Muramyl dipeptide and its derivatives: peptide adjuvant in immunological disorders and cancer therapy

Abstract

Muramyl dipeptide (MDP) is a synthetic immunoreactive peptide consisting of N-acetyl muramic acid attached to a short amino acid chain of L-Ala-D-isoGln. It was first identified in bacterial cell wall peptidoglycan as an active component in Freund’s complete adjuvant. In the cell, MDP is detected by NOD2, a cytoplasmic receptor belonging to the human innate immune system. NOD2 mutations are frequently observed in patients with Crohn’s disease, an autoimmune disorder, suggesting the significance of the MDP-NOD2 pathway in activating immunity. For this reason, structural modifications of MDP and its derivatives have been extensively studied in an attempt to increase adjuvant activity and boost the immune response effectively for clinical use in the treatment of cancer and other diseases. This review summarizes the synthetic chemistry of MDP and its derivatives and discusses their pharmacological action and stereoselective synthesis.

1. Introduction

Peptidoglycan is found in the bacterial cell wall as a thin layer in Gram-negative and as a thick layer in Gram-positive bacteria. The presence of peptidoglycan serves not only to preserve cell integrity and to maintain a defined cell shape but also as an important scaffold for anchoring other components such as lipoproteins ¹. Peptidoglycan consists of an N-acetylyglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide chain and intercalating amino acid chains linked from the lactyl group of one N-acetylmuramic acid to the other. This chain is typically composed of four to five amino acids starting with L-Ala and D-Glu as the first and the second amino acids, respectively. L-Lys or DAP (diaminopimeric acid) often follows as the third amino acid (Fig. 1).

Fig. 1.

MDP is a component of bacterial cell wall peptidoglycan

Smaller products of peptidoglycan containing MurNAc are called muropeptides. The minimum component that remains biologically potent is muramyl dipeptide (MDP), which consists of MurNAc and two amino acids, D-Ala and D-isoGln (or D-Glu). While MDP is recognized by the NOD2 protein immune receptor, muropeptides containing DAP activate the related protein NOD1 ^{2, 3}. Synthetic immunoactive peptides that activate NOD1 include FK-156 (D-lactoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(L)-glycine), which will be described later.

2. Discovery of MDP

In 1974, MDP was discovered to be the minimal structure required for the efficacy of Freund’s Complete Adjuvant (FCA), one of the most potent and widely used adjuvants in animal experimental models to date ². FCA was developed in 1937 by Freund and colleagues ³. Composed of heat-killed mycobacterial components in an oil emulsion, FCA can strongly elicit both humoral and cellular immune responses. Unfortunately, its strong toxicity hampers the possibility of its use in a clinical setting. A search for smaller yet biologically active components in FCA resulted in the discovery of a tripeptide-monosaccharide by Lederer’s laboratory at the Université Paris-Sud ². A series of similar peptide-monosaccharides were synthesized and tested in rabbits for adjuvant activity through their ability to elicit immunoglobulin production ^{4, 5}. These peptides included MDP as well as DAP (diaminopimeric acid)-containing peptides, which we know today is a ligand for NOD1 ^{6, 7}. MDP was the smallest compound found to elicit adjuvant activity and could thus replace FCA for its ability to induce both humoral and cellular activity. However, it did not induce immunoglobulin production as it is a pure adjuvant lacking the antigens contained in the FCA complex ^{2, 5, 8}.

3. Medical and research applications

3.1. Biological activity

3.1.1. Adjuvant activity of MDP

An adjuvant is an agent that enhances the stimulatory response elicited by compounds having few if any direct effects on their own. MDP and other muropeptides are effective adjuvants and may be used for boosting the potency of drugs and vaccines. They do so by enhancing the expression surface markers necessary for cell adhesion and antigen presentation, thereby increasing phagocytic and anti-microbial activity and facilitating antibody-mediated cytotoxicity ^{9–12, 13, 14}. Moreover, MDP and other muropeptides (tripeptides and disaccharide tri- and tetrapeptides) induce immune responses by increasing IFN-γ and other cytokine production, stimulating the differentiation and proliferation of lymphocytes, a subset of white blood cells that play and integral role in the body’s defense against foreign intruders ^15–17. MDP has also been shown in vitro and in vivo to be the minimal structure required for the priming of cells, where pre-exposure to the peptide augments immune responses to a later challenge ^{18, 19}. Analogues where the D-isoglutamine residue is replaced by D-glutamine, D-glutamic acid, or D-isoasparagine have a reduced priming effect, whereas analogues replaced with L-glutamic acid, L-glutamine, or L-isoglutamine are inactive ^{15, 19}. Furthermore, muropeptides express strong synergy with other ligands, where together they elicit a greater immune response than each alone would. For example, MDP has been shown to have a synergistic effect with LPS (lipopolysaccharides), found in the outer membrane of Gram-negative bacteria and recognized by the cell-surface receptor Toll-like receptor-4 (TLR4). This synergy was observed in vitro in human primary cells, including whole blood, peripheral blood mononuclear cells (PBMCs), purified monocytes, and various human monocytic and rodent cell lines, and in vivo in a rat model for anorexia ^20–28.

