Bioconjugate Chem. 1995, 6, 395-400
395
Tethered Benzophenone Reagents for the Synthesis of Photoactivatable Ligands J o h n D. Olszewski,' Gyorgy Dorman,+J o h n T. Elliott,' Yang Hang,* David G. Ahern,* a n d Glenn D. Prestwich",' Department of Chemistry, University at Stony Brook, Stony Brook, New York 11794-3400, and E. I. DuPont de Nemours & Co. (Inc.), Biomedical Products Division, 549 Albany Street, Boston, Massachusetts 02118. Received January 17, 1995@
A new radiolabeled, bifunctional photoaffinity cross-linking reagent, N-succinimidyl p-benzoyl-[2,33H21dihydrocinnamate,has been synthesized in high yield and with high specific activity. This reagent can be used to append the benzophenone photophore to amino groups of small molecules, such as 0-aminoalkylinositol polyphosphates and polypeptides. The resulting tritiated photoaffinity labels can be purified and manipulated in ambient light and can be activated a t 360 nm.
INTRODUCTION
0
Although many mono- and bifunctional reagents incorporating photoactivatable groups have been described, only arylazide and diazoester photophores have been commercially available. Moreover, few existing photophore-containing heterobifunctional reagents have been available in a radioactively labeled form. In connection with our efforts in mapping binding sites for peptide hormones and for inositol polyphosphates, we required a versatile, high specific activity, tritium-labeled reagent that was chemically robust and stable in ambient light. The benzophenone (BPI photophore appeared well-suited to this task, and BP-containing photoaffinity labels have been employed to circumvent many of the problems associated with the use of nitrene- and carbene-producing photoaffinity labels (DormBn & Prestwich, 1994). In particular, (i) BPs can be activated at wavelengths (> 320 nm) that are not associated with protein damage, (ii) BPs are reported to give highly efficient covalent labeling via insertion into unreactive C-H bonds, (iii) BP photolabeling shows no interference from water or bulk nucleophiles, and (iv) no special lighting conditions are necessary during synthesis and biochemical manipulations. Recently, the BP moiety has been used in polypeptides as 4-benzoylphenylalanine (ONeil & DeGrado, 1989; ONeil et al., 1989; Boyd et al., 1991; McNicoll et al., 1992; Shoelson et al., 1993; Williams & Shoelson, 1993) and as 4-benzoylbenzoyl esters and amides in nucleotides (Mahmood et al., 1989; Boyer et al., 1990; Chavan et al., 1990; Gonzalez et al., 1990; Pal & Coleman, 1990; Aloise et al., 1991; Bar-Zvi et al., 1992; Pal et al., 1992; Rajagopalan et al., 1993; Salvucci et al., 1993; Zarka & Shoshan-Barmatz, 19931, in sugars (Holman et al., 1988), in phospholipids (Ishidate et al., 1992), and in proteins (Leszyk et al., 1988; Agarwal et al., 1991; Rajasekharan et al., 1991; Combeau et al., 1992; Thiele & Fahrenholz, 1993). Interactions within lipid bilayers have also been probed using BP-lipid analogs (Yamamoto et al., 1993). We report herein the development of a versatile BPbased heterobifunctional cross-linking reagent that may be used to modify small molecules or peptides for photoaffinity labeling studies. Figure 1 shows this new reagent, 4-benzoyldihydrocinnamicacid N-hydroxysuccinimidate (BZDC-NHS) (la),which is readily prepared +
@
University at Stony Brook. E. I. DuPont de Nemours & CO. (Inc.). Abstract published in Advance ACS Abstracts, June 1,1995.
l a X=H l b X4H
0
Pa XsH 2b X d H
Figure 1.
