Enhancement in the rate of conversion of peptide Cys-Pro esters to peptide thioesters by structural modification Toru Kawakami ⇑ , Akitaka Kamauchi, Emi Harada, Saburo Aimoto Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan article info Article history: Received 8 September 2013 Revised 22 October 2013 Accepted 25 October 2013 Available online 4 November 2013 Keywords: Chemical ligation CPE peptide Diketopiperazine N–S acyl shift Peptide thioester abstract We previously reported that the peptide containing a Cys-Pro ester (CPE) moiety is spontaneously trans- formed into a peptide thioester via an N to S acyl shift followed by diketopiperazine formation. In an attempt to identify more reactive structures for the formation of a peptide thioester, we modified the CPE structure, in which the Pro residue in the CPE moiety was replaced with N-substituted glycine deriv- atives. These peptides were transformed into a peptide thioester more rapidly. Alternatively, the addition of an amino acid residue at the C-terminus of the CPE moiety also accelerated thioester formation. Ó 2013 Elsevier Ltd. All rights reserved. The peptide thioester is one of key building blocks for achieving protein synthesis by the ligation method. 1 It is used in the thioester method 2 and in native chemical ligation (NCL). 3 In a previous study, we reported that a peptide containing a Cys-Pro ester (CPE) sequence at the C-terminus, denoted as a CPE peptide 1a, is spontaneously transformed into a peptide diketopiperazine (DKP) thioester 2a, and the ligation product 4 is produced in the presence of the Cys peptide 3 (Scheme 1). 4 In this study, we mod- ified the structure of the CPE group, in an attempt to design a more reactive structure for the formation of a peptide thioester. The CPE peptide 1a is transformed into the thioester 2a via an N–S acyl shift, followed by DKP formation (Scheme 2). We focused on improving the DKP formation step in producing the thioester. The amide bond of the Cys-Pro moiety of the S-peptide intermediate 5a should adopt a cis conformation to form a DKP structure. 5 The cis conformation at the N-substituted glycine residue is preferred to that at the Pro residue. 6 Therefore, the Pro residue in the CPE moiety was replaced with a series of N- substituted glycine derivatives. We prepared several peptides 1b–d (peptide = Ala-Lys-Leu-Arg-Phe-Gly), with different types of substitutions (Scheme 2). 7 These peptides would be trans- formed into the peptide DKP thioester 2 via an N–S acyl shift reaction, followed by DKP formation, in which the cis conforma- tion intermediate cis-5 would be favored for DKP formation. A mixture of these peptides 1a–d (2 mM each) was treated in 0.1 M sodium phosphate buffer (pH 7.8) at 37 °C in the presence of 50 mM sodium mercaptoethanesulfonate (MESNa), 20 mM tris(2-carboxyethyl)phosphine (TCEP), 6 M guanidine (Gdn), and 3 mM Boc-His(Bom)-OH (as an internal standard), in which DKP thioester 2 was further transformed into the thioester, Ala-Lys- Leu-Arg-Phe-Gly-SCH 2 CH 2 SO 3 H(6a) 8 (Scheme 2). The conversion of these peptides 1a–d, relative to the internal standard, was monitored by RP–HPLC, in which the thioester 6a was produced and the DKP thioesters 2a–d were not observed (Figs. 1A and 2A). The results showed that peptide 1d was consumed within 6 h, and the order of the reaction rate was 1d > 1c > 1b > 1a. This result indicates that the replacement of the Pro residue by N- substituted glycine residues accelerates the formation of the DKP thioester, and that the bulky substituent at the amide tends to increase the concentration of the cis conformation of 5, thus enhancing the rate of DKP formation. 0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2013.10.121 ⇑ Corresponding author. E-mail address: [email protected](T. Kawakami). N H N O O HS O R 1 O peptide 1 S O HN peptide 1 N O O 1a 2a H 2 N HS O peptide 2 N H HS O peptide 2 O peptide 1 3 4 Scheme 1. Peptide thioester formation and its ligation via the use of CPE peptide 1a. Tetrahedron Letters 55 (2014) 79–81 Contents lists available at ScienceDirect Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Transcript
Tetrahedron Letters 55 (2014) 79–81
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier .com/ locate/ tet le t
Enhancement in the rate of conversion of peptide Cys-Pro estersto peptide thioesters by structural modification
0040-4039/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tetlet.2013.10.121
⇑ Corresponding author.E-mail address: [email protected] (T. Kawakami).
