Synthesis of orthogonally protected thioamide dipeptides for use in solid-phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2016.10.036

Orthogonally protected thioamide-containing dipeptides were efficiently and cleanly prepared from the precursor dipeptides using Curphey's method (P₄S₁₀, hexamethyldisiloxane (HMDO), reflux, DCM) in 67–96% isolated yield. This was in

Full text of DOI:10.1016/j.tetlet.2016.10.036

Tetrahedron Letters 57 (2016) 5237–5239
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of orthogonally protected thioamide dipeptides for use
in solid-phase peptide synthesis
⇑
Kim Manzor, Fintan Kelleher
Department of Science, Molecular Design & Synthesis Group, Centre of Applied Science for Health, Institute of Technology Tallaght, Dublin 24, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Orthogonally protected thioamide-containing dipeptides were efficiently and cleanly prepared from the
precursor dipeptides using Curphey’s method (P4S10, hexamethyldisiloxane (HMDO), reflux, DCM) in 67–
96% isolated yield. This was in contrast to the use of Lawesson’s or Berzelius’ reagents where significant
issues with reaction non-completion, decomposition and purification were observed. Subsequent clean
removal of the dipeptides’ t-butyl ester protecting groups gave thioamide dipeptide acids which were
suitable for use in solid-phase peptide synthesis (SPPS).
Received 17 August 2016
Revised 4 October 2016
Accepted 10 October 2016
Available online 12 October 2016
Keywords:
Thioamide
Ó 2016 Elsevier Ltd. All rights reserved.
Thionation
Curphey’s method
Thiopeptides
The WHO has recognised antimicrobial resistance (AMR) as one
of the greatest challenges facing humankind, with some bacterial
species already becoming resistant to all currently used antibi-
otics.1 A recent report has predicted that, if the situation is left
unchecked, by 2050 up to 10 million people annually will die from
bacterial infections, with an overall cost of US$100 trillion.2 How-
ever, the pipeline of new antibiotics for tackling this crisis is very
low since most of the world’s major pharmaceutical companies
no longer have antibiotic discovery programmes. In order to
address this critical issue, there is an urgent global need for the
development of a wide range of new antibiotic classes. The world-
wide market for peptide-based therapeutics is forecasted to grow
to $23.7 billion by 2020,3 where chemically synthesised peptides
will be a significant, and growing, proportion of this figure. Pep-
tide-based antimicrobials are a class that has significant potential
in combatting AMR, and include lantibiotics and antimicrobial pep-
tide (AMPs).4 A further class is the thiopeptide antibiotics, which
include thiostrepton, siomycins and thiopeptins, amongst many
others.5 The chemical synthesis of many thiopeptides requires
the efficient preparation of thioamides, in place of amides, as the
peptide link. These thioamides can then be left with the thioamide
peptide bond intact as bioisosteres, or further converted to the thi-
azoline and thiazole heterocyclic moieties, which are common in
thiopeptide antibiotics. Peptides containing thioamides are known
in some cases to improve aqueous solubility and pharmacokinetic
properties, such as half-life.6 The synthesis of thioamides has been
reviewed, and one of the principal methods used is the thionation
of amides.7 A large number of methods have been developed for
the thionation of carbonyl groups to their thiocarbonyl analogues,
where the main reagents include Lawesson’s reagent,8 Berzelius’
reagent,9 more recently Curphey’s method.10 The use of a P4S10
–
pyridine complex was also reported by Bergman et al. in 2011.11
As part of a project for preparing antimicrobial peptides con-
taining specific thioamide isosteres in place of amides by solid-
phase peptide synthesis (SPPS), there was a need for the efficient
synthesis of specific peptidic thioamide moieties. It is known that
it is not possible to conduct the thionation reaction of an amide
bond when the peptide is already on a solid-phase resin. It is also
not possible to selectively thionate a particular amide in an
oligopeptide or polypeptide sequence. Therefore, we required the
solution-phase synthesis of orthogonally protected dipeptide frag-
ments containing the thioamide peptide bond, where the fragment
could then be incorporated into a peptide sequence using SPPS. Our
initial targets required the preparation of dipeptides of type (A)
Fmoc-NH-Ile-Xaa-COOBut, where Xaa was glycine, alanine, pheny-
lalanine and type (B) Fmoc-NH-Xaa-Leu-COOBut where Xaa were
similar residues to type A (Fig. 1). Fmoc SPPS was selected as the
method of choice for preparation of the required peptides.
A review of the literature showed that Strømgaard and co-
workers12 recently reported the successful use of Lawesson’s
reagent in tetrahydrofuran at room temperature for the prepara-
tion of thioamide-containing dipeptides in good isolated yields of
>80%. Before attempting the amide thionation reaction, the dipep-
tide precursors for the type A fragments were efficiently prepared
in acetonitrile, in 93–97% isolated yield, by the coupling of
⇑
Corresponding author. Tel.: +353 1 404 2869.
E-mail address: fintan.kelleher@ittdublin.ie (F. Kelleher).
http://dx.doi.org/10.1016/j.tetlet.2016.10.036
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

