One-pot synthesis of alternating peptides exploiting a new polymerization technique based on Ugi's 4CC reaction

Article abstract of DOI:10.1039/c6cc09379e

We developed a one-pot synthetic technique for alternating peptides. Central to this technique is a new, catalyst-free polymerization based on Ugi's 4CC reaction. The treatment of imines with the ambident molecules bearing both an isocyanide and a carboxylic acid afforded alternating peptides.

Full text of DOI:10.1039/c6cc09379e

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
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One-Pot Synthesis of Alternating Peptides Exploiting a New
Polymerization Technique based on Ugi’s 4CC Reaction
Yasuhito Koyamaa* and Prashant G. Gudeangadia
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We developed a one-pot synthetic technique of alternating itself is highly challenging, which strictly restricts the industrial
peptides. Central to the technique is a new, catalyst-free applications.
polymerization based on Ugi’s 4CC reaction. Treatment of imines
with the ambident molecules bearing both an isocyanide and technique of alternating peptides. The technique is a catalyst-
a carboxylic acid afforded alternating peptides. components
free polymerization10 analogous to Ugi’s
We have recently developed a new, one-pot synthetic
4
condensation (Ugi’s 4CC) for the synthesis of dipeptides.11
Scheme 1 shows the monomer structures for the alternating
peptide conceived by the reaction mechanism of Ugi’s 4CC.
Polypeptides with
particular attention as a promising candidate of innovative
structural materials.1 Fibrous proteins such as fibroin,2
a repeating sequence have attracted
collagen,3 and elastin4 are the representative class among
natural polypeptides. Fibroin is the main ingredient of silk and
spider silk, whose fiber has been adopted to textile industry,
owing to its luster, favourable mechanical properties, and good
gas and moisture permeability. Collagen and elastin are
structural proteins in a vertebrate body and are included in the
extracellular matrix. Such proteins have a specific repeating
sequence, which form a higher-order-structure related to the
expression of fibrous functions.
For the creation of new structural materials, we became
intrigued by the evaluation of polypeptide sequence−property
relationship based on the development of practical synthetic
method for alternating peptides.
The ring-opening
polymerization of α-amino acid N-carboxyanhydrides (NCA)5 or
chemoenzymatic synthesis of polypeptide6 enable the synthesis
of homo poly(α-amino acid)s and block copolypeptide with
controlled molecular weight and narrow polydispersity index in
a living fashion. Poly(α-amino acid)s can be also prepared by
the polymerization of activated urethane derivatives of α-
Scheme 1. Reaction mechanism of Ugi’s 4CC and monomer
structures for alternating peptide.
In a typical Ugi’s 4CC reaction, a stoichiometric mixture of
aldehyde, amine, isonitrile, and carboxylic acid produces the
corresponding dipeptide (Scheme 1). The reaction starts from
the condensation between an aldehyde (R1CHO) and an amine
(R2NH2) to give an imine. Nucleophilic addition of an isonitrile
(R3NC) to the imine is followed by the addition of carboxylic
acid (R4COOH) to I to afford an intermediate II. Subsequent
acyl migration and tautomerization give the dipeptide. These
amino acid7 or N-thiocarboxyanhydrides.8
However, the
alternating copolymerization technique for polypeptide has not
been sufficiently developed. Although the polycondensation of
dipeptide as
a monomer can afford the corresponding
alternating peptide,9 the preparation of the dipeptide monomer
a.Department of Biotechnology, Faculty of Engineering, Toyama Prefectural
University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Electronic Supplementary Information (ESI) available: [Experimental procedures,
and characterization data for all reactions and products, including 1H NMR, 13C NMR,
IR, and DOSY spectra and DLS profiles]. See DOI: 10.1039/x0xx00000x
steps require no catalyst.
