Caged luciferins and related compounds and methods

Abstract

The present invention relates to substrate molecules suitable for monitoring enzymatic activity via chemiluminescence. The substrates are provided via a new synthetic method that uses new intermediate products, which are more robust, generate fewer side products, and lead to higher yields. The precursors can be converted to caged luciferins such as peptide-linked aminoluciferins or glycosyl luciferins, useful for assessing enzymatic activity such as protease or glycosidase activity.

Description

[0001] Caged luciferins and related compounds and methods

[0002] Field of the invention

[0003] The present invention relates to substrate molecules suitable for monitoring enzymatic activity via chemiluminescence. The substrates are provided via a new synthetic method that uses new intermediate products, which are more robust, generate fewer side products, and lead to higher yields. The precursors can be converted to caged luciferins such as peptide-linked aminoluciferins or glycosyl luciferins, useful for assessing enzymatic activity such as protease or glycosidase activity.

[0004] Background art

[0005] In the recent decades luminescent imaging has emerged as a powerful tool to monitor enzymatic activity. Luminescent imaging generally occurs via the oxidation of small molecules, where no external light source is needed, a high signal-to-noise ratio can be obtained, and the background signal is usually low as compared to fluorescence. Numerous methods have been reported for luminescent imaging, as for instance for sensing reactive oxygen and nitrogen species, imaging of cancer cells, and the in vivo quantification of glucose uptake.

[0006] For instance, White and co-workers reported on the use of the D-luciferin derivative aminoluciferin (aLuc), which can be used in proteolytic assays since the amine can be coupled to the C-terminus of a peptide (E. H. White et al., J. Am. Chem. Soc. 1966, 88, 2015-2019.) The free 6'-amino group of aLuc appeared to be crucial to preserve its luminescent properties (as similar for the 6'-hydroxy in D-luciferin), modification of this moiety usually results in quenching of the luminescence. These quenched luminescent probes have also been referred to as caged luciferins and can be used in luminescent enzymatic assays, as upon enzymatic activity the free luciferin is released which can be quantified. Luciferin is oxidized into the excited oxyluciferin by the catalytic action of luciferase and subsequent relaxation back into the ground state releases photons. Numerous luminescent probes have been developed according to this uncaging principle and are widely used with a variety of enzymes among which hydrolases and proteases, see for instance W02020 / 079155. Podsiadly et al. describe progress in the synthesis of firefly luciferin derivatives (2019, dyes and pigments, DOI: 10.1016 / J.DYEPIG.2019.107627).

[0007] Glycosylation, the cell surface expression of carbohydrate structures on proteins and lipids, is found in all the domains of life. The glycosylation pattern of cells is produced by glycosyltransferases, detected by lectins, and can be degraded by glycosidases, of which many have already been identified. The breakdown of glycans is achieved by glycosidase enzymes that are specific for their monosaccharide substrate and are expressed by a human host, by bacteria, and by viruses. Aberrations in glycosidase expression and / or activity can be used to diagnose various pathologies. For example, in various cancer types, the overexpression of specific glycosidases can be used as a prodrug targeting strategy or as biomarker. Furthermore, in infectious diseases such as influenza, glycosidase activity assays may be used as a diagnostic tool to detect upregulated neuraminidase activity.

[0008] The monitoring ofglycosidase activity forvarious diagnostic applications has been achieved by the preparation of synthetic substrates that produce a detectable signal upon enzymatic conversion, as described by Wu et al., Curr. Opin. Chem. Biol. 2019, 53, 25-36. The use of luminescent glycosidase substrates could facilitate miniaturization of the assays since no external light source is required which may ultimately make point-of-care (POC) or fast clinical interceptions feasible. To this end, various synthetic substrates have been developed to monitor enzymatic activity with a luminescent output, as also demonstrated for proteases such as in recent work on the main protease (MPro) of SARS-CoV-2 (see US20210371461A1).

[0009] The use of glycosyl luciferins has been explored by Goode and co-workers in 1990, demonstrating that the coupling of D-luciferin to a monosaccharide can be used to monitor glycosidase activity. Key in this approach is that the glycosyl luciferin is not a substrate for the luciferase and hence does not produce a light signal. Only when this substrate is hydrolyzed by a glycosidase does it liberate D-luciferin which can be converted into a light signal that is proportional to the glycosidase activity. This occurs through the action of firefly luciferase. However, the chemical lability of the luciferin moiety makes the synthesis of glycosyl luciferins synthetically challenging (see Amess et a., Carbohydr. Res. 1990, 205, 225-233.) The luciferin moiety is known to be prone to oxidation to produce dehydroluciferin. In addition, the chiral center on luciferin is prone to racemization to afford L-luciferin. Both dehydroluciferin and L-luciferin inhibit luciferase activity and interfere with the monitoring of glycosidase activity. The luciferin moiety is typically constructed by the condensation of a benzothiazole-nitrile precursor with D-cysteine. However, due to the electrophilic nature of the nitrile moiety, it is prone to react with water to form the amide, or with methanol to afford the corresponding imidate. These factors all complicate the design of a synthetic scheme that can provide glycosyl luciferins in a high yield an purity.

[0010] Measuring glycosidase activity is important to monitor any aberrations in carbohydrate hydrolase activity, but also for the screening of potential glycosidase inhibitors. To this end, synthetic substrates are needed which provide an enzyme-dependent read-out upon hydrolysis by the glycosidase. There is a need for improved caged luciferins. There is a need for improved methods for the provision of caged luciferins. There is a need for more pure caged luciferins.

[0011] Summary of the invention

[0012] Herein, we present new routes for the synthesis of caged luminescent compounds such as carbohydrates, which can be used for determining glycosidase activity with a luminescent reporter molecule. Peptide analogues can be used for determining protease activity. The substrates were validated with glycosidase and revealed a clear linear range and enzyme-dependent signal upon in situ generation of the luciferin moiety from the corresponding nitrile precursors. It was shown that these compounds could directly be synthesized from unprotected glycosyl-a-fluorides in a two-step procedure (Fig. 1 B) with good yields (up to 75%). The intermediate methyl imidate was a key intermediate which also reacted with D-cysteine to give the corresponding D-luciferin substrate, rendering this a highly attractive method for synthesizing glycosyl luciferins.

[0013] In some embodiments temporary hydration of the luciferin nitrile precursor is used, leading to the less reactive, but more stable, amide, which can undergo subsequent glycosylation, for instance with protected glycosyl a-bromides (Fig. 1 B). In other embodiments, unprotected a- glycosyl fluorides are used to introduce the luciferin moiety, which under base-promoted glycosylation conditions leads to the corresponding methyl imidate. This intermediate can react with D-cysteine to give the corresponding D-luciferin moiety. Thus the invention allows the avoidance of side product formation through the provision of improved synthetic strategies and of more stable intermediates.

[0014] Provided is a method for producing a caged luciferin compound, the method comprising the steps of: i) providing an intermediate that is a compound of general formula (I): wherein X is O or NH; Q is O or NH; R is -NH2, -0-Ci-io(cyclo)alkyl, -S-Ci-io(cyclo)alkyl, -O-(5-10 membered (hetero) ary I), or -S-(5-10 membered (hetero) ary I); and cage is a glycosyl moiety or a peptide; ii) optionally converting the provided compound of general formula (I) to a compound of general formula (l-CN): iii) contacting the compound with cysteine to provide a caged luciferin compound of formula (I- probe):

[0015] In preferred embodiments the intermediate of general formula (I) is of general formula (l-imidate): wherein c is Ci-4alkyl, preferably -CH3 or -CH2CH3, more preferably -CH3. In other preferred embodiments the intermediate of general formula (I) is of general formula (l-amide), wherein preferably step ii) is not optional:

[0016] In such cases step ii) preferably comprises treatment of the compound of general formula (l-amide) with an acid anhydride such as trifluoroacetic anhydride.

[0017] In some embodiments step i) comprises one or more of the following sub-steps: i-a) hydrolyzing hydroxybenzo[c / |thiazole-2-carbonitrile or aminobenzo[c / |thiazole-2-carbonitrile to form hydroxybenzo[c / |thiazole-2- carboxamide or aminobenzo[c / |thiazole-2-carboxamide, such as through acid hydrolysis using sulfuric acid; i-b) reacting the benzo[c / |thiazole-2-carboxamide with a protected glycosyl a- halide such as an a-bromide to obtain a protected compound of general formula (I); i-c) optionally deprotecting the protected compound of general formula (I) to obtain a deprotected compound of general formula (I).

[0018] In preferred embodiments step i) comprises reacting hydroxybenzo[c / ]thiazole-2-carbonitrile with a glycosyl a-fluoride to provide a compound of general formula (l-imidate). cage can be of general formula (II): wherein m is 0 or 1 ; n is 1 , 2, or 3; oh is H, -OH, -Oh’, -NH2, -NHh’, -Nh’2, -NHSO3H, -NHSO3h’, - COOH, -COOh’, -OSO3H, or -OSO3I1’; q is H, -CH2-OH, -CH2-OI1’, -CH2OSO3H, -CH2OSO3I1’, - COOH, -COOh’, -CH2-(CH(OH))I-6-H, or -CH2-(CH(Oh’))i-6-H; h is H, -OH, -CH2OH, -Oh’, - CH20h’,-NH2, -NHh’, -Nh’2, -COOH, or -COOh’; and h’ is in each instance independently p’ or a C1-8 alkyl or acyl that is optionally cyclic and that is optionally unsaturated and that is optionally halogenated; p' is in each instance independently a protecting group; wherein an instance of q or oh or h may be a further moiety of general formula (II) to form an oligosaccharide-type glycosyl moiety. Preferably m is 1 ; and / or n is 2 or 3; and / or oh is H, -OH or -NH-C(=O)CH3; and / or q is H or -CH2-OH or -CH2-CH(OH)-CH(OH)-CH(OH)-H; and / or h is H, -COOH, or -COOCH3. Suitable examples of cage are: galactopyranosyl

[0019] Step iii) can comprise contacting the compound with from 1 -10 equivalents of cysteine such as 2 equivalents of cysteine, wherein the contacting is preferably in an aqueous buffer such as 25 mM HEPES, 125 mM NaCI, and 0.5 wt.-% BSA, at pH 7.4 or 7.2, wherein the cysteine is preferably D-cysteine, wherein the cysteine is preferably an acid addition salt such as cysteine HCI.

[0020] Also provided is a compound of general formula (I) or a salt thereof: wherein X is O or NH; R is -NH2, -OCH3, or -OCH2CH3; and cage is a glycosyl moiety or a peptide, wherein the compound is not compound 21 or 22 as shown in the examples. Preferred compounds are compounds 6, 7, 8, 9, 10, 11 , 23, 29, and 30 as shown in the examples. Also provided is a kit of parts comprising i) such a compound; and ii) cysteine, preferably D-cysteine.

[0021] Also provided is a method for determining glycosidase or protease activity, the method comprising the steps of: i) providing one or more compounds as described above; ii) contacting the provided compounds with cysteine to provide caged luciferin compounds of formula (l-probe); iii) contacting the caged luciferin compounds with the glycosidase or protease to provide free luciferin or aminoluciferin, wherein the glycosidase is preferably glucosidase, galactosidase, xylosidase, or neuraminidase; iv) contacting the free luciferin or aminoluciferin with luciferase to generate a luminescent signal.

