Methods and compounds related to beta-ethynylserine

Abstract

The non-canonical amino acid beta-ethynyl serine (BES) is a close structural analogue of canonical amino acid threonine and it has applications in metabolic protein labelling. The invention relates to a synthetic strategy that provides BES in a good overall yield. The strategy allows a high degree of optical purity. The strategy involves ring-opening of a 2,2-dialkyloxazolidine-4-carboxaldehyde, and oxidation of the resulting alcohol to form a carboxylic acid.

Description

[0001] Methods and compounds related to beta-ethynylserine

[0002] Field of the invention

[0003] The non-canonical amino acid beta-ethynyl serine (BES) is a close structural analogue of canonical amino acid threonine and it has applications in metabolic protein labelling. The invention relates to a synthetic strategy that provides BES in a good overall yield. The strategy allows a high degree of optical purity. The strategy involves ring-opening of a 2,2-dialkyloxazolidine-4- carboxaldehyde, and oxidation of the resulting alcohol to form a carboxylic acid.

[0004] Background art

[0005] The non-canonical amino acid beta-ethynyl serine (also known as BES, p-ethynyl serine, or pES) is a close structural analog of the canonical amino acid threonine. As a result, it is efficiently incorporated into proteins that are synthesized by the endogenous biosynthetic machinery of living cells. Due to this potent metabolic incorporation and owing to its alkyne chemical handle that can be used for bioorthogonal chemistry, BES is suited for metabolic protein labeling experiments to study protein synthesis. Particularly the L-threo (2S,3R) isomer of BES is incorporated into newly synthesized proteins (Ignacio et al., Nature Communications, volume 14, Article number: 3367 (2023)). However, BES is not commercially available, hindering its widespread use.

[0006] BES can be isolated as a natural product. So far, BES has been isolated from Athelia rolfsii and from Streptomyces cattleya. Isolation from fungi can yield optically pure BES, but requires large, specialized fermentation tanks while offering only moderate yields (< 50 mg / l) after extensive chromatography steps (Sanada et al., J. Antibiot. (Tokyo) 39, 304-305 (1986)).

[0007] BES can also be acquired through chemical synthesis and it has been synthesized through a non-stereoselective aldol condensation. Potgieter et al. (Phytochemistry 16, 1757-1759 (1977)) used an aldol condensation of copper glycinate with propiolaldehyde to provide BES via a short chemical synthesis of a single synthetic step, but as a diastereomeric mixture of racemates in only a 12% yield. Ignacio et al. (see above) improved on this by providing a stereoselective synthesis route using an asymmetric aminohydroxylation as a key step. BES could be obtained in four steps in a 22% overall yield.

[0008] There is a need for a synthetic strategy that provides BES in an improved overall yield. There is a need for a strategy that provides an improved degree of optical purity. There is a need for a strategy that can be efficiently scaled.

[0009] Summary of the invention

[0010] A synthetic strategy was designed that could provide protected or unprotected beta- ethynylserine (BES) in a good overall yield and with a high degree of optical purity. Alkylation on Garner’s aldehyde was found to provide a-amino-p-hydroxy acids with a high degree of control over stereochemistry. The inventors surprisingly identified this pathway towards highly pure L-threo BES, using a few synthetic steps from Garner’s aldehyde or analogues, employing a scalable synthetic route. Thus the invention provides a method for providing a protected or unprotected beta- ethynylserine or a salt thereof, the method comprising the steps of:

[0011] I) ring-opening a compound of general formula (i) to obtain an alcohol compound of general formula (ii) wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1-8alkyl or a C2-8 alkenyl; c2 is H or a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure;

[0012] II) oxidation ofthe alcohol compound of general formula (ii) to obtain a carboxylic acid compound of general formula (iii)

[0013] In some embodiments step I) is preceded by step la): alkylating a 2,2-dialkyloxazolidine-4- carboxaldehyde to obtain the compound of general formula (i). In some embodiments step I) is preceded by step lb): protecting a compound of general formula (i) wherein po is H to obtain a compound of general formula (i) wherein po is a protecting group. In some embodiments step la) is followed by step lb), which is then followed by step I). In some embodiments at least one of pn, pc, or po is a protecting group, wherein the method further comprises step III): deprotecting all protecting groups of the carboxylic acid compound of general formula (iii) to obtain a compound of general formula (BES)

[0014] Preferably pc is a silyl such as trimethylsilyl (TMS), Triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tri- isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); preferably po 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), ethoxyethyl (EE), or a silyl such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); preferably pn is trityl, allyl, benzyl (Bn), 9-fluorenylmethyl oxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), 2-trimethylsilylethyloxycarbonyl, tosyl (Ts), acetyl (Ac), trifluoroacetyl, phthalimide, benzylideneamine, or allyloxycarbonyl (Alloc). More preferably pc is a silyl such as trimethylsilyl (TMS); po is acetyl (Ac) or a silyl such as tert- butyldiphenylsilyl (TBDPS); and pn is t-butyloxycarbonyl (Boc).

[0015] In some embodiments step I) comprises ring-opening the compound of general formula (i) by contacting it with a strong nucleophile such as sodium amide, an organic acid such as a sulfonic acid, preferably tosylic acid, or such as a carboxylic acid, preferably acetic acid or trifluoroacetic acid, or with a Lewis acid salt such as a bismuth salt, preferably BiBrs. In some embodiments step II) comprises oxidation using a chlorite salt such as sodium chlorite, a hypochlorite salt such as sodium hypochlorite, pyridinium chromate such as dichromate or chlorochromate, a chromium salt such as chromium trioxide, a ruthenium salt such as RuO4, a permanganate such as KMnC , 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) or a derivative thereof, phenyliodine(lll) diacetate (PIDA), or a combination thereof, preferably using TEMPO, or wherein the oxidation is a Swern oxidation or a Dess-Martin oxidation.

[0016] Also provided is a compound of general formula (i), (2), or (3), or a salt thereof: wherein pc is H or a protecting group; po is H or a protecting group; and pn is in each instance independently H or a protecting group; c1 is H or a C1 -8alkyl or a C2-8 alkenyl; c2 is H or a C1 -8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure; wherein for formula (3) at least one of pc, po, or pn is a protecting group.

[0017] In some embodiments the compound is of general formula (i) wherein po is a protecting group, or wherein the compound is of general formula (2). In some embodiments the compound is of general formula (3) wherein po is a protecting group. Preferably the compound is selected from tert-butyl- 4-(-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3-carboxylate; 4-((tert- butoxycarbonyl)amino)-5-hydroxy-1 -(tri methy Isi ly I) pent- 1 -yn-3-yl acetate; 3-acetoxy-2-((tert- butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid; 2-((tert-butoxycarbonyl)amino)-3- hydroxypent-4-ynoic acid; tert-butyl (1 ,3-dihydroxypent-4-yn-2-yl)carbamate; 3-hydroxy-2-((tert- butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid; tert-butyl-4-(1-((tert- butyldiphenylsilyl)oxy)-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3-carboxylate; tert- butyl(3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2-yl)carbamate; 2-((tert- butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5-(trimethylsilyl)pent-4-ynoic acid; tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3-carboxylate;

[0018] (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1 -(tri methy Isi ly I) pent-1 -yn-3-yl acetate; (2S,3R)- 3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid; (2S,3R)-2-((tert- butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid; tert-butyl ((2R,3R)-1 ,3-dihydroxypent-4-yn-2- yl)carbamate; tert-butyl (R)-4-((R)-1-((tert-butyldiphenylsilyl)oxy)-3-(trimethylsilyl)prop-2-yn-1-yl)- 2,2-dimethyloxazolidine-3-carboxylate; tert-butyl ((2R,3R)-3-((tert-butyldiphenylsilyl)oxy)-1- hydroxy-5-(trimethylsilyl)pent-4-yn-2-yl)carbamate; (2S,3R)-2-((tert-butoxycarbonyl)amino)-3- ((tert-butyldiphenylsilyl)oxy)-5-(trimethylsilyl)pent-4-ynoic acid.

