SUGAR-LINKED AMINO ACIDS FOR SOLID-PHASE PEPTIDE SYNTHESIS

Abstract

The present disclosure relates to sugar-linked amino acids and processes for preparing the same. The sugar-linked amino acids may be used for solid-phase peptide synthesis. A sugar compound and an amino acid compound having a nucleophilic side chain are reacted in a heated halogenated solvent. The reaction is catalyst by a Lewis acid, such as InBr.sub.3. The reaction is performed as a batch or continuous process.

Claims

1. A process for preparing a sugar-linked amino acid of the formula I ##STR00028## wherein each R.sup.1 is independently a hydroxyl protecting group; X is O or N; each R.sup.2 is H or a protecting group; provided that when X is O, R.sup.2 is a protecting group; and when X is N, at least one R.sup.2 is a protecting group; Z is O or S; R.sup.3 is an N-terminal protecting group; and n is 1 when X is O and n is 2 when X is N; the process comprising contacting a compound of the formula II ##STR00029## wherein each R.sup.1 is independently a hydroxyl protecting group; X is O or N; each R.sup.2 is H or a protecting group; provided that when X is O, R.sup.2 is a protecting group; and when X is N, at least one R.sup.2 is a protecting group; LG is a leaving group; and n is 1 when X is O and n is 2 when X is N; with a compound of the formula III ##STR00030## wherein R.sup.3 is an N-terminal protecting group; and Z is O or S; in the presence of a Lewis acid catalyst.

2. The process of claim 1, wherein X is N and one R.sup.2 is hydrogen.

3. The process of claim 1, wherein Z is S.

4. The process of claim 1, wherein each R.sup.1 is acetyl.

5. The process of claim 1, wherein one R.sup.2 is acetyl.

6. The process of claim 1, wherein R.sup.3 is Fmoc or Boc.

7. (canceled)

8. The process of claim 1, wherein the Lewis acid catalyst comprises indium.

9. The process of claim 1, wherein the Lewis acid catalyst is InBr.sub.3.

10. (canceled)

11. (canceled)

12. The process of claim 1, further comprising heating a reaction mixture of the compound of the formula II and the compound of the formula III.

13.-14. (canceled)

15. The process of claim 1, wherein a reaction mixture of the compound formula II and the compound of the formula III are contacted in a continuous flow process.

16.-20. (canceled)

21. A process for forming a sugar-linked polypeptide, the process comprising deprotecting one or more sugar-linked amino acids of formula I ##STR00031## wherein each R.sup.1 is independently a hydroxyl protecting group; X is O or N; each R.sup.2 is H or a protecting group; provided that when X is O, R.sup.2 is a protecting group; and when X is N, at least one R.sup.2 is a protecting group; Z is O or S; R.sup.3 is an N-terminal protecting group; and n is 1 when X is O and n is 2 when X is N; the process comprising removing R.sup.3.

22. The process of claim 1 wherein the compound of formula I is of formula IVa, IVb, or IVc ##STR00032## wherein each R.sup.1 is independently a hydroxyl protecting group; X is O or N; each R.sup.2 is H or a protecting group; provided that when X is O, R.sup.2 is a protecting group; and when X is N, at least one R.sup.2 is a protecting group; R.sup.3 is an N-terminal protecting group; Z is O or S; and n is 1 when X is O and n is 2 when X is N; the process comprising contacting a compound of the formula Va, Vb, or Vc ##STR00033## wherein each R.sup.1 is independently a hydroxyl protecting group; X is O or N; each R.sup.2 is H or a protecting group; provided that when X is O, R.sup.2 is a protecting group; and when X is N, at least one R.sup.2 is a protecting group; LG is a leaving group; and n is 1 when X is O and n is 2 when X is N; with a compound of the formula VI ##STR00034## wherein R.sup.3 is an N-terminal protecting group and Z is O or S; in the presence of a Lewis acid catalyst.

23.-28. (canceled)

29. The process of claim 22, wherein the Lewis acid catalyst comprises indium.

30. The process of claim 22, wherein the Lewis acid catalyst is InBr.sub.3.

31.-33. (canceled)

34. The process of claim 22, further comprising heating a reaction mixture of the compound of the formula V and the compound of the formula VI.

35.-43. (canceled)

44. The sugar-linked amino acid of claim 46, having the structure of formula VIIa, VIIb, or VIIc, ##STR00035## wherein Z is O or S.

45. The compound of claim 44, wherein Z is S.

46. A sugar-linked amino acid of the formula VIII ##STR00036## wherein R.sup.1 is a hydroxyl protecting group; Z is O or S; and each R.sup.2 is a protecting group.

