Next Article in Journal
The Impact of Production Technology on the Quality of Potato Spirit
Previous Article in Journal
Unlocking the Therapeutic Potential of Ellagitannins: A Comprehensive Review of Key Representatives
Previous Article in Special Issue
Synthesis and Docking Studies of Glycolipids Inspired by Bacteroides fragilis Lipid A
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 30
Issue 22
10.3390/molecules30224329
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Exploring the Scope of Functionalized N-Acylneuraminic Acid β-Methyl Glycosides as Inhibitors of Neisseria meningitidis CMP-Sialic Acid Synthetase

by
Pradeep Chopra
1,
Jana Führing
2,
Preston Ng
1,
Thomas Haselhorst
1,
Jeffrey C. Dyason
1,
Faith J. Rose
1,
Robin J. Thomson
1,
Rita Gerardy-Schahn
2,
I. Darren Grice
1,
Michael P. Jennings
1,*,
Anja K. Münster-Kühnel
2,* and
Mark von Itzstein
1,*
1
Institute for Biomedicine and Glycomics, Griffith University, Gold Coast, QLD 4222, Australia
2
Institut für Klinische Biochemie, Medizinische Hochschule, Carl-Neuberg-Straße 1, 30625 Hannover, Germany
*
Authors to whom correspondence should be addressed.
Molecules 2025, 30(22), 4329; https://doi.org/10.3390/molecules30224329
Submission received: 1 July 2025 / Revised: 16 October 2025 / Accepted: 16 October 2025 / Published: 7 November 2025
(This article belongs to the Special Issue Contemporary Synthetic Glycoscience: A Theme Issue Dedicated to the Memory of Hans Paulsen)
Browse Figures
Versions Notes

Abstract

Cell surface sialylation is utilized by a number of pathogenic bacteria to evade the host immune system through molecular mimicry of host sialoglycoconjugates. Human pathogen Neisseria meningitidis serotype B (NmB) expresses both sialylated capsule and surface lipooligosaccharides as pivotal virulence factors. An essential enzyme in the sialylation pathway of NmB is CMP-sialic acid synthetase (CSS), which produces the activated nucleotide sugar necessary for sialic acid transfer. In this work, novel C-4, -5, -7, and -9 functionalized derivatives of neuraminic acid β-methyl glycoside (Neuβ2Me) were synthesized as candidate CSS inhibitors. A number of these were found to reduce the activity of NmB CSS in vitro. The highest inhibition of NmB CSS, in a mixed mode manner, was observed with a Neu5Acβ2Me C-9 serine carboxamide. Direct interaction with the enzyme was confirmed by Saturation Transfer Difference (STD) NMR. Supplementation of growth media with this compound reduced lipooligosaccharide (LOS) sialylation of living N. meningitidis, thus providing an interesting starting point for the development of specific NmB CSS inhibitors as an alternative treatment strategy to fight bacterial infections.

Graphical Abstract

1. Introduction

Neisseria meningitidis serogroup B (NmB) is an obligate human pathogen and a leading cause of meningitis and septicemia in developed countries [1]. Vaccines are currently available for protection against N. meningitidis serogroups A, B, C, W, and Y [2,3]. The Men A, C, W, and Y vaccines are based on bacterial capsular polysaccharide antigens [2,3,4]. In contrast, the MenB vaccine uses subcapsular membrane proteins and outer membrane vesicle proteins [2,5] due to the similarity of its capsular polysaccharides to human neuronal glycans [6,7,8]. NmB expresses sialic acid (Sia) on its surface, both in its capsule, which is composed of an α2,8-linked homopolymer of Sia (polySia), and as the terminal sugar on lipooligosaccharides (LOS) [9,10]. Sialic acids are 9-carbon acidic sugars [11], incorporated into glycoconjugates as α-linked sialosides, which are negatively charged under physiological conditions. The highly charged polySia capsule of serogroup B meningococci, as well as LOS terminated by Sia, are pivotal virulence factors [12]. Sia not only mediates interactions with the host [13] but also promotes survival and dissemination of this pathogen. Sialylated LOS has been shown to be involved in immune evasion of NmB, resistance against phagocytosis by human dendritic cells [14,15], and hindrance of complement attack by interfering with the alternative pathway of complement activation [16,17,18]. Genetic interruption of the NmB sialylation pathway abolishes capsule formation as well as LOS sialylation, and results in rapid bacterial killing by human serum [19]. Interference with NmB cell surface sialylation may therefore provide an attractive antimicrobial strategy.
A prerequisite for incorporation of Sia into sialylated glycans in both pro- and eukaryotes is the enzymatic conversion of free Sia to the activated nucleotide sugar CMP-Sia. Exclusively CMP-Sia is used by sialyltransferases for the transfer of Sia onto nascent sialoglycoconjugates. The sialic acid present in NmB polysaccharides is N-acetylneuraminic acid, Neu5Ac 1. Activation of Neu5Ac to CMP-Neu5Ac 2 through reaction with cytidine 5′-triphosphate (CTP) [20,21] is catalyzed by CMP-sialic acid synthetase (N-acylneuraminate cytidylyltransferase, CSS, EC 2.7.7.43) (Figure 1A) [20,21,22,23,24,25]. Interference with CSS activity in mammalian cells abolishes cell surface sialylation [26], suggesting the enzyme as a potential target for manipulation of cell surface sialylation in NmB.
Tolerance of NmB CSS to structural variation on the substrate Neu5Ac at carbon atoms C-5 [29,30,31,32], C-7 [33], C-8 [34], and C-9 [30,31,35], and indeed to replacement of the glycerol side chain [36], has been reported. This substrate tolerance has been exploited for the generation of modified sialoglycans [29,37], including in multi-enzyme and chemoenzymatic syntheses [30,31,32,33,36,38,39]. In contrast, comparatively little research has to date been reported on the inhibition of CSS. Only a relatively small number of studies have examined the inhibitory effect of modified sialic acids [27,28,40,41] or nucleotide isosteres [42] on CSS activity. Removal of the C-2 hydroxyl group of Neu5Ac (the proposed nucleophile in coupling Neu5Ac 1 to CTP [43,44]) was found to abolish recognition by mammalian CSS [27]. Retaining the C-2 oxygen but blocking it with a methyl group—giving the methyl glycoside in the β-configuration to mimic the β-configuration of CMP-Neu5Ac 2—however, led to inhibition of mammalian CSS, albeit at millimolar concentration [Neu5Acβ2Me (3): Ki = 2.5 mM [27]; 15 mM [28]. As part of our continuing work in sialic acid and CSS biology [13,18,24,45,46,47,48], we sought to explore the potential for inhibition of CSS from pathogenic NmB using novel Neu5Ac derivatives. As the inhibitor template, we chose the above-mentioned β-methyl glycoside of Neu5Ac (Neu5Acβ2Me 3, Figure 1B), based on its known inhibitory effect on CSS activity [27,28].

2. Results and Discussion

2.1. Structure Analysis of NmB CSS

To decide on a rational basis which positions of Neu5Acβ2Me (3) would be most promising for modification, to potentially increase binding affinity and inhibitory effect, we began our study with an analysis of the available X-ray structural data for CSS. At the beginning of our studies, the X-ray crystal structure of NmB CSS in complex with CDP as a substrate mimetic (PDB: 1EYR) was available [49]. This structure showed the enzyme as an asymmetric homodimer, with the active site present at the interface of the core domain of one subunit and the dimerization domain of the other subunit [49]. In 2020, X-ray crystal structures of NmB CSS in complex with substrate CTP, and with the product of reaction CMP-Neu5Ac 2 (PDB: 6CKM; Figure 2) were reported [44], providing further insights into the catalytic cycle of the enzyme. From kinetic studies with active-site mutants and the X-ray crystallographic studies, it is apparent that the CSS active site switches between an open form for substrate binding (and product release), and a catalytically active ‘closed’ form [43,44,49].
It is clear that in the open form of the enzyme, there would be significant space around bound Neu5Ac. Substitutions on the Neu5Ac template, however, would no doubt have implications for the enzyme being able to close to give the correct positioning of the sialic acid and CTP for catalytic activity. The apparent extensive flexibility of the protein as it performs its function may limit a traditional molecular docking study. Instead, more complex techniques would need to be employed that would take into account protein flexibility, e.g., molecular dynamics simulations [51,52]. Our approach was therefore to systematically explore changes in spatial and functional group characteristics on the Neu5Ac template, based on analysis of contacts around Neu5Ac in the available models and crystal structures.
In our original modelling study, and subsequently in the NmB CSS–CMP-Neu5Ac (2) complex [44] (Figure 2), the glycerol side-chain (C-7 to C-9 hydroxyl groups) of the Neu5Ac moiety is seen to project into a spacious channel or cleft. The potential binding environment of the Neu5Ac glycerol side chain contains a number of charged amino acid residues, including positively charged residues on one side of the channel. This suggested that the introduction of a negatively charged substituent at the terminus of the glycerol side chain was of particular interest. The scope for this modification could be explored through the synthesis of a C-9 carboxylic acid derivative that could then be further elaborated with various simple neutral amines and with amino acids to generate a series of C-9 carboxamide derivatives of Neu5Acβ2Me (Figure 3, Series I).
Activation of 7-deoxy-Neu5Ac by Nm CSS [33] indicates that the C-7 hydroxyl group is not essential for substrate binding. Inspection of Nm CSS crystal structures revealed that a phenylalanine residue is in proximity to the Neu5Ac C-7 hydroxyl group, suggesting that an opportunity for possible π-π interaction exists in this region. With this in mind, we chose to synthesize a number of 7-O-aryl (e.g., benzyl) and unsaturated-alkyl (e.g., allyl) ethers, as well as the ethyl and propyl ethers, to explore this possible binding domain (Figure 3, Series II).
Apparent spatial constraints around C-4 and C-5 of Neu5Ac in Nm CSS structures seem to imply there is little potential for further functionalization at these positions. However, there are extensive protein dynamics involved in the function of CSS [44,45,53], and reports of successful activation of various N-acylated neuraminic acids by NmB CSS [29,30,31,32], and a number of C-4 modified Neu5Ac derivatives (including 4-acetamido-4-deoxy-Neu5Ac [54]) by mammalian CSS. Together, these indicate that some structural variation at positions C-5 and C-4 can be tolerated. We therefore chose to introduce separately at each position, amides with varying functionality, ranging from small alkyl chains to large hydrophobic groups, in order to investigate structure–activity relationships around these positions (Figure 3, Series III and IV). In summary, four series of novel functionalized Neu5Ac derivatives, as the β-methyl glycosides (Figure 3), were proposed to probe interactions with Nm CSS, introducing carboxamides at C-9, ethers at C-7, and amides at C-5 and C-4.

