Total Synthesis of a Stereochemically Pure "Topoisomer."

[NNadir]

The paper I'll discuss in this post is this one: Total synthesis reveals atypical atropisomerism in a small-molecule natural product, tryptorubin A (Solomon H. Reisberg1, Yang Gao1, Allison S. Walker2, Eric J. N. Helfrich2, Jon Clardy2,*, Phil S. Baran1, Science, Vol. 367, Issue 6476, pp. 458-463.

One may say "Life is unfair," because there is asymmetry in the way people are treated, an orange lunatic might with no personal merits, low intelligence and no integrity whatsoever might end up living in the White House, supported by a criminal rabble, while a person like Raoul Wallenberg might die alone, possibly under horrific conditions, in a Soviet Prison.

But life is asymmetric both in a moral sense and also in a physical sense.

This is the science section of a website devoted mostly to the issue of political ethics, and so here, we limit discussion to physical realities.

The physical asymmetry of life involves chirality, the property of objects that are not superimposable on their mirror images, the most common evocation of which are the human hand because the left hand is (more or less) the mirror image of the right, but the two hands cannot be superimposed upon each other. In fact, a word often used, even by scientists, to describe chirality is "handedness."

Most of the organic molecules in living systems possess this property of chirality, with some exceptions, for example the common amino acid glycine, and the acid pyruvic acid, but the other 19 coded proteogenic amino acids, all sugars, and all of the nucleic acids possess chirality.

In almost every case, the chirality is associated with one or more "chiral centers" where the chirality derives from the tetrahedral arrangement of bonds to saturated carbon, if these bonds are attached to four different types of groups, the molecule is chiral. Some amino acids, threonine and isoleucine have two chiral centers, and others, like sugars (which also cause the asymmetry of nucleic acids of which they are a constituent) can have many chiral centers.

However there is a somewhat unusual type of chirality that can be present without a chiral center that derives from rigid bonds to carbons that are lacking in chiral centers. Most organic chemists will be familiar with well known chiral catalysts - in order to synthetically generate a chiral center, one must introduce a chiral molecule into the synthetic pathway somewhere - based on "Binap" which has this property:



Although the molecule here is a peptide, and possesses amino acids having chiral centers, including isoleucine having two chiral centers, it also possesses the other kind of chirality. The molecule is tryptorubin A, a cyclic peptide, with non-amino acid moieties in it (that clearly can be distinguished as having been biosynthesized from amino acids. Tryptorubin A was discovered in the bacteria associated with the fungus that is in a symbiotic relationship with a species of ants.



Similar molecules, modified cyclic peptides, have proven to be important medications; vancomycin, an antibiotic that is a "antibiotic of last resort" for treating bacterial infections caused by organisms that have evolved resistance to many other antibiotics, is in this class.

Anyway, the authors of this paper have discovered interesting stereochemical properties of this molecule, tryptorubin A as a result of working on its total synthesis.

The introduction to the paper is well written, and should be accessible to some non-chemists:

In 1894, Emil Fischer proposed a lock-and-key analogy for how biological molecules interact to carry out biological functions, and the three-dimensional (3D) shapes of molecules have been a major focus of biological chemistry ever since (1). Accordingly, the structure of small molecules has been assumed to be defined solely by atomic connectivity and point or axial chirality. For example, the steroid hormones all have the same basic carbon skeleton—a rigid assembly of four rings fused one to another—and their different biological roles depend on the modifications to the periphery of this basic skeleton. In contrast, large molecules such as proteins can reversibly self-organize into well-defined 3D structures, and the rules governing this ability are increasingly well understood (2). This structural feature of biological macromolecules encodes many of the functions that form the basis of life (1). For example, hydrogen-bonding, hydrophobic, arene-?, and solvation interactions drive proteins to fold into specific tertiary structures that render them operable (2). Molecular shapes (i.e., tertiary structures) for most macromolecules are derived from atomic connectivity but are fundamentally separate from it; that is, many proteins can be folded and unfolded without breaking or forming covalent bonds (3).

