Introduction
The enormous molecular diversity of natural products is achieved through elaborate biosynthetic pathways, and secondary metabolites are a particularly fascinating and rich source of biological activity. Natural products have undergone extensive optimization throughout evolution. The structures of natural products have been well tuned to allow optimal interaction with biomolecular targets, a characteristic that makes them blueprints for drug discovery. In fact, close to 50% of all drugs originate from natural products1. Not surprisingly, higher hit rates are typically seen in the biological screening of libraries containing natural product–like molecules than in the screening of libraries of purely synthetic molecules2. The privileged skeletons3 of steroids, alkaloids and polypropionates are present in molecules that are recognized by their biological targets with high specificity. Lately, improving the skeletal diversity of synthetic compound collections has been an important goal of synthesis4. The balance of heteroatom substituents in a molecule is crucial to both its biological activity and its potential as a drug5. In this commentary, we trace the origin of some of the key heteroatoms that are present in biologically active molecules and explore the ways in which biological and chemical syntheses differ in their approaches to carbon-nitrogen bond formation.
Special traits of nitrogen
From a chemist's perspective, organic molecules are best viewed as largely carbon-based entities equipped with functional groups that contain heteroatoms such as nitrogen, oxygen, halogens, sulfur and phosphorus. From this list, the nitrogen- and oxygen-containing functional groups are ubiquitous components of biologically active molecules. The nitrogen-containing fragments are particularly important because both the basic character of the nitrogen lone pair and the hydrogen bond–donating capacity of the NH group can be modulated by substitution. Perhaps the most celebrated example of integration of these donor and acceptor properties is found in DNA (Fig. 1a). Nitrogen's capacity to carry positive charge has also been instrumental in molecules that are used as electron sinks in biological systems. The pyridoxal phosphate coenzyme uses a pyridinium ion as an electron sink in enzymatic processes such as transamination and decarboxylation of amino acids (Fig. 1b). Furthermore, modulation of nitrogen substitution can have marked effects on drug action within the body. Simple methylation of the nitrogen in normorphine produces the narcotic morphine, which has a four-fold greater analgesic activity than normorphine (Fig. 1c)6. This delicate balance of steric and electronic factors makes nitrogen the heteroatom of choice for chemical synthesis and for studies of the structure and activity of biologically active molecules7.
Figure 1: Nitrogen-containing fragments are key to biological activity.
(a) A-T base pairs in DNA associate through hydrogen bonds. dRib, D-deoxyribose. (b) Pyridoxal phosphate acts as an electron sink in decarboxylase-catalyzed reactions. (c) The poor analgesic activity of normorphine is attributed to the lack of a methyl substituent on nitrogen.
Full size image (30 KB)Catalysis
The essence of chemical synthesis is to transform simple starting materials into complex targets. The so-called catalytic reactions are at the center of attention for those who seek to discover molecules with novel biological function. Typically, a catalyst selectively transforms a molecule by introducing new functional groups into its structure. There is a wealth of reactions available to practitioners of synthesis, yet all of them belong to one of two broad classes: redox and nonredox reactions. The redox reactions affect the number of covalent bonds that a carbon makes to a heteroatom substituent, such as nitrogen and oxygen. Each redox state of carbon (Fig. 2) is associated with a class of nonredox reactions specific for that particular redox state. For example, alcohols can undergo nucleophilic displacements of the hydroxyl functionality, carboxaldehydes can participate in condensations with primary amines to form enamines and carboxylic acids can undergo decarboxylations, resulting in loss of carbon dioxide. Nature's redox enzymes are catalysts proficient in adjusting the oxidation state of carbons. Redox transformations can be followed by a nonredox process that establishes key connections en route to a complex target. Enzymes achieve unparalleled levels of chemoselectivity—differentiation among the functional groups present in a molecule—through shape recognition of a precursor molecule.
Non-natural catalysts that mediate chemical synthesis of biologically active molecules are often compared to and measured against enzymatic systems. Although they do not have the substrate limitations typically imposed on enzymes, their Achilles' heel is slow turnover and low chemoselectivity. Because many functional groups interfere with the adjustment of carbon's oxidation state, chemists must address the challenges of chemoselectivity by resorting to an essential evil of synthesis: protecting-group manipulations. On the surface, these deficiencies are seemingly insurmountable, but as a growing number of small-molecule catalysts continue to rival the high enantioselectivities of enzymatic systems8, the poor chemoselectivity observed in non-natural catalysis may also one day be overcome.
The inspiration for many non-natural catalysts has come from the emulation of enzyme active sites. For instance, many useful catalysts that mediate selective carbon-oxygen bond formation have been inspired by the mechanism of oxidation seen in the monooxygenase family of enzymes (Fig. 3a). In their pioneering work, Breslow et al. used synthetic porphyrins (porphyrins are essential constituents of monooxygenases) for oxygen atom transfer to both saturated and unsaturated systems (Fig. 3b)9. The chiral salen epoxidation catalysts, which were first developed by Jacobsen, contain a manganese ion in the center of a small-molecule ligand (Fig. 3c)10. The parallels with the monooxygenase active site can be seen from the structures of these synthetic catalysts.
