Synthesis and anti-osteoporosis activity of novel Teriparatide glycosylation derivatives

Abstract

Osteoporosis is a metabolic bone disease that is characterized by low bone mass and micro-architectural deterioration of bones. The mechanism underlying this disease implicates an imbalance between bone resorption and bone remodeling. In 2002, the US Food and Drug Administration (FDA) approved Teriparatide for the treatment of osteoporosis, and so far, this compound is the only permitted osteoanabolic. However, as a structurally flexible linear peptide, this drug may be further optimized. In this study, we develop a series of novel N-acetyl glucosamine glycosylation derivatives of Teriparatide and examine their characteristics. Of the analyzed compounds, PTHG-9 exhibits enhanced helicity, greater protease stability, and increased osteoblast differentiation promoting ability compared with the original Teriparatide. Accordingly, PTHG-9 is suggested as a therapeutic candidate for postmenopausal osteoporosis (PMOP) and other related diseases. The successful development of an enhanced osteoporosis drug proves that the method proposed herein can be used to effectively enhance the chemical and biological properties of linear peptides with various biological functions.

Fig. 1. Structures of Teriparatide (PTHG-1) and its N-acetyl glucosamine glycosylation derivatives (PTHG-2–9).

Scheme 1. Synthetic route of Fmoc-Asn(Ac₃GlcNAc)-OH (4) and Fmoc-Ser(Ac₃GlcNAc)-OH (6). Reagents and conditions: (a) (i) acetyl chloride, r.t., 4 days; (ii) NaN₃, Bu₄NI, DCM/water, r.t., 2 h, 82.4% in 2 steps; (b) (i) H₂, Pd/C, MeOH, r.t., overnight; (ii) Fmoc-Asp-OtBu, HOBt, DIC, DMF, r.t., overnight, 71.2% in 2 steps; (c) DCM/TFA (3 : 1, v/v), r.t., 2 h, 95.3%; (d) (i) 4 Å molecular sieves, BF₃·Et₂O, DCM, 0 °C, 12 h; (ii) Et₃N, Fmoc-Ser-OH, DCM/MeCN (1 : 2, v/v), r.t., 3 days, 32% in 2 steps.

Scheme 2. Synthetic route of PTHG-2. Reagents and conditions: (a) 10% hydrazine hydrate/DMF, r.t., overnight; (b) TFA/EDT/TIPS/water (95 : 2 : 2 : 1, v/v/v/v), r.t., 2 h, 25%; the resin-bound peptides were protected on side chains at asterisk sites. The following protecting groups for amino acid side chains were used: tert-butyl (tBu; for Glu and Ser), 2,2,4,6,7-pentamethyldihydrobenzo-furane-5-sulfonyl (pbf; for Arg), tert-butyloxycarbonyl (tBoc; for Lys and His) and trityl (Trt; for Asn).

Fig. 2. (A) CD spectra of the glycosylation derivatives in 50.0% TFE aqueous solution at 20 °C. (B) Proteolytic stability of glycosylation derivatives under α-chymotrypsin treatment. Data points are displayed as the mean value SEM of duplicate independent experiments. The percent residual peptide was monitored via HPLC.

Fig. 3. Activity of the glycosylation derivatives at 50 nM concentrations as assessed by alkaline phosphatase (ALP) activity in MC3T3-E1 cells. Data represent the mean ± SEM from three independent experiments: *p < 0.01 as compared with PTHG-1.