In solid-phase peptide synthesis, a key factor for achieving complete coupling is the diffusion of acylating reagents within the matrix. Only when the peptide resin is adequately solvated can a satisfactory reaction be achieved. However, for peptide chains with lengths ranging from 6 to 16 residues, almost every coupling step becomes challenging. These peptide segments are referred to as "difficult sequences" in the context of coupling reactions. The presence of difficult sequences results in incomplete coupling reactions, sometimes preventing further synthesis altogether. Even when reaction conditions are improved-such as by changing coupling reagents, extending reaction times, repeating couplings, or using blocking agents-there is often no significant improvement. In some cases, larger steric hindrance from the amino acids or a higher resin loading can even worsen the outcome. This situation is more closely related to the length of the peptide chain that has already been synthesized, rather than to the amino acids to be coupled.
"Difficult sequences" can be categorized into two types: random and non-random.
In 1983, Mutter M. and colleagues pointed out that extensive hydrogen bonding and molecular aggregation between the peptide chain and the resin leads to tight packing of the peptide chain, poor solvation, and reduced solubility. This is the fundamental reason for incomplete coupling. In 1989, Deber C. M. discovered that peptides exhibiting molecular aggregation always contain secondary structures predominantly composed of β-sheets. In contrast, unordered coils are more conducive to chemical reactions.
In solid-phase synthesis, peptides with the same chain length but different structures may exhibit varying coupling efficiencies. Peptide chains that are difficult to couple typically feature β-sheet structures, while those that are easy to couple do not. Therefore, predicting the relationship between peptide sequences and β-sheet formation is crucial for designing easily synthesizable target peptides and optimizing coupling conditions. There are two methods for predicting the solubility of peptide sequences:
1. Calculating the Contribution Parameters of Amino Acids to Secondary Structures: These parameters predict the tendency of each amino acid to form three secondary structures (α-helix, β-sheet, and unordered coil). The parameters include Pα(for α-helix formation), Pβ(for β-sheet formation), and PC(for unordered coil formation). This helps in designing peptides that are easier to synthesize or in improving coupling conditions.
Note:
2. Average Value of PC for All Residues in a Peptide: The average value
For example, based on the prediction of peptide solubility, thymopoietin α1 has
Detection of β-Sheet Formation: The formation of β-sheets can be detected using Fourier transform infrared (FTIR) spectroscopy.
The presence of difficult sequences is a significant obstacle in the smooth progression of peptide synthesis. To ensure effective peptide chain coupling, it is essential to disrupt the β-sheet secondary structures induced by these difficult sequences. Based on over 40 years of scientific research and literature, several strategies have been identified to disrupt β-sheet structures and improve coupling efficiency in peptide synthesis. These measures include choosing the appropriate synthesis strategy and resin, introducing auxiliary sequences and precursor sequences, using fragment coupling, temporary substitution of amide N, changing coupling reagents, selecting solvents, adding high-ion-exchange-sequence additives, and applying microwave and ultrasound techniques.
In the Boc synthesis strategy, peptide chain aggregation is not a severe issue because it can be alleviated by repeatedly using trifluoroacetic acid (TFA) to remove temporary protecting groups. In this strategy, TFA is frequently used to remove the Boc protecting groups, and when the peptide is cleaved from the resin, hydrofluoric acid (HF) is employed. However, HF requires specialized equipment for safe handling, and side reactions can occur during the cleavage process, making it increasingly less commonly used due to experimental limitations.
The resins commonly used in peptide synthesis include PAM, MBHA, Wang, 2-Cl-Trt, and Rink-Amide-MBHA, among others. PAM and MBHA are used in the Boc strategy because they are highly resistant to acids and require strong acids like HF or TFMSA for cleavage. In contrast, Fmoc synthesis typically uses weak acid-sensitive resins, such as Wang, Rink, or HMAP resins. To facilitate coupling reactions, it is beneficial to reduce the resin substitution value (0.05-0.2 mmol/g) and decrease the resin loading. This helps reduce the interactions between peptide chains, making the coupling process more efficient. Additionally, using resins with better swelling properties, such as PEG-PS resins (polyethylene glycol-polystyrene crosslinked copolymer carriers), can reduce peptide chain interactions, further enhancing the coupling reaction.
