Traceless reductively cleavable linker molecules for peptide purification

文档序号：788643 发布日期：2021-04-09 浏览：35次中文

阅读说明：本技术 用于肽纯化的无痕还原性可裂解接头分子 (Traceless reductively cleavable linker molecules for peptide purification ) 是由罗伯特·齐特巴特奥利弗·赖曼于 2019-08-27 设计创作，主要内容包括：本发明涉及式(1)：X-T-b-V-a-U-Y-Z(1)的接头分子以及使用所述接头分子纯化肽的方法。接头分子可以经由部分X偶联至纯化树脂并且在释放离去基团Z下经由部分Y偶联至肽。T是任选的间隔基部分并且V是任选的吸电子部分。U是结合至至少一个吸电子部分V、W或E的芳基或5元或6元杂芳基部分。接头在酸性条件下是稳定的并且经添加还原剂释放肽。(The invention relates to a compound of formula (1): X-T b ‑V a -U-Y-Z (1) and a method for purifying a peptide using said linker molecule. The linker molecule may be coupled to the purification resin via moiety X and to the peptide via moiety Y upon release of the leaving group Z. T is an optional spacer moiety and V is an optional electron withdrawing moiety. U is bound to at least oneAn aryl or 5-or 6-membered heteroaryl moiety of the electron-withdrawing moiety V, W or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.)

1. A method of formula 1: X-T_b-V_a-U-Y-Z (1), wherein,

-X is selected from a moiety of formula 2, 2a, 3a or 4, in particular a moiety of formula 2, 2a, 3 or 3a, more in particular a moiety of formula 2 or 2a,

wherein the content of the first and second substances,

each R¹And R²Independently of one another, selected from H or B, wherein at least R¹Or R²Is a compound of formula (I) and (II),

- R³is selected from the group consisting of H and B,

- R⁴selected from H, C₁-C₁₂An alkyl or aryl group, wherein the aldehyde or ketone group may be protected by an acid-labile protecting group,

-B is an acid-labile amine protecting group,

-T is a linear or branched spacer comprising at least one, in particular 1 to 5, of the following moieties: -C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, phenyl, 5-or 6-membered heteroaryl, where,

j is CH or N, in particular N,

in particular T is a spacer selected from

-C₁-C₁₂-alkyl-, in particular C_1-6Alkyl, more particularly C_1-3-alkyl, -R⁵-C(＝O)-，-R⁵-C(＝O)-NR⁹-R⁶-，-R⁵-C(＝O)-NR⁹-，-C(＝O)-NR⁹-R⁶-，-R⁵-NR⁹-C(＝O)-R⁶-，-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-，-R⁵-NR⁹-，-R⁵-NR⁹-R⁶-，-R⁵-NR⁹-R^5’-NR^9’-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-R⁶-，-R⁵-C(＝O)-O-R⁶-，-C(＝O)-O-R⁶-，-R⁵-phenyl-R⁶-，-R⁵-phenyl-, -phenyl-R⁶-, - (O-phenyl-) -, -R⁵-pyrrolyl, -R⁵-pyrazolyl, -R⁵Imidazolyl radical, R⁵-piperazinyl-, -R⁵-pyridyl, -R⁵-pyrimidinyl, -R⁵-pyrazinyl, -R⁵-pyridazinyl, -R⁵-pyrrolyl-R⁶-，-R⁵-pyrazolyl-R⁶-，-R⁵-imidazolyl-R⁶-，-R⁵-piperazinyl-R⁶-，-R⁵-pyridyl-R⁶-，-R⁵-pyrimidinyl-R⁶-，-R⁵-pyrazinyl-R⁶-，-R⁵-pyridazinyl-R⁶-, pyrrolyl-R⁶-, pyrazolyl-R⁶-, imidazolyl-R⁶-piperazinyl-R⁶-, pyridinyl-R⁶-, pyrimidinyl-R⁶-, pyrazinyl-R⁶-, pyridazinyl-R⁶Pyrrolyl, pyrazolyl, imidazolyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, wherein,

R⁵、R^5’and R⁶Independently of one another, from C₁-C₁₂-alkyl or (-C)₂H₄O-)_1-12In particular C₁-C₆Alkyl, especially C₁-C₃An alkyl group, and wherein,

R⁹and R^9’Are independently selected from H, C_1-4-alkyl, -C_1-6-alkyl-NH₂、-C_1-6alkyl-NHB, -C_1-6-alkyl-NB₂、-R¹⁵、-C_1-6-alkyl-R¹⁵、-C_1-6-alkyl-NH-R¹⁵In particular selected from H and C_1-2-alkyl, more particularly R⁹Is a compound of formula (I) wherein,

b is an independently selected acid-labile amine protecting group,

R¹⁵are blocking agents capable of reacting with aldehyde moieties, in particular R¹⁵Selected from the group consisting of cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more in particular cysteinyl, wherein,

the amine and/or thiol moiety of the blocking agent may be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and/or an acid-labile thiol protecting group, particularly trityl,

b is 0 or 1, in particular 1,

-V is an electron withdrawing moiety selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-, -pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, and the like,-C (═ O) -, -C (═ O) -O-, in particular-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-, - (pyridyl) -pyrimidinyl, more particularly chosen from-NH-C (═ O) -, -C (═ O) -NH-, -N- (CH) -, -C (═ O) -NH-, -C- (CH) -, and₃) -, -piperazinyl- (CH)₂)_p-, -pyridyl-, pyrimidyl, wherein,

R¹¹selected from H and C_1-4-alkyl, especiallySelected from H and C_1-2-alkyl, more particularly R¹¹Is a compound of formula (I) wherein the compound is H,

R¹²selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is a methyl group, and the compound is,

p is 0, 1 or 2, in particular 0 or 1,

a is 0 or 1, where the sum of a and b is 1 or 2,

-U is phenyl or a five or six membered heteroaryl moiety, in particular phenyl or a six membered heteroaryl moiety, more in particular phenyl, which is bound to moiety V, W_qAnd E_nAnd may optionally be C_1-6Alkyl, especially C_1-3-alkyl substitution, wherein,

v is defined as described above in the specification,

w is selected from-N₃、-NO₂、-S(＝O)-R⁸、-S-S-R⁸、-O-CH₂-N₃、-O-C(＝O)-O-CH₂-N₃-N ═ N-phenyl, -N ═ N-R⁸、 In particular-N₃、-N＝N-R⁸、-O-CH₂-N₃、-S-S-R⁸Wherein, in the step (A),

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4,

e is an electron withdrawing group under acidic conditions,

n is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 or 1, and q is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 and 1, wherein the sum of n and q is equal to or less than 4, and wherein,

in which U is a phenyl moiety and Y is-(CH₂)_m-O-C (═ O) -, the sum of the Hammett constants of V, W, E under acidic conditions is greater than 0.45, and wherein,

w is in the ortho or para position relative to Y,

-Y is- (CH)₂)_m-C (═ O) -or- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more particularly 1,

-Z is an electron withdrawing leaving group.

2. The compound of claim 1, wherein E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N (C)₂H₄)₂NH₂、-N(C₂H₄)₂N-B, -N ═ N-phenyl, -N ═ N-R⁸、-(CH₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, especially pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N-phenyl, -N-R⁸、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H, more particularly pyridyl, pyrimidinyl, pyridazinyl or-Br, wherein,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4, and

b is an acid-labile amine protecting group, and

r is 0, 1,2, 3 or 4, in particular 0, 1 or 2.

3. The compound of any one of the preceding claims, wherein,

-B is selected from Boc (-C (═ C)O) OtBu), Eei (═ cmoet, 1-ethoxyethylidene), trityl (-c (ph)₃)、-C(＝O)CPh₃、Mmt(-C(Ph)₂C₆H₄OMe)、DMT(-C(Ph)(C₆H₄OMe)₂)、Cbz(-C(＝O)OCH₂Ph), benzal amine (═ CPh), phthalimide (═ CO)₂C₆H₄) P-toluenesulfonamide (-SO)₂C₆H₄Me), benzylamine (-CH)₂Ph), acetamide (-COMe), trifluoroacetamide (-COCF)₃) Dde (1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-ethyl) and 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-methylbutyl (ivDde), wherein especially B is Boc or Eei, wherein more especially B is Boc, and/or

-acetal-or ketal-protecting groups are selected from

Wherein r is 0 to 12, especially 0 to 6, more especially 0, 1 or 2, and

R¹⁰is-C₁-C₁₂-alkyl-, in particular C_1-6-alkyl, more particularly C_1-3-an alkyl group.

4. A compound according to any preceding claim, wherein T is selected from

-C₁-C₁₂-alkyl-, in particular C_1-6Alkyl, more particularly C_1-3-alkyl, -R⁵-C(＝O)-，-R⁵-C(＝O)-NR⁹-，-R⁵-NR⁹-C(＝O)-R⁶-，-R⁵-C(＝O)-NR⁹-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’C(＝O)-R⁶-, in particular C_1-3-alkyl, -R⁵-C(＝O)-NR⁹-、-R⁵-NR⁹-C(＝O)-R⁶-, more particularly C_1-3-alkyl or-R⁵-C(＝O)-NR⁹-, wherein R⁵、R^5*、R⁶、R^9’And R⁹As defined above.

5. A compound according to any one of the preceding claims, wherein the five-or six-membered heteroaryl moiety of U comprises 1 or 2 heteroatoms, in particular the five-membered heteroaryl moiety of the moiety U is selected from pyrazole, imidazole and the six-membered heteroaryl moiety of the moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine, in particular pyridine.

6. A compound according to any one of the preceding claims, wherein U is selected from a moiety of formula 5, 6, 7 or 8, in particular of formula 5 or 6,

wherein the content of the first and second substances,

t, V, Y, W and E are defined as described above,

in the case of formulas 5 and 6, U is bound to the moiety T or V,

in the case of formulas 7 and 8, U is bound to the moiety V,

A¹、A²、A³、A⁴and D¹、D²、D³、D⁴Independently of one another, from C, N, S and O, in particular from C and N, where A¹、A²、A³And A⁴Or D¹、D²、D³And D⁴2 to 4 moieties of (A) are C, especially A¹、A²、A³And A⁴Or D¹、D²、D³And D⁴Is C, more particularly all moieties A¹、A²、A³And A⁴Or D¹、D²、D³And D⁴Is a compound of formula (I) and (II),

n is

In the case of formulae 5 and 6, an integer between 0 and 3, in particular an integer between 0 and 2,

in the case of formulae 7 and 8, an integer between 0 and 4, particularly an integer between 0 and 2, more particularly 0 and 1,

q is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 and 1, where the sum of n and q is equal to or less than 4.

7. A compound according to claim 5, wherein U is selected from a moiety of formula 9, 10, 11 or 12, in particular of formula 9 or 10,

wherein the content of the first and second substances,

t, V, Y, W, E, q and n are defined as described above,

in the case of formulas 9 and 10, U is bound to the moiety T or V,

in the case of formulas 11 and 12, U is bonded to the portion V,

all parts A²、A³And A⁴Is C or A²、A³And A⁴Are C and A²、A³And A⁴The other two of (A) are N, in particular A²And A³Are all C, and

D²is C or N, in particular C.

