Bloconjugate Chem. 1993, 4, 314-318
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Protected Thiol-Polyethylene Glycol: A New Activated Polymer for Reversible Protein Modification Clement Woghiren,’,t Basant Sharma,* and Stanley Steint Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854. Received February 17,1993
Monomethoxypolyethylene glycol (mPEG) of average molecular weight 5000 was transformed in a series of synthetic steps to a new activated form of PEG, a stable thiol-protected intermediary, for reaction with cysteine residues in proteins under mild conditions to produce PEGprotein conjugates as possible candidates for therapeutics. The modified protein has PEG polymer molecules attached to the backbone by the newly formed disulfide bonds, which are readily cleaved to regenerate the native protein under mild reducing conditions. The model protein papain, which has seven cysteine residues including a lone cysteine in ita active site, was modified through the free available thiol. The resulting PEGpapain was characterized by matrix-assisted laser desorption mass spectrometry, SDS-PAGE, and high performance gel filtration chromatography. The major modified PEGpapain variant was demonstrated to be 5000 Da larger than the unreacted papain.
INTRODUCTION Polyethylene glycol (PEG) has been used to modify enzymes and other proteins for the purpose of preparing improved therapeutic agents (11. The conjugated protein acquires longer circulatory life in the body due to increased resistance to proteolytic digestion (2-5). Generally, succinimidyl succinate-activatedPEG is allowed to react with amino groups on the protein. Other studies have been based on alternative activation chemistries for PEG (6, 7),but they share a similar mode of attachment to the protein, through amino groups. In many instances, this is undesirable, such as when derivatization of a specific lysine residue may inactivate the protein. For example, although many enzymeshave been shown to retain activity after PEG modification (81, other proteins such as monoclonal antibodies may become inactivated (9). Furthermore, more controlled and specific modification of a protein by PEG may be achievable by using another target amino acid, such as cysteine, which in the reduced form is typically less abundant than lysine in proteins. In our studies, we have prepared a new activated form of PEG that is a stable reagent, but readily reacts with the thiol group of cysteine to form a disulfide-linked PEG adduct. The usefulness of this reagent has been demonstrated by modification of papain. EXPERIMENTAL PROCEDURES
Materials. mPEG 5000 and potassium thioacetate were obtained from Fluka Chemicals, Ronkonkoma, NY. Tosyl chloride, 4,4-dipyridyl disulfide, dry methanol, dry pyridine, and sodium methoxide (methanolic 25 % ) were the products of Aldrich Chemical, Milwaukee, WI. Dithiothreitol (DTT), Ellman’s reagent [5,5’- dithiobis(2-nitrobenzoic acid)], bovine pancreatic trypsin inhibitor (BPTI), papain (from papaya latex), and cysteine were obtained from Sigma Chemicals, St. Louis, MO. Sephadex G-25 (coarse) was from Pharmacia LKB Biotechnology,
* To whom correspondenceshould be addressed. Phone: (908) 235-5320. Fax: (908) 235-4850. + Department of Chemistry, Rutgers-The State University of New Jersey, Piscataway, NJ 08854. f R.W. Johnson PRI, R t 202, Raritan, N J 08869. 1043-1802/93/2904-0314$04.00/0
