Peptide Modifications: Linkers, Spacers and PEGylation

Peptide Modifications: Linkers, Spacers and PEGylation

LifeTein provides custom PEGylated conjugates and fatty acid conjugation services for peptides, and small molecules. PEGylation is the process of covalently attaching polyethylene glycol (PEG) polymer chains to peptides. By increasing their molecular mass and shielding them from proteolytic enzymes, PEGylation improves the pharmacokinetics of peptides and proteins. PEGylation reduces renal clearance and results in more sustained absorption after subcutaneous administration, as well as restricted distribution. PEGylations have been shown to significantly improve water solubility, biocompatibility, immunogenicity, and other physico-chemical properties. It is an established method for the delivery of biopharmaceuticals.

The Glutathione (GSH), a tripeptide formed from glutamic acid, cysteine and glycine, is active in many biological redox reactions. The conjugation of glutathione to its PEGylated liposome can be transported through the blood brain barrier in vivo via a sodium-dependent transporter. The GSH PEGylated liposomes enhance delivery of a fluorescent marker in the brain.

LifeTein will PEGylate your peptide, or small molecule, and deliver your PEGylated product with a certificate of analysis, as a regular end-product, for further testing at your site.

As a global partner, we can supply commercial quantities of high quality functionalized PEGs, which are essential for your PEGylated therapeutic proteins.

Our PEGylation services and fatty acid conjugation include:

Multiple choices: amine reactive PEG acid, carbonyl reactive aminooxy PEG, click chemistry alkyne PEG and PEG azide, DBCO PEG, lipid PEG in drug delivery DSPE PEG, thiol reactive PEG Maleimide, fatty acids of C18, C20 and more

PEGylation feasibility studies

Pilot production and process development of PEG-drug conjugate

Development of PEGylated biosimilars

Discover more about Peptide Drug Conjugation and linkers and spacers.

Pharmacological advantages of peptide PEGylation:

Improved peptide solubility and enhanced protection from proteolytic degradation. The PEG polymer, along with its associated water molecules, acts like a shield to protect the attached peptide drug from enzyme degradation, thereby limiting adverse immunological effects. PEGylated peptides are more stable over a range of pH and temperature changes compared with their un-PEGylated counterparts.

Reduced dosing frequency with potentially reduced toxicity. PEG exhibits little toxicity, and is eliminated intact from the body by either the kidneys (for PEGylated drugs <30 kDa) or in the feces (for PEGylated drugs >20 kDa).

Increased peptide or drug stability and extended circulating life. PEG lacks immunogenicity, and antibodies against PEG in rabbits are generated only if it is combined with highly immunogenic proteins.

LifeTein can attach the PEGs to peptides. Some examples are listed below:

Monofunctional PEG Maleimide, Molecular Weight of 1K, 2K, 5K, 10K, 20K, or 30K

Bifunctional Maleimide PEG Maleimide, Molecular Weight of 1K, 2K, 5K, 10K, 20K, or 30K

DSPE-PEG-MAL, Maleimide functionalized PEG Lipid,Molecular Weight of 1K, 2K, or 5K

Biotin PEG Maleimide, FITC, PEG Maleimide, Multi-arm-4/8 arm-PEG Maleimide

Fmoc-ε-Ahx-OH, or N-ε-Fmoc-ε-aminocaproic acid; or Fmoc-6-aminohexanoic acid; CAS number: 88574-06-5; Molecular weight: 353.42 g/mol; Molecular Formula: C21H23NO4. Ahx or b-Ala can be used successfully as spacers during the generation of FITC-labeled peptides, which increases the stability of the fluorescent label. FITC can also be linked easily to a cysteine thiol moiety or to the amino group of lysine at any position.Synthesis using an Ahx linker in the lysine core resulted in better yields. Ahx increases the flexibility of peptide chains, which might help keep peptide chains properly solvated during synthesis, thereby preventing aggregation and increasing the amount of viable growing peptide sequences.

