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Microwave-Assisted Peptide Synthesis

Wed, 12/05/2012 - 3:40pm
Grace S. Vanier, PhD, Product Manager, Life Science Division, CEM Corp., Matthews, N.C.

Figure 1: 0.1 mmol coupling reaction performed with both thermal and microwave heating. (All figures: CEM Corp.) Due to the flexibility of microwave technology, new methods and applications are continuously being developed.

In 1986, organic chemists first reported the use of microwave energy to accelerate small-molecule synthesis transformations. It was not long after this seminal work that peptide chemists were using microwave technology to improve the solid-phase synthesis of more complex biomolecules. While the early work in this field focused on applying microwave energy only to the coupling reaction, the scope of microwave technology as a tool for enhancing peptide synthesis now covers the entire process, including both the coupling and deprotection reactions, as well as the post-synthesis cleavage step to remove the peptide from the resin and remove the side chain-protecting groups. 
 
Microwave energy has some unique advantages compared to thermal heating techniques that make it especially beneficial to peptide synthesis. In a peptide synthesis reaction there are many polar and ionic species present that can rapidly be heated by microwave energy. These include polar solvents, the peptide backbone, the terminal amine group, bases for deprotection, and polar/ionic activators. The resulting temperature increase can help break up chain aggregation due to intra- and interchain association and allow for easier access to the growing end of the chain. Microwave energy is the most efficient way to heat peptide synthesis reactions, which typically utilize temperatures of about 75 C. To demonstrate the efficiency of microwave heating, a 0.1 mmol scale coupling was performed both with microwave and thermal heating (Figure 1). The thermal heating experiment was performed with a water bath set to 80 C, while the microwave experiment was performed with optimized power input to ensure rapid heating. The microwave method reached the desired temperature in 60 seconds with very little temperature overshoot, while the thermal method took over 3 minutes to reach 75 C. 
 
Figure 2: Synthesis of WFTTLISTIM-NH2 under standard microwave peptide synthesis conditions. The use of microwave energy for peptide synthesis has become a widely accepted method for producing high-quality peptides in a short period of time. There are now more than 100 publications per year that specifically indicate that microwave technology was used for the peptide synthesis discussed and there are likely dozens more per year that do not call specific attention to the technology, since it has become such a routine method. Microwave energy has been applied to the synthesis of a variety of different types of peptides, including ß-amyloid; cyclic peptides; glycopeptides; peptide dendrimers and polymers; and peptidomimetics, such as ß-peptides and peptoids. Due to the flexibility of microwave technology, new methods and applications are continuously being developed.
 
One recent development with microwave technology for peptide synthesis is the application of a UV detector to monitor the progress of the Fmoc removal step. The byproduct from this reaction absorbs at 301 nm, and can be used as a diagnostic to determine how well the Fmoc removal has proceeded. To demonstrate the utility of this feature, a 10-mer peptide derived from the C-terminal portion of the AKR/Gross MuLV CTL epitope2 (WFTTLISTIM-NH2) was synthesized. Under standard microwave synthesis conditions, a crude purity of 37% was obtained with deletions of F, W, T, and WT observed (Figure 2).
 
Using the UV monitoring feature, it was observed that the Fmoc removal step did not proceed to completion prior to the coupling of Thr, Phe, and Trp (Figure 3). TheFigure 3: Synthesis of WFTTLISTIM-NH2 with UV monitoring modified conditions. failure to remove all the Fmoc resulted in deletions of these amino acids. The UV monitoring feature also allows the synthesis conditions to be modified in situ, so the system automatically repeated the Fmoc removal step to achieve full deprotection. Also, the assumption can be made that if an amino acid suffers from a difficult Fmoc removal, the subsequent coupling may be difficult, as well. Therefore, the software can also modify the coupling conditions to improve the outcome for this potential difficulty. In the case of this 10-mer peptide, 15-minute double couplings were selected, which in addition to the ancillary deprotection step, improved the synthesis quality to 88% with minimal deletions observed.
 
The application of microwave energy to solid phase peptide synthesis is not limited to the coupling and deprotection steps. As mentioned earlier, the post-synthetic cleavage from the resin and side-protecting group removal can be performed in the microwave at 38 C for 30 minutes. In addition, microwave technology can be used to improve C-terminal peptide labeling or modification, side chain manipulations—such as metathesis and copper-catalyzed azide-alkyne couplings—and the synthesis of peptidomimetics—including beta-peptides, peptoids, and peptide nucleic acids. 
 
Scheme 1: Microwave synthesis of head-to-tail cyclized peptide. [a] 20% piperidine, 5 min, 75 C. [b] Fmoc-AA-OH/HBTU/DIEA, 5 min, 75 C.  [c] 5% hydrazine, 2 x 3 min, 75 C. [d] DIC/HOBt, 3 x 10 min, 75 C. [e] TFA/H2O/TIS/DODT, 40 min, 38 C.New methods have been developed for the synthesis of head-to-tail cyclic peptides on resin. Cyclic peptides exhibit improved metabolic stability and increased potency and bioavailability, as compared to their linear counterparts. The head-to-tail on-resin cyclization strategy is an important tool in solid phase peptide synthesis that takes advantage of the resin-induced pseudo-dilution effects, thereby limiting undesirable dimerization. Such cyclizations often require long reaction times under conventional conditions and result in a low crude purity of the cyclized peptide. Scheme 1 outlines the on-resin microwave-enhanced synthesis of a head-to-tail cyclized peptide. The peptide backbone (Gly-Val-Tyr-Leu-His-Ile-Glu) was synthesized on Fmoc-Glu(Wang resin)-ODmab with the side chain γ-carboxyl group anchored to the resin and the α-carboxyl protected by the Dmab orthogonal protecting group in 91% crude purity. Selective on-resin removal of Dmab protection was performed with 5% hydrazine in DMF (2 x 3 min at 75 C) to give the linear precursor quantitatively. Head-to-tail cyclization of the resin-bound peptide was accomplished using DIC/HOBt (3 x 10 min at 75 C) followed by cleavage of the cyclic peptide from the resin provided crude product in overall 77% purity.
 
Microwave technology is a versatile tool for peptide synthesis that can be utilized for the routine Fmoc removal and coupling steps of solid phase peptide synthesis, as well as a variety of other types of chemistries related to peptide synthesis. Future techniques will allow for the synthesis of even more complex peptides, small proteins, and peptides conjugates in larger amounts. Hardware improvements will make microwave technology a viable tool for addressing these market needs, as more and more peptide targets begin clinical trials in the coming years.
 
About the author
Grace Vanier is responsible for the research and development of new methods and instrumentation, customer support, and publications for the entire product line.
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