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This work aims to synthesize new trehalase inhibitors selective towards the insect trehalase versus the porcine trehalase, in view of their application as potentially non-toxic insecticides and fungicides. The synthesis of a new pseudodisaccharide mimetic 8 , by means of a stereoselective α-glucosylation of the key pyrrolizidine intermediate 13 , was accomplished. The activity of compound 8 as trehalase inhibitor towards C. riparius trehalase was evaluated and the results showed that 8 was active in the μM range and showed a good selectivity towards the insect trehalase. To reduce the overall number of synthetic steps, simpler and more flexible disaccharide mimetics 9 - 11 bearing a pyrrolidine nucleus instead of the pyrrolizidine core were synthesized. The biological data showed the key role of the linker chain's length in inducing inhibitory properties, since only compounds 9 (α,β-mixture), bearing a two-carbon atom linker chain, maintained activity as trehalase inhibitors. A proper change in the glucosyl donor-protecting groups allowed the stereoselective synthesis of the β-glucoside 9 β, which was active in the low micromolar range (IC 50 = 0.78 μM) and 12-fold more potent (and more selective) than 9 α towards the insect trehalase., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Trehalase (EC 3.2.1.28) hydrolyzes the main haemolymph sugar of insects, trehalose, into the essential cellular substrate glucose. Trehalase in locust flight muscle is bound to membranes that appear in the microsomal fraction upon tissue fractionation, but the exact location in vivo has remained elusive. Trehalase has been proposed to be regulated by a novel type of activity control that is based on the reversible transformation of a latent (inactive) form into an overt (active) form. Most trehalase activity from saline-injected controls was membrane-bound (95%) and comprised an overt form (∼25%) and a latent form (75%). Latent trehalase could be assayed only after the integrity of membranes had been destroyed. Trehazolin, a potent tight-binding inhibitor of trehalase, is confined to the extracellular space and has been used as a tool to gather information on the relationship between latent and overt trehalase. Trehazolin was injected into the haemolymph of locusts, and the trehalase activity of the flight muscle was determined at different times over a 30-day period. Total trehalase activity in locust flight muscle was markedly inhibited during the first half of the interval, but reappeared during the second half. Inhibition of the overt form preceded inhibition of the latent form, and the time course suggested a reversible precursor-product relation (cycling) between the two forms. The results support the working hypothesis that trehalase functions as an ectoenzyme, the activity of which is regulated by reversible transformation of latent into overt trehalase.

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 microg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis., (Copyright 2009 Elsevier Ltd. All rights reserved.)

[reaction: see text] A strategy for the concise synthesis of trehazolamine, the aminocyclitol core of the potent trehalase inhibitor trehazolin, has been developed. The methodology takes advantage of photocyclization reaction of 1-methoxyethoxymethyl-3-pivaloxymethylpyridinium perchlorate to generate a bicyclic-aziridine intermediate, which is transformed under aziridine ring opening conditions to the key intermediate, 3,5-diacetoxy-3-pivaloxymethyl-4-(N-acetylamino)cyclopentene. In addition, the strategy is used in an enantio-divergent sequence for preparation of the natural (+)-trehazolamine and its unnatural (-)-enantiomer. In this route, the chiral auxiliary containing 1-(tetracetyl-alpha-D-glucosyl)-3-pivaloxymethylpyridinium perchlorate undergoes photocyclization to generate separable, diastereomeric bicyclic-aziridines, which are then independently transformed to enantiomeric 3,5-diacetoxy-3-pivaloxymethyl-4-(N-acetylamino)cyclopentenes.

A new trehazolin analogue, 1-thiatrehazolin, has been synthesized from carbohydrate precursors by a highly efficient route based on our previously developed ketone/oxime ether reductive carbocyclization reaction for the construction of the cyclitol ring and an intramolecular nucleophilic displacement reaction for the construction of the thiazoline ring. 1-Thiatrehazolin is a very potent, slow, tight-binding trehalase inhibitor. A structural model for trehalase inhibition by trehazolin and its analogues, based on the experimental results and supported by theoretical calculations, is proposed.