For certain macromolecules, however, shape is directly tied to atomic connectivity rather than to conformational changes (Fig. 1A, left). In the case of cyclic DNA, for example, the wound and unwound topologies are interconvertible only by the scission and reformation of phosphate linkages (4). Likewise, molecular catenanes have been synthesized with defined topology (5). Such nonsuperimposable and noninterconvertible topologies are called topoisomers. Two molecules are topoisomers of each other if they have identical connectivity but nonidentical molecular graphs—that is, molecular pairs that are noninterconvertible without the breaking and reformation of chemical bonds (6).

The next parts may be less accessible to non specialists:

This type of defined topoisomerism is conspicuously absent from small-molecule natural products. A distinct, if seemingly analogous, isomerism in a small-molecule context is atropisomerism (i.e., shape isomerism through hindered bond rotation). Canonically, atropisomerism involves a single torsionally hindered bond that bestows axial chirality; hindered biaryls (Fig. 1A, right) represent a prototypical example.

In contrast to both canonical (singly axially chiral) atropisomerism and topoisomerism, there exist a variety of shape-defined molecules that are theoretically interconvertible by bond rotation but are categorically distinct from canonical atropisomers because of the multiple and nonphysical bond torsions required for their interconversion. Many mechanically interlocked molecules fit into this middle ground; for example, both rotaxanes (7) and lasso peptides (8) (Fig. 1A, center) are topologically trivial and should formally be considered atropisomers with their unthreaded counterparts, but are clearly categorically distinct from simple prototypical examples of atropisomerism. [For another compelling case of noncanonical atropisomerism, see (9).] In a physical (rather than theoretical) sense, most members of the lasso peptide class of natural products can be interconverted from unthreaded to threaded shapes only by breakage and repair of the peptide backbone...

Figure 1:



It's caption:

Fig. 1 Shape isomerism in macro- and small molecules.
(A) Shape-based isomerism in synthetic and natural products spans a broad range. At one end (left), defined topology encodes topoisomers. At the other end (right), canonical atropisomerism is defined by simple axial differences (i.e., torsion of a single bond). Under the broad umbrella of atropisomerism, but distinct from more canonical examples, are noncanonical atropisomers (center) that are formally topologically trivial, but whose interconversion requires complex multibond rotations and unphysical torsions. Historically, this area has been occupied only by macromolecules; in this work, we disclose a small-molecule natural product that presents this type of noncanonical atropisomerism. Structures obtained from PDB and/or CCDC database: circular DNA, reproduced from (30); lasso peptide, PDB 5TJ1 (8); catenane, CCDC #1835146 (5); rotaxane, CCDC #1576710 (7). (B) Left: Originally proposed structure of tryptorubin A. Right: Two noncanonical atropisomers are possible within the limits of the originally proposed 2D structure. Note that 3D structures of 1a and 1b are computed, not crystallographic, and their terminal residues are truncated for clarity.

The point of the paper is described here:

...We have found that tryptorubin A (1), as a result of chirality and connectivity alone, could theoretically present as two possible noncanonical atropisomers. We describe an atroposelective synthesis of atrop-tryptorubin A (1b), the discovery of its atypical atropisomerism, and a hypothesis-driven atropospecific strategy that led to the synthesis of the natural product (1a) and its unambiguous atropisomeric assignment. Additionally, we report genomic data that help to clarify the biogenesis of 1a; these data suggest a biosynthetic pathway involving ribosomal peptide synthesis followed by atroposelective posttranslational modification...