8. The compound according to any one of the preceding claims, wherein U is selected from a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20 or 21, in particular a moiety of formula 13 to 19, more in particular a moiety of formula 15 or 19,

wherein the content of the first and second substances,

t, V, Y, W, E, q and n are defined as described above,

in the case of formulae 13, 14 and 15, U is bound to the moiety T or V,

in the case of formulae 16, 17 and 18, U is bound to the moiety V,

A²、A³and A⁴Is a group of carbon atoms of C or N, in particular C,

D²is C or N.

9. A compound according to any one of claims 1 to 3, wherein Z is selected from: -F, -Cl, -Br, -I, -N₃、-OH、-O(C＝O)CH₂(C＝O)OH、-SR¹⁴、-OCF₃、-OCH₂CF₃、-OSO₂CF₃、-SO₂C₆H₄CH₃、-SO₂CF₃、-SO₂CH₃

In particular-OH, -Cl,In particular-OH,

Wherein R is¹⁴Is C₁-C₆-alkyl-, aryl-or benzyl substituents.

10. A method for purifying a peptide, the method comprising the steps of:

-providing a linker modified crude peptide, wherein the crude peptide is covalently bound to the linker molecule according to claim 1,

-coupling the linker modified peptide to a solid support in a coupling step resulting in an immobilized linker modified peptide,

-in a release step, releasing the peptide, in particular by adding a reducing agent under acidic conditions.

11. The method according to claim 10, wherein in the release step a reduced intermediate of the reduced linker moiety characterized by the immobilized linker modified peptide is achieved and the peptide is released from the reduced intermediate by a trigger, in particular a change in temperature and/or pH, more in particular by increasing pH to pH > pKa of the most basic heteroatom of the linker moiety of the reduced intermediate in case of an amine switch or by decreasing pH to pH < pKa of the carbamate in case of a carbamate switch.

12. The method according to any one of claims 10 to 11, wherein the linker modified peptide is additionally bound to a synthetic resin and the synthetic resin is cleaved off before the coupling step is performed.

13. A method according to any one of claims 10 to 12 wherein the linker molecule according to claim 1 comprises moieties W and/or E which comprise azido moieties.

14. The process according to any one of claims 10 to 13, wherein after performing step (b) the unreacted aldehyde moiety of the solid support is blocked with a blocking agent, in particular by using a blocking agent selected from cysteine, threonine, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, more in particular from cysteine and N-methylhydroxylamine.

15. A process according to any one of claims 10 to 14, wherein the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris (2-carboxyethyl) phosphine, trimethyl phosphite, triethyl phosphite, tributylphosphine, diethyl phosphite, 5' -dithiobis (2-nitrobenzoic acid), sodium dithionite (Na), sodium dithionite (t), sodium dithionite₂S₂O₄) Ethanedithiol, propanedithiol, dithioerythritol, dithiothreitol, Na₂S, NaSH glutathione, 2' -dithiodipyridine, BH₃4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane, catechol borane, borane tetrahydrofuran, borane dimethyl sulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH₄、LiBH₄、NaBH₄、NaBH₃CN、NaBH(OMe)₃、NaBH(OCCH₃)₃、LiAlH(OCMe₃)₃Hydroquinone, sodium ascorbate, ascorbic acid with KI, hydrazine, NH ═ NH, formaldehyde, in particular dithioerythritol, dithiothreitol, triphenylphosphine, ascorbic acid with KI, tributylphosphine, trimethylphosphine, tris (2-carboxyethyl) phosphine, sodium dithionite (Na)₂S₂O₄) Borane dimethylsulfide, borane triphenylphosphine complexes, NaBH₄Ascorbic acid, more particularly triphenylphosphine and trimethylphosphine.

Technical Field

The present invention relates to a method of purifying a peptide or peptide nucleic acid produced by Solid Phase Peptide Synthesis (SPPS) and a linker molecule for use in said purification.

Background

Solid phase peptide synthesis is a well-established method for synthesizing peptides. The standard procedure is to couple the first N-terminally protected amino acid to a synthetic resin, followed by repeated cycles of N-terminal deprotection, coupling the next N-terminally protected amino acid, and capping of unreacted peptide sequences. Finally, the synthesized peptide is cleaved from the synthetic resin and purified.

A widely used method for purifying peptides is preparative High Performance Liquid Chromatography (HPLC). This method is disadvantageous in that it is poorly scalable with respect to desired production volumes, and thus different volumes cannot be produced with the same system. This results in a relatively high acquisition cost for the correspondingly complex arrangement. Another disadvantage is that relatively extensive knowledge is required to correctly analytically evaluate the individual fractions. In addition, HPLC purification involves high consumption of solvent and occasionally column material (solid phase) during operation.

Thus, a less expensive and less prone to failure process would be advantageous to reduce the cost of peptide production.

An alternative approach uses a linker molecule that can be attached to the peptide and subsequently coupled to a functionalized solid phase used during purification.

EP 0552368 a1 describes linker molecules with thiols that can be covalently bound to a purification support. However, this method is not applicable to thiol-containing peptides, such as peptides comprising the amino acid cysteine or penicillamine.

A similar approach is proposed in EP 2501711B 1, in which the linker is via the azido group (-N)₃) And a1, 3-dipolar cycloaddition between alkyne to a solid phase, which requires the presence of copper. However, methionine, cysteine; arginine or lysinePeptides of amino acids can complex copper, making removal difficult. Due to the toxicity of copper, such peptides are not suitable for all applications, such as pharmaceutical use.

WO 2017129818(a1) discloses linker molecules that can be coupled to peptides that remain bound to the synthetic resin after SPPS. The peptide was then cleaved from synthesis by applying commonly used TFA conditions. However, a disadvantage of linker molecules forming benzyl carbamates with peptides, as disclosed in WO 2017129818(a1), is their instability towards acid treatment (TFA > 50%, pH <0 in the presence of water). Premature decay of the linker molecule results in a significant decrease in the yield of purified peptide.

Peptides comprising Thr, Ser or Cys at the N-terminus of the peptide also undergo unwanted side reactions due to nucleophilic attack of the β -hydroxy or β -thiol group on the sulfo-vinyl carbamate (thio) moiety (moley) of the linker under alkaline conditions (pH >9) for releasing the peptide. Further side reactions of the linker molecule disclosed in WO 2017129818(a1) are asparagine (aspartimide) formation under basic conditions and conversion of arginine to citrulline at the Arg-Glu sequence, as well as disulfide formation and nucleophilic side reactions through internal Cys residues.

Furthermore, the sulfone linker described in WO 2017129818(a1) is affected by a reactive vinyl sulfone moiety that remains on the solid support and requires an additional quenching step.

To overcome the disadvantages of side reactions under basic conditions and premature decay of the linker under acidic conditions, the present invention provides linker molecules that are stable under TFA conditions and allow release of the peptide under mildly acidic conditions (especially at pH 7).

Description of the invention

According to a first aspect of the present invention, there is provided formula 1: X-T_b-V_a-U-Y-Z (1), wherein,

-X is selected from a moiety of formula 2, 2a, 3a or 4 (moiety), in particular of formula 2, 2a, 3 or 3a, more in particular of formula 2 or 2a,

wherein the content of the first and second substances,

each R¹And R²Independently of one another, selected from H or B, wherein at least R¹Or R²Is a compound of formula (I) and (II),

-R³is selected from the group consisting of H and B,

-R⁴selected from H, C₁-C₁₂An alkyl or aryl group, wherein the aldehyde or ketone group may be protected by an acid-labile protecting group,

-B is an acid-labile amine protecting group,

-T is a linear or branched spacer comprising at least one, in particular from 1 to 5, more in particular from 1 to 3, of the following moieties: -C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, phenyl, 5-or 6-membered heteroaryl, in particular-C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, in which,

j is CH or N, in particular N,

in particular T is a spacer selected from

-C₁-C₁₂-alkyl-, in particular-C_1-6-alkyl-, more particularly-C_1-3-alkyl-, -R⁵-C(＝O)-，-R⁵-C(＝O)-NR⁹-R⁶-，-R⁵-C(＝O)-NR⁹-，-C(＝O)-NR⁹-R⁶-，-R⁵-NR⁹-C(＝O)-R⁶-，-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-，-R⁵-NR⁹-，-R⁵-NR⁹-R⁶-，-R⁵-NR⁹-R^5’-NR^9’-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-R⁶-，-R⁵-C(＝O)-O-R⁶-，-C(＝O)-O-R⁶-，-R⁵-phenyl-R⁶-，-R⁵-phenyl-, -phenyl-R⁶-, - (O-phenyl-) -, -R⁵-pyrrolyl, -R⁵-pyrazolyl, -R⁵Imidazolyl radical, R⁵-piperazinyl-, -R⁵-pyridyl, -R⁵-pyrimidinyl, -R⁵-pyrazinyl, -R⁵-pyridazinyl, -R⁵-pyrrolyl-R⁶-，-R⁵-pyrazolyl-R⁶-，-R⁵-imidazolyl-R⁶-，-R⁵-piperazinyl-R⁶-，-R⁵-pyridyl-R⁶-，-R⁵-pyrimidinyl-R⁶-，-R⁵-pyrazinyl-R⁶-，-R⁵-pyridazinyl-R⁶-, pyrrolyl-R⁶-, pyrazolyl-R⁶-, imidazolyl-R⁶-piperazinyl-R⁶-, pyridinyl-R⁶-, pyrimidinyl-R⁶-, pyrazinyl-R⁶-, pyridazinyl-R⁶Pyrrolyl, pyrazolyl, imidazolyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, wherein,

R⁵、R^5’and R⁶Independently of one another, from C₁-C₁₂-alkyl or (-C)₂H₄O-)_1-12In particular C₁-C₆Alkyl, especially C₁-C₃An alkyl group, and wherein,

b is an independently selected acid-labile amine protecting group,

the amine and/or thiol moieties of the blocking agent may be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and/or an acid-labile thiol protecting group, particularly trityl.

B is 0 or 1, in particular 1,

R¹¹selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is a compound of formula (I) wherein the compound is H,

R¹²selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is a methyl group, and the compound is,

p is 0, 1 or 2, in particular 0 or 1,

a is 0 or 1, where the sum of a and b is 1 or 2,

v is defined as described above in the specification,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4,

e is an electron withdrawing group under acidic conditions,

where U is a phenyl moiety and Y is- (CH)₂)_m-O-C (═ O) -, the sum of the Hammett constants of V, W, E under acidic conditions is greater than 0.45, and wherein,

w is in the ortho or para position relative to Y,

-Y is- (CH)₂)_m-C (═ O) -or- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more particularly 1,

-Z is an electron withdrawing leaving group.

