Industrial application of coupling reagents in peptides OLEG MARDER 1 FERNANDO ALBERICIO 2,3 ABSTRACT In the actual pharmaceutical market, peptides Peptides play a key role in the post-genomic and are not only considered as hormones, as in the PEPTIDES proteomic era. From an industrial basis, the past, but also as active pharmaceutical formation of the amide bond is crucial for ingredients (API), in antibiotics, antiviral and in obtaining an efficient and economic process. other therapeutic areas such as cancer as Mechanisms associated with the different methods immunomodulators and anti-angiogenesis agents, are reviewed. Cl-HOBt as additive and CNS and neurogogical disorders as analgetics HCTU/TCTU are excellent alternatives to the most and anti-obesity drugs, immune disorders for the classic coupling methods. treatment of allergy, asthma and autoimmune disease (1). Furthermore, over 200 new peptide-based drugs are under different stages of development with INTRODUCTION 50% of them under clinical trials and prior to approval (2). Nowadays, peptides represent 1% of There is no doubt that the last years are recognised total API with a market of US$ 300-500 M per year as the renaissance of the peptide world. The and a growth rate of 15-25% annually (1), with the important advances in molecular biology and gene expectation of a 100% increase in the next two technology are bringing significant changes in years when generic and recently approved new biomedical sciences. The elucidation of the human chemical entities enter the market (3). This is genome, followed by advancements in functional reflected by the fact that over ten known bulk genomics and proteomics, will revolutionize our peptide producers and over twenty companies are understanding of the detailed molecular offering custom peptides synthesis and these mechanisms, underlying a broad spectrum of numbers, as well as capacity are growing (4). This diseases. These developments will also identify trend has subsequently affected all raw material new therapeutic targets and suggest novel manufacturers and the entire peptide industry mechanism-based therapeutic paradigms. Peptides supply chain. will play a key role in all these processes. Today, manufacturing companies face the There has been a notable increase in research unprecedented challenge of production of hundred institutions and pharmaceutical companies kilograms to tons quantities of complex peptides involved in the development of peptide-based involving modern technologies. Thus, the customer drugs, resulting in an increase in the number of supplier relationships are becoming more complex, university core facilities and spin-offs, commercial involving basic producers with their extensive peptide suppliers, contract peptide manufacturers, knowledge and experience in the reagents field and and other outsourcing institutions. increasingly into a process development at different stages of production. Akey step in the peptide production process is the formation of the peptide bond. This requires the 1. Luxembourg Industries (Palmol) Ltd. activation of a carboxylic acid, which is usually 27 Hamered Street, P.O. Box 13 carried out using the so-called peptide coupling Tel Aviv, Israel reagents (5,6). In addition to peptides, amide 2. Barcelona Biomedical Research Institute, Barcelona bonds are present in a huge array of other organic Science Park, University of Barcelona compounds of biological interest such as peptoids, Josep Samitier 1 oligocarbamates, oligoamides, β-lactams, E-08028 Barcelona, Spain polyenamides, benzodiazepines, diketopiperazines, 6 and hydantoins. Furthermore, the ester group is 3. Department of Organic Chemistry, University of another important functionality present in many CHIMICAOGGI Barcelona, chemistry today organic compounds and can also be prepared Martí i Franqués 1-11 directly from the carboxylic acid using peptide E-08028 Barcelona, Spain June coupling reagents. 2003 PEPTIDES N-acylurea (2), which is not reactive, or sustain an intramolecular cyclization to give a 5(4H)-oxazolone (3), which is less reactive than 1 and can tautomarize with the corresponding loss of chirality. If activation is carried out in a solvent of low dielectric constant such as CHCl3 or CH2Cl2, the formation of 1 occurs instantaneously, which is absent of a nucleophile or base and can be stable for many hours. However, if the activation is carried out in a more polar solvent such as DMF, no immediate reaction can be detected, and a complex mixture of starting amino acid, symmetrical anhydride (4), and 2 is formed. If the activation is carried out in the presence of an extra equivalent of acid, 4 is Figure 1 - Mechanism of peptide bond formation through carbodiimide activation formed, which is also very