The main blood sugar of locusts is trehalose, which is hydrolysed to two glucose units by trehalase. Homogenates of locust flight muscles are rich in trehalase activity, which is bound to membranes. A minor fraction of trehalase is in an overt form while the remainder is latent, i.e. active only after impairing membrane integrity. Trehazolin, an antibiotic pseudosaccharide, inhibits locust flight muscle trehalase with apparent K(i)- and EC(50) values of 10(-8) mol l(-1) and 10(-7) mol l(-1), respectively. Trehazolin is insecticidal: 50 micro g injected into locusts completely and selectively blocked the overt form of muscle trehalase (with little effect on latent activity) and killed 50% of the insects within 24 h. Here, it is demonstrated for the first time that trehazolin causes dramatic hypoglycaemia. Injection of 10 micro g trehazolin caused glucose levels to fall by over 90% in 24 h, from 2.8 mmol l(-1) to 0.23 mmol l(-1), while trehalose increased from 61 mmol l(-1) to 111 mmol l(-1). Feeding glucose to the locusts fully neutralized the effects of a potentially lethal dose of trehazolin. This indicates that hypertrehalosaemia is not acutely toxic, whereas lack of glucose causes organ failure (presumably of the nervous system), and that sufficient haemolymph glucose can only be generated from trehalose by trehalase. The results also suggest that overt flight muscle trehalase is located in the plasma membrane with the active site accessible to the haemolymph. Trehalase inhibitors are valuable tools for studying the molecular physiology of trehalase function and sugar metabolism in insects.

Bicyclic azasugar glycomimetics related to castanospermine and trehazolin have been prepared from sugar carbodiimides via aminooxazoline intermediates; preliminary enzyme inhibition tests showed a marked dependence of the selectivity and potency towards alpha and beta-glucosidases upon the nature of the exocyclic substituent.

Trehazolin (1) is a trehalase inhibitor produced by Micromonospora coriacea. Biosynthesis of 1 was studied by feeding experiments with a variety of labeled precursors. Feeding experiments with [1-13C]- and [6-13C]-D-glucose revealed that the carbon skeletons of both a glucose residue and a cyclopentane ring moiety in 1 were each derived from glucose, and that C-C bond formation between C-1 and C-5 of glucose occurred during the cyclopentane ring formation. Furthermore, an experiment with [guanidino-13C, 15N2]-L-arginine revealed that two nitrogen atoms and a quaternary carbon atom involved in the aminooxazoline moiety of 1 originated from an amidino group of arginine. Further feeding experiments with [1-2H]-, [2-2H]-, [4-2H]-, [6,6-2H2]- and [1,2,3,4,5,6,6-2H7]-D-glucose as well as [1-13C]-D-fructose showed that deuteriums on C-1, C-3, C-4 and C-6 of glucose were retained during the formation of the cyclopentane ring moiety of 1.

Eight analogues of trehazolin were synthesized and tested for trehalase inhibitors. Deoxygenation of the cyclopentanepolyol moiety all decreased the inhibitory activity. Epimerization at the branching point of the cyclopentane ring did not so affect the potency. The cyclic isourea part was shown to be replaced with guanidine structure with a considerable decrease of activity. The 6'-fluoro-6'-deoxy derivative was still a strong inhibitor. It seems that trehazolin strictly mimies the substrate alpha, alpha-trehalose and any structural change and/or removal of the hydroxyl functions appreciably influence its potency. The present results led to finding 5-aminocyclopentane-1,2,3,4-tetraols to be new lead compounds for glycohydrolase inhibitors.

