Gábor N.
Boross
a,
Satomi
Shimura
a,
Melissa
Besenius
b,
Norbert
Tennagels
b,
Kai
Rossen‡
b,
Michael
Wagner
b and
Jeffrey W.
Bode
*a
aLaboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH Zürich, 8093 Zürich, Switzerland. E-mail: bode@org.chem.ethz.ch; Web: http://www.bode.ethz.ch/
bSanofi-Aventis Deutschland GmbH Industriepark Hoechst, 65926 Frankfurt am Main, Germany Web: http://www.sanofi.com
First published on 11th September 2018
The chemical synthesis of insulin is an enduring challenge due to the hydrophobic peptide chains and construction of the correct intermolecular disulfide pattern. We report a new approach to the chemical synthesis of insulin using a short, traceless, prosthetic C-peptide that facilitates the formation of the correct disulfide pattern during folding and its removal by basic treatment. The linear precursor is assembled by an ester forming α-ketoacid-hydroxylamine (KAHA) ligation that provides access to the linear insulin precursors in good yield from two readily prepared segments. This convergent and flexible route provides access to various human, mouse, and guinea pig insulins containing a single homoserine mutation that shows no detrimental effect on the biological activities.
Early chemical syntheses of insulin by chain combination methods are low yielding.2,3 Stepwise formation of disulfide bonds can bypass the problem of incorrect disulfide pairing but these methods suffer from a long synthetic route and a low overall yield.4–6 To improve synthetic access, Kent has reported a method for the synthesis of insulin Lispro variants, employing oxime-forming ligation for straightforward access to the folding precursors.7 Separately, Kent has successfully utilized a zero length C-peptide (ester bond between the GluA4 and ThrB30 sidechains) for folding of linear insulins prepared by native chemical ligation.8,9 The DiMarchi group has reported elegant methods to access single chain insulins as folding precursors. Oxime-forming ligation combined with a traceless C-peptide surrogate or enzyme-cleavable single-chain precursors resulted in a variety of correctly folded insulins.10,11
Inspired by these pioneering studies, here we report a convergent method to access insulin and its analogues with a traceless, base-labile prosthetic C-peptide that shepherds folding into the correct disulfide pattern (Scheme 1). The modestly sized linear insulin precursor is assembled from two fragments by the α-ketoacid-hydroxylamine (KAHA) ligation.12 The use of the chemoselective coupling of peptide α-ketoacids and (S)-5-oxaproline (Opr) proceeds under acidic conditions that readily solubilize the notoriously hydrophobic insulin chains and forms an ester linked linear insulin that offers improved solubilisation and handling. Tandem folding, linker cleavage, and O → N shift that products ThrB27Hse insulin variants that exhibit identical biological activity to medicinal forms.
The basis for our prosthetic C-peptide is the work of Brandenburg et. al., who showed that an intermolecularly crosslinked (Nα GlyA1, Nε LysB29) native insulin induces the formation of the correct disulfide bonds upon reduction and subsequent refolding.13 Obermeier et. al. have designed a cleavable chemical linker that can mimic the function of the C-peptide of proinsulin and efficiently fold the reduced single-chain insulin into its native structure.14 Although a very promising as a strategy for folding synthetic insulin, this linker was employed only on isolated bovine insulin and – to the best of our knowledge – has not been used for the chemical synthesis insulin.
In our preliminary studies the necessary linear miniproinsulin could not be prepared directly by Fmoc SPPS (See ESI† page S62). We dissected our target into two shorter peptide segments that could be assembled by KAHA ligation. From the ligation product, folding, ester-to-amide rearrangement, and linker cleavage could all occur under basic conditions to access a variety of insulins.
In order to access the key synthetic intermediate of M2 insulin (ThrB27Hse) 9 (Scheme 2), we placed the ligation site on the B chain between TyrB26 and ThrB27. We chose this site because this disconnection resulted in two similarly sized peptide segments and our previous studies have shown that replacement of Ser/Thr residue with homoserine (Hse, T§) do not affect the folding or biological activity of synthetic proteins compared to wild type references.18–20
We prepared both peptide segments 6 and 7 by standard automated Fmoc solid phase peptide synthesis (SPPS). The segments were synthesized on ChemMatrix® resin with a loading of 0.2 mmol g−1 to avoid the aggregation of the growing peptide chains on resin. Due to the notoriously poor solubility of the insulin A chain and the single-chain insulin derivatives, we initiated the (S)-5-oxaproline segment (6) with a base-cleavable hexaarginine solubilizing tag (Arg-tag). The Arg-tag was attached to the C-terminus of the A chain via a base labile ester bond21 that can be concomitantly removed with the base-labile prosthetic C-peptide.
In our preliminary studies we prepared the peptide segments with unprotected Cys residues but observed premature formation and scrambling of disulfides during purification and ligation, regardless the presence of various reducing agents. To circumvent this we elected to keep the cysteine residues protected during purification of the segments and KAHA ligation. After evaluating several possibilities, we selected Acm protection for all of the Cys residues and prepared the requisite peptides without deviation from our optimized conditions.
The KAHA ligation of the two segments worked well in aqueous DMSO in the presence of oxalic acid. It was nearly complete after 18 hours without the formation of decomposition products. The ligated depsipeptide was separated by preparative reversed phase (RP) HPLC in 41% isolated yield. We retained the ester bond until the end of the synthesis to benefit from an expected increase in solubility from the additional unprotected primary amine in the linear insulin.22
The six Acm groups of ligated peptide 8 were removed by AgOAc under acidic conditions,23 which were ideal for solubilizing the linear, unfolded insulin 9. Although the starting material was completely converted after one hour, the isolated yield was modest, most likely due to the poor recovery from the preparative RP-HPLC.