NH
NO
O
HS
OR1
O
peptide 1S
O
HNpeptide 1 N
O
O1a 2a
H2N
HS
O
peptide 2
NH
HS
O
peptide 2O
peptide 1
3
4
Scheme 1. Peptide thioester formation and its ligation via the usepeptide 1a.
Toru Kawakami ⇑, Akitaka Kamauchi, Emi Harada, Saburo AimotoInstitute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
Article history:Received 8 September 2013Revised 22 October 2013Accepted 25 October 2013Available online 4 November 2013
We previously reported that the peptide containing a Cys-Pro ester (CPE) moiety is spontaneously trans-formed into a peptide thioester via an N to S acyl shift followed by diketopiperazine formation. In anattempt to identify more reactive structures for the formation of a peptide thioester, we modified theCPE structure, in which the Pro residue in the CPE moiety was replaced with N-substituted glycine deriv-atives. These peptides were transformed into a peptide thioester more rapidly. Alternatively, the additionof an amino acid residue at the C-terminus of the CPE moiety also accelerated thioester formation.
� 2013 Elsevier Ltd. All rights reserved.
of CPE
The peptide thioester is one of key building blocks for achievingprotein synthesis by the ligation method.1 It is used in the thioestermethod2 and in native chemical ligation (NCL).3 In a previousstudy, we reported that a peptide containing a Cys-Pro ester(CPE) sequence at the C-terminus, denoted as a CPE peptide 1a,is spontaneously transformed into a peptide diketopiperazine(DKP) thioester 2a, and the ligation product 4 is produced in thepresence of the Cys peptide 3 (Scheme 1).4 In this study, we mod-ified the structure of the CPE group, in an attempt to design a morereactive structure for the formation of a peptide thioester.
The CPE peptide 1a is transformed into the thioester 2a via anN–S acyl shift, followed by DKP formation (Scheme 2). Wefocused on improving the DKP formation step in producing thethioester. The amide bond of the Cys-Pro moiety of the S-peptideintermediate 5a should adopt a cis conformation to form a DKPstructure.5 The cis conformation at the N-substituted glycineresidue is preferred to that at the Pro residue.6 Therefore, thePro residue in the CPE moiety was replaced with a series of N-substituted glycine derivatives. We prepared several peptides1b–d (peptide = Ala-Lys-Leu-Arg-Phe-Gly), with different typesof substitutions (Scheme 2).7 These peptides would be trans-formed into the peptide DKP thioester 2 via an N–S acyl shiftreaction, followed by DKP formation, in which the cis conforma-tion intermediate cis-5 would be favored for DKP formation. Amixture of these peptides 1a–d (2 mM each) was treated in0.1 M sodium phosphate buffer (pH 7.8) at 37 �C in the presence
of 50 mM sodium mercaptoethanesulfonate (MESNa), 20 mMtris(2-carboxyethyl)phosphine (TCEP), 6 M guanidine (Gdn), and3 mM Boc-His(Bom)-OH (as an internal standard), in which DKPthioester 2 was further transformed into the thioester, Ala-Lys-Leu-Arg-Phe-Gly-SCH2CH2SO3H (6a)8 (Scheme 2). The conversionof these peptides 1a–d, relative to the internal standard, wasmonitored by RP–HPLC, in which the thioester 6a was producedand the DKP thioesters 2a–d were not observed (Figs. 1A and2A). The results showed that peptide 1d was consumed within6 h, and the order of the reaction rate was 1d > 1c > 1b > 1a. Thisresult indicates that the replacement of the Pro residue by N-substituted glycine residues accelerates the formation of theDKP thioester, and that the bulky substituent at the amide tendsto increase the concentration of the cis conformation of 5, thusenhancing the rate of DKP formation.