In some embodiments of the present invention, the substrate is,

-X is selected from a moiety of formula 2, 2a, 3a or 4, in particular a moiety of formula 2, 2a, 3 or 3a, more in particular a moiety of formula 2 or 2a,

wherein the content of the first and second substances,

each R¹And R²Independently of one another, selected from H or B, wherein at least R¹Or R²Is a compound of formula (I) and (II),

-R³is selected from the group consisting of H and B,

-R⁴selected from H, C₁-C₁₂An alkyl or aryl group, wherein the aldehyde or ketone group may be protected by an acid-labile protecting group,

-B is an acid-labile amine protecting group,

-T is a linear or branched spacer comprising at least one of the following moieties: -C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, in which,

j is C or N, in particular N,

in particular T is a spacer selected from

-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-、-R⁵-C(＝O)-NR⁹-R⁶-、-R⁵-C(＝O)-NR⁹-、-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-、-R⁵-NR⁹-、-R⁵-NR⁹-R⁶-、-R⁵-NR⁹-R^5’-NR^9’-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-R⁶-、-R⁵-C(＝O)-O-R⁶-、-C(＝O)-O-R⁶-、-R⁵-phenyl-R⁶-、-R⁵-phenyl-, -phenyl-R⁶-, -phenyl-, wherein,

R⁵、R^5’and R⁶Independently of one another, from C₁-C₁₂-alkyl or (-C)₂H₄O-)_1-12In particular C₁-C₆Alkyl, especially C₁-C₃An alkyl group, and wherein,

b is an independently selected acid-labile amine protecting group,

b is 0 or 1, in particular 1,

-V is an electron withdrawing moiety selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, -piperazinyl-, -pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,-C (═ O) -, -C (═ O) -O-, in particular-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly selected from-NH-C (═ O) -, -C (═ O) -NH-, -N- (CH) — NH₃) -, -piperazinyl-, -pyridinyl-, pyrimidinyl, wherein,

R¹¹selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is a compound of formula (I) wherein the compound is H,

R¹²selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is a methyl group, and the compound is,

a is 0 or 1, where the sum of a and b is 1 or 2,

v is defined as described above in the specification,

w is selected from-N₃、-S(＝O)-R⁸、-S-S-R⁸、-O-CH₂-N₃、-O-C(＝O)-O-CH₂-N₃-N ═ N-phenyl, -N ═ N-R⁸、 In particular-N₃、-N＝N-R⁸、-O-CH₂-N₃、-S-S-R⁸Wherein, in the step (A),

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4,

e is an electron withdrawing group under acidic conditions,

where U is a phenyl moiety and Y is- (CH)₂)_m-O-C (═ O) -, the sum of the Hammett constants of V, W, E under acidic conditions is greater than 0.45,

-Y is- (CH)₂)_m-C (═ O) -or- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more particularly 1,

z is an electron withdrawing leaving group.

In some embodiments of the present invention, the substrate is,

-X is selected from a moiety of formula 2, 2a, 3a or 4, in particular a moiety of formula 2, 2a, 3 or 3a, more in particular a moiety of formula 2 or 2a,

wherein the content of the first and second substances,

each R¹And R²Independently of one another, selected from H or B, wherein at least R¹Or R²Is a compound of formula (I) and (II),

-R³is selected from the group consisting of H and B,

-R⁴selected from H, C₁-C₁₂An alkyl or aryl group, wherein the aldehyde or ketone group may be protected by an acid-labile protecting group,

-B is an acid-labile amine protecting group,

-T is a spacer selected from

-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-、-R⁵-C(＝O)-NR⁹-R⁶-、-R⁵-C(＝O)-NR⁹-、-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-、-R⁵-C(＝O)-O-R⁶-、-C(＝O)-O-R⁶-、-R⁵-phenyl-R⁶-、-R⁵-phenyl-, -phenyl-R⁶-, -phenyl-, wherein,

R⁵、R^5’and R⁶Independently of one another, from C₁-C₁₂Alkyl, especially C₁-C₆Alkyl, especially C₁-C₃Alkyl, and, wherein R⁹And R^9’Independently of one another, selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R⁹Is a compound of formula (I) wherein the compound is H,

b is 0 or 1, in particular 1,

R¹¹selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is a compound of formula (I) wherein the compound is H,

R¹²selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is a methyl group, and the compound is,

a is 0 or 1, where the sum of a and b is 1 or 2,

-U is phenyl or a penta-or hexa-membered radicalA membered heteroaryl moiety, particularly a phenyl or six membered heteroaryl moiety, more particularly a phenyl, which is bound to moiety V, W_qAnd E_nAnd may optionally be C_1-6Alkyl, especially C_1-3-alkyl substitution, wherein,

v is defined as described above in the specification,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4,

e is an electron withdrawing group under acidic conditions,

where U is a phenyl moiety and Y is- (CH)₂)_m-O-C (═ O) -, the sum of the Hammett constants of V, W, E under acidic conditions is greater than 0.45, and wherein,

in particular W is in ortho or para position with respect to Y,

-Y is- (CH)₂)_m-C (═ O) -or- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more particularly 1,

-Z is an electron withdrawing leaving group.

In some embodiments of the present invention, the substrate is,

-X is selected from a moiety of formula 2, 2a, 3a or 4, in particular a moiety of formula 2, 2a, 3 or 3a, more in particular a moiety of formula 2 or 2a, most in particular a moiety selected from 2,

wherein the content of the first and second substances,

each R¹And R²Independently of one another, selected from H or B, wherein at least R¹Or R²Is a compound of formula (I) and (II),

-R³is selected from the group consisting of H and B,

-R⁴selected from H, C₁-C₁₂An alkyl or aryl group, wherein the aldehyde or ketone group may be protected by an acid-labile protecting group,

-B is an acid-labile amine protecting group,

-T is a spacer selected from

-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-NR⁹-R⁶-、-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-R⁵-NR⁹C(＝O)-R⁶-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-C(＝O)-O-R⁶-、-C(＝O)-O-R⁶-、-R⁵-phenyl-R⁶-、-R⁵-phenyl-, -phenyl-R⁶-, -phenyl-, wherein,

R⁵and R⁶Independently of one another, from C₁-C₁₂Alkyl, especially C₁-C₆Alkyl, especially C₁-C₃Alkyl, and wherein R⁹Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R⁹Is a compound of formula (I) wherein the compound is H,

b is 0 or 1, in particular 1,

-V is an electron withdrawing moiety selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C (-O) -O-, in particular-NR¹¹-C(＝O)-、-S(＝O)-、-NR¹²-, -pyridyl-, pyrimidyl, wherein,

R¹¹selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is a compound of formula (I) wherein the compound is H,

R¹²selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is a methyl group, and the compound is,

a is 0 or 1, where the sum of a and b is 1 or 2,

-U is a phenyl or a five or six membered heteroaryl moiety, in particular a phenyl or six membered heteroaryl moiety, which is bound to at least one of moieties V, W and E, wherein,

v is defined as described above in the specification,

w is selected from-N₃、-S(＝O)-R⁸、-SSR⁸、-OCH₂N₃、-OC(＝O)OCH₂-N₃-N ═ N-phenyl, -N ═ N-pyridine, and, Wherein the content of the first and second substances,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂Wherein p is 1,2, 3 or 4,

e is selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO₂、-N₃、-CF₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, TeIn particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO₂、-N₃、-CF₃、-SO₃H、-CO₂H, wherein,

R¹³selected from-F, -Cl, -Br, -I, -PF₆And wherein the first and second end portions of the first and second,

where U is a phenyl moiety and Y is- (CH)₂)_m-O-C (═ O) -, the sum of the Hammett constants of V, W, E under acidic conditions is greater than 0.45, and wherein,

in particular W is in ortho or para position with respect to Y,

-Y is- (CH)₂)_m-C (═ O) -or- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more particularly 1,

-Z is an electron withdrawing leaving group.

The linker molecule of formula 1 is suitable for purification of peptides after Solid Phase Peptide Synthesis (SPPS).

A common method for purifying the peptide after SPPS using a linker molecule is to couple the linker molecule to the N-terminus of the peptide in a final coupling step. In this coupling step, the N-terminus of the peptide will nucleophilically attack the linker molecule to form a covalent carbamate or amide bond with the moiety Y of the linker, while releasing the electron-withdrawing leaving group Z. Subsequently, the peptide-linker-construct was cleaved from the synthetic resin by addition of TFA.

The linker according to the invention is stable under acidic conditions when the peptide-linker construct is cleaved off, e.g. by using TFA.

The moiety X of the linker may be coupled to a functionalized solid phase, such as a resin, used during purification. Moiety X may form a hydrazone or oxime linkage by reaction with an appropriate moiety of a functionalized solid phase, such as an aldehyde, ketone, amino-oxy, or hydrazine.

The moiety T represents a spacer that is non-reactive under the purification conditions typically employed.

The moiety T is bonded to the moiety U directly or via the moiety V.

The moiety U contributes to the stability of the linker molecule under acidic conditions (in particular TFA > 50%, in the presence of water, pH < 0). This is achieved by using a heterocyclic moiety or a phenyl moiety that is bound to at least one of the electron withdrawing moieties E, W and V.

If the electron-withdrawing moiety (E, W, V) is attached to the phenyl moiety, the benzyl position of the linker molecule to which the peptide is attached attains a smaller electron density and is therefore less susceptible to acid-catalyzed degradation. In order for the linker molecules of the invention to have sufficient stability under acidic conditions, a certain electron-withdrawing threshold must be met. The threshold is expressed as a Hammett constant (σ) of V, W, E under acidic conditions_mAnd σ_p) The sum is greater than 0.45. Hammett's constants were calculated according to Hansch and Taft (1991), chem. Rev.91: 165-. A positive hammett constant reflects the ability of a substituent to exert an electron withdrawing effect on a phenyl moiety, while a negative value indicates that the substituent exerts an electron withdrawing effect.

Hammett's constant is the meditope (. sigma.) of benzoic acid derivatives_m) And alignment (σ)_p) The substituents of the phenyl nucleus lead to empirically determined constants of different acidity (pKa). In the context of the present invention, the position is determined relative to the bound moiety Y. The Hammett value of the para position is a good approximation for the ortho position substituent and is therefore used in the context of the present invention to calculate the sum of the Hammett values of substituents V, W and E.

Notably, the Hammett constants of substituents V, W and E were calculated under acidic conditions. For example, the amine moiety is characterized by a Hammett constant σ at neutral pH_m0.16 and σ_p-0.66, and thus is a donating substituent. Under acidic conditions, the amine moiety is protonated. For protonated amines, the Hammett constant is σ_m0.86 and σ_p+0.60, indicating that the protonated amine is an electron withdrawing substituent, this also includes aromatic amines that are capable of withdrawing electrons from U in their protonated form through the conjugated pi system, either directly as a substituent on U or in the pi conjugation of U.

A threshold value of greater than 0.45 for the sum of the Hammett constants is applicable to U being a phenyl moiety and Y being- (CH)₂)_m-O-C (═ O) -, where m is 1, since-O-C (═ O) -constitutes benzylA good leaving group in the radical position, contributes to the acid-catalyzed degradation. Therefore, the electron density in the aromatic ring should be low enough to prevent the cation stabilization of the benzyl position in Y.

TABLE 1

GetSubstitute for Chinese traditional medicineBase of	σ_m ^[a]	σ_p ^[a]	GetSubstitute for Chinese traditional medicineBase of	σ_m ^[a]	σ_p ^[a]
						-Br	0.39	0.23	-Cl	0.37	0.23
-N₃	0.37	0.08	-NO₂	0.71	0.78
						-CONHMe	0.35	0.36	-N＝NC₆H₅	0.32	0.39
-SH	0.25	0.15	-S^-	-0.36	-1.21
						-OH	0.12	-0.35	-O^-	-0.47	(-0.81)
-NH₃ ⁺	0.86	0.60	-NH₂	-0.16	-0.66
						-NMe₃ ^+[b]	0.88	0.82	-NMe₂	-0.16	-0.83
-CH₂NH₃ ⁺	0.59	0.53	-CH₂NH₂	-0.03	-0.11
						-CH₂NMe₂H⁺	0.40	0.43	-CH₂NMe₂	0.00	0.01
- (4-pyrimidines)Base of )	0.30	0.63	- (4-pyridine)Pyridine (I)Base of )	0.27	0.44
						-SOMe	0.52	0.49	-SMe	0.15	0.00
-SO₂Me	0.60	0.72	-SSMe	0.22	0.00
						-OCH₂CH₃	0.10	-0.24	Et	-0.07	-0.15

[a]σ_mSigma for meta and para substituents relative to the benzylic carbon_pValue σ_pConsidered as good approximations of the values of sigma for ortho-substituents (all values according to Hansch and Taft (1991), chem. Rev.91:165-]-NMe³⁺is-NMe₂H⁺Good approximation of

If the moiety Y is- (CH)₂)_m-C (═ O) -, then this threshold is not necessary, since-C (═ O) -is not a good leaving group at the benzyl position.

