Prebiotic Synthesis of N-Formylaminonitriles and Derivatives in
Formamide
Nicholas J. Green,* David A. Russell, Sasha H. Tanner, and John D. Sutherland*

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ABSTRACT: Amino acids and their derivatives were probably
instrumental in the transition of prebiotic chemistry to early
biology. Accordingly, amino acid formation under prebiotic
conditions has been intensively investigated. Unsurprisingly, most
of these studies have taken place with water as the solvent. Herein,
we describe an investigation into the formation and subsequent
reactions of aminonitriles and their formylated derivatives in
formamide. We find that N-formylaminonitriles form readily from
aldehydes and cyanide in formamide, even in the absence of added
ammonia, suggesting a potentially prebiotic source of amino acid
derivatives. Alkaline processing of N-formylaminonitriles proceeds
with hydration at the nitrile group faster than deformylation,
protecting aminonitrile derivatives from reversion of the Strecker
condensation equilibrium during hydration/hydrolysis and furnishing mixtures of N-formylated and unformylated amino acid
derivatives. Furthermore, the facile synthesis of N-formyldehydroalanine nitrile is observed in formamide from glycolaldehyde and
cyanide without intervention. Dehydroalanine derivatives have been proposed as important compounds for prebiotic peptide
synthesis, and we demonstrate both a synthesis suggesting that they are potentially plausible components of a prebiotic inventory,
and reactions showing their utility as abiotic precursors to a range of compounds of prebiological interest.

■ INTRODUCTION
Amino acids and their derivatives are indispensable to biology
and a likely cornerstone of any prebiotic inventory supporting
the advent of life.1 Chemistries potentially leading to the
endogenous production of amino acid derivatives include the
Strecker reaction,2 Bucherer−Bergs reaction,3,4 reductive
amination of α-ketoacids,5,6 and hydrolytic processing of
oligomers of HCN and its derivatives.7−10 None of these
pathways toward the accumulation of amino acid derivatives is
without potential drawbacks in a prebiotic context: the
Strecker reaction requires relatively high concentrations of
ammonia,11 is pH-sensitive,12,13 and furnishes varied products
after hydration/hydrolysis;14 reductive amination under pre-
biotic conditions also requires high ammonia concentration
and pH control and is low-yielding and limited in scope;15,16

the Bucherer−Bergs chemistry under an atmosphere of CO2
provides relatively refractory hydantoins and carbamoyl amino
acids,13 and pathways based on the oligomerization of HCN
and its derivatives provide complex mixtures.7,17 While these
aqueous chemistries have been extensively investigated,
surprisingly, the synthesis of amino acid derivatives in
formamide, a medium commonly invoked in prebiotic
chemistry, has not progressed beyond parts per million�
yielding thermal and photochemical decompositions of
formamide itself.18−20 Although the occurrence of primordial
terrestrial formamide is debated,21 mechanisms for its potential

accumulation exist.22 Formamide could also be accumulated by
the reaction of hydrogen cyanide with hydrosulfide to give
thioformamide,23 which is hydrolyzed to formamide more
rapidly than formamide itself is hydrolyzed to ammonium
formate. Given the relationship between HCN, its hydration
product (formamide), and aldehydic products of its reduction
and homologation,24,25 the co-occurrence and reactions of the
constituents of aminonitriles (HCN, aldehydes, and ammonia)
in formamide-rich media are of potential prebiotic relevance.
Also, given a major problem in biomimetic peptide synthesis
(diketopiperazine formation26) and its solution in bacteria
(initiation of peptide synthesis with an N-formyl-protected
amino acid27), we wondered if amino acid derivative synthesis
in formamide and subsequent aqueous processing might
provide mixtures of formylated and unformylated amino
acids, as a potential basis for a similar, prebiotic solution to
the problem of diketopiperazine formation. Thus, we set out to

Received: December 13, 2022
Published: May 5, 2023

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explore the formation and reactivity of amino acid building
blocks in formamide.

