Reactivity and chemical synthesis of L-pyrrolysine- the 22(nd) genetically encoded amino acid

L-pyrrolysine, the 22(nd) genetically encoded amino acid, was previously deduced to be (4R, 5R)-4-substituted-pyrroline-5-carboxylate attached to the epsilon-nitrogen of lysine based on the crystal structure of the M. barkeri monomethylamine methyltransferase (MtmB). To confirm L-pyrrolysine's identity, structures of MtmB have been determined following treatment with hydroxylamine, N-methylhydroxylamine, or dithionite. Analysis of these structures has provided additional support for the presence of the pyrroline ring and, together with previous mass spectroscopy data, has led us to assign the C(4)-substituent to a methyl group. Based on this assignment, synthetic L-pyrrolysine was prepared by chemical methods. Detailed study of this chemically synthesized L-pyrrolysine has allowed us to characterize its physical properties, to study its chemical stability, and to elucidate the role of its C(4) substituent. Future applications of this synthetic L-pyrrolysine include its in vivo incorporation into recombinant proteins.     

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.     

Mimicking/Extracting Structure and Functions of Natural Products: Synthetic Approaches that Address Unexplored Needs in Chemical Biology

Natural products are often attractive and challenging targets for synthetic chemists, and many have interesting biological activities. However, synthetic chemists need to be more than simply suppliers of compounds to biologists. Therefore, we have been seeking ways to actively apply organic synthetic methods to chemical biology studies of natural products and their activities. In this personal review, I would like to introduce our work on the development of new biologically active compounds inspired by, or extracted from, the structures of natural products, focusing on enhancement of functional activity and specificity and overcoming various drawbacks of the parent natural products.     

Encrypting Chemical Reactivity in Protein Sequences toward Information‐Coded Reactions†

Controlling chemical reactivity has been the central theme in chemistry. Herein, we review the recent progress on the development of genetically encoded protein coupling reactions and their potential applications. The chemical reactivity is encoded in the protein sequences. The information is read out by folding and molecular recognition between two reactive components and subsequently translated into chemical bonding via autocatalysis. It has emerged as a unique way to tune the chemical reactivity and is regarded as one type of information‐coded reactions. Not only has it received many applications such as protein topology engineering, bioconjugation, biomaterials and synthetic biology, but also its principle may be extended beyond protein chemistry to enable new modes of supramolecular interactions that promote chemical bonding and that are simultaneously reinforced by covalent bonds.     

SLAP: Small Labeling Pair for Single‐Molecule Super‐Resolution Imaging

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small‐molecule recognition unit (Ni‐trisNTA) and the genetically encoded minimal protein His_6‐10‐tag. High‐density protein tracing by SLAP was demonstrated. This technique allows super‐resolution fluorescence imaging and fulfills the necessary sampling criteria for single‐molecule localization‐based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).     

Rational design,properties, and applications of biosurfactants: a short review of recent advances

Biosurfactants combine physicochemical properties with biological activities. Although biosurfactants are often expressed by microorganisms, an increasing amount is produced by chemical synthesis. As many exist in the form of homologous compounds, it is often difficult to purify biosurfactants. But this has not limited the efforts to develop their commercial applications. In this short review, we have featured the recent advances in three important types of biosurfactants, lipopeptides, nucleolipids, and glycolipids. We have focused on comparing some of the key properties and functionalities between modern synthetic versions and their corresponding natural counterparts. We end the review by outlining the needs for not only strengthening their basic structure–property relationships through further research but also developing better technologies, irrespective of direct chemical synthesis or biological synthesis of biosurfactants through constructions of genetically engineered strains, to help advance the commercial use of biosurfactants.     

生物正交反应法制备含有赖氨酸翻译后修饰类似物的蛋白质

翻译后修饰一直是表观遗传学的重要研究内容,尤其是近年来多种新型天然蛋白质中翻译后修饰被发现广泛存在于蛋白质组中。细胞生物学证明这些翻译后修饰对染色体结构和基因转录功能有关,但是其中具体的分子生物学机制还处于未知状态。为了后续的进一步研究,人们需要发展制备方法以求获取足量具有特定翻译后修饰的蛋白质。本文将讨论利用生物正交反应的手段制备含有这些新型赖氨酸翻译后修饰的蛋白的探索,期对教学与科研有助。     

Diverse organo-peptide macrocycles via a fast and catalyst-free oxime/intein-mediated dual ligation

Macrocyclic Organo-Peptide Hybrids (MOrPHs) can be prepared from genetically encoded polypeptides via a chemoselective and catalyst-free reaction between a trifunctional oxyamino/amino-thiol synthetic precursor and an intein-fusion protein incorporating a bioorthogonal keto group.     

A Challenging Pie to Splice: Drugging the Spliceosome

Since its discovery in 1977, the study of alternative RNA splicing has revealed a plethora of mechanisms that had never before been documented in nature. Understanding these transitions and their outcome at the level of the cell and organism has become one of the great frontiers of modern chemical biology. Until 2007, this field remained in the hands of RNA biologists. However, the recent identification of natural product and synthetic modulators of RNA splicing has opened new access to this field, allowing for the first time a chemical‐based interrogation of RNA splicing processes. Simultaneously, we have begun to understand the vital importance of splicing in disease, which offers a new platform for molecular discovery and therapy. As with many natural systems, gaining clear mechanistic detail at the molecular level is key towards understanding the operation of any biological machine. This minireview presents recent lessons learned in this emerging field of RNA splicing chemistry and chemical biology.     