Such additional thresholds for selection of specific moieties V, W and E are not necessary for the stability of the linker molecule comprising heterocyclic moiety U under acidic conditions. Since the heterocyclic moiety itself is more electron deficient than the phenyl moiety, any combination of V, W and E appears to be sufficient for the stability of the linker molecule under acidic conditions. Especially when U is a nitrogen-containing heterocycle, the nitrogen will be protonated during acidic release of the peptide, making the electron density of the U's aromatic system especially low. Therefore, the benzyl cation cannot be stabilized.

In addition to modulating the stability of the linker molecule under acidic conditions, moieties V, W and E are also important for the release mechanism of the peptide. Furthermore, they may contribute to the solubility of the linker molecule under acidic conditions.

The moiety W is a reducible substituent which triggers decay of the linker and thus release of the peptide. Linkers comprising a reduced moiety W are also referred to as reduced intermediates. In contrast to stable linker molecules, reduced intermediates are unstable. The instability of the reduced intermediate is pH dependent. If the moiety W is protonatable, e.g., pyridyl, it also contributes to the solubility of the linker molecule under acidic conditions.

Part E is an electron-withdrawing substituent which exhibits electron withdrawing action under acidic conditions. For example, moieties having a positive Hammett constant under acidic conditions are electron withdrawing, particularly at pH3 to 6, more particularly at pH 4.5. If moiety E is protonatable, it also contributes to the solubility of the linker molecule under acidic conditions.

In addition to or instead of part E, part V may exhibit electron withdrawing action under acidic conditions. Part V may also contribute to the stability and solubility of the linker under acidic conditions.

Linker molecules according to the invention may release peptides via an amine switching mechanism (see fig. 5, 6 and 7) or via a carbamate switching mechanism (see fig. 8).

The moiety Y is a-C (═ O) -or-O-C (═ O) -moiety. Upon coupling of the linker molecule, an amide (-C (═ O) -NH-) or carbamate (-O-C (═ O) -NH-) moiety is formed between the linker molecule and the N-terminus of the peptide. After purification, the peptide is released from the linker molecule, and thus from the purification medium, under reducing conditions by 1.4 or 1.6 elimination or nucleophilic attack. The reductive stimulus converts the W to its reduced version, now acting as an electron donating group and a nucleophile, thereby enabling the peptide to be released.

In certain embodiments, X is selected from a moiety of formula 2 or 3.

In certain embodiments, X is selected from a moiety of formula 2.

The reaction time required to couple moiety X to the functionalized solid phase by forming a hydrazone or oxime bond is longer than when a linker having a moiety of formula 4 is used and shorter than when a linker having a moiety of formula 2 or 3 is used. The formation of a hydrazone bond between the aldehyde or ketone moiety of the solid support and the moiety X of formula 3 is reversible. Due to this reversibility, the inventors observed a loss of peptide material up to about 10% after each washing step during purification. In contrast, when a linker having a moiety of formula 2 is used, little loss of peptide material is observed.

In certain embodiments, U is C_1-6-alkyl substitution.

In certain embodiments, U is C_1-3-alkyl substitution.

In certain embodiments, U is substituted with methyl.

If U is further substituted with one or more alkyl moieties, the Hammett value of the alkyl moiety is considered. In the case where U is further substituted with a phenyl moiety or heteroatom, the sum of the hammett values of V, W, E and the optional alkyl substituent is greater than 0.45. In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N (C)₂H₄)₂NH₂、-N(C₂H₄)₂N-B, -N ═ N-phenyl, -N ═ N-R⁸、-(CH₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, wherein,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4, and

b is an acid-labile amine protecting group as defined herein, in particular-C (═ O) otbu (boc) or C (═ O) CPh₃And is and

r is 0, 1,2, 3 or 4, in particular 0, 1 or 2. In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, - (a)CH₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-, -N ═ N-phenyl, -N ═ N-R⁸、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H, wherein r is 0, 1,2, 3 or 4, in particular 0, 1 or 2.

In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyridazinyl, -NH-C_1-6-alkyl, -N (C)_1-6-alkyl groups)₂-, -N ═ N-pyridyl or-Br.

In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyridazinyl, -NH-C_1-6-alkyl, -N (C)_1-6-alkyl groups)₂-or-Br.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl, -NH-C_1-6-alkyl, -N (C)_1-6-alkyl groups)₂-or-Br.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N ═ N-phenyl, -N ═ N-R⁸、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, wherein R⁸Is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N ═ N-phenyl, -N ═ N-R⁸、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H, wherein R⁸Is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl radicals, whichWherein p is 1,2, 3 or 4.

In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, - (CH)₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-, in particular- (CH)₂)_r-NH-C_1-3-alkyl, - (CH)₂)_r-N(C_1-3-alkyl groups)₂-、-F、-Cl、-Br、-I、-CN、-NO₂、-N₃、-CF₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, where r is 0, 1 or 2, in particular 0.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO₂、-N₃、-CF₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et、-SOEt。

In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO₂、-N₃、-CF₃、-SO₃H、-CO₂H, wherein r is 0, 1 or 2, in particular 0.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO₂、-N₃、-CF₃、-SO₃H、-CO₂H。

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl, -NH-C_1-6-alkyl, -N (C)_1-6-alkyl groups)₂-, especially-NH-C_1-3-alkyl, -N (C)_1-3-alkyl groups)₂-or-Br.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl, or-Br.

In certain embodiments, n is 0, 1, or 2.

In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl and n is 1 or E is-Br and n is 1 or 2.

Peptide release via amine switch (fig. 5, 6 and 7) requires a basic nitrogen that can be protonated. Such basic nitrogen may be provided by moiety E.

In certain embodiments, E is selected from- (CH)₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-, in particular- (CH)₂)_r-NH-C_1-3-alkyl, - (CH)₂)_r-N(C_1-3-alkyl groups)₂-, piperidinyl, piperazinyl, pyridinyl, pyrimidinyl and pyridazinyl, wherein r is 0, 1 or 2, especially 0, wherein especially n is 1.

In certain embodiments, E is selected from- (CH)₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-, in particular- (CH)₂)_r-NH-C_1-3-alkyl, - (CH)₂)_r-N(C_1-3-alkyl groups)₂-, pyridinyl, pyrimidinyl and pyridazinyl, r is 0, 1 or 2, in particular 0, and n is 1.

In particular, the linker molecule that releases the peptide through the carbamate switch (fig. 8) includes an electron-withdrawing moiety that is not reduced by the reducing agent used to reduce moiety W. Suitable substituents E for urethane switches may be non-protonatable, such as halogen or nitro substituents. Likewise, substituents characterized by pKa <0 of the corresponding acid are suitable substituents E for urethane switches.

In certain embodiments, E is-F, -Cl, -Br, -I, -NO₂、-CF₃、-CN、-NC、BF₂、-PF₄、-OCF₃、-SOCF₃、-SOR₈、-SO₂R₈。

In certain embodiments, E is-Br.

In certain embodiments, E is-Br and n is 1 or 2.

If the solubility of the linker under acidic conditions should be increased, for example for purification of hydrophobic peptides, E may be selected from protonatable moieties. In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N (C)₂H₄)₂NH₂、-N(C₂H₄)₂N-B、-N＝N-R⁸、-(CH₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, wherein R⁸Selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, - (CH)₂)_p-NMe₂Wherein p is 1,2, 3 or 4, especially 1 or 2.

In certain embodiments, B is selected from

-Boc (-C (═ O) OtBu), Eei (═ cmoet, 1-ethoxyethylidene), trityl (-C (ph)₃)、-C(＝O)CPh₃、Mmt(-C(Ph)₂C₆H₄OMe)、DMT(-C(Ph)(C₆H₄OMe)₂)、Cbz(-C(＝O)OCH₂Ph), benzal amine (═ CPh), phthalimide (═ CO)₂C₆H₄) P-toluenesulfonamide (-SO)₂C₆H₄Me), benzylamine (-CH)₂Ph), acetamide (-COMe), trifluoroacetamide (-COCF)₃) Dde (1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-ethyl) and 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-methylbutyl (ivDde), wherein especially B is Boc or Eei, wherein more especially B is Boc, or

-acetal-or ketal-protecting groups are selected from

Wherein r is 0 to 12, especially 0 to 6, more especially 0, 1 or 2, and

R¹⁰is-C₁-C₁₂-alkyl-, in particular C_1-6-alkyl, more particularly C_1-3-an alkyl group.

In certain embodiments, B is selected from

-Boc (-C (═ O) OtBu), Eei (═ cmoet, 1-ethoxyethylidene), trityl (-C (ph)₃)、Mmt(-C(Ph)₂C₆H₄OMe)、DMT(-C(Ph)(C₆H₄OMe)₂)、Cbz(-C(＝O)OCH₂Ph), benzal amine (═ CPh), phthalimide (═ CO)₂C₆H₄) P-toluenesulfonamide (-SO)₂C₆H₄Me), benzylamine (-CH)₂Ph), acetamide (-COMe), trifluoroacetamide (-COCF)₃) Dde (1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-ethyl) and 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-methylbutyl (ivDde), wherein especially B is Boc or Eei, wherein more especially B is Boc, or

-acetal-or ketal-protecting groups are selected from

Wherein r is 0 to 12, especially 0 to 6, more especially 0, 1 or 2, and

R¹⁰is-C₁-C₁₂-alkyl-, in particular C_1-6-alkyl, more particularly C_1-3-an alkyl group.

In certain embodiments, B is selected from Boc (-C (═ O) OtBu), Eei (═ cmoet, 1-ethoxyethylidene), trityl (-C (ph)₃)、Mmt(-C(Ph)₂C₆H₄OMe)、DMT(-C(Ph)(C₆H₄OMe)₂)、Cbz(-C(＝O)OCH₂Ph), benzal amine (═ CPh), phthalimide (═ CO)₂C₆H₄) P-toluenesulfonamide (-SO)₂C₆H₄Me), benzylamine (-CH)₂Ph), acetamide (-COMe), trifluoroacetamide (-COCF)₃) Dde (1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-ethyl) and 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-methylbutyl (ivDde).

In certain embodiments, B is Boc or Eei (═ cmoet, 1-ethoxyethylidene).

In certain embodiments, B is Boc.

In certain embodiments, B is selected from acetal-or ketal-protecting groups selected from

Wherein R is 0 to 12, especially 0 to 6, more especially 0, 1 or 2, and R¹⁰is-C₁-C₁₂-alkyl-, in particular C_1-6-an alkyl group.

In certain embodiments, T is a linear or branched spacer comprising at least one of the following moieties: -C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, phenyl, 5-or 6-membered heteroaryl, where J is CH or N, in particular N, where R⁹Independently selected from H, C_1-4-alkyl, -C_1-6-alkyl-NH₂、-C_1-6alkyl-NHB, -C_1-6-alkyl-NB₂In particular selected from H and C_1-2-alkyl, more particularly R⁹Is H, wherein B is an independently selected acid-labile amine protecting group.