in tritiated form, specific activity > 30 Ci/mmol, with the label in non-solvent-exchangeable positions (lb). A second reagent, 4-benzoyldihydrocinnamic acid sulfo-Nhydroxysuccinimidate (BZDC-sulfo-NHS, 2a,b) was prepared for modification of proteins in aqueous solutions. The amide linkage between reagent and polypeptide (or other ligand) is stable under standard conditions for HPLC purification of peptides, Edman degradation, CNBr digestion, and proteolysis, thus facilitating mapping of the covalently modified binding site. EXPERIMENTAL PROCEDURES
Materials and Methods. All NMR spectral data were obtained on either QE-300 or AC-250 instruments. Melting points are uncorrected. Elemental analyses were performed by M-H-W Laboratories (Phoenix, AZ). All reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI), except sulfo-NHS, which was obtained from Pierce Chemical Co. (Rockford, IL). All solvents used were reagent grade or HPLC grade. CH2Clz was distilled from CaH2, and DMF was dried by storage over molecular sieves. The normal workup procedure involved extraction three times with the appropriate solvent, washing the combined organic extracts once with brine, drying over anhydrous MgS04, filtration, and concentration. Chromatographic purification was performed over "flash" grade Si02. HPLC retention times are denoted as t ~ . p-BenzoylcinnamicAcid (4) (Moureyet al., 1993). The following detailed description contains modifications
1043-1802/95/2906-0395$09.00/00 1995 American Chemical Society
Olszewski et al.
396 Bioconjugate Chem., Vol. 6,No. 4, 1995
&:
- f0
.'O~PO '
2
~
3
~
N H
&yfJ \
o
~
7 9
-9\
\
Y
C - N - ~ - C - N - ~Y- CO -N-~-COOH
x
X
H H
H H
7H2 y
X = H or3H
X = H or3H
BZDC-IPg BZDC-GGR
LSDDMPATPADQEMYRQDPEQIDSRT-NH
2
7H2 YH C=NH
Y9
7 -C-YFSPRL-NHz y
2
yH2
&JJ:
NH
x
o
X=Hor3H Lys27.BZDC-PBAN
Figure 2.
to the original procedure to ensure optimal yield. To a three-necked round-bottomed flask was added p-aminobenzophenone (3)(800 mg, 4.06 mmol), 15 equiv of 48% HBr (3.2 mL, 58.86 mmol), and 8-10 mL of acetone. The mixture was stirred a t 0 "C in a n icehalt bath while 5 M aqueous sodium nitrite was added dropwise until a dark brown color was achieved. After the mixture was stirred an additional 5 min a t 0 "C, 30 equiv of acrylic acid (8.34 mL, 121.8 mmol) was added while the temperature was maintained a t 0-5 "C, and the mixture was immediately placed under a steady stream of nitrogen and stirred a t 0 "C for 15 min. Cuprous bromide (2 mol %, 12 mg, 0.08 mmol) was added, the flow of nitrogen was removed, and the mixture was stirred a t 0 "C (10 min) and a t ambient temperature (30 min, or until gas evolution had ceased). The mixture was then poured into 5 vol of water and allowed to sit overnight. A yellow-orange oil that separated out was extracted with CH2C12, the normal workup procedure was followed, and the product was eluted from Si02with CHC13, followed by 50% MeOH in CHC13. The product (cu. 94%) containing traces of acrylic acid was used without further purification. Crude a-bromoacid (1.2 g, 3.60 mmol) was dissolved in 40 mL of MeOH, powdered NaOH (2.25 g, 56.3 mmol) was added, and the mixture was stirred for 40 min a t ambient temperature as a white precipitate formed. The reaction mixture was concentrated in vucuo, the resulting solid was dissolved in water, and the mixture was slowly acidified with concentrated hydrobromic acid while it was stirred. The cinnamate 4 precipitated as an off-white solid after being