■ RESULTS AND DISCUSSION
N-Formylaminonitrile Formation. Initially, we inves-

tigated the behavior of aminonitriles 1 (AA-CN, AA = amino
acid) in formamide. Unsurprisingly, given synthetic formyla-
tion procedures,28,29 glycine nitrile (Gly-CN 1a), alanine
nitrile (Ala-CN 1b), and valine nitrile (Val-CN 1c) were
readily N-formylated in good yield by heating their acid salts in
formamide in an open vessel exposed to air and moisture
(Table 1, entries 1−3; Graph S1; and Figures S1−S9). Gly-CN
1a formylated at an appreciable rate at room temperature
(RT), while Ala-CN 1b and Val-CN 1c required heating to 80
°C to achieve high conversion after16 h. Formylated
aminonitriles (FoAA-CN 2) FoGly-CN 2a, FoAla-CN 2b,
and FoVal-CN 2c were stable when heated to 100 °C in
formamide, without any additional products being observed.
Experiments employing a 15N-labeled aminonitrile demon-
strated that the major mechanism of formylation is trans-
amidation between the aminonitrile and formamide, rather
than a pathway involving initial reversion of the Strecker
condensation (see Table S5 and Figures S145−S147). Serine
nitrile (Ser-CN 1d) was also formylated relatively quickly
(Table 1, entries 4), but at temperatures above 50 °C, we

observed its disappearance as it was converted to other
products. This effect was especially apparent in the presence of
MgCl2 (5 equiv), with either the free base or HNO3 salt of Ser-
CN (Table 1, entries 4b and 4c; Figures S10−S13).
Further investigation of the formylation of Ser-CN 1d led to

the identification of N-formyldehydroalanine nitrile (FoDHA-
CN, 3) and (N,N′-diformyl)-β-aminoalanine nitrile (Fo(β-
FoNH)Ala-CN, 2e) as additional products (Table 2 and
Figures S10−S28). Although we observed no intermediate, we
postulate that FoDHA-CN 3 is formed via N- and O-
formylation of Ser-CN 1d followed by rapid elimination of
formic acid to provide FoDHA-CN 3. Given the simplicity and
likely prebiotic availability of both Ser-CN 1d and
formamide,22 the simple heating of a mixture of the two
presents an expedient route to a dehydroalanine derivative of
notable recent prebiotic interest.30 In the presence of MgCl2 (5
equiv), we observed yields of up to 18% for the formation of
FoDHA-CN 3 just by heating Ser-CN 1d in formamide (Table
2, entry 3).
Intrigued by the facile production of FoDHA-CN 3 as a

byproduct of Ser-CN 1d formylation, we wondered if the
Strecker condensation itself, which produces aminonitriles
such as Ser-CN 1d, could be carried out in formamide starting
from an aldehyde, cyanide, and ammonia. We began by using
similar conditions to those used for aminonitrile formation in

Table 1. Formylation of α-Aminonitriles (AA-CN) in Formamide

conversion (%)a

entry AA-CN RT,e 1 h RT, +16 h 50 °C, +4 h 50 °C, +16 h 80 °C, +5 h 80 °C, +16 h 80 °C, +20 h

1a Gly-CN·HCl 6 67 ∼90b ndb ndb ∼90b ∼90b

1b Gly-CN·HClc nd 28 77 ∼90b ∼90b ∼90b ∼90b

2a Ala-CN·HCl 1 13 20 25 37 57 65
2b Ala-CN·HClc nd 6 9 15 20 48 59
3a Val-CN·HCl 0 9 28 35 47 87 88
3b Val-CN·HClc 0 6 15 26 38 85 87
4a Ser-CN·HNO3 0 11 31 53 82 77 74
4b Ser-CN·HNO3

c 6 28 55 51 37 29 20
4c Ser-CNc,d nd 40 53 58 47 31 18

aMeasured by integration relative to a 1H NMR standard. Conditions applied successively. bAccurate integration not possible due to signal overlap
with the water peak, but only a single product was observed by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy. cMgCl2 (5
equiv) added. dPrepared by lyophilizing an aqueous solution of Ser-CN·HNO3 at pH 9.2. eRT = room temperature.

