Internal cysteine accelerates thioester-based peptide ligation

Article abstract of DOI:10.1002/ejoc.200900024

Thioester-mediated peptide coupling reactions are powerful tools in protein synthesis. The fragment coupling occurs extremely fast at ligation sites that contain an N-terminal cyste- ine residue. This native chemical ligation involves a capture step that

Full text of DOI:10.1002/ejoc.200900024

FULL PAPER
DOI: 10.1002/ejoc.200900024
Internal Cysteine Accelerates Thioester-Based Peptide Ligation
Christian Haase[a] and Oliver Seitz*[a]
Keywords: Coupling / Peptides / Macrocycles / Synthetic methods / Thioester
Thioester-mediated peptide coupling reactions are powerful
tools in protein synthesis. The fragment coupling occurs ex-
tremely fast at ligation sites that contain an N-terminal cyste-
ine residue. This native chemical ligation involves a capture
step that affixes the acyl component in the immediate vicinity
of the N-terminal amino group. The subsequent intramolecu-
lar S,N-acyl transfer step proceeds via an entropically fa-
vored five-membered ring intermediate. In this study we in-
vestigated whether sequence internal cysteine residues that
lead to the formation of macrocyclic intermediates are also
able to accelerate the rate of thioester-based fragment coup-
lings. It was the aim to identify distance requirements that
enable internal cysteine-mediated ligation reactions to pro-
ceed at synthetically useful rates. It was found that appropri-
ately positioned cysteine residues can induce up to 25-fold
rate enhancements compared to a cysteine-lacking control
peptide. Highest ligation rates and yields were obtained
when the internal thiol amino acid was incorporated at the
fifth or sixth position from the N-terminus of the C-terminal
coupling segment. The findings reveal a correlation between
the size of the macrocyclic ring intermediate and the ease of
the peptide ligation. Internal cysteine ligation may provide
the opportunity to shift amino acid cysteine bonds that are
difficult to access through native chemical ligation (such as
Pro–Cys bonds) by 4–5 amino acids to the N-terminus.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
The recent developments in peptide chemistry have sig-
nificantly extended the repertoire available for the total syn-
thesis of proteins. Among the techniques employed, the
native chemical ligation of unprotected fragments has
proven exceptionally useful.[1] In this reaction a peptide thi-
oester fragment A is allowed to react with a cysteine peptide
fragment B (Scheme 1). The mercapto group of B initially
replaces the thiol component of thioester A in a reversible
manner. The formed thioester intermediate C reacts in a
favoured 5-exo-trig ring closing reaction to a tetrahedral
intermediate. The release of the cysteine mercapto function
from the cyclic intermediate state establishes the native pep-
tide bond in product D.
In its original form native chemical ligation is restricted
to nucleophiles that have cysteine at the N-terminus. How-
ever, cysteine is a relatively rare amino acid (1.3% average
content).[2] Various methods have been developed with the
aim to extend the repertoire of native chemical ligation. For
example, involved cysteine residues have been converted to
alanine,[3] serine[4] and mimics of glutamine.[5] A homocys-
teine-ligation/methylation sequence has provided access to
methionine-containing ligation sites.[6] Ligation was per-
Scheme 1. Native chemical ligation. In the reversible initial step the
cysteine replaces the thiol in thioester peptide A to form intermedi-
ate C. The subsequent SǞN acyl transfer furnishes the peptide
bond in D.
formed at β-mercapto-phenylalanine.[7] Subsequent desul-
furization furnished phenylalanine. Penicillamine, the β-
mercapto derivative of valine (6.6%), is commercially avail-
able and allows the synthesis of crowded leucine–valine
conjunctions.[8] A γ-thiolated valine building block has been
used as alternative precursor to valine.[9] Though useful in
many cases, these methods have one drawback; the need for
protection of non-participating cysteine residues.
