branched poly(ethylene glycol) linkers

Macromol. Chem. Phys. 198,2489-2498 (1997) 2489

Branched poly(ethy1ene glycol) linkers

Anthony Martinez, Annapuma Pendri, Jing Xia, Richard B. Greenwald*

ENZON, Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854, U.S.A.

(Received: November 1, 1996; revised manuscript of January 23, 1997)

SUMMARY Novel types of methoxy poly(ethy1ene glycol) (PEG) linkers (U-PEG linkers) have

been synthesized. These PEG linkers are linear polymers that attach to bioactive agents via a functional group, derived from a 2” alcohol, located in the center of the polymer chain versus the traditional terminal attachment site. These new types of linkers can be prepared with different functional groups (e. g. active ester, succinimidyl carbonate, and carbazate) for selected point of attachment, including ethylene oxide oligomers to provide “stems” when steric factors need to be addressed. Conversion of p-nitrophenyl carbonates to the more desirable succinimidyl carbonates has also been accomplished by a novel nucleophilic displacement procedure. Modification of proteins with these reagents is easily accomplished and is illustrated by the conjugation of a U-PEG linker with L-

asparaginase.

Introduction

Current trends in enzyme therapy utilize the activated linear polymer, methoxy poly(ethy1ene glycol) (PEG)’), e. g., 1, to routinely modify various therapeutic proteins*). The resulting bioconjugate imparts desirable properties such as reduced toxi- city, increased circulating half-life, enhanced plasma solubility, and reduced antigenicity to the substrate. Generally, activated esters or carbonates are employed when conjugation with &-amino groups is desired, while PEG hydrazides are used with the oxidized portions (aldehydes) of glyc~proteins~). This technology has resulted in the commercialization of PEG modified adenosine deaminase (Adage@) and asparaginase (OncaspaQ), and continues to be employed in the development of new and unique products. In particular, attachment of PEG to bovine hemoglobin (bHb)4) has provided a possible adjunct for radiation therapy of solid tumors.

In order to accomplish effective PEG modification of a bioactive agent, a balance must be maintained between the number of PEG chains (of a given molecular weight) which are required to prevent immunogenicity, and the decrease in protein activity resulting from the number of PEGS bound to, and in proximity of, active sites. This can be especially difficult in situations where there are limited sites available for attachment and in this case the size of the PEG units necessary to convey the desired properties to the bioactive species becomes of greater importance. The use of higher molecular weight polymers has, in some instances, demonstrated this point, as was shown by the conjugation of CFS-I and Ricin A5), but generally PEGS of high molecular weight (>12 kDa) are difficult to obtain in high purity with low polydispersity. The purity of PEG with a size greater than 10 kDa is reported to be diminished by the inclusion of branched structures and hydrophobic linking groups6).

0 1997, Huthig & Wepf Verlag, Zug CCC 1022-1352/97/$10.00

2490 A. Martinez, A. Pendri, J. Xia, R. B. Greenwald

We have successfully developed a new methodology for the preparation of high molecular weight PEG linkers which can solve some of the intrinsic problems men- tioned above. The novelty of these linkers is that the site of nucleophilic attachment is in the center of the PEG chain instead of at the chain terminus and thus results in a branched polymer. Since the polydispersity of the starting linear PEG of 5 kDa was 1.05, the resulting high molecular weight derivative should also exhibit a simi- lar polydispersity since no additional polymerization was done. Fig. 1 a shows a gra- phic description of a traditional PEG modified bioactive agent, while 1 b and 1 c illustrate a centrally attached PEG modified bioactive agent. Inspection of Fig. 1 b and l c reveals that these new linkers may impart an umbrella like covering to the target therapeutic agent. This potential umbrella configuration generated the trivial name U-PEG for these novel polymers. In situations where standard PEG modification may block too many active sites on the target molecule, the use’of the higher molecular weight branched U-PEG would, in concept, require only half the attachment sites to deliver an equal coverage (Fig. lb). Thus this approach may have great applicability in minimizing any loss of activity while providing increased circulating half-life and non-antigenic properties to the bioactive protein. Fig. 1 c represents a U-PEG linker that would allow the conjugation of multiple PEG chains to smaller bioactive molecules having only one site for attachment.

