Polyethylene Glycol Building Blocks for PEGylation

Matthias Junkers

ChemFiles 2010, 10.3, 10

Circulatory half-life is a key success factor for new drugs. In this respect, PEGylation or PEG-ing—the modification of potential candidates ranging from non-peptidic small molecules to peptides and proteins, antibody fragments, aptamers, and saccharides or oligonucleotides with polyethylene glycol chains—offers numerous advantages.

PEGylation is considered one of the most successful techniques to enhance the therapeutic and biotechnological potential of peptides and proteins.¹

• PEGs are non-toxic, non-immunogenic, non-antigenic, highly soluble in water, and FDA approved.²
• The PEGylated conjugates show a decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity as the PEG coating prevents the approach or recognition by proteolytic enzymes and antibodies. (Scheme 1)
• Furthermore, PEGylation may favorably alter drug distribution in the organism.³
• While PEGylation may improve the pharmacokinetic properties of a drug, the drug’s main activity is predominantly preserved.⁴

Since the first therapeutic PEG-protein conjugate (PEG-adenosine deaminase) had been approved by the FDA in 1990, a large number of new PEGylated drugs was introduced to the market representing a multi-billion dollar business.² Table 1 summarizes a selection of FDA approved PEG conjugates including therapeutic peptides, proteins, small molecules, oligonucleotides, and antibodies:

Scheme 1. PEGylation may improve the properties of drug candidates

Table 1. Examples of FDA approved therapeutic PEG conjugates (Ref 5-9)

Year	Disease	PEG conjugate	Tradename
1990	Severe combined  immunodeficiency disease(SCID)	PEG-adenosine deaminase	Oncaspar•
1994	Acute lymphoblastic leukemia	PEG-asparaginase	Adagen•
2000	Hepatitis C	PEG-interferon α2β	PEG-Intron•
2002	Hepatitis C	PEG-interferon α2a5	Pegasys•
2002	Treating of neutropenia during  chemotherapy	PEG-G-CSF(pegfilgratism)	Neulasta•
2002	Acromegaly	PEG-growth hormone receptor antagonist, pegvisomant6	Somavert•
2004	Age-related macular degeneration	Branched PEG-anti-VEGFaptamer, Pegaptanib  (obligonucleotide PEG conjugate)7	Macugen•
2005	Ovarian cancer, Karposi's sarcoma, AIDS related cancer	Pegylated liposome-encapsuled doxorubicin (small molecule drug)8	Doxil•, Caelyx•
2008	Crohn's disease	PEGylated fab fragment of humanized anti-tumor necrosis factor (monoclonar antibody), certolizumab pegol9	Cimzia•

Drug Discovery Applications for PEGs

More recently, researchers have enforced strategies that utilize releasable PEGs in order to control protein release rates.¹⁰ Usually a trigger moiety is introduced between protein and PEG that can be cleaved enzymatically or by aqueous hydrolysis.

Further important applications of functionalized polyethylene glycols apart from drug discovery are:

• Introduction of solubilizing handles in SPPS¹¹
• oluble polymer supports for Peptide Synthesis¹²
• oluble polymer supports for Organic Synthesis¹³
• Introduction of hydrophilic amino acids in Peptide Synthesis¹⁴
• Preparation of PEG-coated surfaces¹⁵
• Linking of macromolecules to surfaces¹⁶
• Preparation of PEG-cofactor adducts for biorectors¹⁷

Reagents for PEGylation

We are proud to offer a comprehensive portfolio of PEG reagents, with molecular weights up to 20 kDa for efficient PEGylations. Numerous homobifunctionalized, heterobifunctionalized, and mono-methoxy endcapped monofunctionalized PEG’s are available in high quality and monodispersity. We provide polyethylene glycols activated for the most widely used conjugation techniques to primary amines or thiols. The amino groups (e.g. lysine) of proteins and peptides are the preferred choice for PEG conjugation by alkylation or acylation. Alkylation maintains a positively charged amine, whereas acylation yields a neutral amide linkage. Acylation can be realized most conveniently with N-hydroxysuccinimide (NHS) activated carboxylated PEGs. Azide functionalized PEGs allow the application of Staudinger ligation and Huisgen dipolar cycloaddition techniques. The introduction of different protecting groups leads to extremely useful macromolecular cross-linking agents and spacers. All common target moieties can be specifically addressed with the appropriate functionalized PEG linker. Please find a collection of our most recent additions to our portfolio of PEGylation products below, and visit us on the web for a full product listing.

References

1. Freitas, D. . SciTopics 2010, April 14th. www.scitopics.com/ PEGylation.

2. Veronese FM, Pasut G. 2005. PEGylation, successful approach to drug delivery. Drug Discovery Today. 10(21):1451-1458. https://doi.org/10.1016/s1359-6446(05)03575-0

3. Veronese FM. 2001. Peptide and protein PEGylation. Biomaterials. 22(5):405-417. https://doi.org/10.1016/s0142-9612(00)00193-9

4. Veronese FM, Mero A. 2008. The Impact of PEGylation on Biological Therapies. BioDrugs. 22(5):315-329. https://doi.org/10.2165/00063030-200822050-00004

5. Bailon, P. et al.. 2001. Bioconjug. Chem., 12, 195. .

6. Trainer, P. J. N. . 2000. Eng. J. Med., 342, 1171. .

7. 2002. The EyeTech Study Group Retina, 22, 143..

8. Pasut G, Guiotto A, Veronese F. 2004. Protein, peptide and non-peptide drug PEGylation for therapeutic application. Expert Opinion on Therapeutic Patents. 14(6):859-894. https://doi.org/10.1517/13543776.14.6.859

9. Chapman AP. 2002. PEGylated antibodies and antibody fragments for improved therapy: a review. Advanced Drug Delivery Reviews. 54(4):531-545. https://doi.org/10.1016/s0169-409x(02)00026-1

10. Zhao, H.; Yang, K.; , Martinez., A. et al. . 2006. Bioconjug. Chem., 17, 341. .

11. Seitz O, Kunz H. 1997. HYCRON, an Allylic Anchor for High-Efficiency Solid Phase Synthesis of Protected Peptides and Glycopeptides. J. Org. Chem.. 62(4):813-826. https://doi.org/10.1021/jo960743w

12. Pillai VNR, Mutter M, Bayer E, Gatfield I. 1980. New, easily removable poly(ethylene glycol) supports for the liquid-phase method of peptide synthesis. J. Org. Chem.. 45(26):5364-5370. https://doi.org/10.1021/jo01314a032

13. Gravert DJ, Janda KD. 1997. Organic Synthesis on Soluble Polymer Supports:  Liquid-Phase Methodologies. Chem. Rev.. 97(2):489-510. https://doi.org/10.1021/cr960064l

14. Koskinen, A. M. P. et al. . 1995. Bioorg. Med. Chem. Lett., 5, 573. .

15. Harris JM, Struck EC, Case MG, Paley MS, Yalpani M, Van Alstine JM, Brooks DE. 1984. Synthesis and characterization of poly(ethylene glycol) derivatives. J. Polym. Sci. Polym. Chem. Ed.. 22(2):341-352. https://doi.org/10.1002/pol.1984.170220207

16. Andreadis JD. 2000. Use of immobilized PCR primers to generate covalently immobilized DNAs for in vitro transcription/translation reactions. 28(2):5e-5. https://doi.org/10.1093/nar/28.2.e5

17. Okada H, Urabe I. 1987. [4] Polymerizable NAD derivative and model enzyme reactor with recycling of polyethylene glycol-bound NAD.34-45. https://doi.org/10.1016/s0076-6879(87)36006-9