[a] Humboldt-Universität zu Berlin, Institut für Chemie,
Brook-Taylor-Straße 2, 12489 Berlin
Fax: +49-30-2093-7266
E-mail: oliver.seitz@chemie.hu-berlin.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Internal Cysteine Accelerates Thioester-Based Peptide Ligation
In the so-called extended native chemical ligation ap- reactions (see G in Schemes 2 and 3). In an internal cysteine
proach, auxiliary groups are attached to the N-terminus of ligation strategy no special building blocks would be re-
the nucleophilic peptide fragment.[10] These groups provide quired as a cysteine within a sequence would offer its side
a cysteine mimic that can be removed in a subsequent reac- chain function for the initial thioester exchange. This ap-
tion to liberate the newly formed amide. Unfortunately, proach would be useful when the C-terminal amino acid of
coupling reactions via N-linked auxiliaries proceed slowly the used peptide thioester hampers ligation. For example,
because the commonly used scaffolds confer steric hin- proline thioesters are known to react reluctantly in native
drance to an already weakly nucleophilic secondary amino chemical ligations.[13] Thioesters of aspartic or glutamic
group.
acid or lysine are prone to side reactions and may form
The above discussed native chemical ligation-like reac- cyclic anhydrides[14] or lactams,[15] respectively. We herein
tions involves a thiol-mediated capture step that affixed the report a systematic study of internal cysteine ligation. This
acyl donor peptide in the immediate vicinity of the amino investigation reveals a sequence-dependence and suggests
group. According to a generally accepted notion the subse- optimal distances between the internal cysteine residue and
quent SǞN acyl shift reactions should proceed via entropi- the N-terminal amino group. It is demonstrated that in-
cally favored 5- and 6-membered ring intermediates in order ternal cysteine residues can provide up to 25-fold accelera-
to occur at useful rates. Recent contributions from the tion of ligation rate compared to cysteine-lacking control
laboratories of Wong and Brik have challenged this para- peptides.
digm.[11a,12] Wong and co-workers attached a removable
thiol handle to O- and N-linked carbohydrates and ob-
served remarkable ligation efficiencies when the ligation site
included at least one glycine unit.[11b] The reaction involved
primary amino groups, which couple more rapidly than sec-
ondary amino groups. Up to five amino acids can be in-
serted between the glycosylated residue and the N-terminal
amino acid of the nucleophilic peptide fragment.[11c] It was
proposed that the acyl transfer proceeded via 14- to 29-
membered ring intermediates such as E (Scheme 2) and
may, thus, become rate limiting.[11] The authors showed evi-
dence that the sugar part critically affects the proximity of
the N-terminal amine to the thioester.[11c] Brik and cowork-
ers attached thiol-containing cyclohexane auxiliaries to
amino acid side-chains such as serine or aspartic/glutamic
acid.[12] In this case, the SǞN acyl transfer had to proceed
via 15- to 16-membered rings such as F.
Scheme 3. Thioester-mediated peptide coupling via a) direct ami-
nolysis and b) internal cysteine ligation. The latter proceeds
through side-chain thioester intermediate I which undergoes a sub-
sequent acyl transfer to form the ligation product D. If the forma-
tion of D proceeds fast, an S-acyl ligation product K can be ob-
served.
Scheme 2. Ring intermediates formed during A) first-generation
sugar-assisted ligation via an α-GlcNAc unit O-linked to serine;
B) side chain-auxiliary ligation, in which 2-aminocyclohexanol is
Results and Discussion
attached to an aspartic acid side chain via an ester linkage and
The ligation partners were designed in analogy to pub-
C) internal cysteine ligation wherein a sequence internal cysteine
lished ligation reactions.[13] The known H-LYRAG-SR thio-
presents the acyl residue to the terminal amino acid.
ester 1 was allowed to react with glycinyl-peptides 2–6
The remarkable results achieved in peptide ligations via (Table 1). In the latter, the number of glycine spacer units
internally appended thiol handles inspired us to explore between the N-terminal glycine and the internal cysteine
whether internal cysteine residues likewise facilitate ligation residue was successively increased from none to four amino
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C. Haase, O. Seitz
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acids. The peptides were dissolved in a degassed buffer con- implies that direct aminolysis of the thioester 7 partially
taining 6  guanidine hydrochloride, 100 m Na2HPO3 and contributes to product formation (see also Scheme 3). Of
25 m tris(carboxyethyl)phosphane (TCEP) at pH = 7.0 in note, peptide 3, which contained the Cys residue in Gly+2
5 m concentration. Thiophenol (5%) was added as sole position had a 0.6-fold lower reactivity than cysteine-free
additive to the reaction mixture. The course of the ligation peptide 7. It can be speculated that the medium sized 11-
reactions of the peptides 2 – 6 is illustrated by the represen- membered ring that occurs upon formation of the tetrava-
tative HPLC-analysis of the coupling reaction between 1 lent intermediate is unfavorable for an intramolecular SǞN
and 4 (Figure 1, A/B). The HPLC trace after 48 h shows acyl transfer. According to this notion the acyl donor seg-
three main peaks, with the ligation product (e in Figure 1, ment would be trapped at the thioester intermediate stage,
B) as major peak, followed by peaks for the excess nucleo- which would effectively decrease the concentration of thio-
phile (a) and the hydrolyzed thioester (b). The HPLC trace esters available for direct aminolysis.
