Synthesis and screening of diverse chemical libraries for and HMBC (11), for determining the 13 C and 1 H resonances.
The assignments can be completed with this method at natu-pharmaceutical-lead discovery has been a topic of great interest in recent years. Oligo-N-substituted glycine (NSG) ral abundance 13 C. However, the initial assignments are considerably facilitated if the NSG peptoid is also available with peptoids have been used as a source of chemical diversity, and submonomer synthesis of these libraries has allowed the 13 C label at each main-chain ac-Ca position. Fortunately, these 13 C-labeled compounds are straightforward to synthe-generation of greater chemical diversity than that available with peptides (1-4). Recently, two trimers, from an NSG-size with 13 C-labeled bromoacetic acid, using the standard submonomer synthesis method (2). The optimal combina-peptoid library of over 3600 compounds, have been identified as specific, nanomolar ligands for two important 7-trans-tion of spectra for assignment of NSG peptoids includes natural abundance COSY (or TOCSY), HMQC, and membrane G-protein coupled receptors, a1-andrenergic and m-opiate (5). In view of the pharmaceutical relevance of HMBC, and 13 C-labeled HMQC and HMBC.
Initially, a COSY or TOCSY of the natural-abundance this new class of compounds, the NMR characterization of NSG peptoids becomes crucial, both for establishing cova-compound is used to establish 1 H-1 H connectivities, and a natural-abundance HMQC provides 1 H-13 C connectivities. lent connectivity and for determining the solution and receptor-bound conformations. A prerequisite for achieving these These two experiments establish the proton spin systems and their associated carbon chemical shifts. At this point, the goals is the chemical-shift assignments. A new method is required for NSG-peptoid resonance assignments because of HMQC of the 13 C-labeled NSG peptoid is particularly useful to identify unambiguously the main-chain ac-Ca/ac-Ha the lack of amide protons on the N-substituted amide bonds.
For peptide sequential NMR assignments, spin systems cross peaks, which occur in the same region at the same peak intensity as the side-chain Nxxx-Ca/Nxxx-Ha cross consisting of HN, Ha, Hb1, Hb2, etc., are identified using homonuclear COSY (6, 7) or TOCSY (8) experiments. peaks in a natural-abundance HMQC spectrum. Then an HMBC is performed with the 13 C-labeled NSG peptoid, in These homonuclear experiments are adequate for peptide systems, because, for an amino-acid residue, the main-chain order to connect the side-chain spin system to the mainchain spin system. The Nxxx-Ha resonances are the only protons and most of the side-chain protons are within three bonds of another proton. Thus, most amino-acid monomers protons connected through three bonds to the 13 C-labeled ac-Ca positions (Fig. 1). Thus, a cross peak observed be-form one spin system. However, with NSG peptoids, the side-chain branch point is shifted from the main-chain alpha tween a main-chain ac-Ca and a side-chain Nxxx-Ha in the 13 C-labeled HMBC experiment unambiguously connects carbon to the main-chain nitrogen. As a result, each monomer unit has two spin systems, the side-chain branching the two spin systems and identifies them as a monomer unit.
This step is general and can be performed with a natural-from the main-chain nitrogen, designated as ''Nxxx,'' and the two-carbon main-chain unit designated as ''ac'' (Fig. abundance HMBC, but the ac-Ca/Nxxx-Ha cross peaks must then be distinguished from the other three-bond cou-1). These subunits are separated by at least four bonds between the side-chain Nxxx-Ha and the main-chain ac-Ha. pling of Nxxx-Ca to ac-Ha.
In peptides, the sequential connection of spin systems is Described here is a method for assigning the 1 H and 13 C resonances of NSG peptoids. This method employs both usually performed using 1 H-1 H NOESY (12) cross peaks from the amide protons of one residue to the amide, alpha, traditional homonuclear sequential assignment techniques (9) and heteronuclear NMR pulse sequences, HMQC (10) or beta protons of the next residue (11). However, with 195