[0019] Also provided is a method for providing a compound of general formula (2) wherein the compound of general formula (2) is as defined above, the method comprising the step of:

[0020] I) ring-opening a compound of general formula (i) to obtain the compound of general formula (2) wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1 -8alkyl or a C2-8 alkenyl; c2 is H or a C1 -8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure. Also provided is a method for providing a compound of general formula (3) wherein the compound of general formula (3) is as defined above, the method comprising the step of:

[0021] II) oxidation of a compound of general formula (2) wherein the compound of general formula (2) is as defined above, to obtain the compound of general formula (3).

[0022] Also provided is the use of a 2,2-dialkyloxazolidine-4-carboxaldehyde in the manufacture of a protected or unprotected beta-ethynylserine or a salt thereof.

[0023] Description of embodiments

[0024] A synthetic strategy was designed that could provide BES in a good overall yield and with a high degree of optical purity. Alkylations on Garner’s aldehyde was found to provide a-amino-p- hydroxy acids with a high degree of control over stereochemistry. The inventors surprisingly identified this pathway towards highly pure L-threo BES, using a few synthetic steps from Garner’s aldehyde or analogues, employing a scalable synthetic route.

[0025] Method for providing BES

[0026] The invention provides a method for providing a protected or unprotected beta- ethynylserine (BES) or a salt thereof, the method comprising the steps of:

[0027] I) ring-opening a compound of general formula (i) to obtain an alcohol compound of general formula (ii) wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1-8alkyl or a C2-8 alkenyl; c2 is H or a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure;

[0028] II) oxidation ofthe alcohol compound of general formula (ii) to obtain a carboxylic acid compound of general formula (iii)

[0029] Such a method is referred to herein as a method according to the invention. The steps are preferably performed in numerical and / or alphabetical order where applicable. In the context of the invention, a salt of a compound 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.

[0030] Compound as described herein 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.

[0031] Preferably the method comprises two steps when po is a protecting group. Preferably the method comprises two steps when po, pn, and pc are each a protecting group. Preferably the method comprises three steps when po is H in the compound of general formula (i). Preferably the method comprises three steps when po is H in the compound of general formula (i) and po is a protecting group in the compound of general formula (iii). Preferably the method comprises three steps when in the compound of general formula (i) pn and pc are each a protecting group and po is H, and in the compound of general formula (iii) po, pn, and pc are each a protecting group. Preferably the method comprises five steps when in the compound of general formula (i) pn and pc are each a protecting group and po is H, and in the compound of general formula (iii) po, pn, and pc are each H. Preferably the method comprises four steps when in the compound of general formula (i) po, pn and pc are each a protecting group, and in the compound of general formula (iii) po, pn, and pc are each H.

[0032] Preferably the yield of the protected or unprotected beta-ethynylserine (BES) or a salt thereof is at least 55%, more preferably at least 56, 57, 58, 59, 60, 61 ,62,63,64,65,66,67,68,69, or 70%. Highly preferred methods have a yield of at least 60%, more preferably at least 62%, most preferably at least 64%. Yield is preferably relative to the amount of compound of general formula (i) wherein pn and pc are each a protecting group and po is H that is provided in step I). Yield as described in this paragraph is preferably calculated as the yield of the compound of general formula (iii) wherein po, pn, and pc are each a protecting group.

[0033] In preferred embodiments at least 3 grams of the compound of general formula (i) is provided in step I), more preferably at least 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or more grams are provided. It is highly preferred that at least 10 grams is provided.

[0034] Compounds of general formula (i) or (ii) or (iii)

[0035] In the method a compound of general formula (i) is ring-opened to obtain an alcohol compound of general formula (ii) wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1 -8alkyl or a C2-8 alkenyl; c2 is H or a C1 -8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure.

[0036] A compound of general formula (i) resembles an alkylate Garner’s aldehyde. A common example of Garner’s aldehyde is shown below. Examples of analogues can have different protecting groups on the nitrogen atom, or can have different substituents instead of the geminal methyl. A compound of general formula (ii) can be referred to as an alcohol compound because it comprises an unprotected hydroxyl moiety. A common form of Garner’s aldehyde

[0037] Herein it could be said that c1 and c2 are both -CH3, and pn is t-butyloxycarbonyl (Boc). It is attractive for c1 and c2 to both be relatively small yet inert moieties. Therefore c1 is H or a C1- 8alkyl or a C2-8 alkenyl; and c2 is H or a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure. Preferably c1 is a C1-8alkyl or a C2-8 alkenyl; and c2 is a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure; more preferably c1 is a C1-8alkyl or a C2-8 alkenyl; and c2 is a C1 -8alkyl or a C2-8 alkenyl.

[0038] Preferably when c1 is a C1-8alkyl or a C2-8 alkenyl, it is a C1-6alkyl or a C2-6 alkenyl, more preferably a C1-4alkyl or a C2-4 alkenyl, even more preferably a C1-2alkyl, most preferably it is -CH3. Preferably when c2 is a C1-8alkyl or a C2-8 alkenyl, it is a C1-6alkyl or a C2-6 alkenyl, more preferably a C1-4alkyl or a C2-4 alkenyl, even more preferably a C1-2alkyl, most preferably it is -CH3. In preferred embodiments the same choice is made for both c1 and c2. Preferably both c1 and c2 are C1-4alkyl, more preferably C1-2alkyl, most preferably -CH3.

[0039] As used herein, C1-8alkyl and C2-8 alkenyl can be linear, cyclic, or branched. In the context of this invention, the number of carbon atoms in a moiety such as a linear, branched, or cyclic alkyl, or alkenyl is indicated as for example C1 -6, in this non-limiting case indicating that from 1 to 6 carbon atoms are envisaged, such as 1 , 2, 3, 4, 5, or 6 carbon atoms. Similarly C2-4alkyl has 2, 3, or 4 carbon atoms. The number of carbon atoms can be expressed as the total number of carbon atoms not counting further substitutions, the total number of carbon atoms, or as the number of carbon atoms that can be found in the longest continuous internal sequence of carbon atoms. Preferably, the number of carbon atoms is expressed as the total number of carbon atoms not counting further substitutions.

[0040] In the context of this invention, unsubstituted alkyl groups have the general formula CnH2n+i and may be linear or branched. Unsubstituted alkyl groups may also be cyclic, and thus have the concomitant general formula CnH2n-i. Optionally, the alkyl groups are substituted by one or more substituents chosen independently from halogen, alkoxy, and haloalkoxy. Examples of suitable alkyl groups include, but are not limited to, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, - CH(CH3)CH2CH3, -CH2CH(CH3)2, -CH2CH2CH2CH3, -C(CH3)3, 1 -hexyl and the like. Preferred alkyl groups are linear or branched, most preferably linear. Preferred cyclic alkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, more preferably cyclopropyl or cyclohexyl, most preferably cyclopropyl.

[0041] In the context of this invention, unsubstituted alkenyl groups have the general formula CnH2n-i, and may be linear or branched. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, pentenyl and the like. Unsubstituted alkenyl groups may also contain a cyclic moiety, and thus have the concomitant general formula CnH2n-3. Preferred alkenyl groups are linear or branched, most preferably, linear. Highly preferred unsaturated cyclic alkyl groups are aryl groups, such as phenyl.

[0042] In some embodiments c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure. This cyclic structure thus comprises c1 and c2 and the carbon atom to which they are linked. The cyclic structure can be said to form a spiro bicyclic compound with the ring of Garner’s aldehyde or the analogue thereof. Preferably, when c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure, they form a 3-8-membered cyclic structure, more preferably a 3-6-membered cyclic structure, even more preferably a 3-5-membered cyclic structure, still more preferably a 3-4-membered cyclic structure such as cyclobutyl or cyclopropyl. The cyclic structure is preferably a spiro carbocyclic structure, and thus it can be described as c1 and c2 together forming -(CH2)3-IO-. It is most preferred that c1 and c2 are both -CH3. pc, po, and pn are each independently H or a protecting group. As used herein, a protecting group has its customary meaning of a group that is linked to the relevant (hetero)atom 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 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. pc is H or a protecting group. When it is a protecting group, pc is preferably a silyl such as trimethylsilyl (TMS), Triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); most preferably pc is trimethylsilyl (TMS). po is H or a protecting group. When it is a protecting group, po is preferably 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), ethoxyethyl (EE), or a silyl such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tri-isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); most preferably po is acetyl (Ac) or a silyl such as tert-butyldiphenylsilyl (TBDPS). pn H or a protecting group. When it is a protecting group, pn is preferably trityl, allyl, benzyl (Bn), 9-fluorenylmethyl oxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), 2- trimethylsilylethyloxycarbonyl, tosyl (Ts), acetyl (Ac), trifluoroacetyl, phthalimide, benzylideneamine, or allyloxycarbonyl (Alloc), and optionally mesitylenesulfonyl (Mts); most preferably pn is t-butyloxycarbonyl (Boc).