Table 2. Reaction of Serine Nitrile (Ser-CN 1d) in Formamide

conversion (%)a

20 h, 50 °C +5 h, 80 °C +16 h, 80 °C +20 h, 80 °C
entry conditions 2d 3 2e 2d 3 2e 2d 3 2e 2d 3 2e

1 Ser-CN·HNO3 53 0 0 82 1 8 77 2 9 74 4 5
2 Ser-CN·HNO3

b 53 0 0 51 9 5 37 12 14 29 14 17
3 Ser-CNb,c 58 3 0 47 18 0 31 16 22 18 8 19

aMeasured by relative integration compared to a 1H NMR spectroscopic standard. Conditions applied successively. bMgCl2 added (5 equiv).
cPrepared by lyophilizing an aqueous solution of Ser-CN·HNO3 at pH 9.2.

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water, which require an excess of ammonia and alkaline pH to
ensure the equilibrium favors aminonitrile formation.11,12

Under these conditions, we observed facile cyanohydrin
formation in formamide and, upon heating, conversion to
aminonitriles 1 and ultimately N-formylaminonitriles 2 of Gly,
Ala, Val, and Ser (Table 3, entries 1−4; Graphs S2−S5; and
Figures S29−S33). When glycolaldehyde 4d, NaCN (3.0
equiv), and NH4Cl (5.0 equiv) were heated in formamide,
FoSer-CN 2d was ultimately produced without any FoDHA-
CN 3 being observed, suggesting that these conditions either
inhibit the formation of FoDHA-CN 3 or lead to its
destruction as it forms. However, FoDHA-CN 3 could be
observed, as a minor product, alongside FoSer-CN 2d and
Fo(β-FoNH)Ala-CN 2e under various other conditions
employing ammonium salts (Tables S1 and S2).

N-Formylaminonitrile Formation without Added
Ammonia. We suspected that FoDHA-CN 3 probably does
form alongside FoSer-CN 2d from the Strecker reaction in
formamide, but the presence of excess ammonia might lead to
its decomposition. This presents a potential obstacle for the
formation of any dehydroalanine derivative, since concen-
trations of ammonia high enough to promote aminonitrile
formation11 are likely to lead to reaction with a dehydroalanine
as it is formed (see also Table 6) and may explain why
reported prebiotically themed syntheses of dehydroalanine
derivatives were performed with isolation of intermediates.
Eschenmoser et al. had previously synthesized dehydroalanine
nitrile from an N-silylated imine precursor,31 and Powner et al.
synthesized N-acetyldehydroalanine nitrile30 (AcDHA-CN 5)
by isolating its precursor, Ser-CN 1d, thereby separating
ammonia-requiring aminonitrile formation from alkene for-

Table 3. Reaction of Aldehydes and Cyanide in Formamide with Added Ammonia

conversiona (%), AA-CN (1)/FoAA-CN (2)

entry R 16 h, RT +16 h, 50 °C +16 h, 80 °C +16 h, 80 °C
1 Hb 0/2 32/7 5/45 0/47
2 Hc 1/0 25/6 1/60 0/52
3 Me 35/0 58/6 32/34 9/51
4 iPr 19/0 46/3 52/20 31/41d

5 CH2OH 7/0 36/0 35/36 0/31
aMeasured by relative integration compared to a 1H NMR spectroscopic standard. Conditions applied successively. bParaformaldehyde used
instead of formaldehyde. cGlycolonitrile used in place of formaldehyde with 2 additional equivalents of NaCN. dYield of FoVal-CN 2c increases to
59% after an additional 80 h heating at 80 °C.

Scheme 1. Comparison of Prebiotic Approaches to Dehydroalanine Nitrile Derivativesa

aNAI, N-acetylimidazole.

