PH-Dependent peptide bond formation by the selective coupling of α-amino acids in water

Article abstract of DOI:10.1039/d0cc06042a

A novel mechanism enabling selective peptide elongation by coupling α-amino acids over other potentially competing prebiotic amines under acidic aqueous condition is suggested. It proceeds via the generation of a carboxylic acid anhydride intermediate with subsequent intramolecular formation of the amide bond. This journal is

Full text of DOI:10.1039/d0cc06042a

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pH-Dependent peptide bond formation by the
selective coupling of a-amino acids in water†
Cite this:DOI: 10.1039/d0cc06042a
Long-Fei Wu, * Ziwei Liu
and John D. Sutherland*
Received 7th September 2020,
Accepted 20th November 2020
DOI: 10.1039/d0cc06042a
rsc.li/chemcomm
A novel mechanism enabling selective peptide elongation by coupling Thus, a model peptide, N-acetyl L-alanine 2 (Ac-Ala, 100 mM),
a-amino acids over other potentially competing prebiotic amines under methyl isonitrile 1 (B130 mM) and one of the different amine
acidic aqueous condition is suggested. It proceeds via the generation nucleophiles 3 (50 mM, the concentration of 2 was higher than
of a carboxylic acid anhydride intermediate with subsequent intra- that of the amine nucleophiles to minimise multiple elongation)
molecular formation of the amide bond.
were mixed in water and left to incubate at pH 3, 4 or 5 (Fig. 1A).
The reaction mixture was monitored by H-NMR spectroscopy at
1
The widespread importance of the synthesis of amides lies not 23 1C until most of the methyl isonitrile has been consumed. After
only in synthetic organic chemistry,1–3 but also in long-standing 15 days at pH 5, 62% of glycine 3a (pKaH = 9.6) was incorporated
efforts aimed at understanding the formation of peptides into amide products, Ac-Ala-Gly 4a and Ac-Ala-Gly-Gly 4c (Fig. 1B
composed of a-amino acids in the origin of life.4–7 Although and Fig. S1, ESI†). Using b-alanine 3b (b-Ala, pKaH = 10.2),
the ‘‘RNA world’’ hypothesis is a popular working model for glycylglycine 3c (Gly-Gly, pKaH = 8.1), glycinamide 3d (pKaH = 8.2),
studying the origin of life,8,9 peptides could have coexisted glycine nitrile 3e (pKaH = 5.2) and methylamine 3f (pKaH = 10.6), we
with RNA at the outset of life. This is suggested by the fact that observed reaction with Ac-Ala 2 to give the elongated amide
a-amino acids and nucleotide building blocks might have had a products, Ac-Ala-b-Ala 4b, Ac-Ala-Gly-Gly 4c (and minor amount
common chemical origin.10–12 We have previously shown that of Ac-Ala-Gly-Gly-Gly-Gly), Ac-Ala-Gly-NH2 4d, Ac-Ala-CN 4e, and
the chemical energy inherent in methyl isonitrile 1 – a poten- Ac-Ala-NHCH3 4f, respectively, in comparable yield to the com-
tially prebiotic activating reagent13 – can be harnessed to activate bined yield of products from 3a (as determined by 1H-NMR
and join phosphate and carboxylate building blocks simultaneously spectroscopy, Fig. 1B and Fig. S2–S6, ESI†). However, Ac-Ala-NH2
under acidic aqueous conditions.14 We have now investigated 4g was only formed in 17% yield when ammonia 3g (pKaH = 9.3)
peptide formation chemistry mediated by methyl isonitrile 1 in a was used as the nucleophile (Fig. S7, ESI†), this is consistent with
systems chemistry scenario. We considered various potentially the lower nucleophilicity of ammonia compared to some amines
prebiotic amine nucleophiles which could have been present with similar pKaH values in water.19,20 At pH 4, the rate of reaction
together in a common pool on early Earth. We found that amide was significantly higher than it was at pH 5 with consumption of
bond formation shows very limited selectivity for all amines methyl isonitrile 1 in 48 hours.14,21,22 The yield of incorporation of
examined at pH 5, regardless of the fact that the pKa values of glycine 3a into peptide product 4a was only slightly lower (58%)
their conjugate acids differ by up to 5.4 units. When the pH of the than the analogous reaction run at pH 5 (Fig. 1B and Fig. S8, ESI†),
reactions was lowered to 3, however, a-amino acids had the highest and the yield of 4b decreased to 47% when using 3b (Fig. 1B and
reactivity amongst all the tested substrates, suggesting a unique Fig. S9, ESI†). However, the conversion of 3c or 3d proceeded in
mechanism and a potential means of selecting a-amino acids for significantly lower yields (approximately 20%, Fig. 1B and Fig. S10,
peptide elongation.