5238
K. Manzor, F. Kelleher / Tetrahedron Letters 57 (2016) 5237–5239
Table 1
Key 1H and 13C NMR chemical shifts for the characterisation of dipeptides and
thioamide derivatives
R'
O
H
N
O
OBut
H
N
FmocHN
Structure
Analogue
Amide/
NH
FmocHN
OBut
O
thioamide (ppm)
C@O/C@S
O
R
(ppm)
Type A
Type B
R = CH2-
Ph (1)
170.6
6.21
R or R' = H, CH3, PhCH2
R = CH3 (2)
R = H (3)
171.5
171.2
6.33
6.52
Figure 1. Structures of the two types of dipeptide precursor targets.
O
H
N
OBut
FmocHN
Fmoc-l-Ile-COOH with the required amino acid protected as its t-
butyl ester, using HATU in the presence of triethylamine
(Scheme 1).12
The type B dipeptides were similarly prepared by the coupling
of Fmoc protected L-amino acids with L-leucine t-butyl ester under
O
R
R
R = CH2-Ph
(4)
R = CH3 (5)
R = H (6)
204.1
8.21
203.9
204.1
8.59
8.14
O
O
similar reaction conditions. In this case the isolated yields were
slightly lower (86–96%).
H
N
OBut
FmocHN
FmocHN
With the dipeptides in hand, the next step involved selective
thionation of the amide group to a thioamide in the presence of
both carbamate and ester functional groups. Although Bergman
and co-workers reported the use of the P4S10–pyridine complex
for amide thionation, the reaction conditions required reflux in
acetonitrile (bp 85 °C), pyridine (bp 115 °C) or in dimethyl sul-
folane at 165–175 °C. In our experience, heating of orthogonally
protected dipeptides at temperatures of 80–90 °C, leads to signifi-
cant decomposition, so it was felt that Bergman’s method was too
harsh for the examined dipeptide substrates. Likewise Shibihara
and Murai’s method for converting amides to thioamides using ele-
mental sulfur required reflux in toluene at 115 °C,13 while Poupaert
and co-workers reported the use of P4S10 in the presence of Al2O3
in 1,4-dioxane at reflux (bp 101 °C).14 Both sets of conditions were
also considered too harsh in our case.
S
R0 = CH2-Ph
(7)
R'
R'
6.13
171.4
H
N
R’ = CH3 (8)
172.1
172.1
6.71
6.63
OBut R’ = H (9)
O
R0 = CH2-Ph
(10)
O
202.3
7.97
H
N
R’ = CH3 (11)
204.9
199.0
8.24
8.35
OBut R0 = H (12)
FmocHN
S
As a result, Strømgaard’s method for selective amide thionation
in the presence of both carbamate and ester carbonyl groups was
used.12 However, in our hands it was found that even after further
multiple additions of molar equivalents of Lawesson’s reagent,
incomplete reaction was observed with significant quantities of
the starting dipeptide still present in each case. It was also found
when using this method, that purification of the required thioa-
mide peptide was laborious due to difficulties in separating the
required compounds from by-products formed from Lawesson’s
O
+
O
O
i
ii
H3N
Cl-
+
OH
OBut
H
N
H
N
FmocHN
OBut
OBut
FmocHN
FmocHN
R
O
O
R
S
R
R
1
= PhCH2- ( ) 97%
= CH3
= H
R
4
= PhCH2- ( ) 96%
= CH3
= H
2
( ) 93%
5
( ) 76%
3
( ) 95%
6
( ) 67%
Scheme 1. Synthesis of Type A dipeptides and their thioamide analogues. Reagents and conditions: (i) Et3N, HBTU, CH3CN, rt, 2 h; ii) P4S10, (Me3Si)2O, DCM, reflux.
R'
O
R'
O
O
R'
+
H
N
H
N
H3N
+
OH
OBut
OBut
OBut
FmocHN
FmocHN
FmocHN
Cl-
i
ii
O
S
O
R
7
R
10
= PhCH2- ( ) 81%
= PhCH2- ( ) 86%
8
11
12
= CH3
= H
( ) 92%
= CH3
= H
(
(
) 95%
) 79%
9
( ) 96%
Scheme 2. Synthesis of Type B dipeptides and their thioamide analogues. Reagents and conditions: (i) Et3N, HBTU, CH3CN, rt, 2 h; (ii) P4S10, (Me3Si)2O, DCM, reflux.

