin in the presence of a nucleophile such as tyrosinamide (22)intercepted the acylated chymotrypsin intermediate and led to the isolation of the desired adduct 22A in 69% yield. Functionalization of CaM with Succinimidyl N’-[N ”-( 3-Azido-5-nitrobenzoyl)tyrosyll-/3-alanate (16C). CaM was treated with reagent 16C in the presence and absence of calcium chloride. In the presence of calcium, CaM underwent specific modification at Lys75. Separation of native and modified CaM was achieved as described by Mann and Vanaman (13) and as shown in Figure 1. Under these conditions, 59% of the CaM was modified by 16C and the Lys-75 derivative (indicated by an arrow in Figure 1)comprised 78% of the modified CaM or 46% of the total CaM used in the experiment. The purified monoadduct of CaM and 16C was digested with S. aureus V8 protease (Sigma) and the single peptide exhibiting 310-nm absorbance characteristic of the nitroaryl azide was sequenced to confirm complete modification of Lys-75 (data not shown). P hotolabeling of Ca2+,Mg2+-ATPase with 1251-Labeled Monoadduct of CaM and 16C. Irradiation of a solution of Ca2+,Mg2+-ATPaseand 1251-labeledmonoadduct of CaM and 16C (0.5 mCi/pmol) in the presence of calcium led, after separation on 7.5% SDS-PAGE and autoradiography, to the gels and autoradiograms in Figure 2. The calcium dependence of cross-linking was determined by substituting 2 mM EGTA for CaC1, in separate experiments. As shown in lanes l and 2, photolysis in the presence of Ca2+ yielded a radiolabeled band in the autoradiogram corresponding to a cross-linked species of CaM and Ca2+,M$+-ATPase having a M,of approximately 150 kDa. In the absence of Ca2+ (lanes 5 and 6) or of photolysis (lanes 3 and 4), the radiolabeled band was absent, demonstrating that the formation of the crosslinked species required both Ca2+and light. Photolabeling of the M13 Peptide. Irradiation of a solution of monoadduct of CaM and 16C and the M13 peptide led, after separation on 15% SDS-PAGE and silver-staining, to the gel in Figure 3. Lane l shows the photolysis of the M13 peptide and the CaM-16C monoadduct in the presence of Ca2+. The higher molecular weight band in Figure 3 is the cross-linked species. On the basis of the computed molecular weights of the cross-linked
iments with M13. Lane 1 corresponds to the photolysis of the CaM-16C monoadduct and M13 in the presence of Ca2+. Lane 2 corresponds to an experiment conducted in the absence of light, and lane 3 corresponds to an experiment conducted in the absence of Ca2+. Molecular weight markers are indicated a t the left of lane 1.
species and CaM, there is a difference of ca. 2000 Da, and since the molecular weight of M13 is 1427 Da, this indicates that only one M13 is cross-linked per CaM molecule. An estimate of cross-linking efficiency was provided by densitometry that showed that ca. 20% of the CaM was cross-linked to M13. The high yield of photocross-linked species obviated, in this case, the need for radioisotopes. Lanes 2 and 3 demonstrate that the crosslinking is light and Ca2+ dependent, respectively. DISCUSSION
New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, an 1251-radiolabel,and an enzymatically cleavable linking arm between the two termini were synthesized and applied to cross-linking studies of calmodulin derivatized at Lys-75 and either the human erythrocyte plasma membrane Ca2+,Mg2+-ATPaseor a synthetic fragment (M13) containing the CaM-binding region of myosin lightas the radiolabel was chain kinase. The selection of 1251 based on the availability of sodium iodide in high specific activity, the reasonable half-life, and the ease with which 1251 could be introduced in the cross-linking reagents late in the synthetic route. The neutral conditions necessary to preserve the succinimidyl ester functionality during radioiodination dictated the need for an activating phenolic hydroxyl group (20,21). This need as well as the desire to incorporate an enzymatically cleavable bond led to the selection of a tyrosine residue as the spacer between the electrophilic and the photoactive termini. Reagents in which the photoactive aryl azide was attached to the N-terminus rather than the C-terminus of tyrosine were preferred on the basis of ease of synthesis and product stability. Model studies involving the successful coupling of one of these reagents (16A) with n-butylamine as a surrogate for a lysine residue, iodination, and chymotrypsin cleavage (22)in the presence of tyrosinamide, as shown in Scheme 111, augured well for the proposed biochemical applications. This result, although promising, does not guarantee success in the biochemical system where steric bulk of the protein cross-linked through the azide may also play a role. Preliminary studies indicated that reagents 16 were viable cross-linking reagents. Exposure of reagent 16C to calmodulin and subsequent HPLC purification provided a principal monoadduct of 16C and calmodulin. Trypsin digestion of this monoadduct and sequencing of the azide-bearing fragments indicated that 16C reacted preferentially a t Lys-75 in calmodulin in a specific, calcium-dependent manner. This Lys-75 monoadduct of CaM and 16C retained 100% of the biological activity of calmodulin as established by activation of human erythrocyte plasma membrane Ca2+,Mg2+-ATPase (data not
