DE10116252A1 - New 2-aryl-2-triorganylsilylether compounds useful for the solid phase synthesis of peptides and peptide conjugates, may be cleaved from the solid phase substrate under practically neutral conditions - Google Patents

Abstract

2-Aryl-2-triorganylsilylether compounds (I) are new. 2-Aryl-2-triorganylsilylether compounds of formula (I) are new; R<1>-R<3> = aryl, alkyl or a benzoid, condensed or heterocyclic aromatic group; X = substituent containing heteroatoms such as N or O and/or functional groups such as carbonyl or carboxyl, and optionally bonded to a polymeric organic or inorganic support; Y = anchoring compound, containing protecting, deblockable or free functional groups at which solid phase synthesis can be carried out. An Independent claim is also included for the preparation of (I).

Description

The invention relates to anchor compounds of the general formula I,

in which R ¹ , R ² and R ^{3 are,} independently of one another, aryl or alkyl groups, preferably methyl groups, aryl is a benzoid or condensed or heterocyclic aromatic radical and X is a substituent attached to them in the p, m or o position, which also contain heteroatoms such as O and N and / or functional groups such as carbonyl or carboxyl groups and can be or can be attached to a polymeric organic or inorganic support; Y is the anchored compound that contains protected, deblockable functional groups on which solid phase syntheses can be performed. As a rule, Y is an acyl group or an aminoacyl group. However, it can also be an aryl or functionalized alkyl group.

The type I structures serve as reversibly releasable anchors for the connection of Substrate molecules on the polymeric carrier. Such anchors are of particular importance in the Solid phase synthesis of peptides and glycopeptides.

Therefore, the advantageous properties of this new anchor should be shown using the example of the 2-phenyl-2-trimethylsilylethyl anchor (PTMSEL, formula I, aryl = phenyl, R ¹ , R ² , R ³ = methyl) in solid-phase syntheses of peptides and glycopeptides.

The solid phase synthesis is for the versatile and fast synthesis of peptides and Peptide conjugates of particular importance. This applies in particular to the combinatorial Synthesis of compound libraries and groups of individual compounds. This combinatorial synthesis principle is meanwhile on the production of a variety of organic substance classes have been expanded (Combinatorial Chemistry, ed. G. Jung, Wiley-VCH, Weinheim, 1999).  

An important structural element for solid phase synthesis of peptides, peptide conjugates and The anchor forms combinatorial syntheses on a solid phase, often in English literature Called left. It must be stable during the generally multi-stage syntheses on the solid phase be, but ultimately under the mildest possible conditions and without affecting the have the manufactured products split. The most common are acid-labile anchors (see e.g. G.B. Fields, R. Noble, Int. J. Pept. Protein Res. 1990, 35, 161), e.g. B. of the p-alkyloxy benzyl ester type (S.S. Wang, J. Am. Chem. Soc. 1973, 95, 1328). When they are split, there are usually others acid-labile protective groups, for example from the amino acid side chains of those on the solid phase built peptides split off simultaneously, so that the products are not in subsequent Fragment condensations (G. Benz, Synthesis 1993, 37) can be used again. at acidic and basic cleavage reactions also occur undesirable side reactions on peptides, among which the aspartimide formation and transacylation on aspartyl peptides often is particularly difficult to avoid (M. Bodanszky, J. Martinez, Synthesis 1981, 333). Avoid most of these side reactions occur when neutrally cleavable anchors, such as the allylic Anker (O. Seitz, H. Kunz, Angew. Chem. Int. Ed. Engl. 1995, 34, 803) can be used. Because of However, the allyl anchor esters are less steric than aminolysis susceptible. Accordingly, they also tend to detach diketopiperazine on the resin-bound Dipeptide level, if you consider the base-labile Fmoc group as an amino protective group at this level used.

The 2- (alkoxyphenyl) -2- corresponding to the compounds of the general formula I trimethylsilyl-ethyl (PTMSEL) anchor is a new type of anchor based on the 2-phenyl-2- trimethylsilyl-ethyl (PTMSE) ester protecting group.

The new PTMSEL anchor has the following advantages:

• a) It can be easily obtained by adding aryl cuprate to trimethylsilyl-oxirane 2 (Example 1).
• b) The attachment of the anchored substrate, in particular of N-protected amino acids functionalized carriers, such as aminomethyl polystyrene (AMPS) or amino-functionalized Tentagel resin, can be achieved with good yields (Example 2).
• c) The cleavage of the PTMSEL anchor and thus the release of the compounds synthesized on the solid phase already succeeds through the action of tetrabutylammonium fluoride trihydrate (TBAF) in dichloromethane, ie under practically neutral conditions according to the mechanism outlined in Scheme 1.
  

  Scheme 1

  Fluoride-induced cleavage of the PTMSEL linker

  In contrast to other silyl anchors (R. Ramage et al., Tetrahedron Lett. 1987, 28, 4105; G. Barany et al., J. Org. Chem. 1988, 53, 5240; M. Bernatowicz et al., J. Am. Chem. Soc. 1994, 116, 1746), which have to be cleaved with TBAF in dimethylformamide, N-methyl-pyrrolidone or also tetrahydrofuran, can, under the mild conditions of cleavage of the PTMSEL anchor, cause undesirable side reactions, such as formation of aspartimides and rearranged peptides in peptides containing aspartic acid are largely suppressed (Example 3).
• d) Under the mild conditions of cleavage of the PTMSEL anchor, most of those in the Peptide chemistry usual protective groups and base-sensitive glycosidic bonds and protecting groups are stable in glycopeptide syntheses (Example 4). So protected too Peptides and glycopeptides are detached from the solid phase, which are directly in Fragment condensations can be used (Examples 4 and 5).
• e) The PTMSEL anchor is sterically so demanding that the Fmoc group is split off no loss of polymer-bound peptide is recorded (Examples 3-5). Also the intramolecular aminolysis to diketopiperazine, which is at the polymer-bound stage Dipeptides are often responsible for losses, especially when the Fmoc group is split off is largely suppressed (Example 5).

The above advantages are demonstrated in the individual examples below and in the associated experimental parts performed.

example 1 Synthesis of the PTMSEL anchor

The synthesis of the anchor is carried out in three stages from trimethylvinylsilane 1, which with m-chloroperbenzoic acid is epoxidized to trimethylsilyloxirane 2. Subsequent reaction with  Lithium di (p-ethoxyethyloxyphenyl) cuprate, which is obtained in situ by lithiation of 1- (4- Bromophenoxy) -1-ethoxyethane with n-butyllithium and reaction of the lithiated compound with Copper (I) iodide is obtained, provides (2-p-ethoxyethyloxyphenyl-2-trimethylsilyl) ethanol 3 in 55% yield. The ethoxethyl protective group is cat. Amounts of p-toluenesulfonic acid Cleaved pyridinium salt in MeOH. The PTMSEL linker, (2-p-hydroxyphenyl-2- trimethylsilyl) ethanol 4, is obtained in 90% yield (50% based on 2- Trimethylsilyloxirane 2).

Scheme 2

Synthesis of the anchor

Example 2 The connection of the anchor loaded with an N-protected amino acid polymeric carriers

The binding to the polymer takes place via the phenolic hydroxyl function. The N-acyl Amino acid is coupled to the anchor in solution. Esterification on a solid phase is often with problems of racemization of the start amino acid to be bound.

(2-p-Hydroxyphenyl-2-trimethylsilyl) ethanol 4 is reacted with allyl chloroacetate in acetone in the presence of K ₂ CO ₃ and potassium iodide to give 4- [2-hydroxy-1- (trimethylsilyl) ethyl] phenoxyacetate 5. Subsequent esterification with the Fmoc-protected amino acid according to Steglich (dicyclohexylcarbodiimide, cat. Amounts of DMAP) provides compound 6, which is protected as an allyl ester (yields: <90%). The allyl ester in 6 is quantitatively determined by allyl transfer using Pd (PPh ₃ ) ₄ and p-toluenesulfinic acid sodium salt (I. Nagakura et al., J. Org. Chem. 1997, 62, 8932) or another allyl scavenger (M. Ciommer, H. Kunz, Synlett 1991, 593) cleaved and gives the anchorable carboxylic acid 7 (yields ≈35% over 5 steps starting from 2-trimethylsilyloxirane 2), which are now using coupling reagents, such as. B. TBTU / HOBt / DIPEA (R. Knorr et al., Tetrahedron Lett. 1989, 30, 1927) in a mixture of DMF / CH ₂ Cl ₂ , can be bound to an amino-functionalized polymer resin.

Scheme 3

Connection of the anchor loaded with an Fmoc amino acid to the polymer

Amino-functionalized polystyrene resin, AMPS (ACT; 200-400 mesh; loading: 1.00 mmol / g), or amino-functionalized tentagel resin (W. Rapp et al. In "Innovation & Perspectives in Solid Phase Synthesis, 1 ^st International Symposium ", Epton (Eds), SPCC UK Ltd., Birmingham, 1990, 205), Nova Syn Tg amino resin (Novabiochem, 110 µm beads, loading: 0.43 mmol / g).

Unreacted amino groups of the polymeric carrier are reacted with a mixture acetylated from acetic anhydride / pyridine (1: 3).

