Asymmetric Catalysis Using Chiral Salen-Metal Complexes: Recent Advances.

Chiral salen-metal complexes are among the most versatile asymmetric catalysts and have found utility in fields ranging from materials chemistry to organic synthesis. These complexes are capable of inducing chirality in products formed from a wide variety of chemical processes, often with close to perfect stereoinduction. Salen ligands are tunable for steric as well as electronic properties, and their ability to coordinate a large number of metals gives the derived chiral salen-metal complex very broad utility in asymmetric catalysis. This review primarily summarizes developments in chiral salen-metal catalysis over the last two decades with particular emphasis on those applications of importance in asymmetric synthesis.

Synthesis and antiproliferative evaluation of new zampanolide mimics.

(-)-Zampanolide is a marine microtubule-stabilizing macrolide that has been shown by in vitro experiments to be a promising anticancer lead compound. Through its unique covalent-binding with ß-tubulin, zampanolide exhibits cytotoxic potency towards multi-drug resistant cancer cells that is superior to paclitaxel. However, the limited availability of zampanolide impedes its further in vivo evaluation as a viable drug candidate. Zampanolide is envisioned to become more drug-like if its chemically fragile side chain can be stabilized; hence, this project aims to develop mimics of zampanolide with a stable side chain using straightforward synthetic methods. To this end, twelve novel zampanolide mimics (51-62) with conjugated and planar side chains have been synthesized via a 24-step sequence for each mimic from commercially available 2-butyn-1-ol as starting material. A Horner-Wadsworth-Emmons reaction incorporates the α,ß-unsaturated ketone side chain and also closes the core macrocycle. WST-1 cell proliferation assays in three docetaxel-sensitive and two docetaxel-resistant human prostate cancer cell models confirm that a suitably designed side chain can serve as a bioisostere for the N-acyl hemiaminal side chain in zampanolide. Mimic 52 with a 17R chiral center was identified as the optimal candidate with IC50 values of 0.29-0.46 µM against both docetaxel-sensitive (PC-3 and DU145) and docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX). Zampanolide mimic 52 exhibited equivalent antiproliferative potency towards both docetaxel-sensitive and docetaxel-resistant cell lines, with relative resistance in the range of 0.9-1.6.

Optimized synthesis and antiproliferative activity of desTHPdactylolides.

Dactylolide and certain analogues are attractive targets for study due to their structural resemblance to zampanolide, a very promising anticancer lead compound and a unique covalent-binding microtubule stabilizing agent. The primary goal of this project is identification and synthesis of simplified analogues of dactylolide that would be easier to prepare and could be investigated for antiproliferative activity in comparison with zampanolide. Extension of Almann's concept of a simplified zampanolide analogue to dactylolide in the form of desTHPdactylolide was attractive not only for reasons of synthetic simplification but also for the prospect that analogues of dactylolide could be prepared in both (17S) and (17R) configurations. Since Altmann's overall yield for the six-step procedure leading to the C9-C18 fragment of desTHPdactylolide was only 8.7%, a study focused on optimized synthesis and antiproliferative evaluation of each enantiomer of desTHPdactylolide was initiated using Altmann's route as a framework. To this end, two optimized approaches to this fragment C9-C18 were successfully developed by us using allyl iodide or allyl tosylate as the starting material for a critical Williamson ether synthesis. Both (17S) and (17R) desTHPdactylolides were readily synthesized in our laboratory using optimized methods in yields of 37-43%. Antiproliferative activity of the pair of enantiomeric desTHPdactylolides, together with their analogues, was evaluated in three docetaxel-sensitive and two docetaxel-resistant prostate cancer cell models using a WST-1 cell proliferation assay. Surprisingly, (17R) desTHPdactylolide was identified as the eutomer in the prostate cancer cell models. It was found that (17S) and (17R) desTHPdactylolide exhibit equivalent antiproliferative potency towards both docetaxel-sensitive (PC-3 and DU145) and docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX).

cis-2,5-Diaminobicyclo[2.2.2]octane, a New Chiral Scaffold for Asymmetric Catalysis.