[0022] Description of embodiments

[0023] The inventors developed advantageous new strategies for obtaining luciferins caged with a carbohydrate or peptide. Accordingly the method according to the invention is a method for producing a caged luciferin compound, the method comprising the steps of: i) providing an intermediate that is a compound of general formula (I): wherein

[0024] X is O or NH;

[0025] Q is O or NH;

[0026] R is -NH2, -0-Ci-io(cyclo)alkyi, -S-Ci-io(cyclo)alkyl, -O-(5-10 membered (hetero) ary I), or -S-(5-10 membered (hetero) ary I); and cage is a glycosyl moiety or a peptide; ii) optionally converting the provided compound of general formula (I) to a compound of general formula (l-CN): iii) contacting the compound with cysteine to provide a caged luciferin compound of formula (l-probe):

[0027] These steps are preferably performed in numerical order. In some embodiments X is O. In some embodiments X is NH. In some embodiments Q is O. In some embodiments Q is NH and X is NH. In some embodiments Q is NH and X is O. In some embodiments Q is O and X is NH. In some embodiments Q is O and X is O.

[0028] R is -NH2, -0-Ci-io(cyclo)alkyl, -S-Ci-io(cyclo)alkyl, -O-(5-10 membered (hetero) ary I), or -S- (5-10 membered (hetero) ary I). In some embodiments R is -NH2. This is preferred for embodiments wherein X is O. When X is NH, R is preferably -0-Ci-io(cyclo)alkyl, -S-Ci-io(cyclo)alkyl, -O-(5-10 membered (hetero) ary I), or -S-(5-10 membered (hetero) ary I). When X is NH, R is preferably -O-C1- io(cyclo)alkyl or -O-(5-10 membered (hetero) ary I), most preferably -0-Ci-io(cyclo)alkyl. C1- io(cyclo)alkyl is preferably Ci-8(cyclo)alkyl, more preferably Ci-6(cyclo)alkyl, more preferably C1- 4alkyl such as methyl, ethyl, propyl, or butyl. (5-10 membered (hetero)aryl) is preferably 5-6 membered heteroaryl such as pyridinyl or imidazolyl, or 6-membered aryl such as phenyl.

[0029] A compound of general formula (I) if preferably of general formula (III):

[0030] Luciferin is well known as a substrate for luciferase. The caging of compounds is a known strategy for rendering such compounds temporarily inaccessible and / or undetectable and / or inactive. For instance caged luminescent compounds are not luminescent while caged, but the removal of the caging moiety, for instance through enzyme activity, can restore luminescence. A caged luciferin is a luciferin that has been derivatized in such a way that is no longer a substrate for luciferase. In the invention, the caging moiety is the glycosyl moiety or the peptide that is represented by cage as depicted in general formula (l-probe), which depicts a caged luciferin. When enzymatic activity, or another suitable reaction, hydrolyses the bond between cage and the remainder of general formula (l-probe), luciferin is released when Q is O, and aminoluciferin is released when Q is NH. Both are good substrates for luciferase enzymes and both can thus generate a luminescent signal. cage is a glycosyl moiety or a peptide. A skilled person understands that cage can be protected using protecting groups, but that cage can also be free, for instance when no groups need protection. Cage is suitably selected in such a way that it is, or that it can easily be converted into, a substrate for a glycosidase enzyme or a protease enzyme. This can help select a suitable cage.

[0031] In some embodiments cage is a peptide. In such cases, the remainder of general formula (I) is preferably linked to the peptide at its C-terminus, so that when Q is NH an amide bond is formed. A skilled person can select a suitable peptide moiety, as the nature of the peptide moiety relates to the intended use - when use of a probe with a certain protease is intended, a matching peptide moiety should be selected. Suitable peptides are tripeptides such as p-Ala-Gly-Arg, p-Ala- Gly-Lys, or tripeptides where the central glycine residue of those two tripeptides is replaced by another amino acid, preferably another proteinogenic amino acid.

[0032] In some embodiments cage is a glycosyl moiety. A glycosyl moiety is a univalent free radical or substituent structure obtained by removing the hemiacetal hydroxyl group from the cyclic form of a saccharide such as a monosaccharide or an oligosaccharide. A skilled person can select a suitable glycosyl moiety, as the nature of the glycosyl moiety relates to the intended use - when use of a probe with a certain glycosidase is intended, a matching glycosyl moiety should be selected. In such cases, the remainder of general formula (I) is preferably linked to the glycosyl moiety at an anomeric position, so that when Q is O a glycosyl bond is formed.

[0033] In preferred embodiments cage is of general formula (II): wherein m is 0 or 1 ; preferably m is 1 ; n is 1 , 2, or 3; preferably n is 2 or 3; when m is 0, n is preferably 1 or 2; oh is H, -OH, -Oh’, -NH2, -NHh’, -Nh’2, -NHSO3H, -NHSO3h’, -COOH, -COOh’, -OSO3H, or -OSO3I1’; preferably oh is H, -OH or -NH-C(=O)CH3; q is H, -CH2-OH, -CH2-Oh’, -CH2OSO3H, -CH2OSO3h’, -COOH, -COOh’, -CH2-(CH(OH))I- 6-H, or -CH2-(CH(Oh’))i-6-H; preferably q is H or -CH2-OH or -CH2-CH(OH)-CH(OH)-CH(OH)-H; preferred -CH2-(CH(OH))I-6-H is -CH2-(CH(OH))2-4-H, more preferably -CH2-(CH(OH))2-3-H. Preferred -CH2-(CH(Oh’))i-6-H is -CH2-(CH(Oh’))2.5-H, more preferably -CH2-(CH(Oh’))2-3-H. h is H, -OH, -CH2OH, -Oh’, -CH20h’,-NH2, -NHh’, -Nh’2, -COOH, or -COOh’; preferably h is H, -COOH, or -COOCH3; h’ is in each instance independently p’ or a C1-8 alkyl or acyl that is optionally cyclic and that is optionally unsaturated and that is optionally halogenated; a preferred C1-8 alkyl or acyl is a C1-6, more preferably a C1 -4 alkyl or acyl; particularly preferred is C1 alkyl or C2 acyl; p' is in each instance independently a protecting group; wherein an instance of q or oh or h may be a further moiety of general formula (II) to form an oligosaccharide-type glycosyl moiety. Preferably the oligosaccharide comprises from 2 to at most 100 moieties of general formula (II), more preferably from 2 to at most 50, still more preferably from 2 to at most 20, more preferably from 2 to at most 10, or from 3 to at most 9, or from 4 to at most 8, or from 5 to at most 7, or about 6. In other preferred embodiments q and oh and h do not represent such a further moiety of general formula (II).

[0034] In a preferred embodiment of general formula (II), m is 1 ; n is 2 or 3; oh is H, -OH or -NH- C(=O)CH3; q is H or -CH2-OH or -CH2-CH(OH)-CH(OH)-CH(OH)-H; and h is H, -COOH, or - COOCH3. p' is a protecting group. Preferably p' is a protecting group that is suitable for SPPS strategies or for solution carbohydrate chemistry. The protecting group is preferably acid-labile. As used herein, a protecting group has its customary meaning of a group that is linked to the heteroatom in such a way as to reduce its reactivity, or to protect it from certain conditions. After having served their function, protecting groups can generally be removed again using routine chemistry. A skilled person is well aware of protecting groups and can select useful protecting groups for p’ for particular groups or atoms to be protected, reaction conditions, storage conditions, purification techniques, or available deprotection techniques. A helpful reference in this regard is Greene's Protective Groups in Organic Synthesis, Wuts & Greene, DOI:10.1002 / 0470053488.

[0035] In preferred embodiments p', when linked to a nitrogen atom, is 9-fluorenylmethyl carbamate (Fmoc), AButyl carbamate (Boc), benzyl carbamate (Cbz), acetamide (Ac), trifluoroacetamide, phthalimide, benzylamine (Bn), triphenylmethylamine (Tr, or Trt), or tosylamide (Ts). Preferred base-labile embodiments for p' are Fmoc, Cbz, Ac, trifluoroacetamide, and phthalimide, more preferably Fmoc or Cbz, most preferably Fmoc. For its reliability in SPPS, it is most preferred that p' be Fmoc.

[0036] In preferred embodiments p', when linked to an oxygen atom, is acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxyethoxymethyl (MEM), dimethoxytrityl, bis-(4-methoxyphenyl)phenylmethyl (DMT), methoxymethyl (MOM), methoxytrityl (MMT), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methylthiomethyl, pivaloyl (Piv), tetra hydro pyranyl (THP), tetrahydrofuranyl (THF), trityl (Tr), silyls such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS), or ethoxyethyl (EE).

[0037] For glycosyl moieties, preferred embodiments of cage are selected from: lactosyl fructosyl

[0038] More preferably cage is glucopyranosyl, galactopyranosyl, xylopyranosyl, or sialyl. In some embodiments cage is glucopyranosyl, galactopyranosyl, or xylopyranosyl. In some embodiments cage is sialyl. Step I) - provision of an intermediate

[0039] In step i) of the method a compound of general formula (I) is provided. Compounds of general formula (I) are synthetically accessible using known techniques, for instance as described in the examples or as described in EP23174019. When Q is O, cage is preferably a glycosyl moiety. When Q is NH, cage is preferably a peptide. In some embodiments, Q is O. In some embodiments, Q is NH. Most preferably Q is O. Such compounds are of general formula (l-gly), more preferably (lll-gly).

[0040] Compounds wherein Q is NH are of general formula (l-pep), preferably (Ill-pep).

[0041] For a compound of general formula (l-gly), cage is preferably a glycosyl moiety. Such a compound is of general formula (l-glycosyl).

[0042] For a compound of general formula (l-pep), cage is preferably a peptide. Such a compound is of general formula (l-peptide).

[0043] In some embodiments the intermediate of general formula (I) is of general formula (I- i mi date): wherein c is Ci-4alkyl, preferably -CH3 or -CH2CH3, more preferably -CH3. These embodiments can be preferred because methods using such intermediates require fewer steps. In preferred methods, step i) comprises reacting hydroxybenzo[c / |thiazole-2-carbonitrile with a glycosyl a-fluoride to provide a compound of general formula (l-imidate), preferably wherein Q is O, more preferably wherein the compound is also of general formula (l-glycosyl). The hydroxybenzo[c / |thiazole-2- carbonitrile is preferably 6-hydroxybenzo[c / ]thiazole-2-carbonitrile to yield a compound of general formula (III), more preferably (lll-gly).

[0044] Glycosyl a-fluorides can advantageously be used without protecting groups, further shortening the procedure. They can be commercially available, or can be formed from peracetylated building blocks, for instance as described in the Examples. Their use was also surprisingly found to improve reaction yields. Preferred glycosyl a-fluorides are of general formula (IV): wherein q, oh, n, m, and h are as defined for general formula (II), and wherein h is equatorial and its adjacent F is axial. Preferably oh is H, -OH or -NH-C(=O)CH3; preferably q is H, -CH2-OH, or -CH2-(CH(OH))I-6-H, more preferably q is H or -CH2-OH or -CH2-CH(OH)-CH(OH)- CH(OH)-H; preferably h is H, -OH, -CH2OH, more preferably h is H. Preferably q and oh and h do not represent a further moiety of general formula (II). Preferred embodiments of general formula (IV) are shown below, of which glucopyranosyl a-fluoride, galactopyranosyl a-fluoride, xylopyranosyl a-fluoride, and sialyl a-fluoride are more preferred, and glucopyranosyl a-fluoride, galactopyranosyl a-fluoride, and xylopyranosyl a-fluoride still more preferred. lactosyl a-fluoride fructosyl a-fluoride

[0045] When reacting a hydroxybenzo[c / |thiazole-2-carbonitrile, preferably 6- hydroxybenzo[c / ]thiazole-2-carbonitrile, with a glycosyl a-fluoride, a compound of general formula (l-imidate) is readily formed. This reacting is preferably performed using an excess of the glycosyl a-fluoride as compared to the hydroxybenzo[c / |thiazole-2-carbonitrile, such as about 2-5 equivalents, preferably about 3 equivalents. The reacting is preferably in the presence of a base, such as a metal hydroxide, for instance CaOH2. The reacting is preferably at about 15 to about 25 °C, such as at about 20 °C, and the reacting is preferably in a protic solvent such as a C1 -C4 alkyl alcohol, optionally mixed with water, for instance in methanol, ethanol, or propanol, preferably comprising about 10-40 vol.% water, such as 25 vol.% water. The reaction is preferably allowed to proceed for about 8 to about 32 hours, such as about 16 hours.

