Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro
- Omer AdOmer AdDepartment of Chemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Omer Ad
- ,
- Kyle S. HoffmanKyle S. HoffmanDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Kyle S. Hoffman
- ,
- Andrew G. CairnsAndrew G. CairnsDepartment of Chemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Andrew G. Cairns
- ,
- Aaron L. FeatherstonAaron L. FeatherstonDepartment of Chemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Aaron L. Featherston
- ,
- Scott J. Miller*Scott J. Miller*E-mail: [email protected]Department of Chemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Scott J. Miller
- ,
- Dieter Söll*Dieter Söll*E-mail: Dieter.Sö[email protected]Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Dieter Söll
- , and
- Alanna Schepartz*Alanna Schepartz*E-mail: [email protected]Department of Chemistry and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United StatesMore by Alanna Schepartz
Copyright © 2022 American Chemical Society. This publication is licensed under these Terms of Use.
This publication is Open Access under the license indicated. Learn More
Abstract
Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.
Synopsis
Wild type E. coli ribosomes elongate initiator tRNAs acylated with multiple benzoic acids and 1,3-dicarbonyl substrates to generate diverse aramid-peptide and polyketide-peptide hybrid molecules.
Introduction
ARTICLE SECTIONS
As far as we know, ribosomes have evolved for billions of years to perform a single reaction—formation of an amide bond between two α-amino acid substrates brought into proximity by tRNAs within the ribosome active site, the peptidyl transferase center (PTC). In cells and extracts, the chemistry possible within a wild type ribosome PTC has expanded to include reactions of more than 200 different nonproteinogenic α-amino and hydroxy acids; (1−4) ribosomes containing remodeled PTCs support amide bond formation to and from a small number of β-amino acids (5−7) and dipeptides (8,9) with limited efficiency. The combination of cell-free in vitro translation systems and ribozyme-catalyzed tRNA acylation reactions offers the opportunity for even greater reaction diversity, including the introduction of multiple N-alkyl, (10)d-α-, (11,12) α-hydroxy, (13) and β-amino acids, (14,15) the precursors of β-peptide foldamers. (16−19)
A second family of foldamer-like molecules are aramids, oligomers of substituted aminobenzoic acids. (20) Aramids possess remarkably varied properties. Kevlar, a polymer of 1,4-phenylenediamine and terephthaloyl chloride, is a strong and heat-resistant fiber, (21) whereas cystobactamids are DNA gyrase inhibitors active against Gram-negative bacteria. (22) Many other aramid foldamers with diverse and significant properties have been reported. (23−25) Recently, wild type E. coli ribosomes were shown to accept and elongate initiator tRNAs precharged with aromatic foldamer-dipeptide appendages. (26) Notably, in this case the foldamer monomers did not themselves react within the PTC, being displaced from the reaction center by a Gly–Phe dipeptide spacer. (26,27) Here, we report that wild type E. coli ribosomes accept and elongate precharged initiator tRNAs acylated directly with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates. The result is a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work provides new knowledge about the generality of flexizyme-promoted tRNA acylation reactions, expands the scope of ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for biosynthesis of polyketide-peptide natural products.
Results and Discussion
ARTICLE SECTIONS
As the first step toward the ribosomal synthesis of aramid-like peptides, we made use of an established microhelix (MH) gel-shift assay (28) and high-resolution mass spectrometry (Figure 1A) to evaluate whether the cyanomethyl esters of unsubstituted aminobenzoic acids were substrates for the flexizyme ribozyme eFx. (29) Incubation of cyanomethyl esters 1–3 (5 mM) with 25 μM microhelix MH and 25 μM eFx (Table S1) in bicine buffer at pH 9 for 48 h showed little or no evidence of MH acylation when the reaction products were evaluated on an acid-urea PAGE gel (Figure 1B). A low level of MH acylation by the o- and om-analogues 1 and 2 (and a trace with 3) could be observed using a highly sensitive RNase A/LC-HRMS assay (30) that detects the acylated adenine nucleoside (Figure 1C). We also investigated the extent of tRNA acylation using the alternative ribozyme dFx (31) and the 1,3-dinitrobenzyl esters of p- and o-aminobenzoic acid (4 and 5, respectively, as shown in Scheme S1). These substrates also failed to yield the expected MH products when incubated with dFx under standard conditions (14) and analyzed using acid-urea gels or RNase A/LC-HRMS (Figure S1), perhaps due to insolubility. Even the more soluble cyanomethyl ester of ortho-aminonicotinic acid analogue 6 reacted poorly in the presence of eFx (Figure S1).
Figure 1
Figure 1. Simple aminobenzoic acid cyanomethyl esters are poor substrates for the eFx ribozyme. (A) Protocol used to detect acylation of microhelix (MH) or tRNA by cyanomethyl esters 1–3. (B) Acid-urea gel-shift analysis of MH acylation by cyanomethyl esters 1–3 in the presence of ribozyme eFx. Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions after RNase A digestion. Adenine nucleosides acylated on the 2′ or 3′ hydroxyl of the 3′ terminal ribose of MH could be detected in eFx-promoted reactions of the cyanomethyl ester of l-phenylalanine (Phe) and aminobenzoic acid esters 1 and 2; trace levels were detected in reactions containing 3. These products were not observed in analogous reactions containing m-aminobenzoic acid (compound C).
The inability to efficiently acylate MH or tRNA with simple aminobenzoic acids in high yields using eFx or dFx led us to consider chemical acylation methods for the preparation of these materials. Isatoic anhydride can acylate the terminal 2′- or 3′-OH group of an unprotected tRNA, and the resulting anthraniloyl-tRNA (o-AN-tRNA) retains the ability to associate productively with EF-Tu-GTP. (32) Inspired by this reactivity, we incubated E. coli tRNAVal (ValT) or initiator tRNA (fMetT) with 8–80 mM isatoic anhydride in 90% CH3CN containing 2–5 mM NaOH for 3 h at 37 °C, digested the products with RNase A, and used LC-HRMS to detect the formation of nucleoside 7 (m/z = 387.1411, Scheme S1); this product will be observed only if reaction occurs at the tRNA 3′-end (Figure S2A). A peak corresponding to this mass was observed only in reactions containing tRNA, isatoic anhydride, and base; in the absence of base, the acylation efficiency dropped by 1–2 orders of magnitude (Figure S2B). Mindful of the fact that isatoic anhydride reagents can also modify RNA on the 2′-OH group of internal ribose residues in SHAPE reactions, (33) we also evaluated the reaction using ultra-performance liquid chromatography (UPLC), which (as expected) showed evidence of multiple reaction products, whereas eFx-promoted reactions did not (Figure S2C).
We next made use of a commercial in vitro translation kit (PURExpress Δ; aa, tRNA) to evaluate if an initiator tRNA (fMetT) acylated with o- (prepared using isatoic anhydride) or m-aminobenzoic acid (prepared using eFx) would be accommodated by the P-site of wild type E. coli ribosomes and initiate translation. We supplemented the kit with the requisite amino acids and tRNAs, precharged initiator tRNA (o- or m-AN-tRNA) (50–100 μM), and a duplex DNA template (0.5–1 μg) encoding the FLAG-containing polypeptide MVFDYKDDDDK (MVF-FLAG). After a 6 h incubation, the reaction mixture was treated with Ni-NTA resin to remove all PURExpress Δ components (which are His6-tagged), and the remaining material was analyzed by LC-HRMS (Figure 2A). If the o- or m-AN-tRNA initiates translation in place of an initiator tRNA charged with formyl methionine (fMet), then a polypeptide product containing the sequence AN-VFDYKDDDDK (AN-VF-FLAG) should be observed. Parallel experiments were performed using the elongator tRNA ValT acylated (using eFx) with β-Phe. (14) Clear evidence for the formation of a peptide carrying an aminobenzoic acid monomer was observed only in the presence of both DNA template and o-AN-tRNA (Figure 2B). The identity of this product was further confirmed by isotope labeling experiments (Figure S3) that showed the expected mass shift when the reaction was supplemented with 13C-labeled Phe. No AN-VF-FLAG polypeptide was detected in the presence of DNA template and m-AN-tRNA.
Figure 2
Figure 2. Initiator tRNA (fMetT) acylated with o-aminobenzoic acid can initiate translation within the PTC of wild type E. coli ribosomes. (A) Protocol used to evaluate whether an initiator tRNA (fMetT) acylated with o- (prepared using isatoic anhydride) or m-aminobenzoic acid (prepared using eFx) (AN-tRNA) could support translation in vitro. (B) LC-HRMS analysis of reaction products showing DNA template-dependent translation of a polypeptide whose mass corresponds to that of o-AN-VFDYKDDDDK (o-AN-VF-FLAG). No such polypeptide is observed in the absence of DNA template or in the presence of l-methionine. LC-HRMS analysis of an analogous β-Phe-containing polypeptide is shown for comparison.
Aminobenzoate esters hydrolyze exceptionally slowly, (34) suggesting that the electron-rich aromatic ring contributes to the low reactivity of 1–3. In addition, the structure of the ethyl ester of l-phenylalanine bound to Fx (as an Fx-tRNA fusion) (35) shows pi-stacking between Fx base guanine 24 and the l-phenylalanine aromatic ring; this stacking would be less favorable with an electron-rich arene. (36,37) To investigate whether reactivity in eFx-promoted reactions was correlated with arene electron density, we prepared a diverse set of substituted benzoic acid cyanomethyl esters (Figure 3A) and evaluated the extent to which eFx reactivity correlated with the sign and magnitude of the relevant sigma factor, which measures the inductive effect of the aromatic substituent. (38) As expected, benzoic acid cyanomethyl esters possessing strong electron-withdrawing substituents, such as penta-fluoro 8, p-nitro 9 (σ = +0.78), or p-Cl 10 (σ = +0.23), were excellent eFx substrates in model MH reactions, with 78-99% yields (Figure 3B,C). However, other factors are clearly important: a benzoic acid cyanomethyl ester possessing a weak electron-withdrawing substituent, such as p-azido 11 (σ = +0.08), was also an excellent substrate (yield of acylated MH = 74%), as were analogues possessing both strong and weak electron-donating substituents, such as p-methoxy 12 (σ = −0.27; yield of acylated MH = 62%) and p-methyl 13 (σ = −0.17; yield of acylated MH = 54%). Notably, the poorest yields were observed in eFx-promoted reactions of substrates 6 (yield of acylated MH = 25%) and 15 (yield of acylated MH = 23%), all of which contain one or more acidic protons/hydrogen bond donors, just like amino benzoic acids 1, 2, and 3. These results imply that the presence of hydrogen bond donors in certain positions contributes to the poor reactivity of amino benzoic acids 1–3. Consistent with this notion, p-hydroxybenzoic acid 16 [pKa = 8.3 (p-hydroxybenzoic acid methyl ester)] was a poor substrate, whereas alcohol 17 [pKa = 15 (benzyl alcohol)] and aldehyde 18 reacted well (Scheme S1 and Figure S4). It is possible that certain hydrogen bond donors alter the position of the aromatic ring in the eFx active site or coordinate and inactivate functional groups involved in catalysis. Determining the exact nature of these interactions is beyond the scope of this discussion but will be essential to effectively engineer new ribozymes that accept diverse foldamer building blocks in vitro and in vivo.
Figure 3
Figure 3. Probing structure–activity relationships for cyanomethyl esters of substituted benzoic acids in eFx-promoted acylation reactions. (A) Substituted benzoic acid cyanomethyl esters studied herein. (B) Acid-urea gel-shift analysis of MH acylation by cyanomethyl esters 6 and 8–15 in the presence of ribozyme eFx. Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions containing cyanomethyl esters 6 and 8–15 after RNase A digestion. Exact masses are reported in Table S2.
With a new set of aramid substrates in hand, we used the PURExpress Δ (aa, tRNA) in vitro translation kit to evaluate if initiator tRNAs acylated with diverse benzoic acids could be accommodated in the ribosomal P-site and initiate translation of an AR-VF-FLAG polypeptide carrying an aramid monomer (AR) at the N-terminus (Figure 4). Every benzoic acid cyanomethyl ester that acylated the microhelix MH with a yield >50% in an eFx-promoted reaction (Figure 3) was used to acylate fMetT, and translation reactions were performed and analyzed as described above. With one exception, every single AR-fMetT initiated translation of an AR-VF-FLAG peptide whose mass corresponded to incorporation of the prescribed substituted benzoic acid. The singular exception was p-azidobenzoic acid 11; in this case the mass of the isolated polypeptide was consistent with in situ reduction of the azide to an amine. These results demonstrate that diverse aramid-like monomers can be accommodated directly within the ribosomal P-site and act as acceptors for a natural α-amino acid in the A-site. They show further that use of p-azidobenzoic acid 11 effectively circumvents the poor reactivity of p-aminobenzoic acid 3 to generate a polypeptide with a p-aminobenzoic acid monomer at the N-terminus. The observation that wild type E. coli ribosomes can initiate translation using tRNAs acylated with diverse aramid-like monomers significantly expands the scope of in vitro translation reactions beyond that of Kawakami (39) and lays the initial groundwork for the biosynthesis of genetically encoded, sequence-defined polyaramid oligomers.
Figure 4
Figure 4. Initiator tRNAs acylated with diverse benzoic acids are accommodated in the ribosomal P-site and are elongated into AR-VF-FLAG polypeptides. LC-HRMS analysis of reaction products whose masses correspond to AR-VFDYKDDDDK (AR-VF-FLAG) polypeptides containing diverse substituted benzoic acid monomers.
We next sought to evaluate the relative efficiency of PURExpress reactions initiated with differentially acylated fMetT derivatives. To begin, we monitored the yield of fMet-VF-FLAG (approximated as the extracted ion abundance) as a function of time in PURExpress Δ reactions supplemented with either 50 μM precharged fMetT-fMet (charged using the dFx substrate fMet-DBE) or 50 μM l-methionine. The bulk of both reactions was complete within 100 min, but the yield of fMet-VF-FLAG in reactions supplemented with precharged fMetT-fMet was 1.5% of that obtained in reactions supplemented with l-methionine (Figure S5A). Next, we compared the extracted ion abundance (after 30–90 min) of the AR-VF-FLAG peptide initiated with fMetT precharged with benzoic acid ester 8. The yield of this AR-VF-FLAG polypeptide (C6F5-VF-FLAG) was 25–30% of the yield of fMet-VF-FLAG (generated in reactions supplemented with precharged fMetT-fMet) (Figure S5B) and within the range observed when translation was initiated with fMetT precharged with natural amino acids. (40) The relative yields of AR-VF-FLAG peptides initiated with other precharged fMetT derivatives were also comparable (Figure S5D), suggesting similar initiation efficiencies. We note that when ValT was precharged with β-Phe, the yield of fMet-β-Phe-F-FLAG was 5-fold higher than the yield of fMet-VF-FLAG generated with precharged fMetT (Figure S5C). As initiation complex assembly is the rate limiting step during translation, (41) the higher yield of fMet-β-Phe-F-FLAG relative to fMet-VF-FLAG is likely due to the difficulty assembling the translation initiation complex using non-natural fMetT derivatives. Benzoic acid monomers that were poor eFx substrates (yields <50%) in model MH reactions, such as 6 and 15 (Figure 3), failed to detectably initiate peptide synthesis from WT ribosomes in vitro. This observation suggests that the ribosome is largely agnostic of aramid structure, and that the concentration of non-natural fMetT derivative, rather than monomer structure, determines the reaction yield in PURExpress reactions. (42)
Like aramid natural products, (22) polyketide-peptide hybrid molecules are biosynthesized by mega-assemblies of complex protein enzymes; (43−45) the combination of peptide and polyketide-based functionality can translate into highly unique biological functions. (46−48) To evaluate whether wild type E. coli ribosomes are capable of biosynthesizing a polyketide-peptide hybrid, we prepared malonate derivatives 19–23 (Figure 5A). Model microhelix MHacylation reactions were analyzed using acid-urea gels (Figure 5B) and RNase A/LC-HRMS (Figure 5C) as described above. Although the malonic acid half esters 19, 20, and 21 were poor substrates for the requisite Fx analogue, cyanomethyl ester 22 was a moderate substrate, acylating the acylated MH in 40% yield. Although no gel-shift was observed in the eFx-promoted MH acylation reaction of cyanomethyl ester 23 (perhaps because of low molecular weight and/or polarity), (49) strong evidence for reaction was observed using RNase A/LC-HRMS (Figure 5C). Indeed, the addition of fMetT derivatives acylated with 22 and 23 (50–100 μM) to PURExpress Δ (aa, tRNA) in vitro translation reactions led to the isolation of polypeptides carrying malonates 22 and 23 (22-VF-FLAG and 23-VF-FLAG, respectively), whose masses were confirmed by LC-HRMS (Figure 5D). The yield of 23-VF-FLAG, estimated as described above, was approximately 20% of the yield of fMet-VF-FLAG produced in reactions supplemented with precharged fMetT (Figure S5A). We conclude that extant E. coli ribosomes have the capacity to biosynthesize simple polyketide-peptide hybrid molecules.
