Adenosine phosphonate inhibitors of lipid II: Alanyl tRNA ligase MurM from Streptococcus pneumoniae Elena Cressina, a Adrian J. Lloyd, b Gianfranco De Pascale, a,b David I. Roper, b Christopher G. Dowson b and Timothy D. H. Bugg a, * a Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK b Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Received 7 May 2007; revised 20 May 2007; accepted 22 May 2007 Available online 25 may 2007 Abstract—Adenosine and 2 0 -deoxyadenosine phosphonate transition state analogues act as the first inhibitors for the MurMN/Fem- ABX family of tRNA-dependent ligases implicated in high-level penicillin resistance in Gram-positive bacteria. Ó 2007 Elsevier Ltd. All rights reserved. High-level penicillin resistance in Streptococcus pneumo- niae is associated with the presence of additional -Ala- Ala- and -Ser-Ala- cross-links in the peptidoglycan structure, 1 as shown in Figure 1. The presence of these cross-links is associated with the presence of particular sequences of the murM and murN genes, 2 related to the femABX genes responsible for formation of the (Gly) 5 cross-link in Staphylococcus aureus. 3 The encoded MurMN proteins catalyse the addition of Ala(Ser) and Ala, respectively, to lipid intermediate II in peptidogly- can biosynthesis, 4 using Ala-tRNA and Ser-tRNA, respectively, analogous to the addition of Gly-tRNA by FemABX in S. aureus. 5 Deletion of the murM gene in S. pneumoniae leads to loss of the high-level resistance phenotype, 2 therefore MurM is a potential target for anti-resistance agents that could be applied synergisti- cally with penicillin, against antibiotic-resistant bacteria. The catalytic mechanism of the MurM-catalysed reac- tion is likely to proceed via a tetrahedral transition state, shown in Figure 2. Since adenosine is found at the 3 0 -ter- minus of tRNA nucleic acids, a possible transition state mimic for this family of enzymes is an adenosine 3 0 - phosphonate, shown in Figure 2. This paper describes the synthesis and in vitro assay of a series of adenosine and 2 0 -deoxy-adenosine phosphonates as MurM inhibitors. In the 2 0 -deoxy series, a protected 2 0 -deoxyadenosine derivative (1) 6 was coupled with aminoethylphosphonic monomethyl ester 2 7 using DCC, in 54% yield. Depro- tection was achieved in 52% yield, to give phosphonate 3, as shown in Figure 3. In the ribo-series, attempts to couple 2 to a 2 0 ,5 0 - TBDMS-protected adenosine to obtain selectively the 3 0 phosphonate (6a) were unsuccessful, using a range of coupling agents. Therefore, an improved version of the procedure reported by Zemlicka 8 to obtain a mixture of 2 0 and 3 0 regioisomers was followed. A 5 0 -TBDMS-protected adenosine (4) 6 was coupled with aminoethylphosphonic acid 5 7 to give a mixture of 2 0 - and 3 0 -phosphonates 6a and 6b, in a 3:1 ratio, 0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2007.05.071 Keywords: MurM; Inhibitor; Peptidoglycan biosynthesis; Antibacte- rial; Streptococcus pneumoniae. * Corresponding author. Tel.: +44 02476 573018; fax: +44 02476 524112; e-mail: T.D.Bugg@warwick.ac.uk MurNAc L-Ala D-Gln L-Lys D-Ala D-Ala MurNAc L-Ala D-Gln L-Lys D-Ala MurNAc L-Ala D-Gln L-Lys D-Ala D-Ala MurNAc L-Ala D-Gln L-Lys D-Ala L-Ala(Ser) L-Ala Figure 1. (a) Direct peptidoglycan cross-link found in normal S. pneumoniae; (b) indirect peptidoglycan cross-link found in penicillin-resistant S. pneumoniae. Bioorganic & Medicinal Chemistry Letters 17 (2007) 4654–4656