Selective inactivation of rabbit muscle glycerophosphate dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Incorporation of a proline analog into collagen polypeptides was studied by incubating matrix-free tendon cells from 17-day-old chick embryos with cis-4-hydroxy-l-proline. Velocity sedimentation of intracellular polypeptides provided further evidence that incorporation of the analog into protein prevented the pro-α- and pro-γ-chains of procollagen from folding into a stable triple-helical conformation. The size of the newly synthesized intracellular and extracellular protein was examined under conditions which prevented proteolysis during processing of the samples. In contrast to previous observations, the results demonstrated that there was little if any intracellular degradation of nonhelical pro-α-and pro-γ-chains containing the proline analog, and a fraction of the nonhelical pro-γ-chains was secreted into the medium without extensive degradation. In further studies, the cells were incubated with ¹⁴C lysine, and the synthesis of glycosylated hydroxylysyl residues was measured in control cells and in cells incubated with cis-4-hydroxy-l-proline. The results demonstrated that the content of glycosylated hydroxylysyl residues in nonhelical pro-γ-chains containing cis-4-hydroxy-l-proline was increased twofold as compared to the triple-helical procollagen in control cells. The results suggested that under control conditions folding into the triplehelical conformation limits the extent of glycosylation of collagen. If folding is prevented or delayed, procollagen polypeptides are more extensively glycosylated.     

Decreased thermal stability of collagens containing analogs of proline or lysine

Fibroblasts were incubated with analogs of proline or lysine and the thermal stability of procollagen molecules containing the analogs was investigated using pepsin digestion at different temperatures as an enzymatic probe of conformation. The procollagens containing either 4-cis-hydroxy-l-proline, 3,4-dehydroproline, or 4,5-trans-dehydrolysine were less stable than normal procollagen and these abnormal collagens were largely in a non-triplehelical conformation within the cells at 37 °C. These results support the idea that procollagen molecules which are not in a triple-helical conformation are not secreted at a normal rate. Procollagens containing both 4,5-trans-dehydrolysine and a proline analog were much less stable than molecules containing a single type of analog. This result suggests that simultaneous administration of both types of analogs may have a greater effect on collagen accumulation in whole-animal experiments than administration of a single analog.     

Cyclic side-chain-linked opioid analogs utilizing cis- and trans-4-aminocyclohexyl-d-alanine

Cyclization of linear sequences is a well recognized tool in opioid peptide chemistry for generating analogs with improved bioactivities. Cyclization can be achieved through various bridging bonds between peptide ends or side-chains. In our earlier paper we have reported the synthesis and biological activity of a cyclic peptide, Tyr-c[d-Lys-Phe-Phe-Asp]NH₂ (1), which can be viewed as an analog of endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂). Cyclization was achieved through an amide bond between side-chains of d-Lys and Asp residues. Here, to increase rigidity of the cyclic structure, we replaced d-Lys with cis- or trans-4-aminocyclohexyl-d-alanine (d-ACAla). Two sets of analogs incorporating either Tyr or Dmt (2′,6′-dimethyltyrosine) residues in position 1 were synthesized. In the binding studies the analog incorporating Dmt and trans-d-ACAla showed high affinity for both, μ- and δ-opioid receptors (MOR and DOR, respectively) and moderate affinity for the κ-opioid receptor (KOR), while analog with Dmt and cis-d-ACAla was exceptionally MOR-selective. Conformational analyses by NMR and molecular docking studies have been performed to investigate the molecular structural features responsible for the noteworthy MOR selectivity.     

Inhibition of tropoelastin secretion by incorporation of DL-3,4-dehydroproline

Cells isolated from embryonic chick aorta were incubated in suspension culture with labeled amino acids and proline analogs. Incorporation of 4-cis-hydroxy-l-proline inhibited the secretion of labeled procollagen but not tropoelastin, while incorporation of dl-3,4-dehydroproline inhibited the secretion of both proteins and caused them to accumulate intracellularly. Protein synthesis did not appear to be significantly diminished during the 2-h incubation period. Incorporation of dl-3,4-dehydroproline may alter the conformation of tropoelastin leading to abnormal intracellular processing and a decreased rate of secretion.     