Piscataway, NJ. Triethylamine (sequenal grade) was obtained from Pierce Chemicals, Rockford, IL. Polymethacrylate (25% ) was obtained from Polysciences, Warrington, PA, and precast polyacrylamide gels (1020 % ) were purchased from Intergrated Separations Systems Inc., Natick, MA. All aqueous solutions were made with deionized water (Milli-Q, Millipore, Bedford, MA). General Procedures. Gel Filtration. The products obtained after each synthetic step were purified through G-25 coarse material. Elution was with degassed 0.1 N acetic acid. PEG Detection. To each 50-pL aliquot of the gel filtration fractions was added 1 mL of 0.02% polymethacrylate (PMA) in 0.02 N HC1 (10).The intensity of light scattering a t 412 nm was directly proportional to the amount of PEG. (Data not shown). Free Thiol Detection. The standard Ellman method (11) was adapted. To each 150-pL aliquot of the gel filtration fractions was added 1 mL of 0.1 M potassium phosphate buffer, pH 8, and 40 pL of 5,5’-dithiobis(2nitrobenzoic acid) a t 5 mg/mL in the phosphate buffer. The solution was mixed and assayed a t 412 nm. Determination of the 4-Thiopyridone Content in PEGSS-4TP. A varying amount of PEGSS-4TP (100, 200, and 400 pL of 2 pmol/mL in water) was reacted separately with an excess of cysteine (200 pL of 91 pmol/ mL in water) and diluted to a final volume of 1.4 mL with 0.1 M potassium phosphate buffer, pH 7. The amount of 4-thiopyridone liberated was quantitated using a molar extinction coefficient of 1.98 X lo4 (12)a t 323 nm. HPLC. A Hitachi liquid chromatography system equipped with an L-6200 pump and L-4200 variable detector was used. A TSK 3000 SWXLgel permeation column was used with a mobile phase of 0.12 M potassium phosphate/0.2 M sodium chloride, pH 6.6. A Supelco C-4 reverse-phase column was used for desalting, with water and acetonitrile as mobile phases. A HEMA CM cation exchage column (15 cm X 4.6 mm) from Alltech (Deerfield, IL) was used with 20 mM sodium acetate, pH 5.8, and 50 mM sodium acetate/0.5 M sodium chloride11 M potassium chloride, pH 6.2, as mobile phases A and B, respectively. Preparation of Tosyl-PEG. Methoxypolyethylene glycol of molecular weight 5000 (5 g, 1mmol) was dissolved in 26 mL of dry pyridine and treated with tosyl chloride 0 1993 American Chemical Society
lhlol-Acthrated Polyethylene Glycol (9 g, 50 mmol). The solution was stirred and brought to 65 "C in a hot water bath. It was allowed to react for 3 h and left under ambient conditions overnight. The pyridine was evaporated under vacuum to dryness. The tosyl-PEG was purified from tosyl chloride by passage through Sephadex G-25 (coarse, 50 cm X 3 cm), with 0.1 N acetic acid as eluant. The void volume, positive to the PMAtest (see above), was collected, frozen, and evaporated to dryness using a vacuum concentrator. PEG recovery was 80%. Preparation of PEGThioacetate. The tosyl-PEG was taken up in 6 mL of dry pyridine with extensive mixing using a vortex mixer. In a separate flask, 1.2 g (10.5 mmol) of potassium thioacetate was dissolved in 15 mL of pyridine/methanol(2:1) mixture. The tosyl-PEG solution was then added to the thioacetate mixture with stirring and 500 pL of triethylamine was added. The flask was then layered with N2, capped, and left to react overnight. It was evaporated to dryness under vacuum. The P E G thioacetate was then purified using Sephadex G-25 as above. The void volume, positive to PMA (see above), was collected, frozen, and evaporated to dryness in the vacuum concentrator. PEG recovery was 70%. The product was brown to dark purple, with a UV maximum a t 260 nm. Alcoholysis of the PEGThioacetate. PEGthioacetate (480 mg) was dissolved in 16 mL of dry methanol. Hydroxylamine (1.5 g) was added to form a saturated solution. Sodium methoxide, 25 5% in methanol (3.64 mL), was also added to make a 1M solution. It was heated in a water bath with stirring a t 60 "C for 1h and then left under ambient conditions overnight (24 h). The solution was neutralized with 9 mL of 5 N acetic acid, frozen, and evaporated to dryness using a vacuum concentrator. The product obtained after purification through the Sephadex column was negative to the Ellman reagent. The product was then reduced by dissolving in 4.2 mL of 0.1 M potassium phosphate buffer, pH 8.4, and adding 155 mg of dithiothreitol (DTT). It was incubated a t 65 "C for 40 min and purified through Sephadex