[0043] In preferred embodiments is provided the method according to the invention, wherein pc is a silyl such as trimethylsilyl (TMS); po is acetyl (Ac) or a silyl such as tert-butyldiphenylsilyl (TBDPS); and pn is t-butyloxycarbonyl (Boc). More preferably pc is trimethylsilyl (TMS); po is acetyl (Ac) or tert-butyldiphenylsily I (TBDPS); and pn is t-butyloxycarbonyl (Boc). In some highly preferred embodiments pc is a silyl such as trimethylsilyl (TMS); po is acetyl (Ac); and pn is t- butyloxycarbonyl (Boc). In other highly preferred embodiments pc is a silyl such as trimethylsilyl (TMS); po is a silyl such as tert-butyldiphenylsi ly I (TBDPS); and pn is t-butyloxycarbonyl (Boc).

[0044] It is preferred that during ring-opening pc is a protecting group. It is preferred that during ring-opening po is a protecting group. It is preferred that during ring-opening pn is a protecting group. It is particularly preferred that during ring-opening pc, po, and pn are each a protecting group. The protecting groups can be removed later as desired. Accordingly, in preferred embodiments at least one of pn, pc, or po is a protecting group, and the method further comprises step III) deprotecting all protecting groups of the carboxylic acid compound of general formula (iii) to obtain a compound of general formula (BES). The compound of general formula BES can also be a salt or hydrate or solvate as described earlier herein.

[0045] A skilled person is aware of suitable methods for deprotection. Deprotection of a protecting group generally refers to removal of the protecting group from the (hetero)atom to regenerate the originally protected moiety. In other words, deprotection of pn when it is a protecting group regenerates an amine, wherein pn is H; pn can generally be deprotected using acid or base treatment; deprotection of po when it is a protecting group regenerates a hydroxyl, wherein po is H; po can generally be deprotected using acid or base treatment; deprotection of pc when it is a protecting group regenerates an alkyne, wherein pc is H; pc can generally be deprotected using acid or base treatment. The examples provide further reductions to practice of deprotections to supplement a skilled person’s common general knowledge.

[0046] Carboxylic acid compounds of general formula (iii) are desirable products of the method of the invention. The compound is referred to as a carboxylic acid compound because it comprises an unprotected carboxylic acid moiety.

[0047] For compounds of general formula (i) or (ii) it is preferred that pc, po, and pn are each a protecting group to facilitate efficient ring-opening and / or oxidation. For compounds of general formula (iii) the nature of pc, po, and pn can depend on which groups were selected for the compounds of general formula (i) and (ii), so these moieties can also each be a protecting group so that the compound of general formula (iii) is a protected beta-ethynylserine. One or more of pc, po, and pn can also be H, for instance as the result of deprotection. In particular all three of pc, po, and pn can be H when the beta-ethynylserine is fully deprotected. Such a compound is of general formula BES as shown above.

[0048] The following are possible embodiments of compounds of general formula (i), (ii), and (iii).

[0049] Compounds of general formula (i) are preferably of general formula (i-D). Compounds of general formula (i) are more preferably of general formula (i-DR). Compounds of general formula (ii) are preferably of general formula (ii-D). Compounds of general formula (ii) are more preferably of general formula (ii-DR). Compounds of general formula (iii) are preferably of general formula (iii- D). Compounds of general formula (iii) are more preferably of general formula (iii-DR).

[0050] Ring opening a compound of general formula (i) to form a compound of general formula (ii) generally does not alter its stereochemistry, nor does oxidation of a compound of general formula (ii) to a compound of general formula (iii). Thus the selection of a compound of general formula (i- DR) will generally lead to a compound of general formula (ii-DR) and to one of general formula (iii- DR) when the method of the invention is followed.

[0051] Details of steps of the method

[0052] Compounds of general formula (i) can be obtained by alkylating a Garner’s aldehyde or analogues thereof. Accordingly, in preferred embodiments, step I) is preceded by step la): alkylating a 2,2-dialkyloxazolidine-4-carboxaldehyde to obtain the compound of general formula (i). The 2,2- dialkyloxazolidine-4-carboxaldehyde is preferably protected at its nitrogen atom. Its stereochemistry is preferably D. The dialkylmoieties are preferably as defined for c1 and c2 above. Accordingly the 2,2-dialkyloxazolidine-4-carboxaldehyde is preferably of general formula (G), more preferably of general formula (G-D). In some embodiments it is of general formula (G-L).

[0053] Because of addition to the aldehyde carbon atom, alkylation as described above leads to a compound of general formula (i) wherein po is H. It was found that improved yields were obtained when this hydroxy group was protected prior to oxidation. To avoid the simultaneous presence of multiple different hydroxy groups such protection prior to ring opening is even more advantageous. Accordingly in preferred embodiments of the method step I) is preceded by step lb): protecting a compound of general formula (i) wherein po is H to obtain a compound of general formula (i) wherein po is a protecting group. A skilled person knows how to protect such a hydroxy compound, and a selection of options for such strategies are provided in the examples section. Protection is preferably by introduction of a moiety for po as described earlier herein.

[0054] Alkylations on Garner’s aldehyde to provide a-amino-p-hydroxy acids with a high degree of control over stereochemistry is known as such. Alkylation of (D)-Garner’s aldehyde can advantageously be performed with a protected ethynyl magnesium halide, such as a protected ethynyl magnesium bromide, for instance trimethylsilyl-protected ethynyl magnesium bromide. The alkylation can take place in a suitable solvent such as an aprotic solvent, for instance an ether, preferably a cyclic ether, more preferably tetra hydrofuran. The reaction can take place at low temperature, for instance at about -100 °C to about -10 °C, preferably at about -80 °C to about -30 °C. During reacting the reaction can be allowed to warm to about 20 °C. The reaction is preferably stirred. The reaction is preferably allowed to proceed for about 2-24 hours, more preferably about 4-18 hours, most preferably about 8-14 hours such as about 12 hours. Additional details are preferably as provided in the examples.

[0055] Ring-opening of the oxazolidine moiety can be performed using methods that are known as such. In preferred embodiments step I) comprises ring-opening the compound of general formula (i) by contacting it with a strong nucleophile such as sodium amide, an organic acid such as a sulfonic acid, preferably tosylic acid, or such as a carboxylic acid, preferably acetic acid or trifluoroacetic acid, or with a Lewis acid salt such as a bismuth salt, preferably BiBrs. The ringopening can be performed in a protic solvent such as a lower alcohol, for instance methanol, or in a mixture of water and a polar aprotic solvent such as acetonitrile. The ring-opening can be performed at a temperature of about 1-60 °C, preferably about 10-40 °C, more preferably about 15- 25 °C such as about 20 °C. The reaction is preferably stirred. The reaction is preferably allowed to proceed for about 2-24 hours, more preferably about 4-20 hours, most preferably about 8-18 hours such as about 16 hours. Additional details are preferably as provided in the examples.

