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New tech­no­logy CF LINK for pro­tein biocon­jug­a­tion and struc­tur­al pro­teo­m­ics from col­lab­or­a­tion of a Brno biotech star­tup and Czech Academy of Sciences

03.12.2019
The recently pub­lished, pat­ent pending tech­no­logy, allows to select­ively pre­pare pro­tein con­jug­ates via their trypto­phan residues and per­form posttrans­la­tion­al modi­fic­a­tion of aro­mat­ic amino acids. Fur­ther­more, it can also be used as a tool for map­ping of pro­tein sur­faces and stud­ies of pro­tein-pro­tein inter­ac­tions. The com­pany CF Plus Chem­ic­als, an ETH Zurich spin off foun­ded in 2014, is based on almost ten years of cooper­a­tion of the group of Dr. Petr Bei­er at the Insti­tute of Organ­ic Chem­istry and Bio­chem­istry of the Czech Academy of Sci­ences (IOCB) and Dr. Václav Matoušek, a Ph.D alum­nus of Prof. Dr. Ant­o­nio Togni at ETH Zurich. Reagents based on cyc­lic hyper­valent iod­ine-per­fluoroal­kyl com­pounds, nowadays also termed as the Togni reagents, have become widely used and pop­u­lar tools in organ­ic syn­thes­is, espe­cially in medi­cin­al chem­istry for pre­par­a­tion of new flu­or­in­ated drug can­did­ates, in-line with the grow­ing demand for flu­or­in­ated scaf­folds in drug design. While in the early 1990s flu­or­in­ated molecules accoun­ted for 5% of the total num­ber of approved drugs, now it is 15% and roughly 30% for newly approved thera­peut­ics. This fam­ily of chem­ic­al com­pounds was sub­sequently expan­ded in 2013 in col­lab­or­a­tion of ETH Zurich and Dr. Petr Bei­er from IOCB to a new gen­er­a­tion of pat­en­ted Togni reagents that carry a more com­plex RCF2CF2 group instead of a simple CF3 group.1 The new fam­ily of these chem­ic­als shares not only the rich react­iv­ity of the first gen­er­a­tion of Togni reagents, but also brings an extra dimen­sion due to the vir­tu­ally unlim­ited struc­tur­al vari­ab­il­ity of the β‑substituted tet­ra­fluoro­ethyl group which they are able to trans­fer to a vari­ety of sub­strates rel­ev­ant to med­ic­al chem­istry of small molecules. In 2017, the applic­a­tion poten­tial of the second gen­er­a­tion of Togni reagents was exten­ded to pro­teins.2 Their high affin­ity towards the thi­ol group allows to per­form select­ive biocon­jug­a­tion through cysteines to form stable con­jug­ates which, unlike maleim­ide con­jug­ates, are not sub­ject to slow decon­jug­a­tion and thi­ol exchange. The present inven­tion3,4 builds on the pre­vi­ous know­ledge of the rad­ic­al nature of Togni reagent-medi­ated reac­tions. In the SME Instru­ment Hori­zon 2020 pro­ject, sup­por­ted by the City of Brno and in cooper­a­tion with IOCB and Dr. Petr Novák from the Insti­tute of Micro­bi­o­logy of the Czech Academy of Sci­ences (IMIC), it was show­cased that Togni reagents, when mixed with sodi­um ascorbate, a cheap, non-tox­ic and biocom­pat­ible redu­cing agent, imme­di­ately gen­er­ate ß‑substituted tet­ra­fluoro­ethyl rad­ic­als that select­ively attach to ster­ic­ally access­ible trypto­phan residues of the pro­tein under trans­ition met­al-free con­di­tions. Once azido­fluoroal­kyl groups are attached to the pro­tein, vari­ous func­tion­al groups, such as fluor­es­cent dyes, radi­o­nuc­lides or ADC-tox­ins for tar­geted onco­ther­apy can be sub­sequently linked via click reac­tion to afford the cor­res­pond­ing pro­tein con­jug­ates. The dis­closed trypto­phan-select­ive biocon­jug­a­tion meth­od does not dis­rupt pro­tein disulf­ide bridges and offers an altern­at­ive solu­tion where con­ven­tion­al cysteine con­jug­a­tion is not pos­sible, for example due to an undesir­able disulf­ide scram­bling. This biocon­jug­a­tion meth­od can also be exten­ded to oth­er aro­mat­ic amino acids and hence to pro­teins lack­ing trypto­phan. Thus, it was pos­sible to suc­cess­fully modi­fy human recom­bin­ant insulin and attach up to 7 modi­fic­a­tions to its aro­mat­ic amino acids, demon­strat­ing the poten­tial for post-trans­la­tion­al modi­fic­a­tion of pro­teins. The extremely rap­id nature of this reac­tion that tar­gets solvent-access­ible aro­mat­ic amino acids makes it a con­veni­ent tool for map­ping pro­tein sur­faces and study­ing pro­tein-pro­tein inter­ac­tions. Using the example of human car­bon anhyd­rase, it has been demon­strated that the res­ults of sur­face map­ping are in excel­lent agree­ment with its pub­lished nat­ive struc­ture. The second gen­er­a­tion of Togni-CF2CF2R reagents is pro­tec­ted by a world­wide pat­ent owned by ETH Zurich and IOCB and are exclus­ively licensed by CF Plus Chem­ic­als. The new CF LINK4 ascorbate tech­no­logy, a sub­ject of anoth­er pat­ent applic­a­tion, fur­ther strengthens the intel­lec­tu­al prop­erty pro­tec­tion and forms a tech­no­logy pack­age which is cur­rently offered to licens­ing to busi­ness part­ners for biocon­jug­a­tion of dia­gnost­ic­ally and thera­peut­ic­ally rel­ev­ant proteins/​antibodies, pre­par­a­tion the cor­res­pond­ing immun­o­con­jug­ates with fluor­es­cence dyes, radi­o­nuc­lides or ADC-tox­ins, post-trans­la­tion­al pro­tein modi­fic­a­tion, struc­tur­al pro­teo­m­ics and study of pro­tein-pro­tein inter­ac­tions. Source: CF Plus Chem­ic­als, Pressemit­teilung, 03.12.2019
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