212
Bioconjugate Chem. 1993, 4,212-218
p-Maleimidophenyl Isocyanate: A Novel Heterobifunctional Linker for Hydroxyl to Thiol Coupling Michael E. Annunziato, Usha S. Patel, Madhuri Ranade, and Paul S. Palumbo’ PB Diagnostic Systems, Inc., 151 University Avenue, Westwood, Massachusetts 02090. Received December 7, 1992
p-Maleimidophenyl isocyanate (PMPI, 1) is a heterobifunctional cross-linking agent useful for thiol to hydroxyl coupling. Several maleimide-activated compounds were prepared and characterized and then shown to be reactive with thiol-containing proteins. Examples include activation of vitamin BIZ, digoxigenin, digitoxigenin, estradiol, progesterone, and some serine-containing peptides.
Progress in immunochemistry has required the preparation of numerous protein-protein or protein-hapten conjugates (1). The attachment of cytotoxic drugs and toxins to tumor-localizing monoclonal antibodies is an approach to chemotherapy which has received much attention over the years (2). Stable, active enzymeantigen and enzymeantibody conjugates are of paramount importance in immunoassays (3). The successful immobilization of proteins or haptens onto a solid phase through the use of chemical agents is also a prerequisite for successful ELISA techniques. As a result, many reagents for the preparation of these conjugates have been reported (4). The so-called chemical cross-linking reagents, an outgrowth of protein modification chemistry, are designed to have specificreactivity with functional groups contained in each reactant. Both homo- and heterobifunctional reagents are available, with the latter having more utility. Since heteroreagents possess two selectively reactive groups that allow coupling to be carried out in a stepwise manner, better control of the conjugation chemistry is attainable. For instance, component A may be activated, purified, and characterized, before combining with component B. By far the most popularly used heterobifunctional agenb contain an amine reactive group (i.e., an N-hydroxysuccinimide ester) and a sulfhydryl reactive group (i.e., a maleimide) connected by a spacer (5) (see, for example, the commercially available G M B S and SMCC). Most proteins and polypeptides contain one or more eaminolysine and/or cysteine residues which serve as convenient linking points via these agents. The spacer group serves several functions, not the least of which is its effect on steric hindrances; linear bridging between protein and/or hapten moieties with minimal steric interaction is often a requirement for maintaining proper immunoreactivity or enzyme activity. Careful selection of the linking group is essential for probing proximity and folding within biomolecules as well as in determining the interactions of biomolecules in complexsystems. As a result, cross-linking agents are available in a host of linker types (4b). Small molecules or haptens often do not possess one of the conventional functional groups, and so the task of
* Author to whom correspondence should be addressed. Abbreviations used: PMPC = N-@-maleimidopheny1)carbamate; GMBS = N - [(y-maleimidobutyryl)oxy]succinimide; SMCC = succinimidyl 4-(N-maleimidomethyl)cyclohexane-lcarboxylate; 2-IT = 24minothiolane (Traut’s reagent); DTNB = 5,5’-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent); DPPA = diphenyl phosphorazidate; CBZ = carbobenzyloxy; AP = alkaline phosphatase; BI2 = vitamin BIZ;TEA = triethylamine; Gly = glycine; Ser = serine; Tyr = tyrosine.
linking becomes increasingly more difficult. One is usually faced with the need to chemically modify the material by more rigorous and formal chemistries. Often total synthesis of a drug analog is necessary in order to incorporate a reactive group. The efficacy and/or immunoreactivity of the resulting drug is then in question. Many haptens do, however, contain hydroxyl functionalities (consider for example the digitalis cardiac glycosides, adrenal hormones, rhodosaminylanthracyclinone-typeanthracyclines, and serine-containing peptides, just to name a few). While linking via ester formation may be a tempting approach in those cases, the resulting ester-linked conjugate may often have only limited stability (6). There is a general need for a hydroxyl-reactive cross-linker which will lead to a stable product. The isocyanate group is a highly reactive, sterically unencumbered functionality which will react with amines to form ureas and with alcohols to form carbamates (7); the resulting products display reasonable stability (8,9). We report the use of p-maleimidophenyl isocyanate (PMPI) for joining hydroxyl-containing compounds such as haptens and peptides to sulfhydryl-containing compounds such as proteins, most notably for us in the preparation of alkaline phosphatase conjugates of vitamin BIZ,progesterone, estradiol, digitoxigenin,digoxigenin,and some serine-enriched peptides (Chart I). This heterobifunctional agent constitutes a net hydroxyl-to-thiol coupling. EXPERIMENTAL PROCEDURES
Materials. Alkaline phosphatase was obtained from Boehringer Mannheim (enzyme immunoassay grade from calf intestine, -3000 IU/mg). Chicken y-globulin was obtained from Jackson Immuno Research Labs, Inc. Cyanocobalamin,digoxigenin, digitoxigenin,progesterone, &estradiol, N-CBZ-Gly-Gly-Ser, and N-CBZ-Tyr-Ser methyl ester were obtained from Sigma. Maleic anhydride, p-aminobenzoic acid, DPPA, and TEA, were obtained from Aldrich. PD-10 columns were obtained from Pharmacia. 2-IT and DTNB were obtained from Pierce. DMSO (J.T. Baker) was stored over 3-A molecular sieves in a sealed container. Toluene (J.T. Baker) was dried by distillation from sodium metal and stored over 3-A molecular sieves in a sealed flask. TSMZ refers to a buffer containing 30 mM triethanolamine, 150 mM sodium chloride, 1 mM magnesium chloride, and 0.1 mM zinc chloride, with the pH adjusted to 7.3. Melting Points. Melting points (mp) were obtained on an Electrothermal melting point apparatus and are uncorrected. NMR Spectroscopy. Proton NMR spectra were
1043-1a o 2 1 ~ ~ 1 2 ~ 0 4 - 0 2 ~ 2 ~ o 4 . 00 0 11993 0 American Chemical Society
Bbtxnjlugte Chem., Vol. 4, No. 3, 1993 219
gMalelmldophenyl Isocyanate as a Novel Linker
&T
Chart I CN
/
H'2NO!:
.CONHz
'
HO
N-CBZ-TYR-SER, methyl ester: R= -OH 21: R= -0-PMPC
H
o
H + N ~ C O ~ H
C B Z N , ) . ~
N-CBZ-GLY-GLY-SER: R= -OH 26: R= -0-PMPC
Vitamin B12: R= -OH
&?'
