Native chemical ligation at a base-labile 4-mercaptobutyrate Nα-auxiliary

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx

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Native chemical ligation at a base-labile 4-mercaptobutyrate Na-auxiliary Ziv Harpaz, Simon Loibl, Oliver Seitz ⇑ Institut für Chemie, Humboldt Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 23 December 2015 Revised 20 January 2016 Accepted 21 January 2016 Available online xxxx Keywords: Native chemical ligation Auxiliary assisted NCL Extending NCL Dermicidine Peptide coupling Radicals Total chemical protein synthesis

a b s t r a c t Native chemical ligation (NCL) proceeds via a S–N acyl shift and, therefore, requires N-terminal cysteine. Na-auxiliaries have long been used to enable NCL beyond cysteine. However, the reversibility of the S–N acyl shift under the acidic conditions used to remove the commonly applied N-benzyl auxiliaries limits the scope of this reaction. Herein, we introduce a new class of Na-auxiliary which is designed for removal under mild basic conditions. The 3-N-linked 4-mercaptobutyrate auxiliary is readily synthesized in a single step and enables introduction on solid phase by means of reductive amination. The usefulness of the new auxiliary was demonstrated in the synthesis of the anti-microbial C-terminal domain of Dermicidine-1L. Ó 2016 Elsevier Ltd. All rights reserved.

Native chemical ligation1 has dramatically advanced the field of peptide and protein synthesis by enabling chemoselective coupling of unprotected peptides under mild conditions. The reaction involves a peptide a-thioester and a peptide carrying an N-terminal cysteine (Scheme 1A). Reversible thiol exchange followed by an irreversible S?N acyl shift yields the native peptide bond. The method has gained broad use and is to date perhaps the method of choice for chemoselective synthesis of peptides and proteins.2 Unfortunately, the requirement for a cysteine at the N-terminus of the C-terminal segment is a limitation. Cysteine is a rare amino acid and may be located at positions unsuitable for NCL chemistry. Efforts to overcome the Cys restriction have led to the development of two main methods,3–6 which rely on desulfurization of thiolated amino acid building blocks (Scheme 1B)7–20 or the use of Na-auxiliaries (Scheme 1C).21–24 While several impressive feats were accomplished through the ligation–desulfurization approach8,25–28 the method requires access to a specific thiol-modified amino acid for every ligation junction. The limited commercial availability of thiolated amino acid building blocks calls for dedicated synthesis efforts which are a burden to non-specialist’s laboratories. The Na-auxiliary method involves the introduction of a thiolbearing scaffold onto the N-terminus of a peptide. Typically, an N-benzyl scaffold (see 9a or 9b) is designed to enable the removal

⇑ Corresponding author.

of the auxiliary once ligation is complete. Though the approach has widened the scope of ligation junctions accessible by NCL, there are problems which limited a broader acceptance. For example, the amide bond established in the ligation step may break under the acidic conditions used for the removal of the existing N-benzyl type auxiliaries.29 This side reaction is caused by an acid promoted N?S acyl shift which forms the thioester intermediate and, subsequently, hydrolysis products. The sterically demanding N-benzyl type auxiliaries typically show low ligation rates at non-glycine ligation junctions. Furthermore, the synthesis of some acid labile auxiliaries is rather tedious and their introduction frequently requires solution steps to prepare preformed amino acid-auxiliary conjugates or special building blocks such as a-bromo-derivatives of amino acids.24 Photolysis is an alternative method used for the removal of o-nitrobenzyl-type auxiliaries, yet the bulky structures cause, again, rather slow ligation kinetics.30–34 Herein, we present a new class of Na-auxiliary. The design is based on a 3-N-linked 4-mercaptobutyrate scaffold (see 9c), which is rapidly accessible by a single step synthesis, facilitates auxiliary introduction via reductive amination directly on the solid-supported peptide and enables auxiliary cleavage under mildly basic conditions. In the new auxiliary (9c), the thiol handle is positioned two carbon atoms away from the N-terminal peptide amino group enabling a five-membered ring transition state as in NCL. The cleavage characteristic of the auxiliary arises from the presence of an ethyl ester group (Scheme 2). Under mildly basic conditions,

http://dx.doi.org/10.1016/j.bmcl.2016.01.060 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Harpaz, Z.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.01.060