While other researchers have explored the use of “doubling” the number of PEG chains to provide bioconjugates of reduced antigenicity, the approach taken utilized aromatic spacer molecules; either sym triazine or benzene’). These groups are inher- ently toxic, as well as being haptens, and as such should be avoided if possible when dealing with proteins.

Chemistry

The first example which demonstrates the concept of aliphatic branched chain PEGS is given by utilizing lysine. As is often the case with chemistry of current inter- est, independent researchers converge on a defined goal at the same time. Thus, our work with branched PEG linkers, (Greenwald, R.B. and Martinez, A., International Publication number WO 95/11924) contains examples utilizing lysine which are vir- tually identical to those recently published by Harris, Veronese and coworker?). The subject of our investigations diverged at that point, so that this paper will present new and facile chemistries which enlarge and add to existing PEG linker technology.

Linear PEG polymer of molecular weight 5 kDa is generally employed for protein modification’b). In the present approach, we utilized two linear PEG strands of MW 5 kDa and connected them through the use of a trifunctional spacer in order to accomplish the synthesis of high molecular weight U-PEGS. SC-PEG9’ (1) is a convenient activated linear PEG, while the spacer group can be designed in any number of ways. It is most convenient to have nucleophilic amino groups as the terminal moieties in the trifunctional spacer group to react with the activated straight chain PEG. The remaining functional group can be chosen from various moieties such as OH, NH,, SH and C02H moieties. After suitable activation these linkers can be utilized for bioconjugation.

Branched poly(ethy1ene glycol) linkers 249 1


In the present approach, we chose commercially available symmetrical trifunctional 1,3-diamino-2-propano1. This compound was utilized very effectively as a spacer with SC-PEG 5 kDa to provide U-PEG-OH (2) in high yield (Scheme I) . Compound 2 was then activated by conversion to the p-nitrophenyl carbonate (3). Reaction of 3 with various proteins containing lysine residues"), surprisingly, afforded little or no product; however, a deep yellow reaction mixture was produced which indicated that formation of p-nitrophenol had taken place. We believc that conjugation did not occur because compound 3 rapidly ionized to a 2" carbonium ion (4) in the highly polar aqueous reaction medium, followed by rapid solvolysis to 2, a situation which cannot occur with linkers derived from I alcohols (viz. linear SC or PNP-PEG). We reasoned that a less reactive leaving group such as succinimidyl would not be as susceptible to hydrolysis as the p-nitrophenyl carbonate (PNP) moiety, but still would be capable of reacting with strong nucleophiles. Attempts to prepare succinimidyl carbonates by conventional methods"', using triphosgene and N-hydroxysuccinimide (NHS) led to decomposition of 2. To circumvent this pro- blem a novel approach was taken. Since it appears that 4 is formed only in polar solvents, the reaction of 3 with NHS in a non-polar solvent, like methylene chloride, could provide an alternate pathway to the desired succinimidyl carbonate derivative 5. In fact reaction of 3 and NHS in the presence of a base, e.g. diisopropylethylamine (DIEA) took place in methylene chloride to produce 5. In contrast to 3, compound 5 reacted with amines and with amino groups of proteins to form carbamates in good yield. We have successfully used PEG 12 kDa in the same fashion, but with 20 kDa polymer a decrease in yields was noted. Reaction of 5 with hydrazine gave a 90% yield of U-PEG carbazate 6 which can be considered for conjugation of oxidized glycoproteins. In compounds 5 and 6 nucleophilic attack in the center of a polymer chain is required for conjugation. Steric hindrance at the point of attachment could potentially lead to longer reaction times, a need for higher temperatures, lower yields or even no reaction with a protein. This consideration was addressed by the synthesis of a U-PEG linker 9 having a sidc chain as outlined in Scheme 2 .