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mation (Scheme 1). In addition to this apparent incompati-
bility between FoDHA-CN 3 and ammonia, the prebiotic
availability of ammonia in concentrations high enough to
promote the Strecker aminonitrile synthesis in useful yields has
been questioned, given ammonia’s modest theoretical
production rates and its volatility, reactivity, and photo-
chemical instability.13,32−34 We thus set out to determine
whether added ammonia is required for a Strecker-type
reaction in formamide.
We next attempted aminonitrile and N-formylaminonitrile

synthesis without added ammonia. Strecker aldehydic
precursors 4 to Gly, Ala, and Val were cleanly converted to
N-formylaminonitriles 2 in good yield when combined with
equimolar quantities of sodium cyanide and formic acid and
heated in formamide (Table 4, entries 1−3; Graph S6; and

Figures S34−S36). Formic acid was used to neutralize the
cyanide salt and better simulate conditions of partially
hydrolyzed primordial formamide. Formamide is thus not
only an excellent medium for the production of N-
formylaminonitriles 2 but also a plausible source of nitrogen
that is readily available for incorporation into amino acid
derivatives, without requiring exogenous ammonia or pH
modulation. In contrast to the reactions in Table 3, which
likely proceed via a Strecker-type mechanism involving the

condensation of ammonia and an aldehyde to form imines, in
the absence of added ammonia, products 2 in Table 4 are likely
formed via N-formyliminia, although we did not observe their
intermediacy by 1H NMR.

N-Formyldehydroalanine Nitrile from Glycolalde-
hyde and Cyanide. When we combined glycoladehyde 4d,
NaCN, and formic acid in formamide without ammonia, at 80
°C, we observed the formation of FoSer-CN 2d (Table 4,
entry 4), but to a lesser extent compared to N-formylaminoni-
triles 2a−c, because under these conditions FoSer-CN 2d is
converted to FoDHA-CN 3 (see Table 5 and Figure S37).
Further experimentation revealed that FoDHA-CN 3 was
observed alongside FoSer-CN 2d and Fo(β-FoNH)Ala-CN 2e
under the majority of conditions tested in the presence of
additives including MgCl2, KH2PO4, NH4H2PO4, and K3PO4
(Tables 5, S1, and S2). While the yield for FoDHA-CN 3 in
some reactions was low, the continuous conversion of these
precursors to FoDHA-CN 3 under varied conditions suggests
that locales producing FoDHA-CN 3 may not have been
uncommon. In some cases, the reaction was remarkably clean.
For example, heating a 0.1 M formamide solution of
glycolaldehyde 4d with 5 equiv of NaCN and formic acid at
80 °C for 36 h resulted in the formation of FoDHA-CN in a
yield of 16% (Table 5, entry 1). The 1H NMR spectrum of this
reaction mixture is shown in Figure 1. Additives such as
KH2PO4 or MgCl2 did not significantly affect this yield (17 and
9%, respectively, Table 5, entries 2 and 3). A similar reaction
using only 1.5 equiv of NaCN, with 5 equiv of MgCl2 present,
resulted in the formation of FoDHA-CN 3 in a 7% yield
(Table 5, entry 4). Thus, we demonstrate that wherever
formamide, glycoladehyde 4d, and cyanide were present, it is
reasonable to expect some flux of these feedstocks to FoDHA-
CN 3.
Our observation of the facile manner of the formation of

FoDHA-CN 3 is interesting because it presents a simple and
prebiotically plausible route to dehydroalanine derivatives.
Dehydroalanine and its derivatives are important motifs in
modern biology,35,36 including the biosynthesis of cysteine37

and α,β-diaminopropionic acid,38 and thus the provision of a
similar molecule in a prebiotic context may have been
important to allow nascent biology to access a variety of
useful building blocks. Eschenmoser et al. had previously used
chemically synthesized dehydroalanine nitrile to study its

Table 4. Reaction of Aldehydes and Cyanide in Formamide
without Added Ammonia

conversiona (%)

entry R 50 °C, 16 h +5 h, 80 °C +16 h, 80 °C
1 Hb 5 51 79
2 Me 0 31 85
3 iPr 10 37 98
4 CH2OH nd nd 46

aMeasured by relative integration compared to a 1H NMR
spectroscopic standard. Conditions applied successively. bGlycoloni-
trile used in place of formaldehyde with 4 additional equivalents of
NaCN.