Before synthesizing the target peptide, adding an auxiliary sequence can effectively increase the distance between the target peptide and the resin, thereby reducing peptide chain aggregation. This approach is known as the peptide synthesis auxiliary sequence (SAPS). In 1998, Due Larsen B. et al. observed that when a certain amino acid sequence was coupled to the C-terminus of the target peptide, using an auxiliary sequence significantly reduced peptide chain aggregation. For example, when synthesizing difficult-to-assemble polyalanine polymers (Ala)n (where n≤20), using [Lys (Boc)]m (m≤6) and [Glu (tBu)]m (m≥6) as precursor sequences, single homogenous target peptide products could be obtained when m≥3. In contrast, without the precursor sequence, complex mixtures containing peptides with missing precursor sequences and Fmoc-protected peptide fragments (n≥6) were produced. The mixed precursor sequence [Lys (Boc) Glu (tBu)]3 also had a similar positive effect on the synthesis results. The side-chain coupling method, using resins such as Rink-MBHA, Sieber amide, Rink-PEG-MBHA, and Rink-ChemMatrix sequentially, also progressively improved the coupling efficiency.
For longer peptides or those containing "difficult sequences", fragment coupling is often employed.
Methods and Advantages of Fragment Coupling
Fragment coupling involves first preparing peptide fragments with protective groups, followed by activation and coupling in either liquid or solid phase to form the protected target peptide or protein. Finally, the protective groups are removed to obtain the target product. In solid-phase synthesis, particularly for difficult-to-synthesize peptides, after a chain length of over twenty amino acids, the content of side products increases significantly and continues to accumulate. These side products are often truncated oligopeptides with missing residues. More troublesome is that these oligopeptides are similar in molecular weight, solubility, polarity, and structure to the target peptide, making them difficult to purify. To overcome this limitation, strategies such as repeated couplings, the use of efficient new coupling reagents, and improved reaction conditions can be employed, alongside fragment synthesis strategies.
Fragment coupling improves both the overall yield and purity of the product because the structural difference between side products and target products is greater than one fragment, making it easy to separate them by solvent precipitation methods. In 1975, a study reported that a chemist successfully completed the solid-phase synthesis of glucagon using a 6+9+5+5+3 segment approach, yielding a crystalline pure product with an overall yield of 25%. This demonstrated the superiority of fragment synthesis for peptide molecular synthesis.
Key Factors in Fragment Coupling
When designing peptide fragments for coupling, two critical factors must be considered: the length of the peptide fragments and the coupling sites. The carboxyl-terminal amino acids of peptide fragments are more prone to racemization than the amino-terminal amino acids, so it is preferable to first select amino acids at the C-terminus of the peptide fragments. Suitable amino acids for the C-terminus include Gly, Pro, Glu, Leu, and Asn.
During fragment design, Pro is often placed at the C-terminus to minimize racemization. However, the greatest challenge of β-structure formation is the inability of the reactive components to dissolve adequately. It is only when Pro residues are present in the middle of the peptide chain that they effectively disrupt the formation of β-structures, improving the solubility of the fragment peptide and facilitating the coupling reaction.
For example, when synthesizing the human proinsulin C-peptide (containing 31 residues), Route A (a successful synthesis path) extended the fragments containing Pro residues, resulting in the target peptide. In contrast, Route B (a failed synthesis path) involved peptide segments without Pro residues, containing as many as 13 amino acid residues, which exhibited poor solubility due to β-sheet structures, thereby preventing successful coupling with the third fragment.
