Preparation of well-defined protein conjugates using enzyme-assisted

Feb 15, 1991 - exists nowhere else on the protein, specificity is assured in a subsequent chemical reaction (formation of a hydrazone bond) of the mod...
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Bioconjugate Chem. 1991, 2, 154-159

Preparation of Well-Defined Protein Conjugates Using Enzyme-Assisted Reverse Proteolysis’ Keith Rose,. L. Antonio Vilaseca, Raymond Werlen, Anne Meunier, Igor Fisch, Robert M. L. Jones, and Robin E. Offord DBpartement de Biochimie MBdicale, Centre MBdical Universitaire, 1 rue Michel Servet, 1211 Geneva 4,Switzerland. Received February 15, 1991

A two-step approach to the production of well-defined protein conjugates is described. In the first step, a linker group, carbohydrazide, having unique reactivity (a hydrazide group) is attached specifically to the carboxyl terminus by using enzyme-catalyzed reverse proteolysis. Since the hydrazide group exists nowhere else on the protein, specificity is assured in a subsequent chemical reaction (formation of a hydrazone bond) of the modified protein with a molecule (chelator, drug, or polypeptide) carrying an aldehyde or keto group. The product is sufficiently stable at neutral pH, no reduction of the hydrazone bond being necessary for the hydrazones described. Protein modification is thus restricted to the carboxyl terminus and a homogeneous product results. With insulin as a model, conditions are described for producing such well-defined conjugates in good yields. The use of other linker groups besides carbohydrazide, and applications of these techniques to antibody fragments, are discussed.

INTRODUCTION

Artificial protein conjugates, consisting of a protein covalently linked to another group (reporter group, radiolabel, cytotoxic agent, polypeptide chain, etc.) are useful in many areas of research. However, most current methods for preparing protein conjugates suffer from two disadvantages. The first disadvantage is that current methods of protein modification tend to produce inhomogeneous material. Even material substituted with only one modifying group is in general itself heterogeneous, being a mixture of species each having one group attached to one of several residues of the type involved in the modification reaction. The use of a heterogeneous preparation can lead to problems of interpretation of results, since different isomers may have different biological properties (e.g. the monoiodoinsulins). A homogeneous product is desirable, particularly for clinical applications. The second disadvantage of current methods is that attempts to reach a high degree of substitution of the protein tend to lead rapidly to loss of protein function. This is illustrated by numerous attempts to increase the degree of labeling of antibodies: in general, a higher degree of labeling leads to a lower degree of antigen binding (I). We believe that both these disadvantages of most current methodologies may be circumvented by attaching groups to proteins in a site-specific manner, ideally far from the functional region of the protein in question (active site, in the case of an enzyme, or antigen binding region, in the case of an antibody or fragment thereof). The advantages of “site-specific” modification, through oxidized carbohydrate, of antibodies of the IgG type have been reported (2,3). The success of such an approach depends upon the existence of appropriate glycosylation,so it is not generally applicable to other classes of polypeptide (nor even to antibody fragments of the F(ab) and F(ab)z types, in general). Since some IgG molecules possess more than

* Author to whom correspondence should be addressed.

Abbreviations used: DAI, des-AlaW-insulin;HPLC, highpressure liquid chromatography; TPCK, ~-1-chloro-4-phenyl-3(tosylamino)butan-2-one;TFA, trifluoroacetic acid; FAB-MS, fast atom bombardment mass spectrometry; EDTA, ethylenediaminetetraaceticacid.