Catalysis of widely used chemical transformations in which the goal is to obtain the product as a pure enantiomer has become a major preoccupation of synthetic organic chemistry over the past three decades. A large number of chiral entities has been deployed to this end, many with considerable success, but one of the simplest and most effective catalytic systems to have emerged from this effort is that based on a chiral diamine, specifically trans-1,2-diaminocyclohexane. While there have been attempts to improve upon this scaffold in asymmetric synthesis, few have gained the recognition needed to take their place alongside this classic diamine. The challenge is to design a scaffold that retains the assets of trans-1,2-diaminocyclohexane while enhancing its intrinsic chirality and maximizing the scope of its applications. It occurred to us that cis-2,5-diaminobicyclo[2.2.2]octane could be such a scaffold. Synthesis of this diamine in enantiopure form was completed from benzoic acid, and the (1R,2R,4R,5R) enantiomer was used in all subsequent experiments in this laboratory. Condensation of the diamine with various salicyl aldehydes generated imine derivatives which proved to be excellent "salen" ligands for encapsulation of transition and other metals. In total, 12 salen-metal complexes were prepared from this ligand, many of which were crystalline and three of which, along with the ligand itself, yielded to X-ray crystallography. An advantage of this ligand is that it can be tuned sterically or electronically to confer specific catalytic properties on the salen-metal complex, and this feature was used in several applications of our salen-metal complexes in asymmetric synthesis. Thus, replacement of one of the tert-butyl groups in each benzenoid ring of the salen ligand by a methoxy substituent enhanced the catalytic efficiency of a cobalt(II)-salen complex used in asymmetric cyclopropanation of 1,1-disubstituted alkenes; the catalyst was employed in an improved synthesis of the cyclopropane-containing drug candidate Synosutine. Reduction of the pair of imine functions of the ligand to secondary amines permitted formation of a copper(I)-salen complex that catalyzed asymmetric Henry ("nitroaldol") condensation with excellent efficiency; this catalyst was applied in an economical synthesis of three drugs of the "beta-blocker" family including (S)-Propanolol. Chromium(II) and chromium(III) complexes were prepared from our bicyclooctane-salen ligand bearing a pair of tert-butyl groups in each benzenoid ring. These complexes were found to catalyze, respectively, enantioselective formation of homoallylic alcohols from Nozaki-Hiyama-Kishi allylation of aromatic aldehydes and dihydropyranones from hetero-Diels-Alder cycloaddition. Plausible reaction models emerging from knowledge of the absolute configuration of products from each of these reactions place the metal-coordinated substrate in a quadrant beneath the bicyclooctane scaffold so that one face of the substrate is blocked by an aryl ring of the salen ligand while the opposite face is left open to attack. The consistent and predictable stereochemical outcome from reactions catalyzed by salen-metal complexes derived from our diaminobicyclo[2.2.2]octane scaffold adds a valuable new dimension to asymmetric synthesis.

Bioengineered functional brain-like cortical tissue.

The brain remains one of the most important but least understood tissues in our body, in part because of its complexity as well as the limitations associated with in vivo studies. Although simpler tissues have yielded to the emerging tools for in vitro 3D tissue cultures, functional brain-like tissues have not. We report the construction of complex functional 3D brain-like cortical tissue, maintained for months in vitro, formed from primary cortical neurons in modular 3D compartmentalized architectures with electrophysiological function. We show that, on injury, this brain-like tissue responds in vitro with biochemical and electrophysiological outcomes that mimic observations in vivo. This modular 3D brain-like tissue is capable of real-time nondestructive assessments, offering previously unidentified directions for studies of brain homeostasis and injury.

Synthesis of two subunits of the macrolide domain of the immunosuppressive agent sanglifehrin a and assembly of a macrolactone precursor. application of masamune anti-aldol condensation.

Asymmetric anti-aldol coupling of a norephedrine-derived ester with an α-chiral aldehyde was used to synthesize a carboxylic acid representing the C13-C19 segment of the macrocyclic domain present in the immunosuppressive agent sanglifehrin A. Felkin addition set configuration at the C14-C17 stereotetrad in this unit in which hydroxyl functions at C15 and C17 were masked as an internal ketal. The carboxyl group of this segment was coupled to the N-terminus of the tripeptide portion (C1-N12) of sanglifehrin A macrolactone to assemble the C1-C19 domain. Synthesis of the C20-C25 subunit of sanglifehrin A containing a (23S) alcohol was completed via asymmetric allylation of (E)-3-iodo-2-methylprop-2-enal followed by oxidative cleavage of the terminal vinyl appendage and a Takai olefination with pinacol dichloromethylboronate. Esterification of this alcohol with a C1-C19 carboxylic acid furnished an open C1-C25 macrolactone precursor, but this substance failed to undergo macrocyclization via intramolecular Suzuki-Miyaura coupling.