In certain embodiments, T is a linear or branched spacer, particularly a linear spacer, comprising 1 to 5 moieties independently selected from: -C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, phenyl, 5-or 6-membered heteroaryl,

in certain embodiments, T is a linear or branched spacer comprising at least one, in particular 1 to 5, of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, phenyl, piperazinyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl,

in certain embodiments, T is a linear or branched spacer comprising at least one, in particular 1 to 5, of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, phenyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl,

in certain embodiments, T is a linear spacer.

In certain embodiments, T is a linear or branched spacer comprising at least one of the following moieties: -C_1-12-alkyl-, (-C)₂H₄O-)_1-12、-C(＝O)-、-C(＝O)-JR⁹-、-JR⁹-C(＝O)-、-JR⁹-, where J is C or N, especially N, where R⁹Independently selected from H, C_1-4-alkyl, -C_1-6-alkyl-NH₂、-C_1-6alkyl-NHB, -C_1-6-alkyl-NB₂In particular selected from H and C_1-2-alkyl, more particularly R⁹Is H, wherein B is an independently selected acid-labile amine protecting group.

In certain embodiments, the total length of the spacer T is between 0.5 and 100 nm.

In certain embodiments, T is selected from-C₁-C₁₂-alkyl-, in particular C_1-6Alkyl, more particularly C_1-3-alkyl, -R⁵-C(＝O)-，-R⁵-C(＝O)-NR⁹-R⁶-，-R⁵-C(＝O)-NR⁹-，-C(＝O)-NR⁹-R⁶-，-R⁵-NR⁹-C(＝O)-R⁶-，-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-，-R⁵-NR⁹-，-R⁵-NR⁹-R⁶-，-R⁵-NR⁹-R^5’-NR^9’-R⁶-，-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-R⁶-，-R⁵-C(＝O)-O-R⁶-，-C(＝O)-O-R⁶-，-R⁵-phenyl-R⁶-，-R⁵-phenyl-, -phenyl-R⁶-, - (O-phenyl-) -, -R⁵-pyrrolyl, -R⁵-pyrazolyl, -R⁵Imidazolyl radical, R⁵-piperazinyl-, -R⁵-pyridyl, -R⁵-pyrimidinyl, -R⁵-pyrazinyl, -R⁵-pyridazinyl, -R⁵-pyrrolyl-R⁶-，-R⁵-pyrazolyl-R⁶-，-R⁵-imidazolyl-R⁶-，-R⁵-piperazinyl-R⁶-，-R⁵-pyridyl-R⁶-，-R⁵-pyrimidinyl-R⁶-，-R⁵-pyrazinyl-R⁶-，-R⁵-pyridazinyl-R⁶-, pyrrolyl-R⁶-, pyrazolyl-R⁶-, imidazolyl-R⁶-piperazinyl-R⁶-, pyridinyl-R⁶-, pyrimidinyl-R⁶-, pyrazinyl-R⁶-, pyridazinyl-R⁶Pyrrolyl, pyrazolyl, imidazolyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl.

In certain embodiments, T is selected from-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-、-R⁵-C(＝O)-NR⁹-R⁶-、-R⁵-C(＝O)-NR⁹-、-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-、-R⁵-NR⁹-、-R⁵-NR⁹-R⁶-、-R⁵-NR⁹-R^5’-NR^9’-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-R⁶-、-R⁵-C(＝O)-O-R⁶-、-C(＝O)-O-R⁶-、-R⁵-phenyl-R⁶-、-R⁵-phenyl-, -phenyl-R⁶-, -phenyl-, -R⁵-imidazolyl, -R⁵-imidazolyl-R⁶-, imidazolyl-R⁶-, imidazolyl.

In certain embodiments, T is selected from-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-、-R⁵-C(＝O)-NR⁹-R⁶-、-R⁵-C(＝O)-NR⁹-、-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-NR⁹-R^5’-NR^9’C(＝O)-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-C(＝O)-R⁶-、-R⁵-NR⁹-、-R⁵-NR⁹-R⁶-、-R⁵-NR⁹-R^5’-NR^9’-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’-R⁶-、-R⁵-C(＝O)-O-R⁶-、-C(＝O)-O-R⁶-、-R⁵-imidazolyl, -R⁵-imidazolyl-R⁶-, imidazolyl-R⁶-, imidazolyl.

In certain embodiments, T is selected from-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-、-R⁵-C(＝O)-NR⁹-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-C(＝O)-NR⁹-R⁶-、-R⁵-C(＝O)-NR⁹-R^5’-NR^9’C(＝O)-R⁶-, in particular C_1-3-alkyl, -R⁵-C(＝O)-NR⁹-、-R⁵-NR⁹-C(＝O)-R⁶-, more particularly C_1-3-alkyl or-R⁵-C(＝O)-NR⁹-, wherein R⁵、R^5*、R⁶、R^9’And R⁹As defined above.

In certain embodiments, T is a linear or branched spacer comprising at least one, in particular 1 to 5, of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, phenyl, 5-to 6-membered heteroaryl, where R⁹Independently selected from H, C_1-4-alkyl, -C_1-6-alkyl-NH₂、-C_1-6alkyl-NHB, -C_1-6-alkyl-NB₂、-R¹⁵、-C_1-6-alkyl-R¹⁵、-C_1-6-alkyl-NH-R¹⁵In particular selected from H and C_1-2-alkyl, more particularly R⁹Is H, wherein B is an independently selected acid-labile amine protecting group, wherein,

R¹⁵is a block capable of reacting with an aldehyde moietyAgents, especially R¹⁵Selected from the group consisting of cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more in particular cysteinyl, wherein,

in certain embodiments, T is a linear or branched spacer comprising at least one, in particular 1 to 5, of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, phenyl, imidazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl,

in certain embodiments, T is a linear or branched spacer comprising at least one of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, wherein R⁹Independently selected from H, C_1-4-alkyl, -C_1-6-alkyl-NH₂、-C_1-6alkyl-NHB, -C_1-6-alkyl-NB₂、-R¹⁵、-C_1-6-alkyl-R¹⁵、-C_1-6-alkyl-NH-R¹⁵In particular selected from H and C_1-2-alkyl, more particularly R⁹Is H, wherein B is an independently selected acid-labile amine protecting group,

R¹⁵are blocking agents capable of reacting with aldehyde moieties, in particular R¹⁵Selected from the group consisting of cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, dithiol, and mixtures thereof,Hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein,

As noted above, the spacer T is generally non-reactive under the purification conditions typically employed, except that the protecting group is removed under acidic conditions. The spacer T may enhance the solubility of the linker molecule. In particular, in R⁹Branched spacers comprising protected or unprotected amine moieties help to enhance solubility. Under acidic conditions, the amine protecting group B is removed and the amine is protonated.

In certain embodiments, T is a linear or branched spacer comprising at least one of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, wherein R⁹Independently selected from H, C_1-4-alkyl, -C_1-6-alkyl-NH₂、-C_1-6alkyl-NHB, -C_1-6-alkyl-NB₂In particular selected from H and C_1-2-alkyl, more particularly R⁹Is H, wherein B is an independently selected acid-labile amine protecting group.

The spacer T may also include a blocking function. In particular, the branched spacer may comprise a blocking agent suitable for binding to an aldehyde moiety. When the solid phase used for peptide purification comprises aldehyde moieties, such as agarose beads, the moiety X of the linker compound may be covalently bound to the solid phase, for example by forming an oxime bond. The unreacted aldehyde moiety may cause unwanted side reactions during subsequent purification. To prevent such side reactions, the unreacted aldehyde portion of the solid phase may be blocked by a blocking function of a spacer, such as a cysteinyl moiety.

In certain embodiments, T is a linear or branched spacer comprising at least one of the following moieties: -C_1-12-alkyl-, -C (═ O) -NR⁹-、-NR⁹-C(＝O)-、-NR⁹-, wherein R⁹Independently selected from H, C_1-4-alkyl, -R¹⁵、-C_1-6-alkyl-R¹⁵、-C_1-6-alkyl-NH-R¹⁵In particular selected from H and C_1-2-alkyl, more particularly R⁹Is H, wherein B is an independently selected acid-labile amine protecting group,

In certain embodiments, T is selected from-C₁-C₁₂-alkyl- (especially C)_1-6Alkyl, more particularly C_1-3-alkyl), -R⁵-C(＝O)-NR⁹-R⁶-、-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-C(＝O)-O-R⁶-、-C(＝O)-O-R⁶-、-R⁵-NR⁹-R⁵-NR⁹C(＝O)-R⁶-, in particular selected from-C₁-C₁₂-alkyl-, in particular C_1-6-alkyl, more particularly C_1-3-alkyl, -R⁵-NR⁹-C(＝O)-R⁶-、-R⁵-C(＝O)-NR⁹-R⁶-、-R⁵-NR⁹-R⁵-NR⁹C(＝O)-R⁶-, most especially-R⁵-NR⁹-C(＝O)-R⁶-, wherein R⁵、R⁶And R⁹As defined above.

As noted above, portions U, V, W and E contribute to the stability of the linker under acidic conditions. For example, when an amine or heterocycle is protonated, a linker comprising a moiety U or V comprising an amine or heterocycle, such as pyridine, is stable under acidic conditions (specifically TFA > 50%, pH <0 in the presence of water). In addition, the protonated linker improves the solubility of the linker-peptide complex. If the pH is above the pKa of the linker, the linker will rapidly break down as a final step during the desired peptide release period.

In certain embodiments, V is an electron withdrawing moiety selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-, -pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyridinyl, and the like,-C (═ O) -, -C (═ O) -O-, wherein,

R¹¹selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is a compound of formula (I) wherein the compound is H,

R¹²selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is a methyl group, and the compound is,

p is 0, 1 or 2, in particular 0 or 1.

In certain embodiments, V is selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-。

In certain embodiments, V is selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-。

In certain embodiments, V is selected from-NH-C (═ O) -, -C (═ O) -NH-, -N- (CH)₃) -, -NH-, -piperazinyl- (CH)₂)_p-。

In certain embodiments, V is selected from-NH-C (═ O) -, -C (═ O) -NH-, -N- (CH)₃) -, -piperazinyl- (CH)₂)_p-。

In certain embodiments, V is selected from-NH-C (═ O) -, -C (═ O) -NH-, -N- (CH)₃) -, -piperazinyl- (CH)₂)_p-, where p is 0 or 1.

In certain embodiments, V is selected from-NH-C (═ O) -, -C (═ O) -NH-, and-piperazinyl- (CH)₂)_p-, where p is 0 or 1, especially p is 0.

The linker molecule, which releases the peptide in particular via a carbamate switch (fig. 8), does not comprise a basic nitrogen atom, wherein such an atom has a pKa of the corresponding acid of less than 2. In certain embodiments, V is selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-and-S (═ O) -, in particular selected from-NH-C (═ O) -and-C (O ═ NH-, more particularly V is-NH-C (═ O) -.

V is an electron withdrawing moiety selected from the group consisting of-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, -piperazinyl-, -pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,-C (═ O) -, -C (═ O) -O-, wherein R is¹¹Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is H, R¹²Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is methyl.

V is an electron withdrawing moiety selected from the group consisting of-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C (═ O) -O-, wherein R¹¹Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is H, R¹²Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is methyl.