Table 5. Reaction of Glycolaldehyde 4d and Cyanide in Formamide without Added Ammonia

conversion (%)a

16 h 36 h

entry additive 2d 3 2e 2d 3 2e

1 none 46 12 17 36 16 21
2 MgCl2 (5 equiv) 37 3 20 34 9 26
3 KH2PO4 (5 equiv) 35 18 16 23 17 21
4 MgCl2 (5 equiv)b 26 5 11 14 7 14

aMeasured by relative integration compared to a 1H NMR spectroscopic standard. bReaction performed using 1.5 equiv NaCN, without added
formic acid.

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potential utility in prebiotic amino acid and cofactor
synthesis.31 Moreover, Powner et al. recently showed that
the related N-acetyldehydroalanine nitrile (AcDHA-CN 5) is a
precursor to cysteine derivatives, which have proven difficult to
access under prebiotic conditions.30 Such derivatives were
shown to be organocatalysts for peptide ligation and both
peptidyl amidine and peptide synthesis.30,39 While Powner et
al. demonstrated routes to AcDHA-CN 5 in water via
acetylation and elimination, our observation of the formation
of FoDHA-CN 3 by simply heating serine nitrile 1d or its
precursors in formamide, without the use of purified
intermediates, synthetic acetylating agents, oxidants, pH
switches, or other interventions, significantly bolsters the
argument that dehydroalanine nitriles would have been present
on the prebiotic earth (Scheme 1). We thus set about accessing
FoDHA-CN 3 synthetically and investigating its reactivity with
other molecules of prebiotic relevance in water and formamide.
Preparative N-Formyldehydroalanine Nitrile Synthe-

sis. FoDHA-CN 3 was prepared synthetically from glyco-
laldehyde 4d in four steps and a 32% overall yield (Scheme 2
and Figures S38−S45). Serine nitrile 1d was prepared using
the Strecker reaction2 and then N-formylated using the
condensing agent 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDCI) and formic acid. Removal of the spent
condensing agent by ion exchange chromatography (Dowex-
Na+) preceded O-acetylation by N-acetylimidazole, which

provided FoSer(Ac)-CN 7 in 50% yield. When attempting to
telescope the subsequent elimination step and the acetylation
step, at alkaline pH, we found that the remaining imidazole
added readily to FoDHA-CN 3 as it was formed, giving 2f (see
Scheme 5). Thus, we purified FoSer(Ac)-CN 6 before
performing the elimination. This observation of cross-reactivity
in our preparative synthesis underscores the importance of our
findings of a single-operation prebiotic synthesis of FoDHA-
CN 3 in formamide, which obviates the requirement to purify
intermediates.
Additional Reactions of FoDHA-CN. With FoDHA-CN

3 in hand, we evaluated its reactivity with a variety of
molecules that might reasonably be expected to have co-
occurred in various primordial settings. FoDHA-CN 3 reacts
readily with ammonia in both formamide and water (Table 6
and Figures S72−S83). When FoDHA-CN 3 and ammonium
chloride (5 equiv) were mixed in water at pH 9.2, clean
addition of ammonia to the alkene was observed to form Fo(β-
NH2)Ala-CN 2g, followed by migration of the formyl group to
form (β-FoNH)Ala-CN 1e in 84% yield. In formamide,
FoDHA-CN 3 reacted at room temperature with NH4OH (5
equiv) to provide Fo(β-NH2)Ala-CN 2g as the major product.
A similar reaction in formamide under approximately neutral
conditions, with ammonium formate (5 equiv), produced the
diformyl ammonia adduct Fo(β-FoNH)Ala-CN 2e after
heating for 36 h at 80 °C. 1e, 2g, and 2e are the formylated

Figure 1. 1H NMR spectrum showing the formation of major products FoSer-CN 2d, FoDHA-CN 3, and Fo(β-FoNH)Ala-CN 2e from the
reaction between NaCN and glycolaldehyde 4d in formamide/formic acid after heating at 80 °C for 36 h. The two sets of alkenic peaks correspond
to the two conformers of FoDHA-CN 2d, presumably due to restricted rotation about the amide C−N bond.

