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Cover
Preface
1 Enzymatic Synthesis of Nucleoside Analogues by Nucleoside Phosphorylases
1. 1.1 Introduction
2. 1.2 Nucleoside Phosphorylases
3. 1.3 Enzymatic Approaches to Produce Nucleoside Analogues Using Nucleoside Phosphorylases
4. 1.4 Approaches to Produce Nucleoside Analogues
5. 1.5 Upscaling Approaches for the Production of Nucleoside Analogues
6. 1.6 Production of Pharmaceutically Active Compounds by Nucleoside Phosphorylases
7. 1.7 Outlook for the Application of Nucleoside Phosphorylase in the Production of Nucleoside Analogues
8. References
2 Enzymatic Phosphorylation of Nucleosides
1. 2.1 Introduction
2. 2.2 Nonspecific Acid Phosphatases (NSAPs)
3. 2.3 Deoxyribonucleoside Kinases (dNKs)
4. 2.4 Conclusion
5. References
3 Enzymatic Synthesis of Nucleic Acid Derivatives Using Whole Cells
1. 3.1 Introduction
2. 3.2 Nucleoside Synthesis Mediated by Microbial Nucleoside Phosphorylases
3. 3.3 Nucleoside Analogues Synthesis by the Combined Action of Microbial Nucleoside Phosphorylases and Other Enzymes
4. 3.4 Chemoenzymatic Preparation of Nonconventional Nucleoside Analogues Involving Whole Cell Biocatalyzed Key Steps
5. 3.5 Nucleoside Prodrugs Preparation by Whole Cell Systems
6. 3.6 Other Nucleoside Derivatives
7. 3.7 Perspectives
8. References
4 Enzymatic Synthesis of Nucleic Acid Derivatives by Immobilized Cells
1. 4.1 Introduction
2. 4.2 Nucleic Acid Derivatives
3. 4.3 Whole Cell Immobilization: Generalities
4. 4.4 Synthesis of Nucleosides by Immobilized Cells
5. 4.5 Conclusion
6. References
5 Enzymatic Synthesis of Nucleic Acid Derivatives by Immobilized Enzymes
1. 5.1 Introduction
2. 5.2 Immobilized Glycosyltransferases
3. 5.3 Immobilized Nucleoside Oxidase
4. 5.4 Immobilized Hydrolases
5. 5.5 Immobilized Phosphopentomutases
6. 5.6 Immobilized Deoxyribonucleoside Kinases
7. References
6 Synthesis of Nucleic Acid Derivatives by Multi‐Enzymatic Systems
1. 6.1 Multi‐Enzymatic Systems in Biosynthesis
2. 6.2 General Overview of Multi‐Enzymatic Synthesis of Nucleic Acid Derivatives
3. 6.3 Multi‐Enzymatic Synthesis of Nucleosides and Their Derivatives
4. 6.4 Multi‐Enzymatic Synthesis of Nucleotides and Their Derivatives
5. 6.5 Conclusion
6. References
7 Enzymatic Synthesis Using Polymerases of Modified Nucleic Acids and Genes
1. 7.1 Introduction
2. 7.2 Types of XNA Biomolecules
3. 7.3 Enzymatic Synthesis of XNA and DNA Polymerases
4. 7.4 Base‐Modified XNAs (Base‐XNAs)
5. 7.5 Sugar‐Modified XNAs (Sugar‐XNAs)
6. 7.6 Phosphodiester Backbone‐XNA
7. 7.7 A Mirror‐Image L‐DNA
8. 7.8 Conclusions
9. References
8 Synthetic Approaches to the Fleximer Class of Nucleosides – A Historic Perspective
1. 8.1 Distal Fleximers
2. 8.2 Proximal Fleximers
3. 8.3 “Reverse” Fleximers
4. 8.4 Acyclic Fleximers
5. 8.5 Conclusion
6. References
9 Synthesis of Oligonucleotides Carrying Nucleic Acid Derivatives of Biomedical and Structural Interest
1. 9.1 Introduction
2. 9.2 Oligonucleotides Carrying the DNA Lesion O ‐Alkylguanine
3. 9.3 The Effect of Chemical Modifications in Non‐Canonical DNA Structures
4. 9.4 Modified siRNAs for Gene Silencing
5. References
10 Synthesis of Carbohydrate–Oligonucleotide Conjugates and Their Applications
1. 10.1 Introduction
2. 10.2 Synthesis of COCs
3. 10.3 Synthesis of Glycocluster Oligonucleotides
4. 10.4 Applications of COCs
5. 10.5 Outlook
6. References
11 Advances in Light‐Directed Synthesis of High‐Density Microarrays and Extension to RNA and 2′F‐ANA Chemistries
1. 11.1 Introduction
2. 11.2 Phosphoramidite Chemistry Applied to the Photolithographic Synthesis of Microarrays
3. 11.3 Recent Improvements in the Synthesis of DNA Microarrays
4. 11.4 Synthesis of RNA Microarrays
5. 11.5 Enzymatic Approaches to RNA Array Synthesis
6. 11.6 Synthesis of 2′F‐ANA Microarrays
7. 11.7 Conclusion and Outlook
8. References
12 SAMHD1‐Mediated Negative Regulation of Cellular dNTP Levels: HIV‐1, Innate Immunity, and Cancers
1. 12.1 Cellular dNTP Concentrations
2. 12.2 SAMHD1 and Negative Regulation of Cellular dNTPs
3. 12.3 SAMHD1 Substrates, Activators, and Inhibitors
4. 12.4 SAMHD1 and HIV‐1 Reverse Transcription
5. 12.5 SAMHD1 Mutations and Innate Immunity
6. 12.6 SAMHD1 and Cancers
7. 12.7 Summary
8. Acknowledgment
9. References
Index
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List of Tables

Chapter 01
1. Table 1.1 Nucleoside phosphorylases with acronyms and EC numbers.
2. Table 1.2 Drugs synthesized by different nucleoside phosphorylases.
Chapter 04
1. Table 4.1 Advantages and disadvantages of the different immobilization techniques.
2. Table 4.2 Summary of different nucleic acid derivatives obtained by the use of immobilized whole cells.
Chapter 06
1. Table 6.1 Multi‐enzymatic synthesis of nucleosides by combination with NPase.
Chapter 07
1. Table 7.1 Thermostable DNA polymerases used for PCR amplification with modified dNTP.a
2. Table 7.2 Different modifications of nucleic acid that were successfully PCR amplified.
Chapter 08
1. Table 8.1 Mechanistic study of the terminal cyclization step of tricyclic guanosine/xanthosine.
2. Table 8.2 Fleximer products via transglycosylation through Ll‐NDT or PNP and their yield percent [63, 65].
3. Table 8.3 Antiviral activites of doubly flexible fleximers (EC₅₀ in µM) [10, 11].
Chapter 10
1. Table 10.1 Carbohydrate receptors used as potential target for COCs (shown in chronological order).
2. Table 10.1
Chapter 11
1. Table 11.1 Sequences synthesized on microarray with single rU inserts and Cy3 terminal labeling.

List of Illustrations

Chapter 01
1. Figure 1.1 Classification of nucleosides and nucleoside analogues. Canonical (unmodified) nucleosides are the building blocks of DNA and RNA. Non‐canonical (naturally modified on pentose moiety, base moiety or both) are mainly occurring in RNA. Synthetic nucleosides are used in the treatment of viral and bacterial infections as well as in cancer treatment.
