Peptide ligation by chemoselective aminonitrile coupling in water

Article abstract of DOI:10.1038/s41586-019-1371-4

Amide bond formation is one of the most important reactions in both chemistry and biology^1–4, but there is currently no chemical method of achieving α-peptide ligation in water that tolerates all of the 20 proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes that the biological role of peptides predates life’s last universal common ancestor and that peptides played an essential part in the origins of life^5–9. The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis point towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of life^5,9–13. However, a robust mechanism for aminoacyl thioester formation has not been demonstrated¹³. Here we report a chemoselective, high-yielding α-aminonitrile ligation that exploits only prebiotically plausible molecules—hydrogen sulfide, thioacetate^12,14 and ferricyanide^12,14–17 or cyanoacetylene^8,14—to yield α-peptides in water. The ligation is extremely selective for α-aminonitrile coupling and tolerates all of the 20 proteinogenic amino acid residues. Two essential features enable peptide ligation in water: the reactivity and pK_aH of α-aminonitriles makes them compatible with ligation at neutral pH and N-acylation stabilizes the peptide product and activates the peptide precursor to (biomimetic) N-to-C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological α-peptides, suggesting that short N-acyl peptide nitriles were plausible substrates during early evolution.

Full text of DOI:10.1038/s41586-019-1371-4

Letter
https://doi.org/10.1038/s41586-019-1371-4
Peptide ligation by chemoselective aminonitrile
coupling in water
Pierre Canavelli1, Saidul Islam1 & Matthew W. Powner1*
Amide bond formation is one of the most important reactions in this ligation would (electronically) activate the nitrile moiety to thiol-
both chemistry and biology1–4, but there is currently no chemical ysis. Accordingly, AA-CN N-acylation, which appears to be essential
method of achieving α-peptide ligation in water that tolerates all to prevent diketopiperazine (DKP)-induced peptide degradation27,28
of the 20 proteinogenic amino acids at the peptide ligation site. The (Fig. 1c), would initiate peptide synthesis by promoting thioacid
universal genetic code establishes that the biological role of peptides synthesis.
predates life’s last universal common ancestor and that peptides
Ferricyanide-mediated acetylation of AA-CN (50 mM) by AcSH (3
played an essential part in the origins of life5–9. The essential role of equiv.)12,14 gave α-amidonitriles (Ac-AA-CN) in near-quantitative yield
sulfur in the citric acid cycle, non-ribosomal peptide synthesis and in water (Table 1). As anticipated, acylation of AA-CN activated the
polyketide biosynthesis point towards thioester-dependent peptide nitrile moiety, and quantitative conversion of Ac-AA-CN to Ac-AA-
ligations preceding RNA-dependent protein synthesis during the SNH2 was observed upon incubation with H2S (10 equiv., pH 9, room
evolution of life5,9–13. However, a robust mechanism for aminoacyl temperature, 1–4 d) (Supplementary Figs. 39–52, 64, 80). Incubation
thioester formation has not been demonstrated13. Here we report of Ac-Gly-CN (50 mM) and Gly-CN (50 mM) or acetonitrile (50 mM)
a chemoselective, high-yielding α-aminonitrile ligation that with H2S (0.25 M, pH 9, room temperature, 24 h) gave smooth conver-
exploits only prebiotically plausible molecules—hydrogen sulfide, sion to Ac-Gly-SNH2 (91%), whereas only 7% of Gly-CN was converted
thioacetate12,14 and ferricyanide12,14–17 or cyanoacetylene8,14—to to Gly-SNH2 (Supplementary Fig. 18) and acetonitrile thiolysis was not
yield α-peptides in water. The ligation is extremely selective for observed (Supplementary Fig. 20). This demonstrates the pronounced
α-aminonitrile coupling and tolerates all of the 20 proteinogenic nitrile activation provided by acylation. Electrophilic activation is also
amino acid residues. Two essential features enable peptide ligation specific to α-amidonitriles; for example, the reaction of Ac-Gly-CN
in water: the reactivity and pKaH of α-aminonitriles makes them and Ac-β-Ala-CN (1:1) with H2S results in almost exclusive Ac-Gly-CN
compatible with ligation at neutral pH and N-acylation stabilizes the thiolysis (Fig. 1e).
peptide product and activates the peptide precursor to (biomimetic)
Notably, we observed hydrolysis of Ac-AA-SNH2 to Ac-AA-SH
N-to-C peptide ligation. Our model unites prebiotic aminonitrile to realize effective synthesis of thioacids (Fig. 1b). This is in stark
synthesis and biological α-peptides, suggesting that short N-acyl contrast to the reactivity of AA-SNH2, for which hydrolysis to the
peptide nitriles were plausible substrates during early evolution.