3.1.2. MDP for therapies of cancer and other diseases

MDP and its derivatives have a variety of clinical uses and therapeutic potential. Murabutide (MB), for example, is a synthetic immunomodulator derived from MDP that enhances non-specific resistance to bacterial and viral infections without fever and decreases the lethality of LPS in mice ^29–32. It has also been observed to synergize with antiviral and anti-inflammatory cytokines such as IFN-α as well as increase the anti-tumor effects of IFN-α and IL-2 in mouse models ^{33, 34}. Most importantly, MB regulates cytokine production without dramatically inducing proinflammatory mediators ³⁵. Studies have shown that injecting it in combination with IL-2 into Meth-A sarcoma-bearing mice resulted in significant tumor inhibition and complete tumor regression in 70% of the treated mice ³³. MB has also been shown to significantly inhibit HIV-1 replication in acutely infected monocyte-derived macrophages and dendritic cells ³⁶. Efforts have already been made to develop other similarly MDP-derived drugs. Macrophages activated by a liposome-encapsulated immunomodulator (MTP-PE, a MDP-derivative) or MDP conjugated by PolyG (a 10-mer polyguanylic acid), have resulted in tumoricidal activity ^{37, 38}. Another reagent, Paclitaxel (Taxol®) conjugated to MDP, has not only antitumor activity but also immunoenhancement effects ³⁹.

3.2. Mechanism of actions

3.2.1. Nod2: MDP receptor and its signaling

MDP and its derivatives are specifically recognized by the pathogen recognition receptor molecule NOD2 (CARD15) that plays a role in both adaptive and innate immune systems by regulating cytokine, chemokine, and antimicrobial peptide production ^40–44. NOD2 belongs to the NLR (nucleotide binding domain-leucine rich repeats) protein family and is characterized by three motifs: (1) An N-terminal effector domain containing a caspase recruitment domain (CARD); (2) An NBD (nucleotide binding domain), which has a binding site for ATP and is required for oligomerization; and (3) A leucine rich repeats (LRR) domain ^45–48. It is expressed in the cytoplasm of several cell types involved in host defense, including macrophages, dendritic cells, peripheral blood mononuclear cells, and intestinal epithelial cells (especially Paneth cells) ^{42, 49–52}.

Three mutations within NOD2 have been identified in 30 – 40% of Crohn’s Disease patients in North American and European populations ^{52, 53}. Inflammatory bowel diseases (IBDs) such as Crohn’s Disease (CD) are due to genetic, epigenetic, and environmental factors leading to the overproduction of cytokines from a chronically activated immune system ^54–58. Mapping of the IBD1 locus has led to the discovery of NOD2 encoded on human chromosome 16q12 as the first gene linked to CD ^{52, 53}. All three CD-associated mutations are restricted to or are in the vicinity of the LRR domain located in the C-terminus of the protein. While the 3020insC frameshift mutation results in a premature stop codon that partially truncates the LRR domain, the R702W and G908R mutants are single nucleotide polymorphisms (SNPs) ^{59, 60}. The precise mechanism underlying how mutations in the NOD2 gene cause CD is not yet fully understood. Proposed hypotheses include an altered immune response by dysregulated Toll-like receptor signaling or a defective function of Paneth cells, which regulate commensal and pathogenic bacteria thorough antimicrobial compounds ^{42, 61–65}.