one site of glycosylation, the carbohydrate oxidation approach may not always be site-specific. Several groups of workers have exploited the specificity of proteases working in reverse to fix, exclusively a t the carboxyl terminus of polypeptides, groups which are not normally found in proteins. In a subsequent chemical reaction, the conjugation step, a bond is formed between this enzymatically introduced group and a molecule of interest carrying an appropriate functionality. (It is not usually possible or desirable to fix enzymically the molecule of interest directly to the polypeptide chain for reasons of enzyme specificity or the large excess of amino component generally required to drive the enzymic coupling to high yield.) Thus, Carpenter’s group attached phenylhydrazine prior to oxidation to a diimide (4),and various workers (5-7) attached hydrazine or protected hydrazine as a prelude to azide coupling. Such approaches, used to join together two peptide fragments by means of a peptide bond, require a strategy involving protection and deprotection in order to obtain coupling to the desired amino group, a strategy which is inappropriate when dealing with proteins which are easily denatured. We (8,9) have used reverse proteolysis to attach amino acid dichlorophenyl esters, which are aminolyzed in a subsequent step, but again protection and deprotection are required if aminolysis is to be attempted in cases where there is no conformational assistance. Wilchek’s group (IO) has used carboxypeptidase Y to attach biocytin amide and “-(maleimidopropiony1)-L-lysinamideto the carboxyl terminus of a variety of proteins and reported yields of 40-75%. The conditions used were rather harsh (waterldimethylformamide/ethanol 2:1:1, with shaking), and “ragged ends” are to be expected since carboxypeptidase Y may remove more than one residue before the reaction is stopped. We are interested in conjugation techniques based on reverse proteolysis which do not require protection followed by deprotection and which yield homogeneous products under very mild conditions; such techniques should be applicable to polypeptides which are relatively easily denatured, such as antibodies and fragments of antibodies. Our initial approach involved reductive alkylation (II, I2),which gave good results with insulin but was less easy to apply to antibody fragments owing to the 0 1991 American Chemical Society

Proteln Conjugates by Reverse Proteolysis Scheme I. Examples of the Formation of a Defined Protein Conjugate.

