В рассмотрении клеточного метаболизма мы достигли теперь как бы поворотного пункта. До сих пор мы знакомились с тем, как главные типы питательных веществ — углеводы, жирные кислоты и аминокислоты, — расщепляясь, включаются по сходящимся катаболическим путям в цикл лимонной кислоты, чтобы передать свои богатые энергией электроны в дыхательную цепь. Перемещаясь по дыхательной цепи к кислороду, эти электроны поставляют энергию для синтеза АТР. Теперь нам предстоит рассмотреть анаболические пути. На этих путях химическая энергия в форме АТР и NADPH используется для синтеза клеточных компонентов из простых предшественников. Катаболизм и анаболизм протекают одновременно; при этом поддерживается динамическое стационарное состояние, так что расщепление клеточных компонентов, обеспечивающее клетки энергией, уравновешивается биосинтетическими процессами, которые создают и поддерживают в живых клетках присущую им упорядоченность.
Здесь уместно вспомнить (гл. 10) и лишний раз подчеркнуть некоторые организационные принципы биосинтеза.
1. Пути биосинтеза и пути расщепления тех или иных биомолекул, как правило, не идентичны. Эти пути могут включать какую-нибудь общую обратимую реакцию или даже несколько таких реакций, но у них всегда имеется хотя бы одна ферментативная стадия, по которой они различаются. Если бы катаболические и анаболические реакции катализировались одним и тем же набором ферментов, действующих обратимо, то никакая биологическая структура независимо от ее сложности попросту не могла бы существовать, потому что число клеточных макромолекул менялось бы в ответ на любые колебания концентраций молекул-предшественников.
2. Биосинтетические пути и соответствующие им катаболические пути контролируются разными регуляторными ферментами. Обычно регуляция соответствующих биосинтетических и катаболических путей осуществляется координированным образом, реципрокно, так что стимулирование биосинтетического пути сопровождается подавлением катаболического пути, и наоборот. Более того, биосинтетические пути регулируются обычно на одном из первых этапов. Это избавляет клетку от непроизводительных трат: она не расходует предшественники на синтез тех промежуточных продуктов, которые ей не понадобятся. Мы вновь убеждаемся на этом примере, что принцип экономии лежит в основе молекулярной логики живых клеток.
3. Требующие затраты энергии биосинтетические процессы обязательно сопряжены с поставляющим энергию расщеплением АТР, вследствие чего весь процесс в целом является практически необратимым, точно так же как в целом необратим катаболизм. Таким образом, общее количество энергии АТР (или NADPH), используемое на данном биосинтетическом пути, всегда превосходит то минимальное количество свободной энергии, которое требуется для превращения предшественника в биосинтетический продукт.
Рассмотрение биосинтетических процессов мы начнем с центрального биосинтетического пути, который в животных тканях приводит к образованию различных углеводов из неуглеводных предшественников. У всех высших животных биосинтез D-глюкозы — абсолютно необходимый процесс, потому что D-глюкоза крови служит единственным или главным источником топлива для нервной системы (в том числе и для мозга), а также для почек, семенников, эритроцитов и для всех тканей эмбриона. У человека один только мозг потребляет более 120 г глюкозы в сутки.
Рис. 20-1. Путь, ведущий от фосфоенолпирувата к глюкозо-6-фосфату, является общим для превращения многих предшественников в различные углеводы в животных тканях.
В организме животных D-глюкоза непрерывно синтезируется в строго регулируемых реакциях из более простых предшественников, таких, как пируват и некоторые аминокислоты, а затем поступает в кровь. Из неуглеводных предшественников образуются также и другие важные углеводы (рис. 20-1). Особенно большое значение имеет биосинтез гликогена, протекающий в печени и мышцах. Гликоген печени служит резервуаром глюкозы: из него образуется глюкоза, которая поступает в кровь. Мышечный же гликоген, распадаясь в процессе гликолиза, служит источником энергии АТР для мышечного сокращения. У животных образование D-глюкозы из неуглеводных предшественников называют глюконеогенезом (образование «нового» сахара). Важными предшественниками D-глюкозы являются у них лактат, пируват, глицерол, большинство аминокислот и промежуточные продукты цикла лимонной кислоты (рис. 20-1). Глюконеогенез протекает у животных главным образом в печени и значительно менее интенсивно — в корковом веществе почек.
