Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and ß-farnesene.

Large-Scale Synthesis of Man9GlcNAc2 High-Mannose Glycan and the Effect of the Glycan Core on Multivalent Recognition by HIV Antibody 2G12.

Access to homogeneous high-mannose glycans in high-mg quantities is necessary for carbohydrate-based HIV vaccine development research. We have used directed evolution to design highly antigenic oligomannose clusters that are recognized in low-nM affinity by HIV antibodies. Herein we report an optimized large-scale synthesis of Man9GlcNAc2 including improved building block synthesis and a fully stereoselective 5 + 6 coupling, yielding 290 mg of glycan. We then use this glycan to study the effect of the GlcNAc2 core on the antigenicity of an evolved 2G12-binding glycopeptide, 10F2.

A Mechanistic Switch from Homoallylation to Cyclopropylcarbinylation of Aldehydes.

Cyclopropanated allylboration reagents participate in homoallylation reactions of aliphatic and aromatic aldehydes, generating allylic-substituted alkenes that are difficult to produce via other methods. In studying the effect of cyclopropane substituents, we discovered that an aryl substituent completely changes the outcome to cyclopropylcarbinylation, as if the cyclopropylcarbinyl fragment were transferred intact. However, density functional theory computation suggested a novel mechanism involving ring opening and reclosure, which is supported by experimental evidence.

Homoallylboration of Aldehydes: Stereoselective Synthesis of Allylic-Substituted Alkenes and E-Alkenes.

Cyclopropanated allylboration reagents participate in the homoallylation of aliphatic and aromatic aldehydes, generating substituted alkenes that are difficult to produce via other methods. In this study, we explored the scope and reactivity of homoallylation with cyclopropylcarbinylboronates bearing various aliphatic and aromatic α- and γ-substituents. α-Alkyl substituted boronates afforded E-disubstituted alkenyl secondary alcohols in high enantiomeric ratios, while aryl substituents promoted rearrangement. γ-Alkyl substituents all resulted in diastereoselective homoallylation, while aryl substitution changed the outcome to cyclopropylcarbinylation.

An Optimized Synthesis of Fmoc-l-Homopropargylglycine-OH.

An efficient multigram synthesis of alkynyl amino acid Fmoc-l-homopropargylglycine-OH is described. A double Boc protection is optimized for high material throughput, and the key Seyferth-Gilbert homologation is optimized to avoid racemization. Eighteen grams of the enantiopure (>98% ee) noncanonical amino acid was readily generated for use in solid phase synthesis to make peptides that can be functionalized by copper-assisted alkyne-azide cycloaddition.

Directed Evolution of 2'-Fluoro-Modified, RNA-Supported Carbohydrate Clusters That Bind Tightly to HIV Antibody 2G12.

Carbohydrate binding proteins (CBPs) are attractive targets in medicine and biology. Multivalency, with several glycans binding to several binding pockets in the CBP, is important for high-affinity interactions. Herein, we describe a novel platform for design of multivalent carbohydrate cluster ligands by directed evolution, in which serum-stable 2'-fluoro modified RNA (F-RNA) backbones evolve to present the glycan in optimal clusters. We have validated this method by the selection of oligomannose (Man9) glycan clusters from a sequence pool of ∼1013 that bind to broadly neutralizing HIV antibody 2G12 with 13 to 36 nM affinities.

Synthesis of Mannosidase-Stable Man3 and Man4 Glycans Containing S-linked Manα1→2Man Termini.

Oligomannose glycans are of interest as HIV vaccine components, but they are subject to mannosidase degradation in vivo. Herein, we report the synthesis of oligosaccharides containing a thio linkage at the nonreducing end. A thio-linked dimannose donor participates in highly stereoselective glycosylations to afford trimannose and tetramannose fragments. Saturation transfer difference nuclear magnetic resonance (STD NMR) studies show that these glycans are recognized by HIV antibody 2G12, and we confirm that the reducing terminal S-linkage confers complete stability against x. manihotis mannosidase.

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase.