2a: R=-0-PMPC
HO
3
P-Estradiol: R= -OH 2f: R= -0-PMPC
0
.. Digoxigenin: Digitoxigenin: 2b: 2C:
A
_BL
-OH -OH
-OH -H -OH -H
-0-PMPC -0-PMPC
obtained using either a Bruker AC300 (300 MHz), Varian XL 300 (300 MHz), or Varian EM390 (90 MHz) NMR spectrometer. The samples were dissolved in DMSO-& (2.50 ppm), DzO (4.80 ppm), or CDC13 (7.25 ppm) and the chemical shifts reported in ppm on the 6 scale using the residual proton absorptions of these solvents as references. Proton assignments and structural determinations were made by analogy to published spectra for digitoxigenin and digoxigenin (10) and cyanocobalamin (11). Infrared Spectroscopy. IR spectra were obtained on a Nicolet 2ODXC FTIR spectrometer as KBr pellets. Mass Spectroscopy. Mass spectral analyses were run on a Hewlett-Packard 5985 spectrometer. Both FAB (M+ + 1)and E1 (M+) techniques were used, and the results are listed as such. Ultraviolet Spectroscopy. UV absorbance spectra of aqueous solutions were measured using a Beckman DU-7 instrument. Analyses. Elemental analyses (C, H, N) were performed by Galbraith Laboratories, Inc. (Knoxville, TN).
1 Progesterone:
JL
-H
-H
1 1a-Hydroxyprogesterone:
-OH
17a-Hydroxyprogesterone: 2e: 2h:
-H
-H -OH
-0-PMPC
-H
-H
-0-PMPC
Thin-Layer Chromatography. Reactions were monitored on TLC using normal-phase, glass-backed silica plates (HLF, 0.25" thickness) and reversed-phase, glass-backed silica plates (RPS-F, 0.25" thickness) obtained from AnalTech. Spots were visualized under a 254-nm UV lamp (Model UVG-11 from UVP,Inc.) or by treatment with iodine vapors. Gel Permeation Chromatography. PD-10 columns (Sephadex G-25) were equilibrated with the eluting buffer by passing five column volumes of the buffer through the column prior to sample application. Fractions (0.5-1.0 mL) were collected and the absorbance a t 280 nm read for each. When plotted (ODzmnmvs fraction number), the chromatograms displayed complete baseline separation of the protein and small molecule components in all cases. Silica Chromatography. Reversed-phase silica (Cle bonded, 35-75-pm particle size, 150-A pore size) and normal phase silica (35-75-pm particle size, 60-A pore size) was obtained in bulk from AnalTech. All silica purifications utilize flash techniques (12)unless otherwisenoted.
214
Bloconlugate Chem., Vol. 4,No. 3, 1993
Fractions were collected (0.5-2 mL) and products identified by TLC. pMaleimidobenzoic Acid (5). This benzoic acid derivative was synthesized according to the procedure of Yoshitake et al. (13). Thus, 4-aminobenzoic acid (4.26 g, 31mmol) was suspended in 30 mL of acetone and solublized by the addition of 5 mL of methanol. A solution of maleic anhydride (3.66 g, 37 mmol) in 10mL of acetone was added dropwise and the resulting precipitate stirred for 20 min. The material was suction filtered, washed with acetone, and vacuum-dried to afford a yellow powder (6.36 g). This material was dissolvedin acetic anhydride (13mL), treated with sodium acetate (1.08 g), and then heated with stirring at 50 "C for 2 h. The volatiles were then removed in vacuo, and the resulting residue was taken up in 150 mL of water and heated at 70 "C for 2.5 h. The resulting white precipitate was suction filtered, washed with water, and vacuum-dried overnight to afford compound 5 (4.7 g, 70%1. Analysis by silica TLC (10% methanol in methylene chloride as eluent, visualized by UV absorption andlor iodine vapor staining) showed one spot at Rf 0.8. It was used without further purification. 'H NMR (CDC13 + DMSO-&): 6 10.0-8.5 (br 8, 1H, C02H), 7.95 (d,J = 9 Hz, 2 H, Ar-H ortho to maleimide), 7.32 (d, J = 9 Hz, 2 H, Ar-H ortho to carboxylicacid),6.8 (s,2H, maleimide vinyl). MS (FAB, 3-nitrobenzyl alcohol): mle 218 (M+ + 1). pMaleimidobenzoy1 Azide (6). A stirred suspension of compound 5 (4.3 g, 20 mmol) in 150 mL of dry toluene was treated with triethylamine (3.04 mL, 22 mmol) and then DPPA (4.7 mL, 22 mmol). After stirring at room temperature for 2 days, the volatiles were removed in vacuo, and the resulting residue was chromatographed on silica with methylene chloride as eluent. The product, 6, elutes as a pale yellow band and is isolated upon evaporation of solvent to afford a pale yellow crystalline mass (3.9 g, 91% 1. A recrystallized sample from methylene chloride showed the following behavior in a melting point apparatus: 115120 "C, sample appears to upop"; 125-130 "C dec with vigorous gas evolution [mp 131-132 "C (1411. lH NMR (CDCl3): 6 8.0 (d, J = 8 Hz, 2 H, Ar-H ortho to maleimide), 7.45 (d, J = 8 Hz, 2 H, Ar-H ortho to acyl azide), 6.75 (8, 2 H, maleimide vinyl). IR (KBr): 3110,2160,1720,1690, 1600,1510,1400,1380,1310,1275,1210,1180,1150,1120, 1070, 1040, 1010, 850, 830, 690 cm-l. MS (FAB, 3-nitrobenzyl alcohol): mle 215 (M+ + 1). pMaleimidopheny1 Isocyanate, PMPI (1). A solution of compound 6 (3.4 g, 14 mmol) in dry toluene (150 mL) is refluxed under nitrogen for 1 h and 20 min and then evaporated in vacuo. This quantitatively affords compound 1 as yellow micro needles (3 g). Mp 121-123 "C [mp115-117°C(14)1. Thiscompoundshouldbestored in a sealed vial, protected from light and moisture, in a freezer. 1H NMR (CDCl3): 6 7.2 (d, J = 9 Hz, 2 H, Ar-H ortho to maleimide), 7.02 (d, J = 9 Hz, 2 H, Ar-H ortho to isocyanate), 6.7 (8, 2 H, maleimide vinyl). IR (KBr): 3380, 3360, 3120, 3080, 2310, 1720, 1690 shoulder, 1520, 1390,1150,830, 690 cm-l. A small sample (38 mg) was dissolved in 1 mL of methylene chloride and treated with excess methanol (0.15 mL). After 1 h the volatiles were removed in vacuo to quantitatively afford methyl N-@-maleimidopheny1)carbamate (2g). 'H NMR (CDC13 + CD30D): 6 8.45 (br s, 1 H, amide), 7.45 (d, J = 9 Hz, 2 H, Ar-H ortho to maleimide), 7.1 (d,J = 9 Hz, 2 H, Ar-H ortho to carbamate), 6.7 (s,2 H, maleimide vinyl), 3.67 (s,3 H, OCH3). MS (EI) mle 246 (M+). N,N-Bis(pmaleimidopheny1)urea (8). A stirred, room temperature solution of PMPI (100 mg, 0.47 mmol)
Annunrlato et al.
