Imaging and targeting LOX-mediated tissue remodeling with a reactive collagen peptide.

Collagens are fibrous proteins that are integral to the strength and stability of connective tissues. During collagen maturation, lysyl oxidases (LOX) initiate the cross-linking of fibers, but abnormal LOX activity is associated with impaired tissue function as seen in fibrotic and malignant diseases. Visualizing and targeting this dynamic process in healthy and diseased tissue is important, but so far not feasible. Here we present a probe for the simultaneous monitoring and targeting of LOX-mediated collagen cross-linking that combines a LOX-activity sensor with a collagen peptide to chemoselectively target endogenous aldehydes generated by LOX. This synergistic probe becomes covalently anchored and lights up in vivo and in situ in response to LOX at the sites where cross-linking occurs, as demonstrated by staining of normal skin and cancer sections. We anticipate that our reactive collagen-based sensor will improve understanding of collagen remodeling and provide opportunities for the diagnosis of fibrotic and malignant diseases.

Alkylation of γ-Azaproline Creates Conformationally Adaptable Proline Derivatives for pH-Responsive Collagen Triple Helices.

Cγ -substituted proline derivatives are valuable tools for developing functionalized collagen peptides for biological and materials investigations, yet the stereochemistry at Cγ can produce undesired steric or stereoelectronic constraints. Alkylated γ-azaproline (γ-azPro) derivatives are proline mimetics that lack a stereogenic center at the γ-position of the ring and can thus utilize the invertibility of nitrogen to adapt their conformation. NMR spectroscopic analyses and DFT calculations highlighted how alkylated γ-azPro derivatives are conformationally dynamic and adopt conformational preferences through ring pucker flip along with nitrogen inversion. Lastly, incorporation of alkylated γ-azPro into collagen peptides produced functionalized pH-responsive triple helices with similar thermal stabilities, regardless of their placement in the Xaa or Yaa position within the characteristic Xaa-Yaa-Gly repeating unit of collagen peptides.

Recent Advances in Bioorthogonal Reactions.

The ability to selectively perform chemical reactions within living systems has transformed the field of Chemical Biology. These chemoselective processes have had a major scientific impact by enabling studies of cellular processes, producing tools for the ligations of large biomolecules, and advancing our ability to image or track small molecular changes such as posttranslational modifications. As more investigators become involved in the development and application of chemical reactions performed within complex biological settings, there have been a rising number of innovations that create improved tools and resources for understanding nature. This perspective highlights some recent achievements that show how innovation and creativity can provide new opportunities in this exciting and rapidly expanding area of chemical research.

γ-Azaproline Confers pH†Responsiveness and Functionalizability on Collagen Triple Helices.

Proline derivatives bearing substituents at Cγ are valuable tools for biological and materials investigations. However, the stereochemistry at Cγ can produce undesired steric or stereoelectronic interactions. Here, we introduce γ-azaproline (γ-azPro), which lacks a stereogenic center at Cγ, as a pH-responsive and functionalizable proline analogue that can adapt to its environment. Conformational analyses by NMR spectroscopy and DFT calculations revealed that the imidazolidine ring of γ-azPro is flexible. Incorporation of γ-azPro into collagen model peptides (CMPs) produced pH-responsive triples helices and triple helices that can be easily functionalized.

Oligoprolines as Molecular Entities for Controlling Distance in Biological and Material Sciences.

Nature utilizes large biomolecules to fulfill tasks that require spatially well-defined arrangements at the molecular level such as electron transfer, ligand-receptor interactions, or catalysis. The creation of synthetic molecules that enable precise control over spacing and functionalization provides opportunities across diverse disciplines. Key requirements of functionalizable oligomeric scaffolds include the specific control of their molecular properties where the correct balance of flexibility and rigidity must be maintained in addition to the prerequisite of defined length. These molecules must ideally be equally applicable in aqueous and organic environments, they must be easy to synthesize in a controlled stepwise fashion, and they must be easily modified with a palette of chemical appendages having diverse functionalities. Oligoproline, a peptidic polymer comprised of repeating units of the amino acid proline, is an ideal platform to meet such challenges. Oligoproline derives its characteristic rigidity and well-defined secondary structure from the innate features of proline. It is the only naturally occurring amino acid that has its side-chain cyclized to its α-amino group, generating often-populated trans and cis conformers around the tertiary amide bonds formed in proline oligomers. Oligoprolines are widely applied to define distance on the molecular level as they are capable of serving as both a "molecular ruler" with a defined length and as a "molecular scaffold" with precisely located and predictably oriented substitutions along the polymeric backbone. Our investigations focus on the use of oligoproline as a molecular scaffold. Toward this end, we have investigated the role of solvent upon helical structure of oligoproline, and the effect that substituents on the pyrrolidine ring and the oligomer termini have on the stability of the helix. We have also further explored the molecular characteristics of oligoproline through spectroscopic and crystallographic methods. All of these structural insights laid the basis for implementation of oligoproline in materials science and chemical biology. Within this Account, we highlight the value of oligoprolines for applications in distinctly different research areas. Toward materials chemistry, we have utilized oligoprolines for the size-controlled generation of noble metal nanoparticles, and to probe the role of spatial preorganization of π-systems for molecular self-assembly. Within the biological realm, we have applied oligoprolines to probe the role of distance on G-protein coupled receptor-mediated ligand uptake by cancerous cells and to investigate the effects of charge preorganization on the efficacy of cationic cell-penetrating peptides.