47. The compound of claim 46, wherein Z is S.

48. The compound of claim 46, wherein each R.sup.1 and R.sup.2 is independently RC(O), wherein R is alkyl, alkenyl, or aryl, and wherein each hydrogen atom on aryl, alkenyl, or aryl is optionally substituted with halo.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0119] The FIGURE shows an NMR spectrum of the compound prepared in Example 1.

DETAILED DESCRIPTION

[0120] The present disclosure is directed to processes for preparing certain sugar-linked amino acids for solid-phase peptide synthesis. It will be understood that in some embodiments, the present disclosure provides processes for preparing sugar-linked serine and threonine compounds for solid-phase peptide synthesis. These compounds may be used as building blocks related to glycosylated serine, threonine, or cysteine derivatives. The processes, which may be accomplished using a batch process of continuously flow chemistry, can be represented the following general Scheme A.

##STR00019##

wherein each of R.sup.1, R.sup.2, R.sup.3, X, Z, LG, and n is as defined herein. In some embodiments, the sugar compound may be a glucose, a mannose, a galactose, or a derivative, such as a C-2 amino, thereof. In some embodiments, the amino acid compound may be a cysteine, a threonine, or a serine.

[0121] In some embodiments, X is O or N. In some embodiments, X is O. In some embodiments, X is N.

[0122] In some embodiments, each R.sup.2 is H or a protecting group. Illustratively, if X is O, the O may be substituted by an acetyl group, a trifluoroacetyl group, a trimethylacetyl group, or a benzoyl group, although other substitutions are contemplated. Additional protecting groups suitable for use here are described in Greene's Protective Groups in Organic Synthesis, 4th ed., published by John Wiley and Sons in 2007, which is hereby incorporated by reference in its entirety.

[0123] In some embodiments, when X is N, at least one R.sup.2 is a protecting group. In some embodiments, when X is N, one R.sup.2 is a protecting group and the other R.sup.2 is H. In some embodiments, when X is N, each R.sup.2 is independently a protecting group. In some embodiments, when X is N, two R.sup.2 groups can combine to form a single protecting groups, such as a phthalate group. Additional protecting groups suitable for use here are described in Greene's Protective Groups in Organic Synthesis, 4th ed., published by John Wiley and Sons in 2007, which is hereby incorporated by reference in its entirety.

[0124] In some embodiments, Z is O or S. In some embodiments, Z is O. In some embodiments, Z is S.

[0125] In some embodiments, n is an integer. In some embodiments, n may be 1 or 2. In some embodiments, n is 1 when X is O. In some embodiments, n is 2 when X is N.

[0126] More particularly, the processes of the present disclosure can be described by the following Scheme B.

##STR00020##

wherein each of R.sup.1, R.sup.2, R.sup.3, X, Z, LG, and n is as defined herein. Although a glucose derivative is shown as the sugar compound, Scheme B is equally applicable for other sugars such as mannose, galactose, and derivatives thereof. In some embodiments, the amino acid compound may be a cysteine, a threonine, or a serine.

[0127] It will be appreciated that the reaction can be carried out according to any of the conditions described herein.

[0128] In some embodiments, the sugar compound is any sugar capable of reacting with any amino acid side chain. The sugar is derivatized to include at least one leaving group. For example, the sugar compound may be glucose or glucosamine, derivatized to include a leaving group. In some embodiments, the sugar compound may be mannose or mannosamine, derivatized to include a leaving group. In some embodiments, the sugar compound may be galactose or galactosamine, derivatized to include a leaving group. The sugar may have any physically stable stereochemistry, including natural or unnatural configurations.

[0129] The leaving group may be any leaving group known to those skilled in the art that can be displaced via an SN1 or an SN2 process. In some embodiments, a hydroxyl group of the sugar is acylated with an acyl group of the formula RCO, where R is an alkyl, alkenyl, or aryl group, wherein each hydrogen on the alkyl, alkenyl, or aryl group is optionally substituted, to form an acyloxy leaving group. In some embodiments, the hydroxyl group is substituted by an acetyl group or a trifluoroacetyl group, although other substitutions are contemplated. In some embodiments, the leaving group is on the anomeric carbon.

[0130] The other alcohols, amines, or similar groups on the sugar may also be functionalized. For example, the groups may by functionalized by reaction with an acyl group of the formula RCO, where R is an alkyl, alkenyl group, or aryl group, wherein each hydrogen on the alkyl, alkenyl, or aryl group is optionally substituted, to form an acyloxy leaving group. In some embodiments, the optional substitution on the alkyl, alkenyl, or aryl group is halo. In some embodiments, the hydroxyl group is substituted by an acetyl group, a trifluoroacetyl group, a trimethylacetyl group, or a benzoyl group, although other substitutions are contemplated. Additional protecting groups suitable for use here are described in Greene's Protective Groups in Organic Synthesis, 4th ed., published by John Wiley and Sons in 2007, which is hereby incorporated by reference in its entirety.