[0046] The intermediate of general formula (I) can also be of general formula (l-amide):

[0047] In such as case, step i) preferably comprises one or more of the following sub-steps: i-a) hydrolyzing hydroxybenzo[c / |thiazole-2-carbonitrile, preferably 6- hydroxybenzo[c / ]thiazole-2-carbonitrile, to form hydroxybenzo[c / |thiazole-2- carboxamide, such as through acid hydrolysis, for instance using sulfuric acid; i-b) reacting the hydroxybenzo[c / |thiazole-2-carboxamide with a protected glycosyl a-halide such as an a-bromide, or with a protected peptide, to obtain a protected compound of general formula (I); i-c) optionally deprotecting the protected compound of general formula (I) to obtain a deprotected compound of general formula (I).

[0048] Preferably, all three sub-steps are comprised, preferably in alphabetical order.

[0049] Hydrolysis of step i-a) is generally quantitative and takes about 1 -5 hours such as about 3 hours. The acid such as sulfuric acid can be used as the solvent.

[0050] In step i-b) the hydroxybenzo[c / |thiazole-2-carboxamide is preferably used in excess such as about 2-20 equivalents, preferably about 3-10 equivalents, such as about 5 equivalents. It is preferably performed in the presence of a basic metal salt such as a carbonate, preferably potassium carbonate, at a small excess as compared to the cage, such as 1 .05-1 .4 equivalents, preferably 1 .2 equivalents. The reaction is preferably allowed to proceed for about 6-48 hours such as about 10-30 hours, preferably about 20 hours. The reaction is preferably performed at about 30- 90 °C such as about 40-70 °C, preferably about 50-65 °C, preferably about 60 °C. The reaction is advantageously performed in a biphasic system, using water and an immiscible organic solvent such as chloroform, in about equal amounts. In such a case, a phase transfer catalyst (preferably about 0.1 equivalents) can be used, such as TBAB.

[0051] In step i-c) the deprotection depends on the protecting groups that were used. For acetylated glycosyl moieties, deprotection can be performed using standard chemistry, for instance using a methoxide such as NaOMe, preferably at catalytic quantity, in a protic solvent such as methanol or ethanol, preferably methanol.

[0052] A protected glycosyl a-halide such as an a-bromide can be any suitable one. Preferred protected glycosyl a- halides are of general formula (V): wherein q, n, m, and h are as defined for general formula (II), and wherein halogen is a halogen, preferably chlorine or bromine, most preferably bromine, h is equatorial and its adjacent halogen is axial, oh is H, -Oh’, -NHh’, -Nh’2, -NHSO3I1’, -COOh’, or -OSOsh’; preferably oh is H, -Oh’, or -NH-C(=O)CH3; q is H, -CH2-OI1’, -CH2OSO3I1’, -COOh’, or-CH2-(CH(Oh’))i-6-H; preferably q is H or -CH2-OI1’ or -CH2-(CH(Oh’))i-6-H . h is H, -Oh’, -CH2OI1’, -NHh’, -Nh’2, or -COOh’; preferably h is H or -COOCH3; h’ is in each instance independently p’ or a C1-8 alkyl or acyl that is optionally cyclic and that is optionally unsaturated and that is optionally halogenated; a preferred C1-8 alkyl or acyl is a C1 -6, more preferably a C1 -4 alkyl or acyl; particularly preferred is C1 alkyl or C2 acyl; preferably h’ is p’; p' is in each instance independently a protecting group.

[0053] Step ii) - conversion to a nitrile

[0054] In step ii) of the method, which is optional, the provided compound of general formula (I) is converted to a compound of general formula (l-CN):

[0055] Step ii) is preferably omitted when the intermediate of general formula (I) is of general formula (l-imidate). Step ii) is preferably not optional when the compound of general formula (I) is of general formula (l-amide). Thus provided is the method according to the invention, wherein the intermediate of general formula (I) is of general formula (l-amide), wherein preferably step ii) is not optional:

[0056] A skilled person is aware of suitable reactions for converting an amide into a nitrile. For instance, in preferred embodiments, step ii) comprises treatment of the compound of general formula (l-amide) with an acid anhydride such as acetic acid anhydride or preferably trifluoroacetic anhydride. The anhydride is preferably used in an excess, such as at about 5-50 equivalents, preferably 10-30, such as about 20 equivalents. The reaction can be performed under mildly basic conditions, for instance using pyridine as a solvent. The reaction can be allowed to proceed for about 6-48 hours, preferably about 10-30 hours, such as about 20 hours. The reaction can be performed at about 15-25 °C such as about 20 °C.

[0057] Step Hi) - reaction with cysteine

[0058] The intermediate provided in step i) can be of general formula (l-imidate) or it can be converted into a nitrile of general formula (l-CN). For nitriles it is known that this group readily reacts with cysteine to form luciferin or aminoluciferin. The inventors surprisingly found that the imidate of general formula (l-imidate) can also readily convert to luciferin or aminoluciferin upon contacting with cysteine. In preferred embodiments, the method further comprises the step of: iii) contacting the compound with cysteine to provide a caged luciferin compound of formula (l-probe):

[0059] The reaction with cysteine can be performed close to the moment of intended use of the caged conjugates, which can even be in situ directly prior to an assay. In preferred embodiments, the compound is reacted with cysteine in situ prior to addition of a protease. Examples of step iii) are provided in the Examples.

[0060] When the compound is contacted with cysteine, this is preferably performed in an aqueous buffer such as 25 mM HEPES, 125 mM NaCI, and 0.5 wt.-% BSA, at pH 7.4. Cysteine is preferably present in excess, such as 10-50 equiv., or 20-40 equiv., or more. Contacting can be performed at room temperature or at slightly elevated temperature, such as at 30-50 °C, preferably at 35-40 °C such as at about 37 °C. Contacting is preferably performed for about 1 -600 minutes, preferably about 5-300 minutes, more preferably about 10-180 minutes, more preferably about 15-120 minutes, more preferably about 20-60 minutes, most preferably about 25-45 minutes such as about 30 minutes. For this contacting, a preferred cysteine is D-cysteine. For this contacting, cysteine is preferably free cysteine, without protecting groups.

[0061] In other preferred embodiments, step iii) comprises contacting the compound with from 1 - 10 equivalents of cysteine such as 2 equivalents of cysteine, wherein the contacting is preferably in an aqueous buffer such as 25 mM HEPES, 125 mM NaCI, and 0.5 wt.-% BSA, at pH 7.4 or 7.2, wherein the cysteine is preferably D-cysteine, wherein the cysteine is preferably an acid addition salt such as cysteine HCI.

[0062] Preferred compounds of formula (l-probe) are of formula (Ill-probe):

[0063] Preferred compounds of formula (l-probe) are of formula (lll-glycosyl-probe): Products

[0064] The invention provides compounds that are useful for performing assays. The compounds can for example be used to convert to suitable probes. Accordingly the invention provides a compound of general formula (I) or a salt thereof: wherein

[0065] X is O or NH; preferably as defined above;

[0066] R is -NH2, -OCH3, or -OCH2CH3; preferably as defined above; and cage is a glycosyl moiety or a peptide; preferably as defined above; wherein the compound is not

[0067] In preferred embodiments of the provided compound, cage is of general formula (II): wherein m is 0 or 1 ; n is 1 , 2, or 3; oh is H, -OH, -Oh’, -NH2, -NHh’, -Nh’2, -COOH, or -COOh’; q is H, -CH2-OH, -CH2-Oh’, -CH2-(CH(OH))I-6-H, or -CH2-(CH(Oh’))i-6-H; h is H, -OH, -Oh’, -NH2, -NHh’, -Nh’2, -COOH, or -COOh’; and h’ is in each instance independently p’ or a C1-8 alkyl or acyl that is optionally cyclic and that is optionally unsaturated and that is optionally halogenated; p' is in each instance independently a protecting group.

[0068] Further features and definitions for cage are preferably as defined above. A salt of a compound according to the invention is preferably a pharmaceutically acceptable salt. Such salts include salts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Zn and Mn; salts of organic bases such as N,N’-diacetylethylenediamine, glucamine, triethylamine, choline, dicyclohexylamine, benzylamine, trialkylamine, thiamine, guanidine, diethanolamine, alpha-phenylethylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, hydroxyethylpiperidine, and the like. Such salts also include amino acid salts such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine, etc. Such salts may include acid addition salts where appropriate, which are for example sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides such as HCI or HBr salts, acetates, trifluoroacetates, tartrates, maleates, citrates, succinates, palmoates, methanesulphonates, tosylates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like. Preferred salts are HCI salts, formic acid salts, acetic acid salts, and trifluoroacetic acid salts. More preferred salts are HCI salts, acetic acid salts and formic acid salts, most preferably HCI salts.

[0069] The compound according to the invention can be a hydrate or a solvate. In the context of the invention a hydrate refers to a solvate wherein the solvent is water. The term solvate, as used herein, refers to a crystal form of a substance which contains solvent. Solvates are preferably pharmaceutically acceptable solvates and may be hydrates or may comprise other solvents of crystallization such as alcohols, ether, and the like. Salts and solvates can also be provided in step i) of the method of the invention.

[0070] Examples of compounds according to the invention are:

[0071] The invention provides a kit of parts comprising a compound as defined above, and cysteine. Preferably, the cysteine is D-cysteine. Such a kit can be useful for performing luciferase assays. The kit can further comprise a luciferase enzyme. The kit can further comprise ATP, or MgCh, or instructions for use.

[0072] Method for determining protease activity

[0073] The compounds can suitably be used in assays for determining glycosidase or protease activity. Proteases of interest can for instance be blood clotting factors. Glycosidases of interest can for instance be glucosidase, galactosidase, xylosidase, or neuraminidase. The method for determining glycosidase or protease activity preferably comprises the steps of: i) providing one or more compounds according to the invention; ii) contacting the provided compounds with cysteine to provide caged luciferin compounds of formula (l-probe); iii) contacting the caged luciferin compounds with the glycosidase or protease to provide free luciferin or aminoluciferin; iv) contacting the free luciferin or aminoluciferin with luciferase to generate a luminescent signal.

[0074] Features and definitions are preferably as defined above. For instance, compounds of general formula (l-peptide) can be incubated with D-cysteine for 30 min at 37 °C in buffer to generate the respective aminoluciferin derivatives prior to the addition of protease, ATP, MgCh and luciferase. Similarly, compounds of general formula (l-glycosyl) can be incubated with D-cysteine for 30 min at 37 °C in buffer to generate the respective luciferin derivatives prior to the addition of glycosidase. Then, luminescence can be recorded at 37 °C for 30 minutes and the area under the curve (AUC) can be calculated. AUC can for instance be calculated via: where t is the time in minutes and y the luminescent signal in RLU / s.