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acid. DAI was dissolved in the carbohydrazide solutions at 1, 2,and 20 mg/mL. Enzyme was added as a freshly H~N-CHRI-CO- (...) -NH-CHR,-CO-OH prepared solution in water (10 mg/mL for Achromobacter protease; 10or 100mg/mL for trypsin), and the samples were incubated a t room temperature for various times. Reversed-Phase High-pressure Liquid Chromatography (HPLC). Samples were generally injected directly H~N-CHRI-CO- (...) -NH-CHRn-CO-NHNH-CO-NHNH2 onto a Waters HPLC system, with monitoring at 214,229, or 280 nm. In the case of incubations performed at a pH greater than 5.5, samples were acidified with 10 volumes of pure acetic acid then diluted with 100 volumes of 0.1 5% trifluoroacetic acid (TFA)prior to injection. For analytical H~N-CHRI-CO- (...) -NH-CHR,-CO-NHNH-CO-NHN=CH-R work, a column 25 cm X 4 mm i.d. (Nucleosil300-A 5-pm Stepi consistsof a protease-catalyzedcoupling step conducted (28, Machery-Nagel, Oensingen, Switzerland) was used at under conditionswhere formation of a 40-NH- bond is favored a flow rate of 0.6 mL/min. Solvent A was prepared by (reverse proteolysis). The nucleophile used in step i is carboadding 1 g of TFA (HPLC grade, AB1 Inc.) to 1000 mL hydrazide, and specificity in this step is assured by the choice of HPLC-grade water (Milli-Q system), followed by of an appropriate protease. After isolation of the product of step vacuum filtration. Solvent B was prepared by adding 1 i, step ii consists of a chemical conjugation reaction resulting in g of TFA to 100 mL of HPLC-grade water (previously formation of a hydrazone. Specificity in step ii is assured by chemical complementarity. degassed by vacuum filtration) then adding 900 mL acetonitrile (gradient grade, Merck, previously degassed by vacuum filtration). For preparative work, a column 25 substantial concentrations of organic solvents necessary cm X 1 cm i.d. of Nucleosil 300-A 5-pm CIS (Macheryto obtain high coupling yields. More recently, we have been able to combine the mild conjugation conditions Nagel) was used at a flow rate of 4 mL/min. For this offered by hydrazone bond formation (see e.g. ref 13)with system, solvent A was prepared by mixing 100 mL of 3 M the specificity of reverse proteolysis (Scheme I). The fact (NH4)2S04with degassed HPLC-grade water to complete that it is not necessary, nor sometimes even desirable, to to 1000 mL. Solvent B was prepared by mixing 100 mL form an amide bond between the protein and the group of 3 M (NH4)2S04 with 350 mL of degassed acetonitrile to be attached removes the difficulty of differential and completing to 1000 mL with degassed HPLC-grade protection which would otherwise exist. water (thorough mixing is required). Before use, the pH of the 3 M (NH4)zS04 had been adjusted to 2.7 with A preliminary presentation of our work has already appeared (14,15). Using insulin as a model, we present concentrated sulfuric acid and the pH was not adjusted after dilution. Samples were recovered after preparative here a study of the range of conditions for attaching carbohydrazide to the carboxyl group of LysBmand conditions HPLC by dilution with an equal volume of 0.1% TFA for producing well-defined protein conjugates from this followed by adsorption to a Sep-Pak Cla (Waters Assomodified species. The use of other linker groups besides ciates) column, previously washed with methanol and hydrazide, and applications of these techniques, are also equilibrated with 0.1 % TFA. After washing thoroughly discussed. with 0.1% TFA, sample was eluted with 4 mL of 0.1% TFA containing 50% acetonitrile and dried in a vacuum EXPERIMENTAL PROCEDURES centrifuge (SpeedVac, Savant Instruments). HCO-m-C6H4CH=NOCH2CO-Ferrioxaminea n d Except where otherwise specified, solvents and reagents Similar Compounds. A solution of isophthalaldehyde were of analytical grade or better, were obtained from was prepared by dissolving 6.7 mg (50pmol) in 0.2 mL of commercial sources, and were used without further puCH3CN and completing to 1 mL with a buffer made by rification. Carbohydrazide was purchased from Fluka bringing a 0.1 M solution of sodium acetate in water to pH (Buchs, Switzerland). Pig insulin (Monocomponent grade) 4.6with glacialacetic acid. To the isophthalaldehyde soluwas obtained from Novo Industri (Bagsvaerd, Denmark). tion was added dropwise, with mixing, a solution of NHzIt was freed from zinc and then converted into des-AlaB30OCHzCO-ferrioxamine (prepared according to ref 18; 8 insulin (DAI), essentially according to the method of mg as isolated, ca. 10 pmol, dissolved in 1 mL of the acetate Morihara et al. (16),by incubating 100 mg of insulin with buffer). After 70 min, the reaction mixture was applied 0.5 mg of lysyl endopeptidase (from Achromobacter lytito a Cla Sep-Pak column, previously washed with methanol c ~ Wako , Pure Chemical Co.) in 20 mL of 0.1 M (10 mL) and equilibrated with 1% acetic acid (10 mL). ammonium bicarbonate solution for 30 h a t 37 "C followed The Sep-Pak was washed with 1% acetic acid/CHgCN by lyophilization. The dipeptide Trp-Phe and porcine (91v/v, 14mL) to remove excess aldehyde and the product trypsin were purchased from Sigma Chemical Co. (St. then eluted with 1 % acetic acid/CH&N (8:2,v/v; 7 mL). Louis, MO). For some experiments the trypsin was treated with ~-l-chloro-4-phenyl-3-(tosylamino)butan-2-oneSolvent was removed in the vacuum centrifuge and the product taken up in 0.1% TFA and purified by reversed(TPCK) (17). All reactions and chromatography were carried out at room temperature (ca. 23 "C) unless phase HPLC (4mm i.d. column, TFA system, monitoring otherwise specified. at 214 nm). Sample was injected at 15% B, and after 5 min a linear gradient of 0.55% /min was applied for 60min. Reverse Proteolysis. All pH values are apparent Product eluted as an orange solution with tR = 41 min. values obtained with a glass electrode calibrated with Solvent was removed in the vacuum centrifuge. Product aqueous standards, no corrections being applied. Solutions was characterized as the expected HCO-m-C6H&H= of carbohydrazide were prepared in water and in diNOCHzCO-ferrioxamine by fast atom bombardment mass methyl sulfoxide/water (1:l v/v) and were generally brought to the required indicated pH by addition of pure spectrometry (FAB-MS) in the positive ion mode using acetic acid. However, in order to obtain a pH below 5.5, glycerol, thioglycerol,acetic acid as matrix. A strong signal was found at m/z 803 (protonated molecular ion) and a the solutions were made 5 mg/mL in sodium acetate and then titrated to the desired pH value with 98% formic weaker signal at m / z 909 (adduct with thioglycerol,