Figure 5
Figure 5. Wild type E. coli ribosomes support the biosynthesis of polyketide-peptide hybrid molecules. (A) Malonic esters 19–23 evaluated as substrates for eFx or dFx. (B) Acid-urea gel-shift analysis of MH acylation by esters 19–23 in the presence of eFx (19, 22) or dFx (20, 21,23). Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions containing esters 19–23 after RNase A digestion. (D) LC-HRMS analysis of reaction products whose masses corresponds to Mal-VFDYKDDDDK (Mal-VF-FLAG) polypeptides containing methyl and nitrobenzyl malonates 23 and 22. ND = not determined due to lack of separation from unacylated microhelix. Exact masses are reported in Table S2.
In summary, here we report that wild type E. coli ribosomes accept precharged initiator tRNAs acylated with multiple substituted benzoic acids, including the monomeric unit of Kevlar, as well as malonyl (1,3-dicarbonyl) substrates. The ribosome then elongates these substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of reactions catalyzed by both flexizyme and wild type ribosomes, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for biosynthesis of polyketide-peptide natural products.
Safety Statement
No unexpected or unusually high safety hazards were encountered during the execution of these experiments.
Supporting Information
ARTICLE SECTIONS
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.9b00460.
Synthesis and characterization of flexizyme substrates; and procedures for formation, characterization, purification, and analysis of tRNA acylation and IVT reactions (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
ARTICLE SECTIONS
- Scott J. Miller - Department of Chemistry and , Yale University, New Haven, Connecticut 06520, United States;
http://orcid.org/0000-0001-7817-1318;
- Dieter Söll - Department of Molecular Biophysics and Biochemistry and , Yale University, New Haven, Connecticut 06520, United States;http://orcid.org/0000-0002-3077-8986;
- Alanna Schepartz - Department of Chemistry and Department of Molecular Biophysics and Biochemistry and , Yale University, New Haven, Connecticut 06520, United States;http://orcid.org/0000-0003-2127-3932;
- Aaron L. Featherston - Department of Chemistry and , Yale University, New Haven, Connecticut 06520, United States;http://orcid.org/0000-0003-0918-067X
Acknowledgments
ARTICLE SECTIONS
This work was supported by the Center for Genetically Encoded Materials, an NSF Center for Chemical Innovation (NSF CHE-1740549). O.A. was supported in part by Agilent Technologies as an Agilent Fellow.
References
ARTICLE SECTIONS
This article references 49 other publications.
- 1Guo, J.; Wang, J.; Anderson, J. C.; Schultz, P. G. Addition of an α-hydroxy acid to the genetic code of bacteria. Angew. Chem., Int. Ed. 2008, 47, 722– 725, DOI: 10.1002/anie.200704074[Crossref], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhslalt74%253D&md5=b12c1b9b6b01e438d5d74943fb677c90Addition of an α-hydroxy acid to the genetic code of bacteriaGuo, Jiantao; Wang, Jiangyun; Anderson, J. Christopher; Schultz, Peter G.Angewandte Chemie, International Edition (2008), 47 (4), 722-725CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The α-hydroxy acid p-hydroxy-L-phenyllactic acid has been genetically incorporated into proteins in E. coli in response to an amber nonsense codon. The site-specific introduction of backbone ester mutations was used for the site-specific hydrolysis of an affinity tag, as well as for detn. of the energetic contributions of backbone hydrogen bonds to protein stability.
- 2Chin, J. W. Expanding and reprogramming the genetic code. Nature 2017, 550, 53– 60, DOI: 10.1038/nature24031[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Sqs73J&md5=2cf1adcbc37e3fd9f077e20b5c3c13c8Expanding and reprogramming the genetic codeChin, Jason W.Nature (London, United Kingdom) (2017), 550 (7674), 53-60CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chem. and phys. properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.
- 3Vargas-Rodriguez, O.; Sevostyanova, A.; Söll, D.; Crnković, A. Upgrading aminoacyl-tRNA synthetases for genetic code expansion. Curr. Opin. Chem. Biol. 2018, 46, 115– 122, DOI: 10.1016/j.cbpa.2018.07.014[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVSmtLfK&md5=4aa6fece6db9278a047a1a3ff6f27911Upgrading aminoacyl-tRNA synthetases for genetic code expansionVargas-Rodriguez, Oscar; Sevostyanova, Anastasia; Soll, Dieter; Crnkovic, AnaCurrent Opinion in Chemical Biology (2018), 46 (), 115-122CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biol. Progress in this field has enabled site-specific incorporation of over 200 chem. and structurally diverse amino acids into proteins in an increasing no. of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the L-alpha-amino acid structure (e.g. D-amino acid or Beta-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient aminoacyl-tRNA synthetases for genetic code expansion.
- 4Young, D. D.; Schultz, P. G. Playing with the molecules of life. ACS Chem. Biol. 2018, 13, 854– 870, DOI: 10.1021/acschembio.7b00974[ACS Full Text
], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2nur0%253D&md5=89996e366e3b9b94a0efde80f89e52cfPlaying with the Molecules of LifeYoung, Douglas D.; Schultz, Peter G.ACS Chemical Biology (2018), 13 (4), 854-870CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Our understanding of the complex mol. processes of living organisms is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of biol. macromols., is allowing chemists to begin to harness and reprogram the remarkable machinery of life. Here we review one such example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins in ways previously unimaginable. - 5Maini, R.; Nguyen, D. T.; Chen, S.; Dedkova, L. M.; Chowdhury, S. R.; Alcala-Torano, R.; Hecht, S. M. Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center. Bioorg. Med. Chem. 2013, 21, 1088– 1096, DOI: 10.1016/j.bmc.2013.01.002[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslChu7c%253D&md5=bfdfbefdebf3a546efa39bc6d858b3b2Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase centerMaini, Rumit; Nguyen, Dan T.; Chen, Shengxi; Dedkova, Larisa M.; Chowdhury, Sandipan Roy; Alcala-Torano, Rafael; Hecht, Sidney M.Bioorganic & Medicinal Chemistry (2013), 21 (5), 1088-1096CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Ribosomes contg. modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 prepns. for in vitro protein biosynthesis expts. When utilized in the presence of mRNAs contg. UAG codons at predetd. positions + β-alanyl-tRNACUA, the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted β-amino acids, and direct evidence for β-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in expts. employing four substituted β-amino acids, including α-methyl-β-alanine, β,β-dimethyl-β-alanine, β-phenylalanine, and β-(p-bromophenyl)alanine. The β-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of β-alanine and representative α-L-amino acids. The isolated proteins contg. the modified β-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the β-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of β,β-dimethyl-β-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the β-amino acid.
- 6Maini, R.; Chowdhury, S. R.; Dedkova, L. M.; Roy, B.; Daskalova, S. M.; Paul, R.; Chen, S.; Hecht, S. M. Protein synthesis with ribosomes selected for the incorporation of β-amino acids. Biochemistry 2015, 54, 3694– 3706, DOI: 10.1021/acs.biochem.5b00389[ACS Full Text], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosFaksLw%253D&md5=9a23376fcfad051c01a6c8b15a523ae6Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino AcidsMaini, Rumit; Chowdhury, Sandipan Roy; Dedkova, Larisa M.; Roy, Basab; Daskalova, Sasha M.; Paul, Rakesh; Chen, Shengxi; Hecht, Sidney M.Biochemistry (2015), 54 (23), 3694-3706CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In an earlier study, β3-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β3-puromycin employed for ribosome selection influences the regio- and stereochem. preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNACUA activated with each of four methyl-β-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochem. as the O-methylated β-tyrosine moiety of β3-puromycin. Also conducted were a selection of clones that are responsive to β2-puromycin and a demonstration of reversal of the regio- and stereochem. preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.
- 7Melo Czekster, C.; Robertson, W. E.; Walker, A. S.; Söll, D.; Schepartz, A. In vivo biosynthesis of a β-amino acid-containing protein. J. Am. Chem. Soc. 2016, 138, 5194– 5197, DOI: 10.1021/jacs.6b01023[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVymsrk%253D&md5=ccf95e341314ea854a164a870a0f28ceIn Vivo Biosynthesis of a β-Amino Acid-Containing ProteinMelo Czekster, Clarissa; Robertson, Wesley E.; Walker, Allison S.; Soll, Dieter; Schepartz, AlannaJournal of the American Chemical Society (2016), 138 (16), 5194-5197CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 exts. and incubated with chem. mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here the authors report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β3-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β3-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.
- 8Maini, R.; Dedkova, L. M.; Paul, R.; Madathil, M. M.; Chowdhury, S. R.; Chen, S.; Hecht, S. M. Ribosome-mediated incorporation of dipeptides and dipeptide analogues into proteins in vitro. J. Am. Chem. Soc. 2015, 137, 11206– 11209, DOI: 10.1021/jacs.5b03135[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlOqu7jO&md5=3be559cec7e2dc8519d0d06ecc5db9c8Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in VitroMaini, Rumit; Dedkova, Larisa M.; Paul, Rakesh; Madathil, Manikandadas M.; Chowdhury, Sandipan Roy; Chen, Shengxi; Hecht, Sidney M.Journal of the American Chemical Society (2015), 137 (35), 11206-11209CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Plasmids contg. 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addn. to the pre-existing wild-type ribosomes. These clones were screened with a deriv. of puromycin, a natural product which acts as an analog of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin deriv. in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analog to recognize dipeptides. Several clones inhibited by the puromycin deriv. were used to make S-30 prepns., and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analog (4) and a fluorescent oxazole (5) having amine and carboxyl groups approx. the same distance apart as in a normal dipeptide. A protein contg. both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogs.
- 9Chen, S.; Ji, X.; Gao, M.; Dedkova, L. M.; Hecht, S. M. In cellulo synthesis of proteins containing a fluorescent oxazole amino acid. J. Am. Chem. Soc. 2019, 141, 5597– 5601, DOI: 10.1021/jacs.8b12767[ACS Full Text], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlt1yksLw%253D&md5=6f793dac4189184d1da4cc22bff25c3cIn Cellulo Synthesis of Proteins Containing a Fluorescent Oxazole Amino AcidChen, Shengxi; Ji, Xun; Gao, Mingxuan; Dedkova, Larisa M.; Hecht, Sidney M.Journal of the American Chemical Society (2019), 141 (14), 5597-5601CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved α-L-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpuromycin analog, were employed to incorporate dipeptides and dipeptidomimetics. Presently, the authors report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asym. center or α-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (PylRS). Initially, a plasmid en-coding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium contg. oxazole 2 resulted in the elaboration of RRM1 contg. the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo contg. oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homolog of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent, and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo.
- 10Subtelny, A. O.; Hartman, M. C. T.; Szostak, J. W. Ribosomal synthesis of N-methyl peptides. J. Am. Chem. Soc. 2008, 130, 6131– 6136, DOI: 10.1021/ja710016v[ACS Full Text], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXksFarsr0%253D&md5=31e1ecb5c2a4c2ba0b88ad281fdb5ae8Ribosomal Synthesis of N-Methyl PeptidesSubtelny, Alexander O.; Hartman, Matthew C. T.; Szostak, Jack W.Journal of the American Chemical Society (2008), 130 (19), 6131-6136CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)N-Me amino acids (N-Me AAs) are a common component of nonribosomal peptides (NRPs), a class of natural products from which many clin. important therapeutics are obtained. N-Me AAs confer peptides with increased conformational rigidity, membrane permeability, and protease resistance. Hence, these analogs are highly desirable building blocks in the ribosomal synthesis of unnatural peptide libraries, from which functional, NRP-like mols. may be identified. By supplementing a reconstituted Escherichia coli translation system with specifically aminoacylated total tRNA that has been chem. methylated, the authors have identified three N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) that are efficiently incorporated into peptides by the ribosome. Moreover, the authors have demonstrated the synthesis of peptides contg. up to three N-Me AAs, a no. comparable to that found in many NRP drugs. With improved incorporation efficiency and translational fidelity, it may be possible to synthesize combinatorial libraries of peptides that contain multiple N-Me AAs. Such libraries could be subjected to in vitro selection methods to identify drug-like, high-affinity ligands for protein targets of interest.
- 11Dedkova, L. M.; Fahmi, N. E.; Golovine, S. Y.; Hecht, S. M. Enhanced d-amino acid incorporation into protein by modified ribosomes. J. Am. Chem. Soc. 2003, 125, 6616– 6617, DOI: 10.1021/ja035141q[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjs12ks7g%253D&md5=932096443b831dd0620db2c301b5b187Enhanced D-Amino Acid Incorporation into Protein by Modified RibosomesDedkova, Larisa M.; Fahmi, Nour Eddine; Golovine, Serguei Y.; Hecht, Sidney M.Journal of the American Chemical Society (2003), 125 (22), 6616-6617CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)By overexpression of modified Escherichia coli 23S rRNAs from multicopy plasmids, ribosomes were prepd. that contained mutations in two regions (2447-2450 and 2457-2462) of 23S rRNA. Following mutagenesis and selection, two clones with mutations in the 2447-2450 region (peptidyltransferase center) and six with mutations in the 2457-2462 region (helix 89) were characterized. The mutations were shown to exhibit a high level of homol. Cell-free protein synthesizing systems prepd. from these clones were found to exhibit significantly enhanced incorporation of D-methionine and D-phenylalanine into protein. The incorporations involved positions 10, 22, and 54 of E. coli dihydrofolate reductase and positions 247 and 250 of Photinus pyralis firefly luciferase. Interestingly, some of the derived proteins contg. the D-amino acids (notably DHFR analogs altered at position 10) functioned as well as those contg. the resp. L-amino acids, while substitution at other positions resulted in proteins having greatly diminished activity.
- 12Goto, Y.; Murakami, H.; Suga, H. Initiating translation with d-amino acids. RNA 2008, 14, 1390– 1398, DOI: 10.1261/rna.1020708[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXot1amtLs%253D&md5=efa90e6f836e03246973e7565ebe37daInitiating translation with D-amino acidsGoto, Yuki; Murakami, Hiroshi; Suga, HiroakiRNA (2008), 14 (7), 1390-1398CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)Here we report exptl. evidence that the translation initiation app. accepts D-amino acids (Daa), as opposed to only L-methionine, as initiators. Nineteen Daa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNAfMetCAU using flexizyme technol. and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to Daa. Remarkably, all Daa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with Daa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding Daa-tRNAfMetCAU. Although the inefficient formylation of Daa-tRNAfMetCAU resulted in modest expression of the corresponding peptide, preacetylation of Daa-tRNAfMetCAU dramatically increased expression level, implying that the formylation efficiency is one of the crit. determinants of initiation efficiency with Daa. Our findings provide not only the exptl. evidence that translation initiation tolerates Daa, but also a new means for the mRNA-directed synthesis of peptides capped with Daa or acyl-Daa at the N terminus.
- 13Ohta, A.; Murakami, H.; Higashimura, E.; Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem. Biol. 2007, 14, 1315– 1322, DOI: 10.1016/j.chembiol.2007.10.015[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhsVOktrfJ&md5=2587638ab5c21965b8c3654721fc437dSynthesis of Polyester by Means of Genetic Code ReprogrammingOhta, Atsushi; Murakami, Hiroshi; Higashimura, Eri; Suga, HiroakiChemistry & Biology (Cambridge, MA, United States) (2007), 14 (12), 1315-1322CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Here we report the ribosomal polymn. of α-hydroxy acids by means of genetic code reprogramming. The flexizyme system, a ribozyme-based tRNA acylation tool, was used to reassign individual codons to seven types of α-hydroxy acids, and then polyesters were synthesized under controls of the reprogrammed genetic code using a reconstituted cell-free translation system. The sequence and length of the polyester segments were specified by the mRNA template, indicating that high-fidelity ribosome expression of polyesters was possible. This work opens a door for the mRNA-directed synthesis of backbone-altered biopolymers.
- 14Fujino, T.; Goto, Y.; Suga, H.; Murakami, H. Ribosomal synthesis of peptides with multiple β-amino acids. J. Am. Chem. Soc. 2016, 138, 1962– 1969, DOI: 10.1021/jacs.5b12482[ACS Full Text], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlWkt7c%253D&md5=f7365b08e95f75738119a38232096814Ribosomal Synthesis of Peptides with Multiple β-Amino AcidsFujino, Tomoshige; Goto, Yuki; Suga, Hiroaki; Murakami, HiroshiJournal of the American Chemical Society (2016), 138 (6), 1962-1969CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The compatibility of β-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various β-amino acids, and whether the ribosome can introduce multiple β-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened β-amino acids that give high single incorporation efficiency and used them to synthesize peptides contg. multiple β-amino acids. The expts. of single β-amino acid incorporation into a peptide revealed that 13 β-amino acids are compatible with ribosomal translation. Six of the tested β-amino acids (βhGly, L-βhAla, L-βhGln, L-βhPhg, L-βhMet, and D-βhPhg) showed high incorporation efficiencies, and seven (L-βhLeu, L-βhIle, L-βhAsn, L-βhPhe, L-βhLys, D-βhAla, and D-βhLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other β-amino acids (L-βhPro, L-βhTrp, and L-βhGlu). Subsequent double-incorporation expts. using β-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive β-amino acids is prohibited. Efficiency of the double-incorporation of the β-amino acids was restored by the insertion of Tyr or Ile between the two β-amino acids. On the basis of these expts., we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides contg. different types of β-amino acids at multiple positions.
