Assembly of Peptidoglycan Fragments-A Synthetic Challenge

Abstract

Peptidoglycan (PGN) is a major constituent of most bacterial cell walls that is recognized as a primary target of the innate immune system. The availability of pure PGN molecules has become key to different biological studies. This review aims to (1) provide an overview of PGN biosynthesis, focusing on the main biosynthetic intermediates; (2) focus on the challenges for chemical synthesis posed by the unique and complex structure of PGN; and (3) cover the synthetic routes of PGN fragments developed to date. The key difficulties in the synthesis of PGN molecules mainly involve stereoselective glycosylation involving NAG derivatives. The complex synthesis of the carbohydrate backbone commonly involves multistep sequences of chemical reactions to install the lactyl moiety at the O-3 position of NAG derivatives and to control enantioselective glycosylation. Recent advances are presented and synthetic routes are described according to the main strategy used: (i) based on the availability of starting materials such as glucosamine derivatives; (ii) based on a particular orthogonal synthesis; and (iii) based on the use of other natural biopolymers as raw materials.

Keywords: NAG-NAM disaccharide; PGN biosynthesis; PGN synthesis; bacterial peptidoglycan.

Figure 1

Representation of the monomeric muropeptides frequently found in the peptidoglycan (PGN) of different bacteria. (a) the structure of the Staphylococcus aureus PGN (a Lys-type PGN) unit is presented. N-acetylglucosamine (NAG)–N-acetylmuramic (NAM) disaccharide units (red) are linked to each other via NAG-(β-1,4)–NAM linkage. Stem peptides (black), of which the composition is specific for each bacteria species, are attached to the d-lactyl (Lac – highlighted in the green circle) moiety of each NAM. The sequence of the peptide stem attached to the lactyl carboxylate of NAM is l-Ala-d-iGln-l-Lys-d-Ala-d-Ala. (b) the structure of a muropeptide of Gram-positive Mycobacterium spp., with an L-Ala-D-iGln-meso-DAP-d-Ala-d-Ala stem peptide sequence. (c) a representative muropeptide of the Gram-negative bacterium Escherichia coli.

Figure 2

Schematic representation of PGN biosynthesis in Gram-negative bacteria: (1) synthesis of a PG precursor in the cytoplasm; (2) synthesis of lipid-linked precursors at the inner leaflet of the cytoplasmatic membrane; (3) flipping to the outer leaflet of the cytoplasmatic membrane and incorporation of the precursor into nascent PGN by transglycosylation (TG) and transpeptidation (TP) reactions. Enzymes shown in blue synthesize substrates added on the depicted reaction: Alr racemases convert l-Ala to d-Ala and Ddl ligases form the d-Ala-d-Ala dipeptide.

Scheme 1

Synthesis of PGN fragment 9 reported by Fukase [55]. (a) (i) AllocCl, NaHCO₃, H₂O; (ii) AllylOH, DOWEX 50W X8 200–400 mesh, 80 °C; (iii) PhCH(OMe)₂, p-TsOH, 57%; (b) NaH then trifluoromethanesulfonyl-l-(S)-2-propionic acid benzyl ester, 98%; (c) Pd(PPh₃)₄, AcOH, DCM, then TrocCl, 58%; (d) Me₃N·BH₃, BF₃·Et₂O, ACN, 87%; (e) BnBr, AgO, DCM, 87%; (f) Ir complex, H₂, THF, then I₂, H₂O, 99%; (g) CCl₃CN, Cs₂CO₃, DCM, quantitative; (h) TMSOTf (0.1 equiv.), MS 4 Å, DCM, −15 °C, 88%; (i) Me₃N·BH₃, BF₃·Et₂O, ACN, 52%; (j) Ir complex, H₂, THF, then I₂, H₂O, 91%; (k) CCl₃CN, Cs2CO3, DCM, 85%; (l) TMSOTf (0.1 equiv.), MS 4 Å, DCM, −15 °C, 79%; (m) Me₃N·BH₃, BF₃·Et₂O, ACN, 60%; (n) Ir complex, H₂, THF, then I₂, H₂O, 81%; (o) CCl₃CN, Cs₂CO₃, DCM, 83%; (p) TMSOTf (0.1 equiv.), MS 4 Å, DCM, −15 °C, 70%; (o) Zn–Cu, AcOH, then Ac₂O, Py, 82%; (r) 1 M NaOMe, THF, H₂O: LiOH, dioxane, THF, H₂O, 97%; (s) HCl·H-l-Ala-d-Glu(OBn)-NH₂, WSCI·HCl, HOBt, TEA, DCM, 31%; (t) Pd(OH)₂, AcOH, H₂ (10 atm), 48%.