respective AA-SH was not observed (Supplementary Discussion and
To improve the efficiency and selectivity of peptide ligation in water Supplementary Fig. 16). Hydrolysis of Ac-AA-SNH2 generally fur-
we sought to develop a mechanism for non-enzymatic peptide syn- nished the respective Ac-AA-SH in good yields (51%–85%; Table 1
thesis, which would operate via biomimetic N → C ligation in water and Supplementary Figs. 53–58, 64, 80). However, the sterically
at near-neutral pH, and we suspected that a combination of sulfur and bulkier Val residue hydrolysed sluggishly to give the corresponding
nitrile chemistry would be required8,9,14,18–21 (Fig. 1a). Proteinogenic α-amidothioacid Ac-Val-SH in poor yield (8%; entry 9 in Table 1
α-aminonitriles (AA-CN) are readily synthesized8,18, and their direct and Supplementary Fig. 59). This amino acid residue is one of several
ligation would provide the simplest prebiotic pathway to peptides. notoriously problematic C-terminal ligation residues observed during
Unfortunately, incubation of AA-CN in water results in extremely inef- the (semi)synthesis of peptides in the related process of thioester-
fective peptide synthesis22. α-Amino acids (AA) are widely assumed to mediated native chemical ligation4,29,30. Future investigation of catalytic
be prebiotic precursors of peptides, but the harsh conditions (typically α-amidothioacid Ac-AA-SH synthesis is warranted; however, we note
strongly acidic or alkaline solutions) required for AA formation from that (uncatalysed) Ac-Val-CN thiolysis already delivers an Ac-Val-SH
AA-CN are incompatible with the integrity of both peptides and elec- yield seven times greater than that of AA-SH analogues synthesized by
trophilic activating agents. Therefore, we sought a more congruent and electrophilic AA activation25. Furthermore, Ac-AA-SH are highly stable
direct pathway from AA-CN to α-peptides. Although the conversion of to the conditions of their formation, whereas AA-SH are destroyed by
AA-CN to α-aminothioacids (AA-SH) has never been reported23, har- the activating agents required for their synthesis25.
nessing the AA-CN nitrile moiety for thioacid synthesis seemed more
We next investigated the ligation of Ac-AA-SH. We observed that
prudent than dissipating its activation through exhaustive hydrolysis. incubation of Ac-Gly-SH (50 mM) with Gly-CN (2 equiv.) and fer-
Maurel and Orgel have previously suggested that AA-SH16 might ricyanide (3 equiv.) gave Ac-Gly2-CN in near-quantitative yield over
offer an interesting alternative to biological thioesters10,11. AA-SH com- a broad pH range (pH 5–9, room temperature). A range of activating
bine excellent aqueous stability with highly selective (electrophilic or agents—including ferricyanide12,14–17, cupric salts8, cyanoacetylene8,14
oxidative) activation12,14,16,24, but their prebiotic synthesis presents and N-cyanoimidazole14—were all found to be effective (Extended
difficulties25 and they undergo inefficient ligation at near-neutral pH Data Table 1), showing that multiple methods of Ac-AA-SH activation
(Supplementary Discussion)16,26. To overcome these problems we towards AA-CN ligation in water are possible.
reconsidered the synthesis of thioacids from nitriles (Fig. 1b). Recently
We then carried out an iterative one-pot AA-CN coupling with-
we reported that high-yielding nucleophilic displacement of sulfides out isolating the intermediate ligation products. The α-amidonitrile
by Gly-CN19 is promoted by the low pKaH of AA-CN in water, and we Ac-Gly-CN was successively homologated to afford the correspond-
hypothesized that coupling AA-CN to the C terminus of a growing ing peptides Ac-Glyn-CN (n = 2–5; n = 2, 71%; n = 3, 71%; n = 4,
peptide would be facile at neutral pH. Importantly, we suspected that 63%; n = 5, 41%; Table 2). After four iterations of the homologation
1Department of Chemistry, University College London, London, UK. *e-mail: matthew.powner@ucl.ac.uk
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reSeArCH Letter
a
O
R
d
N
H
(i)
N
S8
H2S
R
n
H2N
AA-CN
N
Oxidation
Thiolysis
(ii)
Ligation cycle
(iii)
O
R
O
R
NH2
SH
N
H
N
H
S
O
n
(iv)
(v)
n
Hydrolysis
b
R
R
R
H2S
NH2
SH
H2N
H2N
H2N
N
S
O
AA-CN
AA-SH
AA-SNH2
(vi)
AcSH [Fe(CN)6]3–
4.0
3.5
Chemical shift (p.p.m.)
