- Email: [email protected]
Optimal peptide hydrazide ligation with C-terminus Asp, Asn, and Gln hydrazides
Accepted Manuscript Optimal peptide hydrazide ligation with C-terminus Asp, Asn and Gln hydrazides Xiaobo Tian, Jie Li, Wei Huang PII: DOI: Reference:
S0040-4039(16)30969-8 http://dx.doi.org/10.1016/j.tetlet.2016.07.101 TETL 47963
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
16 June 2016 20 July 2016 30 July 2016
Please cite this article as: Tian, X., Li, J., Huang, W., Optimal peptide hydrazide ligation with C-terminus Asp, Asn and Gln hydrazides, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.07.101
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Optimal peptide hydrazide ligation with C-terminus Asp, Asn and Gln hydrazides Xiaobo Tian1,2,*, Jie Li2, Wei Huang2,* 1
College of Pharmacy, Nanchang University, 461 Bayi Road, Nanchang 330006, PR China CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203 2
*Corresponding authors: Xiaobo Tian Tel: +86-791-86360902 Fax: +86-791-86360902 Email: [email protected] Prof. Wei Huang Tel: +86-21-20231000 ext. 2517 Fax: +86-21-50807088 Email: [email protected]
Abstract: Peptide hydrazide ligation is a newly reported approach using C-terminus hydrazide as the precursor of thioester for ligation with N-terminus Cys-peptides. However, peptide hydrazides with C-terminus Asp, Asn, and Gln are difficult to generate because of cyclization side reaction happened during peptide cleavage from Wang resin. To prevent the cyclization, we chose the acid-sensitive CTC-resin as the support and utilized two-step cleavage strategy: 1) release the peptide hydrazide bearing side-chain protection from the support with 1% TFA; 2) remove the side-chain protection with 90% TFA to give the corresponding peptide hydrazides. Peptide ligation using these hydrazides with a Cys-peptide indicated the high efficiency in peptide elongation. The current study expands the scope of the peptide hydrazide ligation to all 20 natural amino acids at C-terminal. Keywords: Peptide hydrazide ligation, C-terminus cyclization, CTC-resin
Chemical synthesis of protein has become an important approach to produce homogeneous proteins especially for proteins with precise modifications or with unnatural tags1-4. Solid-phase synthesis of long peptide beyond 50 amino acids encountered challenges on product purity and purification procedures, therefore peptide ligation is crucial to elongate the polypeptide in protein synthesis5. Peptide ligation methods at various amino acid sites, including cysteine,6 serine/threonine,7 histidine, glutamine,8 lysine,9 were reported. Native chemical ligation,6, 10-12 a key method to elongate peptide chain by chemoselective reaction between a C-terminal peptide thioester and an N-terminal cysteine peptide, was widely employed in protein and peptide chemical synthesis due to its high efficiency. In this method, thioester exchange occurs between the peptide thioester and thiol of the N-terminal cysteine on another peptide, then an irreversible S to N acyl shift results the ligation product with native amide bond. Originally, peptide thioester was synthesized via Boc-protection strategy of solid-phase synthesis13, which employs HF for cleavage with hazardous and environmental concerns. For Fmoc-protection peptide synthesis14, thioester is unstable under the basic condition used in deprotection of Fmoc. To overcome this problem, various thioester preparation methods by post-cleavage conversion were developed.15-23 Recently, Liu group have reported an elegant method of peptide hydrazide ligation for protein synthesis24-27. This method features the excellent stability of hydrazide during solid-phase peptide synthesis and the efficient conversion from hydrazide to thioester. As the thioester precursor, C-terminus peptide hydrazide was converted to carbonyl azide by NaNO2, then formed a thioester with a thiol compound, 2-(4-sulfanylphenyl)acetic acid (MPAA), in almost quantitative yield. The resulted in situ thioester exhibited good ligation with Cys-peptides. This approach was also applied in branched peptide ligation28. In the literature24, peptide hydrazides could be successfully prepared when the C-terminus amino acid was 17 of total 20 native amino acids except Asp, Asn, and Gln. The intramolecular cyclization side reaction happened for these three amino acids during peptide cleavage from Wang resin, thus hampered the ligation reaction in next step.