Disadvantages of Fragment Coupling
A major disadvantage of fragment coupling is that the peptide fragments involved in the reaction often contain many lipophilic protective groups, making it difficult to find suitable solvents for dissolution. As a result, the reaction yields may not be high, and multiple attempts may be required to determine the optimal cleavage points. This presents a barrier to the efficiency of the peptide fragment coupling method.
Based on the principle that Pro residues can terminate β-sheet formation, a substituent group of sufficient size can be introduced at the amide N-atom of a target peptide that does not contain Pro. This converts the residue into a secondary amine-like amino acid (a Pro-like structure), preventing the formation of β-sheets and improving peptide solubility. After synthesis is completed, the substituent group is removed during cleavage of the linker, restoring the original structure of the target peptide. If the target peptide contains Ser or Thr residues, they can be modified into a five-membered oxazoline ring structure, similar to Pro, which also prevents the formation of β-sheets.
Nα-Dmb (dimethoxybenzyl) was the first substituent introduced at the amide N-atom to prevent β-sheet formation. The mechanism of action is that the Dmb substituent provides significant steric hindrance at the N-atom, thereby terminating β-sheet formation and promoting the formation of unordered coils. As a result, peptides with localized N-Dmb substitutions have significantly improved solubility, enabling more efficient coupling reactions. After the coupling reaction, the Dmb group is removed along with the resin during cleavage using TFMSA.
Hmb (2-hydroxy-4-methoxybenzyl) is another substituent that can be removed under mild conditions. The substitution of hydrogen on the amide N-atom is achieved via an O→N transfer mechanism.
The choice of coupling reagent is crucial in peptide synthesis, as its reactivity directly impacts the reaction time, as well as the purity and yield of the peptide. Typically, HBTU has a higher reactivity than DIC, but lower than PyBOP and HATU. Both HATU and HBTU show excellent performance in peptide synthesis, offering fast reaction times, minimal racemization, and high yields. Notably, HATU is especially effective at facilitating the formation of amide bonds, even in the presence of steric hindrance.
In 1970, it was reported that urea molecules could disrupt hydrogen bonding and destroy the β-sheet structure of peptide chains. By adding urea to DMF to a final concentration of 1.5 mol/L, the condensation rates of Asn and Glu were increased from 70% to 100%. High-ion-exchange sequence additives are generally suitable for active ester methods but not for the simple DCC method. Large anions such as CLO4- and CNS-, when combined with monovalent cations (e.g., Na+ and K+), make ideal additives for disrupting β-structures. It is important to note that high-ion-exchange sequence salts should not be added when the coupling solvent is a hydrogen-donating solvent system.
In solid-phase synthesis of difficult peptides, solvents that disrupt the stability of secondary structures can be added, such as 2, 2, 2-trifluoroethanol, 1, 1, 1, 3, 3, 3-hexafluoroisopropanol, DMSO, ethylene carbonate, and Triton X. According to relevant literature, reaction solvents can be categorized into hydrogen-donating solvents and hydrogen-accepting solvents. When two or more solvents are used, they should be mixed in the same direction.
Hydrogen-donating solvents, such as HFIP, nBuOH, iPrOH, and tetrahydronaphthalene, contain O-H groups that can form hydrogen bonds with the C=O of the peptide chain. Hydrogen-accepting solvents, such as DMF, DMSO, and others, contain C=O, S=O, or P=O groups that can form hydrogen bonds with the N-H group of the peptide chain. These solvents significantly enhance the solvation of the peptide chain, reducing aggregation tendencies between peptide chains.
The electron-accepting number (AN) of hydrogen-accepting solvents is as follows: HMPA>DMSO>NMP>DMF. The electron-donating number (DN) of hydrogen-donating solvents is as follows: HFIP>phenol>TFE>AcOH>EtOH. Solvents with higher AN and DN values have a stronger ability to disrupt β-structures and improve peptide solubility.
When two or more solvents are mixed in the same direction, the ability to disrupt β-structures can be maintained or even enhanced. However, if inverse solvents are mixed, the hydrogen bonding between the two solvents can interfere with the peptide chain hydrogen bonds, reducing their disruptive effect.