Scheme 2. Preparative Route to FoDHA-CN

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nitrile precursors to α,β-diaminopropionic acid, which is a
secondary metabolite biosynthesized by the addition of
ammonia to dehydroalanine generated by dehydration of
serine.38

When FoDHA-CN 3 was reacted with aqueous cyanide, a
cascade reaction was observed, resulting in the formation of
imidazole 740 in a 63% yield via the formation of three new
bonds (Scheme 3 and Figures S84−S102). The hydration
product, Fo(β-CN)Ala-NH2 8h, of the cyanide adduct Fo(β-
CN)Ala-CN 2h, also formed, in a 22% yield. Initial addition of
cyanide to FoDHA-CN 3 was observed, but adduct 2h reacted
further, undergoing a second addition of cyanide at the (N-
formylamino)nitrile group, followed by cyclization to form
imidazole 7. This sequence was verified using 13C-isotopically
labeled cyanide, and the same imidazole could be accessed
starting from Fo(β-CN)Ala-CN 2h and cyanide (Figures S85
and S103). When a single equivalent of NaCN was reacted
with FoDHA-CN 3 at pH 9.2, cyanide adduct 2h, its hydration
product 8h, and imidazole derivative 7 were observed in 39,
14, and 16% yields, respectively. Given the important role of
imidazoles in prebiotic chemistry and nonenzymatic nucleic
acid replication,41,42 the properties of this readily formed
heterocycle in related processes are now under investigation.
We also saw a dichotomy of reactivity of FoDHA-CN 3 with

cyanide in water versus formamide. In formamide, the addition
of an equimolar mixture of NaCN and formic acid (3.0 equiv)
at room temperature led to the stable formation of Fo(β-
CN)Ala-CN 2h in an 88% yield (Scheme 4 and Figure S104).
Without formic acid to neutralize the NaCN, we observed
conversion of the starting material to cyanide adduct 2h, but

upon standing, it was slowly converted to another product,
FoAsn-NH2 8i (Figure S105). This in situ hydration of both
nitrile groups is notable given the less activated nature of the β-
nitrile, which is slow to hydrate in aqueous conditions (see
below). This result could be recapitulated by reacting Fo(β-
CN)Ala-CN 2h with NaOH in formamide (Scheme 4 and
Figure S106).
FoDHA-CN 3 reacted cleanly with the model heterocyclic

nucleophile, imidazole, in water at pH 7.5 (87%) and in
formamide (86%) (Scheme 5 and Figures S107−S114). The
addition product 2f was the only product observed by 1H
NMR spectroscopy.
Finally, given the interest in the potentially prebiotic

synthesis of cysteine and its derivatives, we examined the
reactivity of FoDHA-CN 3 with hydrosulfide in water and
formamide (Table 7 and Figures S115−S140). We found that
at neutral pH, a single hydrosulfide molecule was added to two
molecules of FoDHA-CN 3, and H2S subsequently the nitrile
groups, forming sulfur-bridged dimers 9 and 10 (Table 7,
entries 1 and 3). Similar reactivity to provide dithioamide 10
was observed in formamide, where the added NaSH·H2O was
neutralized with formic acid (Table 7, entry 5). In contrast,
under slightly alkaline conditions (pH 9), 9 formed first but
was converted to N-formylcysteine nitrile 2j (entry 2, 56%),
with subsequent addition of hydrosulfide to the nitrile forming
11. Similar reactivity was observed in formamide without
neutralization of the added NaSH, forming 2j (Table 7, entry
4, 83%). Thus, we have disclosed a prebiotically plausible
synthesis of cysteine precursors from glycolaldehyde 4d
requiring just two straightforward steps in formamide, without
the use of oxidants or protecting groups on sulfur.
These additional reactions were all performed using purified

