Thomas Hoeg-Jensen

Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin.

ABSTRACTPurposeBasal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements.MethodsSelf-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection.ResultsWith depletion of phenol, insulin degludec and another fatty diacid–insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T3R3 conformation in presence of phenol and zinc, changing to T6 with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly.ConclusionsInsulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

Total Synthesis of desB30 Insulin Analogues by Biomimetic Folding of Single‐Chain Precursors

Insulin is a peptide hormone consisting of 51 amino acids in two chains with three disulfide bridges. Human insulin and various analogues are used for the treatment of diabetes and are produced recombinantly at ton scale. Herein, we report the chemical synthesis of insulin by the step‐wise, Fmoc‐based, solid‐phase synthesis of single‐chain precursors with solubilising extensions, which under redox conditions, spontaneously fold with the correct pairing of the three disulfide bridges. The folded, single‐chain, insulin precursors can be transformed into bioactive two‐chain desB30 insulin by the simultaneous removal of the solubilising extension (4–5 residues) and the chain‐bridging C‐peptide (3–5 residues) by employing Achromobacter lyticus protease—a process well‐known from the yeast‐based recombinant production of insulin. The overall yields of synthetic insulins were as much as 6 %, and the synthetic process was straightforward and not labour intensive.

Formation of peptide thioamides by use of Fmoc amino monothioacids and PyBOP.

Endothiopeptides have been obtained by using PyBOP® promoted coupling between Fmoc-protected amino monothioacids and amino acid or peptide esters. The protected endothiopeptides (Fmoc-Gly-ψ(CSNH)-Phe-OEt, Fmoc-Tyr(But)-ψ(CSNH)-Gly-Gly-Phe-Leu-OBut and Fmoc-Gln(Trt)-ψ(CSNH)-Phe-OEt) were formed (55, 45 and 37% yield) in admixture with the corresponding oxopeptides, from which they were easily separated chromatographically. Preliminary racemisation studies indicate that products of high optical purity are obtained. The mechanism for endothiopeptide formation is briefly discussed.

Chemo- and Regioselective Ethynylation of Tryptophan-Containing Peptides and Proteins.

Ethynylation of various tryptophan-containing peptides and a single model protein was achieved using Wasers reagent, 1-[(triisopropylsilyl)ethynyl]-1,2-benziodoxol-3(1 H)-one (TIPS-EBX), under gold(I) catalysis. It was demonstrated by NMR that the ethynylation occurred selectively at the C2-position of the indole ring of tryptophan. Further, MS/MS showed that the tryptophan residues could be modified selectively with ethynyl functionalities even when the tryptophan was present as a part of the protein. Finally, the terminal alkyne was used to label a model peptide with a fluorophore by means of copper-catalyzed click chemistry.

Decarboxylation-based traceless linking with aroyl acrylic acids

Abstract β-Keto carboxylic acids are known to decarboxylate readily. In our pursuit to synthesize β-indolinyl propiophenones, we have exploited this chemistry as a mean of establishing a traceless handle. 2-Aroyl acrylic acids have been esterified to a trityl resin, after which Michael-type addition of indolines have been performed. Upon cleavage, the products are decarboxylated, and the β-indolinyl propiophenones are isolated. The reaction conditions have been optimized, and a small library has been prepared.

Masked side-chain aldehyde amino acids for solid-phase synthesis and ligation

Abstract The masked aldehyde amino acid Fmoc-Hyl(Boc-oxazolidine) 1 , has been synthesized from the parent amino acid in five steps (3 pots). The employed protection scheme renders 1 well suited for standard Fmoc-based solid-phase assembly of peptides and similar structures, including TFA-based deprotections. The resulting peptides possess a side-chain 1,2-amino alcohol, and post-TFA treatment, periodate oxidation of the unprotected peptide unmasks the aldehyde function. The given order of transformations circumvents the known, problematic release of reactive aldehydes in TFA solution. The post-TFA generated peptide aldehydes have been utilized in model chemo-selective ligations, with formation of hydrazone constructs. Additionally, Fmoc-Hyl(Alloc-oxazolidine) 10 was synthesized, and used for on-resin aldehyde generation and hydrazone transformation

Preparation and application of O-amino-serine, Ams, a new building block in chemoselective ligation chemistry

The non‐codable amino acid O‐amino‐serine, Ams, has been prepared in both L‐ and D‐forms as the orthogonally protected derivative, Fmoc‐Ams(Boc)‐OH (1 and 2). This new amino acid derivative is useful for chemoselective ligations. Under acidic conditions and in the presence of all other common amino acid functionalities, the oxyamine function selectively forms oxime linkages with aldehydes. The Ams residue has been incorporated into both ends of the peptide sequence Asp‐Leu‐Trp‐Gln‐Lys using standard SPPS. The deprotected peptide has been used for chemical ligation to afford a peptide dimer as well as a glycopeptide. Ams racemization was found to be negligible, as monitored by HPLC separation of Ams dipeptide diastereomers. Copyright

Backbone cyclic insulin

Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single‐chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C‐terminal lysine and proximal N‐terminal. The presence of the ring‐closing bond and the native insulin disulfide patterns were documented by LC–MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity. Copyright

Perfluoroalkyl chains direct novel self-assembly of insulin.

The self-assembly of biopharmaceutical peptides into multimeric, nanoscale objects, as well as their disassembly to monomers, is central for their mode of action. Here, we describe a bioorthogonal strategy, using a non-native recognition principle, for control of protein self-assembly based on intermolecular fluorous interactions and demonstrate it for the small protein insulin. Perfluorinated alkyl chains of varying length were attached to desB30 human insulin by acylation of the ε-amine of the side-chain of LysB29. The insulin analogues were formulated with Zn(II) and phenol to form hexamers. The self-segregation of fluorous groups directed the insulin hexamers to self-assemble. The structures of the systems were investigated by circular dichroism (CD) spectroscopy and synchrotron small-angle X-ray scattering. Also, the binding affinity to the insulin receptor was measured. Interestingly, varying the length of the perfluoroalkyl chain provided three different scenarios for self-assembly; the short chains hardly affected the native hexameric structure, the medium-length chains induced fractal-like structures with the insulin hexamer as the fundamental building block, while the longest chains lead to the formation of structures with local cylindrical geometry. This hierarchical self-assembly system, which combines Zn(II) mediated hexamer formation with fluorous interactions, is a promising tool to control the formation of high molecular weight complexes of insulin and potentially other proteins.