Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin

We have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A20-A21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A20-A21 amide bond can modulate biological activity independently of receptor binding affinity. The A20-A21 amide bond is thus considered as an important element in the "message region" of insulin.     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 2. [21-Asparagine diethylamide-A]insulin

We have synthesized [21-asparagine diethylamide-A]insulin, which differs from the parent molecule in that the free carboxyl group of the C-terminal amino acid residue, asparagine, of the A chain moiety has been converted to a diethylamide group. The analogue displays equivalent potency in receptor binding and biological activity, 48% and 56%, respectively, relative to bovine insulin. In contrast, we have reported previously [Burke, G. T., Chanley, J. D., Okada, Y., Cosmatos, A., Ferderigos, N., & Katsoyannis, P. G. (1980) Biochemistry 19, 4547-4556] that [21-asparaginamide-A]insulin exhibits a divergence in these properties, ca. 60% in receptor binding and ca. 13% in biological activity. The disparity in the biological behavior of these analogues is discussed, and we ascribe the modulation of biological activity independent of receptor binding activity observed between these analogues to the difference in the negativity of the carbonyl oxygen of the A chain moiety C-terminal amino acid residue.     

21-arginine-A] insulin: a biologically active analog.

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.     

A new structural analog of human insulin-- [glutamine-B30] insulin

A new structural analogue of human insulin, [glutamine-B30]insulin, has been prepared by enzymatic-chemical means. This analogue differs from natural hormone by substitution of the ThrB30 residue. Biological activity of the [glutamine-B30]insulin is 100% in the mouse convulsion assay.     

Interaction between the A2 and A19 amino acid residues is of critical importance for high biological activity in insulin: [19-leucine-A]insulin

The replacement of tyrosine at position A19 by leucine in the insulin molecule led to an analogue, [19-leucine-A]insulin [( Leu19-A]insulin), displaying insignificant receptor binding affinity and in vitro biological activity less than 0.1 and 0.05%, respectively, compared to the natural hormone. This analogue along with the previously reported [2-glycine-A]-, [2-alanine-A]-, and [2-norleucine-A]insulins is the least potent insulin analogue we have examined. Circular dichroic studies showed that all these analogues are monomeric at concentrations at which insulin is primarily dimeric. We conclude that an aromatic ring at position A19 and the presence of the side chain of isoleucine at position A2 are each of critical importance for high biological activity in insulin. It appears that the van der Waals interaction between the side chain of isoleucine A2 and tyrosine A19, present in crystalline insulin, is among the most important determinants for high biological activity in insulin.     

Synthesis of an insulin analogue embodying a strongly fluorescent moiety, [19-Tryptophan-A]insulin

As part of our aim to study the conformation of insulin in solution by time-resolved fluorescence spectroscopy, we have synthesized the analogue [19-Tryptophan-A]insulin. In this compound, the tyrosine residue at position 19 of the A-chain of insulin, one of the most strongly conserved residues in insulins from various species, is substituted with the strongly fluorescent tryptophan residue. [19-Tryptophan-A]insulin displays 4.1±1.9% of the potency of natural insulin in binding to the insulin receptor from rat liver plasma membranes, 5.0±2.3% in stimulating lipogenesis in rat adipocytes, and 75.7±4% of the potency of insulin in radioimmunoassay. In connection with our previous work, these data indicate that an aromatic side chain at position A¹⁹ of insulin seems necessary but not sufficient for high biological activity. We further conclude that in regard to the immunogenic determinants of insulin, tryptophan in position A¹⁹ is an essentially neutral substitution for tyrosine in that position, in sharp contrast to the situation with regard to biological activity.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of ～17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays ～50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.     

Effect of N-methylation of selected peptide bonds on the biological activity of insulin. [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin-receptor interaction. Our previous work suggested that the A2-A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin-receptor interaction the [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin ([MeIle2-A]- and [MeVal3-A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2-A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2-A]insulin displayed a potency of 5.4 +/- 0.3% in stimulating lipogenesis and 4.6 +/- 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3-A]insulin displayed a potency of 2.1 +/- 0.2% in lipogenesis and 1.0 +/- 0.3% in receptor binding assays. In radioimmunoassays [MeIle2-A]- and [MeVal3-A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2-A8 segment brought about by N-methylation of the A1-A2 or A2-A3 peptide bonds.     

An insulin analogue with gamma-amino butyric acid substitution for A13Leu-A14Tyr.

An insulin A chain analogue, [A13-14 GABA, A21 Ala]A chain, for which the dipeptide Leu-Try at A13-A14 was substituted by a non-coded amino acid, gamma-amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid-phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13-14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50% in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C-N peptide bond by an unnatural C-C bond may have general meaning in structure and function studies of other proteins.     

B22-D-arginine]insulin: synthesis and biological properties

An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone.     

