Tandem deprotection/coupling for peptide synthesis in water at room temperature

Article abstract of DOI:10.1039/c7gc01575e

A tandem deprotection/coupling sequence is reported for solution-phase peptide synthesis in water under micellar catalysis conditions using the designer surfactant TPGS-750-M. Cbz deprotection followed by peptide coupling in the presence of COMU and 2,6-lutidine afforded polypeptides containing up to 10 amino acid residues. A broad scope characterizes this new technology. No epimerization has been detected. The associated E Factors, as a measure of "greenness" and known to be extremely high for peptide couplings, have been reduced to less than 10 due to the step-economy and minimal amounts of organic solvent needed for product extraction.

Full text of DOI:10.1039/c7gc01575e

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Tandem deprotection/coupling for peptide synthesis in water at
room temperature
Margery Cortes-Clerget,a Jean-Yves Berthon,b Isabelle Krolikiewicz-Renimel,b Laurent
Chaisemartin,b and Bruce H. Lipshutza
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
tandem deprotection/coupling sequence is reported for jeopardize the stereointegrity of sensitive amino acids,17
solution-phase peptide synthesis in water under micellar catalysis microwaves were also proposed as a source of energy to
conditions using the designer surfactant TPGS-750-M. Cbz accelerate the coupling step in water, or in the absence of
deprotection followed by peptide coupling in the presence of solvent.18–20 Nonetheless, in most of these reports the scope of
COMU and 2.6-lutidine afforded polypeptides containing up to 10 the reaction is limited and large amounts of co-solvents are
amino acid residues.
A broad scope characterizes this new often needed to prevent aggregation.
technology. No epimerization has been detected. The associated E
Factors, as a measure of “greenness” and known to be extremely Another approach to reduce waste creation during peptide
high for peptide couplings, have been reduced to less than 10 due synthesis is to rely on step-economy. Katoh et al. described a
to the step-economy and minimal amounts of organic solvent one-pot tripeptide synthesis where the Fmoc protecting group
needed for product extraction.
was removed by tetrabutylammonium fluoride (TBAF) hydrate,
followed by in-situ peptide bond elongation using the HOBt
analog TBTU. The reaction takes place in either THF or in DMF,
along with a thiol to scavenge the resulting dibenzofulvene.21
Zorn et al. applied a 1-pot process to arrive at an Alloc-
protected peptide. Deprotection was performed by Pd(PPh3)4
and DABCO, and was followed by in-situ coupling in the
presence of either a Boc- or Fmoc-protected amino acid and
EDC/HOBt in dichloromethane.22
Introduction
For decades, amide bond formation especially in medicinal
chemistry has been among the most heavily utilized reactions.
1,2
Peptides alone will account for an estimated USD 25.4
billion by 2018 on the global therapeutic market.3 But with
respect to the choice of reaction solvent, peptide synthesis in
terms of its environmental footprint is far from benign.
Attempts have been made to replace harmful DMF or DCM by
less egregious organic solvents such as ethyl acetate, 2-methyl
tetrahydrofuran, or N-methylpyrrolidine.4,5,6 However, massive
consumption of organic solvents is still widespread due to the
step-by-step nature of these sequences, even though water is
the natural peptide biosynthetic medium. Few examples have
been described in this “solvent”, mainly because of the low
solubility of protected amino acids. Various approaches have
been designed to mimic Nature; among them, the introduction
of water-soluble activating reagents,7,8 the development of
hydrosoluble protecting groups,9–12 and the pulverization of
protected amino-acid to form hydrosoluble nanoparticles.13–16
And although use of elevated temperatures is known to
Our group has previously introduced an environmentally
friendly method for amide/peptide bond formation in an
aqueous micellar medium.23 The reaction takes place within
the core of nanomicelles, formed by a 2 wt % aqueous solution
of TPGS-750-M (Figure 1). COMU and 2,6-lutidine were found
to be the most efficient combination for the coupling step
between two protected amino acids at room temperature.
