Synthesis of uniformly deuterated n‐dodecyl‐β‐d‐maltoside (d₃₉‐DDM) for solubilization of membrane proteins in TROSY NMR experiments

Abstract

This work reports the first synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d 39 -DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d 39 -DDM for solubilizing large α-helical membrane proteins in samples for [ 15 N-1 H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d 39 -DDM used d 7 -D-glucose and d 25 -n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. 1 H NMR spectra were used to confirm a high level of deuteration in the synthesized d 39 -DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52-kDa sugar transport protein solubilized in DDM.

Solution Properties of Alkyl β‐D‐Maltosides

“…Furthermore, the chemical shifts (the coupling constant) of β‐D‐maltoside ( 6i ) are 5.00 ppm ( J 1,2 = 3.0 Hz) for H‐1′ and 4.14 ppm ( J 1,2 = 7.7 Hz) for H‐1 in DMSO‐d6. All data indicate that the newly formed glycosyl bond ( 6a – 6i ) is in a 1,2‐trans β‐configuration because the configuration is the 1,2‐trans β‐anomer as 4 Hz < J 1,2 ≤ 8 Hz and the configuration is the 1,2‐cis α‐anomer as J 1,2 < 4 Hz (Aripin et al, ; Boyd et al, ; Chen and Kong, ; Hiruma‐Shimizu et al, ).…”

“…They can be prepared with SnCl 4 or BF 3 ·Et 2 O as Lewis acid catalysts of the condensation reaction, but the condensation time is rather difficult to control to some extent and the yield was low for the condensation step as concurrent decomposition and serious α‐anomerization take place (Aripin et al, ). Alkyl β‐D‐maltoside (Pillon et al, ), deuterated dodecyl β‐D‐maltoside (d 39 ‐DDM) (Hiruma‐Shimizu et al, ), and dodecyl β‐D‐melibioside (Hutchison et al, ) were stereoselectively prepared by the Koenigs and Knorr procedure with silver carbonate or silver triflate (AgOTf) as an expensive promoter in the coupling reaction. However, the Koenigs and Knorr procedure usually is uneconomic and/or may easily cause the accumulation of toxic heavy metal and the related environmental pollution in spite of its fairly high efficiency.…”

Solid-state NMR structures of integral membrane proteins

“…There is a limit to the size of the particles that can be analysed, however, and elevated sample temperatures (20-50 C) usually have to be used to accelerate particle tumbling, which can introduce issues of sample stability. Detergent micelles are most often used for solubilization (Arora, 2013;Hiruma-Shimizu et al, 2014Kim et al, 2009;Klammt et al, 2012;Maslennikov & Choe, 2013;Nietlispach & Gautier, 2011;Page et al, 2006;Patching, 2011;Reckel & Hiller, 2013) as well as other membrane mimetics such as organic solvents, lipids, bicelles, nanodiscs, fluorinated surfactants and amphipols (Bayburt & Sligar, 2010;Dürr et al, 2012Dürr et al, , 2013Inagaki et al, 2013;Popot, 2010;Warschawski et al, 2011;Zhou & Cross, 2013a;Zoonens & Popot, 2014). For larger membrane proteins that tumble more slowly and also have higher spectral crowding, deuteration of the protein and detergent, selective isotope labelling and TROSY-type NMR experiments are also used to achieve NMR spectra of the required quality (Patching, 2011;Hiruma-Shimizu et al, 2014Kalverda et al, 2014).…”

Micellar Catalysis in Fischer Glycosidation: En Route to Diverse Functional Glycosides

“…1‐Octanol was selected as representative example of aliphatic alcohol. Long and medium chain alkyl maltoside and lactoside can be obtained by enzymatic methods or in Koenigs–Knorr reaction . Although these glycoside are often used in biochemical applications, liquids crystals technologies and in food industry, both routes of synthesis have their limitations.…”