Intramolecular Lactones of Sialic Acids

The so-called “sialo-chemical-biology” has become an attractive research area, as an increasing number of natural products containing a sialic acid moiety have been shown to play important roles in biological, pathological, and immunological processes. The intramolecular lactones of sialic acids are a subclass from this crucial family that could have central functions in the discrimination of physiological and pathological conditions. In this review, we report an in-depth analysis of the synthetic achievements in the preparation of the intramolecular lactones of sialic acids (1,4-, 1,7- and γ-lactones), in their free and/or protected form. In particular, recent advances in the synthesis of the 1,7-lactones have allowed the preparation of key sialic acid derivatives. These compounds could be used as authentic reference standards for their correct determination in biological samples, thus overcoming some of the limitations of the previous analytical procedures.

These carbohydrates possess several unique characteristics. First, they usually occupy the distal end of the glycoconjugate glycan chains to which they are attached, which is the ideal location to mediate the interaction of the cell with the external environment, including other cells and pathogens. Therefore, they play important roles in physiological, pathological, and immunological processes [1,2,[4][5][6]. Second, Sias-containing natural compounds are often susceptible to structural biological modifications, generating a family of more than eighty distinct members [1,4,5]. Third, due to their peculiar structures, they present unique biochemical characteristics as compared to common monosaccharides [1,3,4,7]. Indeed, they are biosynthesized by condensation of a neutral six-carbon unit sugar with pyruvate, and they are activated in the form of CMP-Sias (i.e., 2a-c) [1,4].  (Neu5Ac 1a, Neu5Gc 1b, and KDN 1c), the general structure of modified Sias linked to the glycoconjugate chain, and the three cytidine mononucleotide Sias (CMP)-Sias (2a-c).

Synthesis of 1,4-and 1,7-bicyclic Lactones of Neu5Ac
The first synthetic route to achieve free or protected intramolecular bicyclic lactones of Neu5Ac was published by Dereviskaya et al. in 1966 [16], affording the 1,4-bicyclic lactone 3a. The authors claimed to obtain this molecule by treating Neu5Ac 1a with N,N′-dicyclohexylcarbodiimide (DCC), as a condensing agent, in pyridine followed by electrophoretic purification (Scheme 1); however, the structure of the obtained lactone was not sufficiently supported by physico-chemical data. In fact, after this initial report, the formation of this 1,4-bicyclic lactone 3a has never been observed again, nor could the compound be detected in any biological sample. Scheme 1. Synthesis of the free 1,4-bicyclic lactone 3a starting from Neu5Ac 1a [16].

Synthesis of 1,4-and 1,7-bicyclic Lactones of Neu5Ac
The first synthetic route to achieve free or protected intramolecular bicyclic lactones of Neu5Ac was published by Dereviskaya et al. in 1966 [16], affording the 1,4-bicyclic lactone 3a. The authors claimed to obtain this molecule by treating Neu5Ac 1a with N,N -dicyclohexylcarbodiimide (DCC), as a condensing agent, in pyridine followed by electrophoretic purification (Scheme 1); however, the structure of the obtained lactone was not sufficiently supported by physico-chemical data. In fact, after this initial report, the formation of this 1,4-bicyclic lactone 3a has never been observed again, nor could the compound be detected in any biological sample.

