JPH06145195A - Method for preventing side-reaction in peptide synthesis - Google Patents

Abstract

PURPOSE:To obtain a peptide while suppressing the side reactions by reacting an amino acid having non-protected side chain with a free amino acid in the presence of a specific alcohol component using a condensation agent containing benzotriazole, oxo-benzotriazine, imide compound, etc. CONSTITUTION:A peptide containing asparagine and glutamine is easily produced in high yield while suppressing side reactions by activating the carboxyl group of an asparagine having non-protected side chain or a glutamine having non-protected side chain with a condensation agent having benzotriazole, oxo- benzotriazine or dialkylimide group and expressed by the formula {A-O-B}X [A is group of formula I (R is halogen, alkyl, aryl, etc.; (n) is 0-4), formula II or formula III (R1 to R4 are H, alkyl or aryl; R1 and R3, or R2 and R4 may together form a ring); B is group of formula IV (R5 and R6 are alkyl, aryl, etc.), etc.; X is halogen ion, (PF6)<->, etc.] and condensing the activated group to free amino group in the presence of an alcohol component expressed by the formula A-OH.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for introducing a side chain-unprotected asparagine or glutamine into a peptide chain in a simple peptide synthesis in a high yield while suppressing side reactions derived from asparagine or glutamine in chemical synthesis of peptides. .

[0002]

2. Description of the Related Art It is known that dehydration occurs when asparagine or glutamine without side chain protection is used in the chemical synthesis of peptides to produce nitriles (Int.
J. Peptide Protein Res. , 3
4, 287 (1989)). Benzotriazol-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate (Benzotriazol-), which is said to have a high reaction rate as a condensing agent
1-yl-oxy-tris (dimethylami
no) phosphoniumhexafluorop
It has been known that the use of such a condensing agent is a problem, although the use thereof is particularly increasing in recent years. In particular, it has been pointed out that the by-product of β-cyanoalanine from asparagine is remarkable and the yield is greatly reduced (Peptides, 74 (1)
991)). Further, when such a side reaction occurs, there is a problem that the produced β-cyanoalanine is further activated by the condensing agent and is introduced into the peptide chain during synthesis.

As a protecting group for α-amino group, 9-fluorenylmethoxycarbonyl (9-Fluorenylmethyl)
Fmo using a thycarbonyl, Fmoc) group
In the c-strategy, it has become common practice to use a protecting group for the side chain amide, but side reactions and cost increase when removing the protecting group pose problems. However, t-butoxycarbonyl (ter
t. In the Boc strategy using a —Butoxycarbonyl, Boc) group, this side reaction is not so important and has been used in a state in which the side chain amide is not protected for the following reasons. 1) When dicyclohexylcarbodiimide (DCC) is used as the condensing agent, the nitrile by-product itself due to the dehydration reaction of the side chain amide group is very small. 2) By-product nitrile returns to the original amide group almost quantitatively in the final hydrogen fluoride treatment. In the DCC method, which has been mainly used as an activator in the past, only a slight amount of a nitrile by-product is used as an additive for 1-hydroxybenzotriazole (1-Hydr).
It is known that it can be almost completely suppressed by adding equimolar amount of oxybenzotriazole, HOBt (Chem. Ber., 103, 788 (197).
0)). However, in the method using BOP, the circumstances are completely different and no effective solution has been found other than the method of introducing a protecting group into the asparagine side chain. However, even when this protecting group is used, side chain-protected asparagine is particularly expensive and there is a problem of side reaction when removing the protecting group.

[0004]

The present inventors have found that when peptide synthesis is carried out on such a side chain-unprotected asparagine using a condensing agent which has been actively developed in recent years and has a high reaction rate, side chain-free The nitrile, even slightly generated from the protected asparagine, is stable to hydrogen fluoride during the subsequent de-Boc reaction and is stable to N-tert. -Butyramide (N-tBu amide). The side chain N-tBu amide group incorporated in the main chain of the peptide cannot be returned to the original amide group like the amide bond of the main chain, which causes a decrease in the yield of the desired peptide. , Hinders purification. Further, in the Fmoc strategy, it is a conventional method to already use a protecting group for the side chain amide as described above, but in this case the cost increase becomes a problem, and it is also possible to remove the protecting group efficiently. It's a problem. Therefore, in the present invention, even when a condensing agent which does not have such a problem and has a faster activation and reaction rate than conventional DCC is used, side chain amide group-unprotected asparagine or glutamine can be simply operated. The purpose is to achieve condensation without or with little nitrile by-product.