Mechanisms of enzyme inhibition by trehazolin, a new inhibitor of trehalase (Ando et al. (1991) J. Antibiot. 44, 1165), were investigated using purified soluble silkworm trehalase and other glycosidases. Trehazolin inhibited trehalase with an IC50 value of 27 nM, whereas some other exo-alpha-glucosidases were inhibited only weakly, with IC50 values ranging from 7 to 370 microM. Other glycosidases tested were not inhibited by 500 microM trehazolin. The inhibition of trehalase by trehazolin was competitive with respect to trehalose. A notable feature of the inhibition was a slow progression of the association and dissociation of the enzyme-inhibitor complex. Preincubation of the enzyme and the inhibitor at 37 degrees C potentiated the inhibition by 10-times in a time-dependent manner up to 6 h. Dialysis of the inactivated enzyme recovered the enzymatic activity very slowly, and the rate constant for the dissociation at 37 degrees C was (7.3).10(-2) h-1. Trehalamine, a deglucosylated form of trehazolin, inhibited both silkworm trehalase and exo-alpha-glucosidases only weakly. The inhibition of trehalase by trehalamine was reversible. Rat isomaltase inhibition by trehazolin and sucrase inhibition by trehalamine were also reversible. Taken together, trehazolin is a specific slow, tight-binding inhibitor of trehalase, and the glucose moiety of the inhibitor is essential to the tight binding.

Trehazolin, a new trehalase inhibitor isolated from the culture broth of Micromonospora, was reported to be a highly specific inhibitor for porcine and silk worm trehalases with IC50 values of 5.5 x 10(-9) and 3.7 x 10(-9) M, respectively (O. Ando, H. Satake, K. Itoi, A. Sato, M. Nakajima, S. Takashi, H. Haruyama, Y. Ohkuma, T. Kinoshita, and R. Enokita (1991) J. Antibiot. 44, 1165-1168). We also found that trehazolin is a very powerful and quite specific inhibitor against purified pig kidney trehalase, giving an IC50 value of 1.9 x 10(-8) M. Lineweaver-Burk plots showed that this compound was a competitive inhibitor of the trehalase. However, even at concentrations of 200 micrograms/ml, trehazolin did not inhibit the rat intestinal maltase or sucrase, yeast alpha-glucosidase or almond beta-glucosidase. Validoxylamine A and validamycin A, two other trehalase inhibitors, showed potent competitive inhibition against purified pig kidney trehalase, with IC50 values of 2.4 x 10(-9) and 2.5 x 10(-4) M, respectively. On the other hand, validoxylamine A was almost inactive against rat intestinal sucrase and maltase, with some inhibition being observed at millimolar concentration. A number of other glucosidase inhibitors, such as MDL 25637, castanospermine, and deoxynojirimycin were also tested against the purified trehalase and showed reasonable inhibitory activity.

Synthesis of 5-epi-trehazolin (trehalostatin) (2) was accomplished via the crucial intermediate, epoxide (6 alpha), from D-glucose. The stereochemistry of epoxide (6 alpha) and its isomer (6 beta) which were obtained from Sharpless epoxidation, was determined by comparison between the NMR relaxation times of relevant protons.

Synthesis of trehazolin beta-anomer (3) from a D-glucose derived azido alcohol (4), was accomplished. 2-Chloro-1-methylpyridinium iodide was used in place of 2-chloro-3-ethylbenzoxazolium tetrafluoroborate as a means of preventing concomitant anomerization. Evaluation of compound (3) reveals that the stereochemistry of the anomeric position is significant for generation of inhibitory activities towards trehalases.

The 3-keto derivatives of trehalose and sucrose respectively were prepared by a one-step enzymatic route using D-glucoside 3-dehydrogenase from Flavobacterium saccharophilum. Reductive amination of 3-ketotrehalose with ammonium acetate and sodium cyanoborohydride gave three compounds, 3-amino-3-deoxy-alpha-D-allopyranosyl alpha-D-glucopyranoside, 3-amino-3-deoxy-alpha-D-glucopyranosyl alpha-D-glucopyranoside (3-trehalosamine) and 3-amino-3-deoxy-alpha-D-mannopyranosyl alpha-D-glucopyranoside. From the reductive amination of 3-ketosucrose, 3-amino-3-deoxy-alpha-D-allopyranosyl beta-D-fructofuranoside and 3-amino-3-deoxy-alpha-D-glucopyranosyl beta-D-fructofuranoside were obtained. The antibiotic and glycohydrolase inhibitory activities of these 3-amino-3-deoxy derivatives were determined.