2.2. Synthesis of Functionalized Neu5Acβ2Me Derivatives

2.2.1. Synthesis of C-9 Functionalized Neu5Acβ2Me Derivatives (Series I)

The starting point for the current studies was the synthesis of the β-methyl glycoside of N-acetylneuraminic acid methyl ester [(Neu5Ac1,β2Me2, 4) from Neu5Ac 1 (Scheme 1). Methyl esterification of 1 and concomitant formation of the β-methyl glycoside can be achieved by heating 1 under reflux [55], or under microwave irradiation [56], with dry acidic ion exchange resin in anhydrous methanol. We have found [56] that while both approaches give comparable yields, microwave irradiation provides a more rapid reaction, and is more economical in terms of solvent and resin requirement than reaction under conventional heating. Saponification of 4 provided the benchmark β-methyl glycoside derivative, Neu5Acβ2Me, 3 [27,57,58].
To pursue modification at the C-9 position, we took advantage of the efficient synthesis of the C-9 carboxylic acid derivative of Neu5Ac2βMe [59], which could then be elaborated to generate a series of C-9 carboxamides. Regioselective TEMPO-mediated oxidation of the primary C-9 hydroxyl group of Neu5Ac2βMe methyl ester 4 was followed by peracetylation to assist in isolation and purification (Scheme 1). This gave the key intermediate 9-carboxy derivative 5 [59] in 66% yield over two steps. Compound 5 ultimately provides an interesting probe of CSS in its own right (see 8), while the C-9 carboxy group of 5 provided us with the opportunity to further modify this position through coupling with amines of varying functionality. Elaboration of the carboxylic acid was carried out by reaction with neutral amines, as well as carboxy-protected amino acids. The latter would, upon deprotection, provide a negative charge at the end of the glycerol side-chain that may engage binding with positively charged protein residues.
The amide coupling was achieved using a slight modification of an established method for coupling the C-1 carboxyl group of Neu5Ac to amino acid esters [60,61], with the addition of 1-hydroxybenztriazole (HOBt) to minimize racemization and side reactions. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), which gives a water-soluble urea byproduct that can be easily removed by aqueous work-up, was used in place of (benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) because of the potential hazard of HMPA formation associated with coupling using the latter reagent [62]. 9-Carboxy derivative 5 was coupled with a series of aliphatic amines, and with a range of L-amino acid methyl esters (with the exception of D-/L-serine methyl ester), to give carboxamides 6a–d and 7a–h, respectively, in moderate yields (Scheme 1). De-O-acetylation followed by saponification yielded the 1,9-dicarboxylic acid derivative 8 [59], and carboxamides 9a–d and 10a–h. For the serine amino acid carboxamide 10d, prepared from racemic D-/L-serine methyl ester, two isomers were separable by HPLC (denoted 10d-1 and 10d-2); however, the relative stereochemistries at the serine a-carbon were not determined.

2.2.2. Synthesis of C-7 Functionalized Neu5Acβ2Me Derivatives (Series II)

For the synthesis of the target Neu5Acβ2Me C-7 ethers (Series II), we utilized a route analogous to that reported by Masuda and coworkers [63,64,65] (Scheme 2). To isolate the relatively unreactive C-7 hydroxyl group, Neu5Ac1,β2Me2 4 was reacted with 2,2-dimethoxypropane under acid catalysis to give the 8,9-O-isopropylidenated derivative 11 [66] in 90% yield. Regioselective monosilylation of the C-4 hydroxyl group then afforded 4-O-t-butyldimethylsilyl-8,9-O-isopropylidene derivative 12 [67]. The C-7 hydroxyl group of 12 was successfully alkylated using ethyl iodide (13a), allyl bromide (13b), propargyl bromide (13d), and benzyl bromide (13e) in the presence of NaH in DMF, to afford the corresponding alkyl ethers in moderate yield (36–60%). Use of ethyl iodide gave a comparable yield of 13a to that reported when using diethylsulfate as the alkylating agent [64]. Higher equivalents of base were used to overcome the low reactivity of the C-7 hydroxyl group, which resulted in partial to complete transesterification in each reaction. Advantage was taken of the 7-O-allyl derivative 13b, to prepare the 7-O-propyl derivative 13c [63,64] by hydrogenation. Finally, simultaneous deprotection of both acid-labile groups (isopropylidene and TBDMS) of compounds 13a–e by heating with 80% acetic acid at 80 °C, followed by saponification, gave the target 7-O-alkylated Neu5Acβ2Me derivatives 14a–e.
The C-7 propyne ether derivative 13d provided us with the opportunity to utilize Sonogashira coupling to further diversify the C-7 substituent. In order to achieve coupling selectively at the C-7 propyne ether, a sequence of deprotection and re-protection was employed on 13d to remove the propyne ester functionality, and subsequently produce the acetylated methyl ester derivative 15 (Scheme 2). Following the method of Sato and coworkers [68], compound 15 was treated with aryl iodides in the presence of PdCl2(PPh3)2, CuI, and Et3N in anhydrous CH3CN to yield the disubstituted alkyne derivatives 16a–c in good yield (60–87%). Upon saponification, these afforded the target compounds 17a–c.

2.2.3. Synthesis of C-5 Functionalized Neuβ2Me Derivatives (Series III)

For the synthesis of compounds of Series III, which have alternative acyl groups on the amine at C-5, basic hydrolysis of the C-5 acetamido group of 4 was utilized to obtain key 5-amino derivative 18 (Scheme 3). Instead of using conventional heating, which requires long reaction times [69,70], in this work, we took advantage of reaction under microwave irradiation to provide a method for rapid de-N-acetylation of 4 [71]. Under microwave irradiation (max. 100 W) at 120 °C, NaOH-promoted de-N-acetylation of 4 was complete in only 15 min, with the 5-amino derivative 18 obtained cleanly in 80% yield. The carboxylate group of 18 was then re-esterified under acidic conditions to give methyl ester 19. N-Acylation of the C-5 amino group of 19 was performed with acid chlorides of varying functionality using triethylamine as base in water/dioxane (1:5) to provide the desired C-5 amide derivatives 20a–i in high yields (78–91%). In contrast to an earlier reported method [72], no bis-acylation was observed. Finally, saponification afforded the target variously N-acylated Neuβ2Me derivatives 21a–i.

2.2.4. Synthesis of C-4 Functionalized Neu5Acβ2Me Derivatives (Series IV)

The synthesis of the C-4 amide derivatives of Neu5Acβ2Me (Series IV) required the introduction of an amino group at C-4 in the equatorial configuration. Nitrogen functionality can be introduced at C-4 on a suitably protected Neu5Ac template via a 3-step sequence of oxidation of the equatorially substituted C-4 hydroxyl group, reduction in the 4-oxo derivative to the 4-epi-OH derivative, and finally introduction of azide in the equatorial configuration via a Mitsunobu reaction using HN3.[54] An alternative approach is to proceed via the 2,3-unsaturated sialic acid, Neu5Ac2en, scaffold, where the 4-azido derivative 22 [73,74,75] can be produced in good yield over four steps from Neu5Ac 1 [75]. Having 22 at hand, we chose the second approach (Scheme 4). Bromo-methoxylation [76] of the 2,3 double bond of 4-azido-4-deoxy-Neu5Ac2en derivative 22 gives rise to an approximately 1:1 isomeric mixture of 2,3-diaxial (23) and 2,3-diequatorial (24) products [77]. Treatment of the 2,3-di-axial bromomethoxylated isomer (the β-methyl glycoside) 23 with tributyltin hydride in the presence of AIBN [77] resulted in both debromination at C-3 and reduction in the C-4 azide, to give 4-amino Neu5Acβ2Me derivative 25. Treatment of 25 with a range of acid chlorides afforded the C-4 amide derivatives 26a–i, which, after global deprotection, gave the target 4-acylamino-4-deoxy-Neu5Acβ2Me derivatives 27a–i.

2.3. Screening of Functionalized Neu5Acβ2Me Derivatives for Inhibition of NmB CSS

To analyze the effect of the compounds on NmB CSS activity in vitro, we recombinantly expressed and purified the enzyme to homogeneity. The EnzChek™ pyrophosphate assay kit (Life Technologies, Darmstadt, Germany) was used to determine NmB CSS activity in the forward reaction (i.e., formation of CMP-Neu5Ac and PPi), revealing Km values of 22 and 37 µM for CTP and Neu5Ac, respectively. After confirming that the test compounds did not show any inhibitory effect on the assay components, we screened all functionalized Neu5acylβ2Me derivatives and the parent compound 3 at a concentration of 100 μM (Figure 4). Interestingly, the benchmark parent compound, unsubstituted β-methyl glycoside 3, previously reported [27] to be a modest inhibitor of mammalian CSS (Ki = 2.5 mM), showed no significant inhibitory effect on NmB CSS in this in vitro assay (Figure 4). This difference might be attributed to differences in the employed CSS species and/or in assay conditions: while Zbiral and coworkers [27] utilized the Warren assay [78] under CTP-limiting conditions, we provided both substrates in saturating concentrations of 1 mM each. Three of the C-9 modified Neu5Acβ2Me derivatives—parent 9-carboxy-Neu5Acβ2Me 8 [59], benzamide 9c, and serine carboxamide 10d-1—and one of the C-7 ethers—7-O-benzyl-Neu5Acβ2Me 14e, however, showed inhibition of NmB CSS activity by more than 20% in this screen. Detailed kinetic analysis of these four compounds suggests a mixed-mode inhibition that most closely resembles the non-competitive mode, as indicated by α-values close to 1 and supported by the appearance of Lineweaver-Burk plots (Supplementary Figure S1). As all compounds were designed to target the active site and compete with the natural substrate Neu5Ac 1 for binding, the finding that none of the four characterized inhibitors display competitive kinetics may be somewhat unexpected, but does not necessarily indicate binding outside of the catalytic center. Indeed, multiple mechanisms exist that may account for non-competitive or “mixed-mode mimicking” inhibition patterns of inhibitors that in fact bind to the active site [79,80]. One of these mechanisms applies to multisubstrate/-product enzymes that follow a sequential ordered mechanism, which is the case for CSS, where CTP binds prior to Neu5Ac [44]. With inhibitory constants (Ki) in the range between 100 and 500 μM, these compounds appear to be stronger inhibitors than the previously reported sulfone-based nucleotide isosteres, which inhibited NmB CSS activity up to 55% at a concentration of 1 mM [42].

