Copper(ii) mediated facile and ultra fast peptide synthesis in methanol

Article abstract of DOI:10.1039/c2cc32581k

A novel, ultrafast, mild and scalable amide bond formation strategy in methanol using simple thioacids and amines is described. The mechanism suggests that the coupling reactions are initially mediated by CuSO₄· 5H₂O and subsequently catalyzed by in situ generated copper sulfide. The pure peptides were isolated in satisfactory yields in less than 5 minutes. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc32581k

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Copper(II) mediated facile and ultra fast peptide synthesis in methanolw
Sachitanand M. Mali, Sandip V. Jadhav and Hosahudya N. Gopi*
Received 11th April 2012, Accepted 24th May 2012
DOI: 10.1039/c2cc32581k
A novel, ultrafast, mild and scalable amide bond formation
strategy in methanol using simple thioacids and amines is
described. The mechanism suggests that the coupling reactions
are initially mediated by CuSO4Á5H2O and subsequently catalyzed
Scheme 2 Schematic representation of the metal mediated amide
by in situ generated copper sulfide. The pure peptides were isolated
bond formation.
in satisfactory yields in less than 5 minutes.
were observed in Cu(I) and Cu(II) compounds. In comparison,
Cu(II) complexes CuSO4Á5H2O and Cu(OAc)2ÁH2O gave
Amide bond formation is one of the most important reactions in
better yields than Cu(I)I. We continued our work with
contemporary organic chemistry. Numerous strategies and reagents
CuSO4Á5H2O as it is cheap and commonly available in any
undergraduate laboratory. The slow reactivity and the longer
time duration of CuSO4Á5H2O [also Cu(OAc)2ÁH2O] in both
DMF and DMSO, as compared to the standard coupling
reagents such as HBTU and HATU, led us to search for a
solvent in which both reactants and the metal complex are
soluble. As CuSO4Á5H2O has better solubility in methanol, we
carried out the same coupling reaction in methanol with a
suspicion, since methanol has never been used as a common
solvent in any acylation reactions. Surprisingly, the amide
bond formation was observed in good yield within 5 min
and no other byproduct was observed except the insoluble
metal sulfide. As the metal complex is insoluble in EtOH,
EtOAc and THF, we did not further pursue the amide bond
synthesis in these solvents.
have been developed for the synthesis of amide–peptide bonds.1,2
Recently, the thioacid mediated amide bond coupling reactions
have been gaining momentum due to their unique reactivity
and selectivity compared to those of carboxylic acids.3 The
reactivity of thioacids with azides,4 isonitriles,5 sulphonamides,6
nitroso derivatives,7 thioisocyanates,8 isocyanates,8 dinitrofluoro-
benzene9 and aziridines10 has been effectively utilized in the direct
peptide bond formation or native chemical ligation. Herein we
disclose a novel, ultrafast, mild and scalable copper sulfate
mediated and subsequently copper sulfide catalyzed amide bond
formation involving thioacids and amines in methanol.
Inspired by the thioester strategies in the acylation reactions
of coenzyme, acetyl-CoA (Scheme 1), and in non-ribosomal
peptide synthesis,11 we anticipate that the coenzyme in the
thioester is replaced with sulfur loving metal (M) which would
readily react with amines to form highly stable metal sulfides
(Scheme 2).
We began our search for a suitable metal salt or metal
complex to mediate the amide bond synthesis using thioacid
Boc-Ala-SH and benzylamine in DMF. Various metal salts
and complexes including Ag(I), Au(III), Fe(II), Fe(III), Cu(I),
Cu(II), Mo(IV) and Zn(II) have been screened and better yields
Scheme 3 Amide bond synthesis using thioacids. A comparison of
various metal salts and complexes in the acylation reaction. Time is
given in minutes.
Entry
M
Time (min)
%Yield
a
b
c
Cu(OAc)2
CuSO4Á5H2O
CuI
5
57
71
41
14
38
14
14
20
5
30
30
5
Scheme 1 Acetyl transfer reactions by acetyl coenzyme A through
d
e
ZnCl2
thioesters.
AgOAc
FeCl2
f
420
420
30
Department of Chemistry, Indian Institute of Science Education and
Research, Dr Homi Bhabha Road, Pashan, Pune-411008, India.
E-mail: hn.gopi@iiserpune.ac.in; Fax: +91-20-2589 9790;
Tel: +91-20-2590 8075
g
h
FeCl3
AuCl3
w Electronic supplementary information (ESI) available: Experimental
details, NMR spectra, mass spectra and X-ray crystallography. CCDC
876063. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2cc32581k
Encouraging results of copper sulfate mediated coupling
reactions in methanol promoted us to probe the amide bond
synthesis using other metal salts and complexes (Scheme 3).
c
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Chem. Commun., 2012, 48, 7085–7087 7085

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the product was isolated after 24 h. We speculate that the
oxidative dimerization of thioacids in solution may result in the
formation of the product.3,13 Further, we continued peptide
coupling reactions using 30 mol% of CuSO4Á5H2O (Scheme 4).
