Dendritic macromolecules (dendrimers) are monodisperse macromolecules with a highly branched, regular structure. The first dendritic macromolecules that attracted widespread attention were the polyamide-amine (PAMAM) series synthesized by Tomalia in 1985 and Newkome's "arborol" systems. Early dendrimer synthesis primarily used the divergent approach, until 1990, when Fréchet introduced the convergent approach in dendrimer synthesis, forming the two main methods for creating dendritic macromolecules. Thanks to their unique tree-like structure, dendrimers have many potential applications in fields such as biomolecular modeling, catalysts, immunodiagnostic reagents, drug and gene delivery systems, and surfactants. In recent years, both domestic and international research on dendritic macromolecules has received increasing attention, with the number of related publications growing exponentially.
Dendritic peptides (peptide dendrimers) are broadly defined as dendritic macromolecules containing peptides. Dendritic peptides possess general characteristics of dendritic macromolecules, such as regular, multi-branch spherical structures, dense surface groups, and larger internal cavities. Compared to linear peptides, dendritic peptides have better water solubility, stronger resistance to enzymatic hydrolysis, and lower toxicity to cells. Additionally, their numerous binding sites enable them to bind a larger number of useful functional groups. As a result, dendritic peptides have vast potential applications in biochemistry, molecular biology, and chemical biology. Broadly speaking, dendritic peptides can fall into two categories: one involves attaching peptide segments to the ends of the traditional dendrimer core, and the other directly uses branched peptides as the core of the dendrimer. This article primarily discusses the latter case. In 1988, Tam introduced the first dendritic peptide with lysine as the core.
There are two main methods for synthesizing dendritic peptides: the traditional stepwise solid-phase synthesis method and the chemoselective and orthogonal ligation method. These methods correspond to the divergent and convergent approaches in dendrimer synthesis. The divergent approach builds the dendritic structure from the core outward, while the convergent approach first synthesizes the necessary functional groups, purifies them, and then links them to the dendritic core using specific chemical methods.
In 1963, Merrifield first introduced the solid-phase peptide synthesis method, which is simple and versatile. It has since become a general method for synthesizing peptides, oligonucleotides, and even certain organic small molecules. The divergent method is often used in the synthesis of dendritic peptides, and the specific synthesis steps are similar to those of linear peptides. Typically, synthesis begins with the formation of secondary or tertiary branch structures using di-Boc or di-Fmoc protected lysine, followed by stepwise addition of other peptide sequences onto the lysine backbone. After synthesis, the dendritic peptide is cleaved from the resin, followed by further purification and characterization.
As the number of lysine units increases, the tendency for interchain aggregation also rises. To mitigate this effect, the divergent method often uses a lower resin loading, typically around 0.1 mmol/g, compared to the usual 0.3-0.8 mmol/g resin loading used in conventional solid-phase synthesis. Additionally, the formation of interchain hydrogen bonds that hinder connection reactions can be reduced by using special solvent combinations and increasing reaction temperatures. Besides lysine, Crespo and colleagues introduced dendritic peptides with cis-4-amino-L-proline (Amp) as the branching unit. A variant of the stepwise divergent synthesis method involves connecting different peptide chains onto a lysine backbone, which is achieved by using different protecting groups on the α- and ε-amino ends of lysine, typically Boc and Fmoc, respectively.
In addition to solid-phase synthesis, traditional divergent liquid-phase peptide synthesis has also been applied in dendritic peptide synthesis. Rodriguez-Hemández et al. used N-carboxyanhydrides (NCAs) of amino acids to initiate ring-opening polymerization, yielding dendritic peptides with long poly-lysine chains and high yields. Baigude et al. synthesized dendritic peptides with polysaccharide outer layers for use in AIDS vaccines, using di-Boc protected lysine in a liquid-phase synthesis approach. Choi et al. employed liquid-phase peptide synthesis to create dendritic peptide molecules with lysine backbones at both ends of a polyethylene glycol (PEG) polymer, resulting in dumbbell-shaped dendritic peptide molecules.
The divergent synthesis method is advantageous for its simplicity, speed, high reliability, and the fact that intermediate products do not need to be purified. However, as the size of the dendritic peptides increases, the overall yield declines exponentially with the dendrimer generation. Even with high yields at each step, the total yield after 4-5 generations becomes unacceptable. Particularly when the molecular weight of the target molecule exceeds 20,000, it becomes difficult to separate the final product from defective by-products, and the convergent synthesis method is usually employed in such cases.
The convergent synthesis method differs from the divergent synthesis method in that, in the former, the dendritic core and the surrounding peptides of the dendritic peptide are synthesized separately and then connected via chemical bonds. There are two main strategies for this connection: one involves using protected peptides at the ends, while the other uses unprotected peptides. The choice of which strategy to employ depends primarily on the properties of the peptide monomer, the complexity of the dendritic core, and the technical capabilities available in the laboratory. For methodological considerations regarding different connection strategies, one can refer to the relevant review by Tam et al. (1999).