FoDHA-CN 3 to demonstrate model reactivity in formamide
and water. Now that the outcome of these reactions is
characterized, further studies will investigate which may occur
continuously (with all starting materials present from the
outset) and which would require mutually exclusive chem-
istries occurring separately before a geochemically plausible
combination of reagents.
Hydrolysis of N-Formylaminonitriles. Having shown the

potentially prebiotic synthesis of N-formylaminonitriles 2 in
formamide, we evaluated the hydrolysis pathways that
subsequent aqueous processing might bring about (Tables 8,

Table 6. Reactions of Ammonia with FoDHA-CN 3

conversion (%)

entry conditions 2g 1e 2e

1 NH4Cl; H2O, pH 9.2, RT, 96 h 3 84
2 NH4OH; H2NCHO, RT, 20 h 55 9 2
3 NH4O2CH; H2NCHO, 80 °C, 36 h 77

Scheme 3. Reactions of FoDHA-CN 3 and Aqueous Cyanide

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S3, and S4; Graphs S7 and S8; and Figures S141−S144). We
found that alkaline hydrolysis did not proceed via initial
deformylation to give aminonitriles. Instead, at pH 10, FoGly-
CN 2a underwent hydration of the nitrile moiety and
hydrolysis of the newly formed terminal amide faster than N-
formyl hydrolysis, furnishing a mixture with FoGly-OH 12a
and FoGly-NH2 8a as major products (32 and 30%,
respectively) alongside smaller amounts of Gly-NH2 13a and
Gly-OH 14a (16 and 12%, respectively; Table 8, entry 1). For
FoAla-CN 2b, overlapping 1H NMR signals prevented a
precise quantitative analysis, but the same general trend in
reactivity was observed, i.e., hydration and subsequent
hydrolysis of the newly formed terminal amide were faster

than formyl hydrolysis. After 11 days, the major products were
FoAla-NH2 8b (∼30%), FoAla-OH 12b (∼30%), and FoAla-
CN 2b (∼15%), with Ala-NH2 13b (∼5%), Ala-CN 1b
(∼2%), and Ala-OH 14b (∼1%) as minor products (Table 8,
entry 2). FoSer-CN 2d underwent fairly rapid hydration of its
nitrile group, providing a mixture of FoSer-NH2 8d, FoSer-OH
12d, and Ser-NH2 13d as major products (Table 8, entry 4).
Since the addition of cyanide to FoDHA-CN 3 provides the
dinitrile precursor 2h to N-formylamino acids Asn and Asp
(Schemes 3 and 4), we also investigated the hydrolysis of 2h
(Table 7, entry 4). After 11 days at pH 10 and 40 °C,
hydration of the α-nitrile and hydrolysis of the newly formed
α-amide had occurred faster than both N-formyl hydrolysis and
β-nitrile hydration, furnishing a mixture of FoAsn-OH 12i and
Fo(β-CN)Ala-OH 12h as major products, alongside Asn-OH
14i. Further hydrolysis proceeds more quickly at the N-formyl
group than the β-amide moiety, and thus Asp-OH 14k was
observed with only traces of FoAsp-OH 12k. These results
show that when exposed to alkaline conditions, N-formylami-
nonitriles undergo hydration at the α-nitrile position fastest
and provide mixtures of N-formylamino amides/acids and
amino amides/acids en route to the terminal products of
hydration and hydrolysis, amino acids. Such mixtures may have
been advantageous for prebiotic peptide synthesis, providing
N-terminal protection from the major pathway of peptide
degradation26 (diketopiperazine formation), which is indeed
the strategy used for bacterial peptide biosynthesis.27 The
investigation of hydrolytic processing in the presence of
various potential prebiotic catalysts is ongoing.