Example 3 Solid phase synthesis of a heptapeptide containing aspartic acid on PTMSEL anchor

With the help of the new anchor, a protected heptapeptide is synthesized on the solid phase the side reaction of aspartimide formation which often occurs in peptide synthesis and -examine rearrangement. Aspartimide formations are found both under acidic and under basic conditions observed. Most of the silyl anchors described have that Disadvantage that the conditions used for cleavage (TBAF in DMF or THF) are so basic are that aspartimide formation cannot be suppressed, even in most cases leads to an undesirable main product.

The heptapeptide Boc-Leu-Asp (OtBu) -Ala-Asn (Trt) -Gly-Ile-Ile-OH is automated in a Synthesis made on the PTMSEL anchor.

The Fmoc cleavages are carried out in four to five 4-minute cycles with piperidine (30% piperidine in NMP). The couplings are carried out with 10 equivalents of Fmoc or Boc amino acids and coupling reagents (HBTU, HOBt, DIPEA). After each coupling, a capping step is carried out with a mixture of acetic anhydride / DIPEA / HOBt in NMP. After synthesis is complete, the synthesizer is washed extensively with NMP and then with CH ₂ Cl ₂ . This is important because there is no NMP or DMF present in the fluoride-induced cleavage. TBAF.3H ₂ O is strongly basic in polar, aprotic solvents such as DMF, NMP or THF, but only very weakly basic (almost neutral) in CH ₂ Cl ₂ . The fluoride-induced cleavage from the polymeric carrier is carried out manually. For this, the resin is mixed with a solution of 2 equiv. TBAF.3H ₂ O (0.2 mmol) in CH ₂ Cl ₂ , shaken for approx. 25 minutes, filtered and the organic solution washed with water to remove any fluoride still present. The splitting off process is carried out again with approx. 0.7 equiv. TBAF.3H ₂ O (0.07 mmol, approx. 2 equiv. Based on the product still remaining on the resin) is repeated. Details and processing are described in the experimental part.

Scheme 4

Synthesis of a protected heptapeptide containing aspartic acid

The CH ₂ Cl ₂ solutions obtained from the two cleavages are worked up separately. Both fractions show the same composition in the HPLC spectra. Such an HPLC spectrum is shown in Fig. 1. The individual peaks are identified by HPLC-MS analysis.

Fig. 1

HPLC chromatogram of the crude product 9

As can be seen from the HPLC-ESI spectra ( Fig. 1), the aspartimide formation can be reduced to approx. 3%. After purification, the desired, fully protected heptapeptide is obtained in 90% yield.

It turns out that the separation conditions used are so mild that even otherwise under basic conditions, especially in the case of an Asp-Gly sequence Aspartimide formation is observed to a very limited extent.  

Example 4 Solid phase synthesis of a glycodecapeptide on the PTMSEL anchor

According to the principle described in the previous example, a glycodecapeptide synthesized to the stability of base-labile O-glycopeptides or of base-labile Hydroxy protective groups (acetyl) under the elimination conditions used investigate.

The glycodecapeptide Boc-Leu-Ala-Ala-Leu-Asp (OtBu) -Ser (αAc ₃ GalNAc) -Gln (Trt) -Gly-Ala-Ile-OH is synthesized in a peptide synthesizer like the heptapeptide. However, the glycosylated building block Fmoc-Ser (αAc ₃ GalNAc) -OH is coupled manually. Only 3 equiv. used, but the more reactive coupling mixture HATU / HOAt (LA Carpino et al., J. Chem. Soc. Chem. Commun. 1994, 201) is used for this.

Scheme 5

Solid phase synthesis of a glycodecapeptide on the PTMSEL anchor

Splitting off and working up are again carried out analogously to the procedure for the synthesis of the heptapeptide. Again, both cleavage fractions show the same chemical composition in HPLC. Such an HPLC chromatogram of the crude product is shown in Fig. 2. The individual peaks were identified by HPLC-MS analysis.

Fig. 2

HPLC chromatogram of the crude product 11

Again the main peak (92%) corresponds to the desired product. As a by-product can peptide 12 (2%) containing aspartimide and also various deletion sequences (together about 4%) can be identified.

However, no by-products are observed that are caused by a β-elimination on the glycosylated serine fragment or by splitting off individual acetyl protective groups are. The by-products described can be separated off by preparative HPLC. The glycodecapeptide is isolated in 66% yield based on the initial loading of the resin (Polymer 8a).  

Example 5 Synthesis and cleavage of an Fmoc peptide with the diketopiperazine Prolyl-glycine-forming sequence

Another frequently observed side reaction in peptide solid phase synthesis is Diketopiperazine. When using sterically not demanding anchor systems it can be at the dipeptide level after the Fmoc protecting group has been split off intramolecular aminolysis of the ester bond by the N-terminal released amino function come. Such diketopiperazine formations are particularly common in sequences such as -Pro-Pro-O- Anchor, -Pro-Gly-O-anchor or -Tyr-Pro-O-anchor observed. On the other hand could show be that through sterically demanding linkers such. B. the trityl linker, the Diketopiperazine formation can be suppressed.

In the following it is shown on the synthesis of the tripeptide Fmoc-Phe-Pro-Gly-OH that at PTMSEL anchor prevents such diketopiperazine formation. This is done by the over the PTMSEL anchor to polymer 8b bound Fmoc-glycine and the synthesis done manually. It is coupled with only 6.5 equiv. on coupling reagents.

Scheme 6

Synthesis of a tripeptide to investigate possible diketopiperazine formation

After the second Fmoc cleavage, which was carried out with a 20% solution of piperidine in DMF by shaking for 25 minutes (normally less than 10 minutes are sufficient), the solution obtained is examined for diketopiperazine (→ thin layer chromatography, HPLC-MS-coupling). However, no diketopiperazine can be detected. Moreover a photometric load determination is carried out after each coupled amino acid carried out in order to make a statement about possible difficulties with the Obtain amino acid couplings (Table 1).

Table 1

Load determination at the level of each coupled amino acid

The loading determinations also show no formation of diketopiperazine. After the synthesis of the tripeptide, the fluoride-induced cleavage from the polymeric carrier was carried out in the usual way. Fig. 3 shows the HPLC spectrum of the crude product obtained.

Fig. 3

HPLC chromatogram of the Fmoc tripeptide 13

Subsequent column chromatographic purification on silica gel provides the desired N- terminal Fmoc-protected tripeptide in 77% yield. Taking that into account Amount of resin taken from the load determination, a yield of approx. 90% based on the initial loading of polymer 8b.

In summary, it can be stated that the PTMSEL linker according to the invention in excellent way for the solid phase synthesis of protected peptides and glycopeptides according to the Fmoc strategy. The products can be created using the new linker system produce in high yield. The cleavage from the polymeric carrier is simple, without absolute solvents or expensive chemicals and usually delivers the desired ones Peptides in a very pure form. Most of the protecting groups used in peptide chemistry (Fmoc, Boc, Z, Aloc, tert. Butyl ester, benzyl ester, allyl ester, trityl, acetyl, etc.) are among the used, mild conditions of cleavage of the PTMSEL anchor stable, so that protected Peptides of all kinds (three-dimensional protecting group strategies for N-terminus or Side chain functionalities are conceivable) can be synthesized. The used Detachment conditions are so mild (almost neutral) that even basic ones Conditions of easy aspartimide formation can be largely suppressed and no side reactions were observed even with the base-labile O-glycopeptides. Is too the new linker sterically so demanding that no diketopiperazine formation at the level of second amino acid bound to the polymer can be observed.

A solid phase synthesis according to the Boc strategy is not possible, however, because the PTMSEL Linker is cleaved by the action of TFA (to the C-terminal free acid). This instability  was already under acidolytic conditions when the PTMSE protective group was introduced (M. Wagner, H. Kunz, Synlett 2000, 400).

Experimental part 2-trimethylsilyloxirane 2

In a three-necked flask, 24.51 g (0.1008 mmol) of m-chloroperbenzoic acid in approx. 235 ml of chloroform are cooled to 0 ° C. using an ice bath. 15.5 ml (0.1008 mmol) of trimethylvinylsilane in 50 ml of chloroform are added dropwise via a dropping funnel. After stirring for 60 hours at room temperature. If the precipitated colorless precipitate is filtered off and the filtrate is saturated with 200 ml. NaHCO ₃ solution. shaken. After washing with 200 ml sat. NaHSO ₃ solution. and sat with 200 ml. NaHCO ₃ solution. the organic phase is dried over MgSO ₄ . The chloroform is distilled off. The residue is i. vak. (370 mbar) distilled.
Yield: 7.50 g (64%); colorless liquid; Bp _{370 mbar} : 75-79 ° C (lit bp: 109-110 ° C); n _D ²⁵ = 1.414 (Lit .: CT Dong, Chem. Abstr. 1959, 54, 471; n _D ²⁵ = 1.414).
200 MHz ¹ H NMR (CDCl ₃ ): δ (ppm): 2.88 (t, 1H, β-CH, J = 5.6 Hz); 2.50-2.56 (m, 1H, β-CH); 2.17 (t, 1H, α-CH, J = 4.6 Hz); 0:04 (s, 9H, Si (CH _{3) 3).}

(2-p-ethoxyethyloxyphenyl-2-trimethylsilyl) ethanol 3 1. Lithiation

To 33.09 g (135.0 mmol) of 1- (4-bromophenoxy) -1-ethoxyethane in 150 ml of absolute. Diethyl ether under Argon are 84.37 ml (135.0 mmol) of an n-butyllithium solution at -45 ° C. (1.6 M solution in hexane) added dropwise in the course of 30 minutes. The mixture is then stirred at -40 ° C for 20 minutes. Then lets the mixture is brought to + 10 ° C. within 45 minutes and stirred for a further 90 minutes.