Synthesis of 1,4-and 1,7-bicyclic Lactones of Neu5Ac
The first synthetic route to achieve free or protected intramolecular bicyclic lactones of Neu5Ac was published by Dereviskaya et al. in 1966 [16], affording the 1,4-bicyclic lactone 3a. The authors claimed to obtain this molecule by treating Neu5Ac 1a with N,N′-dicyclohexylcarbodiimide (DCC), as a condensing agent, in pyridine followed by electrophoretic purification (Scheme 1); however, the structure of the obtained lactone was not sufficiently supported by physico-chemical data. In fact, after this initial report, the formation of this 1,4-bicyclic lactone 3a has never been observed again, nor could the compound be detected in any biological sample. Scheme 1. Synthesis of the free 1,4-bicyclic lactone 3a starting from Neu5Ac 1a [16].
In the 1990s, in light of Ogura's work, Gervay's group attempted the synthesis of these bicyclic lactones, but they obtained them only in the protected form (Scheme 4). In particular, they proposed [67] the use of the 1,4-protected compounds as starting material to synthesize a new class of molecules, the amino acid-NeuAc saccharopeptides (Scheme 4A). For this purpose, starting from Neu5Ac 1a, or from its 2-O-methyl derivative 15, they synthesized mixtures of 1,4-and 1,7-lactones, 10 and 11a, or 16 and 17, respectively. Then, lactones 10 and 16 were treated with protected glycine to give the desired saccharopeptides 18 or 19. Importantly, this synthetic strategy also allowed the obtainment of several sialyl donors. A second application that was proposed [68] concerned the use of protected 13 C-labeled intramolecular 1,4-and 1,7-lactones, 10-13 C and 11a-13 C or 16-13 C and 17-13 C, obtained from Sias 1a-13 C and 15-13 C, respectively. They are suitable model compounds to study more complex sialyllactones by the isotope edited NMR spectroscopy technique (Scheme 4B). Notably, the authors concluded that further investigations would have been necessary to confirm the applicability of the proposed method also on the unprotected 1,4-or 1,7-lactones, as they failed to obtain them in free form. Interestingly, Gervay's group performed a computational study [69] to evaluate whether the sialyllactones, or their derivatives, could be a valid scaffold in the design of high-affinity neuraminidase inhibitors. For this purpose, they used virtual free 1,4-and 1,7-bicyclic lactones to compare the ability of different computational methods to reproduce the NMR constant coupling of these molecules.    13 and 14a-c starting from Neu5Ac 1a [70]; (B) proposed mechanism of intramolecular lactonization [70].
In the 1990s, in light of Ogura's work, Gervay's group attempted the synthesis of these bicyclic lactones, but they obtained them only in the protected form (Scheme 4). In particular, they proposed [67] the use of the 1,4-protected compounds as starting material to synthesize a new class of molecules, the amino acid-NeuAc saccharopeptides (Scheme 4A). For this purpose, starting from Neu5Ac 1a, or from its 2-O-methyl derivative 15, they synthesized mixtures of 1,4-and 1,7-lactones, 10 and 11a, or 16 and 17, respectively. Then, lactones 10 and 16 were treated with protected glycine to give the desired saccharopeptides 18 or 19. Importantly, this synthetic strategy also allowed the obtainment of several sialyl donors. A second application that was proposed [68] concerned the use of protected 13 C-labeled intramolecular 1,4-and 1,7-lactones, 10-13 C and 11a-13 C or 16-13 C and 17-13 C, obtained from Sias 1a-13 C and 15-13 C, respectively. They are suitable model compounds to study more complex sialyllactones by the isotope edited NMR spectroscopy technique (Scheme 4B). Notably, the authors concluded that further investigations would have been necessary to confirm the applicability of the proposed method also on the unprotected 1,4-or 1,7-lactones, as they failed to obtain them in free form. Interestingly, Gervay's group performed a computational study [69] to evaluate whether the sialyllactones, or their derivatives, could be a valid scaffold in the design of high-affinity neuraminidase inhibitors. For this purpose, they used virtual free 1,4-and 1,7-bicyclic lactones to compare the ability of different computational methods to reproduce the NMR constant coupling of these molecules. Richard R. Schmidt's group reported a biologically interesting study concerning the synthesis of some protected Neu5Ac 1,7-bicyclic lactones (20a-c and 21a-c; Scheme 5) [75]. They used the lactones 21a-c, as sialyl acceptors, and the phosphite 22, as a donor, for the synthesis of β-2,8-unnatural saccharides 23a-c. This synthetic strategy represented a novel and interesting approach for the preparation of unusual gangliosides starting from these 1,7-lactonic compounds.
Overall, these seminal studies reached several important goals, as they: (i) established synthetic routes for the synthesis of protected 1,4-and 1,7-lactones, yet still in as mixtures; (ii) elucidated their correct structures, which was not obvious; (iii) clarified the molecular mechanism leading to their formation, which was also intricate; (iv) showed their application in the biological field. However, the main limit of these studies was that they all failed in delivering the free bicyclic lactones, as only the corresponding protected derivatives could be obtained. Indeed, to date, the synthesis of the free 1,4-bicyclic lactone 3a remains elusive. On the other hand, the synthesis of the free 1,7-lactones (4a, 4e, and 4f) was accomplished by Allevi  Richard R. Schmidt's group reported a biologically interesting study concerning the synthesis of some protected Neu5Ac 1,7-bicyclic lactones (20a-c and 21a-c; Scheme 5) [75]. They used the lactones 21a-c, as sialyl acceptors, and the phosphite 22, as a donor, for the synthesis of β-2,8-unnatural saccharides 23a-c. This synthetic strategy represented a novel and interesting approach for the preparation of unusual gangliosides starting from these 1,7-lactonic compounds.
compound 24 in anhydrous ethyl acetate. The structure of the intermediate 24 was attributed by performing a complete NMR analysis and its synthetic conversion into the peracetylated derivative 25 [17]. Moreover, the low stability of this 1,7-lactone 4a, under neutral or acidic aqueous solutions, was also suggested [17][18][19][20][21]66,78] (see Section 3.4). Notably, in this way, they could validate the mechanism of the intramolecular lactonization, which was previously hypothesized by Ogura and co-workers [70].   Overall, these seminal studies reached several important goals, as they: (i) established synthetic routes for the synthesis of protected 1,4-and 1,7-lactones, yet still in as mixtures; (ii) elucidated their correct structures, which was not obvious; (iii) clarified the molecular mechanism leading to their formation, which was also intricate; (iv) showed their application in the biological field. However, the main limit of these studies was that they all failed in delivering the free bicyclic lactones, as only the corresponding protected derivatives could be obtained. Indeed, to date, the synthesis of the free 1,4-bicyclic lactone 3a remains elusive. On the other hand, the synthesis of the free 1,7-lactones (4a, 4e, and 4f) was accomplished by Allevi's group in the 2000s [17][18][19][20][21]66,76], starting from Neu5Ac, Neu5Gc, and KDN, respectively (for the synthesis of 1,7-lactones of Neu5Gc and KDN, 4e and 4f, see Section 2.2). Actually, the effective idea for the synthesis of lactone 4a was simply the use of a bulky and easily removable acylating agent. For this purpose, Neu5Ac 1a was treated with benzyl chloroformate in a dimethylformamide-tetrahydrofuran (DMF-THF) mixture containing triethylamine, affording the 1,7-lactone 24, exclusively protected at the anomeric hydroxylic function, as reported in Scheme 6. The synthesis of the desired free N-acetylneuraminic acid 1,7-lactone (Neu5Ac1,7L) 4a [77] was accomplished by hydrogenolysis of the benzyloxy carbonyl function of compound 24 in anhydrous ethyl acetate. The structure of the intermediate 24 was attributed by performing a complete NMR analysis and its synthetic conversion into the peracetylated derivative 25 [17]. Moreover, the low stability of this 1,7-lactone 4a, under neutral or acidic aqueous solutions, was also suggested [17][18][19][20][21]66,78] (see Section 3.4). Notably, in this way, they could validate the mechanism of the intramolecular lactonization, which was previously hypothesized by Ogura and co-workers [70].
Furthermore, the same authors also synthesized the deuterated isotopologues of the 1,7-lactone 4a for its use as an authentic internal standard for high-performance liquid chromatography (HPLC) and GC analyses, coupled with mass spectrometry [18]. The isotopically labeled lactones 4a-d3, 4a-d5 have been synthesized, under similar reaction conditions as for 4a, starting from the isotopically labeled Sias 1a-d3, 1a-d5, which were labeled with deuterium at the amido group alone or both at the amido-function and the C3-position.