in DMSO (2 mL) was treated dropwise with 100 pL water. After 5 min, excess water was added (10 mL) to precipitate the product, which was isolated by suction filtration. After vacuum-drying, compound 8 was obtained as a yellow powder (175 g, 93% yield). lH NMR (DMSO-&): 6 8.8 (br s, 1H, amide H), 7.5 (d, J = 9 Hz, 2 H, Ar-H ortho to maleimide), 7.15 (d, J = 9 Hz, 2 H, Ar-H ortho to urea), 7.1 (s,2 H, maleimidevinyl). MS (EI) mle 402 (M+).Anal. Calcd for C21H14N40~0.3H~O: C, 61.85; H, 3.61; N, 13.74. Found: C, 62.05; H, 3.77; N, 13.61. PMPI-Activated Vitamin BQ (2a). A solution of cyanocobalamin (54 mg, 0.04 mmol) in 0.5 mL of DMSO was treated with PMPI (97.3 mg, 0.45 mmol) and the resultant red solution stirred under argon, at room temperature, protected from light, overnight. The mixture was treated with ether (10 mL) and stirred vigorously to extract the DMSO. After decanting, the residue was stirred in ether (10 mL). After decanting again, the red oil was then triturated with methylene chloride to afford a red powder which was suction filtered, washed with excess methylene chloride, and vacuum-dried. The product was purified via flash chromatography on C-18 silica using methanol-water (2:3) as eluent. The product elutes as a red band and is isolated upon evaporation of solvent in vacuo, affording 2a (41 mg, 66%)as a red powder. partial lH NMR (DzO): 6 7.5 (d, 2 H, Ar-H ortho to maleimide), 7.26 (d, 2 H, Ar-H ortho to carbamate), 7.2 ( 8 , 1 H, benzimidazole C-4 H), 7.1 ( 8 , 1H, benzimidazole C-2 H), 7.0 (8, 2 H, maleimide vinyl), 6.48 (s, 1H, benzimidazole C-7 H), 6.3 (br s, 1H, ribose C-1 H), 6.02 (s,1H, C-10 H), 4.3 (m, 4 H, ribose (2-5, C-5', C-4, and C-2 H's), 4.18 (d, 1H), 4.1 (d, 1H), 3.6 (d, 1H, propanolamine C-1 H), 3.4 (m, 1 H, C-8 H), 3.3 (d, 1 H, C-13 H), 3.01 (m, 1 H, propanolamine C-1' H), 2.5 (8, 3 H, C-35 CH3), 2.48 (8, 3 H, C-53 CH3), 2.2 (8, 3 H, benzimidazole CH3), 2.18 (8, 3 H, benzimidazole CHd, 1.8 (s,3 H, C-25 CH3), 1.34 (br s, 9 H, (2-47 CH3, (2-54 CH3, (2-36 CH3), 1.22 (d, 3 H, propanolamine CH3), 1.24 (s,3 H, (2-46 CH3), 1.0 (m, 2 H, (2-41 H, C-60 H), 0.42 (e, 3 H, C-20 CH3). MS (FAB): mle 1570 (M++ 1). UV (H2O): X 280 (e 16 626), 361 ( E 26 410), 548 nm (t 7950). 38-DigoxigeninN-(pMaleimidopheny1)carbamate (2b). A solution of digoxigenin (53 mg, 0.136 mmol) in DMSO (1mL) was treatedwithPMP1(29mg,O.l36mmol), and the resultant yellow solution was stirred at room temperature, under argon, and protected from light for 4 h. Dropwise addition of water (6 mL) affords a precipitate which was suction filtered, washed with excess water, and vacuum-dried. This crude residue was chromatographed on silica with 3% methanol in methylene chloride aseluent. The product elutes as a pale yellow band after two impurity bands. Evaporation of solvent invacuo affords the product as a pale yellow precipitate (25 mg, 30 ?61. Analysis by 300-MHz proton NMR suggests that the point of attachment is through the 38-hydroxyl group (the equatorial C3-H is shifted downfield to 6 5.1; the axial C12-H remains at 6 3.4). lH NMR (CDC13): 6 7.5 (d, 2 H, Ar-H ortho to maleimide), 7.27 (d, 2 H, Ar-H ortho to carbamate), 6.84 (s,2 H, maleimide vinyl), 6.7 (br s, 1H, amide H), 5.93 (br s, 1 H, C-22 H), 5.10 (br s, 1H, C-3 H), 4.9 (d, 1 H, C-21 H), 4.8 (d, 1 H, C-21' H), 3.4 (dd, 1 H, C-12 H), 3.3 (dd, 1H, C-17 H), 2.15 (m, 1H, C-168 H), 2.0-1.0 (complex m, 18 H), 0.98 ( ~ , H, 3 C-19 CH3), 0.8 ( ~ , H, 3 C-18 CH3). MS (FAB, 3-nitrobenzyl alcohol): mle 606 (M+ + 1). UV 246 nm (e 16 680). (TSMZ, pH 7.3, + 1%DMF): ,A, 38-Digitoxigenin N(pMaleimidopheny1)carbamate (2c) was prepared as in 2b from digitoxigenin (77.39 mg, 0.2066 mmol). Progress of the reaction was followed