2. Cuprate formation and cuprate addition

12.29 g (65.5 mmol) Cu (I) J are made absolute in 40 ml. Diethyl ether suspended. Now the solution of the lithiated compound described above is cannulated into a 500 ml dropping funnel. At 0 ° C., the solution of the lithiated compound is added dropwise to the Cu iodide suspension within 70 minutes. The mixture is stirred for 30 minutes, cooled to -50 ° C. and 2.7 g (23.2 mmol) of 2-trimethylsilyloxirane are added dropwise at -50 ° C. using a syringe. The mixture is stirred for 5 hours at this temperature. Then you replace the acetone / dry ice cooling with an ice-salt cooling (ratio 3: 1 → -19 ° C) and stir for 14 hours at -19 ° C. 200 ml of a sat. NH ₄ Cl solution. and stirred for 10 minutes at 0 ° C. The mixture is placed in a separatory funnel, with 300 ml of ethyl acetate and a further 200 ml of a sat. NH ₄ Cl solution. offset and shaken vigorously. The organic phase is saturated twice with 250 ml. NH ₄ Cl solution. washed. The aq. Phases are shaken out with 300 ml of ethyl acetate. The combined organic phases are dried over MgSO ₄ and i. Vak. freed from the solvent. The residue (approx. 30 g) is purified by chromatography on 650 g of silica gel in the eluent PE / EE 7: 1 (addition of 0.1% triethylamine). The product fractions are mixed with toluene and concentrated. The product is co-distilled four times with toluene to remove residues of the triethylamine.
Yield: 3.6 g (55%); brown oil; R _f = 0.23 (PE / EE 6: 1); [α] _D ²² = 0 ° (c = 1.00, acetone).
200 MHz ¹ H NMR (acetone-d ⁶ ): δ (ppm): 7.03 (d, 2H, 2 aromatic H, J = 8.79 Hz); 6.89 (d, 2H, 2 aromatic H, J = 8.30 Hz); 5.35 (q, 1H, O-CH-O, J = 5.21 Hz); 3.92-4.07 (m, 2H, CH ₂ -OH); 3.65-3.77 (m, 1H, OH); 3:45 to 3:58 (m, 2H, CH ₃ -CH ₂ -O); 2.31-2.38 (dd, 1H, TMSCH, J ₁ = 3.41 Hz, J ₂ = 5.37 Hz); 1:39 (d, 3H, CH-CH _3, J = 5:37 Hz); 1.13 (t, 3H, CH ₂ -CH ₃ , J = 6.84 Hz); -0.04 (s, 9H, Si (CH _{3) 3).}
50.3 MHz ¹³ C NMR (acetone-d ⁶ ; BB, DEPT): δ (ppm): 155.42 (1C, C _ipsoAr-O ); 136.51 (1C, C _{ipso Ar-CH} ); 129.48 (2C, 2 aromatic C); 118.02 (2C, 2 aroma C); 100.37 (1C, O-CH-O); 63.60 (1C, CH ₂ - O); 61.79 (1C, CH ₃ -CH ₂ -O); 41.00 (1C, Si (CH _{3) 3} -CH); 20.71 (1C, CH ₃ -CH); 15:52 (1C, CH ₃ - CH _2); -2.16 (3C, Si (CH ₃ ) ₃ ).

(2-p-hydroxyphenyl-2-trimethylsilyl) ethanol 4

A solution of 68.1 mg PPTS (0.27 mmol, 0.03 equiv.) In 3 ml methanol is added to 2.55 g (9.03 mmol) (2-p-ethoxyethyloxyphenyl-2-trimethylsilyl) ethanol 3 in 40 ml methanol (pa) under an argon atmosphere. The mixture is stirred at room temperature for 2 hours. When the reaction is complete (TLC control), the solvent is i. Vak. away. The residue is purified by chromatography on 140 g of silica gel in a PE / EE 4: 1 eluent mixture.
Yield: 1.70 g (90%); colorless, crystalline solid; Mp 138-141 ° C, R _f = 0.18 (PE / EE 3: 1); [α] _D ²² = 0 ° (c = 1.00 g / 100 ml, acetone).
FD-MS (m / z) = 210.4 [M] ⁺ .
200 MHz ¹ H NMR (acetone-d ⁶ ): δ (ppm): 7.94 (s _b , 1H, OH phenol); 6.95 (d, 2H, 2 aromatic H, J = 8.79 Hz); 6.72 (d, 2H, 2 aromatic H, J = 8.79 Hz); 3.86-4.07 (m, 2H, CH ₂ -OH); 3.37 (t, 1H, OH, J = 5.37 Hz); 2.28 (dd, 1H, TMS-CH, J ₁ = 3.42 Hz, J ₂ = 8.79 Hz); -0.05 (s, 9H, Si (CH _{3) 3).}
50.3 MHz ¹³ C NMR (acetone-d ⁶ ; BB, DEPT): δ (ppm): 155.53 (1C, C _ipsoAr-OH ); 133.74 (1C, C _{ipso Ar-CH} ); 129.60 (2C, 2 arom. C); 115.80 (2C, 2 aroma C); 63.85 (1C, CH ₂ -O); 41.00 (1C, TMS-CH); 20.71 (1C, CH ₃ -CH); -2.11 (3C, Si (CH ₃ ) ₃ ).

4- [2-Hydroxy-1- (trimethylsilyl) ethyl] phenoxyacetic acid allyl ester 5

To 1.55 g (7.37 mmol) of [2- (p-hydroxyphenyl) -2-trimethylsilyl] ethanol 4 in 40 ml of acetone, 1,325 g (9.59 mmol) of K ₂ CO ₃ , 160 mg (0.96 mmol) of KI and 1,027 are added in succession under argon ml (8.85 mmol, d = 1,159 g / ml) added chloroacetate and at room temperature. touched. After 18 h, the conversion is ≈60% (DC control PE / EE 3: 1). A further 0.795 g (5.75 mmol) K ₂ CO ₃ , 96 mg (0.5B mmol) KI and 0.617 ml (5.32 mmol) allyl chloroacetate are therefore added, and the mixture is stirred for a further 20 h. It is filtered through Hyflo-Supercel® and washed with approx. 150 ml acetone. The solvent is i. Vak. away. The oily brown residue is purified on 180 g of silica gel in the eluent PE / EE 5: 1. A colorless, slightly yellowish oil is obtained, which crystallizes after drying in a high vacuum and storage at 4 ° C.
Yield: 1.76 g (78%); colorless, crystalline solid; Mp 27-28 ° C; R _f = 0.29 (PE / EE 3: 1).
FD-MS (m / z) = 308.4 [M] ⁺ .
200 MHz ¹ H NMR (CDCl ₃ ): δ (ppm): 7.01 (d, 2H, 2 aromatic H, J = 8.31 Hz); 6.83 (d, 2H, 2 aromatic H, J = 8.79 Hz); 5.81-6.00 (m, 1H, CH = _CH2); 5.31 (dd, 1H, CH = CH _A H _B , J ₁ = 16.32 Hz, J ₂ = 1.47 Hz); 5.24 (dd, 1H, CH = CH _A H _B , J ₁ = 10.52 Hz, J ₂ = 1.46 Hz); 4.69 (d, 2H, CH ₂ -CH = CH _2, J = 5:45 Hz); 4.61 (s, 2H, O-CH ₂ -C = O); 3.89-4.12 (m, 2H, CH-CH ₂ -OH); 2.37 (dd, 1H, TMSCH, J ₁ = 4.39 Hz, J ₂ = 11.23 Hz); 1.50 (s _b , 1H, OH); -0.06 (s, 9H, Si (CH _{3) 3).}
50.3 MHz ¹³ C NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 168.80 (1C, C = O ester); 155.74 (1C, C _ipsoAr-O ); 133.61 (1C, C _ipsoAr-CH ); 131.47 (1C, CH = CH _2); 128.82 (2C, 2 arom. C); 119.02 (1C, CH = CH _2); 115.03 (2C, 2 aroma C); 65.79, 65.61 (2C, CH ₂ = CH-CH ₂ -O, O = C-CH ₂ -O); 63.22 (1C, TMS-CH-CH ₂ -O); 40.81 (1C, TMS-CH); -2.11 (3C, Si (CH ₃ ) ₃ ).