2.4. Interaction of Neu5Acβ2Me C-9 Serine Carboxamide 10d-1 with NmB CSS as Determined by STD NMR

To confirm direct binding and to investigate the interactions of the most promising C9 carboxamide derivative 10d-1 with CSS, we used Saturation Transfer Difference (STD) NMR spectroscopy [46,81,82]. This powerful tool allows evaluation of ligand–biomolecule interactions by mapping the binding epitope of the ligand, essentially through screening the protons of the ligand for their proximity to the protein upon binding.
The binding epitope of 10d-1 in complex with NmB CSS (Figure 5iii) was determined from the relative strength of transferred saturation (with the effect at the C-5 NHAc methyl group set to 100%). It is evident from the STD NMR spectrum (Figure 5ii) that the carbohydrate ring of 10d-1 is in close contact with the enzyme. Relatively strong STD NMR signals were observed for the equatorial proton at C-3 (H-3eq 217%), with more moderate signal intensities observed for the axial C-3 proton (H-3ax 165%) and the glycoside methyl group (126%). Interaction of the serine ‘tail’ could not be unequivocally determined due to the signal overlap, for example, of H-2′ with H-5; however, any interaction would appear to be only weak.

2.5. Neu5Acβ2Me C-9 Serine Carboxamide 10d-1 as a Modulator of NmB LOS Sialylation

To determine whether Neu5Acβ2Me C-9 serine carboxamide 10d-1 was capable of inhibiting CSS (encoded by the gene neuA) in living bacteria, we established an ELISA-based assay utilizing a sialic acid-recognizing lectin to detect LOS sialylation on the cell surface of NmB. The capsule-deficient N. meningitidis strain ¢3 (¢3 WT) [13] expressed sialylated LOS at high levels (defined as 100%, Figure 6). To define the background signal in the absence of neuA activity, we generated a corresponding neuA knockout strain (¢3neuA::kan) and observed a reduction in LOS sialylation to about 2%. Complementation of the neuA knockout mutant by reintroducing the neuA gene (¢3neuA::kan, NeuA+) confirmed that loss of signal in the assay was due to inactivity of CSS and thus, loss of sialylation in this mutant (Supplementary Figure S2). To evaluate whether the results of this assay can be interpreted in a quantitative manner, we mixed ¢3 wild type strain and ¢3neuA::kan knockout mutant in a 1:1 ratio. This resulted in a ~44% ± 6% reduction in signal intensity, indicating a corresponding reduction in LOS sialylation of wild-type Nm, which was close to ~50%. Given that not all ¢3 wild-type LOS molecules are sialylated, a value of under 50% is reasonable (Supplementary Figure S2). We then tested the effect of C-9 serine carboxamide 10d-1 on LOS sialylation by supplementing the growth medium with 1 mM of the compound. The ELISA results indicated that bacterial growth in the presence of 10d-1 resulted in a reduction in NmB LOS sialylation to ~66% of ¢3 wild wild-type level (Figure 6). This suggests that inhibitor 10d-1 is able to cross the cell membranes and act on CSS, leading to a decrease in cell surface sialylation of whole cells.

3. Materials and Methods

3.1. Modelling of Neu5Ac 3 in the Active Site of NmB CSS

The PyMOL Molecular Graphics System (Version 1.7.x Schrödinger, LLC, New York, NY, USA) was used for modelling.

3.2. Chemical Synthesis—Materials and Methods

N-Acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid) (1) was obtained from Jülich Chiral Solutions GmbH (Jülich, Germany) and Carbosynth Ltd. (Compton, UK). Reagents and dry solvents purchased from commercial sources were used without further purification. Anhydrous reactions were carried out under an atmosphere of nitrogen or argon, using oven-dried glassware. Microwave reactions were conducted using a CEM Discover® SP Explorer Hybrid-12 (CEM Corporation, Matthews, NC, USA) microwave system with a single-mode cavity, and were carried out in a 10 mL pressure tube, sealed with a Teflon septum.
Reactions were monitored using thin-layer chromatography (TLC) on aluminum plates precoated with Silica Gel 60 F254 (E. Merck, Darmstadt, Germany). Developed plates were observed under UV light at 254 nm and then visualized after application of a solution of H2SO4 in EtOH (5% v/v) or ninhydrin in EtOH (0.2% v/v), as appropriate, and heating. Flash chromatography was performed on silica gel 60 (0.040–0.063 mm) or using a Reveleris® flash chromatography system (Grace Davison, Columbia, MD, USA) (as indicated) using distilled solvents.
1H and 13C NMR spectra were recorded either at 600 or 300 MHz and 150 or 75.5 MHz, respectively, on a Bruker Avance 600 or 300 MHz spectrometer (Bruker, Rheinstetten, Germany; as indicated). Low-resolution mass spectra (LRMS) were recorded, in electrospray ionization mode, on a Bruker Daltonics Esquire 3000 ESI spectrometer (Bruker, Bremen, Germany), using positive or negative mode (as indicated). High-resolution mass spectrometry (HRMS) was carried out by the Griffith University FTMS Facility on a Bruker Daltonics Apex III 4.7e Fourier Transform MS, fitted with an Apollo ESI source, or by the University of Queensland MS Facility on a Bruker MicrOTOF-Q with a Bruker ESI source (Bruker, Bremen, Germany).
HPLC purification was performed on an Agilent HP1100 instrument (Agilent, Santa Clara, CA, USA) using a Phenomenex Aqua 5 μ C18 124 Å column (250 × 10 mm) (Phenomenex, Torrance, CA, USA) at a flow rate of 3 mL/min and column temperature of 40 °C using isocratic elution with solvents as indicated. The purities of all synthetic intermediates after chromatographic purification were judged to be >90% by 1H and 13C NMR. The purity of tested compounds was ≥95% by HPLC analysis or by 1H and 13C NMR.
Detailed synthetic methods for all test compounds and intermediates [83] are provided in the Supporting Information—Part 1. NMR spectra (1H and 13C) of all test compounds and intermediates [83] are provided in the Supporting Information—Part 2.

3.3. Protein Expression and Purification (For Screening Assay and STD NMR)

A modified pET22b-Strep vector (IBA Lifesciences, Göttingen, Germany) allowing the prokaryotic expression of NmB CSS (accession No. M95053) was kindly provided by PD Dr. Martina Mühlenhoff, MHH, Germany. Recombinant NmB CSS with an N-terminal StrepII-tag was expressed in E. coli BL21(DE3) (Novagen, registered trademark of Merck, Darmstadt, Germany) at 15 °C and purified by affinity chromatography utilizing the StrepII-Tag. Peak fractions were desalted (HiPrep 26/10, GE Healthcare/now Cytiva, Freiburg, Germany) and concentrated in buffer containing 50 mM Tris-HCl, pH 8, 20 mM MgCl2, 150 mM NaCl, and 1 mM DTT to 2.5 mg/mL. Protein concentrations were determined by measuring the absorption at 280 nm under consideration of the specific extinction coefficients calculated at https://web.expasy.org/protparam/ (accessed on 15 January 2020). Purified protein samples were flash-frozen in liquid nitrogen and stored at −80 °C until required.

3.4. In Vitro Inhibition Assay and Kinetic Characterization

For initial screening of the synthesized compounds, CSS in vitro activity in the absence or presence of synthesized compounds was measured in 96-well half-area plates (Greiner Bio-One, Frickenhausen, Germany) in a reaction volume of 100 μL, utilizing the EnzChek pyrophosphate assay kit (Life Technologies, Darmstadt, Germany). Briefly, the inorganic pyrophosphate (PPi), produced by CSS as a byproduct, is cleaved by inorganic pyrophosphatase (IPP), yielding two molecules of phosphate (Pi), which subsequently react with 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) in a reaction catalyzed by purine nucleoside phosphorylase (PNP). Detection of the reaction product 2-amino-6-mercapto-7-methylpurine at 360 nm allows quantitation of the initially generated Pi. The assay reaction mixture contained 50 mM Tris-HCl, pH 8.0, 25 mM MgCl2, 1 mM Neu5Ac (Nacalai Tesque, Kyoto, Japan), 1 mM CTP (Sigma-Aldrich, Taufkirchen, Germany), as well as 0.22 mM MESG, 1 U/mL PNP, and 0.03 U/mL IPP (all from Life Technologies), and 0.1 mM of the compounds to be tested. To exclude an effect of the compounds on the coupling enzymes PNP and IPP, a control assay without Neu5Ac, CTP, and CSS was performed, and the reaction was instead initiated by the addition of inorganic pyrophosphate (Sigma-Aldrich) at 20 μM final concentration. For CSS activity measurements, the reaction was initiated by the addition of purified NmB CSS at a final concentration of 157.5 ng/mL. The enzyme was diluted in 50 mM Tris-HCl, pH 8.0, 25 mM MgCl2, and briefly pre-incubated with the compounds before transferring the CSS/compound mixture to the reaction mixture with a 96-well pipette. Product formation was monitored at 360 nm in a 96-well plate reader at 25 °C for 4 min. As a reference, CSS activity without compounds was measured and defined as 100% activity. Activity in the presence of each compound was calculated as a percentage of the reference activity.
For kinetic characterization, the CSS in vitro activity assay was carried out as described above, but with Neu5Ac and effector compound concentrations varied between 0.01 and 1.0 mM and 0–0.4 mM, respectively, while CTP concentration was maintained at 1 mM. All measurements were performed in duplicate. Uninhibited CSS activity was used to calculate kinetic parameters for the NmB enzyme by nonlinear regression according to the Michaelis–Menten model. Inhibitory constants were determined by plotting CSS activity in the presence of different compound concentrations against Neu5Ac concentration, and performing nonlinear regression curve fitting according to different inhibition modes (competitive, non-competitive, uncompetitive, and mixed-mode). Lineweaver–Burk plots were generated by plotting the reciprocal values of enzyme activity (1/v) against substrate concentration (1/[s]). The displayed linear regression lines with x- and y-intercepts at −1/Km and 1/Vmax, respectively, were constructed based on the corresponding kinetic parameters obtained from nonlinear regression analysis of the original data. All kinetic analyses were carried out using GraphPad Prism version 5.01 for Windows (GraphPad Software, Boston, MA, USA, www.graphpad.com).