[0075] Luciferase is a generic term for the class of oxidative enzymes that produce (bio)luminescence using luciferin as a substrate. Luciferases do not require an external light source, but do require luciferin and O2, and often also ATP. Mg2+is known to increase luminescent yield of some luciferases. Luciferases and their assays are known in the art, as are suitable conditions for their activity. Suitable luciferases are Enhanced Beetle Luciferase (ELuc), Click Beetle Green Luciferase (CBG), R. ohbai Luciferase (RoLuc), Firefly Luciferase (FLuc), Red Firefly Luciferase (RedF), P. hirtus Red Luciferase (RedLuc), Nano Luciferase (NLuc), Renilla Luciferase (Renilla), Metridia Luciferase (MetLuc), Lucia Luciferase (Lucia) Gaussia Luciferase (GLuc), or Green Renilla Luciferase (GrRenilla), most preferably it has the activity of NLuc. Many luciferases can be commercially obtained from manufacturers such as Promega, Sigma, and the like.

[0076] General definitions

[0077] In preferred embodiments, compounds and compositions according to the invention are for use in methods according to the invention, or are for use according to the invention. Each embodiment as identified herein may be combined together unless otherwise indicated.

[0078] Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components. It is understood that charge is often associated with equilibrium. A moiety that is said to carry or bear a charge is a moiety that will be found in a state where it bears or carries such a charge more often than that it does not bear or carry such a charge. As such, an atom that is indicated in this disclosure to be charged could be non-charged under specific conditions, and a neutral moiety could be charged under specific conditions, as is understood by a person skilled in the art.

[0079] In the context of this invention, a decrease or increase of a parameter to be assessed means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter.

[0080] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. “Hemostasis” and “Haemostasis” can be used interchangeably herein. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value more or less 1 % of the value. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention.

[0081] When a structural formula or chemical name is understood by the skilled person to have chiral centers, yet no chirality is indicated, for each chiral center individual reference is made to all three of either the racemic mixture (having any enantiomeric excess), the pure R enantiomer, and the pure S enantiomer. Whenever a fragment of a molecule, often referred to as a moiety, is represented, a dotted or wavy line indicates which bond links it to the entirety of the molecule; alternately, an asterisk (*) indicates where the represented moiety is linked to the rest of the molecule. This asterisk does not imply an atom, and neither does a bond that is crossed by a dotted or wavy line convey information about which atom is at the non-moiety side of the bond. All this is known in the art, and is routine practice.

[0082] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0083] Description of Drawings

[0084] Fig. 1A - General principle of a glycosidase luminescence-based assay using glycosyl luciferins. The glycosidase cleaves D-luciferin from the caged monosaccharide substrate. The released D- luciferin is converted by luciferase (Luc) in the presence of ATP and Mg2+to produce a light signal proportional to the glycosidase activity.

[0085] Fig. 1 B - Synthetic routes to prepare glycosyl luciferins from either protected glycosyl-a-bromides (top route) or unprotected glycosyl-a-fluorides (bottom route) and subsequent luciferin-induced generation of light.

[0086] Fig. 2A - Reaction scheme for the luminescent glycosidase assay via in situ generation of the D- luciferin substrates, i. D-cysteine HCI (2 equiv), K2CO3 (2 equiv), H2O (125 mM NaCI, 25 mM HEPES, 0.1 % w / w BSA, pH = 7.2), 1 h, 37 °C. ii. glycosidase (gluco-, galacto- or xylosidase), MgCL (125 equiv), ATP (50 equiv), firefly luciferase (final cone. 0.147 mg / mL).

[0087] Fig. 2B - The linear range of the luminescent assay (triplo) and Michaelis-Menten kinetic plots of substrates 12-13 (quintuple) corresponding to Fig. 2A.

[0088] Fig. 3A - Reaction scheme for the luminescent neuraminidase assay via the hydrolysis of D-luciferin from substrate 32. i. neuraminidase, MgCL (125 equiv), ATP (50 equiv), firefly luciferase (final cone. 0.147 mg / mL).

[0089] Fig. 3B - The respective linear range of the luminescent assay (triplo) and Michaelis-Menten kinetic plot of substrate 32 (quintuple). Examples

[0090] Example 1 - improved methods and intermediates for the provision of glycosyl Luciferins

[0091] 1.1 - amides as precursors for luciferin-cacied molecules

[0092] A challenge while preparing luciferin-caged molecules is the inherent low nucleophilicity and chemical instability of D-luciferin and its precursor 6-hydroxybenzo[d]thiazole-2-carbonitrile (6- HBTC, structure 1). Especially during glycosylation reactions and the subsequent deprotection of the glycoside substrates this instability can cause problems and induce byproduct formation. The direct glycosylation of the luciferin moiety might be problematic due to the reactivity of D-luciferin and the risk of oxidation. Nevertheless, synthetic routes have been reported employing direct glycosylation of D-luciferin, although generally an extensive purification was required (see for instance S. Wu, X. Lin, et al., Sci. Rep. 2019, 9, 1-9.). The use of precursor 1 throughout the synthesis instead of D-luciferin is a viable alternative, however, during methanolysis of the acetyl protecting groups unwanted methyl imidate formation takes place (see Amess et al., cited in the background section).

[0093] Searching for a new more robust synthesis route to glycosyl luciferins, the inventors considered that the more stable amide derivative 2 could circumvent undesired imidate formation during the deprotection conditions. To this end, compound 2 was readily synthesized via acidic hydrolysis of nitrile 1 in sulfuric acid at room temperature in quantitative yield (Scheme E1). The acetylated glycosyl a-bromide donors 3 (glucoside), 4 (galactoside) and 5 (xyloside) were prepared with hydrogen bromide in acetic acid. Subsequent glycosylation reactions of acceptor 2 with donors 3-5 were executed under phase transfer conditions in a water / chloroform mixture (1 :1) with a catalytic amount of tetrabutylammonium bromide (TBAB) and potassium carbonate at 60 °C to furnish the amide intermediates 6-8. These glycosylation reactions provide moderate yields of the desired product, presumably due to the inherently unreactive character of phenol 2 and competing elimination reactions on the glycosyl bromide donors. Multiple attempts to improve the yield of the glycosylation reaction with stronger bases or different solvents were unsuccessful. Neither the in situ phenolate formation of 2 nor BFs-mediated glycosylation of peracetylated glycosides proved to be higher yielding. Nevertheless, we proceeded with the acetyl deprotection of 6-8 with sodium methoxide in methanol to afford the glycosides 9-11. We were able to reintroduce the cysteine reactive nitrile moiety via dehydration of the amides 9-11 with trifluoroacetic anhydride (TFAA) in pyridine in the presence ofthe free hydroxyl groups, to afford the luciferin precursors 12-14 in good yields. y

[0094] The inventors considered that the reaction of D-cysteine with nitriles 12-14 could be executed in situ prior to the enzymatic assay to afford the glycosyl luciferins. The nitrile-bearing substrates appeared to be more stable compared to the corresponding luciferins, making storage and use of these compounds more practical. To demonstrate that our probes could be used as substrates for determining glycosidase activity, we incubated 12-14 with D-cysteine hydrochloride and an equimolar amount of potassium carbonate prior to the addition of glycosidase (Fig. 2A). We subsequently determined the enzymatic hydrolysis of the luciferin conjugates 15-17 upon reacting them with glycosidase in buffer and addition of luciferase, all performed in triplicate. This secondary readout allows for direct measurement of glycosidase activity, since glycosidase cleavage is known to be the rate limiting step in this experiment due to the excess of luciferase.

[0095] The released photons were measured with a luminometer and the area under the curve (AUC) and the subsequent linear range of the assay were determined (Fig. 2B). All three substrates 15-17 revealed a clear linear range and Km values in the low pmolar range (15-30 pM), similar to values found for fluorescent and chromogenic analogues in literature. All control experiments without glycosidase, luciferase, or D-cysteine indicated that the luminescent signal was solely glycosidase-dependent and that the assay was sufficiently robust.

[0096] 1.2 - methyl imidates as precursors for luciferin-caqed molecules

[0097] In the previous Example it was successfully demonstrated that the amide precursors are more stable, can be dehydrated to afford the corresponding nitrile, and can be used to generate glycosyl luciferins in situ. To improve the overall synthesis yields, the synthesis of glycosyl luciferins starting from unprotected glycosides was explored. This would abrogate the need for a base-catalyzed deprotection and reduce overall reaction steps. Initial attempts with glucose and 2-chloro-1 ,3- dimethylimidazolinium chloride (DMC)-mediated glycosylation were unsuccessful. Minimal conversion was observed and a large excess of donor 1 was required. Next, the use of a-glycosyl fluorides was explored. Initially, it was intended to couple nitrile 1 to the a-fluoride acceptors 18-20 (gluco-, galacto- and xyloside), which were either commercially available or synthesized from the corresponding peracetylated building blocks. This protecting-group-free approach would save synthetic steps and it was considered that the yield might also improve due to the relative stability of the a-fluorides towards hydrolysis as compared to the a-bromides. However, during optimization of the O-glycosylation, it was observed that the reaction in water did not give any conversion with nitrile 1 and glucoside 18. Eventually, it was found that the reaction in methanol / water (1 :1) and calcium hydroxide as Ca2+additive gave good conversions. Yet, upon isolation of the product, it was observed that undesired nitrile methanolysis occurred to afford the corresponding methyl imidate 21 , possibly due to the alkaline glycosylation conditions.

[0098] The methyl imidates appeared to be a dead-end intermediate for luciferin synthesis. Surprisingly, it was found that 21 still reacted with D-cysteine to afford the corresponding D-luciferin. The glycosylation reactions of donor 1 with the a-fluoride acceptors 18-20 were executed and afforded the methyl imidate intermediates 21-23 as a mixture of the product with the corresponding methyl-p-D-glycopyranoside. Subsequently, introduction of the thiazoline ring was performed upon reacting compounds 21-23 with D-cysteine and potassium carbonate in methanol to afford the pure glycosyl D-luciferins 24-26 after purification.

[0099] This two-step protecting-group-free synthetic procedure allows for the rapid synthesis of caged luminescent carbohydrate substrates. We improved and shortened the initial dehydration method and showed that the methyl imidates are a useful intermediate to react with D-cysteine to provide pure D-luciferin-caged carbohydrate substrates such as 24-26. The new method significantly improved the yield (28-85% over two steps) as compared to the initial dehydration route (< 5% over four steps). Scheme E2 summarizes this strategy.

[0100] 1.3 - the methods allow access to a broad scope of glycosylation products

[0101] Gluco-, galacto- and xylosidase-substrates have already been described above. To further extend the scope of our synthesis method, we aimed to synthesize a luminescent probe for measuring sialidase activity. Sialic acid is a nine-carbon carbohydrate which plays a key role in immune regulation, viral infection and various types of cancer, making sialidase an exceptionally attractive diagnostic target. There is a need for concise methods for the synthesis of luminescent carbohydrate substrates. The synthesis of Sia-Luc (32, Scheme E3) has been described in literature, but its known synthesis is inefficient due to the need for extensive purification and the formation of byproducts during the direct glycosylation approach. We commenced our synthesis with the fully protected sialic acid 27, which was converted into a-chloride 28 by in situ hydrochloride formation with acetyl chloride in methanol. Compound 28 was directly used without further purification for the O-glycosylation with nitrile 1 and potassium carbonate in acetonitrile, to provide 29 which was directly used in the next reaction step. Deprotection of the acetyl protecting groups furnished product methyl imidate 30 in 50% yield over three steps. Methyl imidate 30 was reacted with D-cysteine to give 31 in a good yield of 85%. Hydrolysis of the methyl ester of 31 was successful with sodium hydroxide to give final product 32 (55% yield), a caged luminescent probe for measuring sialidase activity.