[0005]

The present invention provides the formula (1) {A-0-B} X (I) [wherein A is

[0006]

[Chemical 5]

Or

[0008]

[Chemical 6]

(Wherein R represents a halogen, an alkyl group, an aralkyl group, or an aryl group, and n represents an integer of 0 to 4) or

[0010]

[Chemical 7]

(However, R ₁ , R ₂ , R ₃ , and R ₄ are each independently hydrogen, an alkyl group, an aralkyl group,
Represents an aryl group, R ₁ and R ₃ and R ₂ and R ₄ may each independently form a ring),
B is

[0012]

[Chemical 8]

(However, R ₅ and R ₆ are each independently an alkyl group, an aralkyl group, or an aryl group, and R ₅ may form a ring with R ₆ ) and X is a halogen ion. , (PF ₆ ) ⁻ , or (BF ₄ ) ⁻ ], which activates the carboxyl group of side chain unprotected asparagine or side chain unprotected glutamine and condenses with a free amino group. In the chemical synthesis method of, the alcohol component represented by formula (II) A-OH (II) [wherein A is the same as defined above and is independently selected from A in formula (I)] is added. A peptide synthesis method characterized by the above is provided.

According to the present invention, there is provided a peptide synthesis method capable of suppressing the by-product reaction of nitrile and performing the condensation reaction in a high yield. Specific examples of the condensing agent represented by the formula (I) of the present invention include benzotriazole-1.
-Yloxy tris (dimethylamino) phosphonium (Benzotriazol-1-yl-oxy-tr
is (dimethylamino) phosphon
ium) salt, benzotriazol-1-yloxy trispyrrolidinophosphonium (Benzotriazo)
l-1-yl-oxy-tris-pyrrolidi
nophosphonium) salt, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium (2- (1H-Benzotriazo
l-1-yl) -1,1,3,3-tetrameth
yluronium) salt, 2- (5-norbornene-
2,3-Dicarboximido) tetramethyluronium (2- (5-Norbornene-2,3-dica
rboximidio) tetramethluroni
um) Salts and the like can be mentioned, and examples thereof include those having reactivity similar to these and those participating in the condensation reaction according to the same reaction mechanism. Among them, benzotriazol-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate (Benzotri
azol-1-yl-oxy-tris (dimension
ylamino) phosphonium hexaf
Fluorophosphate, BOP), Benzotriazol-1-yloxy trispyrrolidinophosphonium hexafluorophosphate (Benzotri)
azol-1-yl-oxy-tris-pyrrol
idinophosphonium hexafluo
rophosphate, PyBOP), 2- (1H-
Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (2- (1H-Benzotriazol-1-yl)
-1,1,3,3-Tetramethyluroni
um hexafluorophosphate, HB
TU), 2- (5-norbornene-2,3-dicarboximide) tetramethyluronium tetrafluoroborate (2- (5-Norbornene-2,3-d
icarboximido) tetramethylu
ronium tetrafluoroborate,
TNTU), 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (2- (1H-Benzotria
zol-1-yl) -1,1,3,3-tetrame
thyluronium tetrafluorobo
rate, TBTU) is preferably used.