The convergent synthesis method using protected peptides typically employs traditional coupling strategies and reagents. The main difference from linear peptide synthesis lies in the need to connect two or more protected peptides to the dendritic core simultaneously. In 1989, Sasaki and Kaiser used this method to synthesize ferroheme proteins. A challenge with using protected peptides is that the protecting groups often make the peptide fragments difficult to dissolve and result in lower coupling yields. Therefore, methods involving unprotected peptides have rapidly developed in recent years.
The unprotected peptide coupling method, also known as orthogonal ligation, chemoselective coupling, capture-activation, in situ reaction, intramolecular, or biomimetic ligation, relies on specific chemical reactions between the terminal peptides of the peripheral peptides and the dendritic core. These reactions can be nucleophilic-electrophilic reactions or reactions that form amide bonds. Since both the peptide fragments and dendritic cores are unprotected, they generally exhibit better water solubility and can be more easily purified via reverse-phase high-performance liquid chromatography (RP-HPLC), leading to improved reaction specificity. Several orthogonal coupling methods have been reported, including those that form thioester bonds, imine and amide linkages, and head-to-tail cyclization reactions. Tam et al. reviewed various unprotected peptide coupling methods.
For dendritic peptides synthesized by solid-phase synthesis, traditional solid-phase peptide methods, such as high-performance liquid chromatography (HPLC), are used for purification. Due to the unique properties of dendritic peptides, a broader peak often appears during reverse-phase HPLC separation, which can complicate the separation process to some extent. For dendritic peptides synthesized by liquid-phase methods, purification steps are performed after each reaction, meaning that the final product usually does not require specialized purification techniques. Most studies characterize dendritic peptides using techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance (NMR). Other methods, including circular dichroism (CD), electrophoresis, atomic force microscopy (AFM), and electrospray ionization mass spectrometry (ESI-MS), have also been employed in the characterization of dendritic peptides. However, some literature reports indicate that certain types of dendritic peptides do not produce stable spectra in ESI or MALDI-TOF MS.
In 1988, Tam first introduced the concept of the multiple antigen peptide (MAP), marking the first application of dendritic peptides. Today, dendritic peptides have a wide range of applications in the field of biochemistry, similar to those of general dendritic macromolecules. This section will briefly outline these various applications.
As mentioned above, dendritic peptides' role as immunogens is their earliest and most well-known application. Traditional antigenic peptides, when used to induce immune responses, typically need to be attached to a larger peptide or protein. However, such immunogens have a low density of epitopes, and the carrier protein may trigger unintended immune responses. Dendritic immunogens overcome these shortcomings by attaching many small peptides to the dendritic core, resulting in more epitopes per unit mass, thereby enhancing immunogenicity. Additionally, dendritic peptides are easy to synthesize, reliably induce epitope-specific antibodies, and can provoke diverse immune responses.
In 1989, Tam and colleagues synthesized a MAP with multiple epitopes, including independent epitopes representing the S and S(2) fragments of the HBV virus, thereby increasing the immunogenicity of the molecule. In 1990, they synthesized another MAP with continuous T-cell and B-cell epitopes, which was used for a malaria vaccine. In 1992, Tam's group synthesized an AIDS vaccine containing HIV-1 surface antigen gp120 and a lipophilic tripalmitoyl-S-glycerol-cysteine ester group.
Beyond Tam's group, de Oliveira et al. synthesized a MAP with a poly-L-lysine core and four cyclic disulfide peptides for use as a foot-and-mouth disease vaccine. Baigude et al. synthesized a MAP with a polysaccharide outer layer for use in an AIDS vaccine. Tallima et al. attached a derivative of the Schistosoma japonicum 3-phosphoglycerate dehydrogenase (G3PDH) main peptide sequence to a MAP, producing excellent immune effects. Bisang et al. synthesized a cyclic peptide fragment of the Plasmodium falciparum sporozoite surface protein and attached it to a four-branched MAP, also linking T-cell epitope peptides to the C-terminal, which was used in malaria vaccine research.
Currently, MAPs are routinely used in antibody production and cytotoxic immune response studies. Comparative studies have shown that MAPs exhibit higher efficacy and faster antibody induction compared to traditional peptide-protein conjugates. A comprehensive review by Sadler et al. summarizes the use of dendritic peptides in this application.
In immunoassays, small peptides that can bind to solid-phase surfaces are often required as antigens. Due to their strong binding ability and easy detectability, dendritic peptides have become the ideal choice for such applications. Adesida et al. studied the stronger binding affinity of dendritic peptides compared to linear peptides and developed direct and indirect peptide-antibody binding assays for immunoglobulins and peptides without epitopes. Gómara et al. linked the VP3 capsid protein of the Hepatitis A virus (HAV) to the end of a MAP, which was used in an enzyme-linked immunosorbent assay (ELISA) to test for HAV antibodies, finding that the two-branched dendritic peptide showed the best performance.