4- [2- (N-fluorenylmethoxycarbonyl-L-isoleucyloxy) -1- (trimethylsilyl) ethyl] -phenoxy allyl acetate 6a

To 1,048 g (2.97 mmol) Fmoc-Ile-OH in 20 ml absolute. CH ₂ Cl ₂ , 0.832 g (2.70 mmol) of 4- [2-hydroxy-1- (trimethylsilyl) ethyl] phenoxyacetic acid allyl ester are added. The mixture is stirred under argon and cooled to about 0 ° C. Then 20 mg DMAP (0.16 mmol) and 0.673 g DCC (3.26 mmol) are added. The mixture is stirred at 0 ° C. under argon for 3 h. It is filtered through Hyflo-Supercel® and washed with approx. 70 ml CH ₂ Cl ₂ . The filtrate is washed twice with 100 ml of a 5% NaHCO ₃ solution, with 100 ml H ₂ O and with 100 ml of a sat. NaCl solution. washed. It is dried over MgSO ₄ and the solvent is removed i. Vakkum. The residue is purified by chromatography on 130 g of silica gel in the mobile phase PE / EE 8.5: 1.
Yield: 1.58 (91%); colorless oil; [α] _D ²⁴ = -7.01 ° (c = 1.00 g / 100 ml, CHCl ₃ ); R _f = 0.23 (PE / EE 5: 1), 0.44 (PE / EE 10: 3).
ESI-MS (m / z) = 666.4 [M + Na] ⁺ .
200 MHz 1H NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 7.74 (d, 2H, H4, H5 Fmoc, J = 7.32 Hz); 7.57 (d, 2H, H1-, H8-Fmoc, J = 7.32 Hz); 7.24-7.42 (m, 4H, H2-, H3, H6-; H7-Fmoc); 6.93 (d, 2H, 2 aromatic H, J = 7.82 Hz); 6.78 (d, 2H, 2 aromatic H, J = 8.31 Hz); 5.81-6.00 (m, 1H, CH = _CH2); 5:17 to 5:31 (m, 3H, CH = CH _2, NH); 4.67 (d, 2H, CH ₂ -CH = CH _2, J = 5.86 Hz); 4:57 (s, 2H, O-CH ₂ -C = O); 4.52-4.77 (m, 1H, α-CH Ile); 4:33 to 4:37 (m, 2H, CH ₂ -Fmoc); 4.04-4.22 (m, 3H, TMS-CH-CH ₂ -O, H9-Fmoc); 2.47-2.55 (m, 1H, TMS-CH); 1.41-1.64 (m, 1H, β-CH Ile); 0.65-1.06 (m, 8H, χ-CH ₂ , χ-CH ₃ , δ-CH ₃ Ile); -0.02 (s, 9H, Si (CH _{3) 3).}
50.3 MHz ¹³ C NMR (CDCl ₃ ): δ (ppm): 172.20 (1C, C = O Ester Ile); 168.73 (1C, C = O allyl ester); 156.02, 155.74 (2C, C = O urethane, C _ipsoAr-O ); 143.98, 143.84 (2C, C4a, C4b-Fmoc); 141.32 (2C, C8a, C9a-Fmoc); 133.37 (1C, C _ipsoAr-CH ); 131.49 (1C, CH = CH _2); 128.39 (2C, 2 arom. C); 127.69 (2C, C3, C6 Fmoc); 127.06 (2C, C2, C7 Fmoc); 125.12 (2C, C1-, C8-Fmoc); 119.98 (2C, C4, C5 Fmoc); 119.01 (1C, CH = CH _2); 114.73 (2C, 2 aromatic C); 66.98, 66.31, 65.75, 65.59 (4C, CH ₂ = CH-CH ₂ -O, O = C-CH ₂ -O, CH ₂ -Fmoc, TMS-CH-CH ₂ -O); 58.33 (1C, α-CH Ile); 47.22 (1C, C9 Fmoc); 37.85 (1C, TMS-CH); 36.52 (1C, β-CH Ile); 24.7 (1C, _χ-CH2-Ile); 15.13 (1C, χ-CH ₃ Ile); 11.56 (1C, δ-CH ₃ Ile); -2.11 (3C, Si (CH ₃ ) ₃ ).

Signal doubling occurs in part because of diastereomers.

4- [2- (N-fluorenylmethoxycarbonyl-L-isoleucyloxy) -1- (trimethylsilyl) ethyl] phenoxy acetic acid 7a

1,454 g (2.26 mmol) of 4- [2- (N-fluorenylmethoxycarbonyl-L-isoleucyloxy) -1- (trimethylsilyl) ethyl] - phenoxyacetic acid 6-allyl ester 6 are dissolved in 20 ml of THF (pa). 156.5 mg (0.14 mmol) of Pd (PPh ₃ ) ₄ and a solution of 522.9 mg (2.94 mmol) of p-toluenesulfinic acid sodium salt in 12 ml of MeOH (pa) are added under argon. The mixture is stirred for 90 minutes under argon at 20 ° C., the solvent is removed i. Vak. and purifies the residue chromatographically on silica gel in the mobile phase mixture CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O 97.5: 2.5: 0.25: 0.25.
Yield: 1,363 g (quant.); colorless amorphous substance; [α] _D ²⁴ = -6.09 ° (c = 0.5 g / 100 ml, CHCl ₃ ); R _f = 0.11 (CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O 97.5: 2.5: 0.25: 0.25).
FD-MS (m / z) = 604.1 [M + H] ⁺ .
200 MHz ¹ H NMR (CDCl ₃ ): δ (ppm): 8.32 (s _b , 1H, COOH); 7.75 (d, 2H, H4-, H5-Fmoc, J = 7.32 Hz); 7.58 (d, 2H, H1-, H8-Fmoc, J = 7.32 Hz); 7.14-7.52 (m, 4H, H2, H3, H6, H7 Fmoc); 6.95 (d, 2H, 2 aromatic H, J = 7.32 Hz); 6.80 (d, 2H, 2 aromatic H, J = 8.79 Hz); 5.28 (t, 1H, NH); 4.54-4.78 (m, 1H, α-CH Ile); 4:58 (s, 2H, O-CH ₂ -C = O); 4:33 to 4:37 (m, 2H, CH ₂ -Fmoc); 4.16-4.22 (m, 3H, TMS-CH-CH ₂ -O, H9-Fmoc); 2.49-2.57 (m, 1H, TMS-CH); 1.55-1.81 (m, 1H, β-CH Ile); 0.66-1.06 (m, 8H, χ-CH ₂ , χ-CH ₃ , δ-CH ₃ Ile); -0.01 (s, 9H, Si (CH _{3) 3).}
100.6 MHz - ¹³ C NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 172.09, 172.03 (2C, COOH; C = O Ester Ile); 156.08, 155.67 (2C, C = O urethane, C _ipsoAr-O ); 143.97, 143.81 (2C, C4a, C4b-Fmoc); 141.31 (2C, C8a, C9a-Fmoc); 132.20 (1C, C _ipsoAr-CH ); 127.04-129.01 (6C, 2 aroma C, C2, C3, C6, C7 Fmoc); 125.26, 125.05 (2C, C1-, C8-Fmoc); 119.94 (2C, C4, C5 Fmoc); 114.82 (2C, 2 aromatic C); 67.02, 66.30, 65.25 (3C, O = C-CH ₂ -O, CH ₂ -Fmoc, TMS-CH-CH ₂ -O); 58.41 (1C, α-CH Ile); 47.22 (1C, C9 Fmoc); 37.82 (1C, TMS-CH); 36.62 (1C, β-CH Ile); 24.7 (1C, _χ-CH2-Ile); 15.21 (1C, χ-CH ₃ Ile); 11.47 (1C, δ-CH ₃ Ile); -2.72 (3C, Si (CH ₃ ) ₃ ).

Signal doubling occurs in part because of diastereomers.

4- [2- (N-Fluorenyhnethoxycarbonyl-glycyloxy) -1- (trimethylsilyl) ethyl] phenoxy-acetic acid allylester 6b

