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Peptide Synthesis 5. Coupling Methods

Author:N/A    | Post time:2012-05-26

5. Coupling Methods
Top of page
1. Introduction
2. Fundamentals of Peptide Synthesis
3. Strategies for Peptide Synthesis
4. Chain-Growing and Side-Chain Protective Groups
5. Coupling Methods
6. Side Reactions
7. Summary and Outlook
Acylation of the amino group of a primary amine with the carboxyl function of a second amino acid is the basic principle in peptide synthesis. A large variety in the chemical composition of the amino acids causes also a large variety in reaction speeds and expected yields, depending on the properties of the molecules. Therefore, numerous activation methods and activation tools are used to enhance both the reaction rate of the amide bond formation and the yield. Most of the activation tools are based on the formation of active esters that are either preformed or generated in situ.
Typically, a 2–10 fold excess of the activated amino acid over the solid-phase-bound residue is used to drive the reaction near completeness. The main factor that determines the coupling yield is the nature of the activated amino acid derivative. However, sequence specific structural effects of the growing peptide chain have an influence on the coupling reaction as well. Therefore, usually the best results are obtained if the used protective groups, the molar excess, and the reaction time are adapted to the specific peptide sequence. However, this cannot be done when synthesizing many different peptides in parallel.
5.1. Diisopropylcarbodiimide (DIC)/N-Hydroxybenzotriazole (HOBt) Activation
A mixture of an Fmoc-protected amino acid, diisopropylcarbodiimide (DIC), and N-hydroxybenzotriazole (HOBt) leads to the formation of the corresponding OBt ester (Fig. 23). The reaction is routinely carried out in DMF, but it is even faster in less polar solvents like DCM [160]. The amino acid is preactivated and then added to the reaction partner or resin. The use of DIC is preferred over DCC because it is liquid at room temperature and the urea adduct generated with DIC is perfectly soluble in DMF. Thereby, some initially insoluble amino acid derivates can be converted into soluble compounds upon activation.
Figure 23. Reaction scheme for the DIC/HOBt activation
5.2. Active-Ester Method
The extent of the activation of the carboxylic C-atom strongly influences the coupling rate. Using esters as activation compounds, the reaction rates are depending on the nature of the substituents. A large variety of active esters have been tested for peptide synthesis. It has been shown that extensively halogenated active esters outperformed other compounds in terms of coupling rate, and, eventually, also in terms of minimized racemization. The most prominent examples are ortho-2,4,5-trichlorophenyl (OTCp), ortho-pentachlorophenyl (OPCp), and ortho-pentafluorophenyl (OPfp) (Fig. 24). OPfp esters are widely applied in Fmoc/tBu-based SPPS routines. Besides the halogenated forms, also 4-nitrophenyl (ONp) and N-succinimidyl (OSu) active ester variants are commonly used. In general, active esters are efficient acylation agents, and the reaction rate is increased when they are used in combination with one equivalent of HOBt. Another advantage of the active-ester method is the low susceptibility to side reactions because of high reaction rates and almost complete conversions.
Figure 24. Active esters
5.3. Symmetrical/Asymmetrical (Mixed)-Anhydride Method
The symmetrical anhydride of a Fmoc-protected amino acid is formed upon adding half of an equivalent of a carbodiimide in DCM to the amino acid derivative. With DIC as the dehydrating agent, the preformed anhydride could be used directly in SPPS. This is a relatively expensive method because only one of two amino acid molecules from the educt side is coupled to the growing peptide chain.
The formation of mixed anhydrides with the amino acid and a second carboxylic acid as “dummy” is, except for the use of H2CO3, not a preferable method because of possible side products. However, in the case of H2CO3, side products are minimized because of an efficient and fast removal of gaseous CO2 from the reaction vessel. Asymmetrical anhydrides are no longer commercially available.
5.4. Aminium and Phosphonium Activation
In modern peptide synthesis, aminium (also: uronium) and phosphonium derivatives gain increasing importance for the in situ activation of carboxylic components. The most common reagents are (Fig. 25) [161, 162]:
PyBOP =  N-(1H-benzotriazol-1-yl)oxytris(pyrrolidino)phosphonium hexafluorophosphate;
TBTU =  N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide, and
HBTU =  N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide.

Figure 25. Commonly used aminium and phosphonium coupling reagents

For TBTU and HATU the guanidinium isomers are shown.
In this activation method, the Fmoc-protected amino acids are converted into the corresponding benzotriazolyl (OBt) esters. Analogous reagents are:
HATU =  N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide and
PyAOP =  7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate

HATU and PyAOP generate 9-azabenzotriazolyl (OAt) esters [163]. It was shown that the latter reagents tend to give better results in terms of coupling efficiency and diminished racemization [164]. Highly reactive species are formed, which results in high yields and in a typical reaction time of less than 30 min. However, complete conversion of hindered residues like  -branched or N-substituted amino acids may take longer [165]. Therefore, double coupling steps are usually performed in SPPS with respect to the chemical variation of the different amino acid compounds. The coupling procedure is done twice in order to increase the yield, without an intermediate capping or Fmoc-deprotection step. In standard protocols, the yield of the coupling reaction is assayed with a Kaiser, chloranil, or TNBS (2,4,6-trinitrobenzenesulfonic acid) test before proceeding to the next step [166].
5.5. Acid Fluorides
Amino acid fluorides are generated with tetramethyl fluoroformamidinium hexafluorophosphate (TFFH, Fig. 25) [167, 172], diethylaminosulfur trifluoride (DAST), or cyanuric fluoride [168]. They are less reactive than acid chlorides [169, 173, 174] but nevertheless extreme powerful acylation agents. Acid fluorides are stable in the presence of tertiary amines, which completely block oxazolone-mediated racemization (Chap. Side Reactions) [170]. The in situ formation of acid fluorides with TFFH is carried out in DMF in the presence of diisopropylethylamine (DIPEA).


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