Synthesis of Liraglutide Through Threonine Ligation†

doi:10.7503/cjcu20180350

Abstract

Abstract:

A new method for the synthesis of liraglutide was reported, using threonine ligation to couple the segment histidine¹-glycine⁴and the segment threonine⁵-glycine³¹. This method addressed the problem of low coupling efficiency at N-terminal residuals of liraglutide. The lysine²⁰ side-chain modification group was directly introduced onto the unprotected linear peptide by controlling pH of the solution. The ligation reaction was achieved with 76.4% overall yield. This new method is featured with the advantages of high chemoselectivity and mild reaction conditions, and has a potential of industrial application.

Key words: Liraglutide, Threonine ligation, Side-chain modification

CLC Number:

O629.72

TrendMD:

FAN Jiahui,BIAN Yanan,SU Xianbin. Synthesis of Liraglutide Through Threonine Ligation^†[J]. Chem. J. Chinese Universities, 2018, 39(12): 2679.

Figures/Tables 8

Fig.1 Structure of liraglutide

Scheme 1 Model reaction of Gly and Thr

Scheme 2 Synthesis routes of liraglutide

Table 1 Appearance, melting points and optical rotation for compounds 1—12

Compd.	Appearance	m. p./℃	[α $] D 20$ /(°) (c 1, CHCl₃)	Compd.	Appearance	m. p./℃	[α $] D 20$ /(°) (c 1, CHCl₃)
1	White solid	129.4—130.3		7	Pale yellow oil
2	Pale yellow solid	78.2—80.0	-33.5	8	White solid
3	White solid	95.6—97.3	-27.2	9	White solid
4	Pale yellow oil			10	White solid
5	White solid			11	Pale yellow oil
6	White solid			12	White solid	64.5—65.7	-15.2

Table 1 Appearance, melting points and optical rotation for compounds 1—12

Compd.	Appearance	m. p./℃	[α $] D 20$ /(°) (c 1, CHCl₃)	Compd.	Appearance	m. p./℃	[α $] D 20$ /(°) (c 1, CHCl₃)
1	White solid	129.4—130.3		7	Pale yellow oil
2	Pale yellow solid	78.2—80.0	-33.5	8	White solid
3	White solid	95.6—97.3	-27.2	9	White solid
4	Pale yellow oil			10	White solid
5	White solid			11	Pale yellow oil
6	White solid			12	White solid	64.5—65.7	-15.2

Table 2 1H NMR and ESI-MS data for compounds 1—12

Compd.	¹H NMR(400 MHz, CDCl₃), δ	ESI-MS, m/z
1	10.05(s, 1H, CHO), 7.90—7.21(m, 12H, PhH), 5.42(s, 1H, NH), 4.46(d,J=6.8 Hz, 2H, OCH₂), 4.39(d, J=6.8 Hz, 2H, NCH₂CO), 4.26(t, J=7.2 Hz, 1H, PhCHPh)	424.6[M+Na]⁺
2	7.75—6.85(m, 17H, PhH), 6.45(s, 1H,NH), 5.54[s, 1H, NCH(Ph)O], 5.28(q, J=11.5 Hz, 2H, PhCH₂O), 4.36—3.77(m, 6H, OCH₂+NCH₂CO+PhCHPh, OCH), 3.14(d, J=17.2 Hz, 1H, NCHCO), 1.49(d, J=6.0 Hz, 3H, CH₃)	593.2[M+H]⁺
3	8.10(br, 3H, CONH+OH), 7.75—7.25(m, 13H, PhH), 5.18(s, 2H, PhCH₂O), 4.64(d, J=7.8 Hz, 1H, NCHCO), 4.36(m, 3H, PhCHPh+NCH₂CO), 4.18(m, 1H, OCH), 3.97(d, J=5.6 Hz, 2H, OCH₂), 1.18(d, J=6.4 Hz, 3H, CH₃)	489.5[M+H]⁺
Compd.	¹H NMR(400 MHz, CDCl₃), δ	ESI-MS, m/z
4		811.1[M+H]⁺
5	10.11(s, 1H, CHO), 8.33(d,J=8.0 Hz, 1H, CONH), 7.86—6.69(m, 21H, ArH), 6.55(s, 1H, CONH), 6.43(s, 1H, CONH), 4.52—4.15(m, 5H, NCH₂CO+3NCHCO), 3.02—2.89(m, 2H, ArCH₂), 2.39—2.20(m, 4H, CH₂COO+CH₂), 1.41—1.37(m, 21H, 7CH₃)	915.4[M+H]⁺
6		997.2[M+3H]³⁺
7		1296.3[M+3H]³⁺
8		1128.6[M+3H]³⁺
9		1251.2[M+3H]³⁺
10		1138.6[M+3H]³⁺
11		1437.4[M+3H]³⁺
12	6.27(d,J=7.6 Hz, 1H, CONH), 4.54(m, 1H, NCHCO), 2.42(m, 2H, CH₂COO), 2.23(m, 3H, CH₂CO+CCH), 1.93(m, 1H, CCH), 1.60(m, 2H, CH₂), 1.47(s, 9H, 3CH₃), 1.32—1.25(m, 24H, 12CH₂), 0.88(t, J=7.6 Hz, 3H, CH₃)	440.2[M-H]^-

Table 3 Effects of solvents on the yield of intermediate 2

Solvent	HPLC Yield of intermediate 2(%)
Solvent	10 min	2 h
V(Py):V(AcOH)=1:1	91	98
V(Py):V(AcOH)=1:3	87	97
DMF		<1
DMSO		<1
V(H₂O):V(MeCN)=1:1	<1	3.6
V(Et₃N):V(AcOH)=1:3	82	91
AcOH	<1	2.3

Fig.2 HPLC and ESI-MS monitoring of side chain reactionCoupling time/min: a. 10; b. 30; c. acidification time: 30; d. standard sample of liraglutide.

Table 4 Effects of pH values on the yield of coupling intermediate

pH	HPLC yield(%)		pH	HPLC yield(%)
pH	10 min	30 min	pH	10 min	30 min
8	16	27	12	68	91
10	71	93	14	18	30

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