Choosing the Right Coupling Reagent for Peptides. A Twenty-Five

Review Cite This: Org. Process Res. Dev. XXXX, XXX, XXX−XXX

pubs.acs.org/OPRD

Choosing the Right Coupling Reagent for Peptides: A Twenty-FiveYear Journey Fernando Albericio*,†,‡,§,∥ and Ayman El-Faham*,‡,⊥ †

School of Chemistry and Physics, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South Africa Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia § Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, Barcelona 08028, Spain ∥ CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, Barcelona 08028, Spain ⊥ Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt

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‡

ABSTRACT: This work is not a typical review that is trying to cover all coupling reagents in-depth. This is a personal accountit is written in the first personseeking to summarize the 25 years of collaboration that has brought the release of several products onto the market, together with strategies for the efficient synthesis of peptides and also other molecules of biological interest, broadly adopted by the scientific community. KEYWORDS: amide, aminium salts, OxymaPure, racemization, solid-phase peptide synthesis, uronium/phosphonium salts

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INTRODUCTION

Without doubt, the consideration of peptides as drugs was driven by the development of the SPPS strategy by the Nobel Laurate Bruce Merrifield in the late 1950s and early 1960s.14,15 Thanks to SPPS methodology, a large number of chemistry laboratories were able to synthesize peptides manually or with the aid of automatic synthesizers using tert-butyloxycarbonyl (Boc)/benzyl (Bzl) chemistry to fuel their research programs. Furthermore, the Boc/Bzl SPPS strategy was also adopted by the API manufacturing industry to produce peptides for the pharmaceutical sector. It is important to bear in mind that the first Nobel Prize in the peptide field was awarded to Vicent du Vigneaud in 1955 for the synthesis of the first active peptide, oxytocin, which contains only eight amino acids.16 The second democratization of peptide synthesis was when Sheppard and Atherton17 in Europe and Chang and Meienhofer18 in the United States independently adapted the fluorenylmethoxycarbonyl (Fmoc) Nα-amino protecting group developed by Carpino19 to the SPPS strategy to develop Fmoc/tert-butyl (tBu) chemistry. Although Boc/Bzl chemistry is possibly more robust and performs better than Fmoc/tBu, the latter can be used by laboratories with very basic knowledge of chemistry, thereby allowing the use of peptides to reach a larger research community. An additional advantage of the Fmoc/tBu strategy is that it allows the straightforward preparation of protected peptides, which are crucial for the synthesis of large peptides on an industrial scale by condensation in solution of protected peptides prepared in the solid phase.20 Again, implementation of the Fmoc/tBu strategy by the API industry has been decisive for peptide drug development.12 The amide/peptide bond is present not only in peptides but also in a large number of non-peptidic drugs. Amide bond formation is possibly the most common chemical trans-

Oligonucleotide and peptide therapeutics (TIDES) are currently recognized as valid alternatives to the two most important classes of drugs, the so-called small molecules and biologicals.1,2 In 2017, a total of 46 new entities32 new chemical entities (NCEs) and 12 biologicalswere approved by the U.S. Food & Drug Administration (FDA). Of these, six were peptides [Angiotensin II (Giapreza), Etelcalcetide (Parsabiv), Plecanatide (Trulance), Abaloparatide (Tymlos), Semaglutide (Ozempic), and Macimorelin (Macrilen)].1−3 Furthermore, Vosevi and Mavyret, which are drug combinations, contain in one of the drugs that forms the combination a couple of peptide bonds each.1 In the previous year, of the 15 NCEs approved, two were peptides [Lixisenatide (Adlyxin) and Lifitegrast (Xiidra)], and each of the molecules, Zepatier and Epclusa, in the two-drug combination contains peptide bonds.1,4,5 At present, almost 100 peptides are on the market and more than 400 are in clinical trials.6−10 Around 10% of the market of active pharmaceutical ingredients (APIs) correspond to peptides. Indeed, in 2025, the peptide market is expected to be worth more than US $47 billion.6 How can the marked increase of peptides in the drug arena be explained? From the chemical perspective, this increase has come about as a result of (i) the development of the solidphase peptide synthesis (SPPS) methodology; (ii) the finetuning of the chemistry associated with the synthetic methodology, mainly protecting groups and coupling reagents; and (iii) the implementation of reverse-phase high-performance liquid chromatography for peptide purification. The combination of these three factors has allowed the production of large peptide-based APIs in multi-kilogram amounts or a degree of peptide complexity that was impossible to envisage only a few decades ago.11−13 © XXXX American Chemical Society

Received: May 15, 2018 Published: June 21, 2018 A

DOI: 10.1021/acs.oprd.8b00159 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

Review

Figure 1. Mechanism of carbodiimide-based activation of carboxylic acids.

concept that introduced N,N′-dicyclohexylcarbodiimide (DCC). Carbodiimides activate the carboxylic group in situ before forming the peptide bond. The use of this reagent allows the use of protected amino acids off the shelf before the coupling, thereby avoiding the need for previous preparation of the active species of carboxylic acids, which, due to their active character, were often difficult to prepare and store. Both Z and DCC were used by Merrifield for the synthesis of the first peptide, H-Leu-Ala-Gly-Val-OH, in solid-phase.14 The reaction of the carboxylic group with carbodiimide renders a superactive species, O-acylisourea (Figure 1). This molecule can undergo rearrangement to become the totally inactive Nacylurea. Although this side reaction takes place in N,Ndimethylformamide (DMF), it is faster in dichloromethane (DCM), which was the solvent used during the first years of SPPS. When the α-amino acid is in the form of an amide or carbamate moiety, O-acylisourea can render the oxazolone, which is much less reactive than the former and can cause epimerization,38 a process that can also be triggered by Oacylisourea itself due to its high reactivity. Konig and Geiger39 proposed the addition of 1-hydroxybenzotriazole (HOBt, Figure 2) during carbodiimide coupling to form the -OBt

