A new method for peptide synthesis in the N→C direction: Amide assembly through silver-promoted reaction of thioamides

Article abstract of DOI:10.1039/c4cc07601j

The Ag(i)-promoted coupling of amino acids and peptides with amino ester thioamides generates peptide imides without epimerisation. The peptide imides undergo regioselective hydrolysis under mild conditions to generate native peptides. This method was employed to prepare the pentapeptide thymopentin in the N→C direction, in high yield and purity.

Full text of DOI:10.1039/c4cc07601j

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A new method for peptide synthesis in the N-C
direction: amide assembly through silver-promoted
reaction of thioamides†
Cite this:DOI: 10.1039/c4cc07601j
Received 26th September 2014,
Accepted 30th October 2014
Aysa Pourvali,‡ James R. Cochrane‡ and Craig A. Hutton*
DOI: 10.1039/c4cc07601j
www.rsc.org/chemcomm
The Ag(I)-promoted coupling of amino acids and peptides with amino
ester thioamides generates peptide imides without epimerisation. The
peptide imides undergo regioselective hydrolysis under mild condi-
tions to generate native peptides. This method was employed to
prepare the pentapeptide thymopentin in the N-C direction, in high
yield and purity.
The quest for rapid and direct synthesis of peptides and proteins
under mild conditions has led to a vast number of modern
methods for the construction of amide bonds.1 The search for
chemoselectivity has led to the development of methods that
incorporate non-traditional coupling partners such as alcohols,2
bromonitroalkanes,3 ketoacids,4 isonitriles5 and others.6 Further,
the development methods amenable to the N-C direction
synthesis of peptides is of interest for the direct preparation of
C-terminally modified peptides.7 Standard coupling methods
involving generation of an active ester at the peptide C-terminus
suffer from epimerisation of the C-terminal residue.7
Danishefsky has developed a peptide coupling strategy
through the reaction of isonitriles with thioacids (Scheme 1,
X = S).5 In this process, the thioacid and isonitrile react to
generate a thioformimidate carboxylate mixed anhydride inter-
mediate 3 (thioFCMA, X = S), which can undergo trapping with
an exogenous amine to generate an amide, or a 1,3-acyl transfer
to generate a thioformimide 5.1d,5 The closely related coupling
of isonitriles with carboxylic acids (Scheme 1, X = O), only
undergoes the 1,3-acyl transfer at temperatures above 150 1C to
generate formimides 5.8 Interestingly, it was found that addition
of thiophenol results in generation of the imide product 5 at
room temperature, albeit in low yield (10–25%).9 A tetrahedral
intermediate 4 was invoked to rationalise the rearrangement
occurring at room temperature.
Scheme 1 Imides from isonitriles and carboxylic acids or thioacids.
In the quest for new methods of amide formation utilising
novel coupling partners, we hypothesised that the reaction of a
thioamide with a carboxylate in the presence of a thiophilic
metal such as Ag(I) would generate a tetrahedral species similar
to 4, which would provide access to imides 5. This approach
would avoid the concurrent formation of thioacetal products,
a major limitation in the isonitrile route.9 Indeed, the reaction of
secondary thioamides with silver carboxylates has been shown
to generate imides.10 The mechanism of this transformation is
proposed to proceed through both a tetrahedral intermediate
40 and a mixed anhydride intermediate 30, closely related to those
observed in the isonitrile route, with final 1,3-acyl transfer
generating the imide 5 (path a, Scheme 2).10 Despite the analogous
reaction pathways, the coupling of thioamides and carboxylic acids
to generate peptides has not been exploited.
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, VIC 3010, Australia. E-mail: chutton@unimelb.edu.au
† Electronic supplementary information (ESI) available: Full experimental details
and NMR spectra for all new compounds. HPLC traces of peptides 10AG and 28.
See DOI: 10.1039/c4cc07601j
‡ These authors contributed equally.
Scheme 2 Proposed mechanism of imide formation from thioamides.10
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Table 1 Optimisation of imide formationa
Table 2 Synthesis of dipeptide imidesa
12 R2
11 R1
H (G)
Me (A)
Bn (F)
iPr (V)
iBu (L)
Stoichiometry
Acid : thioamide : Ag(I) Yield (%)
H (G)
76
84
72
76
70
99
95
86
71
92
94
83
79
71
75
99
95
99
94
90
95
99
81
99
84
Entry Acid Thioamide Ag(I)
Me (A)
Bn (F)
iPr (V)
iBu (L)
1
2
3
4
5
6
7
8
—
8
8
8
11G
11G
11G
11G
11G
9
9
9
9
9
9
9
9
9
AgOAc — : 1 : 2
89
85
40
Ag2CO3 1 : 1 : 2
Ag2CO3 1 : 1 : 1
Ag2SO4 1 : 1 : 2
Ag2CO3 1 : 1 : 2
Ag2CO3 1 : 1 : 2
Ag2CO3 2 : 1 : 2
Ag2CO3 1 : 1 : 4
Ag2CO3 1 : 2 : 4
Ag2CO3 1 : 1 : 2
0 (25b)
a
66 (61c)
60d
32
56
69
Isolated yields (%). Conditions: 1.0 equiv. 11, 1.5 equiv. 12, 1.5 equiv.