V is an electron withdrawing moiety selected from the group consisting of-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, - (piperazinyl-, -pyridyl-, pyrimidinyl wherein R is¹¹Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is H, R¹²Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is methyl.

V is an electron withdrawing moiety selected from the group consisting of-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-NR¹²-, - (piperazinyl-, -pyridyl-, pyrimidinyl wherein R is¹¹Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹¹Is H, R¹²Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is methyl.

V is an electron withdrawing moiety selected from the group consisting of-NH-C (═ O) -, -C (═ O) -HN-, -N- (CH) -, -h₃) -, -piperazinyl-, -pyridinyl-, pyrimidinyl, pyrazinyl, pyridazinyl.

V is an electron withdrawing moiety selected from the group consisting of-NH-C (═ O) -, -C (═ O) -HN-, -N- (CH) -, -h₃) -, -piperazinyl-, -pyridinyl-, pyrimidinyl.

In certain embodiments, V is an electron withdrawing moiety selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-, -pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C (═ O) -O-.

In certain embodiments, V is selected from-NR¹¹-C(＝O)-、-C(＝O)-NR¹¹-、-S(＝O)-、-NR¹²-and-pyridinyl-, pyrimidinyl, wherein R¹¹Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularlyRespectively R¹¹Is H, and R¹²Selected from H and C_1-4-alkyl, in particular selected from H and C_1-2-alkyl, more particularly R¹²Is methyl.

In certain embodiments, V is selected from-NR¹¹-C(＝O)-、S(＝O)-、-NR¹²-, - (Y-O) -pyridinyl-, pyrimidinyl, wherein R is¹¹Selected from H and C_1-4-alkyl, and R¹²Selected from H and C_1-4-an alkyl group.

In certain embodiments, V is selected from-NR¹¹-C(＝O)-、S(＝O)-、-NR¹²-, - (Y-O) -pyridinyl-, pyrimidinyl, wherein R is¹¹Selected from H and C_1-2-alkyl, and R¹²Selected from H and C_1-2-an alkyl group.

In certain embodiments, V is selected from-NH-C (═ O) -, -N- (CH) -₃) -, -pyridinyl-, pyrimidinyl.

In particular, the linker molecule which releases the peptide via an amine switch requires a basic nitrogen atom. The basic nitrogen atom may be provided via the moiety V, E or U (U ═ heteroaryl). If U is phenyl, a basic nitrogen atom may be provided via the moiety V or E.

In certain embodiments, E and V are selected as described above, wherein where U is phenyl, at least one moiety E or V is selected from

-E: piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N (C)₂H₄)₂NH₂、-N(C₂H₄)₂N-B、-N＝N-R⁸、-(CH₂)_r-NH-C_1-6-alkyl, - (CH)₂)_r-N(C_1-6-alkyl groups)₂-、-N₃、-SO₃H、-CO₂H、-C(＝O)NH₂、-SO₂Me、-SOMe、-SO₂Et, -SOEt, wherein R⁸Selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, - (CH)₂)_p-NMe₂P and B are defined as described herein, and

-V：-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-, -pyridinyl, pyrimidinyl,Pyrazinyl, pyridazinyl, and mixtures thereof,In particular-NR¹²-(CH₂)_p-, -piperazinyl- (CH)₂)_p-, - (Y-O) -pyridinyl-, pyrimidinyl, wherein R is¹²And p is defined as described herein.

As noted above, W is a reducible moiety. Upon W reduction, the reduced intermediate further decays in a pH-dependent manner to release the peptide with a free N-terminus.

In certain embodiments, W is selected from-N₃、-NO₂、-S(＝O)-R⁸、-S-S-R⁸、-O-CH₂-N₃、-O-C(＝O)-O-CH₂-N₃-N ═ N-phenyl, -N ═ N-R⁸、 Wherein the content of the first and second substances,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4. C_1-6Alkyl groups may be linear or branched, for example, butyl or tert-butyl.

In certain embodiments, W is selected from-N₃、-NO₂、-S(＝O)-R⁸、-S-S-R⁸、-O-CH₂-N₃、-O-C(＝O)-O-CH₂-N₃-N ═ N-phenyl, -N ═ N-R⁸、

In certain embodiments, W is selected from-N₃、-S(＝O)-R⁸、-S-S-R⁸、-O-CH₂-N₃、-N＝N-R⁸In particular-N₃、-N＝N-R⁸、-O-CH₂-N₃、-S-S-R⁸Wherein R is⁸Is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C₁-C₆-alkyl or- (CH)₂)_p-NMe₂In particular pyridyl or-C₁-C₆-alkyl, wherein p is 1,2, 3 or 4.

At R⁸Is C_1-6In the case of alkyl, the alkyl moiety may be linear or branched, for example tert-butyl. In particular when W is-S-R⁸In the case of (1), R⁸May be pyridyl or-C₁-C₄-alkyl, in particular pyridyl or tert-butyl.

Comprising the moiety W-S-S-R⁸The linker of (2) can be cleaved with a thiol. Thus, when such linkers are used for peptide purification, cleavage of the peptide-linker complex from the synthetic resin used during SPPS under acidic conditions (e.g., TFA > 50%, pH <0 in the presence of water) can be performed in the absence of a thiol.

In certain embodiments, W is selected from-N₃、-S(＝O)-R⁸、-S-S-R⁸、-O-CH₂-N₃、-N＝N-R⁸In particular-N₃、-N＝N-R⁸、-O-CH₂-N₃、-S-S-R⁸Wherein R is⁸Is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl or-C₁-C₆-alkyl, or, in particular, pyrimidinyl, pyridinyl or-C₁-C₆-alkyl, more particularly pyridyl or-C₁-C₆-an alkyl group.

In certain embodiments, W is selected from-N₃、-S-S-R⁸Wherein R is⁸is-C₁-C₆-alkyl and-NO₂。

Linker molecules that release peptides via amine switches with azido reductive safeties (FIG. 5) or through carbamate switches (FIG. 8) include a reducible moiety-N₃. In certain embodiments, W is-N₃。

Linker molecules that release peptides via amine switches with a reductive security lock without an azide group (FIG. 6) include a reducible moiety, such as-NO₂or-S-S-tert-butyl. In certain embodiments, W is selected from-S-R⁸(wherein R is⁸is-C₁-C₆-alkyl), -NO₂、-N＝N-R⁸(wherein R is⁸Is pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl),

in certain embodiments, W is selected from-S-R⁸(wherein R is⁸is-C₁-C₆-alkyl), -NO₂、-N＝N-R⁸(wherein R is⁸Is pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl), in particular-NO₂or-S-S-R⁸(wherein R is⁸is-C₁-C₆-an alkyl group).

Linker molecules releasing peptides via amine switches with nucleophilic release (FIG. 7) include reducible moieties such as-N₃、-S-S-R⁸Wherein R is⁸Is C_1-6Alkyl, especially tert-butyl, or R⁸Is a pyridyl group.

As described above, the linker molecule may contribute to the solubility of the linker-peptide construct under acidic conditions, particularly during purification of the hydrophobic peptide. Solubility can also be adjusted by a fraction of W, i.e., W is selected from groups that introduce enhanced solubility to the peptide to be purified in the pH range of 0-7.

In certain embodiments, W is selected from-S (═ O) -R⁸、-S-S-R⁸、-N＝N-R⁸、Wherein the content of the first and second substances,

R⁸is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, - (CH)₂)_p-NHBoc or- (CH)₂)_p-NMe₂In particular pyridyl or- (CH)₂)_p-NHBoc-, wherein p is 1,2, 3 or 4.

In certain embodiments, U is selected from phenyl or a five or six membered heterocyclic ring, wherein the five or six membered heterocyclic ring includes 1 or 2 heteroatoms.

In certain embodiments, U is selected from phenyl or a five or six membered heterocyclic ring, wherein the five or six membered heterocyclic ring includes 1 heteroatom.

In certain embodiments, U is selected from phenyl or a six membered heterocyclic ring.

In certain embodiments, U is selected from phenyl or a six membered heterocyclic ring, wherein the six membered heterocyclic ring comprises 1 or 2 heteroatoms.

In certain embodiments, the five-or six-membered heteroaryl moiety of U comprises 1 or 2 heteroatoms, in particular the five-membered heteroaryl moiety of part U is selected from pyrazole, imidazole and the six-membered heteroaryl moiety of part U is selected from pyridine, pyridazine, pyrimidine, pyrazine, in particular pyridine.

In certain embodiments, U is selected from phenyl or a six membered heterocyclic ring, wherein the six membered heterocyclic ring includes 1 heteroatom. In certain embodiments, the five-membered heterocyclic ring of the moiety U is selected from pyrazole, imidazole, and the six-membered heterocyclic ring of the moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine.

In certain embodiments, U is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, especially phenyl or pyridine, more especially phenyl.

The linker according to the present invention may release the peptide through an amine switch (fig. 5 to 7) or a carbamate switch (fig. 8). These release mechanisms require the reducible substituent W to be in the ortho or para position relative to the moiety Y,

in certain embodiments, U is selected from a moiety of formula 5 or 6,

wherein the content of the first and second substances,

t, V, Y, W and E are defined as described above,

u is bound to the moiety T or V,

A¹、A²、A³、A⁴and D¹、D²、D³、D⁴Independently of one another from C, N, S and O, in particular from C and N, and

n is an integer between 0 and 3, in particular 0, 1 or 2.

In certain embodiments, A¹、A²、A³And A⁴Or D¹、D²、D³And D⁴2 to 4 moieties of (A) are C, especially A¹、A²、A³And A⁴Or D¹、D²、D³And D⁴3 or 4 moieties in (a) are C.

In certain embodiments, all moieties A¹、A²、A³And A⁴Or D¹、D²、D³And D⁴Is a compound of formula (I) and (II),

in certain embodiments, U is selected from a moiety of formula 5, 6, 7 or 8,

wherein the content of the first and second substances,

t, V, Y, W and E are defined as described above,

in the case of formulas 5 and 6, U is bound to the moiety T or V,

in the case of formulas 7 and 8, U is bound to the moiety V,

In the case of expressions 5 and 6, an integer between 0 and 3,

in the case of equations 7 and 8, an integer between 0 and 4,

q is an integer between 0 and 4, wherein the sum of n and q is equal to or less than 4.

In certain embodiments, U is selected from a moiety of formula 5 or 6.

In certain embodiments, U is selected from a moiety of formula 5, 6, 7, or 8, wherein,

t, V, Y, W and E are defined as described above,

in the case of formulas 5 and 6, U is bound to the moiety T or V,

in the case of formulas 7 and 8, U is bound to the moiety V,

A¹、A²、A³、A⁴and D¹、D²、D³、D⁴Independently of one another, from C, N, S and O, in particular from C and N,

n is

In the case of formulas 5 and 6, an integer between 0 and 2,

in the case of formulae 7 and 8, integers between 0 and 2, in particular 0 and 1,

q is an integer between 0 and 2, in particular 0 and 1.

In certain embodiments, U is selected from a moiety of formula 9, 10, 11 or 12, particularly a moiety of formula 9 or 10,

wherein the content of the first and second substances,

t, V, Y, W, E, q and n are defined as described above,

in the case of formulas 9 and 10, U is bound to the moiety T or V,

in the case of formulas 11 and 12, U is bonded to the portion V,

all parts A²、A³And A⁴Is C or A²、A³And A⁴Are C and A²、A³And A⁴The other two of (A) are N, in particular A²And A³Are all C, and

D²is C or N, in particular C.