Rose et at.

156 Bioconjugate Chem., Vol. 2, No. 3, 1991 100

-

1

OF!

4.5

L pH 5.0

1

loo 80 n

s

Y

E

60 -

40 -

20 pH 5.5

pH 6.5

Figure 1. Yield of DAI-carbohydrazide as a function of time and pH. The conditionswere DAI, 20 mg/mL; carbohydrazide, 9.5 M in DMSO/H& 1:l v/v. Columns refer to time points 15 min, 4 h, and 16 h (left to right, respectively, in groups of three). Horizontally hatched columns refer to catalysis with TPCK-

treated porcine trypsin (enzyme:substrate ratio 1:lO w/w). Open columns refer to catalysiswith Achromobacter protease (enzyme: substrate ratio 1:lO w/w), and slanted hatched columns refer to ccrtalvsia with Achromobacter protease (enzyme:substrate ratio 150 w/w).

-

0 pH 4.7

pH 5.1

pH 5.5

Figure 2. Yield of DAI-carbohydrazide as a function of time and pH. The conditionswere DAI, 20 mg/mL; carbohydrazide, 2.5 M in water. Columns refer to time points 15 min, 4 h, and

16h (left to right, respectively, in groups of three). Horizontally hatched columns refer to catalysis with TPCK-treated porcine trypsin (enzyme:substrateratio 1:lO w/w). Open columns refer to catalysiswith Achromobacter protease(enzyme:substrate ratio 1:50 w/w).