2. Figure 1.2 Classification of nucleoside phosphorylases (NPs) and their substrate affinities. NPs are classified into two main families: NP‐I and NP‐II. NP‐I family is further subdivided into hexameric and Trimeric NPs. NP‐II family is subdivided into two main classes: TP and PyNP. Residues labeled in red are crucial for enzyme–substrate interaction; green and blue labeled substitutions are those accepted by enzymes.
3. Figure 1.3 Catalytic mechanism described for PNP.
4. Figure 1.4 Structural motifs described for members of the NP‐I family. Highlighted in grey are domains that are unique for either MTAP or UP. Trimeric- trimeric PNP, Hexameric‐hexameric PNP.
5. Figure 1.5 Schematic presentation of amino acid residues that define substrate specificity of different nucleoside phosphorylases.
6. Figure 1.6 Approaches for the synthesis of nucleosides and nucleoside analogues. (a) One‐pot transglycosylation reaction. (b(I)) Enzymatic synthesis starting from glyceraldehyde‐3‐phosphate. (b(II)) Chemo‐enzymatic synthesis starting from deoxyribose, ribose, or arabinose. (b(III)) Enzymatic synthesis starting from D‐pentoses (deoxyribose, ribose, or arabinose). PF‐1P: Pentose‐1P
7. Figure 1.7 Immobilization techniques applied for whole cells and purified enzymes.
Chapter 02
1. Scheme 2.1 Regioselective phosphorylation of inosine to 5′‐IMP.
2. Scheme 2.2 Mechanism of NSAP‐catalyzed transphosphorylation and hydrolysis.
3. Scheme 2.3 Regioselective phosphorylation of 2′‐deoxyribonucleosides.
Chapter 03
1. Scheme 3.1 Pharmacologically active nucleoside analogues.
2. Scheme 3.2 Nucleoside synthesis mediated by nucleoside phosphorylases (NPs). R : H, OH; B₁,B₂: pyrimidine, or purine bases.
3. Scheme 3.3 One‐pot AraG synthesis catalyzed by whole cells containing nucleosides phosphorylases (UP and PNP) and adenosine deaminase (ADA) activities.
4. Scheme 3.4 Biocatalyzed strategies for the synthesis of 2‐deoxyribose‐5‐phosphate and its subsequent conversion to pyrimidine 2′‐deoxynucleosides. FBA: fructose‐1,6‐diphosphate aldolase. TIM: triose isomerase. DERA: 2‐deoxy‐5‐phosphate aldolase. PPM: phosphopentomutase. NSAPs: nonspecific acid phosphatases. TP: thymidine phosphorylase. RK: ribokinase.
5. Scheme 3.5 Nonconventional nucleoside analogues.
6. Scheme 3.6 Biocatalyzed approaches to synthesize L‐ribose.
7. Scheme 3.7 Microbial strategies for UMP and UDP synthesis.
8. Scheme 3.8 Synthesis of UDP‐glucose catalyzed by sugar synthases.
9. Scheme 3.9 Biocatalyzed strategies for the synthesis of cytidine 5‐monophosphate N‐acetylneuraminic acid (CMP‐NeuAc).
Chapter 04
1. Figure 4.1 Chemical structure of natural nucleosides that compose DNA (R = H: β‐D‐deoxyribofuranose) and RNA (R = OH: β‐D‐ribofuranose).
2. Figure 4.2 Strategies for the biocatalytic synthesis of nucleoside analogues and their derivatives.
3. Figure 4.3 Nucleic acid derivatives in current clinical use: (a) PNAs and (b)PyNAs.
4. Figure 4.4 Different stabilization methodologies employed for microbial cell immobilization.
5. Figure 4.5 Ribavirin bioconversion using bacterial biocatalysts in different matrices.
6. Figure 4.6 Floxuridine bioconversion using different immobilized biocatalysts.
Chapter 05
1. Figure 5.1 Enzymatic synthesis of 5‐methyluridine catalyzed by immobilized PyNP and PNP from Bacillus stearothermophilus JTS 859. One milliliter of the immobilized biocatalyst was packed into a column reactor (7 × 26 mm) 0.75 mM inosine and thymine and 20 mM potassium phosphate (pH 7.0) was fed 20 ml h⁻¹ continuously at 60 °C for 17 days.
2. Figure 5.2 Enzymatic transglycosylations catalyzed by immobilized UP and PNP from E. coli. Synthesis of 506U78 was carried out in 10 mM phosphate buffer pH 7.4 at 55 °C [10], whereas synthesis of β‐D‐arabinofuranosyl‐2,6‐diaminopurine ( ara‐DAMP) in 30 mM phosphate buffer pH 7.0 at 60 °C.
3. Figure 5.3 Enzymatic syntheses of 2′‐deoxyguanosine and 2′‐deoxyinosine catalyzed by immobilized UP and PNP from B. subtilis. 2′‐Deoxyuridine (50 mM) was used as sugar donor, whereas guanine or hypoxanthine (25 mM) were used as sugar acceptors. Both reactions were performed in 10 mM carbonate pH 10 and 45 °C [15].
4. Figure 5.4 Enzymatic synthesis of ara‐A and ddI catalyzed by immobilized NPs. Reactions were carried out at room temperature, in 50 mM phosphate pH 7.5 containing 10% (v/v) of acetonitrile. Donor concentration was 10 mM and acceptor concentration was 5 mM [14]. Immobilized NPs; CpUP: uridine phosphorylase from Clostridium perfringens ; AhPNPII: purine nucleoside phosphorylase type II from Aeromonas hydrophila ; CkPNP: purine nucleoside phosphorylase type I from Citrobacter koseri [14, 16].
5. Figure 5.5 Enzymatic synthesis of 5‐fluoro‐2′‐deoxyuridine (floxuridine) catalyzed by different immobilized NPs. Best results were afforded in10 mM phosphate pH 7.5 and nucleoside/nucleobase 3 : 1 molar ratio. Examples of immobilized NPs: EcTP, thymidine phosphorylase from E. coli; BsPyNP, pyrimidine nucleoside phosphorylase from Bacillus subtilis.
