Selected bond distances and bond and torsion angles for compound 5 “(Garcia et al., 2009)”.
\r\n\t
\r\n\tAlso, the book deals with the motions and path of charged particles in an electromagnetic field and in a field gradient. Some numerical and unsolved problems are discussed as well.
\r\n\t
\r\n\tFurther discussion is based on the linearized theory for plasma for single and two-fluid species, the dispersion relations for cold plasma, electron plasma wave in warm plasma and ion-acoustic wave. Electromagnetic waves in cold plasma and electrostatic electron oscillation perpendicular to the applied magnetic field are given. Experimental techniques used in plasma physics and diagnostic methods are discussed, as well. The growth rate of plasma fluid stability is calculated for different types of plasma instabilities and conditions.
\r\n\t
\r\n\tThe methods of heating and confinements of plasma will be also used as a topic.
X-ray crystallography is an important method for determination of the arrangement of atoms within a crystal of compound in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From electron density, in the molecule the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds and various other information.
The hexamethylenetetramine as an organic compound was solved in 1923 “(Dickinson & Raymond, 1923)”. Several studies of long-chain fatty acids were followed which are an important component of biological membranes “(Bragg, 1925; de Broglie & Trillat, 1925; Caspari, 1928; Müller, 1923, 1928, 1929; Piper, 1929; Saville & Shearer, 1925; Trillat, 1926)”. In the 1930s, the structures of larger molecules with two-dimensional complexity began to be solved. An important advance was the structure of phthalocyanine “(Robertson, 1936)”, that is closely related to porphyrin molecules important in biology, such as heme, corrin, chlorophyll and etc.
In this chapter, crystal structures of some organic compounds such as organic torsion helicoids, organic compounds consists of intra- and intermolecular hydrogen bond and their some metal complexes and crystal structures of some organic spiro compounds were described.
In recent years, several different interesting organic compounds structures have been found by X-ray crystallography. Helicenes are an extremely attractive and interesting class of conjugated molecules currently investigated for optoelectronic applications “(Groen et al., 1971; Katz, 2000; Rajca et al., 2007; Schmuck, 2003; Urbano, 2003)”. They combine the electronic properties afforded by their conjugated system with the chiroptical properties “(Bossi et al., 2009; Collins & Vachon, 2006; Larsen et al., 1996)” afforded by their interesting and peculiar helix-like structure, resulting from the condensation of aromatic (and/or heteroaromatic) rings, all of them in ortho position. For example, the formula and crystal structures of tetrathia-[7]-helicene 1 are shown in Figures 1 and 2, respectively. The compound 1 has been synthesized in three step by starting material of benzo[1,2-b:4,3-b\']dithiophene and is shown in Scheme 1 “(Maiorana et al., 2003)” and this compound showed second-order non-linear optical (NLO) properties and has been investigated (Clays et al., 2003). In particular, carbohelicenes only include benzene rings, and also in heterohelicenes one or more aromatic rings are heterocyclic (pyridine, thiophene, pyrrole and etc.) “(Miyasaka et al., 2005; Rajca et al., 2004)”. With increasing number of condensed rings (typically, n > 4), the steric interference of the terminal rings forces the molecule to be a helicoidal form. For n > 4 the energetic barrier is such that the two enantiomers can be separated and stored “(Martin, 1974; Newman, et al., 1955, 1967; Newman & Lednicer, 1956; Newman & Chen, 1972)”. Of course, the conjugation of π system decreases with decreasing of planarity; however, in longer helicenes π-stack interactions can also take place between overlapping rings “(Caronna et al., 2001; Liberko et al., 1993)”. All helicenes (generally, n > 4) are chiral molecules and exhibit huge specific optical rotations “(Nuckolls et al., 1996, 1998)” since the chromophore itself, in this case the entire aromatic molecule, is inherently dissymmetric (right-hand or left-hand helix), having a twofold symmetry axis, C2, perpendicular to its cylindrical helix (in carbohelicenes), or inherently asymmetric (in heterohelicenes) “(Wynberg, 1971)”.
Formula structures of 1 and 2.
The helicoid structures of unsubstituted tetrathia-[7]-helicene 1 and unsubstituted hexathia-[11]-helicene 3 “(Caronna et al., 2001)” with the labelling scheme adopted for structural discussion “(Bossi et al., 2009)”.
The synthesis of 1 from benzo[1,2-b:4,3-b\']dithiophene as a starting material.
Reaction mechanism for formation of 5 “Garcia et al., 2009)”.
Tetrathia-[7]-helicene 1 have been used for the synthesis of organometallic complexes “(Garcia et al., 2009)”. A series of organometallic complexes possessing tetrathia-[7]-helicene nitrile derivative ligands 5 as chromophores, has been synthesized and fully characterized by Garcia et al. “(Garcia et al., 2009)”. This compound was analyzed by means of 1H NMR, FT-IR, UV–Vis and X-ray crystallography techniques. The spectroscopic data of this compound was shown with in order to evaluate the existence of electronic delocalization from the metal centre to the coordinated ligand to have some insight on the potentiality of this compound as non-linear optical molecular materials. Slow crystallization of compound 4 revealed an interesting isomerization of the helical ligand with formation of two carbon-carbon bonds between the two terminal thiophenes, leading to the total closure of the helix 5. The reaction mechanism for the formation of 5 is shown in Scheme 2. Crystal structure of 5 is shown in Figure 3. A selected bond length, angles and torsion angles for compound 5 is summarized in Table 1 “Garcia et al., 2009)”.
Another example about helicenes is the hexahelicene 2 and its derivatives that is a chiral molecule “(Noroozi Pesyan, 2006; Smith & March, 2001)”. A convenient route for the synthesis of [7]-helicene (6a) and [7]-bromohelicene (6b) is reported “(Liu et al., 1991)”. The crystal structure of 6b is shown in Fig. 4. The crystal structure of 6b and its unusual oxidation reaction product 7 (as a major product) has been reported “(Fuchter et al., 2012)” (Figure 4 and Scheme 3). Alternatively, compound 6 may be an option for a neutral helicene-derived metallocene complex, since the seven-membered benzenoid rings give rise to a scaffold that completes one full turn of the helix with the two terminal rings being co-facial. It has been theoretically predicted and reported that the 6 has potential to bind some metal cation such as Cr, Mo, W, and Pt in a sandwich model “(Johansson & Patzschke, 2009)”. Fuchter and co-workers “(Fuchter et al., 2012)” also reported the crystal structure of 7 that obtained via unusual oxidation rearrangement of 6. In this structure, The bonds within the pyrenyl unit range between 1.3726(19) and 1.4388(14) Å with the exception of one outlier at 1.3512(18) Å for the C(26)–C(27) bond. The C=C double bonds in rings D and E are 1.3603(15) and 1.3417(16) Å respectively, and the C=O bond is 1.2419(14) Å. The structure of 7 revealed the dominant canonical form to have a pyrenyl group consisting of rings A, B, C and H linked by single bonds to a C–C=C–C=C–C=O unit to form rings D and E (Scheme 3). The pyrenyl unit is flat, the sixteen carbon atoms being coplanar. Ring I has four single bonds and two aromatic bonds, and has a folded conformation with the methylene carbon lying ca. 0.87Å out of the plane of the other five carbons which are coplanar. Aryl ring G forming the five-membered ring J, links to ring I. The planes of the five coplanar atoms of ring I and the four coplanar atoms of ring J are inclined by ca. 108° to each other. The ring of E is slightly distorted in a boat-like fashion with the carbon shared just with ring D and that shared with rings I and J, out of the plane of the other four atoms which are coplanar to within ca. 0.01 Å.
The formula structure of Katz\'s helical ferrocene 8 is shown in Figure 5 “(Katz & Pesti, 1982; Sudhakar & Katz, 1986)”.
Crystal structure of 5.
Bond distances (Å) | |||
Ru(1)–N(1) | 2.030(5) | P(2)–C(221) | 1.850(7) |
Ru(1)–Cpa | 1.8595(6) | P(2)–C(231) | 1.829(6) |
Ru(1)–P(1) | 2.350(2) | N(1)–C(1) | 1.147(8) |
Ru(1)–P(2) | 2.353(2) | C(1)–C(2) | 1.433(9) |
P(1)–C(111) | 1.843(6) | C(2)–C(3) | 1.598(9) |
P(1)–C(121) | 1.831(7) | C(3)–C(4) | 1.535(9) |
P(1)–C(131) | 1.817(7) | C(4)–C(23) | 1.519(9) |
P(2)–C(211) | 1.846(7) | C(23)–C(2) | 1.565(9) |
Bond angles (°) | |||
N(1)–Ru(1)–Cpa | 121.79(14) | C(1)–C(2)–C(3) | 115.3(6) |
N(1)–Ru(1)–P(1) | 87.93(14) | C(2)–C(3)–C(4) | 88.8(5) |
N(1)–Ru(1)–P(2) | 90.83(15) | C(2)–C(23)–C(4) | 90.6(5) |
P(1)–Ru(1)–P(2) | 99.84(6) | C(3)–C(4)–C(23) | 91.2(5) |
P(1)–Ru(1)–Cpa | 124.15(5) | C(3)–C(2)–C(23) | 87.2(5) |
P(2)–Ru(1)–Cpa | 122.98(5) | C(5)–C(4)–C(23) | 106.9(6) |
Ru(1)–N(1)-C(1) | 169.9(5) | C(4)–C(23)–C(22) | 107.7(5) |
N(1)–C(1)–C(2) | 175.7(7) | C(2)–C(3)-S(1) | 119.3(5) |
C(1)–C(2)–C(23) | 116.4(6) | C(3)–C(2)–S(4) | 116.5(5) |
Torsion angles (°) | |||
Ru(1)–N(1)–C(1)–C(2) | 29(11) | N(1)-C(1)–C(2)–C(23) | 62(9) |
N(1)–C(1)–C(2)–C(3) | -38(9) | C(2)–C(3)–C(4)–C(23) | -11.0(5) |
N(1)–C(1)–C(2)–S(4) | -175(9) |
Selected bond distances and bond and torsion angles for compound 5 “(Garcia et al., 2009)”.
The formula structures of 6a and 6b and its unusual reaction for synthesis of 7a (and also its structure).
Crystal structures of 6b and 7a.
The formula structure of Katz\'s helical ferrocene 8.
Two possible different torsion helicoids of 9.
Two independent molecules of 9 in the crystal studied.
Diazepinone dervatives are of pharmaceutical compounds. Another interesting helical diazepinone compound that is discussed in this section, is 1,9-dimethyl-4,5-dihydro-6H-pyrido[3\',2\':4,5]thieno[2,3-f]pyrrolo[1,2-a][1,4]diazepin-6-one (9). This molecule show two crystallographically independent molecules that form the asymmetric unit of the structure are shown in Figure 6. The X-ray crystallographic analysis shows the molecular structure of the compound 9 and reveals an interesting fact that this structure features two stereochemically different molecules (9A and 9B) that can be understood as different torsion helicoids (Figure 6). The compound has two stereoisomers (R and S conformers). In each structure the seven-membered diazepinone ring exhibits a boat conformation. The fused pyrido[3\',2\':4,5]thieno ring moiety has planar geometry. The C3–H3 bond is slightly off the fused pyrido[3\',2\':4,5]thieno ring plane. The hindrance repulsion between the hydrogen atom at C3 on pyridine ring and methyl group on pyrrole ring makes the molecule of 9 essentially non-planar (repulsion of C3–H3A with C15 and C3\'–H3\'B with C15\' of methyl groups) (Scheme 4). The torsion angles between the pyrrole and thiophene rings in 9A and 9B are 45.7(6)° and –49.3(6)°, respectively “(Noroozi Pesyan, 2010)”.
The –NH– group of each molecule (e.g. molecule 9A) makes an intermolecular hydrogen bond to the C=O functional group of the molecule of another kind (molecule 9B), and vice versa. For example, the intermolecular hydrogen bond N3–H3····O1\' involves the N3 atom from molecule 9A and O1\' atom from the carbonyl group of molecule 9B, and vice\n\t\t\t\t\tversa for N3\'–H3\'····O1 (Figure 7). The crystal packing diagram indicates zigzag hydrogen-bonded chains along the crystallographic axes with two distinct hydrogen bonds (Figure 7). The intermolecular hydrogen bonds play a principal and important role in the crystal packing diagram of 9 “(Noroozi Pesyan, 2010)”.
Crystal packing diagram of 9 showing zigzag H-bonds (shown by dashed lines).
One of the most interesting helical primary structure is sown in Figure 8 has been reported by Fitjer et al. “(Fitjer et al., 2003)”. Helical primary structures of spiro annelated rings are unknown in nature but have been artificially produced, both in racemic and enantiomerically pure form. The formula structure of 1-cyclobutylidenespiro[3.3]heptane (10) as a starting material is shown in Scheme 5. The compound 10 yielded enantiomeric mixture of 11 and 12 in the presence of zinc and 2,2,2-trichloroacetyl chloride. Reductive dehalogenation of 11 and 12 then Wolff–Kishner reduction yielded the desired trispiro[3.0.0.3.2.2]tridecane [rac-(15), (symmetry, C2)]. The crystal structure of the camphanic acid derivative of 15 ((1S,5\'S,10\'S)-16) is shown in Figure 8 “(Fitjer et al., 2003)”.
Synthesis of the compounds trispiro[3.0.0.3.2.2]tridecane (15) and the formula structure of its derivative (1S,5\'S,10\'S)-16 “(Fitjer et al., 2003)”.
Crystal structure of (1S,5\'S,10\'S)-16.
Helquats, the family of N-heteroaromatic cations “(Arai & Hida, 1992)”, recently were introduced helical dications that represent a missing structural link between helicenes and viologens“(Casado et al., 2008)”. Specifically, basic [7]-helquat (17) “(Severa et al., 2010)” is a structural hybrid between [7]-helicene and a well-known herbicide diquat (Scheme 6). Synthesis of [7]-helquat (17) starts with bisquaternization of bis-isoquinoline precursor (18) with an excess of 3-butynyltriflate followed by the key metal catalyzed [2+2+2] cycloisomerization of the resulting triyne, formed 17 (Scheme 7).
