A Synthetic Route to Human Insulin Using Isoacyl Peptides

The chemical synthesis of insulin has been a longstanding challenge, mainly because of the notorious hydrophobicity of the A chain and the complicated topology of this 51‐mer peptide hormone consisting of two chains and three disulfide bonds. Reported herein is a new synthetic route utilizing the isoacyl peptide approach to address the hydrophobicity problems. The incorporation of isoacyl dipeptide segments into both A and B chains greatly improved their preparation and purification, and the RP‐HPLC recovery of the chain ligation intermediates. The new route affords human insulin with a yield of 68 % based on the starting purified A chain and an overall yield of 24 % based on the substitution of the resin used for the preparation of A chain. To the best of our knowledge, this represents the most efficient route of human insulin chemical synthesis reported to date.     

Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.     

Solid‐Phase Combinatorial Synthesis and Biological Evaluation of Destruxin E Analogues

The solid‐phase combinatorial synthesis of cyclodepsipeptide destruxin E has been demonstrated. The combinatorial synthesis of cyclization precursors 8 was achieved by using a split and pool method on SynPhase Lanterns. The products were successfully macrolactonized in parallel in the solution phase by using 2‐methyl‐6‐nitrobenzoic anhydride and 4‐(dimethylamino)pyridine N‐oxide to afford macrolactones 9 , and the subsequent formation of an epoxide in the side chain gave 18 member destruxin E analogues 6 . Biological evaluation of analogues 6 indicated that the N‐MeAla residue was crucial to the induction of morphological changes in osteoclast‐like multinuclear cells (OCLs). Based on structure–activity relationships, azido‐containing analogues 15 were then designed for use as a molecular probe. The synthesis and biological evaluation of analogues 15 revealed that 15 b , in which the Ile residue was replaced with a Lys(N₃) residue, induced morphological changes in OCLs at a sufficient concentration, and modification around the Ile residue would be tolerated for attachment of a chemical tag toward the target identification of destruxin E ( 1 ).     

蝴蝶霉素及其类似物的化学合成进展

蝴蝶霉素是一种新型的天然抗生素,由于具有特殊的抗肿瘤活性而引起了人们的关注。然而,由于一些不利因素导致蝴蝶霉素无法直接运用于临床。为了寻找活性更好的药物先导化合物,人们对蝴蝶霉素的结构进行修饰和改造,通过生物方法和化学方法,合成了大量的类似物。本文将从化学合成角度出发,立足蝴蝶霉素及其类似物结构构造的关键步骤,对近年来的合成研究进行综述。     

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, Cys^A6 and Cys^A11 (which form the internal intrachain A6–A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid-phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild-type B chain-di-S-sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long-standing challenge in chemical insulin synthesis. The affinity of the Se-insulin analogue for the lectin-purified insulin receptor was indistinguishable from that of WT-insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔG_u ≈0.8 kcal mol⁻¹). In accordance with such enhanced stability, reductive unfolding of the Se-insulin analogue and resistance to enzymatic cleavage by Glu-C protease occurred four times more slowly than that of WT-insulin. 2D-NMR and X-ray crystallographic studies demonstrated a native-like three-dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.     

Facile Chemoenzymatic Strategies for the Synthesis and Utilization of S‐Adenosyl‐L‐Methionine Analogues

A chemoenzymatic platform for the synthesis of S‐adenosyl‐L ‐methionine (SAM) analogues compatible with downstream SAM‐utilizing enzymes is reported. Forty‐four non‐native S/Se‐alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non‐native SAM analogues. As a proof of concept for the feasibility of natural product “alkylrandomization”, a small set of differentially‐alkylated indolocarbazole analogues was generated by using a coupled hMAT2–RebM system (RebM is the sugar C4′‐O‐methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.     

A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides

Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time‐consuming, and low yielding. Inspired by the cellular synthesis of pro‐insulin in which the two constituent peptide chains are expressed as a single‐chain precursor separated by a connecting C‐peptide, we have developed a novel chemically cleavable bis‐linker tether which allows the convenient assembly of two peptide chains as a single “pro”‐peptide on the same solid support. Following the peptide cleavage and post‐synthetic modifications, this bis‐linker tether can be removed in one‐step by chemical means. This method was used to synthesize a drug delivery‐cargo conjugate, TAT‐PKCi peptide, and a two‐disulfide bridged heterodimeric peptide, thionin (7‐19)‐(24‐32R), a thionin analogue. To our knowledge, this is the first report of a one‐pot chemically cleavable bis‐linker strategy for the facile synthesis of cross‐bridged two‐chain peptides.     

Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding,Stability, and Structure of the ShK Toxin

ShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.     

Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding,Stability, and Structure of the ShK Toxin

ShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.     

Epothilone Analogues with Benzimidazole and Quinoline Side Chains: Chemical Synthesis,Antiproliferative Activity,and Interactions with Tubulin

A series of epothilone B and D analogues bearing isomeric quinoline or functionalized benzimidazole side chains has been prepared by chemical synthesis in a highly convergent manner. All analogues have been found to interact with the tubulin/microtubule system and to inhibit human cancer cell proliferation in vitro, albeit with different potencies (IC₅₀ values between 1 and 150 nM ). The affinity of quinoline‐based epothilone B and D analogues for stabilized microtubules clearly depends on the position of the N‐atom in the quinoline system, while the induction of tubulin polymerization in vitro appears to be less sensitive to N‐positioning. The potent inhibition of human cancer cell growth by epothilone analogues bearing functionalized benzimidazole side chains suggests that these systems might be conjugated with tumor‐targeting moieties to form tumor‐targeted prodrugs.     