6. Figure 5.6 Enzymatic synthesis of nucleoside analogues catalyzed by immobilized nucleoside 2′‐deoxyribosyltransferases (NDTs): NDT from Lactobacillus reuteri (LrNDT) [23]; NDT from Bacillus psychrosaccharolyticus (BpNDT)(21); and NDT from Lactobacillus animalis (LaNDT) [26, 27].
7. Figure 5.7 Two approaches for the enzymatic synthesis of 5‐trifluorothymidine (trifluridine) employing immobilized nucleoside 2′‐deoxyribosyltransferase [21, 28] and immobilized nucleoside phosphorylases [17].
8. Figure 5.8 Enzymatic synthesis of dietary nucleoside‐5′‐monophosphates employing immobilized hypoxanthine–guanine–xanthine phosphoribosyltransferase from Thermus thermophilus HB8 (TtHGXPRT) onto MagReSyn® aldehyde microspheres. Reaction conditions: 12 mM tris/HCl pH 8.0 and 60 °C.
9. Figure 5.9 Enzymatic synthesis of 1‐[2‐chloro‐6‐[(2,2‐diphenylethyl)amino]‐9H‐purin‐9‐yl]‐1‐deoxy‐β‐D‐ribofuranuronic acid from 2‐chloro‐N‐(2,2‐diphenylethyl)‐adenosine, catalyzed by immobilized nucleoside oxidase from Stenotrophomonas maltophilia (FERM BP‐2252). The reaction was carried out in 50 mM potassium phosphate buffer (pH 6), the presence of immobilized beads at 40% (w/v), substrate (20 mg ml⁻¹) and quinol (1 mg ml⁻¹).
10. Figure 5.10 Regioselective acylations of floxuridine catalyzed by immobilized lipase from Burkholderia cepacia (PSL‐C) [41, 42].
11. Figure 5.11 Synthesis of inosine esters catalyzed by immobilized lipase from Mucor miehei in organic solvents.
12. Figure 5.12 Regioselective acylation of ara ‐C catalyzed by immobilized lipase B from Candida antarctica [45–47]. Highest 5′‐regioselectivity (>99.9%) and conversion (97%) was obtained with the mixture hexane–pyridine (1 : 3).
13. Figure 5.13 Enzymatic synthesis of vinyl derivatives of acyclovir and gemcitabine for the preparation of polymerizable prodrugs [52, 53].
14. Figure 5.14 Enzymatic synthesis of gemcitabine‐5′‐O‐lipoate catalyzed by immobilized lipase B from Candida antarctica. This compound shows higher activity compared to its parent counterpart against non‐small cell lung cancer.
15. Figure 5.15 Enzymatic synthesis of valacyclovir catalyzed by immobilized subtilisin A from Bacillus licheniformis.
16. Figure 5.16 Enzymatic resolution of racemic (±) 2′,3′‐dideoxy‐5‐fluoro‐3′‐thiacytidine butyrate using immobilized cholesterol esterase yielding the (–) isomer alcohol (FTC, emtricitabine). Reaction conditions: 5 mM potassium phosphate pH 7.0, 30 °C.
17. Figure 5.17 Synthesis of lamivudine (3TC) by enzymatic resolution of racemic (±) 2′,3′‐dideoxy‐5‐fluoro‐3′‐thiacytidine catalyzed by an immobilized cytidine deaminase from E. coli.
18. Figure 5.18 Enzymatic synthesis of S‐adenosyl‐homocysteine by immobilized CgSAHase from Corynebacterium glutamicum ATCC 13032. Reaction was carried out in 50 mM phosphate buffer pH 7.6 containing adenosine (5–200 μM), (D,L)‐homocysteine (10–400 μM) and 2 g (wet weight) of immobilized enzyme in a final volume of 5 ml.
19. Figure 5.19 Enzymatic synthesis of ribavirin catalyzed by a multi‐enzymatic system containing immobilized phosphopentomutase and immobilized PNP. Reaction conditions: 25 mM phosphate pH 7.0 at 25 °C.
20. Figure 5.20 Enzymatic 5′‐phosphorylation of araA and F‐araA catalyzed by immobilized deoxyribonucleoside kinases (dNKs). DmdNK, deoxyribonucleoside kinase from Drosophila melanogaster; DddAK, deoxyadenosine kinase from Dictyostelium discoideum; ATP, adenosine‐5′‐triphosphate. Reaction conditions: 50 mM ammonium acetate pH 8; MgCl₂ 2 mM; 37 °C [65, 66].
Chapter 06
1. Figure 6.1 Scheme of different multi‐enzymatic reactions. S, substrate; I, intermediate; P, product.
2. Figure 6.2 Mechanism of the biosynthesis of NAs by NPase. B1, B2: purine or pyrimidine base; X: H or OH; E1, E2: NPase (if B1 and B2 are the same type, then E1 and E2 can be the same).
3. Figure 6.3 Multi‐enzymatic synthesis of thymidine in the presence of XODase.
4. Figure 6.4 Multi‐enzymatic synthesis of 2′‐dGR by combining reactions catalyzed by NPase or NDTase and ADase (or AMPDase).
5. Figure 6.5 Multi‐enzymatic synthesis of araG.
6. Figure 6.6 Multi‐enzymatic synthesis of 2′‐F‐2′‐deoxyguanosine and 3′‐deoxyguanosine.
7. Figure 6.7 Multi‐enzymatic synthesis of protected inosine by a combination of CAL and ADase.
8. Figure 6.8 Chemoenzymatic synthesis of ddI.
9. Figure 6.9 Multi‐enzymatic synthesis of 5′‐acetate nelarabine by a combination of transglycosylation and selective esterification.
10. Figure 6.10 Chemoenzymatic synthesis of Doxifluridine.
11. Figure 6.11 Two‐lipase‐catalyzed sequential synthesis of ribavirin derivatives.
12. Figure 6.12 Fluorinase coupled with other enzymes for the synthesis of various ¹⁸F‐labeled compounds.
13. Figure 6.13 Biosynthesis of nucleosides by PPMase and NPase.
14. Figure 6.14 Synthesis of deoxynucleosides by the transglycosylation reaction (path a) and multi‐enzymatic conversion of 2‐deoxy‐D‐ribose (dR) to deoxynucleosides (path b).