Figure 1. Hypothesis on cleavage mechanism of Asn-hydrazide on Wang resin and CTC resin By analysis on the possible cyclization mechanism as shown in Fig.1, we hypothesize that cleavage cocktail containing high percentage of TFA may lead to rapid deprotection of Trt or tBu groups on side chains before peptide cleavage from Wang resin. Therefore, when hydrazine was released from the Wang resin, the transition state nitrogen anion attacks the unprotected side-chain amide or acid to give the cyclization product. Based on this mechanism, we proposed a solution using an acid-sensitive resin, 2-chlorotrityl chloride (CTC) resin, followed by two-step cleavage.
Firstly, the peptide hydrazide is released from CTC resin under a milder acid condition and the hydrazine nitrogen anion prefers protonation but not attacking the protected side-chain amide or acid because of steric hindrance. Next, the side-chain deprotection is performed using strong acid and the protonated hydrazine is less nucleophilic for cyclization therefore giving the target deprotected peptide hydrazide. To test this idea, we synthesized the pentapeptide hydrazide 1a-d (Leu-Tyr-Arg-Ala-Xaa, Xaa=Asp, Asn, or Gln) on a CTC resin. As shown in Fig. 2a, we treated 1a directly with 95% TFA and the HPLC monitoring of the crude peptide indicated two major peaks which were identified by HRMS as target hydrazide 2a and the cyclization by-product 2a′ in a 3:2 ratio. In the reference24, 2a′ is the major product when using the Wang resin that demonstrated the efficacy of our strategy using the acid-sensitive resin to reduce the cyclization. To further control the cyclization, we performed the two-step cleavage with 1% TFA in DCM (Fig. 2b) then with 95% TFA (Fig. 2c). Mild acid condition released the protected peptide hydrazide 2a’’ (Leu-Tyr(OtBu)-Arg(Pbf)-Ala-Asn(Trt)-NHNH2) and 95% TFA then removed the side-chain protection giving 2a with only trace amount of cyclization by-product. Then, we tested the C-terminus Gln and Asp peptide 1b-d bearing acid-sensitive or acid-resistant side-chain protection. Surprisingly, even under 95% TFA, only peptide hyrazide products were observed without cyclization (Fig. 2d-f). These data implicated that the peptide was cleaved from acid-sensitive resin faster than side-chain deprotection therefore avoided the cyclization. The acid-resistant Bn protection on Asp side-chain also hampered the cyclization that further verified our hypothesis on side-chain deprotection-induced cyclization mechanism. These results clearly showed control on velocities of resin cleavage and side-chain deprotection is effective for preparation of peptide hydrazides with C-terminus Asn, Asp, and Gln. O Leu Tyr(OtBu) Arg(Pbf) Ala N H
R1 Cl nH NN H O
O Leu-Tyr-Arg-Ala N H
or i) 1% TFA ii) 95% TFA
a)
2a
n=1 n=2 n=1 n=1
R1=NHTrt R1=NHTrt R1=OtBu R1=OBn
[M+H]+ 650.3721
2a 2b 2c 2d
d)
2b
O
2
1 1a 1b 1c 1d
R1 n NHNH 2
95%TFA
n=1 n=2 n=1 n=1
R1=NH2 R1=NH2 R1=OH R1=OBn
[M+H]+ 664.3889
2a’ [M+H]+ 633.3459
b)
2a’’
e)
2c
[M+H]+ 651.3578
[M+2H]2+ 600.8180
c)
2a
f)
Retention Time (min)
2d
[M+H]+ 741.4044
Retention Time (min)
Figure 2. Cleavage of peptide hydrazide 1a-d from CTC resin and HPLC profiles of released crude peptides. a) 1a treated with 95% TFA in water; b) 1a treated with 1% TFA in dichloromethane; c) 1a treated with 1% TFA then 95% TFA; d) 1b treated with 95% TFA; e) 1c
treated with 95% TFA; f) 1d treated with 95% TFA. The peaks were marked with compound numbers with HRMS characterization. All the MS result is the monoisotope molecular weight. With the peptide hydrazides 2a-d in hands, we were able to investigate their peptide ligation with a model peptide Cys-Lys-Tyr-Met-His-OH (6) following the reported procedure24. As shown in Scheme 1, peptide hydrazide 2a-d were converted to the carbonyl azide 3a-d with NaNO2, then a thiol compound, 4-mercaptophenylacetic acid (MPAA), was added to give the thioester 4a-d in excellent yields (see supplementary data). The ligation between thioester and Cys-peptide 6 was performed in a pH 7 phosphate buffer in the presence of guanidine hydrochloride at room temperature. The reaction was monitored on the consumption of the thioesters and the accumulation of the ligation product by HPLC and HRMS characterization. Scheme 1 O 2a-d