3.5. STD NMR Spectroscopy

Saturation Transfer Difference (STD) NMR experiments were performed on a Bruker Avance 600 MHz NMR spectrometer, equipped with a 5 mm TXI probe with triple-axis gradient at 298 K [83]. The protein was saturated on-resonance at −1 ppm and off-resonance at 33 ppm with a cascade of 40 selective Gaussian-shaped pulses, of 50 ms duration with a 100 μs delay between each pulse in all STD NMR experiments. The total duration of the saturation time was set to 2 s. For the STD NMR experiments, 23.3 μM NmB CSS in deuterated 50 mM TRISD5, 50 mM NaCl, and 5 mM MgCl2 at pH 8.0 was used. The test compound was added in a molecular protein:ligand ratio of 1:100. A total of 1024 scans per STD NMR experiment were acquired, and a WATERGATE sequence was used to suppress the residual HDO signal. A spin-lock filter with a strength of 5 kHz and a duration of 10 ms was applied to suppress protein background.

3.6. ELISA-Based Assay to Monitor Sialylation of NmB LOS

Bacterial strains, plasmids, media, and growth conditions: Neisseria strains were grown on brain heart infusion (BHI, Oxoid; Thermo Scientific, Scoresby, Australia) agar plates made with 1% agar and supplemented with 10% Levinthal base [84] at 37 °C with 5% CO2. E. coli strain DH5α was grown on Luria–Bertani agar plate or broth [85]. Antibiotics ampicillin (amp) and kanamycin (kan) were used at a final concentration of 100 μg/mL where appropriate.
Construction of mutant strains: A PCR product encompassing 1 kb upstream and downstream of the sequence flanking the CMP-Neu5Ac synthase gene (neuA, NMB0069) was amplified from Neisseria strain MC58 using primers containing Neisseria uptake sequence; NeuAMC58_FOR (5′-GCCGTCTGAACAGAACCTACAAGGAAGTAAC-3′) and NeuAMC58_REV (5′- GACGCT GAAGTCTCCATTG-3′) and cloned into pGEM-T-Easy (Promega, Madison, WI, USA). Expression of neuA was disrupted through the insertion of a kanamycin resistance cassette (KanR) from the pUC4Kan plasmid (Cytiva (formerly GE Healthcare, Amersham Biosciences), USA) into a BamHI restriction site. The resulting mutant construct named pGEM-T::neuA::KanR was linearized with NcoI-HF and transformed into Neisseria strain MC58 ¢3 as described previously [86]. The new construct was named (¢3neuA::kan). Orientation of the insert into the vector was verified through sequencing and the generation of a unique PCR product with primers NeuABMC58_REV and KanUP_OUT (5′-AGACGTTTCCCGTTGAATATGGTCAT-3′), a primer located within the KanR cassette.
Complementation of the siaB gene: The neuA mutant was complemented as described previously [87]. A PCR product of siaB with 300 bp upstream and downstream flanking region was amplified using primers with added random bases and restriction enzyme sequence; NeuA_COMP_FOR (5′-ACATCGCTTAAG GCAACTCAAGTGCAGGTATTAG-3′) (AflII restriction enzyme sequence) and NeuA_COMP_REV (5′-GTCGTACCCGGGCTTCTTCATTCAGGGCGCAAC-3′) (SmaI restriction enzyme sequence). This insert was cloned into the corresponding AflII and SmaI digested pCTS32 vector under the control of the vector promoter. The subsequent construct, named ¢3neuA::kan, NeuA+, was linearized with SalI and transformed into ¢3neuA::kan. Clones were selected on BHI plates supplemented with 10% Levinthal base and antibiotics ampicillin (100 μg/mL) and spectinomycin (50 μg/mL), respectively. Correct recombination of siaB into the chromosome was verified by PCR.
ELISA-based LOS sialylation assay: Neisseria strains were plated out on BHI plates (supplemented with antibiotics where necessary) and incubated at 37 °C + 5% CO2 overnight. Cell suspension was prepared from overnight growth in 2X BHI supplemented with levinthal, and normalized to an OD600 of 1.0.
Test compound was suspended in 300 μL of sterile dH2O to give a final concentration of 1 mM and filter sterilized. Standing cultures were grown with the test compound at a starting OD of 0.1 for 4 h at 37 °C, shaken at 200 rpm. After incubation, cells were washed and resuspended in PBS and normalized to an OD600 of 0.2. Cells were heat-killed for 1 h at 60 °C before being used for ELISA. A total of 50 μL of normalized heat-killed cells was added per well (Nunc, Thermo Scientific, Waltham, MA, USA), and the plates were dried overnight at room temperature. Wells were blocked with 5% BSA + 1× TBST overnight, followed by 1 h incubation with 100 μL of Maackia amurensis lectin–alkaline phosphatase conjugate (MAA-AP; EY Laboratories, San Mateo, CA, USA) in 1/1400 dilution. Wells were washed at 5 min intervals with 1× TBST, repeated 4 times, and developed with substrate p-nitrophenyl phosphate (PNPP, Thermo Scientific) for 30 min. Reaction was stopped with 50 μL of 1N NaOH, and absorbance was read at 405 nm. Experiments were performed in triplicate.

4. Conclusions

We have functionalized the C-9, -7, and -4 positions of Neu5Acβ2Me and varied the C-5 N-acyl substituent on Neuβ2Me to produce a series of compounds with varied substitution around the sialic acid framework to probe interaction with N. meningitidis CSS. Evaluation of the compounds for inhibition of NmB CSS activity in vitro identified four compounds that showed an increased inhibition of the recombinant enzyme (Ki range between 100 and 500 μM), compared to the unsubstituted parent compound 3. Importantly, the Neu5Acβ2Me C-9 serine carboxamide 10d-1 was shown to directly interact with NmB CSS, to exhibit mixed mode inhibition of the enzyme, and to reduce the level of sialylation of N. meningitidis LOS. This data provides an intriguing starting point for further development of NmB CSS inhibitors as novel and specific anti-microbial agents for the treatment of bacterial meningitis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules30224329/s1: Supporting Information-S1, which includes: Figure S1, Kinetic analysis of test compounds with NmB CSS; Figure S2, ELISA to measure N. meningitidis LOS sialylation; Detailed experimental procedures for synthesis of the reported functionalized sialic acids and their characterization, including NMR data. Supporting Information-S2: NMR spectra.

Author Contributions

Conceptualization, M.v.I., R.G.-S., J.F., A.K.M.-K.; structure analysis, P.C., J.C.D., A.K.M.-K.; compound synthesis and characterization, P.C., I.D.G.; compound purification, F.J.R.; STD NMR and analysis, P.C., T.H.; protein production and enzymatic assays, J.F., A.K.M.-K.; Nm in vitro assay development and assay analysis, P.N., M.P.J.; Nm assays P.N.; supervision, I.D.G., R.J.T., M.v.I., M.P.J.; writing—original draft preparation, P.C., P.N., J.F., A.K.M.-K.; writing—review and editing, P.C., J.F., P.N., T.H., J.C.D., R.J.T., R.G.-S., I.D.G., M.P.J., A.K.M.-K., M.v.I. All authors have read and agreed to the published version of the manuscript.

Funding

M.v.I. thanks the Australian Research Council (A10017093, FF0345363) and the National Health and Medical Research Council (APP1071659) for financial support. P.C. thanks the Institute for Glycomics and Griffith University for the award of scholarships. R.G.-S. and A.K.M.-K. acknowledge the German Research Foundation (DFG) for financial support (MU-1849/2-1), also in the framework of DFG Research Unit 548 (Ge801/10-2), and J.F. acknowledges the Hannover Medical School for intramural funding (HILF). M.P.J. thanks the National Health and Medical Research Council for a Principal Research Fellowship (1138466).

Institutional Review Board Statement

This study does not involve humans or animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are included in the article and Supplementary Material. Further inquiries can be directed to the corresponding authors.

Acknowledgments

This project is part of the Fraunhofer International Consortium for Antimicrobial Research (iCAIR) initiative. We thank Paul Madge and Ryan Bennet (Institute for Glycomics) for the preparation of synthetic intermediates. We thank Martina Mühlenhoff (MHH) for kindly providing the NmB CSS expression vector and Wiebke Schaper and Friedrich Freiberger (MHH) for their contribution to activity assay design. We thank Plabon Das (IBG) for his contribution to the preparation of figures. Aspects of the work reported in this paper, including the synthesis of functionalized Neu5Acβ2Me derivatives described in Section 2.1, and the STD NMR study described in Section 2.3, are drawn from the doctoral thesis of P.C. [83].