time a t four pH values. At pH 3.85, no product was formed commonly found with aldehydes). Iron was removed from a t all (not shown),most probably due to lack of sufficient a portion of the material (1 mg in 0.1 mL of water) by enzymaticactivity a t this low pH value. A t pH 4.5, porcine incubating with Na2EDTA (0.4 mL of a solution 50 mM trypsin, which is remarkably active at low pH values (6), made up in 1%acetic acid). All color was lost within 30 catalyzes the formation of DAI-carbohydrazide. Coupling min and a white precipitate formed which was washed is favored over cleavage, and less than 2% cleavage at three times with 0.4-mL portions of 1% acetic acid. Drying ArgB22occurs after 16 h as determined by HPLC. A t pH under high vacuum afforded 0.74 mg of white powder, 5, trypsin is still more active, and DAI-carbohydrazide is characterized by positive ion FAB-MS as the expected produced more rapidly (compare 15-min time points). iron-free material ( H C O - ~ - C G H ~ C H = N O C H ~ C OAgain, - ~ ~ less ~ ~than ~ ~ -2% cleavage a t ArgB22occurs after 16 h. rioxamine, M + H, 750, three protons having replaced the At pH 5.5., DAI-carbohydrazide is produced very rapidly trivalent iron). The metal-free material was loaded with indeed and almost quantitative couplingyield (as defined gallium under conditions adapted later for radiolabeling above) is obtained under comparable conditions (horiwith 67Ga. A suspension of 0.36 mg (0.48 pmol) of metalzontal hatching). Cleavage a t ArgBZ2was ca. 1%after 4 free compound in 1mL of the acetate buffer, pH 4.6 (see h but had risen to ca. 45% after 16 h. The yield of above), was added to 0.7 hmol of GaC13 (in solution in 103 recoverable DAI-carbohydrazide is thus compromised at pL of 0.8 M HC1, brought to an indicated pH of 4.6 through long reaction times under conditions of high enzymic the addition of 6.5 M ammonium acetate, ca. 40 pL, just activity, which is a function of pH, substrate concentration, prior to addition of the chelator). Within 2.5 h, the and enzyme:substrate ratio. A t pH 6.5, maximum coupling suspension had dissolved through capture of gallium. yield is attained after 4 h (Figure l),but the yield is not Analytical HPLC showed that the product eluted with as high as may be obtained a t lower pH. Enzymic activity approximately the same retention time as the ironis high, and cleavage a t ArgB22reached 40% after 4 h and containing material. After isolation on a Sep-Pak column 70% after 16 h. as described for the iron-containing material, the product Also shown in Figure 1are results obtained with Achwas characterized as gallium-labeled HCO-m-CsH4romobacter protease a t pH 5.5 at an enzyme:substrate CH-NOCH2CO-desferrioxamine by positive FAB-MS ratio of 1:IO (w/w) (as for trypsin) and 150, open bars and (protonated molecular ions at m / z 816 and 818 due to the slanted hatching, respectively. This protease, which is natural isotopes 69Gaand 71Ga). specific for the carboxyl side of Lys residues, permits the Hydrazone Formation. Des-AlaB30-insulin-carbohy- attainment of high yields without the danger of cleavage drazide (DM-carbohydrazide) and zinc-free insulin control of ArgBZ2. were dissolved separately (0.2 mM) in 0.1 M acetic acid, Couplingof Carbohydrazidein Aqueous Solution. and the pH was adjusted to 4.6 with 5 M NaOH. To a Similar results were obtained whether the porcine trypsin given volume of sample solution (generally 100 pL) was had been treated with TPCK or not. Figure 2 shows the added an equal volume of a solution of aldehyde dissolved yields of DAI-carbohydrazide (ratio of product to reinO.1 M acetate (Na),pH 4.6. Various aldehydesat various maining DAI, as a percentage) obtained with 2.5 M carconcentrations were used (see Results and Discussion); bohydrazide in aqueous solution, expressed as a function for reasons of solubility, 2,4-dihydroxybenzaldehydeand of time at three pH values and with two enzymes: porcine 4-nitrobenzaldehyde were dissolved in acetonitrile and trypsin a t an enzyme:substrate ratio of 1:lO (w/w) (hatched then diluted with 49 volumes of pH 4.6 buffer to obtain bars) and Achromobacter protease at an enzyme:substrate a find concentration of 1 mM. Analysis was by HPLC. ratio of 150 (w/w) (open bars). Achromobacter protease Mass Spectrometry. Equipment and operating conwas not studied at pH 4.7. Comparison with Figure 1 ditions were as previously described (19). shows that couplingwith trypsin is less rapid than in 50 % DMSO, in spite of the higher nucleophile concentration. RESULTS AND DISCUSSION The 15-min and 4-h time points clearly show that coupling Coupling of Carbohydrazidein 50%DMSO. Figure rate rises with increasing pH over the range studied. 1shows the yields of DAI-carbohydrazide (ratio of product Cleavage at ArgBZ2by trypsin was below 2% after 16 h at to remaining DAI, as a percentage) obtained with 0.5 M pH 5.1 but was ca. 25% after 16 h at pH 5.5. Achromocarbohydrazide in 50 76 DMSO, expressed as a function of bacter protease, used at an enzyme:substrate ratio of 1:50