pMaleimMopheny1 Isocyanate as a Novel Linker
by normal phase TLC (2 % CH30H in CHzClZ eluent: 2c
Rf0.48, digitoxigenin Rf 0.31). The product was purified on silica (2% CH30H in CHZClz) to afford 46 mg (38%) of compound 2c as a pale yellow solid. lH NMR (CDCl3): 6 7.43 (d, 2 H, Ar-H ortho to maleimide), 7.2 (d, 2 H, Ar-H ortho to carbamate), 6.8 (8, 2 H, maleimide vinyl), 6.7 (s, 1 H, amide H), 5.8 (8, 1 H, C-22 H), 5.07 (br s, 1 H, C-3 H), 4.9 (d, 1H, C-21 H), 4.74 (d, 1 H, (2-21' H), 2.7 (m, 1 H, C-17 H), 2.1 (m, 2 H, C-15a H, C-160 H), 1.82 (m, 3 H, C-6a H, (2-48 H, C-16a H), 1.7-1.05 (complex m, 16 H), 0.9 ( ~ , H, 3 C-19 CH3), 0.8 ( ~ ,H, 3 (2-18 CH3). MS (FAB, 3-nitrobenzyl alcohol): m / e 590 (M++ l),611 (M+ + Na). UV (TSMZ, pH 7.3, + 1%DMF): X 246 nm (e 16 000). N-CBZ-Gly-Gly-Ser N-(pMaleimidopheny1)carbamate (2d) was prepared as in 2b from N-CBZ-GlyGly-Ser (218 mg, 0.62 mmol). The resulting residue was chromatographed on silica with acetic acid-methanolmethylene chloride (1:1089, by volume) to afford the pure product 2d as a pale yellow solid (246 mg, 70 % ). lH NMR (DMSO-de): 6 8.32 (d, 1H, amide H), 8.1 (br t, 1H, amide H), 7.55 (d, 2 H, Ar-H ortho to maleimide), 7.5 (t, 1 H, amide H), 7.34 (br s , 6 H, benzyl-Ar + amide H), 7.2 (d, 2 H, Ar-H ortho to carbamate), 7.12 (8, 2 H, maleimide vinyl), 5.0 ( 8 , 2 H, benzyl-CHz), 4.6 (m, 1H, Ser methine H), 4.45 (dd, 1 H, Ser methylene H), 4.25 (dd, 1 H, Ser methylene H'), 3.8 (s, 2 H), 3.66 (8, 2 H). MS (FAB, 3-nitrobenzyl alcohol): m / e 568 (M+ + 1). 1laProgesterone N-(pmaleimidopheny1)carbamate (28) was prepared as in 2b from llcu-hydroxyprogesterone (94 mg, 0.28 mmol). The product was purified by chromatography on silica using 2 % methanol in methylene chloride as eluent, with isolation of the major highest Rf material to afford 90 mg (58% yield) as a pale yellow crystalline solid. 'H NMR (CDC13): 6 7.74 (br s, 1 H, amide H), 7.47 (d, 2 H, Ar-H ortho to maleimide), 7.2 (d, 2H, Ar-H ortho to carbamate), 6.75 (s, 2 H, maleimide vinyl), 5.7 (s, 1 H, C-4 H), 5.2 (m, 1 H, C-11 H), 2.5-1.0 (complex m, 18 H), 2.07 (s, 3 H, C-21 CH3), 1.25 (s, 3 H, C-18CH3),0.7 (s,3 H, C-19CH3). MS (FAB, 3-nitrobenzyl alcohol): m / e 546 (.M+ + 1). 178-EstradiolN-(pmaleimidopheny1)carbamate (2f) was prepared as in 2b from /%estradiol (35mg, 0.13 mmol). The crude residue was chromatographed on silica with 1%methanol in methylene chloride as eluent to afford the purified product, 2f (29 mg, 46%). Analysis by 300MHz proton NMR suggests that the point of attachment is through the 17fi-hydroxyl group (the 17a-H is shifted downfield to 6 4.65). lH NMR (CDCl3): 6 7.46 (d, 2 H, Ar-H ortho to maleimide), 7.22 (d, 2 H, Ar-H ortho to carbamate), 7.1 (d, 1 H, C-1 H), 6.73 (s, 2 H, maleimide vinyl), 6.62 ( 8 , 1H, amide), 6.54 (dd, 1H, C-2 H), 6.50 (d, 1H, C-4 H), 4.70 (t,1H, aC-17 H), 2.75 (m, 2 H), 2.3-1.2 (complex m, 13 H), 0.8 (s, 3 H, CH3). MS (FAB, 3-nitrobenzyl alcohol): m / e 486 (M+ + 1). N-CBZ-Tyr-Ser-OMeN-(pMaleimidopheny1)carbamate, (2i) was prepared as in 2b from N-CBZ-Tyr-Ser methyl ester (220 mg, 0.53 mmol). The resulting crude yellow precipitate was stirred vigorously in absolute methanol (50 mL) and suction filtered and the filter cake washed with excess methanol. The filtrate was evaporated in vacuo and the resulting residue chromatographed on silica (3% methanol in methylene chloride as eluent) to afford the pure product, 2i, as a pale yellow solid (227 mg, 68%). 'H NMR (CDCl3 and DMSO-dG): 6 7.36 (d, J = 9 Hz, 2 H, Ar-H ortho to maleimide), 7.14 (s, 5 H, CBZ phenyl), 7.07 (d, J = 9 Hz, 2 H, Ar-H ortho to carbamate), 6.87 (d, J = 7 Hz, 2 H, Tyr Ar-H meta to OH), 6.74 (s, 2 H, maleimide vinyl), 6.57 (d, J = 7 Hz, 2 H, Tyr Ar-H