After deprotection, performed in organic solvent, a convergent
[2+2] synthesis led, for example, to the Streptocidin
precursor Cbz-Leu-Phe-Pro-Leu-OEt in good (87%) yield.23
C
H2O
H2O
H2O
O
H2O
H2O
O
O
O
17
O
O
3
vitamin E =
hydrophobic part
succinate
linker
M-PEG-750 =
hydrophilic part
H2O
Hydrophobic core
The reaction takes
place here
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Herein, we describe an investigation into related peptide From this model dipeptide
Journal Name
(
2a), Cbz deprotection was
syntheses in water, focusing now on both N-terminus performed using 10 wt % Pd/C and hydrogen gas immediately
deprotection and subsequent coupling in a 1-pot fashion at followed by the coupling of Cbz-Ala-OH in the presence of
room temperature, together with a greatly enlarged substrate COMU and 2.6-lutidine. To prevent loss of the Cbz group on
scope of both polar and apolar amino acids leading to this third amino acid, the hydrogen gas was removed by argon
elongated peptide lengths.
bubbling through the mixture in between both steps (Figure
2).
O
O
1) H2, Pd/C10% (10 wt %)
2 wt % TPGS-750-M/H2O
H
Results and discussion
H
Cbz
N
OEt
N
OEt
N
N
Cbz
N
H
H
H
2)
O
O
Among the multitude of known amine protecting groups, the
Cbz residue was chosen for study as its hydrogenation is the
preferred method of deprotection, rather than other
alternatives (e.g., Fmoc),24 typically being both clean and
without by-product formation that could affect the
subsequent coupling step. We also took advantage of the
lipophilicity of this commercial and readily available protecting
group, which should enhance its localization inside the
hydrophobic micellar core. To achieve this N-terminus
deprotection, a screening of sources of both palladium as well
as hydrogen donors was conducted on model dipeptide Cbz-L-
Phe-L-Leu-OEt, 2a (Table 1).
O
Cbz
OH
N
H
O
COMU, 2,6-lutidine, rt, 12 h
Cbz-L-Phe-L-Leu-OEt (2a)
Cbz-L-Ala-L-Phe-L-Leu-OEt (3a)
E Factorsa
organic solvent: 10
pH adjustment
non homogeneous: 50%
+ HCl (1.0 equiv) homogeneous: 92%
organic solvent + water: 15
Fig. 2 Importance of pH on the tandem deprotection/coupling sequence for
tripeptide synthesis, and E Factor calculations for the 2-step process. a Single run
on a 3.5 mmol scale; 20 mL of MTBE were used for extraction, isolated yield:
86% (1.53 g)
Initially, the non-homogeneous nature of the reaction during
deprotection led ultimately to the tripeptide in yields as low as
50% over the two steps. The free amine was also suspected of
strong chelation, occupying the active sites of the catalyst. In
this regard, acetic acid is often used as co-solvent to generate
the ammonium salt and prevent loss of palladium activity, as
well as aggregation. Hence, an aqueous solution of HCl (1.0
equiv vs. peptide) was added resulting in a homogeneous
Table 1 Cbz deprotection conditions: screening of catalysts and sources of H2
O
O
H
O
N
OEt
H2N
OEt
Pd source, H2 donor
N
N
H
H
2 wt % TPGS-750-M/H2O
[0.5 M]
O
O
O
reaction mixture (pHt=0 ≈ 1
ꢀ pHt=2h ≈ 4) leading to the desired
tripeptide in a global yield of 92%. To verify the importance of
the surfactant, the coupling step leading to dipeptide 2a was
conducted solely in deionized water. The yield dropped to 40%
compared to a nearly quantitative outcome in aqueous TPGS-
750-M. It should also be noted that the same procedure was
tested in the presence of EDC and HOBt. Thus, in addition to
safety concerns raised by the use of HOBt, slightly lower yields
were obtained confirming our previous results and the
efficiency of the COMU/2,6-lutidine system.