[0056] Oxidation of primary hydroxy groups to form carboxylic acids can be performed using methods that are known as such. In preferred embodiments step II) comprises oxidation using a chlorite salt such as sodium chlorite, a hypochlorite salt such as sodium hypochlorite, pyridinium chromate such as dichromate or chlorochromate, a chromium salt such as chromium trioxide, a ruthenium salt such as RuO4, a permanganate such as KMnC , 2,2,6,6-tetramethylpiperidine-1- oxyl (TEMPO) or a derivative thereof, phenyliodine(lll) diacetate (PIDA), or a combination thereof, preferably using TEMPO, or wherein the oxidation is a Swern oxidation or a Dess-Martin oxidation. Preferred derivatives of TEMPO are 4-carboxy-2,2,6,6-tetramethylpiperidine 1 -oxyl, 4-hydroxy- 2,2,6,6-tetramethylpiperidine 1-oxyl benzoate, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine 1- oxyl, 4-Glycidyloxy-2,2,6,6-tetramethylpiperidine 1 -oxyl, 4-oxo-2,2,6,6-tetramethylpiperidine 1 -oxyl, and 4-cyano-2,2,6,6-tetramethylpiperidine 1-oxyl. Oxidation can also be performed using 1-methyl- 2-azaadamantane-N-oxyl or 2-azaadamantane-N-oxyl. Oxidation is preferably performed in an aprotic solvent, more preferably a polar aprotic solvent such as acetonitrile. Oxidation is preferably performed at low temperature such as in the range -10 to 15 °C, more preferably 0-10 °C, most preferably about 0 °C. Oxidation can be performed in the presence of phenyliodine diacetate (PIDA) combined with at least one further agent as recited above, preferably PIDA and TEMPO or a derivative thereof. The reaction is preferably stirred. The reaction is preferably allowed to proceed for about 0.5-10 hours, more preferably about 1 -8 hours, most preferably about 3-7 hours such as about 5 hours. Additional details are preferably as provided in the examples.

[0057] Compounds and intermediates

[0058] The invention provides a compound of general formula (i), (2), or (3), or a salt thereof: wherein pc is H or a protecting group; po is H or a protecting group; and pn is in each instance independently H or a protecting group; c1 is H or a C1 -8alkyl or a C2-8 alkenyl; c2 is H or a C1 -8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure; wherein for formula (3) at least one of pc, po, or pn is a protecting group. In preferred embodiments for formula (3) at least two of pc, po, or pn are a protecting group. In other preferred embodiments for formula (3) all three of pc, po, or pn are a protecting group.

[0059] Features and definitions for the compound of general formula (i) are preferably as described earlier herein, where the compound is described for use in the method of the invention. Similarly, features and definitions for the compound of general formula (2) are preferably as described earlier herein for general formula (ii). Similarly, features and definitions for the compound of general formula (3) are preferably as described earlier herein for general formula (iii). General formulas (2) and (3) differ from general formulas (ii) and (iii) in that the nitrogen atom in general formulas (2) and (3) bears two instances of pn, where the nitrogen atom in general formulas (ii) and (iii) bears one instance of pn and one H instead. Thus, a compound of general formula (ii) can be seen as a compound of general formula (2) wherein H is selected for at least one instance of pn.

[0060] Further preferred is a compound wherein it is of general formula (i) wherein po is a protecting group, or wherein the compound is of general formula (2). In some preferred embodiments the compound is of general formula (i) wherein po is a protecting group. In some preferred embodiments the compound is of general formula (2), preferably wherein po is a protecting group. In other preferred embodiments the compound is of general formula (3) wherein po is a protecting group, preferably an acyl such as Ac or a silyl such as TBDPS. In some embodiments the compound is of general formula (3) wherein po and pc are a protecting group. In some embodiments the compound is of general formula (3) wherein po and at least one instance of pn are a protecting group. In some embodiments the compound is of general formula (3) wherein po and one instance of pn are a protecting group and the other instance of pn is H. In some embodiments the compound is of general formula (3) wherein po and pc and at least one instance of pn are a protecting group. In some embodiments the compound is of general formula (3) wherein po and pc and one instance of pn are a protecting group and the other instance of pn is H. In highly preferred embodiments for general formula (3), at least one pn is H. In highly preferred embodiments for general formula (3), at least one pn is a protecting group.

[0061] Provided compounds of general formula (i) are preferably of general formula (i-D), more preferably of general formula (i-DR). Provided compounds of general formula (2) are preferably of general formula (2-D), more preferably of general formula (2-DR). Provided compounds of general formula (3) are preferably of general formula (3-D), more preferably of general formula (3-DR).

[0062] Preferred compounds provided by the invention are tert-butyl-4-(-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3-carboxylate; 4-((tert-butoxycarbonyl)amino)-5-hydroxy-1-(trimethylsilyl)pent-1-yn-3-yl acetate;

[0063] 3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;

[0064] 2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid; tert-butyl (1 ,3-dihydroxypent-4-yn-2-yl)carbamate;

[0065] 3-hydroxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid; tert-butyl-4-(1-((tert-butyldiphenylsilyl)oxy)-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2- dimethyloxazolidine-3-carboxylate; tert-butyl(3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2-yl)carbamate; 2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5-(trimethylsilyl)pent-4-ynoic acid; tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate;

[0066] (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1 -(trimethylsilyl) pent-1 -yn-3-yl acetate; (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;

[0067] (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid; tert-butyl ((2R,3R)-1 ,3-dihydroxypent-4-yn-2-yl)carbamate;

[0068] (2S,3R)-3-hydroxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid; tert-butyl (R)-4-((R)-1-((tert-butyldiphenylsilyl)oxy)-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2- dimethyloxazolidine-3-carboxylate; tert-butyl ((2R,3R)-3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2- yl)carbamate;

[0069] (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5-(trimethylsilyl)pent-4- ynoic acid.

[0070] More preferred compounds are tert-butyl-4-(-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3-carboxylate; 4-((tert-butoxycarbonyl)amino)-5-hydroxy-1-(trimethylsilyl)pent-1-yn-3-yl acetate; 3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid; 2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid; tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate;

[0071] (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1 -(trimethylsilyl) pent-1 -yn-3-yl acetate; (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;

[0072] (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid.

[0073] Even more preferred are tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2- dimethyloxazolidine-3-carboxylate; (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1-

[0074] (trimethylsily l)pent-1 -yn-3-yl acetate; (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5- (trimethylsilyl)pent-4-ynoic acid; and (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4- ynoic acid.

[0075] In preferred embodiments the compound is not of general formula (3) wherein pc is H, po is H, one instance of pn is H and the other instance of pn is Boc. In preferred embodiments the compound is not of general formula (2) wherein pc is H, po is H, and each instance of pn is Bn. In preferred embodiments the compound is not of general formula (3) wherein pc is TMS, po is H, and each instance of pn is H. In preferred embodiments the compound is not of general formula (2) wherein pc is TMS, po is H, one instance of pn is H and the other instance of pn is Mts. In preferred embodiments the compound is not of general formula (2) wherein pc is H, po is H, one instance of pn is H and the other instance of pn is Z. In preferred embodiments the compound is not of general formula (2) wherein pc is H, po is H, one instance of pn is H and the other instance of pn is Boc. In preferred embodiments the compound is not of general formula (2) wherein pc is H, po is H, and each instance of pn is H. In preferred embodiments the compound is not of general formula (3-DR) wherein pc is H, po is tBu, one instance of pn is H, and the other instance of pn is Fmoc. In preferred embodiments the compound is not of general formula (2-DR) wherein pc is H, po is tBu, one instance of pn is H, and the other instance of pn is Fmoc. In preferred embodiments the compound is not of general formula (2-DR) wherein pc is H, po is H, one instance of pn is H, and the other instance of pn is Fmoc.

[0076] In preferred embodiments the compound is not of general formula (i) wherein:

[0077] - pc is TMS, po is H, pn is Boc, c1 is -CH3, c2 is -CH3;

[0078] - pc is H, po is H, pn is Boc, c1 is -CH3, c2 is -CH3;

[0079] - pc is H, po is Ac, pn is Boc, c1 is -CH3, c2 is -CH3;

[0080] - pc is TIPS, po is Ac, pn is Boc, c1 is -CH3, c2 is -CH3;

[0081] - pc is TIPS, po is H, pn is Boc, c1 is -CH3, c2 is -CH3;

[0082] - pc is H, po is H, pn is Z, c1 is -CH3, c2 is -CH3;

[0083] - pc is H, po is TBDMS, pn is Boc, c1 is -CH3, c2 is -CH3; or

[0084] - pc is H, po is Bn, pn is Boc, c1 is -CH3, c2 is -CH3.