after 8 h is more informative and provides evidence of
It was our aim to identify distance requirements that en-
formed reaction intermediates such as the non-rearranged able internal cysteine-mediated ligation reactions to proceed
thioester (d in Figure 1, A) and a side chain thioesterified at synthetically useful rates. In Gly–Gly ligation the achiev-
ligation product (f in Figure 1, A) that is amenable to hy- able rates were perturbed by the direct aminolysis reaction,
drolysis. In this series, the N-terminal glycine peptides 5 and which complicated the determination of effects induced by
6 exhibited the fastest ligation rates (Table 1). This sug- the internal cysteine residue. Thus, the steric demand of one
gested that internal cysteine-mediated ligation of glycinyl- ligation partner was increased to suppress interference by
peptides may proceed fastest with cysteine in Gly+4 and direct fragment fusion.
Gly+5 sites. The applied reaction conditions have been re-
The peptides 8–14 were incubated with LYRAG-thioes-
ported to enable chemoselective native chemical ligation re- ter 1 under conditions denoted above. The replacement of
actions.[17] Nonetheless, the coupling of the thiol-lacking al- the N-terminal glycine in 7 with the alanine in 14 dramati-
anine-containing peptide 7 still occurred at a significant cally decreased the rate of direct aminolysis. Only 3.1% of
rate, which was only threefold lower than the ligation of the coupling product 41 were formed after 48 h. The pep-
the most reactive cysteine-containing peptides 4 and 5. This tide H-AGGGCRAEYS-NH2 11 ligated 25-fold faster than
Table 1. Peptides used in the internal cysteine-mediated ligation with thioester H-LYRAG-SR (1).
Nucleophilic peptide
Ligation product
Rate acceleration
Yield[a]/%
H-GnCRAEYS-NH2
2, n = 1
H-LYRAGGnCRAEYS-NH2
29, n = 1
1.0[b]
1.0[b]
0.6[b]
3.0[b]
3.0[b]
/
25.2
29.7
18.0
54.4
56.4
42.8
3, n = 2
30, n = 2
4, n = 3
31, n = 3
5, n = 4
32, n = 4
6, n = 5
33, n = 5
7, H-G3ARAEYS-NH2
H-AGnCRAEYS-NH2
8, n = 0
34, H-LYRAG4ARAEYS-NH2
H-LYRAGAGnCRAEYS-NH2
35, n = 0
1.4[b]
3.7[c]
6.3[c]
25.1[c]
9.2[c]
4.8[c]
/
4.3
6.5
18.0
49.2
20.0
9.1
9, n = 1
36, n = 1
10, n = 2
37, n = 2
11, n = 3
38, n = 3
12, n = 4
39, n = 4
13, n = 5
40, n = 5
14, H-AGGARAEYS-NH2
H-AGnDCRAEYS-NH2
15, n = 1
41, H-LYRAGAG2ARAEYS-NH2
H-LYRAGAGnDCRAEYS-NH2
42, n = 1
3.1
3.3[c]
18.0[c]
5.7[c]
5.3[c]
11.5
33.4
15.5
9.5
16, n = 2
43, n = 2
17, n = 3
44, n = 3
18, n = 4
45, n = 4
H-AGnNCRAEYS-NH2
19, n = 1
H-LYRAGAGnNCRAEYS-NH2
46, n = 1
6.1[c]
21.0[c]
7.2[c]
3.9[c]
18.4
39.8
15.6
10.2
20, n = 2
47, n = 2
21, n = 3
22, n = 4
48, n = 3
49, n = 4
H-AaGnPCRAEYS-NH2
23, a = 0; n = 0
24, a = 1; n = 0
25, a = 1; n = 1
26, a = 1; n = 2
27, a = 1; n = 3
28, a = 1; n = 4
H-LYRAGAaGnPCRAEYS-NH2
50, a = 0; n = 0
51, a = 1; n = 0
52, a = 1; n = 1
53, a = 1; n = 2
54, a = 1; n = 3
55, a = 1; n = 4
0.8[c]
2.8[c]
1.4[c]
4.8[c]
2.4[c]
3.9[c]
3.4 (10.5)[d]
8.5 (15.1)[d]
6.0 (15.0)[d]
12.8 (43.5)[d]
3.7 (13.6)[d]
8.8 (15.7)[d]
[a] Determined by HPLC analysis of aliquots withdrawn after 48 hours, conditions: see Figure 1. [b] Compared to cysteine-lacking control
HGGGARAEYSNH2 (7). [c] Compared to cysteine-lacking control HAGGARAEYSNH2 (14). [d] 10 mM peptides, 100 mM Na2HPO4,
pH 8.0, 35 °C, 72 hours.