Figure 2. Conversion of the modified CPE peptides into thioester 6a in 0.1 Msodium phosphate buffer (pH 7.8) at 37 �C in the presence of 50 mM MESNa, 20 mMTCEP, and 6 M Gdn.9 (A) Modified CPE peptides 1b–d containing N-substituted Glyresidues (see Scheme 2), (B) Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-OCH2CO-Xaa-NH2
Scheme 2. Mechanism of the formation of DKP peptide thioesters.
80 T. Kawakami et al. / Tetrahedron Letters 55 (2014) 79–81
During the search for structures that could be used in thioesterformation, we discovered an alternative possibility, namely thatthe addition of an amino acid residue after ester moiety of theCPE group accelerated thioester formation. A mixture of CPEpeptides containing additional amino acid residues at theC-terminus, Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-OCH2CO-Xaa-NH2
(8) (Xaa = Gly, a; Ala, b; Val, c; Tle (tert-leucine), d; Pro, e)7 wastreated under the same conditions as were used for the reactionof 1a–d (Fig. 1(B), Fig. 2(B)). The CPE peptides 8c, d containing
retention time / min
acet
onitr
ile /
%
rela
tive
abso
rban
ceat
220
nm
0 10 20 30
15
20
37
retention time / min
rela
tive
abso
rban
ceat
220
nm
0 10 20
acet
onitr
ile /
%
15
201a
1b 1d6a
1c
(A)
(B) 6a
8a8b
8c 8d8e
Boc-His(Bom)(internal standard)
Figure 1. RP–HPLC of the reaction mixture derived from a mixture of the modifiedCPE peptides, which were transformed into the thioester 6a, after 1 h in 0.1 Msodium phosphate buffer (pH 7.8) at 37 �C in the presence of 50 mM MESNa, 20 mMTCEP, 6 M Gdn, and 3 mM Boc-His(Bom)-OH. (A) Modified CPE peptides 1b–dcontaining N-substituted Gly residues (see Scheme 2), (B) Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-OCH2CO-Xaa-NH2 (8) (Xaa = Gly, a; Ala, b; Val, c; Tle, d; Pro, e). Column:YMC-Pack ProC18 (4.6 � 150 mm). Eluent: 0.1% TFA in aq acetonitrile, 1.0 mL/min.Gradient of acetonitrile concentration is shown on the chromatogram.
a CPE peptides, Ala-Lys-Leu-Arg-Phe-Xaa-Cys-Pro-OCH2CO-Tle-NH2 (9)(Xaa = Ala, b; Val, c; Ser, d) (Xaa = Gly, 8d), were reacted in 0.1 M sodiumphosphate buffer (pH 7.8) at 37 �C for 6 h in the presence of 50 mM MESNa,20 mM TCEP, and 6 M Gdn.
b Ala-Lys-Leu-Arg-Phe-Xaa-SCH2CH2SO3H (6) (Xaa = Gly, a; Ala, b; Val, c; Ser, d).Ala-Lys-Leu-Arg-Phe-Xaa-SCH2CH2SO3H (d6) (Xaa = Ala, b; Val, c; Ser, d).
c Ala-Lys-Leu-Arg-Phe-Xaa-OH (7) (Xaa = Gly, a; Ala, b; Val, c; Ser, d).d CPE peptide 9d was reacted with Cys in 0.1 M sodium phosphate buffer (pH 7.8)
at 37 �C for 6 h in the presence of 50 mM MESNa, 20 mM TCEP, and 6 M Gdn.Ala-Lys-Leu-Arg-Phe-Ser-Cys (11d). Ala-Lys-Leu-Arg-Phe-D-Ser-Cys (d11d).