Cyclobutane Synthesis and Fragmentation. A Cascade Route to the Lycopodium Alkaloid (-)-Huperzine A.

An asymmetric total synthesis of the nootropic alkaloid (-)-huperzine A was completed using a cascade sequence initiated by an intramolecular aza-Prins reaction and terminated by a stereoelectronically guided fragmentation of a cyclobutylcarbinyl cation as the key step in assembling the bicyclo[3.3.1]nonene core of the natural product. Intramolecular [2 + 2]-photocycloaddition of the crotyl ether of (S)-4-hydroxycyclohex-2-enone afforded a bicyclo[4.2.0]octanone containing an embedded tetrahydrofuran in which the cyclohexanone moiety was converted to a triisopropylsilyl enol ether and functionalized as an allylic azide. The derived primary amine was acylated with α-phenylselenylacrylic acid, and the resulting amide was reacted with trimethylaluminum to give a [2 + 2]-cycloadduct, which underwent retroaldol fission to produce a fused α-phenylselenyl δ-lactam. Periodate oxidation of this lactam led directly to an α-pyridone, which was converted to a fused 2-methoxypyridine. Reductive cleavage of the activated "pyridylic" C-O bond in this tetracycle and elaboration of the resultant hydroxy ketone to a diketone was followed by chemoselective conversion of the methyl ketone in this structure to an endo isopropenyl group. Condensation of the remaining ketone with methyl carbamate in the presence of acid initiated the programmed cascade sequence and furnished a known synthetic precursor to huperzine A. Subsequent demethylation of the carbamate and the methoxypyridine, accompanied by in situ decarboxylation of the intermediate carbamic acid, gave (-)-huperzine A.

A new iron(III)-salen catalyst for enantioselective Conia-ene carbocyclization.

A chiral iron(III)-salen complex based on a cis-2,5-diaminobicyclo[2.2.2]octane scaffold catalyzes asymmetric Conia-ene-type cyclization of α-functionalized ketones containing an unactivated terminal alkyne and produces an exo-methylenecycloalkane possessing a stereodefined quaternary center.

Studies of the synthesis of providencin: construction and assembly of two major subunits.

The "northern" sector of the cembranoid diterpene providencin containing a tetrasubstituted cyclobutane was synthesized from the bis(acetonide) of d-glucose using dicyclopentadienylzirconium(0)-mediated oxygen abstraction from a furanose. Oxidative scission of the vinyl substituent of this cyclobutane gave an aldehyde, which was reacted with an alkynylstannane to provide an allenol. Cyclization of the derived allenone with silver nitrate led to a cyclobutylfuran comprising the northern subunit of providencin. The "southern" sector of the cembranoid skeleton containing a trisubstituted iodoalkene attached to an α-phenylselenyl-γ-lactone was synthesized from (R)-glycidol. Negishi carbometalation-iodination established the (E)-iodoalkene, and addition of the lithio dianion of phenylselenoacetic acid to a tosylate generated the substituted lactone. The two sectors were joined via stannylation of the furan of the northern component followed by Stille cross-coupling of the furylstannane with the iodoalkene of the southern subunit. Linkage of the two segments was also made at C12-C13 of providencin using intermolecular aldol condensation of the enolate from the selenyl lactone of the southern portion with an acetaldehyde appendage on the cyclobutane of the northern sector. Closure of the providencin macrocycle from these conjoined subunits was unsuccessful.

Total synthesis of macrodiolide ionophores aplasmomycin A and boromycin via double ring contraction.

