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This article is providing non-experts with strategic considerations and general information about principles of peptide chemistry. The article is not encyclopedic but rather devoted to the history and bases of peptide synthesis.

Brief History of Peptide Synthesis

A «peptide synthesis» includes a large range of techniques and procedures that enable the preparation of materials ranging from small peptides to large proteins. The pioneering work of Bruce Merrifield , which introduced solid phase peptide synthesis (SPPS), dramatically changed the strategy of peptide synthesis and simplified the tedious and demanding steps of purification associated with solution phase synthesis. Moreover, Merrifield's SPPS allows development of automation and the extensive range of robotic instrumentation. After defining a synthesis strategy and programming the amino acid sequence, machines can automatically perform all synthesis steps to prepare multiple peptide samples. SPPS has now become the method of choice to produce peptides, although solution phase synthesis can still be useful for large-scale production of given peptide.

Peptide chemistry is a sub-discipline of chemistry, developed with considerable delay. Its growth is not comparable to the rapid blossoming of aromatic compounds' chemistry which started with the isolation of benzene from coal gas by Faraday in 1825 and the proposition of its structure by A.Kekule in 1865. Those discoveries built a basis of dyes and medicines manufacture and later followed by petro-chemistry production. The prehistory of peptide chemistry lies hidden in early studies of proteins and in physiological chemistry, e.g. finding a relationship between nutrients and the blood compositions.

The first peptide synthesis, as well as a term «peptide», was reported by Hermann Emil Fischer and Ernest Fourneau in 1901. Fischer and E. Fourneau published preparation of first dipeptide, glycylglycine by partial HCl hydrolysis of glycine diketopiperazine.

At German Scientists and Physicians Society, in Karlsbad in 1902, Emil Fischer summarized the isolation of amino acids and peptides from protein hydrolyzates and discussed the coupling of amino acids in proteins. In his own words (translated from German): «the idea that acidamide - like groups play the principal role comes readily to mind, as Hofmeister also assumed in his general lecture this morning». Fischer had begun experiments to link amino acids to each other by methods of organic chemistry as early as 1900. The two lectures mark the emergence of the so called Fischer-Hofmeister theory of protein structure. Emil Fischer, who in a paper with E. Foumeau had just described the first prototype, glycyl-glycine, proposed in his lecture also the designations «peptide, dipeptide, tripeptide etc.» and later «polypeptides».

Emil Fischer and his co-workers synthesized about 100 peptides within the first decade of the 20th century using the methods developed in his laboratory, peptides containing from 2 to 18 amino acid residues.

Synthetic work with peptides was resumed by Bergmann along the lines of the Fischer school in a systematic way with Joseph S. Fruton, who joined Bergmann in 1934, soon after his arrival at the Rockefeller Institute. Fruton utilized the potential of the new carbobenzoxy method to synthesize inaccessible peptides for testing as substrates for enzymatic hydrolysis. He and Bergmann published a syntheses of simple substrates which were cleaved by papain, chymotrypsin, trypsin, and pepsin at certain peptide bonds.

The history of peptide chemistry stretches over a century, from compounds as simple as diglycine to enzymes with more than a hundred amino acids. Due to the individual character of each amino acid and combination of many amino acids forming large molecules, peptides show an extremely great variety of specific functions. They can act as chemical messengers, hormone, intra- or inter-cellular mediators, highly specific stimulators and inhibitors and as biologically active peptides in the brain and nervous systems. Many antibiotic compounds from bacteria, molds and amphibian skin are peptic in nature and so are compounds toxic not only for pathogens but also for cells of higher organisms. Peptides actively involved in reactions of the immune system are attracting increasing attention from biomedical researchers. Peptide science is still in full development.

Protective Groups for Peptide Synthesis

Because of amino acid is an acid with a basic group at one end and an acid group at the other, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protective groups are used.

Currently, two protecting groups are commonly used in solid-phase peptide synthesis – Fmoc (9-fluorenylmethyl carbamate) and t-Boc (Di-tert-butyl dicarbonate).

Fmoc Protecting Group

The use of Fmoc chemistry for protection of the alpha amino group has become the preferred method for most contemporary solid and solution phase peptide synthetic processes. Fmoc has also been shown to be more reliable and produce higher quality peptides than Boc chemistry.

The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions. After base treatment, the nascent peptide is typically washed and then a mixture including an activated amino acid and coupling co-reagents is placed in contact with the nascent peptide to couple the next amino acid. After coupling, non-coupled reagents can be washed away and then the protecting group on the N-terminus of the nascent peptide can be removed, allowing additional amino acids or peptide material to be added to the nascent peptide in a similar fashion.

Boc Protecting Group

Before the Fmoc group became popular, the t-Boc group was commonly used for protecting the terminal amine of the peptide, requiring the use of more acid stable groups for side chain protection in orthogonal strategies. Boc groups can be added to amino acids with Di-tert-butyl dicarbonate (Boc anhydride) and a suitable base.

The t-Boc protecting group is removed by exposing the Boc-protected residue on the chain to a strong acid. Typical reagents of choice for deprotection in existing methods are trifluoroacetic acid (TFA) in dichloromethane, hydrochloric acid or methanesulfonic acid in dioxane. The acid used to remove the Boc protecting group is typically neutralized with a tertiary amine such as N-methylmorpholine, N-diisopropylethylamine (DIEA) or triethylamine (TEA).

Basic principles and reagents in peptide synthesis

The protection groups (Y) were not meant to be part of amides and remained problematic for a long time. The first peptide derivatives secured by synthesis, benzoylglycyl-glycine and ethoxycarbonylglycyl-glycine ethyl ester carried blocking groups which could not be removed without the destruction of a newly formed peptide bond. It became obvious that for peptide synthesis easily removable protecting groups were needed. A breakthrough toward the solution of this problem was discovery of benzyloxycarbonyl (carbobenzoxy) group (Z or Cbz) by remarkably lasting contribution of Bergmann and Zervas in 1932.

A removal of Z group stimulated further research toward acid-sensitive protecting groups. This led to development of series of blocking groups cleavable under mild conditions.

Coupling through anhydrides, however, became popular among peptide chemists. Due to variability of the components used for activation of protected amino acids, mixed or unsymmetrical anhydrides, were proposed by many investigators as different versions of general approach to peptide bond formation.

Some esters became very popular, e.g. nitrophenyl ester, 2,4,5-trichlorophenyl ester, N-hydroxysuccinimide ester and pentaftuorophenyl ester.

Most of the peptide synthesis are accompanied by minor, undesired side reactions, and the peptides can be contaminated by by-products. There's a solution that avoids by-products formation.

Solid-Phase Peptide Synthesis

Peptide synthesis is much more complicated than simply forming amide bonds by mixing the desired amino acids in a reaction vessel. Twenty natural and endless number of unnatural amino acids give numerous possible combinations formed with this technique.

In a solution of two amino acids mixed together, four different dipeptides as well as other longer peptides will be formed, e.g. in a mixture of glycine and alanine four dipeptides would be Gly-Gly, Gly-Ala, Ala-Gly and Ala-Ala.

The solid-phase peptide synthesis was developed for the purpose of providing rapid, simplified and effective way of peptides and small proteins preparation (Merrifield, 1963).

To ensure the desired peptide is formed, the basic group of amino acid and the acidic group of another must both be made unable to react. Such «deactivation» is known as the protection of reactive groups. A group that is unable to react, is called a protecting group.

In classical organic synthesis the acids are protected, allowed to react and de-protected. Afterward one end of peptide is protected and allowed to undergo a reaction with a new protected acid and so on. In solid-phase peptide synthesis (SPPS) one peptide end is attached to water-insoluble polymer and remains protected throughout the entire peptide formation, meaning both fewer steps and simplified purification, the reagents can be rinsed away without losing the peptide.

Solid-phase peptide synthesis involves the following steps: attaching amino acid to the polymer, protection, coupling, de-protection and polymer removal.

Attaching of amino acid to polymer

The FMOC protected amino acid reacts with the last amino acid attached to the polyamide. The reaction is catalyzed by DCC (N,N'-Dicyclohexylcarbodiimide).

De-protection

DCC excess is washed off by water. FMOC group is removed by piperidine (secondary cyclic amine). The acid protection group is removed from the chain and is able to add another acid molecule. On the step two a new amino acid is protected. The cyclic process continues until a required chain length has been accomplished.

Polymer removal

Once the desired peptide is completed it must be removed from polyamide. It can be reached by using 95% solution of trifluoroacetic acid (TFA). The side-chain protecting groups are removed at this stage as well.

Advantages and disadvantages of SPPS

The main advantage of SPPS is a peptide high yield. As peptide consists of many amino acids and each acid yield is less than 100%, overall peptide yield is negligible. For example, if each amino acid gives 90% an overall yield of peptide containing 100 amino acid is 0,003% only.

Solid-phase peptide synthesis is much faster than classical solution process. SPPS allows a formation of 20 amino acids peptide in 24 hours and longer ones in less than a week.

As modern automated synthesizers and sophisticated analytical and purification equipment are improved, a peptide chemist can make peptides in the range of 20-50 amino acids in length and in amounts from 20-100 milligrams.

The essential advantage of SPPS is efficiency of separation of the intermediates from starting materials, reagents and most of the by-products. The peptide, attached to the insoluble polymer, remains undissolved during solvents' treatment.

Another plus of SPPS is absence of time consuming isolation of intermediates through extraction or crystallization. A problem to find solvents for large molecular weight intermediates no longer exists because it's sufficient using solvents in which polymer just swells.

It becomes rather simple to convert amino salt to free amine, by treating peptidyl resin with tertiary base and following removal of alkylammonium salt formed, by washing.

The SPPS biggest weakness: the intermediates cannot be purified and the possibilities for their characterization are rather limited.

However, numerous instruments for the automatic execution of SPPS have been described and several methods were developed for the continuous analytical control of peptide building.

References 1. M. Bodanszky. Principles of Peptide Synthesis, 2nd ed., Springer, Heidelberg, 1993. [ Book ]. 2. Bernd Gutte. Peptides: Synthesis, Structures, and Applications. Academic Press; 1 edition (November 2, 1995). [ Book ]. 3. Gregg B. Fields. Introduction to Peptide Synthesis. Current Protocols in Protein Science . [ Article ]. 4. T. Wieland, M. Bodanszky. The World of Peptides. A Brief History of Peptide Chemistry. [ Book ]. 5. Fields GB, Noble RL. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res. 1990; 35:161–214 . [ Article ]. 6. Merrifield B. Solid phase synthesis. Science. 1986; 232:341–347 . [ Article ].

This page was last updated: 25 December 2023.

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Introduction to Peptide Synthesis

A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

DEVELOPMENT OF SOLID-PHASE PEPTIDE-SYNTHESIS METHODOLOGY

A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin ( Pontiroli, 1998 ). In the year 2008, the peptide therapeutics market reached the multi-billion dollar level ( Saladin et al., 2009 ). More than 400 peptides have entered clinical studies so far. Rapid, efficient, and reliable methodology for the chemical synthesis of these molecules is therefore of utmost interest. The stepwise assembly of peptides from amino acid precursors has been described for nearly a century. The concept is a straightforward one, whereby peptide elongation proceeds via a coupling reaction between amino acids, followed by removal of a reversible protecting group. The first peptide synthesis, as well as the creation of the term “peptide,” was reported by Fischer and Fourneau ( Fischer and Fourneau, 1901 ). Bergmann and Zervas created the first reversible N α -protecting group for peptide synthesis, the carbobenzoxy (Cbz) group ( Bergmann and Zervas, 1932 ). DuVigneaud successfully applied early “classical” strategies to construct a peptide with oxytocin-like activity ( Vigneaud et al., 1953 ). Classical, or solution-phase methods for peptide synthesis have an elegant history and have been well chronicled. Solution synthesis continues to be especially valuable for large-scale manufacturing and for specialized laboratory applications.