formation used in medicinal chemistry programs. A survey carried out by the three largest pharmaceutical companies (AstraZeneca, GSK, and Pfizer) in 2013 determined that amide formation is the first manipulation performed in their medicinal chemistry programs, accounting for 16% of all reactions, the amide bond being present in 54% of the compounds biologically tested.21,22 Research into peptide/amide formation experienced a breakthrough during the early 1990s. At that time, the two authors of this report were living in Massachusetts. A.E-F. was a Ph.D. student at the University of Massachusetts at Amherst under the supervision of Professor Louis A. Carpino, and F.A. was Director of Peptide Research at Millipore-Waters in Bedford. Carpino’s laboratory was developing the second generation of stand-alone coupling reagents, and MilliporeWaters was involved in developing applications for their commercialization. This was to be the start of a long collaboration between the authors, first catalyzed by Professor Carpino and then maintained through their own joint research. This Review seeks to summarize the 25 years of collaboration that has brought about the release of several products onto the market, together with strategies for the efficient synthesis of peptides and also other molecules of biological interest. This Review does not intend to cover all coupling reagents in-depth, since there are already some excellent reviews in the literature.23−34 Instead, this is a personal account by two researchers who have dedicated a quarter of a century to working together in the intriguing field of peptide/amide formation.

Figure 2. Most important 1-hydroxybenzotriazole derivatives used as coupling additives.

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FROM CARBODIIMIDE TO AMINIUM SALTS: THE IMPORTANCE OF 1-HYDROXYBENZOTRIAZOLE DERIVATIVES From the synthesis of the first peptide carried out by Fischer35 at the beginning of the 20th century until the development of the SPPS strategy carried out by Merrifield in 1963,14 two main milestones were crucial for the implementation of SPPS. The first was the development of the benzyloxycarbonyl group (Z, Cbz) for the protection of the amine function by Bergmann and Zervas.36 The second was the introduction of the concept of coupling reagents by Sheehan and Hess,37 a

ester. Although less reactive than O-acylisourea, OBt esters are more efficient in terms of controlling the racemization and yield. The presence of a second equivalent of the carboxylic acid renders the symmetric anhydride, which is also a highly reactive species and often leads to a “double hit” (double incorporation of the protected amino acid).40 DCC/HOBt has been very often used for the Boc/Bzl-based SPPS strategy. Although the corresponding side product, N,N′dicyclohexylurea (DCU), is very insoluble in the solvents used during the synthesis (DCM, DMF), it is soluble in trifluoroB



Review

shortly after we demonstrated that all “uronium” salts based on HOBt are aminium salts (Figure 4).46,50

acetic acid (TFA) used to remove the Boc group; therefore DCU is removed from the reaction pot during the TFA treatment. For the Fmoc/tBu SPPS strategy, the use of the N,N′-diisopropylcarbodiimide (DIC) is preferred because the N,N′-diisopropylurea is soluble in DMF. For chemistry in solution, the water-soluble carbodiimide N′-ethyl-N′-(3(dimethylamino)propyl)carbodiimide (EDC·HCl) is very often the carbodiimide of choice because it is easy to remove during the workup.27 At the beginning of the 1990s, Carpino41 proposed 7-aza-1hydroxybenzotriazole (HOAt, Figure 2) as a more reactive substitute for HOBt. The electron-withdrawing influence of the N at position 7 of the benzotriazole exacerbates the acidity of HOAt, thereby improving the quality of the leaving group and allowing greater reactivity. This coincided with the flowering of uronium and phosphonium salts (Figure 3) as stand-alone coupling

Figure 4. N-HATU vs O-HATU (N-HATU is the only structure that exists).

After this, several HOBt derivatives with electron-withdrawing groups (Cl, NO2, CF3)27 in position 6 were developed, together with the corresponding aminium and phosphonium salts. Among these derivatives, 7-chloro-1hydroxybenzotriazole (Cl-HOBt) has been the most widely used, as well as HCTU51 and PyClock, developed also by our group.52 These reagents showed a good compromise in terms of reactivity and price when compared with HOBt/HOAt.53 At that time, and although HOAt, HATU, and PyAOP were commercially available, one of the most important obstacles impeding their use was price. The seminal paper on HOAt by Carpino,38 which has been discussed in several reviews,23,24,26,27 points out that, in addition to the presence of N (regardless of its position) in the OBt system, which makes OAt a better leaving group, its placement at position 7 makes it feasible to achieve a classic neighboring group effect, thereby enhancing reactivity and minimizing side reactions (Figure 5). The superior perform-

Figure 3. HOBt-based aminium/phosphonium salt coupling reagents.

reagents, which already incorporated hydroxybenzotriazole moieties. The first salt of uronium N-[(1H-benzotriazol-1yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU) was reported by Dourtoglu et al.42 Around the same time, Castro and coworkers reported an equivalent phosphonium salt, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 43 which had its roots in acyloxyphosphonium salts, developed by Kenner and co-workers.44 Given that after the coupling reaction takes place, BOP gives rise to the highly toxic side product hexamethylphosphoramide, it was replaced by benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP).45 In a joint collaboration between Carpino and Millipore, the corresponding uronium and phosphonium salts of HOAt (Figure 3) were developed. In all cases, HOAt, N[(dimethylamino)-1 H-1,2,3-triazolo[4,5- b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), and [(7-azabenzotriazol-1-yl)oxy]tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) outperformed their HOBt counterparts in terms of yield and control of epimerization.46−48 Intrigued by the superiority of HOAt, we crystallized HATU, and X-ray diffraction showed that it was not a uronium salt (the positive C bonded to the O of HOBt) but rather an aminium salt (guanidinium N-oxides) (the positive C bonded to a heterocyclic N of HOAt).49 We initially believed that this might explain the higher reactivity of HATU, but

Figure 5. Neighboring group effect associated with HOAt.