- 15Katoh, T.; Suga, H. Ribosomal incorporation of consecutive β-amino acids. J. Am. Chem. Soc. 2018, 140, 12159– 12167, DOI: 10.1021/jacs.8b07247[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslWgtL7P&md5=825f4a1be80ef41fecee313f4eddc9bfRibosomal Incorporation of Consecutive β-Amino AcidsKatoh, Takayuki; Suga, HiroakiJournal of the American Chemical Society (2018), 140 (38), 12159-12167CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Due to their unique characteristics, which are not shared by canonical α-peptides, peptides that contain stretches of consecutive β-amino acids are attractive scaffolds for novel peptide drugs and nanomaterials. Although ribosomal incorporation of single or non-consecutive β-amino acids into peptides has previously been reported, the incorporation of consecutive β-amino acids has not yet been accomplished. This is primarily due to their incompatibility with the ribosomal translation system. Here, we took advantage of engineered β-aminoacyl-tRNAs bearing optimized T-stem and D-arm motifs for enhancing binding affinity to EF-Tu and EF-P, resp. Combined with a reconstituted E. coli translation system and optimized translation factor concns., up to seven consecutive β-amino acids could be incorporated into a model peptide. Furthermore, the synthesis of macrocyclic β-peptides closed by a thioether bond between two D-α-amino acids is also demonstrated. This represents the first example of the ribosomal synthesis of peptides contg. stretches of consecutive β-amino acids.
- 16Wang, P. S.; Schepartz, A. β-peptide bundles: Design. Build. Analyze. Biosynthesize.. Chem. Commun. 2016, 52, 7420– 7432, DOI: 10.1039/C6CC01546H[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsVWqs7w%253D&md5=f2b3b1cf34e77aada1ab698e5f84118dβ-Peptide bundles: Design. Build. Analyze. Biosynthesize.Wang, Pam S. P.; Schepartz, AlannaChemical Communications (Cambridge, United Kingdom) (2016), 52 (47), 7420-7432CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. Peptides contg. β-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. Peptides contg. β-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. When composed solely of β-amino acids, the structures formed, defined assemblies of 14-helixes called β-peptide bundles, fold cooperatively in water solvent into unique and discrete quaternary assemblies that are highly thermostable, bind complex substrates and metal ion cofactors, and, in certain cases, catalyze chem. reactions. In this perspective, the authors recount the design and elaboration of β-peptide bundles, and provide an outlook on recent, unexpected discoveries that could influence research on β-peptides and β-peptide bundles (and β-amino acid-contg. proteins) for decades to come.
- 17Checco, J. W.; Gellman, S. H. Targeting recognition surfaces on natural proteins with peptidic foldamers. Curr. Opin. Struct. Biol. 2016, 39, 96– 105, DOI: 10.1016/j.sbi.2016.06.014[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGitr%252FE&md5=59db458d1a2f21a7c60ecf3ff6cdc862Targeting recognition surfaces on natural proteins with peptidic foldamersChecco, James W.; Gellman, Samuel H.Current Opinion in Structural Biology (2016), 39 (), 96-105CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Mols. intended to antagonize protein-protein interactions or augment polypeptide-based signaling must bind tightly to large and sp. surfaces on target proteins. Some types of unnatural oligomers with discrete folding propensities ('foldamers') have recently been shown to display this capability. This review covers important recent advances among several classes of foldamers, including α-peptides with secondary structures stabilized by covalent bonds, D-α-peptides, α/β-peptides and oligo-oxopiperazines. Recent advances in this area have involved enhancing membrane permeability to provide access to intracellular protein targets, improving pharmacokinetics and duration of action in vivo, and developing strategies appropriate for targeting large and irregularly-shaped protein surfaces.
- 18Montalvo, G.; Waegele, M. M.; Shandler, S.; Gai, F.; DeGrado, W. F. Infrared signature and folding dynamics of a helical β-peptide. J. Am. Chem. Soc. 2010, 132, 5616– 8, DOI: 10.1021/ja100459a[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksVansbw%253D&md5=cf5fef952f2e9e57b2fa2a98bf44e6ddInfrared Signature and Folding Dynamics of a Helical β-PeptideMontalvo, Geronda; Waegele, Matthias M.; Shandler, Scott; Gai, Feng; De Grado, William F.Journal of the American Chemical Society (2010), 132 (16), 5616-5618CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Synthetic foldamers consisting of β-amino acids offer excellent model systems for examg. the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding-unfolding kinetics of a β-peptide that folds into a 14-helical structure in water. We find that the T-jump induced relaxation kinetics of this peptide occur on the nanosecond time scale and are noticeably slower than those of alanine-based α-helical peptides, and addnl., the relaxation rates show a weaker dependence on temp. These differences appear to indicate that the folding energy landscapes of these peptides are different. In addn., we find that the amide I' band of this β-peptide exhibits a sharp feature at ∼1612 cm-1, which we believe is a distinct IR reporter of 14-helix.
- 19Seebach, D.; Gardiner, J. Beta-peptidic peptidomimetics. Acc. Chem. Res. 2008, 41, 1366– 1375, DOI: 10.1021/ar700263g[ACS Full Text], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnvVKksrk%253D&md5=e679836f00843a02103caf63773ffa80β-Peptidic peptidomimeticsSeebach, Dieter; Gardiner, JamesAccounts of Chemical Research (2008), 41 (10), 1366-1375CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. For more than a decade now, a search for answers to the following two questions has taken us on a new and exciting journey into the world of β- and γ-peptides: What happens if the O atoms in a 3i-helix of a polymeric chain composed of (R)-3-hydroxybutanoic acid are replaced by NH units. What happens if 1 or 2 CH2 groups are introduced into each amino acid building block in the chain of a peptide or protein, thereby providing homologues of the proteinogenic α-amino acids. Our journey has repeatedly thrown up surprises, continually expanding the potential of these classes of compd. and deepening our understanding of the structures, properties, and multifaceted functions of the natural "models" to which they are related. β-Peptides differ from their natural counterparts, the α-peptides, by having CH2 groups inserted into every amino acid residue, either between the C=O groups and the α-C atoms (β3) or between the α-C and N atoms (β2). The synthesis of these homologated proteinogenic amino acids and their assembly into β-peptides can be performed using known methods. Despite the increased no. of possible conformers, the β-peptides form secondary structures (helixes, turns, sheets) even when the chain lengths are as short as 4 residues. Furthermore, they are stable toward degrading and metabolizing enzymes in living organisms. Linear, helical, and hairpin-type structures of β-peptides can now be designed in such a way that they resemble the characteristic and activity-related structural features ("epitopes") of corresponding natural peptides or protein sections. This account presents examples of β-peptidic compds. binding, as agonists or antagonists (inhibitors), to (i) major histocompatibility complex (MHC) proteins (immune response), (ii) the lipid-transport protein SR-B1 (cholesterol uptake from the small intestine), (iii) the core (1-60) of interleukin-8 (inflammation), (iv) the oncoprotein RDM2, (v) the HIV gp41 fusion protein, (vi) G-protein-coupled somatostatin hsst receptors, (vii) the TNF immune response receptor CD40 (apoptosis), and (viii) DNA. Short-chain β-peptides may be orally bioavailable and excreted from the body of mammals; long-chain β-peptides may require i.v. administration but will have longer half-lives of clearance. It has been said that an interesting field of research distinguishes itself in that the results always throw up new questions; in this sense, the structural and biol. investigation of β-peptides has been a gold mine. We expect that these peptidic peptidomimetics will play an increasing role in biomedical research and drug development in the near future.
- 20García, J. M.; García, F. C.; Serna, F.; de la Peña, J. L. High-performance aromatic polyamides. Prog. Polym. Sci. 2010, 35, 623– 686, DOI: 10.1016/j.progpolymsci.2009.09.002[Crossref], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjvFKitr8%253D&md5=aa7ccb4975af61aef614c6c9b1a26ba3High-performance aromatic polyamidesGarcia, Jose M.; Garcia, Felix C.; Serna, Felipe; de la Pena, Jose L.Progress in Polymer Science (2010), 35 (5), 623-686CODEN: PRPSB8; ISSN:0079-6700. (Elsevier Ltd.)A review. Wholly arom. polyamides (aramids) are considered to be high-performance org. materials due to their outstanding thermal and mech. resistance. Their properties arise from their arom. structure and amide linkages, which result in stiff rod-like macromol. chains that interact with each other via strong and highly directional hydrogen bonds. These bonds create effective cryst. microdomains, resulting in a high-level intermol. packing and cohesive energy. The better known com. aramids, poly(p-phenylene terephthalamide) and poly(m-phenylene isophthalamide), are used in advanced technologies and have been transformed into high-strength and flame resistant fibers and coatings, with applications in the aerospace and armament industry, bullet-proof body armor, protective clothing, sport fabrics, elec. insulation, asbestos substitutes, and industrial filters, among others. Owing to their chem. structure, they exhibit extremely high transition temps. that lie above their decompn. temps., are sparingly sol. in common org. solvents and, accordingly, can only be transformed upon soln. Research efforts are therefore underway to take advantage of their properties, enhance their processability and soly., and incorporate new chem. functionalities in the polyamide backbone or lateral structure, so that their applicability is expanded and remains on the forefront of scientific research.
- 21Tanner, D.; Fitzgerald, J. A.; Phillips, B. R. The kevlar story - an advanced materials case study. Angew. Chem., Int. Ed. Engl. 1989, 28, 649– 654, DOI: 10.1002/anie.198906491
- 22Baumann, S.; Herrmann, J.; Raju, R.; Steinmetz, H.; Mohr, K. I.; Háttel, S.; Harmrolfs, K.; Stadler, M.; Máller, R. Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activity. Angew. Chem., Int. Ed. Engl. 2014, 53, 14605– 14609, DOI: 10.1002/anie.201409964[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MznvVSqsg%253D%253D&md5=c500f536a8abceedd4aaa7ef7fe8bab6Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activityBaumann Sascha; Herrmann Jennifer; Raju Ritesh; Steinmetz Heinrich; Mohr Kathrin I; Huttel Stephan; Harmrolfs Kirsten; Stadler Marc; Muller RolfAngewandte Chemie (International ed. in English) (2014), 53 (52), 14605-9 ISSN:.The development of new antibiotics faces a severe crisis inter alia owing to a lack of innovative chemical scaffolds with activities against Gram-negative and multiresistant pathogens. Herein, we report highly potent novel antibacterial compounds, the myxobacteria-derived cystobactamids 1-3, which were isolated from Cystobacter sp. and show minimum inhibitory concentrations in the low μg mL(-1) range. We describe the isolation and structure elucidation of three congeners as well as the identification and annotation of their biosynthetic gene cluster. By studying the self-resistance mechanism in the natural producer organism, the molecular targets were identified as bacterial type IIa topoisomerases. As quinolones are largely exhausted as a template for new type II topoisomerase inhibitors, the cystobactamids offer exciting alternatives to generate novel antibiotics using medicinal chemistry and biosynthetic engineering.
- 23Saraogi, I.; Incarvito, C. D.; Hamilton, A. D. Controlling curvature in a family of oligoamide α-helix mimetics. Angew. Chem., Int. Ed. 2008, 47, 9691– 9694, DOI: 10.1002/anie.200803778[Crossref], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFanurfL&md5=f8bde9f3cd2a9e89e77004365a578ffdControlling curvature in a family of oligoamide α-helix mimeticsSaraogi, Ishu; Incarvito, Christopher D.; Hamilton, Andrew D.Angewandte Chemie, International Edition (2008), 47 (50), 9691-9694CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The natural curvature found in a majority of α helixes has been mimicked in small synthetic oligoamide scaffolds. Differences in hydrogen-bonding patterns in these scaffolds lead to mimetics with varying degrees of curvature in the backbone. This adds another parameter to the structural and functional mimicry of a helixes.
- 24Meisel, J. W.; Hu, C. T.; Hamilton, A. D. Mimicry of a β-hairpin turn by a nonpeptidic laterally flexible foldamer. Org. Lett. 2018, 20, 3879– 3882, DOI: 10.1021/acs.orglett.8b01463[ACS Full Text], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFWhurfK&md5=aa0642e59d820b9bacca47cf68bfab8cMimicry of a β-Hairpin Turn by a Nonpeptidic Laterally Flexible FoldamerMeisel, Joseph W.; Hu, Chunhua T.; Hamilton, Andrew D.Organic Letters (2018), 20 (13), 3879-3882CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)The design and characterization of a proteomimetic foldamer that displays lateral flexibility endowed by intramol. bifurcated hydrogen bonds is reported. The MAMBA scaffold, derived from meta-aminomethylbenzoic acid, adopts a serpentine conformation that mimics the side chain projection of all four residues in a β-hairpin turn.
- 25Saha, S.; Kauffmann, B.; Ferrand, Y.; Huc, I. Selective encapsulation of disaccharide xylobiose by an aromatic foldamer helical capsule. Angew. Chem., Int. Ed. 2018, 57, 13542– 13546, DOI: 10.1002/anie.201808370[Crossref], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslaiu7%252FP&md5=b912c7c178c6272b9bdddfa5a14e0c7fSelective Encapsulation of Disaccharide Xylobiose by an Aromatic Foldamer Helical CapsuleSaha, Subrata; Kauffmann, Brice; Ferrand, Yann; Huc, IvanAngewandte Chemie, International Edition (2018), 57 (41), 13542-13546CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Xylobiose sequestration in a helical arom. oligoamide capsule was evidenced by CD, NMR spectroscopy, and crystallog. The prepn. of the 5 kDa oligoamide sequence was made possible by the transient use of acid-labile dimethoxybenzyl tertiary amide substituents that disrupt helical folding and prevent double helix formation. Binding of other disaccharides was not detected. Crystallog. data revealed a complex composed of a D-xylobiose α anomer and two water mols. accommodated in the right-handed helix. The disaccharide was found to adopt an unusual all-axial compact conformation. A dense network of 18 hydrogen bonds forms between the guest, the cavity wall, and the two water mols.
- 26Rogers, J. M.; Kwon, S.; Dawson, S. J.; Mandal, P. K.; Suga, H.; Huc, I. Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids. Nat. Chem. 2018, 10, 405– 412, DOI: 10.1038/s41557-018-0007-x[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXos1Smt7s%253D&md5=207db9badb99e896f9f5aeb06181f0eeRibosomal synthesis and folding of peptide-helical aromatic foldamer hybridsRogers, Joseph M.; Kwon, Sunbum; Dawson, Simon J.; Mandal, Pradeep K.; Suga, Hiroaki; Huc, IvanNature Chemistry (2018), 10 (4), 405-412CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Translation, the mRNA-templated synthesis of peptides by the ribosome, can be manipulated to incorporate variants of the 20 cognate amino acids. Such approaches for expanding the range of chem. entities that can be produced by the ribosome may accelerate the discovery of mols. that can perform functions for which poorly folded, short peptidic sequences are ill suited. Here, we show that the ribosome tolerates some artificial helical arom. oligomers, so-called foldamers. Using a flexible tRNA-acylation ribozyme-flexizyme-foldamers were attached to tRNA, and the resulting acylated tRNAs were delivered to the ribosome to initiate the synthesis of non-cyclic and cyclic foldamer-peptide hybrid mols. Passing through the ribosome exit tunnel requires the foldamers to unfold. Yet foldamers encode sufficient folding information to influence the peptide structure once translation is completed. We also show that in cyclic hybrids, the foldamer portion can fold into a helix and force the peptide segment to adopt a constrained and stretched conformation.
- 27Schepartz, A. Foldamers wave to the ribosome. Nat. Chem. 2018, 10, 377– 379, DOI: 10.1038/s41557-018-0036-5[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXos1SktrY%253D&md5=a0dd482713baeb9a88bcaeeda02d2878Foldamers wave to the ribosomeSchepartz, AlannaNature Chemistry (2018), 10 (4), 377-379CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Ribosomes have now been shown to accept certain initiator tRNAs acylated with arom. foldamer-dipeptides thereby enabling the translation of a peptide or protein with a short arom. foldamer at the N-terminus. Some foldamer-peptide hybrids could be cyclized to generate macrocycles that present conformationally restricted peptide loops.