Cbz-L-Phe-L-Leu-OEt (2a)
H-L-Phe-L-Leu-OEt
Entry
Catalyst
Hydrogen donor
TMDS (1.5 equiv.)
Et3SiH (1.5 equiv.)
H2 gas
Time (h)
1
2
3
4
5
6
7
PdCl2 (5 mol %)
PdCl2 (5 mol %)
PdCl2 (5 mol %)
PdCl2 (5 mol %)
Pd/C10% (10 wt %)
Pd(OH)2 (20 mol %)
Pd(OAc)2 (10 mol %)
2
21
2
NaBH4 (1.2 equiv.)
H2 gas
21
2
H2 gas
2
Several dipeptide building blocks were prepared, including
both apolar and polar amino acids (Table 2). Thus, peptides
containing, e.g., ornithine (in peptide 2c), aspartic acid (in
peptide 2m) or tyrosine (in 2r and 2t) could all be fashioned in
water with good-to-excellent yields. Both tyrosine and serine27
(in peptide 2o) were used without a protecting group on the
side chain.
H2 gas
2
We have previously described the in-situ generation of
hydrogen gas during Pd-catalyzed silylations and
hydrodehalogenations of aryl halides from PdCl2 and
tetramethyldisiloxane (TMDS) on water at room
temperature.25 These conditions were first evaluated on
dipeptide 2a (Table 1, entry 1). Total deprotection was
observed within 2 h. Unfortunately, several by-products were
also detected. Another silane, triethylsilane, as well as sodium
borohydride were tested but found to be less efficient (entries
2 and 4; time to completion: 21 h). Hydrogen gas was used
directly and the reaction was fast and clean (entry 3). The
palladium source was then investigated (entries 5-7). In all
cases, deprotection was complete in less than 2 h. From an
economics point of view, palladium-on-charcoal and hydrogen
gas were selected for this tandem process.26
In some cases, especially when the final product was not
readily soluble in the core of the micelle, the media turned
into a paste, affecting stirring and the efficiency of the
reaction. We also observed that glycine-containing peptides
showed lower yields. As previously demonstrated,28 the
addition of a co-solvent could improve both yield and handling
of a given reaction mixture, and potentially facilitate an
industrial process. Addition of 10% THF was noted to increase
the yield significantly from 41% to 75% in the case of Z-
Arg(Pbf)-Ala-OEt (to compound 2s), while the reaction failed
2 | J. Name., 2012, 00, 1-3
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completely under classic conditions (in DCM with EDC and decapeptide 10a could thus be obtained via a [5+5] convergent
HOBt). The co-solvent effect played an even greater role in the strategy in 72% yield over two steps. Likewise, hexapeptide 6a
case of Cbz-Pro-Gly-OEt (compound 2d) and Cbz-Ser-Ile-OMe was prepared using a [4+2] and a [3+3] approach in 89% and
(compound 2o), leading to an increase of approximately 40% 76% yields, respectively.
yield. Thus, the precursor of the C-terminal tripeptide portion
of
α-MSH-13, Lys-Pro-Val (Figure 3; Table 3; peptide 3c),
Table 3 Tri- to deca-peptides synthesized by a deprotection/coupling tandem process
known for its anti-inflammatory activity,29 was obtained in
better yield in the presence of THF (65% vs. 51%). In addition,
the more lipophilic Cbz protecting group leads to better yields
compared to Boc; e.g., in the case of PG-Pro-Leu-OEt
(compounds 2k and 2f; 94% vs. 84%, isolated yields).