Synthesis of 1,4-and 1,7-bicyclic Lactones of Neu5Gc and KDN
As we will explain shortly, the bicycle lactones of Neu5Gc 4d and KDN 4f have been less studied than those of Neu5Ac 4a. The first attempt to isolate and characterize these compounds, in a protected form, was realized by Ogura's group in 1992 [79] on the peracetylated derivative of KDN 26 (Scheme 7A). The protected lactone 26 was reached, as a major compound from KDN 1c, after acylation with acetic anhydride in pyridine and esterification, together with compounds 27a and 27b. The authors clarified the structure of 26 using NMR and X-ray diffraction analyses.
These two articles remain the only sporadic attempts of achieving 1,7-protected lactones of KDN; while there are no papers on obtaining Neu5Gc lactone derivatives, before the articles published by Allevi  Furthermore, the same authors also synthesized the deuterated isotopologues of the 1,7-lactone 4a for its use as an authentic internal standard for high-performance liquid chromatography (HPLC) and GC analyses, coupled with mass spectrometry [18]. The isotopically labeled lactones 4a-d 3 , 4a-d 5 have been synthesized, under similar reaction conditions as for 4a, starting from the isotopically labeled Sias 1a-d 3 , 1a-d 5 , which were labeled with deuterium at the amido group alone or both at the amido-function and the C3-position.

Synthesis of 1,4-and 1,7-bicyclic Lactones of Neu5Gc and KDN
As we will explain shortly, the bicycle lactones of Neu5Gc 4d and KDN 4f have been less studied than those of Neu5Ac 4a. The first attempt to isolate and characterize these compounds, in a protected form, was realized by Ogura's group in 1992 [79] on the peracetylated derivative of KDN 26 (Scheme 7A). The protected lactone 26 was reached, as a major compound from KDN 1c, after acylation with acetic anhydride in pyridine and esterification, together with compounds 27a and 27b. The authors clarified the structure of 26 using NMR and X-ray diffraction analyses.
These two articles remain the only sporadic attempts of achieving 1,7-protected lactones of KDN; while there are no papers on obtaining Neu5Gc lactone derivatives, before the articles published by Allevi  In that year, Allevi et al. [19], in light of the knowledge on Neu5Ac 4a lactone, synthesized the free 1,7-lactones of Neu5Gc 4d and KDN 4f. As depicted in Scheme 8A, Neu5Gc 1b was transformed to the protected 1,7-lactone 30, under similar reaction conditions reported for the achievement of compound 24. Then, the hydrogenolysis of this intermediate, in anhydrous ethyl acetate, afforded the desired lactone of Neu5Gc 4d. A similar route, carried out on the KDN 1c, led to the formation of its protected 1,7-lactone 31, together with the by-product 32. Finally, the hydrogenolysis of 31 gave the free 1,7-bicyclic lactone 4f. A second work, published in 1994 by David et al. [80], described the reaction of the partially protected Neu5Ac derivative 28 with tributyltin oxide, affording the 1,7-lactone 29 in 55% yield (Scheme 7B).
These two articles remain the only sporadic attempts of achieving 1,7-protected lactones of KDN; while there are no papers on obtaining Neu5Gc lactone derivatives, before the articles published by Allevi et al. in the 2010.
In that year, Allevi et al. [19], in light of the knowledge on Neu5Ac 4a lactone, synthesized the free 1,7-lactones of Neu5Gc 4d and KDN 4f. As depicted in Scheme 8A, Neu5Gc 1b was transformed to the protected 1,7-lactone 30, under similar reaction conditions reported for the achievement of compound 24. Then, the hydrogenolysis of this intermediate, in anhydrous ethyl acetate, afforded the desired lactone of Neu5Gc 4d. A similar route, carried out on the KDN 1c, led to the formation of its protected 1,7-lactone 31, together with the by-product 32. Finally, the hydrogenolysis of 31 gave the free 1,7-bicyclic lactone 4f.
Notably, the set-up of an accessible and versatile route to synthesize the 1,7-lactones 4a, 4d, and 4f, in high yields, allows their use as authentic standards for analytical investigations and biological evaluations.
Moreover, the deuterated derivatives of Neu5Gc have been reported as authentic internal standards for the analysis by MS spectrometry techniques [18]. Similarly to that described above for Neu5Ac derivatives, isotopic lactones 4d-d 2 , 4d-d 4 have been accomplished starting from the isotopically labeled Sias 1b-d 2 , 1b-d 4 (Scheme 8B), under analogous reaction conditions to those previously described (see Section 2.1).