Structural relation of [7]-helquat (17) to [7]-helicene 6a and herbicide diquat.
Synthesis of 17 via one-pot bis-quaternization of 18.
Recently, Nakano et al. have been reported the helical structure, λ5-phospha [7]-helicenes 9-phenyl-9H-naphtho[1,2-e]phenanthro[3,4-b]phosphindole-9-oxide (21) and its thio analogue 9-phenyl-9H-naphtho[1,2-e]phenanthro[3,4-b]phosphindole-9-sulfide (22) “(Nakano et al., 2012)”. The formula structure of 21 and 22 and the crystal structure of 21 are shown in Fig. 10. Phospha [7]-helicenes 21 and 22 have more distorted structures than the other heterohelicenes. In the structure of 21, the sums of the five dihedral angles that are derived from the seven C–C bonds [C(17)-C(17a)-C(17b)-C(17c), C(17a)-C(17b)-C(17c)-C-(17d), C(17b)-C(17c)-C(17d)-C(17e), C(17c)-C(17d)-C(17e)-C(17f), and C(17d)-C(17e)-C(17f)-C(1)] are 95.28 for 21 and 99.68 for 22. These angles are larger than those of hetero[7]-helicenes 23–25 (79–88°). This case can be attributed to the large angles between the two double bonds of phosphole oxide (50°) and phosphole sulfide (50°) relative to furan (32°), pyrrole (35°), and thiophene (45°). Owing to the larger angle, a larger overlap of the two terminal benzene rings was occurred in the λ5-phospha[7]-helicenes, therefore, a stronger steric repulsion. These larger distortions in 21 and 22 explain the higher tolerance of 21 and 22 towards racemization.
Formula and X-ray single crystal structure of compounds 19 and 20 (Triflate counterions are omitted for clarity) “(Severa et al., 2010)”.
Formula structures of λ5-Phospha[7]-helicenes 21 and 22 and crystal structure of 21 as representative.
Hydrogen bond plays a key and major role in the biological and pharmaceutical systems and remains a topic of intense current interest. Few selected recent articles exemplify the general scope of the topic, ranging from the role of H-bonding such as in: weak interaction in gas phase “(Nishio, 2005; Wang et al., 2005)”, supramolecular assemblies “(McKinlay et al., 2005)”, helical structures “(Azumaya et al., 2004; Noroozi Pesyan, 2010)”. Important consequences of both inter- and intra-molecular H-bonding have long been recognized in the physicochemical behavior of DNA and RNA “(Jeffery & Saenger, 1991)”.
Several kinds of hydrogen bond have been reported. If the donor-acceptor distance to be in the range of; 2.50 ≤ d (O O) ≤ 2.65, this kind of hydrogen bond is strong and when shorter than 2.50 Å (d(O O)≤ 2.50), to be very strong hydrogen bond “(Gilli et al., 1994)”.
In very short O H O bonds (2.40-2.45 Å) the major distribution of the proton are as follows:
The proton is closer to one of the O atoms (asymmetric hydrogen bond).
The proton is located precisely at the centre (symmetric or centred hydrogen bond).
There is statistically disorder of the proton between two positions on either side of the centre (the proton is closer to one or the other side in different domains of the crystal).
There is a dynamical disorder between two positions as in (iii); the proton jumps between the two positions in the same hydrogen bond “(Gilli et al., 1994; P. Gilli & G. Gilli, 2000; Olovsson et al., 2001; Steiner, 2002)”.
For instance, the structure of the potassium hydrogen dichloromaleate (26) has been studied by neutron diffraction at 30 and 295 K, with the emphasis on the location of the protons. There are two crystallographically independent hydrogen atoms in two very short hydrogen bonds, 2.437(2) and 2.442(2) Å at 30 K. For the centrosymmetric space group P1, with the hydrogen atoms located at the centres of symmetry, the structure could be refined successfully. Olovsson et al. have then been applied several different types of refinements on this structure, including unconventional models; with all atoms except hydrogen constrained in P1, but with hydrogen allowed to refine without any constraints in P1, anisotropic refinement of all atoms resulted in clearly off-centred hydrogen positions. The shifts of the two hydrogen atoms from the centres of symmetry are 0.15(1) and 0.12(1) Å, respectively, at 30 K, and 0.15(1) Å for both hydrogen atoms at room temperature. At 30 K: R(F) = 0.036 for 1485 reflections; at 295 K: R(F) = 0.035 for 1349 reflections (Olovsson et al., 2001)” (Fig. 11).
One of the most interesting example about intermolecular hydrogen bond is the heptan-4-yl (2\'-hydroxy-[1,1\'-binaphthalen]-2-yl) phosphonate (27a) “(Dabbagh et al., 2007)”. The phosphonate 27a was existed in dimmer form via two strong intermolecular hydrogen bonds with centrocymmetric (Ci) 18-membered dimmer form consisting of two monomers strongly hydrogen-bonded between the oxygen of P=O units and hydroxyl hydrogen atoms (Fig. 12). The crystal structure of 27a was determined by X-ray crystallography and is shown in Fig. 12. The selected bond lengthes, angles and torsion angles of 27a are summarized in Tables 2-4, respectively. Crystal data indicated the torsion angles (φ) between two naphthalenic rings moieties in BINOL species are 95.28(16)° and are transoid forms (Fig. 13). The intermolecular hydrogen bond distance in the structure of 27a was obtained 2.70 Å (strong hydrogen bond) and comparised with other hydrogen bonds P-containing systems (Table 5).
Crystal structure of potassium hydrogen dichloromaleate (26).
Crystal structure of 27a.
Representatively, two strong intermolecular hydrogen bonds with centrocymmetric 18-membered dimmer form in 27a and 27b.
Entry | Bond | length (Å) |
1 | P(1) – O(3) | 1.4578(11) |
2 | P(1) – O(2) | 1.5544(12) |
3 | P(1) – O(1) | 1.5865(11) |
4 | P(1) – H(1) | 1.295(16) |
5 | O(1) – C(1) | 1.4073(17) |
6 | O(2) – C(21) | 1.5006(19) |
7 | O(4) – C(12) | 1.3634(18) |
8 | O(4) – H(4) | 0.89(2) |
9 | O(3) – H(4) | 1.81(2) |
10 | C(10) – C(11) | 1.4942(19) |
Selected bond length (Å) of dimmer 27a.
Entry | Bond | Angle (θ, °) |
1 | O(3) – P(1) – O(2) | 118.32(7) |
2 | O(3) – P(1) – O(1) | 113.50(7) |
3 | O(2) – P(1) – O(1) | 102.16(6) |
4 | O(3) – P(1) – H(1) | 112.7(7) |
5 | O(2) – P(1) – H(1) | 103.9(7) |
6 | O(1) – P(1) – H(1) | 104.7(8) |
7 | C(1) – O(1) – P(1) | 122.24(9) |
8 | C(21) – O(2) – P(1) | 122.27(10) |
9 | C(12) – O(4) – H(4) | 114.4(14) |
10 | C(10) – C(1) – C(2) | 123.47(14) |
11 | C(10) – C(1) – O(1) | 117.78(13) |
12 | C(2) – C(1) – O(1) | 118.66(13) |
13 | C(9) – C(10) – C(11) | 120.98(12) |
14 | O(4) – C(12) – C(11) | 124.08(14) |
15 | O(4) – C(12) – C(13) | 114.68(13) |
16 | O(2) – C(21) – C(22) | 107.92(12) |
17 | O(2) – C(21) – C(25) | 111.86(18) |
18 | C(22) – C(21) – C(25) | 108.84(18) |
19 | O(2) – C(21) – H(21) | 109.4 |
20 | C(22) – C(21) – H(21) | 109.4 |
21 | C(25) – C(21) – H(21) | 109.4 |
Selected bond angle of dimmer 27a.
Entry | Bond | Torsion angles (Φ, °) |
1 | O(3) – P(1) – O(1) – C(1) | 51.11(13) |
2 | O(2) – P(1) – O(1) – C(1) | 179.64(11) |
3 | O(3) – P(1) – O(2) – C(21) | 58.93(13) |
4 | O(1) – P(1) – O(2) – C(21) | -66.49(12) |
5 | P(1) – O(1) – C(1) – C(10) | 110.03(13) |
6 | P(1) – O(1) – C(1) – C(2) | -73.13(15) |
7 | O(1) – C(1) – C(2) – C(3) | -177.12(12) |
8 | C(7) – C(8) – C(9) – C(10) | 178.21(14) |
9 | C(2) – C(1) – C(10) – C(11) | -178.59(12) |
10 | O(1) – C(1) – C(10) – C(11) | -2.11(19) |
11 | C(1) – C(10) – C(11) – C(12) | -97.30(17) |
12 | C(9) – C(10) – C(11) – C(12) | 83.69(18) |
13 | C(1) – C(10) – C(11) – C(20) | 83.73(17) |
14 | C(9) – C(10) – C(11) – C(20) | -95.28(16) |
15 | C(20) – C(11) – C(12) – O(4) | 179.63(140 |
16 | C(10) – C(11) – C(12) – O(4) | 0.6(2) |
17 | P(1) – O(2) – C(21) – C(22) | -119.33(13) |
18 | P(1) – O(2) – C(21) – C(25) | 120.97(19) |
19 | O(2) – C(21) – C(22) – C(23) | 62.75(18) |
20 | C(25) – C(21) – C(22) – C(23) | -175.66(19) |
21 | C(21) – C(22) – C(23) – C(24) | 166.78(15) |
22 | O(2) – C(21) – C(25) – C(26) | -68.8(3) |
23 | C(22) – C(21) – C(25) – C(26) | 172.0(2) |
24 | C(21) – C(25) – C(26) – C(27) | -173.4(3) |
Selected torsion angles of dimmer 27a.
Linkages | Bond distance [Range, donor….H….acceptor] (Å) | Strength |
P–O–H....O–P | 2.39-2.50 | Very strong |
P–O–H....O–P | 2.50-2.65 | Strong |
P–O–H....O–C | 2.41-2.82 | Strong |
P–H....OH2 | 2.56-3.15 | Moderate |
P–O....H–N | 2.65-3.10 | Moderate |
Dimmer 27 | 2.70 | Strong |
Classification of hydrogen bonds within P-containing systems.
Dimeric centrosymmetric ring structures are quite common within phosphorous chemistry: for example; the structures of 28 and 29 are of 12- and 8-membered structures, respectively “(Corbridge, 1990)”. According to spectroscopic evidence, esters of (trichloroacetyl) amidophosphoric acid (29) exist as 29I rather than 29II, which suggests that the hydrogen bond in N-H O=P is stable than that of in N-H O=C “(Corbridge, 1990)”.
Formula structures of 30a and 30b.
Crystal structure of 30a.
Possible tautomeric forms of 30a.
There is hydrogen bonding between the acrylate O(3) and the pyridine N(2) atoms; the distance between these two atoms is 2.483 Å, and the O(3)-H(12)-N(2) angle is 171.3°. The O(3)-H(12) distance is 1.345 Å (the theoretical distance is 0.920 Å for general carboxyl O-H bond), which is longer than the N(2)-H(12) distance of 1.145 Å (the general distance is 0.960 Å). The distance difference revealed that H(12) is closer to the pyridine N(2) than it is to the acrylate O(3). The O(2)-C(9) and O(3)-C(9) distances are 1.233 Å and 1.272 Å, respectively. These results show that H(12) is involved in a strong intramolecular hydrogen bonding. N(2)-H(12)····O(3), in which the H(12) interaction with the pyridine N(2) is stronger than that with O(3) atom. The carboxylic acid proton moves to the pyridine N atom, while an electron delocalizes across O(2), O(3), and C(9) to form two almost equivalent carbonyl groups. These results provide further evidence that compound 30a exists mainly as a tautomeric form 30a (NH) in the solid state (30a[II]) form “(Guo, et al. (2011)”.
Resorcarene derivatives are used as units in self-assembled capsules via hydrogen bonds. Like to calixarenes, resorcarenes are the core to which specific functional groups are attached. These groups are responsible for the hydrogen bonds while the resorcarenes offer the right spatial arrangement of them. McGillivray and Atwood found that 31 forms in the crystalline state a hexameric capsule with the internal volume of about 1375Å3. There are 60 hydrogen bonds in hexameric with the help of eight molecules of water (Fig. 16) “(McGillivray & Atwood, 1997)”.
Formula structure of 31 unit and crystal structure of (31)6·8H2O.
Yoshida et al. have also been reported the formation of a three-dimensional hydrogen bonding network by self-assembly of the Cu(II) complex of a semi-bidentate Schiff base “(Yoshida et al., 1997)”. The crystal structure of the Cu(II) complex of Shiff base 32 is shown in Fig. 17. The infinite overall structure of 32 is found to be organized by a three-dimensional hydrogen-bonding network in which the –NH2 O2S– type intermolecular hydrogen bonds play an important role, as shown in Fig. 18. One complex molecule is surrounded by four adjacent complexed molecules through four –NH2 O2S– hydrogen bonds. These hydrogen bonds would be strong judging from the NH O distances in the range 2.032–2.941 Å. From the neutron diffraction study of sulfamic acid (NH3+SO3-), a comparably strong hydrogen bond has been observed (–N+H -O–S– distances in the range 1.95–2.56 Å) “(Jeffrey & Saenger, 1991)”. Similar hydrogen bonds between sulfone and hydroxyl groups [2.898(6) Å] have been found in a supramolecular carpet formed via self-assembly of bis(4,4’-dihydroxyphenyl) sulfone “(Davies et al., 1997)”. Furthermore, four weak Br H hydrogen bonds may participate in the hydrogen-bonding arrays “(Yoshida et al., 1997)”.