NaNO2 Leu-Tyr-Arg-Ala N H
O
R1
n N 3
MPAA Leu-Tyr-Arg-Ala N H
O
3a-d
R1 n S CO2H
O
4a-d
HS H2N
O
Lys-Tyr-Met-His-OH O 6 PB, pH7, GnHCl
Leu-Tyr-Arg-Ala N H
R1 H O n N
O
Lys-Tyr-Met-His-OH
SH
5a 5b 5c 5d
n=1 n=2 n=1 n=1
R1=NH2 R1=NH2 R1=OH R1=OBn
As shown in Fig. 3a, the ligation between Asn thioester 4a and 6 was expeditious. After 5 min, the ligation product 5a was observed as the major new formed peak implicating the efficient ligation. The thioester 4a was all consumed after 2h to give 5a as the key product. HRMS data of 5a is in high accordance with the calculated molecular weight. Gln thioester 4b indicated the similar ligation reactivity as Asn (Fig. 3b). For Asp, there was an observed isomer of the thioester 4c as marked 4c’ in Fig. 3c, indicating an identical MS data as 4c, which may be due to the side reaction on the side-chain carboxylic acid. This isomer the thioester was also reported in previous work29,30. The corresponding isomer 5c’ of ligation product 5c was also observed (Fig. 3c and supplementary data). Then, we employed the Bn protected Asp hydrazide 2d for the ligation and the thioester 4d was obtained as a single peak without isomer (supplementary data). Ligation of 4d and 6 also generated the single product 5d (Fig. 3d) that indicated the side-chain protection could prevent the isomer formation. Debenzylation of 5d by mild base of 20 mM K2CO331 provided 5c that was identical with the ligation product from 4c in HPLC retention time and HRMS characterization (Fig. 3d and supplementary data). These results demonstrated the efficient peptide hydrazide ligation could be successfully achieved for C-terminal Asn, Gln, and Asp. In addition, side-chain protection with acid-resistant group such as Bn, could be an optional choice to avoid cyclization during peptide cleavage and isomer formation during ligation.
a)
c)
6
4a + 6 5 min
*
6
4c + 6 5 min
5a 4a
MPAA
*
4c 4c’
4a + 6 2 hr
*
b)
MPAA
6
* 5c’
d)
4b + 6 5 min
*
6
4c + 6 2 hr
5a
5b 4b
MPAA
5c MPAA
6
4d + 6 5 min MPAA
6
*
5d
*
5d
4d MPAA
6
4b + 6 2 hr
4d + 6 2 hr
5b
*
Retention Time (min)
MPAA
MPAA
Retention Time (min)
Figure 3. HPLC profiles of peptide ligation between thioester 4a-d with Cys-peptide 6. a) Ligation of 4a and 6 at 5 min and 2h; HRMS of ligation product 5a: calculated mass, 1298.6138, found, 1298.6098; b) Ligation of 4b and 6 at 5 min and 2h; HRMS of ligation product 5b: calculated mass, 1312.6259, found, 1312.6244; c) Ligation of 4c and 6 at 5 min and 2h; HRMS of ligation product 5c: calculated mass, 1299.5978, found, 1299.5938; d) Ligation of 4d and 6 at 5 min and 2h; HRMS of ligation product 5d: calculated mass, 1389.6448, found, 1389.6404. All the MS result is the monoisotope molecular weight. The internal standard (benzamide) peak was marked with asterisk (*) in the spectra. In conclusion, we have optimized the peptide hydrazide ligation with C-terminal Asp, Asn, and Gln hydrazides. By replacing the Wang resin with the acid-sensitive CTC resin, intramolecular cyclization was prevented because of faster peptide cleavage than side-chain deprotection under one-step or two-step cleavage procedures. The resulted peptide hydrazides, which were unable be obtained by the reported method, were successfully ligated with a Cys-peptide. Bn protection on Asp side chain provided an alternative solution for optimal peptide hydrazide ligation to avoid cyclization and isomerization during the peptide cleavage and ligation. The current work expands the scope of the peptide hydrazide ligation to all 20 natural amino acids at C-terminal for peptide elongation and protein synthesis. Acknowledgements This work was supported by the National Natural Science Foundation of China (NNSFC, No.21372238 and 21572244) and SIMM institute fund (CASIMM0120153004). We thank massspec facility of iHuman Institute for providing us the LC-MS and peptide synthesizer instruments. We thank Dr Raymond Stevens, Dr Zhijie Liu, Dr Houchao Tao, and other colleagues in iHuman and SIMM for helpful discussion.