Scheme 2

Synthesis of the Lipid II unit 16 (undecaprenyl phosphate, highlighted in blue) reported by Wang [57]. (a) HAlaOTMSE, DIEA, HOBt, PyBOP, 1:1 THF:DCM, r.t. (room temperature), 2 h, 81%. (b) Catalyst TsOH, MeOH, 75 °C, 30 min, 89%. (c) Pyridine, 1 equiv. of AcCl, −30 °C, 15 min, then warm to r.t., 80%. (d) AgOTf (3 equiv.), donor (2 equiv.), MS 4 Å, DCM, −40 °C, drybox, 3 h, then overnight, 60%. (e) Amino-modified resin, MS 4 Å, butanol, 85 °C, 24 h, then pyridine, Ac₂O, r.t., overnight, 53%. (f) 10%Pd/C, H₂, MeOH, 1 h, 93%. (g) Tetrazole, (BnO)₂PN(i-Pr)₂, DCM, −30 °C to r.t., 1 h, then m-CPBA, −40 °C to r.t., 1 h, 55%. (h) TBAF, THF, r.t., 45 min, 87%. (i) H-γ-d-Glu(OTMSE)-Lys(TEOC)-d-Ala-d-AlaOTMSE, DIEA, HOBt, PyBOP, 1:1 THF:DCM, r.t., 2 h, 61%. (j) 10%Pd/C, H₂, MeOH, 1 h, 100%. (k) CDI, dry DMF, r.t., 4 h, then MeOH, then undecaprenyl phosphate, r.t., 48 h, 39%. (l) TBAF, DMF, r.t., 24 h, then 3% NaOMe, MeOH, 0 °C, 1 h, 35%.

Scheme 3

Synthesis of the Lipid II unit 16 reported by Blaszczak [58,59]. (a) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, DCM, 2 h, 95%; (b) Et₃SiH (3 equiv.), TFA (6 equiv.), DCM, 0 °C, 5 h, 61%; (c) AgOTf (3 equiv.), donor (3 equiv.), MS 4 Å (5.5 wt equiv.), DCM, 25 °C, 20 h 84%; (d) (1) anhydrous ZnCl₂,Ac₂O/AcOH (2:1), 25 °C, 20 h; (2) Zn dust (20 reducing equiv.), 25 °C, 4 h, 67%. (e) H₂, Pd/C, MeOH/THF, 94% yield; (f) (1) dibenzyl-N,N-diethylphosphoramidite, 1H-tetrazole, DCM; (2) 30% H₂O₂, THF, −78 °C to room temperature, 78% yield; (g) (1) DBU, DCM; (2) EDCI, NHS, DMF, then peptide and iPr₂NEt, 46% yield; (h) H₂, Pd/C, MeOH, then pyridine, 91% yield; (i) (1) CDI/DMF/THF; (2) undecaprenyl mono-phosphate (bis-amonium salt); (j) NaOH/H₂O/1,4-dioxane, 24% overall yield from 10.

Scheme 4

Synthesis of the disaccharides 26 and 27—precursors of NAM-NAG and NAG-NAM reported by Boons [60]. (a) TBDMSOTf, 2,6-lutidine, DCM, 96%; (b) PMBCl, NaH, TBAI, THF, reflux, 84%; (c) Me₃N·BH₃, AlCl₃, THF, 72%; (d) HO(CH₂)₃N₃, NIS, TMSOTf, 67%; (e) NIS, TMSOTf, 62%; (f) H₂N(CH₂)₂NH₂, EtOH then Ac₂O, MeOH, 70%; (g) Bu₄NF, THF, 75%; (h) NaH, (S)-2-chloropropionic acid, dioxane, 14 (76%) and 15 (66%); (i) DDQ, DCM, H₂O, 79%.