[0085] Uses of compounds and of intermediate compounds

[0086] The invention also provides methods wherein the above intermediates and compounds can be used. These methods can be for the provision of further intermediates. For instance, the invention provides a method for providing a compound of general formula (2) wherein the compound of general formula (2) is as defined above, the method comprising the step of:

[0087] I) ring-opening a compound of general formula (i) to obtain the compound of general formula (2) wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1 -8alkyl or a C2-8 alkenyl; c2 is H or a C1 -8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure.

[0088] Features and definitions for the compounds and their moieties, as well as for ring-opening, are preferably as described above. Preferably the compound of general formula (2) as obtained by this method has one instance of pn as H. Preferably pc, po, and pn are the same for the compound of general formula (i) and for the compound of general formula (2). It is of course possible that the method comprises steps where protecting groups are added or removed or replaced by different protecting groups. Another useful application of intermediate compounds as described above is in a method for providing a compound of general formula (3) wherein the compound of general formula (3) is as defined above, the method comprising the step of:

[0089] II) oxidation of a compound of general formula (2) wherein the compound of general formula (2) is as defined above, to obtain the compound of general formula (3).

[0090] Features and definitions for the compounds and their moieties, as well as for oxidation, are preferably as described above. Preferably the compound of general formula (3) as obtained by this method has one instance of pn as H. Preferably pc, po, and pn are the same for the compound of general formula (2) and for the compound of general formula (3). It is of course possible that the method comprises steps where protecting groups are added or removed or replaced by different protecting groups.

[0091] As described herein the invention provides the use of a 2,2-dialkyloxazolidine-4- carboxaldehyde in the manufacture of a protected or unprotected beta-ethynylserine or a salt thereof. The 2,2-dialkyloxazolidine-4-carboxaldehyde is preferably as earlier herein. The use is preferably in a method as defined earlier herein. Other features and definitions are preferably as defined elsewhere herein.

[0092] General Definitions

[0093] 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. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a combination or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. 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".

[0094] 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, the pure R enantiomer, or the pure S enantiomer. Stereochemistry is represented in accordance with IUPAC nomenclature.

[0095] 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. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value.

[0096] Each embodiment as identified herein may be combined together unless otherwise indicated. The invention has been described above with reference to a number of embodiments. A skilled person could envision trivial variations for some elements of the embodiments. These are included in the scope of protection as defined in the appended claims. All patent and literature references cited are hereby incorporated by reference in their entirety.

[0097] Examples

[0098] Example 1 - improved synthesis of non-canonical threonine analogues

[0099] 1. 1 1ntroduction

[0100] Non-canonical amino acid p-ethynyl serine (BES) is a close structural analog of canonical amino acid threonine. As a result, it is efficiently incorporated into proteins synthesized by the endogenous biosynthetic machinery of living cells. Due to BES’ potent metabolic incorporation and due its alkyne chemical handle that can be used for bioorthogonal chemistry, it is uniquely suited for metabolic protein labeling experiments to study protein synthesis. However, BES is not conveniently available, prohibiting its widespread use as a metabolic protein labeling probe.

[0101] BES can be isolated as a natural product or it can be acquired through chemical synthesis: so far, BES has been isolated as a natural product from Athelia rolfsii and Streptomyces cattleya and it has been synthesized through a non-stereoselective aldol condensation. While isolation from fungi yielded optically pure BES, it required large, specialized fermentation tanks and provided only moderate yields (< 50 mg / l) after extensive chromatography steps. On the other hand, Potgieter et al. used an aldol condensation of copper glycinate with propiolaldehyde to provide BES via a chemical synthesis of a single synthetic step, but as a diastereomeric mixture of racemates in only a 12% yield. This is unattractive because only the L-threo (2S,3R) isomer of BES is incorporated into newly synthesized proteins. To obtain the pure, bio-active isomer of BES, a synthetic strategy was designed that could provide BES in a good overall yield and with a high degree of optical purity.

[0102] 1.2 Improved synthesis

[0103] 1.2.1 Efficient synthesis using Garner’s aldehydes or analogues

[0104] Alkylations on Garner’s aldehyde or analogues can provide a-amino-p-hydroxy acids with a high degree of control over stereochemistry. Alkylation of (D)-Garner’s aldehyde with trimethylsilyl (TMS)-protected ethynyl magnesium bromide has previously been reported to proceed with almost complete (> 20:1) syn-selectivity (Scheme S1). The inventors envisaged highly pure L-threo BES in a few synthetic steps from Garner’s aldehyde, via a scalable synthetic route.

[0105] D-Garner's aldehyde

[0106] Scheme S1. Alkylation of D-Garner’s aldehyde with TMS-ethynylMgBr is known to give > 20:1 syn / anti selectivity. The envisaged synthetic route required deprotection such as TMS-deprotection and subsequent oxazolidine ring opening (Scheme S2). Next, we envisioned an oxidation such as a TEMPO-based oxidation of the primary alcohol of 2, which we anticipated could be performed in the presence of unprotected p-hydroxyl, avoiding potentially unnecessary (de-)protection steps. Boc-deprotection would provide the product BES as an HCI salt.

[0107] Scheme S2. synthetic route towards BES, starting from alkylated D-Garner’s aldehyde.

[0108] 1.2.2 protection of the 13-hydroxy group increases yield

[0109] TMS-deprotection using TBAF and oxazolidine ring-opening with p-TsOH of 1 proceeded smoothly on test-scale and up to decagram scale. However, TEMPO oxidation of 2 gave only trace amount of product 3, despite extensive efforts to optimize the reaction conditions. We hypothesized that we could obtain a higher yield by oxidizing TMS-protected alcohol 5, which was obtained by BiBrs- catalyzed oxazolidine ring-opening of 1 , but again only trace amounts of carboxylic acid 6 were obtained (scheme S3).

[0110] Scheme S3. Protection of terminal alkyne with TMS does not increase yield of TEMPO-based oxidation of 5 to carboxylic acid 6.

[0111] Gratifyingly, after protection of the p-hydroxy group, for example by acetylation or with a silyl ether (TBDPS), oxidation with TEMPO or pyridinium dichromate (PDC) proceeded smoothly to provide protected pES in good yields (scheme S4). Acetylation of alkylated Garner’s aldehyde 1 gave protected intermediate 7, which was ring-opened using p-TsOH or BiBrs to give alcohol 8. TEMPO or PDC oxidation proceeded with high yields, affording carboxylic acid 9. TMS-deprotection using for example either TBAF or fcCOs / MeOH gave Boc-protected BES 3 and Boc-deprotection using 6M HCI afforded BES (4). Alternatively, TBDPS-protection of 1 gave 10, which was ring-opened to give alcohol 11. Oxidation again proceeded smoothly giving carboxylic acid 11 , which could be deprotected to give pES.

[0112] Scheme S4. Synthesis routes towards BES. HCI from alkylated D-Garner’s aldehyde. Protection of the (3-hydroxy group improved oxidation efficiency of primary alcohol to carboxylic acid.

[0113] 1.2.3 various oxidants perform well

[0114] As discussed above several oxidation strategies were successful. TEMPO oxidation is a preferred oxidation method here; using cheap and readily available sodium chlorite as stoichiometric oxidant and catalytic amounts of TEMPO (0.4 eq) and phenyliodine(lll) diacetate (PIDA, 0.2 eq) we obtained full conversion to the desired carboxylic acid in 2.5h at 0° C, with no detectable side products. The workup involves an extraction to obtain product that is > 90% pure, which can be further purified using column chromatography. Since residual TEMPO and PIDA byproduct are the only impurities present in the crude product, reactions with even lower amounts of these reagents could be attempted to further simplify the workup and thus improve the scalability of the reaction. Indeed, TEMPO oxidation with PIDA as the stoichiometric oxidant also gave good conversion to the desired product, but some more product was lost during column chromatography that was needed to purify the product. Similarly, oxidation with PDC gave good conversion to product, but PDC is not always attractive because it is carcinogenic and complicates the workup, especially with (deca-)gram scale reactions.

[0115] 1.2.4 various protection and deprotection strategies perform well

[0116] Next, TMS / Ac protecting groups were removed in a single step using either foCOs / MeOH or NaOMe / MeOH, and Boc-group removal with 6M HCI provided BES as an HCI salt. Alternatively, TMS / TBDPS protecting groups were removed with TBAF and Boc-group removal with 6M HCI provided BES as an HCI salt.