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Sohma, Y.; Hua, Q. X.; Whittaker, J.; Weiss, M. A.; Kent, S. B. Angew. Chem. Int. Ed. 2010, 49, 5489.
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Hojo, H. Curr. Opin. Struc. Biol. 2014, 26, 16.
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Philip E. Dawson, T. W. M., lan Clark-Lewis,Stephen B. H. Kent Science 1994.
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Lee, C. L.; Li, X. Curr. Opin. Chem. Biol. 2014, 22, 108.
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Siman, P.; Brik, A. Org. Lett. 2012, 14, 1520.
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Yang, R.; Pasunooti, K. K.; Li, F.; Liu, X. W.; Liu, C. F. J. Am. Chem. Soc. 2009, 131, 13592.
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Dawson, P. E.; Kent, S. B. H. Annu. Rev. Biochem. 2000, 69, 923.
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Malins, L. R.; Payne, R. J. Curr. Opin. Chem. Biol. 2014, 22, 70.
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Johnson, E. C. B.; Kent, S. B. H. J. Am. Chem. Soc. 2006, 128, 6640.
13 Schnölzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. Int. J. Pept. Protein Res. 1992, 40, 180. 14 Mende, F.; Seitz, O. Angew. Chem. Int. Ed. 2011, 50, 1232. 15 Ingenito, R.; Bianchi, E.; Daniela Fattori, A.; Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369. 16
Kawakami, T.; Aimoto, S. Chem. Lett. 2007, 36, 76.
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Kawakami, T. Top. Curr. Chem. 2015, 362, 107.
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Graphical Abstract
Optimal peptide hydrazide ligation with
Leave this area blank for abstract info.
C-terminus Asp, Asn and Gln hydrazides Xiaobo Tian1,2,*, Jie Li2, Wei Huang2,* 1College of Pharmacy, Nanchang University, 461 Bayi Road, Nanchang 330006, PR China 2CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203
Highlights 1. Optimization of the peptide hydrazide ligation with C-terminal Asp, Asn, and Gln hydrazides. 2. Replacing the Wang resin with the acid-sensitive CTC resin, intramolecular cyclization is prevented. 3. Extension of peptide hydrazide ligation to all 20 natural amino acids at C-terminal for peptide elongation and protein synthesis.