- 28Goto, Y.; Suga, H. Flexizymes as a tRNA acylation tool facilitating genetic code reprogramming. Methods Mol. Biol. 2012, 848, 465– 478, DOI: 10.1007/978-1-61779-545-9_29[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1agsLjM&md5=300684998389c280029bee28f646fc7bFlexizymes as a tRNA acylation tool facilitating genetic code reprogrammingGoto, Yuki; Suga, HiroakiMethods in Molecular Biology (New York, NY, United States) (2012), 848 (Ribozymes), 465-478CODEN: MMBIED; ISSN:1064-3745. (Springer)A review. Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. We here describe a protocol that facilitates the genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation app., referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the prepn. of a diverse array of non-proteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-acyl groups, D-amino acids, N-Me amino acids, and physiol. stable macrocyclic scaffolds. Facility of the protocol allows for a wide variety of applications in the synthesis of new classes of nonstandard peptides with biol. functions.
- 29Murakami, H.; Saito, H.; Suga, H. A versatile tRNA aminoacylation catalyst based on RNA. Chem. Biol. 2003, 10, 655– 662, DOI: 10.1016/S1074-5521(03)00145-5[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlvVGjs7w%253D&md5=0b42c261bbce87009ddaddcc47ac897cA Versatile tRNA Aminoacylation Catalyst Based on RNAMurakami, Hiroshi; Saito, Hirohide; Suga, HiroakiChemistry & Biology (2003), 10 (7), 655-662CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognizes arom. amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNAAsnCCCG). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and nonnatural amino acids.
- 30McMurry, J. L.; Chang, M. C. Y. Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 11920– 11925, DOI: 10.1073/pnas.1711482114[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslSktrfJ&md5=70a570ad8451738c6e156330a9fd03d3Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleyaMcMurry, Jonathan L.; Chang, Michelle C. Y.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (45), 11920-11925CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small mols., ranging from therapeutics to materials. In contrast, only a few fluorinated compds. made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in Streptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochem., genetic, physiol., and proteomic studies, we show that FthB is a trans-acting tRNA editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While trans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metab. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.
- 31Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 2006, 3, 357– 359, DOI: 10.1038/nmeth877[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjslSktrw%253D&md5=d6694f40ba3b0f5791ac361fa6d29a38A highly flexible tRNA acylation method for non-natural polypeptide synthesisMurakami, Hiroshi; Ohta, Atsushi; Ashigai, Hiroshi; Suga, HiroakiNature Methods (2006), 3 (5), 357-359CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Here the authors describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the prepn. of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows the authors to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple nonnatural amino acids.
- 32Nawrot, B.; Sprinzl, M. Aminoacyl-tRNA analogues; synthesis, purification and properties of 3-anthraniloyl oligoribonucleotides. Nucleosides Nucleotides 1998, 17, 815– 829, DOI: 10.1080/07328319808004677[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitFWqtbY%253D&md5=c419d507c871431a0afba116cc4ccf17Aminoacyl-tRNA analogs; synthesis, purification and properties of 3'-anthraniloyl oligoribonucleotidesNawrot, B.; Sprinzl, M.Nucleosides & Nucleotides (1998), 17 (4), 815-829CODEN: NUNUD5; ISSN:0732-8311. (Marcel Dekker, Inc.)Reaction of isatoic anhydride with adenosine, adenosine 5'-phosphate, oligoribonucleotides or with the E. coli tRNAVal led to attachment of an anthraniloyl residue at 2'- or 3'-OH groups of 3'-terminal ribose residue. No protection of the 5'-hydroxyl group or internal 2'-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analog of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.
- 33Mortimer, S. A.; Weeks, K. M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 2007, 129, 4144– 4145, DOI: 10.1021/ja0704028[ACS Full Text], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXivF2jtr0%253D&md5=d0b0d3d7582cd98cd801077ade6f42c5A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE ChemistryMortimer, Stefanie A.; Weeks, Kevin M.Journal of the American Chemical Society (2007), 129 (14), 4144-4145CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chem. allows local nucleotide flexibility to be quant. assessed at single nucleotide resoln. in any RNA. SHAPE chem. exploits structure-based gating of the nucleophilic reactivity of the ribose 2'-hydroxyl group by the extent to which a nucleotide is constrained or flexible. SHAPE chem. was developed using N-methylisatoic anhydride (NMIA), which is only moderately electrophilic and requires tens of minutes to form ribose 2'-O-adducts. Here, we design and evaluate a significantly more useful, fast-acting, reagent for SHAPE chem. Introduction of a nitro group para to the reactive carbonyl to form 1-methyl-7-nitroisatoic anhydride (1M7) yields a reagent that both reacts significantly more rapidly with RNA to form 2'-O-adducts and is also more labile toward advantageous, self-limiting, hydrolysis. With 1M7, the single nucleotide resoln. interrogation of the RNA structure is complete in 70 s. SHAPE anal. performed with 1M7 accurately reports the secondary and tertiary structure of the RNase P specificity domain and allows the secondary structure of this RNA to be predicted with up to 91% accuracy.
- 34Drossman, H.; Johnson, H.; Mill, T. Structure activity relationships for environmental processes 1: Hydrolysis of esters and carbamates. Chemosphere 1988, 17, 1509– 1530, DOI: 10.1016/0045-6535(88)90204-4[Crossref], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXmtFGltro%253D&md5=ed9e54b4a2522d09572a225555c6363bStructure activity relationships for environmental processes 1: hydrolysis of esters and carbamatesDrossman, Howard; Johnson, Howard; Mill, TheodoreChemosphere (1988), 17 (8), 1509-30CODEN: CMSHAF; ISSN:0045-6535.Structure activity relationships (SAR) for base-promoted hydrolysis of esters and carbamates were developed using Hammett and Taft parameters. A master SAR equation correlates base promoted hydrolysis rate const. (kB) values for 103 esters with a range of reactivity of 1010. Two SAR equations are needed to correlate kB value for 80 carbamates.
- 35Xiao, H.; Murakami, H.; Suga, H.; Ferré-D’Amaré, A. R. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 2008, 454, 358– 361, DOI: 10.1038/nature07033[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXosFCqtLc%253D&md5=a6a47091d470d51e4cfd92ce0e4df78aStructural basis of specific tRNA aminoacylation by a small in vitro selected ribozymeXiao, Hong; Murakami, Hiroshi; Suga, Hiroaki; Ferre-D'Amare, Adrian R.Nature (London, United Kingdom) (2008), 454 (7202), 358-361CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In modern organisms, protein enzymes are solely responsible for the aminoacylation of tRNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalyzing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated L-phenylalanine derivs. In addn. to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-Å resoln. structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA mol., flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallog. independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.
- 36Hunter, C. A.; Lawson, K. R.; Perkins, J.; Urch, C. J. Aromatic interactions. J. Chem. Soc. Perkins Trans. 2 2001, 2, 651– 669, DOI: 10.1039/b008495f
- 37Meyer, E. A.; Castellano, R. K.; Diederich, F. Interactions with aromatic rings in chemical and biological recognition. Angew. Chem., Int. Ed. 2003, 42, 1210– 1250, DOI: 10.1002/anie.200390319[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXivVSrtbw%253D&md5=e91e6a8d115d56229e1660697a286b04Interactions with aromatic rings in chemical and biological recognitionMeyer, Emmanuel A.; Castellano, Ronald K.; Diederich, FrancoisAngewandte Chemie, International Edition (2003), 42 (11), 1210-1250CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Intermol. interactions involving arom. rings are key processes in both chem. and biol. recognition. Their understanding is essential for rational drug design and lead optimization in medicinal chem. Different approaches-biol. studies, mol. recognition studies with artificial receptors, crystallog. database mining, gas-phase studies, and theor. calcns. - are pursued to generate a profound understanding of the structural and energetic parameters of individual recognition modes involving arom. rings. This review attempts to combine and summarize the knowledge gained from these investigations. The review focuses mainly on examples with biol. relevance since one of its aims it to enhance the knowledge of mol. recognition forces that is essential for drug development.
- 38Hansch, C.; Leo, A.; Taft, R. W. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 1991, 91, 165– 195, DOI: 10.1021/cr00002a004[ACS Full Text], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhs1ehsLo%253D&md5=9fc814cd57c47680a5213f3438037800A survey of Hammett substituent constants and resonance and field parametersHansch, Corwin; Leo, A.; Taft, R. W.Chemical Reviews (Washington, DC, United States) (1991), 91 (2), 165-95CODEN: CHREAY; ISSN:0009-2665.Included in this review is an anal. of newer methods which can supplant this classic procedure for detn. of the title consts., 283 refs.
- 39Kawakami, T.; Ogawa, K.; Hatta, T.; Goshima, N.; Natsume, T. Directed evolution of a cyclized peptoid-peptide chimera against a cell-free expressed protein and proteomic profiling of the interacting proteins to create a protein-protein interaction inhibitor. ACS Chem. Biol. 2016, 11, 1569– 1577, DOI: 10.1021/acschembio.5b01014[ACS Full Text], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xks12gsr8%253D&md5=94544c0a684d5784920cbce562d4de87Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction InhibitorKawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, TohruACS Chemical Biology (2016), 11 (6), 1569-1577CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liq. chromatog.-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.
- 40Goto, Y.; Ohta, A.; Sako, Y.; Yamagishi, Y.; Murakami, H.; Suga, H. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol. 2008, 3, 120– 129, DOI: 10.1021/cb700233t[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXptl2isA%253D%253D&md5=1627a925839f35c767df198041d7bc2cReprogramming the Translation Initiation for the Synthesis of Physiologically Stable Cyclic PeptidesGoto, Yuki; Ohta, Atsushi; Sako, Yusuke; Yamagishi, Yusuke; Murakami, Hiroshi; Suga, HiroakiACS Chemical Biology (2008), 3 (2), 120-129CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report a new technol. to reprogram the initiation event, where various amino acids and those bearing Nα-acyl groups can be used as an initiator for peptide synthesis. The technol. is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technol. to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiol. stable bond, and it is also extended to the custom synthesis of its analogs with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodol., we also prepd. a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technol. offers a new means to prep. a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.
- 41Gualerzi, C. O.; Pon, C. L. Initiation of mRNA translation in bacteria: structural and dynamic aspects. Cell. Mol. Life Sci. 2015, 72, 4341– 4367, DOI: 10.1007/s00018-015-2010-3[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlelurvN&md5=a35b5f76a4d78a90f17b28601b9f5b96Initiation of mRNA translation in bacteria: structural and dynamic aspectsGualerzi, Claudio O.; Pon, Cynthia L.Cellular and Molecular Life Sciences (2015), 72 (22), 4341-4367CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)Initiation of mRNA translation is a major checkpoint for regulating level and fidelity of protein synthesis. Being rate limiting in protein synthesis, translation initiation also represents the target of many post-transcriptional mechanisms regulating gene expression. The process begins with the formation of an unstable 30S pre-initiation complex (30S pre-IC) contg. initiation factors (IFs) IF1, IF2 and IF3, the translation initiation region of an mRNA and initiator fMet-tRNA whose codon and anticodon pair in the P-site following a first-order rearrangement of the 30S pre-IC produces a locked 30S initiation complex (30SIC); this is docked by the 50S subunit to form a 70S complex that, following several conformational changes, positional readjustments of its ligands and ejection of the IFs, becomes a 70S initiation complex productive in initiation dipeptide formation. The first EF-G-dependent translocation marks the beginning of the elongation phase of translation. Here, we review structural, mechanistic and dynamical aspects of this process.
- 42Goto, Y.; Katoh, T.; Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 2011, 6, 779– 790, DOI: 10.1038/nprot.2011.331[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmvFyqu78%253D&md5=fbfd338c7f874ac9871c0f3cfd84b3edFlexizymes for genetic code reprogrammingGoto, Yuki; Katoh, Takayuki; Suga, HiroakiNature Protocols (2011), 6 (6), 779-790CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation app., referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the prepn. of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-Me amino acids, D-amino acids and physiol. stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biol. functions. Prepn. of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 wk. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.
- 43Staunton, J.; Weissman, K. J. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep. 2001, 18, 380– 416, DOI: 10.1039/a909079g[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXltlyqsbo%253D&md5=2f5a7eaf4d900456bb89d2e75721e2dfPolyketide biosynthesis: A millennium reviewStaunton, James; Weissman, Kira J.Natural Product Reports (2001), 18 (4), 380-416CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review with refs. on the developments of the past 100 yr in the study of polyketides, which have various medicinally important activities, including antibiotic, anticancer, antifungal, antiparasitic, and immunosuppressive properties. The speculation on the future of polyketide research is also discussed.
- 44Dutta, S.; Whicher, J. R.; Hansen, D. A.; Hale, W. A.; Chemler, J. A.; Congdon, G. R.; Narayan, A. R. H.; Håkansson, K.; Sherman, D. H.; Smith, J. L.; Skiniotis, G. Structure of a modular polyketide synthase. Nature 2014, 510, 512– 517, DOI: 10.1038/nature13423[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVGjsr%252FO&md5=371c994e2b89ae3618118bf6bc5593dfStructure of a modular polyketide synthaseDutta, Somnath; Whicher, Jonathan R.; Hansen, Douglas A.; Hale, Wendi A.; Chemler, Joseph A.; Congdon, Grady R.; Narayan, Alison R. H.; Hakansson, Kristina; Sherman, David H.; Smith, Janet L.; Skiniotis, GeorgiosNature (London, United Kingdom) (2014), 510 (7506), 512-517CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Polyketide natural products constitute a broad class of compds. with diverse structural features and biol. activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyze two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to det. sub-nanometer-resoln. three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a sep. entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for mol. dissection of these multifunctional enzyme systems.
- 45Robbins, T.; Liu, Y.-C.; Cane, D. E.; Khosla, C. Structure and mechanism of assembly line polyketide synthases. Curr. Opin. Struct. Biol. 2016, 41, 10– 18, DOI: 10.1016/j.sbi.2016.05.009[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptVWkt74%253D&md5=5a94a7bde2d0d80b4d916f2aa14f57aaStructure and mechanism of assembly line polyketide synthasesRobbins, Thomas; Liu, Yu-Chen; Cane, David E.; Khosla, ChaitanCurrent Opinion in Structural Biology (2016), 41 (), 10-18CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Assembly line polyketide synthases (PKSs) are remarkable biosynthetic machines with considerable potential for structure-based engineering. Several types of protein-protein interactions, both within and between PKS modules, play important roles in the catalytic cycle of a multimodular PKS. Addnl., vectorial biosynthesis is enabled by the energetic coupling of polyketide chain elongation to the channeling of intermediates between successive modules. A combination of high-resoln. anal. of smaller PKS components and lower resoln. characterization of intact modules and bimodules has yielded insights into the structure and organization of a prototypical assembly line PKS. This review discusses our understanding of key structure-function relationships in this family of megasynthases, along with a recap of key unanswered questions in the field.
- 46Du, L.; Sanchez, C.; Shen, B. Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules. Metab. Eng. 2001, 3, 78– 95, DOI: 10.1006/mben.2000.0171[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXnvFOnsg%253D%253D&md5=7e332359fb3b6b2236fcfee96bbc3259Hybrid Peptide-Polyketide Natural Products: Biosynthesis and Prospects toward Engineering Novel MoleculesDu, Liangcheng; Sanchez, Cesar; Shen, BenMetabolic Engineering (2001), 3 (1), 78-95CODEN: MEENFM; ISSN:1096-7176. (Academic Press)A review, with 84 refs. The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examg. the biochem. and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a crit. role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products. (c) 2001 Academic Press.
- 47Silakowski, B.; Nordsiek, G.; Kunze, B.; Blöcker, H.; Máller, R. Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga151. Chem. Biol. 2001, 8, 59– 69, DOI: 10.1016/S1074-5521(00)00056-9[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhslGku78%253D&md5=36ea779d9d0a51aaa01ff0f264e2c66aNovel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15Silakowski, Barbara; Nordsiek, Gabriele; Kunze, Brigitte; Blocker, Helmut; Muller, RolfChemistry & Biology (2001), 8 (1), 59-69CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)Myxobacteria have been well established as a potent source for natural products with biol. activity. They produce a considerable variety of compds. which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Mol. cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. A DNA region of approx. 50 000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence anal. reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homol. to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the anal. and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Addnl., a new type of reductive release from PKS/NRPS systems is described.
- 48Walsh, C. T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 2004, 303, 1805– 1810, DOI: 10.1126/science.1094318[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXitFehu74%253D&md5=5c71b6744699c5335b450124c2f31595Polyketide and Nonribosomal Peptide Antibiotics: Modularity and VersatilityWalsh, Christopher T.Science (Washington, DC, United States) (2004), 303 (5665), 1805-1810CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Polyketide (PK) and nonribosomal peptides (NRP), constructed on multimodular enzymic assembly lines, often attain the conformations that establish biol. activity by cyclization constraints introduced by tailoring enzymes. The dedicated tailoring enzymes are encoded by genes clustered with the assembly line genes for coordinated regulation. NRP heterocyclizations to thiazoles and oxazoles can occur on the elongating framework of acyl-S enzyme intermediates, whereas tandem cyclic PK polyether formation of furans and pyrans can be initiated by post-assembly line epoxidases. Macrocyclizations of NRP, PK, and hybrid NRP-PK scaffolds occur in assembly line chain termination steps. Post-assembly line cascades of enzymic oxidns. also create cross-linked and cyclized architectures that generate the mature scaffolds of natural product antibiotics. The modularity of the natural product assembly lines and permissivity of tailoring enzymes offer prospects for reprogramming to create novel antibiotics with optimized properties.