2.0
S
e
O
R
O
R
O
R
N
H2S
H2S
H2O
NH2
NH2
SH
AcHN
AcHN
NH2
AcHN
AcHN
N
H
N
H
N
H
N
N
S
S
O
Ac-Gly-CN
Ac-β-Ala-CN
Ac-Gly-SNH2
(91%)
Ac-β-Ala-SNH2
Ac-AA-SNH2
Ac-AA-SH
Ac-AA-CN
(7%)
c
R1
O
O
H
N
R2
O
HN
R1
H2N
N
H
Peptide
O
O
R2
+ H2N Peptide
NH
R1
H
N
O
4.0
3.5
3.0
Chemical shift (p.p.m.)
2.5
2.0
AcHN
N
H
Peptide
DKP
R2
reaction of Ac-Gly-CN (50 mM) with H2S (10 equiv.) in water at pH 9
at room temperature after 1 d; (iv) Ac-Gly-SH (81%; blue) synthesized
by hydrolysis of Ac-Gly-SNH2 (50 mM) at pH 9 and 60 °C after 1 d;
(v) Ac-Gly2-CN (quant.; green) synthesized by the reaction of Ac-Gly-SH
(50 mM) with Gly-CN (2 equiv.; magenta) and K3[Fe(CN)6] (3 equiv.) in
water at pH 9 and room temperature after 20 min; (vi) pure Ac-Gly2-CN.
e, 1H NMR spectrum (600 MHz, H2O:D2O = 98:2, 25 °C) showing the
reaction of homologous amidonitriles Ac-Gly-CN (red) and Ac-β-Ala-CN
(brown) with H2S (10 equiv., pH 9, room temperature, 1 d), which strongly
favours thiolysis of the proteinogenic glycyl residue to yield Ac-Gly-SNH2
(orange) (Supplementary Fig. 19).
Fig. 1 | Sulfide-mediated α-aminonitrile ligation a, Iterative AA-CN
ligation to give N-acetyl peptide nitriles (Ac-AAn-CN; green) by
sequential thiolysis, hydrolysis and AA-CN ligation. b, The thiolysis
of AA-CN (magenta) to yield AA-SH (black) is not observed, whereas
the thiolysis of N-acetyl aminonitrile (Ac-AA-CN; red) to α-amidoacyl
thioacid (Ac-AA-SH; blue) is facile. c, Iterative truncation of peptides
by DKP excision is blocked by N-acylation. d, 1H nuclear magnetic
resonance (NMR) spectra (600 MHz, H2O:D2O = 98:2, 25 °C), showing:
(i) Gly-CN (magenta); (ii) Ac-Gly-CN (quantitative yield, quant.; red)
synthesized by the reaction of Gly-CN (50 mM) with thioacetic acid (3
equiv.) and K3[Fe(CN)6] (9 equiv.) in water at pH 9 at room temperature
after 10 min; (iii) Ac-Gly-SNH2 (quant.; orange) synthesized by the
cycle, partial precipitation of Ac-Gly4-CN reduced the overall coupling Gly-CN proceeds with retention of stereochemistry (Supplementary
yield for Ac-Gly5-CN synthesis (13% overall yield of Ac-Gly5-CN from Figs. 292–294). This demonstrates that enantiomeric enrichment is
Ac-Gly-CN; Supplementary Figs. 209–211). This demonstrates that preserved during our peptide ligation, which is a testament to the mild
iterative ligation of AA-CN can be achieved in good yield in water ligation conditions.
without purification, within the limits of peptide solubility. Our ligation
We next investigated the chemoselectivity and robustness of AA-CN
is highly robust and tolerates monomer-by-monomer peptide growth ligation. Stoichiometric (1:1) competition reactions between Gly-CN
and fragment ligations to produce oligomers in high yield, even at low (50 mM) and ammonia, glycine (Gly), glycine amide (Gly-NH2), β-
concentrations (3.1 mM; entry 17 in Table 2) and with stoichiometric alanine (β-Ala), β-alanine nitrile (β-Ala-CN), phosphate, propylamine,
(1:1) coupling partners (entries 5–17 in Table 2). To our knowledge, cytosine, cytidine-5′-phosphate and adenosine-5′-phosphate across a
these are the first examples of fragment ligations with prebiotic sub- broad pH range (pH 5–9; Extended Data Table 2) were investigated. All
strates in water.
competition reactions demonstrated outstanding selectivity for Gly-CN
Activation of the C terminus of peptides and amino acids (such ligation (>80% yield) at neutral pH (Supplementary Figs. 235–244).