The alcohol represented by the formula (II) is specifically 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (3-
Hydroxy-3,4-dihydro-4-oxo
-1,2,3-benzotriazine, HOOB
t), N-hydroxy-5-norbornene-2,3-dicarboximide (N-Hydroxy-5-norb)
ornene-2,3-dicarboximide,
HONB), N-hydroxyphthalimide (N-Hyd
roxyphthalimide), 1-hydroxybenzotriazole (1-Hydroxybenzotr)
iazole, HOBt) and the like, and HOOBt and HONB are particularly preferably used. Formula (I
The alcohol represented by I) can be used without limitation as long as it has the same action as that described above. The condensing agent represented by the formula (I) and the formula (II)
It is preferable that A in formula (I) and A in formula (II) are different from each other, and specifically, for example, BOP and HONB, TNTU and HOBt, TNTU and HOOBt, etc. Is preferably used. The amount of the alcohol component represented by the formula (II) used is 0.5 to 100 times mol of the amount of the condensing agent represented by the formula (I). If it is less than 0.5 times by mole, the effect is insufficient, and if it exceeds 100 times by mole, the viscosity of the reaction solution becomes too high and it causes a cost increase, which is not preferable. The amount used is preferably 1 to 50 times mol, more preferably 2 to 10 times mol. Formula (I
The alcohol component represented by I) is added before the condensation reaction is completed, and preferably before the condensation reaction is started.

The present invention is directed to asparagine or glutamine without side chain amide group protection, and is most effective in the condensation of side chain amide group unprotected asparagine, which is particularly prone to nitrile by-product. It can also be applied to amino acids having similar side chain amide groups. The protective group for the α-amino group of the amino acid used in the present invention includes
For example, there are Fmoc group and Boc group, but the present invention is not limited thereto. It can be selected and used from various protecting groups usually used in peptide synthesis. Benzyloxycarbonyl (Benzyloxyc
Although amino acids protected with various other protecting groups such as arbonyl, Z) groups can be used, the present invention is more effective, particularly when the protecting group is the Boc group. Further, the present invention is effective for both the liquid phase method and the solid phase method, and the reagents, compounds, devices and the like which are commonly used for them can be used. According to the invention, the Boc strategy, Fmo
Regardless of the c-strategy, asparagine or glutamine can be introduced without side chain protection, and it can be synthesized more easily, without side reactions, and at a lower cost. Furthermore, it is not necessary to consider the yield of side chain protecting group removal and side reactions, and it is possible to synthesize a peptide of higher purity.

[0017]

EXAMPLES The present invention will be described in detail and specifically below with reference to examples, but the present invention is not limited thereto.

Comparative Example 1. "BOP-based Boc-Asn
And Gly-OBzl condensation reaction "t-butoxycarbonyl-L-asparagine (Boc-Asn) 100 mg,
And (glycine benzyl ester p-toluenesulfonate (Gly-OBzlTos, Glycin
e benzyl ester p-toluenes
145 mg of sulfonate) was dissolved in 1 ml of DMF, 300 μl of DIEA was added, and the mixture was stirred until the solution became uniform. 190 mg of BOP was added as a powder, and the mixture was stirred at room temperature for 1 hour. When a part of the reaction solution was analyzed by reverse phase HPLC, two signals derived from the peptide were 2
It appeared at 0 minutes (I peak) and 23 minutes (II peak) (area ratio = 5: 2). Prep and analyze by HPLC, NM
From R and MS, the I peak is N-α-t-butoxycarbonylasparaginylglycine benzyl ester (α-B
oc-Asn-Gly-OBzl), II peak is t-.
Butoxycarbonyl-β-cyanoalanylglycine benzyl ester (Boc-Ala (CN) -Gly-O
Bzl) was revealed.

I peak: MS (EI method): m / z 380 ((M + 1) ⁺ ) HPLC: retention time: 19.5 minutes Column: YMC R-ODS-5 S-5 120A
ODS (registered trademark, YMC Co.) Flow rate: 1.5 ml / min. Mobile phase: A = 0.1% TFA / water B = 0.1% TFA / CH ₃ CN Gradient: B conc. 5 to 65% (30
min. )

II peak: HPLC: retention time: 23.5 minutes NMR (CDCl ₃ solution): δ 1.45 (9H,
s), 2.8 to 2.9 (2H, d), 4.0 to 4.2
(2H, d), 5.2 (2H, s), 7.4 (5H,
m) IR (KBr): ν 2300 cm ^-1 MS (EI method): m / z 361 (M ⁺ ).