[0131] The amino acid compound is any amino acid having a nucleophilic side chain. The amino acid compound may be a natural or unnatural amino acid. In some embodiments, the amino acid has a thiol or hydroxyl side chain. The amino acid compound may be serine or threonine. In some embodiments, the amino acid is cysteine.

[0132] It is to be understood that amino acid compound refers to both protected and unprotected amino acids that are capable of undergoing the reactions described herein. In some embodiments, the amino acid compound has a free carboxyl group in a C-terminus position and a protected amino group in an N-terminus position. The amino group is protected by any group useful in solid phase peptide synthesis (SPPS), such as a fluorenylmethyloxycarbonyl (Fmoc) group or a tert-butyloxycarbonyl (Boc) group. It is contemplated that after reaction of the sugar compound and the amino acid compound, the amino protecting group may be cleaved such that the amino group is able to react with a free carboxyl group on another amino acid to form a peptide bond. In some embodiments, the carboxyl group on the other amino acid is activated according to traditional peptide chemistry. Additional protecting groups suitable for use here are described in Greene's Protective Groups in Organic Synthesis, 4th ed., published by John Wiley and Sons in 2007, which is hereby incorporated by reference in its entirety.

[0133] The reaction between the sugar compound and the amino acid compound takes place in the presence of a Lewis acid catalyst. It is contemplated that the Lewis acid catalyst may be a Lewis acid catalyst useful in any nucleophilic substitution reaction described herein. In some embodiments, the Lewis acid catalyst is an indium catalyst, such as an indium (III) catalyst. Illustratively, the indium catalyst may be InBr.sub.3. In some embodiments, the amount of Lewis acid present is sub-stoichiometric.

[0134] The reactions described herein may take place in any suitable solvent. In some embodiments, the solvent is a halogenated solvent. For example, the solvent may be dichloromethane (CH.sub.2Cl.sub.2) or chloroform (CHCl.sub.3). In some embodiments, the solvent comprises a non-halogenated. In some embodiments, the non-halogenated solvent is acetonitrile or ethyl ether. In some embodiments, the solvent comprises a halogenated solvent and a non-halogenated solvent. In some embodiments, the solvent is a mixture of chloroform and acetonitrile. In some embodiments, the solvent is a mixture of chloroform and ethyl ether.

[0135] The reactions between the sugar compound and the amino acid compound described herein preferably take place at elevated temperatures. The temperature may be about 30 C. to about 150 C., about 50 C. to about 125 C., about 50 C. to about 100 C., about 70 C. to about 100 C., or about 75 C. to about 95 C. In some embodiments, the temperature may be the boiling temperature of the solvent in which the reaction takes place.

[0136] When the reaction occurs in a bath process, the reaction may occur over about 5 hours to about 30 hours, about 10 hours to about 30 hours, about 15 hours to about 30 hours, about 5 hours to about 25 hours, about 10 hours to about 25 hours, about 15 hours to about 25 hours, or about 15 hours to about 22 hours.

[0137] When the reaction occurs in a continuous process, the reaction may occur with a residence time from about 30 seconds to about 5 minutes, about 30 seconds to about 4 minutes, about 30 seconds to about 3 minutes, about 1 minute to about 5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 3 minutes, about 2 minutes to about 5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 3 minutes, or about 2.5 minutes. The flow rate may be about 50 L/min to about 500 L/min, about 100 L/min to about 500 L/min, about 200 L/min to about 500 L/min, about 50 L/min to about 400 L/min, about 100 L/min to about 400 L/min, about 200 L/min to about 400 L/min, about 50 L/min to about 300 L/min, about 100 L/min to about 300 L/min, about 200 L/min to about 300 L/min, or about 200 L/min.

[0138] The process described herein provides the sugar-amino acid compound (compound formula VII) in a particular yield. In some embodiments, the yield is at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, or at least 85%.

[0139] According to one aspect, a sugar-linked amino acid may have the formula VIII

##STR00021##

wherein each R.sup.1 is independently a hydroxyl protecting group; Z is O or S; and each R.sup.2 is a protecting group. In some embodiments, Z is S. In some embodiments, each R.sup.1 and R.sup.2 is independently RC(O), wherein R is alkyl, alkenyl, or aryl, and wherein each hydrogen atom on aryl, alkenyl, or aryl is optionally substituted. In some embodiments, the optional substitution is halo.