Synthesis of γ-Lactones of Sias
The free γ-lactone of Neu5Ac 33 was initially studied only as a synthetic synthon, as it had never been detected in biological samples. However, more recently, its formation was shown during the degradation of 1,7 Sias lactones, which could be present in natural matrices [78]. Historically, the synthesis of this lactone was instrumental for the attribution of the correct Neu5Ac 1a C4 configuration [80][81][82] (for a complete discussion of this topic, see recent book chapter "Exploration of the Sialic Acid World" by R. Schauer and J. P. Kamerling) [1]. Moreover, other protected γ-lactones have been shown to be key intermediates in the synthesis of several natural compounds and enzymatic inhibitors.
Kuhn et al. led some pioneering studies in the field in the late 1950s and 1960s. In particular, in 1962, they proposed a synthetic route to Neu5Ac 1a via the free γ-lactone of Neu5Ac 35 (Scheme 9A), which also allowed reassessment and reassignment of its stereochemistry [83]. Briefly, the condensation of the N-acetylmannosamine 34 and tert-butyloxaloacetate in methanol (MeOH) yielded the 3-carboxy-t-butyl γ-lactone of Neu5Ac 35 in 48% yield. Successively, lactone 35 was decarboxylated, affording the free γ-lactone 33, which was directly hydrolyzed in 2M sodium hydroxide (NaOH) to give the pure Neu5Ac 1a (34% yield). Comparable results were obtained starting from the 4,6-O-benzylidene derivative of N-acetylglucosamine 36, giving the protected lactone 38, eventually affording lactone 33 after deprotection and decarboxylation. Finally, compound 33 was directly hydrolyzed to Neu5Ac 1a in 30% yield (Scheme 9B).    A few years later, this synthetic method was applied by Faillard for the synthesis of N-glycolylneuraminic acid 1b (Neu5Gc), starting from N-glycolylglucosamine 37 via its protected and free γ-lactones, 39 and 40, respectively [84]. The free γ-lactone 40 could be converted into the pure Neu5Gc 1b under basic conditions (Scheme 9B).
The free γ-lactone of Neu5Ac was obtained also by Dereviskaya [16], although without any chemical-physical properties elucidation. In fact, the complete chemical-physical characterization (NMR, MS, ect.) of this molecule was reported only several decades later, when two independent synthetic routes were published [77,85], as discussed later in this review.
Between 1966 and 2010, major efforts in the field were directed to study some protected γ-lactones, as they were suitable intermediates to synthesize various Neu5Ac substituted derivatives. In those years, two independent studies by Khorlin [86] and by Holmqusit [87] are of particular interest, as they described the synthesis of several sialidase inhibitors. Successively, Zbiral and Schmid obtained the acetylated γ-lactone of Neu5Ac 41 in good yield (52% overall yield) [88]. This γ-lactone was synthesized from compound 42, via a classical acetylation reaction to afford the intermediate 43, A few years later, this synthetic method was applied by Faillard for the synthesis of Nglycolylneuraminic acid 1b (Neu5Gc), starting from N-glycolylglucosamine 37 via its protected and free γ-lactones, 39 and 40, respectively [84]. The free γ-lactone 40 could be converted into the pure Neu5Gc 1b under basic conditions (Scheme 9B).
The free γ-lactone of Neu5Ac was obtained also by Dereviskaya [16], although without any chemical-physical properties elucidation. In fact, the complete chemical-physical characterization (NMR, MS, ect.) of this molecule was reported only several decades later, when two independent synthetic routes were published [77,85], as discussed later in this review.
Between 1966 and 2010, major efforts in the field were directed to study some protected γlactones, as they were suitable intermediates to synthesize various Neu5Ac substituted derivatives. In those years, two independent studies by Khorlin [86] and by Holmqusit [87] are of particular interest, as they described the synthesis of several sialidase inhibitors. Successively, Zbiral and Schmid obtained the acetylated γ-lactone of Neu5Ac 41 in good yield (52% overall yield) [88]. This γ-lactone was synthesized from compound 42, via a classical acetylation reaction to afford the Very significant results in this field were reported in the 1980s by Ogura's group [70,88], concerning the synthesis of some differently protected γ-lactones of Neu5Ac of relevant biological interest. To this purpose, Neu5Ac 1a was first dissolved in DMF and then treated with an excess of cesium carbonate (Cs2CO3), together with the appropriate reagent: (i) methyl iodide, (ii) benzyl Very significant results in this field were reported in the 1980s by Ogura's group [70,88], concerning the synthesis of some differently protected γ-lactones of Neu5Ac of relevant biological interest. To this purpose, Neu5Ac 1a was first dissolved in DMF and then treated with an excess of cesium carbonate (Cs 2 CO 3 ), together with the appropriate reagent: (i) methyl iodide, (ii) benzyl bromide and (iii) allyl bromide, respectively. After 1.5-3 h at 40 • C, the corresponding protected lactones 44-46 where obtained in 42-78% yield (Scheme 11A). Additionally, the methylated γ-lactone 44 could be obtained treating the methyl ester 47 in methanol solution with diazomethane (overnight at r.t.) in 29% yield (Scheme 11B) [70].

Scheme 10.
Synthesis of protected γ-lactone 41 [88]. Very significant results in this field were reported in the 1980s by Ogura's group [70,88], concerning the synthesis of some differently protected γ-lactones of Neu5Ac of relevant biological interest. To this purpose, Neu5Ac 1a was first dissolved in DMF and then treated with an excess of cesium carbonate (Cs2CO3), together with the appropriate reagent: (i) methyl iodide, (ii) benzyl bromide and (iii) allyl bromide, respectively. After 1.5-3h at 40 °C, the corresponding protected lactones 44-46 where obtained in 42-78% yield (Scheme 11A). Additionally, the methylated γ-lactone 44 could be obtained treating the methyl ester 47 in methanol solution with diazomethane (overnight at r.t.) in 29% yield (Scheme 11B) [70]. In the same studies, the authors hypothesized a complex mechanism for the formation of the γlactone, via a 2 C5 to 5 C2 conformation-switch, followed by an intramolecular ester exchange reaction with retention of the C4 stereochemistry (Scheme 11A) [89]. Remarkably, the proposed mechanism could only be experimentally confirmed twenty-seven years later, by Allevi's group, as described below. Surprisingly, no further studies on γ-lactones were published for over two decades, with the exception of the highly efficient synthesis of KDN 1c by Warwel in 2000 [90], which involved the free γ-lactone of KDN as key intermediate.
Only in 2010 Sawada and coworkers [85] were able to isolate the free form of the γ-lactone of Neu5Ac 33 (62%), upon treatment of Neu5Ac methyl ester 47 under basic conditions using sodium methoxide (NaOMe 0.5 M in methanol for 0.5 h at 25 °C), followed by a neutralization and a final recrystallization step (Scheme 12).  In the same studies, the authors hypothesized a complex mechanism for the formation of the γ-lactone, via a 2 C 5 to 5 C 2 conformation-switch, followed by an intramolecular ester exchange reaction with retention of the C4 stereochemistry (Scheme 11A) [89]. Remarkably, the proposed mechanism could only be experimentally confirmed twenty-seven years later, by Allevi's group, as described below. Surprisingly, no further studies on γ-lactones were published for over two decades, with the exception of the highly efficient synthesis of KDN 1c by Warwel in 2000 [90], which involved the free γ-lactone of KDN as key intermediate.