To 212 mg (0.71 mmol) Fmoc-Gly-OH in 20 ml absolute. CH ₂ Cl ₂ , 200 mg (0.65 mmol) of 4- [2-hydroxy-1- (trimethylsilyl) ethyl] phenoxyacetic acid allyl ester 5 are added. The mixture is stirred under argon and cooled to 0 ° C. Then 5 mg DMAP (0.04 mmol) and 162 g DCC (0.785 mmol) are added. The mixture is left at 0 ° C. for 10 minutes and then at room temperature for 4 hours. stir under argon. It is filtered through Hyflo-Supercel® and washed with approx. 100 ml CH ₂ Cl ₂ . The filtrate is washed twice with 80 ml of a 5% NaHCO ₃ solution. and with 100 ml of a sat. NaCl solution. washed. It is dried over MgSO ₄ and removed i. Vak. the solvent. The residue is purified by chromatography on 50 g of silica gel in the mobile phase PE / EE 5: 1.
Yield: 362 mg (95%); colorless oil; [α] _D ²² = 0 ° (c = 1.00 g / 100 ml, CHCl ₃ ); R _f = 0.23 (PE / EE 10: 3).
ESI-MS (m / z) = 610.3 [M + Na] ⁺ .
400 MHz ¹ H NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 7.74 (d, 2H, H4, H5 Fmoc, J = 7.33 Hz); 7.57 (d, 2H, H1-, H8-Fmoc, J = 7.33 Hz); 7.38 (t, 2H, H2-, H7-Fmoc, J = 7.33 Hz); 7.29 (t, 2H, H3-, H6- Fmoc, J = 7.34 Hz); 6.93 (d, 2H, 2 aromatic H, J = 8.51 Hz); 6.79 (d, 2H, 2 aromatic H, J = 8.51 Hz); 5.81- 5.94 (m, 1H, CH = _CH2); 5:18 to 5:32 (m, 3H, CH = CH _2, NH); 4.68 (d, 2H, CH ₂ -CH = CH _2, J = 5.87 Hz); 4:58 (s, 2H, O-CH ₂ -C = O); 4.48-4.63 (m, 2H, TMS-CH-CH ₂ -O); 4:35 (d, 2H, CH ₂ - Fmoc, J = 7.05 Hz); 4.19 (t, 1H, H9-Fmoc, J = 7.34 Hz); 3.87 (dd, 1H, α-CH _a Gly, J _gem = 18.20 Hz, J _vic = 5.57 Hz); 3.80 (dd, 1H, α-CH _b Gly, J _gem = 18.33 Hz, J _vic = 5.28 Hz); 2.48 (dd, 1H, TMS-CH, J ₁ = 11.32 Hz, J ₂ = 4.71 Hz); -0.03 (s, 9H, Si (CH _{3) 3).}
100.6 MHz ¹³ C NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 170.14 (1C, C = O Ester Gly); 168.68 (1C, C = O allyl ester); 156.15, 155.77 (2C, C = O urethane, C _ipsoAr-O ); 143.87 (2C, C4a, C4b-Fmoc); 141.31 (2C, C8a, C9a-Fmoc); 133.46 (1C, C _ipsoAr-CH ); 131.51 (1C, CH = CH _2); 128.40 (2C, 2 arom. C); 127.68 (2C, C3, C6 Fmoc); 127.04 (2C, C2, C7 Fmoc); 125.06 (2C, C1-, C8-Fmoc); 119.94 (2C, C4, C5 Fmoc); 118.87 (1C, CH = CH _2); 114.73 (2C, 2 aromatic C); 67.18, 66.74, 65.67, 65.62 (4C, CH ₂ = CH-CH ₂ -O, O = C-CH ₂ -O, CH ₂ -Fmoc, TMS-CH-CH ₂ -O); 47.17 (1C, C9-Fmoc); 42.79 (1C, α-CH Gly); 36.38 (1C, TMS-CH); -2.63 (3C, Si (CH ₃ ) ₃ ).

4- [2- (N-fluorenylmethoxycarbonylglycyloxy) -1- (trimethylsilyl) ethyl] phenoxyacetic acid 7b

To 321 mg (0.55 mmol) of 4- [2- (N-fluorenylmethoxycarbonylglycyloxy) -1- (trimethylsilyl) ethyl] - phenoxyacetic acid 6b in 10 ml of THF, 38 mg (0.03 mmol) of Pd (PPh ₃ ) ₄ and a solution of 126.5 mg (0.71 mmol) of p-toluenesulfinic acid sodium salt in 6 ml of MeOH was added. The mixture is stirred for 90 minutes under argon and with the exclusion of light at room temperature. The solvent is removed i. Vak. and purifies the residue chromatographically on 60 g of silica gel in a CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O eluent mixture 97.5: 2.5: 0.25: 0.25.
Yield: 299 mg (quant.); colorless amorphous substance; [α] _D ²² = 0 ° (c = 1.00 g / 100 ml, CHCl ₃ ); R _f = 0.09 (CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O 97.5: 2.5: 0.25: 0.25).
ESI-MS (m / z) = 570.4 [M + Na] ⁺ , 586.6 [M + K] ⁺ .
400 MHz ¹ H NMR (CDCl ₃ ): δ (ppm): 7.74 (d, 2H, H4, H5 Fmoc, J = 7.62 Hz); 7.56 (d, 2H, H1-, H8-Fmoc, J = 7.63 Hz); 7.38 (t, 2H, H2, H7 Fmoc, J = 7.63 Hz); 7.29 (t, 2H, H3-, H6-Fmoc, J = 7.64 Hz); 6.94 (d, 2H, 2 aromatic H, J = 8.51 Hz); 6.80 (d, 2H, 2 aromatic H, J = 8.51 Hz); 5.26 (t, 1H, NH); 4:58 (s, 2H, O-CH ₂ -C = O); 4.48-4.64 (m, 2H, TMS-CH-CH ₂ -O); 4:35 (d, 2H, _CH2 -Fmoc, J = 7:04 Hz); 4.18 (t, 1H, H9-Fmoc, J = 7.04 Hz); 3.85 (dd, 1H, α-CH _a Gly, J _gem = 18.19 Hz, J _vic = 5.58 Hz); 3.80 (dd, 1H, α-CH _b Gly, J _gem = 18.33 Hz, J _vic = 5.58 Hz); 2.48 (dd, 1H, TMS-CH, J ₁ = 11.30 Hz, J ₂ = 4.69 Hz); -0.02 (s, 9H, Si (CH _{3) 3).}
100.6 MHz - ¹³ C NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 171.90 (1C, COOH); 170.18 (1C, C = O Ester Gly); 156.30, 155.36 (2C, C = O urethane, C _ipsoAr-O ); 143.81 (2C, C4a, C4b-Fmoc); 141.31 (2C, C8a, C9a-Fmoc); 133.83 (1C, C _ipsoAr-CH ); 127.04-128.99 (6C, 2 ar. C, C2, C3, C6, C7 Fmoc); 125.27, 125.02 (2C, C1-, C8-Fmoc); 119.94 (2C, C4, C5 Fmoc); 114.87 (2C, 2 aromatic C); 67.27, 66.69, 65.21 (3C, O = C-CH ₂ -O, CH ₂ -Fmoc, TMS-CH-CH ₂ -O); 47.15 (1C, C9-Fmoc); 42.79 (1C, α-CH Gly); 36.48 (1C, TMS-CH); -2.63 (3C, Si (CH ₃ ) ₃ ).

Loading of aminomethyl polystyrene (AMPS) with 4- [2- (N-fluorenylmethoxycarbonyl-L- isoleucyloxy) -1- (trimethylsilyl) ethyl] phenoxyacetic acid 8a

1.39 g AMPS (ACT; 200-400 mesh; 1.00 mmol / g; 1.39 mmol) are weighed into a reactor and pre-swollen in approx. 20 ml CH ₂ Cl _{2 for} 30 min. 673.0 mg (1.11 mmol) of 4- [2- (N-fluorenylmethoxycarbonyl-L-isoleucyloxy) -1- (trimethylsilyl) ethyl] -phenoxyacetic acid 7a are dissolved in a mixture of 40 ml of CH ₂ Cl ₂ and 5 ml of DMF. 171.2 mg (1.11 mmol) of HOBt, 0.246 ml (225.6 mg, 2.23 mmol) of N-methylmorpholine and finally 358.0 mg (1.11 mmol) of TBTU are added to this solution in succession. It is left for about 20 minutes at room temperature. pre-react and then transfer this solution to the solid phase reactor. Shake at room temperature for 18 hours. and then filters off. The resin is washed three times with 20 ml of DMF and three times with 20 ml of CH ₂ Cl ₂ . 20 ml of a solution consisting of pyridine / acetic anhydride (3: 1) are added and the mixture is shaken for 20 minutes. After the unreacted amino functions have been capped, the mixture is washed four times with 20 ml of DMF, once with 20 ml of MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ and finally six times with 20 ml of diethyl ether and in High vacuum dried. 2.33 g of loaded resin are obtained. The loading is determined photometrically on the basis of the UV absorption of the fluorenylmethyl-piperidine adduct, which is obtained by adding 20 mg of the resin loaded with an Fmoc-amino acid to piperidine. Loading: c = 0.365 mmol / g, which corresponds to a coupling yield of 74%.

Loading of AMPS with 4- [2- (N-fluorenylmethoxycarbonyl-glycyloxy) -1- (trimethylsilyl) - ethyl] phenoxyacetic acid 8b

593 mg AMPS (ACT; 200-400 mesh; 1.00 mmol / g; 0.593 mmol) are weighed into a reactor and pre-swollen in approx. 10 ml CH ₂ Cl _{2 for} 30 min. 260 mg (0.475 mmol) of 4- [2- (N-fluorenylmethoxycarbonyl-glycyloxy) -1- (trimethylsilyl) -ethyl] phenoxy-acetic acid 7b are dissolved in a mixture of 20 ml of CH ₂ Cl ₂ and 3 ml of DMF. 72.9 mg (0.475 mmol) of HOBt, 0.105 ml (96.0 mg, 0.95 mmol) of N-methylmorpholine and 152.4 mg (0.475 mmol) of TBTU are added to this solution in succession. It is left for about 20 minutes at room temperature. pre-react and then transfer this solution to the solid phase reactor. Shake at room temperature for 18 hours. and filtered off. The resin is washed three times with 20 ml of DMF and three times with 20 ml of CH ₂ Cl ₂ . 20 ml of a solution consisting of pyridine / acetic anhydride (3: 1) are added and the mixture is shaken for 20 minutes. After the unreacted amino functions have been capped, the mixture is washed four times with 20 ml of DMF, once with 20 ml of MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ and finally six times with 20 ml of diethyl ether and in high vacuum. dried. 870 mg of loaded resin are obtained. The loading is determined photometrically on the basis of the UV absorption of the fluorenylmethyl-piperidine adduct, which is obtained by adding 20 mg of the resin loaded with an Fmoc-amino acid to piperidine. Loading: c = 0.474 mmol / g, which corresponds to a coupling yield of 87%.