[0140] According to another aspect a sugar-linked amino acid of the formula VII

##STR00022##

wherein Z is O or S.

EXAMPLES

Example 1

Manual Batch Synthesis of AcGlcNAc-S-Cys

[0141] ##STR00023##

[0142] Peracetylated GlcNAc (2.0 eq) and Fmoc-Cys (1.0 eq) were taken up in chloroform to which was added indium bromide (0.5 eq). The mixture was refluxed for 15-22 hours. Upon completion of the reaction as monitored by thin layer chromatography, the reaction mixture was evaporated and purified using silica gel chromatography. The yield of AcGlcNAc-S-Cys was about 73%. The .sup.1H NMR is shown in the FIGURE.

Example 2

Continuous Flow Production of AcGlcNAc-S-Cys

[0143] ##STR00024##

[0144] All starting materials including indium bromide were dissolved in chloroform and the solution was pumped through tubing with a 200 L/min flow rate and 2.5 min residence time at elevated temperature and pressure. Data show at least 30% conversion to the desired product under these conditions.

Example 3

Synthesis of N.SUP..-Fluoren-9-ylmethoxycarbonyl-S-(3,4,6-tetra-O-acetyl-2-acetamido-2-deoxy)--D-glucopyranosyl)-L-cysteine

[0145] ##STR00025##

[0146] Peracetyl--D-GlcNAc (850 mg, 2.18 mmol, 1.5 eq), InBr.sub.3 (232 mg, 0.65 mmol, 0.3 eq), and N.sup.-fluoren-9-ylmethoxycarbonyl-L-cysteine (500 mg, 1.47 mmol, 1 eq) were suspended in 1:1 CHCl.sub.3:diethyl ether (30 mL). The reaction mixture was stirred at reflux for 3 hours. Gradually precipitation was observed and the precipitate was filtered and washed with diethyl ether. The crude was slurred with ether for 1 hr, filtered and washed with ether to afford 3 as an off-white powder (744 mg, 76%). R.sub.f 0.4 (CH.sub.2Cl.sub.2/methanol, 9:1 v/v with 0.5% Acetic acid).

[0147] .sup.1H NMR (500 MHz, Methanol-d.sub.4) 7.82 (d, J=7.5 Hz, 2H), 7.70 (t, J=6.5 Hz, 2H), 7.41 (t, J=7.4 Hz, 2H), 7.34 (ddd, J=8.2, 6.8, 2.8 Hz, 2H), 5.22 (t, J=9.7 Hz, 1H), 5.01 (t, J=9.7 Hz, 1H), 4.80 (d, J=10.5 Hz, 1H), 4.46 (dd, J=8.9, 4.2 Hz, 1H), 4.40-4.36 (m, 2H), 4.27 (t, J=7.0 Hz, 1H), 4.22-4.11 (m, 2H), 4.02 (d, J=10.3 Hz, 1H), 3.84-3.73 (m, 1H), 3.38 (dd, J=14.3, 4.2 Hz, 1H), 2.94-2.85 (m, 1H), 2.03 (s, 2H), 2.02 (s, 3H), 2.00 (s, 3H), 1.86 (s, 2H).

[0148] .sup.13C NMR (126 MHz, Methanol-d4) 171.3, 171.1, 171.0, 169.5, 143.5, 141.1, 127.6, 127.0, 124.8, 119.8, 83.4, 75.6, 73.7, 68.4, 67.0, 62.1, 52.6, 52.6, 46.9, 30.5, 22.3, 20.3, 20.3, 20.3.

[0149] HRMS-ESI-TOF (m/z): Calcd. for [(C.sub.32H.sub.37N.sub.2O.sub.12S+]673.2067 (M+H.sup.+), Found 673.2067 (100%).

Example 4

Synthesis of N.SUP..-Fluoren-9-ylmethoxycarbonyl-S-(3,4,6-tetra-O-acetyl-2-acetamido-2-deoxy)--D-galactopyranosyl)-L-cysteine

[0150] ##STR00026##

[0151] -D-galactosamine pentaacetate (850 mg, 2.18 mmol, 1.5 eq), InBr.sub.3 (232 mg, 0.65 mmol, 0.3 eq), and N.sup.-fluoren-9-ylmethoxycarbonyl-L-cysteine (500 mg, 1.47 mmol, 1 eq) were suspended in 1:1 CHCl.sub.3:ethyl ether (30 mL). The reaction mixture was stirred at reflux for 3 hours. The solvent was evaporated and ether was added to the crude solid. The mixture was stirred for 1 hr at 0 C., filtered and washed with cold ether, Again the crude product was slurred with dichloromethane for 30 min and filtered and washed with cold DCM to afford 5 as an off-white powder (725 mg, 74%). R.sub.f 0.4 (CH.sub.2Cl.sub.2/methanol, 9:1 v/v with 0.5% Acetic acid)