HO
Only in 2010 Sawada and coworkers [85] were able to isolate the free form of the γ-lactone of Neu5Ac 33 (62%), upon treatment of Neu5Ac methyl ester 47 under basic conditions using sodium methoxide (NaOMe 0.5 M in methanol for 0.5 h at 25 • C), followed by a neutralization and a final re-crystallization step (Scheme 12).
below. Surprisingly, no further studies on γ-lactones were published for over two decades, with the exception of the highly efficient synthesis of KDN 1c by Warwel in 2000 [90], which involved the free γ-lactone of KDN as key intermediate.
Only in 2010 Sawada and coworkers [85] were able to isolate the free form of the γ-lactone of Neu5Ac 33 (62%), upon treatment of Neu5Ac methyl ester 47 under basic conditions using sodium methoxide (NaOMe 0.5 M in methanol for 0.5 h at 25 °C), followed by a neutralization and a final recrystallization step (Scheme 12). Scheme 12. Synthesis of the free γ-lactone 33 starting from the methyl ester 47 [85].
Curiously, two possible intermediates (I or II) involved in the reaction mechanism were proposed: a bicyclo-1,4 lactone I or a ring-opened intermediate structure II (Scheme 12). Moreover, to confirm the correct structure, the obtained γ-lactone 33 had to be transformed into the corresponding methyl ether derivative 44 by treatment with diazomethane. More recently, in 2015, Allevi's group described a novel synthetic route to obtain the free γ-lactone 33 in very high yields (83-91%), starting from the bicyclic 1,7-lactone 4a and using different acidic and basic reaction conditions in a definite reaction time [78] (Scheme 13) (for detail about reaction conditions, see Section 3.4). Curiously, two possible intermediates (I or II) involved in the reaction mechanism were proposed: a bicyclo-1,4 lactone I or a ring-opened intermediate structure II (Scheme 12). Moreover, to confirm the correct structure, the obtained γ-lactone 33 had to be transformed into the corresponding methyl ether derivative 44 by treatment with diazomethane. More recently, in 2015, Allevi's group described a novel synthetic route to obtain the free γ-lactone 33 in very high yields (83-91%), starting from the bicyclic 1,7-lactone 4a and using different acidic and basic reaction conditions in a definite reaction time [78]  Scheme 13. Synthesis of the free γ-lactone 33 starting from the bicyclic 1,7-lactone 4a [78].
Interestingly, they discovered that the bicyclic 1,7-lactone 4a could evolve into the γ-lactone 33. These could be rationalized, taking into account the pioneering work of Ogura [89]. In particular, the formation of the γ-lactone, from different esters of Neu5Ac, goes through an intermediate flipped structure, obtained by an inversion from a 2 C5 to a 5 C2 conformation, the latter being similar to that of the 1,7-lactone. In light of this unexpected rearrangement, Allevi and coworkers proposed a possible mechanism for the formation of the γ-lactone, involving the intramolecular transesterification of the free bicyclic 1,7-lactone 4a, mediated by the attack of the C4 hydroxy group on the carboxyl function at C-1, under acidic or basic conditions (Scheme 13). According to the proposed mechanism [78], the 9-acetylated γ-lactone 48 could also easily be obtained, starting from the corresponding 1,7-lactone 4b. Conversely, the protection of the C4 hydroxyl group, as shown in the compound 4c, prevented the rearrangement, thus blocking the formation of the corresponding γ-lactone (Scheme 14; for detail about reaction conditions, see Section 3.4). Interestingly, they discovered that the bicyclic 1,7-lactone 4a could evolve into the γ-lactone 33. These could be rationalized, taking into account the pioneering work of Ogura [89]. In particular, the formation of the γ-lactone, from different esters of Neu5Ac, goes through an intermediate flipped structure, obtained by an inversion from a 2 C 5 to a 5 C 2 conformation, the latter being similar to that of the 1,7-lactone. In light of this unexpected rearrangement, Allevi and coworkers proposed a possible mechanism for the formation of the γ-lactone, involving the intramolecular transesterification of the free bicyclic 1,7-lactone 4a, mediated by the attack of the C4 hydroxy group on the carboxyl function at C-1, under acidic or basic conditions (Scheme 13). According to the proposed mechanism [78], the 9-acetylated γ-lactone 48 could also easily be obtained, starting from the corresponding 1,7-lactone 4b. Conversely, the protection of the C4 hydroxyl group, as shown in the compound 4c, prevented the rearrangement, thus blocking the formation of the corresponding γ-lactone (Scheme 14; for detail about reaction conditions, see Section 3.4). mechanism for the formation of the γ-lactone, involving the intramolecular transesterification of the free bicyclic 1,7-lactone 4a, mediated by the attack of the C4 hydroxy group on the carboxyl function at C-1, under acidic or basic conditions (Scheme 13). According to the proposed mechanism [78], the 9-acetylated γ-lactone 48 could also easily be obtained, starting from the corresponding 1,7-lactone 4b. Conversely, the protection of the C4 hydroxyl group, as shown in the compound 4c, prevented the rearrangement, thus blocking the formation of the corresponding γ-lactone (Scheme 14; for detail about reaction conditions, see Section 3.4).

Biological Relevance of Neu5ac 1,7-lactones
To date, the only intramolecular lactones identified in biological matrices, and related to physiopathological states, are the 1,7-lactones of Sias 4a-e. Instead, to the best of our knowledge, 1,4-or γlactone derivatives have not been identified in glycoconjugates.
In this section, before reviewing the biological functions of the 1,7-lactones of Sias, we will briefly touch on the analytical method used for their identification in biological samples [60]. Indeed, these two aspects are closely related, as the identification of these molecules, which has been carried out almost exclusively with the gas chromatography-mass spectrometry (GC-MS) method developed by Zanetta and collaborators in 2001 [60], could have generated incorrect results due to some recently discovered glitches in the analytical method [66]. However, recently, the use of biochemical assays (differential scanning fluorometric, kinetic studies) performed with authentic standards of these lactones has overcome the limitations of the GC-MS method by Zanetta [63,64].

