Paper 4: Biomolecules and Their Interactions Module 12: Bases, Sugars, Nucleosides and Nucleotides Introduction Nucleic acids are involved in storage

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1 Paper 4: Biomolecules and Their Interactions Module 12: Bases, Sugars, Nucleosides and Nucleotides Introduction Nucleic acids are involved in storage and transfer of genetic information. The double helical structure of DNA (Deoxyribo Nucleic Acid) by Francis Crick and James D. Watson in 1953 accounts for one of the greatest discoveries of the twentieth century (refer to Time line of Scientific discoveries). The reason being, it initialised the great Human Genome Project (HGP) with spending between amounting to $3.8 billion. However, this was nothing, compared with the economic impact of $796 billion (Jonathan Max, 2011). I am always fascinated by the beauty of the structure of DNA, its stability vs. flexibility, its information content and capacity to transfer this information. It is said that we are what our proteins are. However, what controls the synthesis, structure, architecture and function of body s vital proteins, is the three dimensional structure of DNA. Objectives The objective of the present module is to: a) Enumerate basic building blocks of nucleic acids, b) Describe nomenclature, occurrence and importance of nucleic acid bases, c) Give structural formula, nomenclature and conformation of ribose and deoxyribose sugars, d) Distinguish between nucleotides and nucleosides, e) Elaborate on the conformation around glycosydic bond and, f) Discuss implications of sugar pucker and glycosyl conformation in DNA structure Basic building blocks of DNA and RNA The DNA molecule is made up of four nitrogenous bases adenine, guanine, thymine and cytosine (generally we refer these only as bases and use one letter symbols A, G, T and C to designate them), phosphate and a sugar (deoxyribose) group. The polymer is built by attaching these bases to sugar phosphate backbone. This necessitates us to understand also the structures of sugars, basesugar complexes (nucleosides) and base-sugar-phosphate (nucleotide) moieties. The DNA molecule stores the genetic information through the sequence of four heterocyclic bases (A), (G), (T) and (C). The sequence information in DNA molecule is copied into a complementary sequence, like a carbon copy and 1

2 passed to subsequent cell cycles through the process of replication. Sequence of nucleic acid bases in DNA controls the sequence of amino acids in a protein chain. It is passed as a three letter code (codon) to ribosomes through the process of transcription by messenger RNA (ribonucleic acid) or simply mrna and translated with the help of transfer RNA (trna). The chemical structure of RNA molecule is similar to that of DNA. It consists of heterocyclic bases adenine (A), guanine (G), cytosine (C) and uracil (U). It has ribose sugar instead of deoxyribose. Before going through the details of historic developments leading to the discovery of DNA structure, intricacies in the three dimensional (3D) structure of DNA, chemical, short range and long range (local and global) structural modifications and their biological implications, let us first look into the basic building blocks the bases Nucleic acid bases Nucleic acid Bases are nitrogen containing aromatic biological compounds. The primary or canonical bases are cytosine, guanine, thymine, adenine and uracil (in RNA). Adenine and guanine belong to the double-ringed (five-six fused ring) class of molecules called purines and abbreviated as R. Thymine, uracil and cytosine have six member aromatic rings called pyrimidines and are abbreviated as Y. Uracil (U) lacks a methyl group at position 5. The DNA bases can exist in at least two tautomers. Adenine and cytosine (cyclic amides) can exist in amino or imino forms, guanine, thymine and uracil (cyclic aldehydes) can exist either in lactam (keto) or lactim (enol) forms (figure 12.1). Although tautomeric forms of each form, exist in equilibrium with other, amino and lactim forms are more stable. Based on the meteorites found on earth, it has been recently reported by NASA that complex organic compounds of life such as: nucleobases in DNA and RNA (adenine, guanine, xanthyne, hypoxanthine, 2-6 diaminopurine and 6-8 diaminopurine), are formed in outer space and can be formed in laboratory under outer space conditions. Cytosine was discovered by Albrecht Kossel and Albert Newman in 1894 when it was hydrolyzed from calf thymus. The structure was proposed in the same year. It has been found to have use in quantum computing. Cytosine is a six member heterocyclic aromatic ring with two nitrogen atoms at positions 1 and 3. During the glycosyl bond formation the proton at N1 is replaced by deoxyribose sugar. There is an amino group at the fourth position and two hydogens H41 and H42 2