Bioconjugate Chem., Vol. 4, No. 3, 1993 215
ortho to OH), 4.9 (s, 2 H, CBZ methylene), 4.67 (t, 1 H, Ser methine), 4.3 (m, 3 H, Tyr methine and Ser methylene), 3.67 (e, 3 H, Ser OCH3), 2.90 (t,2 H, Tyr methylene). MS (FAB, 3-nitrobenzyl alcohol): m / e 631 (M+ + 1). Maleimide Stability Study. According to the procedure of Kitagawa (4a), a 20 pL-aliquot of a 10 mM solution of 2d in DMF was incubated in 0.5 mL of 0.05 M phosphate buffer (pH 6,7, or 8) or 0.05 M acetate buffer (pH 5) for 30 min at 30 "C (run in duplicate). Next, 200 pL of 1 mM mercaptoethanol in 0.05 M phosphate (pH 6) was added, mixed well for 1-2 min, and then treated with 1.1mL 0.2 M Tris-HC1 buffer (pH 8.2) containing 0.02 M EDTA.Na4, and then 0.2 mL of 10 mM methanolic DTNB was added. After 10 min the absorbance values at 412 nm of each test sample (A), a zero blank (B) (without the incubation), and the sample blank value (C) (20 pL of DMF instead of the sample solution, incubated at 30 "C/ 30 min) were obtained. The percent decomposition of the maleimide residue is calculated as [(A - B)/(C- B)] X 100. The results are presented in Table 11. Preparation of a Vitamin BIZ-Alkaline Phosphatase Conjugate (3a). The general procedure for alkaline phosphatase conjugation is illustrated below for vitamin Biz. Alkaline phosphatase is thiolated with 5 mM Traut's reagent for 20 min in a preliminary step and then treated with 30 equiv of the maleimido-activated compound of interest (dissolved in either buffer or a minimum amount of DMF). Thus the thiolation of alkaline phosphatase was carried out by treating alkaline phosphatase (1 mg, 7.1 nmol) in 1 mL TSMZ buffer (pH 7.3) with 2-iminothiolane hydrochloride (50.0 pL of a 100 mM solution in distilled water) and incubating for 20 min at room temperature. After treating with 10 pL of 1 M glycine, the excess reagents were removed using a PD-10 column, eluting withTSMZ buffer (pH 7.3). The fractions containing the activated alkaline phosphatase [analysis for free thiol groups using DTNB reagent (15) reveals 6-7 thiols/APl were pooled (absorbance reading at 280 nm) and then treated immediately with compound 2a (5-fold excess/mol of thiol). The solution was stored at room temperature for 1 h and then at 4 "C overnight. Gel filtration in a PD-10 column using TSMZ (pH 7.3) as an eluent separated the conjugate from the low molecular weight reactants. The UV spectrum of the conjugate showed the following absorbances: X 277.4 (OD = 0.418), 361 (OD = 0.30), and 548.3 nm (OD = 0.097). UV Determination of B12 Incorporation into AP. The extent of incorporation of BIZ(conjugation ratio) was determined via spectral analysis of the resulting conjugate. Thus, relating absorbance at 361 and 280 nm for the conjugate relative to each component's respective absorbance at 361 and 280 nm gives a calculated BIZ: protein ratio of 6.9:l (a 1 mg/mL solution of AP gives an OD = 1.0 a t X 280 nm and an OD = 0 at X 361 nm). This ratio agrees reasonably well with the DTNB results above and confirms the success of conjugation. Preparation of a Panel of Digoxigenin-Alkaline Phosphatase Conjugates (3b). Alkaline phosphatase in TSMZ (pH 7.3) was adjusted to 5 mg/mL and reacted with 3 mM 2-IT at 28 "C. At 10,20, and 120 min, aliquots were removed and quenched with a 1/100 volume of 1M glycine (pH 7.3). Gel filtration chromatography over a PD-10 column equilibrated with TSMZ (pH 7.3) separated the thiolated protein from residual reagents. The thiol groups were quantified as in 3a and gave thiokprotein values of 1.2:1,1.9:1, and 6.01, respectively. The thiolated AP aliquots were then each reacted with 2b at a mole ratio of 3:l (mol of 2b/mol of thiol). The conjugates were
Annunzlato et al.