S0040-4039(16)30969-8 http://dx.doi.org/10.1016/j.tetlet.2016.07.101 TETL 47963
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
16 June 2016 20 July 2016 30 July 2016
Please cite this article as: Tian, X., Li, J., Huang, W., Optimal peptide hydrazide ligation with C-terminus Asp, Asn and Gln hydrazides, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.07.101
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Optimal peptide hydrazide ligation with C-terminus Asp, Asn and Gln hydrazides Xiaobo Tian1,2,*, Jie Li2, Wei Huang2,* 1
College of Pharmacy, Nanchang University, 461 Bayi Road, Nanchang 330006, PR China CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203 2
*Corresponding authors: Xiaobo Tian Tel: +86-791-86360902 Fax: +86-791-86360902 Email: [email protected] Prof. Wei Huang Tel: +86-21-20231000 ext. 2517 Fax: +86-21-50807088 Email: [email protected]
Abstract: Peptide hydrazide ligation is a newly reported approach using C-terminus hydrazide as the precursor of thioester for ligation with N-terminus Cys-peptides. However, peptide hydrazides with C-terminus Asp, Asn, and Gln are difficult to generate because of cyclization side reaction happened during peptide cleavage from Wang resin. To prevent the cyclization, we chose the acid-sensitive CTC-resin as the support and utilized two-step cleavage strategy: 1) release the peptide hydrazide bearing side-chain protection from the support with 1% TFA; 2) remove the side-chain protection with 90% TFA to give the corresponding peptide hydrazides. Peptide ligation using these hydrazides with a Cys-peptide indicated the high efficiency in peptide elongation. The current study expands the scope of the peptide hydrazide ligation to all 20 natural amino acids at C-terminal. Keywords: Peptide hydrazide ligation, C-terminus cyclization, CTC-resin
Chemical synthesis of protein has become an important approach to produce homogeneous proteins especially for proteins with precise modifications or with unnatural tags1-4. Solid-phase synthesis of long peptide beyond 50 amino acids encountered challenges on product purity and purification procedures, therefore peptide ligation is crucial to elongate the polypeptide in protein synthesis5. Peptide ligation methods at various amino acid sites, including cysteine,6 serine/threonine,7 histidine, glutamine,8 lysine,9 were reported. Native chemical ligation,6, 10-12 a key method to elongate peptide chain by chemoselective reaction between a C-terminal peptide thioester and an N-terminal cysteine peptide, was widely employed in protein and peptide chemical synthesis due to its high efficiency. In this method, thioester exchange occurs between the peptide thioester and thiol of the N-terminal cysteine on another peptide, then an irreversible S to N acyl shift results the ligation product with native amide bond. Originally, peptide thioester was synthesized via Boc-protection strategy of solid-phase synthesis13, which employs HF for cleavage with hazardous and environmental concerns. For Fmoc-protection peptide synthesis14, thioester is unstable under the basic condition used in deprotection of Fmoc. To overcome this problem, various thioester preparation methods by post-cleavage conversion were developed.15-23 Recently, Liu group have reported an elegant method of peptide hydrazide ligation for protein synthesis24-27. This method features the excellent stability of hydrazide during solid-phase peptide synthesis and the efficient conversion from hydrazide to thioester. As the thioester precursor, C-terminus peptide hydrazide was converted to carbonyl azide by NaNO2, then formed a thioester with a thiol compound, 2-(4-sulfanylphenyl)acetic acid (MPAA), in almost quantitative yield. The resulted in situ thioester exhibited good ligation with Cys-peptides. This approach was also applied in branched peptide ligation28. In the literature24, peptide hydrazides could be successfully prepared when the C-terminus amino acid was 17 of total 20 native amino acids except Asp, Asn, and Gln. The intramolecular cyclization side reaction happened for these three amino acids during peptide cleavage from Wang resin, thus hampered the ligation reaction in next step.
Figure 1. Hypothesis on cleavage mechanism of Asn-hydrazide on Wang resin and CTC resin By analysis on the possible cyclization mechanism as shown in Fig.1, we hypothesize that cleavage cocktail containing high percentage of TFA may lead to rapid deprotection of Trt or tBu groups on side chains before peptide cleavage from Wang resin. Therefore, when hydrazine was released from the Wang resin, the transition state nitrogen anion attacks the unprotected side-chain amide or acid to give the cyclization product. Based on this mechanism, we proposed a solution using an acid-sensitive resin, 2-chlorotrityl chloride (CTC) resin, followed by two-step cleavage.