Yang et al. reported the crystal structure of Bis(barbiturato)triwater complex of copper(II). The neutral Cu(H2O)3(barb)2 molecules are held together to form an extensive three- dimensional network via –OH·····O– and –NH·····O– hydrogen-bonded contacts “(Yang et al., 2003)”. Hydrogen bonding motifs in fullerene chemistry have been reported by Martín et al. as a minireviewe. The combination of fullerenes and hydrogen bonding motifs is a new interdisciplinary field in which weak intermolecular forces allow modulation of one-, two-, and three-dimensional fullerene-based architectures and control of their function “(Martín et al., 2005)”.
Crystal structure of 32 unit.
Crystal packing diagram of 32.
Methyl 2,4-dimethoxy salicylate (33) as potential antitumor activity, was synthesized from the reaction of 1,3,5-trimethoxybenzene (the most electron-rich aromatic ring) with 2-methoxycarbonyl-5-(4-nitrophenoxy) tetrazole, under solvent-free conditions, a low yield product was obtained (< 2%), while in the presence of a Lewis acid (AlCl3), the yield was increased to 30% (a kind of trans esterification reaction) “(Dabbagh et al., 2003)”.
Crystal structure of 33 is shown in Fig. 19. The carbon-oxygen framework of the molecular structure of 33 is essentially planar; bond lengths and angles are summarized in Table 6, while a structural diagram is shown also in Fig. 20. Planarity is maintained by a strong intramolecular hydrogen bonding interaction between the carbonyl-oxygen and phenolic-H atom [H(1) O(1) = 1.68(4) Å; O(5) – H(1) = 1.00(4) Å], and a much weaker intramolecular hydrogen bond of distance 2.535 Å between Me hydrogen’s [H(8)] and the C=O group (in what we label a “bisected” conformation with Cs symmetry, Figs. 19 and 20). The orientations of the o-OMe and ester-OMe are such to minimize steric interactions. The structure of 33 was also calculated by semi-empirical ab-initio, PM3 and AM1 methods, and data for bond lengths, angles and torsion angles are in good agreement together with the experimental ones (Tables 6 and 7), while the corresponding calculated H(1) O(1) bond lengths were 1.57, 1.78 and 1.97 Å, and the calculated O(5) – H(1) values were 1.00, 0.980, 0.970 Å, respectively. The ab-initio value for the weaker hydrogen bonding interaction was 2.574 Å. The ab-initio calculation also revealed a 1.40 Kcal higher energy, eclipsed conformation with C1 symmetry (Fig. 21 c and d,\n\t\t\t\t\tFig. 20, Table 7) with an H(8) – carbonyl bond length of 2.14 Å “(Dabbagh et al., 2004)”.
Crystal structure of 33 with 50% probability ellipsoids.
Diagrams showing the favored so-called “bisected” (left) and “eclipsed” (right) conformations of 33.
Molecular structures for 33 from ab-initio analysis [side-view: a (bisected); c (eclipsed), and front-view: b (bisected0; d (eclipsed)].
Atoms | Bond lengths (Å) | Atoms | Bond angles(º) |
O(1) – C(7) | 1.247(4) | C(7) – O(2) – C(8) | 116.1(3) |
O(2) – C(7) | 1.325(4) | C(2) – O(3) – C(9) | 117.1(3) |
O(2) – C(8) | 1.465(4) | C(4) – O(4) – C(10) | 115.8(2) |
O(3) – C(2) | 1.363(4) | C(2) – C(1) – C(6) | 116.9(3) |
O(3) – C(9) | 1.438(4) | C(2) – C(1) – C(7) | 124.7(3) |
O(4) – C(4) | 1.362(4) | C(6) – C(1) – C(7) | 118.3(3) |
O(4) – C(10) | 1.443(4) | O(3) – C(2) – C(1) | 117.1(3) |
C(1) – C(2) | 1.432(4) | O(3) – C(2) – C(3) | 121.9(3) |
C(1) – C(6) | 1.393(4) | C(1) – C(2) – C(3) | 121.03 |
C(1) – C(7) | 1.465(4) | C(2) – C(3) – C(4) | 119.8(3) |
C(2) – C(3) | 1.378(4) | O(4) – C(4) – C(3) | 114.4(3) |
C(3) – C(4) | 1.406(4) | O(4) – C(4) – C(5) | 125.1(3) |
C(4) – C(5) | 1.364(4) | C(3) – C(4) – C(5) | 120.6(3) |
C(5) – C(6) | 1.402(5) | C(4) – C(5) – C(6) | 119.7(3) |
O(5) – C(6) | 1.349(4) | O(5) – C(6) – C(1) | 122.3(3) |
- | - | O(5) – C(6) – C(5) | 115.7(3) |
- | - | C(1) – C(6) – C(5) | 122.0(3) |
- | - | O(1) – C(7) – O(2) | 120.7(3) |
- | - | O(1) – C(7) – C(1) | 122.0(3) |
- | - | O(2) – C(7) – C(1) | 117.3(3) |
Bond lengths (Å) and angles (o) of 33.
Bisected | Eclipsed | Relative Energy | |||||
Method | Etotal | [C=O--H-C] | [C=O--H-O] | Etotal | [C=O--H-C] | [C=O--H-O] | |
(kcal/mol) | Å | Å | (kcal/mol) | Å | Å | (Eeclip -Ebist) | |
X-Ray | - | 2.535 | 1.68(4) | - | - | - | - |
Ab-intio | - 470792.80 | 2.574 | 1.565 | -470791.40 | 2.139 | 1.557 | 1.40 |
PM3 | -2812.50 | 2.647 | 1.780 | -2811.10 | 2.309 | 1.780 | 1.40 |
AM1 | -2813.50 | 2.554 | 1.974 | -2812.50 | 2.186 | 1.969 | 1.0 |
Experimental and calculateda hydrogen bond lengths and energies (kcal/mol) for bisected and eclipsed structure of 33.
Crystal packing diagram of 34 (a) and 35 (b). Intermolecular hydrogen bond assigned by red dashed line (Carbon: grey; hydrogen: white; oxygen: red and nitrogen: blue).
Tetrazole ring can exist to be an equilibrium mixture of two tautomeric forms (1H and 2H-tetrazoles) “(Dabbagh & Lwowski, 2000)”. 5-Aryloxy (1H) and/or (2H)-tetrazoles often show intermolecular hydrogen bond “(Noroozi Pesyan, 2011)”. For instance, the crystal packing diagram of 5-(2,6-dimetylphenoxy)-(1H)-tetrazole (34) and 5-(2,6-diisopropylphenoxy)-(1H)-tetrazole (35) show intermolecular hydrogen bond (Fig. 22). In the compound 34, the crystal structure indicated that the tetrazole and phenyl rings are nearly perpendicular to each other, forming a dihedral angle of 95.5° (versus 92.06° from calcd. B3LYP/6-31G(d) and 6-31+G(d)). Because of the conjugation of O1 with tetrazole ring, the bond distance C1–O1 [1.322 Å] is slightly shorter than O1–C7 [1.399 Å]. These bond distances for C1–O1 and O1–C2 were obtained 1.333 and 1.419 Å with calculation by B3LYP/6-31G(d) method, respectively and also 1.332 and 1.420 Å derived with calculation by B3LYP/6-31+G(d) basis set, respectively. These data are in good agreement with experimental results (Table 8). In the compound 35, the crystal structure indicated that the tetrazole and phenyl rings are nearly perpendicular to each other, forming a dihedral angle of 85.91° (versus 107.2° from calcd. B3LYP/6-31G(d)). Because of the conjugation of O1 with tetrazole ring, the bond distance C2–O1 [1.3266(14) Å] is slightly shorter than O1–C7 [1.4257(13) Å]. These bond distances for C2–O1 and O1–C7 were obtained 1.332 and 1.423 Å with calculation by B3LYP/6-31+G(d) method, respectively and are in good agreement with experimental results. These bond distances were also obtained 1.322 and 1.422 Å with calculation by B3LYP/6-31G(d) method, respectively. The torsion angles between phenyl ring and each of methyl units on two isopropyl groups are -110.70°, 124.18° and 116.15° and 154.12°, respectively (Table 9).The selected parameters of bond length, angles and torsion angles of 34 and 35 derived by experimental and calculated results are shown in Tables 8 and 9.
The crystal packing of 34 exhibits an intermolecular N1–H1 N4 hydrogen bonds and comparized with the calculated at DFT (B3LYP) at 6-31G(d) and 6-31+G(d) basis sets (Table 10). The crystal structure indicated that the bond distance value between donor – hydrogen (N1–H1) and hydrogen-acceptor (H1 N4) were found in results 0.861 and 1.959 Å, respectively. For instance, these bond distances were also found in results 1.033 for (N1–H1) and 1.814 for (H1 N4) by calculated at B3LYP/6-31G(d) and 1.031 for (N1–H1) and 1.809 for (H1 N4) B3LYP/6-31+G(d), respectively. The donor-acceptor distance value (N1 N4) was obtained 2.804 by experimental method. This parameter was found 2.842 and 2.838 Å by calculated methods B3LYP/6-31G(d) and 6-31+G(d), respectively. The angle of N1-H1 N4 was found 169.9, 172.9 and 172.1° by experimental, calculated B3LYP/6-31G(d) and B3LYP/6-31+G(d) basis sets, respectively. The results of calculated method (specially 6-31+G(d) basis set) are in good agreement with experimental results (Table 10).
Compd. 34 | |||
Atom | Ex. | Calcd.a | Calcd.b |
O1-C1 | 1.322 | 1.332 | 1.333 |
O1-C2 | 1.399 | 1.420 | 1.419 |
C1-N1 | 1.327 | 1.348 | 1.348 |
C1-N4 | 1.305 | 1.316 | 1.315 |
N1-N2 | 1.354 | 1.362 | 1.363 |
N1-H1 | 0.861 | 1.01 | 1.01 |
N2-N3 | 1.285 | 1.288 | 1.288 |
N3-N4 | 1.368 | 1.368 | 1.368 |
C2-C3 | 1.349 | 1.397 | 1.396 |
C2-C7 | 1.389 | 1.397 | 1.396 |
C3-C9 | 1.518 | 1.508 | 1.508 |
C7-C8 | 1.495 | 1.508 | 1.508 |
C1-O1-C2 | 117.3 | 117.6 | 117.6 |
O1-C1-N1 | 121.0 | 120.8 | 120.8 |
O1-C1-N4 | 129.3 | 130.05 | 130.05 |
C1-N1-H1 | 126.1 | 130.4 | 130.4 |
O1-C2-C3 | 117.8 | 117.8 | 117.8 |
O1-C2-C7 | 116.3 | 117.8 | 117.8 |
C2-C3-C9 | 120.4 | 121.2 | 121.2 |
C2-C7-C8 | 123.0 | 121.2 | 121.2 |
C2-O1-C1-N1 | 170.0 | -180 | -180 |
O1-C1-N1-H1 | -0.8 | 0.0 | 0.0 |
O1-C2-C3-C9 | 4.4 | 4.9 | 4.9 |
O1-C2-C3-C4 | -175.4 | -175.6 | -175.6 |
O1-C2-C7-C8 | -5.7 | -4.9 | -4.9 |
The selected bond lengths (Å), angles (°) and torsion angles (φ) for 34. Experimental and B3LYP/6-31+G(d) and B3LYP/6-31G(d).
The crystal packing of 35 also exhibits an intermolecular N3–H31 N6 hydrogen bonds and comparized with the calculated at DFT (B3LYP) at 6-31G(d) and 6-31+G(d) basis sets (Table 10). The crystal structure indicated that the bond distance value between donor – hydrogen (N3–H) and hydrogen-acceptor (H31 N6) were found in results 0.926 and 1.919 Å, respectively. For instance, these bond distances were also found in results 1.03 for (N3–H31) and 1.91 for (H31 N6) by calculated at B3LYP/6-31G(d) and 1.01 for (N3–H) and 1.93 for (H N6) B3LYP/6-31+G(d), respectively. The donor-acceptor distance value (N3 N6) was obtained 2.835 by experimental method. This parameter was found 2.941 and 2.912 Å by calculated methods B3LYP/6-31G(d) and 6-31+G(d), respectively. The angle of N3–H31 N6 was found 169.1, 177.0 and 173.0° by experimental, calculated B3LYP/6-31G(d) and B3LYP/6-31+G(d) basis sets, respectively. The results of calculated method (specially 6-31+G(d) basis set) are in good agreement with experimental results (Table 10). Compounds 34 (entry no. CCDC-838541) and 35 (entry no. CCDC-819010) were deposited to the Cambridge Crystallographic Data Center and are available free of charge upon request to CCDC, 12 Union Road, Cambridge, UK (Fax: +44-1223-336033, e-mail: deposit@ccdc.cam.ac.uk).
Compd. 35 | |||
Atom | Ex. | Calcd.a | Calcd.b |
O1-C2 | 1.327 | 1.332 | 1.322 |
O1-C7 | 1.426 | 1.423 | 1.422 |
C2-N3 | 1.327 | 1.349 | 1.348 |
C2-N6 | 1.314 | 1.316 | 1.315 |
N3-N4 | 1.358 | 1.362 | 1.363 |
N3-H31 | 0.926 | 1.01 | 1.010 |
N4-N5 | 1.288 | 1.288 | 1.288 |
N5-N6 | 1.373 | 1.368 | 1.368 |
C7-C8 | 1.392 | 1.401 | 1.400 |
C7-C15 | 1.389 | 1.404 | 1.403 |
C8-C9 | 1.521 | 1.525 | 1.525 |
C15-C16 | 1.528 | 1.527 | 1.527 |
C2-O1-C7 | 114.49 | 118.64 | 118.39 |
O1-C2-N3 | 121.3 | 120.47 | 120.38 |
O1-C2-N6 | 128.5 | 130.51 | 130.57 |
C2-N3-H31 | 129 | 130.43 | 130.33 |
O1-C7-C8 | 116.8 | 118.67 | 118.72 |
O1-C7-C15 | 117.9 | 117.02 | 117.08 |
C7-C8-C9 | 120.8 | 123.14 | 123.08 |
C8-C9-H91 | 106.1 | 108.37 | 108.29 |
C10-C9-C11 | 111.7 | 111.27 | 111.41 |
C7-C15-C16 | 122.2 | 124.82 | 124.6 |
C15-C16-H161 | 106.3 | 104.94 | 105.01 |
C17-C16-C18 | 111.1 | 111.52 | 111.49 |
C7-O1-C2-N3 | -174.8 | 178.5 | 178.7 |
O1-C2-N3-H31 | -7.5 | -0.2 | -0.08 |
O1-C7-C8-C9 | 1.5 | 1.6 | 1.9 |
O1-C7-C8-C12 | -176.5 | -177.3 | -177.3 |
O1-C7-C15-C16 | -2.0 | -1.1 | -1.2 |
C7-C8-C9-C10 | 154.1 | 119.45 | 116.5 |
C7-C8-C9-C11 | -80.2 | -115.5 | -118.5 |
C7-C15-C16-C17 | -110.7 | -63.8 | -63.8 |
C7-C15-C16-C18 | 124.2 | 64.7 | 64.5 |
The selected bond lengths (Å), angles (°) and torsion angles (φ) for 35. Experimental and B3LYP/6-31+G(d) and B3LYP/6-31G(d).