Our idea for the new
polymerization technique is to connect R3 and R4. This
connection between R3 and R4 of the product would indicate
the structure of alternating
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peptide. Therefore, the reaction of imine (R1C=NR2) with the neutralization of the potassium salt (1 and 2). We first selected
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DOI: 10.1039/C6CC09379E
ambident molecule bearing both isonitrile and carboxylic acid N-allyl-1-(4-methoxyphenyl)methanimine (5) as the coupling
could proceed to the unprecedented polymerization to give partner of 3, considering that the proton signals of the resulting
the alternating peptide without catalyst. Herein, we describe polymer would separately appear in the 1H NMR spectrum,
the first
demonstration
of
one-pot
synthesis
of which could be diagnostic for the formation of alternating
alternating peptides.12 We found that the use of i-PrOH peptide.
as a solvent afforded high yield of the polymer. It turned
The ambident molecule 3 was generated by the addition of a
out that the polymerization efficiency can be qualitatively stoichiometric amount of TfOH to 1, which was directly
estimated by the diffusion coefficient (D) obtained in the treated with a stoichiometric amount of imine 5. The mixture
diffusion-ordered NMR spectroscopy (DOSY).13
was stirred at arbitrary temperature and quenched by the
The ambident molecules were prepared by the addition of sat. aq. NaHCO3. After typical workup, CHCl3
saponification of corresponding ethyl esters with KOH and solution of the products was reprecipitated into hexane to give
isolated as a potassium salt (1 and 2) by the evaporation of Poly-5a as a hexane-insoluble part. It is noted that treatment
solvent according to the literatures14 (Scheme S115). Because of 5 with 1 without the neutralization process resulted in no
the preparations of the neutral compounds 3 and 4 with a high reaction.
purity were difficult, 3 and 4 were prepared in situ by the
Table 1. Effects of solvent, temperature, and time on polymerization.a
entry
1
2
3
4
5
6
7
8
solvent
MeOH
MeOH
DMF
DMF
DMF
MeOH
i-PrOH
2,4-dimethyl-3-pentanol
o-chlorophenol
i-PrOH
MeOH−H2O (9:1)
MeOH−H2O (1:1)
MeOH−H2O (1:1)
i-PrOH
additive
temp. & time
rt, 1 d
yield of Poly-5a (%)b
D (m2/S)c
−44 x 10.7−6.34
10
63.2
52.3
68.0
65.6
67.5
54.9
64.4
72.6
50.5
94.7
48.1
53.4
60.6
94.4
37.9
reflux, 1 d
rt, 1 d
−x 10 7.54−6.82
−x 10 9.35−8.39
−x 10 9.19−7.64
10
10
10
−
−
MS 3A
o
MS 3A
MS 3A
60 C, 1 d
100 C, 1 d
−x 10 1.44−1.08
o
9
−x 10 5.97−5.64
−x 10 6.23−5.26
−x 10 7.81−7.40
10
10
10
rt, 3 d
rt, 3 d
rt, 3 d
rt, 3 d
rt, 3 d
rt, 3 d
rt, 3 d
−
−
−
−x 10 1.25−1.15
−x 10 10.6−9.53
−x 10 9.39−8.84
−x 10 6.49−5.64
−x 10 7.97−7.38
−x 10 6.76−5.79
9
9
10
10
10
10
10
−
10
11
12
13
14d
15d,e
MS 3A
−
−
−
−
o
60 C, 3 d
rt, 3 d
rt, 3 d
−x 10 9.21−8.42
10
i-PrOH
a
b
The reaction was performed by using 1 (1.0 mmol), 5 (1.0 mmol), and TfOH (1.0 mmol) in solvent (2.0 M). Hexane-insoluble
part. Diffusion coefficients were determined by the DOSY spectra of Poly-5a (ca. 3 mg) in CDCl3 (0.6 mL). After stirring for 2 d,
solvent was removed. The residue was stirred for the additional 1 d. In place of 1, compound 2 was used to give Poly-5b. DMF
−
c
d
e
= N,N-Dimethylformamide. MS 3A = Molecular sieves 3Å.
To confirm polymerization efficiency, size exclusion column
chromatography (SEC) was not reliable due to the very strong
interaction between the polymer and column stationary
phase. It was found that dynamic light scattering (DLS) was
also not reliable for the measurement of non-optimal
condition mainly consisting of low molecular weight (MW)
polymer. Thus, we utilized the DOSY to obtain the D values of
molecules, which
are related to hydrodynamic radius (Rh) and MW.13
Previously there has been attempts to provide
molecular weight distribution directly from DOSY.13c-e It
has been recently revealed that the logarithm of D (log D)
linearly correlates to the log MW.13f The unit model 6 was
prepared as a reference sample. The D values of 5 and 6
were evaluated to be 1.55−1.63 x 10− and 1.25−1.33 x
9
10− (m2/S), respectively
9
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(Figures S9 and S27).15 Table 1 shows the effects of reaction
solvent, temperature, and time on the yield and the D value.