in 2 wt % aqueous TPGS-750-M
Peptide
Yield (%)
92
Peptide
Yield (%)
70
3a
3b
3c
3d
Cbz-Ala-Phe-Leu-OEt
Cbz-Gly-Phe-Leu-OEt
Cbz-Lys(Cbz)-Pro-Val-OMe
Cbz-Val-Orn(Boc)-Leu-OMe
4a
4b
4c
4d
Cbz-Phe-Leu-Ile-Val-OMe
Cbz-Pro-Leu-Phe-Leu-OEt
Cbz-Val-Gly-Val-Ala-OEt
Cbz-Pro-Val-Pro-Tyrb-OMe
Cbz-D-Phe-Pro-Val-
Orn(Boc)-Leu-OMe
Table 2 Dipeptides synthesized in 2 wt % aqueous TPGS-750-M solution
82
89
51 (65)a
51 (79)a
60
86
3e
Cbz-D-Phe-Pro-Val-OMe
Peptide
66
5a
88
Peptide
Yield (%)
99
Peptide
Yield (%)
94
Yield (%)
2a
2b
Cbz-Phe-Leu-OEt
Cbz-Val-Gly-OEt
2k
2l
Cbz-Pro-Leu-OEt
Cbz-D-Phe-Pro-OMe
26 (95)a
83
5b
6a
Cbz-Ala-Phe-Leu-Asp(tBu)-Ala-OMe
Cbz-Pro-Leu-Phe-Leu-Phe-Ala-OEt
89
89 (76)c
2c
2d
2e
2f
Cbz-Orn(Boc)-Leu-OMe
Cbz-Pro-Gly-OEt
91
2m
2n
2o
2p
Cbz-Asp(tBu)-Ala-OMe
Cbz-Tyrb-Tyrb-OMe
Cbz-Serb-Ile-OMe
Cbz-Phe-Ala-OEt
74 (83)a
42 (85)a
72
84
6b
8a
Cbz-D-Phe-Pro-Val-Orn(Boc)-Leu-D-Phe-OMe
75
Cbz-Val-Ala-OEt
44 (82)a
85
Cbz-D-Phe-Pro-Val-Orn(Boc)-Leu-D-Phe-Pro-Val-OMe
86
10a
Cbz-D-Phe-Pro-Val-Orn(Boc)-Leu-D-Phe-Pro-Val-Orn(Boc)-Leu-OMe
82 (72)d
Boc-Pro-Leu-OEt
84
a
b
c
2g
2h
2i
Cbz-D-Phe-Pro-OMe
Cbz-Ile-Val-OMe
Cbz-Pro-Val-OMe
Cbz-Ala-Phe-OEt
30 (63)a
70
2q
2r
2s
Cbz-Pro-Ala-OEt
65
performed in presence of 10
%
THF as co-solvent, unprotected side chain, performed with
a
[3+3]
Cbz-Pro-Tyrb-OMe
71
convergent approach, d performed with a [5+5] convergent approach
41 (75)a
Nature of the residue: Aliphatic / Acidic / Basic / Aromatic / Hydroxylic
88
Cbz-Arg(Pbf)-Ala-OEt
2j
82
a performed in presence of 10 % THF as co-solvent, b unprotected side chain
Nature of the residue: Aliphatic / Acidic / Basic / Aromatic / Hydroxylic
Attempts to obtain the tripeptide 3a in a coupling/depro-
tection/coupling 1-pot sequence using COMU/2,6-lutidine
gave very poor results (<20 % yield). Addition of up to 80%
Pd/C failed to fully deprotect the newly formed polypeptide.
This m,ay be attributable to the oxime by-product interfering
with the catalyst. The resulting pasty mixture was not optimal
for proper stirring. EDC-HOBt/TEA gave encouraging results
but no more than 50% yield overall was achieved.