ance of HOAt derivatives over Cl-HOBt ones has not been explained to date since their X-ray diffraction has not been resolved. However, the slight difference between the pKa value of HOAt (pKa = 3.38) and that of Cl-HOBt (pKa = 3.35) supports the notion of the active species being present in the O-acyl in the case of HOAt derivatives and the N-acyl in the case of HOBt derivatives. Supporting this fact, 4-HOAt (pKa = 3.14), which shows poorer performance than 7-HOAt, is more acidic.54 Finally, 6-HOAt, whose pKa could not be obtained due to decomposition of the compound under the conditions of the determination but is presumably lower than that of 7HOAt, also shows poorer performance than the 6-isomer in terms of both yield and control of racemization.51 In parallel to the screening of the leaving group, we turned our attention to studying the immonium part. In the first generation, no heteroatoms were introduced. As a general trend, the chemical structure of the immonium part is important for reactivity.55,56 However, high reactivity is also associated with high instability. Thus, the 1,3-dimethylimidazolidine derivatives (HBMDU and HAMDU, Figure 6) were much more reactive but showed considerable instability, which made them unsuitable for practical purposes. The salts derived C



Review

carbocation moiety conferred greater solubility to the reagent when compared with the tetramethyl derivatives (HXTU series). Furthermore, the oxygen atom enhanced coupling yields and decreased racemization, allowing the use of only 1 equiv of base.56,60

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FLUOROFORMAMIDINIUM SALTS Fischer61 had already proposed acyl chlorides as a strategy to facilitate the formation of the peptide bond. However, this strategy was not extensively used in peptide synthesis due to problems of over-activation with double incorporation and loss of configuration and low compatibility with Boc chemistry since the Boc group is unstable in acid conditions.62 Later, Fmoc-acid chloride proved useful in peptide synthesis under certain conditions.63 A logical extension of acyl chloride was the use of fluoride derivatives.60,64−66 Carpino and El-Faham reported N,N,N′,N′-tetramethylfluoroformamidinium hexafluorophosphate (TFFH) (Figure 9),67 which is the

Figure 6. Stand-alone coupling reagents prepared from different immonium structures.

from dimethylamine (HXTU series) were the most stable and, as discussed earlier, showed excellent reactivity. Finally, the pyrrolidino derivatives showed greater reactivity than HXTU and intermediate stability, thereby allowing their practical use. However, 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]-pyridin-1ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU) and its HOBt congener, HBPyU, were not commercially successful because the increase in reactivity was not accompanied by affordability. Again, HAPyU was crystallized in the N-form (guanidinium N-oxides).52 Later, Carpino and El-Faham, in collaboration with their long-term collaborators Beyermann and Bienert, in Berlin, crystallized the Oform (uronium salt).57 In a second generation of reagents, we were interested in introducing a proton acceptor in the immonium part to act as a self-catalyst, thus influencing performance in terms of reactivity and racemization control, as well as solubility and stability. Our first objective was to prepare the immonium salt shown in Figure 7, which contained an N-methylpiperazine moiety.

Figure 9. Structure of TFFH.

stand-alone coupling reagent that renders the fluoride acyl derivative. TFFH is a convenient and stable reagent for in situ acid fluoride formation using the same methods as those used for the aminium and phosphinium salts. As with the immonium/uronium salts, we prepared a family of fluoroform−amidinium salts with distinct chemical structures in the amidinium part. The same trends as before were observed; i.e., the greater the reactivity of these salts, the greater the instability.68,69

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SEARCHING FOR A REPLACEMENT FOR HOBT: THE OXIME FAMILY September 11, 2001, was a day that changed the world.70 The significance of what happened on that day also had repercussions for the chemical world, more specifically for the field of coupling reagents. In 2005, Wehrstedt from Bundesanstalt für Materialforschung and prüfung (Working Group “Explosive Substances of Chemical Industries”) and researchers at Bayer 71 demonstrated that HOBt and derivatives, more importantly HOAt, show explosive properties, according to the Directive 92/69/EEC,72 and should therefore be classified as substances with a risk of explosion. Thus, these derivatives were regrouped under the “Class 1 explosive category”, thereby making their transport difficult (they can be transported only by land and sea). In view of the relevance of these compounds in day-to-day peptide research and the pharmaceutical industry, it became evident that there was a need for another family of safe and efficient additives, based on a different template and less prone to explosion. This posed a challenge when considering that the performance of additives based on other compounds, such as p-nitrophenol (HONp), N-hydroxysuccinimide (HOSu), or pentafluorophenol (HOPfp), is not comparable to that of HOBt and less so to HOAt because active esters of ONp, OSu, or OPfp are less reactive than benzotriazole esters.73,74 In this regard, for several years we worked on synthesizing and screening various N-hydroxylamines as coupling additives.

Figure 7. Left: failed reaction to obtain aminium chlorides. Right: preparation of piperazine-containing active carbamates.

However, during the preparation of this compound a side reaction took place, which led to unsymmetrical bis-ureas, which were found to have anti-HIV properties.58 Next, we prepared new coupling reagents, including a morpholine moiety with HOBt, Cl-HOBt, and HOAt (Figure 8).59 In all cases, the presence of the oxygen atom in the

Figure 8. Stand-alone coupling reagents based on the morpholine moiety. D



Review

from the resin. Furthermore, K-Oxyma shows greater solubility than OxymaPure in a broad range of solvents. Finally, KOxyma shows higher thermal stability and a lower pressure release than OxymaPure and, of course, than HOBt and HOAt.77

To minimize the risk of their being hazardous and/or explosive, we avoided the synthesis of compounds with concatenated N atoms or those that infringe the rule regarding the numbers of C, O, and N present in a molecule.75

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C+O 90% after 5 days) and γE