D-H····A | D-H | H····A | D····A | D-H····A (degree, °) | |
Exp.a (34) | N1-H1····N4b | 0.861 | 1.959 | 2.804 | 166.9 |
Calcd.c (34) | 1.033 | 1.814 | 2.842 | 172.9 | |
Calcd.d (34) | 1.031 | 1.809 | 2.838 | 172.1 | |
Exp.a (35) | N3-H31····N6e | 0.926 | 1.919 | 2.835 | 169.1 |
Calcd.c (35) | 1.03 | 1.91 | 2.941 | 177 | |
Calcd.d (35) | 1.01 | 1.93 | 2.912 | 174 |
Experimental and calculated B3LYP/6-31+G(d) and B3LYP/6-31G(d) levels for hydrogen-bond geometry of 34 and 35 (Å, °)
Formula and crystal structures of the compounds 36a and 36b.
Nickel(II) complexes containing specific phosphorus– oxygen chelating ligands are very efficient catalysts for the oligomerisation of ethylene to linear form “(Braunstein et al., 1994)”. For instance, Nickel(II) diphenylphosphinoenolate complexes have been prepared from (ortho-HX- substituted benzoylmethylene)triphenyl phosphoranes (X = NMe, NPh) and [Ni(1,5-cod)2] in the presence of a tertiary phosphine (PPh3 or P(p-C6H4F)3) and their crystal structures have been studied by Braunstein et al. (structures of 36a and 36b). Formula and crystal structures of the compounds 36a and 36b are shown in Fig. 23. Crystallographic study of the complexes 36a and 36b establishes the presence of strong intramolecular hydrogen bonding between the enolate oxygen and the N–H functional group “(Braunstein et al., 2005)”. The most notable feature in these structures is the strong intramolecular N–H O hydrogen bonding: the calculated distance between the NH hydrogen atom and the oxygen atom of the enolate ligand is short: 2.18(5) Å in 36a and 2.00(5) Å in 36b, respectively “(Taylor & Kennard, 1982)”.
Intramolecular hydrogen bond is also shown in alkoxyamines. These compounds and persistent nitroxide radicals are important regulators of nitroxide mediated radical polymerization (NMP). The formula and crystal structure of β-phosphorylated nitroxide radical (37) is shown in Fig. 24. Compound 37 show an eight-membered intramolecular hydrogen bond between P=O····H-O (versus N-O····H-O). The hydrogen bond distance for two enantiomers of 37 is different. The hydrogen bond distances of P=O····H-O in (R)- and (S)-37 are 1.570 and 2.040 Å, respectively and favored. Instead, the hydrogen bond distance for N-O····H-O in (R)- and (S)-37 are 3.070 and 3.000 Å, respectively and unfavored “(Acerbis et al., 2006)”.
Formula and crystal structure of two enantiomers of compound 37 (O: red, N: blue and P: yellow).
1,8-diaminonaphthalene derivatives such as; N-(8-(dimethylamino)naphthalen-1-yl)-2-fluoro-N-methylbenzamide (38) is a proton sponge. An unusual strong intramolecular hydrogen bond was observed in the protonated 38. In compound 38 in which a protonated amine group (38-H+) can act as a donor suitably positioned to engage in a strong intramolecular hydrogen bond with the amide nitrogen atom rather than with the carbonyl oxygen atom (Scheme 9). Crystal structure of the triflate salt of 38-H+ is shown in Fig. 25. The unit cell consists of two molecules of 38-H+, two triflate counter ions, and one molecule of water. The dashed line indicates the proposed hydrogen bond between H1A and N2A. Selected bond lengths and angles are N2A–H1A = 2.17(4), N1A–N2A = 2.869(5), C14A–N2A = 1.369(5) (Å) and N2A–H1A–N1A = 136(3)° “(Cox et al., 1999)”.
Protonation of 38 in the presence of trifluoromethanesulfonic acid (TfOH).
Crystal structure of the triflate salt of 38-H+ (50% ellipsoids and triflate counter ion is omitted).
2,4,6-Trisubstituted phenolic compounds such as 2,4,6-tri-tert-butyl phenol are as antioxidant “(Jeong et al., 2004)”. Owing to the nature of the catalytic centres of galactose oxidase (GAO) and glyoxal oxidase (GLO), the N,O-bidentate pro-ligand, 2\'-(4\',6\'-di-tert-butylhydroxyphenyl)-4,5-diphenyl imidazole (LH) (39) has been synthesized “(Benisvy et al., 2001)”. The compound 39 possesses no readily oxidisable position (other than the phenol) and involves o- and p-substituents on the phenol ring that prevent radical coupling reactions. The compound 39 undergoes a reversible one-electron oxidation to generate the corresponding [LH]·+ radical cation that possesses phenoxyl radical character. The unusual reversibility of the [LH]/[LH]·+ redox couple is attributed to a stabilisation of [LH]·+ by intramolecular O–H N hydrogen bonding “(Benisvy et al., 2003)”. The formula and crystal structure of 39 are shown in Fig. 26. Crystal structure of 39 shows an intra- and intermolecular hydrogen bonds in 39. In respect of the chemical properties of 39, there is a strong intramolecular hydrogen bond between the phenolic O–H group and N(5) of imidazole ring. The strength of this hydrogen bond, as measured by the O(1) N(5) distance of 2.596(2) Å and the O(1)–H(1) N(5) angle of 150.7°. Also, the N–H group of imidazole ring in 39 is involved in an intermolecular N–H O hydrogen bond [N(2) O(1S) 2.852(2) Å and N(2)–H(2A) O(1S) 168.8°] to an adjacent trapped acetone molecule (39·Me2CO) “(Benisvy et al., 2003)”.
Formula and crystal structure of 39·Me2CO.
The azamacrocyclic ligand 1,4,7-triazacyclononane or TACN, 40, has attracted considerable interest in recent years for its applications in oxidative catalysis. Another application of this compound was discussed by Pulacchini, et al. “(Pulacchini et al., 2003)”. The incorporation of the 1,2-diaminocyclohexane moiety into a 1,4,7-triazacyclononane macrocyclic ligand was done by this research group, as it is an inexpensive starting material and both enantiomers are readily available. Moreover, this chiral framework has been included in a number of ligands that have been successfully applied in a range of asymmetric catalytic processes by Jacobsen et al. in metallosalen complexes “(Jacobsen & Wu, 1999)”.
Crystal structure of 41 is shown in Fig. 27 and revealed the structure of the macrocyclic ligand in which the six-membered ring has chair conformation (Fig. 27). The asymmetric unit is completed by the two chloride ions and a water molecule in which all C–C, C–O and C–N bonds are unexceptional. Two short hydrogen bonding interactions of 2.724(4) Å between N(1)–H(01) O(1) and 2.884(5) between N(2)–H(05) O(1) within the macrocycle are then supplemented by an extensive hydrogen bonding network between the ammonium nitrogen atoms N(1) and N(2) the two chloride ions Cl(1) and Cl(2), as well as the water molecule of crystallisation, as shown in Fig. 28. The roles of the two chloride ions in the network are distinct with Cl(1) acting as a direct bridge between two macrocyclic moieties as well as linking to a third via a water molecule. In contrast, the second chloride ion, appears to essentially serve to template the macrocyclic ligand into the conformation observed via hydrogen bonding interactions with N(1)–H(01) and N(2)–H(05). The second chloride ion also links to other macrocyclic moieties via the water molecules.
The following hydrogen bond lengths (Å) were observed from the polymeric hydrogen bonding array in 41·2HCl·H2O.; N(1)–H(06) Cl(1) 3.099(4), N(1)–H(01) Cl(2) 3.185(4), N(1)–H(01) N(2) 3.043(5), N(1)–H(01) O(1) 2.724(4), N(2)–H(02) Cl(1)#1 3.103(4), N(2)–H(05) Cl(2) 3.108(3), N(2)–H(05) O(1) 2.884(5), O(2)– H(04) Cl(1) 3.271(4), O(2)–H(03) Cl(2)#2 3.217(4) “(Pulacchini, (2003)”.
Formula structures of 40 and 41 and crystal structure of 41·2HCl·H2O.
Polymeric hydrogen bonding network in 41·2HCl·H2O “(Pulacchini, (2003)”.
In all thiohelicene crystals (see also Figs. 1 and 2) specific interactions were found involving sulfur “(Nakagawa et al., 1985; Yamada et al., 1981)” and hydrogen atoms at distances slightly shorter than the sum of van der Waals radii (1.80 Å for S and 1.20 Å for H). They are quite probably attractive, and, in all structures except TH11 (hexathia-[11]-helicene 3) they involve only atoms of terminal rings. In the case of the 5-ring system each molecule has two equivalent S S interactions of 3.544 Å, while each TH7 (tetrathia-[7]-helicene 1) molecule is involved in four equivalent S H contacts measuring 2.89 Å. All these interactions occur between enantiomeric pairs. Crystal structyre of pentathia-[9]-helicene (TH9, 42) and crystal packing diagram of this compound including S H contacts are shown in Figs. 29 and 30, respectively. For 42, each molecule presents four equivalent S H contacts at 2.87 Å, all with homochiral molecules giving rise to a quasi-hexagonal packing of tilted helices in planes parallel to the ab lattice plane. The crystal structure of TH11 (3) is unusual because the asymmetric unit is formed by two complete molecules as opposed to half a molecule in all the lower racemic thiohelicenes. The packing environment of each of the two closely similar but crystallographically independent molecules, and of each of its halves, is unique: thus the C2 axes bisecting the central ring of each TH11 (3) molecule are noncrystallographic. This situation is likely to arise in order to optimize the complex network of specific interactions involving S and H atoms. It leads to larger than expected asymmetric units and lower crystal symmetry, common occurrences in hydrogen bonded molecular systems. In the triclinic TH11 (3) crystals four nonequivalent short S S and an equal number of S H interactions are found “(Caronna et al., 2001)”. The essential geometric features of all these contacts in the racemic thiohelicene series and evidencing a remarkable consistency of the S H interaction with expectations for weak hydrogen bonds have been reported “(Desiraju & Steiner, 2000)”.
Crystal structure of TH9 (42).
Crystal packing diagram of 42 in which each molecule consists of four equivalent S····H contacts.
The fused pyrimidines such as pyrimido[4,5-c]pyridazine-5,7(6H,8H)-diones, which are common sources for the development of new potential therapeutic agents, is well known “(Altomare et al., 1998; Brown, 1984; Hamilton, 1971)”. Some of this class of compounds play new heterocyclizations based on
Recently, the synthesis of 3-arylpyrimido[4,5-c]pyridazine-5,7(6H,8H)-diones (43a–d) and their sulfur analogs 3-aryl-7-thioxo-7,8-dihydropyrimido[4,5-c]pyridazin-5(6H)-ones 44a–d have been reported “(Rimaz et al., 2010)”. One of the most interesting intermolecular hydrogen bond in 43a–d have been reported by our research group “(Rimaz et al., 2010)” (Figure 31). Owing to the less solubility of 43a–d and 44a–d, an attempt to achieve the single crystal of these compounds for investigation of the clustered water in their crystalline structure was failed. The 1H NMR spectra of 43a–d show two broad singlets in the range of δ = 7.00–8.00 ppm that correspond to the protons of clustered water molecule in the 43a-d. The chemical shift values of two variable protons of water in 43a–d in ambient temperature are shown in Table 11. There are some reasons for demonstration and interpretation of this criterion. (i) One of the evidence is the mass spectra. The mass spectra of the compounds 43a–d show not only the molecular ion fragment (M) but also the fragment of M+18. Therefore, the strength of hydrogen bond between the proton of H2O (Ha) and oxygen atom of carbonyl group (C5=O Ha–O) and also hydrogen bond between the N6–H of 43a–d and oxygen atom of H2O (N6–H O–Ha) is considered more than that of the hydrogen bonding in the dimer form of 43a–d (judging by the observation of the M+18 ion) (Fig. 32) “(Rimaz et al., 2010)”. It seems that at least one molecule of water clustered and joined to 43 and 44 by two strong intermolecular hydrogen bonds and dissociated neither by DMSO molecules as a polar aprotic solvent nor in mass ionization chamber. Presumably, this intermolecular hydrogen bond is of quasi-covalent hydrogen bond type. There are some reports on literatures about quasi-covalent hydrogen bonds “(Dabbagh et al., 2007; Gilli et al., 1994, 2000, 2004; G. Gilli & P. Gilli, 2000; Golič et al., 1971; Madsen et al., 1999; Nelson, 2002; Steiner, 2002; Vishweshwar et al., 2004; Wilson, 2000)”.
Formula structures of 43a–d·(H2O) and 44a–d·(H2O).
Representatively, strong intermolecular hydrogen bond and the chemical shifts of two hydrogen bonded protons of clustered water molecule with 43a “(Rimaz et al., 2010)”.