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Proteln Conjugates by Reverse Proteolysis loo

T

0

0

4

8

12

16

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Time (h)

Figure 3. Yield of DAI-carbohydrazide as a function of time, substrate concentration, and Achromobacter protease concentration. Conditions were carbohydrazide, 2.5 M in water, pH 5.5 (adjusted with acetic acid): A, 1mg mL DAI (170 pM), 55 pg/ mL enzyme (1.8 pM); 2 mg/mL AI (340 pM), 110 pg/mL enzyme (3.7 pM);c],2 mg/mL DAI (340 pM),210 pg/mL enzyme

.,

b

(7 PM).

0.4

1 0

80 Time (min)

Figure 4. Separation of insulin and DAI-carbohydrazide. The

above chromatogram was obtained by analytical reversed-phase HPLC ("FA system), with monitoring at 214 nm, AUFS 1.0. After 5 min at 30% solvent B, a linear gradient of 0.5% /min to 50% B was applied. DAI-carbohydrazide elutes as the earlier peak at t R = 38.5 min, and insulin elutes at t R = 40 min.

by weight (compared to 1:lO for trypsin), shows somewhat slower coupling rates but permits the attainment of high yield within 16h at pH 5.5without any hydrolysis at ArgB22. At pH 5.5,equilibrium is almost reached in 16 h and a yield of 75-77 % obtained in fully aqueous medium (Figure 2). On a preparative scale, 10mg of DAI in 5 mL of solution incubated with 0.2 mg of porcine trypsin gave a coupling yield of about 75% after 18 h. These yields are considerably lower than the nearly quantitative yield obtained a t this pH in 50% DMSO. However, some protein substrates, such as antibodies and large fragments of antibodies, are denatured by and even precipitate from 50% DMSO, whereas these problems are not experienced in fully aqueousmedium (data not shown). For this reason, we studied the aqueous coupling in more detail. Figure 3 shows the yield of DAI-carbohydrazide as a function of time, substrate concentration, and enzyme concentration. At equilibrium, the yield is ca. 83%. Higher substrate concentration and higher enzyme concentration both lead to faster reaction (Figure 3). Figure 4 shows an example of the separation between insulin (which elutes at the same retention time as DAI under the conditions used) and DAI-carbohydrazide on analytical reversed-phase HPLC. The hydrazide group is charged a t the low pH of the HPLC system and this probably is responsible for the earlier elution of the modified form of DAI.