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Z. Harpaz et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx

A

B

O SR 1

Pep1

HS

5 HN 2 O

HS

1

O Pep2

H2N

Pep1

6

thiol exchange

Pep2

H2N

O S-N acyl shift

O S

HS O

3 H2N

Pep1 O

Pep2 Pep1

N H

4

O

R Pep2 HN O Aux Pep1 S S-N acyl O shift

Pep1

O desulfurization R

O

SH

O

R

Pep2 N Aux O 11 HS auxiliary cleavage

Pep2

N H

O

10

O Pep2

N H

7

S-N acyl shift

HN Aux HS

R

Pep2

Pep2

1

R

S

2

O

Pep1

R

9

Pep2

+

Pep1

C

R

O

8

R

O Pep1

Pep2

N H

12

O

R HS

SH

Pep2

HN

N H

O

9a

9b

R

SH

R

R

Pep2

R Pep2

N H

O

O

CO2Et

previous work

9c this work

Scheme 1. Mechanism of (A) NCL, (B) ligation–desulfurization and (C) auxiliary assisted NCL.

thiolactone 13 may be formed. We assumed that the electron withdrawing thioester group would allow a-deprotonation under mildly basic conditions and a concomitant elimination of the amide group would form the native peptide. The resulting a,bunsaturated thiolactone product may react further under the reaction conditions. As an alternative to the ionic mechanism and in accordance with the cleavage mechanism proposed for the recently introduced 2-mercapto-2-phenethyl auxiliary,35 oxygen or light may trigger the formation of thiyl radical 14. As reported for cysteine, the reaction with triscarboxyethylphosphine (TCEP) would induce desulfurization.36 In the absence of a powerful hydrogen donor the alkyl radical 15 may undergo a fragmentation reaction, which would deliver the crotonate and the amide radical 16. The latter should be more reactive than the alkyl radical and may therefore be scavenged by rather unreactive hydrogen donors such as amines. R

O Pep1 EtO2 C

N O SH

A

B

O 2 or hν

11c

B

Pep1 EtO2 C

Pep2

N O H

TCEP

R Pep1 EtO2 C

O

H

13

S

R

O

Pep2

N

14

S

base

O Pep1

R

O Pep2

Pep2

N O

O

O

15

Cl

S

base EtO 2C

R

O N H

Pep2 O

12

CO2Et

17

EtO 2C O

Pep1

To enable the introduction of the auxiliary by reductive amination, ketone 18 was synthesized in a single step starting from commercially available ethyl 4-chloro-3-oxobutanoate 17 (Scheme 3). Subsequently, the auxiliary ketone 18 was attached to two resin-bound model peptides 2 and 3 by using trimethoxyorthoformate under acidic conditions. The conjugate was treated with TFA for liberation and removal of side chain protection and the auxiliary S-trityl group. After HPLC purification, peptides 20G and 20A were obtained in 40% and 28% overall yield, respectively (Scheme 3, Fig. S3). The new auxiliary was evaluated in native chemical ligation reactions (Fig. 1A) involving two peptide mercaptopropionamide thioesters 21G and 21A. The first ligations were performed by dissolving the auxiliary peptide 20G and the thioester 21G in a ligation buffer (pH 7.5) and incubating at 25 °C. Thiophenol was included for in situ conversion of the mercaptopropionamide esters into the more reactive mercaptophenyl esters. The formation of the Gly-Gly junction upon reaction of 20G with 21G proceeded smoothly (Fig. 1B and C) and provided the ligation product 22GG in 90% yield after 4 h. After HPLC purification auxiliary-modified ligation product 22G was isolated in 55% yield. Next we assessed the sterically more demanding Ala-Gly (21A + 20G) and Gly-Ala (21G + 20A) ligations (Fig. 1C). The reactions furnished the ligation products 22AG and 22GA albeit at significantly reduced rate. The analysis of the early reaction phase suggests that the Gly-Ala ligation was more challenging for the 4mercaptobutyrate auxiliary than the Ala-Gly ligation. Nevertheless, after 8 h the Ala-Gly ligation product 22AG was isolated in 40% yield. The more challenging establishment of the Gly-Ala junction was less successful and 22GA was obtained in 10% isolated yield after HPLC purification. We also explored the 4-mercaptobutyrate auxiliary in an Ala-Ala ligation (21A + 20A). HPLC/MS analysis suggested that the thiol exchange step succeeded but the initially formed thioester intermediate did not rearrange via the S–N acyl shift (Fig. S8). This is in agreement with the results reported for most other ligation auxiliaries2a but the recently introduced 2-mercapto-2-phenethyl scaffold.35 We noticed that under the slightly basic conditions (pH 7.5) applied, the maximally achievable yields were limited by a side reaction. Ligation competed with cleavage of the 4-mercaptobutyrate auxiliary (Figs. S5–S8). Of note, under the ligation conditions chosen the auxiliary was exclusively released from the starting auxiliary peptides 20 rather than from the ligation products 22. This side reaction reduces the concentration of the available auxiliary peptide in the course of the ligation reaction and, therefore, contributes to the rather inefficient ligation at the Gly-Ala junction. On the other hand, the side reaction suggested that a mild auxiliary cleavage is feasible. We screened several conditions to induce auxiliary cleavage from ligation product 22GG. In the first attempts, we explored various glycine buffers at pH 9.0–10.5. This led to formation of the disulfide without apparent cleavage of the auxiliary (Fig. S9). According to our working hypotheses, a free thiol is