15. Figure 6.15 Microbial synthesis of 2′‐deoxyribonucleosides from triosephosphate, acetaldehyde, and nucleobase.
16. Figure 6.16 Multi‐enzymatic synthesis of purine nucleotides.
17. Figure 6.17 Synthesis of adenine nucleoside analogues by FDP aldolase and phosphatase.
18. Figure 6.18 NTP regeneration by isolated enzymes.
19. Figure 6.19 Multi‐enzymatic synthesis of dNMPs coupled with NTP regeneration by ACKase.
20. Figure 6.20 Coupled reaction scheme for the total biosynthesis of dNTP from dNMP.
21. Figure 6.21 Scheme of multi‐enzymatic synthesis of (a) 5F‐UTP and (b) 5F‐CTP.
22. Figure 6.22 Enzymatic synthesis of NDP‐glucose by sucrose synthase.
23. Figure 6.23 Multi‐enzymatic synthesis of CDP‐choline.
Chapter 07
1. Figure 7.1 Possible chemical alteration of nucleotide structure.
2. Figure 7.2 Examples of modified nucleobases with intact Watson–Crick base pairing (C5‐substituted pyrimidine and N7‐substituted purine analogues) that were successfully applied in PCR amplification. (a) Non‐canonical bases. (b) Analogues bearing amino‐acid‐like groups. (c) Analogues bearing fluorescent tags.
3. Figure 7.3 General scheme of in vivo studies with the R67 DHFR gene containing either natural or different DZA‐modified fragments (DZA₃ or DZA₆) conferring trimethoprim resistance ( TmpR ). The samples were cloned into an ampicillin‐resistant vector ( AmpR ) followed by transformation into E. coli cells. The cultures were grown in media containing antibiotics and the resulting viable cells were counted and analyzed.
4. Figure 7.4 Unnatural base pairs that are not occurring in nature. (a) Hydrogen‐bonding base pairs with changed Watson–Crick base pair formation. (b) Specific base pairs formed by hydrophobic interactions based on steric complementary.
5. Figure 7.5 The examples of sugar, phosphodiester backbone (αS‐DNA), and mirror‐image (L‐DNA) nucleic acid analogues.
Chapter 08
1. Figure 8.1 Example of Seley‐Radtke's fleximers.
2. Figure 8.2 Biological advantages of fleximers [3–6].
3. Figure 8.3 Expanded base nucleosides.
4. Figure 8.4 Retrosynthetic method for fleximer adenosine (numbering included for clarity).
5. Synthesis of thieno‐expanded tricyclic adenosine and hypoxanthosine [1, 2]. Reagents and conditions: a. Br₂, AcOH, NaOAc, rt, three hours (71%); b. EtMgBr, Et₂O, rt, three hours (90%); c. (i) β‐D‐ribofuranose‐1,2,3,5‐tetraacetate, BSA, ACN, rt, six hours and (ii) TMSOTf, 60 °C, 18 hours (66%); d. NH₃, MeOH, 100 °C, three hours (quant.); e. (i) NaH, THF, rt, three hours and (ii) BnBr, TBAI, rt, six hours (87%); f. (i) EtMgBr, Et₂O, rt, four hours and (ii) DMF, rt, three hours; g. hydroxylamine HCl, NaHCO₃, EtOH, H₂O, rt, 18 hours; h. Ac₂O, reflux, two hours; i. NH₂C(O)CH₂SH, K₂CO₃, DMF, 55 °C, 12 hours; j. NaOEt, EtOH, reflux, 1.5 hours (26%, five steps); k. triethyl orthoformate, Ac₂O, reflux, three hours (82%); l. (i) P₂S₅, pyridine, reflux, 24 hours, (ii) K₂CO₃, CH₃I, CH₃OH, rt, 10 minutes, and (iii) NH₃, n‐BuOH, 160 °C, 90 hours (60%, three steps).
6. Synthesis of thieno‐expanded and fleximers guanosine, adenosine, and hypoxanthosine [1, 2]. Reagents and conditions: a. (i) NaOH, CS₂, MeOH, 150 °C, 18 hours, (ii) H₂O₂, MeOH, 0 °C, two hours, and (iii) NH₃, MeOH, 120 °C, 12 hours (57%); b. Raney Nickel, MeOH, H₂O, reflux, 18 hours; c. Pd/C, ammonium formate, MeOH, reflux, 18 hours; d. BF₃·OEt₂, EtSH, CH₂Cl₂.
7. Optimized synthesis of thieno‐expanded purine nucleosides [8, 25]. Reagents and conditions: a. (i) BSA, ACN, four hours and (ii) TMSOTf, 60 °C, 18 hours; b. NH₄OH, EtOH, rt, 18 hours (75%, two steps); c. (i) NaH, THF, rt, three hours and (ii) BnBr, TBAI, rt, 18 hours (80%); d. (i) EtMgBr, THF, five hours and (ii) DMF, 18 hours (60%); e. NH₂OH·HCl, NaHCO₃ or pyridine, EtOH, reflux, 2.5 hours (quant.); f. Ac₂O, reflux, three hours (85%); g. NH₂C(O)CH₂SH, K₂CO₃, DMF, 60 °C, 40 hours (50%); h. EtONa, EtOH, 80 °C, one hour (80%); i. (i) NaOH, CS₂, MeOH, 145 °C, 18 hours, (ii) H₂O₂, MeOH, 0 °C, two hours, and (iii) NH₃, MeOH, 120 °C, 18 hours.
8. Synthesis of 2′‐deoxy fleximers starting with the ribose analogue [25, 27]. Reagents and conditions: a. BF₃·OEt₂, EtSH, CH₂Cl₂, 24 hours; b. (i) TMSCl, pyridine, 0 °C to rt, 2.5 hours and (ii) for adenosine, benzoyl chloride, 0 °C to rt, 24 hours, for guanosine, isobytyryl chloride, 0 °C to rt, 24 hours; c. 1,3‐dichloro‐1,1,3,3‐tetraisopropyldisiloxane, pyridine, rt, 24 hours; d. phenyl chlorothionoforrmate, DMAP, rt, 24 hours; e. AIBN, Bu₃SnH, toluene, reflux, six hours; f. (i) 1 M TBAF THF, rt, four hours and (ii) NH₃, MeOH, 40 °C, 18 hours; g. Raney nickel, MeOH, reflux, 18 hours.
9. Synthesis of Hoffer's chloro sugar [33–36]. Reagents and conditions: Pathway A (Hoffer way): a. HCl(g) in MeOH; b. p‐TolCl, pyridine, 0 °C; c. HCl(g), MeOH. (65%, three steps) Reagents and conditions: Pathway B: a. AcCl, MeOH; b. p‐TolCl, pyridine, 0 °C; c. AcCl, AcOH. (73%, three steps).