as Ac-Ala-OH) with electrophilic reagents (such as 1-ethyl-3-(3- We observed selective Gly-CN ligation in the presence of Gly-NH2
dimethylaminopropyl)carbodiimide, EDC) can result in racemiza- (pKaH = 8.4) and β-Ala-CN (pKaH = 8.0) in neutral and acidic solution,
tion31. However, we observed the formation of chiral α-amidothioacid but selectivity was lost at pH values above their pKaH. The excellent
l-Ac-Ala-SH (from l-Ac-Ala-CN), and its subsequent ligation with selectivity for AA-CN ligation in neutral solution was attributed to
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Letter reSeArCH
Table 1 | α-Amidothioacid synthesis and α-aminonitrile ligation
Table 3 | Chemoselective synthesis of N-acetyl dipeptides
Entry
AA
Yield (%)
Entry
AA
Ac-Gly-AA-OH (%)
b
Ac-AA-CNa
99
Ac-AA-SNH2
Ac-AA-SHc
Ac-AA-Gly-CNd
1
Gly
Ala
Arg
Asn
Asp
Cys
Gln
Glu
His
Ile
94
83
88
81
89
80a
90
92
95
84
86
94b
95
90
89
85
81
71
23c
84
1
2
3
4
5
6
7
8
9
Gly
Ala
99
99
99
99
99
99
99
99
99
81
85
51
77
70
84
72
61
8
99
93
64e
93
80
78
82
87f
92
2
99
3
Arg
Leu
Met
Phe
Pro
Ser
Val
99
4
99
5
99
6
99
7
99
8
99
9
99
10
11
12
13
14
15
16
17
18
19
20
1H NMR yields are determined with an internal NMR standard. See Extended Data Table 5 for
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
further examples of AA-CN ligations.
aAcetylation of AA-CN (50 mM) with AcSH (150 mM) and K3[Fe(CN)6] (450 mM) in water (pH 9;
room temperature; <20 min).
bThiolysis of Ac-AA-CN (50 mM) to Ac-AA-SNH2 in water by H2S (10 equiv., pH 9, room
temperature) (Supplementary Figs. 39–52, 64, 80).
cHydrolysis of Ac-AA-SNH2 (50 mM) to Ac-AA-SH in water with H2S (500 mM; pH 9, 60°C)
(Supplementary Figs. 53–59, 64, 80).
dLigation of Ac-AA-SH (50 mM) to Gly-CN (100 mM) in water with K3[Fe(CN)6] (150 mM; pH 9,
room temperature), unless stated otherwise.
eYield for the coupling of Ac-Arg-SH (46 mM) with Gly-CN (91 mM) and K3[Fe(CN)6] (136 mM).
fYield for the coupling of Ac-Ser-SH (30 mM) with Gly-CN (61 mM) and K3[Fe(CN)6] (92 mM).
their uniquely suppressed pKaH values (for example, pKaH = 5.3 for
Gly-CN)19, which renders them predominantly neutral, and conse-
quently nucleophilic, even in weakly acidic solutions. AA-CN ligation
is also observed across a broad temperature range (T = 3–60°C), as
well as at physiologically relevant concentrations (0.5 mM) (Extended
Data Table 3).
Developing a universal strategy to activate and ligate peptides that
accommodates all proteinogenic amino acids is problematic. Lysine
and cysteine, for example, contain highly nucleophilic moieties that
are incompatible with electrophilic activation2,17,32, and aspartate
Yields are given for the products of oxidative coupling of Ac-Gly-SH (50 mM) with AA (150 mM)
with K3[Fe(CN)6] (150 mM) in water at room temperature and pH 9.5. 1H NMR yields determined
with an internal NMR standard.
aYield observed using K3[Fe(CN)6] (300 mM), followed by methanethiol (600 mM, pH 10.8)
reduction (see Extended Data Fig. 1a and Supplementary Figs. 112–114).
bThe observed ratio of mono- and di-acylated products varies with solution pH (see Extended
Data Fig. 1b for α-selectivity of Lys ligation at pH 7.5 and Supplementary Table 11).
c
l-Tyrosine (Tyr) exhibits extremely low solubility in water (6.5 mM, pH 9.5, room temperature;
see Supplementary Table 8).
and glutamate have β- and γ-carboxylate residues, respectively, in
addition to the α-carboxylate that must be selectively activated and
ligated2,4,30. α-Amidothioacid ligation is highly general and chemose-
lective. All investigated amino acids and their derivatives were cou-
pled in good-to-excellent yields (Tables 1–3, Extended Data Tables 4,
5). Sterically congested and β-branched thioacid ligations were also
highly effective; ligations yielding Ac-Phe-Phe-CN, Ac-Phe-Val-CN
and Ac-Val-Val-CN were all rapid and high-yielding (entries 18–20
in Extended Data Table 5). We observed unprecedented protecting-
group-free ligation for all 20 proteinogenic side-chain residues—
including His, Asp, Lys, Cys, Ser, Thr and Tyr, which are all essential
to enzyme catalysis but notoriously difficult to ligate under previously
reported (prebiotic) conditions2,4,30,32. Although Cys is incompatible
with electrophilic activating agents2,32, it underwent highly selective
ligation under our conditions to furnish Ac-Gly-Cys-OH (80%; entry
6 in Table 3) after thiol exchange (Extended Data Fig. 1a).