S11, ESI†), and no reaction of 3f and 3g with 2 was detected by
A prebiotically plausible mixture would most likely have con- 1H-NMR spectroscopy (Fig. 1B and Fig. S12, S13, ESI†). In contrast,
tained a range of different amine nucleophiles, regardless of quantitative conversion to 4e was obtained when glycine nitrile 3e
whether it was produced endogenously15,16 or exogenously.17,18 was used as the nucleophile (Fig. 1B and Fig. S14, ESI†). We argued
that it might be related to more of the unprotonated form of glycine
nitrile being available because of its low pKaH.6 When reactions
were run at pH 3, methyl isonitrile was consumed in only 6 hours.
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge,
CB2 0QH, UK. E-mail: lfwu@mrc-lmb.cam.ac.uk, johns@mrc-lmb.cam.ac.uk
However, only glycine 3a, b-alanine 3b, glycine nitrile 3d underwent
coupling with 2 to give 4a, 4b and 4d in yields of 46%, 5% and 30%,
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc06042a
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Fig. 2 (A) Model peptide elongation by coupling a mixture of various amine
nucleophiles at pH 3 mediated by methyl isonitrile 1. Concentrations of the
two dominating products 4a and 4d were calculated based on proton signal
1
integration on H-NMR spectroscopy. (B) Multiple rounds of peptide elon-
gation by coupling an a-amino acid with model peptide 2 mediated by
methyl isonitrile at pH 3, 4 or 5. Concentrations of the products were
1
calculated based on proton signal integration on H-NMR spectroscopy.
Considering that progressive peptide elongation is needed to
make potentially functional peptides, a reaction with Ac-Ala 2
(20 mM), glycine 3a (100 mM), and methyl isonitrile 1 (B110 mM)
was incubated at pH 3, 4 or 5 to evaluate multiple rounds of peptide
elongation (Fig. 2B, other amine nucleophiles were excluded to
simplify interpretation of NMR spectra). At pH 3, dipeptide Ac-Ala-
1
Gly 4a and tripeptide Ac-Ala-Gly-Gly 4c were observed by H-NMR
spectroscopy in yields of 46% and 14%, respectively, after 6 hours
(Fig. S23, ESI†), which clearly showed that progressive peptide
elongation can be achieved under a-amino acid-selective conditions.
At pH 4, the yields of 4a and 4c were 54% and 24%, respectively, after
48 hours (Fig. S24, ESI†). Longer peptides could be observed in
minor amounts based on 1H-NMR spectroscopy. At pH 5, after
15 days, the yields of 4a and 4c were 34% and 24% respectively, and
tetrapeptide Ac-Ala-Gly-Gly-Gly 4h was also observed in a yield of
13% (Fig. 2B and Fig. S25, ESI†). Although higher yields and longer
peptides could be achieved at pH 5, other amine nucleophiles
Fig. 1 (A) Formation of amide bond mediated by methyl isonitrile with
different amine nucleophiles in water at acidic pH values. (B) Yields based on
the incorporation of the amine nucleophiles 3 into peptide products 4 at pH 5,
4 and 3 after 15 days, 48 hours and 6 hours, respectively. Reactions were run in
duplicate and average yields are given. n.d.: amide product not detected.
respectively (Fig. 1B and Fig. S15, 17, ESI†). The best yield when would start to compete with a-amino acids to form amide bonds
using glycine 3a as amine nucleophile suggests there must be a as shown in Fig. 1.