Peptide synthesis became a more practical part of present-day scientific research following the advent of solid-phase techniques. The concept of solid-phase peptide synthesis (SPPS) is to retain chemistry that has been proven in solution but to add a covalent attachment step that links the nascent peptide chain to an insoluble polymeric support (resin). Subsequently, the anchored peptide is extended by a series of addition cycles ( Fig. 18.1.1 ). It is the essence of the solid-phase approach that reactions are driven to completion by the use of excess soluble reagents, which can be removed by simple filtration and washing without manipulative losses. Once chain elongation has been completed, the crude peptide is released from the support.

[*Gwen: legend same as original fig]

Generalized approach to solid-phase peptide synthesis.

In the early 1960s, Merrifield proposed the use of a polystyrene-based solid support for peptide synthesis. Peptides could be assembled stepwise from the C to N terminus using N α -protected amino acids. SPPS of a tetrapeptide was achieved by using Cbz as an α-amino-protecting group, coupling with N,N '-dicyclohexylcarbodiimide (DCC), and liberating the peptide from the support by saponification or by use of HBr ( Merrifield, 1963 ). SPPS was later modified to use the t -butyloxycarbonyl (Boc) group for N α protection ( Merrifield, 1967 ) and hydrogen fluoride (HF) as the reagent for removal of the peptide from the resin ( Sakakibara et al., 1967 ). SPPS was thus based on “relative acidolysis,” where the N α -protecting group (Boc) was labile in the presence of moderate acid (trifluoroacetic acid; TFA), while side-chain-protecting benzyl (Bzl)-based groups and the peptide/resin linkage were stable in the presence of moderate acid and labile in the presence of strong acid (HF). The peptide bonds of the assembled chain were stable to these manipulations. The first instrument for automated synthesis of peptides, based on Boc SPPS, was built by Merrifield, Stewart, and Jernberg ( Merrifield et al., 1966 ). From the 1960s through the 1980s, Boc-based SPPS was fine-tuned ( Merrifield, 1986 ). This strategy has been utilized for synthesis of proteins such as interleukin-3 and active enzymes including ribonuclease A and all- l and all- d forms of HIV-1 aspartyl protease.

In 1970, Carpino introduced the 9-fluorenylmethoxycarbonyl (Fmoc) group for N α protection ( Carpino and Han, 1970 ). The Fmoc group requires moderate base for removal, and thus offered a chemically mild alternative to the acid-labile Boc group. In the late 1970s, the Fmoc group was adopted for solid-phase applications. Fmoc-based strategies utilized t -butyl ( t Bu)-based side-chain protection and hydroxymethylphenoxy-based linkers for peptide attachment to the resin. This was thus an “orthogonal” scheme requiring base for removal of the N α -protecting group and acid for removal of the side-chain protecting groups and liberation of the peptide from the resin. The milder conditions of Fmoc chemistry as compared to Boc chemistry, which include elimination of repetitive moderate acidolysis steps and the final strong acidolysis step, were envisioned as being more compatible with the synthesis of peptides that are susceptible to acid-catalyzed side reactions. In particular, the modification of the indole ring of Trp was viewed as a particular problem during Boc-based peptide synthesis ( Barany and Merrifield, 1979 ), which could be alleviated using Fmoc chemistry. One example of the potential advantage of Fmoc chemistry for the synthesis of multiple-Trp-containing peptides was in the synthesis of gramicidin A. Gramicidin A, a pentadecapeptide containing four Trp residues, had been synthesized previously in low yields (5% to 24%) using Boc chemistry. The mild conditions of Fmoc chemistry dramatically improved the yields of gramicidin A, in some cases up to 87% ( Fields et al., 1989 ; Fields et al., 1990 ). A second multiple-Trp-containing peptide, indolicidin, was successfully assembled in high yield by Fmoc chemistry ( King et al., 1990 ). Thus, the mild conditions of Fmoc chemistry appeared to be advantageous for certain peptides, as compared with Boc chemistry.

One of the subsequent challenges for practitioners of Fmoc chemistry was to refine the technique to allow for construction of proteins, in similar fashion to that which had been achieved with Boc chemistry. Fmoc chemistry had its own set of unique problems, including suboptimum solvation of the peptide/resin, slow coupling kinetics, and base-catalyzed side reactions. Improvements in these areas of Fmoc chemistry ( Atherton and Sheppard, 1987 ; Fields et al., 2001 ; Fields and Noble, 1990 ) allowed for the synthesis of proteins such as bovine pancreatic trypsin inhibitor analogs, ubiquitin, yeast actin-binding protein 539–588, human β-chorionic gonadotropin 1–74, mini-collagens, HIV-1 Tat protein, HIV-1 nucleocapsid protein NCp7, and active HIV-1 protease.

The milder conditions of Fmoc chemistry, along with improvements in the basic chemistry, have led to a shift in the chemistry employed by peptide laboratories. This trend is best exemplified by a series of studies ( Angeletti et al., 1997 ) carried out by the Peptide Synthesis Research Committee (PSRC) of the Association of Biomolecular Resource Facilities (ABRF). The PSRC was formed to evaluate the quality of the synthetic methods utilized in its member laboratories for peptide synthesis. The PSRC designed a series of studies from 1991 to 1996 to examine synthetic methods and analytical techniques. A strong shift in the chemistry utilized in core facilities was observed during this time period, i.e., the more senior Boc methodology was replaced by Fmoc chemistry. For example, in 1991 50% of the participating laboratories used Fmoc chemistry, while 50% used Boc-based methods. By 1994, 98% of participating laboratories were using Fmoc chemistry. This percentage remained constant in 1995 and 1996. In addition, the overall quality of the peptides synthesized improved greatly from 1991 to 1994. Possible reasons for the improved results were any combination of the following ( Angeletti et al., 1997 ):

• The greater percentage of peptides synthesized by Fmoc chemistry, where cleavage conditions are less harsh;
• The use of different side-chain protecting group strategies that help reduce side reactions during cleavage;
• The use of cleavage protocols designed to minimize side reactions;
• More rigor and care in laboratory techniques.

The present level of refinement of solid-phase methodology has led to numerous commercially available instruments for peptide synthesis ( Table 18.1.1 ).

Table 18.1.1

Instruments for Solid-Phase Peptide Synthesis currently available on the market.

The next step in the development of solid-phase techniques includes applications for peptides containing non-native amino acids, post-translationally modified amino acids, and pseudoamino acids, as well as for organic molecules in general. Several areas of solid-phase synthesis need to be refined to allow for the successful construction of this next generation of biomolecules. The solid support must be versatile so that a great variety of solvents can be used, particularly for organic-molecule applications. Coupling reagents must be sufficiently rapid so that sterically hindered amino acids can be incorporated. Construction of peptides that contain amino acids bearing post-translational modifications should take advantage of the solid-phase approach. Finally, appropriate analytical techniques are needed to assure the proper composition of products.

THE SOLID SUPPORT

Successful SPPS depends upon the choice of the solid support, linker (between the solid support and the synthesized peptide), appropriately protected amino acids, coupling methodology, and protocol for cleaving the peptide from the solid support ( Fields, 1997 ). Choosing the right solid support is often paramount for successful, non-problematic synthesis of the desired peptide. Currently, there are a vast number of commercially available resins, suitable for complex peptide synthesis. It has to be noted that effective solvation of the peptide/resin is perhaps the most crucial condition for efficient chain assembly during solid-phase synthesis. Swollen resin beads may be reacted and washed batch-wise with agitation, then filtered either with suction or under positive nitrogen pressure. Alternatively, they may be packed in columns and utilized in a continuous-flow mode by pumping reagents and solvents through the resin ( Lukas et al., 1981 ). 1 H, 2 H, 13 C, and 19 F nuclear magnetic resonance (NMR) experiments have shown that, under proper solvation conditions, the linear polystyrene chains of copoly(styrene-1%-divinylbenzene) resin (PS) are nearly as accessible to reagents as if free in solution ( Albericio et al., 1989 ; Ford and Balakrishnan, 1981 ; Live and Kent, 1982 ; Ludwick et al., 1986 ; Manatt et al., 1980 ). 13 C and 19 F NMR studies of Pepsyn (copolymerized dimethylacrylamide, N,N '-bisacryloylethylenediamine, and acryloylsarcosine methyl ester) have shown similar mobilities at resin-reactive sites as PS. Additional supports created by grafting polyethylene glycol (polyoxyethylene) onto PS [either by controlled anionic polymerization of ethylene oxide on tetraethylene glycol–PS (POE-PS) or by coupling N ω -Boc– or Fmoc–polyethylene glycol acid or –polyethylene glycol diacid to amino-functionalized PS (PEG-PS)] combine the advantages of liquid-phase synthesis (i.e., a homogeneous reaction environment) and solid-phase synthesis (an insoluble support). 13 C NMR measurements of POE-PS showed the polyoxyethylene chains to be more mobile than the PS matrix, with the highest T 1 spin-lattice relaxation times observed with POE of molecular weight 2000 to 3000. Other supports that show improved solvation properties and/or are applicable to organic synthesis include polyethylene glycol polyacrylamide (PEGA), cross-linked acrylate ethoxylate resin (CLEAR), and augmented surface polyethylene prepared by chemical transformation (ASPECT). As the solid-phase method has expanded to include organic-molecule and library syntheses, the diversity of supports will enhance the efficiency of these new applications.

Successful syntheses of problematic sequences can be achieved by manipulation of the solid support. In general, the longer the synthesis, the more polar the peptide/resin will become ( Sarin et al., 1980 ). One can alter the solvent environment and enhance coupling efficiencies by adding polar solvents and/or chaotropic agents ( Fields and Fields, 1994 ). Also, using a lower substitution level of resin to avoid interchain crowding can improve the synthesis ( Tam and Lu, 1995 ). During difficult syntheses, deprotection of the Fmoc group can proceed slowly. By spectrophotometrically monitoring deprotection as the synthesis proceeds, one can detect problems and extend base-deprotection times and/or alter solvation conditions as necessary.

Solid phase peptide synthesis is traditionally carried out in the C → N direction. The majority of peptides are being synthesized as C-terminal acids or amides. For synthesis of C-terminal modified peptides one can take advantage of many linkers that are available ( Guillier et al., 2000 ). The use of linkers provides control and flexibility of the synthetic process, e.g., functionalization of the C-terminal amino acid, loading of the C-terminal amino acid, and/or cleavage conditions utilized for liberation of the peptide following synthesis.

COUPLING REAGENTS

The term coupling refers to formation of a peptide bond between two adjacent amino acids. Coupling involves attack of the amino group of one residue at the carbonyl group of the carboxy-containing component that has been activated by an electron withdrawing group. The activated form of the amino acid can be a shelf-stable reagent, compound of intermediate stability, or a transient intermediate which is neither isolable nor detectable ( El-Faham and Albericio, 2011 ).