Based on an Einstein−Stokes equation (Rh = kBT/6ηπD), the
lower D suggests the bigger Rh, indicating the formation of
higher MW polymer. All polymers except for those of entries
5 and 9 exhibited lower D than those of 5 and 6. Through the
comparison of the results in entries 1−5, it was found that (i)
the lower temperature afforded a higher MW of Poly-5a and
(ii) the use of MeOH as a protic solvent fVaiecwiliAtratitcelesOntlihnee
polymerization reaction, similar to the tendency of Ugi’s
DOI: 10.1039/C6CC09379E
4CC.11 The prolonged reaction time from 1 d to 3 d afforded
increased molecular weight of Poly-5a (entries 1 and 6).
For the optimization of a protic solvent, we examined the use
of bulky solvents (entries 7 and 8) and phenolic solvent (entry
9), and the addition of water (entries 10−13).
Table 2. Substrate scope and limitation of polymerization.a
entry
imine
yield of polymer (%)
D (m2/S)
Mwb (Da)
−x 10 7.61−
10
1
Poly-7: 93.2
4,400
7.10
−x 10 7.87−
10
2
3
Poly-8: 65.7
Poly-9: 91.2
6,100
4,400
6.90
−x 10 8.70−
10
6.70
4
5
decomp.
−
−
−x 10 8.27−
10
10
Poly-10: 81.7
Poly-11: 98.2
8,600
6.46
−x 10 5.12−
6
12,200
4,400
4.30
−x 10 8.20−
10
7
8
Poly-12: 57.1
7.64
No reaction
−
−
a
The reaction was performed by using imine (1.0 mmol), 1 (1.0 mmol), and TfOH (1.0 mmol) in i-PrOH (2.0 M) at room
b
After stirring for 2 d, i-PrOH was removed. The residue was stirred for the additional 1 d. Mw was calculated from
temperature.
the calibration curve using the experimental values of D.13f,15
As a result, the use of i-PrOH gave the lowest D value
(entry 7), indicating that polar and less-nucleophilic i-PrOH
affords both high MW and excellent yield. The use of
nucleophilic solvent and high temperature condition
turned out that the hydrodynamic volume of Poly-5a was
almost 26−30 times larger than that of 5. After considerable
efforts, we concluded that the high concentration condition
as shown in entry 14 is optimal from both sides of D and
yield. Poly-5b was also prepared by using 4 in place of 3 at
the same manner (entry 15), although the yield was low,
probably
might lead the isonitrile as
a propagation end to
unfavourable side reactions such as alcoholysis, hydrolysis,
and oligomerization.16 It also
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dependent on the unstability of
4 under the reaction
Notes and references
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DOI: 10.1039/C6CC09379E
condition.16 The polymer structures were confirmed by 1H
NMR, 13C NMR, and IR spectra.15 The 1H NMR spectrum of
Poly-5a appears as broad signals, but consists of all proton
signals from each component with appropriate integral ratio
(Figure S29),15 indicating that the polymer includes both 5
and 3 skeletons. In the 13C NMR spectrum (Figure S30),15
the appearance of carbon signals at around 160−170 ppm
clearly indicates the formation of amide linkages
between the fragments. The chemical shifts are in good
accordance with those of model compound 6. In the IR
1
(a) J. M. van Hest, J. Macromol. Sci., Part C: Polym. Rev. 2007,
47, 63. (b) T. J. Deming, Adv. Mater. 1997, 9, 299. (c) T. J.
Deming, Chem. Rev. 2016, 116, 786. (d) L. A. Smith, P. X. Ma,
Colloid. Surf. B: Biointer. 2004, 39, 125. (e) S. A. Sell, M. J.
McClure, K. Garg, P. S. Wolfe, G. L. Bowlin, Adv. Drug. Deliv.
