A Lead (II) 3D Coordination Polymer Based on a Marine Cyclic Peptide Motif

The crystal structure of a naturally occurring cyclic tetrapeptide cyclo(Gly-l-Ser-l-Pro-l-Glu) [cyclo(GSPE)] was obtained. The conformation of synthesized cyclo(GSPE) fixes the coordination to lead ion in a 1:1 ratio. This cyclo(GSPE)-Pb complex was constructed as an asymmetric 3D network in the crystalline state. The polymerization of a heavy metal ion with a rigid asymmetric cyclic tetrapeptide represents the first example of a new class of macrocyclic complexes.


Introduction
In the area of biomedicinal chemistry much interest has been generated in the design of metalorganic frameworks (MOFs) [1,2]. Due to their chemical variety, biocompatibility and ability to assemble spontaneously, peptides can form excellent simple nanotubes [3][4][5]. Some metal-peptide frameworks (MPFs) [6] have been reported, but very few of them have been characterized by crystallography [6,7]. The size and conformation of cyclic peptides [8,9] and their metal binding side chains provide a novel tool for construction of discrete metal-assembled supermolecules with unique chemical and physical properties. Cyclic tetrapeptides (CTPs) [10][11][12][13][14][15] also possess a definite inner nanocavity of a constrained nature and hence, generated highly entropic advantages in molecular recognition with the potential to confer chirality and biological activity on these compounds [16][17][18].

OPEN ACCESS
Compared with the d-and f-block metals, Pb 2+ ion has a larger radius, variable coordination numbers and unique prospects for the construction of novel polymeric or multi-dimensional supramolecular networks [19][20][21][22][23][24][25]. The chemistry of lead has drawn a lot of interest for its wide applications in fields such as fuel additives, batteries, oil refining and paint manufacturing, but also in biological systems, where Pb metal has been found to interact with many amino acids, peptides and proteins: these binding preferences of Pb(II) have provided the inspiration for the design of a selective chelating therapy agent [26][27][28][29][30][31].
Cyclo(Gly-L-Ser-L-Pro-L-Glu) [cyclo(GSPE), 1] has metal binding sites on the side chains (serine hydroxyl and glutamic carboxylate group) and the carbonyl groups. It was first isolated from the Ruegeria strain of marine bacteria [32] and was found to possess moderate antibacterial activity against Bacillus subtilis. Its structure was elucidated on the basis of 1D and 2D NMR data, followed by the determination of absolute configuration [33]. However, a crystal structure has not been reported. Due to strain of the twelve member ring, binding to metal ions by carbonyls is more difficult than by side chains. Keeping in mind the combination of hydroxyl and carboxylato groups and from a study of natural CTPs-metal complexes, cyclo(GSPE) 1 was selected as a linking ligand to investigate the coordination chemistry of Pb(II) ion. Here the crystal structure of cyclo(GSPE) and {[cyclo(Gly-L-Ser-L-Pro-L-Glu)Pb(NO 3 )]2H 2 O} n 2 are reported for the first time. Further {[cyclo(Gly-L-Ser-L-Pro-L-Glu)Pb(NO 3 )]2H 2 O} n 2 was constructed as an asymmetric 3D network in the crystalline state. Coordination relied on the prearranged conformation of synthesized cyclo(GSPE) 1. The polymerization of a heavy metal ion with a rigid asymmetric cyclic tetrapeptide represents the first example of a new class of complexes.

Synthesis of cyclo(GSPE) 1 and {[cyclo(Gly-L-Ser-L-Pro-L-Glu)Pb(NO 3 )]2H 2 O} n 2
Cyclo(GSPE) 1 was synthesized by using standard solution-phase peptide coupling protocols. Its spectral data were identical with those of natural 1 [32,33]. Crystals were prepared for the first time by slow cooling to room temperature in water. The synthesized peptide 1 was subjected to complexation with Pb(II). By heating an aqueous solution of the peptide 1 and the lead(II) salt in a 1:1 ratio, the {[cyclo(Gly-L-Ser-L-Pro-L-Glu)Pb(NO 3 )]2H 2 O} n 2 complex was obtained. Crystals were prepared by slow cooling to room temperature and resulted in a 3D coordination polymer. Several coordination polymers of lead with a variety of synthetic organic ligands have been reported earlier [34][35][36][37][38]. The structure of macrocyclic complex 2 is the first solved marine cyclo(GSPE) 1 lead complex.   Figures S4-S7). All the assignments showed good agreement between the observed and calculated isotopic distributions.