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Internal Cysteine Accelerates Thioester-Based Peptide Ligation
were less reactive. The peptide that terminated at a Cys-4
position was the most reactive nucleophile among the series.
This suggests that 17-20 membered rings have the optimal
size for the cyclic intermediate in the S,N-acyl transfer step
of the internal cysteine ligation. The different reactivity of
asparagine peptide 20, aspartate peptide 16 and proline
peptide 26 suggests that both steric and electronical proper-
ties of the amino acids next to the internal cysteine affect
the ligations rates.
We decided to modify the ligation conditions to improve
reactions of the proline-containing peptides. The optimiza-
tion of native chemical ligation-like reactions has been re-
ported previously.[8,13,17] For example, the use of organic co-
solvents such as NMP and high pH has enabled facile lig-
ation reactions.[11e] We chose comparatively mild ligation
conditions and performed the ligation in a plain phosphate
buffer (100 m Na2HPO4) without guanidine hydrochloride
and tris(carboxyethyl)phosphane (TCEP) with a pH-value
adjusted to 8.0. The temperature was elevated to 35 °C and
the concentration of the peptides was increased from 5 m
to 10 m to increase the probability for effective collisions.
Prior to the final estimation of the ligation yields a solution
of tris(carboxyethyl)phosphane (250 m) was added to the
reaction solutions to ensure the absence of disulfides.
HPLC-MS analysis (Figure 2) revealed two major path-
ways, ligation and competing thioester hydrolysis. The reac-
tions proceeded smoothly at the optimized conditions fur-
nishing up to 3.5-fold increases in ligation yields when com-
pared to reactions performed at lower pH, temperature and
concentration. In particular, ligation product 53 was formed
at synthetically useful 44% yield with hydrolyzed thioester
and starting material 26 as sole by-products. Again, the
Figure 1. A) Ligation of 1 and 4 after 8 h and B) after 48 h. a: H-
GGGCRAEYS-NH2; b: thioester hydrolysis; c: H-LYRAG-
SCH2CH2CONHCH2CONH2 (1); d: non-rearranged reaction
product; e: ligation product H-LYRAGGGGCRAEYS-NH2 (31);
f: side chain thioester adduct of 31 and 1. Conditons: 5 m peptide
concentrations, 6  GnHCl, 100 m Na2HPO4, 25 mM tris(car-
boxyethyl)phosphane (TCEP), pH = 7.0, 25 °C, 5% thiophenol. C)
Kinetics of product formation of the ligations of the thioester H-
LYRAG-SCH2CH2CONHCH2CONH2 (1) with the peptides 6, 11,
17, 20 and 26.
cysteine-lacking control 14. This confirms a trend that was peptide 26, which displayed the N-terminus at Cys-4 posi-
observed in ligation of glycinyl-peptides 2–7 where three tion, reacted faster than peptides 23, 24, 25, 27 and 28. This
spacer amino acids conferred highest ligation accelerations. suggests that appropriately placed internal cysteine residues
The positive effect of internal cysteine residues was attenu- can accelerate ligation reactions regardless of the reaction
ated when the alanine terminus was positioned in Cys-3 (10) conditions applied.
and Cys-5 (12) sites. Nevertheless, the internal cysteine still
provided six- and ninefold rate acceleration, respectively.
The other peptides, which offered internal cysteine residues
at Cys-1 (8), Cys-2 (9) and Cys-6 (13) sites showed low li-
gation rates, which were too small to provide useful synthe-
sis yields.