Comparative Example 2. "BOP-based Boc-Asn
And Gly-OBzl Condensation Reaction "

Experiments were carried out using the same reagents as in Example 1. 100 mg of Boc-Asn was dissolved in 1 ml of DMF, 300 μl of DIEA and 390 mg of BOP.
Was added and stirred at 0 ° C. for 5 minutes. Gly-OBzl.Tos (150 mg) dissolved in 500 μl of DMF was added, and the mixture was stirred at 0 ° C. for 30 minutes. HPLC of a part of the reaction mixture
As a result of analysis, it was confirmed that the I peak hardly appeared and that the β-cyanoalanine derivative (II peak) was quantitatively produced. 2.0 mg of 1 was added to 10 mg of the obtained II peak component.
0% ethanedithiol / 40% TFA / dichloromethane was added and stirred at room temperature for 20 minutes. The solvent was removed by reduced pressure, and the residue was dissolved in 50% methanol / water and subjected to HPLC analysis. Two peak components appearing at 12 minutes (III peak) and 17 minutes (IV peak) were collected and analyzed. From NMR and FAB-MS, the III peak is β-cyanoalanylglycine benzyl ester (Ala (C
N) -Gly-OBzl), and the IV peak was confirmed to be γ-N-tert-butylasparaginylglycine benzyl ester (Asn (tBu) -Gly-OBzl).

III peak: NMR (CDCl ₃ solution): δ 2.9 to 3.1 (2
H, d), 4.1 (2H, br), 5.2 (2H,
s), 7.3 (5H, m) FABMS: m / z 262 ((M + H) ⁺ ) IV peak: NMR (CDCl ₃ solution): δ 1.3 (9H, s),
4.0-4.2 (1H, br), 5.2 (2H, s),
6.6 (1H, br), 7.3 (5H, m) FABMS: m / z 336 ((M + H) ⁺ ).

Example 1. Boc-As by BOP
Reaction of n with Gly-OBzl ”Boc-Asn 100 mg, and Gly-OBzl
・ Tos 145mg dissolved in DMF1ml, HON
B (280 mg) and DIEA (300 μl) were added, and the mixture was stirred at room temperature for 30 minutes. BOP (190 mg) was added, and the mixture was stirred at room temperature for 3 days.
Stir for 0 minutes. A part of the reaction solution was analyzed by reverse phase HPLC to give Example 1. It was confirmed that the area of the II peak appearing in 1. was 1% or less.

Example 2. "BOP-based Boc-Asn
And Gly-OBHzl condensation reaction "HOOBt 280m instead of HONB as an additive
Example 1 with the addition of g. The same reaction was carried out. HPL
From C, it was confirmed that the area of the II peak was 1% or less.

Example 3. The same reaction as in Comparative Example 2 was conducted under different conditions. The analysis was performed in the same manner. The results are shown in Table 1.

[0027]

[Table 1]

Example 4. The same procedure as in Example 3 was used to study the effect of alcohol additives on various condensing agents. The analysis was performed in the same manner. The results are shown in Table 2.

[0029]

[Table 2]

Comparative Example 3. "Acyl carrier protein by the solid phase method using BOP as a condensing agent (65-74)
(Synthesis of ACP (65-74)) "Merrifield Res
sin) was used as the starting material for stepwise synthesis.

1) De-Boc reaction Boc-Gly-O-resin (Resin) 0.79 g
(0.63 mmol / g) was placed in an eggplant-shaped flask, and 4
0% trifluoroacetic acid / dichloromethane was added and gently stirred for 30 minutes. Pour the solution into a glass filter,
The solution was removed by filtration. The residual resin was washed twice with dichloromethane and twice with 10% DIEA / dichloromethane, alternately washed with dichloromethane and diethyl ether, and then dried in air to obtain Gly-O-resin.

2) Condensation reaction of asparagine Boc-Asn 232 mg was dissolved in DMF 20 ml. DIEA (0.7 ml) was added subsequently to the resin obtained in the previous section, and the mixture was gently stirred at room temperature. 900 mg of BOP dissolved in 10 ml DMF was added, and the mixture was stirred at room temperature for 30 minutes. A part of the resin was taken, and it was confirmed by a Kaiser test that the reaction was quantitatively proceeding. After stirring for another 10 minutes, the solution was poured into a glass filter, and the resin was washed with dichloromethane and diethyl ether alternately and dried in air. Thereafter, the above operations 1) and 2) were repeated. The condensation reaction was performed 9 times in total to obtain 1.4 g of resin.