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CSSCMP-sialic acid synthetase
LOSlipooligosaccharide
NmBNeisseria meningitidis serotype B
STDSaturation Transfer Difference (STD) NMR

References

  1. Harrison, L.H.; Trotter, C.L.; Ramsay, M.E. Global epidemiology of meningococcal disease. Vaccine 2009, 27 (Suppl. 2), B51–B63. [Google Scholar] [CrossRef]
  2. Dull, P.M.; McIntosh, E.D. Meningococcal vaccine development—From glycoconjugates against MenACWY to proteins against MenB—Potential for broad protection against meningococcal disease. Vaccine 2012, 30, B18–B25. [Google Scholar] [CrossRef]
  3. Berti, F.; Romano, M.R.; Micoli, F.; Adamo, R. Carbohydrate based meningococcal vaccines: Past and present overview. Glycoconj. J. 2021, 38, 401–409. [Google Scholar] [CrossRef] [PubMed]
  4. Basu, N.; Mukherjee, M.M.; Ghosh, R. Recent advances on the syntheses of oligosaccharides and the corresponding glycoconjugates towards development of vaccine candidates against invasive Neisseria meningitidis strains. Carbohydr. Res. 2025, 555, 109545. [Google Scholar] [CrossRef] [PubMed]
  5. Serruto, D.; Bottomley, M.J.; Ram, S.; Giuliani, M.M.; Rappuoli, R. The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: Immunological, functional and structural characterization of the antigens. Vaccine 2012, 30, B87–B97. [Google Scholar] [CrossRef]
  6. Finne, J.; Bitter-Suermann, D.; Goridis, C.; Finne, U. An IgG monoclonal antibody to group B meningococci cross-reacts with developmentally regulated polysialic acid units of glycoproteins in neural and extraneural tissues. J. Immunol. 1987, 138, 4402–4407. [Google Scholar] [CrossRef]
  7. Häyrinen, J.; Jennings, H.; Raff, H.V.; Rougon, G.; Hanai, N.; Gerardy-Schahn, R.; Finne, J. Antibodies to polysialic acid and its N-propyl derivative: Binding properties and interaction with human embryonal brain glycopeptides. J. Infect. Dis. 1995, 171, 1481–1490. [Google Scholar] [CrossRef]
  8. Granoff, D.M.; Bartoloni, A.; Ricci, S.; Gallo, E.; Rosa, D.; Ravenscroft, N.; Guarnieri, V.; Seid, R.C.; Shan, A.; Usinger, W.R.; et al. Bactericidal monoclonal antibodies that define unique meningococcal B polysaccharide epitopes that do not cross-react with human polysialic acid. J. Immunol. 1998, 160, 5028–5036. [Google Scholar] [CrossRef] [PubMed]
  9. Frosch, M.; Weisgerber, C.; Meyer, T.F. Molecular characterization and expression in Escherichia coli of the gene complex encoding the polysaccharide capsule of Neisseria meningitidis group B. Proc. Natl. Acad. Sci. USA 1989, 86, 1669–1673. [Google Scholar] [CrossRef]
  10. Mandrell, R.E.; Kim, J.J.; John, C.M.; Gibson, B.W.; Sugai, J.V.; Apicella, M.A.; Griffiss, J.M.; Yamasaki, R. Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis. J. Bacteriol. 1991, 173, 2823–2832. [Google Scholar] [CrossRef]
  11. Schauer, R.; Kamerling, J.P. Exploration of the sialic acid world. Adv. Carbohydr. Chem. Biochem. 2018, 75, 1–213. [Google Scholar] [CrossRef] [PubMed]
  12. Vogel, U.; Hammerschmidt, S.; Frosch, M. Sialic acids of both the capsule and the sialylated lipooligosaccharide of Neisseria meningitis serogroup B are prerequisites for virulence of meningococci in the infant rat. Med. Microbiol. Immunol. 1996, 185, 81–87. [Google Scholar] [CrossRef]
  13. Virji, M.; Makepeace, K.; Peak, I.R.A.; Ferguson, D.J.P.; Jennings, M.P.; Moxon, E.R. Opc- and pilus-dependent interactions of meningococci with human endothelial cells: Mechanisms and modulation by surface polysaccharides. Mol. Microbiol. 1995, 18, 741–754. [Google Scholar] [CrossRef]
  14. Unkmeir, A.; Kämmerer, U.; Stade, A.; Hübner, C.; Haller, S.; Kolb-Mäurer, A.; Frosch, M.; Dietrich, G. Lipooligosaccharide and polysaccharide capsule: Virulence factors of Neisseria meningitidis that determine meningococcal interaction with human dendritic cells. Infect. Immun. 2002, 70, 2454–2462. [Google Scholar] [CrossRef]
  15. Kurzai, O.; Schmitt, C.; Claus, H.; Vogel, U.; Frosch, M.; Kolb-Mäurer, A. Carbohydrate composition of meningococcal lipopolysaccharide modulates the interaction of Neisseria meningitidis with human dendritic cells. Cell Microbiol. 2005, 7, 1319–1334. [Google Scholar] [CrossRef]
  16. Jarvis, G.A.; Vedros, N.A. Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation. Infect. Immun. 1987, 55, 174–180. [Google Scholar] [CrossRef]
  17. Jarvis, G.A. Recognition and control of neisserial infection by antibody and complement. Trends Microbiol. 1995, 3, 198–201. [Google Scholar] [CrossRef]
  18. Hammerschmidt, S.; Hilse, R.; van Putten, J.P.; Gerardy-Schahn, R.; Unkmeir, A.; Frosch, M. Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element. EMBO J. 1996, 15, 192–198. [Google Scholar] [CrossRef] [PubMed]
  19. Kahler, C.M.; Martin, L.E.; Shih, G.C.; Rahman, M.M.; Carlson, R.W.; Stephens, D.S. The (alpha 2–>8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum. Infect. Immun. 1998, 66, 5939–5947. [Google Scholar] [CrossRef]
  20. Warren, L.; Blacklow, R.S. The biosynthesis of cytidine 5′-monophospho-N-acetylneuraminic acid by an enzyme from Neisseria meningitidis. J. Biol. Chem. 1962, 237, 3527–3534. [Google Scholar] [CrossRef] [PubMed]
  21. Roseman, S. Enzymatic synthesis of cytidine 5′-mono-phospho-sialic acids. Proc. Natl. Acad. Sci. USA 1962, 48, 437–441. [Google Scholar] [CrossRef]
  22. Kean, E.L. Sialic acid activation. Glycobiology 1991, 1, 441–447. [Google Scholar] [CrossRef] [PubMed]
  23. Masson, L.; Holbein, B.E. Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis. J. Bacteriol. 1983, 154, 728–736. [Google Scholar] [CrossRef]
  24. Münster-Kühnel, A.K.; Tiralongo, J.; Krapp, S.; Weinhold, B.; Ritz-sedlacek, V.; Jacob, U.; Gerardy-Schahn, R. Structure and function of vertebrate CMP-sialic acid synthetase. Glycobiology 2004, 14, 43R–51R. [Google Scholar] [CrossRef]
  25. Severi, E.; Hood, D.W.; Thomas, G.H. Sialic acid utilization by bacterial pathogens. Microbiology 2007, 153, 2817–2822. [Google Scholar] [CrossRef]
  26. Abeln, M.; Borst, K.M.; Cajic, S.; Thiesler, H.; Kats, E.; Albers, I.; Kuhn, M.; Kaever, V.; Rapp, E.; Münster-Kühnel, A.; et al. Sialylation is dispensable for early murine embryonic development in vitro. ChemBioChem 2017, 18, 1305–1316. [Google Scholar] [CrossRef] [PubMed]
  27. Schmid, W.; Christian, R.; Zbiral, E. Synthesis of both epimeric 2-deoxy-N-acetylneuraminic acids and their behaviour towards CMP-sialate synthetase—A comparison with 2-β-methylketoside of N-acetylneuraminic acid. Tetrahedron Lett. 1988, 29, 3643–3646. [Google Scholar] [CrossRef]
  28. Petrie, C.R.I.; Sharma, M.; Simmons, O.D.; Korytnyk, W. Synthesis of analogs of N-acetylneuraminic acid and their effect on CMP-sialate synthase. Carbohydr. Res. 1989, 186, 326–334. [Google Scholar] [CrossRef]
  29. Knorst, M.; Fessner, W.-D. CMP-Sialate synthetase from Neisseria meningitidis—Overexpression and application to the synthesis of oligosaccharides containing modified sialic acids. Adv. Synth. Catal. 2001, 343, 698–710. [Google Scholar] [CrossRef]
  30. Yu, H.; Yu, H.; Karpel, R.; Chen, X. Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: Comparison of substrate flexibility of three microbial CMP-sialic acid synthetases. Bioorg. Med. Chem. 2004, 12, 6427–6435. [Google Scholar] [CrossRef]
  31. He, N.; Yi, D.; Fessner, W.-D. Flexibility of substrate binding of cytosine-5′-monophosphate-N-acetylneuraminate synthetase (CMP-Sialate Synthetase) from Neisseria meningitidis: An enabling catalyst for the synthesis of neo-sialoconjugates. Adv. Synth. Catal. 2011, 353, 2384–2398. [Google Scholar] [CrossRef]
  32. Yi, D.; He, N.; Kickstein, M.; Metzner, J.; Weiß, M.; Berry, A.; Fessner, W.-D. Engineering of a cytidine 5′-monophosphate-sialic acid synthetase for improved tolerance to functional sialic acids. Adv. Synth. Catal. 2013, 355, 3597–3612. [Google Scholar] [CrossRef]
  33. Khedri, Z.; Li, Y.; Muthana, S.; Muthana, M.M.; Hsiao, C.-W.; Yu, H.; Chen, X. Chemoenzymatic synthesis of sialosides containing C7-modified sialic acids and their application in sialidase substrate specificity studies. Carbohydr. Res. 2014, 389, 100–111. [Google Scholar] [CrossRef] [PubMed]
  34. Morley, T.J.; Withers, S.G. Chemoenzymatic synthesis and enzymatic analysis of 8-modified cytidine monophosphate-sialic acid and sialyl lactose derivatives. J. Am. Chem. Soc. 2010, 132, 9430–9437. [Google Scholar] [CrossRef] [PubMed]
  35. Rauvolfova, J.; Venot, A.; Boons, G.-J. Chemo-enzymatic synthesis of C-9 acetylated sialosides. Carbohydr. Res. 2008, 343, 1605–1611. [Google Scholar] [CrossRef] [PubMed]
  36. Mertsch, A.; Poschenrieder, S.; Fessner, W.-D. Semi-synthetic sialic acid probes for challenging the substrate promiscuity of enzymes in the sialoconjugation pathway. Adv. Synth. Catal. 2020, 362, 5485–5495. [Google Scholar] [CrossRef]
  37. Mizanur, R.M.; Pohl, N.L. Bacterial CMP-sialic acid synthetases: Production, properties, and applications. Appl. Microbiol. Biotechnol. 2008, 80, 757–765. [Google Scholar] [CrossRef]
  38. Yu, H.; Chokhawala, H.A.; Huang, S.; Chen, X. One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities. Nat. Protoc. 2006, 1, 2485–2492. [Google Scholar] [CrossRef]
  39. Malekan, H.; Fung, G.; Thon, V.; Khedri, Z.; Yu, H.; Qu, J.; Li, Y.; Ding, L.; Lam, K.S.; Chen, X. One-pot multi-enzyme (OPME) chemoenzymatic synthesis of sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides containing natural or non-natural sialic acid. Bioorg. Med. Chem. 2013, 21, 4778–4785. [Google Scholar] [CrossRef]
  40. Hartmann, M.; Christian, R.; Zbiral, E. Structural variations of N-acetylneuraminic acid, 14. Synthesis of the β-methyl ketosides of 4-oxo-, 7-oxo-, and 8-oxo-N-acetylneuraminic acid and the corresponding 7,7- and 8,8-dimethoxy derivatives their behaviour towards CMP-sialate synthase. Liebigs Ann. Chem. 1990, 83–91. [Google Scholar] [CrossRef]
  41. Hartmann, M.; Christian, R.; Zbiral, E. Structural variations of N-acetylneuraminic acid. Part 19. Synthesis of both epimeric pairs of the 4-C-methyl- and 4-deoxy-4-C-methyl- as well as of the β-methylketoside of 4-deoxy-4-C-methylene-N-acetylneuraminic acid. Behavior towards CMP-sialate synthase. Monatsh. Chem. 1991, 122, 111–125. [Google Scholar] [CrossRef]