stored overnight a t 4 "C (88% yield). Sometimes a slightly yellow product was obtained, which could be purified by suspending it in hot acetone and collecting the insoluble material after cooling to give 4: mp = 229-230 "C. Anal. Calcd for C~H1203:C, 76.18; H, 4.79. Found: C, 75.93; H, 5.00. p-Benzoyldihydrocinnamic Acid (5,x = 1). Cinnamate 4 (200 mg, 0.793 mmol) was suspended in 20 mL of 95% ethanol, and 5% palladium on carbon (14 mg, 7% by weight) was added. The flask was evacuated and filled with hydrogen to atmospheric pressure. The mixture was stirred vigorously for 110 min, filtered over a bed of Celite, and washed several times with MeOH. The filtrate was concentrated, and the product was eluted from Si02 with 3:l:l Hex/EtOAc/CH&l2 containing 1% acetic acid to give a white solid in 82.5% yield of 5: mp
= 99-100 "C. 'H-NMR (CDC13,300 MHz) 6 2.74 (t, 2H, J = 7.5 Hz), 3.05 (t, 2H, J = 7.5 Hz), 7.33 (d, 2H, J = 8.1 Hz), 7.47 (t, 2H, J = 7.2 Hz), 7.58 (t, lH, J = 7.2 Hz), 7.77 (t, 4H, J = 8.4, 8.7 Hz), 11.45 (br s, COOH). 13CNMR (CDCl3, 63 MHz) 6 30.49, 35.09, 128.28, 130.00, 130.56, 132.37, 135.91, 137.73, 145.23, 178.64, 196.59. Anal. Calcd for C16H1403: C, 75.57; H, 5.55. Found: C, 75.34; H, 5.69. N-Succinimidylp-Benzoylcinnamate(6) was prepared according to known procedures. The product could be recrystallized from EtzO/Hex to result in fine white needles in 84.5% yield: mp = 176-177 "C. Anal. Calcd for C20H15N05: C, 68.76; H, 4.33; N, 4.01. Found: C, 68.92; H, 4.17; N, 4.14. N-Succinimidylp-Benzoyldihydrocinnamate(la) was prepared as described (Estevez, 1991). The product was crystallized from EbO/Hex and isolated as fine white needles in 84% yield: mp = 100-102 "C. 'H-NMR (CDC13,300 MHz) 6 2.85 (s,4H), 2.98 (t, 2H, J = 7.8 Hz), 3.16 (t, 2H, J = 7.8 Hz), 7.36 (d, 2H, J = 8.1 Hz), 7.48 (t, 2H, J = 7.2 Hz), 7.59 (t, l H , J = 7.2 Hz), 7.79 (m, 4H). 13C-NMR (CDCl3, 63 MHz) 6 169.04, 167.70, 143.92, 137.65, 136.13, 132.35, 130.62, 129.98, 128.28, 32.16, 30.37, 25.59. Anal. Calcd for C20H18N05.5 (partially hydrated): C, 66.66; H, 5.03; N, 3.89. Found: C, 67.02; H, 5.28; N, 4.05. N-Sulfosuccinimidylp-Benzoyldihydrocinnamate (2a). Acid 5 (69 mg, 0.272 mmol) was dissolved in DMF (6 mL). Sulfo-N-hydroxysuccinimide(65 mg, 0.299 mmol) was added, followed by a solution of DCC (61.6 mg, 0.299 mmol) in 3 mL of DMF, and the mixture was stirred for 24 h a t ambient temperature. An additional 0.5 equiv of both sulfo-NHS and DCC were added, and stirring was continued for another 24 h. The flask was stored a t -4 "C overnight, and the cyclohexylurea byproduct was removed by filtration. ARer being washed with 5 mL of cold DMF, the filtrate was concentrated under reduced pressure to give a light yellow solid. The solid was suspended in CHzClz, filtered, and washed repeatedly with CHzClz and EtOAc. The filtrate, which had become cloudy with voluminous precipitate, was filtered and washed with a small amount of cold EtOAc to give the product a s a n off-white solid in 79% yield: mp = 254-255 "C (dec). IH-NMR (DMSO-~B, 250 MHz) 6 2.83 (s, lH), 2.90 (8,lH), 3.07 (s, 4H), 3.96 (br d, lH), 7.47-7.71 (m, 9H). 13C-NMR (DMSO-d6, 63 MHz) 6
Tritiated Benzophenone Phootlabel