R

O

X-H Pep1

N

Pep2 O 16

Scheme 2. Hypothetical mechanisms for cleavage of 3-amide-linked 4-mercaptobutyrate auxiliaries under basic conditions via (A) ionic mechanism or (B) radical fragmentation.

XRAEYSGLG

19G, X=G 19A, X=A

O

TrtSH, K2CO3, DMF

TrtS

CO2Et

80%

18

1. 18, NaCNBH3, NMP, MeOH, HC(OMe)3, AcOH 2. TFA, TIS, H2O

R RAEYSGLG-NH2

HN HS

O CO2Et

20G, R=H 20A, R=CH3

Scheme 3. Synthesis of auxiliary ketone 18 and reductive amination with resin-bound peptides 19G and 19A to yield auxiliary peptides 20G and 20A. (NMP, N-methylpyrrolidone; TIS, triisopropylsilane; Trt, trityl.)

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A HS

21G, X=G 21A, X=A

CO 2Et

B

LYRAX ZRAEYSGLG NH2

ZRAEYSGLG NH2

LYRAX SR

HS

20G, Z=G 20A, Z=A

CO2Et 22GG, X=Z=G, 22AG, X=A, Z=G 22GA, X=G, Z=A, 22AA, X=Z=A

21G

22GG

20G

a

* 1,0

1,5

C

2,5

3,0

1,0

3,5

1,5

21G+20G 21A+20G 21G+20A 21A+20A

50% 40%

2,0

2,5

3,0

3,5

tR /min

tR /min 60%

Yield

2,0

b

100% 80% 60%

30% 40%

20%

20%

10% 0%

0% 0

5

10

15

t/ min

0

60

120 180 240 300 360 420 480

t/ min

Figure 1. (A) Ligation between thioesters 21G and 21A and auxiliary peptides 20G and 20A; (B) UPLC analysis (k = 210 nm) of ligation between thioester 21G and auxiliary peptide 21G at 0 h (left) and after 4 h (right), peak ‘a’ corresponds to hydrolysis of thioester 21G; peak ‘b’ corresponds to double acylated product; (C) formation of ligation product over time for the four different ligation junctions. The left figure shows the early reaction phase.