Following the excellent selectivity of AA-CN ligation, we challenged
the α-NH2 selectivity with lysine, which possesses two amine nucleo-
philes. We observed poor selectivity for α-coupling of Lys (1.2:1 α/ε)
and Lys-NH2 (2.7:1 α/ε), but Lys-CN ligated with exceptional α-selec-
tivity (>80:1 α/ε; Extended Data Fig. 1b; Supplementary Fig. 149). We
then turned our attention to the coupling of AA-CN to a C-terminal
lysine residue, which requires intermolecular AA-CN coupling to out-
compete cyclization (Extended Data Fig. 1c). We first demonstrated that
activation of Ac-α-Lys-SH (30 mM) by ferricyanide (90 mM) at pH 9.0
led to rapid lactamization (92%). This was not surprising, given the
close proximity of the ε-NH2 and thioacid moieties of Ac-α-Lys-SH.
However, we found that Gly-CN (64 mM) successfully coupled with
Ac-α-Lys-SH (32 mM). The intermolecular coupling of Gly-CN out-
competed lactamization across a broad pH range to give Ac-α-Lys-
Gly-CN (88%–95%, pH 6.5–9.0; Supplementary Figs. 70–71). The
chemoselective coupling of lysine residues at the C and N termini
of peptides underscores that AA-CN ligation is predisposed to yield
Table 2 | Synthesis of oligomeric N-acetyl peptides and peptide
nitriles by oxidative fragment ligation
Entry
(AA1)n
(AA2)m-X
Ac-(AA1)n–(AA2)m-X (%)
1
Gly
Gly-CN
71a
2
Gly2
Gly3
Gly4
Gly3
Gly3
Gly3
Gly3
Gly3
Gly3
Gly3
Gly3
Gly5
Gly5
Gly6
Gly5
Gly6
Gly-CN
71b
63c
41d
3
Gly-CN
4
Gly-CN
5
Ala3-OH
65
6
Arg-Gly-Asp-OH
Gly3-OH
76
7
90
8
Gly3-CN
>95
90e
9
Gly2-His-OH
Leu3-OH
Met-Ala-Ser-OH
Phe-Gly2-OH
Ala3-OH
10
11
12
13
14
15
16
17
70
75
74
74
Gly2-His-OH
Gly3-CN
80
43f
79 (66g)
>95h (92g)
Gly5-OH
Gly5-OH
Ferricyanide-mediated oxidative coupling of Ac-(AA1)n-SH with (AA2)m-X (X = CN or CO2H) to
produce oligopeptides Ac-(AA1)n–(AA2)m-X. Yields of the oxidative coupling of thioacid Ac-(AA1)n-
SH (25 mM) with peptide (AA2)m-X (25 mM, pD 9.5) and K3[Fe(CN)6] (75 mM) in D2O at room
temperature, unless stated otherwise. See Supplementary Table 15 for further details.
a–dAc-Glyn-CN synthesis by iterative ligation after two, three, four and fve cycles of thiolysis,
hydrolysis and ligation (Supplementary Figs. 209–211).
eCoupling of Ac-Gly3-SH (30 mM) with Gly2-His-OH (25 mM, pD 9.5) with K3[Fe(CN)6] (75 mM).
fYield of Ac-Gly9-CN is given after four sequential steps from Ac-Gly3-SH.
gYield determined by product isolation.
hCoupling of Ac-Gly6-SH (3.13 mM) and Gly5-OH (6.25 mM).
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Letter reSeArCH
MeThodS
resonance during the original NMR analysis in H2O/D2O (98:2). Yields and HRMS
General and safety information. Reagents and solvents were obtained and used data are given in Table 3, Supplementary Table 8 (Ac-Gly–AA-OH), Extended Data
without further purification, unless specified. Sodium hydrosulfide hydrate Table 4 and Supplementary Table 9 (Ac-Gly–AA-NH2), and characterization data
(NaSH∙xH2O; 50% purity) and sodium sulfide (Na2S; >97%) were used without are provided in Supplementary Information.