Characterization of {[cyclo(Gly
IR Spectra. The IR spectrum of macrocycle 1 showed a characteristic ν(C=O) absorption band for the -COOH group at 1,718 cm −1 , which vanishes in the case of metal complex 2. For the free peptide the ν as (COO) and ν s (COO) stretching vibrations of the carboxylate group appear at 1,545 and 1,444 cm −1 respectively, while for the lead complex 2, the first absorption region became broad and the second was obscured by the nitrate ion absorption, which displays a quite intense band at 1,384 cm −1 ( Figures S8 and S9). The backbone of the cyclic peptide adopts a two-fold symmetric conformation which is very common for cyclic tetrapeptides containing alternate 'cis' and 'trans' peptide units and the two side chains (glutamic and serine) are away from the ring. The glutamic residue assumes the gauche − conformation to the main-chain amino group (χ 1 = −54.6°). In the prolyl residue, C α , C β , C δ and N form an approximate plane with C γ deviating from the plane by 0.56 Å towards the side of the carbonyl group. The torsion angles that define the conformation of the molecule are listed in Table 2. The conformation of the cyclic tetrapeptide 1 is illustrated in the Figure 1 (top, left). The overall shape of the backbone is a distorted boat with the cis amide bonds at the two ends which could facilitate ion binding.

Crystal Structures
Each discrete molecule of cyclo(GSPE) 1 is strongly hydrogen bonded to a neighboring molecule through the amide N-H of glutamic acid moiety to a carboxyl O-atom of proline (d N-HO 2.066 Å and θ N-HO 176.6°) moiety and amide N-H of serine to a carboxyl O-atom of a glycine moiety (d N-HO 1.997 Å and θ N-HO 145.3°), to form a 1D-chain along the a-axis. An oriented CTP nanotube (yellow) is thus assembled. The structure of this nanotube is expanded into a three-dimensional network (5.4 × 2.8 Å 2 ), which is devoid of water molecules (Figure 1, top and right).
The carboxylic acid group of glutamic acid and the hydroxyl group of serine are flanked in opposite directions and are hydrogen bonded to a neighboring 1D-chain to form an overall 3D-network.

Crystal Structure of Complex 2
A single-crystal X-ray diffraction study reveals that complex 2 is a 3D-coordinated polymer, crystallized in the monoclinic system with P 2(1) space group. Crystallographic data of 2 are listed in Table 1. As shown in Figure 2, the asymmetric unit of 2 is composed of one Pb(II) center, one deprotonated cyclo(GSPE) ligand, one nitrate molecule and two lattice water molecules. The Pb(II) center is in a seven coordination environment ( Figure 2) with five oxygen atoms [O1(proline carbonyl), O3(glutamic carbonyl), O5(serine hydroxyl) O6 and O7(glutamic carboxylate)] of three distinct cyclo(GSPE) 1 ligands and two μ 1,1 -oxygen atoms (O8) of nitrate molecules from two EO bridges (joining it to a neighboring Pb atom). The torsion angles that define the conformation of the complex 2 are listed in Table 3.  (15) The geometry around the Pb center appears holodirected [39]. The bidentate carboxylate oxygen atoms (O6 and O7) provided the asymmetric chelation (Pb-O 2.463(6) and 2.643 (6) Å). The Pb-O bond distances (Table 3) are in the range of 2.463(6)-2.833(5) Å and the O-Pb-O bond angles vary from 50.55(17)-145.03 (18)° and are comparable to those reported earlier for lead-oxygen donor complexes [24,[40][41][42]. 1D linear chain around the Pb center is formed via carbonyl and hydroxyl oxygen atoms of two distinct cyclo(GSPE) ligands along [001] direction ( Figure 3). Three molecules of cyclo(GSPE) 1 binds with three Pb(II) ions to form one cyclic ring and the same unit repeats along b-axis to form a 2D layer structure (Figure 4a,b). When viewed along the [100] direction, these sheets are further cross-linked by NO 3 − molecules giving rise to a three-dimensional structure. The size and the shape of the CTP nanotube remains unchanged ( Figure 5).   Out of three nitrate anion, one nitrate anion lying inside the cage is further coordinated to a water molecule and the remaining two nitrate anions lying inside two neighboring cages. For clarity, each layer is differentiated from neighboring layer by different color.