Inhibition of Hsp90 with Resorcylic Acid Macrolactones: Synthesis and Binding Studies

A series of resorcylic acid macrolactones, analogues of the natural product radicicol has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves acylation of an ortho‐toluic acid dianion, esterification, followed by a ring‐closing metathesis to form the macrocycle. Subsequent manipulation of the protected hydroxymethyl side chain allows access to a range of new analogues following deprotection of the two phenolic groups. Co‐crystallization of one of the new macrolactones with the N‐terminal domain of yeast Hsp90 confirms that it binds in a similar way to the natural product radicicol and to our previous synthetic analogues, but that the introduction of the additional hydroxymethyl substituent appears to result in an unexpected change in conformation of the macrocyclic ring. As a result of this conformational change, the compounds bound less favorably to Hsp90.     

A convergent solution-phase synthesis of the macrocycle Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg], a potent new antiinflammatory drug

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-d-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn(Boc)-Pro-OH and H-d-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boc-d-Cha-OH was obtained very efficiently via hydrogenation of d-Phe with PtO(2) in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50-100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.     

Modular Chemoenzymatic Synthesis of Terpenes and their Analogues

Non‐natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large‐scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)‐FDP and (2Z,6Z)‐FDP, were isolated in greater than 80 % yields. The synthesis of 14,15‐dimethyl‐FDP, 12‐methyl‐FDP, 12‐hydroxy‐FDP, homo‐FDP, and 15‐methyl‐FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)‐14,15‐dimethylgermacrene D and (S)‐12‐methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non‐natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions.     

Insulin capture by an insulin-linked polymorphic region G-quadruplex DNA oligonucleotide

Insulin capture by a G-quadruplex DNA oligonucleotide containing a two-repeat sequence of the insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region is reported. The immobilized oligonucleotide was demonstrated to capture human insulin from standard solutions and from nuclear extracts of pancreatic cells with high selectivity, using affinity MALDI mass spectrometry and affinity capillary chromatography. Insulin was preferentially captured by the two-repeat ILPR oligonucleotide over another G-quadruplex-forming oligonucleotide, the thrombin-binding aptamer, as well as over a single repeat of the ILPR sequence that is not capable of forming the G-quadruplex architecture. Binding was shown to involve the beta chain of insulin. The discovery raises the possibility that insulin may bind to G-quadruplex DNA formed in the ILPR in vivo and thereby play a role in modulation of insulin gene expression, and it provides a basis for design of insulin analogues to probe this hypothesis. The availability of a DNA ligand to human insulin has analytical importance as well, offering an alternative to antibodies for in vitro or in vivo detection and sensing of insulin as well as its isolation and purification from biological samples.     

Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF,an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

In this paper, we describe the structure‐based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non‐mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C₅ precursors to isoprenoids, isopentenyl diphosphate (IPP, 1 ) and dimethylallyl diphosphate (DMAPP, 2 ; Scheme 1). The non‐mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non‐mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug‐like inhibitors of IspF protein, and synthesized (Schemes 2–5). Determination of the enzyme activity by ¹³C‐NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine‐2,5‐diamine derivatives in the upper micromolar range (IC₅₀ values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56′, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub‐pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to ‘Pocket III’) and rings that occupy the flexible hydrophobic region of ‘Pocket II’. The proposed binding mode remains to be further validated by X‐ray crystallography.     

Single-Shot Solid-Phase Synthesis of Full-Length H2 Relaxin Disulfide Surrogates

Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next-generation drugs. Separate synthesis of multiple peptide fragments and tedious chain-to-chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre-made diaminodiacid bridge at A-B chain terminal disulfide can be easily and rapidly synthesized by a single-shot automated solid-phase synthesis and expedient one-step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.     

Liquid‐Phase Synthesis of 2′‐Methyl‐RNA on a Homostar Support through Organic‐Solvent Nanofiltration

Due to the discovery of RNAi, oligonucleotides (oligos) have re‐emerged as a major pharmaceutical target that may soon be required in ton quantities. However, it is questionable whether solid‐phase oligo synthesis (SPOS) methods can provide a scalable synthesis. Liquid‐phase oligo synthesis (LPOS) is intrinsically scalable and amenable to standard industrial batch synthesis techniques. However, most reported LPOS strategies rely upon at least one precipitation per chain extension cycle to separate the growing oligonucleotide from reaction debris. Precipitation can be difficult to develop and control on an industrial scale and, because many precipitations would be required to prepare a therapeutic oligonucleotide, we contend that this approach is not viable for large‐scale industrial preparation. We are developing an LPOS synthetic strategy for 2′‐methyl RNA phosphorothioate that is more amenable to standard batch production techniques, using organic solvent nanofiltration (OSN) as the critical scalable separation technology. We report the first LPOS‐OSN preparation of a 2′‐Me RNA phosphorothioate 9‐mer, using commercial phosphoramidite monomers, and monitoring all reactions by HPLC, ³¹P NMR spectroscopy and MS.     

Synthesis of Bioactive Side‐Chain Analogues of TAN‐2483B

The fungal metabolite TAN‐2483B has a 2,6‐trans‐relationship across the pyran ring of its furo[3,4‐b]pyran‐5‐one core, which has thwarted previous attempts at its synthesis. We have now developed a chiral pool approach to this core and prepared side‐chain analogues of TAN‐2483B. The synthesis relies on ring expansion of a reactive furan ring‐fused dibromocyclopropane and alkynylation of the resulting pyran. The furan ring is constructed by palladium‐catalysed carbonylative lactonisation. Various side‐chains are appended through Wittig‐type chemistry. The prepared analogues showed micromolar activity towards cancer cell lines HL‐60, 1A9 and MCF‐7 and certain human disease‐relevant kinases, including Bruton's tyrosine kinase (Btk).     

Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry

Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.