3.2.2. Signaling cascades of MDP stimulation

Upon detection of MDP, NOD2 binds to the kinase RIP2 via CARD-CARD homophilic interactions, a step required in order for downstream signaling to proceed ^66–68. Signaling to RIP2 leads to NF-kB transcriptional activity through the IKK (IkB kinase) complex as well as other cascades involving MAP kinases that result in the production of pro-inflammatory cytokines and chemokines such as interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), IL-12, and IL-8 (Fig. 2) ^{42, 69, 70}.

Fig. 2.

A simplified schematic of the NOD2 signaling pathway

Several proteins are postulated to regulate NOD2 signaling, including Erbin ^{58, 71–74}. Currently, it has been shown that Erbin serves as a negative regulator of NOD2 by binding to the protein via its CARDs and thus inhibiting its ability to induce NF-kB activity upon MDP stimulation ^{58, 73}. NOD2 and RIP2 are also involved in the regulation of cell death and inflammation through the caspase-1-dependent maturation of IL-1β and IL-18. It has been shown in vitro and in vivo that upon MDP stimulation NOD2 and RIP2 are both required for Caspase-1 activation and IL-1β production ⁷⁵.

4. Synthetic approach of MDP analogs

4.1. Background of MDP synthesis

The first synthesis of N-acetylmuramic acid was reported by Jeanloz and Flowers in 1963 ⁷⁶. Since then, several reviews have followed ^77–81. In general, MDP (N-acetylmuramyl- L-alanyl-D-isoglutamine) can be synthesized by the coupling reaction of 3 subunits; N-acetyl-D-glucosamine, lactic acid or its equivalent and dipepide. In 4.1) and 4.2), a synthetic approach of the N-acetylmuramic acid (MurNAc) moiety is mainly discussed. MDP has (R)-configuration at the lactic acid moiety, while N-acetylisomuramyl-L-alanyl-D-isoglutamine has (S)-configuration. Treatment of the N-acetylisomuramyl-derivative ((S)-isomer) in acetic acid at 80 °C for 1 h caused decomposition to give the intramolecular ester and the dipeptide in about 50% yield through an N to O migration of the amide bond. In comparison, MDP ((R)-isomer) decomposed only 5%, suggesting that the (S)-isomer undergoes migration more easily than its (R)-isomer (Scheme 1). Furthermore, a biological assay for the induction of delayed-type hypersensitivity to N-acetyl-3-(4-arsonophenylazo)-L-tyrosine in guinea pigs revealed that the (S)-isomer dramatically reduced its adjuvant activity ⁸². It should be noted that this chiral center has an impact on chemical stability as well as biological activity. Therefore, it is important to obtain a diastereochemically pure MurNAc moiety.

Scheme 1.

Stability of N-acetylisomuramyl-L-alanyl-D-isoglutamine

4.2. Synthesis of MurNAc moiety

Two examples of synthetic procedures are described in Schemes 2 and 3^83–87. To achieve diastereomerically pure MurNAc derivatives, D-Glucosamine and its equivalents are often used as starting materials. Anomeric isomers are separable by chromatography. In the following introduction step of the lactic acid moiety to the masked-form of D-Glucosamine, the desirable (R) isomer 2 in Scheme 2 or 2′ in Scheme 3, is also separable respectively. In general, the syntheses of MurNAc in protected forms for the preparation of MDP analogues involve laborious multi-stage procedures. 2 is an important intermediate for modifying the C4 and C6 positions, whereas 2′ acts to more easily diversify the synthetic analogs.

Scheme 2.

Synthesis of MurNAc precursor (1)

Scheme 3.

Synthesis of MurNAc precursor (2)

On the other hand, 2-methyl-(1,2-dideoxy-5,6-O-isopropylidene-α-D-glucofurano-[2, 1-d])-2-oxazolin 3 (Scheme 4) is accessible from 2-acetamido-2-deoxy-D-glucose in one step on a large scale ⁸⁸. The oxazoline moiety already contains the NAc group of MurNAc in a masked form. Therefore, under the treatment of sodium hydride, a lactate side-chain can be introduced at the HO-3 selectively without further protection (Scheme 4) ^{89, 90}.

Scheme 4.

Synthesis of methyl-β-glucoside of N-acetylmuramic acid

Protected MurNAc is coupled with dipeptide ester (L-Ala-D-Glu(OR)-NH₂, R = Bzl, ^tBu, CH₃) ⁸⁵, followed by the de-protection to afford MDP (Scheme 5). A number of synthetic approaches to MDP analogs have been made to improve its pharmacological properties by changing the peptide chain or sugar parts.