[0102] Having substrate 32 in hand, we validated the compound for measuring sialidase activity. The linear range upon reacting 32 with neuraminidase was determined and a Km of 40.5 pM was found, which is similar to values reported in literature for fluorescent analogues. These results (shown in Fig. 3) indicate that our method is robust and is compatible with complex saccharides.

[0103] 1.4 - conclusions

[0104] New routes for the synthesis of caged (glycosyl or peptide) luciferins were developed. Applying temporary hydration of the nitrile precursor of luciferin is an appealing method to eliminate the risk of unwanted byproducts that can otherwise form during glycosylation and subsequent deprotection reactions. It was demonstrated that the in situ formation of the D-luciferin moiety, prior to the enzymatic assay, is an attractive synthetic approach. The nitrile moiety is more stable, which facilitates its storage, offering a longer shelf-life. The gluco-, galacto- and xylosidase-substrates appeared excellent substrates for determining glycosidase activity and the linear range and Michaelis-Menten kinetics were determined, providing an overall robust and enzyme-dependent assay.

[0105] It was also demonstrated that unprotected glycosyl-a-fluorides can be used to generate the glycosyl D-luciferins in good yields in two single steps. The methyl imidate intermediates that were generated upon the glycosylation reactions surprisingly reacted with D-cysteine to provide the D- luciferin-caged carbohydrates. This methodology was shown to also be useful for complex carbohydrates, because a sialidase probe was synthesized that was successfully validated giving an enzyme-dependent signal.

[0106] The newly developed chemistry approach, the probe compounds, and the intermediate products for the further provision of probe compounds all offer innovative new options and improved access to diagnostic technology. The invention benefits the synthesis of luminescent caged carbohydrate substrates, offering considerable opportunities for diagnostics, for inhibitor design, or for potential point-of-care applications.

[0107] Example 2 - Material and Methods

[0108] 2.1 - General

[0109] NMR spectra were recorded on a Bruker 400 MHz or 500 MHz or a JEOL 400 MHz or 500 MHz spectrometer and the compounds were assigned using1H NMR,13C NMR, COSY, HSQCED and HMBC spectra. Chemical shifts were reported in parts per million (ppm.) relative to reference (CDCb:1H: 7.26 ppm. and13C 77.16 ppm.; CD3OD:1H: 3.31 ppm. and13C 49.00 ppm.; D2O:1H: 4.79 ppm.; (CD3)2SO:1H: 2.50 ppm. and13C 39.52 ppm.). NMR data are presented in the following way: chemical shift, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dtd = doublet of triplet of doublets h = heptet, m = multiplet and / or multiple resonances) and coupling constants J in Hz. Mass spectra were recorded on a JEOL AccuTOF CS JMS-T100CS (ESI) mass spectrometer. Automatic flash column chromatography was executed on a Biotage Isolera Spektra One using SNAP or Silicycle cartridges (Biotage, 30-100 pm, 60A) 4-50 g. Reactions were monitored using TLC F254 (Merck KGaA) by UV absorption detection (A = 254 nm) and by spraying them with either 5% cone. H2SO4 in MeOH stain, cerium ammonium molybdate (Hannesian’s) stain or with potassium permanganate stain followed by heating with a heat gun. Preparative HPLC was performed on a Phenomenex® Gemini-NX 3u C18 1 10A reversed-phase column (150 x 21.2 mm) using gradient elution with a constant flow of 10 mL / min at 30 °C. MiliQ (0.1 % TFA) and CH3CN (0.1 % TFA) were used as the solvents. The pure fractions containing product were combined and lyophilized overnight to yield the target compounds. Reactions under protective atmosphere were performed under positive N2 flow using flame-dried glassware. Adenosine 5’-triphosphate (ATP), bovine serum albumin (BSA), 4-(2- hydroxyethyl)-1 -piperazine-ethane-sulfonic acid (HEPES), magnesium chloride hexahydrate (MgCh) and sodium chloride (NaCI) were obtained from Merck KGaA (Germany). D-Cysteine was obtained from Fluorochem (UK). Compound 1 was obtained from Fluorochem (UK) and compound 18 from Ambeed (USA). p-Glucosidase (Agrobacterium sp.) and p-xylosidase (Selenomonas ruminantium) were obtained from Megazyme Ltd. (UK), p-galactosidase (E coli) from Merck KGaA (Germany) and neuraminidase (Clostridium perfringens) from Sigma Aldrich (NL). White Greiner F- bottom 384 well plates were obtained from VWR (NL) and luminescence was measured with a Varioskan™ Lux (Thermo Scientific).

[0110] 2.2 - Synthesis

[0111] 6-hydroxybenzo[d]thiazole-2-carboxamide (2): Concentrated H2SO4 (2.5 mL, 46 mmol) was added to 6-hydroxybenzo[c / |thiazole-2- carbonitrile (1 , 259 mg, 1 .47 mmol) and the mixture was stirred at rt for

[0112] 3 h. Hereafter, the product was precipitated by adding it to water (25 mL). The crude product was isolated by filtration and lyophilized to afford 2 (285 mg, quant) as a yellow solid. TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.27.1H NMR (400 MHz, DMSO-D6) 5 10.1 1 (bs, 1 H), 8.30 (s, 2H), 7.95-7.88 (m, 2H), 7.45 (d, J = 2.4 Hz, 1 H), 7.07 (dd, J = 8.9, 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO-D6) 6 162.1 , 161.4, 157.4, 146.8, 138.7, 125.2, 1 17.6, 107.3. HRMS (m / z): [M + Na]+calcd. for C8H6N2O2SNa: 217.0048, found 217.0060.

[0113] Penta / tetra-O-acetyl-d-carbohydrate (25.67 mmol) was dissolved in dry DCM (75 mL) and HBr (0.36 mol, 60 mL, 33 wt. %) was added. The mixture was stirred for 3 h under N2 at rt. Hereafter, the mixture was diluted with DCM (100 mL) and water (100 mL). The aqueous layer was extracted with DCM (3 x 100 mL) and washed with saturated aqueous NaHCOs (100 mL) and brine (100 mL). The combined organic layers were dried (MgSO4) and concentrated in vacuo to afford products 3-5.

[0114] 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide (3): synthesized according to general procedure A. After concentration in vacuo, the product was crystallized from EtOAc / pentane to afford 3 (8.24 g, 78%) as a white crystalline solid. TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.68.1H NMR (400 MHz, CDCb) 6 6.63 (d, J = 4.0

[0115] Hz, 1 H), 5.58 (t, J = 9.7 Hz, 1 H), 5.23-5.13 (m, 1 H), 4.86 (dd, J = 10.0, 4.0 Hz, 1 H), 4.39-4.28 (m, 2H), 4.18-4.12 (m, 1 H), 2.12 (d, J = 1.8 Hz, 9H), 2.06 (d, J = 7.0 Hz, 6H).13C NMR (101 MHz, CDCb) 6 170.5, 169.9, 169.8, 169.5, 86.6, 72.1 , 70.6, 70.2, 67.2, 61 .0, 20.7, 20.7, 20.6, 20.6.

[0116] 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (4): Was synthesized according to general procedure A and not further purified to afford 4 (8.08 g, 77%) as an oil. TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.55.1H NMR (400 MHz, CDCb) 6 6.70 (d, J = 4.0 Hz, 1 H), 5.53 (dd, J = 3.4, 1 .3 Hz, 1 H), 5.41 (dd, J = 10.9, 3.3, 0.9

[0117] Hz, 1 H), 5.05 (dd, J = 10.6, 3.9, 0.9 Hz, 1 H), 4.49 (t, J = 7.8, 6.2, 1 .2 Hz, 1 H), 4.24-4.08 (m, 2H),

[0118] 2.16 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H).13C NMR (101 MHz, CDCb) 6 170.3, 170.1 ,

[0119] 169.9, 169.7, 88.1 , 71.1 , 68.0, 67.8, 67.0, 60.8, 20.7, 20.6, 20.6, 20.5. 2,3,4-tri-O-acetyl-a-D-xylopyranosyl bromide (5): Was synthesized according to general procedure A and purified with silica gel column chromatography (50% EtOAc in heptane) to afford 5 (2.06 g, 39%) as a white solid. TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.74.1H NMR (500 MHz, CDCb) 6 6.60 (d, J = 3.5 Hz,

[0120] 1 H), 5.58 (t, J = 9.8, 2.8 Hz, 1 H), 5.10-5.01 (m, 1 H), 4.82-4.75 (m, 1 H), 4.10-4.03 (m, 1 H), 3.90 (t, J = 11 .1 , 2.7 Hz, 1 H), 2.11 (d, J = 3.0 Hz, 3H), 2.07 (d, J = 1 .3 Hz, 6H).13C NMR (126 MHz, CDCb) 6 169.9, 169.8, 87.6, 70.9, 69.5, 68.1 , 62.5, 20.7, 20.7, 20.6.

[0121] TBAB (33 mg, 103 pmol) was dissolved in a 1 :1 water / CHCb mixture (30 mL) and stirred and heated to 40 °C. Hereafter, 3, 4, or 5 (1 .03 mmol), 2 (100 mg, 514 pmol) and K2CO3 (179 mg, 1 .292 mmol) were dissolved in water (0.5 mL) and added to the reaction. The reaction was stirred for 20 h at 60 °C. The organic layer was separated and washed with aqueous NaOH (3 x 20 mL, 1.25 M), dried with Na2SO4, concentrated in vacuo and purified with silica gel column chromatography (25 — 70% EtOAc in heptane) to afford 6-8 as white crystalline solids.

[0122] 14%). TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.1 1.1H NMR (400 MHz, CDCb) 6 7.98 (dd, J = 9.0, 1.5

[0123] Hz, 1 H), 7.56 (t, J = 2.0 Hz, 1 H), 7.21 (dt, J = 9.0, 2.0 Hz, 1 H), 5.37-5.28 (m, 2H), 5.22-5.13 (m, 2H), 4.32-4.18 (m, 2H), 3.94 (ddt, J = 9.9, 5.7, 2.1 Hz, 1 H), 2.08 (dd, J = 3.6, 1 .5 Hz, 6H), 2.05 (dd, J = 6.1 , 1.5 Hz, 6H).13C NMR (101 MHz, CDCb) 6 170.5, 170.2, 169.4, 169.3, 161.8, 161.7, 155.9,

[0124] 149.1 , 138.7, 125.4, 118.6, 108.7, 99.2, 72.6, 72.3, 71.1 , 68.2, 62.0, 29.7, 20.7, 20.7, 20.6, 20.6.

[0125] HRMS (m / z): [M + Na]+calcd. for C22H24N20iiSNa: 547.0999, found 547.0990.

[0126] 33%). TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.15.1H NMR (500 MHz, CDCb) 6 8.00 (d, J = 9.0 Hz,

[0127] 1 H), 7.60 (d, J = 2.4 Hz, 1 H), 7.24 (dd, J = 9.0, 2.5 Hz, 1 H), 5.55 (dd, J = 10.4, 7.9 Hz, 2H), 5.50 (dd, J = 3.5, 1.1 Hz, 1 H), 5.19-5.15 (m, 2H), 4.29-4.18 (m, 2H), 2.10 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H).13C NMR (126 MHz, CDCb) 6 170.3, 170.2, 170.1 , 169.4, 161.8, 161 .8, 156.0, 149.1 , 138.7, 125.3, 118.5, 108.6, 99.7, 71.4, 70.7, 68.6, 66.9, 61.5, 20.7, 20.7, 20.6, 20.6. HRMS (m / z): [M + Na]+calcd. for C22H24N20iiSNa: 547.0999, found 547.0999.