Rev. 2009, 61, 1007.
2
(a) B. Lotz, Biochim. 1979, 61, 205. (b) C. Vepari, D. L. Kaplan,
Prog. Polym. Sci. 2007, 32, 991. (c) Y. Wang, H.-J. Kim, G.
Vunjak-Novakovic, D. L. Kaplan, Biomater. 2006, 27, 6064.
(d) E. Wenk, H. P. Merkle, L. Meinel, J. Contr. Rel. 2011, 150,
128.
3
4
K. A. Piez, Encycl. Polym. Sci. Eng. 1985, 3, 699.
spectrum (Figure S32), amide absorption bands are observed
(a) J. Rosenbloom, W. R. Abrams, R. Mecham, FASEB J. 1993,
7, 1208. (b) D. R. Eyre, Ann. Rev. Biochem. 1984, 53, 717.
(a) T. J. Deming, J. Am. Chem. Soc. 1997, 119, 2759. (b) J.
Huang, A. Heise, Chem. Soc. Rev. 2013, 42, 7373. (c) T. J.
at around 1650 cm−
,
also
1 15
in the same region of
6
supporting the formation of amide linkages. The results
clearly indicate that the reaction led to Ugi’s 4CC-based
polymerization to form Poly-5a as an alternating
copolymer. In the 1H and 13C NMR spectra of Poly-5b
(Figures S33 and S34),15 all signals separately appear,
whose chemical shifts were well agreed with those of 6. The
methylene carbon at the α-position of amide is observable as
a couple of signal at 35.7 and 35.6 ppm in Figure S34, which
could be attributed to the cis-trans rotamers of N,N-
disubstituted amide bond. From the above, it is indicated
that the broadening of 1H NMR spectrum of Poly-5a would
come from the proximity of both chiral methyne centers
and the N,N-disubstituted amides between the repeating
unit. The weight-average MW (Mw) were estimated by
the linear correlation between log D and log M, to be 8,600
Da for Poly-5a and 2,200 Da for Poly-5b, respectively.13f,15
With the optimal condition (Table 1, entry 14), we next
investigated the scope and limitation of the polymerization
(Table 2). As for the substituent on the nitrogen atom as a R2
described in Scheme 1, bulkier functional groups such as
benzyl, isopropyl, and phenyl groups were also available for
the polymerization to give Poly-7−Poly-9 (entries 1−3). When
the aliphatic group was introduced to the R1 moiety of imine,
the polymerization gave a complex mixture (entry 4), mainly
because of the degradation of 10. On the other hand, the
preparation of 10 in situ dramatically improved the
polymerization results, which gave Poly-10 in a good yield
(entry 5). It is noted that the benzyl deprotection of Poly-10
can produce Poly(Gly-Val), although the chirality on the Val
structure is not controlled. The reaction of cyclic imine 11
and ketimine 12 with 3 gave the corresponding polymers
(Poly-11 and Poly-12) (entries 6 and 7). However, O-
methyloxime 12 was not reactive under the polymerization
condition (entry 8).
5
Deming, Adv. Polym. Sci. 2006, 202, 1.
6
(a) S. Nitta, A. Komatsu, T. Ishii, H. Iwamoto, K. Numata,
Polym. J. 2016, 48, 955. (b) J. Fagerland, A. Finne-Wistrand, K.
Numata, Biomacromol. 2014, 15, 735. (c) P. J. Baker, K.
Numata, Biomacromol. 2012, 13, 947.
(a) Y. Kamei, A. Sudo, H. Nishida, K. Kikukawa, T. Endo, J.
Polym. Sci. Part A: Polym. Chem. 2008, 46, 2525. (b) Y. Kamei,
A. Nagai, A. Sudo, H. Nishida, K. Kikukawa, T. Endo, J. Polym.
Sci. Part A: Polym. Chem. 2008, 46, 2649.
7
8
9
X. Tao, B. Zheng, H. R. Kricheldorf, J. Ling, J. Polym. Sci. Part
A: Polym. Chem. 2017, 55, 404.
(a) W. L. Mattice, L. Mandelkern, Biochem. 1971, 10, 1926.