reactive. Although the synthesis of medium-large At the beginning of the 70’s, peptide for basic research is a well established 1-hydroxybenzotriazole (HOBt) (9) was proposed procedure, the combination of the 20 as an additive to DCC to reduce racemization and proteinogenic amino acids and growing number from then on other benzotriazole derivatives such of unnatural amino acids makes each peptide as 1-hydroxy-5-chlorobenzotriazole (Cl-HOBt) synthesis at the industrial level unique, requiring (10) or 1-hydroxy-7-azabenzotriazole (HOAt) (11) closer attention to each amino acid coupling. have also been used. The OBt active esters (5) are Some of the rules for coupling reagents less reactive than 1, but are more stable and less validated in the research scale can be applied at prone to racemize. All these factors make the industrial level, but the results are still hardly addition of benzotriazole derivatives almost predictable. Therefore, although peptides are mandatory to preserve the peptide bond formation produced in the industry in hundred kilograms by carbodiimide activation of low yields and to tons scale, there is still a need to search for undesired side reactions. the ultimate coupling reagent. In the last decade onium (phosphonium and The peptide coupling reagent field has clearly aminium/uronium) salts of evolved in the last decade from carbodiimides to hydroxybenzotriazole derivatives have been onium (phosphonium and uronium) salts. The introduced. Although, they have been rapidly era of industrial coupling reagents began in adapted for research purposes, only a few of 1955 with the introduction of them have been found compatible with current dicyclohexylcarbodiimide (DCC) (7), which at industrial requirements and synthetic strategies that time was already known and well studied, and therefore adopted by the industry. as a reagent for formation of amide bond (8). The specie that reacts with onium salts is the Unfortunately, carbodiimides did not comply carboxylate (Figure 2) and therefore the presence with the concept of ultimate coupling reagents because its high reactivity provokes racemization and side reactions during the coupling reaction (Figure 1). The mechanism of the carbodiimide activation, which is complex and depending on the solvent, starts by a proton transfer, followed by addition of the carboxylate to form the O-acylisourea (1). This is the most reactive specie that can attack the amino component to 7 give the corresponding amide. CHIMICAOGGI However, the O-acylisourea chemistry today (1) can undergo a rearrangement to give the Figure 2 - Mechanism of peptide bond formation through onium salts activation June 2003 of at least one THERE ARE equivalent base is MORE THAN 80 essential. The REAGENTS intermediate species KNOWN TODAY, acyloxy-phosphonium HOWEVER ONLY or amidinium salts have A FEW HAVE not been detected and FOUND THEIR react immediately with WAY TO THE the benzotriazole INDUSTRY derivative (an extra equivalent of it is added Analyzing the bulk in some synthetic coupling reagent protocols) to give 5, market, we can see which react with the that is shared amino component to between three main give the corresponding groups of coupling amide. reagents in addition to the additives (Figure 3). WHAT IS A GOOD PEPTIDES COUPLER FROM Additives THE INDUSTRIAL As was outlined in POINT OF VIEW? the introduction, the addition of Effective Coupling benzotriazoles Reagent [HOBt (6) and • Works with high Cl-HOBt (7)] to the efficiency for a wide carbodiimides based variety of peptide coupling reagents sequences. leads to the • Works in formation of the steochimetric benzotriazole active quantities. esters that are less reactive than the O-acylisourea • Has a high conversion rate at room temperature. (5), reducing racemization of the protected amino • Works for both solution and solid-phase peptide acid and avoiding the formation of other synthesis. derivatives less reactive. Cl-HOBt (7) performs at • Is soluble in all the currently used solvents and least as well as HOBt (6), but since it is more can be used at high concentrations. acidic (pKa: 3.35 for Cl-HOBt and 4.60 for HOBt) • Solutions are stable for several days at room it is a better leaving group and its active esters are temperature. more reactive than OBt esters. As discussed in • Allows monitoring of coupling reagents. section 5, another advantage of Cl-HOBt is that the • Shows few side reactions. chlorine atom stabilized the structure, making • Its secondary products after coupling can be Figure 3 - The most completely removed by common coupling solvent extraction. reagents and additives used in the industry Cost-Effective Reagent Can Be Produced in Large Quantities • Conventional raw material available. • Technology is known and does