NMR Structure of Two Novel Polyethylene Glycol Conjugates of the Human Growth Hormone-Releasing Factor, hGRF(1−29)−NH₂

Abstract

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.

Versatile Route to Synthesize Heterobifunctional Poly(ethylene glycol) of Variable Functionality for Subsequent Pegylation

“…Subsequently, the tosyl end group was converted into a variety of functional end groups, either directly or via intermediate steps as shown in Scheme I. Synthesis efficiency, final yields and molecular characteristics are summarized in Table 1. PEG (1). Tosylate groups are known as good substrate for substitution reactions.…”

“…Here, α-thiol-ω-hydroxyl PEG (5) was prepared by reaction of α-tosyl-ω-hydroxyl PEG (1) with an excess of sodium hydrosulfide hydrate in water. The downfield tosylate aryl peaks were not detectable by 1 Figure S10) confirming the complete displacement of the tosylate group by the hydrosulfide ion. However, the GPC chromatogram presented in Figure 1(b) shows two peaks, one with similar retention time as the starting α-tosyl-ω-hydroxyl PEG and a second peak with shorter retention time corresponding to twice the molar mass.…”

“…Other chemicals and their suppliers were toluene (VWR, Switzerland), dichloromethane (DCM), methanol (MeOH) and diethyl ether (Fisher Scientific, Switzerland), filter cell cake (Macherey-Nagel, Switzerland), and sodium sulfate anhydrous (AppliChem, Germany). 1 H and 13 C-NMR spectra were recorded on a Bruker AV-400 NMR spectrometer. NMR data were processed by Mnova software (Mestrelab Research, Spain).…”

“…The matrix solution was alpha-cyano-4-hydroxycinnamic acid (7 mg/mL in ACN/0.1%TFA (1:1 v/v)). (1). PEG (1450 g/mol, 20 g, 13.8 mmol, previously dried by azeotropic distillation in toluene using a Dean-Stark trap) was dissolved in 250 mL of dry toluene.…”

Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

“…Therefore, the structural characterization of the protein component, the identification of the conjugation sites, and the evaluation of the conjugation degree at different lysine sites are a compelling requirement towards structure‐based development15, 16, 17, 18 of glycoconjugate vaccines. The presence of polysaccharide chains prevents the crystallization of the protein conjugates, and extends the molecular weight of the system beyond the limits of solution NMR spectroscopy,19 similarly to what happens upon PEGylation 20, 21, 22. Solution NMR spectroscopy is currently used to characterize both the polysaccharide23, 24 and the protein components separately before the conjugation 25.…”