The pH measurement studies suggest that the reaction mixture
was almost neutral (pH 7.76) during the amide bond coupling.
The elemental analysis of the black material obtained as a
byproduct in the reaction gave 31.5% of sulfur, which is nearly
equivalent to the CuS. However, the MALDI-TOF analysis
suggests the complex mixture of copper and sulfur (see ESIw).
It is also evident from the literature that the copper sulfide may exist
in the complex of [Cu(I)–Cu(II)]nSx.14 As solubility experiments
suggest that the metal sulfide is insoluble in all solvents
including methanol (except con. HNO3), we subjected it to
the powder XRD analysis. Results from the X-ray analysis
reveal the formation of CuS (covellite) as a byproduct in the
amide bond coupling reaction. The powder XRD data of the
byproduct along with the standard CuS (covellite) are shown
in Fig. 1a and b, respectively.14 Further, the UV absorption
measurements of Boc-Ala-SH and H-Leu-OMe with and
without copper sulfate in time course experiments suggest
the complete disappearance of thiol absorption within 1 min,
indicating the formation of an amide bond within a minute. In
addition, to investigate the racemization during the amide
bond synthesis, the two diastereomeric dipeptides 1 and 2
(Table 1) were synthesized using Boc-Ala-SH, and Boc-DAla-SH
along with the racemic mixture of Boc-(Æ)-Ala-SH. The chiral
HPLC results indicate that no racemization occurred during the
amide bond formation (see ESIw). Additionally, it has also
been reported that the presence of Cu(II) complexes reduces
the racemization during the peptide coupling reactions.15
Further, to understand the compatibility of this protocol with
other N-terminal protecting groups, a series of N-protected
thioacids were synthesized and subjected to coupling reactions
in methanol using 30 mol% of copper sulfate. The list of
dipeptides (1–10) synthesized from this protocol is given in
Table 1. All N-protected thioacids were synthesized using the
reported procedure.16 Instructively, no methyl esters or free
carboxylic acids of the corresponding thioacids were observed
in the acylation reactions. Out of all the thioacids, we were
able to obtain single crystals of Boc-Leu-SH and its X-ray
structure is shown in the ESI.w All dipeptides were isolated in
satisfactory yields including sterically hindered amino acids,
Val(3, 6), Aib(7) and Ile(8), in less than 5 min. Though the
reaction proceeds with the coupling efficiencies more than
90% (HPLC analysis), the isolated yields after the column
purification are given in Table 1. Inspired by the clean, efficient
and fast amide bond coupling reaction, we further extended
this strategy to synthesize tri and tetrapeptides. The dipeptide
amine 1 was coupled to the Boc-Ser(OBut)-SH, as anticipated
the tripeptide 11 was isolated in good yield in less than 5 min.
Similarly, other tripeptides 12 and 13 were isolated with
satisfactory yields. The tetrapeptides (14–16) were synthesized
using a 2+2 convergent strategy. The corresponding dipeptide
thioacids were synthesized similar to the N-protected thioacids
and subjected to the coupling reactions with dipeptide amines.
Peptide 15 with highly b-branched valine residues was isolated
in satisfactory yield within 5 min. To ensure the compatibility
of the thioacid coupling reactions in the presence of electron
Scheme 4 Synthesis of peptides using 30 mol% of CuSO4Á5H2O in
MeOH.
Table 1 List of peptides synthesized using 30 mol% of CuSO4Á5H2O
Entry
Xxa
Xxb
Xxa–Xxb
% Yield
Pg = Boc, n = 1, m = 0, X = OMe
1
2
3
4
5
Ala
Leu
Leu
Ala
Leu
Val
Ala-Leu
DAla-Leu
Val-Ala
68
62
76
72
63
DAla
Val
Ser(OBut)
Trp
Ser(OBut)-Leu
Trp-Val
Pg = Fmoc, X = OMe
6
7
8
9
Val
Aib
Ile
Leu
Phe
Val
Val
Val-Lue
Aib-Phe
Ile-Val
77
69
70
65
Pro
Pro-Val
Pg = Cbz, X = OMe
10 Leu
Trp
Leu-Trp
60
n = 1, m = 1, Pg = Boc, X = OMe
Xxa
Ser(OBut)
Ser(OBut)
Xxb–Xxc
Ala-Leu
Ala-Val
Xxa–Xxb–Xxc
Ser(OBut)-Ala-Leu
Ser(OBut)-Ala-Val
11
12
64
61
Pg = Cbz, X = OMe
13 Leu
Val-Val
Leu-Val-Val
64
n = 2, m = 1, Pg = Boc, X = OMe
14
15
Ala-Val
Val-Leu
Leu-Leu
Val-Val
Ala-Val-Leu-Leu
Val-Leu-Val-Val
74
72
Pg = Boc, X = OEt
16 Aib-Ala
dgL-dgL
Aib-Ala-dgL-dgL
69
(dgL = a,b-unsaturated g-leucine).