Loading of Nova-Syn-Tg-amino-resin HL with 4- [2- (N-fluorenylmethoxycarbonyl-L- isoleucyloxy) -1- (trimethylsilyl) ethyl] phenoxyacetic acid 8c

3.24 g of Nova Syn Tg amino-resin HL (Novabiochem; 10 µm beads; 0.43 mmol / g; 1,393 mmol) are weighed into a reactor for solid phase synthesis and pre-swollen in approx. 20 ml of CH ₂ Cl ₂ for 30 min. 673.0 mg (1.11 mmol) of 4- [2- (N-fluorenylmethoxycarbonyl-L-isoleucyloxy) -1- (trimethylsilyl) ethyl] phenoxyacetic acid 7a are dissolved in a mixture of 40 ml of CH ₂ Cl ₂ and 5 ml of DMF. 171.2 mg (1.11 mmol) of HOBt, 0.246 ml (225.6 mg, 2.23 mmol) of N-methylmorpholine and finally 358.0 mg (1.11 mmol) of TBTU are added to this solution in succession. It is left for about 20 minutes at room temperature. pre-react and then transfer this solution to the solid phase reactor. Shake at room temperature for 18 hours. and filtered off. The resin is washed three times with 30 ml of DMF and three times with 30 ml of CH ₂ Cl ₂ . 35 ml of a solution consisting of pyridine / acetic anhydride (3: 1) are added and the mixture is shaken for 20 minutes. After the unreacted amino functions have been capped, the mixture is washed four times with 30 ml of DMF, once with 30 ml of MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ and finally six times with 30 ml of diethyl ether and in high vacuum. dried. 3.72 g of loaded resin are obtained. The loading is determined photometrically on the basis of the UV absorption of the fluorenylmethyl-piperidine adduct, which is obtained by adding 20 mg of the resin loaded with an Fmoc-amino acid to piperidine. Loading: c = 0.278 mmol / g, which corresponds to a coupling yield of 93%.

Solid phase synthesis of a protected heptapeptide to investigate a potential Aspartimide Education N ^α -Tert.-butyloxycarbonyl-L-leucyl-O-tert.-butyl-L-aspartyl-L-alanyl-N ^ω -trityl-L-aspariginyl-glycyl-L-isoleucyl-L-isoleucine 9 (Boc-Leu-Asp (OtBu) -Ala-Asn (Trt) -Gly-Ile-Ile-OH)

In a Perkin-Elmer ABI 433A peptide synthesizer, 274.1 mg of the ammomethyl polystyrene resin 8a loaded with Fmoc-Ile-OPTMSEL are used (loading: c = 0.365 mmol / g). Fmoc cleavages were performed in four to five four minute cycles with piperidine (30% piperidine in NMP). The couplings were carried out with 10 equivalents of Fmoc or Boc amino acids and coupling reagents (HBTU, HOBt, DIPEA) within 34-36 min. After each coupling, a capping step was carried out with a mixture of acetic anhydride / DIPEA / HOBt in NMM. After the synthesis was complete, the synthesizer was washed extensively with NMP and then with CH ₂ Cl ₂ . To split off the protected heptapeptide from the polymeric carrier, the resin is transferred to a reactor and i. High vacuum. dried. It is then washed three times with 10 ml of CH ₂ Cl ₂ . A solution of 63.1 mg of tetrabutylammonium fluoride trihydrate (TBAF.3H ₂ O, 0.2 mmol, 2 equiv.) In 10 ml of CH ₂ Cl ₂ is then added to the resin and the mixture is stirred at room temperature for 25 minutes. shaken. It is filtered off and washed with 10 ml of CH ₂ Cl ₂ . This cleavage process is repeated once more with a solution of 18.9 mg of TBAF.3H ₂ O (0.06 mmol) in 10 ml of CH ₂ Cl ₂ . The two cleavage solutions are worked up separately: 10 ml of H ₂ O are added and the mixture is washed twice with 10 ml of H ₂ O. The combined organic phases are dried over MgSO ₄ and i. Vak. freed from the solvent.

1st fraction: 89 mg, colorless, crystalline solid; 2nd fraction: 37 mg, colorless, crystalline solid. Both fractions are analyzed by HPLC-MS examination. Both fractions have the same composition and consist of 97% of the desired heptapeptide and 3% of the aspartimide-containing peptide. A purification of the crude product, ie the separation of the peptide containing aspartimide (approx. 3%), is possible by preparative HPLC. (Column: C8 Eurosphere, gradient: 30% CH ₃ CN in H ₂ O → 60% CH ₃ CN in H ₂ O in 100 min). After freeze-drying, a colorless, amorphous solid is obtained.
Yield: 100 mg (90%); colorless, amorphous solid; [α] _D ²³ = -22.29 ° (c = 1.01 g / 100 ml, MeOH); R _f = 0.39 (CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O 90: 10: 1: 1).
M: 1113.34 (C ₅₉ H ₈₄ N ₈ O ₁₃ )
HPLC-MS (column A, gradient A): R _t , m / z = 6.53 min (3.3%; 1083.5, [aspartimide + 2Na] ⁺ ; 1077.5 [aspartimide + K] ⁺ ; 1061.4 [aspartimide + Na] ⁺ ); 7.28 min (96.7%; 1157.4, [M + 2Na] ⁺ ; 1151.4, [M + K] ⁺ ; 1135.4, [M + Na] ⁺ ).
ESI-MS (m / z) = 1173.4 [M + 2K] ⁺ ; 1157.4 [M + 2Na] ⁺ ; 1151.4 [M + K] ⁺ ; 1135.5 [M + Na] ⁺ ; 1095.5 [M + K + H-tBu] ⁺ ; 1079.5 [M + Na + H-tBu] ⁺ ; 995.4 [M + K-BoctBu + 2H] ⁺ ; 979.4 [M + Na-Boc-tBu + 2H] ⁺ .
400 MHz ¹ H NMR (DMSO-d ⁶ , ¹ H, ¹ H-COZY): δ (ppm): 8.72 (s _b , 1H, β-NH Asn); 8.18-8.35 (m, 2H, α-NH Asn, Asp); 7.78-7.93 (m, 4H, NH Ala, Gly, 2x Ile); 7.14-7.25 (m, 15H, Trt); 7.02 (d, 1H, NH Leu); 4.51-4.58 (m, 2H, α-CH Asn, Asp); 4.23-4.27 (m, 2H, α-CH Ala, Ile); 3.99-4.01 (m, 1H, α-CH Ile); 3.93-3.96 (m, 1H, α-CH Leu); 3.65-3.77 (m, 2H, α-CH ₂ Gly); 2.41-2.71 (m, 4H, 2x β-CH ₂ Asn, Asp); 1.68-1.81 (m, 2H, 2x β-CH Ile); 1.52-1.61 (m, 1H, χ-CH Leu); 1.35 (s, 9H, 3x CH ₃ Boc); 1.34 (s, 9H, 3x CH ₃ tBu); 1.29-1.41 (m, 4H, β-CH ₂ Leu, 2x χ-CH _A Ile); 1.20 (d, 3H, CH ₃ Ala, J = 7:04 Hz); 1.04-1.20 (m, 2H, 2x χ-CHB Ile); 0.75-0.84 (m, 18H, 2x χ-CH ₃ Ile, 2x δ-CH ₃ Ile, 2x δ-CH ₃ Leu).
100.6 MHz- ¹³ C-NMR (DMSO-d ⁶ ; BB, DEPT): δ (ppm): 172.56, 171.62, 170.91, 170.38, 169.83, 169.35, 169.01, 168.83, 168.11 (9C, 1x COOH Ile, 1x COOtBu Asp , 7x C = O Amid Leu, α-Asn, β-Asn, Ala, Asp, Gly, Ile); 144.69 (3C, C _ipso -Trt); 128.47 (3C, C _para -Trt); 127.31 (6C, C _ortho -Trt); 126.19 (6C, C _meta -Trt); 80.05 (1C, C _quart Boc); 77.91 (1C, C _quart tBu); 69.36 (1C, C _quart ); 56.89 56.75 (2C, α-CH Leu, α-CH Ile); 52.72 (1C, α-CH Asn); 50.26 (1C, α-CH Ile); 49.26 (1C, α-CH Asp); 48.29 (1C, α-CH Ala); 42.04 (1C, α-CH ₂ Gly); 40.70 (1C, β-CH ₂ Leu); 38.30 (1C, β-CH ₂ Asn); 36.92 (1C, β-CH ₂ Asp); 36.79; 36.51 (2C, 2x β-C _H Ile); 28.07 (3C, 3x CH ₃ Boc); 27.59 (3C, 3x CH ₃ tBu); 24.77, 24.07 (2C, 2x χ-CH ₂ Ile); 22.83 (1C, χ-CH Leu); 21.40 (2C, 2x δ-CH ₃ Leu); 18:21 (1C, CH ₃ ala); 15.53, 15.22 (2C, 2x χ-CH ₃ Ile); 11.47, 10.96 (2C, 2x δ-CH ₃ Ile).