Biological Relevance of Neu5ac 1,7-Lactones
To date, the only intramolecular lactones identified in biological matrices, and related to physio-pathological states, are the 1,7-lactones of Sias 4a-e. Instead, to the best of our knowledge, 1,4-or γ-lactone derivatives have not been identified in glycoconjugates.
In this section, before reviewing the biological functions of the 1,7-lactones of Sias, we will briefly touch on the analytical method used for their identification in biological samples [60]. Indeed, these two aspects are closely related, as the identification of these molecules, which has been carried out almost exclusively with the gas chromatography-mass spectrometry (GC-MS) method developed by Zanetta and collaborators in 2001 [60], could have generated incorrect results due to some recently discovered glitches in the analytical method [66]. However, recently, the use of biochemical assays (differential scanning fluorometric, kinetic studies) performed with authentic standards of these lactones has overcome the limitations of the GC-MS method by Zanetta [63,64].

Analytical and Biochemical Techniques Applied to Identify Neu5Ac 1,7-Lactones
The GC-MS method, reported by Zanetta et al. in 2001 [60], was used to identify several sialic acids derivatives in biological samples. The three fundamental steps of the method are depicted in Scheme 15, and consist of: (i) the release of sialic acids from the glycoconjugates; (ii) the derivatization of their carboxylic function with diazomethane and (iii) the derivatization of the hydroxyl functions with heptafluorobutyric anhydride (HFBAA) (except the semi-acetal one at C2, see Scheme 15 for details). It should be noted that the 1,7-lactones of Sias were the only subclass of compounds that could be determined without the derivatization step with diazomethane.
The main features of this method, which quickly became the preferred analytical method in the field, were: (i) the possibility to analyze different classes of compounds, including oligosaccharides, glycolipids and glycoproteins; (ii) the low interference of peptides and amino acids, which are present in the sample and that can be simultaneously analyzed with the Sias; (iii) the lack of intermediate purification steps of Sias after the initial hydrolysis; (iv) the prompt identification of the different analytes as heptafluorobutyrate (HFB) derivatives using their specific ions in the MS profile; (v) the stability of the HFB derivatives.
The GC-MS method, reported by Zanetta et al. in 2001 [60], was used to identify several sialic acids derivatives in biological samples. The three fundamental steps of the method are depicted in Scheme 15, and consist of: (i) the release of sialic acids from the glycoconjugates; (ii) the derivatization of their carboxylic function with diazomethane and (iii) the derivatization of the hydroxyl functions with heptafluorobutyric anhydride (HFBAA) (except the semi-acetal one at C2, see Scheme 15 for details). It should be noted that the 1,7-lactones of Sias were the only subclass of compounds that could be determined without the derivatization step with diazomethane. Scheme 15. The three fundamental steps of Zanetta's gas chromatography-mass spectrometry (GC-MS) method. From the hydrolysis of the glycoconjugates with acetic acid (AcOH), different Sias and their lactones are produced. Here, by way of example, the structures of Neu5Ac 1a and its lactone 4a have been reported [60].
The main features of this method, which quickly became the preferred analytical method in the field, were: (i) the possibility to analyze different classes of compounds, including oligosaccharides, glycolipids and glycoproteins; (ii) the low interference of peptides and amino acids, which are present in the sample and that can be simultaneously analyzed with the Sias; (iii) the lack of intermediate purification steps of Sias after the initial hydrolysis; (iv) the prompt identification of the different analytes as heptafluorobutyrate (HFB) derivatives using their specific ions in the MS profile; (v) the stability of the HFB derivatives.
However, a critical pitfall of the analytical method was overlooked for almost two decades [60]. In fact, due to the lack of authentic reference samples for most Sias, including the 1,7-lactones, their structure was established only by the mass fragmentation profile, after a required multistep isolation and derivatization process that could alter their original structure. Indeed, it was recently shown [66] that the 1,7-bicyclic lactones, including the three derivatives of N-acetylneuraminic acid 4a-c and the two derivatives of N-glycolylneuraminic 4d-e, cannot be determined using the current analytical methodology (see Section 3.4) [60].
The GC-MS method of Zanetta et al. was also used in a study investigating the physiological role played by the 1,7-lactones in the interleukin-4 (IL-4) signaling modulation [59]. In particular, the  However, a critical pitfall of the analytical method was overlooked for almost two decades [60]. In fact, due to the lack of authentic reference samples for most Sias, including the 1,7-lactones, their structure was established only by the mass fragmentation profile, after a required multistep isolation and derivatization process that could alter their original structure. Indeed, it was recently shown [66] that the 1,7-bicyclic lactones, including the three derivatives of N-acetylneuraminic acid 4a-c and the two derivatives of N-glycolylneuraminic 4d-e, cannot be determined using the current analytical methodology (see Section 3.4) [60].
The GC-MS method of Zanetta et al. was also used in a study investigating the physiological role played by the 1,7-lactones in the interleukin-4 (IL-4) signaling modulation [59]. In particular, the authors combined the GC-MS approach with more traditional biochemical techniques, including the use of glycoconjugates immobilization methods coupled with electrophoresis, blotting techniques, and enzymatic inhibition assays using specific oligosaccharides.

1,7-Lactones of Sias in Biological Samples
The GC-MS analyses performed by Zanetta et al.'s method revealed the presence of 1,7-lactones in mucins of different physiological and pathological tissues from different species.

Animal Tissues
The 1,7-lactones of Sias have been initially detected in mucins of different animal species, such as in the bovine (1%) and ovine (0.5%) submandibular glands, in eggs of B. bufo, and in the skin of A. Anguilla. Among them, the mucins of B. bufo eggs were very rich in these lactones, containing all the known compounds 4a-e, where the derivative 4a was the most abundant [60].
Successively, the 1,7-lactone 4a was detected in mouse tissues in a study aimed at evaluating the distribution of the different sialic acids as endogenous substrates of coronavirus hemagglutinin-esterases [50]. In particular, the authors evaluated the distribution of 13 sialic acids, including lactone 4a, linked to glycoproteins and glycolipids at different stages of mouse brain development. They found that the 1,7-lactone 4a was not expressed in glycoproteins of mouse embryonic tissues nor in the newborn or the adult mouse brain. On the other hand, they found modest quantities (0.35% of total Sias) of 4a in seven-day-old mice. Additionally, they found that, among the extracerebral tissues, only the liver contained this lactone (3.54% of total Sias). In this study, the 1,7 lactone 4a was only found in the initial embryonic state (on the thirteenth day), then it could not be detected until the mouse birth. Then, at seven days from birth, it was widely expressed (57.73% of total Sias), although its content was reduced to minor quantities (0.20% of total Sias) in the adult mouse. Curiously, a high concentration of this lactone was detected in the metencephalon of adult mouse tissue; on the other hand, it was absent in other cerebral or extracerebral compartments. Unfortunately, the biological significance of the occurrence of Neu5Ac1,7L in large quantities in the brain and in the liver is still puzzling to date [50].