The authors began their synthesis with the protected version of a the dipeptide Tryptophan-3-iodotyrosine methyl ester and went through a number of (fairly low yielding) steps:



The caption:

ig. 2 Tryptorubin A’s noncanonical atropisomerism: Discovery and synthesis of the unnatural atropisomer.
(A) Synthetic route to atrop-tryptorubin A (1b). (B) Strategic hypothesis to use point chirality to drive an atropospecific synthesis of tryptorubin A. Piv, pivalate; PMB, para-methoxybenzyl; Ns, nosyl; DTBMP, 2,6-di-tert-butyl-4-methylpyridine; HATU, hexafluorophosphate azabenzotriazole tetramethyl uronium; PyAOP, (7-azabenzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate; nOe, nuclear Overhauser effect.

This represented, I'm sure, a huge amount of work for graduate students and/or postdocs.

And then they discovered that this was a case, as someone - I forget who - said of the origin of advances in basic science, where the scientists said, "Hey, that's funny..."

This is a somewhat esoteric description of "Hey, that's funny..." but trust me, that's what it is:

At this juncture, characterization by nuclear magnetic resonance (NMR) spectroscopy became challenging (even at high temperature), presumably because of cis/trans amide isomerization of the tertiary pyrroloindolinyl amide, various rotameric populations, and conformational equilibrium between 8a and 8b. Nonetheless, 8 appeared as a single sharp peak in high-performance liquid chromatography (HPLC) and exhibited a high-resolution mass spectrum (HRMS) consistent with the postulated structure. After extensive experimentation (13), this structure could be cyclized in low yield to a bis(macrocycle). Global deprotection yielded 1b, with HRMS data indicating the same molecular formula as the natural isolate (1). Unfortunately, the NMR data [1H, 13C, heteronuclear multiple bond correlation (HMBC), heteronuclear single quantum coherence (HSQC), rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY)] and LC retention of 1b were distinct from the natural product (1) [see below and (13)].

With these contrasts in spectral data in mind, we began to consider possible explanations for the structural discrepancy between 1 and 1b. We considered the possibilities of stereochemical misassignment (e.g., a D–amino acid) or regiochemical misassignment (e.g., alternate regiochemistry in the indole-pyrroloindoline C-C bond) in the natural and/or synthetic products. After exhaustive review of natural 1 and synthetic 1b’s respective spectral data as well as a separate total synthesis of C26-epimeric species epi-8 [see (13) for this additional synthesis], we confirmed that natural 1 and synthetic 1b had the same connectivity and point-stereochemistry (13). It was only upon careful analysis of the two compounds’ ROESY spectra that a key insight was discovered: Although the natural product (1a) showed strong nuclear Overhauser effect correlations from H9 and H10 to H42 (Fig. 2B), the analogous H9 and H10 protons in the synthetic (1b) compound’s ROESY spectrum showed correlations to H40 (Fig. 2A). This key geometric constraint, combined with additional spectral evidence [1b and 1a in Fig. 2, A and B; see (13) for additional details and full skeletal numbering system], illuminated our understanding that even within the limits of identical connectivity and stereochemistry, 1 could potentially exist as two noncanonical atropisomers (“bridge above,” 1a; “bridge below,” 1b)...

... We hypothesized that by geometrically locking the cyclization precursor into the “bridge above” conformation, we could achieve inversion of atroposelectivity. Combining this hypothesis with crystallographic evidence of the geometry of indoline 7, we recognized that in a substrate such as indoline 9, the point chirality at indoline (Fig. 2B, purple methine) would geometrically preclude the “bridge below” conformer (9b); indeed, geometric limitations of 9 would render the cyclization atropospecific for the “bridge above” atropisomer 1a (resulting from cyclization of 9a). Such a strategy is reminiscent of methods to control more canonical atroposelectivity by point-to-axial chirality transfer (18).

Figure 3A describes our successful execution of the atropospecific strategy laid out in Fig. 2B and the subsequent total synthesis of the natural isomer of tryptorubin A (1a)...

