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The University of Manchester Research
How to name atoms in phosphates, polyphosphates, theirderivatives and mimics, and transition state analogues forenzyme-catalysed phosphoryl transfer reactions (IUPACRecommendations 2016)DOI:10.1515/pac-2016-0202
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Citation for published version (APA):Blackburn, G. M., Cherfils, J., Moss, G. P., Richards, N. G. J., Waltho, J. P., Williams, N. H., & Wittinghofer, A.(2017). How to name atoms in phosphates, polyphosphates, their derivatives and mimics, and transition stateanalogues for enzyme-catalysed phosphoryl transfer reactions (IUPAC Recommendations 2016). Pure andApplied Chemistry, 89(5), 653-675. https://doi.org/10.1515/pac-2016-0202Published in:Pure and Applied Chemistry
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Journal: PureandAppliedChemistry
ManuscriptID:
ManuscriptType: Recommendation
Date Submitted by the Author: 18 December 2015, Revised 6 January 2017
Complete List of Authors Blackburn,G.Michael,KrebsInstitute,UniversityofSheffield;Cherfils,Jacqueline,CNRSandEcoleNormalSupérieur,
Cachan;Moss,GerardP.,QueenMaryUniversityofLondon;Richards,NigelJ.,DepartmentofChemistry,IUPUI,
Indianapolis;Waltho,JonathanP.,BiosciencesInstitute,Universityof
Manchester;Williams,NicholasH.,ChemistryDepartment,Universityof
Sheffield;Wittinghofer,Alfred,GroupforStructuralBiology,Max-
Planck-InstitutfürMolekularePhysiologie,Dortmund. Keywords:
Author-suppliedKeywords Phosphatenomenclature,recommendations,N,O,Patomlabels,phosphatestereochemicalnaming,polyphosphates,phosphoryltransfer,atomlabelsfortransitionstates.
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INTERNATIONALUNIONOFPUREANDAPPLIEDCHEMISTRY
2
December 18, 2015 11
INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY 12
ORGANIC AND BIOMOLECULAR CHEMISTRY DIVISION 13
14
HOW TO NAME ATOMS IN PHOSPHATES, POLYPHOSPHATES, THEIR 15 DERIVATIVES AND MIMICS, AND TRANSITION STATE ANALOGUES FOR 16
ENZYME-CATALYSED PHOSPHORYL TRANSFER REACTIONS 17
IUPAC Recommendations 2016 18 19 20
G. Michael Blackburn,a∗ Jacqueline Cherfils,b Gerard P. Moss,c Nigel J. Richards,d Jonathan P. 21 Waltho,e Nicholas H. Williams,f Alfred Wittinghoferg 22 a) Department of Molecular Biology, Krebs Institute, University of Sheffield, S10 2TN, UK 23 b) Laboratoire de Biologie et Pharmacologie Appliquée, CNRS - École Normale Supérieure de 24
Cachan, Cachan, France 25
c) Queen Mary University of London, School of Biological and Chemical Sciences, London E1 26 4NS, UK 27
d) Department of Chemistry, Indiana University Purdue University Indianapolis, IL 46202, 28 USA, and School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK 29
e) Biosciences Institute, University of Manchester, M1 7DN, UK 30 f) Chemistry Department, University of Sheffield, Sheffield S3 7HF, UK 31 g) Group for Structural Biology, Max-Planck-Institut für Molekulare Physiologie, 44227 32
Dortmund, Deutschland 33 34 35 36 Publication of this document by any means is permitted on the condition that it is 37 whole and unchanged. Copyright © IUPAC & De Gruyter38
∗ Corresponding authors: [email protected], [email protected]
3
HOW TO NAME ATOMS IN PHOSPHATES, POLYPHOSPHATES, THEIR 39 DERIVATIVES AND MIMICS, AND TRANSITION STATE ANALOGUES FOR 40 ENZYME-CATALYSED PHOSPHORYL TRANSFER REACTIONS 41
(IUPAC Recommendations 2016) 42
43
Abstract: Procedures are proposed for the naming of individual atoms, P, O, F, N, and S in 44 phosphate esters, amidates, thiophosphates, polyphosphates, their mimics, and analogues of 45 transition states for enzyme-catalyzed phosphoryl transfer reactions. Their purpose is to enable 46 scientists in very different fields, e.g. biochemistry, biophysics, chemistry, computational 47 chemistry, crystallography, and molecular biology, to share standard protocols for the labelling of 48 individual atoms in complex molecules. This will facilitate clear and unambiguous descriptions 49 of structural results and scientific