Human Healthy and Diseased Tissues
The physiological presence of the 1,7-lactone 4a and its interaction with interleukin 4 (IL-4) in human tissues was investigated, suggesting the possible clinical use of this Neu5Ac derivative as a potential modulator of IL-4-mediated immune response. More specifically, in 2001, Zanetta's group disclosed that the 1,7-lactone 4a was the ligand of human interleukin 4 [56,58,59], thus proposing a "lectin" activity of IL-4. In fact, they found that this interleukin could bind to the mucins, extracted from B. bufo eggs, which are glycoproteins enriched in sialylated 1,7-lactones. Moreover, the authors excluded that the 1,7-lactone of Neu5Gc could be the ligand of IL-4 in the B. bufo mucins, as they performed analogous experiments on the mucins of the ovine submandibular gland, where this lactone is known to be absent [60]. Importantly, the 1,7-lactone 4a could modulate the signal transduction events initiated by IL-4 via a 63 kDa protein (p63) on resting lymphocytes, supporting that the pathway depends on the lectin activity. Overall, the study suggests that specific carbohydrates, such as the 1,7-lactones of Sias, could be instrumental to modulate the immune system, as an alternative to therapies employing antibodies against interleukins or interleukin receptors. In fact, the involvement of the 1,7-lactone 4a in immunomodulation has been also supported by some studies performed on human purified glycoproteins from pregnant women [53]. Specifically, this molecule is highly expressed in both the mucin MUC2, having a high degree of O-linked glycans, and the uromodulin, holding essentially N-linked ones. Interestingly, the 1,7-lactone 4a accounted for 7.7% and 35% of the overall sialic acids present in MUC2 and in the uromodulin protein, respectively. Indeed, the urine glycoproteins of pregnant women are known to specifically bind IL-4 in a Sias carbohydrate-dependent way, although the actual ligand(s) have yet to be identified [56,58,59]. Thus, these results [53] seem to support that lactone 4a could be the ligand of IL-4, although further studies to confirm this hypothesis are still needed.
The different expression of 1,7-lactone in pathological and healthy human tissues gave a particular relevance to this molecule, suggesting its possible role as a biomarker. For example, a high amount of 1,7-Neu5Ac lactone 4a has been detected in glycoproteins derived from the human colon-rectum tissues, supporting its use as a possible biomarker of colon cancer, if overexpressed in pathological tissues [53]. Moreover, the presence of Neu5ac1,7L in mucins and oligosaccharides isolated from the human gut has also been reported [55]. The study underlined the difference in glycosylation of distinctive parts of the intestine. Consistent levels of lactone 4a were found in different gut compartments, the highest being found in the ileum (20-21% of total sialic acids in each sample) and in the rectum (19-26%), whereas minor quantities were detected in the cecum (11-20%), in the transverse (8-14%), and in the sigmoid (6-9%).
Interestingly, the GC-MS analytical method of Zanetta et al. was applied to evaluate human erythrocyte membrane glycoprotein-and glycolipid-containing sialic acids of different serotypes [54]. Remarkably, these lactones were absent from the red blood cell (RBC) samples of healthy people, but were present, sometimes even at a high level, in several malignant tumors. In 2007, the Neu5Ac 1,7-lactone 4a was found in the erythrocyte membranes of patients affected by polycythemia vera [49]. In particular, a drastic reduction in the Sias diversity was observed, as well as the absence of Neu5Gc and its derivatives. Despite that, the 1,7-lactone 4a became a predominant compound, suggesting a crucial role of this molecule to discriminate between healthy and diseased RBC samples. Furthermore, in a study on malignant melanocytes [65], lactone 4a was found as a minor component in healthy tissues, but the dominant Sia in some malignant cells. Specifically, it was found that the Sia of monosialodihexosylganglioside (GM3) is mainly in the form of the internal 1,7-lactone 4a. Based on the known interaction between IL-4 and 1,7-lactone, the authors proposed that the high expression of GM3 in malignant melanocytes was instrumental to "disturb" the activity of the immune system near the tumors [65].

Recent Insights into the Biological Role of Neu5Ac 1,7-Lactone
The biological role of 4a has been recently investigated in the colonization of the human intestine by commensal bacteria, such as E. coli [63,64]. In fact, these 1,7-lactones are relatively common in the large intestine, supporting the role of this molecule as an essential source of bacterial nutrition. In this regard, a recent work by Horne et al. [63] investigated the role of the E. coli YjhC enzyme, and its involvement in sialic acid catabolism. Notably, differential scanning fluorimetry experiments, coupled with Phenotype MicroArray, revealed that N-acetylneuraminic acid 1a and its 1,7-lactone 4a, along with reduced nicotinamide adenine dinucleotide (NADH), could be the biologically relevant substrates of YjhC. Notably, the 1,7-lactone 4a was selected as a model compound for in silico docking experiments, based on the obtained crystal structure of YjhC, to support the binding of this molecule in the enzyme catalytic pocket.