Мы знаем, что в растительном мире огромные количества глюкозы, а также других углеводов образуются путем восстановления двуокиси углерода в процессе фотосинтеза (гл. 23). В отличие от растений у животных не происходит реального (net) превращения в новые молекулы глюкозы.
Carbohydrate Synthesis
Alternatively, carbohydrate synthesis by glycosidases is especially useful where a glycosyltransferase is not available or difficult to obtain.
Related terms:
CARBOHYDRATE BIOSYNTHESIS
Publisher Summary
This chapter discusses carbohydrate biosynthesis. Many bacteria have the ability to grow in media with acetate as the sole carbon source, and it follows that carbohydrate and other cellular components are synthesized from acetate. The scheme for the biosynthesis of hexose from acetate occurs in bacteria. The first step in the assimilation of acetate is the formation of acetyl-CoA either by the acetokinase-phosphotransacetylase pathway or by the action of acetyl-CoA synthetase that has been reported in a few species of bacteria. Acetate is incorporated into citrate and isocitrate by tricarboxylic acid cycle reactions. The key reaction in the assimilation of acetate is catalyzed by isocitritase. Isocitritase has been found in many species of bacteria, yeast, and fungi but not in higher animals. Another reaction unique to assimilation of acetate by microorganisms is the condensation of glyoxylate and acetyl-CoA to malate catalyzed by malate synthetase.
Extracellular polymeric substances in microbial biofilms
Thomas R. Neu , John R. Lawrence , in Microbial Glycobiology , 2010
5.2. Microbial carbohydrate metabolism
The biochemistry of carbohydrate synthesis and metabolism is well known for the intracellular space. Many of the cycles and pathways have been studied in detail and are part of microbial physiology textbooks. Recent investigations have indicated mechanisms of transport and excretion of polymers through the membrane and cell wall ( Whitfield, 2006 ; Whitfield and Naismith, 2008 ). However, not much is known about the fate of released EPS, their lifetime, turnover and possible recycling. The question posed is: for how long (time) or how far (distance) can the microorganisms control the polymers they have produced?
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Carbohydrates, Nucleosides & Nucleic Acids
Wei Zhao , . Wanyi Guan , in Comprehensive Natural Products II , 2010
6.02.2.3 Conclusion
A rule of thumb in classical complex saccharide synthesis is that the addition of each monosaccharide unit requires on an average seven steps. Although there have been striking improvements in synthetic methods in recent years, the synthesis of a trisaccharide, for example, can still take 10 or more steps. 87 The application of glycosidases to the synthesis of complex hetero-oligosaccharides represents a considerable saving in time and materials, and hence in overall cost.
The need for efficient synthesis of complex saccharides has stimulated major advances in glycosidase research. The more classical approach of substrate engineering aptly complements the construction of ‘superactive’ mutant glycosidases by means of rational design, or through directed evolution strategies using ingenious screening methods, such as a yeast three-hybrid system. The portfolio of genetically engineered glycosidases has expanded with novel activities, and the original glycosynthase concept was broadened to include mutants exercising other catalytic mechanisms. Particular carbohydrate structures that are in high demand can be produced by selective modification of sugar structures using specific glycosidases. The future multidisciplinary research on the interface of biochemistry, synthetic chemistry, and modeling will hopefully result in engineering new glycosidases for industrial uses and in overcoming more glycosidase-induced pathologies, which will be spurred by intensive structural and mechanistic studies on selected enzymes of interest.
