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 , with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1-C11 bond.

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 , 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.

Large-Scale Synthesis of ManGlcNAc 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 ManGlcNAc including improved building block synthesis and a fully stereoselective 5 + 6 coupling, yielding 290 mg of glycan.

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.

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.

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.

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

Oligomannose glycans are of interest as HIV vaccine components, but they are subject to mannosidase degradation . 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.

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 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 Man resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1→2Man tips of Man glycans. A possible reason could be processing of our immunogen by host serum mannosidases.

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 -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.

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 selection, that bind tightly to anti-HMP antibody 2G12.

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.

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 K values of 2.

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.

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.

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.

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.

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.

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).

Directed evolution of multivalent glycopeptides tightly recognized by HIV antibody 2G12.

Herein, we report a method for in vitro selection of multivalent glycopeptides, combining mRNA display with incorporation of unnatural amino acids and "click" chemistry. We have demonstrated the use of this method to design potential glycopeptide vaccines against HIV. From libraries of ~10(13) glycopeptides containing multiple Man9 glycan(s), we selected variants that bind to HIV broadly neutralizing antibody 2G12 with picomolar to low nanomolar affinity.

High temperature SELMA: evolution of DNA-supported oligomannose clusters which are tightly recognized by HIV bnAb 2G12.

SELMA (SELection with Modified Aptamers) is a directed evolution method which can be used to develop DNA-supported clusters of carbohydrates in which the geometry of clustering is optimized for strong recognition by a lectin of interest. Herein, we report a modification of SELMA which results in glycoclusters which achieve dramatically stronger target recognition (100-fold) with dramatically fewer glycans (2-3-fold). Our first applications of SELMA yielded clusters of 5-10 oligomannose glycans which were recognized by broadly neutralizing HIV antibody 2G12 with moderate affinities (150-500 nM Kd's).

Directed evolution of 2G12-targeted nonamannose glycoclusters by SELMA.

Clusters of Man glycans which are recognized by broadly-neutralizing anti-HIV antibody 2G12 have potential as HIV vaccines. However, optimal recognition by 2G12 requires optimal clustering of glycans. Using our recently described SELMA technique (SELection with Modified Aptamers), we have developed Man clusters in which glycans are supported by DNA sequences selected from among 2 × 10 variants.

Enantioselective homocrotylboration of aliphatic aldehydes.

A practical route to optically pure syn-homocrotylation reagents is described, including highly diastereo- and enantioselective preparation of numerous syn-homocrotyl products, as well as several matched mismatched pairs. NMR experiments suggest that the active homocrotylating species is a cyclopropylcarbinyldichloroborane generated by chloride exchange from the PhBCl(2) activator. Computational studies support the intermediacy of chloroboranes and suggest that homoallyl/homocrotyl transfers occur through Zimmerman-Traxler transition states.