To highlight the robustness of this 2-step, 1-pot process,
longer peptides were also investigated (Table 3). To access a
peptide of more than four residues, we first adopted a [x+2]
convergent strategy where only 10 wt % Pd/C10% was needed
for deprotection of the first dipeptide. As the length and the
polarity of the peptide increased, the global concentration had
The stereointegrity of the process was evaluated based on a
representative coupling step. The mild conditions employed
favored maintenance of optical purity, as epimerization was
not observed in the formation of dipeptide Z-L-Phe-L-Leu-OEt
to be reduced to 0.25
M to achieve better stirring and
solubility. Cbz-Val-Gly-Val-Ala-OEt (Figure 3; peptide 4c), a
tetrapeptide precursor of the anti-microbial Dermaseptin30
was thus obtained by a [2+2] tandem sequence in 60% yield
over two steps. This process also tolerated both polar and
nonpolar amino acids and allowed the preparation of
decapeptide Cbz-D-Phe-Pro-Val-Orn(Boc)-Leu-D-Phe-Pro-Val-
Orn(Boc)-Leu-OMe, the linear precursor of the antibiotic
gramicidin S via a convergent [8+2] approach (Figure 4;
peptide 10a, 82% isolated yield). For polypeptides longer than
two units, 50 wt % of Pd/C10% was required. The same
(
2a) (Table 4, entry 1).31 Likewise, the tandem deprotection/
coupling sequence applied to the preparation of the same
dipeptide (2a), starting from Cbz-L-Leu-OEt, led to the same
conclusion: that the chiral integrity was not compromised
(Table 4, entry 2).
Fig. 4 Synthesis of gramicidin S precursor 10a via a convergent [5+5] or [8+2], 2-steps,
Fig 3 Precursors of biologically active peptides via a 2-step, 1-pot process
1-pot approach
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Journal Name
4
5
6
Y. E. Jad, G. A. Acosta, S. N. Khattab, B. G. de la Torre, T.
Govender, H. G. Kruger, A. El-Faham and F. Albericio, Amino
Acids, 2016, 48, 419.
Table 4 Determination of extent of epimerization under optimized conditions
O
O
H
H
N
O
Y. E. Jad, G. A. Acosta, S. N. Khattab, B. G. de la Torre, T.
Govender, H. G. Kruger, A. El-Faham and F. Albericio, Org.
Biomol. Chem., 2015, 13, 2393.
N
H
Cbz
OH
Cbz
N
R
OEt
+
N
OEt
H
O
(2a)
S. Lawrenson, M. North, F. Peigneguy and A. Routledge, Green
Chem., 2017, 19, 952.
Entry
1
-R
Conditions
eea
7
8
9
A. El-Faham and F. Albericio, J. Pept. Sci., 2010, 16, 6.
J. C. Sheehan and J. J. Hlavka, J. Org. Chem., 1956, 21, 439.
G. I. Tesser and I. C. Balvert-Geers, Int. J. Pept. Res. Ther., 1975,
7, 295.
H·HCl
CBz
COMU, 2.6-lutidine, TPGS-750-M/H2O
> 99 %
> 99 %
1) Pd/C, H2, HCl, TPGS-750-M/H2O
2) COMU, 2.6-lutidine
2
a Determined by chiral HPLC analysis
10 K. Hojo, M. Maeda and K. Kawasaki, Tetrahedron Lett., 2004, 45,
9293.
While E Factors for pharmaceuticals are typically between 25
and 100, due to the stepwise nature of chemical peptide
synthesis, values are estimated to be 100 times larger.32 Here,
the reduced amounts of organic solvent used for extraction,
and the step-economy involved, led to E Factors of 15 and 10
based on organic solvent used both with, and without, water
in the calculations, respectively (Figure 2).
11 K. Hojo, M. Maeda and K. Kawasaki, Tetrahedron, 2004, 60,
1875.
12 H. Kunz, Angew. Chem. Int. Ed. Engl., 1978, 17, 67.
13 K. Hojo, H. Ichikawa, M. Onishi, Y. Fukumori and K. Kawasaki, J.
Pept. Sci., 2011, 17, 487.
14 K. Hojo, H. Kitai, M. Onishi, H. Ichikawa, Y. Fukumori and K.
Kawasaki, Chem. Cent. J., 2011, 5, 49.