10. Synthesis of 2′‐deoxy thieno bicyclic nucleoside [9, 27]. Reagents and conditions: a. (i) NaH, CH₃CN, rt, 30 minutes and (ii) 4,5‐diiodoimidazole, rt, 18 hours (84%); b. NaOMe, MeOH, rt, four hours (87%); c. (i) NaH, THF, rt, three hours and (ii) TBAI, p‐methoxybenzylchloride, rt, 18 hours (64%); d. (i) EtMgBr, THF, 0 °C, 15 minutes and (ii) DMF, 0 °C, 20 minutes (90%); e. NH₂OH·HCl, NaHCO₃, H₂O, EtOH, rt, 18 hours (79%); f. CDI, THF, 60 °C, 18 hours (60%); g. NH₂C(O)CH₂SH, K₂CO₃, DMF, 60 °C, 19 hours; h. EtONa, EtOH, 80 °C, six to nine hours (62% in two steps).
11. Optimized synthesis of the thieno‐expanded tricyclic and fleximer nucleosides [9, 27]. Reagents and conditions: a. (i) KSC(S)OEt, DMF, reflux, four hours, (ii) t‐butylhydroperoxide, MeOH, 0 °C two hours, and (iii) NH₃, MeOH, 125 °C, 18 hours. (53%); b. CH(OEt)₃, 4 Å molecular sieves, 150 °C, six hours (88%); c. (i) TPSCl, DMAP, NEt₃, MeCN, three hours and (ii) NH₃, MeOH, 24 hours (65%); d. Raney Ni, MeOH, 65 °C, 72 hours; e. Pd/C, ammonium formate, EtOH, reflux, 18 hours; f. BF₃·OEt₂, EtSH, CH₂Cl₂.
12. Figure 8.5 Retrosynthetic outline for constructing the xanthosine fleximer.
13. Failed attempts to achieve the xanthosine fleximer. Reagents and conditions: a. (i) EtMgBr, THF, rt, three hours and (ii) n‐BuLi, C₂Cl₆, −78 °C to rt, 2.5 hours (50%); b. TMEDA, n‐BuLi, bis(pinacolato) diboron, −78 °C to rt, 18 hours; c. (i) EtMgBr, THF, rt, three hours and (ii) TMEDA, n‐BuLi, I₂ −78 °C to rt, 18 hours; d. n‐BuLi or EtMgBr, THF, rt, three hours.
14. Deiodination of 3.3 using sodium sulfite. Reagents and conditions: a. Na₂SO₃, DMF, H₂O, reflux, 18 hours.
15. Coupling of 4(5)‐iodoimidazole to Hoffer's chlorosugar [9]. Reagents and conditions: a. (i) NaH, MeCN, 30 minutes and, (ii) 4(5)‐iodoimidazole, 24 hours, rt; b. NaOMe, MeOh, four hours, rt; c. TBDMSCI, imidazole, DMF, 18 hours.
16. Synthesis of 2′‐deoxyxanthosine distal fleximer [9]. Reagents and conditions: a. POBr₃, N,N‐dimethylaniline, toluene, reflux, three hours (97%); b. BnOH, NaOBn, benzene, rt, 18 hours (79%); c. (i) triisopropylborate, THF/toluene (1 : 4), −78 °C, (ii) n‐BuLi, 18 hours, and (iii) HCl; d. (i) 10.4, Pd(PPh₃)₄, DME, 10 minutes and (ii) NaHCO₃, reflux, four hours (75%); e. (i) ammonium formate, Pd/C, EtOH, reflux, 18 hours and (ii) TBAF, THF (53%).
17. Synthesis of 2′‐OH/CH₃ tricyclic nucleosides [26]. Reagents and conditions: a. 1,3‐dichloro‐1,1,3,3‐tetraisopropyldisiloxane, pyridine, rt, 14 hours (81%); b. PCC, CH₂Cl₂, 4 Å molecular sieves, rt, 12 hours (67%); c. (CH₃)₃Al, CH₂Cl₂, 0 °C to rt, two hours (73%); d. TBAF, THF, rt, five hours (76%); e. (i) NaH, THF, rt, three hours and (ii) TBAI, BnBr, rt, 18 hours (65%).
18. Synthesis of 2′‐CH₃/F Flex‐G and its McGuigan Protide [40]. Reagents and conditions: a. BzCl, pyridine, 0 °C, 10 minutes, rt, 30 minutes (80%); b. LiAl(OtBu)₃H, THF, −20 °C, six hours (85%); c. 4,5‐diiodoimidazole, Ph₃P, DIAD, THF, 0 °C to rt, 24 hours (20%); d. MeONa, MeOH, 0 °C, one hour (85%); e. (i) NaH, DMF, 0 °C, one hour and (ii) TBAI, BnBr, rt, four hours (81%); f. t‐BuMgCl, DMF, 0 °C to rt, 18 hours.
19. Figure 8.6 Retrosynthetic outline for synthesizing proximal analogues.
20. First synthesis of the guanosine proximal fleximer [8]. Reagents and conditions: a. (CH₃)₂SO₄, H₂O, reflux, two hours (80%); b. (SnBu₃)₂, PdCl₂(PPh₃)₂, toluene, 90 °C, four hours (62%); c. 7.1, Pd catalysts; d. 7.1, CuTC, NMP, rt, 18 hours (20%); e. guanidine, NaOEt, EtOH, reflux, 24 hours (trace).
21. Synthesis of xanthosine and 2,6‐diaminopurine proximal fleximers [8]. Reagents and conditions: a. NaH, BnOH, toluene, rt, 18 hours (91%); b. (i) triisopropylborate, THF/toluene (1 : 4), −78 °C, (ii) n‐BuLi, rt, 18 hours, and (iii) HCl (95%); c. (i) 13.1, Pd(PPh₃)₄, DME, 10 minutes and (ii) NaHCO₃, reflux, four hours (67%); d. NH₃, CH₃OH, 170 °C, 96 hours (40%); e. Pd/C, ammonium formate, EtOH, reflux, two hours.
22. Synthesis of isoguanosine and guanosine proximal fleximers [8]. Reagents and conditions: a. NaNO₂, AcOH, H₂O/THF (1 : 1), 60 °C, two hours; b. Pd/C, ammonium formate, EtOH, reflux, two hours (41%, two steps); c. NaHSO₃, H₂O, 60 °C, 10 hours (88%).
23. Synthesis of 4‐ or 5‐cyanomethylimidazole nucleoside from histidine [7]. Reagents and conditions: a. NaOCI, H₂O, 10–20 °C, three hours, then rt, 18 hours (70%); b. (i) β‐d‐ribofuranose‐1,2,3,5‐O‐tetraacetate, BSA, ACN, three hours and (ii) TMSOTf, 60 °C, 18 hours.
