Rapid access to Asp/Glu sidechain hydrazides as thioester precursors for peptide cyclization and glycosylation

Article abstract of DOI:10.1039/d0cc07404g

Head-to-sidechain macrocylic peptides, and neoglycopeptides, were readily prepared by site-specific amidation of aspartic and glutamic acid sidechain hydrazides. Hydrazides, serving as latent thioesters, were introduced through regioselective opening of the corresponding N_α-Fmoc protected anhydride precursors.

Full text of DOI:10.1039/d0cc07404g

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Rapid access to Asp/Glu sidechain hydrazides as
thioester precursors for peptide cyclization and
glycosylation†
Cite this: Chem. Commun., 2021,
57, 1006
Received 11th November 2020,
Accepted 22nd December 2020
Natalie G. Barnes, Kudakwashe Nyandoro, Hanzhang Jin and Derek Macmillan
*
DOI: 10.1039/d0cc07404g
rsc.li/chemcomm
Head-to-sidechain macrocylic peptides, and neoglycopeptides, precursors.7 Some protected Cb/g-hydrazide building blocks of
were readily prepared by site-specific amidation of aspartic and Asp7a and Glu8 respectively, suitable for use in Fmoc-based SPPS,
glutamic acid sidechain hydrazides. Hydrazides, serving as latent have previously been prepared albeit by disparate, and often
thioesters, were introduced through regioselective opening of the protracted, synthetic pathways.
corresponding Na-Fmoc protected anhydride precursors.
Here we show how various sidechain protected Asp and Glu
hydrazides can be accessed rapidly in only two steps from the
The use of peptide Ca-thioesters as building blocks for synthetic corresponding Na-Fmoc protected amino acid, via regioselec-
proteins using Native Chemical Ligation (NCL) is well established.1 tive opening of the cyclic anhydride.9 Protected analogues were
These thioesters have additionally functioned as precursors for introduced to model synthetic peptides to demonstrate their
NCL-mediated head-to-tail peptide cyclisation.2 Both C-terminal utility in head-to-sidechain macrocyclisation and NCL-like
thioesters and hydrazides (which function as stable thioester glycosylation (Fig. 1).
precursors) can be readily prepared from synthetic or bacterially
Table 1 shows that several protected Asp/Glu building blocks
produced materials which, in part, overcomes the size limitations were prepared via regioselective opening of the corresponding
associated with traditional stepwise solid phase peptide synthesis Fmoc-protected amino acid anhydride in as little as 1 h.
(SPPS).3 Meanwhile, selective formation of thioesters at the side-
chain carboxyl groups of aspartic (Asp) and glutamic (Glu) acids
within fully unprotected peptides and proteins is much less
common, yet valuable for the production of branched and cyclic
peptides possessing novel architectures relevant to natural
products, therapeutics, and new biomaterials. For example, bio-
active lariat peptides contain a head-to-sidechain macrocycle
connecting their N-terminal amino and sidechain carboxyl groups
of specific Asp or Glu residues.4 Sidechain amidation has also
served as a key macrocyclisation strategy for preparation of
synthetic stapled peptides, invariably employing fully protected
precursors.5 Sidechain thioesters are difficult to incorporate into
synthetic peptides by Fmoc-based SPPS due to their instability in
the presence of piperidine. Consequently, alternative building
blocks possessing a latent thioester activated via an N–S(e) acyl
transfer have been described.6 Sidechain acyl hydrazides are also
potentially valuable here as air and moisture stable alternatives, but
there are only few reports of their use as sidechain thioester
Department of Chemistry, University College London, 20 Gordon Street,
Fig. 1 (a) Rapid synthesis of various sidechain protected Glu and Asp
hydrazides by anhydride opening, by reaction with a suitable carbazate or
hydrazine (b) Head-to sidechain cyclisation, typical of lariat peptides (inset)
and (c) site-selective bioconjugation via NCL.
London, WC1H0AJ, UK. E-mail: d.macmillan@ucl.ac.uk
† Electronic supplementary information (ESI) available: Details of all synthetic
procedures, and NMR spectra, HPLC traces and LC-MS analysis for selected
compounds is available. See DOI: 10.1039/d0cc07404g
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Table 1 Synthesis of protected hydrazides from the corresponding anhydrides
Hydrazide
n
Ra
hydrazine/carbazate equiv. Time/h Yieldb/%
1
2
3
4
5
6
1
1
1
0
0
0
Boc
Cbz
2-ClTrt 1.5
Boc
Cbz
2-ClTrt 1.5
1.5
1.5
2
2
16
1
1
16
62
88
73
77
63
66
1.5
1.5
a
tert-Butyloxycarbonyl (Boc), Benzyloxycarbonyl (Cbz), and 2-Chlorotrityl
b
(2-ClTrt). Isolated yields following column chromatography, although
crude amino acids were often sufficiently pure to be employed directly in
peptide synthesis.
Fig. 2 (a) Acyl hydrazide activation to form macrocycle 9, monitored by
HPLC. (bi) Upper trace shows initial hydrazide 7 while the lower trace
shows the reaction mixture after NaNO2 activation (20 min at pH3, 0 1C),
followed by addition of sodium 2-mercaptoethane sulfonate (MESNa), 1 h,
RT, pH 7.3c (bii) Activation of 7 with acetyl acetone (acac)/MESNa (pH 3–4,
Hydrazide building blocks were then introduced to short model
peptides derived from microcin J25 (H-GGAGHVPEF-NH2, 7),10
À
and lassomycin (H-GLRRLFADQ-NH2, 8).11 To assess their
À
performance in NCL-type cyclisation, the N-terminal Gly residues
were replaced with Cys, and the target sidechain carboxyl group 2 h, upper trace) leads to a mix of MESNa thioester (*) and acyl pyrazole (+)