Compd. | δ (ppm) | ||
Ha | Hb | ||
43a | 7.76 | 7.57 | |
43b | 7.75 | 7.61 | |
43c | 7.74 | 7.61 | |
43d | 7.75 | 7.61 | |
44a | 4.89 | ||
44b | 4.90 | ||
44c | 4.90 | ||
44d | 4.89 |
The chemical shift values of the two protons of a clustered water molecule in 43a–d and 44a–da at ambient temperature “(Rimaz et al., 2010)”.
The proton/deuterium exchange was examined on 43a–d by adding one drop of D2O. Interestingly, from hydrogen to fluorine substituent on phenyl ring in 43a–d the exchange rate was decreased, and no deuterium exchanging of Ha and Hb was observed in 43d while the amide protons were easily exchanged (Fig. 33). This phenomenon attributed to the fluorine atom that has made new intermolecular hydrogen bond with Ha and Hb of clustered water molecule in another molecule of 43d. The intermolecular hydrogen bond of fluorine with the proton of clustered water (–F····Ha– and –F····Hb–) in 43d inhibited the proton/deuterium exchanging of the clustered water protons. However, the electronegativity of fluorine atom caused deshielding of Ha and Hb on 43d and blocked the proton/deuterium exchange (Fig. 33 and Scheme 10). Two conformational forms of IA and IB in 43d are equivalent because of free rotation of phenyl ring about the C3–C9 and C12–F single bonds (Scheme 10) “(Rimaz et al., 2010)”.
Spiro compounds are very important and useful compounds and versatile applications. Many of heterocyclic spirobarbituric acids “(Kotha et al., 2005)”, furo[2,3-d]pyrimidines “(Campaigne et al., 1969)” and fused uracils “(Katritzky & Rees, 1997; Naya et al., 2003)” are well known for their pharmaceutical and biological effects.
Recently, we have reported new spiro compound based on barbiturates; 5-alkyl and/or 5-aryl-1H, 1’H-spiro[furo[2,3-d]pyrimidine-6,5’-pyrimidine]2,2’,4,4’,6’(3H, 3’H, 5H)-pentaones which are dimeric forms of barbiturate (uracil and thiouracil derivatives) “(Jalilzadeh et al., 2011)”. Reaction of 1,3-dimethyl barbituric acid (DMBA) with cyanogen bromide (BrCN) and acetaldehyde in the presence of triethylamine afforded 1,1’,3,3’,5-pentamethyl-1H,1’H-spiro[furo[2,3-d]pyrimidine-6,5’-pyrimidine]-2,2’,4,4’,6’(3H,3’H,5H)-pentaone (46) in excellent yield “(Jalilzadeh et al., 2011)”. The formula structures of spiro compounds derived from barbituric acid (BA, 45), DMBA 46 and 1,3-thiobarbituric acid (TBA, 47) is shown in Fig. 34. Attempt for single crystallization of spiro compounds 45 and 47 were unsuccessful. The crystal structure and crystal packing diagram of 46 are shown in Figs. 35 and 36. This compound was crystalized in triclinic system. Selected crystallographic data for 46 is summarized at Table 12.
Proton/deuterium exchangeability of the Ha and Hb of clustered H2O molecule in 1H NMR spectra of 43a (A), 43b (B), 43c (C) and 43d (D). The assigned spectra are shown before (a) and after added D2O (b). No exchange occurred in 43d of clustered H2O protons (D) “(Rimaz et al., 2010)”.
Possible various types of intermolecular hydrogen bond of fluorine with a proton of a clustered were (-F····Hb- and -F····Ha-) in 43d. This phenomenon presumably inhibited the proton/deuterium exchangeability of the clustered water protons.
Formula structures of 45-47.
Crystal structure of 46.
Crystal packing diagram of 46.
Crystal data | |
Emprical formula | C14H16N4O6 |
M | 336.30 |
T | 298 K |
a (Å) | 8.974 (5) |
b (Å) | 9.539 (5) |
c (Å) | 10.314 (5) |
α ( ◦) | 64.782 (5) |
β ( ◦) | 69.349 (5) |
γ ( ◦) | 69.349 (5) |
V (Å3) | 725.8 (7) |
Z | 2 |
F(000) | 352 |
Dx (mg m−3) | 1.539 |
λ (Å) | 0.71073 |
μ(mm−1) | 0.12 |
Data collection | |
Rint | 0.062 |
θmax | 29.0 ◦ |
θmin | 2.3 ◦ |
Refinement | |
R[F2 "/> 2σ(F2)] | 0.067 |
wR(F2) | 0.203 |
S | 1.04 |
Selected crystallographic data for 46.
Another spiro barbiturate compound derived from the reaction of DMBA with BrCN and acetone in the presence of triethylamine is 1,1’,3,3’,5,5’-Hexamethylspiro[furo-[2,3-d]pyrimidine-6(5H),5’-pyrimidine]-2,2’,4,4’,6’(1H,3H,1’H,3’H,5H)-pentaone (48) “(Noroozi Pesyan et al., 2009)”. Reaction of aldehydes with (thio)barbiturates is faster than ketones due to the reactivity and less hindrance in aldehydes. The formula and crystal structure of 48 is shown in Figs. 37 and 38, respectively. In Fig. 38, the fused 2,3-dihydrofurane ring has an envaloped conformation, and spiro pyrimidine ring has a half-chair conformation. Spiro pyrimidine ring is nearly perpendicular to 2,3-dihydro furan ring moiety as was observed earlier in the related compound. Torsion angles C2-C1-O4-C7 and C2-C1-C5-C6 are -99.39(3)° and 94.87(3) °, respectively. In the crystal, short intermolecular interaction O C contacts between the carbonyl groups prove an existing of electrostatic interactions, which link the molecules into corrugated sheets parallel to ab plane (Table 13).
C8····O2i | 2.835 (4) | C3····O5ii | 2.868 (4) |
Selected interatomic distances (Å) in 48.
Formula structure of 48.
Crystal structure of 48.
One of another interesting spiro barbiturate compound is the trimeric form of 1,3- DMBA; 5,6-dihydro - 1,3-dimethyl - 5,6 – bis - [l’,3’-dimethyl-2’,4’,6’-trioxo-pyrimid(5’,5’)yl]furo[2,3-d]uracil (49). This compound was first reported by electrochemical method “(Kato et al., 1974; Kato & Dryhurst, 1975; Poling & van der Helm, 1976)” and it has been reported the synthesis of 49 by chemical method for a first time two years ago “(Hosseini et al., 2011)”. The formula and crystal structures of 49 are shown in Figs. 39 and 40, respectively. Crystals of 49 were obtained by slow evaporation of a solution of 49 in acetone at room temperature. The data were acquired using a STOE IPDS II diffractometer, data collection and cell refinement were processed using STOE X-AREA “(Stoe & Cie, 2002)” and data reduction was processed using STOE X-RED “(Stoe & Cie, 2002)” program. Program(s) used to refine structure was SHELXL97 “(Sheldrick, 1997). Crystal data for 49: Orthorhombic; C18H18N6O9; M = 462.38; Unit cell parameters at 293(2) K: a = 13.2422(4), b = 15.9176(6), c = 19.5817(6) Å; α = β = γ = 90°; V = 4127.5(2) Å3; Z = 8; μ = 0.122 mm–1; Total reflection number 4275; 304 parameters; λ = 0.71073 Å; 2916 reflections with I > 2σ(I); Rint = 0.056; θmax = 26.49°; R[F2 > 2 σ(F2)] = 0.048; wR(F2) = 0.112; S = 1.02, F000 = 1920 “(Hosseini et al., 2011)”.
Formula structure of 49.
Crystal structure of 49.
Amino acids derived from sugar are of extensive family of peptidomimetics “(Baron et al., 2004; Chakraborty et al., 2004)”, an important sub-class of which incorporate an α-amino acid with a carbohydrate has anomeric effect. Such sugar amino acids may form spiro derivatives, some of which have been demonstrated to possess significant biological activity. For instance, the formula and crystal structure of (2\'S,3aR,6S,6aR)-2,2,6-trimethyldihydro-3aH-spiro[furo[3,4-d][1,3]dioxole-4,2\'-piperazine]-3\',6\'-dione (50) are shown in Figs. 41 and 42 “(Watkin et al., 2004)”. This molecule show hydrogen bonds between N-H….O=C groups and are shown in crystal packing diagram, viewed along the c axis as dashed lines (Fig. 43).
Formula structure of compound 50.
Crystal structure of compound 50 (Green: C, blue: N and red: O atom).
Crystal packing diagram of 50.
Another interesting spiro linked barbituric acid to the cyclopentane ring moiety (spiro-nucleoside) possessing of hydroxyl and hydroxymethyl groups is (3S,2R)-3-hydroxy-2-hydroxymethyl-7,9-diazaspiro[4.5]decane-6,8,10-trione (51) (Figs. 44 and 45). Crystal structure of 51 shows trans stereochemical relationship of the two substituents hydroxyl and hydroxymethyl on cyclopentane ring moiety. The barbituric acid ring is almost planar, while the cyclopentane moiety adopts the C3\'-endo-type conformation. Molecules of 51 interconnected by a two-dimensional network of hydrogen bonds build layers parallel to the ab plane. The hydrogen bond data for 51 is outlined at Table 14 “(Averbuch-Pouchot et al., 2002)”.
Formula structure of 51.
Crystal structure of 51.
D—H····A | D—H | H····A | D····A | D—H····A |
O11—H12····O6v | 0.81 | 2.00 | 2.809 (2) | 173 |
N7—H8····O10iv | 0.86 | 1.99 | 2.840 (2) | 170 |
N9—H9····O2vii | 0.85 | 2.04 | 2.8620 (10) | 161 |
Hydrogen-bond geometry in 51 (Å, º).
Hydantoins are very useful compounds due to their pharmaceutical behaviour such as; antitumor “(Kumar et al., 2009)”, anticonvulsant “(Sadarangani et al., 2012)” and antidiabetic activity “(Hussain et al., 2009)”. In the molecules of 52 and 53 (Figs. 46 and 47), the atoms in the hydantoin ring are coplanar. The crystal structures of 52 and 53 are stabilized by intermolecular N—H····O=C hydrogen bonds. The hydrogen bond lengthes and angles for 52 and 53 are summarized at Table 15. Crystal packing diagram of these molecules show the molecules are centrosymmetric dimer forms. The dihedral angle subtended by the 4-chloro- and 4-bromophenyl groups with the plane passing through the hydantoin unit are 82.98(4)° and 83.29(5)°, respectively. The cyclohexyl ring in both molecules adopts an ideal chair conformation and methyl group in an equatorial position “(Kashif et al., 2009)”.
D—H····A | D—H | H····A | D····A | D—H····A | |
52 | N2—H2····O4i | 0.84 (2) | 2.04 (2) | 2.8763 (15) | 171.5 (19) |
53 | N2—H2····O4i | 0.82 (3) | 2.06 (3) | 2.871 (2) | 171 (3) |
Hydrogen-bond geometry in 52 (Å, º).
Formula structures of 52 and 53.
Crystal structures of 52 (top) and 53 (bottom).
Dihydropyridine are interesting and important systems because of their exceptional properties as calcium channel antagonists “(Si et al., 2006)” and as powerful arteriolar vasodilators “(Kiowski et al., 1990)”. 4\',4\'-Dimethyl-2-methylsulfanyl-3,4,5,6,7,8-hexahydropyrido-[2,3-d]pyrimidine-6-spiro-1\'-cyclohexane-2\',4,6\'-trione, (54), has a markedly polarized molecular electronic structure, and the molecules are linked into a three-dimensional framework by a combination of N–H O, C–H O and C–H л hydrogen bonds (Table 16). Two independent N–H O hydrogen bonds generate a one-dimensional substructure in the form of a chain of rings; these chains are linked into sheets by the C–H O hydrogen bonds, and the sheets are linked by C–H л hydrogen bonds. Crystal packing diagram of 54 show four types of centrosymmetric ring. “(Low et al., 2004)” (Fig. 48). Compound 54 can exist in two zwitterionic forms of 54I and 54II (Scheme 11). For example, the bond lengths of N3–C4 and C4–O4 are both long for their types, the C4–C4A and C4A–C8A bonds are too similar in length to be characterized as single and double bonds, respectively. Also, the C8A–N8 bond, involving a three-coordinate N atom, is much shorter than the C8A–N1 bond, which involves a two-coordinate N atom. These observations, taken together, effectively preclude the polarized form (54I) as an effective contributor to the overall molecular electronic structure, instead pointing to the importance of the polarized vinylogous amide form (54II) “(Low et al., 2004)”.
Zwitterionic forms of 54.
Crystal structure of 54.
D–H····A | D–H | H····A | D····A | D–H····A |
N3–H3····O4i | 0.88 | 1.84 | 2.715 (2) | 176 |
N8–H8····O65ii | 0.88 | 2.10 | 2.965 (2) | 166 |
C5–H5B....O61iii | 0.99 | 2.46 | 3.389 (2) | 155 |
C64–H64A....Cg1iv | 0.99 | 2.87 | 3.854 (2) | 173 |
Hydrogen-bonding geometry (Å, °) for 54.
In summary, X-ray single crystal diffraction analysis of the some helicenes and other helix molecules were discussed. In continuation, the crystal structure of some organic and organometallic compounds consists of intra- and/or intermolecular hydrogen bond were described. Finally, crystal structures of some new spiro compounds were analyzed.