168.75, 165.39, 144.65, 135.19, 132.54, 129.85, 129.51, 128.64,128.55,56.25,31.14,30.85,29.68.FAB-MS Calcd for CzoH17NO&3 (sulfonic acid form): 431.42. Found: 431.0. p-[3HlBenzoyldihydrocinnamicAcid (5, x = 3). Hydrogenation using carrier-free tritium gas was performed a t DuPont-New England Nuclear (Boston, MA). A mixture of 5 (15 mg, 0.06 mmol) and 5% palladium on carbon (1.2 mg, 8% by weight), suspended in 2 mL of EtOH, was stirred under a n atmosphere of carrier-free tritium gas for 90 min. Labile tritium was removed, and the mixture was filtered through a 5 x 25 mm bed of Celite and washed with 10 mL of MeOH. After concentration, the product was purified by preparative TLC; first with Hex/EtOAc/CHzClz(3:l:l) on a Whatman PL& silica plate and then with MeOH/H20/HOAc (70:30:0.1) on a Whatman PKCls plate. The product was isolated in 30% yield (800 mCi) with specific activity = 44.8 Ci/ mmol as determined by MS. N-[3H]Succinimidylp-Benzoyldihydrocinnamate (lb). Hydrogenation using carrier-free tritium gas was performed a t DuPont-New England Nuclear. Unsaturated ester 6 (18 mg, 0.05 mmol) and 5 mg of 5% palladium on carbon were suspended in 2 mL of EtOAc, placed under an atmosphere of carrier-free tritium gas, and stirred a t atmospheric pressure and ambient temperature for 4 h. The catalyst was removed by filtration and washed twice with 2 mL of EtOAc. The filtrate was concentrated, and labile tritium was removed by rotary evaporation twice using MeOH as a carrier. The product was purified by preparative HPLC using a Zorbax silica column with detection a t 280 nm. For analytical scale separations, the elution solvent was 97.5:2.5 CHzC12/ EtOAc, and the flow rate was 1 mumin; t~ = 29.45 min for the starting material and 48.52 min for the product. The product may be stored in H e a t O A c a t or below -20 "C (I 5 mCi/mL) for several months without chemical or radiochemical decomposition. Specific activity of different lots of L3H1BZDC-NHSranged from 30 to 60 CUmmol. [3H]-N-Sulfosuccinimidyl p-Benzoyldihydrocinnamate (2b). From a stock solution of p-L3H1benzoyldihydrocinnamic acid in methanol, an aliquot containing 2 mCi was placed in a 5-mL reaction vessel and evaporated to dryness under a stream of argon. Dry DMF (20 pL) and solutions of sulfo-NHS (3 pL of a 5 mg/ mL stock) and DCC (3 pL of a 5 mg/mL stock) in DMF were added, and the mixture was stirred a t ambient temperature for 48 h. Due to slow progression of the reaction (analysis by TLC and autoradiography), additional sulfo-NHS (3pL of stock) and DCC (3pL of stock) were added and stirring was continued for a n additional 48 h. These additions were repeated and stirring was continued for another 48 h. The crude reaction mixture was applied to a Si02 column (0.8 x 8 cm), and the product was eluted with 4:l CHClfleOH in 8% radiochemical yield. Analysis by TLC and autoradiography showed that the product Rf matched that of the cold standard and had a purity > 95%. General Procedure for p-[2,3-3H21Benzoyldihy. drocinnamyl Derivatives of P-140-Aminoalkyl) Tethered Inositol Polyphosphates. An example for the Ins(1,4,5)P3 derivative is described (Mourey et al., 1993). The r3H1BZDC-NHSester (0.5 mL of stock solution containing 2 mCi/mL in ethyl acetatehexane, 1:l) was carefully concentrated under nitrogen. The residue was redissolved in 50 pL of DMF and added to 50 pL of a stirred solution (0.42 mM in 0.25 M triethylammonium bicarbonate, TEAB) of the P-l-(O-aminopropyl)-Ins(1,4,5)P3 (Prestwich et al., 1991). The reaction mixture was stirred for 48 h a t ambient temperature and then
Bioconjugate Chem., Vol. 6,No. 4, 1995 397