With the reduced linear insulin 9 in hand, we proceeded to establish folding conditions using the prosthetic linker to direct the formation of the correct disulfide bond pattern. Unfolded, linear insulin variants are known to be poorly soluble in aqueous buffers and in the complete absence of denaturing agents and buffering salts, are very prone to form aggregates and precipitate.24
Based on folding conditions reported by Kent8 and DiMarchi,11 we decided to perform the folding in two steps. First, a strongly denaturing buffer containing 0.3 M Tris with 6 M guanidine hydrochloride (Gn·HCl) at lower pH (6.6) was used to solubilize the peptide (0.5 mg ml−1) at room temperature. Cysteine hydrochloride was added, as reducing agent to allow the formation of the thermodynamically most stable native disulfide pattern.
Once the peptide was completely dissolved, the buffer was rapidly diluted with deionized water to a final peptide concentration of 0.25 mg ml−1, the pH was set to 8.2 and the reaction was incubated at 4 °C. After 12 hours the solution was incubated for 4 hours at room temperature, in order to induce the O → N acyl shift. The folded peptide 10 was separated by preparative RP-HPLC. The purified folded peptide was treated with 0.1 M NaOH at 0 °C for 10 minutes in order to cleave C-peptide and the Arg-tag. After quenching the reaction with acetic acid the final peptide was isolated by preparative RP-HPLC. The denaturing, folding, O → N acyl shift, base mediated cleavage of the prosthetic C-peptide, Arg-tag removal and two RP-HPLC purification steps gave M2 insulin (ThrB27Hse) (11) in 10% overall yield from the unprotected linear precursor. The purity and identity of the isolated product was confirmed by analytical RP-HPLC and high resolution (HR) MS. The tertiary structure of the peptide was characterized by circular dichroism (CD) and compared with authentic recombinant insulin. The synthetic M2 insulin variant exhibits similar negative bands at 208 nm and 222 nm and a positive band at 193 nm as the reference compound; indicative of the presence of α-helices (Scheme 3).25
The recombinant production of guinea pig insulin has long posed a challenged and a successful expression in yeast has only very recently been described.26 Guinea pig insulin is known to lack the ability to form hexamers, rending it of importance for studying the oligomer forming properties of different insulin variants. In guinea pig insulin the key structural motive of the three disulfide bonds is conserved, but the primary amino acid sequence differs from human insulin in 18 residues out of the 51.
We could successfully apply our workflow on the synthesis of a modified guinea pig insulin (ThrB27Hse, AsnA21Gly), demonstrating that our approach readily tolerates changes in the sequence of the targeted peptides and offers a reliable approach to distinct insulin variants.
We prepared the (S)-5-oxaproline segment 12 with the photolabile solubilizing tag (Scheme 4) by following our established protocol. The ThrB30 residue was introduced with an appropriately modified linker (5d, Scheme 1). The KAHA ligation preceded smoothly, and after 18 h 97 mg of ligated depsi peptide (13) was isolated by preparative RP-HPLC in 61% yield. The Acm protecting groups were removed as described above and the folding precursor was isolated in 46% yield. The folding, subsequent O → N acyl shift were performed and the protein purified as described for M2 insulin. During folding the peptide behaved similar to the base labile analogues. The correctly folded peptide was observed as the main peak on the analytical RP-HPLC and purified by preparative RP-HPLC. We did not observe premature cleavage of the photolabile Arg tag during any of the purification, handling, or coupling steps.
The combined preparative RP-HPLC fractions containing pure folded insulin (15) were irradiated with a hand held UV lamp at 365 nm. The progress of the reaction was followed by MALDI-MS. After 4 hours the sample was lyophilized and treated with 0.1 M NaOH at 0 °C for 10 min in order to cleave the C-peptide. After neutralizing the mixture with acetic acid, the final product (16) was isolated by preparative RP-HPLC. The folding, O → N acyl shift, photorelease of the Arg tag and base mediated cleavage of the C-peptide and two HPLC purification steps gave human insulin (ThrB27Hse) in 10% yield.
The purity and identity of the isolated human insulin was confirmed by analytical RP-HPLC and HRMS. In the CD spectra negative bands at 208 nm and 222 nm and a positive band at 193 nm were detected as indicative for the presence of α-helices and characteristic for authentic recombinant human insulin.
With the folded, human insulin (ThrB27Hse) in hand the disulfide bond pattern could be confirmed by endoproteinase Glu-C disulfide mapping (ESI† S55). The fragments obtained from digesting the synthetic insulin by Glu-C enzyme corresponded to the ones from commercial recombinant insulin.10
This convergent approach tolerates numerous mutations on the insulin sequence and can be applied to other members of the insulin protein family. It allows the synthesis of insulin variants, such as guinea pig insulin, that are difficult to produce by recombinant methods. A fully synthetic approach will also facilitate the generation of new insulin analogues with improved therapeutic utility.
Footnotes |
† Electronic supplementary information (ESI) available: Experimental procedures and analytical data of all new compounds. See DOI: 10.1039/c8sc03738h |
‡ Current address: H. Lundbeck A/S Ottiliavej 9, 2500 Valby, Denmark, Web: http://www.lundbeck.com |
This journal is © The Royal Society of Chemistry 2018 |