Bloconlugate Chem., Vol. 4, No. 3, 1993
216
Scheme I. Hydroxyl to Thiol Cross-Linking Using PMPI
Scheme I11 r
II
U
c
YHZ A
-
I
I
N
4
R-OH
DMSO
o*o 1
PM PI"
I'
0
a R'-SH (pH 5-7.5)
SR'
2
3
Scheme 11. Synthesis of PMPI CO2H 1.) Ref. 13 I
I
2.)DPPA TEA
NH2
4
3.) Toluene Reflux / . N2
(Curtius Rearrangement)
-
1
"eo 5: X=OH 6: X=N3
incubated for 18 h at 4 "C and then purified over PD-10 columns equilibrated with TSMZ (pH 7.3). The conjugation ratio was determined spectrally for each case and gave l.l:l, 2.0:1, and 7.4:1,respectively. A panel of digitoxigenin-antibody conjugates (3c) was prepared as in 3b from chicken IgG, diluted to 4 mg/ mL in TSMZ (pH 7.3). The thiol groups were quantified as in 3a and gave thio1:protein values of 1.3:1, 3.81,and 6.6:l. Thiolated chicken IgG was reacted with 2c a t a ratio of 3:l (mol of 2c/mol of thiol). The conjugates were incubated for 18 h at 4 "C and then purified over PD-10 columns equilibrated with TSMZ (pH 7.3). The conjugation ratio was determined spectrally for each case and gave 0.6:1,3.1:1,and 7.81,respectively. RESULTS AND DISCUSSION
The preparation of PMPI involves the three-step sequence shown in Scheme 11. Thus, p-maleimidobenzoic acid, 6, prepared as described elsewhere (131,is treated at room temperature in toluene with 1 equivof triethylamine followed by 1 equiv of DPPA. The resulting acyl azide, 6, is isolated and purified on silica to afford yellow crystals, in 91% yield. This product undergoes a Curtius rearrangement with smooth release of nitrogen gas upon heating at reflux in dry toluene. The resulting isocyanate
(PMPI) is obtained in quantitative yield upon evaporation of solvent in vacuo. The use of DPPA alleviates the need for intermediate acid chloride formation as reported by Rao (14). PMPI is stable for months when stored dessicated and protected from light at CO "C. For the reaction of PMPI with hydroxyl-containing haptens, we have found DMSO to be the solvent of choice, especially in light of its catalytic effect on isocyanate reactivity (16). The hydroxyl compound is thus combined with 1equiv of PMPI and stirred at room temperature in DMSO, under dry nitrogen, until TLC confirms the quantitative conversion to product. Isocyanates are extremely unstable in water and therefore PMPI activation of alcohols must only be performed in dry organic solvents. In those cases where water might be present in the reaction mixture (either surreptitiously present or in the form of hydrated reactants), excess PMPI must be added. Water reacts quickly and quantitatively with PMPI to afford N,ZV'-bis@-maleimidophenyl)urea, 8, a novel homobifunctional cross-linking reagent in its own right, presumably by the route shown in Scheme 111. Once all water is consumed in this way, the additional PMPI is then free to react with the alcohol moiety present in the reactant. Because of this solvent limitation, PMPI will be mostly useful for activation of small molecules. The carbamates formed, 2, are conveniently purified by flash chromatography on either normal or reversed-phase silica. In this way the activated hapten can be characterized (MS, NMR, UV/vis, etc.) and stored as a purified material for later use. When ready, these products are coupled to sulfhydryl-containing proteins in the appropriate buffers (pH 5-7.5). Where water solubility is a problem, a DMF stock solution of the activated hapten is first prepared and added to the protein-buffer solution. At the end of the conjugation reaction, residual uncoupled material is conveniently removed by Sephadex G-25 chromatography. We have found PMPI to undergo a wide scope of reactivity with various alcohol types (see Table I). In general, reaction with primary and secondary alcohols is facile, while tertiary alcohols are often incomplete or unsuccessful. Phenols also react with isocyanates; however, the hydrolytic stability of the resulting aryl carbamates may not be suitable for all applications (17).Two phenol examples that were tried (pestradio1 and N-CBZTyr-Ser methyl ester) failed to afford any identifiable phenyl carbamate product, possibly due to instability in the chromatography. We have successfully reacted the secondary alcohol groups of digoxigenin (to give 2b), digitoxigenin (togive 2c), 8-estradiol (togive 20, and lla-
pMalelmMopheny1 Isocyanate as a Novel Linker
Table I. Reaction of Various Hydroxylic Compounds with PMPI and 'H NMR Shift Data of Corresponding Products isolated maleimide yield IH NMR data: (9%) 6 (ppm), solvent reactant product 7.1, DMSO-& water 8 93 100 6.7, CDC13lCD3OD methanol 2g vitamin BIZ 66 7.0, D20 2a 30 6.84, CDC13 digoxigenin 2b 38 6.8, CDC13 digitoxigenin 2c 7.12, DMSO-& 70 N-CBZ-Gly-Gly-Ser 2d lla-hydroxyprogesterone 58 6.75, CDC13 2e 17b-estradiol 46 6.73, CDC13 2f 68 6.74, CDC13/ 2i N-CBZ-Tyr-Ser, methyl ester DMSO-& Table 11.8 Percent Decomposition of Maleimide Groups in Related Cross-Linkers after 30 min at 30 "C in pH 5.0, 6.0, 7.0, and 8.0 Buffers sampleb pH 5.0 pH 6.0e pH 7.0e pH 8.0" 69.0 3.1' 6.2 21.4 OBS 7.1 43.8 MBS 2.9' 2.5 32.0 52.0 3.8' 6.6 PBS 56.0 Od 2.3 14.0 2d a Data for OBS, MBS, and PBS taken from ref 4a. OBS,MBS, and PBS are 0-(0-, -(m,and -@-maleimidobenzoy1)-N-hydroxysuccinimide, respectively. Citrate Buffer. 0.05 M acetate buffer. e 0.05 M phosphate.
hydroxyprogesterone (to give 2e) and the primary alcohol groups of serine peptides (to give 2d and 2i) and vitamin B12 (to give 2a). Several tertiary alcohol examples, including 17cu-hydroxyprogesterone,failed to give any appreciable product (i.e., 2h). The aqueous stability of the model compound 2d was studied and shown to be comparable to similar compounds (4a). The results, shown in Table 11, indicate that the stability of the maleimide moiety decreases at pH >7. Maleimides, of course, undergo a facile Michael addition with free thiol groups under slightly acidic to neutral conditions (18). When activated haptens are dissolved in the appropriate buffers just prior to reaction, no significant decomposition is observed. The desired conjugation chemistry is obtained even when near stoichiometric reactant mole ratios are used. PMPI methodology provides a stable, well-defined, vitamin BIZ-alkaline phosphatase conjugate. Previous workers had shown that mild acid hydrolysis of cyanocobalamin yields a mixture of mono- and dicarboxylicacids and one tricarboxylic acid that is derived from the propionamide side chains (19). We felt that this approach to a BIZconjugate would certainly suffer from an obvious isomer problem and therefore require a tedious purification scheme. Yields are low and the product characterizations ambiguous (20). We instead chose to pursue the lone primary hydroxyl group located in the ribose ring as a potential point of attachment since we knew that this C-5 hydroxyl group was reported to react with anhydrides (21). Reaction with PMPI, indeed, afforded a single isomer, 2a, in >60% yield after purification. Characterization by highfield NMR confirmed that we had successfully targeted the primary hydroxyl group [a 0.5 ppm downfield shift in the ribose C-5 methylene protons was observed (1111;no reaction with the sterically hindered secondary alcohol group (ribose C-2 H) was seen, even under excess conditions (10 equiv of PMPI). Fortunately, attachment through the C-5hydroxyl group provides maximal distance between the linking point and the porphyrin ring [3D models show that the activating group points down and away from the main body of the molecule (2211.