24. Synthesis of the proximal adenosine fleximer through a Diels–Alders cycloaddition [7]. Reagents and conditions: a. 1,3,5‐triazine, NaOMe, MeOH, 40 °C, 18 hours (15%); b. NaNO₂, AcOH, H₂O/THF (1 : 1), 80 °C, five hours (68%).
25. Synthesis of boronic acid pyrimidine precursors [9]. Reagents and conditions: a. (i) triisopropylborate, THF/toluene (1 : 4), −78 °C, (ii) n‐BuLi, rt, 18 hours, and (iii) HCl; b. NaOH, CH₃I, H₂O, 0 °C, four hours; c. Br₂, CHCl₃, MeOH, 12 hours; d. DIAD, PPh₃, BnOH, DMF, 1.5 hours; e. (i) NBS, CHCl₃, rt, 12 hours and (ii) CH₂Cl₂, 1 M NaOH; f. AcCl, pyridine, 0 °C, two hours; g. TMSCl, pyridine; h. (i) Br₂, H₂O, rt, two hours and (ii) 0 °C, 18 hours; i. DMF–DMAc, DMF, rt, 18 hours.
26. Synthesis of tributylstannyl pyrimidine precursors [9]. Reagents and conditions: a. H₂, Pd/C, DIPEA, EtOAc, EtOH, rt, three hours; b. NIS, AcOH, 80 °C, two hours; c. (SnBu₃)₂, Pd₂dba₃·CHCl₃, 65 °C, three hours; d. NaOH, I₂, 80 °C, 18 hours; e. DIAD, PPh₃, BnOH, DMF, 1.5 hours.
27. Synthesis of 4‐iodoimidazole 2′‐deoxynucleoside precursor [9, 27]. Reagents and conditions: a. (i) NaH, CH₃CN, rt, 30 minutes and (ii) 4,5‐diiodo‐imidazole, rt, 24 hours, rt.; b. NaOMe, MeOH, four hours, rt; c. (i) NaH, THF, rt, three hours and (ii) TBAI, BnBr, rt, 18 hours (85%); d. EtMgBr, THF, 0 °C, three hours (80%).
28. Suzuki–Miyaura and Stille couplings to achieve proximal fleximers guanosine, adenosine, hypoxanthosin, and xanthosine [9]. Reagents and conditions: a. 15.3, Pd(PPh₃)₄, DME, NaHCO₃, reflux, four hours; b. ammonium formate, Pd/C, reflux, 18 hours; c. TBSMSCI, imidazole, DMF, rt, 18 hours; d. (i) TBSCI, DMAP, TEA, CH₃CN, rt, 18 hours and (ii) NH₃, THF, 110 °C, 72 hours; e. TBAF, THF, rt, 18 hours; f. Pd₂dba₃·CHCl₃, DMF, 100 °C, 18 hours.
29. Synthesis of 5′‐truncated 1‐deazaneplanocin fleximer derivatives [57]. Reagents and conditions: a. NaBH₄, MeOH, CeCl₃·7H₂O, rt, one hour (quant.); b. 4,5‐diiodoimidazole, DPPE, DIAD, THF, rt, 18 hours (41%); c. EtMgBr, THF, 0 °C, one hour (85%); d. 2‐chloro‐3‐stannylpyridine, Pd(PPh₃)₄, CuBr, 1,4‐dioxane, 120 °C, 12 hours (64%); e. (i) hydrazine, 80 °C, one hour and (ii) TiCl₃, THF, 70 °C, four hours; f. AcOH, 120 °C (56%); g. TFA/H₂O/THF (1 : 1 : 5), 18 hours.
30. Transglycosylation products [63, 65]. Reagents and conditions: a. Pd(PPh₃)₄, Na₂CO₃, H₂O, DMF, 110–120 °C microwave, one to two hours; b. thymidine, L/‐NDT, citrate buffer pH 6.5, 50 °C; c. adenosine, E. coli PNP, phosphate buffer pH 7.4, 50 °C.
31. Robert Hudson's “click” fleximers [66]. Reagents and conditions: a. TMS‐acetylene, Pd(PPh₃)₄, Cul, DMF or THF, NEt₃; b. (i) 1 M NaOH and (ii) HCl; c. Cul, DMEDA, THF, NEt₃, reflux; d. NH₃, MeOH, 0 °C to rt.
32. Figure 8.7 Retrosynthetic outline for synthesizing reverse fleximer analogues.
33. Synthesis of thophene, furan, and thiazole organostannanes [12, 13]. Reagents and conditions: a. n‐BuLi, THF, Bu₃SnCl, −78 °C; b. n‐BuLi, THF, TMSCI, −78 °C.
34. Synthesis of neplanocin and aristeromycin reverse fleximers [13]. Reagents and conditions: a. N ³‐benzoyluracil or N ³‐benzoyl‐5‐bromouracil, DPPE, DIAD, CH₃CN, rt, 18 hours; b. NH₃, MeOH, rt, 12 hours; c. for thiophene, 26.1, 1,4‐dioxane, PdCl₂(PPh₃)₂, 90 °C, 18 hours, for furan, 26.2, 1,4‐dioxane, PdCl₂(PPh₃)2, 90 °C, 18 hours; for thiazole, 26.4, THF, Pd(PPh₃)₄, reflux, 72 hours; d. TFA/H₂O (2 : 1), rt, six hours; e. Pd/C, MeOH, H₂, 25 psi; f. NBS, NaN₃, DME, H₂O, rt, 24 hours.
35. Carbocyclic 5′‐nor “reverse” fleximers [12]. Reagents and conditions: a. peracetic acid, NaOAc, NaHCO₃, CH₂Cl₂, four hours; b. AcOH, Pd(PPh₃)₄, THF, 0 °C to rt, 18 hours (39%, two steps); c. Pseudomonas cepacia lipase, potassium phosphate buffer pH 7.4, acetone, 1 N NaOH, four hours, (49%); d. NaH, DMF, Pd₂dba₃, DPPP, N ³‐benzoylated‐5‐bromouracil, 55 °C, 72 hours (31%); e. NH₃, MeOH, rt, three hours (77%); f. for thiophene, 26.1, 1,4‐dioxane, PdCl₂(PPh₃)₂, 120 °C, 18 hours; for furan, 26.2, 1,4‐dioxane, PdCl₂(PPh₃)₂, 120 °C, 18 hours; for thiazole, 26.4, THF, Pd(PPh₃)₄, reflux, 72 hours.
