Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues.

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

In the cell, most tasks are performed by big molecules called proteins, which behave like molecular machines. Although proteins are often described as having one job each, this is not always true, and many proteins can perform different roles. Enzymes are a type of protein that facilitate chemical reactions. They are often specialised to one reaction, but they can also accelerate other side-reactions. During evolution, these side-reactions can become more useful and, as a result, the role of the enzyme may change over time. The main role of the enzyme called NfsA in Escherichia coli bacteria is thought to be to convert molecules called quinones into hydroquinones, which can protect the cell from toxic molecules produced in oxidation reactions. As a side-reaction, NfsA has the potential to protect bacteria from an antibiotic called chloramphenicol, but it generally does this with such low efficacy that the effects are negligible. Producing hydroquinones is helpful to the cell in some situations, but if bacteria are regularly exposed to chloramphenicol, NfsA's role aiding antibiotic resistance could become more important. Over time, the enzyme could evolve to become better at neutralising chloramphenicol. Therefore, NfsA provides an opportunity to study the evolution of proteins and how bacteria adapt to antibiotics. To see how evolution might affect the activity of NfsA, Hall et al. generated 250 million E. coli with either random or targeted changes to the gene that codes for the NfsA enzyme. The resulting variants of NfsA that were most effective against chloramphenicol all had a change that eliminated the enzyme's ability to convert quinones. This result demonstrates a key trade-off between roles for NfsA, where one must be lost for the other to improve. These results demonstrate the interplay between a protein's different roles and provide insight into bacterial drug resistance. Additionally, the experiments showed that the bacteria with improved resistance to chloramphenicol also became more sensitive to another antibiotic, metronidazole. These findings could inform the fight against drug-resistant bacterial infections and may also be helpful in guiding the design of proteins with different roles.

Comparison of disaccharide donors for heparan sulfate synthesis: uronic acids vs. their pyranose equivalents.

Late oxidation of hexose based building blocks or the use of uronic acid containing building blocks are two complementary strategies in the synthesis of glycosaminoglycans, the latter simplifiying the later stages of the process. Here we report the synthesis and evaluation of various disaccharide donors-uronic acids and their pyranose equivalents-for the synthesis of heparan sulfate, using an established protective group strategy. Hexose based "imidate" type donors perform well in the studied glycosylations, while their corresponding uronate esters fall short; a uronate ester thioglycoside performs equal to, if not better than, a hexose thioglycoside equivalent.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides.

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a "Shotgun" Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.

Using automated glycan assembly (AGA) for the practical synthesis of heparan sulfate oligosaccharide precursors.

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography.

Composition, sequencing and ion mobility mass spectrometry of heparan sulfate-like octasaccharide isomers differing in glucuronic and iduronic acid content.

Here we report ion mobility mass spectrometry (IMMS) separation and tandem mass spectrometry (MS(2)) sequencing methods used to analyze and differentiate six synthetically produced heparin/heparan sulfate (HS)-like octasaccharide (dp8) isomeric structures. These structures are isomeric with regard to either glucuronic acid (GlcA) or iduronic acid (IdoA) residues at various positions. IMMS analysis showed that a fully GlcA structure exhibited a more compact conformation, whereas the fully IdoA structure was more extended. Interestingly, the change from IdoA to GlcA in specific locations resulted in strong conformational distortions. MS(2) of the six isomers showed very different spectra with unique sets of diagnostic product ions. Analysis of MS(2) product ion spectra suggests that the GlcA group correlated with the formation of a glycosidic product ion under lower energy conditions. This resulted in an earlier product ion formation and more intense product ions. Importantly, this knowledge enabled a complete sequencing of the positions of GlcA and IdoA in each of the four positions located in each unique dp8 structure.

Crystal packing in three related disaccharides: precursors to heparan sulfate oligosaccharides.

The three title compounds form part of a set of important precursor dissacharides which lead to novel therapeutics, in particular for Alzheimer's disease. All three crystallize as poorly diffracting crystals with one independent mol-ecule in the asymmetric unit. Two of them are isostructural: 4-meth-oxy-phenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-de-oxy-4-O-(9-fluor-en-yl-methyl-oxycarbon-yl)-α-d-gluco-pyranos-yl]-2-O-benzoyl-3-O-benzyl-6-O-chloro-acetyl-α-l-ido-pyran-oside, C59H56ClN3O16, (I), the ido-relative of a reported gluco-disaccharide [Gainsford et al., 2013 ▶). Acta Cryst. C69, 679-682] and 4-meth-oxy-phenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-de-oxy-4-O-(9-fluorenyl-methyl-oxycarbon-yl)-α-d-gluco-pyranos-yl]-2-O-benzoyl-3-O-benzyl-6-O-meth-oxy-acetyl-α-l-ido-pyran-oside, C60H59N3O17, (II). Both exhibit similar conformational disorder of pendant groups. The third compound 4-meth-oxy-phenyl 4-O-[6-O-acetyl-2-azido-3,4-di-O-benzyl-2-de-oxy-α-d-gluco-pyranos-yl]-2-O-benzoyl-3-O-benzyl-6-O-meth-oxy-oacetyl-ß-d-gluco-pyran-oside, C52H55N3O15, (III), illustrates that a slightly larger set of weak inter-molecular inter-actions can result in a less disordered mol-ecular arrangement. The mol-ecules are bound by weak C-H⋯O(ether) hydrogen bonds in (I) and (II), augmented by C-H⋯π inter-actions in (III). The absolute configurations were determined, although at varying levels of significance from the limited observed data.