Scheme 5

Synthesis of the tetrasaccharide with pentapeptides attached to NAM units, 33, reported by Mobashery [61]. (a) TfOH, 71%. (b) (1) NaOMe, Amberlite IR-120 (H+), (2) benzaldehyde dimethylacetal, p-TsOH, 60% in two steps; (c) Ac₂O, 85%; (d) n-Bu₄NF, 75%; (e) Cl₃CCN, DBU; (f) acceptor A, TfOH, 55% in two steps; (g) BH₃.NMe₃, BF₃.OEt₂, 62%; (h) donor B, TfOH, 68%; (i) (1) NaOH, dioxane/water (4:1); HCl, pH 3.0; (2) Ac₂O, 48% in two steps; (j) NaOMe, 74%; (k) NaH, (S)-2-chloropropionic acid, 46%; (m) 4-nitrophenyl trifluoroacetate, pyridine; peptide (prepared as CF₃CO₂H salt), 41%; (l) 60% AcOH; H₂, Pd/C, 76%.

Scheme 6

Synthesis of the heptaprenyl—Lipid IV 43b reported by Walker [63]. (a) Bu₂SnO, toluene, reflux, then Bu₄NI, BnBr, reflux, 51%; (b) TBSOTf, 2,6-lutidine, CH₂Cl₂, 91%; (c) mCPBA, CH₂Cl₂, −78 °C to −60 °C, 93%; (d) (1) (Bu₃Sn)₂O, MeOH, reflux; (2) Bu₄NI, BnBr, toluene, 91 °C, 75%; (e) mCPBA, CH₂Cl₂, −78 °C to −60 °C, 91%; (f) Tf₂O, DTBMP, ADMB, MS 4Å, CH₂Cl₂, −60 °C to −40 °C, 58%; (g) Tf₂O, DTBMP, ADMB, MS 4Å, CH₂Cl₂, −60 °C to −30 °C, 75%; (h) mCPBA, CH₂Cl₂, −78 °C to −60 °C, 86%; (i) Tf₂O, DTBMP, ADMB, MS 4Å, CH₂Cl₂, −40 °C, 77%; (j) (NH₂CH₂)₂, THF/CH₃CN/EtOH (1:2:1), 60 °C; (k) Ac₂O, MeOH/H₂O (5:1), r.t., 75% two steps; (l) NaH, S-(−)-2-bromo-propionic acid, THF, 0 °C to r.t.; (m) TMSCHN₂, benzene/MeOH (3:1), 0 °C, 70% two steps; (n) NIS, CH₃CN/H₂O (5:1), r.t., 75%; o) 1H-tetrazole, (i-Pr)₂NP(OBn)₂, CH₂Cl₂, −40 to −20 °C; (p) mCPBA, CH₂Cl₂, −40 °C to r.t., 84% two steps; (q) TBAF, THF, 0 °C to r.t.; (r) 1.3 M KOH, THF/H₂O (10:1), r.t., 64% two steps; (s) HATU, DIEA, DMF, r.t., 60%; (t) Pd(OH)₂/C, H₂, 44%; (u) ammonium heptaprenyl phosphate, 1,1′-carbonyl diimidazole, THF, r.t., then 30, SnCl₂, DMF, r.t., 50%; (v) TBAF, DMF, r.t., 69%; (w) (CH₃[14]CO)₂O, toluene/16 mM NaOH in MeOH (1:1), sonication, 37 °C, 50%.

Scheme 7

Synthesis of cross-linked PGN fragment 46 reported by Fukase [65]. (a) LiOH, THF/1,4-dioxane/H₂O = 4/2/1, quantitative; (b) TFA; (c) HCl.Et₂O; (d) Peptide, HATU, Et₃N, DMF/DMSO (1:1), 75%, three steps; (e) 10% TFA/DCM; (f) 20% piperidine/DMSO; (g) H₂, Pd(OH)₂, AcOH.