  42. Wong, J.H.; Sahni, U.; Li, Y.; Chen, X.; Gervay-Hague, J. Synthesis of sulfone-based nucleotide isosteres: Identification of CMP-sialic acid synthetase inhibitors. Org. Biomol. Chem. 2009, 7, 27–29. [Google Scholar] [CrossRef]
  43. Horsfall, L.E.; Nelson, A.; Berry, A. Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis. FEBS J. 2010, 277, 2779–2790. [Google Scholar] [CrossRef] [PubMed]
  44. Matthews, M.M.; McArthur, J.B.; Li, Y.; Yu, H.; Chen, X.; Fisher, A.J. Catalytic cycle of Neisseria meningitidis CMP-sialic acid synthetase illustrated by high-resolution protein crystallography. Biochemistry 2020, 59, 3157–3168. [Google Scholar] [CrossRef] [PubMed]
  45. Krapp, S.; Münster-Kühnel, A.K.; Kaiser, J.T.; Huber, R.; Tiralongo, J.; Gerardy-Schahn, R.; Jacob, U. The crystal structure of murine CMP-5-N-acetylneuraminic acid synthetase. J. Mol. Biol. 2003, 334, 625–637. [Google Scholar] [CrossRef]
  46. Haselhorst, T.; Münster-Kühnel, A.K.; Stolz, A.; Oschlies, M.; Tiralongo, J.; Kitajima, K.; Gerardy-Schahn, R.; von Itzstein, M. Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy. Biochem. Biophy. Res. Commun. 2005, 327, 565–570. [Google Scholar] [CrossRef]
  47. Sellmeier, M.; Weinhold, B.; Münster-Kühnel, A. CMP-Sialic acid synthetase: The point of constriction in the sialylation pathway. In SialoGlyco Chemistry and Biology I: Biosynthesis, Structural Diversity and Sialoglycopathologies; Gerardy-Schahn, R., Delannoy, P., von Itzstein, M., Eds.; Springer: Berlin, Heidelberg, 2015; pp. 139–167. [Google Scholar]
  48. Jen, F.E.-C.; Ketterer, M.R.; Semchenko, E.A.; Day, C.J.; Seib, K.L.; Apicella, M.A.; Jennings, M.P. The Lst sialyltransferase of Neisseria gonorrhoeae can transfer keto-deoxyoctanoate as the terminal sugar of lipooligosaccharide: A glyco-achilles heel that provides a new strategy for vaccines to prevent Gonorrhea. mBio 2021, 12, e03666-20. [Google Scholar] [CrossRef] [PubMed]
  49. Mosimann, S.C.; Gilbert, M.; Dombroswki, D.; To, R.; Wakarchuk, W.; Strynadka, N.C.J. Structure of a sialic acid-activating synthetase, CMP-acylneuraminate synthetase in the presence and absence of CDP. J. Biol. Chem. 2001, 276, 8190–8196. [Google Scholar] [CrossRef]
  50. Hartshorn, M.J. AstexViewer: A visualisation aid for structure-based drug design. J. Comp. Aided Mol. Des. 2002, 16, 871–881. [Google Scholar] [CrossRef]
  51. Yuan, Y.; Bleile, D.W.; Wen, X.; Sanders, D.A.R.; Itoh, K.; Liu, H.-w.; Pinto, B.M. Investigation of binding of UDP-Galf and UDP-[3-F]Galf to UDPgalactopyranose mutase by STD-NMR spectroscopy, molecular dynamics, and CORCEMA-ST calculations. J. Am. Chem. Soc. 2008, 130, 3157–3168. [Google Scholar] [CrossRef]
  52. Winger, M.; von Itzstein, M. Exposing the flexibility of human parainfluenza virus hemagglutinin-neuraminidase. J. Am. Chem. Soc. 2012, 134, 18447–18452. [Google Scholar] [CrossRef]
  53. Bose, S.; Purkait, D.; Joseph, D.; Nayak, V.; Subramanian, R. Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae. Acta Crystallogr. D Struct. Biol. 2019, 75, 564–577. [Google Scholar] [CrossRef]
  54. Zbiral, E.; Schreiner, E.; Christian, R. Synthesis of the 4-acetamido-4-deoxy analogue of N-acetylneuraminic acid and its behaviour towards CMP-sialate synthase. Carbohydr. Res. 1989, 194, c15–c18. [Google Scholar] [CrossRef]
  55. Kuhn, R.; Lutz, P.; MacDonald, D.L. Syntheses anomerer sialinsaure-methylketoside. Chem. Ber. 1966, 99, 611–617. [Google Scholar] [CrossRef]
  56. Chopra, P.; Thomson, R.J.; Grice, I.D.; von Itzstein, M. Rapid and clean microwave-assisted synthesis of N-acetylneuraminic acid methyl ester and its β-methyl glycoside. Tetrahedron Lett. 2012, 53, 6254–6256. [Google Scholar] [CrossRef]
  57. Byramova, N.E.; Tuzikov, A.B.; Bovin, N.V. A simple procedure for the synthesis of the methyl and benzyl glycosides of Neu5Ac and 4-epi-Neu5Ac. Conversion of the benzyl and methyl glycosides of Neu5Ac into N-trifluoroacetylneuraminic acid benzyl glycosides. Carbohydr. Res. 1992, 237, 161–175. [Google Scholar] [CrossRef]
  58. Czarniecki, M.F.; Thornton, E.R. Carbon-13 nuclear magnetic resonance spin-lattice relaxation in the N-acylneuraminic acids. Probes for internal dynamics and conformational analysis. J. Am. Chem. Soc. 1977, 99, 8273. [Google Scholar] [CrossRef]
  59. Kiefel, M.J.; Chopra, P.; Madge, P.D.; Szyczew, A.; Thomson, R.J.; Grice, I.D.; von Itzstein, M. Synthesis of C-9 oxidised N-acetylneuraminic acid derivatives as biological probes. Tetrahedron Lett. 2011, 52, 98–100. [Google Scholar] [CrossRef]
  60. Ramamoorthy, P.S.; Gervay, J. Solution phase synthesis of amide-linked N-acetyl neuraminic acid, α-amino acid, and sugar amino acid conjugates. J. Org. Chem. 1997, 62, 7801–7805. [Google Scholar] [CrossRef]
  61. Gregar, T.Q.; Gervay-Hague, J. Synthesis of oligomers derived from amide-linked neuraminic acid analogues. J. Org. Chem. 2003, 69, 1001–1009. [Google Scholar] [CrossRef]
  62. Valeur, E.; Bradley, M. Amide bond formation: Beyond the myth of coupling reagents. Chem. Soc. Rev. 2009, 38, 606–631. [Google Scholar] [CrossRef]
  63. Honda, T.; Masuda, T.; Yoshida, S.; Arai, M.; Kaneko, S.; Yamashita, M. Synthesis and anti-Influenza virus activity of 7-O-alkylated derivatives related to zanamivir. Bioorg. Med. Chem. Lett. 2002, 12, 1925–1928. [Google Scholar] [CrossRef]
  64. Masuda, T.; Yoshida, S.; Arai, M.; Kaneko, S.; Yamashita, M.; Honda, T. Synthesis and anti-influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino-Neu5Ac2en derivatives. Chem. Pharm. Bull. 2003, 51, 1386–1398. [Google Scholar] [CrossRef]
  65. Masuda, T.; Shibuya, S.; Arai, M.; Yoshida, S.; Tomozawa, T.; Ohno, A.; Yamashita, M.; Honda, T. Synthesis and anti-influenza evaluation of orally active bicyclic ether derivatives related to zanamivir. Bioorg. Med. Chem. Lett. 2003, 13, 669–673. [Google Scholar] [CrossRef]
  66. Brandstetter, H.H.; Zbiral, E. Strukturelle Abwandlungen an N-Acetylneuraminsäure, 2 [Transformations with N-acetylneuraminic acid. 2]. Liebigs Ann. Chem. 1983, 2055–2065. [Google Scholar] [CrossRef]
  67. Zbiral, E.; Phadtare, S.; Schmid, W. Strukturelle Abwandlungen an N-Acetylneuraminsäure, 6. Synthesen von 7-Oxo- und 8-Oxo-N-acetylneuraminsäure-Derivaten. Liebigs Ann. Chem. 1987, 39–43. [Google Scholar] [CrossRef]
  68. Sato, K.; Ikeda, K.; Suzuki, T.; Aoyama, S.; Maki, N.; Suzuki, Y.; Sato, M. Synthesis of 4-O-[3-(aryl)prop-2-ynyl]-Neu5Ac2en and its 4-epi-analogs modified at C-4 by Sonogashira coupling reaction. Tetrahedron 2007, 63, 7571–7581. [Google Scholar] [CrossRef]
  69. Isecke, R.; Brossmer, R. Synthesis of 5-N- and 9-N-thioacylated sialic acids. Tetrahedron 1994, 50, 7445–7460. [Google Scholar] [CrossRef]
  70. Roy, R.; Pon, R.A. Efficient synthesis of α(2-8)-linked N-acetyl and N-glycolylneuraminic acid disaccharides from colominic acid. Glycoconj. J. 1990, 7, 3–12. [Google Scholar] [CrossRef]
  71. Chopra, P.; Madge, P.D.; Thomson, R.J.; Grice, I.D.; von Itzstein, M. Microwave-assisted synthesis of N-glycolylneuraminic acid derivatives. Tetrahedron Lett. 2013, 54, 5558–5561. [Google Scholar] [CrossRef]
  72. Sparks, M.A.; Williams, K.W.; Lukacs, C.; Schrell, A.; Priebe, G.; Spaltenstein, A.; Whitesides, G.M. Synthesis of potential inhibitors of hemagglutination by influenza virus: Chemoenzymic preparation of N-5 analogs of N-acetylneuraminic acid. Tetrahedron 1993, 49, 1–12. [Google Scholar] [CrossRef]
  73. Schreiner, E.; Zbiral, E.; Kleineidam, R.G.; Schauer, R. Structural variations on N-acetylneuraminic acid, 20. Synthesis of some 2,3-didehydro-2-deoxysialic acids structurally varied at C-4 and their behavior towards sialidase from Vibrio cholerae. Liebigs Ann. Chem. 1991, 129–134. [Google Scholar] [CrossRef]
  74. von Itzstein, M.; Jin, B.; Wu, W.Y.; Chandler, M. A convenient method for the introduction of nitrogen and sulfur at C-4 on a sialic acid analog. Carbohydr. Res. 1993, 244, 181–185. [Google Scholar] [CrossRef]
  75. Chandler, M.; Bamford, M.J.; Conroy, R.; Lamont, B.; Patel, B.; Patel, V.K.; Steeples, I.P.; Storer, R.; Weir, N.G.; Wright, M.; et al. Synthesis of the potent influenza neuraminidase inhibitor 4-guanidino-Neu5Ac2en. X-Ray molecular structure of 5-acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-d-erythro-l-gluco-nononic acid. J. Chem. Soc. Perkin Trans. 1 1995, 1173–1180. [Google Scholar] [CrossRef]
  76. Okamoto, K.; Kondo, T.; Goto, T. Functionalization of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid methyl ester. Bull. Chem. Soc. Jpn. 1987, 60, 631–636. [Google Scholar] [CrossRef]
  77. Ciccotosto, S.; von Itzstein, M. Synthesis of methyl 5-acetamido-3,4,5-trideoxy-4-guanidinyl-d-glycero-d-galacto-2-nonulopyranosidonic acid (4-deoxy-4-guanidino-Neu5Acα2Me). Tetrahedron Lett. 1995, 36, 5405–5408. [Google Scholar] [CrossRef]
  78. Warren, L. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 1959, 234, 1971–1975. [Google Scholar] [CrossRef]
  79. Blat, Y. Non-competitive inhibition by active site binders. Chem. Biol. Drug Des. 2010, 75, 535–540. [Google Scholar] [CrossRef]
  80. Pesaresi, A. Mixed and non-competitive enzyme inhibition: Underlying mechanisms and mechanistic irrelevance of the formal two-site model. J. Enzym. Inhib. Med. Chem. 2023, 38, 2245168. [Google Scholar] [CrossRef]
  81. Mayer, M.; Meyer, B. Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew. Chem. Int. Ed. Engl. 1999, 38, 1784–1788. [Google Scholar] [CrossRef]
  82. Meyer, B.; Peters, T. NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew. Chem. Int. Ed. Engl. 2003, 42, 864–890. [Google Scholar] [CrossRef] [PubMed]
  83. Chopra, P. The Development of Carbohydrate-Based Chemical Probes of CMP-N-Acetylneuraminate Synthetase. Ph.D. Thesis, Griffith Univeristy, Gold Coast, Australia, 2013. Available online: http://hdl.handle.net/10072/366420 (accessed on 14 July 2025).
  84. Alexander, H.E. The haemophilus group. In Bacterial and Mycotic Infections of Man, 4th ed.; Dubos, R.J., Hirsch, J.G., Eds.; Pitman Medical Publishing Co.: London, UK, 1965; pp. 724–741. [Google Scholar]
  85. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, USA, 1989. [Google Scholar]
  86. Power, P.M.; Roddam, L.F.; Dieckelmann, M.; Srikhanta, Y.N.; Tan, Y.C.; Berrington, A.W.; Jennings, M.P. Genetic characterization of pilin glycosylation in Neisseria meningitidis. Microbiology 2000, 146, 967–979. [Google Scholar] [CrossRef] [PubMed]