concentrated in vacuo. The residue was taken up in 50 pL of water and concentrated to dryness again to remove any remaining TEAB and DMF. The residue was taken up in 0.5 mL of water and applied to a 4 x 0.5 cm column of DEAE-cellulose (HC03- form). The column was washed with 1 mL of water and eluted sequentially with 1-mL volumes of 0.1 M TEAB, 0.2 M TEAB, 0.3 M TEAB, and 0.4 M TEAB followed by 2-mL volumes of 0.5 M TEAB, 0.6 M TEAB, and 0.8 M TEAB. A 1-pL aliquot of each fraction was monitored by liquid scintillation counting. The high-activity fractions (generally 0.4 and 0.5 M TEAB) were analyzed by reversed-phase HPLC (RPHPLC, Aquapore RP-300) (15% CH3CN in 0.05 M KHzPO4 buffer, pH = 4.4) using UV 220, 254, and 280 nm and BetaRam radiochemical detectors; the pure fractions were pooled to obtain a 30-40% radiochemical yield. General Procedure for Unlabeled andp-[2,3JH21Benzoyldihydrocinnamyl Derivatives of Peptides. Probes synthesized include derivatives of Gly-Gly-Arg and pheromone biosynthesis activating neuropeptide (PBAN, amino acid sequence LSDDMPATPADQEMYRQDPEQIDSRTKYFSPRL-amide derivatized a t LysZ7). The synthesis and purification of the radiolabeled peptides were carried out in the same manner as for the cold peptides. Representative protocols for N-terminal or Lys modification are illustrated. BZDC-GGR. To a solution of 1.2 mg (3.5 mmol) of Gly-Gly-Arg acetate in 0.1 mL of water was added 175 pL of 0.05 M BZDC-NHS ester in DMF (8.75 mmol) and 160 pL of 0.5 M triethylamine (70 mmol) in DMF/water (1:l). The reaction mixture was left for 48 h a t ambient temperature in darkness. A blank reaction, containing all the reagents except for the peptide, was performed in parallel. Both reactions were quenched with 0.35 mL of 0.1 M ethanolamine in MeOH, concentrated in vacuo, redissolved in 0.5 mL of phosphate buffer (0.2 M, pH 7.2) and purified by HPLC (Aquapore RP-300): mobile phase, A = 0.1% TFA in water, and B = 80% CH3CN, 19.9% water, and 0.1% TFA, gradient 0-80% B (40 min), 80% B (15 min); t~ = 25.4 min for BZDC-Gly-Gly-Argand 32.5 min for BZDC-ethanolamide. Alternatively, rapid purification could be achieved using a NENSORB-20 cartridge. Thus, an aliquot (10 pL from the phosphate-buffered solution) was diluted to 200 pL in a Tris buffer (reagent A) and applied to the cartridge. A solvent consisting of 20% propanol in water was used for elution, and the first 200-pL fraction showed the majority of the pure peptide as determined by HPLC (see above). IH-NMR (DzO, 600 MHz) 6 7.63-7.72 (m, 5H), 7.50 (t, 2H), 7.36 (d, 2H), 4.23 (m, lH), 3.59 (m, 4H), 3.05 (t, 2H), 2.97 (t, 2H), 2.27 (t, 2H), 1.78 (m, 2H), 1.64 (m, 2H), 1.48 (m, 2H). L~s~~-BZDC-PBAN. Synthetic PBAN (3 mg, 0.75 pmol) was dissolved in 100 pL of DMF, and 12 pL of 0.05 M BZDC-NHS ester in DMF was added, followed by 9 pL of 0.7 M triethylamine in DMF. The reaction mixture was stored for 16 h a t ambient temperature, and the products were first analyzed and then purified by RPHPLC using the system as described above for BZDCGGR; t~ = 28 min for LysZ7-BZDC-PBAN,30 min for N-terminal-BZDC-PBAN,33 min for doubly-labeled BZDCPBAN, and 36 min for BZDC-NHS ester. Verification of L y P Modification. PBAN (10 was mixed with 0.1 nmol of nmol) or LysZ7-BZDC-PBAN Lys-C in 20 pL of 50 mM Tris-HC1, pH 9.0, and incubated for 6 h a t 30 "C. Cleavage of unmodified PBAN occurred a t LysZ7as detected by the shift of the peak corresponding to PBAN (1-33) in the HPLC spectrum profile to a shorter t~ peak assigned as PBAN(1-27). In contrast, LysZ7-BZDC-PBANwas insensitive to the Lys-C protease,
Olszewski et al.