Stability of the Lactones of Biological Interest and Questioning on the Real Presence of 1,7-Lactones of Sias in Biological Tissues
As outlined in this review, the 1,7-lactones of Neu5Ac and Neu5Gc have been shown to play important roles both physiologically and pathologically. However, the relative stability of these compounds is a key feature that needs to be addressed, especially when assessing them in biological samples using multi-step extraction and purification procedures. In this regard, during their synthesis in free form (see Sections 2.1 and 2.2) [17][18][19][20][21]66,78], Allevi's group clarified significant features regarding the stability of these molecules. In particular, lactone 4a was found to be unstable in protic solvents, such as methanol and water, eventually affording the corresponding Neu5Ac methyl ester or the free Neu5Ac [66,78]. The same authors also elucidated the behavior of these molecules in different acidic and basic environments. In particular, treatment with a strong acidic resin (for 1 h at r.t.) led to the formation of the γ-lactone 33, which was transformed in free Neu5Ac 1a, after some additional time (i.e., 120 h at 25 • C or 8 h at 80 • C-Scheme 16 and Table 1, entries 1-2). Analogous results were obtained after treatment with hydrochloric acid (HCl) 1M for 1 h at 80 • C of (Entry 3). Alternatively, treatment of 4a for 24 h at RT with a weak acid, such as AcOH 2M, led to the complete transformation into the γ-lactone 33 (Entry 4). A similar behavior was observed when the stability of 4a was tested under basic conditions. In particular, treatment with NaOMe for 30 min at 30 • C led to the formation of the γ-lactone 33 in very high yields (92%, entry 6). Similarly, the use of 1,1,3,3-tetramethylguanidine (TMG) 0.05 M at 25 • C for 9 min afforded the same compound, which was quantitatively transformed into Neu5Ac (Entry 7 and 8), after an additional time of 72 h at 25 • C (or 3h at 60 • C).
(Entry 3). Alternatively, treatment of 4a for 24h at RT with a weak acid, such as AcOH 2M, led to the complete transformation into the γ-lactone 33 (Entry 4). A similar behavior was observed when the stability of 4a was tested under basic conditions. In particular, treatment with NaOMe for 30 min at 30 °C led to the formation of the γ-lactone 33 in very high yields (92%, entry 6). Similarly, the use of 1,1,3,3-tetramethylguanidine (TMG) 0.05 M at 25 °C for 9 min afforded the same compound, which was quantitatively transformed into Neu5Ac (Entry 7 and 8), after an additional time of 72 h at 25 °C (or 3h at 60 °C). Scheme 16. General two-steps hydrolysis of compound 4a (see Table 1 for reaction conditions) [78]. Surprisingly, when the authors investigated the standard hydrolytic conditions used to cleave Sias derivatives from glycoconjugates [60], they found that treatment of 4a with AcOH 2M at 80 °C for 15 min yielded exclusively the γ-lactone 33 (entry 5 and scheme 17), which eventually evolved in Neu5Ac 1a after an additional time of 3h. Similarly, the C9 monoacetylated 1,7-lactone 4b, after 30 min at 80 °C in AcOH 2M, rapidly evolved in the corresponding γ-lactone 48 (>95% yield, quantification by NMR) [78]. On the other hand, the C4, C9 diacetylated 1,7-lactone 4c was stable under these conditions and, only after 105 min, a partial degradation occurred, which was accompanied by the formation of the sialic acid derivative 49 (around 30% yield, quantification by NMR) (Scheme 17).
Overall, these results support that these 1,7-lactones could not survive under the acidic hydrolysis conditions required for their cleavage from glycoconjugates in Zanetta's method [60], calling for a complete reevaluation of their presence in biological samples and of their physiological and pathological roles.
Moreover, in the same article, Allevi's group clarified some critical flaws of the GC-MS analytical protocol used to detect Sias, including the 1,7-lactonized derivatives, as heptafluorobutyrates [66]. Performing GC-MS and NMR analyses, using the authentic reference standards of free Sias (Neu5Ac 1a; 9-acetyl Neu5Ac 50; and the 4,9-diacetyl Neu5Ac 49 derivatives) and their 1,7-lactonized analogues (4a, 4b and 4c), they discovered interesting features of these compounds (Scheme 18  Table 1 for reaction conditions) [78]. Surprisingly, when the authors investigated the standard hydrolytic conditions used to cleave Sias derivatives from glycoconjugates [60], they found that treatment of 4a with AcOH 2M at 80 • C for 15 min yielded exclusively the γ-lactone 33 (entry 5 and Scheme 17), which eventually evolved in Neu5Ac 1a after an additional time of 3 h. Similarly, the C9 monoacetylated 1,7-lactone 4b, after 30 min at 80 • C in AcOH 2M, rapidly evolved in the corresponding γ-lactone 48 (>95% yield, quantification by NMR) [78]. On the other hand, the C4, C9 diacetylated 1,7-lactone 4c was stable under these conditions and, only after 105 min, a partial degradation occurred, which was accompanied by the formation of the sialic acid derivative 49 (around 30% yield, quantification by NMR) (Scheme 17). Overall, these results support that these 1,7-lactones could not survive under the acidic hydrolysis conditions required for their cleavage from glycoconjugates in Zanetta's method [60], calling for a complete reevaluation of their presence in biological samples and of their physiological and pathological roles.
Overall, they substantiated that 1,7-lactones of Sias, if present in biological samples, decompose under the acidic hydrolysis conditions used for their cleavage and they are accidentally generated as unexpected artifacts during the derivatizing step from their homologs acids [66].
Overall, they substantiated that 1,7-lactones of Sias, if present in biological samples, decompose under the acidic hydrolysis conditions used for their cleavage and they are accidentally generated as unexpected artifacts during the derivatizing step from their homologs acids [66].

Conclusions and Future Perspectives
As detailed in this review, the internal lactones of sialic acids play a central role in many physiological and pathological processes. However, recent advances in understanding their reactivity and stability have also exposed the limits of their identification and quantification from biological samples [66]. Clearly, milder hydrolytic strategies are needed to remove Sias from glycoconjugates and overcome the lability of 1,7-lactone derivatives in free form. This, together with the recent advances in the synthesis of authentic standard samples, could allow the development of new analytical methods to confirm their presence in biological matrices, eventually reassessing their physiological and pathological roles.