The classical examples of in situ coupling reagents are N,N '-dicyclohexylcarbodiimide (DCC) and the related N,N '-diisopropylcarbodiimide ( Rich and Singh, 1979 ). The generality of carbodiimide-mediated couplings is extended significantly by the use of either 1-hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt) as an additive, either of which accelerates carbodiimide-mediated couplings, suppresses racemization, and inhibits dehydration of the carboxamide side chains of Asn and Gln to the corresponding nitriles. Protocols involving benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), O -benzotriazol-1-yl- N,N,N ', N '-tetramethyluronium hexafluorophosphate (HBTU), O -(7-azabenzotriazol-1-yl)- N,N,N ' N '-tetramethyluronium hexafluorophosphate (HATU), O-(6-Chlorobenzotriazol-1-yl)-N, N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), and O -benzotriazol-1-yl- N,N,N',N '-tetramethyluronium tetrafluoroborate (TBTU) result in coupling kinetics even more rapid than that obtained with carbodiimides. Amino acid halides have also been applied to SPPS. N α -protected amino acid chlorides have a long history of use in solution synthesis. Fmoc–amino acid chlorides and fluorides react rapidly under SPPS conditions in the presence of HOBt/ N,N -diisopropylethylamine (DIEA) and DIEA, respectively, with very low levels of racemization. For convenience, tetramethylfluoroformamidinium hexafluorophosphate (TFFH) can be used for automated preparation of Fmoc–amino acid fluorides. Amino acid fluorides have been found to be especially useful for the preparation of peptides containing sterically hindered amino acids, such as peptaibols. Other coupling agents that result in low levels of epimerization, and thus are particularly useful for head-to-tail peptide cyclizations and fragment condensations, include O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N, N',N'-tetramethyluronium tetrafluoroborate (TDBTU) and 3-(diethylphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT). All of the coupling reagents and additives discussed here are commercially available ( Table 18.1.2 ).

Table 18.1.2

Peptide synthesis reagents suppliers list. [*Gwen: remove boxes so it's just a simple list.]

SYNTHESIS OF MODIFIED RESIDUES AND STRUCTURES

Peptides of biological interest often include structural elements beyond the 20 genetically encoded amino acids. Particular emphasis has been placed on peptides containing phosphorylated or glycosylated residues or disulfide bridges. Incorporation of side-chain-phosphorylated Ser and Thr by SPPS is especially challenging, as the phosphate group is decomposed by strong acid and lost with base in a β-elimination process. Boc-Ser(PO 3 phenyl 2 ) and Boc-Thr(PO 3 phenyl 2 ) have been found to be useful derivatives, where hydrogen fluoride (HF) or hydrogenolysis cleaves the peptide/resin and hydrogenolysis removes the phenyl groups. Fmoc-Ser(PO 3 Bzl,H) and Fmoc-Thr(PO 3 Bzl,H) can be used in conjunction with Fmoc chemistry with some care ( Perich et al., 1999 ; Wakamiya et al., 1994 ). Alternatively, peptide/resins that were built up by Fmoc chemistry to include unprotected Ser or Thr side chains may be subject to “global” or post-assembly phosphorylation ( Otvös et al., 1989a ). Side-chain-phosphorylated Tyr is less susceptible to strong-acid decomposition and is not at all base-labile. Thus, SPPS has been used to incorporate directly Fmoc-Tyr(PO 3 methyl 2 ) ( Kitas et al., 1989 ), Fmoc-Tyr(PO 3 t Bu 2 ) ( Perich and Reynolds, 1991 ), Fmoc-Tyr(PO 3 H 2 ) ( Ottinger et al., 1993 ), and Boc-Tyr(PO 3 H 2 ) ( Zardeneta et al., 1990 ). Phosphorylation may also be accomplished on-line, directly after incorporation of the Tyr, Ser, or Thr residue but prior to assembly of the whole peptide ( Perich, 1997 ).

Methodology for site-specific incorporation of carbohydrates during chemical synthesis of peptides has developed rapidly. The mild conditions of Fmoc chemistry are more suited for glycopeptide syntheses than Boc chemistry, as repetitive acid treatments can be detrimental to sugar linkages. Fmoc-Ser, -Thr, -5-hydroxylysine (-Hyl), -4-hydroxyproline (-Hyp), and -Asn have all been incorporated successfully with glycosylated side chains ( Cudic and Burstein, 2008 ). The side-chain glycosyl is usually hydroxyl-protected by either benzoyl or acetyl groups, although some SPPSs have been successful with no protection of glycosyl hydroxyl groups ( Otvös et al., 1989b ). Deacetylation and debenzylation are performed with hydrazine/methanol prior to glycopeptide/resin cleavage or in solution with catalytic methoxide in methanol ( Sjölin et al., 1996 ).

Disulfide-bond formation has been achieved on the solid-phase by air, K 3 Fe(CN) 6 , dithiobis(2-nitrobenzoic acid), or diiodoethane oxidation of free sulfhydryls, by direct deprotection/oxidation of Cys(acetamidomethyl) residues using thallium trifluoroacetate or I 2 , by direct conversion of Cys(9-fluorenylmethyl) residues using piperidine, and by nucleophilic attack by a free sulfhydryl on either Cys(3-nitro-2-pyridinesulfenyl) or Cys( S -carboxymethylsulfenyl). The most generally applicable and efficient of these methods is direct conversion of Cys(acetamidomethyl) residues by thallium trifluoroacetate. In solution, disulfide formation may be mediated by a lengthy catalogue of reagents, the most straightforward of which are molecular O 2 (from air) and DMSO ( Tam et al., 1991 ).

Intra-chain lactams are formed between the side-chains of Lys or Orn and Asp or Glu to conformationally restrain synthetic peptides, with the goal of increasing biological potency and/or specificity. Lactams can also be formed via side-chain-to-head, side-chain-to-tail, or head-to-tail cyclization ( Kates et al., 1994 ). The residues used to form intra-chain lactams must be selectively side-chain deprotected, while all side-chain protecting groups of other residues remain intact. Selective deprotection is best achieved by using orthogonal side-chain protection, such as allyloxycarbonyl or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl protection for Lys and allyl or N -[1-(4,4,-dimethyl-2,6-dioxocyclohexylidene)-3-methyl butyl]aminobenzyl protection for Asp/Glu in combination with an Fmoc/ t Bu strategy. Cyclization is carried out most efficiently with BOP in the presence of DIEA while the peptide is still attached to the resin ( Felix et al., 1988 ; Plaue, 1990 ).

The three-dimensional orthogonal protection scheme of Fmoc/ t Bu/allyl protecting groups is the strategy of choice for head-to-tail cyclizations. An amide linker is used for side-chain attachment of a C-terminal Asp/Glu (which are converted to Asn/Gln) and the α-carboxyl group is protected as an allyl ester ( Stawikowski and Cudic, 2006 ). For side-chain-to-head cyclizations, the N-terminal amino acid (head) can simply be introduced as an N α -Fmoc derivative while the peptide-resin linkage and the other side-chain protecting groups are stable to dilute acid or carry a third dimension of orthogonality.

PROTEIN SYNTHESIS

There are three general chemical approaches for constructing proteins. First is stepwise synthesis, in which the entire protein is synthesized one amino acid at a time. Second is “fragment assembly,” in which individual peptide strands are initially constructed stepwise, purified, and finally covalently linked to create the desired protein. Fragment assembly can be divided into two distinct approaches: (1) convergent synthesis of fully protected fragments, and (2) chemoselective ligation of unprotected fragments. Third is “directed assembly,” in which individual peptide strands are constructed stepwise, purified, and then noncovalently driven to associate into protein-like structures. Combinations of the three general chemical approaches may also be employed for protein construction.

Convergent synthesis utilizes protected peptide fragments for protein construction ( Albericio et al., 1997 ). The advantage of convergent protein synthesis is that fragments of the desired protein are first synthesized, purified, and characterized, ensuring that each fragment is of high integrity; these fragments are then assembled into the complete protein. Thus, cumulative effects of stepwise synthetic errors are minimized. Convergent synthesis requires ready access to pure, partially protected peptide segments, which are needed as building blocks. The application of solid-phase synthesis to prepare the requisite intermediates depends on several levels of selectively cleavable protecting groups and linkers. Methods for subsequent solubilization and purification of the protected segments are nontrivial. Individual rates for coupling segments are substantially lower than for activated amino acid species by stepwise synthesis, and there is always a risk of racemization at the C-terminus of each segment. Careful attention to synthetic design and execution may minimize these problems.

As an alternative to the segment condensation approach, methods have been developed by which unprotected peptide fragments may be linked. “Native chemical ligation” results in an amide bond being generated between peptide fragments (see UNIT 18.4 ) ( Muir et al., 1997 ). A peptide bearing a C-terminal thioacid is converted to a 5-thio-2-nitrobenzoic acid ester and then reacted with a peptide bearing an N-terminal Cys residue ( Dawson et al., 1994 ). The initial thioester ligation product undergoes spontaneous rearrangement, leading to an amide bond and regeneration of the free sulfhydryl on Cys. The method was later refined so that a relatively unreactive thioester can be used in the ligation reaction ( Ayers et al., 1999 ; Dawson et al., 1997 ). “Safety-catch” linkers are used in conjunction with Fmoc chemistry to produce the necessary peptide thioester ( Shin et al., 1999 ). Safety-catch linkers anchor the nascent peptide to the resin and are stable throughout the synthesis. These linkers then allow the release of a C -terminally modified peptide from the solid support under mild conditions following an additional activation step.

SIDE-REACTIONS

The free N α -amino group of an anchored dipeptide is poised for a base-catalyzed intramolecular attack of the C-terminal carbonyl. Base deprotection of the Fmoc group can thus release a cyclic diketopiperazine while a hydroxymethyl-handle leaving group remains on the resin. With residues that can form cis peptide bonds, e.g., Gly, Pro, N -methylamino acids, or d -amino acids, in either the first or second position of the ( C → N ) synthesis, diketopiperazine formation can be substantial. The steric hindrance of the 2-chlorotrityl linker may minimize diketopiperazine formation of susceptible sequences during Fmoc chemistry.

The conversion of side-chain protected Asp residues to aspartimide residues can occur by repetitive base treatments. The cyclic aspartimide can then react with piperidine to form the α- or β-piperidide or α- or β-peptide. Aspartimide formation can be rapid, and is dependent upon the Asp side-chain protecting group. Sequence dependence studies of Asp(O t Bu)-X peptides revealed that piperidine could induce aspartimide formation when X = Arg(2,2,5,7,8-pentamethylchroman-6-sulfonyl; Pmc), Asn(triphenylmethyl; Trt), Asp(O t Bu), Cys(Acm), Gly, Ser, Thr, and Thr( t Bu) ( Lauer et al., 1995 ). Aspartimide formation can also be conformation-dependent. This side-reaction can be minimized by including 0.1 M HOBt in the piperidine solution ( Lauer et al., 1995 ), or by using an amide backbone protecting group (i.e., 2-hydroxy-4-methoxybenzyl) for the residue in the X position of an Asp-X sequence ( Quibell et al., 1994 ).

Cys residues are racemized by repeated piperidine deprotection treatments during Fmoc SPPS. Racemization of esterified (C-terminal) Cys can be reduced by using 1% 1,8-diazabicyclo[5.4.0]undec-7-ene in N,N -dimethylformamide (DMF). Additionally, the steric hindrance of the 2-chlorotrityl linker minimizes racemization of C-terminal Cys residues. When applying protocols for Cys internal (not C-terminal) incorporation which include phosphonium and aminium salts as coupling agents, as well as preactivation in the presence of suitable additives and tertiary amine bases, significant racemization is observed. Racemization is generally reduced by avoiding preactivation, using a weaker base (such as collidine), and switching to the solvent mixture DMF-dichloromethane (DCM) (1:1). Alternatively, the pentafluorophenyl ester of a suitable Fmoc-Cys derivative can be used.