Figure 3:



It's caption:

Fig. 3 Total synthesis of tryptorubin A.
(A) Atropospecific synthesis of tryptorubin A (1a). (B) Top: A RiPP sequence that encodes tryptorubin A’s linear peptide sequence. Bottom: Proposed biosynthetic pathway to 1a. Amino acid abbreviations: A, Ala; F, Phe; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; Q, Gln; R, Arg; S, Ser; W, Trp; Y, Tyr.

A graphical cartoon ("thought experiment" ) from the paper:

Fig. 4 Graphical thought experiment considering putative interconversion of tryptorubin (1a) and its noncanonical atropisomer (1b).

Top: Theoretically, interconversion would require an unphysical inside-out flipping of the molecule, in which one macrocycle passed through the other. Center: This is analogous to atropisomeric inversion of a rotaxane, which would require unphysical stretching of the ring (green) over the dumbbell. Bottom: Such noncanonical atropisomers are contrasted with prototypical atropisomers such as binaphthol, which can interconvert through simple bond torsion.

Some commentary of the synthetic biology of this interesting molecule:

The discovery of tryptorubin A’s geometric isomerism in the total synthesis effort prompted a reexamination of its biosynthesis. The original bioinformatic analysis identified 18 biosynthetic gene clusters (BGCs), none of which could be confidently predicted to encode the biosynthesis of tryptorubin A (12). The most plausible candidate was a modular nonribosomal peptide synthetase by which the hexapeptide chain would be assembled sequentially by dedicated enzymes. However, the selectivity of the module-encoded adenylation domains did not convincingly match the tryptorubin A peptide sequence, and additional genes involved in the biosynthesis of amino acids that are not incorporated into tryptorubin A were present in the direct vicinity (22, 23). We decided to evaluate other possible biosynthetic origins and thus considered the possibility that tryptorubin A is a ribosomally synthesized and posttranslationally modified peptide (RiPP) that is missed by conventional bioinformatic analysis tools because of its small size, its lack of homology to characterized ribosomal peptides, and the presence of noncanonical tailoring genes involved in carbon-carbon bond formation...

...Screening the translated Streptomyces sp. CLI2509 genome sequence for the tryptorubin core peptide sequence (Ala-Trp-Tyr-Ile-Trp-Tyr) resulted in a single hit. Close inspection of the unannotated region revealed a ribosomal binding site followed by a transcriptional start site, a putative RiPP precursor gene encoding a 20–amino acid leader, a core peptide, and a stop codon downstream of the core sequence (Fig. 3B and fig. S17). This sequence is followed by a gene encoding a cytochrome P450 enzyme that is likely involved in the formation of the nonproteogenic carbon-carbon and carbon-nitrogen bridges. Although cytochrome P450 enzymes that catalyze carbon-carbon bond formation in ribosomal peptides have not been reported (24), analogous carbon-carbon linkages between the aromatic residues in the nonribosomal peptide vancomycin have been shown to be installed by cytochrome P450 enzymes (25–28)

Thus spake Vancomycin.

A concluding remark:

Despite the extensive vernacular to describe regio-, stereo-, and atropisomers, the nuances of molecular shape can be lost within the realm of small-molecule natural product chemistry. Although most practicing synthetic chemists are intimately familiar with the canonical examples of biaryl atropisomerism, the much more complex examples of atropisomerism in polycyclic and mechanically interlocked molecules often remain underexamined. Indeed, the possibility of noncanonical atropisomerism was initially missed during both the isolation and synthesis of tryptorubin A. We present this case as a cautionary tale in structural definition, a demonstration of the power of transferring point chirality to molecular shape, and a reminder that small-molecule organic chemists can greatly benefit from the deep understanding of 3D structure known in the biomacromolecular and supramolecular literature.

I don't know what the "use" of this science might be, but irrespective of its use, it is beautiful, and its wonderful to contemplate a beautiful thing on a Sunday afternoon.

I hope your Sunday afternoon is as wonderful as mine. First life is wonderful, and then you die.