purification. Deionized water was obtained from an Elga Option 3 purification Oxidative coupling of α-aminoacetyl thioacids with α-aminonitriles. AA2-CN
system. NMR spectra were recorded on Bruker NMR spectrometers (Avance Neo (100 mM) was dissolved in degassed H2O/D2O (98:2; 2 ml) and the solution was
700, Avance III 600, Avance III 400 and Avance 300), equipped with a Bruker adjusted to pH 9.0 with HCl/NaOH. Ac-AA1-SH (50 mM) was added and the
room-temperature 5-mm multinuclear gradient probe (700 MHz), a 5-mm DCH total volume was adjusted to 2 ml with H2O/D2O (98:2). Potassium hexacyano-
cryoprobe (600 MHz) and a gradient probe (400 and 300 MHz). Where noted, a ferrate(iii) (K3[Fe(CN)6]; 150 mM) was added and the solution was stirred at
solvent suppression pulse sequence with presaturation and spoil gradients was used room temperature for 20 min. The pH was readjusted to pH 9.0 using NaOH.
to obtain 1H NMR spectra (noesygppr1d, Bruker) and 1H–13C heteronuclear multi- The resulting suspension was centrifuged and the supernatant was analysed by
ple bond correlation (HMBC) NMR spectra (hmbcgplpndprqf, Bruker). Coupling one- and two-dimensional NMR spectroscopy (1H–1H COSY, 1H–13C HSQC and
constants are reported in hertz. Spectra were recorded at 298 K. Infrared spectra 1H–13C HMBC) in H2O/D2O (98:2). The reaction mixture was quantified using
(IR) were recorded on a Shimadzu IR Tracer 100 Fourier transform (FT)-IR spec- MSM as an internal standard. The ligation product Ac-AA1–AA2-CN was con-
trometer as a solid or liquid. Absorption maxima are reported as wavenumbers (in firmed by 1H–13C HMBC NMR spectral analysis and HRMS. Reaction mixtures
cm–1). Mass spectra and accurate mass measurements were recorded on a Waters were diluted with DMSO-d6 (1:49:50; D2O/H2O/DMSO-d6) or lyophilized and
LCT Premier quadrupole time-of-flight (QTOF) mass spectrometer connected dissolved in DMSO-d6 or CD3OD for further NMR spectral analysis if 1H–13
C
to a Waters Autosampler Manager 2777C, Thermo Finnigan MAT900, and an HMBC cross-correlation peaks were obscured by the HOD resonance during the
Agilent liquid chromatography (LC) system connected to an Agilent 6510 QTOF original NMR analysis in H2O/D2O (98:2). Yields and HRMS data are given in
mass spectrometer. High-performance liquid chromatography (HPLC) analysis Table 1, Extended Data Table 5 and Supplementary Table 7, and characterization
was carried out using an Agilent Infinity 1260 LC system. Solution pH values were data are reported in Supplementary Information.
measured using a Mettler Toledo Seven Compact pH meter with a Mettler Toledo Preparative oxidative coupling of α-aminoacetyl thioacids with α-aminonitriles.
InLab semi-micro pH probe. The pH readings for H2O and H2O/D2O (9:1) solu- AA2-CN (100 mM) was dissolved in degassed H2O (5 ml) and the solution pH
tions are reported uncorrected. Warning: hydrogen cyanide (HCN) and hydrogen was adjusted to pH 9.0 with NaOH. Ac-AA1-SH (50 mmol) was added and the
sulfide (H2S) are highly toxic poisons by inhalation and ingestion. They generate total volume was adjusted to 10 ml with H2O. Potassium hexacyanoferrate(iii)
poisonous gas at neutral or acidic pH (HCN, pKa = 9.2; H2S, pKa = 7.1). Solutions (K3[Fe(CN)6]; 150 mM) was added and the solution was stirred at room temper-
containing cyanide, (hydro)sulfide or compounds that may generate these should ature for 20 min. The solution was then extracted with ethyl acetate (3 × 25 ml).
be handled in a well ventilated fume hood equipped with appropriate chemical The combined organic layers were washed with HCl (0.1 M, 25 ml), NaHCO3
quenches, such as sodium hypochlorite (bleach) or iron(ii) sulfate solution.
(saturated; 25 ml) and brine (saturated; 25 ml), dried over MgSO4, filtered and
General procedures. Acetylation of α-aminonitriles with thioacetate. α- concentrated in vacuo. The crude residue was purified by flash column chroma-
Aminonitrile hydrochloride (AA-CN∙HCl; 50 mM) and potassium thioacetate tography to give the ligation product (Ac-AA1–AA2-CN) as a white solid. Isolated
(AcSK; 150 mM) were dissolved in H2O (2 ml) and the solution was adjusted to yields and HRMS data are given in Extended Data Table 5 and Supplementary
pH 9.0 with NaOH. Potassium hexacyanoferrate(iii) (K3[Fe(CN)6]; 450 mM) was Table 7, and characterization data are provided in Supplementary Information.
added, and the solution was stirred at room temperature for 20 min. The solution Oxidative peptide fragment ligations. Ac-(AA1)n-SH (3.1–30.0 mM) and (AA2)m-X
was adjusted to pH 9.0 and centrifuged, and NMR spectra of the supernatant were (X = CO2H or CN; 1–2 equiv.) were dissolved in degassed D2O and the solution
acquired. Yields are reported in Table 1 and characterization data in Supplementary was adjusted to pD 9.5 with NaOH. Potassium hexacyanoferrate(iii) (K3[Fe(CN)6];
Information.