398 Bioconjugafe Chem., Vol. 6,No. 4, 1995 Scheme 1 0
1. HBr 2. NaNO? 3. acrylic acid, CuBr * O H NH2 4. NaOH,MeOH
NHS, DCC
DMF
3
0
0
8
0
0 0
Scheme 2
5% PdIC, 'H2
O H -
*
NHS.DCC CH2C12
O H -
'H
0
Wo+ la
'H
0
0
0
x=1
X=1,3
0
showing no peak shift as monitored by HPLC and supporting the modification a t the lysine residue. RESULTS AND DISCUSSION
The BZDC photophore was first employed in mapping the inositol 1,4,5-trisphosphate binding site of the IPS receptor (Mourey et al., 1993). However, in this study the unsaturated cinnamyl NHS ester 6 was attached to a pendant amino group prior to tritium gas reduction to the [2,3-3Hz]BZDCamide. Relatively low specific activity materials ( 5 4 CUmmol) were obtained by this route, since the reductive tritiation was carried out in aqueous solution, We have improved this process by preparing [3H]BZDC-NHS, a heterobifunctional cross-linking reagent of general utility, which allows removal of reduction byproducts prior to coupling to the ligand of interest. Scheme 1 shows the preparation of the reagent. Aminobenzophenone (3) was diazotized, and acrylic acid and cuprous bromide were added to achieve a Meerwein arylation (Cleland, 1961, 1971; Rondestvedt, 1976; Doyle et al., 1977). The resulting (a-bromobenzoy1)hydrocinnamic acid was isolated and then dehydrobrominated. Acidification resulted in precipitation of 4-benzoylcinnamic acid (BZC, 4), which was converted to active ester, BZC-NHS (6). Hydrogenation a t atmospheric pressure afforded BZDC-NHS ester (la). Tritium gas reduction of BZC-NHS (6) with 5% PdK under carrier-free tritium gas gave [3H]BZDC-NHS (lb), which was purified by preparative HPLC (flash chromatography failed to separate 6 and lb). A small amount of the benzhydrol byproduct was occasionally produced in different lots. To prepare the sulfo-NHS derivative, 4-benzoylcinnamic acid (4) was hydrogenated or tritiated using 5% palladium on carbon. The purified dihydro acid ( 5 )was coupled to sulfo-N-hydroxysuccinimideusing DCC. The product (2a) was crystallized from the reaction mixture (Scheme 2). Derivatization of four different 0-aminoalkyl tethered inositol polyphosphates [P-1 tethered Ins(1,4,5)P3 and Ins(1,3,4,5)Pd,P-5 tethered Ins(1,2,5,6)P4,(Chaudhary et
al., 1994) and P-2-0-aminohexyl tethered Ins(1,2,3,4,5,6)PSI with l b has been accomplished (A. A. Profit, G. DormBn, J. D. Olszewski, and G. D. Prestwich, unpublished results). This reaction may be performed in organic or mixed organic-aqueous solvent systems. For these derivatives, l b in DMF was added to a solution of the P-1-0-aminoalkyl polyphosphate in triethylammonium bicarbonate and allowed to react for 48 h. After removal of all solvents and buffer, purification was achieved via a DEAE-cellulose column (HC03- form). Fractions were analyzed by RP-HPLC using W and radiochemical detection. The new reagent provides specific activities in the range of 30-60 CUmmol, improving sensitivity of detection by an order of magnitude over the previous method (Mourey et al., 1993). We have also modified the N-terminal amino group and internal Lys amino groups of polypeptides to prepare peptide photoaffinity labels (Figure 2). The derivatization can be performed in either a mixed aqueous-organic system (DMF/water) or in a n entirely organic system (DMF) a t room temperature. Reaction times ranged from 6 to 48 h with purification and analysis performed by RP-HPLC using W and radiochemical detection. In one example, the simple tripeptide