  87. Steichen, C.T.; Shao, J.Q.; Ketterer, M.R.; Apicella, M.A. Gonococcal Cervicitis: A role for biofilm in pathogenesis. J. Infect. Dis. 2008, 198, 1856–1861. [Google Scholar] [CrossRef] [PubMed]
Molecules 30 04329 g001
Figure 1. (A). Activation of the Sia N-acetylneuraminic acid (Neu5Ac) 1 to the nucleotide sugar form CMP-Neu5Ac 2, catalyzed by CMP-sialic acid synthetase (CSS). (B). Neu5Acβ2Me (3), an inhibitor of mammalian CSS [27,28].
Figure 1. (A). Activation of the Sia N-acetylneuraminic acid (Neu5Ac) 1 to the nucleotide sugar form CMP-Neu5Ac 2, catalyzed by CMP-sialic acid synthetase (CSS). (B). Neu5Acβ2Me (3), an inhibitor of mammalian CSS [27,28].
Molecules 30 04329 g001
Molecules 30 04329 g002
Figure 2. X-Ray crystal structure of CMP-Neu5Ac 2 in the active site of Nm CSS in an open form (PDB 6CKM) [44]. Solvent accessible surfaces are coloured according to charge (red, negative charge; blue, positive charge; grey, neutral regions). CMP-Neu5Ac is shown in stick representation (green). Bound Ca2+ (grey sphere). Visualized using OpenAstexViewer (Version 3.0) [50].
Figure 2. X-Ray crystal structure of CMP-Neu5Ac 2 in the active site of Nm CSS in an open form (PDB 6CKM) [44]. Solvent accessible surfaces are coloured according to charge (red, negative charge; blue, positive charge; grey, neutral regions). CMP-Neu5Ac is shown in stick representation (green). Bound Ca2+ (grey sphere). Visualized using OpenAstexViewer (Version 3.0) [50].
Molecules 30 04329 g002
Molecules 30 04329 g003
Figure 3. Overview of modifications incorporated onto the Neu5Acβ2Me (3) scaffold to explore interactions with Nm CSS.
Figure 3. Overview of modifications incorporated onto the Neu5Acβ2Me (3) scaffold to explore interactions with Nm CSS.
Molecules 30 04329 g003
Molecules 30 04329 sch001
Scheme 1. Synthesis of C-9 carboxamide derivatives of Neu5Acβ2Me. Reagents and conditions: (a) MeOH, H+ ion exchange resin, (i) reflux, 48 h (62%), or (ii) MW (100 W), 120 °C, 15 min (63%); (b) i. TEMPO, DCM, sat. aq. NaHCO3, KBr, Bu4NBr, aq. NaOCl, 0 °C to 5 °C, 1 h; ii. Ac2O, pyridine, rt, 16 h (66% over 2 steps) [59]; (c) R’NH2, HOBt, EDC, DIPEA, DCM/DMF (4:1), rt, 16 h (6a 60%, 6b 60%, 6c 56%, 6d 55%); (d) R’C(NH2)CO2CH3, HOBt, EDC, DIPEA, DCM/DMF (4:1), rt, 16 h (7a 55%, 7b 60%, 7c 60%, 7d 62%, 7e 55%, 7f 60%, 7g 40%, 7h 45%); (e) NaOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h.
Scheme 1. Synthesis of C-9 carboxamide derivatives of Neu5Acβ2Me. Reagents and conditions: (a) MeOH, H+ ion exchange resin, (i) reflux, 48 h (62%), or (ii) MW (100 W), 120 °C, 15 min (63%); (b) i. TEMPO, DCM, sat. aq. NaHCO3, KBr, Bu4NBr, aq. NaOCl, 0 °C to 5 °C, 1 h; ii. Ac2O, pyridine, rt, 16 h (66% over 2 steps) [59]; (c) R’NH2, HOBt, EDC, DIPEA, DCM/DMF (4:1), rt, 16 h (6a 60%, 6b 60%, 6c 56%, 6d 55%); (d) R’C(NH2)CO2CH3, HOBt, EDC, DIPEA, DCM/DMF (4:1), rt, 16 h (7a 55%, 7b 60%, 7c 60%, 7d 62%, 7e 55%, 7f 60%, 7g 40%, 7h 45%); (e) NaOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h.
Molecules 30 04329 sch001
Molecules 30 04329 sch002
Scheme 2. Synthesis of 7-O-alkylated derivatives of Neu5Acβ2Me. Reagents and conditions: (a) 2,2-dimethoxypropane, H+ ion exchange resin, acetone, rt, 16 h (90%); (b) t-BuMe2SiCl, pyridine, 0 °C-rt, 16 h (58%); (c) R1X, NaH, TBAI, DMF, 0 °C-rt, 16 h (13a 56%, 13b 60%, 13d 60%, 13e 36%); (d) H2, 10% Pd/C, MeOH, rt, 16 h (13c 87%); (e) 80% aq. AcOH, 80 °C, 2 h; (f) NaOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h; (g) (i) MeOH, H+ ion exchange resin, MW (100 W), 80 °C, 15 min; (ii) Ac2O, pyridine, 16 h (88% over 4 steps from 13d); (h) R’I, PdCl2(PPh3)2, CuI, Et3N, CH3CN, rt, 2–6 h (16a 60%, 16b 87%, 16c 85%). In 16,17a–c, the sphere indicates the point of attachment of R' to the alkyne carbon.
Scheme 2. Synthesis of 7-O-alkylated derivatives of Neu5Acβ2Me. Reagents and conditions: (a) 2,2-dimethoxypropane, H+ ion exchange resin, acetone, rt, 16 h (90%); (b) t-BuMe2SiCl, pyridine, 0 °C-rt, 16 h (58%); (c) R1X, NaH, TBAI, DMF, 0 °C-rt, 16 h (13a 56%, 13b 60%, 13d 60%, 13e 36%); (d) H2, 10% Pd/C, MeOH, rt, 16 h (13c 87%); (e) 80% aq. AcOH, 80 °C, 2 h; (f) NaOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h; (g) (i) MeOH, H+ ion exchange resin, MW (100 W), 80 °C, 15 min; (ii) Ac2O, pyridine, 16 h (88% over 4 steps from 13d); (h) R’I, PdCl2(PPh3)2, CuI, Et3N, CH3CN, rt, 2–6 h (16a 60%, 16b 87%, 16c 85%). In 16,17a–c, the sphere indicates the point of attachment of R' to the alkyne carbon.
Molecules 30 04329 sch002
Molecules 30 04329 sch003
Scheme 3. Synthesis of C-5 amide derivatives of Neuβ2Me. Reagents and conditions: (a) NaOH (2.0 M), MW (100 W), 120 °C, 15 min (80%) [71]; (b) SOCl2, MeOH, 0 °C-rt, 16 h (92%); (c) R’COCl, Et3N, 1,4-dioxane/water (5:1), 0 °C–40 °C, 2 h (20a 87%, 20b 90%, 20c 91%, 20d 91%, 20e 78%, 20f 80%, 20g 82%, 20h 80% and 20i 88%); d) NaOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h. In 20,21h,i, the sphere indicates the point of attachment of R' to the amide carbon.
Scheme 3. Synthesis of C-5 amide derivatives of Neuβ2Me. Reagents and conditions: (a) NaOH (2.0 M), MW (100 W), 120 °C, 15 min (80%) [71]; (b) SOCl2, MeOH, 0 °C-rt, 16 h (92%); (c) R’COCl, Et3N, 1,4-dioxane/water (5:1), 0 °C–40 °C, 2 h (20a 87%, 20b 90%, 20c 91%, 20d 91%, 20e 78%, 20f 80%, 20g 82%, 20h 80% and 20i 88%); d) NaOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h. In 20,21h,i, the sphere indicates the point of attachment of R' to the amide carbon.
Molecules 30 04329 sch003
Molecules 30 04329 sch004
Scheme 4. Synthesis of C-4 amide derivatives of 4-deoxy-Neu5Acβ2Me. Reagents and conditions: (a) over 4 steps according to the reported procedures: (i) MeOH, H+ ion exchange resin, rt, 16 h; (ii) Ac2O, pyridine, rt, 16 h; (iii) TMSOTf, EtOAc, 50 °C, 2 h; (iv) TMSN3, t-BuOH, reflux, 6 h [75]; (b) according to the reported procedure: NBS, MeOH, 0–5 °C, 72 h (23 + 24, 87%) [77]; (c) Bu3SnH, AIBN, dioxane, 65 °C, 16 h (77%) [77]; (d) R’COCl, Et3N, DCM, 0 °C-rt, 2 h (26a 55%, 26b 67%, 26c 60%, 26d 65%, 26e 65%, 26f 65%, 26g 65%, 26h 60%, 26i 65%); (e) LiOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h. In 26,27e,h,i, the sphere indicates the point of attachment of R' to the amide carbon.
Scheme 4. Synthesis of C-4 amide derivatives of 4-deoxy-Neu5Acβ2Me. Reagents and conditions: (a) over 4 steps according to the reported procedures: (i) MeOH, H+ ion exchange resin, rt, 16 h; (ii) Ac2O, pyridine, rt, 16 h; (iii) TMSOTf, EtOAc, 50 °C, 2 h; (iv) TMSN3, t-BuOH, reflux, 6 h [75]; (b) according to the reported procedure: NBS, MeOH, 0–5 °C, 72 h (23 + 24, 87%) [77]; (c) Bu3SnH, AIBN, dioxane, 65 °C, 16 h (77%) [77]; (d) R’COCl, Et3N, DCM, 0 °C-rt, 2 h (26a 55%, 26b 67%, 26c 60%, 26d 65%, 26e 65%, 26f 65%, 26g 65%, 26h 60%, 26i 65%); (e) LiOH, MeOH/H2O (1:1), pH 13, 0 °C-rt, 16 h. In 26,27e,h,i, the sphere indicates the point of attachment of R' to the amide carbon.
Molecules 30 04329 sch004
Molecules 30 04329 g004
Figure 4. Relative activity of NmB CSS in the presence of C-9, C-7, C-5, and C-4 functionalized Neu5acylβ2Me derivatives (at 100 μM) in the EnzChek® pyrophosphate assay (Life Technologies, Darmstadt, Germany). Bar graphs represent the means of two individual experiments, with error bars representing the standard deviation. Activity of NmB CSS in the absence of functionalized compounds was defined as 100% (Ctrl).
Figure 4. Relative activity of NmB CSS in the presence of C-9, C-7, C-5, and C-4 functionalized Neu5acylβ2Me derivatives (at 100 μM) in the EnzChek® pyrophosphate assay (Life Technologies, Darmstadt, Germany). Bar graphs represent the means of two individual experiments, with error bars representing the standard deviation. Activity of NmB CSS in the absence of functionalized compounds was defined as 100% (Ctrl).
Molecules 30 04329 g004
Molecules 30 04329 g005
Figure 5. 1H NMR and STD NMR spectra of Neu5Acβ2Me C-9 serine carboxamide 10d-1 with NmB CSS. (i) 1H NMR spectrum of 10d-1; (ii) STD NMR of 10d-1 with NmB CSS (ligand to protein ratio of 100:1); (iii) epitope map of 10d-1.
Figure 5. 1H NMR and STD NMR spectra of Neu5Acβ2Me C-9 serine carboxamide 10d-1 with NmB CSS. (i) 1H NMR spectrum of 10d-1; (ii) STD NMR of 10d-1 with NmB CSS (ligand to protein ratio of 100:1); (iii) epitope map of 10d-1.
Molecules 30 04329 g005
Molecules 30 04329 g006
Figure 6. ELISA to measure the effect of 10d-1 on N. meningitidis LOS sialylation. ¢3 WT (capsule–deficient N. meningitidis wild type), ¢3neuA::kan: (CSS-deficient ¢3), and a 1:1 ratio of ¢3 WT and ¢3neuA::kan (50::50) were coated on microtiter plates and surface sialic acid detected photometrically (405 nm) with the sialic-acid-specific lectin MAA-AP conjugate. Similarly, ¢3 wild type was incubated with 10d-1 at 1 mM final concentration for 4 h, after which surface sialic acid was detected. Absorbance at 405 nM was measured to detect p-nitrophenol released from the alkaline phosphatase substrate p-nitrophenyl phosphate (PNPP). Experiments were performed in triplicate.
Figure 6. ELISA to measure the effect of 10d-1 on N. meningitidis LOS sialylation. ¢3 WT (capsule–deficient N. meningitidis wild type), ¢3neuA::kan: (CSS-deficient ¢3), and a 1:1 ratio of ¢3 WT and ¢3neuA::kan (50::50) were coated on microtiter plates and surface sialic acid detected photometrically (405 nm) with the sialic-acid-specific lectin MAA-AP conjugate. Similarly, ¢3 wild type was incubated with 10d-1 at 1 mM final concentration for 4 h, after which surface sialic acid was detected. Absorbance at 405 nM was measured to detect p-nitrophenol released from the alkaline phosphatase substrate p-nitrophenyl phosphate (PNPP). Experiments were performed in triplicate.
Molecules 30 04329 g006
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Chopra, P.; Führing, J.; Ng, P.; Haselhorst, T.; Dyason, J.C.; Rose, F.J.; Thomson, R.J.; Gerardy-Schahn, R.; Grice, I.D.; Jennings, M.P.; et al. Exploring the Scope of Functionalized N-Acylneuraminic Acid β-Methyl Glycosides as Inhibitors of Neisseria meningitidis CMP-Sialic Acid Synthetase. Molecules 2025, 30, 4329. https://doi.org/10.3390/molecules30224329