The combination of side-chain protecting groups and anchoring linkages commonly used in Fmoc chemistry are simultaneously deprotected and cleaved by TFA. Cleavage of these groups and linkers results in liberation of reactive species that can modify susceptible residues, such as Trp, Tyr, and Met. Modifications can be minimized during TFA cleavage by utilizing effective scavengers. Three efficient cleavage “cocktails” quenching reactive species and preserving amino acid integrity, are (1) TFA-phenolthioanisole-1,2-ethanedithiol-H 2 O (82.5:5:5:2.5:5) (reagent K) ( King et al., 1990 ), (2) TFA-thioanisole-1,2-ethanedithiol-anisole (90:5:3:2) (reagent R) ( Albericio et al., 1990 ), and (3) TFA-phenol-H 2 O-triisopropylsilane (88:5:5:2) (reagent B) ( Solé and Barany, 1992 ). The use of Boc side-chain protection of Trp also significantly reduces alkylation by Pmc or 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf) groups. For a list of common problems encountered during peptide synthesis refer to Table 18.1.3 .

Table 18.1.3

Common problems encountered during peptide synthesis.

PURIFICATION AND ANALYSIS OF SYNTHETIC PEPTIDES

Each synthetic procedure has limitations, and even in the hands of highly experienced workers, certain sequences defy facile preparation. The maturation of high-performance liquid chromatography (HPLC) has been a major boon to modern peptide synthesis, because the resolving power of this technique facilitates removal of many of the systematic low-level by-products that accrue during chain assembly and upon cleavage. Peptide purification is most commonly achieved by reversed-phase HPLC (RP-HPLC; UNIT 11.6 ). Either alternatively to or in tandem with RP-HPLC, ion-exchange HPLC ( UNIT 8.2 ) and gel-filtration HPLC ( UNIT 8.3 ) can be used for isolation of desired peptide products. The progress of peptide purification can be monitored rapidly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS; UNIT 16.2 & 16.3 ) or ion-trap electrospray MS ( UNIT 16.8 ).

The homogeneity of synthetic materials should be checked by at least two chromatographic or electrophoretic techniques, e.g., RP-HPLC ( UNIT 11.6 ), ion-exchange HPLC ( UNIT 8.2 ), and capillary zone electrophoresis ( UNIT 10.9 ). Also, determination of a molecular ion by MS (see Chapter 16) using a mild ionization method is important for proof of structure. Synthetic peptides must be checked routinely for the proper amino acid composition, and in some cases sequencing data are helpful. The PSRC studies (see discussion of Development of Solid-Phase Peptide Synthesis Methodology) have allowed for a side-by-side comparison of a variety of analytical techniques. Efficient characterization of synthetic peptides best been obtained by a combination of RP-HPLC and MS, with sequencing by either Edman degradation sequence analysis or tandem MS ( UNIT 16.1 ) being used to identify the positions of modifications and deletions. Proper peptide characterization by multiple techniques is essential.

LITERATURE CITED

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Key References

• Atherton E, Sheppard RC. Solid Phase Peptide Synthesis: A Practical Approach. IRL Press; Oxford: 1989. [ Google Scholar ] An extensive collection of Fmoc-based synthetic methods and techniques.
• Barany G, Merrifield RB. 1979. See above. [ PubMed ] The definitive, comprehensive overview of the solid-phase method.
• Fields GB, editor. Methods In Enzymology. Vol. 289. Academic Press; Orlando, FL: 1997. Solid-phase peptide synthesis. [ Google Scholar ] A collection of SPPS techniques and applications.
• Houben-Weyl Methods of Organic Chemistry . Synthesis of Peptides and Peptidomimetics. Vol 22a–e. Thieme Chemistry; New York: 2004. [ Google Scholar ] A comprehensive description of peptide synthesis methods.
• Chan WC, White PD. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press; 2000. [ Google Scholar ] A contemporary collection of Fmoc-based synthetic methods and techniques.

Home / Introduction to Peptide Synthesis

By James Ashenhurst

• Introduction to Peptide Synthesis

Last updated: February 9th, 2023 |

Peptide bonds: Forming peptides from amino acids with the use of protecting groups

Today we’ll go deeper on how to synthesize the most important amides of all – peptides – with an important contribution from protecting group chemistry.

Table of Contents

• What Are Peptide Bonds?
• The “Proteinogenic” Amino Acids
• Synthesis of a Dipeptide Without Protecting Groups 
• Synthesis of a Dipeptide Using A Protecting Group Strategy
• Synthesis of Longer Peptides – Tripeptides and Tetrapeptides
• Bonus Topic: Solid-Phase Peptide Synthesis

(Advanced) References and Further Reading

1. what are peptide bonds.

A “peptide bond” is an amide linkage (see Amides: Properties. Synthesis, and Nomenclature )  that connects two amino acids, as in the “dipeptides” L-phenylalanyl-L-valine (below left) and L-leucyl-L-alanine (below right):

2. The “Proteinogenic” Amino Acids

Proteinogenic amino acids are the building blocks of proteins. In addition to the 20 amino acids directly encoded by the genome, two other amino acids are coded into proteins under special circumstances: selenocysteine (present in eukaryotes, including humans) and pyrrolysine (found only in methane-producing bacteria).

With the exception of (achiral) glycine, all proteinogenic amino acids are L-amino acids, where the “L-” prefix relates the stereochemistry of the amino acid relative to that of  L-glyceraldehyde  [See post: D and L  Sugars ] .

Of the chiral amino acids, all are  S , with the exception of cysteine and selenocysteine [ Note 1 ] (because sulfur and selenium have a higher priority under the CIP system. )

3. Synthesis of A Simple Dipeptide Without Protecting Groups (is not advisable!)

Let’s build a simple dipeptide between two of these amino acids. For simplicity’s sake, we’ll pick two from the “hydrophobic sidechain” group,  alanine (Ala) and leucine (Leu), since their sidechains don’t need additional protecting groups.

What do we need to do to make L-Ala-L-Leu ?

Surveying the methods previously covered to make amides, it might seem simple.

Why not take 1 equivalent each of L-alanine and L-phenylalanine, add a coupling agent like N,N -dicyclohexylcarbodiimide (DCC) and patiently wait for our product to appear?

What could possibly go wrong?

Well, this  will give us some of our desired product. But it won’t do so efficiently!

That’s because each amino acid has two reactive termini – an amine and a carboxylic acid – and they can bond together in multiple ways.

Just like the letters A and L can combine to make the words AL and LA, in addition to Ala-Leu (our desired product) we will also get Leu-Ala.

Furthermore, since we’re not adding single molecules together but molar quantities (even a millionth of a mole (a “micromole”) has 10 17 molecules in it) we also have the possibility of forming the “homo-dipeptides” AA (Ala-Ala) and LL (Leu-Leu).

And that’s just a start. No matter how you slice it you’re looking at a low yield (<25%)  of the desired material.

That’s inefficient, wasteful, and expensive! Isn’t there a better way?

4. Synthesis Of A Dipeptide Using A Protecting Group Strategy

Yes. Rather than using the native amino acids and just praying for a good yield, we can use protected versions of L-Alanine and L-Leucine.

If we protect the carboxylic acid of Leucine as an ester (e.g. a methyl ester) and protect the amine of L-Alanine as a carbamate (See: Carbamates as protecting groups ) then we set up a situation where we have a single nucleophile and a single electrophile.

This results in a high yield (>95%) of a single product!

[Note: the Boc group is a popular carbamate protecting group for amines;  “Boc” stands for t-butyloxycarbonyl]

5. Synthesis of Longer Peptides – Tripeptides and Tetrapeptides

The good news is that we don’t have to stop at the dipeptide. If we choose protecting groups that can be removed selectively (and the carbamate / ester pair qualifies) then we can then deprotect the carbamate, and add a third amino acid.

The choice of carbamate protecting group here was  t -butoxycarbonyl (Boc) which is removed with strong acid (trifluoroacetic acid, abbreviated as TFA).

Treatment with TFA removes the Boc group but leaves the methyl ester alone.

So if we treat the dipeptide with TFA, we liberate the amine nitrogen, and can react with another Boc-protected amino acid in the presence of DCC to get a tripeptide.

If we’re keen, we can even extend the same method to build a tetrapeptide, a pentapeptide… or beyond!

It’s not unreasonable to consider this method for longer peptides.

For instance, take something like  bradykinin , a 9-peptide chain that causes dilation of blood vessels leading to a rapid drop in blood pressure. (Your body releases bradykinin in response to snake bites, which is how it was originally discovered.)

It might be interesting to synthesize variants of bradykinin where some of the amino acids are swapped out for other ones. In order to do that, we’d need to be able to synthesize it.

So how effective could it be?

If each peptide coupling step has a yield of about 95%, then our overall yield for making bradykinin would be (0.95) 9 , or 63%. That’s actually pretty good! A lot of chemists would be happy to get a yield of 63% for a single reaction, let me tell you.

If the yields are high enough, one can even imagine building something crazy like insulin (51 peptide residues). That’s 7% yield for 51 steps.

Is this possible?

Yes… but it requires a clever modification that won its inventor, Bruce Merrifield, the 1984 Nobel Prize in Chemistry.

6. Bonus Topic: Solid-Phase Peptide Synthesis

What follows below is more supplemental than anything else, but given the importance of the topic, both interesting and useful.

In 1963 a chemist at Rockefeller University named Bruce Merrifield published a paper that would revolutionize how peptides were synthesized, and eventually make the synthesis of long peptides routine.

It was entitled: “ Solid-Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide “.

Here’s the key idea.

Recall that in our original scheme (above) we protected the carboxylic acid as a methyl ester, which stays the same throughout the whole peptide synthesis.

Merrifield’s idea was: what if we find a way to attach the carboxylic acid to a functional group that is itself linked to a polymer bead ? Not only would this also protect the carboxylic acid, it would drastically improve the ease of separations.

Why? Because instead of having to purify the final product by crystallization or column chromatography, you purify by filtering off the polymer beads (each 200-500 μm) and washing them to remove excess reagents.

The polymer beads themselves are pretty small. A typical size is 200 micrometers. Each bead can load about 4 nanomoles of amino acid.

[ Brandon Finlay from ChemTips shows a picture here ]

The video posted below is not mine, but it gives you an idea of the process.

At about 0:34 you can see how small the beads are.

The starting point for the Merrifield process is crosslinked polystyrene. which behaves like one big interlinked molecule. Polystyrene is then attached to a linker, which usually terminates with an NH 2 group. This itself is usually protected; in order to activate the linker, you need to remove the protecting group cap.

The polymer bead needs to swell in a solvent in order for functional groups on the solid support to undergo reactions efficiently.

The essential procedure is: swell –> add reagents –> wait –> filter –> wash, and repeat. Beads stay in the reaction vessel the whole time. There’s also usually some kind of capping step to make sure any unreacted amines don’t participate in the next reaction.

It’s possible to make peptides up to about 50 units this way. In highly automated systems one can be even more ambitious.

Merrifield started knocking off peptides in the 1960s. Bradykinin  was made in 8 days and 68% overall yield.  one example. Insulin was made two years later. The crowning achievement of this initial period was probably  ribonuclease A , which has 150 amino acid residues.

The original Merrifield process has been significantly modified and improved. Originally, removal of the linker required harsh conditions (strong acid). Today, procedures usually employ  FMOC  protecting groups instead of BOC, which allow for deprotection with mild amine base (piperidine). A galaxy of new resins, linkers, and coupling procedures have been subsequently developed. The Wikipedia article on solid-phase peptide synthesis is an OK place to start.