Bloconjugate Chem., Vol. 4, No. 3, 1993 217
Table 111. Comparison of Conjugation Ratio (Spectrophotometrically Determined) to Thiols Introduced for Alkaline PhosDhatase (AP) and Chicken Antibody (I&) thiolation conjugation methodltime conjugation thiolslmol ratio (min)a components (titration)b (spectroscopic)c AI20 6-7 6.9 AP/2a B/10 1.2 1.1 AP/2b B/20 1.9 2.0 AP/2b Bl120 6.0 7.4 API2b B/10 1.3 0.6 IgG12c Bl20 3.8 3.1 IgG/2c B/120 6.6 7.8 IgG/2c Refer to Experimental Section for details; see example 3a for method A and example 3b and 3c for method B. See ref 15. See ref IC.
Thiol groups were introduced into alkaline phosphatase via the use of 2-IT (23). In one example, alkaline phosphatase, adjusted to 1 mg/mL in TSMZ (pH 7.31, was treated with 5 mM 2-ITfor 20 min. The resulting thiolated enzyme was purified by gel filtration chromatography on PD-10 and the number of thiols per AP molecule determined to be 6-7 [quantitated by the DTNB method (E)]. The resulting thiolated protein in TSMZ was then treated with a slight excess of compound 2a (-5 mol/mol of thiol) and the conjugation reaction allowed to proceed overnight at 4 "C. Gel filtration on Sephadex G-25 (PD-10) with TSMZ separates the conjugate, 3a, from residual reactants. Spectral analysis (IC), relating the absorbances at 361 and 280 nm, confirmed the success of conjugation. Digoxigenin, the aglycon of digoxin, presented us with yet another regioselectivity challenge, since we wished to link this molecule to alkaline phosphatase through the 3-position. [Classicaldigoxin linking methodology targets the terminal digitoxose residue (24),but we had experienced unacceptable variability and low conjugation ratios using that chemistry.] From earlier work with digitoxigenin we knew that the tertiary hydroxyl group in the 14-position was essentially nonreactive with PMPI and would pose no threat to the activation selectivity. For example, digitoxigenin activation leads exclusively to the 3B-carbamate derivative, 2c. Competitive reaction with the secondary hydroxyl group in the 12-position, however, was perceived as a real threat. To our delight, the major product obtained from the reaction of PMPI and digoxigenin was the desired 3-isomer, 2b. While a small side product was observed on TLC, the 12-isomer was never isolated. A panel of digoxigenin-AP conjugates (3b with varying conjugation ratios) were prepared. Similarly, we prepared a set of digitoxigenin-antibody conjugates (3c). At 3 mM 2-IT we found it convenient to quench aliquots at 10,20, and 120 min and thereby introduce 1-7 mol of thiol/mol of protein from the same thiol activation vessel. In most cases the spectrally determined conjugation ratio values agree reasonably well with the thiol values obtained by titration with DTNB (see Table 111). However, at the high end of the panels, conjugation appears to go further than theoretically possible. Conjugation ratios can indeed be higher than the degree of thiolation if there are other reactive groups present in the macromolecule (i.e. amino groups). More than likely, however, the apparent inconsistency between the thiols introduced and the conjugation ratio is probably due to the inherent error present in both methods of determination (we have seen variability in both determinations to be as high as &lo%). In cases where the absorbance maxima of the reagent is close to or overlaps with the absorbance maxima of the macromolecule (examples 3b
218
Bioconlugate Chem., Vol. 4, No. 3, 1993
and 3c),the UV determination of the conjugation ratio is not reliable and we tend to trust in the thiol titration value more. Indeed, in the vitamin B12 example, the absorbances are further removed from each other and the agreement between methods is better. Also, in the cases of the more hydrophobic examples (2b and 2c), there is always a chance that residual reactant may bind noncovalently to the protein. When this is the case, more careful chromatography should be performed. Protein conjugates that we have obtained by the methodology described above were found to be useful as elements in immunoassays. For example, the digoxigeninAP conjugate (3b)is used in the OPUS digoxin assay (25). ACKNOWLEDGMENT
We wish to thank Catherine M. Palumbo (Polaroid Corp.) for high-field NMR Spectroscopy and Dr. Gerald Dudek (Polaroid Corp.) for Mass Spectrometry. LITERATURE CITED (1) (a) Means, G. E., and Feeney, R.E. (1990) Chemical Modifications of Proteins: History and Applications. Bioconjugate Chem. 1, 2-12. (b) Means, G. E., and Feeney, R. E. (1971) Chemical Modification of Proteins. Holden-Day, San Francisco, CA. (c) Brinkley, M. (1992) A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-Linking Reagents. Bioconjugate Chem. 3, 2-13. (2) (a) Koppel, G. A. (1990) Recent Advances with Monoclonal Antibody Drug Targeting for the Treatment of Human Cancer. Bioconjugate Chem., 1,13-23. (b) Ghose, T., and Blair, A. H. (1987) CRC Crit. Rev. Ther. Drug