Evaluation of geranylazide and farnesylazide diphosphate for incorporation of prenylazides into a CAAX box‐containing peptide using protein farnesyltransferase*

Abstract

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide (C10) or farnesylazide (C15) moiety from the corresponding prenyldiphosphates to a model peptide substrate, N-dansyl-Gly-Cys-Val-Ile-Ala-OH. The rates of incorporation for these two substrate analogs are comparable and approximately twofold lower than that using the natural substrate farnesyl diphosphate (FPP). Reaction of N-dansyl-Gly-Cys(S-farnesylazide)-Val-Ile-Ala-OH with 2-diphenylphosphanylbenzoic acid methyl ester then gives a stable alkoxy-imidate linked product. This result suggests future generations whereby azide groups introduced using this enzymatic approach are functionalized using a broad range of azide-reactive reagents. Thus, chemistry has been developed that could be used to achieve highly specific peptide and protein modification. The farnesylazide analog may be useful in certain biological studies, whereas the geranylazide group may be more useful for general protein modification and immobilization.

An Efficient Chemoenzymatic Synthesis of Dihydroartemisinic Aldehyde

“…The short synthesis involved low‐temperature acid‐catalyzed selenium dioxide oxidation of 10 14 and diphosphorylation of the resulting chloride 11 15 (Tables S1, S2 and Figures S13–S16). For 1 H NMR and GC–MS comparison, an authentic sample of aldehyde 4 was prepared from commercially available (11 R )‐dihydroartemisinic acid (11 R )‐ 6 (Figures S7, S8, S26, S27) 6b, 16…”

“…To further investigate the capability of ADS to act on unnatural substrates, 12‐acetoxyfarnesyl diphosphate ( 13 ) was prepared in three steps (32 % overall yield) from known 1‐ O ‐THP‐protected 12‐hydroxyfarnesol14 (Figures S17, S18) and incubated with ADS. GC–MS analysis revealed that the same three products were obtained as from incubations with diphosphate 7 (Figure 2 and Figure S9).…”

The Protein Lipidation and Its Analysis

“…Later, the authors used a biotin-geranylgeranyl (GerGer) pyrophosphate analog and an engineered protein prenyltransferases to analyse the in vivo effects of protein prenyltransferase inhibitors by quantifying by MS the amount of biotin-GerGer-tagged RabGTPases after treatment with inhibitors [115]. Synthesis of additional farnesyl diphosphate analogs bearing alkyne or azide groups [116,117] as well as in vitro studies to characterize them as substrates for the corresponding prenyl transferases have also been described by other groups [118,119]. Recently, Tate and co-workers reported an azido-derived GerGer pyrophosphate that could serve as a substrate for both GGTase I and RabGGTase and be subsequently modified by CuAAC to incorporate a double labeled tag [120].…”

Effiziente chemoenzymatische Synthese von Dihydroartemisinaldehyd

“…Die kurze Synthese erforderte eine SelendioxidOxidation von 10 [14] bei niedriger Te mperatur mit tBuOOH und eine Diphosphorylierung des entstandenen Chlorids 11 [15] (siehe die Tabellen S1, S2 und S13-S16). Fürd en 1 H-NMRund GC-MS-Vergleich wurde eine authentische Probe des Aldehyds 4 aus handelsüblicher (11R)-Dihydroartemisinsäu-re (11R)-6 hergestellt (siehe Abbildungen S7, S8, S26, S27).…”

“…Fürd en 1 H-NMRund GC-MS-Vergleich wurde eine authentische Probe des Aldehyds 4 aus handelsüblicher (11R)-Dihydroartemisinsäu-re (11R)-6 hergestellt (siehe Abbildungen S7, S8, S26, S27). Um die Fähigkeit von ADS zur Umsetzung nichtnatürli-cher Substrate weiter zu untersuchen, wurde 12-Acetoxyfarnesyldiphosphat (13)i nd rei Schritten (32 %G esamtausbeute) aus dem bekannten 1-O-THP-geschützten 12-Hydroxyfarnesol [14] hergestellt (siehe die Abbildungen S17, S18) und mit ADS inkubiert. Die GC-MS-Analyse ergab,dass die gleichen drei Produkte wie durch Inkubation mit Diphosphat 7 erhalten wurden (siehe Abbildung 2u nd Abbildung S9).…”