Temporary ether protecting groups at the anomeric center in complex carbohydrate synthesis
1 Introduction
Anomeric protecting groups play important roles in carbohydrate synthesis . 1,2 These groups are generally introduced at the beginning of the synthesis to block the vulnerable anomeric center and are selectively removed at a late stage in the synthesis for subsequent transformation into glycosylation reagents. Therefore, the anomeric, temporary protecting groups are required to tolerate all the chemical manipulations during the synthesis of carbohydrate building blocks up to the glycosylation step. Thus precautious should be taken with choices of only delicate anomeric temporary protecting groups before initiation of the carbohydrate synthesis with a view to:
Good stability and tolerance under various conditions.
Regio- and stereoselective formation in a concise way to distinguish the anomeric hydroxyl groups from others. The formation of only one anomer is preferred.
Mild and orthogonal cleavage without affecting other protecting and functional groups.
Although a great variety of protecting groups are utilized in carbohydrate synthesis, only a few of them have been frequently used as anomeric temporary protecting groups. Apart from those that can be directly or latently activated for glycosylation, such as thioether, 3–8 pentenyl ether, 9,10 2-(2-propylthiol)benzyl ether, 11,12 o-iodobenzyl ether, 13 o-iodophenyl ether, 14 2′-iodo-2,6-dimethylbiphenyl-4-yl ether, 15 etc., the majority of the anomeric protecting groups are eventually removed to result in the free 1-OH for subsequent condensation with exotic leaving groups for further glycosylation. 16–23 Among them the ether type of protecting groups are most commonly used in the chemical synthesis of complex carbohydrates, mainly including allyl ether, p-methoxyphenyl (MP) ether, benzyl ether, p-methoxybenzyl (PMB) ether, silyl ether, etc. Although several reviews on the application of these ether protecting groups in carbohydrate chemistry have been published, 24–28 this chapter focuses on their performance as anomeric, temporary protecting groups. In this chapter, the most widely used allyl, p-methoxyphenyl (MP), benzyl, p-methoxybenzyl (PMB), and silyl ethers at the anomeric center are reviewed as comprehensively as possible, with a focus on their formation from stable or commercially available sugar substrates (i.e., free sugars A, acetates B, lactols C, and glycals D), their cleavage protocols, and the functional groups that are stable under these conditions. Selected examples of their application in the synthesis of complex carbohydrates are given in order to provide a better understanding on the choice of these anomeric ether protecting groups. a
Cell Glycobiology and Development; Health and Disease in Glycomedicine
T. Kawasaki , . Y. Kizuka , in Comprehensive Glycoscience , 2007
4.16.2 Biosynthesis of the HNK-1 Carbohydrate
The key step of the HNK-1 carbohydrate biosynthesis is GlcA transfer, since the inner structure, N-acetyllactosamine, is commonly found in a number of glycoconjugates ( Figure 1b ). Moreover, GlcA, only found in glycosaminoglycan chains, does not exist in N- and O-glycans except for the HNK-1 carbohydrate. The glucuronyltransferases that play a key role in the HNK-1 carbohydrate biosynthesis were cloned and well characterized. Terayama et al. had succeeded in cloning of glucuronyltransferase from rat brain and had named it GlcAT-P. 17 Soon after that, the second glucuronyltransferase, GlcAT-S, was cloned. 18 By Northern blot analysis using several tissues from adult rat, it is revealed that both GlcAT-P and GlcAT-S mRNA are expressed only in the nervous system. Moreover, these enzymes are able to transfer GlcA to the nonreducing end of N-acetyllactosamine, and when these genes are overexpressed in cultured COS-1 cells, the HNK-1 carbohydrate is actually expressed at the cell surface. However, GlcAT-P, compared with GlcAT-S, has wider expression range in brain and higher enzymatic activity, suggesting that GlcAT-P plays a major role in the HNK-1 carbohydrate biosynthesis. We investigated the detailed substrate specificity using purified native GlcAT-P from rat brain and recombinant soluble form of GlcAT-P and GlcAT-S 19 and revealed that these enzymes transfer GlcA not only to a glycoprotein acceptor, asialo-orosomucoid, but also to a glycolipid acceptor, paragloboside. Interestingly, the activity of GlcAT-P toward glycoprotein is markedly enhanced in the presence of sphingomyelin (SM), but that of GlcAT-S does not depend on the presence of phospholipids. In the case of glycolipid acceptor substrate, GlcAT-P essentially requires the presence of phospholipids such as phosphatidylinositol (PI) for its enzymatic activity. PI also enhanced the activity of GlcAT-S toward glycolipids, but its specific activity is much lower than that of GlcAT-P. The activity of these two enzymes toward various kinds of oligosaccharides also revealed interesting results. GlcAT-P strictly recognizes N-acetyllactosamine structure (Galβ1-4GlcNAc) as an acceptor substrate, whereas GlcAT-S can use other oligosaccharides such as lacto-N-biose (Galβ1-3GlcNAc), lactose (Galβ1-4Glc), or Galβ1-4Gal as a substrate. Moreover, among bi-, tri-, and tetraantennary N-linked oligosaccharides, GlcAT-P increasingly recognizes these oligosaccharide acceptors in proportion to the number of branches, whereas GlcAT-S strongly prefers triantennary structure. These results indicate that GlcAT-P and GlcAT-S may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system.