[0117] 1.2.5 products and intermediates have good purity

[0118] Of note, many of the pure synthetic intermediates gave two distinct sets of signals in1H and13C NMR, indicating the presence of 2 rotamers or conformers. Variable temperature (VT-)NMR experiments revealed that the distinct sets of peaks belonged to a single species, confirming that the distinct sets of peaks originated from different conformations of the same compound.

[0119] 1.3 other synthetic routes underperform

[0120] 1.3. 1 Aldol condensation with Ni(ll)-complex is suboptimal

[0121] BES was prepared via asymmetric aldol condensation reactions between glycine complexes and propynal (Scheme S5). A chiral auxiliary 14 was synthesized and used to create a Ni(ll)-glycine complex 15, which provides control over the stereochemistry of the final aldol product 16. Various aldehydes had been explored for aldol condensation with this specific Ni(ll)-glycine complex in the past, and we extended the scope of this reaction with aldehyde TMS-propynal, yielding Ni(ll)- complex 16. While this synthetic strategy allowed for gram-scale synthesis of crude nickel complex 16, its purification proved to be extremely difficult, giving an inseparable mixture of Ni(ll)-complexed with glycine, p-ethynyl serine (16) and other unidentified amino acids. Thus, purification of the Ni(ll)- p-ethynylserine complex gave very low yields, indicating that the use of this synthesis route for large scale synthesis of BES is not as efficient as the route of Example 1 .2.

[0122] 1. 'PrOH, KOH 1. CH2CI2, SOCI2

[0123] 2.BnCI 2. o-aminobenzophenone

[0124] 3. aq. H^ 3. aq. Na2CO3 overnight, RT overnight, -10 °C -> RT

[0125] 73% 61%

[0126] Scheme S5. Synthesis of BES via aldol condensation between a Ni(ll)-glycine complex and TMS- propynal is not as efficient as scheme S4. 1.3.2 Alkylation on OBO-serinal is suboptimal

[0127] BES was synthesized via alkylation of serine aldehyde (serinal) derivatives. Oxidation of commercially available protected serine to the aldehyde, followed by alkylation with ethynyl magnesium bromide seemed like a facile and straightforward synthetic route towards BES (scheme S6). The aldehyde derivative of serine, serinal, was found to be prone to epimerization at the Caposition, which would give a mixture of D- and L-p-ethynyl serine upon alkylation with Grignard reagents. It is known that protection of serine with a 4-methyl-2,6,7-trioxa-bicyclo[2.2.2]octan-1-yl (OBO) ester reduces the acidity of the Ca-proton of serinal, making it amenable to alkylation with Grignard reagents. The OBO-ester protected serinal 21 was prepared following known methods, extending the scope of alkylations with ethynylmagnesium bromide to give OBO-ester protected BES 22. We attempted to deprotect 22 with TMS-iodide, as usually performed on such compounds, but this converted the alkyne of 22 to a vinyl iodide. Deprotection of the Cbz (Z) group of 22 by hydrogenation was no option due to incompatibility the alkyne with these conditions. Changing the protecting group strategy from Cbz to Boc, low yields in the synthesis towards the serinal OBO ester were obtained, primarily in the BF3. Et20 catalyzed OBO-ester formation step (19 to 20, but with Boc instead of Cbz) prompting us to abandon this synthetic strategy altogether.

[0128] Scheme S6. Synthesis offt-ethynyl serine via alkylation of OBO-ester protected serinal.

[0129] Example 2 - methods and analysis tert-butyl (R)-4-((R)-1-hydroxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate (1) Ethyl magnesiumbromide (1 .0 M in THF, 98.0 mL, 98.0 mmol) was added dropwise to a solution of trimethylsilylacetylene (15.4 mL, 110 mmol) in THF (300 mL) at 0 °C. The resulting solution was refluxed for 1 h, cooled to room temperature and then transferred via canula to a solution of Cui (28.1 g, 147 mmol) in THF (300 mL) and dimethyl sulfide

[0130] (60 mL) at - 78 °C over a period of 10 minutes. The resulting yellow suspension was warmed to - 30 °C and stirred for 30 minutes, after which the reaction mixture was cooled to - 78 °C and a solution of D-Garner’s aldehyde 23 (15.9 g, 69.3 mmol) in THF (70 mL) was added. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched by addition of sat. aqueous NH4CI and subsequently diluted with water. The aqueous layer was extracted with diethyl ether (2x) and the combined organic layers were washed with 0.5M HCI and brine. After drying with MgSO4, filtration and concentration in vacuo, the resulting crude residue was purified by flash column chromatography on silica gel (10-20% EtOAc in n-heptane) to yield 1 as a clear oil (18.1 g, 80%).1H NMR (400 MHz, CDCI3): 6 4.56 - 4.47 (m, 2H), 4.20 - 3.98 (m, 4H), 1 .59 (s, 3H), 1 .49 (m, 12H), 0.16 (s, 9H). tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate (7) Pyridine (9.51 mL, 1 18 mmol), AC2O (11.1 mL, 118 mmol) and DMAP (957 mg, 7.84 mmol) were added consecutively to a stirring solution of alcohol 1 (12.8 g, 39.2 mmol) in DCM (100 mL) at room temperature. The reaction was stirred for 1 .5h when TLC indicated complete conversion of starting material. The reaction mixture was diluted with DCM (50 mL) and washed with sat. aqueous NH4CI (2x), water and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo to yield the crude product (14.5g, quant.) as a clear oil. The product was used crude in the next step without additional purification.

[0131] (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1 -(trimethylsilyl)pent-1-yn-3-yl acetate (8)

[0132] Crude oxazolidine 7 (14.5 g, 39.2 mmol) was dissolved dry MeCN (125

[0133] OAc mL) and to it was added BiBrs (3.52 g, 7.84 mmol) and water (3 mL) while stirring at room temperature. The cloudy yellow reaction mixture

[0134] .. , , . . .ater whic , was stirred for 4h at room temperature, af h . it. was quenc ,hed , , by addition of sat. aqueous NaHCOs (50 mL). The aqueous layer was extracted with EtOAc (3x) and combined organic layers were washed with water and brine. Drying over MgSO4, filtration and concentration in vacuo yielded 8 as a white crystalline solid (10.5 g, 81 %).1H NMR (400 MHz, CDCI3): 6 5.62 (d, J = 5.4 Hz, 1 H), 4.99 (s, 1 H), 3.98 (s, 1 H), 3.91 (dd, J = 11 .2, 5.4 Hz, 1 H), 3.69 (dt, J = 11.7, 6.0 Hz, 1 H), 2.17 (s, 1 H), 2.14 (s, 3H), 1 .48 (s, 9H), 0.20 (s, 9H).

[0135] (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid (9)

[0136] Alcohol 8 (10.6 g, 32.2 mmol) was dissolved in MeCN (80 mL) and the solution was diluted with an aqueous PBS solution (40 mL, 0.67M, pH

[0137] 7.0) while stirring at room temperature. Next, the solution was cooled to 0 °C and stirred vigorously and TEMPO (2.00 g, 12.8 mmol) was added. After stirring at 0 °C for 5 min, PIDA (21.6 g, 67.1 mmol) dissolved in MeCN / PBS (2:1 , 30 mL) was added and the reaction mixture was stirred at 0 °C for 2h, when TLC indicated complete conversion of 8. The reaction was quenched by dropwise addition of sat. aqueous Na2S2O3, acidified to pH = 2 with 1 M HCI and extracted with EtOAc (3x). Combined organic layers were washed with water and brine, dried over MgSO4 and concentrated in vacuo. Flash column chromatography on silica gel (0 - 70% EtOAc in n-heptane, with 0.1 % AcOH) yielded product 9 as a clear oil (8.55 g, 78%).1H NMR (400 MHz, CDCh): (mixture of rotamers) 5 6.21 (s, 0.18H), 5.87- 5.83 (m, 1 H), 5.40 (d, J = 8.8 Hz, 0.82H), 4.80 (d, J = 8.9 Hz, 0.82H), 4.56 (s, 0.18H), 2.14 (s, 0.54H), 2.11 (s, 2.46H), 1.51 (s, 9H). tert-butyl (R)-4-((R)-1 -((tert-butyldiphenylsilyl)oxy)-3-(trimethylsilyl)prop-2-yn-1 -y l)-2, 2- dimethyloxazolidine-3-carboxylate (10) Triethylamine (1 17 pL, 840 pmol), te / Y-butyldiphenylchlorosilane (214 pL, 840 pmol) and imidazole