- 49Fujino, T.; Kondo, T.; Suga, H.; Murakami, H. Exploring of minimal RNA substrate of flexizymes. ChemBioChem. 2019, in press. DOI: 10.1002/cbic.201900150
Cited By
This article is cited by 41 publications.
- Chandrima Majumdar, Joshua A. Walker, Matthew B. Francis, Alanna Schepartz, Jamie H. D. Cate. Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome. ACS Central Science 2023, 9 (6) , 1160-1169. https://doi.org/10.1021/acscentsci.3c00153
- Kosuke Seki, Joey L. Galindo, Ashty S. Karim, Michael C. Jewett. A Cell-Free Gene Expression Platform for Discovering and Characterizing Stop Codon Suppressing tRNAs. ACS Chemical Biology 2023, 18 (6) , 1324-1334. https://doi.org/10.1021/acschembio.3c00051
- Joshua A. Walker, Noah Hamlish, Avery Tytla, Daniel D. Brauer, Matthew B. Francis, Alanna Schepartz. Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates. ACS Central Science 2022, 8 (4) , 473-482. https://doi.org/10.1021/acscentsci.1c01577
- Sebasthian Santiago González, Omer Ad, Bhavana Shah, Zhongqi Zhang, Xizi Zhang, Abhishek Chatterjee, Alanna Schepartz. Genetic Code Expansion in the Engineered Organism Vmax X2: High Yield and Exceptional Fidelity. ACS Central Science 2021, 7 (9) , 1500-1507. https://doi.org/10.1021/acscentsci.1c00499
- Yuki Goto, Hiroaki Suga. The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides. Accounts of Chemical Research 2021, 54 (18) , 3604-3617. https://doi.org/10.1021/acs.accounts.1c00391
- Cangjie Yang, Kevin B. Wu, Yu Deng, Jingsong Yuan, Jia Niu. Geared Toward Applications: A Perspective on Functional Sequence-Controlled Polymers. ACS Macro Letters 2021, 10 (2) , 243-257. https://doi.org/10.1021/acsmacrolett.0c00855
- Yi Liu, Roderick G. Davis, Paul M. Thomas, Neil L. Kelleher, Michael C. Jewett. In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins. Biochemistry 2021, 60 (3) , 161-169. https://doi.org/10.1021/acs.biochem.0c00829
- Sabrina E. Iskandar, Victoria A. Haberman, Albert A. Bowers. Expanding the Chemical Diversity of Genetically Encoded Libraries. ACS Combinatorial Science 2020, 22 (12) , 712-733. https://doi.org/10.1021/acscombsci.0c00179
- Takayuki Katoh, Hiroaki Suga. Ribosomal Elongation of Aminobenzoic Acid Derivatives. Journal of the American Chemical Society 2020, 142 (39) , 16518-16522. https://doi.org/10.1021/jacs.0c05765
- Fred R. Ward, Zoe L. Watson, Omer Ad, Alanna Schepartz, Jamie H. D. Cate. Defects in the Assembly of Ribosomes Selected for β-Amino Acid Incorporation. Biochemistry 2019, 58 (45) , 4494-4504. https://doi.org/10.1021/acs.biochem.9b00746
- Riley Fricke, Cameron V. Swenson, Leah Tang Roe, Noah Xue Hamlish, Bhavana Shah, Zhongqi Zhang, Elise Ficaretta, Omer Ad, Sarah Smaga, Christine L. Gee, Abhishek Chatterjee, Alanna Schepartz. Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo. Nature Chemistry 2023, 15 (7) , 960-971. https://doi.org/10.1038/s41557-023-01224-y
- Zoe L. Watson, Isaac J. Knudson, Fred R. Ward, Scott J. Miller, Jamie H. D. Cate, Alanna Schepartz, Ara M. Abramyan. Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates. Nature Chemistry 2023, 15 (7) , 913-921. https://doi.org/10.1038/s41557-023-01226-w
- Andreea Stan, Clemens Mayer. Tethered Ribosomes: Toward the Synthesis of Nonproteinogenic Polymers in Bacteria. ChemBioChem 2023, 24 (12) https://doi.org/10.1002/cbic.202200578
- Kanghun Lee, Jessica A. Willi, Namjin Cho, Inseon Kim, Michael C. Jewett, Joongoo Lee. Cell-free Biosynthesis of Peptidomimetics. Biotechnology and Bioprocess Engineering 2023, 494 https://doi.org/10.1007/s12257-022-0268-5
- Yun‐Nam Choi, Namjin Cho, Kanghun Lee, Da‐ae Gwon, Jeong Wook Lee, Joongoo Lee. Programmable Synthesis of Biobased Materials Using Cell‐Free Systems. Advanced Materials 2023, 35 (4) https://doi.org/10.1002/adma.202203433
- Joongoo Lee, Jaime N. Coronado, Namjin Cho, Jongdoo Lim, Brandon M. Hosford, Sangwon Seo, Do Soon Kim, Camila Kofman, Jeffrey S. Moore, Andrew D. Ellington, Eric V. Anslyn, Michael C. Jewett. Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro. Nature Communications 2022, 13 (1) https://doi.org/10.1038/s41467-022-33701-2
- Tomohiro Kuroda, Yichao Huang, Soichiro Nishio, Yuki Goto, Hiroaki Suga. Post-translational backbone-acyl shift yields natural product-like peptides bearing hydroxyhydrocarbon units. Nature Chemistry 2022, 14 (12) , 1413-1420. https://doi.org/10.1038/s41557-022-01065-1
- Jaime N. Coronado, Phuoc Ngo, Eric V. Anslyn, Andrew D. Ellington. Chemical insights into flexizyme-mediated tRNA acylation. Cell Chemical Biology 2022, 29 (7) , 1071-1112. https://doi.org/10.1016/j.chembiol.2022.03.012
- Takayuki Katoh, Hiroaki Suga. In Vitro Genetic Code Reprogramming for the Expansion of Usable Noncanonical Amino Acids. Annual Review of Biochemistry 2022, 91 (1) , 221-243. https://doi.org/10.1146/annurev-biochem-040320-103817
- Tania Xavier, Sylvie Condon, Christophe Pichon, Erwan Le Gall, Marc Presset. Substituted Malonic Acid Half Oxyesters (SMAHOs): Green Nucleophiles for Organic Synthesis. European Journal of Organic Chemistry 2022, 2022 (5) https://doi.org/10.1002/ejoc.202101392
- Haruka Tsutsumi, Tomohiro Kuroda, Hiroyuki Kimura, Yuki Goto, Hiroaki Suga. Posttranslational chemical installation of azoles into translated peptides. Nature Communications 2021, 12 (1) https://doi.org/10.1038/s41467-021-20992-0
- Erika A. DeBenedictis, Gavriela D. Carver, Christina Z. Chung, Dieter Söll, Ahmed H. Badran. Multiplex suppression of four quadruplet codons via tRNA directed evolution. Nature Communications 2021, 12 (1) https://doi.org/10.1038/s41467-021-25948-y
- Camila Kofman, Joongoo Lee, Michael C. Jewett. Engineering molecular translation systems. Cell Systems 2021, 12 (6) , 593-607. https://doi.org/10.1016/j.cels.2021.04.001
- Joongoo Lee, Kevin J. Schwarz, Hao Yu, Antje Krüger, Eric V. Anslyn, Andrew D. Ellington, Jeffrey S. Moore, Michael C. Jewett. Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro. Chemical Communications 2021, 57 (21) , 2661-2664. https://doi.org/10.1039/D0CC07740B
- Tania Xavier, Sylvie Condon, Christophe Pichon, Erwan Le Gall, Marc Presset. Preparation of mono-substituted malonic acid half oxyesters (SMAHOs). Beilstein Journal of Organic Chemistry 2021, 17 , 2085-2094. https://doi.org/10.3762/bjoc.17.135
- Jeffery M. Tharp, Joshua A. Walker, Dieter Söll, Alanna Schepartz. Initiating protein synthesis with noncanonical monomers in vitro and in vivo. 2021, 495-519. https://doi.org/10.1016/bs.mie.2021.05.002
- Joongoo Lee, Kevin J. Schwarz, Do Soon Kim, Jeffrey S. Moore, Michael C. Jewett. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro. Nature Communications 2020, 11 (1) https://doi.org/10.1038/s41467-020-18001-x
- Zhenling Cui, Wayne A. Johnston, Kirill Alexandrov. Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides. Frontiers in Bioengineering and Biotechnology 2020, 8 https://doi.org/10.3389/fbioe.2020.01031
- Zoe L Watson, Fred R Ward, Raphaël Méheust, Omer Ad, Alanna Schepartz, Jillian F Banfield, Jamie HD Cate. Structure of the bacterial ribosome at 2 Å resolution. eLife 2020, 9 https://doi.org/10.7554/eLife.60482
- Rika Okuma, Tomoki Kuwahara, Takafumi Yoshikane, Mizuki Watanabe, Patricia Dranchak, James Inglese, Satoshi Shuto, Yuki Goto, Hiroaki Suga. A Macrocyclic Peptide Library with a Structurally Constrained Cyclopropane‐containing Building Block Leads to Thiol‐independent Inhibitors of Phosphoglycerate Mutase. Chemistry – An Asian Journal 2020, 15 (17) , 2631-2636. https://doi.org/10.1002/asia.202000700
- Timothy A. Whitehead, Scott Banta, William E. Bentley, Michael J. Betenbaugh, Christina Chan, Douglas S. Clark, Corinne A. Hoesli, Michael C. Jewett, Beth Junker, Mattheos Koffas, Rashmi Kshirsagar, Amanda Lewis, Chien‐Ting Li, Costas Maranas, E. Terry Papoutsakis, Kristala L. J. Prather, Steffen Schaffer, Laura Segatori, Ian Wheeldon. The importance and future of biochemical engineering. Biotechnology and Bioengineering 2020, 117 (8) , 2305-2318. https://doi.org/10.1002/bit.27364
- Joongoo Lee, Rafael Torres, Do Soon Kim, Michelle Byrom, Andrew D. Ellington, Michael C. Jewett. Ribosomal incorporation of cyclic β-amino acids into peptides using in vitro translation. Chemical Communications 2020, 56 (42) , 5597-5600. https://doi.org/10.1039/D0CC02121K
- Jeffery M. Tharp, Natalie Krahn, Umesh Varshney, Dieter Söll. Hijacking Translation Initiation for Synthetic Biology. ChemBioChem 2020, 21 (10) , 1387-1396. https://doi.org/10.1002/cbic.202000017
- Christos Tsiamantas, Joseph M. Rogers, Hiroaki Suga. Initiating ribosomal peptide synthesis with exotic building blocks. Chemical Communications 2020, 56 (31) , 4265-4272. https://doi.org/10.1039/D0CC01291B
- Christos Tsiamantas, Sunbum Kwon, Joseph M. Rogers, Céline Douat, Ivan Huc, Hiroaki Suga. Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages. Angewandte Chemie International Edition 2020, 59 (12) , 4860-4864. https://doi.org/10.1002/anie.201914654
- Christos Tsiamantas, Sunbum Kwon, Joseph M. Rogers, Céline Douat, Ivan Huc, Hiroaki Suga. Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages. Angewandte Chemie 2020, 132 (12) , 4890-4894. https://doi.org/10.1002/ange.201914654
- Jeffery M. Tharp, Omer Ad, Kazuaki Amikura, Fred R. Ward, Emma M. Garcia, Jamie H. D. Cate, Alanna Schepartz, Dieter Söll. Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo. Angewandte Chemie 2020, 132 (8) , 3146-3150. https://doi.org/10.1002/ange.201914671
- Jeffery M. Tharp, Omer Ad, Kazuaki Amikura, Fred R. Ward, Emma M. Garcia, Jamie H. D. Cate, Alanna Schepartz, Dieter Söll. Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo. Angewandte Chemie International Edition 2020, 59 (8) , 3122-3126. https://doi.org/10.1002/anie.201914671
- Michael J Hammerling, Danielle J Yoesep, Michael C Jewett. Single enzyme RT-PCR of full-length ribosomal RNA. Synthetic Biology 2020, 5 (1) https://doi.org/10.1093/synbio/ysaa028
- Willem A. Velema, Eric T. Kool. The chemistry and applications of RNA 2′-OH acylation. Nature Reviews Chemistry 2020, 4 (1) , 22-37. https://doi.org/10.1038/s41570-019-0147-6
- Joongoo Lee, Kenneth E. Schwieter, Andrew M. Watkins, Do Soon Kim, Hao Yu, Kevin J. Schwarz, Jongdoo Lim, Jaime Coronado, Michelle Byrom, Eric V. Anslyn, Andrew D. Ellington, Jeffrey S. Moore, Michael C. Jewett. Expanding the limits of the second genetic code with ribozymes. Nature Communications 2019, 10 (1) https://doi.org/10.1038/s41467-019-12916-w
Abstract
Figure 1
Figure 1. Simple aminobenzoic acid cyanomethyl esters are poor substrates for the eFx ribozyme. (A) Protocol used to detect acylation of microhelix (MH) or tRNA by cyanomethyl esters 1–3. (B) Acid-urea gel-shift analysis of MH acylation by cyanomethyl esters 1–3 in the presence of ribozyme eFx. Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions after RNase A digestion. Adenine nucleosides acylated on the 2′ or 3′ hydroxyl of the 3′ terminal ribose of MH could be detected in eFx-promoted reactions of the cyanomethyl ester of l-phenylalanine (Phe) and aminobenzoic acid esters 1 and 2; trace levels were detected in reactions containing 3. These products were not observed in analogous reactions containing m-aminobenzoic acid (compound C).
Figure 2
Figure 2. Initiator tRNA (fMetT) acylated with o-aminobenzoic acid can initiate translation within the PTC of wild type E. coli ribosomes. (A) Protocol used to evaluate whether an initiator tRNA (fMetT) acylated with o- (prepared using isatoic anhydride) or m-aminobenzoic acid (prepared using eFx) (AN-tRNA) could support translation in vitro. (B) LC-HRMS analysis of reaction products showing DNA template-dependent translation of a polypeptide whose mass corresponds to that of o-AN-VFDYKDDDDK (o-AN-VF-FLAG). No such polypeptide is observed in the absence of DNA template or in the presence of l-methionine. LC-HRMS analysis of an analogous β-Phe-containing polypeptide is shown for comparison.
Figure 3
Figure 3. Probing structure–activity relationships for cyanomethyl esters of substituted benzoic acids in eFx-promoted acylation reactions. (A) Substituted benzoic acid cyanomethyl esters studied herein. (B) Acid-urea gel-shift analysis of MH acylation by cyanomethyl esters 6 and 8–15 in the presence of ribozyme eFx. Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions containing cyanomethyl esters 6 and 8–15 after RNase A digestion. Exact masses are reported in Table S2.
Figure 4
Figure 4. Initiator tRNAs acylated with diverse benzoic acids are accommodated in the ribosomal P-site and are elongated into AR-VF-FLAG polypeptides. LC-HRMS analysis of reaction products whose masses correspond to AR-VFDYKDDDDK (AR-VF-FLAG) polypeptides containing diverse substituted benzoic acid monomers.
Figure 5
Figure 5. Wild type E. coli ribosomes support the biosynthesis of polyketide-peptide hybrid molecules. (A) Malonic esters 19–23 evaluated as substrates for eFx or dFx. (B) Acid-urea gel-shift analysis of MH acylation by esters 19–23 in the presence of eFx (19, 22) or dFx (20, 21,23). Yield was estimated by UV densitometry. (C) LC-HRMS analysis of MH acylation reactions containing esters 19–23 after RNase A digestion. (D) LC-HRMS analysis of reaction products whose masses corresponds to Mal-VFDYKDDDDK (Mal-VF-FLAG) polypeptides containing methyl and nitrobenzyl malonates 23 and 22. ND = not determined due to lack of separation from unacylated microhelix. Exact masses are reported in Table S2.
References
ARTICLE SECTIONSThis article references 49 other publications.