In certain embodiments, U is selected from a moiety of formula 9, 10, 11 or 12, particularly a moiety of formula 9 or 10,

wherein the content of the first and second substances,

t, V, Y, W, E, q and n are defined as described above,

in the case of formulas 9 and 10, U is bound to the moiety T or V,

in the case of formulas 11 and 12, U is bonded to the portion V,

A²and A³Are all C or A²And A³Is C and A²And A³Is N, D²Is C or N.

If the moiety U includes a N-containing heteroaryl group, the N atom is protonated under acidic conditions and thus increases the solubility of the linker molecule.

In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20 or 21, particularly a moiety of formulae 13 to 19, more particularly a moiety of formula 15 or 19,

wherein the content of the first and second substances,

t, V, Y, W, E, q and n are defined as described above,

in the case of formulae 13, 14 and 15, U is bound to the moiety T or V,

in the case of formulae 16, 17 and 18, U is bound to the moiety V,

A²、A³and A⁴Is C, N or N⁺The molar ratio of Me, in particular C,

D²is C or N.

In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22,

wherein T, V, Y, W, E, q and n are defined as described above,

in the case of formulae 13, 14, 15, 19, 20, 21 and 22, U is bound to the moiety T or V,

in the case of formulae 16, 17 and 18, U is bound to the moiety V,

A²、A³and A⁴Is a group of carbon atoms of C or N, in particular C,

D²is C or N. In some embodimentsWherein U is selected from a moiety of formula 13, 14, 15, 16, 17 or 18,

wherein the content of the first and second substances,

t, V, Y, W, E, q and n are defined as described above,

in the case of formulae 13, 14 and 15, U is bound to the moiety T or V,

in the case of formulae 16, 17 and 18, U is bound to the moiety V,

A³is a group of C or N, or a group of N,

D²is C or N.

In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 21, or 22, wherein,

t, V, Y, W, E and n are defined as described above,

u is bound to the moiety T or V,

A³and A⁴Is a group of carbon atoms of C or N, in particular C,

D²is C or N.

In certain embodiments, U is selected from a moiety of formula 13, 21, or 22.

In particular for linker molecules that release the peptide via a carbamate switch, U is phenyl. In certain embodiments, U is selected as described above and all a and all D are C.

For amine switches (fig. 5 and 6) and carbamate switches (fig. 8) with a reductive security lock, part Y is in particular- (CH)₂)_m-O-C (═ O) -to allow release of CO when decay of the linker is triggered by a pH change to release the peptide with free N-terminus₂。

In certain embodiments, U is a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 and Y is- (CH)₂)_m-O-C(＝O)-。

In some instancesIn an embodiment, U is a moiety of formula 13, 14, 15, 21, or 22, and Y is- (CH)₂)_m-O-C(＝O)-。

In certain embodiments, U is a moiety of formula 13 or 21, and Y is- (CH)₂)_m-O-C(＝O)-。

For an amine switch with nucleophilic release (fig. 7), the linker is only stable under acidic conditions if moiety Y forms an amide bond with the peptide. In certain embodiments, Y is- (CH)₂)_m-C(＝O)-。

If the linker molecule ends with-COOH, the peptide can be coupled to the peptide via a common amino acid coupling. In certain embodiments, Y is- (CH)₂)_m-C (═ O) -and Z is-OH.

For amine switches with nucleophilic release, in particular U is a moiety of formula 22 and Y is- (CH)₂)_m-C (═ O) -and Z is-OH.

In certain embodiments, Z is selected from: -F, -Cl, -Br, -I, -N₃、-OH、-O(C＝O)CH₂(C＝O)OH-SR¹⁴、-OCF₃、-OCH₂CF₃、-OSO₂CF₃、-SO₂C₆H₄CH₃、-SO₂CF₃、-SO₂CH₃

In particular-OH, -Cl,In particular-OH,

Wherein R is¹⁴Is C₁-C₆-alkyl-, aryl-or benzyl substituents.

A linker molecule of type 1 suitable for releasing peptides by an amine switch with azido reductive security lock (fig. 5) may be composed of the following moieties:

u is phenyl or pyrimidinyl, in particular U is of formula 13, 14, 15, 21 or 22,

w is-N₃，

N of E is 1 and E is selected from the group consisting of pyridyl, pyrimidinyl, pyridazinyl, -N (CH)₃)₂N-pyridyl or N is 0, in particular N is 0,

a of V is 1 and V is selected from-piperazinyl-, -piperazinyl-CH₂-、-N(CH₃) -, pyrimidinyl, pyridinyl, in particular selected from-piperazinyl-, -piperazinyl-CH₂-、-N(CH₃) -, and

y is- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more in particular 1.

The pyridyl moiety is attached to U at position 3 or 5.

In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 21, or 22,

a linker molecule of type 2 suitable for releasing peptides by an amine switch without an azide group (fig. 6) may be composed of:

u is pyridyl or phenyl, in particular U is of formula 13, 14 or 21,

w is selected from-S-S-tert-butyl, -NO₂-N ═ N-pyridyl,In particular selected from-S-S-tert-butyl, -NO₂，

N of E is 0 and n of E is 0,

a of V is 1 and V is selected from-C (═ O) -NH-and piperazinyl, and

y is- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more in particular 1.

In certain embodiments, U is selected from a moiety of formula 13, 14 or 21,

a linker molecule of type 3 suitable for releasing peptides by an amine switch with nucleophilic release (fig. 7) may be composed of:

u is phenyl or pyridyl, especially phenyl, more especially U is a moiety of formula 13 or 14,

w is-N₃-S-S-tert-butyl, -S-S-pyridyl, especially-N₃，

N of E is 0 and n of E is 0,

a of V is 1 and V is piperazinyl, -NH-, -C (═ O) -NH-, in particular piperazinyl, and

y is- (CH)₂)_m-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more in particular 1.

In certain embodiments, U is selected from moieties of formula 13 or 14, wherein specifically all moieties D and a are C.

A linker molecule of type 4 suitable for releasing peptides by a carbamate switch (fig. 8) may be composed of:

u is phenyl, in particular U is of formula 13 or 15, in which in particular the moiety A is C,

w is-N₃，

N of E is 1 or 2 and E is-Br,

a of V is 1 and V is-NH-C (═ O) -, and

y is- (CH)₂)_m-O-C (═ O) -, where m is 1,2 or 3, in particular 1 or 2, more in particular 1.

In certain embodiments, U is selected from a moiety of formula 13 or 15, wherein in particular moiety a is C,

in certain embodiments, Z is selected from: -F, -Cl, -Br, -I, -N₃、-OH、-SR¹⁴、-OCF₃、-OCH₂CF₃、-OSO₂CF₃、-SO₂C₆H₄CH₃、-SO₂CF₃、-SO₂CH₃

In particular-OH, -Cl,In particular-OH,

Wherein R is¹⁴Is C₁-C₆-alkyl-, aryl-or benzyl substituents.

Where Z is-OH, the-OH moiety is activated by a coupling reagent as is commonly used, for example, in solid phase peptide synthesis, and serves as a leaving group.

In certain embodiments, the compound of formula 1 is selected from compounds of formula X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, or X47.

The second aspect of the invention is directed to a method for purifying a peptide.

According to a second aspect of the invention, a method for purifying a peptide is provided. The method comprises the following steps:

-providing a linker modified crude peptide, wherein the crude peptide is covalently bound to a linker molecule according to the first aspect of the invention,

-coupling the linker modified peptide to a solid support in a coupling step resulting in an immobilized linker modified peptide,

-in a release step, releasing the peptide by adding a reducing agent under acidic conditions.

The linker molecule according to the first aspect of the invention may be used in a method of purifying a peptide. In a first step, the crude peptide mixture is contacted with a linker molecule according to the first aspect of the invention and the crude peptide is coupled to the linker molecule, resulting in a linker modified crude peptide. The crude peptide is coupled to the linker molecule by standard methods generally known to an expert of ordinary skill in the chemical, biochemical or pharmaceutical arts.

The stability of the linker molecule can be addressed by adjusting the pH during purification. Under acidic conditions (especially at pH lower than pKa of the most basic heteroatom of the linker molecule), the linker molecule is stable due to the fact that this heteroatom is protonated and draws out an electron (fig. 5, 6, 7). When the linker is bound to the peptide and immobilized on a solid support, the peptide is released via a reduced intermediate under reducing conditions, e.g., a reducing agent such as triphenylphosphine is added under acidic conditions. The reduced intermediate is characterized by a reduced linker moiety, such as azalide. The linker moiety of the reduced intermediate decays over time or upon triggering (such as increasing the pH, particularly to a pH > pKa of the most basic heteroatom of the linker moiety of the reduced intermediate). Thus, all steps prior to peptide release (coupling, optional washing and reduction of the linker) are performed under acidic conditions. When the pH is increased to a pH above the pKa of the most basic heteroatom of the linker moiety of the reduced intermediate, the reduced intermediate linker moiety decays by a 1.4/1.6 elimination reaction or nucleophilic attack and releases the peptide.

The most basic heteroatoms of the linker molecule/linker moiety of the reduced intermediate relate to the following: for example, part of U is composed of-N₃Substituted pyridines. The heteroatom N of pyridine has a pK of-5_aHigher than-N₃Partially or reduced-N₃Partial pK_a(-NH₃pK of (2)_aAbout 4.6). Thus, the most basic heteroatom is N of the pyridine moiety. By shifting the pH to pH>5, the linker moiety of the reduced intermediate undergoes an elimination reaction and releases the peptide. This mechanism is called amine switching.

Alternatively, the linker molecule may decay through a carbamate switch (fig. 8). Suitable linker molecules include electron-withdrawing moieties (such as-Br) and reducible moieties (e.g. -N)₃). The linker molecule is stable under TFA conditions due to electron withdrawing. Upon reduction, the linker molecule is stable at a pH above the pKa of the carbamate. Finally, by lowering the pH to pH<The pKa of the carbamate, releases the peptide via 1.6 elimination.

In certain embodiments, the pH in all steps prior to addition of the reducing agent is pH < pKa of the most basic heteroatom of the linker moiety.

In certain embodiments, the peptide is released via an intermediate.

In certain embodiments, the peptide is released via a reduced intermediate characterized by a reduced linker moiety of the immobilized linker-modified peptide by addition of a reducing agent under acidic conditions.

In certain embodiments, in the release step, a reduced intermediate of the reduced linker moiety characterized by the immobilized linker modified peptide is achieved and the peptide is released from said reduced intermediate by a trigger, in particular a change in temperature and/or pH, more in particular by increasing the pH to pH > pKa of the most basic heteroatom of the linker moiety of the reduced intermediate in case of an amine switch or by decreasing the pH to pH < pKa of carbamate in case of a carbamate switch.

In certain embodiments, in the releasing step, a reduced intermediate of the reduced linker moiety characterized by the immobilized linker-modified peptide is achieved and the peptide is released from said reduced intermediate by a trigger, in particular a change in temperature and/or pH, more in particular by increasing the pH to pH > pKa of the most basic heteroatom of the linker moiety of the reduced intermediate.

The peptide is released from the intermediate either spontaneously or by triggering.

In certain embodiments, the trigger is a change in temperature and/or pH.

In certain embodiments, the change in temperature is an increase in temperature from ambient temperature (20 ℃ to 30 ℃) to a higher temperature, wherein the higher temperature does not exceed 100 ℃, particularly 70 ℃, more particularly 50 ℃.