36. Synthesis of acyclic halogenated imidazole nucleosides [10, 11]. Reagents and conditions: a. Ac₂O, H₂SO₄, −15 °C to rt, 18 hours (52%); b. 4,5‐diiodoimidazole, BSA, TMSOTf, CH₃CN, rt to 90 °C, 18 hours (47%); c. NH₄OH, EtOH, rt, 16 hours (74%); d. (i) NaH, THF, rt, three hours and (ii) TBAI, BnBr, rt, 12 hours (70%); e. EtMgBr, THF, −15 °C to rt, four hours (78%); f. Na₂SO₃, 30% EtOH/H₂O, 120 °C, 18 hours (84%); g. Ac₂O, TEA, DMAP, CH₂Cl₂, rt, two hours (97%).
37. Acyclovir fleximers' construction through palladium‐ catalyzed cross‐coupling reactions [10, 11]. Reagents and conditions: a. for Bn, Pd(PPh₃)₄, Cul, TBAF, DMF, 45 °C, 18 hours (42%); for Ac and OH, b. Pd₂dba₃·CHCI₃, PPh₃, Cul, TBAF, DMF, 50 °C, 18 hours (20%); c. for Bn, Pd/C, ammonium formate, EtOH, 120 °C, 18 hours; d. for Ac, NH₄OH, EtOH, rt, 18 hours; e. BBr₃, CH₂Cl₂, −15 °C to rt, 18 hours (49%); f. Pd(PPh₃)₄, NaHCO₃, 1,2‐dimethoxyethane, reflux, four hours (21%); g. NH₃, MeOH, 210 °C; h. 30.5, t‐BuMgCl, THF, rt, 2.5 hours.
Chapter 09
1. Figure 9.1 O ⁶‐alkyl guanine derivatives and methods for the evaluation of hAGT activity. (a) Chemical structure of O ⁶‐alkyl 2′‐deoxyguaninosine derivatives. (b) Fluorescence assay based on the conformational changes of the thrombin‐binding aptamer. (c) DNA origami as sensor for hAGT activity. (d) dsDNA FRET method for the quantification of hAGT activity.
2. Figure 9.2 Non‐canonical nucleic acids structures. (a) the parallel triplex (b) the i‐motif, and (C) the G‐quadruplex. In the top, is shown a scheme of base‐pairing units, T‐A·T and C‐G·C+ triads is present in parallel triplex, C:C+ in i‐motif and G‐tetrads in quadruplex. In the bottom are depicted schemes of the corresponding tridimensional structures.
3. Figure 9.3 Scheme of modified nucleoside backbones used in the study of non‐canonical DNA structures.
4. Figure 9.4 (a) Scheme of the RNA interference pathway. (b) Scheme of Ago2 and small interfering RNA (siRNA) molecule. Top strand is the passenger or sense and bottom strand is the guide or antisense. (c) Chemical structure of some of the modifications described for the 3′‐overhang and 5′‐end.
Chapter 10
1. Figure 10.1 Nucleoside J.
2. Figure 10.2 Three different types of COCs depending on the location of the carbohydrate on the oligonucleotide.
3. Figure 10.3 Examples of carbohydrate phosphoramidite derivatives showing the different positions of the sugar for the conjugation to the oligonucleotide. C1–C6 indicate the position of the pyranose ring to be linked to the oligonucleotide.
4. Figure 10.4 Examples of carbohydrates attached to the solid support and ready for oligonucleotide synthesis.
5. Figure 10.5 Examples of carbohydrates attached to the nucleoside and ready for oligonucleotide synthesis.
6. Figure 10.6 Schematic synthesis of HA₆‐COC via oxime chemistry.
7. Figure 10.7 Synthesis of COC using alkynyl sugar and azide modified ODN for the coupling CuAAC reaction.
8. Figure 10.8 Schematic synthesis of mannose‐BSA‐TFO conjugate.
9. Figure 10.9 Schematic synthesis of COC combining enzymatic incorporation of alkynyl modified nucleosides and posterior CuAAC reaction with azidosugars.
10. Figure 10.10 (a) COC using a dsDNA scaffold. (b) COC using a G‐quadruplex scaffold. (c) COC using a three‐way‐junction scaffold.
11. Figure 10.11 Schematic representation of the synthesis of a COC with a dendrimeric scaffold.
12. Figure 10.12 (a) COC based on a peptoid scaffold. (b) COC based on a mannose scaffold. (c) COC based on a uracil nucleoside scaffold.
13. Figure 10.13 (a) Triantennary GalNAc‐siRNA conjugate and its schematic representation. (b) Schematic representation of sequentially linked GalNAc. (c) Schematic representation of GalNAc linked to the sense strand.
14. Figure 10.14 Schematic representation of: (a) dangling‐end DNA model system to study carbohydrate–aromatic interactions; (b) G‐quadruplex carbohydrate conjugate; and (c) DNA double helix containing a carbohydrate–nucleobase pair.
15. Figure 10.15 (a) Cellobiose stacking on the terminal T–A pair of a DNA duplex. (b) Top view of fucose–thrombin binding aptamer (TBA) quadruplex conjugate. Fucose (red) stacks on the G‐tetrad and interacts with the bases of the loop (green). (c) Side view (top) of DNA helix containing a 6‐deoxyglucose–guanine pair (bottom).
Chapter 11
1. Figure 11.1 Schematic representation of the Maskless Array Synthesis system, where UV light is reflected on the Digital Micromirror Device. “ON” mirrors (black lines tilted toward the surface) reflect light onto the DNA chip. “OFF” mirrors (black lines tilted away from the surface) reflect light away from the chip.
2. Figure 11.2 Synthetic cycle for the incorporation of an NPPOC‐protected phosphoramidite in the in situ synthesis of microarrays by photolithography. B = adenine, guanine, cytosine or thymine (B₁ → B_n in the 3′ → 5′ direction). R = base protecting group (tert‐butylPac for adenine, iPrPac for guanine, isobutyryl for cytosine).
3. Figure 11.3 3D representation of the MAS optical system.
4. Figure 11.4 Exploded and section view of the new reaction cell allowing for the synthesis of two identical microarrays in parallel.
5. Figure 11.5 Chemical structures of the NPPOC, Bz‐NPPOC, and SPh‐NPPOC 5′‐photoprotected deoxynucleotides and illustrated representation of the amount of light necessary to complete the photocleavage reaction.
6. Figure 11.6 Synthesis time for high‐density gene expression arrays (60mers) according to various fabrication protocols. Parameters were optimized and resulted in the elaboration of the “express” protocol.
7. Figure 11.7 Exploded and assembled view of the UV LED as a source of UV light. The LED is soldered through a copper block to a heat sink, which is attached to a standard CPU fan‐based cooler.
8. Scheme 11.1 Synthesis of the 5′‐NPPOC 2′‐O‐ALE RNA phosphoramidites. Ade, adenine; Cyt, cytosine; Gua, guanine; Ura, uracil; lev, levulinyl; dmf, dimethylformamidine; CNEt, cyanoethyl.