Tyrosine-Based Cross-Linking Reagents
shown). Radioiodination of the this monoadduct and photolysis in the presence of Ca2+,Mg2+-ATPaseled to successful, calcium-dependent cross-linking as indicated by the gels and autoradiograms in Figure 2. A similar crosslinking experiment using the lZ5I-labeledmonoadduct of CaM and 16C and M13, a synthetic fragment of myosin light-chain kinase containing the CaM-binding region, also led to a successful, calcium-dependent cross-linking as shown in Figure 3. A detailed report of these latter studies will appear in due course. In summary, this study defined the structural parameters necessary for an enzymatically cleavable, heterobifunctional cross-linkingreagent and demonstrated, in a preliminary fashion, the success of such a rationally designed reagent in two cross-linking experiments. ACKNOWLEDGMENT
We thank the National Science Foundation (Grant CHE8607441) for their financial support, K. Draw and A. Bernd of Degussa AG for a generous gift of amino acids, NATO for a travel grant (Grant RG #0346/88) to D.S.W. and A.D., and the University of Kentucky for the purchase of bond issue equipment and for Faculty Grant Initiative Awards (to D.S.W. and T.C.V.). Supplementary Material Available: Synthetic procedures and characteristic data (melting point, 'H NMR, IR, MS, and analysis) for 9-11,12A,B, 13B, 16A,B, 17A, MA, 21A, and 22A (9 pages). Ordering information is given on any current masthead page. LITERATURE CITED (1) Vanaman, T. C. (1980) Calcium and Cell Function (W. Y. Cheung, Ed.) Vol. l., Chapter 3, Academic Press, New York. (2) Klee, C. B., and Vanaman, T. C. (1982) Calmodulin. Adu. Protein Chem. 35, 213-321. (3) Vanaman, T. C. (1983) Chemical approaches to the calmodulin system. Methods Enzymol. 102, 296-310. (4) Peters, K., and Richards, F. M. (1977) Chemical crosslinking: reagents and problems in studies of membrane structure. Annu. Rev. Biochem. 46, 523-551. (5) Bayley, H. (1983) Photogenerated Reagents in Biochemistry and Molecular Biology Elsevier, New York. (6) Crampton, M. R., and Khan, H. A. (1972) A spectroscopic study of the formation of isomeric Meisenheimer complexes from l-methoxycarbonyl-3,5-dinitrobenzene. J. Chem. SOC., Perkin Trans. 2, 733-736. ( 7 ) Terpko, M. O., and Heck, R. F. (1980) Palladium-catalyzed triethylammonium formate reductions. 3. Selective reduction of dinitroaromatic compounds. J . Org. Chem. 45,49924993.
Bioconjugate Chem., Vol. 1, No. 2, 1990
143
(8) Ayyangar, N. R., Kalkote, U. R., Lugade, A. G., Nikrad, P. V., and Sharma, V. K. (1983) Partial reduction of dinitroarenes to nitroanilines with hydrazine hydrate. Bull. Chem. SOC.Jpn. 56,3159-3164. (9) Myers, D. E., and Utter, M. F. (1981) The enzymatic synthesis of some potential photoaffinity analogs of benzoylcoenzyme A. Anal. Biochem. 112, 23-29. (10) Jamieson, G. A., and Vanaman, T. C. (1979) Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine. Biochem. Biophys. Res. Commun. 90,1048-1056. (11) Niggli, V., Zurini, M., and Carafoli, E. (1987) Purification, reconstitution, and molecular characterization of the Ca2+ pump of plasma membranes. Methods Enzymol. 139, 791808. (12) Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191. (13) Mann, D. M., and Vanaman, T. C. (1988) Modification of calmodulin on Lys-75 by carbamoylating nitrosoureas. J. Biol. Chem. 263, 11284-11290. (14) Smith, P. A. S., Hall, J. H., and Kan, R. 0. (1962) The electronic character of the azido group attached to benzene rings. J . Am. Chem. SOC.84, 485-489. (15) Cline, G. W., and Hanna, S.B. (1987) The aminolysis of N-hydroxysuccinimide esters. A structure-reactivity study. J. Am. Chem. SOC.109,3087-3091. (16) Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1964) The use of esters of N-hydroxysuccinimide in peptide synthesis. J . Am. Chem. SOC.86, 1839-1842. (17) Takeda, K., Sawada, I., Suzuki, A., and Ogura, H. (1983) A convenient synthesis of peptides using oxalates. Tetrahedron Lett. 24,4451-4454. (18) Kometani, T., Watt, D. S., and Fitz, T. (1988) An esterification procedure using N-hydroxysuccinimidyl esters. Toyama Kogyo Koto Senmon Gakko Kiyo 22, 7-10 [(1989) Chem. Abstr. 110, 153854~1. (19) Counsell, R. E., Smith, T. D., Ranade, V. V., Noronha, 0. P. D., and Desai, P. (1973) Potential organ or tumor imaging agents. 11. Radioiodinated tyramines. J. Med. Chem. 16, 684-687. (20) Kometani, T., Watt, D. S., and Ji, T. (1985) Iodination of phenols using chloramine T and sodium iodide. Tetrahedron Lett. 2043-2046. (21) Kometani, T., Watt, D. S., Ji, T., and Fitz, T. (1985) An improved procedure for the iodination of phenols using sodium iodide and tert-butyl hypochlorite. J . Org. Chem. 50, 53845387. (22) Fink, A. L. (1973) The a-chymotrypsin-catalyzed hydrolysis of N-acetyl-L-tryptophan p-nitrophenyl esters in dimethyl sulfoxide a t subzero temperatures. Biochemistry 12,17361742.