The author gratefully acknowledge financial support by Research Council of Urmia University
Non-typhoidal Salmonella (NTS), a major cause of diarrheal disease globally, is estimated to cause 93 million enteric infections and 155,000 diarrheal deaths each year and is a leading cause of foodborne infections worldwide [1]. In Canada, 88,000 people are estimated to fall ill from foodborne NTS each year (90% credible intervals: 58,532–125,525) [2] with a mean hospitalization of about 925 individuals and 17 deaths [3]. An estimated 1 million cases of NTS infections occur annually in the United States alone, resulting in 19,000 hospitalizations and 380 deaths (
Some pathogenic Salmonella serovars are restricted to particular host species and are not found in other species. Examples of host-restricted Salmonella are serovars Typhi, Gallinarum, and Abortusovis, and they predictably cause systemic infection in their hosts namely, humans, fowls and ovines, respectively [7]. Another group of serovars are host-adapted including Dublin and Choleraesuis and primarily cause disease in cattle and pigs respectively, but infrequently cause opportunistic disease in another host species especially humans [7, 8]. The most common non-adapted Salmonella are serovars Typhimurium and Enteritidis and they have been studied in live animal models such as mice and cattle, leading to a better understanding of the pathogenesis of NTS and the development of diarrhea [7]. S. typhimurium causes a systemic infection in mice that resembles typhoid fever caused by S. enterica serovar Typhi in humans [9]. While a vast majority of cases in otherwise healthy, Salmonella-infected humans present clinically as a self-limiting gastroenteritis, S. typhimurium can cause life-threatening systemic, invasive disease and bacteremia in some patients [10] but the reasons and mechanisms dictating the different disease manifestations in infected humans are not clear.
\nThe advent of microbial whole genome sequencing promises to provide insights to better understand the biology of virulence determinants and mechanisms of NTS pathogenesis. Genomes of Salmonella are generated increasingly at a faster rate and deposited in public databases [11]. Further understanding of genome diversity and variation of bacterial pathogens has the potential to improve quantitative risk assessment and assess the evolution of Salmonella and emergence of new strains [12]. Mining of the repository of genomes should provide new information expected to complement existing knowledge on virulence genes derived from host infection studies especially involving Salmonella mutants. The Salmonella Foodborne Syst-OMICS database (SalFoS) was developed as a platform to improve diagnostic accuracy, to develop control methods in the field and to identify prognostic markers in epidemiology and surveillance [13]. Bioinformatics analyses of genomes are expected to reveal the mechanisms of action of virulence genes and help decipher whether there is a dichotomy in the genes contributing to invasive disease compared to restricted pathogenesis in the intestinal tract [14].
\nThis review provides an overview of the genetic regulation of over 200 virulence determinants highlighting their involvement in each of the four steps of Salmonella pathogenesis, namely: attachment, invasion, macrophage survival and replication, and systemic dissemination (Figure 1). Further analysis of virulence genes will provide us insights in to understanding the mechanisms of invasive disease which appear distinct from gastroenteritis. For instance, the organisms which are responsible for invasive disease have fewer genes because of pseudogenization. Many of these virulence genes have redundant functions; however two Salmonella molecules are known to exert a dominant effect in pathogenesis, namely: lipopolysaccharide (LPS) and invasion protein A (invA). Many virulence factors have distinct and unique functions but cooperative crosstalk has been documented at the different steps of infection, e.g., protein products of genes encoded on two Salmonella pathogenicity islands (SPI), SPI-2 and SPI-4.
\nPathogenesis of Salmonella following contact with gut epithelium. (I) Salmonella cells attach to the epithelium mainly via adhesins, the representative virulence genes involved are fim, Saf, Bcf, stf, csg, lpf, Pef, sti, sth, hof, as well as a negative regulator of STM0551 (purple circles). (II) Three invasion methods are illustrated: M cells uptake bacteria cells through receptor mediated endocytosis, membrane ruffling and cytoskeletal rearrangement resulting in engulfment; alternatively, bacterial cells can be directly taken up by dendritic cells by phagocytosis. The main virulence factors involved are inv, pip, pag, prg, sap, sip, spa, spv, sop, rop, hil and sii (pink triangles). (III) Salmonella cells taken up by macrophages are localized within a Salmonella containing vacuole (SCV). The representative virulence genes involved in this process are mgt, Ssa, Sse, Ssr, CsrA and Hfq (light red star highlighted). (IV) Phagocyte-mediated systemic dissemination through blood system, mainly to liver, spleen and bone marrow. The virulence genes involved are iro, rfa, rfb, fes, Fhu, fep, ent, wzx and wzz (yellow diamond highlighted).
In a majority of cases, infection occurs following ingestion of Salmonella by the host. Before Salmonella can gain entry into the epithelial cell lining the host’s gut mucosa, it first needs to attach to the cell. NTS attachment is facilitated by fimbriae, non-fimbriae factors of autotransporter and outer-membrane proteins, which serve as adhesions; up to 20 adhesion molecules have been described so far and it has been demonstrated that the entire adhesiome of S. enterica serotype Typhimurium can be expressed [15], which facilitates understanding such a large repertoire of adhesions contributing to colonization of a broad range of host species and adaptation to various environment within the host.
\nFimbriae, also known as pili, are thin, filamentous appendages protruding on the bacterial surface and consist of polymerized aggregates of small molecular weight monomers of the fimbrin protein [16]. Characteristically, fimbriae mediate the initial attachment of Gram-negative bacterial pathogens to host cells and surfaces [17]. In Salmonella, the initial contact results in relatively weak adherence of the bacteria to intestinal epithelial cells but soon induces de novo bacterial protein synthesis which increases the strength and intimacy of the attachment [18]. This process is also accompanied by the development and assembly of a unique secretion apparatus called the Type 3 Secretion System (T3SS) which is required for Salmonella to invade epithelial cells [19]. The chromosome of S. typhimurium contains 13 fimbrial operons, afg (csg), bcf, fim, lpf, pef, saf, stb, stc, std, stf, sth, sti, and stj [20, 21, 22] (Table 1 and Figure 1). Eight types of fimbriae which have been experimentally investigated [23] are outlined below.
\nVirulence genes | \nLocation* | \nFunctions | \n
---|---|---|
BcfABCDEFGH | \nChromosome | \nContribute to long-term intestinal carriage and bovine colonization | \n
csgABCDEFG | \nChromosome | \nCurlin subunit; assembly and transport component in curli production; DNA-binding transcriptional regulator | \n
fimCDFHIWYZ | \nChromosome | \nAdhesion to epithelial cells; biofilm formation | \n
hofBC | \nChromosome | \nType IV pilin biogenesis protein | \n
lpfABCDE | \nChromosome | \nBiofilm formation, contribute to long-term intestinal carriage | \n
misL | \nSPI-3 | \nAn extracellular matrix adhesion involved in intestinal colonization | \n
pefA | \nPlasmid | \nAdhesion to crypt epithelial cells; induction of proinflammatory response | \n
ppdD | \nChromosome | \nPutative major pilin subunit | \n
SafC | \nChromosome | \nSalmonella atypical fimbria outer membrane usher | \n
ShdA | \nCS54 | \nOuter membrane | \n
StdB | \nChromosome | \nContribute to long-term intestinal carriage | \n
stfACDEFG | \nChromosome | \nNot required for long-term intestinal carriage of mice | \n
sthABD | \nChromosome | \nOuter membrane fimbrial usher. Putative fimbrial subunit and chaperone protein | \n
StiABC | \nChromosome | \nPutative fimbrial subunit/usher/chaparone | \n
STM0551 | \nChromosome | \nDownregulates fimbrae protein expression and acts as a negative regulator of virulence | \n
STM4595 | \nChromosome | \nUnknown function | \n
Location and function of the major proteins and virulence determinants contributing to Salmonella attachment.
SPI-3 and CS54 are genomic islands on Salmonella chromosome.
Mannose-sensitive Type I fimbriae (Fim) are encoded by the fim ACDHIFZYW operon and bind to D-mannose-containing receptors on host cell surface as well as the glycoprotein laminin of the extracellular matrix [24]. Type I fimbriae promoted bacterial attachment to epithelial cells, facilitated the invasion of HEp-2 cells and HeLa cells and the colonization of the gut mucosa in chicken, mouse, rat and swine [25, 26]. An immunization experiment using purified Fim protein led to the protection of laying hens against egg contamination and colonization of the reproductive organs by S. enteritidis [27]. FimA, FimF, and FimH are necessary for the assembly of Type 1 fimbriae on S. typhimurium [24]. Differently, STM0551 gene plays a negative regulatory role in the regulation of type 1 fimbriae in S. typhimurium [28].
\nPlasmid-encoded fimbriae (Pef) participate in the attachment of bacteria to the surface of murine small intestine and are necessary for fluid production in the infant mouse similar to the observation with the fimbriae of enterotoxigenic Escherichia coli and Vibrio cholerae [29]. Expression of pef gene is regulated by DNA methylation [30]. Purified Pef specifically binds the trisaccharide Galβ1-4(Fucα1-3) GlcNAc (also known as the Lewis X blood group antigen or Lex), which are preponderant on the surface of human erythrocytes, skin epithelium and mucosal surfaces [31].
\nLong polar fimbriae (Lpf) encoded by the lpfABCDE fimbrial operon is involved in the colonization of murine Peyer’s patches by mediating adherence to M cells, a preferred port of entry for Salmonella in mice [32]. Mutation of the lpfC gene which encodes the fimbrial outer membrane usher attenuated the virulence of Salmonella typhimurium in orally exposed mice as shown by a 5-fold increase in the number of organisms needed to kill 50% of test animals (i.e., LD50) when compared to the wild type organism. Lpf is also involved in the early stages of biofilm formation on host epithelial cells [33] and participate in intestinal persistence in mice [34]. Lpf synthesis is regulated by an on–off switch mechanism (phase variation) to avoid host immune responses [35].
\nThin aggregative fimbriae also known as curli [36] with the designation Agf/Csg, are encoded by the agf/csgBAC gene cluster [37]. The thin aggregative fimbriae for Enteritidis which is known as SEF 17 is responsible not only for the auto-aggregative phenotype of the bacteria, but for fibronectin binding [38] and has been shown in vitro to bind immortalized small intestinal epithelial cells from mice [36]. Mutation in agfB resulted in a 3- to 5-fold increase in the oral LD50 of Typhimurium for mice [39].
\nBovine colonization factor (Bcf) is encoded by genes in the bcf gene cluster. The fimbrial usher protein encoded by bcfC is required for colonization of bovine but not murine Peyer’s patches in oral infection models of calves and mice [40]. The bcf gene together with five other fimbrial operons—lpf, stb, stc, std, and sth—are reported to be required for long-term intestinal carriage of Typhimurium in genetically resistant mice [34].
\nSalmonella atypical fimbriae (Saf) are encoded by the chromosomal safABD operon. A group of BALB/c mice immunized subcutaneously with SafB/D- and recombinant cholera toxin B subunit (rCTB)-conjugated micro-particles had significantly lower CFU counts than the untreated control group [41]. Two additional functions - poly-adhesive and self-associating activities – were attributed to the Saf pili and appear to contribute to host recognition and biofilm formation [42].
\nStd operon is required for adherence to human colonic epithelial cells and for cecal colonization in the mouse by binding to cecal mucosa receptors containing α(1, 2) fucose residues [34, 43]. Stf fimbriae share homology with the MR/P fimbriae of Proteus mirabilis and E. coli Pap fimbriae [44]. StfA expression is induced during infection of bovine ileal loops [45].
\nEnteritidis fimbrial SEF14 contributes to colonization of chicken intestine, liver, spleen and reproductive organs [46, 47]. The fragment encoding genes responsible for SEF14 biosynthesis contain three genes, sefABC. The putative adhesion subunit encoded by sefD is essential for efficient uptake or survival of Enteritidis in macrophages, as the sefD mutants were not readily internalized by peritoneal macrophages compared with the wild-type bacteria soon after intraperitoneal infection of mice [48]. The sefD mutant was severely attenuated after both oral and intraperitoneal infection of BALB/c mice (approximate LD50: >104 (mutant) vs. <10 (wild type)) [48]. In the mouse model, egg-yolk derived anti-SEF14 antibodies afforded passive protection [49].
\nFour distinct non-fimbrial intestinal colonization factors have been identified:
\nMisL encoded within the SPI-3, is an outer membrane fibronectin-binding autotransporter protein which is induced upon bacterial contact with the intestinal epithelial cells, and is required for colonization of the murine cecum and for intestinal persistence. MisL binds fibronectin and collagen IV via its passenger domain [50].
\nShdA gene is located in the 25-kb pathogenicity island called CS54 which is present only in S. enterica subspecies enterica [51]. ShdA is a large fibronectin/collagen I-binding outer membrane protein which is induced in vivo in the murine caecum [52]. It is required for Typhimurium colonization in the murine caecum and Peyer’s patches of the terminal ileum [53] and for efficient and prolonged shedding of the organism in feces [51].
\nBapA is a huge surface-associated protein and secreted via its downstream type I secretion system, BapBCD. BapA contributes to murine intestinal colonization and subsequent organ invasion. Mice orally inoculated with BapA-deficient strain survived longer and have a significant reduction in mortality rate than those inoculated with the wild-type strain [54].
\nSiiE is a SPI4-encoded protein and works as the substrate protein of the T1SS. SiiE is secreted into the culture medium but mediates contact-dependent adhesion to epithelial cell surfaces. SiiE codes for a giant non-fimbrial adhesion of 600 kDa and consists of 53 repeats of immunoglobulin domains; this is a T1SS-secreted protein that functions as a non-fimbrial adhesion in binding to eukaryotic cells [55].
\nShortly after adhesion to a host cell, Salmonella invasion proceeds as a consequence of the activation of host cell signaling pathways leading to profound cytoskeletal rearrangements [56]. These internal modifications dislocate the normal epithelial brush border and induce the subsequent formation of membrane ruffles that engulf adherent bacteria in barge vesicles called Salmonella containing vacuoles (SCVs), which is the only intracellular compartment where Salmonella cells survive and replicate [57, 58]. Simultaneously, induction of secretory response in the intestinal epithelium initiates recruitment and transmigration of phagocytes from the submucosal space into the intestinal lumen. Alternatively, Salmonella cells may be directly engulfed by dendritic cells from the submucosa. Taken up During SCV maturation, Salmonella induces de novo formation of an F-actin meshwork around bacterial vacuoles, a process which is termed vacuole-associated action polymerization (VAP) and is important for maintenance of the integrity of the vacuole membrane [59]. Furthermore, intracellular Salmonella can induce the formation of long filamentous membrane structure called Salmonella-induced filaments (SIFs) [60], which may lead to an increased availability of nutrients within the SCV [61]. A fraction of SCVs transcytose to the basolateral membrane. Once across the intestinal epithelium, Salmonella are engulfed by phagocytes and internalized again with SCVs, triggering a response similar to that reported inside epithelial and M cells to ensure bacterial survival and replication [62]. The pathogenic bacterium must at this stage employ many virulence strategies to evade the host defense mechanisms (Figure 1).