crucial for the auxiliary removal (see Scheme 2). Therefore, 40 mM TCEP was included in the auxiliary cleavage reaction. In addition to the glycine/NaOH mixture (pH 10.0), piperidine (pH 10.0), morpholine (pH 8.5) and ethyldimethylamine (pH 9.5) were tested. The use of piperidine and morpholine both enabled quantitative removal to give product 23GG within 5 h at 80 °C (Fig. 2). Of note, the use of ethyldimethylamine or glycine/NaOH failed to yield the desired product. Saponification of the ethyl butyrate was not observed. Considering that the ionic elimination mechanism should proceed faster under the more basic conditions (ethyldimethylamine rather than piperidine and morpholine), this hints to the radical pathway. Secondary amines are known to be able to quench amide radicals but not lower energy carbon centered radicals, again supporting a radical mechanism.37 Thiophenol, a known radical scavenger, was found to inhibit the auxiliary cleavage (Fig. S11). Though strict proof is lacking, the evidences point to a radical-triggered cleavage of the 4-mercaptobutyrate auxiliary. Regardless of the mechanism involved, we applied optimized auxiliary cleavage conditions (2 mM peptide, 40 mM TCEP, 160 mM morpholine, pH 8.5, 60 °C) to the Gly-Gly and Gly-Ala ligation products 22GG and 22GA, respectively. After 6 h reaction time the deprotected ligation products 23GG and 23GA were isolated by HPLC in 45% yield each (Fig. S12). We next assessed the c-mercaptobutyrate auxiliary in the total chemical synthesis of the antimicrobial domain of Dermicidine-1L. This protein is expressed by sweat glands and provides first line protection against bacteria. The domain is 47 amino acids in length, lacks a cysteine residue and has a Gly-Gly junction at position 78–79 making it an appropriate target for auxiliary assisted

Figure 2. (A) Removal of auxiliary from ligation product 22GG to give native peptide 23GG; (B) UPLC analysis (k = 210 nm) of reactions attempted with glycine/ NaOH (pH 10.0), piperidine (pH 9.5), dimethylethylamine (pH 9.0) and morpholine (pH 8.5).

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auxiliary (ligation beyond glycine feasible), the 3-N-linked 4-mercaptobutyrate offers the advantage of being available in a single step synthesis. The synthesis of a 47 aa long antimicrobial domain of Dermicidine-1L demonstrated the usefulness of the new auxiliary. The possibility to induce auxiliary cleavage under mild basic conditions should make the auxiliary especially attractive for the synthesis of cysteine-free proteins that carry base acid labile posttranslational modifications such as sulfonated proteins or glycoproteins. Acknowledgments This work was financially supported by the Deutsche Forschungsgemeinschaft (Se819-15-1, SPP 1623). We acknowledge Luxembourg Bio Technologies Ltd for providing OxymaPure. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.01. 060. References and notes

Figure 3. (A) Synthesis of Dermicidine-1L (DCD-1L) and UPLC analysis (k = 210 nm) of (B) ligation (Der1 + Der2 ? Der12) and auxiliary removal (Der12 ? DCD-1L) and (C) purified Der12 and DCD-1L.

NCL. The N-terminal fragment Der1 (Fig. 3A) was synthesized as benzylthioester in 35% overall yield on the sulfonamide SafetyCatch linker by using Fmoc-protected amino acids (Fig. S13).33 The auxiliary peptide Der2 was prepared on a Rink-Amide resin, again by using the Fmoc method. After completion of the peptide assembly the auxiliary was introduced on the solid phase as described earlier for the model peptides. TFA cleavage and HPLC purification delivered the auxiliary modified peptide Der2 in 10% overall yield (Fig. S14). In the event of the ligation (Fig. 3A) the two fragments Der1 and Der2 reacted smoothly and provided quantitative conversion after 7 h reaction time (Fig. 3B). After HPLC purification the auxiliary modified ligation product Der12 was obtained in 50% yield. The auxiliary was subsequently removed by applying the mildly basic conditions (100 mM TCEP, 400 mM morpholine, pH 8.5). After 6 h UPLC (Fig. 3B) and MS analysis (Figs. S15 and S16) suggested complete removal of the auxiliary with no apparent side reactions. The protein was purified to give the pure native Dermicidine-1L in 40% yield. In conclusion, we have presented a new Na-auxiliary scaffold for native chemical ligation beyond cysteine. The auxiliary is based on a 3-N-linked 4-mercaptobutyric ethyl ester. Ligation rates are in the range of previously published N-benzyl auxiliaries. In contrast to cleavage procedures reported for the N-benzyl type auxiliaries, the 3-N-linked 4-mercaptobutyrate is readily cleaved under mildly basic conditions (pH 8.5) presumably by means of a radical-triggered fragmentation reaction. The auxiliary, therefore, expands the class of the previously introduced base labile auxiliaries.35 Though clearly not as efficient as the 2-mercapto-2-phenethyl

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