Firstly, the peptide hydrazide is released from CTC resin under a milder acid condition and the hydrazine nitrogen anion prefers protonation but not attacking the protected side-chain amide or acid because of steric hindrance. Next, the side-chain deprotection is performed using strong acid and the protonated hydrazine is less nucleophilic for cyclization therefore giving the target deprotected peptide hydrazide. To test this idea, we synthesized the pentapeptide hydrazide 1a-d (Leu-Tyr-Arg-Ala-Xaa, Xaa=Asp, Asn, or Gln) on a CTC resin. As shown in Fig. 2a, we treated 1a directly with 95% TFA and the HPLC monitoring of the crude peptide indicated two major peaks which were identified by HRMS as target hydrazide 2a and the cyclization by-product 2a′ in a 3:2 ratio. In the reference24, 2a′ is the major product when using the Wang resin that demonstrated the efficacy of our strategy using the acid-sensitive resin to reduce the cyclization. To further control the cyclization, we performed the two-step cleavage with 1% TFA in DCM (Fig. 2b) then with 95% TFA (Fig. 2c). Mild acid condition released the protected peptide hydrazide 2a’’ (Leu-Tyr(OtBu)-Arg(Pbf)-Ala-Asn(Trt)-NHNH2) and 95% TFA then removed the side-chain protection giving 2a with only trace amount of cyclization by-product. Then, we tested the C-terminus Gln and Asp peptide 1b-d bearing acid-sensitive or acid-resistant side-chain protection. Surprisingly, even under 95% TFA, only peptide hyrazide products were observed without cyclization (Fig. 2d-f). These data implicated that the peptide was cleaved from acid-sensitive resin faster than side-chain deprotection therefore avoided the cyclization. The acid-resistant Bn protection on Asp side-chain also hampered the cyclization that further verified our hypothesis on side-chain deprotection-induced cyclization mechanism. These results clearly showed control on velocities of resin cleavage and side-chain deprotection is effective for preparation of peptide hydrazides with C-terminus Asn, Asp, and Gln. O Leu Tyr(OtBu) Arg(Pbf) Ala N H
R1 Cl nH NN H O
O Leu-Tyr-Arg-Ala N H
or i) 1% TFA ii) 95% TFA
a)
2a
n=1 n=2 n=1 n=1
R1=NHTrt R1=NHTrt R1=OtBu R1=OBn
[M+H]+ 650.3721
2a 2b 2c 2d
d)
2b
O
2
1 1a 1b 1c 1d
R1 n NHNH 2
95%TFA
n=1 n=2 n=1 n=1
R1=NH2 R1=NH2 R1=OH R1=OBn
[M+H]+ 664.3889
2a’ [M+H]+ 633.3459
b)
2a’’
e)
2c
[M+H]+ 651.3578
[M+2H]2+ 600.8180
c)
2a
f)
Retention Time (min)
2d
[M+H]+ 741.4044
Retention Time (min)
Figure 2. Cleavage of peptide hydrazide 1a-d from CTC resin and HPLC profiles of released crude peptides. a) 1a treated with 95% TFA in water; b) 1a treated with 1% TFA in dichloromethane; c) 1a treated with 1% TFA then 95% TFA; d) 1b treated with 95% TFA; e) 1c
treated with 95% TFA; f) 1d treated with 95% TFA. The peaks were marked with compound numbers with HRMS characterization. All the MS result is the monoisotope molecular weight. With the peptide hydrazides 2a-d in hands, we were able to investigate their peptide ligation with a model peptide Cys-Lys-Tyr-Met-His-OH (6) following the reported procedure24. As shown in Scheme 1, peptide hydrazide 2a-d were converted to the carbonyl azide 3a-d with NaNO2, then a thiol compound, 4-mercaptophenylacetic acid (MPAA), was added to give the thioester 4a-d in excellent yields (see supplementary data). The ligation between thioester and Cys-peptide 6 was performed in a pH 7 phosphate buffer in the presence of guanidine hydrochloride at room temperature. The reaction was monitored on the consumption of the thioesters and the accumulation of the ligation product by HPLC and HRMS characterization. Scheme 1 O 2a-d
NaNO2 Leu-Tyr-Arg-Ala N H
O
R1
n N 3
MPAA Leu-Tyr-Arg-Ala N H
O
3a-d
R1 n S CO2H
O
4a-d
HS H2N
O
Lys-Tyr-Met-His-OH O 6 PB, pH7, GnHCl
Leu-Tyr-Arg-Ala N H
R1 H O n N
O
Lys-Tyr-Met-His-OH
SH
5a 5b 5c 5d
n=1 n=2 n=1 n=1
R1=NH2 R1=NH2 R1=OH R1=OBn
As shown in Fig. 3a, the ligation between Asn thioester 4a and 6 was expeditious. After 5 min, the ligation product 5a was observed as the major new formed peak implicating the efficient ligation. The thioester 4a was all consumed after 2h to give 5a as the key product. HRMS data of 5a is in high accordance with the calculated molecular weight. Gln thioester 4b indicated the similar ligation reactivity as Asn (Fig. 3b). For Asp, there was an observed isomer of the thioester 4c as marked 4c’ in Fig. 3c, indicating an identical MS data as 4c, which may be due to the side reaction on the side-chain carboxylic acid. This isomer the thioester was also reported in previous work29,30. The corresponding isomer 5c’ of ligation product 5c was also observed (Fig. 3c and supplementary data). Then, we employed the Bn protected Asp hydrazide 2d for the ligation and the thioester 4d was obtained as a single peak without isomer (supplementary data). Ligation of 4d and 6 also generated the single product 5d (Fig. 3d) that indicated the side-chain protection could prevent the isomer formation. Debenzylation of 5d by mild base of 20 mM K2CO331 provided 5c that was identical with the ligation product from 4c in HPLC retention time and HRMS characterization (Fig. 3d and supplementary data). These results demonstrated the efficient peptide hydrazide ligation could be successfully achieved for C-terminal Asn, Gln, and Asp. In addition, side-chain protection with acid-resistant group such as Bn, could be an optional choice to avoid cyclization during peptide cleavage and isomer formation during ligation.