These results suggest that internal cysteine residues may
significantly accelerate thioester-mediated ligation reactions
when positioned 3–5 amino acids apart from the nucleo-
philic ligation site. Bearing this in mind, we turned our at-
tention to a set of nucleophiles, in which asparagine, as-
partic acid and proline were the N-terminal neighbours of
cysteine. The Asn-Cys, Asp-Cys and Pro-Cys ligation sites
would be difficult to access by conventional native chemical
ligation.[14,15] Thus, the ligation sites were shifted to the N-
termini by 2–5 amino acids. Among this series, peptides ter-
Figure 2. Ligation of 1 and 26 under optimized conditions after
72 h a: thioester hydrolysis; b: H-AGGPCRAEYS-NH2 (26); c: li-
gation product H-LYRAGAGGPCRAEYS-NH2 (53).
Conclusions
The work of Blake,[18] Aimoto[19] and Wong[16] has indi-
minating in Cys-4 positions were found to provide the high- cated the usefulness of thioester-mediated fragment coup-
est ligation accelerations. The peptides H-AGGDCRAEYS- lings in peptide and protein synthesis. Dawson and Kent
NH2 (16) and H-AGGNCRAEYS-NH2 (20) reacted demonstrated extremely fast and chemoselective segment
18–20-fold faster than the cysteine-lacking peptide H- couplings in thioester-based ligation reactions that involved
AGGARAEYS-NH2 (14). The proline-containing peptides N-terminal cysteine.[1a] The investigation described here
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C. Haase, O. Seitz
FULL PAPER
(containing 1% acetonitrile and 0.1% TFA) (A) and acetonitrile
(containing 1% water and 0.1% TFA) (B) were used. A linear gra-
dient (3% B – 30% B in 20 min at 55 °C) with a flow rate of 1 mL/
min was employed to separate the reaction components. Detection
was achieved with a UV/Vis detector (λ = 280 nm). Yields are cal-
culated based upon the integration of the HPLC traces under con-
sideration of the extinction coefficients. [ε280 (thioester) = ε280 (cys-
teine peptides) = 1280 Lmol–1 cm–1; ε280 (ligation products) =
2560 Lmol–1 cm–1].
suggests that internal cysteine residues can also confer rate
accelerations. Appropriately positioned cysteine residues
were found to induce up to 25-fold acceleration of ligation
rates. Highest ligation rates and yields were obtained when
the internal thiol amino acid was incorporated at the fifth
or sixth position from the N-terminus of the C-terminal
coupling segment. Cysteine-mediated ligations must pro-
ceed via the formation of cyclic intermediates. The observed
distance dependence of internal cysteine-mediated ligations
suggests that 17-membered peptide macrocycles are formed Supporting Information (see footnote on the first page of this arti-
cle): Details regarding materials and methods, synthesis protocols
and analytical data of synthesized peptides, HPLC analysis and
reaction time course of all ligation reactions.
most readily. By contrast, peptide nucleophiles which con-
tained cysteine at the second or third position showed only
modest rate accelerations. This implies that 8- or 11-mem-
bered peptide rings are difficult to form. A similar behav-
iour is known from attempted syntheses of the 9- and 12-
membered rings in cyclotri- and cyclotetrapeptides, which
have to accommodate unfavourable strain.[20]
The findings of this investigation may have practical im-
plications for the convergent total synthesis of peptides and
proteins. The native chemical ligation will, in most cases, be
the first choice. Thus, the selected ligation site will predomi-
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size of the macrocyclic ring formed during the SǞN acyl
transfer and the reaction rates. The present study was re-
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Future investigations will reveal the sequence context re-
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Experimental Section
General Procedure for Fragment Coupling of Peptides with Se-
quence-Internal Cysteines and C-Terminal Glycine Peptide Thio-
esters: The freeze-dried peptides were dissolved in 5 m concentra-
tion in a degassed buffer containing 6  guanidine hydrochloride,
100 m NaH2PO4, 25 m tris(carboxyethyl)phosphane at pH =
7.0. To accelerate the ligation thiophenol (5 vol.-%) was added as
sole additive. The mixture was agitated at 25 °C. For the HPLC
monitoring aliquots of the reaction mixture (5 µL) were dissolved
in water (95 µL) containing 0.5% TFA. The resulting mixture
(98 µL) was injected into a Merck–Hitachi Elite LaChrom chroma-
tograph (column: Varian Polaris C18 A 5µ 250/4). As eluents water
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