Commercially available 2-(2-aminoethoxy)ethanol reacted smoothly with 5 to give 7 in 84439% yield. Activation of the w-hydroxy group was accomplished by reacting 7 with p-nitrophenyl chloroformate in pyridine to give the PNP derivative 8. Com- pound 8 is derived from a 1" alcohol and therefore no solvolysis would be expected to occur in this case as it did for 3. Thus, while 8 can probably be utilized for protein conjugation, it was nonetheless converted to the preferable succinimidyl carbonate 9 in the same manner as 5 to demonstrate the generality of the displacement reaction. The conversion of PNP derivatives into SC derivatives provides a method that can be utilized when chlorine sensitive groups such as carbamates are present, or when reagents such as disuccinimidyl carbonatel*) are ineffective. The side chain length in 9 can be easily adjusted as needed to address steric considerations, and also utilizes short PEG chains in order to minimize introduction of potential haptens.

Higher molecular weight doubly branched U-PEGS like compound 10 (Scheme 3 ) were also prepared from the key intermediate 5 on reaction with 1,3-diamino-2-propanol, and can be used after further activation (compound 11) to conjugate multiple PEG chains with smaller bioactive molecules where there is only one active site.

Branched poly(ethy1ene glycol) linkers 2493

N

t

I" z 1 z

n o

$ I I O=Y

" 7 O='i

0

I L D

z - O=f O='i O=Y O'Y

9r (I w (I w

n n E E

9r u w n n

E E

1 F ti

(D

2494

Scheme 2:

A. Martinez, A. Pendri, J. Xia, R. B. Greenwald

Pyridin.TToiwno

0 II

mPEG&J-c -NH

0 0 II

mPEGd-C-NH

NHWCHzClz Diiaopropylethylamine I 0

II mPEGm-O- C -NH

0 -C -NHCHZCH~OCH$H

0 mPEGd-C-NH 0 I1 t! 9

The facile use of these U-PEG linkers was demonstrated by modification of L-

asparaginase with U-PEGS 5 and 9. Specific activity values of native protein and its conjugates are presented in Tab. 1 . The results show that U-PEG modified L-asparaginase with fewer attachments contains more weight of PEG per unit weight of protein when compared to linear (SS-PEG)’’ modified enzyme. At the same time the corresponding enzymatic activity is maintained at a higher level. Further evaluation of these linkers, which should define the immunoreactivity profiles of their conjugates will be addressed in future work.

Branched high molecular weight PEG linkers, with various reactive groups located in the center of the polymer backbone, and capable of achieving novel con- figurations, have been synthesized using the readily available trifunctional spacer group, 1,3-diamino-2-propano1, and easily obtained linear poly(ethy1ene glycol) derivatives such as SC-PEG. These new compounds can also be modified with linear stems. Additionally, synthesis of succinimidyl carbonates by displacement of p- nitrophenyl carbonates with NHS offers a new and mild procedure for preparing this class of active linkers.


Scheme 3: 5

0 I I

mPEG&-C -NH /

0 II

H W C - h P E G w \

Tab. 1. nase employing various PEG linkers

Relation between enzymatic activity and degree of modification of L-asparagi-

Modifying reagent Molar ratioa) Degree of grams PEG/ Specific modificationb) grams protein enzymatic in % activity in

IU/mg

Non modified 200 (L)-asparaginasec) SS-peg 5000 300 83 2.7 129 compound 5 300 63 4.1 132 compound 5 70 60 3.9 166 compound 9 300 66 4.3 127

Molar ratio in this case is the amount of mmols of the PEG linker used per mmol of (L)-asparaginase. Degree of modification represents the moles PEG/mole protein after modification. Enzyme modification reactions are done in triplicate and the variance in enzymatic activity is do%. L-Asparaginase is a globular protein of MW 141000 D made up of 4 identical sub units with a total of 92 free amino groups.