As described above, linkers suitable for amine switches decay if they are reduced when the pH is increased to a pH above the pKa of the most basic heteroatom.

In certain embodiments, the trigger is a shift in pH, in particular an increase in pH.

In certain embodiments, the peptide is released from the reduced intermediate by increasing the pH to > the pKa of the most basic heteroatom of the linker moiety of the reduced intermediate.

When the pH is lowered to a pH below the pKa of the carbamate, the linkers suitable for carbamate switches decay if they are reduced.

In certain embodiments, the trigger is a shift in pH, in particular a decrease in pH.

In certain embodiments, the peptide is released from the reduced intermediate by lowering the pH to a pH < pKa of the carbamate of the linker moiety.

If the reduced intermediate is stable under acidic conditions (amine switch) or at a pH > pKa of the carbamate (carbamate switch), an additional washing step may be performed to remove excess reducing agent. Since the reducing agent may react not only with the linker moiety but also with the peptide, unwanted side reactions between the reducing agent and the peptide are reduced by the additional washing step. Furthermore, the non-volatile reducing agent or its use products are also impurities, which would have to be removed by an additional purification step.

In certain embodiments, the reducing agent is removed by washing after formation of the reduced intermediate and before release of the peptide from the reduced intermediate.

In certain embodiments, the reducing agent is removed using MeCN.

In certain embodiments, a linker molecule according to the first aspect of the invention comprises moieties W and/or E which comprise an azido group (-N)₃) And (4) partial.

In certain embodiments, the linker modified peptide is additionally bound to a synthetic resin, and the synthetic resin is cleaved off before the coupling step is performed, in particular at a pH < pKa of the most basic heteroatom of the linker molecule.

Cleavage of the peptide from the synthetic support is achieved by using TFA, which is terminated by precipitation of the peptide from a TFA mixture (e.g. after 2-8 h) to provide a crude peptide mixture. For precipitation, cold ether (Et) can be used₂O、iPr₂O, MeOtBu, THF/hexane (1: 1)).

The crude peptide mixture is dissolved in a suitable organic solvent, in particular DMSO, and a buffer system, in particular 10 volume percent 0.1M sodium citrate buffer at pH 4.5, is added.

The solid support used for the coupling step is an aldehyde-modified solid support, in particular agarose beads, polylysine, polyethylene glycol, polyamide, polystyrene and copolymers thereof, to which the dissolved crude peptide mixture is added.

In certain embodiments, the solid support comprises an aldehyde moiety.

In certain embodiments, after performing the coupling step, the unreacted aldehyde portion of the solid support is blocked using a blocking agent.

In certain embodiments, the blocking agent reacts with the aldehyde moiety of the solid support and includes a thiol and/or amine moiety.

In certain embodiments, the blocking agent is selected from cysteine, threonine, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine.

In certain embodiments, the blocking agent is selected from cysteine and N-methylhydroxylamine.

In particular, the coupled product was washed with DMSO, 6M guanidine hydrochloride (guanidinium hydrochloride), EtOH/water with 0.1M NaCl (7:3), water, MeCN.

In certain embodiments, the releasing step is performed at a pH < pKa of the most basic heteroatom of the linker molecule.

In certain embodiments, the releasing step is performed at a pH < pKa of the carbamate of the linker molecule.

In certain embodiments, the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris (2-carboxyethyl) phosphine, trimethyl phosphite, triethyl phosphite, tributylphosphine, diethyl phosphite, 5' -dithiobis (2-nitrobenzoic acid), sodium dithionite (Na₂S₂O₄) Ethanedithiol, propanedithiol, dithioerythritol, dithiothreitol, Na₂S, NaSH glutathione, 2' -dithiodipyridine, BH₃4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane, catechol borane, borane tetrahydrofuran, borane dimethyl sulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH₄、LiBH₄、NaBH₄、NaBH₃CN、NaBH(OMe)₃、NaBH(OCCH₃)₃、LiAlH(OCMe₃)₃Hydroquinone, sodium ascorbate, ascorbic acid with KI, hydrazine, NH ═ NH, formaldehyde.

In certain embodiments, the reducing agent is selected from dithioerythritol, dithiothreitol, triphenylphosphine, ascorbic acid with KI, tributylphosphine, trimethylphosphine, tris (2-carboxyethyl) phosphine, sodium dithionite (Na)₂S₂O₄) Borane dimethylsulfide, borane triphenylphosphine complexes, NaBH₄Ascorbic acid

In certain embodiments, the reducing agent is selected from dithioerythritol, dithiothreitol, ascorbic acid with KI, triphenylphosphine, and trimethylphosphine.

In certain embodiments, the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris (2-carboxyethyl) phosphine, trimethyl phosphite, triethyl phosphite, tributylphosphine, diethyl phosphite, 5' -dithiobis (2-nitrobenzoic acid), sodium dithionite (Na₂S₂O₄) Ethanedithiol, propanedithiol, dithiothreitol and Na₂S, NaSH glutathione, 2' -dithiodipyridine, BH₃4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane, catechol borane, borane tetrahydrofuran, borane dimethyl sulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH₄、LiBH₄、NaBH₄、NaBH₃CN、NaBH(OMe)₃、NaBH(OCCH₃)₃、LiAlH(OCMe₃)₃Hydroquinone, sodium ascorbate, ascorbic acid, hydrazine, NH ═ NH, formaldehyde.

In certain embodiments, the reducing agent is selected from triphenylphosphine, tributylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, sodium dithionite (Na)₂S₂O₄) Borane dimethylsulfide, borane triphenylphosphine complexes, NaBH₄And ascorbic acid.

In certain embodiments, the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine, or tris (2-carboxyethyl) phosphine, sodium dithionite (Na)₂S₂O₄) Borane dimethylsulfide, borane triphenylphosphine complexes, NaBH₄And ascorbic acid.

In certain embodiments, the reducing agent is selected from triphenylphosphine, sodium dithionite (Na)₂S₂O₄) Borane dimethylsulfide, borane triphenylphosphine complexes, NaBH₄And ascorbic acid.

In certain embodiments, the reducing agent is selected from triphenylphosphine and trimethylphosphine.

In certain embodiments, the reducing agent is triphenylphosphine.

In certain embodiments, the method comprises the steps of:

-providing a linker modified crude peptide, wherein the crude peptide is covalently bound to a linker molecule according to the first aspect of the invention, wherein the linker molecule comprises moieties W and/or E comprising an azido moiety,

-coupling the linker modified peptide to a solid support in a coupling step resulting in an immobilized linker modified peptide,

-in a release step, releasing the peptide by adding a reducing agent under acidic conditions.

In certain embodiments, the method comprises the steps of:

-coupling the linker modified peptide to a solid support in a coupling step resulting in an immobilized linker modified peptide,

-in a release step, releasing the peptide by adding a reducing agent under acidic conditions to produce a reduced intermediate, followed by increasing the pH to a pH > pKa of the most basic heteroatom of the linker moiety of the reduced intermediate.

In certain embodiments, the method comprises the steps of:

-coupling the linker modified peptide to a solid support in a coupling step resulting in an immobilized linker modified peptide,

-in a release step, releasing the peptide by adding triphenylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, in particular triphenylphosphine, under acidic conditions to produce a reduced intermediate, followed by increasing the pH to a pH > pKa of the most basic heteroatom of the linker moiety of the reduced intermediate.

According to a subsidiary aspect of the second aspect of the invention, there is provided a method for purifying a crude peptide prepared by solid phase peptide synthesis.

In certain embodiments, the method for purifying a peptide comprises the steps of:

a) providing a peptide bound to a synthetic resin, wherein the peptide is further covalently bound to a linker molecule according to claim 1, wherein the linker molecule comprises moieties W and/or E comprising an azido moiety,

b) cleaving the peptide from the synthetic resin

c) Coupling of the cleaved peptide mixture to a solid support

d) The peptides are released with triphenylphosphine or trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, in particular triphenylphosphine.

The crude peptide mixture is dissolved in a suitable organic solvent, in particular DMSO, and a buffer system, in particular 10 volume percent 0.1M sodium citrate buffer at pH 4.5, is added.

In particular, the coupling product was washed with DMSO, 6M guanidine hydrochloride, EtOH/water with 0.1M NaCl (7:3), water, MeCN.

In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, in particular triphenylphosphine, is added in MeCN/AcOH (9: 1). In certain embodiments, the addition is performed for 15 min.

In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine, or tris (2-carboxyethyl) phosphine is added at MeCN/AcOH/H₂O (90:5:5), and/or wherein after addition of triphenylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, in particular triphenylphosphine, in particular MeCN or MeCN/H₂O (9:1) washes away the formed azaylide.

In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, in particular triphenylphosphine, is added in MeCN/AcOH/H₂O (90:5: 5). In certain embodiments, the addition is performed for 15 min.

In certain embodiments, after addition of triphenylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, use is made in particular of MeCN or MeCN/H₂O (9:1) washes the azaylide formed.

In certain embodiments, after addition of triphenylphosphine, trimethylphosphine, triethylphosphine or tris (2-carboxyethyl) phosphine, the azaylide formed is washed, in particular with MeCN.

In certain embodiments, H is especially useful₂O/TFA hydrolyzes the azaylide formed. The basis weight may be 99.95% water to 50% water.

When the linker, particularly U, E, W or V, is a nitrogen-containing heterocycle, the pH should be above the pKa of the heterocyclic moiety, as may already be the case in TFA-water hydrolysis mixtures or by addition of buffer solutions having the desired pH.

In certain embodiments, the hydrolysis is followed by an elution step, particularly with TFA/H₂O, more particularly in a ratio of 9: 1.

In certain embodiments, the hydrolysis is followed by an elution step, particularly with TFA/H₂O, more particularly in a ratio of 95: 5.

In certain embodiments, the hydrolysate is precipitated, in particular by addition of a cold ether, more particularly Et₂O、iPr₂O, MeOtBu, THF/hexane (1: 1).

Terms and definitions

In the context of the present invention, an "electron withdrawing group" or "EWG" is any chemical group capable of withdrawing an electron from the atom or aryl system to which it is attached by an inductive or mediated effect.

In the context of the present invention, Hammett's constants are constants as calculated and described in Hansch and Taft (1991), chem.Rev.91: 165-. A positive hammett constant reflects the ability of a substituent to exert an electron withdrawing effect on a phenyl moiety, while a negative value indicates that the substituent exerts an electron withdrawing effect. The stronger the electron-withdrawing action, the larger the Hammett constant. Hammett's constant is the phenyl moiety in the meta position (. sigma.)_m) And alignment (σ)_p) Is a constant determined empirically. In the context of the present invention, the position is determined relative to the bound moiety Y. The Hammett value of the para position is a good approximation for the ortho position substituent and is therefore used in the context of the present invention to calculate the sum of the Hammett values of substituents V, W and E.

In the context of the present invention, the term "under acidic conditions" relates to a pH below pH 7, in particular a pH below the pKa of the linker, more in particular TFA > 50%, in the presence of water, pH < 0.

In the context of the present invention, the term alkyl refers to a straight or branched chain saturated hydrocarbon. For example, the term C_1-12Alkyl represents a saturated, straight-chain or branched hydrocarbon having 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. C₁-C₄Non-limiting examples of alkyl groups are methyl, ethyl, propyl, n-butyl, 2-methylpropyl and tert-butyl.

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Traceless reductively cleavable linker molecules for peptide purification

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