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Chapter 27
β-Substituted D-Leucines and Their Relevance in the Total Synthesis of Natural and Unnatural Aeruginosins 1
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Juan R. Del Valle , Xiaotian Wang , Karolina Ersmark , and Stephen Hanessian * 2,
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Department of Chemistry and Biochemistry, New Mexico State University, 1175 North Horseshoe Drive, MSC 3C, Las Cruces, NM 88003 Department of Chemistry, Université de Montréal, P.O. Box 6128, Station Centre-ville, Montréal, Québec H3C 3J7, Canada Medivir AB, P.O. Box 1086, SE-141 22, Huddinge, Sweden 2
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Methods for the synthesis of diastereomerically pure β-chloro and β-methyl-D-leucines and their derivatives are described. The incorporation of these residues in natural and non-natural analogues is shown with relevance to their in vitro thrombin inhibitory activities.
A large number of natural products contain unusual amino acid moieties as part of their oftentimes intricate structures. Among these are a select group of biologically active peptides that contain a chlorine atom in the side-chain of an amino acid, or as a substituent in the carbon framework. In rare instances, Nature has produced peptide structures that harbor a β-chloroamino acid residue. The paucity of natural products bearing these residues may be testament to the susceptibility of β-chloro, and related amino acids to undergo elimination across the α and β carbons. Indeed, dehydroamino acids are abundant in Nature and play an important role in the biosynthesis of various non-proteinogenic amino acids, and D-amino acids. To date, β-chloroamino acids have only been found 1
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© 2009 American Chemical Society
In Asymmetric Synthesis and Application of -Amino Acids; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
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FR900148
Chlorodysinosin A
Figure 1. Structures of astin A, FR900148, FR225659, and chlorodysinosin A.
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in a select number of natural product families which include the astins, FR900148, FR225659, and chlorodysinosin A , a new member of the aeruginosas (Figure 1). The aeruginosas, derived from cyanobacterial waterblooms and marine sponges, belong to a family of so-called linear peptides incorporating novel nonproteinogenic α-amino acids. Several efforts have been made to synthesize members of this class of natural products and details of seven total syntheses of various aeruginosins have been summarized in a recent review. The first aeruginosin to be discovered was aeruginosin 298A (Table 1), which was isolated from Microcystis aeruginosa by Murakami and coworkers in 1994. This finding was followed by the isolation and characterization of so far 20 more natural compounds belonging to the same structural family gathered from geographically different aquatic sources. Most of the aeruginosins are comprised of four distinct subunits: a C-terminal arginine mimetic, a 6-mono or 5,6-dihydroxy-2-octahydroindole carboxamide (Choi or OHChoi), a bulky hydrophobic amino acid, and an jV-terminal hydroxyl or acidic group. This pharmacophore combination of subunits is in part responsible for the in vitro inhibitory activities against serine proteases, especially those with trypsin-like substrate specificity. Particular attention has been directed to the inhibition of 4
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In Asymmetric Synthesis and Application of -Amino Acids; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
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thrombin and other blood coagulation factors (Table l). Nearly all aeruginosins incorporate a D-configured α-amino acid with a bulky hydrophobic side-chain in the P3 position. Interestingly, it was found that the presence of a 3-chloro substituent in this position, as in the (3/?)-chloro-D-leucyl residue in chlorodysinosin A, gave a several-fold increase in inhibitory activity against some coagulation factors compared to the hydrogen-substituted dysinosin A (Table 1). In fact, chlorodysinosin A is the most potent inhibitor of thrombin in vitro, among all other members of the aeruginosin family. So far, four X-ray crystal structures of aeruginosins in complex with thrombin have been solved. A hirugen-thrombin complex with aeruginosin 298A was reported in 1998, followed by co-crystal structures of thrombin with dysinosin A , oscillarin, and chlorodysinosin A (Table 1). They all show similar binding modes to the thrombin active site (Figure 2). The basic PI sidechain in oscillarin, dysinosin A, and chlorodysinosin A, is positioned in the SI pocket forming a strong interaction from the terminal guanidino group to an aspartic acid residue at the bottom of the SI pocket. The P2 position in the octahydroindole subunit is well accommodated in the S2 pocket, but without any hydrogen bonds formed between the enzyme and the 6- (Choi) or 5,6- (OHChoi) hydroxyl groups. The P3 D-a-amino acid occupies the so-called D-S3 subsite, which is a relatively large hydrophobic pocket. Moreover, the P3-P4 amide of the aeruginosins binds to the backbone of a glycine residue in the thrombin active site in an antiparallel β-strand fashion, and the ^-terminal acidic sulfate group in dysinosin A and chlorodysinosin A form hydrogen bonds to arginine residues on the surface of thrombin. The overall co-crystal structures and binding modes of dysinosin A and chlorodysinosin A, differing only in the presence of the P3 β-chlorine atom in chlorodysinosin A, are very similar. A subtle difference is a shift in orientation of the side-chains of the two active site residues Glu 192 and Arg 173 in thrombin. The conformation of the D-S3 pocket, as well as the binding mode of the P3 leucine and 3-chloroleucine residues is identical in the dysinosin A and chlorodysinosin A complexes respectively (Figure 3A). The enhanced binding of chlorodysinosin A in the DS3 subsite has been suggested to arise from a favorable position of the chlorine atom compared to the same D-Leu residue in dysinosin A (Figure 3A). Molecular dynamics simulation suggested that the chlorine substituent stabilizes the χ dihedral angle in the β-chloro-D-leucyl side-chain in the bioactive conformation (Figure 3B). Additionally, the accommodation of the hydrophobic D-S3 site could also be accompanied by a release of water resulting in a gain in entropy of binding. 9
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Synthetic Approaches to β-Chloroleucine The identification of a new amino acid, β-chloro-D-leucine (Cleu), as a component of chlorodysinosin A , prompted us to explore efforts toward the 6
In Asymmetric Synthesis and Application of -Amino Acids; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
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Table 1. In Vitro Enzyme Inhibitory Activities of Natural and Synthetic Aeruginosins α
Enzyme IC (μΜ) Thrombin „ /τ?ιι \ FVIIa FXa (Flla) 50
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Compound
Structure
r
H O ' ° ^ i T N HN-S.
Aeruginosin 298A
0.5
nd
nd
Oscillarin
0.028
3.9
nd
Dysinosin A
0.046
0.326
5
0.0057
0.039
1.54
0.022
nd
nd
\
NH 0
)=NH
A.>OH
HN 2
^OSOâ
Chlorodysinosin
A
\
Γ
NH 0
Γ
H
...ΟΜβ
>
N
OSO3
Ctv
