Suppression of Simultaneous Fmoc-His(Trt)-OH Racemization and N^α-DIC-Endcapping in Solid-Phase Peptide Synthesis through Design of Experiments and Its Implication for an Amino Acid Activation Strategy in Peptide Synthesis

Abstract

Coupling Fmoc-His­(Trt)-OH in solid-phase peptide synthesis is frequently accompanied by significant racemization. Histidine is among the most susceptible amino acid residues inclined to racemize in peptide syntheses. In this study, the His racemized impurity could not be effectively purged by the applied chromatographic purification. Consequently, a Taguchi design of experiment (DOE) was first applied to screen the critical process parameters affecting the histidine racemization. Optimization of the DOE is subsequently performed to search for the optimum. The derived DOE models reveal that the conditions of Fmoc-His­(Trt)-OH carboxylate pre-activation prior to its coupling to growing peptide chains are critical for the subject histidine racemization. Intensive Fmoc-His­(Trt)-OH pre-activation stimulates this side reaction. On the other hand, without the amino acid pre-activation, by adopting the in situ Fmoc-Xaa-OH activation, another side reaction, that is, peptide N α endcapping by N,N-diisopropylcarbodiimide, is boosted. A conflicting relationship between histidine racemization and peptide N α endcapping has been detected through the DOE investigation. Significant models are established for the histidine racemization and peptide N α endcapping, and reconciliation to balance these two side reactions is accomplished on this basis.

Elevating 1-tert-Butyl-3-ethylcarbodiimide (TBEC) as a Reagent for Sustainable Peptide Synthesis: Quality Assessment and Minimizing Racemization, Precipitation, and Radical-Induced Side Reactions by TBEC/Oxyma Couplings in an Environmentally Sensible Solvent

“…In fact, this His derivative was used by the de la Torre and Albericio group in DIC/Oxyma vs TBEC/Oxyma comparison synthesizing the G H F tripeptide, and we thus chose to examine Fmoc-His­(Trt)-OH in the synthesis of this tripeptide as well. Although GHF is a widely used substrate for studying His racemization, the studies in the art typically only focus on determining the content of D-His, while the contents of other possible byproducts, such as GF (des His) as a result of incomplete coupling or GHHF (endo His) as a result of overcoupling, are not considered . As the aim of our His coupling assessment was to evaluate not only H-CN formation, coupling conversion, and His racemization but also the incomplete conversion and His overcoupling, we synthesized the pertinent GhF, GF, and GHHF reference peptides (ESI, section 10) and developed an LC-MS method capable of separating these impurities from the GHF product (ESI, Figures S129–S130).…”

“…The reasons for the minimal racemization in the coupling of Fmoc-His(Trt)-OH using TBEC/Oxyma in NBP/EtOAc (1:4) notwithstanding, the 0.43% D-His we achieved in the entry 8 coupling is to our knowledge the lowest D-His content for a CDI-mediated solidphase coupling of Fmoc-His(Trt)-OH using Oxyma. In fact, notably lower than in the coupling of Fmoc-His(Trt)-OH using DIC/Oxyma in NBP (2.5% D-His), 51k DIC/Oxyma in DMSO/2-MeTHF (3:7) (2.0% D-His), 51m TBEC/Oxyma in DMF (1.1% D-His), 45 and DIC/Oxyma in DMF (1.0% D-His) 80 while being comparable to our protocol using DIC/ HONB/HOBt in NBP/EtOAc (1:1) (0.3% D-His). 51h Based on the outcome of the solid-phase Fmoc-His(Trt)-OH couplings in Table 7, we envisage that engaging TBEC/ Oxyma in NBP/EtOAc (1:4), or in any other solvent which compares favorably to NBP/EtOAc (1:4), will not only increase coupling rates, it will also significantly reduce racemization.…”

“…Although GHF is a widely used substrate for studying His racemization, 79 the studies in the art typically only focus on determining the content of D-His, while the contents of other possible byproducts, such as GF (des His) as a result of incomplete coupling or GHHF (endo His) as a result of overcoupling, are not considered. 80 As the aim of our His coupling assessment was to evaluate not only H-CN formation, coupling conversion, and His racemization but also the incomplete conversion and His overcoupling, we synthesized the pertinent GhF, GF, and GHHF reference peptides (ESI, section 10) and developed an LC-MS method capable of separating these impurities from the GHF product (ESI, Figures S129−S130). With a suitable analytical system in hand, we carried out a series of Fmoc-His(Trt)-OH + H-Phe resin couplings, in which DIC and TBEC were evaluated as CDIs, and DMF and NBP/EtOAc (1:4) were assessed as solvents.…”

Development of a Mechanistic Amino Acid Activation Reaction Kinetics Model for Safe and Efficient Solid Phase Peptide Synthesis

“…16,24 The activation was conducted using a 1:3:2.5 molar ratio of His/Oxyma/DIC at 20 and 30 °C to ensure 100% activation conversion at the end of the reaction. 17 The rate of the activation reaction was simulated using the rate constants derived from Leucine activation above, and the kinetics parameters for Histidine racemization, k 8 and Ea 8 in eq 8, were obtained by fitting the experimental data. The results from the model fitting are shown in Figure 6 and it can be seen that the rate of racemization increases at least two-fold when the temperature increases by 10 °C.…”

N-Acetyl-l-phenylalanine Racemization during TBTU Amidation: An In-Depth Study for the Synthesis of Anti-Inflammatory 2-(N-Acetyl)-l-phenylalanylamido-2-deoxy-d-glucose (NAPA)

“…In fact, the latter could self-cyclize into an azlactone, characterized by an acid α-proton (pK a ≈ 9), which is responsible for the abundant epimerization/racemization generally occurring during the amidation reaction [ 3 ]. Among other strategies, to reduce or avoid racemization, an in situ activation strategy or a fast activation process are generally proposed to effectively shorten the existence period of the activated amino acid species [ 4 ].…”