The A14 position of insulin tolerates considerable structural alterations with modest effects on the biological behavior of the hormone

As part of our aim to investigate the contribution of the tyrosine residue found in the 14 position of the A-chain to the biological activity of insulin, we have synthesized six insulin analogues in which the A14 Tyr has been substituted by a variety of amino acid residues. We have selected three hydrophilic and charged residues—glutamic acid, histidine, and lysine—as well as three hydrophobic residues—cycloleucine, cyclohexylalanine, and naphthyl-(1)-alanine—to replace the A14 Tyr. All six analogues exhibit full agonist activity, reaching the same maximum stimulation of lipogenesis as is achieved with procine insulin. The potency for five of the six analogues, [A14 Glu]-, [A14 His]-, [A14 Lys]-, [A14 cycloleucine]-, and [A14 naphthyl-(1)-alanine]-insulins in receptor binding assays ranges from 40–71% and in stimulation of lipogenesis ranges from 35-120% relative to porcine insulin. In contrast, the potency of the sixth analogue, [A14 cyclohexylalanine]insulin, in both types of assays is less than 1% of the natural hormone. The retention time on reversed-phase high-performance liquid chromatography for the first five analogues is similar to that of bovine insulin, whereas for the sixth analogue, [A14 cyclohexylalanine]insulin, it is approximately 11 min longer than that of the natural hormone. This suggests a profound change in conformation of the latter analogue. Apparently, the A14 position of insulin can tolerate a wide latitude of structural alterations without substantial decrease in potency. This suggests that the A14 position does not participate directly in insulin receptor interaction. Only when a substitution which has the potential to disrupt the conformation of the molecule is made at this position, is the affinity for the receptor, and hence the biological potency, greatly reduced.     

Structure-function studies on red pigment-concentrating hormone, II. The significance of the C-terminal tryptophan amide

The significance of the C-terminal tryptophan residue of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) regulating the blanching of the crustacean chromatophores has been investigated. RPCH and a number of analogues that differ only in the C-terminal part of the hormone, have been synthesized and assayed for biological activity on the shrimp Leander adspersus. It has been shown that the indole skeleton of tryptophan is an absolute requirement for the biological activity of the hormone. To provide maximum response the tryptophan must be blocked as the amide. The activity of synthetic [Tyr4]RPCH and adipokinetic hormone (AKH) purified from Schistocerca gregaria has been compared with the activity of synthetic RPCH.     

[A_(15)Asn,A_(17)Pro,A_(21)Ala]-胰岛素的体内外生物活性

对胰岛素的研究使得许多生命科学中的重要问题被认识．然而对于胰岛素结构和功能关系,仍有许多不清楚的地方．在胰岛素Ａ链的Ｎ端和Ｃ端各有一个螺旋区：Ａ２－８和Ａ１２－１８．杨士珍［１］的研究表明：缺失Ａ１２－１８肽段的Ａ链类似物虽然能和天然Ｂ链重组,但其重组产物只保留了天然胰岛素的约６％的体内外生物活性．这说明Ａ１２－１８对胰岛素的生物活性十分重要．但是这一结果还不能说明是Ａ１２－１８螺旋还是Ａ１２－１８肽段对胰岛素的生物活性十分重要．根据Ｃｈｕｏ－Ｆａｓｍａｎ法,在含有Ａｓｎ和Ｐｒｏ的肽段中不能形成…     

The effect of modifications of the A5 and A19 amino acid residues on the biological activity of insulin. [Leu5-A] and [Phe19-A] sheep insulins

Two analogs of sheep insulin, both differing from the native material by a single amino acid in the A chain, have been synthesized and isolated in highly purified form by procedures developed in this laboratory. In one case, the glutamine residue in position A⁵ was replaced by leucine ([Leu⁵-A]); in the other, the tyrosine residue in position A¹⁹ was replaced by phenylalanine ([Phe¹⁹-A]). The biological behavior of these analogs was compared with natural bovine insulin inin vitro tests and in receptor-binding assays, as well as in radioimmunoassay. In the stimulation of glucose oxidation by rat adipocytes, the analogs gave relative potencies of 30% and 7.8% for [Leu⁵-A] and [Phe¹⁹-A], respectively. Receptor-binding assays in rat liver plasma membranes showed similar behavior for both analogs. In radioimmunoassay, [Leu⁵-A] displayed a relative potency of 27.9%, while [Phe¹⁹-A] showed a relative potency of 19–27%, compared with bovine insulin. At high concentration, both analogs displayed the same maximal activity as bovine insulin, and the dose-response curves are essentially parallel. It is speculated that the interaction between the glutamine residue in position 5 and the tyrosine residue in position 19 of the A chain of insulin are important in maintaining a three-dimensional structure commensurate with high biological activity. The full intrinsic activity of both analogs at high concentrations and the similarity of the potency figures in receptor-binding and glucose-oxidation assays permit the further conclusion that the reduced potency in the latter assay can be ascribed wholly to the reduced binding affinity toward insulin receptors caused by the substitutions made in the analogs. The receptor-analog complexes are fully capable of triggering the next event in the chain leading to the biological response.     