AMA Style

Chopra P, Führing J, Ng P, Haselhorst T, Dyason JC, Rose FJ, Thomson RJ, Gerardy-Schahn R, Grice ID, Jennings MP, et al. Exploring the Scope of Functionalized N-Acylneuraminic Acid β-Methyl Glycosides as Inhibitors of Neisseria meningitidis CMP-Sialic Acid Synthetase. Molecules. 2025; 30(22):4329. https://doi.org/10.3390/molecules30224329

Chicago/Turabian Style

Chopra, Pradeep, Jana Führing, Preston Ng, Thomas Haselhorst, Jeffrey C. Dyason, Faith J. Rose, Robin J. Thomson, Rita Gerardy-Schahn, I. Darren Grice, Michael P. Jennings, and et al. 2025. "Exploring the Scope of Functionalized N-Acylneuraminic Acid β-Methyl Glycosides as Inhibitors of Neisseria meningitidis CMP-Sialic Acid Synthetase" Molecules 30, no. 22: 4329. https://doi.org/10.3390/molecules30224329

APA Style

Chopra, P., Führing, J., Ng, P., Haselhorst, T., Dyason, J. C., Rose, F. J., Thomson, R. J., Gerardy-Schahn, R., Grice, I. D., Jennings, M. P., Münster-Kühnel, A. K., & von Itzstein, M. (2025). Exploring the Scope of Functionalized N-Acylneuraminic Acid β-Methyl Glycosides as Inhibitors of Neisseria meningitidis CMP-Sialic Acid Synthetase. Molecules, 30(22), 4329. https://doi.org/10.3390/molecules30224329

Article Metrics

Back to TopTop