Review

valerolactone (GVL) (>80% after 5 days).87 ACN is compatible with ChemMatrix resin,88,89 and GVL, which is a green solvent, is compatible with both polystyrene and ChemMatrix resin.90 As COMU is commercially available, it is widely used in both research programs and production processes. Examples of the latest applications of COMU are described below. COMU has been used for the final step of lactamization of the Nannocystin Ax, which is a polyketide-based cyclodepsipeptide with antiproliferative activity in the nanomolar range against various cancer cell lines.91 COMU also finds applications in the preparation of Weinreb amide92 in solution and even for the esterification of tertiary alcohols.93 In the solid-phase mode, this reagent has proved optimal for the preparation of both peptoids94 and aza-peptides.95 Recently, Withey and Bajic from MacEwan University (Edmonton, Alberta) proposed a second-year undergraduate laboratory experiment in which the students prepared the insect repellent, N,N-diethyl-3-methylbenzamide (DEET), from 3-methylbenzoic acid and N,N-diethylamine using COMU as coupling reagent.96 According to this report, the advantage of this experiment from an educational perspective is that the reaction can be monitored visually by virtue of the color change associated with the conversion, and all byproducts are conveniently water-soluble. This tendency of COMU and its byproducts to be soluble in water makes it one of the coupling reagents of choice for green chemistry. Thus, Lipshutz and co-workers97 recently reported amide bond formation in aqueous media at room temperature. After screening various coupling reagents and surfactants, they concluded the optimal conditions were COMU as coupling reagent, TPGS-750-M as surfactant, and 2,6-lutidine as base. More recently, this group reported a tandem deprotection/ coupling sequence in water under micellar catalysis conditions using the surfactant TPGS-750-M. In this strategy, Z removal was followed by peptide coupling in the presence of COMU and 2,6-lutidine, rendering peptides containing up to 10 amino acid residues.98 In the same field of green chemistry, EDC-OxymaPure and COMU-TMP have proven effective for the coupling of carboxylic acids with amines in the presence of water. The second cocktail is specially indicated for the acylation of anilines.99 Finally, Watson and Coll100 screened a range of green solvents to replace DMF and DCM in solution. In their study, various aryl and alkyl acids and amines were coupled in solution. It was concluded that COMU is the best coupling reagent, as it is compatible with 2-methyltetrahydrofuran (2MeTHF), dimethyl carbonate, and ethyl acetate (EtOAc), which are recognized as green solvents and are called to substitute DMF and DCM, especially when COMU was used as coupling reagent.

Figure 12. Structure of PyOxim.

was attributed to steric hindrance caused by the presence of the three morpholine rings bound to the P. Thus, the pyrrolidinio derivative, O-[(cyano(ethoxycarbonyl)methyliden)amino]yloxytripyrrolidinophosphonium hexafluorophosphate (PyOxim, Figure 12), similar to PyBOP, PyAOP, and PyClock, was prepared.101 On this occasion, PyOxim performed as expected, proving to be more soluble in DMF and showing better coupling yields and less racemization than its congeners. Like other phosphonium-based reagents, PyOxim proved efficient for cyclization using H-Ala-AlaNMeAla-Ala-Ala-OH as a model peptide.95 Bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride (BOP-Cl, Figure 13),102 which is not related to the

Figure 13. Organophosphine-based coupling reagents.

phosphonium salt BOP, is the most important member of the organophosphinic family of coupling reagents, which have shown broad applications, mainly for the solution synthesis of N-Me-containing peptides.103,104 The modest acceptance of BOP-Cl in the solid-phase mode is probably a result of its poor performance. Such performance may be because of the fact that the activation of the urethane-protected amino acid with BOP-Cl renders first the corresponding acid chloride and then to the oxazolone, which as discussed earlier (Figure 1) is a poor acylating derivative, and in addition is accompanied by racemization. With the aim to prepare an organophosphinebased coupling agent useful in both solution and solid phase, we prepared the OxymePure, HOAt, and HOBt derivatives of BOP-Cl. As expected, BOP-OXy [ethyl 2-(bis(2-oxooxazolidin-3-yl)phosphoryloxyimino)-2-cyanoacetate, Figure 13] was superior to BOP-OAt [3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl bis(2-oxooxazolidin-3-yl)phosphinate], and even more clearly superior to BOP-OBt [1H-benzo[d][1,2,3]triazol1-yl bis(2-oxooxazolidin-3-yl)phosphinate]. Furthermore, BOP-OXy showed much greater solubility than the rest of the members of the series in DMF. Another important class of phosphorus coupling reagents is the dialkoxyphosphoryloxy family, whose best representative is DEPBT [3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin4(3H)-one, Figure 14], developed jointly by the groups of Ye and Goodman.105 Although DEPBT is highly reactive, it contains the presumably explosive 3-hydroxy-1,2,3-benzo-

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PYOXIM AND OTHER PHOSPHORUS STAND-ALONE OXYMAPURE-BASED COUPLING REAGENTS The development of phosphonium salts occurred always in parallel to that of uronium/aminium. As with COMU, our first idea was to combine OxymaPure and the morpholine moiety in the same molecule. Thus, the corresponding derivative PyOxym-M (Figure 12) was prepared and screened as a coupling reagent. To our surprise, this reagent showed very low reactivity (results not published). This poor performance F



Review

related dimethyl malonate (pKa = 5 vs 13), and second to exploit the presence of the 6-member ring scaffold in order to facilitate the catalyst through a neighboring group effect. Surprisingly, the results achieved with DIC-HONM as coupling cocktail were disappointing. In this regard, coupling yields were extremely low, in fact the lowest ever obtained with this family of coupling reagents. Further studies have indicated that, due to its high reactivity, HONM reacted with DIC to produce an adduct and therefore deactivates DIC (Figure 16).103 This confirmed our initial thoughts about the high

Figure 14. Diethoxyphosphoryloxy family of stand-alone coupling reagents.

triazin-4(3H)-one (HOOBt, HODht), whose use is not exempt from chemical side reactions. Kurosu’s group reported the preparation two oxime-based derivatives.106 The one corresponding to OxymaPure was named diethylphosphoryl-Oxyma (DPOx) by this group, and a second one based on a glyceroacetonide oxime was called diethylphosphoryl-glyceroacetonide-Oxyma (DPGOx).107 These reagents have given excellent results for the formation of amides,100,101 fragment peptide coupling with minimal racemization, and even esterification in aqueous solution media.108 Its compatibility with aqueous media reinforces the notion that OxymaPure-based reagents are highly suitable for use in green chemistry. As DPOx and DPGOx are not solid, we very recently prepared a solid derivative of the same family (DEPAOx) using Amox (2-amino-N-hydroxy-2-oxoacetimidoyl cyanide) as oxime component (unpublished results). As Amox is less reactive than OxymaPure, this derivative is also less reactive, and therefore it is recommended that it be used in the presence of 1 equiv of OxymaPure to improve the reactivity of the coupling cocktail.