3 equiv.) was added and the solution was stirred at room temperature for 20 min
Thiolysis of N-acetylaminonitriles. N-Acetylaminonitrile (Ac-AA-CN; 50 mM) and while being maintained at pD 9.5 with NaOH. The resulting suspension was cen-
NaSH∙xH2O (10 equiv.) were dissolved in degassed H2O/D2O (98:2, 50 ml). The trifuged and the supernatant was analysed by one- and two-dimensional NMR
solution was adjusted to pH 9.0 and stirred at room temperature for 24 h. NMR spectroscopy (1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC). The ligation
spectra were periodically acquired, until complete conversion of Ac-AA-CN to product (Ac-(AA1)n–(AA2)m-X; X = CO2H or CN) was quantified using relative
Ac-AA-SNH2 was observed. The solution was sparged with argon for 15 min at integral analysis by 1H, 1H–13C HMBC NMR spectral analysis and HRMS. Yields
pH 5.0 and concentrated in vacuo. The residue was purified using flash column and HRMS data are given in Table 2 and Supplementary Table 15, and character-
chromatography to afford Ac-AA-SNH2. Yields are reported in Table 1 and char- ization data are reported in Supplementary Information.
acterization data in Supplementary Information.
Hydrolysis of N-acetylaminothioamide to N-acetylaminoacyl thioacids. Ac-AA-SNH2
Data availability
(50 mM), NaSH∙xH2O (10 equiv.) and methylsulfonylmethane (MSM; 50 mM)
All data supporting the findings of this study are available within the main
were dissolved in degassed H2O/D2O (98:2, 1 ml), and the solution was adjusted
text, Extended Data Tables 1–5, Extended Data Fig. 1 and the Supplementary
to pH 9.0 with NaOH/HCl. The solution was incubated at 60°C while being main-
Information (which contains Supplementary Discussion, Supplementary Figs. 1–296,
tained at pH 9.0 with NaOH/HCl, and NMR spectra were periodically acquired
Supplementary Tables 1–16, experimental details and compound characterization data).
until complete consumption of Ac-AA-SNH2 was observed. The Ac-AA-SH was
confirmed by 1H–13C HMBC NMR analysis, spiking or comparison of NMR data
Acknowledgements We thank the Engineering and Physical Sciences Research
Council (EP/K004980/1, EP/P020410/1), the Simons Foundation (318881,
493895) and the Volkswagen Foundation (94743) for financial support. The
authors thank K. Karu (UCL Mass Spectrometry Facility), E. Samuel (Mass
Spectrometry, UCL School of Pharmacy) and A. E. Aliev (NMR spectroscopy) for
assistance.
with pure synthetic standards. The reaction mixture was quantified using MSM as
an internal standard. Yields are reported in Table 1 and characterization data are
given in Supplementary Information.
Oxidative coupling of Ac-Gly-SH with α-amino acids or α-amino amides. AA
or α-amino amide (AA-NH2) (150 mM) was dissolved in degassed H2O/D2O
(98:2; 1 ml) and the solution was adjusted to pH 9.5 with HCl/NaOH. Ac-Gly-SH
(50 mM) was added and the total volume was adjusted to 2 ml with H2O/D2O
(98:2). Potassium hexacyanoferrate(iii) (K3[Fe(CN)6], 150 mM) was added and
the solution was stirred at room temperature for 20 min while maintaining the
solution at pH 9.5 with NaOH. The resulting suspension was centrifuged and
the supernatant was analysed by one- and two-dimensional NMR spectroscopy
(1H–1H correlated spectroscopy (COSY), 1H–13C heteronuclear single quantum
coherence spectroscopy (HSQC) and 1H–13C HMBC in H2O/D2O (98:2). The
yield was quantified using MSM as an internal standard. The ligation product
(Ac-Gly–AA-X; X = OH or NH2) was confirmed by 1H–13C HMBC NMR spec-
tral analysis and high-resolution mass spectrometry (HRMS). Reaction mixtures
were lyophilized and dissolved in DMSO-d6 or CD3OD for further NMR spectral
analysis if 1H–13C HMBC cross-correlation peaks were obscured by the HOD
Author contributions M.W.P. conceived the research. P.C., S.I. and M.W.P.
designed and analysed the experiments. P.C. and S.I. contributed equally to the
experiments. S.I. wrote the Supplementary Information. M.W.P and S.I. wrote the
paper and Supplementary Discussion.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-019-1371-4.