an additional amino acid residue with bulky side chains, such asVal and Tle, were converted into the thioester more rapidly, andthe reaction rate was similar to that for peptide 1d. A peptide con-taining a Pro residue at the C-terminus was converted to the thio-ester in poor yields, and the CPE peptide with a C-terminalcarboxylic acid, Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-OCH2CO2H(1a0) was not transformed into the thioester 6a (data not shown).These results suggest that the presence of an amide proton at theposition after the glycolic acid moiety is required for a successfulreaction, although the mechanism for this acceleration is not clearat this point. A CPE peptide with an additional amino acid residuecan be easily prepared by Fmoc-based solid phase peptide synthe-sis (SPPS).4,7
Figure 3. Reaction of Ser CPE peptides, Ala-Lys-Leu-Arg-Phe-Ser-Cys-Pro-OCH2CO-Xaa-NH2 (Xaa = Tle, 9d; none, 10d) to thioester 6d in 0.1 M sodium phosphatebuffer at 37 �C in the presence of 50 mM MESNa, 20 mM TCEP, and 6 M Gdn.9 Theratio of the D-Ser thioester, Ala-Lys-Leu-Arg-Phe-D-Ser-SCH2CH2SO3H (d6d) in % isshown in brackets, as determined by the area under the RP–HPLC peak.
T. Kawakami et al. / Tetrahedron Letters 55 (2014) 79–81 81
Next, thioesterification at the chiral amino acid residues wascarried out using the model peptide, Ala-Lys-Leu-Arg-Phe-Xaa-Cys-Pro-OCH2CO-Tle-NH2 (9) (Xaa = Ala, b; Val, c; Ser, d).7 The pep-tide thioesters, Ala-Lys-Leu-Arg-Phe-Xaa-SCH2CH2SO3H (6)(Xaa = Gly, a; Ala, b; Val, c; Ser, d)8 were isolated by RP–HPLC afterthe reaction of the CPE peptides 8d and 9b–d with MESNa at pH 7.8for 6 h (Table 1). In all cases, peptide thioesters 6 were obtained ingood yields, although small amounts of the hydrolysis product,Ala-Lys-Leu-Arg-Phe-Xaa-OH (7) (Xaa = Gly, a; Ala, b; Ser, d), wereproduced. Epimerization of the amino acid residue at the thioesterwas also observed. These hydrolysis and epimerization reactionsoccurred after thioester formation, and not during the thioesterifi-cation step.4 In the case of the Val thioester 6c, with a bulky sidechain, these side reactions were not observed under the conditionsused, whereas the Gly thioester 6a was easily hydrolyzed (11%)and the Ser thioester 6d was easily epimerized to give Ala-Lys-Leu-Arg-Phe-D-Ser-SCH2CH2SO3H (d6d) (25%).
The Ser residue underwent more extensive epimerization thanamino acids containing aliphatic side chains. We previously re-ported that the gradual epimerization of the Ser thioester wasdependent on the reaction time and pH of the solution used inthe reaction of the original CPE peptide.4 The Ser CPE peptide 9dwas reacted with MESNa under the several pH conditions to pro-duce the peptide thioester 6d, in which the epimerized thioesterd6d was also observed. The reaction rate and the extent of epimer-ization were compared with the reaction of the original CPE pep-tide without Tle, Ala-Lys-Leu-Arg-Phe-Ser-Cys-Pro-OCH2CO-NH2
(10d) (Fig. 3). The conversion rate was enhanced, when the Tlewas added at the C-terminus and when the pH of the reaction solu-tion was high. The epimerization at the Ser residue of the thioesterwas relatively suppressed at the point of similar conversion, whenthe reaction time was short and when the pH was low. It is note-worthy that the epimerization occurs after the formation of thethioester, not during the thioesterification step.4 In addition,hydrolysis was reduced in the reaction of CPE peptide 9d comparedwith the original CPE peptide 10d. When peptides 9d and 10d wasreacted at pH 7.5 and 8.0, respectively, hydrolyzed peptide 7d wasobserved in 7% and 53%, respectively, as determined by the areaunder the RP–HPLC peak, after 8-h reaction at the point of similar
conversion. Furthermore, when the Ser CPE peptide 9d was reactedin the presence of Cys, in which a Ser thioester is formed, the reac-tion with Cys was immediate and the ligated product, Ala-Lys-Leu-Arg-Phe-Ser-Cys (11d)10 was obtained in high yields (Table 1, entry5). No evidence for the formation of the hydrolysis product 7d wasfound, and minimal levels of the epimerization product (2%), Ala-Lys-Leu-Arg-Phe-D-Ser-Cys (d11d), were found, as evidenced byRP–HPLC.