Scheme 5.

Coupling of N-acetylmuramic acid with dipeptide

4.3. Peptide modifications and their biological activities

4.3.1. Peptide effect on adjuvant activity

Many peptide variations were introduced on N-acetyl muramic acid and their impact on adjuvant activity was evaluated in two different guinea pig models: Immunization with MDP-supplemented water in mineral oil emulsions containing (1) heterologous protein antigens or azobenzenearsonate-N-acetyl-L-tyrosine (induction of delayed type hyper sensitivity) or (2) encephalitogenic proteins and peptides (induction of experimental allergic encephalomyelitis). A study of the structure-activity relationship suggested that the L-configuration of an amino acid linked to the muramyl part and D-configuration of the glutamic acid residue was important in retaining or increasing biological activity. L-Alanine of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP-L-D) could be replaced with another L amino acid such as L-serine, while replacement of L-alanine with D-alanine (MDP-D-D) dramatically decreased adjuvant activity. As for the replacement of D-isoglutamine, the functionality of D-glutamic acid is important and the α-amide is not essential. For example, the D-aspartic, D-norleucine as well as the L-isoglutamine analogs (MDP-D-L) were inactive, while the D-glutamic acid α,γ-dimethyl ester analogs showed high adjuvant activity ^{77, 91, 92}.

4.3.2. Lipophilic peptides as prodrugs

Given that it can be modified to eliminate the drawbacks posed by poor macrophage penetration and rapid elimination, MDP has the potential of becoming a useful immunomodulator. From a synthetic standpoint, the peptide moiety is the easiest part of the MDP structure to modify.

One important parameter to consider in improving the pharmacological properties of MDP is lipophilicity. The lipophilic MDP analogs are described in Figure 3. MTP-Cholesterol contains a hydrolyzable ester, while MTP-octadecane and MTP-heptadecafluorooctadecane have non-hydrolyzable ethers. MTP-Cholesterol was active as free MDP in the stimulation of RAW264.7 cells, measured by nitrite production as an indication of NO-synthase activity, a major effector of macrophage-mediated cytostatic activity in rodent systems responsible for antimicrobial, antiparasitic and antitumoral effects. On the other hand, the lipophilic ether derivatives were not active, suggesting that lipophilic MDP analogs need to be hydrolyzed inside the cells to produce a hydrophilic metabolite in order to activate macrophages ^{85, 93}.

Figure 3.

Lipophilic MDP analogs

4.3.3. Methods to derivatize MDP structures: Solid-phase synthesis

Solid-phase is conventionally used for peptide, oligosaccharide, DNA and RNA syntheses. The advantages of using solid phase are easy handling and simple product separation from the reaction mixture, although there are several drawbacks such as difficulty in monitoring the reactions or the requirement of an excess amount of reagents. Solid-phase synthesis would be the best way to make a diverse MDP derivative library with potential application for drug screening. The use of macro crowns with a loading capacity of 5–8 mmol/pin from Chiron Mimotopes for MDP analog synthesis is described in Scheme 6^{94, 95}.

Scheme 6.

MDP analog synthesis on solid support (1)

N²-[α-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutamyl]-N⁶-trans-(m-nitrocynnamoyl)-L-lysine; MDP-C could be synthesized in a similar way on an MBHA amide resin, and it induced strong cytolytic activity by macrophages on P388 leukemia cells and cytotoxic activity by cytotoxic T lymphocytes on P815 mastocytoma cells (Scheme 7) ⁹⁶. Furthermore, a hyper acid-sensitive Sieber amide resin was used for the synthesis of MDP and spacer modified MDP 4. The neoglycopeptide polymers 5 could be prepared from 4 and pre-activated poly(N- acryloyloxysuccnimide) (pNAS). 5 increased the production of NF-α compared to monomeric MDP (Scheme 8) ⁹⁷.

Scheme 7.

MDP analog synthesis on solid support (2)

Scheme 8.

MDP analog synthesis on solid support (3)

4.4. Sugar modifications

4.4.1. Effect of lactic acid moiety (C3)

As described in 4.1), the chirality of MDP at the lactic acid moiety has an impact on stability as well as activity. In contrast, nor-MDP, which does not have a methyl group at the same position, is known to exhibit comparable biological activity and less toxicity ⁹⁸.