Related Articles

• Reactions of Diazonium Salts: Sandmeyer and Related Reactions
• Protecting Groups for Amines – Carbamates
• The Amide Functional Group: Properties, Synthesis, and Nomenclature
• Formation of Amides Using DCC (MOC Reaction Guide)
• Amine Protection and Deprotection (MOC Reaction Guide)
• Amine Practice Questions (MOC Membership)
• Isoelectric Points of Amino Acids (and How To Calculate Them)

Note 1 . Cysteine (and selenocysteine) are L, but their stereocenters are  (R) , because sulfur and selenium have higher priority within the Cahn-Ingold-Prelog system.

This is a major topic, as the synthesis of peptides is a global billion-dollar industry.

• THE SYNTHESIS OF AN OCTAPEPTIDE AMIDE WITH THE HORMONAL ACTIVITY OF OXYTOCIN Vincent du Vigneaud, Charlotte Ressler, College John M. Swan, Carleton W. Roberts, Panayotis G. Katsoyannis, and Samuel Gordon Journal of the American Chemical Society 1953, 75 (19), 4879-4880 DOI: 1021/ja01115a553
• The Synthesis of Oxytocin Vincent du Vigneaud, Charlotte Ressler, John M. Swan, Carleton W. Roberts, and Panayotis G. Katsoyannis Journal of the American Chemical Society 1954, 76 (12), 3115-3121 DOI: 1021/ja01641a004
• A Method of Synthesis of Long Peptide Chains Using a Synthesis of Oxytocin as an Example Miklos Bodanszky and Vincent du Vigneaud Journal of the American Chemical Society 1959, 81 (21), 5688-5691 DOI: 1021/ja01530a040 In the first half of the 20 th century, peptide synthesis was done using standard organic chemistry solution phase techniques. This is now known as LPPS (liquid-phase peptide synthesis). du Vigneaud received the Nobel Prize in chemistry in 1955 for his work in showing that peptide synthesis could be achieved, using the correct choice of protecting groups and synthetic strategies.In 1963, Prof. Robert Bruce Merrifield (Rockefeller U., New York) revolutionized peptide synthesis by coming up with the SPPS (Solid-Phase Peptide Synthesis) method, making the synthesis of long peptide chains much more feasible. The C-terminus is bound to a polymer resin, and the amino acids are added one at a time following the same cycle: deprotect, wash, couple the next amino acid (with a peptide-coupling reagent such as DCC), wash, deprotect the N-terminus again, and so on. Merrifield’s method came to be called the Boc/Bzl strategy due to the protecting groups employed (Boc for the nitrogen atoms, and Bzl (benzyl) for the side chains). The catch is that final cleavage of the peptide from the resin using this method requires anhydrous HF, which is not easy to handle.
• Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide R. B. Merrifield Journal of the American Chemical Society 1963, 85 (14), 2149-2154 DOI : 10.1021/ja00897a025 This is the paper that started it all – a single-author publication by Prof. Merrifield using the SPPS method to make a tetrapeptide. This remains one of the most highly-viewed and highly-cited papers in JACS , even today.
• The Synthesis of Bovine Insulin by the Solid Phase Method Marglin and R. B. Merrifield Journal of the American Chemical Society 1966, 88 (21), 5051-5052 DOI: 10.1021/ja00973a068 Insulin is a billion-dollar hormone as its dysregulation is what causes diabetes. This paper shows that insulin can be made through SPPS methods. Insulin is tricky to make as it has 2 chains (the A and B chain) connected through disulfide bonds. Interestingly, Merrifield synthesized the active hormone by combining both chains with protected thiols (protected as sulfonates), which he then reduced to the thiol, and then oxidized in air in a basic medium (pH 10). This is called undirected or air oxidation since the disulfide bonds are not being formed selectively; luckily they formed correctly in this case. Insulin today is not manufactured by the SPPS method due to these complications; instead it is made through a recombinant process.
• The Synthesis of Ribonuclease A Bernd Gutte and R. B. Merrifield Journal of Biological Chemistry 1971 , 246 (6), 1922-1941 DOI: 10.1016/S0021-9258(18)62396-8 This is the crowning achievement of SPPS – the synthesis of a 124-mer peptide (or protein, at this point), RNAse A.
• The Solid Phase Synthesis of Ribonuclease A by Robert Bruce Merrifield Nicole Kresge, Robert D. Simoni and Robert L. Hill Journal of Biological Chemistry 2006 , 281 (26), e21 DOI: 10.1016/S0021-9258(20)55702-5 A short biographical account of Prof. Merrifield’s life. This mentions that he developed the first prototype of an automated peptide synthesizer working in the basement of his house in 1965!
• Solid Phase Synthesis Nobel Lecture , December 8, 1984 by Bruce Merrifield Merrifield’s Nobel Lecture upon receiving the Nobel Prize in Chemistry in 1984. This describes his life, how he conceived of and developed the SPPS process, and all the breakthroughs it has enabled.
• 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group Louis A. Carpino and Grace Y. Han Journal of the American Chemical Society 1970, 92 (19), 5748-5749 DOI : 10.1021/ja00722a043
• 9-Fluorenylmethoxycarbonyl amino-protecting group Louis A. Carpino and Grace Y. Han The Journal of Organic Chemistry 1972 37 (22), 3404-3409 DOI : 10.1021/jo00795a005 The discovery and development of the Fmoc (9-fluorenylmethoxycarbonyl) group as a protecting group for amines has also improved the practice of peptide synthesis and SPPS.
• A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonylamino-acids Atherton, Hazel Fox, Diana Harkiss, C. J. Logan, R. C. Sheppard and B. J. Williams J. Chem. Soc. Chem. Comm. 1978 , 537-539 DOI : 10.1039/C39780000537 This is the first paper describing what is now known as the Fmoc/tBu SPPS process, which has largely supplanted the original Boc/Bzl process developed by Prof. Merrifield. The advantages with Fmoc-SPPS are multifold, but the main ones are simplicity and orthogonality. Amine deprotection is done with a base (20% piperidine in DMF is sufficient to deprotect an Fmoc-amine), and final cleavage of the peptide from the resin can be done with TFA (trifluoroacetic acid), which is much easier to handle than HF.
• Advances in Fmoc solid-phase peptide synthesis Raymond Behrendt, Peter White, and John Offer Peptide Sci. 2016, 22 , 4-27 DOI: 10.1002/psc.2836 A modern review on Fmoc-SPPS that describes how far we have come and some of the challenges that remain. For instance, aggregation of peptide chains on the resin is a major issue in Fmoc-SPPS when synthesizing especially hydrophobic peptides, and there are ways to deal with it, such as the introduction of ‘kinking’ residues, like Pro or the use of pseudoprolines, which will revert back to the desired amino acids when the peptide is cleaved with TFA. Native Chemical Ligation is also used nowadays for the synthesis of especially long peptides, like Merrifield’s RNAse A.

00 General Chemistry Review

• Lewis Structures
• Ionic and Covalent Bonding
• Chemical Kinetics
• Chemical Equilibria
• Valence Electrons of the First Row Elements
• How Concepts Build Up In Org 1 ("The Pyramid")

01 Bonding, Structure, and Resonance

• How Do We Know Methane (CH4) Is Tetrahedral?
• Hybrid Orbitals and Hybridization
• How To Determine Hybridization: A Shortcut
• Orbital Hybridization And Bond Strengths
• Sigma bonds come in six varieties: Pi bonds come in one
• A Key Skill: How to Calculate Formal Charge
• The Four Intermolecular Forces and How They Affect Boiling Points
• 3 Trends That Affect Boiling Points
• How To Use Electronegativity To Determine Electron Density (and why NOT to trust formal charge)
• Introduction to Resonance
• How To Use Curved Arrows To Interchange Resonance Forms
• Evaluating Resonance Forms (1) - The Rule of Least Charges
• How To Find The Best Resonance Structure By Applying Electronegativity
• Evaluating Resonance Structures With Negative Charges
• Evaluating Resonance Structures With Positive Charge
• Exploring Resonance: Pi-Donation
• Exploring Resonance: Pi-acceptors
• In Summary: Evaluating Resonance Structures
• Drawing Resonance Structures: 3 Common Mistakes To Avoid
• How to apply electronegativity and resonance to understand reactivity
• Bond Hybridization Practice
• Structure and Bonding Practice Quizzes
• Resonance Structures Practice

02 Acid Base Reactions

• Introduction to Acid-Base Reactions
• Acid Base Reactions In Organic Chemistry
• The Stronger The Acid, The Weaker The Conjugate Base
• Walkthrough of Acid-Base Reactions (3) - Acidity Trends
• Five Key Factors That Influence Acidity
• Acid-Base Reactions: Introducing Ka and pKa
• How to Use a pKa Table
• The pKa Table Is Your Friend
• A Handy Rule of Thumb for Acid-Base Reactions
• Acid Base Reactions Are Fast
• pKa Values Span 60 Orders Of Magnitude
• How Protonation and Deprotonation Affect Reactivity
• Acid Base Practice Problems

03 Alkanes and Nomenclature

• Meet the (Most Important) Functional Groups
• Condensed Formulas: Deciphering What the Brackets Mean
• Hidden Hydrogens, Hidden Lone Pairs, Hidden Counterions
• Don't Be Futyl, Learn The Butyls
• Primary, Secondary, Tertiary, Quaternary In Organic Chemistry
• Branching, and Its Affect On Melting and Boiling Points
• The Many, Many Ways of Drawing Butane
• Wedge And Dash Convention For Tetrahedral Carbon
• Common Mistakes in Organic Chemistry: Pentavalent Carbon
• Table of Functional Group Priorities for Nomenclature
• Summary Sheet - Alkane Nomenclature
• Organic Chemistry IUPAC Nomenclature Demystified With A Simple Puzzle Piece Approach
• Boiling Point Quizzes
• Organic Chemistry Nomenclature Quizzes

04 Conformations and Cycloalkanes

• Staggered vs Eclipsed Conformations of Ethane
• Conformational Isomers of Propane
• Newman Projection of Butane (and Gauche Conformation)
• Introduction to Cycloalkanes (1)
• Geometric Isomers In Small Rings: Cis And Trans Cycloalkanes
• Calculation of Ring Strain In Cycloalkanes
• Cycloalkanes - Ring Strain In Cyclopropane And Cyclobutane
• Cyclohexane Conformations
• Cyclohexane Chair Conformation: An Aerial Tour
• How To Draw The Cyclohexane Chair Conformation
• The Cyclohexane Chair Flip
• The Cyclohexane Chair Flip - Energy Diagram
• Substituted Cyclohexanes - Axial vs Equatorial
• Ranking The Bulkiness Of Substituents On Cyclohexanes: "A-Values"
• Cyclohexane Chair Conformation Stability: Which One Is Lower Energy?
• Fused Rings - Cis-Decalin and Trans-Decalin
• Naming Bicyclic Compounds - Fused, Bridged, and Spiro
• Bredt's Rule (And Summary of Cycloalkanes)
• Newman Projection Practice
• Cycloalkanes Practice Problems

05 A Primer On Organic Reactions

• The Most Important Question To Ask When Learning a New Reaction
• Learning New Reactions: How Do The Electrons Move?
• The Third Most Important Question to Ask When Learning A New Reaction
• 7 Factors that stabilize negative charge in organic chemistry
• 7 Factors That Stabilize Positive Charge in Organic Chemistry
• Nucleophiles and Electrophiles
• Curved Arrows (for reactions)
• Curved Arrows (2): Initial Tails and Final Heads
• Nucleophilicity vs. Basicity
• The Three Classes of Nucleophiles
• What Makes A Good Nucleophile?
• What makes a good leaving group?
• 3 Factors That Stabilize Carbocations
• Equilibrium and Energy Relationships
• What's a Transition State?
• Hammond's Postulate
• Learning Organic Chemistry Reactions: A Checklist (PDF)
• Introduction to Free Radical Substitution Reactions
• Introduction to Oxidative Cleavage Reactions