When synthesizing peptides with difficult sequences, solvent combinations for coupling need to be selected carefully, considering solvent directionality, polarity, swelling index of the resin, toxicity, cost, and mixing ratios to find the most suitable solvent. For instance, in the synthesis of bovine ribonuclease A peptides (containing 115 residues), as the peptide chain length increases, the solvent system gradually shifts from a single solvent to a binary or even ternary co-solvent system, successfully overcoming the issue of long-chain peptide coupling difficulty.
The Need to Improve Resin Swelling
The swelling and gelation of resin are prerequisites for the reaction reagents to enter the resin and react with functional groups. During the reaction, full swelling of the solid-phase carrier enhances the free movement of reaction reagent molecules and soluble component molecules, thus achieving effective reactions.
Under certain conditions, resin swelling is determined by both the resin's polarity and the solvent's polarity. In peptide synthesis, as the peptide chain extends, it may alter the resin's polarity. On the other hand, peptide chains with large protecting groups may have decreasing solubility (affinity) in organic solvents as they lengthen, potentially causing the resin to no longer swell adequately, thus losing the ideal conditions for further peptide synthesis. Therefore, as the peptide chain extends, different solvents should be used to ensure the resin swells sufficiently, improving the coupling rate. For example, when the symmetrical anhydride method fails, adding 20% by volume of trifluoroethanol (TFE) can help improve coupling rates.
Factors Influencing Dynamic Changes in Resin Swelling
The primary factors affecting the dynamic changes in resin swelling are the charge state and molecular weight. Generally, as the reaction steps increase and intermediate product molecular weight increases, the resin's swelling in non-polar solvents decreases, while it increases in polar solvents.
The non-polar sequence of common solvents is: isopentane>hexane>trichloroethylene>diethyl ether>dichloromethane>butanol>chloroform>acetonitrile>dimethylformamide>methanol>dimethyl sulfoxide>water.
The polarity sequence of conventional solvents mixed in certain ratios is as follows: chloroform-acetone (95+5) Solvent Swelling Index Functionalized resins with different structures exhibit different swelling behaviors in the same solvent. Similarly, the same resin behaves differently in various solvents. Based on the swelling index, solvents can be classified as good, moderate, or poor solvents. Solvents like NMP, pyridine, THF, DMF, and DCM exhibit good swelling effects for most resins. Although poor solvents are not suitable for the synthesis reaction, alternating their use with good solvents during the post-reaction washing process can create a squeezing effect, effectively removing excess reagents and by-products. Since 1986, the study of microwave-assisted organic reactions has evolved into a compelling new field-Microwave-Induced Organic Reaction Enhancement (MORE) Chemistry. Microwave-assisted solid-phase peptide synthesis involves using microwave radiation to enhance the peptide synthesis process on solid-phase resins. The steps in microwave-assisted solid-phase peptide synthesis are as follows: Compared to traditional methods, the application of microwaves in deprotection, coupling, and cleavage reactions offers several clear advantages: The presence of difficult sequences represents the biggest challenge in traditional solid-phase synthesis. To overcome the formation of secondary bonds, expensive resins are often used, but these approaches are typically not very effective. After absorbing microwave energy, peptide molecules reduce chain aggregation, enabling the synthesis of long and difficult peptides. This also greatly improves the coupling efficiency of the resin and addresses steric hindrance issues. In 2002, Mate Erdelyi and colleagues used a novel single-mode microwave reactor, where both reaction temperature and pressure were controlled online. When applied to the solid-phase synthesis of sterically hindered Fmoc-amino acids (such as Val, Thr, and Ile), the study found that optimal coupling conditions—though varying depending on the coupling reagent (HATU, TBTU, PYBOP, Mukaiyama's reagents)—were achieved within 1.5 to 20 minutes (reaction temperatures typically above 110°C), and no amino acid racemization occurred. Compared to conventional solid-phase peptide coupling reactions (which typically take over 30 minutes), the reaction speed was increased by 2 to 4 times, with complete coupling