15 K. Hojo, H. Ichikawa, Y. Fukumori and K. Kawasaki, Int. J. Pept.
Res. Ther., 2008, 14, 373.
Conclusion
16 K. Hojo, H. Ichikawa, M. Maeda, S. Kida, Y. Fukumori and K.
Kawasaki, J. Pept. Sci., 2007, 13, 493.
An efficient technology has been developed that dramatically
reduces the environmental impact of traditional solution-
based polypeptide synthesis that relies on
a tandem
17 B. Bacsa, K. Horvati, S. Bosze, F. Andreae and C. O. Kappe, J. Org.
Chem., 2008, 73, 7532.
deprotection/peptide coupling under mild aqueous micellar
conditions. This approach is broadly applicable to several types
of amino acids that contain differing polarities, as well as an
array of varying substitution patterns on their side chains,
including phenyl or alkyl moieties, protected amine or
carboxylic acid, and unprotected alcohol and phenolic groups.
The polypeptide length has been extended to ten amino acid
residues without significant loss of efficiency. Elimination of
two environmentally egregious organic solvents (DCM or DMF)
has been demonstrated, while the associated E Factors as a
measure of waste generated have dropped to 10 for this 2-
step sequence. Access to cyclic peptides by this methodology
is under active investigation.
18 A. S. Galanis, F. Albericio and M. Grotli, Org. Lett., 2009, 11,
4488.
19 K. Hojo, N. Shinozaki, K. Hidaka, Y. Tsuda, Y. Fukumori, H.
Ichikawa and J. D. Wade, Amino Acids, 2014, 46, 2347.
20 A. Mahindra, N. Patel, N. Bagra and R. Jain, RSC Adv., 2014, 4,
3065.
21 M. Ueki, N. Nishigaki, H. Aoki, T. Tsurusaki and T. Katoh, Chem.
Lett., 1993, 721.
22 C. Zorn, F. Gnad, S. Salmen, T. Herpin and O. Reiser, Tetrahedron
Lett., 2001, 42, 7049.
23 C. M. Gabriel, M. Keener, F. Gallou and B. Lipshutz H., Org. Lett.,
2015, 17, 3968.
24. While the Fmoc group might well be amenable to this
Acknowledgments
deprotection/coupling sequence, the methylenefluorene by-
product becomes organic waste, in addition to the base and
resulting salts that end up in the aqueous phase, also as waste.
Hence, the Cbz group appears to be the greenest option.
The financial support provided by Greentech as well as the
instrumentation support from NIH (1S10OD012077) are
gratefully acknowledged.
25 A. Bhattacharjya, P. Klumphu and B. Lipshutz H., Org. Lett.,
2015, 17, 1122.
Notes and references
26 Sigma Aldrich (01/17/17): Pd/C 10%: $26.5/g vs. PdCl2 $56.2/g.
27 For serine, performed with NMM to avoid the formation of an
acyl-pyridinium intermediate. For more details, see: Org.
Process Res. Dev., 2016, 20, 1104.
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Peptide Therapeutics Market - Global Industry Analysis, Size,
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28 C. M. Gabriel, N. R. Lee, F. Bigorne, P. Klumphu, M. Parmentier,
F. Gallou and B. Lipshutz H., Org. Lett., 2017, 19, 194.
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29 A. Macaluso, D. McCoy, G. Ceriani, T. Watanabe, J. Biltz, A.
Catania and J. M. Lipton, J. Neurosci., 1994, 14, 2377.
30 G. D. Brand, J. R. S. A. Leite, S. M. de Sa Mandel, D. A. Mesquita,
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Journal Name
ToC Graphic
Procedures for peptide synthesis in water at rt
Fragment 2
Fragment 1
+
Cbz
OH
Cbz
OMe
1
2
Pd/C + H2 + HCl
COMU + 2,6-lutidine
PG
OMe
up to 10 amino acids
yields as high as 99%
all types of amino acids
no racemization
6 | J. Name., 2012, 00, 1-3
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