[0128] (2S,3R,4S,5R)-2-((2-carbamoylbenzo[d]thiazol-6- yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (8):

[0129] Was synthesized according to general procedure B to afford 8 (11 mg, 5%). TLC (EtOAc / heptane, 1 :1 v / v) RF = 0.15.1H NMR (500 MHz, CDCb) 6 8.01

[0130] (d, J = 9.0 Hz, 1 H), 7.60 (d, J = 2.4 Hz, 1 H), 7.25 (dd, J = 9.0, 2.6 Hz, 2H), 5.73 (s, 1 H), 5.34-5.20 (m, 3H), 5.09-5.02 (m, 1 H), 4.28 (dd, J = 12.2, 4.6 Hz, 1 H), 3.62 (dd, J = 12.1 , 7.3 Hz, 1 H), 2.14- 2.11 (m, 9H).13C NMR (126 MHz, CDCb) 6 169.9, 169.8, 169.4, 161.7, 155.7, 149.0, 138.8, 125.4,

[0131] 118.4, 108.6, 98.7, 70.4, 69.9, 68.3, 61.9, 20.8, 20.8, 20.7. HRMS (m / z): [M + Na]+calcd. for Ci9H2oN209SNa: 475.0787, found 475.0809.

[0132] Protected carbohydrate 6, 7, or 8 (96 pmol) was dissolved in dry MeOH (10 mL) and to this mixture NaOMe in MeOH (19 pmol, 30 wt. %) was added and the reaction was stirred for 20 h at rt. Hereafter, the reaction was neutralized with Dowex®, filtered and concentrated in vacuo, to afford the products 9-11 as white solids.

[0133] 6-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-

[0134] 9.0, 1 .1 Hz, 1 H), 7.99 (s, 1 H), 7.84 (d, 1 H), 7.33-7.27 (m, 1 H), 5.37 (d, J = 4.9 Hz, 1 H), 5.11 (d, J = 4.0 Hz, 1 H), 5.04 (dd, J = 5.3, 1.1 Hz, 1 H), 4.99 (d, J = 6.6 Hz, 1 H), 4.57 (t, 1 H), 3.72 (dd, J =

[0135] 11.6, 5.5 Hz, 1 H), 3.53-3.45 (m, 1 H), 3.44-3.37 (m, 1 H), 3.30-3.26 (m, 1 H), 3.23-3.14 (m, 2H).13C NMR (126 MHz, DMSO-D6) 6 163.4, 161.9, 156.9, 148.5, 138.3, 125.1 , 118.4, 108.9, 101.2,

[0136] 77.6, 77.0, 73.7, 70.1 , 61.1. LRMS (m / z): [M + Na]+calcd. for Ci4Hi6N2O7SNa: 379.06, found 379.11.

[0137] 6-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- l)ox )benzo[d]thiazole-2-carboxamide (10): Was 1 H), 7.99 (s, 1 H), 7.84 (d, J = 2.4 Hz, 1 H), 7.30 (dd, J = 8.9, 2.5 Hz, 1 H), 5.22 (d, J = 5.2 Hz, 1 H), 4.95 (d, J = 7.7 Hz, 1 H), 4.89 (d, J = 5.7 Hz, 1 H), 4.67 (t, J =

[0138] 5.5 Hz, 1 H), 4.53 (d, J = 4.7 Hz, 1 H), 3.73 (t, J = 4.2 Hz, 1 H), 3.68-3.49 (m, 4H), 3.46-3.40 (m, 1 H).13C NMR (126 MHz, DMSO-D6) 6 163.3, 161.9, 157.0, 148.5, 138.3, 125.1 , 118.5, 108.8, 101.9, 76.1 , 73.7, 70.7, 68.6, 60.8. HRMS (m / z): [M + Na]+calcd. for Ci4Hi6N2O7SNa: 379.0576, found 379.0575.

[0139] 6-(((2S,3 / ?,4S,5 / ?)-3,4,5-trihydroxytetrahydro-2H- pyran-2-yl)oxy)benzo[d]thiazole-2 -carboxamide (11):

[0140] Was synthesized according to general procedure C to afford 11 (5 mg, 82%). TLC (MeCN / H2O, 9:1 v / v) RF= 0.52.1H NMR (500 MHz, DMSO-D6) 6 8.39

[0141] (s, 1 H), 8.02 (d, J = 9.0 Hz, 1 H), 7.99 (s, 1 H), 7.85 (d, J = 2.5 Hz, 1 H), 7.32-7.23 (m, 1 H), 5.40 (d, J = 4.9 Hz, 1 H), 5.15 (d, J = 4.5 Hz, 1 H), 5.09 (d, J = 4.6 Hz, 1 H), 5.00 (d, J = 7.2 Hz, 1 H), 3.78 (dd, J = 10.2, 4.3 Hz, 1 H), 3.42-3.25 (m, 4H).13C NMR (126 MHz, DMSO-D6) 6 163.5, 161.9, 156.6, 148.6, 138.3, 125.1 , 118.5, 108.8, 101.6, 76.9, 73.5, 69.8, 66.3. HRMS (m / z): [M + Na]+calcd. for Ci3Hi4N2O6SNa: 349.0470, found 349.0470.

[0142] Compound 9-11 (22 pmol) was dissolved in dry dioxane (5 mL) under N2. Dry pyridine (0.87 mmol) was added and the mixture was stirred for 10 min. at rt Hereafter, TFAA (0.45 mmol) was added and the reaction was stirred for 20 h at rt. The mixture was quenched with water (1 mL) and the solution was diluted with MeOH (5 mL). The reaction was concentrated in vacuo and the crude product was purified by RP-HPLC (0 -> 100 % MeCN (0.1 % HCO2H) in MiliQ (0.1 % HCO2H)) and lyophilized to afford the product 12-14 as white solids.

[0143] 6-(((2S,3 / ?,4S,5S,6 / ?)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)ox )benzo[d]thiazole-2 -carbonitrile

[0144] (d, J = 2.4 Hz, 1 H), 7.45 (dd, J = 9.1 , 2.4 Hz, 1 H), 5.06-5.03 (m, 1 H), 3.94 (dd, J = 12.1 , 2.2 Hz,

[0145] 1 H), 3.71 (dd, J = 12.1 , 5.9 Hz, 1 H), 3.59-3.46 (m, 3H), 3.42-3.37 (m, 1 H).13C NMR (126 MHz,

[0146] CD3OD) 6 158.2, 147.6, 137.2, 134.8, 125.0, 119.4, 112.7, 107.4, 101.0, 77.0, 76.4, 73.4, 69.9,

[0147] 61.1. LRMS (m / z): [M + H]+calcd. for Ci4Hi5N2O6S: 339.06, found 379.09. 6-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-

[0148] Hz, 1 H), 7.40 (dd, J = 9.1 , 2.5 Hz, 1 H), 4.96 (d, J = 7.6 Hz, 1 H), 3.74 (d, J = 3.3 Hz, 1 H), 3.67-3.61 (m, 2H), 3.57 (dd, J = 10.8, 6.0 Hz, 1 H), 3.51 (dd, J = 10.8, 6.4 Hz, 1 H), 3.43 (dd, J = 9.5, 3.3 Hz, 1 H).13C NMR (126 MHz, DMSO-D6) 6 158.3, 147.3, 137.7, 135.0, 125.7, 1 19.7, 114.0, 108.6, 101.7, 76.1 , 73.8, 70.7, 68.4, 60.6. HRMS (m / z): [M + Na]+calcd. for Ci4Hi4N2O6SNa: 361.0470, found 361.0472.

[0149] 6-(((2S,3R,4S,5R)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzo[d]thiazole-2- carbonitrile (14): Was synthesized according to general procedure D to afford 14 (3 mg, 60%). TLC (MeCN / H2O, 9:1 v / v) RF = 0.65.1H NMR (500 MHz, DMSO-De) 6 8.17 (d, J =

[0150] 9.1 Hz, 1 H), 7.98 (d, J = 2.5 Hz, 1 H), 7.38 (dd, J = 9.1 , 2.5 Hz, 1 H), 5.47 (s, 1 H), 5.23 (s, 1 H), 5.14 (s, 1 H), 5.00 (d, J = 7.2 Hz, 1 H), 3.79 (dd, J = 10.6, 4.6 Hz, 1 H), 3.43-3.21 (m, 4H).13C NMR (126 MHz, DMSO-De) 6 158.0, 147.4, 137.7, 135.2, 125.8, 1 19.7, 114.0, 108.5, 101.5, 76.9, 73.4, 69.7, 66.4. LRMS (m / z): [M + H]+calcd. for CI3HI3N2O5S: 309.05, found 309.64.

[0151] 2.2.4. 1 - fluorinated building blocks

[0152] (2R,3R,4S,5R,6R)-2-fluoro-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0153] (19): p-d-galactose peracetate (2.0 g, 5.1 mmol) was weight to a Teflon 100 mL round bottomed flask and the solids were cooled to 0 °C. A 70% HF in pyridine solution (3.4 mL) was added carefully to the flask via a plastic syringe. The reaction mixture was stirred for 16 h before water (15 mL) and DCM (15 mL) were added. The organic phase was separated and the aqueous phase was extracted with DCM (2 x 15 mL). The combined organic phases were washed with brine (20 mL), dried with Na2SO4and concentrated in vacuo to afford 2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl fluoride as a colorless oil which was used without further purification in the next step. The resulting oil was dissolved in dry MeOH (20 mL) under inert atmosphere. The solution was cooled to 0 °C at which temperature a 5.4 M solution of NaOMe in MeOH (0.1 mL, 0.51 mmol) was added dropwise. The reaction was stirred for 10 minutes at 0 °C followed by 1.5 h at rt. The reaction was subsequently cooled to 0 °C, silica gel (3 g) was added and the solution was stirred for 5 minutes. The solvent was evaporated in vacuo and the resulting solids were taken up in EtOAc / MeOH (7:3, 20 mL), stirred for 5 minutes, and the solution was filtrated. The filtrate was evaporated in vacuo to afford 19 (884 mg, 94% over two steps) as a white solid which was used in the next steps without purification.1H NMR (500 MHz, DMSO-De) 6 5.45 (dd, J = 54.6, 2.8 Hz, 1 H), 5.02 (d, J = 6.2 Hz, 1 H), 4.77 (d, J = 5.7 Hz, 1 H), 4.62-4.58 (m, 2H), 3.73 (ddd, J = 4.4, 3.1 , 1.2 Hz, 1 H), 3.70 (t, J = 6.2 Hz, 1 H), 3.64-3.53 (m, 1 H), 3.53-3.48 (m, 2H), 3.48-3.43 (m, 1 H).13C NMR (126 MHz, DMSO-De) 6 108.3, 74.0, 69.1 , 68.3, 67.7, 60.4.19F NMR (470 MHz, DMSO-De) 6 -150.18 (dd, J = 54.6, 27.1 Hz).

[0154] (2 / ?,3 / ?,4S,5 / ?)-2-fluorotetrahydro-2H-pyran-3,4,5-triol (20): Xylose (1.5 g, 10 mmol) was dissolved in pyridine (15 mL). The solution was cooled to 0 °C at which temperature AC2O (9.0 mL, 100 mmol) was added. The reaction was stirred for 16 h allowing it to warm up to rt. Subsequently, the solvent was evaporated in vacuo, the resulting liquid was taken up in EtOAc (80 mL), washed with 0.1 M HCI (3 x 100 mL) and brine (100 mL). The organic phase was dried with Na2SO4, filtrated, and concentrated in vacuo to obtain tetra-O- acetyl-p-d-xylopyranose which was used without further purification in the next step.