- 1Guo, J.; Wang, J.; Anderson, J. C.; Schultz, P. G. Addition of an α-hydroxy acid to the genetic code of bacteria. Angew. Chem., Int. Ed. 2008, 47, 722– 725, DOI: 10.1002/anie.200704074[Crossref], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhslalt74%253D&md5=b12c1b9b6b01e438d5d74943fb677c90Addition of an α-hydroxy acid to the genetic code of bacteriaGuo, Jiantao; Wang, Jiangyun; Anderson, J. Christopher; Schultz, Peter G.Angewandte Chemie, International Edition (2008), 47 (4), 722-725CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The α-hydroxy acid p-hydroxy-L-phenyllactic acid has been genetically incorporated into proteins in E. coli in response to an amber nonsense codon. The site-specific introduction of backbone ester mutations was used for the site-specific hydrolysis of an affinity tag, as well as for detn. of the energetic contributions of backbone hydrogen bonds to protein stability.
- 2Chin, J. W. Expanding and reprogramming the genetic code. Nature 2017, 550, 53– 60, DOI: 10.1038/nature24031[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Sqs73J&md5=2cf1adcbc37e3fd9f077e20b5c3c13c8Expanding and reprogramming the genetic codeChin, Jason W.Nature (London, United Kingdom) (2017), 550 (7674), 53-60CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chem. and phys. properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.
- 3Vargas-Rodriguez, O.; Sevostyanova, A.; Söll, D.; Crnković, A. Upgrading aminoacyl-tRNA synthetases for genetic code expansion. Curr. Opin. Chem. Biol. 2018, 46, 115– 122, DOI: 10.1016/j.cbpa.2018.07.014[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVSmtLfK&md5=4aa6fece6db9278a047a1a3ff6f27911Upgrading aminoacyl-tRNA synthetases for genetic code expansionVargas-Rodriguez, Oscar; Sevostyanova, Anastasia; Soll, Dieter; Crnkovic, AnaCurrent Opinion in Chemical Biology (2018), 46 (), 115-122CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biol. Progress in this field has enabled site-specific incorporation of over 200 chem. and structurally diverse amino acids into proteins in an increasing no. of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the L-alpha-amino acid structure (e.g. D-amino acid or Beta-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient aminoacyl-tRNA synthetases for genetic code expansion.
- 4Young, D. D.; Schultz, P. G. Playing with the molecules of life. ACS Chem. Biol. 2018, 13, 854– 870, DOI: 10.1021/acschembio.7b00974[ACS Full Text], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtF2nur0%253D&md5=89996e366e3b9b94a0efde80f89e52cfPlaying with the Molecules of LifeYoung, Douglas D.; Schultz, Peter G.ACS Chemical Biology (2018), 13 (4), 854-870CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A review. Our understanding of the complex mol. processes of living organisms is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of biol. macromols., is allowing chemists to begin to harness and reprogram the remarkable machinery of life. Here we review one such example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins in ways previously unimaginable.
- 5Maini, R.; Nguyen, D. T.; Chen, S.; Dedkova, L. M.; Chowdhury, S. R.; Alcala-Torano, R.; Hecht, S. M. Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center. Bioorg. Med. Chem. 2013, 21, 1088– 1096, DOI: 10.1016/j.bmc.2013.01.002[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslChu7c%253D&md5=bfdfbefdebf3a546efa39bc6d858b3b2Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase centerMaini, Rumit; Nguyen, Dan T.; Chen, Shengxi; Dedkova, Larisa M.; Chowdhury, Sandipan Roy; Alcala-Torano, Rafael; Hecht, Sidney M.Bioorganic & Medicinal Chemistry (2013), 21 (5), 1088-1096CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)Ribosomes contg. modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 prepns. for in vitro protein biosynthesis expts. When utilized in the presence of mRNAs contg. UAG codons at predetd. positions + β-alanyl-tRNACUA, the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted β-amino acids, and direct evidence for β-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in expts. employing four substituted β-amino acids, including α-methyl-β-alanine, β,β-dimethyl-β-alanine, β-phenylalanine, and β-(p-bromophenyl)alanine. The β-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of β-alanine and representative α-L-amino acids. The isolated proteins contg. the modified β-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the β-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of β,β-dimethyl-β-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the β-amino acid.
- 6Maini, R.; Chowdhury, S. R.; Dedkova, L. M.; Roy, B.; Daskalova, S. M.; Paul, R.; Chen, S.; Hecht, S. M. Protein synthesis with ribosomes selected for the incorporation of β-amino acids. Biochemistry 2015, 54, 3694– 3706, DOI: 10.1021/acs.biochem.5b00389[ACS Full Text], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosFaksLw%253D&md5=9a23376fcfad051c01a6c8b15a523ae6Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino AcidsMaini, Rumit; Chowdhury, Sandipan Roy; Dedkova, Larisa M.; Roy, Basab; Daskalova, Sasha M.; Paul, Rakesh; Chen, Shengxi; Hecht, Sidney M.Biochemistry (2015), 54 (23), 3694-3706CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In an earlier study, β3-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β3-puromycin employed for ribosome selection influences the regio- and stereochem. preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNACUA activated with each of four methyl-β-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochem. as the O-methylated β-tyrosine moiety of β3-puromycin. Also conducted were a selection of clones that are responsive to β2-puromycin and a demonstration of reversal of the regio- and stereochem. preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.
- 7Melo Czekster, C.; Robertson, W. E.; Walker, A. S.; Söll, D.; Schepartz, A. In vivo biosynthesis of a β-amino acid-containing protein. J. Am. Chem. Soc. 2016, 138, 5194– 5197, DOI: 10.1021/jacs.6b01023[ACS Full Text], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtVymsrk%253D&md5=ccf95e341314ea854a164a870a0f28ceIn Vivo Biosynthesis of a β-Amino Acid-Containing ProteinMelo Czekster, Clarissa; Robertson, Wesley E.; Walker, Allison S.; Soll, Dieter; Schepartz, AlannaJournal of the American Chemical Society (2016), 138 (16), 5194-5197CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 exts. and incubated with chem. mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here the authors report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β3-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β3-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.
- 8Maini, R.; Dedkova, L. M.; Paul, R.; Madathil, M. M.; Chowdhury, S. R.; Chen, S.; Hecht, S. M. Ribosome-mediated incorporation of dipeptides and dipeptide analogues into proteins in vitro. J. Am. Chem. Soc. 2015, 137, 11206– 11209, DOI: 10.1021/jacs.5b03135[ACS Full Text], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlOqu7jO&md5=3be559cec7e2dc8519d0d06ecc5db9c8Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in VitroMaini, Rumit; Dedkova, Larisa M.; Paul, Rakesh; Madathil, Manikandadas M.; Chowdhury, Sandipan Roy; Chen, Shengxi; Hecht, Sidney M.Journal of the American Chemical Society (2015), 137 (35), 11206-11209CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Plasmids contg. 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addn. to the pre-existing wild-type ribosomes. These clones were screened with a deriv. of puromycin, a natural product which acts as an analog of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin deriv. in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analog to recognize dipeptides. Several clones inhibited by the puromycin deriv. were used to make S-30 prepns., and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analog (4) and a fluorescent oxazole (5) having amine and carboxyl groups approx. the same distance apart as in a normal dipeptide. A protein contg. both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogs.
- 9Chen, S.; Ji, X.; Gao, M.; Dedkova, L. M.; Hecht, S. M. In cellulo synthesis of proteins containing a fluorescent oxazole amino acid. J. Am. Chem. Soc. 2019, 141, 5597– 5601, DOI: 10.1021/jacs.8b12767[ACS Full Text], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlt1yksLw%253D&md5=6f793dac4189184d1da4cc22bff25c3cIn Cellulo Synthesis of Proteins Containing a Fluorescent Oxazole Amino AcidChen, Shengxi; Ji, Xun; Gao, Mingxuan; Dedkova, Larisa M.; Hecht, Sidney M.Journal of the American Chemical Society (2019), 141 (14), 5597-5601CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Genetic code expansion has enabled many noncanonical amino acids to be incorporated into proteins in vitro and in cellulo. These have largely involved α-L-amino acids, reflecting the substrate specificity of natural aminoacyl-tRNA synthetases and ribosomes. Recently, modified E. coli ribosomes, selected using a dipeptidylpuromycin analog, were employed to incorporate dipeptides and dipeptidomimetics. Presently, the authors report the in cellulo incorporation of a strongly fluorescent oxazole amino acid (lacking an asym. center or α-amino group) by using modified ribosomes and pyrrolysyl-tRNA synthetase (PylRS). Initially, a plasmid en-coding the RRM1 domain of putative transcription factor hnRNP LL was cotransformed with plasmid pTECH-Pyl-OP in E. coli cells, having modified ribosomes able to incorporate dipeptides. Cell incubation in a medium contg. oxazole 2 resulted in the elaboration of RRM1 contg. the oxazole. Green fluorescent protein, previously expressed in vitro with several different oxazole amino acids at position 66, was also expressed in cellulo contg. oxazole 2; the incorporation was verified by mass spectrometry. Finally, oxazole 2 was incorporated into position 13 of MreB, a bacterial homolog of eukaryotic cytoskeletal protein actin F. Modified MreB expressed in vitro and in cellulo comigrated with wild type. E. coli cells expressing the modified MreB were strongly fluorescent, and retained the E. coli cell rod-like phenotype. For each protein studied, the incorporation of oxazole 2 strongly increased oxazole fluorescence, suggesting its potential utility as a protein tag. These findings also suggest the feasibility of dramatically increasing the repertoire of amino acids that can be genetically encoded for protein incorporation in cellulo.
- 10Subtelny, A. O.; Hartman, M. C. T.; Szostak, J. W. Ribosomal synthesis of N-methyl peptides. J. Am. Chem. Soc. 2008, 130, 6131– 6136, DOI: 10.1021/ja710016v[ACS Full Text], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXksFarsr0%253D&md5=31e1ecb5c2a4c2ba0b88ad281fdb5ae8Ribosomal Synthesis of N-Methyl PeptidesSubtelny, Alexander O.; Hartman, Matthew C. T.; Szostak, Jack W.Journal of the American Chemical Society (2008), 130 (19), 6131-6136CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)N-Me amino acids (N-Me AAs) are a common component of nonribosomal peptides (NRPs), a class of natural products from which many clin. important therapeutics are obtained. N-Me AAs confer peptides with increased conformational rigidity, membrane permeability, and protease resistance. Hence, these analogs are highly desirable building blocks in the ribosomal synthesis of unnatural peptide libraries, from which functional, NRP-like mols. may be identified. By supplementing a reconstituted Escherichia coli translation system with specifically aminoacylated total tRNA that has been chem. methylated, the authors have identified three N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) that are efficiently incorporated into peptides by the ribosome. Moreover, the authors have demonstrated the synthesis of peptides contg. up to three N-Me AAs, a no. comparable to that found in many NRP drugs. With improved incorporation efficiency and translational fidelity, it may be possible to synthesize combinatorial libraries of peptides that contain multiple N-Me AAs. Such libraries could be subjected to in vitro selection methods to identify drug-like, high-affinity ligands for protein targets of interest.
- 11Dedkova, L. M.; Fahmi, N. E.; Golovine, S. Y.; Hecht, S. M. Enhanced d-amino acid incorporation into protein by modified ribosomes. J. Am. Chem. Soc. 2003, 125, 6616– 6617, DOI: 10.1021/ja035141q[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjs12ks7g%253D&md5=932096443b831dd0620db2c301b5b187Enhanced D-Amino Acid Incorporation into Protein by Modified RibosomesDedkova, Larisa M.; Fahmi, Nour Eddine; Golovine, Serguei Y.; Hecht, Sidney M.Journal of the American Chemical Society (2003), 125 (22), 6616-6617CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)By overexpression of modified Escherichia coli 23S rRNAs from multicopy plasmids, ribosomes were prepd. that contained mutations in two regions (2447-2450 and 2457-2462) of 23S rRNA. Following mutagenesis and selection, two clones with mutations in the 2447-2450 region (peptidyltransferase center) and six with mutations in the 2457-2462 region (helix 89) were characterized. The mutations were shown to exhibit a high level of homol. Cell-free protein synthesizing systems prepd. from these clones were found to exhibit significantly enhanced incorporation of D-methionine and D-phenylalanine into protein. The incorporations involved positions 10, 22, and 54 of E. coli dihydrofolate reductase and positions 247 and 250 of Photinus pyralis firefly luciferase. Interestingly, some of the derived proteins contg. the D-amino acids (notably DHFR analogs altered at position 10) functioned as well as those contg. the resp. L-amino acids, while substitution at other positions resulted in proteins having greatly diminished activity.
- 12Goto, Y.; Murakami, H.; Suga, H. Initiating translation with d-amino acids. RNA 2008, 14, 1390– 1398, DOI: 10.1261/rna.1020708[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXot1amtLs%253D&md5=efa90e6f836e03246973e7565ebe37daInitiating translation with D-amino acidsGoto, Yuki; Murakami, Hiroshi; Suga, HiroakiRNA (2008), 14 (7), 1390-1398CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)Here we report exptl. evidence that the translation initiation app. accepts D-amino acids (Daa), as opposed to only L-methionine, as initiators. Nineteen Daa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNAfMetCAU using flexizyme technol. and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to Daa. Remarkably, all Daa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with Daa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding Daa-tRNAfMetCAU. Although the inefficient formylation of Daa-tRNAfMetCAU resulted in modest expression of the corresponding peptide, preacetylation of Daa-tRNAfMetCAU dramatically increased expression level, implying that the formylation efficiency is one of the crit. determinants of initiation efficiency with Daa. Our findings provide not only the exptl. evidence that translation initiation tolerates Daa, but also a new means for the mRNA-directed synthesis of peptides capped with Daa or acyl-Daa at the N terminus.
- 13Ohta, A.; Murakami, H.; Higashimura, E.; Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem. Biol. 2007, 14, 1315– 1322, DOI: 10.1016/j.chembiol.2007.10.015[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhsVOktrfJ&md5=2587638ab5c21965b8c3654721fc437dSynthesis of Polyester by Means of Genetic Code ReprogrammingOhta, Atsushi; Murakami, Hiroshi; Higashimura, Eri; Suga, HiroakiChemistry & Biology (Cambridge, MA, United States) (2007), 14 (12), 1315-1322CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Here we report the ribosomal polymn. of α-hydroxy acids by means of genetic code reprogramming. The flexizyme system, a ribozyme-based tRNA acylation tool, was used to reassign individual codons to seven types of α-hydroxy acids, and then polyesters were synthesized under controls of the reprogrammed genetic code using a reconstituted cell-free translation system. The sequence and length of the polyester segments were specified by the mRNA template, indicating that high-fidelity ribosome expression of polyesters was possible. This work opens a door for the mRNA-directed synthesis of backbone-altered biopolymers.
- 14Fujino, T.; Goto, Y.; Suga, H.; Murakami, H. Ribosomal synthesis of peptides with multiple β-amino acids. J. Am. Chem. Soc. 2016, 138, 1962– 1969, DOI: 10.1021/jacs.5b12482[ACS Full Text], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlWkt7c%253D&md5=f7365b08e95f75738119a38232096814Ribosomal Synthesis of Peptides with Multiple β-Amino AcidsFujino, Tomoshige; Goto, Yuki; Suga, Hiroaki; Murakami, HiroshiJournal of the American Chemical Society (2016), 138 (6), 1962-1969CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The compatibility of β-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various β-amino acids, and whether the ribosome can introduce multiple β-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened β-amino acids that give high single incorporation efficiency and used them to synthesize peptides contg. multiple β-amino acids. The expts. of single β-amino acid incorporation into a peptide revealed that 13 β-amino acids are compatible with ribosomal translation. Six of the tested β-amino acids (βhGly, L-βhAla, L-βhGln, L-βhPhg, L-βhMet, and D-βhPhg) showed high incorporation efficiencies, and seven (L-βhLeu, L-βhIle, L-βhAsn, L-βhPhe, L-βhLys, D-βhAla, and D-βhLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other β-amino acids (L-βhPro, L-βhTrp, and L-βhGlu). Subsequent double-incorporation expts. using β-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive β-amino acids is prohibited. Efficiency of the double-incorporation of the β-amino acids was restored by the insertion of Tyr or Ile between the two β-amino acids. On the basis of these expts., we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides contg. different types of β-amino acids at multiple positions.
- 15Katoh, T.; Suga, H. Ribosomal incorporation of consecutive β-amino acids. J. Am. Chem. Soc. 2018, 140, 12159– 12167, DOI: 10.1021/jacs.8b07247[ACS Full Text], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslWgtL7P&md5=825f4a1be80ef41fecee313f4eddc9bfRibosomal Incorporation of Consecutive β-Amino AcidsKatoh, Takayuki; Suga, HiroakiJournal of the American Chemical Society (2018), 140 (38), 12159-12167CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Due to their unique characteristics, which are not shared by canonical α-peptides, peptides that contain stretches of consecutive β-amino acids are attractive scaffolds for novel peptide drugs and nanomaterials. Although ribosomal incorporation of single or non-consecutive β-amino acids into peptides has previously been reported, the incorporation of consecutive β-amino acids has not yet been accomplished. This is primarily due to their incompatibility with the ribosomal translation system. Here, we took advantage of engineered β-aminoacyl-tRNAs bearing optimized T-stem and D-arm motifs for enhancing binding affinity to EF-Tu and EF-P, resp. Combined with a reconstituted E. coli translation system and optimized translation factor concns., up to seven consecutive β-amino acids could be incorporated into a model peptide. Furthermore, the synthesis of macrocyclic β-peptides closed by a thioether bond between two D-α-amino acids is also demonstrated. This represents the first example of the ribosomal synthesis of peptides contg. stretches of consecutive β-amino acids.
