FMOC-L-Valine: Peptide Synthesis & Spectroscopic/Solubility Studies

FMOC-L-valine is an N-terminally protected form of the amino acid L-valine, where the protecting group is fluorenylmethyloxycarbonyl (Fmoc). This reversible protection is fundamental to the stepwise elongation of peptide chains in SPPS. This preserves the natural configuration of L-valine while enhancing chemical stability through the protective group, thereby preventing side reactions involving the amino group in subsequent processes. This material appears as a white or off-white crystalline powder. Its melting point ranges from approximately 150–154°C, and it demonstrates good stability, resisting decomposition under standard storage conditions. As a pivotal intermediate in peptide synthesis, FMOC-L-valine primarily serves to protect amino groups during solid-phase peptide synthesis. Leveraging the FMOC protecting group's advantages of easy removal and minimal side reactions, it enables precise control over the synthesis sequence of peptide chains. FMOC-L-Valine facilitates the preparation of diverse peptide therapeutics, bioactive peptides, and peptide probes, serving as a vital bridge between fundamental amino acids and complex peptide molecules.

NMR Correlation Spectroscopy study of FMOC-L-Valine

The essential role of oxygen in hydrogen bonding in determining the chemistry, structure, and function of peptides and proteins is well known. It is also axiomatic that 17O magic-angle spinning (MAS) nuclear magnetic resonance (NMR) experiments can in principle elucidate the details of these interactions via site-specific measurements of the chemical shift and quadrupole tensors. Furthermore, if a method of dipolar recoupling is included in the experimental protocol, then 13C─17O and 15N─17O and 1H─17O distance measurements and 3D structures are possible. Building on previous 17O NMR experiments, we first describe an efficient procedure for 17O labeling FMOC-protected amino acids using H217O as the source. The approach utilizes a nonequilibrium multiple-turnover reaction under mild and selective conditions. Two FMOC-protected amino acid precursors, FMOC-l-valine and FMOC-l-lecuine, were 17O enriched to 40 and 70%, with an efficiency of >95%, and used to prepare [17O/13C/15N]-N-Ac-VL, a model dipeptide used to develop dipole recoupling experiments. We envision that these labeled amino acids can be used in contemporary peptide synthesis or in cell-free synthesis protocols for 17O labeling of peptides and proteins. Second, we recorded 1D MAS spectra at ω0H/2π = 750, 800, 900, and 1500 MHz and simulated the rotational sideband patterns to extract the chemical shift and electric field gradient (EFG) tensors for the three 17O moieties. Utilizing this approach, FMOC-l-valine and FMOC-l-leucine were prepared using 40% H217O (Cambridge Isotope Laboratories, Andover, MA) for study via 17O NMR spectroscopy. A dipeptide sample, [17O/13C/15N]-N-Ac-VL, was prepared using the same approach with U-13C,15N amino acids and 70% H217O (Cambridge Isotope Laboratories, Andover, MA).[1]

Utilizing this multiple-turnover exchange reaction, FMOC-l-leucine and FMOC-l-valine were enriched with 40% 17O-labeled H2O (vide supra). The 17O isotopic enrichment of the samples was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) (Koch Institute MIT). While the MALDI matrix appears at similar m/z to that of the FMOC amino acids these peaks did not interfere with the ability to determine the 17O labeling of the samples. The MALDI MS results yielded the protected amino acids with a M+ m/z of 376.4 Da for FMOC-l-leucine and 362.4 Da for FMOC-l-valine, the addition of a Na+ ion to the molecule is the cause for the larger than expected m/z. The structure of two protected amino acids, FMOC-l-leucine and FMOC-l-valine, and a dipeptide, N-acetyl-l-valyl-l-leucine (N-Ac-VL), were studied via one- and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy. Utilizing 17O magic-angle spinning (MAS) NMR at multiple magnetic fields (17.6–35.2 T/750–1500 MHz for 1H) the 17O quadrupolar and chemical shift parameters were determined for the two oxygen sites of each FMOC-protected amino acids and the three distinct oxygen environments of the dipeptide.

Re-evaluation of the Solubility and Activation of FMOC-L-Valine

In line with the observations of Lawrenson et al. (vide supra), solvent mixtures generally gave improved swelling compared to neat solvents, and furthermore, the linker appeared to affect resin swelling. For the PS resin for example, more swelling was observed for the Wang over the Rink Amide linker, while for the ChemMatrix® resin the opposite was generally observed. When comparing Wang and Rink Amide linkers on TentaGel™ resin, most swelling was observed in 8:2 and 9:1 anisole/DMC (6.0 mL g−1, entries 21 and 22), and 5:5 anisole/DMC (6.4 mL g−1, entry 24) respectively. The best swelling for the Wang ChemMatrix® resin was obtained with 9:1 sulfolane/DEC (6.8 mL g−1, entry 14) while the Rink Amide ChemMatrix® resin had the highest swelling volume in 7:3 anisole/DMC (10.0 mL g−1, entry 23). Overall, Ferrazzano et al. found, as anticipated, that DMF display good swelling properties, but also that mixtures of anisole/DMC were able to match the swelling profile of DMF very well (entries 21 and 1, respectively). In addition to swelling, Ferrazzano et al. performed solubility experiments with the most promising solvent mixtures (Cyrene™/DEC (3:7), sulfolane/DEC (3:7) and anisole/DMC (7:3)). Dissolving FMOC-L-Valine  at a concentration of 0.2 M was only possible in sulfolane/DEC (3:7).[2]

Activated amino acids are typically more soluble, and so the solubility of FMOC-L-Valine  was reassessed with the activating reagents (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU®)/N,N-diisopropylethylamine (DIPEA), N,N′-diisopropylcarbodiimide (DIC)/ethyl (hydroxyimino)cyanoacetate (Oxyma Pure), DIC/hydroxybenzotriazole (HOBt), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), and 2-(1I-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). The best result was obtained with DIC/Oxyma Pure that was able to dissolve FMOC-L-Valine  in all solvent systems in 5 min. Next, DIC/Oxyma Pure in the three different solvent systems was evaluated for its ability to solubilise 18 representative Fmoc-protected amino acid building blocks. In all cases, the activated Fmoc-amino acid was fully soluble at a concentration of 0.2 M. In recent years a growing number of publications has been addressing the substitution of environmentally problematic solvents such as CH2Cl2, DMF and NMP in SPPS. However, in order to ensure that newly developed methods will be amenable to large scale production, an industrial perspective on this issue has been missing.

References

[1]Keeler EG, Michaelis VK, Colvin MT, Hung I, Gor'kov PL, Cross TA, Gan Z, Griffin RG. 17O MAS NMR Correlation Spectroscopy at High Magnetic Fields. J Am Chem Soc. 2017 Dec 13;139(49):17953-17963. doi: 10.1021/jacs.7b08989. Epub 2017 Nov 30. PMID: 29111706; PMCID: PMC8256432.

[2]Martin, V., Egelund, P. H. G., Johansson, H., Le Quement, S. T., Wojcik, F., & Pedersen, D. S. (2020). Greening the synthesis of peptide therapeutics: an industrial perspective. RSC Advances, 10(63), 42457–42492.

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