■ CONCLUSIONS
In summary, we have investigated the formation and
subsequent reactions of aminonitriles and N-formylaminoni-
triles in formamide, a medium of prebiotic relevance. We
found that N-formylaminonitriles are produced by thermally
promoted reactions between aldehydes and cyanide in
formamide with or without added ammonia. In the case of
glycolaldehyde, reaction with cyanide in formamide generated
not only FoSer-CN 2d but also the elimination product
FoDHA-CN 2 and the formylated α,β-diaminoacid 3. This
facile synthesis of a dehydroalanine derivative significantly
bolsters the prebiotic plausibility of such compounds, which
are precursors to cysteine derivatives that have important
catalytic properties in the context of prebiotic peptide

Scheme 4. Reactions between FoDHA-CN 3 and Cyanide in Formamide

Scheme 5. Reactions between Imidazole and FoDHA-CN 3
in Water and Formamide

Table 7. Reactions of FoDHA-CN 3 and Hydrosulfide in
Water and Formamide

entry conditions outcome

1 1.5 equiv NaSH, pH 7, 1 h 90% 9
2 1.5 equiv NaSH, pH 9, 2 h 56% 2j (via 9), 20%

11
3 5.0 equiv NaSH, pH 7, 16 h 81% 10
4 3.0 equiv NaSH, formamide, 6 h 83% 2j
5 3.0 equiv NaSH + formic acid, formamide,

18 h
61% 10

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synthesis. FoDHA-CN was also shown to react with a variety
of other compounds of prebiotic interest, generating precursors
of Asp and Asn and an imidazole derivative by an unexpected
cascade reaction. The contrasting reactivities of FoDHA-CN in
water and formamide suggest that prebiotic access to some
compounds from dehydroalanine derivatives may have
required formamide as a solvent, which follows naturally
from the synthesis of FoDHA-CN in formamide. Finally, the
alkaline processing of N-formylaminonitriles was shown to
proceed by hydration of the nitrile group, preventing reversion
of the Strecker equilibrium, which would occur upon
deformylation,10,14 and generating mixtures of N-formylated
and unformylated amino amides and acids prior to their
complete hydrolysis to amino acids. Thus, N-formylaminoni-
trile formation from cyanide and aldehydes in formamide, and
the subsequent reactivity of FoDHA-CN, may have been
integral to the provision of chemical building blocks for
peptide synthesis and other processes at the origin of life.

■ EXPERIMENTAL SECTION
All experimental details, as well as characterization data and spectra
collected on all compounds, are available in the Supporting
Information.

■ ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.2c13306.

Methods, supporting data, and NMR spectra (PDF)

■ AUTHOR INFORMATION
Corresponding Authors

Nicholas J. Green − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, U.K.; Department of Chemistry,
University of Otago, Dunedin 9054, New Zealand;
orcid.org/0000-0002-3874-7359; Email: nick.green@

otago.ac.nz
John D. Sutherland − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, U.K.; orcid.org/0000-0001-7099-
4731; Email: johns@mrc-lmb.cam.ac.uk

Authors
David A. Russell − MRC Laboratory of Molecular Biology,
Cambridge CB2 0QH, U.K.; orcid.org/0000-0002-2182-
4178

Sasha H. Tanner − Department of Chemistry, University of
Otago, Dunedin 9054, New Zealand

Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.2c13306

Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
The authors thank Dr. Dougal Ritson for valuable input. This
research was supported by the Medical Research Council
(MC_UP_A024_1009), the Simons Foundation (290362),
and a University of Otago Research Grant (N.J.G.).

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Table 8. Partial Hydrolysis Outcomes of N-Formylaminonitriles

hydrolysis outcome

entry R group (FoAA-CN 2) time (days) FoAA-CN (2) FoAA-NH2 (8) FoAA-OH (12) AA-CN (1) AA-NH2 (13) AA-OH (14)

1 H (FoGly-CN 2a) 14 1% 30% 32% 2% 16% 12%
2 Me (FoAla-CN 2b) 11 ∼15% ∼30% ∼30% ∼2% ∼5% ∼2%
3 CH2OH (FoSer-CN 2d) 10 3% 41% 22% 23%
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Fo(β-CN)Ala-OH 12h 25%
FoAsp-OH 12k trace Asn-OH 14i 6%

(β-CN)Ala-OH 14h 3%

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