(b) W. L. Mattice, L. Mandelkern, Biochem. 1971, 10, 1934.
(c) D. A. Rabenold, W. L. Mattice, L. Mandelkern,
Macromolecule 1974, 7, 43. (d) J. E. Scheffler, L. J. Berliner,
Biophys. Chem. 2004, 112, 285.
10 (a) Click chemistry: fundamentals and practical technologies,
T. Takata, Y. Koyama, K. Fukase Eds. CMC Publishing Co. Ltd.,
Tokyo, 2014. (b) S. Cheawchan, S. Uchida, H. Sogawa, Y.
Koyama, T. Takata, Langmuir, 2016, 32, 309. (c) C.-G. Wang,
Y. Koyama, S. Uchida, T. Takata, ACS Macro Lett. 2014, 3, 286.
(d) S. Cheawchan, Y. Koyama, S. Uchida, T. Takata, Polymer,
2013, 54, 4501. (e) Y.G. Lee, Y. Koyama, M. Yonekawa, T.
Takata, Macromolecules, 2010, 43, 4070.
11 (a) I. Ugi, R. Meyr, U. Fetzer, C. Steinbrückner, Angew. Chem.
1959, 71, 386. (b) A. Dömling, I. Ugi, Angew. Chem., Int. Ed.
2000, 39, 3168. (c) I. Ugi, B. Werner, A. Dömling, Molecules,
2003, 8, 53. (d) U. K. Sharma, N. Sharma, D. D. Vachhani, E.
V. van der Eycken, Chem. Soc. Rev. 2015, 44, 1836. (e) J. G.
Rudick, J. Polym. Sci. Part A: Polym. Chem. 2013, 51, 3985.
12 For selected reviews concerning alternating copolymer from
vinyl monomers, see: (a) A. Sommazzi, F. Garbassi, Prog.
Polym. Sci. 1997, 22, 1547. (b) I. Cho, Prog. Polym. Sci. 2000, 25,
1043. (c) D. Braun, F. Hu, Prog. Polym. Sci. 2006, 31, 239.
(d) B. M. Culbertson, Encycl. Polym. Sci. Eng. 1987, 9, 225. (e)
K. A. Parker, N. S. Sampson, Acc. Chem. Res. 2016, 49, 408.
13 (a) K. F. Morris, C. S. Jr. Johnson, J. Am. Chem. Soc. 1992, 114,
3139. (b) C. S. Jr. Johnson, Prog. NMR Spectrosc. 1999, 34,
203. (c) A. Chen, D. Wu, C. S. Johnson, J. Am. Chem. Soc. 1995,
117, 7965. (d) A. Jerschow, N. Müller, Macromolecules 1998,
31, 6573. (e) J. Viéville, M. Tanty, M.-A. Delsuc, J. Magn.
Reson. 2011, 212, 169. (f) W. Li, H. Chung, C. Daeffler, J. A.
Johnson, R. H. Grubbs, Macromolecules 2012, 45, 9595.
14 (a) R. J. Spandl, H. Rudyk, D. R. Spring, Chem. Commun. 2008,
44, 3001. (b) B. R. Galan, K. P. Kalbarczyk, S. Szczepankiewicz, J.
B. Keister, S. T. Diver, Org. Lett. 2007, 9, 1203. (c) D. Bonne,
M. Dekhane, J. Zhu, Org. Lett. 2004, 6, 4771.
In conclusion, we developed a new, one-pot, and practical
synthetic method for alternating peptides on the basis of
Ugi’s 4CC.
The reaction also features catalyst-free
polymerization. The present method may provide several
insights into not only material innovation exploiting
alternating
polymerization technique in future.
peptide skeleton but also new click
The applications
15 See ESI.
directed toward the synthesis of natural polypeptides
and the asymmetric polymerization using a chiral auxiliary or
catalyst are currently underway.
16 For a selected review, see: F. Millich, Chem. Rev. 1972, 72,
101.
This work was financially supported by JSPS KAKENHI
Grant Numbers JP24685023 and JP15H00718 and by the
Tokuyama Science Foundation.
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Graphical Abstract
Catalyst-free, one-pot synthetic technique for alternating peptide was developed on the basis of Ugi’s
4 component condensation reaction.