The half structure of the symmetrical macrodiolide aplasmomycin A was synthesized by alkylation of a C3-C10 α-sulfonyl ketone subunit, prepared from (R)-pulegone and protected as a C3 ortholactone with (2R,3R)-butanediol, by a protected 15,16-dihydroxy (12E)-allylic chloride representing C11-C17. The latter was obtained from (2S,3R)-1,2-epoxy-3-butanol and propargyl alcohol. Regio- and stereoselective 5-exo-trig cyclization of the ene diol moiety in this segment, mediated by N-bromosuccinimide, led to the (2R,3S,5R)-tetrahydrofuran substructure of aplasmomycin A. Attachment of an α-acetic ester at the C3 carboxylic acid and esterification of the 3'-hydroxyl group of the tetrahydrofuran as its α-bromoacetate enabled coupling of two aplasmomycin half structures as an α-acyloxy acetate. Mukaiyama macrolactonization of this hydroxy acid afforded a symmetrical 36-membered diolide. Base-mediated double Chan rearrangement of this bis α-acyloxy dilactone caused ring contraction to the 34-membered macrocycle of desboroaplasmomycin A while generating the transannular 2-hydroxy-3-hemiketal motif of the natural product in the correct configuration. Final incorporation of boron into the tetraol core produced aplasmomycin A, isolated as its sodium borate. Extension of this route to the unsymmetrical macrodiolide boromycin was accomplished by modifications that included reversal of C12-C13 olefin geometry to (Z) for the southern half structure along with stereoselective hydride reductions of the C9 ketone that produced (9R) and (9S) alcohols for northern and southern half structures, respectively. Coupling of these half structures was made using an α-acyloxy ester linkage as for aplasmomycin A, but ring closure in this case was orchestrated via a blocked C16 alcohol that left open the C15 hydroxyl group of the southern half for Mukaiyama macrolactonization. A double Chan rearrangement of the resulting 35-membered macrocycle produced the 33-membered diolide of desborodesvalinylboromycin which had been obtained previously by degradation of natural boromycin. Insertion of boron into the tetraol core followed by esterification of the C16 alcohol with a masked d-valine and final deprotection furnished boromycin as its zwitterionic (Böeseken) complex.

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability.

Despite recent advances in nucleic acid delivery systems with the success of LNP vehicles, adeno-associated virus (AAV) remains the leading platform for targeted gene delivery due to its low immunogenicity to humans, high transduction efficiency, and range of serotypes with varying tropisms. Depending on the therapeutic goals and serotype used, different production conditions may be more amenable, generating an ever-growing need for rapid yet robust analytical techniques to support the high-quality manufacturing of AAV. A critical bottleneck exists for assessing full capsids where rapid, high-throughput techniques capable of analyzing a range of serotypes are needed. Here, we present a rapid, high-throughput analytical technique, microfluidic electrophoresis, for the assessment of full capsids compatible with AAV1, AAV2, AAV6, AAV8, and AAV9 without the need for assay modifications or optimizations, and AAV5 with some constraints. The method presented in this study uses a mathematical formulation we developed previously with a reference standard to combine the independently obtained capsid protein and single-stranded DNA (ssDNA) profiles to estimate the percentage of full capsids in a sample of unknown concentration. We assessed the ability to use a single serotype (AAV8) as the reference standard regardless of the serotype of the sample being analyzed so long as the melting temperature (Tm) of the capsids is within 12 °C from the Tm of AAV8. Using this method, we are able to characterize samples ±6.1% with an average analytical turnaround time of <5 min/sample, using only 10 µL/sample at a concentration of 2.5 × 1012 VG/mL.

Synthesis of the tripeptide domain of sanglifehrins using asymmetric phase-transfer catalysis.

The tripeptide (S)-valinyl-(S)-m-hydroxyphenylalanyl-(3S)-piperazate common to immunosuppressant sanglifehrins was synthesized from the constituent amino acid residues in nine steps and 42% overall yield. A key construction was the installation of (S) absolute configuration in m-hydroxyphenylalanine using asymmetric phase-transfer catalysis in the presence of N-(1-naphthyl)cinchonidinium bromide. Cbz-protected (S)-valine was first coupled to the amino group of (S)-m-triisopropylsilyloxyphenylalanine tert-butyl ester, and the resulting dipeptide after ester cleavage was linked to (3S)-methyl piperazate.

High-resolution infrared and electron-diffraction studies of trimethylenecyclopropane ([3]-radialene).