It is important to note that the reverse proteolysis reaction must be halted before separating excess carbohydrazide from the protein substrate; otherwise, particularly a t neutral pH, the enzyme will rapidly remove the C-terminal carbohydrazide group. When working with DAI, we stop the reaction by injection onto an HPLC, which is operated a t very low pH (2-3). When working with antibody fragments, however, it is preferable to add sufficient enzyme inhibitor prior to a separation by gel filtration at more moderate pH (4.6). Characterizationof DAI-Carbohydrazideand Hydrazone Formation. DAI-carbohydrazide, isolated by preparative HPLC, was found to react with a number of aldehydes (2,4-dihydroxybenzaldehyde, hitrobenzaldehyde, and several derivatives of ferrioxamine-see below) under very mild conditions (pH 4.6, fully aqueous solution, room temperature, 1-5 equiv of aldehyde) to give a corresponding product eluting later than DAI-carbohydrazide on reversed-phase HPLC. Under similar conditions, native insulin (zinc free) did not react at all. This lack of reaction between aldehyde and the amino groups of insulin is as expected, since the corresponding Schiff bases are unstable at pH 4.6 and would require reduction to an alkylamine if a product is to be isolated. Figure 5 shows the results of incubation with 5 equiv of 2,4-dihydroxybenzaldehyde. A very clean and specific reaction occurs with DAI-carbohydrazide (panels c and d of Figure 5) while no reaction at all occurs with native insulin (panel a). Panel b shows that DAI-carbohydrazide is stable in the absence of aldehyde. In the case of 4-nitrobenzaldehyde, the latereluting product was characterized as the expected hydrazone by isolation (HPLC), digestion with V8 enzyme, and analysis by FAB-MS (not shown). In addition to the known V8 fragments of insulin (20),the C-terminal fragment of the B-chain was shifted in mass from m/z 1015 (for DAI) to 1220,consistent with condensation with carbohydrazide followed by hydrazone formation with 4 4 trobenzaldehyde. In accord with previous observations concerning hydrazone formation (13), we found that the yield of hydrazone at equilibrium depends on reagent concentrations and chemical structure. When using as aldehyde HCOm-CsH&H=NOCH2CO-ferrioxamine at pH 4.6 and with a 0.1 mM final concentration of DAI-carbohydrazide, 1 equiv of aldehyde led to hydrazone yields of ca. 74 % after 78 min and 91 % after 13 h 17 min (Figure 6, panels b and c, respectively). Even with this reactive aldehyde, no trace of reaction occurred with native insulin (Figure 6, panel a), demonstrating the specificity of the proposed two-step process. Similar results were obtained with the galliumcontaining derivative and with the paraisomers (HCOp-CsH&H=NOCH&O-ferrioxamine or the corresponding gallium derivative). It is thus possible to attach a chelator specifically to the C-terminal carboxy group of a protein. The known stability of unreduced hydrazones (13)at neutral pH was confirmed with polypeptides other than DAI (which is relatively insoluble a t pH 7) and radiolabeled (66Feand s7Ga)ferrioxamine derivatives (data not shown). Since aldehydes are known to react with tryptophan residues under acid conditions (the Ehrlich reaction, occurring in strong HC1) and since insulin contains no tryptophan residues, it was important to establish whether, under mild conditions, any reaction occurred with Trp residues. Upon incubation at pH 4.6 of the peptide TrpPhe (0.1mM) with HCO-m-CsH4CH=NOCH2COrrioxamine (0.5mM), analytical HPLC showed no trace of reaction even after 12 h (results not shown).

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I

a

A214 nm a

-------

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Figure 5. Analytical reversed-phase HPLC analysis of a conjugation reaction at pH 4.6 with 2,4-dihydroxybenzaldehyde. Chromatographicconditionewere as for Figure 4. Panel a, control incubation of native insulin, 0.1 mM, with 2,4-dihydroxybenzaldehyde, 0.5 mM, after 8 h 54 min. The aldehyde elutes just after the front. Panel b, control incubation of DAI-carbohydrazide, 0.1 mM, after 14 h 28 min. Panel c, incubation of DAIcarbohydrazide,0.1 mM, with 2,4-dihydroxybenzaldehyde,0.5 mM, after 50 min. Panel d, as for c but after a 5h 23 min reaction.

Reverse Proteolysis with Other Nucleophiles. Reverse proteolysis has been used to couple to oxygen nucleophiles (alcohols (6)) but high yields are normally obtained only by coupling to NH2 groups. As referred to in the Introduction, reverse proteolysis may be used to couple nucleophiles other than hydrazides; for example, good results may be obtained with aldehyde precursors such as NH&H&H(OR)z or NH2CH[CH2SCH2CH(OH)CH20HICONH2 ( I I , I 2 ) . However, substantial proportions of organic solvent (at least 50%) are necessary in order to obtain high yields during reverse proteolysis with amines as nucleophiles, owing to their high pK (alkylamines) or low water solubility (aromatic amines). If reverse proteolysis is to be performed in fully aqueous medium, hydrazides permit high yields to be obtained (6). We have investigated the use of hydrazides other than carbohydrazide. Adipodihydrazide is not as soluble (0.5 M in water) and leads to yields of ca. 27?6 a t equilibrium. Oxalodihydrazide is almost totally insoluble in water and