- 16Wang, P. S.; Schepartz, A. β-peptide bundles: Design. Build. Analyze. Biosynthesize.. Chem. Commun. 2016, 52, 7420– 7432, DOI: 10.1039/C6CC01546H[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsVWqs7w%253D&md5=f2b3b1cf34e77aada1ab698e5f84118dβ-Peptide bundles: Design. Build. Analyze. Biosynthesize.Wang, Pam S. P.; Schepartz, AlannaChemical Communications (Cambridge, United Kingdom) (2016), 52 (47), 7420-7432CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A review. Peptides contg. β-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. Peptides contg. β-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. When composed solely of β-amino acids, the structures formed, defined assemblies of 14-helixes called β-peptide bundles, fold cooperatively in water solvent into unique and discrete quaternary assemblies that are highly thermostable, bind complex substrates and metal ion cofactors, and, in certain cases, catalyze chem. reactions. In this perspective, the authors recount the design and elaboration of β-peptide bundles, and provide an outlook on recent, unexpected discoveries that could influence research on β-peptides and β-peptide bundles (and β-amino acid-contg. proteins) for decades to come.
- 17Checco, J. W.; Gellman, S. H. Targeting recognition surfaces on natural proteins with peptidic foldamers. Curr. Opin. Struct. Biol. 2016, 39, 96– 105, DOI: 10.1016/j.sbi.2016.06.014[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGitr%252FE&md5=59db458d1a2f21a7c60ecf3ff6cdc862Targeting recognition surfaces on natural proteins with peptidic foldamersChecco, James W.; Gellman, Samuel H.Current Opinion in Structural Biology (2016), 39 (), 96-105CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Mols. intended to antagonize protein-protein interactions or augment polypeptide-based signaling must bind tightly to large and sp. surfaces on target proteins. Some types of unnatural oligomers with discrete folding propensities ('foldamers') have recently been shown to display this capability. This review covers important recent advances among several classes of foldamers, including α-peptides with secondary structures stabilized by covalent bonds, D-α-peptides, α/β-peptides and oligo-oxopiperazines. Recent advances in this area have involved enhancing membrane permeability to provide access to intracellular protein targets, improving pharmacokinetics and duration of action in vivo, and developing strategies appropriate for targeting large and irregularly-shaped protein surfaces.
- 18Montalvo, G.; Waegele, M. M.; Shandler, S.; Gai, F.; DeGrado, W. F. Infrared signature and folding dynamics of a helical β-peptide. J. Am. Chem. Soc. 2010, 132, 5616– 8, DOI: 10.1021/ja100459a[ACS Full Text], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksVansbw%253D&md5=cf5fef952f2e9e57b2fa2a98bf44e6ddInfrared Signature and Folding Dynamics of a Helical β-PeptideMontalvo, Geronda; Waegele, Matthias M.; Shandler, Scott; Gai, Feng; De Grado, William F.Journal of the American Chemical Society (2010), 132 (16), 5616-5618CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Synthetic foldamers consisting of β-amino acids offer excellent model systems for examg. the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding-unfolding kinetics of a β-peptide that folds into a 14-helical structure in water. We find that the T-jump induced relaxation kinetics of this peptide occur on the nanosecond time scale and are noticeably slower than those of alanine-based α-helical peptides, and addnl., the relaxation rates show a weaker dependence on temp. These differences appear to indicate that the folding energy landscapes of these peptides are different. In addn., we find that the amide I' band of this β-peptide exhibits a sharp feature at ∼1612 cm-1, which we believe is a distinct IR reporter of 14-helix.
- 19Seebach, D.; Gardiner, J. Beta-peptidic peptidomimetics. Acc. Chem. Res. 2008, 41, 1366– 1375, DOI: 10.1021/ar700263g[ACS Full Text], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnvVKksrk%253D&md5=e679836f00843a02103caf63773ffa80β-Peptidic peptidomimeticsSeebach, Dieter; Gardiner, JamesAccounts of Chemical Research (2008), 41 (10), 1366-1375CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. For more than a decade now, a search for answers to the following two questions has taken us on a new and exciting journey into the world of β- and γ-peptides: What happens if the O atoms in a 3i-helix of a polymeric chain composed of (R)-3-hydroxybutanoic acid are replaced by NH units. What happens if 1 or 2 CH2 groups are introduced into each amino acid building block in the chain of a peptide or protein, thereby providing homologues of the proteinogenic α-amino acids. Our journey has repeatedly thrown up surprises, continually expanding the potential of these classes of compd. and deepening our understanding of the structures, properties, and multifaceted functions of the natural "models" to which they are related. β-Peptides differ from their natural counterparts, the α-peptides, by having CH2 groups inserted into every amino acid residue, either between the C=O groups and the α-C atoms (β3) or between the α-C and N atoms (β2). The synthesis of these homologated proteinogenic amino acids and their assembly into β-peptides can be performed using known methods. Despite the increased no. of possible conformers, the β-peptides form secondary structures (helixes, turns, sheets) even when the chain lengths are as short as 4 residues. Furthermore, they are stable toward degrading and metabolizing enzymes in living organisms. Linear, helical, and hairpin-type structures of β-peptides can now be designed in such a way that they resemble the characteristic and activity-related structural features ("epitopes") of corresponding natural peptides or protein sections. This account presents examples of β-peptidic compds. binding, as agonists or antagonists (inhibitors), to (i) major histocompatibility complex (MHC) proteins (immune response), (ii) the lipid-transport protein SR-B1 (cholesterol uptake from the small intestine), (iii) the core (1-60) of interleukin-8 (inflammation), (iv) the oncoprotein RDM2, (v) the HIV gp41 fusion protein, (vi) G-protein-coupled somatostatin hsst receptors, (vii) the TNF immune response receptor CD40 (apoptosis), and (viii) DNA. Short-chain β-peptides may be orally bioavailable and excreted from the body of mammals; long-chain β-peptides may require i.v. administration but will have longer half-lives of clearance. It has been said that an interesting field of research distinguishes itself in that the results always throw up new questions; in this sense, the structural and biol. investigation of β-peptides has been a gold mine. We expect that these peptidic peptidomimetics will play an increasing role in biomedical research and drug development in the near future.
- 20García, J. M.; García, F. C.; Serna, F.; de la Peña, J. L. High-performance aromatic polyamides. Prog. Polym. Sci. 2010, 35, 623– 686, DOI: 10.1016/j.progpolymsci.2009.09.002[Crossref], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjvFKitr8%253D&md5=aa7ccb4975af61aef614c6c9b1a26ba3High-performance aromatic polyamidesGarcia, Jose M.; Garcia, Felix C.; Serna, Felipe; de la Pena, Jose L.Progress in Polymer Science (2010), 35 (5), 623-686CODEN: PRPSB8; ISSN:0079-6700. (Elsevier Ltd.)A review. Wholly arom. polyamides (aramids) are considered to be high-performance org. materials due to their outstanding thermal and mech. resistance. Their properties arise from their arom. structure and amide linkages, which result in stiff rod-like macromol. chains that interact with each other via strong and highly directional hydrogen bonds. These bonds create effective cryst. microdomains, resulting in a high-level intermol. packing and cohesive energy. The better known com. aramids, poly(p-phenylene terephthalamide) and poly(m-phenylene isophthalamide), are used in advanced technologies and have been transformed into high-strength and flame resistant fibers and coatings, with applications in the aerospace and armament industry, bullet-proof body armor, protective clothing, sport fabrics, elec. insulation, asbestos substitutes, and industrial filters, among others. Owing to their chem. structure, they exhibit extremely high transition temps. that lie above their decompn. temps., are sparingly sol. in common org. solvents and, accordingly, can only be transformed upon soln. Research efforts are therefore underway to take advantage of their properties, enhance their processability and soly., and incorporate new chem. functionalities in the polyamide backbone or lateral structure, so that their applicability is expanded and remains on the forefront of scientific research.
- 21Tanner, D.; Fitzgerald, J. A.; Phillips, B. R. The kevlar story - an advanced materials case study. Angew. Chem., Int. Ed. Engl. 1989, 28, 649– 654, DOI: 10.1002/anie.198906491
- 22Baumann, S.; Herrmann, J.; Raju, R.; Steinmetz, H.; Mohr, K. I.; Háttel, S.; Harmrolfs, K.; Stadler, M.; Máller, R. Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activity. Angew. Chem., Int. Ed. Engl. 2014, 53, 14605– 14609, DOI: 10.1002/anie.201409964[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MznvVSqsg%253D%253D&md5=c500f536a8abceedd4aaa7ef7fe8bab6Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activityBaumann Sascha; Herrmann Jennifer; Raju Ritesh; Steinmetz Heinrich; Mohr Kathrin I; Huttel Stephan; Harmrolfs Kirsten; Stadler Marc; Muller RolfAngewandte Chemie (International ed. in English) (2014), 53 (52), 14605-9 ISSN:.The development of new antibiotics faces a severe crisis inter alia owing to a lack of innovative chemical scaffolds with activities against Gram-negative and multiresistant pathogens. Herein, we report highly potent novel antibacterial compounds, the myxobacteria-derived cystobactamids 1-3, which were isolated from Cystobacter sp. and show minimum inhibitory concentrations in the low μg mL(-1) range. We describe the isolation and structure elucidation of three congeners as well as the identification and annotation of their biosynthetic gene cluster. By studying the self-resistance mechanism in the natural producer organism, the molecular targets were identified as bacterial type IIa topoisomerases. As quinolones are largely exhausted as a template for new type II topoisomerase inhibitors, the cystobactamids offer exciting alternatives to generate novel antibiotics using medicinal chemistry and biosynthetic engineering.
- 23Saraogi, I.; Incarvito, C. D.; Hamilton, A. D. Controlling curvature in a family of oligoamide α-helix mimetics. Angew. Chem., Int. Ed. 2008, 47, 9691– 9694, DOI: 10.1002/anie.200803778[Crossref], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFanurfL&md5=f8bde9f3cd2a9e89e77004365a578ffdControlling curvature in a family of oligoamide α-helix mimeticsSaraogi, Ishu; Incarvito, Christopher D.; Hamilton, Andrew D.Angewandte Chemie, International Edition (2008), 47 (50), 9691-9694CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The natural curvature found in a majority of α helixes has been mimicked in small synthetic oligoamide scaffolds. Differences in hydrogen-bonding patterns in these scaffolds lead to mimetics with varying degrees of curvature in the backbone. This adds another parameter to the structural and functional mimicry of a helixes.
- 24Meisel, J. W.; Hu, C. T.; Hamilton, A. D. Mimicry of a β-hairpin turn by a nonpeptidic laterally flexible foldamer. Org. Lett. 2018, 20, 3879– 3882, DOI: 10.1021/acs.orglett.8b01463[ACS Full Text], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFWhurfK&md5=aa0642e59d820b9bacca47cf68bfab8cMimicry of a β-Hairpin Turn by a Nonpeptidic Laterally Flexible FoldamerMeisel, Joseph W.; Hu, Chunhua T.; Hamilton, Andrew D.Organic Letters (2018), 20 (13), 3879-3882CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)The design and characterization of a proteomimetic foldamer that displays lateral flexibility endowed by intramol. bifurcated hydrogen bonds is reported. The MAMBA scaffold, derived from meta-aminomethylbenzoic acid, adopts a serpentine conformation that mimics the side chain projection of all four residues in a β-hairpin turn.
- 25Saha, S.; Kauffmann, B.; Ferrand, Y.; Huc, I. Selective encapsulation of disaccharide xylobiose by an aromatic foldamer helical capsule. Angew. Chem., Int. Ed. 2018, 57, 13542– 13546, DOI: 10.1002/anie.201808370[Crossref], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslaiu7%252FP&md5=b912c7c178c6272b9bdddfa5a14e0c7fSelective Encapsulation of Disaccharide Xylobiose by an Aromatic Foldamer Helical CapsuleSaha, Subrata; Kauffmann, Brice; Ferrand, Yann; Huc, IvanAngewandte Chemie, International Edition (2018), 57 (41), 13542-13546CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Xylobiose sequestration in a helical arom. oligoamide capsule was evidenced by CD, NMR spectroscopy, and crystallog. The prepn. of the 5 kDa oligoamide sequence was made possible by the transient use of acid-labile dimethoxybenzyl tertiary amide substituents that disrupt helical folding and prevent double helix formation. Binding of other disaccharides was not detected. Crystallog. data revealed a complex composed of a D-xylobiose α anomer and two water mols. accommodated in the right-handed helix. The disaccharide was found to adopt an unusual all-axial compact conformation. A dense network of 18 hydrogen bonds forms between the guest, the cavity wall, and the two water mols.
- 26Rogers, J. M.; Kwon, S.; Dawson, S. J.; Mandal, P. K.; Suga, H.; Huc, I. Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids. Nat. Chem. 2018, 10, 405– 412, DOI: 10.1038/s41557-018-0007-x[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXos1Smt7s%253D&md5=207db9badb99e896f9f5aeb06181f0eeRibosomal synthesis and folding of peptide-helical aromatic foldamer hybridsRogers, Joseph M.; Kwon, Sunbum; Dawson, Simon J.; Mandal, Pradeep K.; Suga, Hiroaki; Huc, IvanNature Chemistry (2018), 10 (4), 405-412CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Translation, the mRNA-templated synthesis of peptides by the ribosome, can be manipulated to incorporate variants of the 20 cognate amino acids. Such approaches for expanding the range of chem. entities that can be produced by the ribosome may accelerate the discovery of mols. that can perform functions for which poorly folded, short peptidic sequences are ill suited. Here, we show that the ribosome tolerates some artificial helical arom. oligomers, so-called foldamers. Using a flexible tRNA-acylation ribozyme-flexizyme-foldamers were attached to tRNA, and the resulting acylated tRNAs were delivered to the ribosome to initiate the synthesis of non-cyclic and cyclic foldamer-peptide hybrid mols. Passing through the ribosome exit tunnel requires the foldamers to unfold. Yet foldamers encode sufficient folding information to influence the peptide structure once translation is completed. We also show that in cyclic hybrids, the foldamer portion can fold into a helix and force the peptide segment to adopt a constrained and stretched conformation.
- 27Schepartz, A. Foldamers wave to the ribosome. Nat. Chem. 2018, 10, 377– 379, DOI: 10.1038/s41557-018-0036-5[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXos1SktrY%253D&md5=a0dd482713baeb9a88bcaeeda02d2878Foldamers wave to the ribosomeSchepartz, AlannaNature Chemistry (2018), 10 (4), 377-379CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Ribosomes have now been shown to accept certain initiator tRNAs acylated with arom. foldamer-dipeptides thereby enabling the translation of a peptide or protein with a short arom. foldamer at the N-terminus. Some foldamer-peptide hybrids could be cyclized to generate macrocycles that present conformationally restricted peptide loops.
- 28Goto, Y.; Suga, H. Flexizymes as a tRNA acylation tool facilitating genetic code reprogramming. Methods Mol. Biol. 2012, 848, 465– 478, DOI: 10.1007/978-1-61779-545-9_29[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1agsLjM&md5=300684998389c280029bee28f646fc7bFlexizymes as a tRNA acylation tool facilitating genetic code reprogrammingGoto, Yuki; Suga, HiroakiMethods in Molecular Biology (New York, NY, United States) (2012), 848 (Ribozymes), 465-478CODEN: MMBIED; ISSN:1064-3745. (Springer)A review. Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. We here describe a protocol that facilitates the genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation app., referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the prepn. of a diverse array of non-proteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-acyl groups, D-amino acids, N-Me amino acids, and physiol. stable macrocyclic scaffolds. Facility of the protocol allows for a wide variety of applications in the synthesis of new classes of nonstandard peptides with biol. functions.
- 29Murakami, H.; Saito, H.; Suga, H. A versatile tRNA aminoacylation catalyst based on RNA. Chem. Biol. 2003, 10, 655– 662, DOI: 10.1016/S1074-5521(03)00145-5[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlvVGjs7w%253D&md5=0b42c261bbce87009ddaddcc47ac897cA Versatile tRNA Aminoacylation Catalyst Based on RNAMurakami, Hiroshi; Saito, Hirohide; Suga, HiroakiChemistry & Biology (2003), 10 (7), 655-662CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognizes arom. amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNAAsnCCCG). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and nonnatural amino acids.