Figure 16. Side reaction when HONM is used as DIC additive.

reactivity of these derivatives. It is important to bear in mind that similar adducts of DIC with HOBt/HOAt have not been reported. Next we consider the corresponding uronium salt, 1-[1-(2,2dimethyl-4,6-dioxo-1,3-dioxan-5-ylideneaminooxy)dimethylaminomorpholinomethylene]methanaminium hexafluorophosphate (HMMU).103 HMMU proved highly reactive and, for instance, outperformed COMU, HATU, and HBTU, among others, by achieving the acylation of p-chloroaniline (PCA) in the presence of just 1 equiv of base. In terms of control of racemization, HMMU performed better than COMU and HATU when TMP or just 1 equiv of DIEA was used. For the synthesis of peptides containing hindered amino acids, HMMU did not match COMU. This could be attributed to the greater activity of HMMU, which would lead to higher instability.103 Playing with the same idea of having a 6-member scaffold, we prepared 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (Oxyma-B, Figure 15),110 which is a derivative of barbituric acid. Oxyma-B performed extremely well in terms of minimizing racemization.104 On the other hand, the Oxyma-T (Figure 15) built on the scaffold of the thiobarbituric derivative111 showed poorer performance. These observations thus confirm that small variations in the structure can have an impact on performance. Low reactivity is not desired. However, high reactivity is often accompanied by undesirable side reactions, and in this case it is preferable to use a less reactive coupling additive/ reagent. A paradigmatic example of this is illustrated by the preparation of Fmoc-amino acids. To sum up, commercial Fmoc-amino acids were initially prepared following Schotten− Baumann conditions by the reaction of Fmoc-Cl with amino acids in basic pH. In the early 1980s, our112 group and that of Verlander and Goodman113 independently reported that the use of the highly reactive Fmoc-Cl leads to the formation of dipeptides and even tripeptides (Figure 17). Although we showed that the use of Fmoc-N3 minimized the side reaction at the research scale, this method was not acceptable in terms of safety for the production of Fmoc-amino acids. In parallel, after performing a broad screening of various leaving groups, Verlander and Goodman proposed the Nhydroxysuccinimide derivative (Fmoc-OSu) as the optimal

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OTHER MEMBERS OF THE OXYMAPURE FAMILY Malonic acid provides the scaffold for the design of this family of coupling reagents. Therefore, malonic acid derivatives facilitate the synthesis of other members of this family. Figure 15 shows most of the oxime coupling reagents synthesized over

Figure 15. Other Oxyma derivative-based coupling reagents.

the past few years. Although OxymaPure is the reagent that showed the best performance, some members of this family deserve further discussion. Using Meldrum’s acid as base, we prepared the corresponding oxime [isonitroso Meldrum’s acid (HONM)].109 The idea behind this design was twofold: first to take advantage of the high acidity of this derivative when compared with the highly G



Review

taking into account the epimerization of two piperidides: aspartimide, β-peptide, α-piperidide, β-piperidide) and the fact that one, the β-peptide, has the same mass as the target peptide make this side reaction extremely troublesome (Figure 19). In the Fmoc/tBu strategy, aspartimide formation occurs mainly during the piperidine−DMF treatment used for Fmoc removal. It was previously demonstrated that N-hydroxy compounds such as HOBt buffer the piperidine solution and therefore decrease the extent of the side reaction. OxymaPure has been demonstrated to have the same effect as HOBt/ HOAt in preventing the side reaction, 1 M OxymaPure in 20% piperidine-DMF being the best conditions to minimize the reaction.122 Fmoc protection is compatible with coupling conditions that use COMU or PyOxim, which involves the presence of DIEA (pKa = 10.1), a reagent that is less basic than the piperidine used for the removal of the Fmoc group (pKa = 11.1). However, during the incorporation of Fmoc-amino acids on Cterminal Pro resins, a double incorporation of the incoming amino acid due to the removal of its Fmoc by the Pro (pKa = 10.6) has been detected (Figure 20). To minimize this side reaction, the Pro resin is washed with 0.1 M OxymaPure in DMF (3×), and then the coupling of the next residue is performed as usual.116 Finally, the chapter devoted to side reactions reports that a 0.1 M solution of OxymaPure in DMF causes less cleavage of a peptide from CTC resin than same solution of HOAt/ HOBt.116 In the absence of a carboxylic group, COMU, like other aminium/uronium coupling reagents, reacts with amines to form guanidinium. This is, in principle, an undesired side reaction because it terminates the peptide chain (Figure 21).52 This reaction does not have practical negative consequences when the coupling reagent is not in excess with respect to the carboxylic component. Thus, it is recommended to use a slightly defect of coupling reagent. However, in the cyclization steps, where both components, the amine and the carboxylic acid, are in equimolar proportion, the side reaction can take place. Another favorable case is during fragment coupling, where the excess of one of the two fragments is tiny. In these cases, the use of phosphonium salts, which do not give the side reaction, is recommended. However, it is possible to take advantage of guanidinium formation for preparing compounds with this moiety. This approach has been applied for the introduction of diversity into combinatorial libraries,123 as well as for the synthesis of modified natural peptides, such as the preparation of analogues of Teixobactin,124,125 an antimicrobial peptide that has raised great expectations.126