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The final step of the HNK-1 carbohydrate biosynthesis is sulfation of GlcA at 3-position ( Figure 1b ). One sulfotransferase involved in this sulfation step was cloned and named as HNK-1 ST. 20 The following results suggest that sulfation is necessary for HNK-1 carbohydrate functions. One, HNK-1-laminin interaction disappeared after removing the sulfate group from the HNK-1 carbohydrate. 9 Two, all the HNK-1 structure so far examined in the nervous system is in the sulfated form, and the nonsulfated form of this epitope has not been identified. 21 Curiously, HNK-1 ST mRNA is expressed not only in the nervous system but also in other tissues where the HNK-1 carbohydrate epitope does not exist, suggesting the possibility that HNK-1 ST may play some other roles in these tissues.
Synthesis: Carbon With Two Heteroatoms Each Attached by a Single Bond
4.05.1.1.2.(iii) By phase transfer catalyst
Phase transfer catalysis (PTC) is a convenient and useful method for carbohydrate synthesis because mild conditions can be used and reactions can be performed on large scale. Anomeric nucleophilic substitutions have been reported using mild PTC conditions for stereospecific entry to 1-thio-β- d -mannosides and 1-thio-β- l -rhamnosides . Glycosyl bromides and chlorides are treated with thiophenol as the nucleophile and tetrabutylammonium hydrogen sulfate (TBAHS) as the phase transfer reagent to afford the corresponding phenyl thioglycosides. Ethyl acetate is shown to be a superior solvent in comparison to dichloromethane because the thiols can react with the latter to form bis(4-nitrophenylthio)methane. Allyl mercaptan can also serve as a nucleophile .
Tetrabutylammonium thiocyanate and tetrabutylphosphonium bromide are also used in PTC for the synthesis of alkyl and aryl thioglycosides . The phosphonium salt is more effective than the ammonium salt.
Glycosylation and Posttranslational Processing of Thyroid-Stimulating Hormone: Clinical Implications
VII Regulation of TSH Biosynthesis and Glycosylation
We have shown that thyroid hormone deficiency caused increases in both TSH apoprotein and carbohydrate biosynthesis in cultured rat pituitaries ( Taylor and Weintraub, 1985a ). In hypothyroid pituitaries plus media [ 14 C]alanine incorporation in combined and free β subunits was 26 times normal and considerably greater than the 3.4‐fold increase seen in total protein; combined and free α showed no specific increase in apoprotein synthesis. [ 3 H]Glucosamine incorporation in combined α and βsubunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9‐fold increase seen in total protein; free α subunit showed no specific increase in carbohydrate synthesis ( Fig. 8 ). The GlcN/Ala ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined α but not for combined β, free α subunits or total proteins.