Combined high-resolution spectroscopic, electron-diffraction, and quantum theoretical methods are particularly advantageous for small molecules of high symmetry and can yield accurate structures that reveal subtle effects of electron delocalization on molecular bonds. The smallest of the radialene compounds, trimethylenecyclopropane, [3]-radialene, has been synthesized and examined by these methods. The first high-resolution infrared spectra have been obtained for this molecule of D3h symmetry, leading to an accurate B0 rotational constant value of 0.1378629(8) cm(-1), within 0.5% of the value obtained from electronic structure calculations (density functional theory (DFT) B3LYP/cc-pVTZ). This result is employed in an analysis of electron-diffraction data to obtain the rz bond lengths (in Å): C-H = 1.072(17), C-C = 1.437(4), and C═C = 1.330(4). The results indicate that the effects of rehybridization and π-electron delocalization affects each result in a shortening of about 0.05 Å for the C-C bond in radialene compared to ethane. The analysis does not lead to an accurate value of the HCH angle; however, from comparisons of theoretical and experimental angles for similar compounds, the theoretical prediction of 117.5° is believed to be reliable to within 2°.

Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy.

Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1-2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1-8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons.

Polymer composition and substrate influences on the adhesive bonding of a biomimetic, cross-linking polymer.

Hierarchical biological materials such as bone, sea shells, and marine bioadhesives are providing inspiration for the assembly of synthetic molecules into complex structures. The adhesive system of marine mussels has been the focus of much attention in recent years. Several catechol-containing polymers are being developed to mimic the cross-linking of proteins containing 3,4-dihydroxyphenylalanine (DOPA) used by shellfish for sticking to rocks. Many of these biomimetic polymer systems have been shown to form surface coatings or hydrogels; however, bulk adhesion is demonstrated less often. Developing adhesives requires addressing design issues including finding a good balance between cohesive and adhesive bonding interactions. Despite the growing number of mussel-mimicking polymers, there has been little effort to generate structure-property relations and gain insights on what chemical traits give rise to the best glues. In this report, we examine the simplest of these biomimetic polymers, poly[(3,4-dihydroxystyrene)-co-styrene]. Pendant catechol groups (i.e., 3,4-dihydroxystyrene) are distributed throughout a polystyrene backbone. Several polymer derivatives were prepared, each with a different 3,4-dihyroxystyrene content. Bulk adhesion testing showed where the optimal middle ground of cohesive and adhesive bonding resides. Adhesive performance was benchmarked against commercial glues as well as the genuine material produced by live mussels. In the best case, bonding was similar to that obtained with cyanoacrylate "Krazy Glue". Performance was also examined using low- (e.g., plastics) and high-energy (e.g., metals, wood) surfaces. The adhesive bonding of poly[(3,4-dihydroxystyrene)-co-styrene] may be the strongest of reported mussel protein mimics. These insights should help us to design future biomimetic systems, thereby bringing us closer to development of bone cements, dental composites, and surgical glues.

Total synthesis of the marine toxin phorboxazole A using palladium(II)-mediated intramolecular alkoxycarbonylation for tetrahydropyran synthesis.

The potent antitumor agent phorboxazole A was synthesized from six subunits comprising C1-C2 (115), C3-C8 (98), C9-C19 (74), C20-C32 (52), C33-C41 (84) and C42-C46 (85). Tetrahydropyrans B and C containing cis-2,6-disubstitution were fabricated via palladium(II)-mediated intramolecular alkoxycarbonylation which, in the case of tetrahydropyran C, was carried out with catalytic palladium(II) and p-benzoquinone as the stoichiometric re-oxidant. Tetrahydropyran D was obtained by a stereoselective tin(IV)-catalyzed coupling of a C9 aldehyde with an allylsilane, and the C19-C20 connection was made using a completely stereoselective Wittig-Schlosser (E) olefination. Coupling of the oxazole C32 methyl substituent with the intact C33-C46 δ-lactone 3was accompanied by elimination of the vinyl bromide to a terminal alkyne, but the C32-C33 linkage was implemented successfully with 83 and C33-C41 lactone 84. The C42-C46 segment of the side chain was then appended via Julia-Kocienski olefination. The macrolide portion of phorboxazole A was completed by means of an Ando-Still-Gennari intramolecular (Z)-selective olefination at C2-C3 which required placement of a (dimethoxyphosphinyl)acetate moiety at C24. Final deprotection led to phorboxazole A via a route in which the longest linear sequence is 37 steps and the overall yield is 0.36%.