New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins.     

Reassignments and Corroborations of Oxo‐Bridged Natural Products Directed by OSE and DU8+ NMR Computation

Structural misassignments of natural products are prevalent in the literature. Developing methods and theoretical concepts to assist those undertaking structural elucidation is therefore of paramount importance, such that biologists and synthetic chemists avoid pursuing phantom chemical entities. Herein described is a strategy for predicting the isolabilities of oxygen‐substituted bridgehead natural products based on calculations of olefin strain energies, NMR chemical shifts and coupling constants (DU8+). This approach provides corroborating evidence for the structures of certain bridgehead alkene natural products while leading to the reassignment of several other structures.     

Chasing Synthetic Life: A Tale of Forms,Chemical Fossils,and Biomorphs

This Essay focuses briefly on early studies elaborated by natural and chemical philosophers, and the once-called synthetic biologists, who postulated the transition from inanimate to animate matter and even foresaw the possibility of creating artificial life on the basis of physical and chemical principles only. Such ideas and speculations, ranging from soundness to weirdness, paved however the way to current developments in areas like abiotic pattern formation, cell compartmentalization, biomineralization, or the origin of life itself. In particular, the generation of biomorphs and their relationship to microfossils represents an active research domain and seems to be the logical way to bring the historical work up to the future, as some scientists are trying to make artificial cells. The last sections of this essay will also highlight modern science aimed at understanding what life is and, whether or not, it can be redefined in chemical terms.     

Chasing Synthetic Life: A Tale of Forms,Chemical Fossils,and Biomorphs

This Essay focuses briefly on early studies elaborated by natural and chemical philosophers, and the once‐called synthetic biologists, who postulated the transition from inanimate to animate matter and even foresaw the possibility of creating artificial life on the basis of physical and chemical principles only. Such ideas and speculations, ranging from soundness to weirdness, paved however the way to current developments in areas like abiotic pattern formation, cell compartmentalization, biomineralization, or the origin of life itself. In particular, the generation of biomorphs and their relationship to microfossils represents an active research domain and seems to be the logical way to bring the historical work up to the future, as some scientists are trying to make artificial cells. The last sections of this essay will also highlight modern science aimed at understanding what life is and, whether or not, it can be redefined in chemical terms.     

Catalytic Antibodies: A New Class of Transition-State Analogues Used to Elicit Hydrolytic Antibodies

The design and generation of selective catalysts is an important aim of chemists and biologists. A number of successful strategies have emerged, including the synthesis and derivatization of synthetic hosts, the chemical modification and site-directed mutagenesis of enzymes, and the attenuation of natural enzyme activities in organic solvents. Since 1986 several laboratories have exploited the immune system to generate selective catalysts capable of catalyzing a wide range of chemical transformations. These include acyl transfer, β-elimination, carbon—carbon bond-forming, carbon—carbon bond-cleaving, porphyrin metalation, peroxidation, and redox reactions. The variety and number of transformations catalyzed by antibodies in this short period of time is testament to the versatility and power of the method in generating selective catalysts for applications in chemistry, biology, and medicine. Here we report the use of a new class of uncharged transition-state analogues for generating antibodies capable of catalyzing ester and carbonate hydrolysis. These antibodies are compared to those raised against tetrahedral phosphate and phosphonate transition-state analogues.     

A DNA‐Encoded Chemical Library Incorporating Elements of Natural Macrocycles

Here we show a seven‐step chemical synthesis of a DNA‐encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide‐polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA‐encoded libraries, while revealing areas where new DNA synthetic methods are needed.     

Unleashing chemical power from protein sequence space toward genetically encoded “click” chemistry

We propose the concept of genetically encoded “click” chemistry (GECC) to describe the “perfect” peptide-protein reactive partners and use SpyTag/SpyCatcher chemistry as a prototype to illustrate their structural plasticity, robust interaction, and versatile applications.     

Functional characterization and NMR spectroscopy on full-length Vpu from HIV-1 prepared by total chemical synthesis

Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically (15)N-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.     

Synthetic compound libraries displayed on the surface of encoded bacteriophage

We describe a technology for attaching libraries of synthetic compounds to coat proteins of bacteriophage particles such that the identity of the chemical structure is encoded in the genome of the phage, analogous to peptides displayed on phage surfaces by conventional phage-display techniques. This format allows a library of synthetic compounds to be screened very efficiently as a single pool. Encoded phage serve as extremely robust reporters of the presence of each compound, providing exquisite sensitivity for identification of active compounds engaged in complex biological processes such as receptor-mediated endocytosis and transcytosis. To evaluate this approach, we constructed a library of 980 analogs of folic acid displayed on T7 phage, and demonstrated rapid identification of compounds that bind to folate receptor and direct endocytosis of associated phage particles into cells that express the targeted receptor.     

Synthesis of bispecific antibodies using genetically encoded unnatural amino acids

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.