[0138] (114 mg, 1.68 mmol) were added consecutively to a stirring solution of alcohol 1 (110 mg, 336 pmol) in dry DMF (3 mL) at room temperature. The reaction was stirred under inert atmosphere for 40h when TLC indicated complete conversion of starting material. The reaction mixture was quenched with 5 mL of sat. aqueous NH4CI and extracted with EtOAc (3 x 20 mL). Combined organic layers were washed with 5% LiCI (2x), water and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. Flash column chromatography on silica gel (0 - 5% EtOAc in n-heptane) yielded product 10 (127.2 mg, 67%).1H NMR (400 MHz, CDCh): (mixture of rotamers) 5 7.76 - 7.62 (m, 4H), 7.47 - 7.32 (m, 6H), 5.00 (d, J = 5.0 Hz, 0.3H), 4.84 (d, J = 5.0 Hz, 0.7H), 4.39 (dd, J = 9.3, 5.0 Hz, 0.7H), 4.35 - 4.31 (m, 0.3H), 4.19 - 4.05 (m, 1 H), 3.94 (dt, J = 6.8, 5.0 Hz, 1 H), 1 .61 (s, 2.1 H), 1 .55 (s, 0.9H), 1 .51 (s, 2.1 H), 1.48 (s, 0.9H), 1 .44 (s, 2.7H), 1 .16 (s, 6.3H), 1.06 (d, J = 4.1 Hz, 9H), 0.05 (s, 2.7H), 0.02 (s, 6.3H). tert-butyl ((2R,3R)-3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2- yl)carbamate (11) Oxazolidine 10 (127.2 mg, 225 pmol) was OTBDPS dissolved in dry MeCN (3 mL) and to it was added BiBrs (12.5 mg, 28.0 pmol) and water (40 pL, 2.25 mmol) while stirring at room temperature.

[0139] The cloudy yellow reaction mixture was stirred for 16h at room temperature, after which it was quenched by addition of sat. aqueous NaHCOs (5 mL). The aqueous layer was extracted with EtOAc (3x) and combined organic layers were washed with sat. aqueous NH4CL, water and brine. Drying over MgSO4, filtration and concentration in vacuo yielded 11 as a white crystalline solid (97.3 mg, 82%).1H NMR (400 MHz, CDCh): 6 7.70 (ddt, J = 15.1 , 6.7, 1.5 Hz, 4H), 7.47 - 7.34 (m, 6H), 4.96 (s, 1 H), 4.48 (d, J = 4.3 Hz, 1 H), 3.95 - 3.80 (m, 2H), 3.76 (dd,

[0140] J = 10.7, 4.8 Hz, 1 H), 1 .43 (s, 9H), 1 .08 (s, 9H), 0.07 (d, J = 3.3 Hz, 9H). (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5- (trimethylsilyl)pent-4-ynoic acid (12) Alcohol 11 (97.3 mg, 295 pmol) TBDPSO O was dissolved in MeCN (1 mL) and the solution was diluted with an aqueous PBS solution (0.4 mL, 0.67M, pH 7.0) while stirring at room temperature. Next, the solution was cooled to 0 °C and stirred vigorously and TEMPO (0.83 mg, 5.3 pmol) was added. After stirring at 0 °C for 5 min, PIDA (75.7 mg, 650 pmol) dissolved in MeCN / PBS (2:1 , 30 mL) was added and the reaction mixture was stirred at 0 °C for 5h, when TLC indicated complete conversion of 11. The reaction was quenched by dropwise addition of sat. aqueous Na2S2O3, acidified to pH = 2 with 1 M HCI and extracted with EtOAc (3x). Combined organic layers were washed with water and brine, dried over MgSO4 and concentrated in vacuo. Flash column chromatography on silica gel (0 - 50% EtOAc in n-heptane, with 0.1 % AcOH) yielded product 12 as a crystalline solid (73.2 mg, 72%).1H NMR (400 MHz, CDCh): 6 7.74 - 7.69 (m, 2H), 7.68 - 7.63 (m, 2H), 7.48 - 7.41 (m, 2H), 7.37 (dd, J = 7.9, 6.5 Hz, 4H), 5.38 (d, J = 9.1 Hz, 1 H), 4.82 (d, J = 3.2 Hz, 1 H), 4.55 (d, J = 8.7 Hz, 1 H), 1 .49 (d, J = 7.0 Hz, 9H), 1 .06 (s, 9H), 0.00 (d, J = 1.0 Hz, 9H).

[0141] (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid (3) QHQ

[0142] Anhydrous K2CO3 (17.2 g, 125 mmol) was added to a stirring solution of 9 (8.55 g, 24.9 mmol) in dry MeOH (125 mL) at 0 °C. the reaction mixture was warmed to room temperature and stirred for 2h, when TLC indicated full conversion of 3. The reaction was quenched by the addition of sat. aqueous NH4CI and the pH adjusted to = 2 with 1 M HCI. The resulting aqueous solution was extracted with EtOAc (4x) and combined organic layers were washed with water and brine. Drying over MgSC , filtration and concentration in vacuo yielded 3 as a clear oil (5.45 g, 95%).1H NMR (400 MHz, CDCh): (mixture of rotamers) 5 6.35 (d, J = 8.9 Hz, 0.74H), 5 5.65 (d, J = 8.8 Hz, 0.26H), 4.90 (m, 1 H), 4.61 (dd, J = 9.0, 3.2 Hz, 0.74H), 4.48 (s, 0.26H) 2.53 (m, 1 H), 1.48 (s, 9H).

[0143] P-ethynyl serine . HCI salt (4) Boc-protected p-ethynyl serine 3 (5.45 g, 23.8

[0144] OH O mmol) was dissolved in 6M aqueous HCI (30 mL) at room temperature and stirred overnight. Then, the reaction mixture was concentrated in vacuo and the crude residue dissolved in water (15 mL). The solution was lyophilized to yield

[0145] 4 (3.80 g, 96%).1H NMR (400 MHz, D2O): 5 5.1 1 (dd, J = 3.1 , 2.2 Hz, 1 H), 4.26 (d, J = 3.1 Hz, 1 H),

[0146] 3.10 (d, J = 2.2 Hz, 1 H) tert-butyl ( R) -4-((R) -1 -hydroxy-3-(trimethylsilyl)prop-2-yn-1 -y I )-2 ,2- dimethyloxazolidine-3-carboxylate -1H NMR (500 MHz, CDCh) 6 4.52 (dd, J = 22.7, 6.3 Hz, 2H), 4.18 - 3.96 (m, 3H), 1.57 (s, 3H), 1.49 (s, 12H), 0.16 (s, 9H).13C NMR (126 MHz, CDCh) 6 66.83, 65.42, 62.39, 31.89,

[0147] 29.02, 28.37, 27.26, 24.32, 22.69, 14.11 , -0.21. tert-butyl ((2R,3R)-1,3-dihydroxypent-4-yn-2-yl)carbamate -1H NMR (500 MHz, CDCh) 6 5.26 (d, J = 7.8 Hz, 1 H), 4.66 (q, J = 4.0 Hz, 1 H), 3.92 - 3.80 (m, 3H), 3.08 (s, 1 H), 2.53 (d, J = 2.2 Hz, 1 H), 2.06 (d, J = 4.0 Hz, 1 H), 1 .47 (s, 9H).13C NMR (126 MHz, CDCh) 6 156.50, 82.11 , 74.48, 63.01 , 63.00, 62.37,

[0148] 55.57, 28.33.