K. Manzor, F. Kelleher / Tetrahedron Letters 57 (2016) 5237–5239
5239
O
O
H
N
H
N
i
OBut
FmocHN
FmocHN
OH
S
R
S
R
R
4
= PhCH2- ( )
R
13
= PhCH2- ( ) 90%
= H (14) 88%
= H (6)
Scheme 3. Representative examples for deprotection of the t-butyl ester group to give thioamide dipeptides 13 and 14, ready for SPPS. Reagents and conditions: (i) 50% TFA
in DCM, Ph3SiH, rt, 2 h.
reagent. Significant time and effort was expended trying to opti-
mise this reaction by changing the solvent, temperature, reaction
time, and the use of new batches of Lawesson’s reagent, however,
to no avail. A significant amount of the starting dipeptide was pre-
sent, in each case, as well decomposition products were observed.
Moving on, Berzelius’ reagent (P4S10) was used, but as for
Lawesson’s reagent after many attempts, using a variety of reaction
conditions, incomplete reaction and/or decomposition was always
observed. As for the use of Lawesson’s reagent, significant difficul-
ties with purification also arose.
into peptide sequences by SPPS. The thioamide dipeptides are
now being used for this purpose and the results of these studies
will be reported in due course.
Acknowledgements
We are grateful to Science Foundation Ireland for funding for
KM (Grant number 11/RFP.1/CHS/3306).
Supplementary data
Finally Curphey’s method,10 using P4S10 (1.1 molar equiv) in the
presence of hexamethyldisiloxane (HMDO, 5.5 M equiv) was
attempted. After some optimisation it was found that the use of
dichloromethane as solvent at reflux, as well as the further addi-
tion of the same amount of P4S10 and HMDO after 5 h, and close
monitoring of the reaction by TLC, consistently gave the desired
thioamide products in 67–96% isolated yields, after purification
by column chromatography (Schemes 1 and 2). No evidence of
any remaining starting dipeptides was observed by TLC and the
chromatographic purification was straightforward.
The dipeptides and their thioamide analogues were fully char-
acterised using a range of spectroscopic techniques, where 1H
and 13C NMR spectroscopy showed the diagnostic downfield move-
ment in the chemical shifts for both the carbon atom of the amide
carbonyl group and the amide N–H proton, when the amides were
converted to their thioamide derivatives12 (Table 1).
With the orthogonally protected thioamide dipeptides in hand
it was then required that the t-butyl ester be efficiently removed,
so that the Fmoc-protected thioamide dipeptides with a free car-
boxylic acid could be used for SPPS. This was readily achieved
(ꢀ90% yield) by treating the orthogonally protected thioamide
dipeptides with 50% TFA in DCM in the presence of triphenylsilane
as a scavenger (Scheme 3).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.10.
036.
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In conclusion the use of P4S10 and HMDO to cleanly and selec-
tively thionate the peptide bond of orthogonally protected dipep-
tides was achieved to consistently give the desired compounds in
good yields. This was in contrast to the use of Lawesson’s reagent
or Berzelius’ reagent, where incomplete reaction and/or decompo-
sition, as well as difficulties with purification, was observed in
every case. Subsequent C-terminus deprotection gave the thioa-
mide dipeptide fragments (13 and 14) suitable for incorporation