Ag2CO3.
and Ag2CO3 gave optimum yield (entry 9). Employing the glycine
thioacetamide 12G, as little as 1.5 equiv. thioamide and Ag2CO3
furnished the imide in optimum yield (entries 13 and 14). While
these reactions are typically complete in 10–15 minutes at room
temperature, they were routinely left for two hours.
9
10
11
12
13
14
11G 12G
11G 12G
11G 12G
11G 12G
11G 12G
66
68
HgO
1 : 1 : 1.5e
CuCO3 1 : 1 : 1.5 f
Ag2CO3 1 : 1.5 : 3
Ag2CO3 1 : 2 : 4
0
76 (82g)
78
The synthesis of protected dipeptides from N-phthaloyl
amino acids 11 and thioacetylated amino esters 12 was inves-
tigated under the optimised conditions (Table 2). In all cases
the yields obtained for the imide products were good to
a
b
Conditions: acid, thioamide, Ag(I) salt in CH2Cl2, RT, 2 h. With
c
d
e
3 equiv. Et3N. With 10 equiv. H2O. Solvent; CH3CN. 1.5 equiv. HgO.
f
g
1.5 equiv. CuCO3. Solvent; DMF.
We herein describe the coupling of thioamides and carboxylic excellent (71–100%), with the only byproducts being silver
acids to generate imides and ultimately amide products, including sulfide and the amide precursor. Notably, excellent yields were
application to the preparation of peptides. This silver-promoted obtained even when sterically hindered amino acids such as
reaction proceeds rapidly at room temperature, without epimeri- valine and leucine were employed.
sation. We show that selective imide hydrolysis is feasible to
The dipeptide imides 10 were isolated as single stereoisomers,
furnish amide products. The application of this process to the with no evidence of epimerisation. Chiral HPLC analysis of
N-C direction synthesis of peptides is highlighted through dipeptide imide 10AG confirmed the absence of epimerisation
preparation of the pentapeptide thymopentin.
during this coupling, with o0.1% of the enantiomeric product
Synthesis of the thioamide coupling partners was achieved detected (see ESI†).
in good to excellent yields (70–92%) through several methods:
Given that the imides 10 are generated rapidly at room
by acylation of the corresponding amine followed by treatment temperature, the findings of Danishefsky et al.8 would suggest
Lawesson’s reagent, or alternatively by direct thioacetylation imide formation does not proceed via the mixed anhydride-type
of the amine with Rapoport’s reagent11 or ethyldithioacetate intermediate, and that instead the 1,3-acyl migration occurs from
(see ESI†).
the tetrahedral intermediate 40 generating the imide directly with
A model coupling of N-benzylthioacetamide 9 with silver expulsion of silver sulfide (see Scheme 2, path b). This mechanism
acetate generated the imide 10 (R1 = Me, R2 = Bn) in excellent is also consistent with the observed lack of epimerisation, as no
yield (Table 1, entry 1). The combination of acetic acid and epimerisation-prone ‘active ester’ species 30 is formed.
silver carbonate gave similar results to the use of silver acetate
Use of other standard amino acid protecting groups was well
(entry 2), demonstrating that pre-formed silver carboxylates are tolerated, with Boc and Cbz-protected amino acids 13 and 16
not necessary for this process. Non-basic Ag(I) salts such as undergoing the imide ligation in high yield (Scheme 3, eqn (1)
Ag2SO4 were only effective if base was added (entry 4). Hg(II) and (2)). Fmoc protecting groups were not well tolerated,
was similarly effective as Ag(I), while Cu(II) was less effective presumably due to the slightly basic conditions. Intriguingly,
(entries 11 and 12).10 Use of N-phthaloyl glycine 11G as the acid rearrangement of the carbamate-protected dipeptide imides
component gave the glycine imide 10 (R1 = PhthCH2, R2 = Bn) in 14a and 17a was observed, with significant 1,3-acyl migration
reasonable yield (entry 5). Optimization of this reaction was generating a mixture of the initially formed imides and the
undertaken with regard to solvent and stoichiometry. CH2Cl2, isomeric species 14b and 17b, respectively.12
acetonitrile and DMF were all suitable solvents for the reaction
The imide coupling can only be considered useful in the
(entries 5 and 13). The reaction was tolerant of water, with context of peptide synthesis if regioselective hydrolysis of the
addition of 10 equiv. of water having no noticeable effect on imide can be achieved to generate the native amide bond.
the yield. At least one equivalent of Ag2CO3 (2 equiv. AgI) was Accordingly, treatment of the Boc- and Cbz-protected dipeptide
required for good yields, while excess carboxylic acid was imides 14a/b and 17a/b under mildly basic methanolysis condi-
detrimental (entries 2 and 3; 6 and 7). An excess of both thioamide tions (NaHCO3, MeOH)5e proceeded to give the desired peptides
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ester were detected, along with trace amounts of phthaloyl-group
methanolysis. The selectivity for solvolysis of the acetyl group in
10GA, 14 and 17 over cleaving the dipeptide into its constituent
amino acids is presumably due to a steric effect, with attack at
the less hindered acetyl group.