In conclusion, the rate of thioesterification of the CPE peptidewas enhanced by two types of structural modifications: One isthe substitution of the Pro residue by an N-substituted Gly residue,and another is the addition of an amino acid residue at the C-ter-minus of the CPE structure. In both cases the bulky substituentsat the N-substituted Gly residue or the side chain of the additionalamino acid residues efficiently enhance thioester formation. In thelatter case, the addition of an amino acid residue, such modifiedCPE peptides can be easily prepared by the Fmoc-based SPPS pro-cedure. The newly modified CPE structures can be used to mediatethe formation of the peptide thioesters under milder conditionsthan are used for the original CPE structure, and side reactions,such as epimerization and hydrolysis, which proceed under basicconditions, are suppressed.
Acknowledgments
This research was supported, in part, by Grants-in-Aid for Scien-tific Research from the Ministry of Education, Culture, Sports, Sci-ence and Technology, Japan.
Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.tetlet.2013.10.121.
References and notes
1. (a) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030–10074; (b) Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249–289; (c) Aimoto, S.Curr. Org. Chem. 2001, 5, 45–87; (d) Dawson, P. E.; Kent, S. B. H. Annu. Rev.Biochem. 2000, 69, 923–960; (e) Aimoto, S. Biopolymers (Pept. Sci.) 1999, 51,247–265.
3. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776–779.
4. (a) Kawakami, T.; Aimoto, S. Chem. Lett. 2007, 36, 76–77; (b) Kawakami, T.;Aimoto, S. Tetrahedron Lett. 2007, 48, 1903–1905; (c) Kawakami, T.; Aimoto, S.Tetrahedron 2009, 65, 3871–3877.
5. Fischer, P. M. J. Pept. Sci. 2003, 9, 9–35.6. Sui, Q.; Borchardt, D.; Rabenstein, D. L. J. Am. Chem. Soc. 2007, 129, 12042–
12048.7. See Supplementary data.8. Thioester 6a: MS (MALDI): found m/z 816.0, calcd for (M+H)+ 815.4; amino acid
analysis: Gly0.99Ala1.0Leu1Phe0.93Lys1.0Arg1.0. Compound 6b: MS (MALDI):found m/z 829.8, calcd for (M+H)+ 829.4; amino acid analysis: Ala2.0Leu1
Phe0.91Lys1.0Arg1.0. Compound 6c: MS (MALDI): found m/z 857.7, calcd for(M+H)+ 857.4; amino acid analysis: Ala1.0Val0.99Leu1Phe0.92Lys1.0Arg1.0.Compound 6d: MS (MALDI): found m/z 845.6, calcd for (M+H)+ 845.4; aminoacid analysis: Ser0.89Ala1.0Leu1Phe0.96Lys1.0Arg1.0.
9. In the reaction mixture hydrolysis product 7 of the thioester was observed afterthe prolonged reaction time, but in the plots only the conversion of themodified CPE peptides is shown.
10. Compound 11d: MS (MALDI): found m/z 825.2, calcd for (M+H)+ 824.5; aminoacid analysis: Ser0.77Ala1.0CysndLeu1Phe0.92Lys1.0Arg1.0.