Most terpene synthase reactions follow Markovnikov rules for formation of high-energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 atom of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide the regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the nonreactive substrate analogue 12,13-difluorofarnesyl diphosphate, and determined the X-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is perfectly positioned to make a C-H···π interaction with the side chain benzene ring of residue F76; this would enhance hyperconjugation to stabilize a developing cation at C10 and thus support the anti-Markovnikov regioselectivity of the cyclization. The benzene ring is also positioned to catalyze the migration of H to C10 and stabilize a C9 carbocation. On the opposite face of C9, further cation stabilization is possible via interactions with the main chain carbonyl of I177 and the neighboring intramolecular C6═C7 bond. Mutagenesis experiments also support a role for residue 76 in these interactions, but most interesting is the F76W mutant, whose crystal structure clearly shows C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

The Impact of Sustained Immunization Regimens on the Antibody Response to Oligomannose Glycans.

The high mannose patch (HMP) of the HIV envelope protein (Env) is the structure most frequently targeted by broadly neutralizing antibodies; therefore, many researchers have attempted to use mimics of this region as a vaccine immunogen. In our previous efforts, vaccinating rabbits with evolved HMP mimic glycopeptides containing Man9 resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1→2Man tips of Man9 glycans. A possible reason could be processing of our immunogen by host serum mannosidases. We sought to test whether more prolonged dosing could increase the antibody response to intact glycans, possibly by increasing the availability of intact Man9 to germinal centers. Here, we describe a study investigating the impact of immunization regimen on antibody response by testing immunogen delivery through bolus, an exponential series of mini doses, or a continuously infusing mini-osmotic pump. Our results indicate that, with our glycopeptide immunogens, standard bolus immunization elicited the strongest HIV Env-binding antibody response, even though higher overall titers to the glycopeptide were elicited by the exponential and pump regimens. Antibody selectivity for intact glycan was, if anything, slightly better in the bolus-immunized animals.

Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.

Oligomannose Glycopeptide Conjugates Elicit Antibodies Targeting the Glycan Core Rather than Its Extremities.

Up to ∼20% of HIV-infected individuals eventually develop broadly neutralizing antibodies (bnAbs), and many of these antibodies (∼40%) target a region of dense high-mannose glycosylation on gp120 of the HIV envelope protein, known as the "high-mannose patch" (HMP). Thus, there have been numerous attempts to develop glycoconjugate vaccine immunogens that structurally mimic the HMP and might elicit bnAbs targeting this conserved neutralization epitope. Herein, we report on the immunogenicity of glycopeptides, designed by in vitro selection, that bind tightly to anti-HMP antibody 2G12. By analyzing the fine carbohydrate specificity of rabbit antibodies elicited by these immunogens, we found that they differ from some natural human bnAbs, such as 2G12 and PGT128, in that they bind primarily to the core structures within the glycan, rather than to the Manα1 → 2Man termini (2G12) or to the whole glycan (PGT128). Antibody specificity for the glycan core may result from extensive serum mannosidase trimming of the immunogen in the vaccinated animals. This finding has broad implications for vaccine design aiming to target glycan-dependent HIV neutralizing antibodies.

Stereoselective Homocrotylation of Aldehydes: Enantioselective Synthesis of Allylic-Substituted Z/ E-Alkenes.

Cyclopropanated allyl- and crotylboron reagents participate in homoallylation and homocrotylation reactions that enable enantioselective access to motifs that otherwise require many steps to synthesize. In this study, we investigated the effect of substituents α- to boron, predicted either to counteract or reinforce the 1,3- selectivity of the parent reagents. We then investigated the transformation of the substituted homocrotylation products in intramolecular photocycloadditions to produce stereochemically complex natural-product-like scaffolds, finding that flow conditions enhanced the regioselectivity and yield.

Directed Evolution of Glycopeptides Using mRNA Display.

Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein. Although yeast, mammalian and Escherichia coli cells can be engineered to produce a homogenous glycoform at all glycosylation sites, there are just a few specific glycan structures that can readily be accessed in this manner. Recently, we reported a novel system for the directed evolution of glycopeptide libraries, which could in principle be decorated with any desired glycan. Our method combines in vitro peptide selection by mRNA display with unnatural amino acid incorporation and chemical attachment of synthetic oligosaccharides. Here, we provide an updated and optimized protocol for this method, which is designed to create glycopeptide mRNA display libraries containing ~1013 sequences and select them for target binding. The target described here is the HIV broadly neutralizing monoclonal antibody 2G12; 2G12 binds to cluster of high-mannose oligosaccharides on the HIV envelope glycoprotein gp120; and glycopeptides that mimic this epitope may be useful in HIV vaccine applications. This method is expected to be readily applicable for other types of glycans and targets of interest in glycobiology.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Antibody recognition of HIV and dengue glycoproteins.