\nThe majority of the virulence determinants are located within highly conserved SPIs on the chromosome, while others are either on a virulence plasmid (pSLT) or elsewhere in the chromosome. To date, 21 SPIs have been identified in Salmonella, and the generalist S. typhimurium and the invasive S. typhi genomes share 11 (SPIs-1 to 6, 9, 11, 12, 13 and 16). Two SPIs namely SPI-8 and 10 were initially found in S. typhi and without counterparts in S. typhimurium chromosome; SPI-14 is specific to S. typhimurium, while SPIs-7, 15, 17 and 18 are specific to S. typhi; and SPIs-19, 20 and 21 are absent in both of them [63]. Because of the prominence of the SPIs in pathogenesis, the virulence factors encoded on the major SPIs, SPI-1 to SPI-5 are described below, and their respective functions summarized (Tables 2 and 3).
\nVirulence genes | \nLocation* | \nFunctions | \n
---|---|---|
Crp | \nChromosome | \ncAMP-regulatory protein | \n
hilACD | \nSPI-1 | \nPromote phop-repressed prgHIJK, sipA, sipC, invF, and orgA; activates the expression of the hilA gene | \n
Hnr | \nSPI-2 | \nSPI-2 regulator (transcriptional and post-transcriptional) | \n
HtrA | \n\n | Resistance to periplasmic stress | \n
IacP | \nSPI-1 | \nPosttranslational modification | \n
iagB | \nSPI-1 | \nInvasion | \n
invABCEFGIJ | \nSPI-1 | \nSecretion and chaperone; promote sipBCDA, sigD and sicA | \n
msgA | \nChromosome | \nUnknown function | \n
ompR/envZ | \nSPI-2 | \nRegulates ssrAB expression | \n
orgABC | \nSPI-1 | \nPathogenesis; secretion | \n
phoR/Q | \nSPI-2 | \nRegulates ssrAB expression; down-regulates the transcription of its master regulator HilA, control mgtC | \n
pagACDP | \nSPI-11 | \nResistance to AMP, macrophage cytotoxicity | \n
pipABB2CD pipC (sigE) | \nSPI-5 | \nPathogenesis, effector protein; sif extension; SCV maturation and positioning | \n
prgHIJK | \nSPI-1 | \nSecretion | \n
Prc | \n\n | Resistance to periplasmic stress | \n
rpoES rpoS (katF) | \nSPI-2 | \nSPI-2 regulator (transcriptional and post-transcriptional); controls the transcription of the regulatory gene spvR; expression of rpoS is induced after entry of Salmonella into macrophages or epithelial cells, or in vitro during the stationary growth phase | \n
rtsA | \nChromosome | \nActivates the expression of the hilA gene | \n
sapABCDF | \n\n | Resistance to AMP, macrophage cytotoxicity | \n
sifA | \nSPI-2 | \nSif formation in epithelial cells and maintenance of SCV membrane integrity | \n
siiCDEF | \nSPI-4 | \nTranslocation; adhesion to apical side of polarized epithelial cells; involved in T3SS-1 dependent invasion | \n
sicAP | \nSPI-1 | \nChaperone for sipBC | \n
sipA (sspA) | \nSPI-1 | \nStabilization and localization of actin filaments during invasion, stabilization of VAP, correct localization of SifA and PipB2, SCV perinuclear migration and morphology, promote inflammatory response and fluid secretion | \n
sipBCD (sspBCD) | \nSPI-1 | \nAdhesion to epithelial cells, early macrophage pyroptosis, macrophage autophagy; Adhesion to epithelial cells | \n
SpaSRQPO | \nSPI-1 | \nEscU/YscU/HrcU family type III secretion system export apparatus switch protein; antigen presentation protein SpaO | \n
sptP | \nSPI-1 | \nDisruption of the actin cytoskeleton rearrangements by antagonizing SopE, SopE2, and SigD, downregulate inflammatory response | \n
sirA | \nSPI-1 | \nSirA/BarA encoded outside SPI-1 activates HilA | \n
slrP | \nChromosome | \nAdhesion to epithelial cells | \n
slyA | \nSPI-2 | \nRegulates resistance to oxidative stress | \n
sspH1H2 | \nPhage | \nLocalize to the mammalian nucleus and inhibits NF-κB-dependent gene expression; SCV maturation and positioning | \n
sodABD | \n\n | Resistance to oxidative stress | \n
SopABDD2EE2 sopB (sigD) | \nSPI-5 | \nChloride secretion; promote actin cytoskeletal rearrangements, invasion and inhibition of apoptosis of epithelial cells, induction of proinflammatory response and fluid secretion, SCV size, instability, maturation and positioning, nitrate respiration, outgrowth in the intestine; inhibition of vesicular trafficking; replication inside macrophages; sif formation | \n
spaOPQRS | \nSPI-1 | \nSecretion | \n
SprB | \nSPI-1 | \nRegulation of transcription, DNA-templated | \n
spvABCD | \nPlasmid | \nModifies actin and destabilizes the cytoskeleton of infected cells; SCV maturation and positioning; induction of apoptosis; Host cell signaling | \n
SsJ | \n\n | Resistance to oxidative stress | \n
STM2231 | \nSPI-2 | \nSPI-2 regulator (transcriptional and post-transcriptional) | \n
YejABEF | \nChromosome | \nResistance to AMP, macrophage cytotoxicity | \n
ymdA | \nChromosome | \nStress response | \n
Location and function of the major proteins and virulence determinants contributing to Salmonella invasion.
SPI1–5 are genomic islands on Salmonella chromosome.
Virulence genes | \nLocation | \nFunctions | \n
---|---|---|
CsrA | \n\n | RNA chaperones | \n
Hfq | \nSPI-2 | \nSPI-2 regulator (transcriptional and post-transcriptional), RNA chaperones | \n
mgtABCD | \nSPI-3 | \nA hydrophobic membrane protein; Mg2+ transporter (Mg2+-transporting P-type ATPase) | \n
SsaABCDEFGHIJKLMNOPQRSTUV ssaB (spiC), ssaC (spiA), ssaD (spiB), ssaR (yscR). | \nSPI-2 | \nRegulate the secretion of translocon proteins under conditions that simulate the vacuolar environment; interferes with vesicular trafficking; intracellular bacterial proliferation; secretion | \n
sscAB | \nChromosome | \nPutative type III secretion system chaperone protein or pathogenicity island effector protein | \n
sseABCDFGIJL | \nSPI-2 | \nTranslocation; sif formation in epithelial cells; SCV maturation and positioning; SCV membrane dynamics; nuclear response-gene expression; | \n
ssrAB (ssrA/SpiR) | \nSPI-2 | \nRegulates SPI-2 gene expression | \n
Location and function of the major proteins and virulence determinants contributing to Salmonella macrophage survival and replication.
SPI-1 codes for several effector proteins that trigger invasion of epithelial cells by mediating actin cytoskeletal rearrangements and hence internalization of the bacteria. These effectors are translocated into host cell by means of a Type III Secretory System or T3SS-1 [64], which is made up of proteins encoded by the SPI-1, such as inv, spa, prg and org [65]. Naturally occurring mutants of Salmonella have been found in the environment with a deletion of a vast DNA segment of SPI-1 locus and are deficient for inv, spa, and hil hindering their ability to enter cultured epithelial cells [66]. Mutations leading to a defective secretory function of T3SS-1 led to a 50-fold increase in LD50 following oral administration of Typhimurium in the mouse model [67]. The prg/org and inv/spa operons encode the needle complex, whereas the sic/sip operons encode the effector proteins and the translocon (SipBCD), a pore-forming structure that embeds in the host cell membrane and delivers these effectors to the host cytosol. In addition, several chaperones are also encoded within SPI-1. For example, SlrP mediate ubiquitination of ubiquitin and thioredoxin [68] and one of the SPI-1 regulons, STM4315 (rtsA) interferes with the interactions of S. typhimurium and host cells [69]. In general, the expression of SPI-1 genes is subject to control by complex regulatory mechanisms involving local regulators such as HilA, iagB and InvF which are necessary for host invasion by Salmonella and induction of gastroenteritis [70, 71]. For example, prgHIJK, invA, invJ, and orgA are primarily regulated by HilA [71]. In addition, two major global regulatory networks, SirA/BarA and PhoP/PhoQ , indirectly regulate the expression of the invasion-associated genes via HilA [72, 73].
\nThe SPI-2 is composed of two segments. The smaller portion contains the ttrRSBCA operon, which is involved in tetrathionate reduction, and seven open reading frames (ORFs) of unknown function. The expression of these genes may contribute a growth advantage over the microbiota [74]. The larger portion of this island was shown to be critical for the ability of Salmonella to survive and replicate inside host cells—both epithelia cells and macrophages—within the SCV [75]. Non-functional SPI-2 mutants are unable to colonize internal target organs such as spleen and liver of mice, although they penetrate the intestinal barrier as efficiently as the wild type strain [76]. These mutants were attenuated by at least five orders of magnitude compared with the wild type strain after either oral or intraperitoneal inoculation of mice [75]. The SPI-2 related events are triggered by the action of effector proteins with its own T3SS known as T3SS-2, which also encodes its proper translocon machinery named SseBCD [77]. Gene sequence similarity to the known components of other T3SS has been used to propose functions for SsaN, SsaR, SsaS, SsaT, SsaU and SsaV as coding for putative proto-channel components, SsaD/SpiB, SsaJ, SsaK and SsaQ appear to code for basal components, whereas SsaC/SpiA may code for an outer ring protein [78]. Generally, SPI-2 contains four types of virulence genes: ssa encodes T3SS-2 apparatus; ssr encodes regulators; ssc encodes the chaperones and sse encodes the effectors (Table 2) [79, 80].
\nUnlike SPI-1 and SPI-2, only four ORFs within SPI-3 have been shown to contribute to replication in macrophages via a high-affinity Mg2+ uptake system [81]. The mgtC gene encoding a 22.5-kDa hydrophobic membrane protein, is the major virulence gene factor found within this locus, and is responsible for growth in Mg2+ limiting environment, intramacrophage survival, and systematic virulence in mice [82]. The transcription of mgtC is followed by activation of PhoP-PhoQ in response to low Mg2+ levels [81].
\nThe fourth SPI contributes to Salmonella colonization in the intestine of cattle, but not of chicks [83]. Loss of SPI-4 attenuates the oral but not intraperitoneal virulence of serovars Typhimurium and Enteritidis in mice [84]. Three genes namely SiiC, SiiD, and SiiF produce proteins that form the type 1 secretion system (T1SS); the fourth gene, siiE codes for a giant non-fimbrial adhesion exported by the T1SS and mediates contact-dependent adhesion to polarized epithelial cells rather than to non-polarized cells. In contrast, SiiA and SiiB are not secreted but represent inner membrane proteins whose function is unknown [55, 85]. Recently, transmembrane mucin MUC1 was shown to be required for Salmonella siiE-mediated entry of enterocytes via the apical route [86].
\nThe SPI-5 locus is well characterized in the serovar Dublin infection in calves. This bovine-adapted serovar primarily causes bacteremia rather than gastroenteritis in humans. This region comprises six genes namely, pipD, orfX, sopB (also known as sigD), pipC (also known as sigE), pipB, and pipA [87]. Four gene products which include three SPI-5 Pip proteins (PipD, PipB, PipA) and one SPI-1 SopB protein are involved in secretory and inflammatory responses in bovine ligated ileal loops but they do not appear to play a significant role in the development of systemic infection in mice inoculated by the intraperitoneal route [87, 88]. Furthermore, it has been found that SigE serves as a chaperone for the S. typhimurium invasion protein, SigD [89].
\nThe SPI-2 genes are activated after Salmonella gains access into the SCV [76]. T3SS-2 secretes multiple effector proteins into different subcellular fractions where they interfere with various host cellular functions to establish a replication-permissive environment [90]. The identified effectors are encoded within SPI-2 (e.g., SpiC, SseF and SseG) and outside SPI-2 (e.g., SifA, SseI, SseJ and SspH 2) [23]. These SPI-2-encoded effectors together with some of SPI-1-encoded effectors (e.g., SipA, SipD, SopA, SopE, SopB) that persist in the host cytosol after invasion, are distributed in different cellular compartments including the vascular membrane of SCV and Sif, host cytosol, cytoskeleton, Golgi apparatus, and nucleus. These molecules influence distinct intracellular events and collectively contribute to establish a Salmonella replicative niche in macrophages [91]. These intracellular events include: inhibition of endocytic trafficking, evasion of NADPH oxidase-dependent killing [92, 93], induction of a delayed apoptosis-like host cell death [94], assembly of a meshwork of F-actin around the SCV [59], accumulation of cholesterol in the SCV [95], and interference with the localization of inducible nitric oxide synthase to the SCV [96]. Efficient replication has been found to be associated with two phenotypes involving host microtubule cytoskeleton and its motor proteins, Golgi apparatus-associated juxtanuclear positioning of SCV [97, 98, 99] and Sifs formation which appear as tubular membrane extensions of SCVs enriched in lysosomal glycan proteins [100].
\nThe functional relatedness between SPI-1 and SPI-4 is reflected by their co-regulation by the same set of key regulators, for example, a transcriptional activator SprB encoded within SPI-1 and regulated by HilA under similar environmental conditions; SprB directly activates SPI-4 gene expression and weakly represses SPI-1 gene expression through HilD [101].