A criterion to determine whether cis-4-hydroxyproline is produced in animal tissues

Hydrolyzates of tissues that had been labeled with [¹⁴C]proline often contain significant amounts of cis-4-hydroxy[¹⁴C]proline. Since animal cells do not contain an enzyme which can effect formation of cis-4-hydroxyproline, there are only two possible explanations for its presence. Either it is formed during acid hydrolysis of trans-4-hydroxyproline (which is synthesized by cells and is a common constituent of connective tissues), or it is produced by a nonenzymatic mechanism such as attack by oxygen radicals. It is important to resolve this issue because if a nonenzymatic mechanism is active in connective tissues, then it will be necessary to reevaluate currently accepted ideas about production of hydroxyproline. This communication describes a method for distinguishing between the two alternate explanations. Tissues or cells are labeled with [¹⁴C]proline, and then a known amount of trans-4-hydroxy[³H]proline is added to each sample before hydrolysis; the relative amounts of [¹⁴C]- and [³H]-cis-4-hydroxyproline are compared after hydrolysis. It is known from a separate series of measurements with mixtures of [¹⁴C]- and [³H]-trans-4-hydroxyproline standards that there is a very high correlation (r = 0.998) between acid-induced formation of the [¹⁴C]- and [³H]-cis epimers. One can thus compare the amount of cis-4-hydroxy[¹⁴C]proline in a hydrolyzate from a biological system with the amount that would be expected if it were all formed during acid hydrolysis. This method was used to show that fibroblasts cultured under conditions commonly used to study collagen metabolism do not produce cis-4-hydroxyproline. This result strongly suggests that nonenzymatic hydroxylation does not normally occur in cell culture systems.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Identification of cis-5-methylproline in hydrolysates of actinomycin Z 5

Actinomycins of the Z series, synthesized by $\text{Streptomyces}$$\text{fradiae}$, contain the unusual amino acid, N-methylalanine, but no proline. Hydrolysates of actinomycin Z₅ were investigated using paper, gas and ion-exchange chromatographic procedures. Identification of an unknown amino acid in actinomycin Z₅ as 5-methylproline was confirmed by mass spectrometry. Configuration of the imino acid was defined as $\text{cis}$.     

Imino acid biosynthesis in Delonix regia

The biosynthesis of l-azetidine-2-carboxylic acid and trans-3-hydroxy-l-proline has been studied in Delonix regia seedlings by labelled precursor feeding techniques. α,γ-Diaminobutyric acid was incorporated into azetidine-2-carboxylic acid more efficiently than homoserine, methionine or aspartic acid. More radioactivity from proline was found in trans-3-hydroxyproline after 2 day's than after 4-day's metabolism, indicating a continuous turnover of the hydroxyimino acid in seedlings.     

Potent MCH-1 receptor antagonists from cis-1,4-diaminocyclohexane-derived indane analogs

Benzimidazole and indane are the two key fragments in our potent and selective MCH-1 receptor (MCHR1) antagonists. To identify novel linkers connecting the two fragments, we investigated diamino-cycloalkane-derived analogs and discovered highly potent antagonists with cis-1,4-diaminocyclohexane as a unique spacer in this chemical class. Structural overlay suggested that cis-1-substituted-4-aminocyclohexane functions as a bioisostere of 4-substituted-piperidine and that the active conformation adopts a U-shaped orientation.     

Synthesis and characterization of cis- and trans-2,3-epoxybutane-1,4-diol 1,4-bisphosphate,potential affinity labels for enzymes that bind sugar bisphosphates

cis- and trans-2,3-Epoxybutane-1,4-diol 1,4-bisphosphate, which can be considered reactive analogs of several sugar bisphosphates, have been synthesized in a continuing effort to develop new and diverse affinity labeling reagents for enzymes which bind phosphorylated substrates. cis-2,3-Epoxybutane-1,4-diol was obtained by epoxidation of commercially available cis-2-butene-1,4-diol with m-chloroperbenzoic acid; the trans epoxide was obtained by reduction of 2-butyne-1,4-diol with LiAlH₄ followed by epoxidation with m-chloroperbenzoic acid. The diols were phosphorylated with diphenyl chlorophosphate, and the phenyl blocking groups were then removed by Pt-catalyzed hydrogenation. By the criterion of their reaction with the sulfhydryl group of glutathione, the phosphorylated epoxides are 6000 times less electrophilic than the previously described and structurally similar reagent 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.     

Selective inhibition of proline hydroxylation by 3,4-dehydroproline

The effect of proline analogs on peptidyl proline hydroxylation has been studied in vivo using aerated root slices of Daucus carota. One analog, 3,4-dehydroproline, acted at micromolar concentrations to rapidly and selectively inhibit peptidyl proline hydroxylation. A structurally altered hydroxyproline-rich cell wall glycoprotein was synthesized and secreted by dehydroproline-treated tissue. The capacity to hydroxylate proline recovered slowly following a short pulse treatment with the analog, with a halftime for recovery of about 24 hours. Recovery was not altered by supplying exogenous proline. Dehydroproline had little effect on the induction of nitrate reductase by nitrate, nor on wound-induced increases in amino acid uptake and protein synthesis. In contrast, other proline analogs inhibit proline hydroxylation only at millimolar concentrations. It is hypothesized that dehydroproline acts as an enzyme-activated suicide inhibitor of prolyl hydroxylase. This analog should become a useful tool for elucidating the functional significance of hydroxyproline-rich glycoproteins.     

Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the μ-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-β-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies.     

Cis-4-hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid from Calliandra

cis-4-Hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid were isolated from leaves of Calliandra pittieri. A system for resolving the eight imino acids isolated from Calliandra is described.     

Synthetic biology of proteins: tuning GFPs folding and stability with fluoroproline

Background

Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C^γ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. C^γ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.

Methodology/Principal Findings

In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the C^γ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.

Conclusions/Significance

We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the C^γ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.     

Biological and conformational studies of [Val4]morphiceptin and [D-Val4]morphiceptin analogs incorporating cis-2-aminocyclopentane carboxylic acid as a peptidomimetic for proline

We report the synthesis, biological activity, and conformational analysis of tetrapeptide analogs related to [Val4]morphiceptin and [D-Val4]morphiceptin in which the proline at the second position has been replaced with cis-2-aminocyclopentane carboxylic acid (cis-2-Ac5c). Since the cis-2-Ac5c residue contains a normal amide, only the trans form has been observed about the amide bond between the first and second residues. The cis-2-Ac5c is a beta amino acid with two chiral centers resulting in two possible configurational isomers, namely (1S, 2R) and 1R, 2S) forms. The analogs containing the (1S, 2R)-Ac5c residue show activity at the mu-receptor but are inactive at the delta-receptor, resulting in a high selectivity for the mu-receptor. The (1R,2S)-Ac5c containing analogs are completely inactive at both the mu- and delta-receptors. The conformational analysis indicates that the separation of the aromatic rings of the tyrosine and phenylalanine residues, as expressed by the center-to-center distance, is 10.1-12.7 A for the preferred conformations of the bioactive analogs containing the (1S,2R)-Ac5c residue while a range of 4.8-7.0 A is observed for the preferred conformations of the inactive analogs with the (IR,2S)-Ac5c residue. A comparison of the findings from the conformational analysis and biological assays establishes the fact that a relatively large separation of the two aromatic side chains is required for the mu-opioid receptor activity of these molecules. Since the tetrapeptide amides studied in this investigation show similar biological profiles to those of the morphiceptin-related analogs, we have compared the preferred conformations estimated for the cis-2-Ac5c containing analogs with those of morphiceptin. One of the low energy conformations calculated for morphiceptin with the cis form about the tyrosine and proline residues has considerable topological similarity with the bioactive analogs containing the (1S,2R)-Ac5c residue, indicating that the cis from about these two residues is required for the biological activity of the morphiceptin-related analogs containing the proline at the second position.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Enzymatic preparation of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines

The lipase catalyzed resolution of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines have been studied. For all the heterocycles, the best enantioselectivity was obtained using Candida antarctica lipases A and B as catalysts in hydrolytic processes. The absolute configuration of the optically pure obtained heterocycles has been assigned.     

Competitive antagonism of insect GABA receptors by 4-substituted 5-(4-piperidyl)-3-isothiazolols

γ-Aminobutyric acid (GABA) receptors are important targets of parasiticides/insecticides. Several 4-substituted analogs of the partial GABA_A receptor agonist 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) were synthesized and examined for their antagonism of insect GABA receptors expressed in Drosophila S2 cells or Xenopus oocytes. Thio-4-PIOL showed weak antagonism of three insect GABA receptors. The antagonistic activity of Thio-4-PIOL was enhanced by introducing bicyclic aromatic substituents into the 4-position of the isothiazole ring. The 2-naphthyl and the 3-biphenylyl analogs displayed antagonist potencies with half maximal inhibitory concentrations in the low micromolar range. The 2-naphthyl analog induced a parallel rightward shift of the GABA concentration–response curve, suggesting competitive antagonism by these analogs. Both compounds exhibited weak insecticidal activities against houseflies. Thus, the orthosteric site of insect GABA receptors might be a potential target site of insecticides.     

Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l-azetidine-2-carboxyl³-bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l-glutamyl residue to the amino terminus of 3,4-dehydro-l-prolyl³-bradykinin and trans-4-hydroxy-l-prolyl³-bradykinin resulted in competitive inhibitors of increased effectiveness with K_i, values approximately 10^?4m. One of the peptides, l-3,4-dehydro-l-prolyl³-bradykinin, appeared to serve as a substrate for prolyl hydroxylase.     

Synthesis,receptor binding studies,optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis‐4‐amino‐L‐proline residues

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis‐4‐aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.