Figure 17. Formation of Fmoc-Gly-Gly-OH during the synthesis of Fmoc-Gly-OH.

reagent. Although the use of the so-called Bolin method,114,115 which consists of the in situ protection of the carboxylic group with a silyl-based protecting group, was also adapted, FmocOSu was continuously used for industrial purposes. However, the use of Fmoc-OSu was also questioned when Hlebowicz et al.116 showed the presence of side products Fmoc-β-Ala-OH and Fmoc-β-Ala-AA-OH when Fmoc-AA-OH’s were prepared from Fmoc-OSu.117 These side products were formed through a Lossen rearrangement after the successive attack of OSu on one of the carbonyls of the HOSu moiety present in FmocOSu (Figure 18).117 Our group and others have extensively worked toward identifying convenient leaving groups which will make the corresponding Fmoc derivative suitable for this purpose. Key characteristics of these leaving groups are (i) absence of high reactivity, thus avoiding the formation of oligopeptides, and (ii) easily removable during the aqueous workup. Taking advantage that we had synthesized several members of the oxime family, we screened those less reactive for the introduction of Fmoc.118,119 From these previous studies, we concluded that Fmoc-Amox was the most suitable compound because it showed a low reactivity profile and the byproduct was easily removable during the workup. Using Fmoc-Amox, we successfully synthesized Fmoc-Gly-OH, which is the amino acid most susceptible to oligomerization, and others, with total absence of the side reaction or Amox in the final product.120

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APPLICATIONS BEYOND AMIDE/PEPTIDE BOND FORMATION OxymaPure is finding applications in other aspects of peptide synthesis, specifically in avoiding base-mediated side reactions, namely aspartimide formation and double hit incorporation on Pro-peptide resins. Aspartimide is perhaps one of the most negative side reactions during the synthesis of Asp-containing peptides.121 It is highly sequence-dependent, and therefore some sequences such as Asp-Gly, Asp-Ser, or Asp-Asn are more prone to this reaction. However, the number of side products (four without

Figure 18. Formation of β-Ala through a Lossen rearrangement during the preparation of Fmoc-AA-OH with Fmoc-OSu116,117 H



Review

Figure 19. Aspartimide formation during peptide synthesis.

The many years that we have devoted to research into coupling additives and reagents have taught us that the most reactive compounds do not always give the best performance. High reactivity is often associated with instability, and therefore sometimes the compound decomposes before reacting. Furthermore, high reactivity can be linked to side reactions, as clearly exemplified by the introduction of the Fmoc protecting group with Fmoc-Cl. We are frequently asked about our favorite coupling strategy. It is then that we feel like chefs, and our answer is that each peptide may require a different coupling strategy. Briefly, for microwave-assisted automatic SPPS, DIC and OxymaPure are globally unbeatable. For manual SPPS, COMU shows an excellent balance between efficiency and price, thus making it very attractive. For cyclization, our favorite is PyOxim, which is powerful and exempt of side reactions. For solution synthesis, EDC/OxymaPure or again COMU would be our choice. For the introduction of Fmoc and other protecting groups, the corresponding carbonates derived from Amox would be appropriate, as they are reactive but exempt of over-activation. In green chemistry, again DIC/OxymaPure and COMU are compatible with the green solvents most widely used. Finally, we draw the readers’ attention to the reach of OxymaPure derivatives, which extends from their use in undergraduate student laboratories to the production of APIs on multi-kilogram scales.

Figure 20. Double incorporation of the amino acid after a Pro residue.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] or [email protected]. *E-mail: [email protected] or aymanel_faham@hotmail. com.

Figure 21. Peptide termination with COMU and other aminium/ uronium salts.

ORCID

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Fernando Albericio: 0000-0002-8946-0462

CONCLUSIONS, OR THERE IS MORE THAN ONE WAY TO SKIN A CAT After 25 years of working in collaboration and developing more than 50 coupling reagents and additives, we are most pleased that OxymaPure and its derivatives have become the reagents of choice for the synthesis of the majority of peptides as it has been demonstrated independently by another colleagues.81,127−129 In addition, in our hands, OxymaPure, COMU, and PyOxim, not being as hazardous as HOBt and derivatives, have consistently outperformed HOBt, HBTU, and PyBOP, and very often even show a similar performance to HOAt, HATU, and PyAOP, also developed by us and considered the most powerful coupling reagents available.

Author Contributions

Both authors have contributed equally to the preparation of the manuscript and have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS These 25 years of research would not have been possible without the most valuable help of many collaborators, who rapidly became our friends. In addition to Professor Louis Carpino, we would like to mention, in alphabetical order, those whose participation was most significant: Gerardo Acosta, I