- 30McMurry, J. L.; Chang, M. C. Y. Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 11920– 11925, DOI: 10.1073/pnas.1711482114[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslSktrfJ&md5=70a570ad8451738c6e156330a9fd03d3Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleyaMcMurry, Jonathan L.; Chang, Michelle C. Y.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (45), 11920-11925CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small mols., ranging from therapeutics to materials. In contrast, only a few fluorinated compds. made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in Streptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochem., genetic, physiol., and proteomic studies, we show that FthB is a trans-acting tRNA editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While trans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metab. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.
- 31Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 2006, 3, 357– 359, DOI: 10.1038/nmeth877[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjslSktrw%253D&md5=d6694f40ba3b0f5791ac361fa6d29a38A highly flexible tRNA acylation method for non-natural polypeptide synthesisMurakami, Hiroshi; Ohta, Atsushi; Ashigai, Hiroshi; Suga, HiroakiNature Methods (2006), 3 (5), 357-359CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Here the authors describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the prepn. of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows the authors to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple nonnatural amino acids.
- 32Nawrot, B.; Sprinzl, M. Aminoacyl-tRNA analogues; synthesis, purification and properties of 3-anthraniloyl oligoribonucleotides. Nucleosides Nucleotides 1998, 17, 815– 829, DOI: 10.1080/07328319808004677[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXitFWqtbY%253D&md5=c419d507c871431a0afba116cc4ccf17Aminoacyl-tRNA analogs; synthesis, purification and properties of 3'-anthraniloyl oligoribonucleotidesNawrot, B.; Sprinzl, M.Nucleosides & Nucleotides (1998), 17 (4), 815-829CODEN: NUNUD5; ISSN:0732-8311. (Marcel Dekker, Inc.)Reaction of isatoic anhydride with adenosine, adenosine 5'-phosphate, oligoribonucleotides or with the E. coli tRNAVal led to attachment of an anthraniloyl residue at 2'- or 3'-OH groups of 3'-terminal ribose residue. No protection of the 5'-hydroxyl group or internal 2'-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analog of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.
- 33Mortimer, S. A.; Weeks, K. M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 2007, 129, 4144– 4145, DOI: 10.1021/ja0704028[ACS Full Text], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXivF2jtr0%253D&md5=d0b0d3d7582cd98cd801077ade6f42c5A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE ChemistryMortimer, Stefanie A.; Weeks, Kevin M.Journal of the American Chemical Society (2007), 129 (14), 4144-4145CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chem. allows local nucleotide flexibility to be quant. assessed at single nucleotide resoln. in any RNA. SHAPE chem. exploits structure-based gating of the nucleophilic reactivity of the ribose 2'-hydroxyl group by the extent to which a nucleotide is constrained or flexible. SHAPE chem. was developed using N-methylisatoic anhydride (NMIA), which is only moderately electrophilic and requires tens of minutes to form ribose 2'-O-adducts. Here, we design and evaluate a significantly more useful, fast-acting, reagent for SHAPE chem. Introduction of a nitro group para to the reactive carbonyl to form 1-methyl-7-nitroisatoic anhydride (1M7) yields a reagent that both reacts significantly more rapidly with RNA to form 2'-O-adducts and is also more labile toward advantageous, self-limiting, hydrolysis. With 1M7, the single nucleotide resoln. interrogation of the RNA structure is complete in 70 s. SHAPE anal. performed with 1M7 accurately reports the secondary and tertiary structure of the RNase P specificity domain and allows the secondary structure of this RNA to be predicted with up to 91% accuracy.
- 34Drossman, H.; Johnson, H.; Mill, T. Structure activity relationships for environmental processes 1: Hydrolysis of esters and carbamates. Chemosphere 1988, 17, 1509– 1530, DOI: 10.1016/0045-6535(88)90204-4[Crossref], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXmtFGltro%253D&md5=ed9e54b4a2522d09572a225555c6363bStructure activity relationships for environmental processes 1: hydrolysis of esters and carbamatesDrossman, Howard; Johnson, Howard; Mill, TheodoreChemosphere (1988), 17 (8), 1509-30CODEN: CMSHAF; ISSN:0045-6535.Structure activity relationships (SAR) for base-promoted hydrolysis of esters and carbamates were developed using Hammett and Taft parameters. A master SAR equation correlates base promoted hydrolysis rate const. (kB) values for 103 esters with a range of reactivity of 1010. Two SAR equations are needed to correlate kB value for 80 carbamates.
- 35Xiao, H.; Murakami, H.; Suga, H.; Ferré-D’Amaré, A. R. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 2008, 454, 358– 361, DOI: 10.1038/nature07033[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXosFCqtLc%253D&md5=a6a47091d470d51e4cfd92ce0e4df78aStructural basis of specific tRNA aminoacylation by a small in vitro selected ribozymeXiao, Hong; Murakami, Hiroshi; Suga, Hiroaki; Ferre-D'Amare, Adrian R.Nature (London, United Kingdom) (2008), 454 (7202), 358-361CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In modern organisms, protein enzymes are solely responsible for the aminoacylation of tRNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalyzing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated L-phenylalanine derivs. In addn. to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-Å resoln. structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA mol., flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallog. independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.
- 36Hunter, C. A.; Lawson, K. R.; Perkins, J.; Urch, C. J. Aromatic interactions. J. Chem. Soc. Perkins Trans. 2 2001, 2, 651– 669, DOI: 10.1039/b008495f
- 37Meyer, E. A.; Castellano, R. K.; Diederich, F. Interactions with aromatic rings in chemical and biological recognition. Angew. Chem., Int. Ed. 2003, 42, 1210– 1250, DOI: 10.1002/anie.200390319[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXivVSrtbw%253D&md5=e91e6a8d115d56229e1660697a286b04Interactions with aromatic rings in chemical and biological recognitionMeyer, Emmanuel A.; Castellano, Ronald K.; Diederich, FrancoisAngewandte Chemie, International Edition (2003), 42 (11), 1210-1250CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Intermol. interactions involving arom. rings are key processes in both chem. and biol. recognition. Their understanding is essential for rational drug design and lead optimization in medicinal chem. Different approaches-biol. studies, mol. recognition studies with artificial receptors, crystallog. database mining, gas-phase studies, and theor. calcns. - are pursued to generate a profound understanding of the structural and energetic parameters of individual recognition modes involving arom. rings. This review attempts to combine and summarize the knowledge gained from these investigations. The review focuses mainly on examples with biol. relevance since one of its aims it to enhance the knowledge of mol. recognition forces that is essential for drug development.
- 38Hansch, C.; Leo, A.; Taft, R. W. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 1991, 91, 165– 195, DOI: 10.1021/cr00002a004[ACS Full Text], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhs1ehsLo%253D&md5=9fc814cd57c47680a5213f3438037800A survey of Hammett substituent constants and resonance and field parametersHansch, Corwin; Leo, A.; Taft, R. W.Chemical Reviews (Washington, DC, United States) (1991), 91 (2), 165-95CODEN: CHREAY; ISSN:0009-2665.Included in this review is an anal. of newer methods which can supplant this classic procedure for detn. of the title consts., 283 refs.
- 39Kawakami, T.; Ogawa, K.; Hatta, T.; Goshima, N.; Natsume, T. Directed evolution of a cyclized peptoid-peptide chimera against a cell-free expressed protein and proteomic profiling of the interacting proteins to create a protein-protein interaction inhibitor. ACS Chem. Biol. 2016, 11, 1569– 1577, DOI: 10.1021/acschembio.5b01014[ACS Full Text], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xks12gsr8%253D&md5=94544c0a684d5784920cbce562d4de87Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction InhibitorKawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, TohruACS Chemical Biology (2016), 11 (6), 1569-1577CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liq. chromatog.-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.
- 40Goto, Y.; Ohta, A.; Sako, Y.; Yamagishi, Y.; Murakami, H.; Suga, H. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol. 2008, 3, 120– 129, DOI: 10.1021/cb700233t[ACS Full Text], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXptl2isA%253D%253D&md5=1627a925839f35c767df198041d7bc2cReprogramming the Translation Initiation for the Synthesis of Physiologically Stable Cyclic PeptidesGoto, Yuki; Ohta, Atsushi; Sako, Yusuke; Yamagishi, Yusuke; Murakami, Hiroshi; Suga, HiroakiACS Chemical Biology (2008), 3 (2), 120-129CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)The initiation codon dictates that the translation initiation event exclusively begins with methionine. We report a new technol. to reprogram the initiation event, where various amino acids and those bearing Nα-acyl groups can be used as an initiator for peptide synthesis. The technol. is built upon the concept of genetic code reprogramming, where methionine is depleted from the translation system and the initiation codon is reassigned to the desired amino acid. We have applied this technol. to the synthesis of an antitumor cyclic peptide, G7-18NATE, closed by a physiol. stable bond, and it is also extended to the custom synthesis of its analogs with various ring sizes. Significantly, cyclization occurs spontaneously upon translation of the precursor linear peptides. To demonstrate the practicality of this methodol., we also prepd. a small cyclic peptide library designated by 160 distinct mRNAs. Thus, this technol. offers a new means to prep. a wide array of in vivo compatible cyclic peptide libraries for the discovery of peptidic drug candidates against various therapeutic targets.
- 41Gualerzi, C. O.; Pon, C. L. Initiation of mRNA translation in bacteria: structural and dynamic aspects. Cell. Mol. Life Sci. 2015, 72, 4341– 4367, DOI: 10.1007/s00018-015-2010-3[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlelurvN&md5=a35b5f76a4d78a90f17b28601b9f5b96Initiation of mRNA translation in bacteria: structural and dynamic aspectsGualerzi, Claudio O.; Pon, Cynthia L.Cellular and Molecular Life Sciences (2015), 72 (22), 4341-4367CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)Initiation of mRNA translation is a major checkpoint for regulating level and fidelity of protein synthesis. Being rate limiting in protein synthesis, translation initiation also represents the target of many post-transcriptional mechanisms regulating gene expression. The process begins with the formation of an unstable 30S pre-initiation complex (30S pre-IC) contg. initiation factors (IFs) IF1, IF2 and IF3, the translation initiation region of an mRNA and initiator fMet-tRNA whose codon and anticodon pair in the P-site following a first-order rearrangement of the 30S pre-IC produces a locked 30S initiation complex (30SIC); this is docked by the 50S subunit to form a 70S complex that, following several conformational changes, positional readjustments of its ligands and ejection of the IFs, becomes a 70S initiation complex productive in initiation dipeptide formation. The first EF-G-dependent translocation marks the beginning of the elongation phase of translation. Here, we review structural, mechanistic and dynamical aspects of this process.
- 42Goto, Y.; Katoh, T.; Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 2011, 6, 779– 790, DOI: 10.1038/nprot.2011.331[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXmvFyqu78%253D&md5=fbfd338c7f874ac9871c0f3cfd84b3edFlexizymes for genetic code reprogrammingGoto, Yuki; Katoh, Takayuki; Suga, HiroakiNature Protocols (2011), 6 (6), 779-790CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation app., referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the prepn. of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-Me amino acids, D-amino acids and physiol. stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biol. functions. Prepn. of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 wk. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.
- 43Staunton, J.; Weissman, K. J. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep. 2001, 18, 380– 416, DOI: 10.1039/a909079g[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXltlyqsbo%253D&md5=2f5a7eaf4d900456bb89d2e75721e2dfPolyketide biosynthesis: A millennium reviewStaunton, James; Weissman, Kira J.Natural Product Reports (2001), 18 (4), 380-416CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review with refs. on the developments of the past 100 yr in the study of polyketides, which have various medicinally important activities, including antibiotic, anticancer, antifungal, antiparasitic, and immunosuppressive properties. The speculation on the future of polyketide research is also discussed.
- 44Dutta, S.; Whicher, J. R.; Hansen, D. A.; Hale, W. A.; Chemler, J. A.; Congdon, G. R.; Narayan, A. R. H.; Håkansson, K.; Sherman, D. H.; Smith, J. L.; Skiniotis, G. Structure of a modular polyketide synthase. Nature 2014, 510, 512– 517, DOI: 10.1038/nature13423[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVGjsr%252FO&md5=371c994e2b89ae3618118bf6bc5593dfStructure of a modular polyketide synthaseDutta, Somnath; Whicher, Jonathan R.; Hansen, Douglas A.; Hale, Wendi A.; Chemler, Joseph A.; Congdon, Grady R.; Narayan, Alison R. H.; Hakansson, Kristina; Sherman, David H.; Smith, Janet L.; Skiniotis, GeorgiosNature (London, United Kingdom) (2014), 510 (7506), 512-517CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Polyketide natural products constitute a broad class of compds. with diverse structural features and biol. activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyze two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to det. sub-nanometer-resoln. three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a sep. entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for mol. dissection of these multifunctional enzyme systems.
- 45Robbins, T.; Liu, Y.-C.; Cane, D. E.; Khosla, C. Structure and mechanism of assembly line polyketide synthases. Curr. Opin. Struct. Biol. 2016, 41, 10– 18, DOI: 10.1016/j.sbi.2016.05.009[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptVWkt74%253D&md5=5a94a7bde2d0d80b4d916f2aa14f57aaStructure and mechanism of assembly line polyketide synthasesRobbins, Thomas; Liu, Yu-Chen; Cane, David E.; Khosla, ChaitanCurrent Opinion in Structural Biology (2016), 41 (), 10-18CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Assembly line polyketide synthases (PKSs) are remarkable biosynthetic machines with considerable potential for structure-based engineering. Several types of protein-protein interactions, both within and between PKS modules, play important roles in the catalytic cycle of a multimodular PKS. Addnl., vectorial biosynthesis is enabled by the energetic coupling of polyketide chain elongation to the channeling of intermediates between successive modules. A combination of high-resoln. anal. of smaller PKS components and lower resoln. characterization of intact modules and bimodules has yielded insights into the structure and organization of a prototypical assembly line PKS. This review discusses our understanding of key structure-function relationships in this family of megasynthases, along with a recap of key unanswered questions in the field.
- 46Du, L.; Sanchez, C.; Shen, B. Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules. Metab. Eng. 2001, 3, 78– 95, DOI: 10.1006/mben.2000.0171[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXnvFOnsg%253D%253D&md5=7e332359fb3b6b2236fcfee96bbc3259Hybrid Peptide-Polyketide Natural Products: Biosynthesis and Prospects toward Engineering Novel MoleculesDu, Liangcheng; Sanchez, Cesar; Shen, BenMetabolic Engineering (2001), 3 (1), 78-95CODEN: MEENFM; ISSN:1096-7176. (Academic Press)A review, with 84 refs. The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examg. the biochem. and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a crit. role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products. (c) 2001 Academic Press.
- 47Silakowski, B.; Nordsiek, G.; Kunze, B.; Blöcker, H.; Máller, R. Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga151. Chem. Biol. 2001, 8, 59– 69, DOI: 10.1016/S1074-5521(00)00056-9[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXhslGku78%253D&md5=36ea779d9d0a51aaa01ff0f264e2c66aNovel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15Silakowski, Barbara; Nordsiek, Gabriele; Kunze, Brigitte; Blocker, Helmut; Muller, RolfChemistry & Biology (2001), 8 (1), 59-69CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Science Ltd.)Myxobacteria have been well established as a potent source for natural products with biol. activity. They produce a considerable variety of compds. which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Mol. cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. A DNA region of approx. 50 000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence anal. reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homol. to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the anal. and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Addnl., a new type of reductive release from PKS/NRPS systems is described.
- 48Walsh, C. T. Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 2004, 303, 1805– 1810, DOI: 10.1126/science.1094318[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXitFehu74%253D&md5=5c71b6744699c5335b450124c2f31595Polyketide and Nonribosomal Peptide Antibiotics: Modularity and VersatilityWalsh, Christopher T.Science (Washington, DC, United States) (2004), 303 (5665), 1805-1810CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Polyketide (PK) and nonribosomal peptides (NRP), constructed on multimodular enzymic assembly lines, often attain the conformations that establish biol. activity by cyclization constraints introduced by tailoring enzymes. The dedicated tailoring enzymes are encoded by genes clustered with the assembly line genes for coordinated regulation. NRP heterocyclizations to thiazoles and oxazoles can occur on the elongating framework of acyl-S enzyme intermediates, whereas tandem cyclic PK polyether formation of furans and pyrans can be initiated by post-assembly line epoxidases. Macrocyclizations of NRP, PK, and hybrid NRP-PK scaffolds occur in assembly line chain termination steps. Post-assembly line cascades of enzymic oxidns. also create cross-linked and cyclized architectures that generate the mature scaffolds of natural product antibiotics. The modularity of the natural product assembly lines and permissivity of tailoring enzymes offer prospects for reprogramming to create novel antibiotics with optimized properties.
- 49Fujino, T.; Kondo, T.; Suga, H.; Murakami, H. Exploring of minimal RNA substrate of flexizymes. ChemBioChem. 2019, in press. DOI: 10.1002/cbic.201900150
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.9b00460.
Synthesis and characterization of flexizyme substrates; and procedures for formation, characterization, purification, and analysis of tRNA acylation and IVT reactions (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.