[0149] (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1-

[0150] OAc

[0151] (trimethylsilyl)pent-l -yn-3-yl acetate -1H NMR (400 MHz, CDCh) 5 5.62 (d, J = 5.4 Hz, 1 H), 4.99 (s, 1 H), 3.98 (s, 1 H), 3.91 (dd, J = 11.2, 5.4 Hz, 1 H), 3.69 (dt, J = 11.7, 6.0 Hz, 1 H), 2.17 (s, 1 H), 2.14 (s, 3H), 1.48 (s, 9H),

[0152] 0.20 (s, 9H).

[0153] (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5- (trimethylsilyl)pent-4-ynoic acid -1H NMR (500 MHz, CDCh) 6 5.82 (d,

[0154] J = 3.2 Hz, 1 H), 5.31 (d, J = 9.7 Hz, 1 H), 4.78 - 4.70 (m, 1 H), 2.09 (d, J = 5.0 Hz, 3H), 1.48 (s, 9H), 0.18 (s, 9H).

Claims

Claims1. A method for providing a protected or unprotected beta-ethynylserine or a salt thereof, the method comprising the steps of:I) ring-opening a compound of general formula (i) to obtain an alcohol compound of general formula (ii)wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1-8alkyl or a C2-8 alkenyl; c2 is H or a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure;II) oxidation of the alcohol compound of general formula (ii) to obtain a carboxylic acid compound of general formula (iii)2. The method according to claim 1 , wherein step I) is preceded by step la): la) alkylating a 2,2-dialkyloxazolidine-4-carboxaldehyde to obtain the compound of general formula (i).

3. The method of claim 1 or 2, wherein step I) is preceded by step lb):lb) protecting a compound of general formula (i) wherein po is H to obtain a compound of general formula (i) wherein po is a protecting group.

4. The method according to any one of claims 1-3, wherein at least one of pn, pc, or po is a protecting group, wherein the method further comprises step III)III) deprotecting all protecting groups of the carboxylic acid compound of general formula (iii) to obtain a compound of general formula (BES)(BES).

5. The method according to any one of claims 1-4, wherein pc is a silyl such as trimethylsilyl (TMS), Triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), tert-buty Idimethylsilyl (TBDMS), tert-buty Idiphenylsilyl (TBDPS), tri- isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); po 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), tetrahydropyranyl (THP), tetra hydrofuranyl (THF), trityl (Tr), ethoxyethyl (EE), or a silyl such as trimethylsilyl (TMS), tertbutyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tri- isopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS); pn is trityl, allyl, benzyl (Bn), 9-fluorenylmethyl oxycarbonyl (Fmoc), t- butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), 2-trimethylsilylethyloxycarbonyl, tosyl (Ts), acetyl (Ac), trifluoroacetyl, phthalimide, benzylideneamine, or allyloxycarbonyl (Alloc).

6. The method according to any one of claims 1-4, wherein pc is a silyl such as trimethylsilyl (TMS); po is acetyl (Ac) or a silyl such as tert-butyldiphenylsilyl (TBDPS); and pn is t-butyloxycarbonyl (Boc).

7. The method according to any one of claims 1 -6, wherein step I) comprises ring-opening the compound of general formula (i) by contacting it with a strong nucleophile such as sodium amide, an organic acid such as a sulfonic acid, preferably tosylic acid, or such as a carboxylic acid, preferably acetic acid or trifluoroacetic acid, or with a Lewis acid salt such as a bismuth salt, preferably BiBrs.

8. The method according to any one of claims 1-7, wherein step II) comprises oxidation using a chlorite salt such as sodium chlorite, a hypochlorite salt such as sodium hypochlorite, pyridinium chromate such as dichromate or chlorochromate, a chromium salt such as chromium trioxide, a ruthenium salt such as RuO4, a permanganate such as KMnC , 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) or a derivative thereof, phenyliodine(lll) diacetate (PIDA), or a combination thereof, preferably using TEMPO, or wherein the oxidation is a Swern oxidation or a Dess-Martin oxidation.

9. A compound of general formula (i), (2), or (3), or a salt thereof:wherein pc is H or a protecting group; po is H or a protecting group; and pn is in each instance independently H or a protecting group; c1 is H or a C1 -8alkyl or a C2-8 alkenyl; c2 is H or a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure; wherein for formula (3) at least one of pc, po, or pn is a protecting group.

10. The compound of claim 9, wherein it is of general formula (i) wherein po is a protecting group, or wherein the compound is of general formula (2).

11. The compound of claim 9, wherein it is of general formula (3) wherein po is a protecting group.

12. The compound of any one of claim 9-11 , wherein it is selected from• tert-butyl-4-(-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate;• 4-((tert-butoxycarbonyl)amino)-5-hydroxy-1-(trimethylsilyl)pent-1-yn-3-yl acetate;• 3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• 2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid;• tert-butyl (1 ,3-dihydroxypent-4-yn-2-yl)carbamate;• 3-hydroxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• tert-butyl-4-(1-((tert-butyldiphenylsilyl)oxy)-3-(tnmethylsilyl)prop-2-yn-1-yl)-2,2- dimethyloxazolidine-3-carboxylate;• tert-butyl(3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2- yl)carbamate;• 2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5-(trimethylsilyl)pent-4-ynoic acid;• tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate;• (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1 -(tri methy Isi ly I) pent-1 -yn-3-yl acetate;• (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid;• tert-butyl ((2R,3R)-1 ,3-dihydroxypent-4-yn-2-yl)carbamate;• (2S,3R)-3-hydroxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• tert-butyl (R)-4-((R)-1 -((te rt-buty Idi phenyls i ly I) oxy)-3-(tri methy Isi ly l)prop-2-yn-1 -y l)-2 , 2- dimethyloxazolidine-3-carboxylate;• tert-butyl ((2R,3R)-3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2- yl)carbamate;• (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5- (trimethylsilyl)pent-4-ynoic acid.

13. Method for providing a compound of general formula (2) wherein the compound of general formula (2) is as defined in any one of claims 9, 10, or 12, the method comprising the step of:I) ring-opening a compound of general formula (i) to obtain the compound of general formula (2)wherein pc is H or a protecting group; po is H or a protecting group; pn is H or a protecting group; c1 is H or a C1-8alkyl or a C2-8 alkenyl; c2 is H or a C1-8alkyl or a C2-8 alkenyl; or c1 and c2 together form a bridging moiety to form a 3-10-membered cyclic structure.

14. Method for providing a compound of general formula (3) wherein the compound of general formula (3) is as defined in any one of claims 9, 11 , or 12, the method comprising the step of:II) oxidation of a compound of general formula (2) wherein the compound of general formula (2) is as defined in any one of claims 9, 10, or 12, to obtain the compound of general formula (3).

15. Use of a 2,2-dialkyloxazolidine-4-carboxaldehyde in the manufacture of a protected or unprotected beta-ethynylserine or a salt thereof.

16. The compound according to claim 12, wherein it is selected from• tert-butyl-4-(-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate;• 4-((tert-butoxycarbonyl)amino)-5-hydroxy-1-(trimethylsilyl)pent-1-yn-3-yl acetate;• 3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• 2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid;• 3-hydroxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• tert-butyl-4-(1-((tert-butyldiphenylsilyl)oxy)-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2- dimethyloxazolidine-3-carboxylate;• tert-butyl(3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2- yl)carbamate;• 2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5-(trimethylsilyl)pent-4-ynoic acid;• tert-butyl (R)-4-((R)-1-acetoxy-3-(trimethylsilyl)prop-2-yn-1-yl)-2,2-dimethyloxazolidine-3- carboxylate;• (3R,4R)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-1 -(tri methy Isi ly I) pent-1 -yn-3-yl acetate;• (2S,3R)-3-acetoxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxypent-4-ynoic acid;• (2S,3R)-3-hydroxy-2-((tert-butoxycarbonyl)amino)-5-(trimethylsilyl)pent-4-ynoic acid;• tert-butyl (R)-4-((R)-1 -((te rt-buty Idi phenyls i ly I) oxy)-3-(tri methy Isi ly l)prop-2-yn-1 -y l)-2 , 2- dimethyloxazolidine-3-carboxylate;• tert-butyl ((2R,3R)-3-((tert-butyldiphenylsilyl)oxy)-1-hydroxy-5-(trimethylsilyl)pent-4-yn-2- yl)carbamate;• (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)-5- (trimethylsilyl)pent-4-ynoic acid.

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