Synthesis and Application of a Hydrophobic Polyglutamate Bearing a Triphenylphosphine Group for the Orientation of Pharmaceutically Active Compounds and the Measurement of Residual Dipolar Couplings

If dissolved in a suitable, helicogenic solvent like chloroform, the homopolypeptide poly-γ-benzyl-l/d-glutamate (PB(L/D)G) is known to adopt an α-helical conformation, stabilized by intramolecular hydrogen bonding [1, 2]. These helices exhibit a screw sense that depends on the centrochirality of the amino acid in the polymer backbone with the γ-esters of l-glutamic acid forming right-handed (P) helices and the γ-esters of d-glutamic acid forming left-handed (M) helices [3, 4]. This α-helical secondary structure leads to a rigid rod-like behavior causing shape anisotropy, which allows dissolved PB(L/D)G—and other polypeptides and polymer classes [5, 6]—to form lyotropic liquid crystalline (LLC) phases above a system-specific critical concentration in helicogenic solvents [7-10]. These LLC phases [11-13] have been studied as alignment media [10, 14-25]. The mesogens of LLC-based alignment media align relative to the external magnetic field [11, 12] and—if an analyte is added—can interact with this compound transferring this alignment partially onto the analyte [26]. This induces anisotropy in the tumbling and rotation of the analyte, making anisotropic NMR observables accessible for structure elucidation [27]. These anisotropic observables yield complementary global structural information to the established local isotropic observables, nuclear Overhauser effect (nOe) [28-30] or scalar coupling (J) [31-33]. The anisotropic NMR observables are the residual chemical shift anisotropy (RCSA) [34-36], residual quadrupolar couplings (RQCs) [24, 37], and residual dipolar couplings (RDCs) [5, 27, 38, 39], with this publication focusing on the latter.

Especially sought-after media are compatible with a wide range of analytes that allow the extraction of all possible one-bond carbon-hydrogen (¹D_CH) RDCs of a given compound and exhibit excellent spectral quality with line widths comparable to the isotropic state. In this work, we present the synthesis of the new polymers diphenylphosphine sulfide poly-γ-benzyl-l/d-glutamate (DPPS-PB(L/D)G), in which the benzyl ring in the sidechain of the previously mentioned PB(L/D)G is extended to a sulfur-protected triphenylphosphine unit (see Figure 1).

By expanding the benzyl ring to triphenylphosphine, we hope to retain the necessary α-helical conformation of PB(L/D)G in solution but alter the alignment properties compared with the parent polymer. The polyglutamate DPPS-PB(L/D)G (Figure 1, bottom left) presented herein is the first result of a hybrid synthesis strategy in which we aim to synthesize polymers that can act not only as an alignment medium but potentially as a polymeric ligand for asymmetric transition metal catalysis [40-43]. The latter could be achieved by removing the sulfur protection group (red boxes in Figure 1) from DPPS-PB(L/D)G to yield the polymer diphenylphosphine poly-γ-benzyl-l/d-glutamate (DPP-PB(L/D)G; Figure 1, bottom right). The sulfur-protected motif in DPPS-PB(L/D)G is chosen to avoid three potential challenges when synthesizing or applying the free triphenylphosphine, which is the unwanted oxidation of the phosphorus atom [44, 45], the high and diverse reactivity with analytes and reagents [46], and the inhibition of the ring-opening polymerization of the N-carboxy anhydride monomers (NCAs) [47, 48].

To demonstrate the broad applicability of the new polymers as an alignment medium for different compound classes, we measured RDCs not only for α-santonin and isopinocampheol (IPC), which we use as a “proof-of-principle” compound, but also for three more complex pharmaceutical compounds. These are artemisinin, which is utilized to treat malaria [49, 50] and has been investigated using RDCs before [51-54], vincamine, which is a cerebral vasodilator and a potential antitumor agent [55], and galantamine, which is used to treat Alzheimer's disease [56]. The latter is exciting because of its conformational flexibility, which we want to investigate using our new alignment medium.

We tested the polyglutamates containing sulfur-protected triphenylphosphine (DPPS-PB(L/D)G) as a novel LLC alignment medium for structure elucidation. After synthesis and confirmation that the helical polymers DPPS-PB(L/D)G form stable LLC phases in chloroform, we found excellent spectral quality using the terpene IPC in “proof-of-principle” RDC measurements. Furthermore, we showed our new alignment medium's broad applicability with three rigid, more challenging, and pharmaceutically relevant compounds: α-santonin, artemisinin, and vincamine. For each of them, all possible ¹D_CH-RDCs were obtained with high accuracy. Since more flexible small-molecule drugs are increasingly interesting for pharmaceutical research, we applied our alignment medium for the compound galantamine, a drug used to treat Alzheimer's disease. Exploration of the conformational space using CREST yielded two relevant conformers, which mainly differ in ring conformations. ¹D_CH RDCs of galantamine could be extracted from spectra showing high spectral quality. The ensemble fit (MCST) of these RDCs improved the correlation of experimental and back-calculated RDCs compared with the individual single-conformer fits. The populations determined experimentally match the populations predicted by CREST.

The results presented herein highlight the properties of DPPS-PB(L/D)G/CDCl₃ as an excellent new alignment medium: It is compatible with an apolar organic solvent and multiple analytes of different functional groups, shapes, and degrees of conformational flexibility. Measurements in the anisotropic samples exhibit excellent spectral quality necessary to extract as many ¹D_CH RDCs as possible and to facilitate advanced RDC analyses. This will become increasingly important when RDC-data-driven algorithms for de novo [107, 108] or refinement-based [109-116] structure elucidation become the norm.

Experimental details regarding the synthesis of the polymer, NMR measurements, RDC fits, coordinates, abbreviations/symbols, and computational details can be found in the Supporting Information.