4.4.2. D-Glucosamine with various aglycones (C1)

The hydroxy group at the C1 position can be removed ⁹⁹ and replaced by thiol or substituted by α- or β-benzyl-glycoside. Lipophilic 1-O-acyl and 1-S-acyl groups do change MDP adjuvant activity ¹⁰⁰. A recent synthetic approach of O-glycoside is described in Scheme 9^101–105. It was reported that as the aglycone carbon number increased (R= 6d<6a<6b<6c), the ability of MDP derivatives to stimulate NK cytotoxic activity also increased.

Scheme 9.

An example of the synthesis of O-glycoside MDP analogs

MDP containing S-glycoside could be synthesized in a similar way (Scheme 10). 1-O-aryl and 1-S-aryl analogs stimulated antibacterial resistance. The substitution on the aromaic ring may change the cytolytic activity on E-562 cells, since non-substituted phenyl thioglycoside did not display significant cytolytic effect toward K-562, whereas substituted analogs did ¹⁰⁶.

Scheme 10.

An example of the synthesis of S-glycoside MDP analogs

The methyl β-glycoside of MDP was reported to be more adjuvant-active than the corresponding methyl α-glycoside ¹⁰⁷. The size and orientation of the aglycon in MDP also influence its biological activities. For example, loss of activity occurred when a p-aminophenyl group was introduced at the anomeric center (Figure 4). However, when the inert p-aminophenyl β-glycoside was cross-linked with glutaldehyde, several biological activities could be recovered and the cross-linked oligomer was more active than MDP in protecting mice nonspecifically against bacterial challenge ¹⁰⁸.

Figure 4.

p-Aminophenyl β-glycoside of MDP

Biological evaluation of MDP analogs indicated that lipophilicity of the molecule caused various important effects on biological activity by increasing adjuvant activity and decreasing pyrogenicity, which is one of the major side effects of MDP ^{93, 109}. As an example, introduction of a perfluoroalkyl group at the anomeric position of the sugar moiety is described (Scheme 11) ¹¹⁰.

Scheme 11.

Perfluoroalkylated MDP

4.4.3. Effect of N-acetyl moiety (C2)

The acetoamide group could be replaced by an OH, NH₂ or N-methylacetoamide group, but deamino/deoxy compounds lost the ability to induce delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine as examined in guinea pigs. In contrast, the introduction of a lipophilic acylamide group increased adjuvant activity ^111,112,

4.4.4. 4, 6-O-Substitution of D-glucosamine (C4, C6)

N-Acetyl-β-D-glucosaminyl-(1–4)-N-acetylmuramyl-L-alanyl-D-isoglutamine was more potent than MDP for the induction of delayed-type hypersensitivity and circulating antibodies to ovalbumin in guinea pigs ^{113, 114}.

At C-6, the hydroxyl could be replaced by a thiol, amino, or a non-acylated amino function ¹¹². In contrast to lipophilic 6-S-acyl-MDP derivatives, lipophilic 6-O-acyl-MDP derivatives were potent compounds ¹¹⁵. Acylation at 6-position of the carbohydrate with several mycolic acids, hydroxy fatty acids and quinonylalkanoic acids enhanced anti-HIV-1 and antitumor activity ^{116, 117}. B30-MDP displayed adjuvant activity on the induction of antibody response antigens and vaccines (Scheme 12) ^{114, 118}. Acridine N-substituted w-aminoalkanocarboxylic acid derived MDP (Scheme 13) showed an immunostimulating effect on the cytotoxic activity of the NK cells obtained from the spleen of healthy and Abmelanoma bearing animals ^{119, 120}.

Scheme 12.

Synthesis of long chain fatty acid esters of MDP

Scheme 13.

Acridine N-substituted w-aminoalkanocarboxylic acid derived MDP

4.4.5. Desmuramylpeptides

A carbocyclic MDP analog (7) and (8) (Figure 5), which have cyclohexanol moieties instead of N-acetylmuramic acid, were inactive as adjuvants for the induction of delayed-type hypersensitiviy to azobenzenearsonate N-acetyl-L-tyrosine in guinea pigs ¹¹². A carbocyclic MDP analog (9) was synthesized via Ferrier rearrangement as a key step ^{121, 122} and its derivatives exhibit the common activity of MDP, especially the stimulation of unspecific resistance against bacterial and viral infections, liberation of colony-stimulating factors, induction of IL-1 production in macrophages and antitumor activity (Scheme 14) ¹²³.