Linker/Spacer (Examples)

Fmoc-Glycine

2 Carbons

3-Amino-3-(2-Nitrophenyl) Propanoic Acid (ANP Linker)

3 Carbons

Fmoc-beta-Ala-OH

4-Aminobutyric Acid (GABA)

Fmoc-GABA-OH

4 Carbons

5-Aminovaleric Acid (Ava)

5 Carbons

Aminohexanoic Acid (Ahx)

6 Carbons

mini-PEG or AEEA

Fmoc-NH-PEG2-CH2COOH

Length of Bonds: 9

mini-PEG2 or AEEP

Fmoc-NH-PEG2-CH2CH2COOH

Length of Bonds: 10

AEEEA

Fmoc-NH-PEG3-CH2COOH

Length of Bonds: 12

AEEEP, or PEG3

Fmoc-NH-PEG3-CH2CH2COOH

Length of Bonds: 13

AEEEEP, PEG4

Fmoc-NH-PEG4-CH2CH2COOH

Length of Bonds: 16

AEEEEEP, PEG5

Fmoc-NH-PEG5-CH2CH2COOH

Length of Bonds: 19

1. Short-Chain Fatty Acids (Usually fewer than 6 carbon atoms)

Formic acid

C1

HCOOH

Saturated

Acetic acid

C2

CH₃COOH

Propionic acid

C3

CH₃CH₂COOH

Butyric acid

C4

CH₃(CH₂)₂COOH

Valeric acid

C5

CH₃(CH₂)₃COOH

2. Medium-Chain Fatty Acids (6 to 12 carbon atoms)

Caproic acid

C6

CH₃(CH₂)₄COOH

Caprylic acid

C8

CH₃(CH₂)₆COOH

Capric acid

C10

CH₃(CH₂)₈COOH

Lauric acid

C12

CH₃(CH₂)₁₀COOH

3. Long-Chain Fatty Acids (13 to 21 carbon atoms)

Myristic acid

C14

CH₃(CH₂)₁₂COOH

Palmitic acid

C16

CH₃(CH₂)₁₄COOH

Stearic acid

C18

CH₃(CH₂)₁₆COOH

Oleic acid

C18:1

CH₃(CH₂)₇CH=CH(CH₂)₇COOH

Monounsaturated

Linoleic acid

C18:2

CH₃(CH₂)₄(CH=CHCH₂)₂(CH₂)₆COOH

Polyunsaturated

Arachidic acid

C20

CH₃(CH₂)₁₈COOH

4. Very-Long-Chain Fatty Acids (22 or more carbon atoms)

Behenic acid

C22

CH₃(CH₂)₂₀COOH

Lignoceric acid

C24

CH₃(CH₂)₂₂COOH

Cerotic acid

C26

CH₃(CH₂)₂₄COOH

{PEGn}: Fmoc-NH-PEGn-CH2CH2COOH

{mini-PEG}, or {AEEA}, Fmoc-NH-PEG2-CH2COOH, CAS number: 166108-71-0; 2-[2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid; Molecular weight: 385.42 g/mol; C21H23NO6

{mini-PEG2}, {AEEP}: Fmoc-NH-PEG2-CH2CH2COOH, CAS number: 872679-70-4; Fmoc-NH-PEG2-Propionic Acid; 9-(Fmoc-amino)-4,7-dioxanonanoic acid; Molecular weight: 399.5 g/mol; C22H25NO6

{AEEEA}: Fmoc-NH-PEG3-CH2COOH, CAS number: 139338-72-0; Molecular weight: 429.46 g/mol; C23H27NO7

{AEEEP}, or {PEG3}: Fmoc-NH-PEG3-CH2CH2COOH, CAS number: 867062-95-1; Molecular weight: 443.50 g/mol; C24H29NO7

{AEEEEP}, or {PEG4}: Fmoc-NH-PEG4-CH2CH2COOH, CAS number: 557756-85-1; Fmoc-NH-PEG4-Propionic Acid, or Fmoc-15-amino-4,7,10,13-tetraoxapentadecacanoic acid; Molecular weight: 487.6 g/mol; C26H33NO8

{AEEEEEP}, or {PEG5}: Fmoc-NH-PEG5-CH2CH2COOH, CAS number: 882847-32-7; Fmoc-NH-PEG5-Propionic acid, or Fmoc-18-amino-4,7,10,13,16-pentaoxaoctadecanoic acid; Molecular weight: 531.6 g/mol; C28H37NO9