The need for high-quality synthetic peptides has been greatly increased due to the expansion of life-science research. Production of polypeptides by genetic engineering has been more and more popular. However, chemical synthesis is still indispensable for production of peptides containing unusual amino acids or peptides with a C-terminal amide. At the present time chemical synthesis is definitely preferable for the preparation of peptides with up to 30 amino acid residues. This article will focus on highly efficient synthesis, which involves rapid synthesis using automated simultaneous multiple solid-phase synthesis with high yield and high homogeneity of synthetic products, safe operation with a small amount of waste, low cost hardware and low running cost. Reagents, solid-supports, a novel synthesizer without cross-contamination, simultaneous cleavage for highly efficient synthesis and characterization of synthetic peptides, and additionally the construction of peptide library are reviewed.
Monellin, an intensely sweet protein, was isolated from the fruit of the West African plant, Dioscoreophyllum cumminsii Monellin consists of two noncovalently associated polypeptide chains, the A chain with 44 amino acid residues and the B chain with 50 residues. The tertiary structure has been determined, and the native conformation is essential for the sweet taste. Recently, the primary structure of monellin was unambiguously determined, and monellin was synthesized by the Fmoc solid-phase method. In an attempt to delineate the active site(s) of monellin, amino acid residues within potential active sites were replaced. Replacement of CysB41 by Ser led to a small change in sweetness. Replacement of IleB6, AspB7 or IleB8 by different amino acids resulted in a complete or marked loss of sweetness. Replacement of AspA16 or TyrA13 by other amino acids did not reduce the substantial sweetness. Replacement of each Lys residue by L-2-aminohexanoic acid (L-norleucine) significantly diminished the sweetness potency, but did not completely remove the sweetness. Analysis of the synthetic analogs by circular dichroism (CD) showed that the major structural features of monellin were not significantly altered. These findings suggest that IleB6, AspB7 and IleB8 are responsible for eliciting a sweet taste, and that one or more basic residues may also be important for eliciting a sweet taste. In the course of the investigation, the D-enantiomer of monellin was synthesized by the Fmoc solid-phase method, and crystallized by the hanging drop vapor diffusion method. The D-isomer was shown by CD to be the mirror image of the synthetic L-monellin, and was devoid of any sweetness (essentially tasteless). DL-Monellin was crystallized from a 1 : 1 mixture of the synthetic L- and D-monellin, and its CD spectrum did not show any absorption. DL-Monellin was 2000 times sweeter than sucrose on a weight basis (cf., monellin is 4000 times sweeter than sucrose) These results indicate that the crystals consist of the racemates of DL-monellin.
In 1980, E. T. Kaiser developed a 4-nitrobenzophenone oxime resin which is useful for the preparation of protected peptides by solid-phase synthesis followed by cleavage from the resin. Standing on this merit, he has also proposed “segment synthesis and condensation” strategy for the synthesis of long chain polypeptides. In past 10 years, several improvements have been achieved by various his successors for the practical application of his strategy for the synthesis of proteins. To increase the applicability, the introduction of various starting amino acids, coupling reagents and protecting groups were examined and selected. Now Kaiser's oxime resin finds special advantages in the synthesis of artificial proteins by de novo design, in which peptides are hybridized with various non-peptidyl compounds such as porphyrins and organic metal complexes. In addition, extreme usefulness was discovered for the convenient synthesis of cyclic peptides by solid-phase synthesis and cyclization-cleavage with the oxime resin. Since increasing contribution of Kaiser's oxime resin is expected for the variously designed syntheses of polypeptide-related compounds, we review here the recent progress in the peptide synthesis based on the oxime resin to provide a manual for a number of young synthetic organic chemists.
Three dimensional structures of peptides and proteins are directly related to their biochemical functions. Since protein tertiary structures are considered to be an assembly of several secondary structures, considerable efforts have been directed toward the synthesis of short peptides that adopt a stable secondary structure. In recent years, various modeling and synthetic techniques for polypeptide structures have become easily accessible for synthetic organic chemists. The peptide design combined with synthetic organic chemistry has established a new class of molecules that judiciously use organic template molecules to assemble predetermined secondary structures of peptides. Functional aspects of these designed peptide/protein mimetics are also discussed.
Synthetic studies on dolastatin 10, an antitumor marine peptide containing unusual amino acids, are reviewed. Strategies for the synthesis of the C-terminal dolaphenine (Doe) are first described, and then the stereoselective synthesis of the two unusual amino acids, dolaproine (Dap) and dolaisoleuine (Dil), is discussed. Finally, the method for the construction of the whole molecule by assembling each amino acid unit is described.