3) Deprotection with hydrogen fluoride Using 500 mg of the resin obtained in the preceding section, deprotection was carried out according to a conventional method. Extraction with 20% acetic acid / water and lyophilization gave 250 mg of crude product. Part of HPL after drying
C analysis was performed. In addition to 14 minutes (V peak) corresponding to the target compound, a signal (V peak) corresponding to tBu amide at 17.5 minutes.
I peak) appeared. (Area ratio 2: 1)

Example 5. "Synthesis of ACP (65-74) Using HOOBt as Additive" Comparative Example 3 in which HOOBt was added during the condensation reaction of asparagine. The same reaction was carried out. 1) De-Boc reaction The same treatment as in Comparative Example 3 was performed. 2) Condensation reaction of asparagine Boc-Asn 232mg, HOOBt 280mg
Was dissolved in 20 ml of DMF. DIEA (0.7 ml) was added subsequently to the resin obtained in the previous section, and the mixture was gently stirred at room temperature. BOP 900 mg dissolved in 10 ml DMF
Was added and the mixture was stirred at room temperature for 30 minutes. Hereinafter, Comparative Example 3. The same process was performed. Thereafter, the above operations 1) and 2) were repeated, but the condensation reaction other than asparagine was carried out without adding an additive. Conducted a total of 9 condensation reactions,
1.4 g of resin was obtained. 3) Deprotection with hydrogen fluoride Deprotection was performed according to Comparative Example 3. Part of HP after drying
As a result of LC analysis, only the target V peak appeared and the by-product VI peak was not detected.

Claims (4)

[Claims] 1. Formula (I) {A-0-B} X (I) [wherein A is Or [Chemical 2] (However, R represents a halogen, an alkyl group, an aralkyl group, or an aryl group, and n represents an integer of 0 to 4) or (However, R ₁ , R ₂ , R ₃ , and R ₄ each independently represent hydrogen, an alkyl group, an aralkyl group, or an aryl group, and R ₁ and R ₃ and R ₂ and R ₄ are independent of each other. A ring may be formed), and B is (However, R ₅ and R ₆ are each independently an alkyl group, an aralkyl group, or an aryl group, R ₅ may form a ring with R ₆ ), X is a halogen ion, and (PF ₆ ) ⁻ or (BF ₄ ) ⁻ ]], a chemical synthesis of a peptide which activates the carboxyl group of side chain unprotected asparagine or side chain unprotected glutamine and condenses with a free amino group. In law,
A peptide comprising the addition of an alcohol component represented by the formula (II) A-OH (II) [wherein A is the same as defined above and is independently selected from the group A of the formula (I)]. Synthetic method. 2. The condensing agent represented by the formula (I) is benzotriazol-1-yloxy tris (dimethylamino) phosphonium (Benzotriazol-1-y).
l-oxy-tris (dimethylamino)
phosphonium) salt, benzotriazole-1
-Iloxy trispyrrolidinophosphonium (Ben
zotriazol-1-yl-oxy-tris-p
yrrolidinophosphonium) salt, 2
-(1H-benzotriazol-1-yl) -1,1,
3,3-Tetramethyluronium (2- (1H-Ben
zotriazol-1-yl) -1,1,3,3-t
Etramethylurium) salt, or 2-
(5-Norbornene-2,3-dicarboximide) tetramethyluronium (2- (5-Norbornen
e-2,3-dicarboximido) tetra
2. The method according to claim 1, which is a (methyluronium) salt. 3. The alcohol component represented by formula (II) is 3-hydroxy-3,4-dihydro-4-oxo-
1,2,3-benzotriazine (3-Hydroxy-
3,4-dihydro-4-oxo-1,2,3-b
enzotriazine), N-hydroxy-5-norbornene-2,3-dicarboximide (N-Hyd)
roxy-5-norbornene-2,3-dic
arboximide), N-hydroxyphthalimide (N-hydroxyphthalimide), 1-
Hydroxybenzotriazole (1-Hydroxyb
The method according to claim 1, which is an enzotriazole). 4. The method according to claim 1, wherein the protecting group for the α-amino group of the amino acid used is a t-butoxycarbonyl group.