9. Figure 11.8 (Left) Coupling efficiencies of poly‐rU (a) and poly‐rA (c) microarrays. For each sequence length (1–12), three features are synthesized: one with a terminal Cy3 label, one without terminal labeling, and one coupling reference. (Right) hybridization of poly‐rU (b) and poly‐rA (d) sequences (1–12 nt) to their complementary Cy5‐dA₂₀ and Cy5‐dT₂₀, respectively.
10. Figure 11.9 Gradient of exposure on a rU₂₀ microarray. (a) Cy5 emission scan of the hybridized rU₂₀:dA₂₀‐Cy5 duplex. (b) Plot of the measured fluorescence intensity as a function of radiant exposure.
11. Figure 11.10 Fluorescence intensity of array‐bound Cy3‐labeled oligonucleotides in contact with RNase A and taken at various points in time.
12. Figure 11.11 Schematic representation of the microarray cleavage procedure leading to LC–MS analysis of the cleaved, deprotected RNA oligonucleotide (rU₁₂).
13. Figure 11.12 Schematic representation of the in situ RNA transcription of dsDNA clusters on a sequencing flow cell. The complementary strand of the ssDNA is first polymerized from a biotin‐labeled primer. Streptavidin is then added to create a biotin–streptavidin block. In a third step, RNA transcription is initiated with RNAP and proceeds until it reaches the roadblock, where RNAP stalls while still being bound to the dsDNA and to the transcribed RNA (in red).
14. Figure 11.13 (a) Schematic structure of the antiparallel G‐quadruplex TBA1 (b) Chemical structure of 2′F‐ANA. (c) Overview of the binding assay on G‐tetrad TBA1 microarrays to thrombin bound to a secondary, Cy3‐labeled aptamer, TBA2.
15. Scheme 11.2 Synthesis of the 2′F‐araT and G phosphoramidites. Thy, thymine. Reagents and conditions: (a) TMS‐Cl, iPrPac‐Cl, pyridine, 5 °C, 45 minutes, 88%; (b) NPPOC‐Cl, THF/pyridine 1 : 1 (v/v), 0 °C, 3 hours, 61% (4); (c) NPPOC‐Cl, pyridine 1 : 1 (v/v), 0 °C, three hours, 45% (4); (d) CEP‐Cl, DIPEA, THF, 1 hour, r.t., 79% (6) and 69% (7).
16. Figure 11.14 Recorded fluorescence intensities of the TBA1 library at a 200 nM thrombin concentration. (a) Higher fluorescence intensities correspond to higher binding affinity. The DNA‐only TBA1 is depicted as “control TBA1.” The double arrow indicates the subrange of fluorescence intensities that was used for K _d measurements. A range of 178 sequences with high thrombin‐binding affinity were identified in this subrange. (b) Average fluorescence intensities for single‐modified TBA1 sequences.
Chapter 12
1. Figure 12.1 Human SAMHD1 as a dNTPase. (a) SAMHD1 is a dNTPase (triphosphohydrolase) that hydrolyzes dNTPs into deoxyribonucleosides (dNs) and inorganic triphosphates. (b) A schematic representation of SAMHD1 protein. Sterile Alpha Motif (SAM) and Histidine‐Aspartic (HD) domain are shown in colored oval (blue and red, respectively) with respective residue numbers. An NMR structure of N‐terminal construct containing SAM domain is shown on the left (PDB accession number 2E8O) (unpublished) and an example of HD domain containing structure determined through X‐ray crystallography is shown on the right (PDB accession number 4mz7) [6]. Both structures are colored according to the schematics below, highlighting corresponding SAM or HD domain.
2. Figure 12.2 Structure of human SAMHD1 tetramer. (a) Active form of SAMHD1 is a tetrameric complex, shown with each monomer represented in a different color in this model (PDB accession number 4mz7) [6]. Allosteric sites (A1 and A2) and catalytic sites are shown with bound nucleotide (dGTP or dATP), colored blue (A1), green (A2), and red (catalytic sites). (b) A detailed view of an active site with bound substrate (dGTP shown) with key catalytic residues His206 and Asp207 shown in sticks. A divalent cation is required for the triphosphohydrolase reaction (Zn shown) and shown in gray sphere. Figures were generated and redered using the program PyMol [21].
3. Figure 12.3 Substrates and activators of Human SAMHD1. (a) Close‐up view of SAMHD1 catalytic site containing two key residues, L150 and Y374, that select against rNTP by inducing molecular clash with the 2′ OH of rGTP (see arrows). This figure was modeled for the rGTP binding to the SAMHD1 catalytic site. SAMHD1 protein is shown in cartoon, with key amino acid residues (L150 and Y374) shown in sticks. GTP is shown in sticks, with the sphere drawn at the position of 2′‐O with a radius of 2 Å, highlighting its proximity to both L150 and Y374 side chains. The measured distance between 2′‐O of the modeled GTP to L150 and Y374 side chains are approximately 2.4 Å at the closest point. The GTP molecule was modeled in SAMHD1 active site (PDB accession number 4MZ7) using the program coot [20]. Figure was generated using the program PyMol [21]. (b) Chemical moieties of dNTPs recognized by human SAMHD1 protein. The moieties and chemical positions recognized by the catalytic site and A1 regulatory site of SAMHD1 are marked with rectangular and ovals, respectively.
4. Figure 12.4 Replication of HIV‐1, HIV‐2, and SIV in macrophages and Vpx‐mediated dNTP elevation in macrophages. (a) SAMHD1‐mediated low dNTP pools restrict HIV‐1 replication in macrophages. Vpx encoded in HIV‐2 and SIV enhances viral infectivity in macrophages by proteosomally degrading SAMHD1 through DCAF1/Cul4A/E3 ligase and elevating cellular dNTPs, ultimately leading to the permissive infection. (b) Intracellular dNTP concentrations in human primary monocyte‐derived macrophages (−Vpx) and activated CD4⁺ T cells. Treatment of macrophages with Vpx‐containing virus‐like particles (+Vpx) elevates dNTP concentration close to that of activated CD4⁺ T cells.
5. Figure 12.5 Mutations found in AGS and human cancers. (a) Summary of SAMHD1 mutations found in AGS (top) and cancers (bottom) reported previously [48, 52, 53]. (b) A representation of selected AGS‐related mutations in SAMHD1. SAMHD1 tetramer (left) and dimer (right) are shown, with locations of mutations indicated by the sphere. Respective positions of allosteric sites (A1 and A2) and catalytic sites are outlined in black and red, respectively. (c) Same as (b), but for cancers. Figures were generated and rendered using the program Pymol [21].

Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives

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