3 attached to N4 serve as hydrogen bond donors, whereas the nitrogen at N3 and carbonyl oxygen at C2 (named as O2) serve as hydrogen bond acceptor groups. Cytosine can be found in DNA, RNA, nucleotide tri-phosphates, act as a cofactor to enzymes and transfer of phosphate group from adenosine diphosphate (ADP) to adenosine tri phosphate (ATP). The atom numbering and structure of cytosine is shown in the figure Figure 12.1 Atom numbering scheme for purines and pyrimidines Guanine has two heterocyclic (having 5 and 6 members each) fused rings with nitrogen atoms at positions 1 and 3 in the six member ring and 7 and 9 of the five member ring (see figure 12.1 nomenclature). It has a C-6 carbonyl group that acts as the hydrogen bond acceptor. While hydrogen atom at N1 (generally named as H1) and two hydrogen atoms of amino group at C-2 (generally referred as H21 and H22 and are attached to N2) act as hydrogen bond donors. The Guanine is linked to sugar through N9 at the five member ring. The proton at N9 is replaced by deoxyribose sugar and a glycosyl linkage is formed (discussed in section 12.3). Guanine has two tautamers. The major form is keto form. The name keto is derived from ketone the aldehyde. The minor form is enol form. The name enol is derived from alcohol. The two forms exist in chemical equilibrium and referred as tautamers of each other. The inter conversion between two forms involves movement of alpha hydrogen from N1 to O6. This process is known as keto-enol transformation. 3

4 The first isolation of guanine was reported in 1844 from the excreta of sea birds known as asguano used as fertilizer and named as guanine in The structure was determined by Fischer who showed that it can be converted into uric acid. Guanine can be hydrolyzed using strong acids as to glycine, ammonia carbon dioxide and carbon monoxide. Guanine was also found in meteorite in Adenine has variety of roles in biochemistry and cellular respiration in the form of energy rich adenosine triphosphate (ATP) and cofactor in nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). It also has function in protein synthesis. Adenine is found in several tautamers which can interchange into each other. However in an inert gas matrix and in gas phase amino tautomer is found most often. Both adenine and guanine are derived from ionosine monophosphate (IMP) which is synthesized from pre-existing ribose phosphate through a complex process from atoms of glycine, glutamine and aspartic acid fused with tetrahydrofolate. The structure of adenine is similar to that of guanine with the only difference of having an amino group at 6 th position instead of a carbonyl group. Also, it does not have hydrogen attached to N1. The amino group at C6 has two hydrogen atoms (H61 and H62) attached to N6 which serve as hydrogen bond donors. The nitrogen atom N1 serves as a hydrogen bond acceptor (figure 12.1). Adenine has also been found in outer space. Very recently physicists found that adenine has an extraordinary variable range of ionization energies along its reaction pathway. How adenine survives UV radiation makes it important for spectroscopic measurements. Thymine similar to cytosine is a six member heterocyclic aromatic ring compound with a methy group attached at position 5. It is also known as 5 methyl uracil. Thymine was first isolated by Albrecht Kossel and Abert Newman in Thymine has a carbonyl oxygen at fourth position instead of amino group. N3 is protonated and hydrogen at N3 (H3) serves as an electron donor while O4 serves as an electron acceptor. The linkage to sugar is through N1 (figure 12.1). Uracil structurally is very similar to thymine. The only difference is it does not have a methyl group at position 5 (For more information see Nucleobase) (figure 12.2). 4

5 Figure12. 2 Uracil There are many naturally occurine purines as: Xantrhine, Hypoxanthine, Threobromine, Uric Acid, Caffeine, Isoguanine which are not part of double helical DNA Ribose and deoxyribose sugars The sugars found in nucleic acids are pentose sugars (five carbon atoms) Ribose sugar Ribose found in RNA is a normal sugar with one oxygen attached to each carbon atom (figure 12.3). Figure 12. 3Ribose sugar Figure 12.4Deoxyribose sugar It is a monosacsharide with the formula H (C=O) (CHOH) 4 H. It has many forms and exists in equilibrium with five forms Deoxyribose sugar Deoxyribose sugar found in DNA lacks one oxygen atom. Hence it gets the name deoxy. It is a mono saccharide with the formula H-(C=O)-(CH 2 )-(CHOH) 3 -H. During the glycosylation there is a loss of one water molecule. Several isomers of 5