Highly extensible bio-nanocomposite fibers.

Here, we show that a poly(ethylene oxide) polymer can be physically cross-linked with silicate nanoparticles (Laponite) to yield highly extensible, bio-nanocomposite fibers that, upon pulling, stretch to extreme lengths and crystallize polymer chains. We find that both, nanometer structures and mechanical properties of the fibers respond to mechanical deformation by exhibiting strain-induced crystallization and high elongation. We explore the structural characteristics using X-ray scattering and the mechanical properties of the dried fibers made from hydrogels in order to determine feasibility for eventual biomedical use and to map out directions for further materials development.

Ecogenomics: using massively parallel pyrosequencing to understand virus ecology.

Environmental samples have been analysed for viruses in metagenomic studies, but these studies have not linked individual viruses to their hosts. We designed a strategy to isolate double-stranded RNA, a hallmark of RNA virus infection, from individual plants and convert this to cDNA with a unique four nucleotide Tag at each end. Using 96 different Tags allowed us to pool samples and still retain the link to the original sample. We then analysed the sequence of pooled samples using massively parallel sequencing with Roche 454 pyrosequencing such that 384 samples could be assessed per picotiter plate. Using this method we have been able to analyse thousands of plants, and we have discovered several thousand new plant viruses, all linked to their specific plant hosts. Here we describe the method in detail, including the results and analysis for eight pools of samples. This technology will be extremely useful in understanding the full scope of plant virus biodiversity.

Tandem intramolecular photocycloaddition-retro-Mannich fragmentation as a route to spiro[pyrrolidine-3,3'-oxindoles]. Total synthesis of (+/-)-coerulescine, (+/-)-horsfiline, (+/-)-elacomine, and (+/-)-6-deoxyelacomine.

Irradiation of a tryptamine linked through its side-chain nitrogen to an alkylidene malonate residue results in an intramolecular [2 + 2] cycloaddition to the indole 2,3-double bond. The resultant cyclobutane undergoes spontaneous retro-Mannich fission to produce a spiro[indoline-3,3'-pyrrolenine] with relative configuration defined by the orientation of substituents in the transient cyclobutane. The tandem intramolecular photocycloaddition-retro-Mannich process, abbreviated as TIPCARM, leads to a spiropyrrolidine which is poised to undergo a second retro-Mannich fragmentation that expels the malonate unit and generates transiently an indolenine. The latter undergoes rearrangement to a beta-carboline, which upon brominative oxidation undergoes further rearrangement to an oxindole. With tryptamine as starting material, the entire sequence leads to the alkaloid (+/-)-coerulescine. Starting from 5-methoxytryptamine, a parallel series affords (+/-)-horsfiline. Modification of the malonylidene unit to include an isobutyl substituent at C3 affords a photosubstrate which also undergoes the TIPCARM process. In this case, a 2'-isobutyl-substituted spiro[indoline-3,3'-pyrrolenine] results. This undergoes stereoselective hydride reduction to give a product with relative orientation at the spiro carbon and the new stereocenter bearing the isobutyl appendage corresponding to that of the alkaloid elacomine. From tryptamine, the sequence paralleling that leading to coerulescine and horsfiline terminates at 6-deoxyelacomine, whereas 6-methoxytryptamine as starting material affords (+/-)-elacomine itself.

Insight into the substrate length restriction of M32 carboxypeptidases: characterization of two distinct subfamilies.

M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted. Structural and sequence analyses implicate a highly conserved Arg at the back of the groove as being critical for this length restriction. However, the Thermus thermophilus and Bacillus subtilis M32 members lack this conserved Arg. Herein, we present the biochemical and structural characterization of these two proteins. Our findings support the important role of the conserved Arg in maintaining the length restriction, and reveal a proline-rich loop as an alternate blocking strategy. Based on our results, we propose that M32 carboxypeptidases from Bacilli belong to a separate subfamily.