Rapid cycloaddition of a diazo group with an unstrained dipolarophile.

The cycloaddition of a diazoacetamide with ethyl 4,4,4-trifluorocrotonate proceeds with k = 0.1 M-1s-1. This second-order rate constant rivals those of optimized strain-promoted azide- alkyne cycloadditions, even though the reaction does not release strain. The regioselectivity and a computational distortion/interaction analysis of the reaction energetics are consistent with the formation of an N-H…F-C hydrogen bond in the transition state and the electronic character of the trifluorocrotonate. Analogous reactions with an azidoacetamide dipole or with an acrylate or crotonate dipolarophile were much slower. These findings suggest a new strategy for the design of diazo-selective reagents for chemical biology.

Diazo Compounds: Versatile Tools for Chemical Biology.

Diazo groups have broad and tunable reactivity. That and other attributes endow diazo compounds with the potential to be valuable reagents for chemical biologists. The presence of diazo groups in natural products underscores their metabolic stability and anticipates their utility in a biological context. The chemoselectivity of diazo groups, even in the presence of azido groups, presents many opportunities. Already, diazo compounds have served as chemical probes and elicited novel modifications of proteins and nucleic acids. Here, we review advances that have facilitated the chemical synthesis of diazo compounds, and we highlight applications of diazo compounds in the detection and modification of biomolecules.

1,3-Dipolar Cycloaddition with Diazo Groups: Noncovalent Interactions Overwhelm Strain.

Like azides, diazoacetamides undergo 1,3-dipolar cycloadditions with oxanorbornadienes (OND) in a reaction that is accelerated by the relief of strain in the transition state. The cycloaddition of a diazoacetamide with unstrained ethyl 4,4,4-trifluoro-2-butynoate is, however, 35-fold faster than with the analogous OND because of favorable interactions with the fluoro groups. Its rate constant (k = 0.53 M(-1) s(-1) in methanol) is comparable to those of strain-promoted azide-cyclooctyne cycloadditions.

Decreasing Distortion Energies without Strain: Diazo-Selective 1,3-Dipolar Cycloadditions.

The diazo group has attributes that complement those of the azido group for applications in chemical biology. Here, we use computational analyses to provide insights into the chemoselectivity of the diazo group in 1,3-dipolar cycloadditions. Dipole distortion energies are responsible for ∼80% of the overall energetic barrier for these reactions. Here, we show that diazo compounds, unlike azides, provide an opportunity to decrease that barrier substantially without introducing strain into the dipolarophile. The ensuing rate enhancement is due to the greater nucleophilic character of a diazo group compared to that of an azido group, which can accommodate decreased distortion energies without predistortion. The tuning of distortion energies with substituents in a diazo compound or dipolarophile can enhance reactivity and selectivity in a predictable manner. Notably, these advantages of diazo groups are amplified in water. Our findings provide a theoretical framework that can guide the design and application of both diazo compounds and azides in "orthogonal" contexts, especially for biological investigations.

1,3-Dipolar Cycloadditions of Diazo Compounds in the Presence of Azides.

The diazo group has untapped utility in chemical biology. The tolerance of stabilized diazo groups to cellular metabolism is comparable to that of azido groups. However, chemoselectivity has been elusive, as both groups undergo 1,3-dipolar cycloadditions with strained alkynes. Removing strain and tuning dipolarophile electronics yields diazo group selective 1,3-dipolar cycloadditions that can be performed in the presence of an azido group. For example, diazoacetamide but not its azido congener react with dehydroalanine residues, as in the natural product nisin.

Diazo groups endure metabolism and enable chemoselectivity in cellulo.

We introduce a stabilized diazo group as a reporter for chemical biology. ManDiaz, which is a diazo derivative of N-acetylmannosamine, is found to endure cellular metabolism and label the surface of a mammalian cell. There its diazo group can undergo a 1,3-dipolar cycloaddition with a strained alkyne, providing a signal comparable to that from the azido congener, ManNAz. The chemoselectivity of diazo and alkynyl groups enables dual labeling of cells that is not possible with azido and alkynyl groups. Thus, the diazo group, which is approximately half the size of an azido group, provides unique opportunities for orthogonal labeling of cellular components.

Detection of boronic acids through excited-state intramolecular proton-transfer fluorescence.

Boronic acids are versatile reagents for the chemical synthesis of organic molecules. They and other boron-containing compounds can be detected readily by the interruption of the excited-state intramolecular proton transfer (ESIPT) of 10-hydroxybenzo[h]quinolone. This method is highly sensitive and selective, and useful for monitoring synthetic reactions and detecting boron-containing compounds on a solid support.

A Divalent Protecting Group for Benzoxaboroles.

1-Dimethylamino-8-methylaminonaphthalene is put forth as a protecting group for benzoxaboroles. The ensuing complex is fluorescent, charge-neutral, highly stable under basic conditions, stable to anhydrous acid, and readily cleavable in aqueous acid to return the free benzoxaborole.