Solid phase synthesis of a protected glycodecapeptide N ^α -Tert.-butyloxycarbonyl-L-leucyl-L-alanyl-L-alanyl-L-leucyl-O-tert.-butyl-L-aspartyl-O- (2-acetamido-3,4,6-tri- O-acetyl-2-deoxy-α-D-galactopyranosyl) -L-seryl-N ^ω -trityl-L-glutaminyl-glycyl-L-alanyl-L-isoleucine 11 (Boc-Leu-Ala-Ala-Leu-Asp (OtBu) -Ser (αAc ₃ GalAc) -Gln (Trt) -Gly-Ala-Ile-OH)

In a Perkin-Elmer ABI 433A peptide synthesizer, 274.1 mg of the ammomethyl polystyrene resin 8a loaded with Fmoc-Ile-OPTMSEL are used (loading: c = 0.365 mmol / g). The Fmoc cleavages are carried out in four to five 4 minute cycles with piperidine (30% piperidine in NMP). The couplings are carried out with 10 equivalents of Fmoc or Boc amino acids and coupling reagents (HBTU, HOBt, DIPEA) within 34-36 min. The glycosylated building block was coupled manually with only 3 equivalents of Fmoc-Ser (αAc ₃ GalNAc) -OH. HATU (3 equiv.), HOAt (3 equiv.) And DIPEA (6 equiv.) Are used as coupling reagents. Due to the lower reagent excess, the reaction time for this coupling is extended to 5 h. After each coupling, a capping step was carried out with a mixture of acetic anhydride / DIPEA / HOBt in NMM. After synthesis is complete, the synthesizer is washed extensively with NMP and then with CH ₂ Cl ₂ . To split off the protected glycodecapeptide from the polymeric support, the resin is first transferred to a reactor and i. High vacuum. dried. It is then washed three times with 10 ml of CH ₂ Cl ₂ . A solution of 63.1 mg of TBAF.3H ₂ O (0.2 mmol, 2 equiv.) In 10 ml of CH ₂ Cl ₂ is then added to the resin and the mixture is at room temperature for 25 minutes. shaken. It is filtered off and washed four times with 10 ml of CH ₂ Cl ₂ . This cleavage process is repeated once more with a solution of 23 mg TBAF.3H ₂ O (0.07) in 10 ml CH ₂ Cl ₂ . The two cleavage solutions are worked up separately: 20 ml of H ₂ O are added and the mixture is washed twice with 20 ml of H ₂ O. (Due to the poor solubility of the product in CH ₂ Cl ₂ , problems occur with the phase separations.) The combined organic phases are i. Vak. freed from the solvent. 1st fraction: 109 mg, colorless, crystalline solid; 2nd fraction: 47 mg, colorless, crystalline solid. Both fractions are analyzed by HPLC-MS examination. The product is approximately 90% contained in both fractions (after integration via UV or ELS chromatogram). The solid obtained can be purified by preparative HPLC. (Column: Eurosphere C8, gradient: 30% CH ₃ CN in H ₂ O → 80% CH ₃ CN in H ₂ O in 100 min). After freeze-drying, a colorless, amorphous solid is obtained.
Yield: 111 mg (66%); colorless, amorphous solid; [α] _D ²³ = 1.96 ° (c = 0.90 g / 100 ml, MeOH); R _f = 0.39 (CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O 90: 10: 1: 1).
M: 1685.95 C ₈₃ H ₁₂₀ N ₁₂ O ₂₅
HPLC-MS (column A, gradient B): R _t , m / z = 4.45 min (2.7%); 5.25 min (3.8%; deletion sequences: last or penultimate amino acid not coupled); 5.80 min (1.6%; 839.6, ([Aspartimid + 3Na] ⁺ / 2); 836.7, ([Aspartimid + Na + K] ⁺ / 2); 828.9, ([Aspartimid + 2Na] ⁺ / 2; 825.9 ([Aspartimid + K] ⁺ / 2)); 6.82 min (92.0%; 876.8, ([M + 3Na] ⁺ / 2); 873.8, ([M + Na + K] ⁺ / 2); 865.8, ([M + 2Na ] ⁺ / 2); 862.8, ([M + K] ⁺ / 2)).
ESI-MS (m / z) = 884.7 ([M + 3Na] ⁺ / 2; 876.8 ([M + Na + K] ⁺ / 2); 873.8 ([M + 2Na] ⁺ / 2); 865.8 ([M + 2Na] ⁺ / 2); 854.7 ([M + Na] ⁺ / 2); 848.8 ([M + 3Na-tBu + H] ⁺ / 2); 845.7 ([M + Na + K-tBu + H] ⁺ / 2); 798.8 ([M + 3Na-tBu-Boc + 2H] ⁺ / 2); 795.8 ([M + Na + K-tBu-Boc + 2H] ⁺ / 2); 787.8 ([M + 2Na-tBu - Boc + 2H] ⁺ / 2).
MALDI-TOF-MS (cca, pos) (m / z) = 1709.3 [M + Na] ⁺ ; 1725.2 [M + K] ⁺ ; 1731 [M + 2Na] ⁺ ; 1747.2 [M + Na + K] ⁺ .
400 MHz ¹ H NMR (DMSO-d ⁶ ; ¹ H, ¹ H-COZY): δ (ppm): 8.53 (s _b , 1H, ω-NH Gln); 8.08-8.14 (m, 3H, 3x NH Asp, Ser, Ala); 7.85-7.98 (m, 5H, 5x NH 2x Ala, Gln, Leu, Gly); 7.61 (d, 1H, NH GalNAc, J = 8.6 Hz); 7.14-7.26 (m, 15H, Trt); 6.85 (d, 1H, NH-urethane Leu, J = 7.43 Hz); 5.28 (s, 1H, H4-Gal); 5.00 (dd, 1H, H3-Gal, J _{H3, H4} = 2.74 Hz, J _{H3, H2} = 11.74 Hz); 4.85 (d, 1H, H1-Gal, J _{H1, H2} = 3.13 Hz); 4.56-4.64 (m, 1H, α-CH Asp); 4.47-4.52 (m, 1H, α-CH Ser); 3.94-4.40 (m, 9H, α-CH 3x Ala, Ile, Gln, H2-Gal, H5-Gal, 2x H6-Gal); 3.88-3.92 (m, 2H, 2x α-CH Leu); 3.72-3.76 (m, 2H, α-CH ₂ Gly); 3.60-3.64 (m, 2H, β-CH ₂ Ser); 2.71-2.73 (m, 1H, β-CH _A Asp,); 2.45-2.49 (m, 1H, β-CH _B Asp); 2.62-2.68 (m, 2H, χ-CH ₂ Gln); 2:07 (s, 3H, _CH3 NHAc); 1.71-1.94 (m, 10H, 3x CH ₃ OAc, β-CH Ile); 1.52-1.59 (m, 2H, 2x χ-CH Leu); 1:36 (s, 9H, 3x CH ₃ Boc); 1.33 (s, 9H, 3x CH ₃ tBu); 1.33-1.42 (m, 6H, 2x β-CH ₂ Leu, β-CH ₂ Gln); 1.11-1.18 (m, 11H, 3x CH ₃ Ala, χ-CH ₂ Ile); 0.79-0.86 (m, 18H, 4x δ-CH ₃ Leu, 2x δ-CH ₃ Leu).
100.6 MHz ¹³ C NMR (DMSO-d ⁶ ; BB, DEPT): δ (ppm): 172.86 (1C, COOH); 172.69 (1C, COOtBu); 172.18, 172.11, 172.02, 171.53, 170.39, 170.19, 170.04, 169.97, 169.91, 169.71, 169.34, 169.05, 168.22, 168.11 (14C, 3x C = O ester, 11x C = O amide, N-acetyl, 3x Ala, 2x Leu, Asp, Ser, Gly, α- and ω-Gln); 145.00 (1C, C _ipso -Trt); 128.61 (3C, C _para -Trt); 127.54 (6C, C _ortho - Trt); 126.41 (6C, C _meta -Trt); 97.29 (1C, C1-Gal); 80.39 (1C, C _quart Boc); 78.34 (1C, C _quart tBu); 69.37 (1C, C _quart Trt); 67.51 (1C, C3-Gal); 66.96 (1C, β-CH ₂ ser); 66.83 (1C, C4-Gal); 65.92 (1C, C5-Gal); 61.14 (1C, C6-Gal); 56.04 (1C, α-CH Leu); 52.66, 52.39, 52.20 (3C, α-CH Leu, Ser, Ile); 50.87, 49.28, 48.08, 47.95, 47.59, 46.57 (6C, α-CH Asp, Gln, 3x Ala, C2-Gal); 41.61 (1C, α-CH ₂ Gly); 40.37, 40.22 (2C, 2x β-CH ₂ Leu); 36.92 (1C, β-CH ₂ Asp; 36.08 (1C, β-CH Ile); 32.39 (1C, χ-CH ₂ Gln); 27.89 (3C, 3x CH ₃ Boc); 27.66 (1C, β-CH ₂ Gln ); 27.37 (3C, 3x CH ₃ tBu); 24.46 (1C, χ-CH ₂ Ile); 23.94, 23.82 (2C, 2x χ-CH Leu); 22.69, 22.33, 21.21, 20.22 (4C, 4x CH ₃ acetyl ); 20.18, 20.13, 18.05 (3C, CH ₃ Ala); 17.67, 17.38 (4C, 4x δ-CH ₃ Leu); 15.24 (1C, χ-CH ₃ Ile); 11.08 (1C, δ-CH ₃ Ile) ,