Figure 3: Oxygen transfer: monooxygenase versus synthetic catalysts.
(a) Protoporphyrin IX in a human cytochrome P450 enzyme17; PDB ID, 1W0E. (b) Synthetic porphyrin catalyst used by Breslow for oxygen transfer reactions9. (c) Manganese (salen) epoxidation catalyst developed by Jacobsen for oxygen transfer to olefins10.
Full size image (50 KB)Oxygen versus nitrogen
As opposed to the many natural mechanistic modes of oxygen transfer, nature does not provide much inspiration for direct oxidative nitrogen transfer to organic molecules. Rather, the majority of biosynthetic carbon-nitrogen bonds are generated downstream of carbon-oxygen bonds, typically via condensation reactions. The placement of nitrogen in natural products is therefore determined by the monooxygenase's preference for oxygen incorporation. The complex oxidation machinery is based on molecular shape recognition and enables highly unstable species and reactive functional groups such as amines and carbonyls to be set in a position to generate new carbon-nitrogen bonds via elaborate condensation and reduction cascades. Retronecine biosynthesis serves as an instructive case: the diamine oxidase chemoselectively transforms only one of the primary amines into an aldehyde, which undergoes condensation to yield an iminium ion, which is central to subsequent skeletal transformations (Scheme 1)11.
In contrast to the apparent lack of biosynthetic schemes for oxidative nitrogen incorporation, non-natural oxidation systems, which deliver carbon-nitrogen fragments to organic molecules in a direct fashion, do exist. For instance, Du Bois recently developed rhodium nitrene C-H insertion chemistry for use in the synthesis of naturally occurring (–)-tetrodotoxin12 (Scheme 2). The stereospecific installation of the nitrogen-containing carbamate functional group was achieved on a complex intermediate in the late stages of synthesis.
![Scheme 2 : C-H amination in the total synthesis of (|[ndash]|)-tetrodotoxin. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com](/nchembio/journal/v2/n6/thumbs/nchembio0606-284-sc2.gif)
This example and related non-natural oxidative nitrogen transfer reactions bypass the requirement for a carbon-oxygen bond–containing progenitor, nature's prerequisite to making nitrogen-containing molecules. As a result, synthetic methodology decreases the number of steps necessary for nitrogen transfer and facilitates nitrogen placement into strategically significant positions within precursor molecules. Thus, a synthetic catalyst can generate a molecule for which no biosynthetic pathway exists. This expands molecular diversity while preserving a privileged scaffold (Scheme 3)13. Most importantly, there is no limitation as to where a synthetic catalyst can install a carbon-nitrogen bond onto a scaffold having privileged status. By default, many molecules created using synthetic catalysts would have eluded biosynthesis; no inducible biosynthetic pathways14 could have been designed for their synthesis either.
It is intriguing to speculate on the underlying reason for the apparent lack of nature-inspired oxidative nitrogen incorporation. Molecular oxygen is an abundant oxidant that fuels all monooxygenase-assisted oxidation reactions15, most notably hydroxylations and epoxidations of aromatic and aliphatic systems (Scheme 4). This is in stark contrast to the unavailability of a similar reagent for nitrogen incorporation. With the emergence of directed evolution, limitations on substrate specificity for a range of enzymes are being lifted. For example, Reetz has recently shown that simultaneous randomization of specific residue pairs within the active site of a lipase from Pseudomonas aeruginosa results in mutant lipases that catalyze the hydrolysis of sterically encumbered carboxylic acid esters, a function not normally achieved by the wild-type enzyme16. However, the fundamental mechanistic basis for chemical transformations catalyzed by enzymes remains the same. The evolution of new mechanisms has not been exploited thus far, and oxidative carbon-nitrogen bond formation is likely to remain in the realm of synthetic endeavors.
Scheme 4: Mechanistic basis for enzymatic oxygen transfer.
The nitrogens coordinated to iron represent the heme prosthetic group.
Full size image (14 KB)Outlook
The search for complex skeletons that facilitate discovery of biologically active molecules will continue to draw inspiration from sources of natural as well as synthetic origin. Special attention must be paid to maintaining the delicate balance of heteroatom constituents, especially for nitrogen and oxygen moieties. The natural product scaffolds that have evolved over millions of years in response to external stimuli are intricately designed through complex biosynthetic pathways. These pathways involve molecular shape recognition as the basis for selective oxidation and subsequent skeletal transformations. Because these pathways are seemingly fixed, a substantial proportion of the chemistry space leading to the new biologically active molecules of the future will be charted by non-natural oxidative carbon-nitrogen bond–forming protocols. Though synthetic catalysts do not accomplish their goals by molecular shape recognition, they can operate at higher oxidation potentials and accommodate higher-energy nitrogen-transfer intermediates that are not available via biosynthetic pathways. Thus, chemists will be left to fulfill the need for more structurally diverse skeletons having new biological function through late-stage elaboration of nature's privileged molecules. However, it is through learning from nature's insights about oxidation-state adjustment in complex synthesis that further expansion of efficient synthetic sequences to molecules with rich skeletal diversity and deep-seated structural alterations will be realized.