06 Free Radical Reactions

• Bond Dissociation Energies = Homolytic Cleavage
• Free Radical Reactions
• 3 Factors That Stabilize Free Radicals
• What Factors Destabilize Free Radicals?
• Bond Strengths And Radical Stability
• Free Radical Initiation: Why Is "Light" Or "Heat" Required?
• Initiation, Propagation, Termination
• Monochlorination Products Of Propane, Pentane, And Other Alkanes
• Selectivity In Free Radical Reactions
• Selectivity in Free Radical Reactions: Bromination vs. Chlorination
• Halogenation At Tiffany's
• Allylic Bromination
• Bonus Topic: Allylic Rearrangements
• In Summary: Free Radicals
• Synthesis (2) - Reactions of Alkanes
• Free Radicals Practice Quizzes

07 Stereochemistry and Chirality

• Types of Isomers: Constitutional Isomers, Stereoisomers, Enantiomers, and Diastereomers
• How To Draw The Enantiomer Of A Chiral Molecule
• How To Draw A Bond Rotation
• Introduction to Assigning (R) and (S): The Cahn-Ingold-Prelog Rules
• Assigning Cahn-Ingold-Prelog (CIP) Priorities (2) - The Method of Dots
• Enantiomers vs Diastereomers vs The Same? Two Methods For Solving Problems
• Assigning R/S To Newman Projections (And Converting Newman To Line Diagrams)
• How To Determine R and S Configurations On A Fischer Projection
• The Meso Trap
• Optical Rotation, Optical Activity, and Specific Rotation
• Optical Purity and Enantiomeric Excess
• What's a Racemic Mixture?
• Chiral Allenes And Chiral Axes
• Stereochemistry Practice Problems and Quizzes

08 Substitution Reactions

• Introduction to Nucleophilic Substitution Reactions
• Walkthrough of Substitution Reactions (1) - Introduction
• Two Types of Nucleophilic Substitution Reactions
• The SN2 Mechanism
• Why the SN2 Reaction Is Powerful
• The SN1 Mechanism
• The Conjugate Acid Is A Better Leaving Group
• Comparing the SN1 and SN2 Reactions
• Polar Protic? Polar Aprotic? Nonpolar? All About Solvents
• Steric Hindrance is Like a Fat Goalie
• Common Blind Spot: Intramolecular Reactions
• The Conjugate Base is Always a Stronger Nucleophile
• Substitution Practice - SN1
• Substitution Practice - SN2

09 Elimination Reactions

• Elimination Reactions (1): Introduction And The Key Pattern
• Elimination Reactions (2): The Zaitsev Rule
• Elimination Reactions Are Favored By Heat
• Two Elimination Reaction Patterns
• The E1 Reaction
• The E2 Mechanism
• E1 vs E2: Comparing the E1 and E2 Reactions
• Antiperiplanar Relationships: The E2 Reaction and Cyclohexane Rings
• Bulky Bases in Elimination Reactions
• Comparing the E1 vs SN1 Reactions
• Elimination (E1) Reactions With Rearrangements
• E1cB - Elimination (Unimolecular) Conjugate Base
• Elimination (E1) Practice Problems And Solutions
• Elimination (E2) Practice Problems and Solutions

10 Rearrangements

• Introduction to Rearrangement Reactions
• Rearrangement Reactions (1) - Hydride Shifts
• Carbocation Rearrangement Reactions (2) - Alkyl Shifts
• Pinacol Rearrangement
• The SN1, E1, and Alkene Addition Reactions All Pass Through A Carbocation Intermediate

11 SN1/SN2/E1/E2 Decision

• Identifying Where Substitution and Elimination Reactions Happen
• Deciding SN1/SN2/E1/E2 (1) - The Substrate
• Deciding SN1/SN2/E1/E2 (2) - The Nucleophile/Base
• SN1 vs E1 and SN2 vs E2 : The Temperature
• Deciding SN1/SN2/E1/E2 - The Solvent
• Wrapup: The Key Factors For Determining SN1/SN2/E1/E2
• Alkyl Halide Reaction Map And Summary
• SN1 SN2 E1 E2 Practice Problems

12 Alkene Reactions

• E and Z Notation For Alkenes (+ Cis/Trans)
• Alkene Stability
• Alkene Addition Reactions: "Regioselectivity" and "Stereoselectivity" (Syn/Anti)
• Stereoselective and Stereospecific Reactions
• Hydrohalogenation of Alkenes and Markovnikov's Rule
• Hydration of Alkenes With Aqueous Acid
• Rearrangements in Alkene Addition Reactions
• Halogenation of Alkenes and Halohydrin Formation
• Oxymercuration Demercuration of Alkenes
• Hydroboration Oxidation of Alkenes
• m-CPBA (meta-chloroperoxybenzoic acid)
• OsO4 (Osmium Tetroxide) for Dihydroxylation of Alkenes
• Palladium on Carbon (Pd/C) for Catalytic Hydrogenation of Alkenes
• Cyclopropanation of Alkenes
• A Fourth Alkene Addition Pattern - Free Radical Addition
• Alkene Reactions: Ozonolysis
• Summary: Three Key Families Of Alkene Reaction Mechanisms
• Synthesis (4) - Alkene Reaction Map, Including Alkyl Halide Reactions
• Alkene Reactions Practice Problems

13 Alkyne Reactions

• Acetylides from Alkynes, And Substitution Reactions of Acetylides
• Partial Reduction of Alkynes With Lindlar's Catalyst
• Partial Reduction of Alkynes With Na/NH3 To Obtain Trans Alkenes
• Alkyne Hydroboration With "R2BH"
• Hydration and Oxymercuration of Alkynes
• Hydrohalogenation of Alkynes
• Alkyne Halogenation: Bromination, Chlorination, and Iodination of Alkynes
• Alkyne Reactions - The "Concerted" Pathway
• Alkenes To Alkynes Via Halogenation And Elimination Reactions
• Alkynes Are A Blank Canvas
• Synthesis (5) - Reactions of Alkynes
• Alkyne Reactions Practice Problems With Answers

14 Alcohols, Epoxides and Ethers

• Alcohols - Nomenclature and Properties
• Alcohols Can Act As Acids Or Bases (And Why It Matters)
• Alcohols - Acidity and Basicity
• The Williamson Ether Synthesis
• Ethers From Alkenes, Tertiary Alkyl Halides and Alkoxymercuration
• Alcohols To Ethers via Acid Catalysis
• Cleavage Of Ethers With Acid
• Epoxides - The Outlier Of The Ether Family
• Opening of Epoxides With Acid
• Epoxide Ring Opening With Base
• Making Alkyl Halides From Alcohols
• Tosylates And Mesylates
• PBr3 and SOCl2
• Elimination Reactions of Alcohols
• Elimination of Alcohols To Alkenes With POCl3
• Alcohol Oxidation: "Strong" and "Weak" Oxidants
• Demystifying The Mechanisms of Alcohol Oxidations
• Protecting Groups For Alcohols
• Thiols And Thioethers
• Calculating the oxidation state of a carbon
• Oxidation and Reduction in Organic Chemistry
• Oxidation Ladders
• SOCl2 Mechanism For Alcohols To Alkyl Halides: SN2 versus SNi
• Alcohol Reactions Roadmap (PDF)
• Alcohol Reaction Practice Problems
• Epoxide Reaction Quizzes
• Oxidation and Reduction Practice Quizzes

15 Organometallics

• What's An Organometallic?
• Formation of Grignard and Organolithium Reagents
• Organometallics Are Strong Bases
• Reactions of Grignard Reagents
• Protecting Groups In Grignard Reactions
• Synthesis Problems Involving Grignard Reagents
• Grignard Reactions And Synthesis (2)
• Organocuprates (Gilman Reagents): How They're Made
• Gilman Reagents (Organocuprates): What They're Used For
• The Heck, Suzuki, and Olefin Metathesis Reactions (And Why They Don't Belong In Most Introductory Organic Chemistry Courses)
• Reaction Map: Reactions of Organometallics
• Grignard Practice Problems

16 Spectroscopy

• Degrees of Unsaturation (or IHD, Index of Hydrogen Deficiency)
• Conjugation And Color (+ How Bleach Works)
• Introduction To UV-Vis Spectroscopy
• UV-Vis Spectroscopy: Absorbance of Carbonyls
• UV-Vis Spectroscopy: Practice Questions
• Bond Vibrations, Infrared Spectroscopy, and the "Ball and Spring" Model
• Infrared Spectroscopy: A Quick Primer On Interpreting Spectra
• IR Spectroscopy: 4 Practice Problems
• 1H NMR: How Many Signals?
• Homotopic, Enantiotopic, Diastereotopic
• Diastereotopic Protons in 1H NMR Spectroscopy: Examples
• C13 NMR - How Many Signals
• Liquid Gold: Pheromones In Doe Urine
• Natural Product Isolation (1) - Extraction
• Natural Product Isolation (2) - Purification Techniques, An Overview
• Structure Determination Case Study: Deer Tarsal Gland Pheromone

17 Dienes and MO Theory

• What To Expect In Organic Chemistry 2
• Are these molecules conjugated?
• Conjugation And Resonance In Organic Chemistry
• Bonding And Antibonding Pi Orbitals
• Molecular Orbitals of The Allyl Cation, Allyl Radical, and Allyl Anion
• Pi Molecular Orbitals of Butadiene
• Reactions of Dienes: 1,2 and 1,4 Addition
• Thermodynamic and Kinetic Products
• More On 1,2 and 1,4 Additions To Dienes
• s-cis and s-trans
• The Diels-Alder Reaction
• Cyclic Dienes and Dienophiles in the Diels-Alder Reaction
• Stereochemistry of the Diels-Alder Reaction
• Exo vs Endo Products In The Diels Alder: How To Tell Them Apart
• HOMO and LUMO In the Diels Alder Reaction
• Why Are Endo vs Exo Products Favored in the Diels-Alder Reaction?
• Diels-Alder Reaction: Kinetic and Thermodynamic Control
• The Retro Diels-Alder Reaction
• The Intramolecular Diels Alder Reaction
• Regiochemistry In The Diels-Alder Reaction
• The Cope and Claisen Rearrangements
• Electrocyclic Reactions
• Electrocyclic Ring Opening And Closure (2) - Six (or Eight) Pi Electrons
• Diels Alder Practice Problems
• Molecular Orbital Theory Practice

18 Aromaticity

• Introduction To Aromaticity
• Rules For Aromaticity
• Huckel's Rule: What Does 4n+2 Mean?
• Aromatic, Non-Aromatic, or Antiaromatic? Some Practice Problems
• Antiaromatic Compounds and Antiaromaticity
• The Pi Molecular Orbitals of Benzene
• The Pi Molecular Orbitals of Cyclobutadiene
• Frost Circles
• Aromaticity Practice Quizzes