Gly-Gly-Arg (GGR) was modified on the N-terminal glycine amino group using l b in 1:l DMF/water with excess triethylamine added. Purification was achieved via RP-HPLC o r by using step gradient elution from a NENSORB 20 cartridge. In the next example, selection of reaction conditions allowed differentiation between an N-terminus and a n internal of amine (Myers et al., 1991). The unique internal PBAN, a 33 amino acid polypeptide important in activating insect pheromone biosynthesis (Raina & Menn, 1993), was selectively modified in the presence of the unblocked N-terminal amino group. The site of modification was verified by digestion of both modified and unmodified peptides with endoproteinase Lys-C. CONCLUSION In summary, we prepared a new heterobifunctional tritium-labeled reagent for conversion of a variety of
Tritiated Benzophenone Phootlabel
biological ligands to photoaffinity labels. The selectivity and ease of use of both unlabeled BZDC-NHS la and the high specific activity tritium-labeled reagent lb are discussed. The development of lb now allows access by biochemical laboratories to a wide range of new photoprobes for receptor site characterization. The successful use (Mourey et al., 1993) of [3H]BZDC-IP3 can now be extended to identification of peptide carrier proteins, other inositol polyphosphate receptors, targets for anticancer agents, odorant receptors, and other systems.
Bioconjugafe Chem., Vol. 6,No. 4, 1995 399
Doyle, M. P., Siegfried, B. and Dellaria, J. F., Jr. (1977) Alkyl Nitrite-Metal Halide Deamination Reactions. 2. Substitutive Deamination of Arylamines by Alkyl Nitrites and Copper (11) Halides. A Direct and Remarkably Efficient Conversion of Arylamines to Aryl Halides. J. Org. Chem. 42, 2426-2436. Estevez, V. A. (1991) Synthesis and Biochemical Applications of Affinity Labels for Inositol Polyphosphate Receptors, Ph.D. Dissertation, State University of New York at Stony Brook. Gonzalez, F. A., Wand, D.-J., Huang, N.-N., and Heppel, L. A. (1990) Activation of Early Events of the Mitogenic Response by a P2Y Purinoceptor with Covalently-Bound 3’-0-(4-Benzoy1)-benzoyladenosine 5’-Triphosphate. Proc. Natl. Acad. Sci. U.S.A. 87, 9717-9721. ACKNOWLEDGMENT Holman, G. D., Karim, A. R., and Karim, B. (1988) Photolabeling We thank the NIH (Grants NS 29632 to G.D.P. and of Erythrocyte and Adipocyte Hexose Transporters Using a Benzophenone Derivative of bis(D-Mannose) Biochim. BioAI 32498 to W.L. Roelofs, with subcontract to G.D.P.), phys. Acta 946, 75-84. DuPont-New England Nuclear, and the Herman Frasch Ishidate, K., Matsuo, R., and Nakazawa, Y. (1992) CDPFoundation for financial support. Dr. J. F. Marecek Choline: 1,2-Diacyglycerol Cholinephosphotransferase from provided synthetic advice in development of the syntheRat Liver Microsomes. 11. Photoaffinity Labeling by Radioacsis. PBAN was synthesized by T. Fischer of the Center tive CDP-Choline Analogs. Biochim. Biophys. Acta 1124, 36for Analysis and Synthesis of Macromolecules, and PBAN 44. reduction and purification were optimized by K. BhatKuechler, E., Steiner, G., and Barta, A. (1988) Photoaffinity nagar. Labeling of Peptidyltransferase. Methods in Enzymology (Noller, H. F., Jr., & Moldave, K., Eds.) Academic Press, New York, 164, 361-372. LITERATURE CITED Leszyk, J., Collins, J. H., Leavis, P. C., and Tao, T. (1987) CrossLinking of Rabbit Skeletal Muscle Troponin with the PhotoAgarwal, R., Rajasekharan, K. 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