Figure 5.

Carbocyclic MDP analogs

Scheme 14.

Synthesis of 9

A carbocyclic nor-MDP analog (10) in which the N-acetylmuramyl moiety was replaced by a trans-2-[[2′-(acetylamino)cyclohexyl]oxy]acetyl group and D-isoglutamine by D-glutamic acid retained the immunostimulating properties of MDP and also displayed antitumor activity. In contrast to MDP, compound 10 was neither pyrogenic nor toxic ¹²⁴. Moreover, cyclopropanoid analog 11 and its analogs have been reported, although no biological data is available ¹²⁵.

In addition to NOD2, the related protein NOD1 is also activated by muropeptides. FK-156 activates NOD1 but not NOD2 and is a potent stimulant of antibody production. It is free of pyrogenicity. Its analog FK-565 is a strong anticancer reagent (Figure 6) ^{126, 127}.

Figure 6.

Desmuramylpeptides (1)

N-Acetylmuramic acid can also be replaced by various N-phthaloylated amino acids or phthalimido substituted aminoethoxyacetic acid (Figure 7) ^{128, 129}.

Figure 7.

Desmuramylpeptides (2)

LK423 augments the capacity to produce IL-10 in the spleen cells of cyclophosphoramide treated mice and alleviates dextran sulfate sodium-induced colitis in rodents. Thus, LK423 is a candidate substance for the development of an anti-inflammatory pharmaceutical agent. Furthermore, LK423 stimulated the production of tumor necrosis factor in vitro phorol 12-myristate 13-acetate and ionomycin-stimulated cultures of human peripheral blood mononuclear cells ^130–134. Adamantane substituted analogs LK 415 and LK 517 as well as LK 423 are strong regulators of IL-12 synthesis and IFN-γ synthesis. The phosphonate moiety introduced in LK 415 plays a key role for augmented T-cell cytokine production (Schemes 15) ^{134, 135, 136}.

Scheme 15.

Synthesis of LK 423 related analogs

5. Concluding remarks

MDP derivatives have a variety of clinical uses and therapeutic potential. Murabutide, for example, has been used to boost immune responses as a form of cancer therapy. MDP is the smallest compound found to elicit adjuvant activity and its multiple functional groups provide a platform to vary its structure, as each functional group can be synthetically modified to improve chemical as well as biological properties. Most studies thus far have been focused on generating derivatives with a higher level of adjuvant activity, but the development of derivatives that suppress rather than enhance immune responses is also a promising area of study. We recently discovered DFK1012, an anti-inflammatory MDP derivative that acts to suppress proinflammatory cytokine production in macrophages upon stimulation of innate immune receptors such as TLR (Toll-like receptor) or NLR (Nucleotide binding domain, Leucine rich repeats) proteins¹³⁷. Together with these recent findings, the synthetic approaches outlined in this paper will help us diversify the chemical structure of MDP and study the relationship between its structure and function in an effort to optimize its desirable biological activity.

Abbreviation

MDP

muramyl dipeptide

TLR

Toll-like receptor

NLR

Nucleotide-binding domain, leucine-rich repeat protein

LDH

lactose dehydrogenase

IKK

IκB kinase

HBBTU

O-benzotriazol-1-yl-N, N, N′, N′-bis(tetramethylene)uronium hexafluorophosphate

BOP

benzotriazoloxy-tris-(dimethylamino)phosphonium hexafluorophosphate

DCC

N,N′-dicyclohexylcarbodiimide

DIEA

N, N-diisopropylethylamine

DMF

N, N-dimethyl formamide

NMM

N-methylmorpholine

HOSu

N-hydroxysuccinimide

HOBt

1-hydroxybenzotriazole hydrate

TFA

trifluoroacetic acid

AcOH

acetic acid

TfOH

trifluoromethanesulfonic acid

Boc-Ala-NCA

Boc-alanine N-caboxyanhydride

Fmoc

9-fluoromethoxycarbonyl

Boc

tert-butoxycarbonyl

Dde

1-(4,4-dimethyl-2,6-dioxocyclohexyidene)ethyl

Pin

macro crown with a loading capacity of 5–8 mmol/pin from Chiron Mimotopes

Bzl

Bn, benzyl

Bz

benzoyl

Ts

p-toluenesulfonyl

AIBN

2,2′-azoisobutyronitrile