19 Reactions of Aromatic Molecules

• Electrophilic Aromatic Substitution: Introduction
• Activating and Deactivating Groups In Electrophilic Aromatic Substitution
• Electrophilic Aromatic Substitution - The Mechanism
• Ortho-, Para- and Meta- Directors in Electrophilic Aromatic Substitution
• Understanding Ortho, Para, and Meta Directors
• Why are halogens ortho- para- directors?
• Disubstituted Benzenes: The Strongest Electron-Donor "Wins"
• Electrophilic Aromatic Substitutions (1) - Halogenation of Benzene
• Electrophilic Aromatic Substitutions (2) - Nitration and Sulfonation
• EAS Reactions (3) - Friedel-Crafts Acylation and Friedel-Crafts Alkylation
• Intramolecular Friedel-Crafts Reactions
• Nucleophilic Aromatic Substitution (NAS)
• Nucleophilic Aromatic Substitution (2) - The Benzyne Mechanism
• Reactions on the "Benzylic" Carbon: Bromination And Oxidation
• The Wolff-Kishner, Clemmensen, And Other Carbonyl Reductions
• More Reactions on the Aromatic Sidechain: Reduction of Nitro Groups and the Baeyer Villiger
• Aromatic Synthesis (1) - "Order Of Operations"
• Synthesis of Benzene Derivatives (2) - Polarity Reversal
• Aromatic Synthesis (3) - Sulfonyl Blocking Groups
• Birch Reduction
• Synthesis (7): Reaction Map of Benzene and Related Aromatic Compounds
• Aromatic Reactions and Synthesis Practice
• Electrophilic Aromatic Substitution Practice Problems

20 Aldehydes and Ketones

• What's The Alpha Carbon In Carbonyl Compounds?
• Nucleophilic Addition To Carbonyls
• Aldehydes and Ketones: 14 Reactions With The Same Mechanism
• Sodium Borohydride (NaBH4) Reduction of Aldehydes and Ketones
• Grignard Reagents For Addition To Aldehydes and Ketones
• Wittig Reaction
• Hydrates, Hemiacetals, and Acetals
• Imines - Properties, Formation, Reactions, and Mechanisms
• All About Enamines
• Breaking Down Carbonyl Reaction Mechanisms: Reactions of Anionic Nucleophiles (Part 2)
• Aldehydes Ketones Reaction Practice

21 Carboxylic Acid Derivatives

• Nucleophilic Acyl Substitution (With Negatively Charged Nucleophiles)
• Addition-Elimination Mechanisms With Neutral Nucleophiles (Including Acid Catalysis)
• Basic Hydrolysis of Esters - Saponification
• Transesterification
• Proton Transfer
• Fischer Esterification - Carboxylic Acid to Ester Under Acidic Conditions
• Lithium Aluminum Hydride (LiAlH4) For Reduction of Carboxylic Acid Derivatives
• LiAlH[Ot-Bu]3 For The Reduction of Acid Halides To Aldehydes
• Di-isobutyl Aluminum Hydride (DIBAL) For The Partial Reduction of Esters and Nitriles
• Amide Hydrolysis
• Thionyl Chloride (SOCl2)
• Diazomethane (CH2N2)
• Carbonyl Chemistry: Learn Six Mechanisms For the Price Of One
• Making Music With Mechanisms (PADPED)
• Carboxylic Acid Derivatives Practice Questions

22 Enols and Enolates

• Keto-Enol Tautomerism
• Enolates - Formation, Stability, and Simple Reactions
• Kinetic Versus Thermodynamic Enolates
• Aldol Addition and Condensation Reactions
• Reactions of Enols - Acid-Catalyzed Aldol, Halogenation, and Mannich Reactions
• Claisen Condensation and Dieckmann Condensation
• Decarboxylation
• The Malonic Ester and Acetoacetic Ester Synthesis
• The Michael Addition Reaction and Conjugate Addition
• The Robinson Annulation
• Haloform Reaction
• The Hell–Volhard–Zelinsky Reaction
• Enols and Enolates Practice Quizzes
• Basicity of Amines And pKaH
• 5 Key Basicity Trends of Amines
• The Mesomeric Effect And Aromatic Amines
• Nucleophilicity of Amines
• Alkylation of Amines (Sucks!)
• Reductive Amination
• The Gabriel Synthesis
• Some Reactions of Azides
• The Hofmann Elimination
• The Hofmann and Curtius Rearrangements
• The Cope Elimination
• Protecting Groups for Amines - Carbamates
• The Strecker Synthesis of Amino Acids
• Amine Practice Questions

24 Carbohydrates

• D and L Notation For Sugars
• Pyranoses and Furanoses: Ring-Chain Tautomerism In Sugars
• What is Mutarotation?
• Reducing Sugars
• The Big Damn Post Of Carbohydrate-Related Chemistry Definitions
• The Haworth Projection
• Converting a Fischer Projection To A Haworth (And Vice Versa)
• Reactions of Sugars: Glycosylation and Protection
• The Ruff Degradation and Kiliani-Fischer Synthesis
• Carbohydrates Practice
• Amino Acid Quizzes

25 Fun and Miscellaneous

• A Gallery of Some Interesting Molecules From Nature
• Screw Organic Chemistry, I'm Just Going To Write About Cats
• On Cats, Part 1: Conformations and Configurations
• On Cats, Part 2: Cat Line Diagrams
• On Cats, Part 4: Enantiocats
• On Cats, Part 6: Stereocenters
• Organic Chemistry Is Shit
• The Organic Chemistry Behind "The Pill"
• Maybe they should call them, "Formal Wins" ?
• Why Do Organic Chemists Use Kilocalories?
• The Principle of Least Effort
• Organic Chemistry GIFS - Resonance Forms
• Reproducibility In Organic Chemistry
• What Holds The Nucleus Together?
• How Reactions Are Like Music
• Organic Chemistry and the New MCAT

26 Organic Chemistry Tips and Tricks

• Common Mistakes: Formal Charges Can Mislead
• Partial Charges Give Clues About Electron Flow
• Draw The Ugly Version First
• Organic Chemistry Study Tips: Learn the Trends
• The 8 Types of Arrows In Organic Chemistry, Explained
• Top 10 Skills To Master Before An Organic Chemistry 2 Final
• Common Mistakes with Carbonyls: Carboxylic Acids... Are Acids!
• Planning Organic Synthesis With "Reaction Maps"
• Alkene Addition Pattern #1: The "Carbocation Pathway"
• Alkene Addition Pattern #2: The "Three-Membered Ring" Pathway
• Alkene Addition Pattern #3: The "Concerted" Pathway
• Number Your Carbons!
• The 4 Major Classes of Reactions in Org 1
• How (and why) electrons flow
• Grossman's Rule
• Three Exam Tips
• A 3-Step Method For Thinking Through Synthesis Problems
• Putting It Together
• Putting Diels-Alder Products in Perspective
• The Ups and Downs of Cyclohexanes
• The Most Annoying Exceptions in Org 1 (Part 1)
• The Most Annoying Exceptions in Org 1 (Part 2)
• The Marriage May Be Bad, But the Divorce Still Costs Money
• 9 Nomenclature Conventions To Know
• Nucleophile attacks Electrophile

27 Case Studies of Successful O-Chem Students

• Success Stories: How Corina Got The The "Hard" Professor - And Got An A+ Anyway
• How Helena Aced Organic Chemistry
• From a "Drop" To B+ in Org 2 – How A Hard Working Student Turned It Around
• How Serge Aced Organic Chemistry
• Success Stories: How Zach Aced Organic Chemistry 1
• Success Stories: How Kari Went From C– to B+
• How Esther Bounced Back From a "C" To Get A's In Organic Chemistry 1 And 2
• How Tyrell Got The Highest Grade In Her Organic Chemistry Course
• This Is Why Students Use Flashcards
• Success Stories: How Stu Aced Organic Chemistry
• How John Pulled Up His Organic Chemistry Exam Grades
• Success Stories: How Nathan Aced Organic Chemistry (Without It Taking Over His Life)
• How Chris Aced Org 1 and Org 2
• Interview: How Jay Got an A+ In Organic Chemistry
• How to Do Well in Organic Chemistry: One Student's Advice
• "America's Top TA" Shares His Secrets For Teaching O-Chem
• "Organic Chemistry Is Like..." - A Few Metaphors
• How To Do Well In Organic Chemistry: Advice From A Tutor
• Guest post: "I went from being afraid of tests to actually looking forward to them".

Comment section

6 thoughts on “ introduction to peptide synthesis ”.

Thanks for very good explanation and excellent step-by-step video!!

These tips are awesome, I was looking for something like that. Thanks!

OK. Do you guys actually do peptide synthesis or do you just drop ship?

We, or least, in my school do cover this topic extensively. We need to learn fischer esterfication of them, acylation, protecting group although it was different than the protecting group you used It had two benzene rings and a five member ring in the middle.

Good to know! Sounds like you’re talking about FMOC. https://en.wikipedia.org/wiki/Fluorenylmethyloxycarbonyl_protecting_group

I should update to include that.

Thank you very much!

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Peptide Synthesis

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Page contents

• Introduction peptide synthesis
• Applications for synthetic peptides

Process of synthesizing peptides

• Common protecting scheme-specific solvents
• Peptide synthesis strategies
• Peptide purification

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Introduction to peptide synthesis

Peptide synthesis is characterized as the formation of a peptide bond between two amino acids. While there is no definitive definition of a peptide, it usually refers to flexible (little secondary structure) chains of up to 30-50 amino acids.

The ability to form peptide bonds to link amino acids together is over 100 years old, although the first peptides to be synthesized, including oxytocin and insulin, did not occur for another 50-60 years, demonstrating the difficult task of chemically synthesizing chains of amino acids (1). In the last 50 years, advances in protein synthesis chemistry and methods have developed to the point where peptide synthesis today is a common approach in even high-throughput biological research and product and drug development (2).

The benefit of peptide synthesis strategies today is that besides having the ability to make peptides that are found in biological specimens, creativity and imagination can be tapped to generate unique peptides to optimize a desired biological response or other result. This page highlights the important aspects of peptide synthesis, the most common methods of synthesis and purification and the strengths and limitations of the respective strategies.

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Applications of synthetic peptides.

The invention of peptide synthesis in the fifties and sixties spurred the development of different application areas in which synthetic peptides are now used, including the development of epitope-specific antibodies against pathogenic proteins, the study of protein functions and the identification and characterization of proteins. Furthermore, synthetic peptides are used to study enzyme-substrate interactions within important enzyme classes such as kinases and proteases, which play a crucial role in cell signaling.

In cell biology, receptor binding or the substrate recognition specificity of newly discovered enzymes can often be studied using sets of homologous synthetic peptides. Synthetic peptides can resemble naturally occurring peptides and act as drugs against cancer and other major diseases. Finally, synthetic peptides are used as standards and reagents in mass spectrometry (MS)-based applications. Synthetic peptides play a central role in MS-based discovery, characterization and quantitation of proteins, especially those that serve as early biomarkers for diseases.

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Peptide synthesis most often occurs by coupling the carboxyl group of the incoming amino acid to the N-terminus of the growing peptide chain. This C-to-N synthesis is opposite from protein biosynthesis, during which the N-terminus of the incoming amino acid is linked to the C-terminus of the protein chain (N-to-C). Due to the complex nature of in vitro protein synthesis, the addition of amino acids to the growing peptide chain occurs in a precise, step-wise and cyclic manner. And while the common methods of peptide synthesis have some critical differences, they all follow the same step-wise method to add amino acids one-at-a-time to the growing peptide chain.

Peptide deprotection

Because amino acids have multiple reactive groups, peptide synthesis must be carefully performed to avoid side reactions that can reduce the length and cause branching of the peptide chain. To facilitate peptide formation with minimal side reactions, chemical groups have been developed that bind to the amino acid reactive groups and block, or protect, the functional group from nonspecific reaction.