unique mechanism related to glycine 3a in amide bond formation
Next, we investigated the underlying mechanism of the
mediated by methyl isonitirle 1 in low pH solution. To further unique property of a-amino acids in the amide bond formation
evaluate if this behaviour also applied to other a-amino acids, we chemistry mediated by methyl isonitrile 1. Considering the
tested arginine, serine, valine and proline with Ac-Ala 2 at pH 4. structural features of all the amine nucleophiles, 3a to 3g, we
Yields of incorporation of these amino acids into peptides ranging reasoned that the carboxylate group of glycine 3a might be
from 44% to 66% (comparable yields to glycine 3a) were observed implicated in the special reactivity of a-amino acids. We
in 48 hours as expected (Fig. S18–S21, ESI†).
suggest that the carboxylate group of Ac-Ala 2 was activated
Accordingly, we wondered if the selectivity we had observed by methyl isonitrile 1 to give intermediate imidoyl acetyl
could play out in the sort of mixed system that might have existed alanine 5 via acid catalysis.14 Imidoyl acetyl alanine 5 can give
on early Earth. A reaction mixture with Ac-Ala 2 (100 mM), glycine oxazolone 6 via intramolecular cyclization.14,23,24 Both 5 and 6
3a (25 mM), b-alanine 3b (25 mM), glycylglycine 3c (25 mM), can be attacked by free amines, if they are sufficiently abun-
glycinamide 3d (25 mM), glycinenitrile 3e (25 mM), methylamine 3f dant, to give the amide products intermolecularly (Fig. 3A,
(25 mM), ammonia 3g (25 mM) and methyl isonitrile 1 (B130 mM) direct pathway). Under more acidic condition, more protona-
was incubated at pH 3 and monitored by 1H-NMR spectroscopy at tion of the amines will inhibit their reactivities as nucleophiles.
23 1C (Fig. 2A). Peptides 4a and 4e were the two major products with However, we assumed that the carboxylate/carboxylic acid
yields of 55% and 42%, respectively, based on 1H-NMR spectroscopy group of glycine 3a (pKaH = 2.3) also acts as a nucleophile to
(Fig. S12, ESI†), and this is in line with the results of the separate attack intermediates 5 and/or 6 to give mixed anhydride 7. 7
experiments described above. Interestingly, 4b was not observed by could afford the desired peptide elongation product 4a by an
1H-NMR spectroscopy (Fig. S18, ESI†), which suggests that the O - N acyl transfer intramolecularly via a 5-membered ring
reactivity of 3b was suppressed under a competing scenario.
transition state (Fig. 3A, indirect pathway). The pKaH of 7 is
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side products were observed (Fig. S1, S8 and S15, ESI†), and were
assigned as cis- and trans-N-imidoyl glycine, 13-cis and 13-trans,
based on NMR spectroscopy (Fig. 3C and Fig. S29–S33, ESI†). We
suggest that the formation of 13-cis and 13-trans is due to the
imidoylation of the amino group presumably via intermediate 14
followed by an O - N imidoyl transfer. 13 can hydrolyse to give
N-formyl glycine.26 And N-formyl alanine was also shown to
elongate effectively by coupling glycine 3a to give N-formylated
peptides (Fig. S34, ESI†). These products are reminiscent of the
formylated N-terminal residue in extant protein biosynthesis.
Prebiotic chemistry is most likely to give a mixed pool of
various plausible species on early Earth.10–12,15–18 Exploring
whether there is selectivity among those mixed species in the
process of forming oligomers is of great importance to under-
stand the transition from prebiotic chemistry to biochemistry.
Poor selectivity among various amine nucleophiles was
observed when amide bond formation was mediated by methyl
isonitrile 1 at only slightly acidic aqueous solution (e.g. pH 5).