[0155] The resulting oil was transferred to a Teflon 100 mL round bottomed flask, cooled to 0 °C, and a 70% HF in pyridine solution (3.4 mL) was added carefully to the flask via a plastic syringe. The reaction mixture was stirred for 16 h before water (25 mL) and DCM (25 mL) were added. The organic phase was separated and the aqueous phase was extracted with DCM (2 x 25 mL). The combined organic phases were washed with brine (20 mL), dried with Na2SO4 and concentrated in vacuo to afford 2,3,4-tri-O-acetyl-D-xylopyranosyl fluoride as a colorless oil which was used without further purification in the next step. The resulting oil was dissolved in dry MeOH (30 mL) under inert atmosphere. The solution was cooled to 0 °C and a 5.4 M solution of NaOMe in MeOH (0.2 mL, 1 .0 mmol) was added dropwise. The reaction was stirred for 10 minutes at 0 °C followed by 1 .5 h at rt. The reaction was subsequently cooled to 0 °C, silica gel (4 g) was added and the solution was stirred for 5 minutes. The solvent was evaporated in vacuo and the resulting solids were taken up in EtOAc / MeOH (7 / 3, 30 mL), stirred for 5 minutes, and the solution was filtrated. The filtrate was evaporated in vacuo to afford 20 (1 .2 g, 80% over three steps) as a white solid which was used in the next steps without purification.1H NMR (500 MHz, DMSO-De) 6 5.44 (dd, J = 53.9, 2.7 Hz, 1 H), 5.26 (d, J = 6.0 Hz, 1 H), 5.10 (d, J = 3.6 Hz, 1 H), 5.06 (d, J = 3.9 Hz, 1 H), 3.65-3.59 (m, 1 H), 3.41- 3.35 (m, 3H), 3.30-3.20 (m, 1 H).13C NMR (126 MHz, DMSO-De) 6 109.0, 107.2, 73.0, 71.4, 68.8, 63.8.19F NMR (471 MHz, DMSO-De) 6 -150.97 (dd, J = 53.9, 27.2 Hz).

[0156] 2.2.5 - General procedure E

[0157] 2 - 23

[0158] 6-Hydroxybenzo[c / |thiazole-2-carbonitrile (32 mg, 0.18 mmol), Ca(OH)2 (40 mg, 0.54 mmol) and 18-20 (0.54 mmol) was dissolved in MeOH (600 pL) and H2O (200 pL). The suspension was stirred vigorously for 16 h. Hereafter, the reaction mixture was concentrated in vacuo and purified with silica gel column chromatography (0 — 10% H2O in MeCN) to afford the products 21-23 as a white solid mixed with a minor amount of the methyl-p-d-glycopyranoside impurity which was directly used in the next reaction step.

[0159] Methyl 6-(((2S,3 / ?,4S,5S,6 / ?)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- was directly used in the next reaction step.

[0160] Methyl 6-(((2S,3 / ?,4S,6 / ?)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- l b d thi l 2 bi id t 22 W methyl-p-d-galactopyranoside. which was directly continued in the next reaction step.

[0161] Methyl 6-(((2S,3 / ?,4S,5 / ?)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzo[d]thiazole-2- carbimidate (23): Was synthesized according to general procedure E starting from fluoride 20 to afford 23 as a white solid mixed with minor amount of methyl-p-d- xylopyranoside. which was directly continued in the next reaction step.

[0162] 2.2.6 - General procedure F

[0163] Imidate 21-23 (1 equiv) was used including its minor amount of corresponding methyl-p-d- glycopyranoside impurity (this mixture is a product of general procedure E described above) and this was dissolved in MeOH (4 mL) and K2CO3 (2 equiv) and D-cysteine HCI (2 equiv) were added as step ii. shown above. The suspension was stirred for 16 h in which the reaction turned yellow and a precipitate was formed. Hereafter the reaction mixture was concentrated in vacuo and purified with silica gel column chromatography (0 — 20% H2O in MeCN) to afford products 24-26 as white solids. (S)-2-(6-(((2S,3 / ?,4S,5S,6 / ?)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-

[0164] TLC (MeCN / H2O, 4:1 v / v) RF = 0.52.1H NMR (500 MHz, D2O) 5 7.74 (d, J = 9.1 Hz, 1 H), 7.56 (d, J = 2.5 Hz, 1 H), 7.18 (dd, J = 9.1 , 2.5 Hz, 1 H), 5.11 (dd, J = 9.9, 8.1 Hz, 1 H), 5.07 (d, J = 7.4 Hz, 1 H), 3.86 (dd, J = 12.4, 2.2 Hz, 1 H), 3.75-3.65 (m, 2H), 3.61-3.49 (m, 4H), 3.43 (dd, J = 9.9, 8.6 Hz, 1 H).13C NMR (126 MHz, D2O) 6 177.3, 165.8, 160.2, 156.0, 147.9, 136.8, 124.3, 118.2, 108.4, 100.5, 80.0, 76.3, 75.5, 73.0, 69.5, 60.6, 36.4. LRMS (m / z): [M + H]+calcd. for C17H19N2O8S2: 443.06, found 443.00.

[0165] (S)-2-(6-(((2S,3 / ?,4S,5 / ?,6 / ?)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- , ,

[0166] 9:1 v / v) RF = 0.15.1H NMR (500 MHz, D2O) 6 7.90 (dq, J = 8.1 , 4.1 Hz, 1 H), 7.74-7.69 (m, 1 H),

[0167] 7.37-7.31 (m, 1 H), 5.24 (t, J = 8.9 Hz, 1 H), 5.14 (d, J = 7.5 Hz, 1 H), 4.07-4.05 (m, 1 H), 3.95 (t, J = 5.6 Hz, 1 H), 3.84 (dt, J = 9.8, 3.0 Hz, 5H), 3.65 (dd, J = 11 .2, 8.0 Hz, 1 H).13C NMR (126 MHz, D2O) 6 172.4, 160.2, 156.2, 147.9, 136.9, 124.3, 118.3, 108.3, 101 .1 , 80.1 , 75.6, 72.56, 70.6, 68.5, 60.8,

[0168] 36.5. LRMS (m / z): [M + H]+calcd. for C17H19N2O8S2: 443.06, found 443.33.

[0169] (S)-2-(6-(((2S,3 / ?,4S,5 / ?)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)benzo[d]thiazol-2-yl)- , .

[0170] (MeCN / H2O, 9:1 v / v) R = 0.14.1H NMR (500 MHz, CD3OD) 6 7.98 (d, J = 9.0 Hz, 1 H), 7.70 (d, J

[0171] = 2.5 Hz, 1 H), 7.30 (dd, J = 9.0, 2.4 Hz, 1 H), 5.23 (t, J = 9.3 Hz, 1 H), 4.99 (d, J = 7.0 Hz, 1 H), 3.96 (dd, J = 11.4, 5.2 Hz, 1 H), 3.76-3.71 (m, 2H), 3.60 (ddd, J = 9.9, 8.3, 5.2 Hz, 1 H), 3.51-3.41 (m, 3H).13C NMR (126 MHz, CD3OD) 6 173.3, 158.3, 149.9, 141.8, 138.5, 125.6, 119.3, 109.3, 103.0, 82.6, 77.6, 74.7, 71.0, 67.0, 37.1. LRMS (m / z): [M + H]+calcd. for C16H17N2O7S2: 413.05, found 413.27.

[0172] 2.2.7 - Sialidase substrate

[0173] Scheme S3. Synthesis of sialic acid luciferin substrate via imidate formation, i. AcCI (56 equiv), MeOH (30 equiv), DCM, 17 h, rt. ii. 1 (1.2 equiv), K2CO3 (1.7 equiv), MeCN, 16 h, rt. Hi.

[0174] Methyl (2S,4S,5 / ?,6 / ?)-5-acetamido-4-hydroxy-2-((2-(imino(methoxy)methyl)benzo[d]thiazol-

[0175] 6-yl)oxy)-6-((1 / ?,2 / ?)-1 ,2,3-trihydroxypropyl)tetrahydro-2H-pyran-2-carboxylate (30): Sialic reaction was stirred for 1 h, the ice bath was removed, and the reaction was subsequently stirred for 16 h at rt. Subsequently, the solvent was evaporated in vacuo and the crude product 28 was used without further purification in the next step. Chloride 28 was dissolved in dry MeCN (10 mL) under inert atmosphere and 6-hydroxybenzo[c / |thiazole-2-carbonitrile (1 , 400 mg, 2.3 mmol) and K2CO3 (440 mg, 3.2 mmol) were also dissolved in dry MeCN (20 mL) under inert atmosphere. The solution of 28 in was added slowly to the benzothiazole solution and the reaction was stirred for 16 h upon which it became a brown suspension. The reaction was evaporated in vacuo, redissolved in EtOAc (100 mL), and washed with water (50 mL) and brine (50 mL). The organic phase was dried with MgSO4, concentrated in vacuo and purified with silica gel column chromatography (50 — 100% EtOAc in heptane) to afford 29 (1.10 g) as an inseparable mixture with starting material (RF = 0.35) which was used directly in the next step. Sialic acid 29 was dissolved in dry MeOH (9 mL) under inert atmosphere. To the mixture was added a 5.4 M NaOMe solution in MeOH (cat). The reaction turned yellow and was stirred for 16 h. Subsequently, the reaction was concentrated in vacuo and purified with silica gel column chromatography (0 — 15% MeOH in DCM) to afford 30 (484 mg, 50% over 3-steps) as a white solid. TLC RF = 0.27 (MeOH / DCM, 15 / 85 v / v).1H NMR (500 MHz, CD3OD) 5 7.99 (d, J = 8.9 Hz, 1 H), 7.86 (d, J = 2.3 Hz, 1 H), 7.38 (ddd, J = 9.0, 2.4, 0.8 Hz, 1 H), 3.98 (s, 2H), 3.92-3.78 (m, 4H), 3.76-3.67 (m, 4H), 3.63 (dd, J = 11 .6, 5.7 Hz, 1 H), 3.57-3.49 (m, 1 H), 2.82 (dd, J = 12.8, 4.6 Hz, 1 H), 2.03-1.97 (m, 4H).13C NMR (126 MHz, CD3OD) 5 175.2, 170.0, 154.3, 150.9, 138.3, 125.6, 123.4, 1 15.7, 102.6, 75.9, 72.4, 70.1 , 68.3, 64.9, 53.7, 53.5, 41.4.

[0176] LRMS (m / z): [M + H]+calcd. for C21H28N3O10S: 514.15, found 514.07. (S)-2-(6-(((2S,4S,5R,6R)-5-acetamido-4-hydroxy-2-(methoxycarbonyl)-6-((1 ,2R)-1 ,2,3- trihydroxypropyl) tetrahydro-2H-pyran-2-yl)oxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4- mg, 0.44 mmol). The reaction was stirred for 16 h after which the solvent was concentrated in vacuo and purified with silica gel column chromatography (0 —>■ 20% H2O in MeCN) to afford 31 (112 mg, 85%) as a white solid. TLC (MeCN / H2O, 4:1 v / v) RF = 0.45.1H NMR (500 MHz, D2O) 5 7.77 (d, J = 8.9 Hz, 1 H), 7.63 (d, J = 2.5 Hz, 1 H), 7.21 (dd, J = 9.0, 2.4 Hz, 1 H), 5.17 (dd, J = 9.9, 8.1 Hz, 1 H), 3.96-3.84 (m, 4H), 3.75 (tdd, J = 10.1 , 7.6, 4.7 Hz, 3H), 3.60-3.54 (m, 2H), 3.51 (dd, J = 8.9, 1 .2 Hz, 1 H), 3.01 (dd, J = 15.0, 5.7 Hz, 1 H), 2.96-2.87 (m, 1 H), 2.80 (dd, J = 12.8, 4.5 Hz, 1 H), 1.96 (d, J = 4.8 Hz, 4H).13C NMR (126 MHz, D2O) 6 181.6, 175.1 , 169.4, 161.3, 152.3, 149.1 , 136.3, 124.1 , 121.9, 114.1 , 101.3, 79.8, 73.9, 70.8, 68.3, 67.1 , 63.2, 55.9, 53.6, 51.7, 39.8, 36.4, 24.8,

[0177] 22.2. LRMS (m / z): [M + H]+calcd. for C23H28N3O11S2: 586.12, found 586.12.