Review

(16) du Vigneaud, V.; Ressler, C.; Swan, J. M.; Roberts, C. W.; Katsoyannis, P. G.; Gordon, S. The synthesis of an octapeptide with hormonal activity of oxytocin. J. Am. Chem. Soc. 1953, 75, 4879− 4880. (17) Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J. A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonylamino acids. J. Chem. Soc., Chem. Commun. 1978, 537−539. (18) Chang, C.-D.; Meienhofer, J. Solid-phase peptide synthesis using mild base cleavage of Nα- fluorenylmethoxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostain. Int. J. Pept. Protein Res. 1978, 11, 246−249. (19) Carpino, L. A.; Han, G. Y. 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J. Am. Chem. Soc. 1970, 92, 5748−5749. (20) Bray, B. L. Innovation: large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rev. Drug Discovery 2003, 2, 587−593. (21) MacMillan, D. S.; Murray, J.; Sneddon, H. F.; Jamieson, C.; Watson, A. J. B. Evaluation of alternative solvents in common amide coupling reactions: replacement of dichloromethane and N,Ndimethylformamide. Green Chem. 2013, 15, 596−600. (22) Roughley, S. D.; Jordan, A. M. The medicinal chemist’s toolbox: an analysis of reactions used in the pursuit of drug candidates. J. Med. Chem. 2011, 54, 3451−3479. (23) Albericio, F.; Chinchilla, R.; Dodsworth, D. J.; Najera, C. New trends in peptide coupling reagents. Org. Prep. Proced. Int. 2001, 33, 203−303. (24) Bray, B. L. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rev. Drug Discovery 2003, 2, 587−593. (25) Montalbetti, C. A. G. N.; Falque, V. Amide bond formation and peptide coupling. Tetrahedron 2005, 61, 10827−10852. (26) Bode, J. W. Emerging methods in amide- and peptide-bond formation. Curr. Opin. Drug Discovery Develop. 2006, 9, 765−775. (27) Valeur, E.; Bradley, M. Amide bond formation: beyond the myth of coupling reagents. Chem. Soc. Rev. 2009, 38, 606−631. (28) El-Faham, A.; Albericio, F. Peptide Coupling Reagents, More than a Letter Soup. Chem. Rev. 2011, 111, 6557−6602. (29) White, C. J.; Yudin, A. K. Contemporary strategies for peptide macrocyclization. Nat. Chem. 2011, 3, 509−524. (30) Al-Warhi, T. I.; Al-Hazimi, H. M. A.; El-Faham, A. Recent development in peptide coupling reagents. J. Saudi Chem. Soc. 2012, 16, 97−116. (31) Cherkupally, P.; Ramesh, S.; de la Torre, B. G.; Govender, T.; Kruger, H. G.; Albericio, F. Immobilized coupling reagents: synthesis of amides/peptides. ACS Comb. Sci. 2014, 16, 579−601. (32) Dunetz, J. R.; Magano, J.; Weisenburger, G. A. Large-scale application of amide coupling reagents for the synthesis of pharmaceuticals. Org. Process Res. Dev. 2016, 20, 140−177. (33) Paradis-Bas, M.; Tulla-Puche, J.; Albericio, F. Making Easy the Synthesis of ″Difficult Peptides″: Solubilizing Strategies. Chem. Soc. Rev. 2016, 45, 631−654. (34) Jaradat, D. M. M. Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation. Amino Acids 2018, 50, 39−68. (35) Fischer, E.; Fourneau, E. Ueber einige Derivate des Glykocolls. Ber. Dtsch. Chem. Ges. 1901, 34, 2868−2877. (36) Bergmann, M.; Zervas, L. A general process for the synthesis of peptides. Ber. Dtsch. Chem. Ges. B 1932, 65, 1192−1201. (37) Sheehan, J. C.; Hess, G. P. A New Method of Forming Peptide Bonds. J. Am. Chem. Soc. 1955, 77, 1067−1068. (38) Carpino, L. A.; Ionescu, D.; El-Faham, A.; Henklein, P.; Wenschuh, H.; Bienert, M.; Beyermann, M. Protected amino acid chlorides vs protected amino acid fluorides: reactivity comparisons. Tetrahedron Lett. 1998, 39, 241−244. (39) Konig, W.; Geiger, R. A new method for synthesis of peptides: activation of the carboxyl group with dicyclohexylcarbodiimide using 1-hydroxybenzotriazoles as additives. Chem. Ber. 1970, 103, 788−798.

Jordi Alsina, Josep M. Bofill, Beatriz G. de la Torre, Monte del Fresno, Bruce Foxman, Yahya Jad, Knud Jensen, Jack Johansen, Steve Kates, Sherine Khattab, Ashish Kumar, Oleg Marder, Zainab Marhoon, Chuck Minor, Lidia NietoRodriguez, Rafel Prohens, Miriam Royo, Anamika Sharma, Hitesh Shroff, Nuria Sole, Ramon Subiros-Funosas, Salvatore Triolo, and Holger Wenschuh. Special thanks to Yoav Luxembourg for supporting this research. We apologize to those whose names have been missed. Finally, the work in the laboratories of the authors was funded in part by the International Scientific Partnership Program ISPP at King Saud University (ISPP no. 0061) (Saudi Arabia); the National Research Foundation (NRF) and the University of KwaZuluNatal (South Africa); and the Generalitat de Catalunya (2017 SGR 1439) and MINECO (CTQ2015-67870-P) (Spain).

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DEDICATION

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REFERENCES

Dedicated to Professor Louis A. Carpino (University of Massachusetts), our mentor, for together with Professor R. Bruce MerrifieldLaurate Nobel Prize in Chemistry, 1984 making possibly the greatest contributions to the field of peptide science.

(1) U.S. Food and Drug Administration (FDA). Drugs@FDA: FDA Approved Drug Products, http://www.accessdata.fda.gov/scripts/ cder/daf/. (2) Mullard, A. 2017 FDA drug approvals. Nat. Rev. Drug Discovery 2018, 17, 81−85 and previous articles by the same author published yearly in the same journal (February issue).. (3) de la Torre, B. G.; Albericio, F. The Pharmaceutical Industry in 2017. An Analysis of FDA Drug Approvals from the Perspective of the Molecules. Molecules 2018, 23, 533. (4) Mullard, A. 2016 FDA drug approvals. Nat. Rev. Drug Discovery 2017, 16, 73−76. (5) de la Torre, B. G.; Albericio, F. The Pharmaceutical Industry in 2016. An Analysis of FDA Drug Approvals from a Perspective of the Molecules. Molecules 2017, 22, 368. (6) Fosgerau, K.; Hoffmann, T. Peptide therapeutics: current status and future directions. Drug Discovery Today 2015, 20, 122−128. (7) Albericio, F.; Kruger, H. G. Therapeutic Peptides. Future Med. Chem. 2012, 4, 1527−1531. (8) Rafferty, J.; Nagaraj, H.; McCloskey, A. P.; Huwaitat, R.; Porter, S.; Albadr, A.; Laverty, G. Peptide therapeutics and the pharmaceutical industry: barriers encountered translating from the laboratory to patients. Curr. Med. Chem. 2016, 23, 4231−4259. (9) Ghosh, S. Peptide therapeutics market: forecast and analysis 2015−2025. Chim. Oggi Chem. Today 2016, 34, V−VII. (10) Lau, J. L.; Dunn, M. K. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorg. Med. Chem. 2018, 26, 2700−2707. (11) Bruckdorfer, T.; Marder, O.; Albericio, F. From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future. Curr. Pharm. Biotechnol. 2004, 5, 29−43. (12) Zompra, A. A.; Galanis, A. S.; Werbitzky, O.; Albericio, F. Manufacturing peptides as active pharmaceutical ingredients. Future Med. Chem. 2009, 1, 361−377. (13) Jad, Y. E., El-Faham, A., de la Torre, B. G., Albericio, F. SolidPhase Peptide Synthesis, the State of the Art. Challenges and Opportunities. In Peptide-Based Drug Discovery: Challenges and Opportunities, Discovery Series No. 5X; V. Srivastava, Ed.; Royal Society of Chemistry: London, UK, 2017; pp 518−550. (14) Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149−2154. (15) Merrifield, R. B. Solid phase synthesis (Nobel lecture). Angew. Chem., Int. Ed. Engl. 1985, 24, 799−810. J