As shown in Scheme 3, CuSO4Á5H2O gave better yield (71%)
followed by Cu(OAc)2ÁH2O (57%) and AgOAc gave only 38%
in 5 min. Other metal complexes gave low yields with longer
time durations. In their pioneering work, Blake and Li
reported the Ag(I) mediated fragment coupling reactions of
peptide thioacids in 50% aqueous DMF.12 The results observed
for the Ag(I) mediated coupling reaction in methanol are in
accordance with the reported results. In the optimization of the
concentration of copper sulfate from 1 to 100 mol%, we found
that reaction with 30 mol% of copper sulfate was comparable
with that with the 100 mol%, however even 10 mol% also
mediated the amide bond formation with longer time duration
(3 h). In a control reaction without the metal complex, 5% of
Fig. 1 Powder XRD pattern of the byproduct CuS (covellite).
7086 Chem. Commun., 2012, 48, 7085–7087
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deficient unsaturated amides and esters,17 which are subjected to
Michael addition reaction,18 the tetrapeptide 16 was synthesized.
Instructively, the tetrapeptide was isolated without any side
products. All tetrapeptides were isolated with satisfactory
yields within 5 min and are given in Table 1.
of a mixture of elemental sulphur (S7, S18 etc.) in the reaction
mixture, the detailed mechanism of the liberation of sulfur in the
reaction needs to be investigated.
In conclusion, we have successfully demonstrated a novel,
ultrafast, scalable amide bond synthesis in methanol with a range
of amides and peptides. The coupling products of sterically
hindered amino acids, tri and tetrapeptides were isolated in less
than few minutes. This protocol can be utilized for the synthesis
of synthetically challenging membrane peptides and peptides
containing sterically hindered amino acids.
In hindsight, the comparable results of 30 mol% of copper
sulfate with that of 100 mol% provoked us to investigate the
role of insoluble byproduct CuS in the coupling reactions. To
understand whether or not CuS is involved in the amide bond
formation, a control reaction with Boc-Ala-SH and benzylamine
was carried out in the presence of 30 mol% of CuS. Surprisingly,
the formation of an amide bond was observed with the same rate
as that of copper sulfate. To verify its compatibility, we further
synthesized dipeptides 2, 3, 10, tripeptide 13 and the tetrapeptide
14 (Table 1). We found no difference between the insoluble CuS
and the soluble CuSO4. Further, we carried out the amide bond
formation using Boc-Ala-SH and H-Leu-OMe in neat methanol
after filtering the CuS to verify whether or not the CuS treated
methanol will accelerate reaction. Results suggest that there was
no acceleration of amide bond formation even after 24 h,
indicating the need of CuS to accelerate the reaction. Albeit it
is contradicting to our initial assumption regarding the solubility
of copper sulfate, the serendipity provides insight into the role of
metal sulfides as catalysts in the peptide bond formation using
thioacids.19 In contrast to the model experiments on the fixation
of the carbon monoxide and the activation of thioacetic acid on
metal sulfides under primordial conditions,19 we observed only
amide bond formation in the CuS catalyzed reaction. Based on
these observations, we propose the possible mechanism for the
amide bond synthesis (Scheme 5). We anticipate that the initial
step proceeds with direct involvement of the copper complex
(CuSO4Á5H2O) in the reaction leading to the formation of CuS
and the amide bond. Subsequently, the in situ generated CuS acts
as a catalyst to activate the thioacid through coordination. The
reaction between the activated intermediate and the free amine leads
to the formation of an amide bond along with the regeneration of
CuS (Scheme 5). Though the LC-MS analysis suggests the presence
We are thankful to DST, Govt of India, for financial
support. S.M.M. and S.V.J. are thankful to CSIR, for a
research fellowship.
Notes and references
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Scheme 5 Proposed mechanism for the CuSO4Á5H2O mediated and
CuS catalyzed amide bond synthesis.
19 C. Huber and G. Wachtershauser, Science, 1997, 276, 245–247.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 7085–7087 7087