6 deoxyribose exist(figure 12.4). However in Fisher projection all OH are on the same side. The OH group from the aromatic portion of the sugars and H from the nucleic acid bases (N1H from pyrimidines or N9H from purines) combine to form a water molecule (figure 12.5). Figure12.5Glycosyl bond formation With the ribose sugar we get adenosine, guanosine, cytidine, uridine Conformation of ribose and deoxyribose sugar The four carbon atoms (C1, C2, C3, C4 and oxygen O4) are not in the same plane. The sugar rings in is puckered. If we consider a plane through C1 -O4 -C4 then the base nitrogen and C5 form a boat like structure. C3 and C2 can lie on the same side or opposite sides of this plane. These two conformations of the sugar ring are named as envelop and twist. If C2 lie on the side of N (N9 of purine or N1 of pyrimidine) and C5, it is called C2 endo sugar pucker (figure 12.6). 6

7 Figure 12.6 C2' endo deoxyadenosine. C2 shown as a yellow ball Figure12.7 C3' endo deoxycytidine C3 shown as a green ball If it is C3 is towards N1/N9 of bases then it is named as C3 endo sugar pucker (figure 12.7). This is discussed in more details in module Nucleosides and nucleotides Nucleosides are N glycosides of ribose and deoxyribose. We show in figure 12.8 adenosine, guanosine, cytidine and uridine where bases are linked to ribose sugar. 7

8 Figure 12.8a adenosine Figure 12.8b guanosine Figure 12.8c cytidine Figure 12.8d uridine Pseudouridine is an exception to the linkage between nucleic acid bases and sugars. Here the ribose sugar C1 is directly linked to C5 of uracil (figure 12.9). It is found in trna. Figure 12.9 Pseudouridine When deoxyribose sugar is connected nucleic acid bases, we get deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine (also called as thymidine) (figure 12.10). 8

9 Figure 12.10a deoxyadenosine Figure 12.10b deoxyguanosine Figure 12.10c deoxy cytidine Figure 12.10d thymidine Nucleotides are defined as compounds where nucleoside is connected to a phosphate group. The building blocks of DNA and RNA are nucleotides. Nucleotides are generally considered as nucleoside monophosphates. However nucleoside diphosphates and nucleoside triphosphates are also nucleotides. Nucleotides carry a packet of energy from nucleoside tri phosphate (ATP, GTP, CTP and UTP) playing a central role in metabolism. 9

10 Figure 12.11a Figure12. 11b Nucleotides participate in cell signaling (cgmp and camp) and are incorporated as cofactors in enzymatic reactions (coenzmye A, FAD, FMN, NAD and NADP+). They can also be radiolabelled. For more information refere to nucleotides. Nucleosides and nucleotides have been studied extensively using different theoretical techniques as: conformation energy calculations with empirical potential energy functions and quantum chemical methods; different physicochemical techniques (DTA, UV-Vis, X-ray, CD, ORD etc) and single crystal-x ray diffraction methods. These studies gave a wealth of information relating to their structures and conformational. It is beyond the scope of this module to go through all these details. One can refer to Pullman and Saran (1976). From the structural point of view rotation around the glycosyl bond is very important for nucleic acids, nucleotides and nucleosides. Relative to the sugar moiety the bases can adopt two main orientations about glycosyl link. These are called syn and anti. In anti conformation the phosphate group is turned away from the bases (figure 12.11a). In the syn orientation phosphate oxygen is near the N3 of purines (figure 12.11b). Anti conformation of bases is favored in DNA. Nucleotides by themselves are found both in syn and anti conformation. Theoretical calculations using molecular orbital techniques gave conflicting reports. 10

11 Summary The module introduces reader with the basic building blocks of nucleic acids: the nucleic acid bases also named as nucleobases or bases, ribose and deoxyribose sugars. The chemical structure of bases and atom numbering is given. We also describe different tautameric forms. The nomenclature for five member sugar ring is given. The basic concepts in the sugar ring puckering are introduced. The difference between nucleosides and nucleotides is given along with the mechanism of glycosyl bond formation. The conformation around the glycosyl bond is elaborated. This introductory module is expected to familiarize the students with the properties of the basic building blocks of nucleic acids to further their knowledge of DNA structure and its intricacies. 11