achieved in a single reaction step. Mafia Caterina Daga and colleagues employed microwave-assisted deprotection to remove the amino acid protecting groups N-Cbz and N-Bn from peptides. Using isopropanol as the solvent, Pd/C as the catalyst, and ammonium formate as the proton donor, the reaction was irradiated at 600 W for three 1-minute intervals. The deprotection was completed without the formation of diketopiperazine side products. The principle behind ultrasound-enhanced chemical reactions is quite complex, but one widely accepted view is that cavitation plays a key role in these reactions. Cavitation refers to the formation and collapse of microbubbles in a liquid medium, accompanied by the release of energy. The instantaneous implosion generated by cavitation creates intense shock waves and a transient high-temperature environment, which can be used to break chemical bonds and promote reactions. Additionally, secondary effects, such as emulsification reactions and macroscopic heating effects, can further facilitate chemical reactions due to sound absorption, as well as the resonance properties of the medium and container. Compared to mechanical stirring, which can only achieve macroscopic-level homogeneous mixing, ultrasound acts on the molecules themselves. Solid-phase peptide synthesis is a heterogeneous reaction, and ultrasound can continuously expose new reaction surfaces by breaking up substrate and reactant aggregates at phase interfaces, effectively promoting the reaction. Proper application of ultrasound can significantly shorten reaction times, increase product yields, and reduce the formation of by-products. In 1995, Anuradha M V and colleagues used ultrasound to study the esterification reaction of Boc-amino acid zinc salts with Merrifield resin. The results showed that ultrasound significantly accelerated the synthesis process, reducing the reaction time from 24 hours to 45 minutes. When peptide chain contraction is not severe, conventional methods such as extending the reaction time, increasing amino acid quantities, heating during coupling reactions, and performing double or even triple couplings can also be convenient and effective approaches. In summary, the key to synthesizing difficult peptides lies in disrupting the β-sheet structures caused by difficult sequences. Various measures to break these β-sheet structures have different levels of feasibility-some are straightforward and effective, while others are more complex or expensive, making them unsuitable for large-scale industrial production. The Boc synthesis strategy, which requires TFA to remove the Boc protecting group, increases synthesis costs and results in significant contamination. The need for specialized equipment for peptide cleavage and the potential for side reactions have led to a gradual decrease in its use. Methods such as using PEG-PS resins or temporary substitution on the amide N atom can significantly improve synthesis efficiency, but due to their high cost, they are not suitable for large-scale industrial production. Microwave synthesis is still in the experimental stage and is not yet feasible for large-scale manufacturing. Introducing auxiliary and precursor sequences in peptide synthesis is a simple and effective method. Fragment coupling is a crucial technique for preparing difficult peptides, and its success depends on fragment design, which requires theoretical predictions and extensive experimental validation. Selecting appropriate coupling reagents can significantly improve peptide purity and yield. When using active ester methods for difficult peptide synthesis, high-ion-exchange sequence additives can be added to assist with coupling and deprotection. The use of co-solvent mixtures requires careful consideration of solvent compatibility, polarity, swelling indices of the resin, toxicity, cost, and mixing ratios. Solvent screening experiments can help determine the most effective solvent combinations. When peptide chain aggregation is not severe, conventional approaches such as extending reaction times, increasing amino acid quantities, heating, or performing double and triple couplings can also be simple yet effective. When designing a synthesis route for difficult peptides, it's essential to consider factors such as cost, yield, process feasibility, and stability. By wisely employing various techniques and conducting experiments, the optimal synthesis process route can be determined. Peptide Synthesis Services at Creative Peptides10. Microwave-Assisted Synthesis of Difficult Peptides
11. Ultrasonic Treatment Method
12. Extending Reaction Time, Repeating Couplings, and Heating
Conclusion