G-25, as before. The void volume was positive to PMA, indicating the presence of PEG, and also positive to the Ellman reagent, indicating the presence of a free thiol on the PEG. The recovery of PEG was about 70% by weight. Protection of the Free Thiol. P E G S H (284 mg, 56 pmol) in 800 pL water was mixed with 245 mg (1.1mmol) of 4,4'-dipyridyl disulfide in 700 pL of methanol. Then 1mL of 0.1 M Trid0.3 M NaC1, pH 10, was added and the reaction mixed. This was incubated a t 65 "C for 30 min with intermittent mixing. The solution was cooled and separated by Sephadex G-25, as before. The void volume was collected, frozen, and evaporated to dryness using a vacuum concentrator . Anal. Calcd for C234H4630114NS2: C, 54.28; H, 8.98; N, 0.27; S, 1.24. Found C, 53.97; H, 9.10; N, 0.26; S, 1.04. Preparation of PEGPapain. Papain (15.5 mg, 674 nmol) was dissolved in 0.5 mL of water (degassed) and added dropwise to a 15-fold excess of PEGSS-4TP (10.4 Mmol) in 0.075 M MOPS buffer, pH 7.5, while being gently mixed. The reaction mixture was kept under ambient conditions and constantly mixed on a rotator for 2 h. It was then diluted to 12 mL with water and chromatographed using weak cation-exchangeHPLC. ExcessP E G SS-4TP was not retained, while the P E G p a p a i n was slightly less retained than unreacted papain. PEGpapain was then purified using high performance gel filtration chromatography. The peaks of modified protein were
Bioconjugete Chem., Vol. 4, No. 5, 1993 315
Scheme I CH,- (OCH,CHhm- OH
- Tosyl C1
mPEG-OH
- Purify
CH,- (OCH,CHJ@-
0-TOS
Tosyl-PEG
- Thioacefafe
1
R
- Purify
CH,- (OCH,CH,),-S-c-cH3 ~
PEG-thioacefafe
- Purify
CH,- (OCH,CH,),-~
Na *
1 CH,- (OCH2CH3,-SH
- Purify
-m - 4.4-dipyridyl disulfide
PEG-SH ~
CHI- (OCH2CHJn- S
Alcoholysis
-S -
Purify
= PEG-SS-4TP
collected and desalted on a reverse-phase C-4 column before application to SDS-PAGE. Formation of PEGBPTI at Varying pH. BPTI, a protein known to contain 18 Cys residues, all disulfide linked, was reduced and reacted with PEGSS-4TP as follows: 113 mg of trypsin inhibitor and 4.8 mg of DTT were dissolved in 5 mL of 0.1 M potassium phosphate, pH 8.4, and incubated a t 65 "C for 35 min. A 3-mL fraction was applied to a gel filtration column, 50 cm X 0.7 cm, packed with Sephadex G-25 (coarse) and eluted with 0.2 N acetic acid. The void volume containing the protein (monitored a t 280 nm) was pooled and lyophilized. The three reactant solutions were prepared in separate containers as follows: (1)2.6 mg of the reduced BPTI in 0.5 mL of 0.2 N acetic acid, (2) 3.6 mg of the native (unreduced) BPTI (control) in 0.5 mL of 0.2 N acetic acid, and (3) 19.3 mg of the PEGSS-4TP in 1mL of water. The conjugation experiment was then carried out in five separate reaction buffers of varying pH: (a) 0.1 M Tris HCU0.3 M NaC1, pH 10; (b) 0.025 M Tris HCU0.1 M NaCl, pH 7.6; (c) 0.1 M Tris HCU0.3 M NaC1, pH 6.5; (d) 0.1 M potassium phosphate, pH 4.7; (e) 0.2 N acetic acid (pH = 2.7). Reagent 1or 2 (100 pL) was mixed with 100pL of reagent 3 and 800 pL of the particular buffer and reacted for 10 min a t room temperature. A scan was taken from 400 to 220 nm. RESULTS AND DISCUSSION The transformation of monomethoxypolyethyleneglycol to the thiol-protected PEGSS-4TP was carried out according to Scheme I. The lone relatively unreactive hydroxyl group on the mPEG was activated by tosylation in dry pyridine (13, 14). The resultant tosyl-PEG was partially purified by Sephadex G-25 and reacted with potassium thioacetate (15, 16) to produce the P E G thioacetate. This was also partially purified by Sephadex G-25 and subsequently treated in basic alcoholysis (17)to give the PEG-SH. The reactive free thiol was then protected using 4,4'-dipyridyl disulfide (12,18).
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Bloconjugate Chem., Vol. 4, No. 5, 1993 -B-
Woghiren et al.
*OD PMA
OD Ellman PEG-SH
E
C
0.6
A
L
1
1 x,;,;,ij, L