Scheme 8

Synthesis of the PGN fragments 54 reported by Fukase [67]. (a) TMSOTf, CH₂Cl₂, −15 °C, MS 4Å, 20 min, 84%; (b) Me₃N BH₃/BF₃·Et₂O, ACN, 1 h, 83%; (c) [Ir(cod)H-(MePh₂P)₂]PF₆, THF, 1.5 h; (d) I₂/H₂O, 30 min, 81%; (e) CCl₃CN/Cs₂CO₃, DCM, 30 min, quantitative; (f) TMSOTf, DCM, MS 4Å, 40 min, 16%; (g) Zn/Cu, AcOH,3 h, Ac₂O/Py, 2 h, 62%; (h) LiOH, quantitative. For the condensation: (i) WSCD/HOBt/DMF/TEA, HCl·l-Ala-d-isoGln(OBn) or HCl·L-Ala-d-isoGln-l-Lys(Z)(OBn); (j) HATU/DMF/TEA, HCl·l-Ala-d-isoGln-l-Lys(Z)-d-Ala(OBn) or HCl·l-Ala-d-isoGln-l-Lys(Z)-d-Ala-d-Ala(OBn); (k) H₂/Pd(OH)₂, AcOH.

Scheme 9

Synthesis of the PGN fragment 62 reported by Marques [70]. (a) (1) PhCHO, ZnCl₂, MS 3Å, r.t., overnight; (2) Ac₂O, pyridine, r.t., overnight 78%; (b) morpholine, EtOAc, r.t., overnight, 87%; (c) CCl₃CN, Cs₂CO₃, CH₂Cl₂, r.t., 2 h, 74%; (d) BnOH, AcCl, 80 °C, 3 h, 61%; (e) PhCHO, ZnCl₂, overnight, 72%; (f) ethyl l-(S)-2- trifluoromethanesulphonyloxy-propionate, NaH, CH₂Cl₂, r.t., 3 h, 68%; (g) Pd(PPh₃)₄, AcOH, TrocCl, r.t., 82%; (h) BH₃Me₂N, BF₃OEt₂, r.t., 3 h, 72%; (i) TMSOTf, DCM, MS 3 Å, -15 °C, 52%; (j) Zn-Cu, THF: AcOH: Ac₂O (1:1:1), then Ac₂O/pyridine, 63%; (l) LiOH, THF:1,4-dioxane:H₂O, 69%; (m) Pd(OH)₂, H₂, AcOH, r.t., quantitative; (n) peptide coupling via Fmoc-solid-phase synthesis; (o) TFA (1% CH₂Cl₂), r.t.

Scheme 10

Reported multistep synthesis of NAG-NAM intermediates via orthogonal routes from glucosamine.

Scheme 11

Synthesis of the NAG-NAM precursor 66 from peracetylated chitobiose 63 reported by Marques group [79]. (a) Ac₂O, H₂SO₄, 55 °C, 3 h, then r.t., 16 h, and 55 °C, 1 h, 13%; (b) TFAA, Pyr, 120 °C, 15 min, 76%; (c) TolSH, BF₃OEt₂, DCM, r.t., overnight, 80%; (d) (1) MeONa, MeOH, r.t., overnight. (2) butane-2,3-dione, p-TsOH, triethylorthoformate, EtOH, 60 °C, 16 h, 58%; (e) TBDMSCl, DMAP, TEA, CH₂Cl₂, 56%; (f) (1) NaOH 2 M, THF, 80 °C, 3 h, (2) Ac₂O, pyridine, CH₂Cl₂, r.t., overnight, 56%; (g) (1) NaH, DCM, 30 min; (2) Ethyl (S)-2-(trifluoromethylsulfonyloxy)propionate, r.t., 16 h, 38%.

Scheme 12

Synthesis of NAG-NAM oligosaccharides (69) by Marques group [83]. (a) Phthalic anhydride, DMF 5% H₂O, 120 °C, overnight; (b) C1 (m = 1) or C2 (m = 2), CDI, DMF, r.t., 24 h; (c) TBDMSCl, imidazole, DMF, N₂, r.t., 72 h; (d) (S)-2-chloropropionic acid, NaH, DMF, r.t., 48 h; (e) (1) NH₂NH₂, H₂O, reflux, 24 h; (2) Ac₂O, pyridine, r.t., overnight; (f) TBAF, pyridine, r.t., overnight.