Lipo-chitin oligosaccharides, plant symbiosis signalling molecules that modulate mammalian angiogenesis in vitro.

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.

Translationally related nearly identical molecules: 4-methoxyphenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(fluoren-9-ylmethoxycarbonyl)-α-D-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-chloroacetyl-ß-D-glucopyranoside.

The title compound, C59H56ClN3O16, is an important dissacharide precursor to novel therapeutics for the treatment of Alzheimer's disease. It crystallizes with two independent enantiomerically identical molecules in almost exactly the same orientation in the cell, being one half cell-edge apart. Apart from the conformation of a single benzyl group, the molecules are superimposable. They are bound efficiently using complementary C-H···O(carbonyl) hydrogen bonds, principally along the `duplicating' axis. The absolute configurations were determined.

Synthesis of a targeted library of heparan sulfate hexa- to dodecasaccharides as inhibitors of ß-secretase: potential therapeutics for Alzheimer's disease.

Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure-activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the ß-site cleaving enzyme ß-secretase (BACE1), a protease responsible for generating the toxic Aß peptides that accumulate in Alzheimer's disease (AD), with 6-O-sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa- to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure-activity relationships for optimal BACE1 inhibition, and yielded a number of potent non-anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aß peptide levels.

2-(Acet-oxy-meth-yl)benzoic acid.

The title compound, C(10)H(10)O(4), crystallizes with the well-known carb-oxy-lic acid dimer-forming R(2) (2)(8) hydrogen-bond motif. Chains approximately parallel to (-1-12) are then built through C(methyl-ene,phen-yl)-H⋯O(carbon-yl) inter-actions [C(6) and C(8) motifs] with one (meth-yl)C-H⋯π inter-action providing inter-planar binding. The weakness of the latter inter-action is consistent with the difficulty experienced in obtaining suitable single crystals.

Asymmetric synthesis and affinity of potent sialyltransferase inhibitors based on transition-state analogues.

Inhibitors that are structurally related to the transition-state model of the proposed SN1-type mechanism of sialyl transfer, exhibit particularly high binding affinities to alpha(2-6)sialyltransferases. Furthermore, replacing the neuraminyl residue with a simple aryl or hetaryl ring and substituting the carboxylate group for a phosphonate moiety, improves both binding affinity and synthetic accessibility. Herein we report on the synthesis and inhibition of a wide range of novel, potent transition-state analogue based alpha(2-6)sialyltransferase inhibitors comprising a planar anomeric carbon, an increased distance between the anomeric carbon and the CMP leaving group, and at least two negative charges. We also present a short, efficient asymmetric synthesis of the most promising benzyl inhibitors, providing rapid access to large quantities of highly potent, stereochemically-pure (>96% de) inhibitors for further biological investigation (e.g.(R)-3b, Ki = 70 nM).

Stereoselective synthesis of phosphoramidate alpha(2-6)sialyltransferase transition-state analogue inhibitors.

The asymmetric synthesis of novel, potent phosphoramidate alpha(2-6)sialyltransferase transition-state analogue inhibitors such as (R)-9 (K(i)=68 microM) is described, via condensation of cytidine phosphitamide 6 with key chiral, non-racemic alpha-aminophosphonates, prepared in >98% ee by Mitsunobu azidation followed by Staudinger reduction of the corresponding chiral, non-racemic alpha-hydroxyphosphonates.

Efficient sialyltransferase inhibitors based on glycosides of N-acetylglucosamine.

D-Glucosamine was transformed into phenyl and 2-benzoyloxyethyl N-acetylglucosamine beta-glycosides 6a and 6b, respectively. Transformation of 6a,b into 6-O-unprotected N-acetylglucosamine derivatives 9a,b permitted the generation of an aldehyde group in the 6-position. Treatment of these intermediates with base afforded unsaturated aldehyde derivatives 10a,b, which are structural mimics of 2,3-dehydroneuraminic acid. H-Phosphonate addition to the aldehyde group and attachment of the cytidine monophosphate residue to the generated hydroxy group gave fully protected transition state analogues of cytidine monophosphate-N-acetylneuraminic acid 14a,b. Liberation of the unprotected compounds 1ah,l and 1bh,l led to excellent inhibitors of alpha(2-6)-sialyltransferase from rat liver. Variation of the protective group cleavage procedure for 14a,b led to formal loss of phosphate, thus resulting in diene derivatives (E)-/(Z)-2a,b, which also exhibited inhibitory properties.