Correspondence and requests for materials should be addressed to M.W.P.
Peer review information Nature thanks Irene Chen and the other anonymous
reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/
reprints.

reSeArCH Letter
Extended Data Fig. 1 | Chemoselective native peptide bond ligations of
cysteine and lysine residues. a, Ligation of Cys is notoriously challenging
owing to its highly nucleophilic thiol side chain, which necessitates
S-protection to prevent it outcompeting C- and/or N-terminal activation
through degradation of the electrophilic activating agents. Protecting-
group-free ligation of Cys (150 mM) is achieved through reaction with
Ac-Gly-SH (50 mM) and K3[Fe(CN)6] (300 mM) in water (pH 9.5, room
temperature), followed by thiol reduction (MeSH, 600 mM, pH 10.8, room
temperature) to give Ac-Gly-Cys-OH in high yield (80%, over two steps)
(Supplementary Figs. 112–114). b, Lys-X coupling partners (X = CN,
CONH2 or CO2H) pose greater chemoselectivity challenges because they
possess two amino groups (α-NH2 and ε-NH2). However, pKa-controlled
native peptide ligation of Lys-CN demonstrates the pivotal role that the
unusually low α-amine pKaH of AA-CN19 can play in selective ligation.
Ligation of Lys-CN (100 mM) with Ac-Gly-SH (50 mM) proceeds with
unprecedented selectivity in neutral water (pH 7.5, room temperature).
Little or no selectivity was observed for the corresponding α-amino amide
(Lys-NH2; 150 mM) and AA (Lys; 150 mM) (Supplementary
Figs. 145–151). c, Selective intermolecular ligation of the C-terminal lysine
residue with AA-CN coupling partner Gly-CN at near-neutral pH
(pH 6.5–9.0, blue; see Supplementary Fig. 70). In the absence of Gly-CN,
highly efficient intramolecular caprolactam formation is observed (red).

Letter reSeArCH
extended data Table 1 | α-Amidothioacid activating agents
Yields of the oxidative coupling of Ac-Gly-SH (50 mM) and Gly-CN (100 mM) with the specifed activating agent (150 mM) after 20 min in water at room temperature. 1H NMR yields were determined
with an internal NMR standard.

reSeArCH Letter
extended data Table 2 | α-Aminonitrile ligation in the presence of nucleophilic competitors
Yields of the oxidative coupling of Ac-Gly-SH (50 mM) and Gly-CN (100 mM) with K3[Fe(CN)6] (150 mM) in the presence of the specifed stoichiometric competitor (100 mM) after 20 min in water at
room temperature. 1H NMR yields were determined with an internal NMR standard. See Supplementary Figs. 235–244 for further details. n.d, not detected.

Letter reSeArCH
extended data Table 3 | α-Aminonitrile ligation at various concentrations and temperatures
Yields of the oxidative coupling of Ac-Val-SH (1 equiv.) and Gly-CN (2 equiv.) with K3[Fe(CN)6] (3 equiv.) at specifed concentration and temperature. 1H NMR yields determined with an internal NMR
standard. (–) = not determined.

reSeArCH Letter
extended data Table 4 | Chemoselective synthesis of N-acetyl dipeptidyl amides
Yields of the oxidative coupling of Ac-Gly-SH (50 mM) and AA-NH2 (150 mM) with K3[Fe(CN)6] (150 mM) in water at room temperature and pH 9.5. 1H NMR yields were determined with an internal
NMR standard.
aThe observed ratio of mono- and di-acylated products varies with solution pH (see Extended Data Fig. 1b for α-selectivity of Lys-NH2 ligation at pH 7.5 and Supplementary Table 12).
bReaction carried out at pH 6.5 (see Supplementary Table 9).

Letter reSeArCH
extended data Table 5 | Chemoselective synthesis of N-acetyl dipeptidyl nitriles
Yields of the oxidative coupling of Ac-AA1-SH (50 mM) and AA2-CN (100 mM) with K3[Fe(CN)6] (150 mM) in water at room temperature and pH 9.0. 1H NMR yields were determined with an internal
NMR standard, unless stated otherwise.
aThe observed ratio of mono- and di-acylated products varies with solution pH (see Extended Data Fig. 1b for α-selectivity of Lys-CN ligation at pH 7.5 and Supplementary Table 13).
bYield for the coupling of Ac-Lys-SH (32 mM) with Gly-CN (64 mM) and K3[Fe(CN)6] (96 mM).
cIsolated yield.