Under conditions of pH 6 or above, peptide elongation will be
very slow because of a lack of acid catalysis. But, a unique
chemical property inherent to a-amino acids could enable the
incorporation of a-amino acids selectively into peptides at pH 3
in the presence of other amine nucleophiles. Also, progressive
peptide elongation by incorporating a-amino acids can still be
achieved in the same set of conditions. A mixed carboxylic acid
Fig. 3 (A) Proposed mechanistic scheme for methyl isonitrile mediated
model peptide elongation by coupling a-amino acids under acidic condi-
tions. The direct pathway represents the mechanism of direct amide bond
formation by attacking of the amine nucleophile on intermediates 5 or 6
intermolecularly, and the indirect pathway represents the mechanism of
amide bond formation intramolecularly following the mixed anhydride
intermediate 7. (B) Supporting evidence for the indirect pathway proposed
in (A). (C) Possible mechanism for the formation of side product N-imidoyl
glycine, 13-cis and 13-trans.
presumably lower than that of glycine 3a and this coupled with anhydride intermediate (e.g. 7a, Fig. 3A) was suggested to be
the lower entropic penalty of an intramolecular reaction25 is the reason contributing to the highest activity of a-amino
why this reaction can occur whilst direct intermolecular amide acids at pH 3. As a-aminonitriles are easily hydrolysed to
formation cannot (e.g. 3a versus 3d in amide bond formation at a-aminoamides (e.g. by organocatalysis by aldehydes27), then
pH 3 in Fig. 1). To test this hypothesis, we devised an experiment to a-aminoacids on a longer time scale (geologically plausible),
to probe the putative formation of mixed anhydride intermediate one can imagine a prebiotic scenario in which a-amino acids
7a. Ac-Ala 2 (100 mM), methyl isonitrile 1 (B130 mM) and existed alongside various other amines, but in the absence of
glycolic acid 8 (50 mM) were mixed at pH 3 or 4, respectively, a-aminonitriles. In such a scenario, peptides comprising only
1
and the reactions were monitored by H-NMR spectroscopy at a-amino acids could have been produced at low pH by the
23 1C (Fig. 3B). The ester product 9 was observed in good yields chemistry described herein. Alternatively, if the hydrolysis of
at both pH values (Fig. S26 and S27, ESI†). In a negative control a-aminonitriles to a-amino acids was incomplete, peptides
reaction, Ac-Ala 2 (100 mM), methyl isonitrile 1 (B130 mM) and based on a-amino acids could still have been produced, but
methyl glycolate 11 (50 mM) were mixed at pH 4 (Fig. 3B), but they would occasionally be terminated by a-aminonitriles.
1
ester 12 was not observed by H-NMR spectroscopy after all the Interestingly, Powner and co-workers recently described a
methyl isonitrile had been consumed (Fig. S28, ESI†). These three-steps process in coupling each a-aminonitrile as
results strongly suggest that the formation of ester 9 proceeds via a-amino acid equivalent in aqueous solution.6 The selective
the generation of mixed anhydride 10 followed by an O - O acyl coupling of a-aminonitrile in their work resulted from the
transfer. Taken together, these results are an indication that corresponding much lower pKaH values of a-aminonitrile than
under the most acidic conditions amide 4a is still able to form in other amino nucleophiles, which was also partially exemplified
good yield because the reaction proceeds through the mixed here. Hud and co-workers have explored the ester product 9
anhydride intermediate 7a followed by an O - N acyl transfer analogues as key intermediates in amide bond formation
(Fig. 3A, indirect pathway). A similar mechanism would be also driven by wet-dry cycles without using activating reagent.28
expected to apply to b-alanine 3b, but the lesser amount of We anticipate that, in solution phase as described here, if the
carboxylate anion of b-alanine 3b relative to that of glycine 3a pH of the solution were brought up by environmental fluctua-
because of the higher pKa of the carboxylic acid group of 3b tion, amines might react with ester 9 slowly and low selectivity
(pKa = 3.6) compared to 3a (pKa = 2.3), the greater degree of would be expected.29 Regarding the low pH conditions,
protonation of the amino group of b-alanine because of its although models predict that the pH of the ocean of early Earth
higher pKaH than glycine 3a, and different ring-size of the was near neutral,30,31 regional lakes/ponds with high acidity
transition states of intramolecular acyl transfer (6-membered would have been possible.32,33
ring versus 5-membered ring) might contribute to its lower yield
This work was supported by the Medical Research Council
of amide product at pH 3 (Fig. 1B). It is worth mentioning that (no. MC_UP_A024_1009 to J. D. S.) and the Simons Foundation
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Conflicts of interest
There are no conflicts to declare.
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