which are both consumed upon adjusting to pH7 for 1 h (lower trace).12
(indicated in bold) was primed as the hydrazide derivative. 7 was
prepared using building block 3 under standard (HBTU/HOBt)
coupling, whilst Boc-protected 1 failed to couple under similar
reaction conditions. Cyclisation at the Glu sidechain via acyl
azide3c and acyl pyrazole12 intermediates was then examined
(Fig. 2).
In both reactions the desired macrocycle 9 was observed
within 1–3 h. In the case of the acyl pyrazole, formed using
acetyl acetone (acac) as activator, a mixture of 2-mercaptoethane
sulfonic acid (MESNa) thioester and acyl pyrazole were initially
observed, both of which were rapidly consumed upon adjusting
the pH to 7. Studies were next extended to Asp derivatives and
model peptide 8. Unfortunately all Asp derivatives 4–6 failed to
couple efficiently under identical reaction conditions employed
to prepare 7. These results underscored the utility of the
Cb-benzyl ester of Asp as a useful alternative hydrazide
precursor.7b Coincidentally, the benzyl ester could also be pre-
pared by regioselective opening of the Fmoc protected aspartic
anhydride with benzyl alcohol (see ESI†).
Encouragingly coupling of 4 or 6 via the symmetrical anhydride
method13 allowed 8 to be isolated in 14% yield from a crude mixture
comprising significant deletion by-product (H-CLRRLFAQ-NH2).
Cyclisation was attempted following NaNO2 activation (Fig. 3). Mind-
À
Fig. 3 (a) Cyclisation of Lassomycin derivative 8. (i) NaNO2 (1.2 equiv. Na
phosphate buffer; pH3, 0 1C, 20 min), followed by addition of MESNa (pH7,
ful that aspartimide formation could potentially compete with NCL
upon hydrazide activation, giving rise to a product of identical mass,
the isolated product was treated with trypsin (0.01 mg mlÀ1 trypsin,
rt, 48 h). (b) LC-MS trace of the reaction mixture after 24 h, showing 10 as
the major species.
pH 8, 30 1C, 1.5 h). Successful formation of 10 was confirmed by the 4 N-terminal residues. Overall the cyclisation product was isolated in
appearance of two major species (see ESI†) with m/z = 1120.6, 64% yield.
corresponding to amide bond hydrolysis at one of the scissile
Sidechain hydrazide activation and NCL is not limited to
arginine (Arg) residues, and m/z = 964.5, corresponding to excision intramolecular reactions. In initial attempts to extend this
of one Arg residue. Had cyclisation been unsuccessful we would process to include convergent N-glycopeptide synthesis, we
have expected to observe cleavage products resulting from loss of all explored the reaction between sugar-linked auxiliary 1114,15
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Fig. 4 (a) Convergent N-neoglycopeptide synthesis: (i) acac (2 equiv.), 0.1 M MPAA, Na phosphate buffer pH 3, 2 h, then, 11 and TCEP (50 mM), pH 7,
48 h. (b) LC-MS analysis of the reaction mixture after 48 h showing predominance of glycoconjugate 14.
and peptides 12–13 (Fig. 4). Auxiliary-mediated ligation removes introduction into synthetic peptide thioester segments, to
the requirement for cysteine to be incorporated at the ligation enable protein backbone assembly via NCL. With recombinant
site.16 Peptides 12 and 13, corresponding to a glycosylated protein sidechain thioesters already available through an
fragment of b-interferon, were prepared as their Glu and Asp expanded genetic code,18 the convergent union of carbohy-
sidechain hydrazides respectively, in a similar manner to 7 and drates with fully recombinant proteins is edging closer.19 It is
8. Alternatively 13 was prepared from the Asp sidechain benzyl likely that genetically encoded hydrazide building blocks will
ester (see ESI†). Whilst conversion from the benzyl ester to the also become available in the future, functioning as versatile
hydrazide proceeded well (96% yield), in our hands much of the intermediates on the path to modified proteins. We envisage
benzyl ester precursor itself (isolated in 19% yield) was lost that fully recombinant proteins modified with 11, or auxiliary-
due to competing aspartimide formation that occurred during linked N-acetyl glucosamine, may provide a viable convergent
peptide synthesis, cleavage and isolation. Consequently, synth- route to glycoprotein therapeutics. Once installed, the monosac-
eses employing the hydrazides 4 or 6, appeared comparable in charide can be extended enzymatically using endo-glucosidases,20
terms of efficiency. 11 was employed in ligation reactions as a dramatically reducing the requirement for protein synthesis by
chemically robust alternative to the naturally occurring glycosy- multi-component NCL.
lamine derived from N-acetyl glucosamine.16 The additional
The authors acknowledge financial support from The Lever-
methylene spacer at the anomeric centre additionally serves to hulme Trust (RPG-2018-133) and UCL.
render eventual glycopeptide products resistant to enzymatic
protein deglycosylation mediated by N-glycanases.17 Peptides 12
or 13 were treated with acac in sodium phosphate buffer at pH
3–4 for 2 h, followed by addition of 11, 4-mercaptophenyl acetic
acid (MPAA) and tris-carboxyethylphosphine (TCEP) at pH 7, and
Conflicts of interest
There are no conflicts to declare.
monitored by LCMS. Ligation products were observed after 1 h
and continued to accumulate over time, emerging as the major
products after 48 h (Fig. 4b). Glycoconjugates 14 and 15 were
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