FIG. 8 . Ratio of hypothyroid to normal labeled precursor incorporation in TSH subunits and total proteins. Shown is the hypothyroid (TX) to normal (NL) ratio for [ 3 H]glucosamine incorporation present in the media (A) and the pituitary (B) and for [ 14 C]alanine incorporation present in the media (C) and the pituitary (D). Free β subunit carbohydrate content could not be determined by the methods used in these experiments and is illustrated by †. Significant differences in the ratios between total protein and TSH subunits were determined by Wilcoxon signed rank sum test (* p Taylor and Weintraub, 1985a ).
In summary, short‐term hypothyroidism selectively stimulated TSH β subunit apoprotein synthesis and carbohydrate synthesis of combined α and β subunits. Hypothyroidism also increased the relative glycosylation of combined α subunit. Thus, thyroid hormone deficiency appears to alter the rate‐limiting step in TSH assembly (i.e., β subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biologic function.
In contrast early biosynthetic studies had suggested that thyrotropin‐releasing hormone (TRH) caused a selective stimulation of TSH glycosylation ( Wilber, 1971 ; Ponsin and Mornex, 1983 ). Using subunit‐specific analytic methods, we have recently shown ( Taylor and Weintraub, 1985b ) that normal rat pituitaries stimulated with TRH for 24 hours showed a 3‐fold stimulation of labeled glucosamine incorporation into secreted TSH but no change in labeled alanine incorporation ( Fig. 9 ). The increased glycosylation was noted in both combined α and β subunits but not in the free α subunit. Preliminary lectin analysis of TSH glycopeptides after TRH treatment of hypothyroid mouse pituitaries revealed increased glucosamine incorporation into biantennary carbohydrate chains (Gesundheit and Weintraub, unpublished data). Moreover, the increased glycosylation of secreted TSH caused by TRH was shown to enhance its intrinsic bioactivity ( Menezes‐Ferreira and Weintraub, 1984 ).
FIG. 9 . TSH subunit incorporation of labeled precursors in normal pituitary incubates at 24 hours ± 1 ng/ml TRH. Demonstrated is [ 3 H]gluCosamine (left panels) and [ 35 S]methionine (right panels) incorporation in combined α (upper panels), combined β (middle panels), and intact TSH (lower panels). Significant differences between control and TRH treated samples are demonstrated by *p Taylor and Weintraub, 1985b ).
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Anomeric-Oxygen Activation for Glycoside Synthesis: The Trichloroacetimidate Method
2 Anomeric O-Alkylation
The direct O-alkylation of the anomeric center ( Scheme 1 , path C) by treatment of furanoses and pyranoses with base and then with simple alkylating agents, for instance an excess of methyl iodide or dimethyl sulfate, has long been known (1,3) . Surprisingly, no studies employing this simple method for syntheses of more-complex glycosides and saccharides have been reported prior to our work (1,37,38) .
In the beginning, direct anomeric O-alkylation seemed very unlikely to fulfill all of the requirements for glycoside and saccharide synthesis. Even when all remaining functional groups (generally hydroxyl groups) are blocked by protecting groups, the ring–chain tautomerism between the anomeric forms and the open-chain form ( Scheme 2 ) already gives three possible sites for attack of the alkylating agent. In addition, base-catalyzed elimination in the open-chain form of the sugar could be a destructive side-reaction. Therefore, the yield, the regioselectivity, and the stereoselectivity of such direct anomeric O-alkylation would not generally be expected to be outstanding. In any event, the process should be governed at least by the following factors:
Scheme 2 . 1-O-Alkylation and 1-O-Acylation (Irreversible Reactions).
the stability of the deprotonated species;
the ring–chain tautomeric equilibrium and its dynamics; and
the relative reactivities (nucleophilicities) of the three O-deprotonated species.
Because of the irreversibility of the O-alkylation reaction, kinetic regio- and stereo-control is required for selective product-formation. Therefore, selective formation of either α or β product seemed to be unattainable.