In serodiagnostics, dendritic peptides can improve the sensitivity of antibody detection, particularly useful for detecting antibodies produced during early infection, such as IgM. In one extreme case, dendritic peptides demonstrated 108 times the sensitivity of conventional peptide antigens in ELISA tests. In the near future, as dendritic peptides can be directly linked to sensor chips and antibody strength can be accurately measured, immunoassays may be replaced by biosensor technology.
Another confirmed application of dendritic peptides is in the design of inhibitors, such as those used to block the entry of Plasmodium into liver cells. A dendritic peptide based on the V3 loop of the HIV-1 surface antigen gp120 has been found to inhibit HIV-1 infection in both CD4+ and CD4- cells. The stronger binding affinity of dendritic peptides makes them highly significant in designing peptides to inhibit tumor cell metastasis. Recent research has shown that a dendritic peptide with 16 laminin branches significantly inhibited tumor growth in in viv. experiments.
Dendritic peptides have been shown to act as substitutes for proteins and even DNA in many autoimmune disease model systems. Dendritic peptides with different peripheral peptides have been used to simulate systemic lupus erythematosus in healthy animal models. Costa et al. used MAP to connect myelin oligodendrocyte glycoprotein (MOG) to induce experimental autoimmune encephalomyelitis in rats. Bracci et al. synthesized a four-branched MAP as a mimic of the nicotinic acetylcholine receptor (nAchR) and demonstrated a significantly stronger in vivo activity when combined with snake venom serum. Mason's experiments showed that specific dendritic peptides could induce antibodies against self-proteins and DNA in animals. Putterman and Diamond identified a double-stranded DNA substitute that induced anti-DNA antibodies in rats, leading to a lupus-like syndrome. Goodman et al. used dendritic peptides to simulate collagen-like molecules with superhelical structures. Kasai et al. synthesized a MAP enriched with arginine at the periphery, simulating the anti-cancer drug vasculostatin.
Azuma et al. discovered that dendritic peptides with a peripheral peptide of 11 residues exhibited antibacterial activity, effective against both Gram-positive and Gram-negative bacteria. Furthermore, antibacterial activity increased with the dendrimer generation. Research by Tam et al. found that a tetrapeptide (RLYR) and an octapeptide (RLYRKVYG) monomer showed no antibacterial activity; however, dendritic peptides incorporating four or eight of these small peptide fragments displayed broad-spectrum antibacterial and antifungal activity.
One of the most significant applications of dendritic macromolecules is as drug and gene carriers, and dendritic peptides have also garnered significant attention in this area. In 1995, Sheldon et al. studied the ability of lysine-based dendritic peptides to cross the cell and nuclear membranes of Chinese hamster ovary (CHO) cells, exploring their potential as drug and gene delivery systems, as well as intracellular fluorescence probes. Cattani-Scholz et al. linked azobenzene to a lysine backbone, creating a photosensitive drug carrier. Brokx et al. connected the chromophore chlorin e6 (Ce6) to the end of a MAP for photodynamic therapy (PDT) of tumor cells, achieving 40-400 times the photodynamic activity of the monomeric chlorin e6. Svarracino et al. attached lysine backbones to DNA molecules, finding that these DNA molecules were more resistant to degradation by nucleases, making them effective DNA carriers. In the field of gene delivery, L-lysine-based dendritic polymers have seen widespread use, with Brown's review on gene delivery dedicating a section to the application of dendritic peptides.
In a recent study, Xu et al. successfully attached calixarene to the end of a lysine backbone, creating a novel receptor. Hahn et al. synthesized a MAP molecule with four peptide branches and found that it exhibited metalloproteinase activity, serving as a prime example of dendritic peptides as artificial enzymes. Kuon et al. studied the interaction between MAP with small peptide fragments and cell surface polysaccharides, discovering that the binding affinity of two-branch and four-branch MAPs was 14 times and 10,000 times greater than that of linear peptides, respectively. Scofield et al. utilized MAPs to study protein interactions of Ro-ribonucleoprotein (Ro-RNP). Moreover, like other dendritic macromolecules, dendritic peptides can also act as surfactants.
In conclusion, dendritic peptides, as the next generation of dendritic macromolecules, not only possess the general properties of dendritic macromolecules but also feature mature synthetic methods, good water solubility, and ease of modification. These characteristics make dendritic peptides highly promising in biochemistry, molecular biology, and chemical biology. As lysine-based dendritic frameworks become commercially available and with further understanding of dendritic peptide properties, research on dendritic peptides is expected to attract increasing attention.
Peptide Synthesis Services at Creative Peptides