The importance of the B10 amino acid residue to the biological activity of insulin. [Lys10-B] human insulin

An analog of human insulin, which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by lysine, has been synthesized and isolated in purified form. This analog, [10-lysine-B] insulin ([Lys¹⁰-B] insulin), in stimulating lipogenesis and in radioimmunoassays, exhibited potencies of 14.2% and 14.7%, respectively, as compared to the natural hormone. In insulin receptor binding in rat liver membranes, [Lys¹⁰-B] insulin was found to possess a potency of 17% compared to insulin. We have shown previously that substitution of the B¹⁰ polar residue histidine with the nonpolar leucine results in an analog exhibiting inin vivo assays 50% of the activity of the parent molecule. It is speculated that in insulin the relative size of the amino acid residue at B¹⁰, rather than its polarity, is the most important factor in maintaining a structure commensurate with high biological activity.For the previous paper in this series see Schwartzet al. (1981).     

Contribution of the B16 and B26 tyrosine residues to the biological activity of insulin

We report the synthesis and biological evaluation of five insulin analogues in which one or both of the B-chain tyrosine residues have been substituted. [B16 Phe]insulin and [B16 Trp]insulin display a very modest reduction in potency (c. 65%) relative to porcine insulin; [B26 Phe]insulin is less active (30–50%), and the doubly substituted [B16 Phe, B26 Phe]insulin displays still lower potency (c. 35%). The further substitution of Asp for B10 His in [B16 Phe, B26 Phe]insulin raises its activity to approximately twofold greater than natural insulin, an increase of approximately fivefold over the parent compound. We conclude that the bulk and/or aromaticity of the amino acid residue at position B16, but not its hydrogen-bonding capacity, contributes to the biological activity of the hormone. We further conclude that hydrogen-bonding capacity or special side-chain packing characteristics are required at the B26 position for insulin to display high biological activity.     

Toward the insulin-IGF-I intermediate structures: functional and structural properties of the [TyrB25NMePheB26] insulin mutant

The origins of differentiation of insulin from insulin-like growth factor I (IGF-I) are still unknown. To address the problem of a structural and biological switch from the mostly metabolic hormonal activity of insulin to the predominant growth factor activities of IGF-I, an insulin analogue with IGF-I-like structural features has been synthesized. Insulin residues Phe(B25) and Tyr(B26) have been swapped with the IGF-I-like Tyr(24) and Phe(25) sequence with a simultaneous methylation of the peptide nitrogen of residue Phe(B26). These modifications were expected to introduce a substantial kink in the main chain, as observed at residue Phe(25) in the IGF-I crystal structure. These alterations should provide insight into the structural origins of insulin-IGF-I structural and functional divergence. The [Tyr(B25)NMePhe(B26)] mutant has been characterized, and its crystal structure has been determined. Surprisingly, all of these changes are well accommodated within an insulin R6 hexamer. Only one molecule of each dimer in the hexamer responds to the structural alterations, the other remaining very similar to wild-type insulin. All alterations, modest in their scale, cumulate in the C-terminal part of the B-chain (residues B23-B30), which moves toward the core of the insulin molecule and is associated with a significant shift of the A1 helix toward the C-terminus of the B-chain. These changes do not produce the expected bend of the main chain, but the fold of the mutant does reflect some structural characteristics of IGF-1, and in addition establishes the CO(A19)-NH(B25) hydrogen bond, which is normally characteristic of T-state insulin.     

Synthetic insulin fragment with insulin-like biological activity.

An insulin fragment, representing the C-terminal functionally important site of its molecule and responsible for receptor binding, was synthesized. The fragment consists of two peptides: a dipeptide (A 20-21) and an octapeptide (B 19-26), linked with a disulfide bond (A20-B19). The biological activity of the newly synthesized fragment relative to insulin was assayed for the influence on glycogenesis and for the ability to stimulate glucose uptake. Comparative tests for the biological activity of the synthesized fragment and of the intact hormone allowed us to conclude that the fragment has insulin-like properties.     

Synthesis and properties of [A19-(p-fluorophenylalanine)] insulin

The synthesis of [Phe(F)A19]insulin (porcine) is described. First the protected [Phe(F)19]A-chain was assembled by segment condensation of [1-12] and [13-21] using the dicyclohexyldiimide/1-hydroxybenzotriazole procedure. [Phe(F)19]A-chain was purified by ion exchange chromatography after removal of all the protecting groups (Boc, But, OBut and S-Trt) and its conversion into the tetra-S-sulfonated derivative. [Phe(F)A19]insulin was prepared by combination with porcine B-chain and purified by gel filtration and ion-exchange chromatography. The in vitro biological activity of this analogue was 60%. CD spectra in the near and far UV are qualitatively very similar to those of insulin.     

D-Met2,Pro5] enkephalin [N1.5-beta-D-glucopyranosyl] amide: a glycosylpeptide with high antinociceptive activity

Different synthetic strategies have been attempted for the synthesis of a glycosylpeptide resulting from the covalent bonding of a sugar residue to the C-terminal carboxyl group of an enkephalin related pentapeptide. The final structure is: Tyr-D-Met-Gly-Phe-Pro [N1.5-beta-D-glucopyranosyl] amide. The in vitro potency on the GPI test of this analogue was IC50 = 64.0 nM. However, its antinociceptive activity by tail immersion tests, after intraperitoneal administration, was 2000 and 200 times higher than morphine in rats and mice, respectively.