Carrier Syst. 3,300-301. (c) Mueller, B. M., Wrasidlo, W. A., and Reisfeld, R. A. (1990) Antibody Conjugates with Morpholinodoxorubicin and AcidCleavable Linkers. Bioconjugate Chem. 1,325-330. (d) Imai, N., Kometani, T., Crocker, P. J., Bowdan, J. B., Demir, A., Dwyer, L. D., Mann, D. M., Vanaman, T. C., and Watt, D. S. (1990) Photoaffinity Heterobifunctional Cross-Linking Reagents Based on N-(Azidobenzoy1)tyrosines. Bioconjugate Chem. 1, 138-143. (3) (a) Kricka, L. J. (1985) Ligand-Binder Assays. Labels and Analytical Strategies, Marcel Dekker, New York. (b) Ngo, T. T., Ed. (1988)Nonisotopic Immunoassay, Plenum Press, New York. (c) Maggio, E. T., Ed. (1980) Enzyme-Immunoassay, CRC Press, Boca Raton, FL. (d) Kurstak, E. (1986) Enzyme Immunodiagnosis, Academic Press, New York. (e) Ngo, T. T., and Lenhoff, H. M., Eds. (1985) Enzyme-Mediated Immunoassay, Plenum Press, New York. (0 Engvall, E., and Pesce, A.J., Eds. (1978) QuantitativeEnzyme Immunoassays, Blackwell Scientific, London, Great Britain. (4) (a) Kitagawa, T., Shimozono, T., Aikawa, T., Yoshida, T., and Nashimura, H. (1981) Preparation and Characterization of Heterobifunctional Cross-Linking Reagents for Protein Modifications. Chem. Pharm. Bull. 29 (4) 1130-1135 and references within. (b)Wong, S. S. (1991) Chemistry ofprotein Conjugationand Cross-Linking,CRC Press, Boca Raton, FL. (5) Haugland, R. P. (1992) Handbook of Fluorescent Probes and Research Chemicals, p54,Molecular Probes, Inc., Eugene, OR. (6) Hermentin,P.,Doenges,R., Gronski,P., Bosslet, K.,Kraemer, H. P., Hoffmann, D., Zilg, H., Steinstraesser, A., Scharwz, A., Kuhlmann, L., Luben, G., and Seiler, F. R. (1990) Attachment
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of Rhodosaminylanthracyclinone-TypeAnthracyclines to the Hinge Region of Monoclonal Antibodies. Bioconjugate Chem. 1, 100-107. (7) For a review of the mechanisms of reactions of isocyanates with various nucleophiles, see: Satchell, D. P. N., and Satchell, R. S. (1975) Acylation by Ketenes and Isocyanates. Mechanistic Comparison. Chem. SOC.Rev., 4, 231-250. (8) Lynn, K. R. (1965) Kinetics of Base-Catalyzed Hydrolysis of Urea. J. Phys. Chem. 69, 687-689. (9) Adams, P., and Baron, F.A. (1965) Esters of Carbamic Acid. Chem. Rev. 65,567-602. (10) Drakenberg, T., Brodelius, P., McIntyre, D. D., and Vogel, H. J. (1989) Structural Studies of Digitoxin and Related Cardenolides by Two-Dimentional NMR. Can. J . Chem. 68, 272-277. (11) Kurumaya, K., and Kajiwara, M. (1989) Proton Nuclear Magnetic Resonance (‘H-NMR) Signal Assignment of Vitamin B12 Based on Normal Two-Dimensional NMR and Feeding Experiments. Chem. Pharm. Bull. 37 (1)9-12. (12) Still, W. C., Kahn, M., and Mitra, A. (1978) Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution. J . Org. Chem. 43, 2923-2925. (13) Yoshitake, S., Yamada, Y., Ishikawa, E., and Masseyeff, R. (1979) Conjugation of Glucose Oxidase from Aspergillus Niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide.Eur. J. Biochem. 101,395-399. (14) Rao, B. S. (1989) Synthesis, Characterization, and Thermal Stability of Bismaleimides Derived from Maleimido Benzoic Acid. J . Polym. Sci.: Part A: Polym. Chem. 27, 2509-2518. (15) Ellman, G. L. (1959) Tissue Sulfhydryl Groups. Arch. Biochem. Biophys. 82, 70-77. (16) Dimethyl Sulfoxide Technical Bulletin, p 106, Crown Zellerbach, Chemical Products Division, Vancouver (Orchards), WA. (17) Williams, A. (1973) Participation of an Elimination Mechanism in Alkaline Hydrolyses of Alkyl N-Phenylcarbamates. J. Chem. SOC.,Perkin Trans. 2 1244-1247. (18) Partis, M. D., Griffiths, D. G., Roberts, G. C., and Beechey, R. B. (1983)Cross-Linkingof Protein byw-Maleimido Alkanoyl N-Hydroxysuccinimido Esters. J . Protein. Chem.2,263-277. (19) Bernhauer, K., Wagner, F., Beisbarth, H., Rietz, P., and Vogelmann, H. (1966) Biochem. 2.344, 289-309. (20) Anton, D. L., Hogenkamp, H. P. C., Walker, T. E., and Matwiyoff, N. A. (1980) Carbon-13 Nuclear Magnetic Resonance Studies of the Monocarboxylic Acids of Cyanocobalamin. Assignments of the b-, d-, and e-Monocarboxylic Acids. J. Am. Chem. SOC.102, (7) 2215-2219. (21) Bernstein, J., Varma, R. K., Vogt, B. R., and Weisenborn, F. L. (1980) Vitamin BIZDerivative Suitable for Radiolabeling. U.S. Patent 4,209,614. (22) Golding, B. T. (1979)Vitamin B12. ComprehensiveOrganic Chemistry (Sir Derek Barton, F. R. S. and W. David Ollie, F. R. S., Eds.) Section 24.4, pp 549-584, Pergamon Press, New York. (23) Jue, R, Lambert, J. M., Pierce,L. R., and Traut, R. R. (1978) Addition of Sulfhydryl Groups of Escherichia Coli Ribosomes by Protein Modification With 2-Iminothiolane (Methyl 4-Mercaptobutyrimidate). Biochemistry 17, 5399-5406. (24) Butler, V. P., Jr., Schmidt, D. H., Watson, J. F., and Gardner, J. D. (1974) Production and Properties of Digoxin-Specific Antibodies. Ann. N . Y. Acad. Sci. 242, 717-730. (25) Olive, C. (1991) P B Diagnostics’ OPUS Immunoassay System. J. Clin. Immunoassay 14, 126-132.