[0178] (S)-2-(6-(((2S,4S,5R,6R)-5-acetamido-2-carboxy-4-hydroxy-6-((1R,2R)-1 ,2,3- trihydroxypropyl)tetrahydro-2H-pyran-2-yl)oxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4- stirred for 1 .5 h after which 1 M HCI was added (0.16 mL, 0.16 mmol). The solvent was concentrated in vacuo and the product was purified with silica gel column chromatography (0 — 30% H2O in MeCN) to afford 32 (17 mg, 55%) as a yellow solid. TLC (MeCN / H2O, 4:1 v / v) RF = 0.45.1H NMR (500 MHz, D2O) 6 7.85 (d, J = 9.0 Hz, 1 H), 7.70 (s, 1 H), 7.30 (d, J = 9.0 Hz, 1 H), 5.17 (t, J = 8.7 Hz, 1 H), 3.89-3.71 (m, 5H), 3.67 (td, J = 10.6, 4.6 Hz, 1 H), 3.53 (qd, J = 11.8, 5.3 Hz, 3H), 2.82 (dd, J = 12.5, 4.7 Hz, 1 H), 1.94 (d, J = 1.5 Hz, 3H), 1.88 (t, J = 12.2 Hz, 1 H).13C NMR (126 MHz, D2O) 6 175.1 , 172.6, 153.2, 149.0, 136.2, 123.8, 122.3, 114.3, 73.5, 71.9, 68.3, 68.2, 62.7, 51.8, 40.7, 22.1 . LRMS (m / z): [M + H]+calcd. for C22H26N3O11S2: 572.10, found 572.33.

[0179] Example 3 - luminescent assays

[0180] 3.1 - Linear titration

[0181] Substrate 12, 13, or 14 (final cone. 20 pM), D-cysteine HCI (final cone. 40 pM), K2CO3 (final cone. 40 pM) and buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.2) were added to a well with a total volume of 23 pL. After incubation for 1 h at 37 °C, 2 pL of corresponding glycosidase (different concentrations) was added and 5 pL of detection mix containing ATP (final cone. 1000 pM), MgCL (final cone. 5.0 mM), firefly luciferase (Quantilum®, Promega, final cone. 0.147 mg / mL) and buffer were added. The luminescence was recorded in Relative Light Units (RLU) for 90 minutes at rt under continuous shaking at 60 rpm with an integration time of 1000 ms per well. Measurements were executed in triple and the Area Under the Curve (AUC) was calculated using Graphpad Prism (version 9.0) and fitted with a linear regression. For each glycosidase, the optimal concentration was determined which was used in the Michaelis-Menten kinetic experiments

[0182] 3.2 - Michaelis-Menten kinetics experiments

[0183] Substrate 12, 13, or 14 (different concentrations), D-cysteine HCI (2 equiv), K2CO3 (2 equiv) and buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.2) were added to a well with a total volume of 23 pL. After incubation for 1 h at 37 °C, 2 pL of corresponding glycosidase was added and 5 pL of detection mix containing ATP (final cone. 1000 pM), MgCh (final cone. 5.0 mM), firefly luciferase (Quantilum®, Promega, final cone. 0.147 mg / mL) and buffer were added. The luminescence was recorded in Relative Light Units (RLU) for 90 minutes at rt under continuous shaking at 60 rpm with an integration time of 1000 ms per well. Measurements were executed in quintuple and the Area Under the Curve (AUC) was calculated using Graphpad Prism (version 9.0) and the data was fitted with the nonlinear regression (curve fit)— Michaelis Menten algorithm.

[0184] 3.3 - Linear titration of sialidase substrate

[0185] Substrate 32 (final cone. 20 pM), and buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.2) were added to a well with a total volume of 23 pL. 2 pL of corresponding glycosidase (different concentrations) was added and 5 pL of detection mix containing ATP (final cone. 1000 pM), MgCh (final cone. 5.0 mM), firefly luciferase (Quantilum®, Promega, final cone. 0.147 mg / mL) and buffer were added. The luminescence was recorded in Relative Light Units (RLU) for 90 minutes at rt under continuous shaking at 60 rpm with an integration time of 1000 ms per well Measurements were executed in triplo and the Area Under the Curve (AUC) was calculated using Graphpad Prism (version 9.0) and fitted with a linear regression.

[0186] 3.4 - Michaelis-Menten kinetics experiment

[0187] Substrate 32 (different concentrations) and buffer (25 mM HEPES, 125 mM NaCI, 0.5% BSA, pH = 7.2) were added to a well with a total volume of 23 pL. 2 pL of neuraminidase was added and 5 pL of detection mix containing ATP (final cone. 1000 pM), MgCL (final cone. 5.0 mM), firefly luciferase (Quantilum®, Promega, final cone. 0.147 mg / mL) and buffer were added. The luminescence was recorded in Relative Light Units (RLU) for 90 minutes at rt under continuous shaking at 60 rpm with an integration time of 1000 ms per well. Measurements were executed in quintuple and the Area Under the Curve (AUC) was calculated using Graphpad Prism (version 9.0) and the data was fitted with the nonlinear regression (curve fit)— Michaelis Menten algorithm.

Claims

ClaimsMethod for producing a caged luciferin compound, the method comprising the steps of: providing an intermediate that is a compound of general formula (I):whereinX is O or NH;Q is O or NH;R is -NH2, -0-Ci-io(cyclo)alkyi, -S-Ci-io(cyclo)alkyl, -O-(5-10 membered (hetero) ary I), or -S-(5-10 membered (hetero) ary I); and cage is a glycosyl moiety or a peptide; optionally converting the provided compound of general formula (I) to a compound of general formula (l-CN):contacting the compound with cysteine to provide a caged luciferin compound of formula (l-probe):

2. The method according to claim 1 , wherein the intermediate of general formula (I) is of general formula (l-imidate):wherein c is Chalky I, preferably -CH3 or -CH2CH3, more preferably -CH3.

3. The method according to claim 1 , wherein the intermediate of general formula (I) is of general formula (l-amide), wherein preferably step ii) is not optional:

4. The method according to claim 3, wherein step ii) comprises treatment of the compound of general formula (l-amide) with an acid anhydride such as trifluoroacetic anhydride.

5. The method according to any one of claims 1 , 3 or 4, wherein step i) comprises one or more of the following sub-steps: i-a) hydrolyzing hydroxybenzo[c / |thiazole-2-carbonitrile or aminobenzo[c / |thiazole-2-carbonitrile to form hydroxybenzo[c / |thiazole-2- carboxamide or aminobenzo[c / |thiazole-2-carboxamide, such as through acid hydrolysis using sulfuric acid; i-b) reacting the benzo[c / |thiazole-2-carboxamide with a protected glycosyl a- halide such as an a-bromide to obtain a protected compound of general formula (I); i-c) optionally deprotecting the protected compound of general formula (I) to obtain a deprotected compound of general formula (I).

6. The method according to claim 1 or 2, wherein step i) comprises reacting hydroxybenzo[c / ]thiazole-2-carbonitrile with a glycosyl a-fluoride to provide a compound of general formula (l-imidate).

7. The method according to any one of claims 1-6, wherein cage is of general formula (II):wherein m is 0 or 1 ; n is 1 , 2, or 3; oh is H, -OH, -Oh’, -NH2, -NHh’, -Nh’2, -NHSO3H, -NHSO3h’, -COOH, - COOh’, -OSO3H, or -OSO3h’; q is H, -CH2-OH, -CH2-Oh’, -CH2OSO3H, -CH2OSO3h’, -COOH, -COOh’, - CH2-(CH(OH))I-6-H, or -CH2-(CH(Oh’))i-6-H; h is H, -OH, -CH2OH, -Oh’, -CH20h’,-NH2, -NHh’, -Nh’2, -COOH, or - COOh’; andh’ is in each instance independently p’ or a C1-8 alkyl or acyl that is optionally cyclic and that is optionally unsaturated and that is optionally halogenated; p' is in each instance independently a protecting group; wherein an instance of q or oh or h may be a further moiety of general formula (II) to form an oligosaccharide-type glycosyl moiety.

8. The method according to claim 7, wherein m is 1 ; n is 2 or 3; oh is H, -OH or -NH-C(=O)CH3; q is H or -CH2-OH or -CH2-CH(OH)-CH(OH)-CH(OH)-H; and / or h is H, -COOH, or -COOCH3.

9. The method according to any one of claims 1 -8, wherein cage is selected from:

10. The method according to any one of claims 1 -9, wherein step iii) comprises contacting the compound with from 1-10 equivalents of cysteine such as 2 equivalents of cysteine, wherein the contacting is preferably in an aqueous buffer such as 25 mM HEPES, 125 mM NaCI, and 0.5 wt.-% BSA, at pH 7.4 or 7.2, wherein the cysteine is preferably D-cysteine, wherein the cysteine is preferably an acid addition salt such as cysteine HCI.11 . Compound of general formula (I) or a salt thereof:whereinX is O or NH;R is -NH2, -OCH3, or -OCH2CH3; and cage is a glycosyl moiety or a peptide, wherein the compound is not12. The compound according to claim 11 , wherein cage is of general formula (II):wherein m is 0 or 1 ; n is 1 , 2, or 3; oh is H, -OH, -Oh’, -NH2, -NHh’, -Nh’2, -NHSO3H, -NHSO3h’, -COOH, - COOh’, -OSO3H, or -OSO3h’; q is H, -CH2-OH, -CH2-Oh’, -CH2OSO3H, -CH2OSO3h’, -COOH, -COOh’, - CH2-(CH(OH))I-6-H, or -CH2-(CH(Oh’))i-6-H; h is H, -OH, -CH2OH, -Oh’, -CH20h’,-NH2, -NHh’, -Nh’2, -COOH, or - COOh’; and h’ is in each instance independently p’ or a C1-8 alkyl or acyl that is optionally cyclic and that is optionally unsaturated and that is optionally halogenated; p' is in each instance independently a protecting group; wherein an instance of q or oh or h may be a further moiety of general formula (II) to form an oligosaccharide-type glycosyl moiety.

13. The compound according to claim 11 or 12, wherein it is:

14. Kit of parts comprising i) a compound according to any one of claims 11-13, or one of compoundsand ii) cysteine, preferably D-cysteine.

15. Method for determining glycosidase or protease activity, the method comprising the steps of: providing one or more compounds according to any one of claims 11 -13, or one of compoundsii) contacting the provided compounds with cysteine to provide caged luciferin compounds of formula (l-probe); iii) contacting the caged luciferin compounds with the glycosidase or protease to provide free luciferin or aminoluciferin, wherein the glycosidase is preferably glucosidase, galactosidase, xylosidase, or neuraminidase; iv) contacting the free luciferin or aminoluciferin with luciferase to generate a luminescent signal.

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