An Inexpensive Adaptation of a Commercial Microwave Reactor for Solid Phase Peptide Synthesis

Summary

A simple, inexpensive, and novel apparatus for performing solid phase peptide syntheses in a commercial microwave reactor is presented.

Abstract

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to conventional SPPS reaction vessels, which drain solvent and byproducts via a frit located at the bottom of the vessel, the presented apparatus employs a gas dispersion tube under vacuum to remove solvent, byproducts, and excess reagents. The same gas dispersion tube supplies nitrogen gas agitation of the SPPS beads during the reaction steps of coupling and deprotection. Microwave heating is beneficial for SPPS couplings of sterically hindered residues, such as alpha-aminoisobutyric acid (Aib), an alpha,alpha-dialkylated amino acid residue. This home-built apparatus has been used to prepare, via manual Fmoc SPPS methods, heptameric and octameric peptides dominated by the Aib residue, which is notoriously difficult to couple under standard room temperature conditions and reagents. Further, typical commercial microwave SPPS reactors are dedicated exclusively to SPPS synthesis rendering them inaccessible to non-SPPS users. In contrast, the presented apparatus preserves the versatility of the microwave reactor for conventional microwave acceleration of chemical reactions, as the apparatus is trivially removed from the commercial microwave reactor.

Introduction

Merrifield's introduction of solid phase peptide synthesis SPPS in the 1960s revolutionized peptide and chemical syntheses and was justly rewarded with a Nobel Prize in Chemistry^1,2. In subsequent decades, many researchers have refined Merrifield's original techniques, leading to two alternatives that dominate SPPS practices: fluorenylmethoxycarbonyl (FMOC)-based versus tert-butyl oxycarbonyl (BOC)-based³. Final cleavage of the peptide from solid resin in FMOC requires a cocktail containing trifluoracetic acid, as compared to HF for BOC techniques, making FMOC-based met....

Protocol

1. Assemble the apparatus (Figure 1)

NOTE: All the components for assembling the apparatus are found in the Table of Materials.

1. Assemble the vacuum outlet.
   1. Connect 2" of 1/8" Teflon tubing to the right side of the "Tee" (Figure 1, Part labeled "1") made of ethylene tetrafluoroethylene (ETFE).
   2. Connect the 1/8" Teflon tube t.......

Discussion

The apparatus presented here invokes a simple, novel, and inexpensive method for removing solvent, excess reagents and waste products, as well as adding nitrogen gas agitation, during microwave-assisted SPPS. In contrast to conventional SPPS vessels, which invoke a frit at the bottom of the vessel, the presented apparatus invokes a gas dispersion tube under vacuum to aspirate the vessel. The reaction vessel is therefore an ordinary test-tube, which is heated at the location recommended by the microwave reactor manufactur.......

Materials

Name	Company	Catalog Number	Comments
CEM Discover SP Microwave Reactor	CEM		Discontinued.  Recently replaced in the product line by the Discover 2.0
Diisopropylcarbodiimide (DCC)	TCI America
Dimethylformamide (DMF)	Thermo Scientific Chemicals
Gas dispersion tube, micro,	Chemglass	CG-207-02	medium porosity
micro septum	ChemGlass	CG-3022-20	"NMR tube" type septum
Morpholine	Thermo Scientific Chemicals
N-Fmoc-protected Amino acids
Oyxma Pure	TCI America
Side-arm Ehrlenmeyer flask	Assorted vendors		Waste collection
"Tee"	Idex	P-713	ETFE
teflon tubing 1/8",	Restek	25306	OD x 0.063" ID, 3 m
Test tube (holder for reaction vessel external to microwave)	Assorted vendors		(30 x 175)
Test tube (reaction vessel)	Corning Glass	9820-25X	Pyrex 25 x 200 mm, rimless
Valve	Idex	P-721	ETFE (2x)
Wang SPPS Resin, 1% crosslinked divinylbenzene, 100-200 mesh	Advanced ChemTech