Experimental part

General methods

Unless stated otherwise, all reagents and solvents were used without further purifica- tion. Asparaginase was purchased from Merck and all buffer components were purchased from Sigma Chemical Company. mPEG (a-hydroxy-w-methoxypoly(oxy-1,2-ethane- diyl): 5.12 and 20 kDa) were obtained from NOF America (New York, NY). These polymers were dried under vacuum or by azeotropic distillation from toluene prior to use. The 'H and 13C NMR spectra were taken using a JEOL CPF-270 NMR spectrometer. The sample was prepared by dissolving 100 mg of PEG compound in 0.5 mL of deuterated chloroform (CDCI,) containing 0.03% tetramethylsilane (TMS) as internal standard. For the 'H NMR, 100 scans at a frequency of 270.05 MHz and a pulse width of 5.4 ps were employed. The peak due to TMS was used as the reference and set to 0 ppm. Recording was done using 30000 scans at a frequency of 270.05 MHz and a pulse width of 2.7 ps for I3C NMR. The center peak of the triplet due to CDCI3 was used as the reference and set to 77.00 ppm. Molecular weight of U-PEGS was determined using a Waters HPLC with PL gel 5uM 10E 4A and 10E 3A GPC columns in series and DMF as mobile phase.

U-PEG-OH, compound 2 To a solution of SC-PEG9' (1, 10.0 g, 2 mmol) in methylene chloride (SO mL) was

added 1,3-diamino-2-propanol (100 mg, 1.1 mmol). This mixture was stirred for 18 h at room temperature, followed by filtration and removal of the solvent in vacuo. The resulting residue was recrystallized from 2-propanol to yield 7.1 g (70%) of 2.

'H NMR: 6 = 1 .O (s), 2.7 (brs), 3.1 (s), 3.2 (s), 3.3-3.9 (PEG, brs), 4.2 (s) and 5.8 (s). 13C NMR: 6 = 43.2, 58.1,63.0,68.7-71 (PEG), 77.5 and 156.3.

Compound 3

A solution of 2 (5.0 g, 0.5 mmol) in toluene (75 mL) was azeotroped with the removal of 25 mL of distillate. The reaction mixture was cooled to 30°C, followed by the addition of p-nitrophenyl chloroformate (120 mg, 0.6 mmol) and pyridine (50 mg, 0.6 mmol). The resulting mixture was stirred for 2 h at 45 "C, and then at room temperature for 18 h. The reaction was filtered through Celite", and the solvent was removed in vacuo. The residue was recrystallized from 2-propanol to yield compound 3 (4.2 g, 81%).

13C NMR: 6 = 39.8,58.3,63.5,69.9-71.3 (PEG), 121.6, 124.6, 146.0, 151.0, 155.0 and 156.5.

U-PEG NHS ester; compound 5

A solution of 3 (5.0 g, 0.5 mmol), N-hydroxysuccinimide (0.6 g, 5 mmol) and diisopropylethylamine (0.13 g, 1 mmol) in methylene chloride (40 mL) was refluxed for 18 h. The solvent was removed in vacuo, and the residue was recrystallized from 2-propanol to yield (4.2 g, 82%) of compound 5.

'H NMR: 6 = 1.0 (s), 2.4 (s), 2.9 (s), 3.3 (s), 3.4-3.8 (PEG), 3.9 (t), 4-4.4 (m). 13C NMR: 6 = 24.8,39.7,58.3,63.5,68.7-71.5 (PEG), 150.2, 156.3 and 168.1.

U-PEG carbazate, compound 6 To a solution of 5 (2.0 g, 0.2 mmol) in methylene chloride (10 mL) at room tempera-

ture was added hydrazine hydrate (0.1 g, 0.3 mmol) and the resulting solution was stirred


for 3 h. Removal of the solvent in V ~ C U O yielded a viscous oil, which was crystallized from 2-propanol to give 6 (1.8 g, 90%).

I3C NMR: 6 = 41.3, 59.1,64.1, 155 and 157.0.

Compound 7 To a solution of 5 (12.0 g, 1.2 mmol) in methylene chloride (100 mL) was added 2-(2-

aminoethoxy)ethanol (0.19 g, 1.8 mmol). The resulting solution was stirred at 40°C for 18 h, followed by removal of the solvent in vucuo. The residue was recrystallized from 2- pro anol to yield 7 (1 I .O g, 89%).