a)
c)
6
4a + 6 5 min
*
6
4c + 6 5 min
5a 4a
MPAA
*
4c 4c’
4a + 6 2 hr
*
b)
MPAA
6
* 5c’
d)
4b + 6 5 min
*
6
4c + 6 2 hr
5a
5b 4b
MPAA
5c MPAA
6
4d + 6 5 min MPAA
6
*
5d
*
5d
4d MPAA
6
4b + 6 2 hr
4d + 6 2 hr
5b
*
Retention Time (min)
MPAA
MPAA
Retention Time (min)
Figure 3. HPLC profiles of peptide ligation between thioester 4a-d with Cys-peptide 6. a) Ligation of 4a and 6 at 5 min and 2h; HRMS of ligation product 5a: calculated mass, 1298.6138, found, 1298.6098; b) Ligation of 4b and 6 at 5 min and 2h; HRMS of ligation product 5b: calculated mass, 1312.6259, found, 1312.6244; c) Ligation of 4c and 6 at 5 min and 2h; HRMS of ligation product 5c: calculated mass, 1299.5978, found, 1299.5938; d) Ligation of 4d and 6 at 5 min and 2h; HRMS of ligation product 5d: calculated mass, 1389.6448, found, 1389.6404. All the MS result is the monoisotope molecular weight. The internal standard (benzamide) peak was marked with asterisk (*) in the spectra. In conclusion, we have optimized the peptide hydrazide ligation with C-terminal Asp, Asn, and Gln hydrazides. By replacing the Wang resin with the acid-sensitive CTC resin, intramolecular cyclization was prevented because of faster peptide cleavage than side-chain deprotection under one-step or two-step cleavage procedures. The resulted peptide hydrazides, which were unable be obtained by the reported method, were successfully ligated with a Cys-peptide. Bn protection on Asp side chain provided an alternative solution for optimal peptide hydrazide ligation to avoid cyclization and isomerization during the peptide cleavage and ligation. The current work expands the scope of the peptide hydrazide ligation to all 20 natural amino acids at C-terminal for peptide elongation and protein synthesis. Acknowledgements This work was supported by the National Natural Science Foundation of China (NNSFC, No.21372238 and 21572244) and SIMM institute fund (CASIMM0120153004). We thank massspec facility of iHuman Institute for providing us the LC-MS and peptide synthesizer instruments. We thank Dr Raymond Stevens, Dr Zhijie Liu, Dr Houchao Tao, and other colleagues in iHuman and SIMM for helpful discussion.
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Graphical Abstract
Optimal peptide hydrazide ligation with
Leave this area blank for abstract info.
C-terminus Asp, Asn and Gln hydrazides Xiaobo Tian1,2,*, Jie Li2, Wei Huang2,* 1College of Pharmacy, Nanchang University, 461 Bayi Road, Nanchang 330006, PR China 2CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203
Highlights 1. Optimization of the peptide hydrazide ligation with C-terminal Asp, Asn, and Gln hydrazides. 2. Replacing the Wang resin with the acid-sensitive CTC resin, intramolecular cyclization is prevented. 3. Extension of peptide hydrazide ligation to all 20 natural amino acids at C-terminal for peptide elongation and protein synthesis.