Review

(40) Merrifield, R. B.; Mitchell, A. R.; Clarke, J. E. Detection and prevention of urethane acylation during solid-phase synthesis by anhydride methods. J. Org. Chem. 1974, 39, 660−668. (41) Carpino, L. A. 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 1993, 115, 4397−4398. (42) Dourtoglou, V.; Ziegler, J. C.; Gross, B. O-Benzotriazolyl-N,Ntetramethyluronium hexafluorophosphate: a new and effective reagent for peptide coupling. Tetrahedron Lett. 1978, 19, 1269−1272. (43) Castro, B.; Dormoy, J. R.; Dourtoglou, B.; Evin, G.; Selve, C.; Ziegler, J. C. Peptide coupling reagents. VI. A novel, cheaper preparation of benzotriazolyloxytris[dimethylamine]phosphonium hexafluorophisphate (BOP reagent). Synthesis 1976, 1976, 751−752. (44) Gawne, G.; Kenner, G. W.; Sheppard, R. C. Acylpxyphosphonium salts as acylating agents. Synthesis of peptides. J. Am. Chem. Soc. 1969, 91, 5669−5671. (45) Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M. N.; Jouin, P. PyBOP and PyBroP: two reagents for the difficult coupling of the α,αdialkyl amino acid. Tetrahedron 1991, 47, 259−70. (46) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. Advantageous applications of azabenzotriazole-based coupling reagents to solid-phase peptide synthesis. J. Chem. Soc., Chem. Commun. 1994, 201−203. (47) Carpino, L. A.; El-Faham, A.; Albericio, F. Racemization studies during solid-phase peptide synthesis using azabenzotriazole-based coupling reagents. Tetrahedron Lett. 1994, 35, 2279−2283. (48) Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.; Kates, S. A. On the Use of PyAOP, a Phosphonium Salt Derived from HOAt, in Solid-Phase Peptide Synthesis. Tetrahedron Lett. 1997, 38, 4853−4856. (49) Abdelmoty, I.; Albericio, F.; Carpino, L. A.; Foxman, B. F.; Kates, S. A. Structural studies of reagents for peptide bond formation: crystal and molecular structures of HBTU and HATU. Lett. Pept. Sci. 1994, 1, 57−67. (50) The phosphonium derivative of BOP was crystallized, and the X-ray diffraction showed that the OBt was linked through the O to the phosphonium: Sieroslawski, K.; Picur, B.; Lis, T. Structure of benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP). J. Mol. Struct. 2003, 657, 93−99. (51) Marder, O.; Shvo, Y.; Albericio, F. HCTU and TCTU. New Coupling Reagents: Development and Industrial Aspects. Chem. Today 2002, 20 (7/8), 37−41. (52) Subiros-Funosas, R.; Moreno, J. A.; Bayó-Puxan, N.; Aburabeah, K.; Ewenson, A.; Albericio, F. PyClocK, the phosphonium salt derived from Cl-HOBt. Chem. Today 2008, 26/4, 10−12. (53) Sabatino, G.; Mulinacci, B.; Alcaro, M. C.; Chelli, M.; Rovero, P.; Papini, A. M. InPeptides 2002: Proceedings of the Twenty-Seventh European Peptide Symposium; Benedettti, E., Ed.; ESCOM: Leiden, The Netherlands, 2002; p 272. (54) Carpino, L. A.; Imazumi, H.; Foxman, B. M.; Vela, M. J.; Henklein, P.; El-Faham, A.; Klose, J.; Bienert, M. Comparison of the Effects of 5- and 6-HOAt on Model Peptide Coupling Reactions Relative to the Cases for the 4- and 7-Isomers. Org. Lett. 2000, 2, 2253−2256. (55) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. K. On the Use of Onium Salt-Based Coupling Reagents in Peptide Synthesis. J. Org. Chem. 1998, 63, 9678−9683. (56) El-Faham, A.; Khattab, S. N.; Abdul-Ghani, M.; Albericio, F. Design and Synthesis of a New Immonium Type Coupling Reagents. Eur. J. Org. Chem. 2006, 6, 1563−1573. (57) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang, C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mügge, C.; Wenschuh, H.; Klose, J.; Beyermann, M.; Bienert, M. The Uronium/ Guanidinium Peptide Coupling Reagents: Finally the True Uronium Salts. Angew. Chem., Int. Ed. 2002, 41, 441−445. (58) El-Faham, A.; Armand-Ugon, M.; Este, J. A.; Albericio, F. Use of N-Methylpiperazine for the Preparation of Piperazine-Based Unsymmetrical Bis-Ureas as Anti-HIV Agents. ChemMedChem 2008, 3, 1034−1037.

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Review

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