The first experiments with iodide derivatives of carbohydrates revealed that better alkylating agents are required (37) . However, excellent reactivity with corresponding trifluoromethanesulfonates (triflates) was observed, providing, for instance, with 2,3-O-isopropylidene- d -ribose and derivatives, depending on the reaction conditions, very high yields of either α— and β-inked disaccharides (37) . Surprisingly, even partial O-protection or, as recently discovered, O-nonprotection was compatible with this reaction (39–44) . The stereocontrol could be effected by intramolecular metal-ion complexation, by steric effects, and by taking advantage of the increased nucleophilicity of the equatorial anomeric oxide over the axial anomeric oxide [kinetic anomeric effect (45,46) ]. This method could even be employed in selective formation of α-glycosides of Kdo (47,48) . Thus, the direct anomeric O-alkylation constitutes an especially simple procedure for glycoside and saccharide synthesis, giving generally high yields and diastereoselectivities. The limitation to primary triflates was a major drawback for the general use of this anomeric O-alkylation in glycoside synthesis. However, this problem was recently overcome, at least in part, by modifying the reaction conditions (49) .
The Synthesis of C-linked Glycosides
Paul Meo , Helen M.I. Osborn , in Carbohydrates , 2003
The radical approach [ 69 ]
The radical approach for C— C bond formation is a popular method within organic chemistry. The use of radical chemistry in carbohydrate synthesis has certain advantages. Firstly the reaction conditions are very mild and tolerant of a range of functional and protecting groups. Anomeric radicals are also stable towards elimination and epimerisation. Most significantly, the chemistry required to incorporate an appropriate substituent at C-1, employed in the initial homolytic cleavage step, is common within carbohydrate chemistry. The use of such radical techniques can be subdivided into two classes, intermolecular and intramolecular reactions.
Yeasts
Yeast sugar metabolism
The principal metabolic fates of sugars in yeasts are the dissimilatory pathways of fermentation and respiration (shown in Figure 3 ) and the assimilatory pathways of gluconeogenesis and carbohydrate biosynthesis. Yeasts described as fermentative are able to use organic substrates (sugars) anaerobically as electron donors, electron acceptors, and carbon sources. During alcoholic fermentation of sugars, S. cerevisiae and other fermentative yeasts reoxidize the reduced coenzyme NADH to NAD (nicotinamide adenine dinucleotide) in terminal step reactions from pyruvate. In the first of these terminal reactions, catalyzed by pyruvate decarboxylase, pyruvate is decarboxylated to acetaldehyde, which is finally reduced by alcohol dehydrogenase to ethanol. The regeneration of NAD is necessary to maintain the redox balance and prevent the stalling of glycolysis. In alcoholic beverage fermentations (e.g., of beer, wine, and distilled spirits), other fermentation metabolites, in addition to ethanol and carbon dioxide, that are very important in the development of flavor are produced by yeast. These metabolites include fusel alcohols (e.g., isoamyl alcohol), polyols (e.g., glycerol), esters (e.g., ethyl acetate), organic acids (e.g., succinate), vicinyl diketones (e.g., diacetyl), and aldehydes (e.g., acetaldehyde). The production of glycerol (an important industrial commodity) can be enhanced in yeast fermentations by the addition of sulfite, which chemically traps acetaldehyde.
Figure 3 . Overview of sugar catabolic pathways in yeast cells. Reproduced from Walker (1998) Yeast Physiology and Biotechnology. Chichester, UK: John Wiley & Sons Limited.
Aerobic respiration of glucose by yeasts is a major energy-yielding metabolic route and involves glycolysis, the citric acid cycle, the electron transport chain, and oxidative phosphorylation. The citric acid cycle (or Krebs cycle) represents the common pathway for the oxidation of sugars and other carbon sources in yeasts and filamentous fungi and results in the complete oxidation of one pyruvate molecule to 2CO2, 3NADH, 1FADH2, 4H + , and 1GTP.
Of the environmental factors that regulate respiration and fermentation in yeast cells, the availability of glucose and oxygen is best understood and is linked to the expression of regulatory phenomena, referred to as the Pasteur effect and the Crabtree effect. A summary of these phenomena is provided in Table 6 .
Table 6 . Summary of regulatory phenomena in yeast sugar metabolism
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