The last 6 years have witnessed an explosion of discoveries at the interface of glycobiology and immunology. Binding of clustered oligosaccharides has turned out to be a very frequent mode by which human antibodies have developed broadly neutralizing activity against HIV. This mini-review will cover many recent developments in the HIV antibody field, as well as emerging data about Dengue broadly neutralizing antibodies.

Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope.

Recently, we reported a directed evolution method which enabled us to discover sequences of glycopeptides that bind with picomolar affinity to HIV antibody 2G12 and are of interest as HIV vaccine candidates. In this manuscript, we describe the syntheses of several of these large (~11-12 kDa) glycopeptides by a combination of fast flow peptide synthesis and click chemistry. We also discuss the optimization of their attachment to carrier protein CRM197, affording antigenic and immunogenic conjugates ready for animal vaccination.

Enantioselective syn and anti Homocrotylation of Aldehydes: Application to the Formal Synthesis of Spongidepsin.

Whereas crotylboration has been a useful method for synthesis of stereochemically complex products, we have shown that homocrotylboration can be achieved with cyclopropanated crotylation reagents, and that the stereoselectivity of the reaction can be predicted by analogous models. This paper presents a full account of this work, including the first examples of asymmetric anti homocrotylation. The scope of this reaction is demonstrated with highly enantioselective homocrotylation of both aliphatic and aromatic aldehydes, as well as double diastereoselection studies. An application of the synthesis of the marine natural product spongidepsin is presented, as well as streamlined syntheses of homocrotylation reagents.

SELMA: Selection with Modified Aptamers.

In vitro selection of nucleic acid aptamers, coined SELEX, has led to the discovery of novel therapeutics and aided in the structural and mechanistic understanding of many ligand-biomolecule interactions. A related method, selection with modified aptamers (SELMA), enables selection of DNA aptamers containing bases with a large modification that cannot undergo PCR. A key application of this method is the evolution of aptamers containing carbohydrate modifications. Carbohydrate-binding proteins normally require several copies of the carbohydrate moiety for strong recognition. Whereas it may be difficult to rationally design synthetic scaffolds that cluster glycans in the optimal spacing and orientation for target recognition, SELMA furnishes glycoaptamers with highly optimized glycan clustering, achieving low-nanomolar recognition. Although numerous applications can be envisioned, the protocols and discussions in this article describe procedures involved in applying SELMA to the discovery glycoDNAs that bind to the HIV broadly neutralizing antibody 2G12.

Recent strategies targeting HIV glycans in vaccine design.

Although efforts to develop a vaccine against HIV have so far met with little success, recent studies of HIV-positive patients with strongly neutralizing sera have shown that the human immune system is capable of producing potent and broadly neutralizing antibodies (bnAbs), some of which neutralize up to 90% of HIV strains. These antibodies bind conserved vulnerable sites on the viral envelope glycoprotein gp120, and identification of these sites has provided exciting clues about the design of potentially effective vaccines. Carbohydrates have a key role in this field, as a large fraction of bnAbs bind carbohydrates or combinations of carbohydrate and peptide elements on gp120. Additionally, carbohydrates partially mask some peptide surfaces recognized by bnAbs. The use of engineered glycoproteins and other glycostructures as vaccines to elicit antibodies with broad neutralizing activity is therefore a key area of interest in HIV vaccine design.

Boron carboxylate catalysis of homoallylboration.

Boron tris(trifluoroacetate) is identified as the first effective catalyst for the homoallyl- and homocrotylboration of aldehydes by cyclopropylcarbinylboronates. NMR spectroscopic studies and theoretical calculations of key intermediates and transition states both suggest that a ligand-exchange mechanism, akin to our previously reported PhBCl2-promoted homoallylations, is operative. Our experimental and theoretical results also suggest that the catalytic activity of boron tris(trifluoroacetate) might originate from more facile catalytic turnover of the trifluoroacetate ligands (in agreement with DFT calculations) or from a lower propensity for formation of off-pathway reservoir intermediates (as observed by (1)H NMR). This work shows that carboxylates are viable catalytic ligands for homoallyl- and homocrotylations of carbonyl compounds and opens the door to the development of catalytic asymmetric versions of this transformation.