To exemplify the utility of this method in peptide synthesis,
we sought to generate pentapeptide thymopentin 28 through an
iterative N-C coupling strategy. Thymopentin 28 is a penta-
peptide fragment of the thymic hormone thymopoeitin, which has
several biological actions, most notably induction of early T-cell
differentiation.13 Thymopentin corresponds to residues 32–36 of
the native protein and retains most of its biological activity.14
The N-C synthesis of thymopentin 28 was performed as
outlined in Scheme 4. While our methodology is tolerant of a
range of N- and C-protecting groups, a Boc/methyl ester strategy
was chosen as this would allow for a one-step concomitant imide
hydrolysis/C-terminal deprotection. Accordingly, Boc-protected
arginine 20 was coupled to lysine thioacetamide methyl ester
12K to give the dipeptide imide 21 in 83% yield. Treatment of the
dipeptide imide 21 with LiOH resulted in regioselective imide
Scheme 3 Dipeptide couplings. Reagents and conditions; (a) Ag2CO3
(1.5 equiv.), CH2Cl2, 25 1C, 2 h. (b) NaHCO3, MeOH: 73% over 2 steps for 15,
74% over 2 steps for 18, 62% for 19.
15 and 18, respectively, in good yield (Scheme 3, eqn (1) and (2)). cleavage together with hydrolysis of the methyl ester to generate
Ultimately, for these carbamate-protected systems, solvolysis of the dipeptide 22. This sequence of thioamide coupling followed
the mixture of imides 14a/b and 17a/b generates a single amide by concomitant imide and ester hydrolysis thus represents a
product, 15 and 18, respectively, which renders the acyl migra- two-step coupling–deprotection protocol suitable for peptide
tion of the imides inconsequential to the use of this method for synthesis via iterative amino acid couplings.
amide bond generation. LiOH (1 equiv.) was also effective for
Coupling of dipeptide 22 to aspartate thioacetamide 12D
chemoselective imide hydrolysis (vide infra). Treatment of the proceeded in good yield to generate 23. Surprisingly, it was
phthaloyl-protected dipeptide imide 10GA under similar condi- found that treatment of tripeptide imide 23 with LiOH resulted
tions proceeded to give the desired dipeptide 19 in reasonable not only in imide and methyl ester hydrolysis, but also cleavage of
yield (62%) (Scheme 3, eqn (3)). Only minor amounts of the the side-chain t-butyl ester. Presumably, upon imide hydrolysis,
undesired imide cleavage to generate N-phthaloylglycine methyl the released amide anion undergoes intramolecular cyclisation to
Scheme 4 N-C synthesis of thymopentin. Reagents and conditions; (a) 12K, Ag2CO3 (1.5 equiv.), CH2Cl2, 40 1C, 16 h, 83%; (b) LiOH (3 equiv.), 70%;
(c) 12D, Ag2CO3 (1.5 equiv.), CH2Cl2, 40 1C, 16 h, 74%; (d) LiOH (3 equiv.), dioxane/H2O, 65% of 24; (e) (1) NaHCO3, MeOH, (2) LiOH (3 equiv.), 70% over
2 steps of 25; (f) (1) 12V, Ag2CO3 (1.5 equiv.), CH2Cl2, 40 1C, 16 h, 73%, (2) NaHCO3, MeOH, (3) LiOH (3 equiv.), 74% over 2 steps; (g) CH3CS-Tyr(tBu)-OtBu,
Ag2CO3 (1.5 equiv.), CH2Cl2, 40 1C, 16 h, 89%; (h) (1) NaHCO3, MeOH, (2) TFA, 63% over 2 steps.
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with TFA gave thymopentin 28 in 10% overall yield (Scheme 4),
with no evidence of epimerisation or other deleterious byproducts.
The synthesized peptide 28 was identical to an authentic sample
by HPLC co-injection and NMR analysis (see ESI†).
In summary, we have developed a method for the coupling
of amino acids and peptides with amino ester thioamides to
generate peptide imides. The imides undergo regioselective
hydrolysis under mild conditions to generate native peptide
bonds. The use of this new method to prepare thymopentin
demonstrates that our method is suitable for N-C direction
peptide synthesis without epimerisation.
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C. J. Duran, F. J. Higes, J. L. Jimenez, I. Lopez and J. C. Palacios,
This research was supported by the Australian Research
Council (DP120101454).
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