\nSimilar mechanisms occur inside epithelial cells after intestinal invasion and once bacteria have been internalized by macrophages. Briefly, Salmonella cells are localized in the SCV once engulfment is completed. Preserving the SCV membrane integrity plays a crucial role in allowing Salmonella replication inside these intracellular niches. These procedures are regulated by T3SS-2 transporting action and its translocon machinery, namely SseBCD complex [77]. Hence, the required effectors which are encoded both inside and outside SPI-2 facilitate the success of Salmonella intramacrophage survival. The SPI-2 gene expression is triggered in response to a number of environmental signals mimicking the vacuolar environment of SCV, including stationary growth phase, low osmolarity [102], low concentrations of Mg2+, Ca2+ or PO3 [103, 104], and low pH [76]. The expression of SPI-2 genes is coordinately regulated at both transcriptional and post-transcriptional levels. During the transcription of SPI-2 genes, many two-component regulatory systems are involved, including SsrA-SsrB, OmpR-EnvZ and PhoP-PhoQ as well as transcriptional regulators, namely SlyA and the alternative sigma factor of RNA polymerase RpoE. The main regulatory proteins that act post-transcriptionally are the RNA chaperons, including Hfq, CsrA, and SmpB. The mgtC gene located in SPI-3 has been shown to contribute to replication in macrophages. All the mentioned virulence determinants can be found in Table 3 and Figure 1.
\nInternalization of the infecting Salmonella within SCV is followed by systemic spread through other target organs, such as the spleen and liver. As a prerequisite for spread, the bacterial cells must evade the innate immune system. During this process, serum resistance or resistance to complement-mediated serum killing is a major virulence factor for the development of systemic salmonellosis. It involves three major factors, namely LPS, outer membrane proteins PagC and Rck and siderophores (Table 4 and Figure 1).
\nVirulence genes | \nLocation | \nFunctions | \n
---|---|---|
cirA | \nChromosome | \nColicin I receptor | \n
entABCDEF | \nChromosome | \nEnterobactin synthase | \n
fepABCDEG | \nChromosome | \nOuter membrane receptor; iron-enterobactin transporter binding protein | \n
Fes | \nChromosome | \nSalmochelin secretion/degradation | \n
FhuABCDE | \nChromosome | \nEnterobactin/ferric enterobactin esterase | \n
foxA | \nChromosome | \nFerrioxamine B receptor precursor | \n
FruR | \nSPI-2 | \nDNA-binding transcriptional regulator | \n
FUR | \nChromosome | \nFerric uptake regulator | \n
iroBCDE | \nChromosome | \nSalmochelin glycosylation, transport and processing | \n
MsbA | \nChromosome | \nLipid transporter ATP-binding/permease protein | \n
rfaBCDFGHIJKLPQYZ | \nChromosome | \nLPS core biosynthesis protein; transcriptional activator; O-antigen ligase | \n
rfbBDFGHIJKMNOPUVX | \nChromosome | \nGlucose biosynthesis pathway; O-chain glycosyltransferase; O-antigen transporter | \n
rfc | \nChromosome | \nO-antigen polymerase | \n
STM0719 | \nChromosome | \nUnknown function | \n
wzxCE | \nChromosome | \nColanic acid exporter; putative LPS biosynthesis protein | \n
wzzBE | \nChromosome | \nLPS chain length regulator and biosynthesis protein | \n
yibR | \nChromosome | \nUnknown function | \n
ybdAB | \nChromosome | \nEnterobactin exporter EntS | \n
Location and function of the major proteins and virulence determinants contributing to Salmonella dissemination.
LPS of Gram-negative bacteria, a major component of the outer membrane, constitute a chemical and physical protective barrier for the cell. LPS consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a long distal repetitive polysaccharide termed O-antigen or O-side chain [105]. Complete LPS is characterized by long O-antigen which confers the smooth (S) phenotype on Salmonella. The O-antigen is a major component associated with serum resistance. Incomplete LPS devoid of O-antigen leads to rough (R) phenotype, which is of low virulence [106]. Naturally occurring infections are caused by S-phenotype Salmonella, which are resistant to complement killing [107, 108]. There is a correlation between the amount, structure, and chain length of the O-antigen and virulence [109]. The long O-antigen of LPS confers on the organism the ability to resist complement-mediated serum killing by sterically hindering the insertion of the membrane attack complement complex (C5b-9) into the bacterial outer membrane [107, 108].
\nSurface expression of O-antigen involves multiple steps: O-antigen biosynthesis in the inner membrane (rfb), translocation across the inner membrane by Wzx flippase (wzx), polymerization (wzz, rfc and rfe) and ligation on to the preformed Core-Lipid A complex by WaaL ligase (rfaL). The Core-Lipid A is translocated independently by the ATP-binding cassette (ABC) transporter MsbA [110, 111]. Complete LPS molecules are then transported to the surface across the periplasm and outer membrane by the Lpt (LPS transport) pathway [111]. Defects in any of the above steps would affect the surface display of the O-antigen and its function. The mutants defective in the biosynthesis of LPS core encoded by the rfa loci or the O side chain by the rfb loci, are significantly attenuated with a LD50 at least 100 times higher than the parental strain in chickens subcutaneously infected with Enteritidis [112].
\nTyphimurium possesses two functional wzz genes responsible for regulating the chain length of the O-antigen [113]. One is wzzST encoding a long LPS with 16–35 O-antigen repeat units and the other fepE gene coding for a very long LPS estimated to contain more than 100 repeat units [113]. Either gene product is sufficient for complement resistance and virulence in the mouse model of infection, which reflects a degree of functional redundancy of these two wzz genes [113]. Double mutation of these two wzz genes resulted in relatively short, random-length O-antigen and the mutant displayed enhanced susceptibility to complement-mediated killing and was highly attenuated in mice [113]. The transcription of wzzST gene is independently activated by two-component systems of Typhimurium, PmrA/PmrB (PmrA, sensor; PmrB, response regulator) and RcsC/YojN/RcsB (RcsC, sensor; YojN, intermediate phosphotransfer protein; RcsB, response regulator) [114]. PmrA/PmrB is activated through two pathways: one is directly activated through its cognate sensor PmrB in response to Fe3+ and the other is dependent on the PhoP/PhoQ two-component system in response to low Mg2+. The RcsC/YojN/RcsB is activated in the presence of low Mg2+ plus Fe3+ [114]. In addition, mutants in a number of genes (rfaG, rfaI, rfaL, rfaQ , rfaP, rfbC, rfbD, rfbJ, rfbM, rfbP, yibR) necessary for LPS biosynthesis/assembly had severely impaired movement on swimming motility agar [115].
\nIn addition to LPS, two outer membrane proteins, the 18-kDa PagC [116] and the 17-kD Rck [117], confer a high level of resistance to the complement-mediated bactericidal activity. These two proteins share homology with virulence-associated outer membrane protein Ail from Yersinia that blocks formation of the complement membrane attack complex on the bacterial surface. Similarly, complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes [117]. One strain of Typhimurium which contains a single mutation in pagC had a virulence defect and decreased survival in cultured murine macrophages and 100-fold reduction in intraperitoneal virulence in mice [118].
\nIron is an essential element for the growth of most bacteria through its involvement in a variety of metabolic and regulatory functions [119]. Studies with different iron concentrations in growth media demonstrated an effect on gene expression of the iron acquisition systems encoded both on the chromosome and plasmids at both transcriptional and translational levels [120]. Siderophores which are bacterial molecules that bind and transport iron are important for bacterial growth in serum in the extracellular stage of Salmonella systemic infection. They are not required after bacteria reside in SCV where siderophore-independent iron acquisition systems are sufficient for iron uptake during intracellular stage. Salmonella produce two major types of siderophores, high-affinity catecholate consisting of salmochelin and enterobactin the latter also known as enterochelin and a low-affinity hydroxamate known as aerobactin which is expressed under iron-restricted conditions [121]. The synthesis, secretion, and uptake of salmochelin requires genes clustered at two genetic loci, the fepA gene cluster and iroBCDEN operon. The fepA gene cluster includes most ent genes for synthesis and export [122]. The iroBCDEN operon encodes gene products for enterobactin glycosylation (IroB, glycosyltransferase), export (IroC, ABC transporter protein), and utilization (IroD, esterase; IroE, hydrolase; IroN, outer membrane receptor) [122]. Mutants deficient in iroB or iroC exhibit reduced virulence during systemic infection of mice via intraperitoneal route, as indicated by lower bacterial load in liver and a delayed time of death [122]. Moreover, the enterobactin metabolite, 2, 3-dihydroxybenzoyl serine (DHBS), can also be used by Salmonella as sources of iron, albeit at much lower affinities, by recognizing the three catechelate receptors, FepA, IroN and Cir. The three receptors demonstrate a significant degree of functional redundancy. The Typhimurium double mutant ΔfepA iroN were similarly virulent to the parental strain after intragastric gavage inoculation of mice, while the triple mutant ΔfepA iroN cir was attenuated as indicated by a significantly reduced cecal colonization and no measurable spread to the liver [123, 124].
\nFurthermore, Salmonella also utilize xenosiderophores as iron sources by utilizing the outer membrane receptors, including FhuA, FhuE, and FoxA. For example, utilization of ferrioxamines B, E, and G by Typhimurium is dependent on the FoxA receptor encoded by the Fur repressible foxA gene. A strain carrying the foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops and was markedly attenuated in mice challenged by either intragastric gavage or intravenously route strain compared to the foxA+ parent [125]. The best characterized regulator for iron uptake is the iron-dependent repressor Fur that acts together with the co[-]repressor ferrous iron (Fe(II)) to regulate genes involved in the iron uptake process in response to iron restriction, including fhuA, fhuB, fepA, fes, fepD, entB, fur, foxA, hemP, and fhuE [126, 127].
\nThe advent of next generation sequencing (NGS) has provided an opportunity to verify or improve on knowledge gained from in vitro and in vivo analyses of Salmonella mutants which were designed for the purpose of understanding gene function and mechanism of action. Recently, Rakov et al. [14] carried out bioinformatics analysis of 500 Salmonella genomes and identified 70 allelic variants virulence factors which were associated with different pathogenesis outcomes, i.e. gastrointestinal vs. invasive disease. However, the causative relationship between a putative virulence factor and disease outcome using a genomics based tool is yet to be attained. To that end, we propose the development of a comprehensive genome based tool such as a NGS AmpliSeq assay that can be used to simultaneously interrogate the presence and potential expression of over 200 virulence genes of Salmonella identified in this communication. The tool can be used to evaluate differences in strains and correlate the output with virulence phenotype derived from epidemiological or experimental observations which can be developed simultaneously or based on historical documentation. The tool could be used in assessing the potential risk posed by a strain of Salmonella given the fact that the serovars obtained from the environment are often distinct with those involved in human diseases. The technology appears suitable for dissecting the complexity associated with the redundancy and pleiotropic nature of some of the currently known virulence genes. In addition, NGS based analysis of virulence genes should provide new insights on Salmonella evolution and a better tool for analyzing epidemiological data that could translate to a reduction in the burden on human health posed by this important foodborne and zoonotic pathogen.
\nThis review provides an outline of over 200 identified virulence determinants and details of their involvement in the four steps of Salmonella pathogenesis, namely: attachment, invasion, intramacrophage survival/replication and systemic dissemination. The genetic regulation of only some of the virulence determinants have been elucidated in live animal models such as mice and cattle, and this has enriched our understanding of the pathogenesis and mechanism of diarrhea and systemic disease. The majority of the current evidence on pathogenesis and virulence determinants of NTS was derived from murine model of serovar Typhimurium infection with and only a few studies focused on NTS infection in humans. For this reason, the relevance of published observations is often called into question. Linking clinical, epidemiological and experimental observations on the nature and severity of diseases caused by Salmonella organisms with the presence of a large number of virulence genes currently may not garner enough predictive ability to infer virulence or pathogenetic potential of a strain. Still, the increasing availability of a large number of Salmonella genomes in the public databases is proving to be a timely resource. Next generation sequencing and the twin subject of bioinformatics represent an unprecedented opportunity to verify past observations and help improve our understanding of Salmonella virulence towards a coherent and comprehensive understanding of the mechanism of Salmonella pathogenesis. What is required is a robust laboratory tool that can be used to analyze the large number of virulence genes in an isolate using the tools of whole genome sequencing. We expect that a tool such as an AmpliSeq assay for Salmonella virulence could be developed to generate accurate and reliable information that can be fed into a quantitative risk assessment framework. This could usher a new era of risk management customized for a Salmonella strain involved in an outbreak and should translate to impactful outcomes in the areas of improved food safety, evaluation of zoonotic diseases and reducing the burden of human salmonellosis.
\nRG is funded by Genome Canada. DO’s research program has received funding support from Genome Research and Development Initiative of the Government of Canada, Ontario Ministry of Agriculture, Food and Rural Affairs, Canadian Security and Science Program of the Department of National Defense and the Canadian Food Inspection Agency.
\nThe authors declare no conflict of interest.
antimicrobial peptides
\ninvasion protein A
\nlipopolysaccharide
\nnon-typhoidal Salmonella
\nnext generation sequencing
\nSalmonella Foodborne Syst-OMICS database
\nSalmonella pathogenicity islands
\nSalmonella-induced filaments
\nSalmonella-containing vacuole
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\\n\\nHumans in Space program aims to remove this barrier and pursue a model under which none of our authors will need to pay for publication and the editors will receive a budget for their editorial work.
\\n\\nWe are currently in the process of collecting sponsorship. If you have any ideas or would like to help sponsor the program, we’d love to hear from you. Contact: Natalia Reinic Babic at natalia@intechopen.com. All of our IntechOpen sponsors are in good company The research in past IntechOpen books and chapters have been funded / sponsored by:
\\n\\nOpen Access is in the heart of the Humans in Space program as it removes barriers and allows everyone to freely access the research published. However, open access publishing fees also pose a barrier to many talented authors who just can’t afford to pay.
\n\nHumans in Space program aims to remove this barrier and pursue a model under which none of our authors will need to pay for publication and the editors will receive a budget for their editorial work.
\n\nWe are currently in the process of collecting sponsorship. If you have any ideas or would like to help sponsor the program, we’d love to hear from you. Contact: Natalia Reinic Babic at natalia@intechopen.com. All of our IntechOpen sponsors are in good company The research in past IntechOpen books and chapters have been funded / sponsored by:
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