Purified, individual amino acids used to synthesize peptides are reacted with these protecting groups prior to synthesis, and then specific protecting groups are removed from the newly added amino acid (a step called deprotection) just after coupling to allow the next incoming amino acid to bind to the growing peptide chain in the proper orientation. Once peptide synthesis is completed, all remaining protecting groups are removed from the nascent peptides. Three types of protecting groups are generally used, depending on the method of peptide synthesis, and are described below.

The amino acid N-termini are protected by groups that are termed "temporary" protecting groups, because they are relatively easily removed to allow peptide bond formation. Two common  N-terminal protecting groups  are tert-butoxycarbonyl ( Boc ) and 9-fluorenylmethoxycarbonyl ( Fmoc ), and each group has distinct characteristics that determine their use. Boc requires a moderately strong acid such as trifluoracetic acid (TFA) to be removed from the newly added amino acid, while Fmoc is a base-labile protecting group that is removed with a mild base such as piperidine.

Boc chemistry was first described in the 1950s and requires acidic conditions for deprotection, while Fmoc, which was not reported for another twenty years, is cleaved under mild, basic conditions (3,4,5,6). Because of the mild deprotection conditions, Fmoc chemistry is more commonly used in commercial settings because of the higher quality and greater yield, while Boc is preferred for complex peptide synthesis or when non-natural peptides or analogs that are base-sensitive are required.

The use of a  C-terminal protecting group  depends on the type of peptide synthesis used; while liquid-phase peptide synthesis requires protection of the C-terminus of the first amino acid (C-terminal amino acid), solid-phase peptide synthesis does not, because a solid support (resin) acts as the protecting group for the only C-terminal amino acid that requires protection (see Protein Synthesis Strategies).

Amino acid side chains represent a broad range of functional groups and are therefore a site of considerable side chain reactivity during peptide synthesis. Because of this, many different protecting groups are required, although they are usually based on the benzyl (Bzl) or tert-butyl (tBu) group. The specific protecting groups used during the synthesis of a given peptide vary depending on the peptide sequence and the type of N-terminal protection used (see next paragraph).  Side chain protecting groups  are known as permanent protecting groups, because they can withstand the multiple cycles of chemical treatment during the synthesis phase and are only removed during treatment with strong acids after synthesis is complete.

Because multiple protecting groups are normally used in peptide synthesis, it is evident that these groups must be compatible to allow deprotection of distinct protecting groups while not affecting other protecting groups.  Protecting schemes  are therefore established to match protecting groups so that deprotection of one protecting group does not affect the binding of the other groups. Because N-terminal deprotection occurs continuously during peptide synthesis, protecting schemes have been established in which the different types of side chain protecting groups (Bzl or tBu) are matched to either Boc or Fmoc, respectively, for optimized deprotection. These protecting schemes also incorporate each of the steps of synthesis and cleavage, as described in the table and in later sections of this page.

Common protecting scheme-specific solvents . 

The act of removing protecting groups, especially under acidic conditions, results in the production of cationic species that can alkylate the functional groups on the peptide chain. Therefore,  scavengers  such as water, anisol or thiol derivatives can be added in excess during the deprotection step to react with any of these free reactive species.

• Amino Acid Physical Properties

Amino acid coupling

Synthetic peptide coupling requires the activation of the C-terminal carboxylic acid on the incoming amino acid using carbodiimides such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC). These coupling reagents react with the carboxyl group to form a highly reactive O-acylisourea intermediate that is quickly displaced by nucleophilic attack from the deprotected primary amino group on the N-terminus of the growing peptide chain to form the nascent peptide bond.

Carbodiimides form such a reactive intermediate that racemization of the amino acid can occur. Therefore, reagents that react with the O-acylisourea intermediate are often added, including 1-hydroxybenzotriazole (HOBt), which forms a less-reactive intermediate that reduces the risk of racemization. Additionally, side reactions caused by carbodiimides have led to the examination of other coupling agents, including benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), which both require activating bases to mediate amino acid coupling.

Peptide cleavage

After successive cycles of amino acid deprotection and coupling, all remaining protecting groups must be removed from the nascent peptide. These groups are cleaved by acidolysis, and the chemical used for cleavage depends on the protection scheme used; strong acids such as hydrogen fluoride (HF), hydrogen bromide (HBr) or trifluoromethane sulfonic acid (TFMSA) are used to cleave Boc and Bzl groups, while a relatively milder acid such as TFA is used to cleave Fmoc and tBut groups. When properly executed, cleavage results in the removal of the N-terminal protecting group of the last amino acid added, the C-terminal protecting group (either chemical or resin) from the first amino acid and any side-chain protecting groups. As with deprotection, scavengers are also included during this step to react with free protecting groups. Because of the importance of cleavage in proper peptide synthesis, this step should be optimized to avoid acid-catalyzed side reactions.

Diagram of peptide cleavage after synthesis. The remaining N-terminal protecting groups, all side-chain protecting groups and the C-terminal protecting group or solid support are removed by strong acid treatment after peptide synthesis is completed.

Liquid-phase peptide synthesis is the classical method that scientists used when first discovering how to generate peptides in vitro and it is still commonly used for large-scale synthesis. This method is slow and labor-intensive, though, because the product has to be manually removed from the reaction solution after each step. Additionally, this approach requires another chemical group to protect the C-terminus of the first amino acid. A benefit of liquid-phase synthesis, though, is that because the product is purified after each step, side reactions are easily detected. Additionally, convergent synthesis can be performed, in which separate peptides are synthesized and then coupled together to create larger peptides.

By far, though, solid-phase peptide synthesis is the most common method of peptide synthesis today. Instead of C-terminal protection with a chemical group, the C-terminus of the first amino acid is coupled to an activated solid support, such as polystyrene or polyacrylamide. This type of approach has a two-fold function: the resin acts as the C-terminal protecting group and provides a rapid method to separate the growing peptide product from the different reaction mixtures during synthesis. As with many different biological manufacturing processes, peptide synthesizers have been developed for automation and high-throughput peptide production.

Although peptide synthesis strategies have been optimized and can be mass-produced, the process to generate peptides is by no means perfect. Events such as incomplete deprotection or reaction with free protecting groups can cause truncated or deletion sequences, isomers or other side products. These events can occur at any step during peptide synthesis, and therefore the longer the peptide sequence, the greater probability that something will negatively affect the synthesis of the target peptide. Thus, peptide yield is inversely correlated with peptide length.

Purification strategies are usually based on a combination of separation methods that exploit the physiochemical characteristics of peptides, including size, charge and hydrophobicity. Purification techniques include:

• Size-exclusion chromatography
• Ion exchange chromatography (IEC)
• Partition chromatography
• High-performance liquid chromatography (HPLC)

Reverse-phase chromatography (RPC) is the most versatile and most widely used method of peptide purification. With traditional methods of HPLC, the stationary phase captures polar, hydrophilic molecules that are then differentially eluted by increasing the concentration of polar solvents in the mobile phase. In RPC, as the name implies, hydrophobic molecules from aqueous solutions are instead captured by the stationary phase using hydrophobic C4, C8 or C18 n-alkyl hydrocarbon ligands, and their retention time is a function of the hydrophobicity of the molecule and that of the mobile phase.

For peptide purification, RPC separates the target peptides from impurities from the synthesis steps, such as isomers, deletion sequences, peptide products from side reactions with free coupling and protecting groups or peptides that have undergone side-chain reactions.

Peptide purity is measured as a percentage of the target peptide to impurities that absorb at the peptide bond absorption wavelength (210-220nm), and varying levels of purity are commercially available based on the application in which the peptides will be used:

• >95% – Quantitative studies such as NMR, receptor-ligand binding studies, ELISA and RIA, monoclonal antibody production, in vivo studies
• >80% – High-throughput screening, non-quantitative blocking in immunohistochemical (IHC) and Western blot analyses, non-quantitative enzyme-substrate studies, antibody affinity purification and plate coating for cell attachment
• >70% – ELISA standards, ELISPOT assays and polyclonal antibody production
• Protein Isolation and Purification Information
• Overview of ELISA
• Antibody Production
• Overview of Immunohistochemistry (IHC)
• Overview of Western Blotting
• Enzyme-linked Immunosorbent Assays (ELISA)
• Immunohistochemistry
• Western Blotting
• Lloyd-Williams P. et al. (1997) Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press. 278
• Merrifield R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 85, 2149-54.
• Carpino L. A. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1, 1-disubstituted-2-arenesulfonhydrazides1-4. Journal of the American Chemical Society. 79, 4427-31.
• McKay F. C. and Albertson N. F. (1957) New amine-masking groups for peptide synthesis. Journal of the American Chemical Society. 79, 4686-90.
• Anderson G. W. and McGregor A. C. (1957) T-butyloxycarbonylamino acids and their use in peptide synthesis. Journal of the American Chemical Society. 79, 6180-3.
• Carpino L. A. and Han G. Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry. 37, 3404-9.

For Research Use Only. Not for use in diagnostic procedures.

26.7 Peptide Synthesis

26.7 • Peptide Synthesis

Once the structure of a peptide is known, its synthesis can be undertaken—perhaps to obtain a larger amount for biological evaluation. A simple amide might be formed by treating an amine and a carboxylic acid with a carbodiimide (either DCC or EDC; Section 21.3 ), but peptide synthesis is a more difficult problem because many different amide bonds must be formed in a specific order, rather than at random.

The solution to the specificity problem is protection ( Section 17.8 ). If we want to couple alanine with leucine to synthesize Ala-Leu, for instance, we could protect the –NH 2 group of alanine and the –CO 2 H group of leucine to shield them from reacting, then form the desired Ala-Leu amide bond by reaction with EDC or DCC, and then remove the protecting groups.

A number of different amino- and carboxyl-protecting groups have been devised, but only a few are used in peptide synthesis. Carboxyl groups are often protected simply by converting them into methyl or benzyl esters. Both groups are easily introduced by standard methods of ester formation ( Section 21.6 ) and are easily removed by mild hydrolysis with aqueous NaOH. Benzyl esters can also be cleaved by catalytic hydrogenolysis of the weak benzylic C–O bond ( RCO 2 –CH 2 Ph + H 2 → RCO 2 H + PhCH 3 RCO 2 –CH 2 Ph + H 2 → RCO 2 H + PhCH 3 ).

Amino groups are sometimes protected as their tert -butyloxycarbonyl amide (Boc) or, more commonly, as their fluorenylmethyloxycarbonyl amide (Fmoc). The Boc protecting group is introduced by reaction of the amino acid with di- tert -butyl dicarbonate in a nucleophilic acyl substitution reaction and is removed by brief treatment with a strong acid such as trifluoroacetic acid, CF 3 CO 2 H. The Fmoc protecting group is introduced by reaction with fluorenylmethyloxycarbonyl chloride and is removed by treatment with a 20% solution of the amine piperidine in dimethylformamide as solvent.

Thus, five steps are needed to synthesize a dipeptide such as Ala-Leu:

These steps can be repeated to add one amino acid at a time to the growing chain or to link two peptide chains together. Many remarkable achievements in peptide synthesis have been reported, including a complete synthesis of human insulin. Insulin is composed of two chains totaling 51 amino acids linked by two disulfide bridges. The three-dimensional structure of insulin, shown previously, was determined by Dorothy Crowfoot Hodgkin, a British chemist who received the 1964 Nobel Prize in Chemistry for her work on this and other complex biological molecules.

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• Authors: John McMurry, Professor Emeritus
• Publisher/website: OpenStax
• Book title: Organic Chemistry
• Publication date: Sep 20, 2023
• Location: Houston, Texas
• Book URL: https://openstax.org/books/organic-chemistry/pages/1-why-this-chapter
• Section URL: https://openstax.org/books/organic-chemistry/pages/26-7-peptide-synthesis

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