"C NMR: 6 = 40.2,40.5,58.3,60.7,63.2,68.8-71.8 (PEG), 155.2 and 156.2.

Compound 9 A solution of 7 (5.0 g, 0.5 mmol) in toluene (50 mL) was azeotroped with the removal

of 10 mL of distillate. The reaction was cooled to 30°C, followed by the addition of p- nitrophenyl chloroformate (0.12 g, 0.6 mmol) and pyridine (0.05 mL, 0.6 mmol) and stirred at 40°C for 18 h. The reaction mixture was filtered through Celite", and the solvent was removed in vucuo. The residue was crystallized from 2-propanol to yield 8 (4.3 g, 86%).

13C NMR: 6 = 40.0, 40.5, 58.3, 63.3, 67.5, 68.1-71.2 (PEG), 121.3, 124.6, 144.7, 151.8, 154.8, 155.2 and 156.2.

Compound 8 was dissolved in methylene chloride (40 mL), followed by the addition of N-hydroxysuccinimide (0.46 g, 4.0 mmol) and diisopropylethylamine (0.14 mL, 0.8 mmol). The reaction mixture was stirred at 40°C for 18 h, followed by filtration through Celite" and removal of the solvent in vucuo. Recrystallization of the residue from 2-propanol gave 9 (2.6 g, 84%).

13C NMR: 6 = 24.8, 40.2, 40.7, 58.4, 63.4, 67.7, 68.5-72.4 (PEG), 151.12, 155.3, 156.2 and 168.7.

Compound 10 To a solution of 5 (3.0 g, 0.3 mmol) in methylene chloride was added 1,3-diamino-2-

propanol (17 mg, 0.19 mmol). The resultant solution was stirred at room temperature for 18 h, followed by removal of the solvent in vacuo to yield a viscous oil which was recrystallized from 2-propanol to yield 2.2 g (71 %) of 10.

13C NMR: 6 = 39.7,42.0,57.3,63.4,67.4-70.5 (PEG), 154.8 and 155.5.

Compound 11 A solution of 10 (10.0 g, 0.5 mmol) in toluene (50 mL) was azeotroped with the

removal of 10 mL of distillate. The reaction was cooled to 30°C, followed by the addition of p-nitrophenyl chloroformate (0.12 g, 0.6 mmol) and pyridine (0.05 mL, 0.6 mmol) and stirred at 40°C for 18 h. The reaction mixture was filtered through Celite", and the solvent was removed in vucuo. The residue was crystallized from 2-propanol to yield the corresponding p-nitrophenyl carbonate derivative 11 (8.6 g, 86%).

13C NMR: 6 = 40.0, 43.0, 57.4, 62.5, 67.5, 68.1-70.5 (PEG), 121.5, 124.0, 144.2, 154.0, 154.8 and 155.5.

Enzyme modi$ication - general procedure Conjugation of U-PEG 5 or 9 to L-asparaginase: U-PEG 5 or 9 (87.5 mg, 0.0085 mmol,

40 eq) was added to native L-asparaginase (30mg, 833 FL, 0.0002 mmol) in 6 mL of

249 8 A. Martinez, A. Pendri, J. Xia, R. B. Greenwald

sodium phosphate buffer (0.1 M, pH 7.8) with gentle stirring. The pH of this solution was monitored and adjusted to 7.8 with 0.5 M sodium hydroxide. This solution was then stirred at 37 "C for 1 h and diluted with 12 mL of formulation buffer (0.05 M sodium phosphate, 0.85% sodium chloride, pH 7.3), and diafiltered with a 50000 centriprep in order to remove the unreacted PEG. Diafilteration was continued with formulation buffer added as needed at 4°C until no more free PEG precipitates were detected by mixing equal amounts of filtrate and 0. I % PMA (poly(methacry1ic acid) in 0.1 M HCI). The specific activity of U-PEG asparaginase was measured using asparaginase assay"'. TNBS assay14' was used to calculate the percent modification of the protein. Biuret assay"' was used to check the protein concentration.

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branched poly(ethylene glycol) linkers

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