[0152] .sup.1H NMR (500 MHz, Methanol-d.sub.4) 7.81 (d, J=7.5 Hz, 2H), 7.71 (dd, J=18.5, 7.5 Hz, 2H), 7.45-7.39 (m, 2H), 7.38-7.31 (m, 2H), 5.37 (d, J=3.3 Hz, 1H), 5.07 (dd, J=10.8, 3.2 Hz, 1H), 4.72 (d, J=10.4 Hz, 1H), 4.47 (dd, J=9.4, 4.0 Hz, 1H), 4.43-4.32 (m, 2H), 4.32-4.24 (m, 2H), 4.17-4.07 (m, 2H), 3.98 (t, J=6.5 Hz, 1H), 3.45 (dd, J=14.4, 4.0 Hz, 1H), 2.88 (dd, J=14.4, 9.4 Hz, 1H), 2.12 (s, 3H), 1.98 (s, 2H), 1.97 (s, 3H), 1.88 (s, 2H).

[0153] .sup.13C NMR (126 MHz, Methanol-d.sub.4) 172.19, 170.80, 170.66, 170.24, 157.03, 143.90, 143.75, 141.16, 127.42, 126.87, 126.83, 124.98, 124.85, 119.55, 119.53, 83.76, 74.34, 71.61, 66.99, 66.76, 61.74, 54.22, 48.48, 46.91, 31.04, 21.34, 19.16, 19.14, 19.13.

[0154] HRMS-ESI-TOF (m/z): Calcd. for [C.sub.32H.sub.37N.sub.2O.sub.12S+]673.2067 (M+H.sup.+), Found 673.2060 (100%).

Example 5

N.SUP..-Fluoren-9-ylmethoxycarbonyl-S-(3,4,6-tetra-O-acetyl-2-acetamido-2-deoxy)--D-manopyranosyl)-L-cysteine

[0155] ##STR00027##

[0156] -D-manosamine pentaacetate (850 mg, 2.18 mmol, 1.5 eq), InBr.sub.3 (232 mg, 0.65 mmol, 0.3 eq), and N.sup.-fluoren-9-ylmethoxycarbonyl-L-cysteine (500 mg, 1.47 mmol, 1 eq) were suspended in CH.sub.3CN:CHCl.sub.3 (15 mL). The reaction mixture was stirred at reflux for 7 hours. The reaction mixture was concentrated in vacuo, and the residue was purified by flash chromatography ISCO (SiO.sub.2, 0-1.0% of methanol in CH.sub.2Cl.sub.2 with 0.5% TFA) to afford 7 as an off-white powder (712 mg, 73%). R.sub.f 0.4 (CH.sub.2Cl.sub.2/methanol, 9:1 v/v with 0.5% Acetic acid).

[0157] .sup.1H NMR (500 MHz, Methanol-d.sub.4) 7.80 (d, J=5.2 Hz, 2H), 7.69 (t, J=7.1 Hz, 2H), 7.39 (t, J=7.8 Hz, 2H), 7.32 (t, J=7.6, 5.3 Hz, 2H), 5.33 (s, 1H), 5.20 (dt, J=9.9, 2.6 Hz, 1H), 5.14 (dd, J=10.1, 4.6 Hz, 1H), 4.64 (s, 1H), 4.50-4.37 (m, 3H), 4.32-4.23 (m, 3H), 4.13 (dt, J=12.1, 2.6 Hz, 1H), 3.22-3.16 (m, 1H), 3.15-3.07 (m, 1H), 2.01 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.95 (s, 3H).

[0158] .sup.13C NMR (126 MHz, Methanol-d.sub.4) 172.09, 171.14, 170.14, 170.12, 156.96, 143.82, 141.17, 127.42, 126.83, 126.83, 124.96, 124.86, 119.52, 84.79, 69.80, 69.18, 66.70, 66.40, 62.64, 54.40, 51.12, 48.10, 47.00, 33.72, 20.93, 19.33, 19.21, 19.17.

[0159] HRMS-ESI-TOF (m/z): Calcd. for [(C.sub.32H.sub.37N.sub.2O.sub.12S+] 673.2067 (M+H.sup.+), Found 673.2062 (100%).