I

r

0

60 Time (min)

Figure 6. Analytical reversed-phase HPLC analysis of a conjugation reaction at pH 4.6 with HCO-m-C&CH=NOCH2CO-ferrioxamine. Chromatographicconditions: the TFA system was used; after 5 min isocratic at 0% B, a linear gradient of solventB,2%/min,wasappliedto100%B,whichwasmaintained for 5 min before descending to 0% B over 5 min. Reaction conditions: protein concentration and aldehyde concentration were both initially 0.1 mM. Panel a, control incubation of aldehyde with unmodified insulin after 14 h 30 min. The reagent and insulin elute at t R = 34.3 and 36.3 min, respectively. Panel b, incubation of aldehyde with DAI-carbohydrazide after a reaction time of 18 min. DAI-carbohydrazide and the hydrazone reaction productelute at t~ =35.5 and 36.8min. respectively. Panel c, incubationof aldehydewith DAI-carbohydrazide after 13 h 17 min. so cannot be used. Succinodihydrazide is also insufficiently soluble in water (less than 0.125 M) to be useful.

CONCLUSION Reverse proteolysis may be used to attach the linker molecule carbohydrazide specificallyto the C-terminal carboxyl group of a protein substrate under mild, fully aqueous conditions. In a second step, not catalyzed by an enzyme, a molecule containing an aldehyde function may be conjugated to the protein through formation of a hydrazone bond, in high yield and under very mild conditions.

Protein Conjugates by Reverse Proteolysis

The two-step procedure described leads to the formation of well-defined, homogeneous protein conjugates with a minimum of side reactions. The results presented should permit the definition of parameters for applying the procedure to other protein substrates, such as antibodies and their fragments, to form conjugates with chelators, reporter groups, or cytotoxic agents. ACKNOWLEDGMENT We thank Ms. Irene Rossitto, Ms. Murielle Haldemann, and Mr. P.-0. Regamey for expert technical assistance. This work was supported by funding from the Fonds National de la Recherche Scientifique, Hoffmann-La Roche, Ligue Suisse Contre le Cancer. We thank the Schmidheiny Foundation for the purchase of some of the HPLC equipment. LITERATURE CITED (1)For example see: Ghose, T., and Blair, A. H. (1987)The design of cytotoxic-agent-antibody conjugates. CRC Crit.Rev. Ther. Drug Carrier Syst. 3,263-359. (2)For example, see: Murayama, A., Shimada, K., and Yamamoto, T. (1978)Modification of immunoglobulin G using specificreactivity of sugar moiety. Immunochemistry 15,523528. (3) For example, see: Rodwell, J. D., Alvarez, V. L., Lee, C., Lopes, A. D., Goers, J. W. F., King, H. D., Powsner, H. J., and McKearn, T. J. (1986)Site-specific covalent modification of monoclonalantibodies: in vitro and in vivo evaluations. Proc. Natl. Acad. Sci. U.S.A. 83,2632-2636. (4)Canova-Davis,E., and Carpenter, F. (1981)Semisynthesis of insulin: specific activation of the arginine carboxyl group of the B chain of desoctapeptide-(B23-30)-insulin (bovine). Biochemistry 20,7053-7058. (5)Jones, R. M. L., and Offord, R. E. (1982)The proteinasecatalysed synthesis of peptide hydrazides. Biochem. J. 203, 125-129. (6)Yagisawa, S. (1981)Studies on protein semisynthesis. I. Formation of esters, hydrazides and substituted hydrazides of peptides by the reverse reaction of trypsin. J. Biochem. (Tokyo) 89,491-501. (7) Zhang, Y. S.,Cao, Q. P., Li, Z. G., and Cui, D. F. (1983) Preparation of [B23-~-alanine]des-(B25-B30)-hexapeptideinsulin by a combination of enzymicand nonenzymicsynthesis. Biochem. J. 215,697-699.

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