The human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), codes for a virus-specific aspartic protease responsible for processing the gag and gag-pol polyproteins and for the proliferation of the retrovirus. The HIV-1 protease functions as a homodimer and can recognize Phe-Pro and Tyr-Pro sequences as the cleavage site, but mammalian aspartic proteases do not have such specificity. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treatment of AIDS and related complex. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing allophenylnorstatine [Apns; (2 S, 3S) -3-amino-2-hydroxy-4-phenylbutyric acid].
This review covers the recent advances in the development of highly potent inhibitors of platelet aggregation as potential therapeutic drugs for thrombosis related to cardiovascular and cerebrovascular diseases. The discovery of RGD sequence-directed cell surface receptors (the integrins) has led to extensive research in the development of small RGD-containing peptides and their mimetics as antithrombotic agents. These agents work by inhibiting platelet aggregation through competitive blocking of fibrinogen to the platelet surface receptor, GPIIb/IIIa. The pharmacophoric nature of the aspartic acid and arginine side chains of the RGD unit has allowed the development of strategies for rational design, largely based on assumed bioactive RGD conformations and lead optimization. Applications of such strategies from RGD peptides to peptide hybrids and then to non-peptide mimetics, are described. Also discussed is the important issue of specificity toward GPIIb/IIIa, keeping in view that the RGD unit is a key recognition signal for a variety of cell surface receptors.
The serological application of synthetic peptides in viral infection can be grouped into two categories;antigenic and immunogenic. In the first category, synthetic peptide antigens are used to bind site-specific antibodies raised against the region corresponding to peptide amino acids sequence of the native protein. This site-directed specificity of peptide antigens can be basis for sensitive and specific immunoassay for diagnosis purposes. EIA using synthetic peptides as antigens have been used successfully for diagnosis of viral infection and discrimination between similar viruses. By using site-directed specificity it is also possible to obtain site-specific antibodies from human plasma by affinity column coupled with antigenic synthetic peptides as ligands These antibodies can be used for analysis of functional domains of the native protein which can neutralize viral infection. In the second category, synthetic peptide immunogens are used to elicite antibodies recognizing the native protein from which the peptide was derived. Antipeptide antibodies have been used for detection, chracterization, localization and purification of proteins. This chapter describes the characteristics of antigenic regions on HTLV- I structural proteins, and the serological application of the antigenic peptides in viral infection.
Biologically active peptide have potential therapeutic applications in the treatment of diseases caused by peptide deficiency. However, there are only a few peptide drugs, Elcitonin (a calcitonin derivatives for osteoporosis, Asahikasei), Hirutonin (TRH tartrate for prolonged disturbance of consciousness, Takeda) and Leuplin (a highly potent LH-RH derivative for prostate cancer/endometriosis, Takeda) There are two serious drawbacks to using peptide derivatives as drugs : their poor oral bioavailability and their short duration of activity. Although the latter can be overcome by studing various derivatives, the development of orally active peptide derivatives is almost impossible. I would like to describe briefly the process of the research and development of “Leuplin Depot”, once-a-month depot type formulation of super agonist of LH-RH.
The creation of antagonists of substance P (SP) has been the focus of intensive research because of their potential as therapeutic agents. A conventional for an SP antagonist is the synthesis of SP analogs in which amino acids are substituted or some peptide bonds are modified. Another useful method is the screening of compounds from a sample file. This method, called random screening, led to the discovery of novel and potent non-peptide antagonists. Apart from these two strategies, we were able to design low-molecular weight antagonists from a known peptide lead. The search for the essential part for receptor binding, improvement of the stability against enzymatic metabolism, and chemical modifications led to a potent tripeptide (4, FR 113680). The molecular size of this tripeptide was reduced to a dipeptide structure such as 13, through newly designed branched tripeptides (510). Further optimization of the lysine part of 13 into (2 S, 4 R) Hyp and subsequent modification of the side chain parts culminated in the potent dipeptide antagonist (15, FK 888). The pharmacological profile of 15 as an antiasthma agent is also presented.
Vasopressin (AVP) plays an important role in the regulation of cardiovascular homeostasis through V1 and V2 receptors. Stimulation of AVP receptors may contribute to the development of congestive heart failure, hypertension and renal failure. Thus, many efforts have been made to discover AVP receptor antagonists and develop them for therapeutic use. All of the antagonists developed thus far have been peptide AVP analogues, so their therapeutic use has been limited because of their low bioavailability and partial agonist activity. It is necessary to discover nonpeptide and orally effective AVP antagonists for therapeutic use. Therefore, we forcused our studies on nonpeptide AVP antagonists. We found a lead compound by means of chemical file screening, and performed optimization of the compound. Finally, we elaborated a V1 receptor selective antagonist (OPC-21268) and a V2 receptor selective antagonist (OPC-31260). The history of the development of our novel nonpeptide AVP receptor antagonists and their pharmacological effects are described herein.