Synthesis of a tripeptide to investigate potential diketopiperazine formation the stage of the second coupled amino acid and to study the stability of the Fmoc group under the conditions of the spin-off N-fluorenylmethoxycarbonyl-L-phenylalanyl-L-prolyl-L-glycine 13 (Fmoc-Phe-Pro-Gly-OH) First Fmoc cleavage and coupling of the first Fmoc amino acid

302.5 mg (0.143 mmol) of the AMPS resin 8b loaded with Fmoc-Gly-OPTMSEL (loading: c = 0.474 mmol / g) are preswollen in a reactor for solid phase synthesis with approx. 10 ml CH ₂ Cl ₂ and with a 20% solution of piperidine in DMF and shaken for 25 min. It is filtered and washed six times with 20 ml of DMF each. 308.7 mg (0.915 mmol) Fmoc-L-Pro-OH are dissolved in 20 ml DMF and successively with 140.5 mg (0.915 mmol) HOBt, 0.313 ml (236.5 mg, 1.83 mmol) N-ethyldiisopropylamine and finally 293.8 mg (0.915 mmol) TBTU transferred. The mixture is left to react for about 10 minutes at room temperature and this solution is then transferred to the solid phase reactor. Shake at room temperature for 18 hours. and then filters off. The resin is washed six times with 20 ml of DMF and then mixed with a solution of 15 ml of pyridine and 5 ml of acetic anhydride. The mixture is shaken for 25 minutes. After the unreacted amino functions have been capped, the mixture is washed four times with 20 ml of DMF, once with 20 ml of MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ and finally six times with 20 ml of diethyl ether and i. High vacuum. dried. The loading is determined photometrically on the basis of the UV absorption of the fluorenylmethyl-piperidine adduct, which is obtained by adding 20 mg of the resin loaded with an Fmoc-amino acid to piperidine. Loading: c = 0.451 mmol / g.

Second Fmoc split off

The resin is mixed with a 20% solution of piperidine in DMF and shaken for 25 minutes. It is filtered and this solution for the presence of the corresponding diketopiperazine examined: To do this, remove the solvent i. Vak. and analyzes the backlog HPLC-MS. No diketopiperazine can be found in the residue, only that Fluorenylmethyl piperidine adduct.

The resin is washed six times with 20 ml DMF each.

Coupling of the second Fmoc amino acid

354.0 mg (0.915 mmol) Fmoc-L-Phe-OH are dissolved in 20 ml DMF and successively with 140.5 mg (0.915 mmol) HOBt, 0.313 ml (236.5 mg, 1.83 mmol) N-ethyldiisopropylamine and finally 293.8 mg (0.915 mmol) TBTU transferred. The mixture is left to react for about 10 minutes at room temperature and this solution is then transferred to the solid phase reactor. Shake at room temperature for 18 hours. and filtered off. The resin is washed five times with 20 ml DMF, once with 20 ml MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ , MeOH, CH ₂ Cl ₂ and finally six times with 20 ml diethyl ether and i. High vacuum. dried. 314 mg of loaded resin are obtained. The loading is determined photometrically on the basis of the UV absorption of the fluorenylmethyl-piperidine adduct, which is obtained by adding 20 mg of the resin loaded with an Fmoc-amino acid to piperidine. Loading: c = 0.419 mmol / g.

Cleavage from the polymeric carrier

The resin is washed three times with 10 ml CH ₂ Cl ₂ . A solution of 85.8 mg of TBAF.3H ₂ O (0.27 mmol, ≈2.2 equiv.) In 10 ml of CH ₂ Cl ₂ is then added to the resin and the mixture is stirred at room temperature for 25 minutes. shaken. It is filtered off and washed with 10 ml of CH ₂ Cl ₂ . This cleavage process is repeated once more with a solution of 30 mg of TBAF.3H ₂ O (0.095 mmol) in 10 ml of CH ₂ Cl ₂ . In the second cleavage, no more product can be detected by thin layer chromatography. The collected filtrates of the first cleavage are mixed with 10 ml H ₂ O and washed twice with a total of 10 ml H ₂ O. The combined organic phases are dried over MgSO ₄ and i. Vak. freed from the solvent. The crude product is purified by chromatography on 20 g of silica gel in a CH ₂ Cl ₂ / MeOH / AcOH / H ₂ O eluent mixture 95: 5: 0.5: 0.5. The solvent is removed i. Vak. and acetic acid still contained by five times co-distillation with about 10 ml of toluene. A colorless, crystalline solid is obtained.
Yield: 60 mg (77%) (60 mg of the loaded resin which was used for loading determinations are not taken into account here, ie the yield is accordingly higher at 90%.); colorless, crystalline solid; [α] _D ²² = -31.58 ° (c = 1.00 g / 100 ml, CHCl ₃ ); R _f = 0.12 (CH ₂ Cl ₂ / MeOH / HOAc / H ₂ O 95: 5: 0.5: 0.5).
400 MHz ¹ H NMR (CDCl ₃ ): δ (ppm): 7.73 (d, 2H, H4, H5 Fmoc, J = 7.34 Hz); 7.57 (d, 2H, H1-, H8-Fmoc, J = 7.63 Hz); 7.12-7.40 (m, 10H, H2-, H3-, H6-, H7-Fmoc, 5 aromatic H Phe, NH Gly); 6.76 (d, 1H, NH urethane, J = 9.09 Hz); 4.76-4.82 (m, 1H, α-CH Pro); 4.56-4.66 (m, 1H, α-CH Phe); 4:11 to 4:35 (m, 3H, CH ₂ Fmoc, Fmoc H9); 4.06 (dd, 2H, α-CH ₂ Gly, J ₁ = 4.99 Hz, J ₂ = 9.68 Hz); 3.65-3.72 (m, 1H, δ-CH _A H _B Pro); 3.05-3.10 (m, 1H, δ-CH _A H _B Pro); 3:04 (d, 2H, CH ₂ Phe, J = 7.34 Hz); 2.25-2.28 (m, 1H, β-CH _A H _B Pro); 1.81-1.89 (m, 3H, β-CHAHB, χ-CH ₂ Pro). 100.6 MHz - ¹³ C NMR (CDCl ₃ ; BB, DEPT): δ (ppm): 172.71 (1C, COOH); 171.51, 170.42 (2 C = O amide, Gly, Pro); 156.08 (1C, C = O urethane); 143.84 (2C, C4a, C4b-Fmoc); 141.30 (2C, C8a, C9a-Fmoc); 135.67 (1C, C _ipso Phe); 127.02-129.31 (9C, C2, C3, C6, C7 Fmoc, 5 aromatic C Phe); 125.06, 125.15 (2C, C1-, C8-Fmoc); 119.92 (2C, C4, C5 Fmoc); 67.22 (1C, CH2-Fmoc); 60.10 (1C, α-CH Pro); 53.90 (1C, α-CH Phe); 47.70 (1C, δ-CH ₂ Pro); 47.13 (1C, C9-Fmoc); 41.80 (1C, α-CH ₂ Gly); 38.91 (1C, CH ₂ Phe); 27.24 (1C, β-CH ₂ Pro); 24.84 (1C, χ-CH ₂ Pro).

Claims (11)

1. Compounds of the general formula I,
in which R ¹ , R ² and R ^{3 are} independently aryl or alkyl groups, aryl is a benzoid, also condensed or heterocyclic aromatic radical and X is a substituent attached to them in the p-, m- or o-position, which is also heteroatoms how O and N and / or functional groups such as carbonyl or carboxyl groups contain and can be or can be attached to a polymeric organic or inorganic carrier; Y is the anchored compound, which contains protected, deblockable or also free functional groups on which solid phase syntheses can be carried out. 2. The syntheses of compounds of general formula I from triorganylsilyloxiranes, in which R ¹ , R ² and R ^{3 have} the meaning indicated in claim 1, and aryl metal compounds, preferably aryl cuprates, in which aryl has a substituent X according to claim 1 with deblockable functional group. 3. Compounds of general formula I according to claim 1, characterized in that R ¹ and R ^{2 are} methyl groups and R ^{3 can be} a long-chain, also branched alkyl radical having 2 to 6 carbon atoms or a phenyl radical. 4. Compounds according to claim 1 and 3, characterized in that aryl is a substituted Is phenyl. 5. Compounds according to claim 1, 3 and 4, characterized in that X is a functionalized alkyl chain, a functionalized alkoxyl substituent or a deblockable phenol group. 6. Compounds according to claim 1, 3, 4 and 5, characterized in that X is a cleavable, is also substituted alkoxy group.   7. Compounds according to claim 1, 3, 4, 5 and 6, characterized in that X is a Carboxyl group or an alkoxy substituent containing a protected carboxyl group. 8. Compounds according to claim 1, 3, 4, 5, 6 and 7, characterized in that Y is an acyl or is an aryl residue. 9. Compounds according to claim 1, 3, 4, 5, 6, 7 and 8, characterized in that Y is an acyl radical, is in particular a free or N-protected aminoacyl radical. 10. Compounds according to claim 1, 2, 3, 4, 5, 6, 7, 8 and 9, characterized in that a organic or inorganic polymer is part of the substituent X. 11. Syntheses of compounds according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, thereby characterized in that 2-aryl-2-triorganylsilylethanols prepared according to claim 2 in ester or aryl ether, deblocked in the substituent X and the resulting Compounds according to the invention according to claims 1 and 3-9 by condensation on a suitably functionalized polymeric carriers can be coupled.