Discussion
There is no report on the solid state structure of a natural (12-membered) cyclic tetrapeptide with a metal ion. The molecular structure of the cyclo(GSPE)-Pb complex 2 is unique as all the complexes reported to date are with higher cyclic peptides or β-cyclic tetrapeptides and are sandwich complexes [43][44][45]. Structure 2 showed that conformation of 1 remains cis-trans-cis-trans and only the side chains and ring carbonyls are pointing towards the Pb metal for binding and, hence, represent an example of a metal complex bound with both a cyclic skeleton and functional groups in side chains. Unlike linear peptides, the retention of conformation of 1 is attributed to the highly constrained nature of CTPs. The rigidity of cyclopeptide backbones ensures the formation of nanotubes [46]. The inability to adjust their conformation makes CTP-metal complexes rare. However, once it formed, the resulting CTP-metal complexes 2 will preserve the nanotubes.

Materials and Physical Measurements
All reagents were commercially available (Aldrich, Saint Louis, MO, USA or Merck, Darmstadt, Germany) and used as supplied. Solvents were dried by standard procedures. The NMR spectra were recorded on a Bruker DRX 400 ( 1 H at 400.13 MHz, 13 C at 100.03 MHz) spectrometer (Bruker Daltonik, Bremen, Germany). MALDI TOF was performed on a Bruker Autoflex MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). High-resolution electrospray ionization mass spectrometry (ESI-MS) was performed on a Shimadzu-LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Elemental analysis for C, H, N was performed on VarioEL-III elementar analyzer (Elementar, Hanau, Germany). Infrared spectra were recorded on a PerkinElmer Spectrum one FT-IR spectrometer (PerkinElmer, Shelton, CT, USA) using KBr pellets (4,000-400 cm −1 ).

Sythesis
Cyclo(Gly-L-Ser-L-Pro-L-Glu) (1). Linear peptide Boc-GS(OBn)PE(OBn) 2 was synthesized by using standard solution-phase peptide coupling protocols starting from Fmoc-L-proline. Subsequently, regioselective enzymatic hydrolysis of the α-benzyl ester on glutamate was achieved [47]. The linear precursor was then activated with pentafluorophenol and cyclized with pyridine to form dibenzyl protected CTP [48].  (2). Pb(NO 3 ) 2 (4.5 mg, 13.5 μmol) was dissolved in water (1 mL). cyclo(GSPE) 1 (5.0 mg, 13.5 μmol) was dissolved in water (1 mL) and added to the metal solution. The pH of solution was adjuted to 5 with diluted HNO 3 solution. The reaction mixture was stirred at RT for 10 minutes and then at 70 °C for 4 days. Crystals suitable for X-ray diffraction were obtained from saturated solution by slow cooling. Anal. Calc. for C 15

X-ray Crystallography
Colorless prism-like crystals of 1 and rod-like crystals of 2 were obtained by slow solvent evaporation at room temperature, respectively. Crystals suitable for X-ray diffraction analysis were selected with size of 0.13 × 0.08 × 0.05 mm for 1 and 0.44 × 0.09 × 0.05 mm for 2. Crystals were mounted on a glass fiber and used for data collection. For both compounds 1 and 2 diffraction data were preliminarily collected with a Bruker APEX-II CCD diffractometer using graphite monochromated MoKα radiation (λ = 0.71073 Å). Absorption corrections for the area detector were performed with the program SADABS. Structures were solved by direct methods and were refined against the least-squares methods on F2 with the SHELXL-97 package, incorporated in SHELXTL/PC V5. Anisotropic thermal factors were used only for all non-hydrogen atoms.

Conclusions
We have developed the synthesis of a CTP cyclo(GSPE) 1. The synthetic building blocks were coupled through hydroxyl, carboxylate and two amide bonds to lead ion and thus created watersoluble, coordination polymers (tris-CTPs) with well-defined three-dimensional structures. This is the first report of a naturally occurring CTP as the host for metal complexation. The CTP retains its conformation through the binding process with metals. The unique crystal structure of this rigid molecule suggests the formation of a designated complex with nanotube properties.