intercommunication concerning them. At the present time, 50 perusal of the Protein Data Bank (PDB) and other sources shows that there is a limited degree of 51 commonality in nomenclature but a large measure of irregularity in more complex structures. The 52 recommendations described herein adhere to established practice as closely as possible, in 53 particular to IUPAC and IUBMB recommendations and to “best practice” in the PDB, especially 54 to its atom labelling of amino acids, and particularly to Cahn-Ingold-Prelog rules for 55 stereochemical nomenclature. They are designed to work in complex enzyme sites for binding 56 phosphates but also to have utility for non-enzymatic systems. 57
Above all, the recommendations are designed to be clear to comprehend and to be user-friendly. 58
59
KEYWORDS: Phosphate nomenclature; recommendations; P, O, F and N atom labels; 60 phosphate stereochemical naming; polyphosphates; phosphate analogues; phosphoryl 61 transfer; atom names for transition states. 62
63
CONTENTS 64
1. Introduction 65
2. Existing Recommendations 66
3. Recommendations for labelling Phosphorus atoms in phosphates 67
4. Recommendations for labelling Oxygen atoms in phosphates 68
5. Recommendations for labelling Fluorine and other atoms in phosphate transition state 69 analogues 70
6. Recommendations for labelling Vanadate and Tungstate analogues of phosphates 71
7. Summary 72
8. References and Notes 73
9. Appendix -A for the use of Cahn-Ingold-Prelog rules for Prochirality 74 75
4
1. INTRODUCTION 76
The advent of stereochemical studies on phosphate esters and diesters with particular reference to 77 their enzyme catalysed reactions, initially through the work of Jeremy Knowles [1] and of 78 Gordon Lowe [2] placed new demands on the nomenclature of the oxygen atoms of the 79 transferring phosphoryl group, PO3
–.a In early work employing thiophosphates made chiral by the 80 specific introduction of oxygen-18 paired with oxygen-16, the direct application of Cahn-Ingold-81 Prelog (CIP) Rules for prochirality [3] resolved the problem by labelling the oxygen atoms (Rp) 82 and (Sp) as appropriate.[4] The more advanced use of 16O, 17O, and 18O bonded to the same 83 phosphorus [5] led to the concept of pro-pro-pro-chirality at phosphorus, which was still capable 84 of CIP identification.[2, 5] However, such isotopic labelling is experimentally demanding and not 85 necessarily applicable to stereochemical problems now more readily amenable to analysis 86 through advances in protein crystallography. The increasing frequency of binary and tertiary 87 structures of proteins in complex with phosphate ester substrates and/or analogues has enabled a 88 rapidly expanding number of enzyme catalysed reactions to be investigated by structural and 89 computational methods.[6, 7, 8] Indeed, there are now over 1600 ligands in the PDB having a 90 phosphoryl group component and they are associated with over 28000 deposited structures. 91 While many of these structures can be, and have been, labelled for their phosphorus and 92 phosphoryl oxygen atoms through current practice, comparative studies of related structures 93 easily identify multiple inconsistencies in labelling that arise from variable methods of naming N, 94 O, and P atoms. 95
This situation has become increasingly complex as a result of the introduction and 96 development of metal fluoride (MFx) analogues of the PO3
– group in studies on transition state 97 analogues (TSA) for phosphoryl transfer enzymes. Trifluoroberyllate (BeF3
–)b is a ground state 98 analogue for phosphate, with characteristic tetrahedral geometry when ligated to anionic oxygen. 99 Tetrafluoroaluminate (AlF4
–)c is a mimic for concerted phosphoryl transfer in multiple enzymes, 100 though it has octahedral geometry. Aluminium trifluoride (AlF3)d forms trigonal bipyramidal 101 (tbp) TSA complexes that have the correct 3-D geometry for concerted PO3
– group transfer but 102 lack its anionic charge. These two values converge in the relatively smaller number of 103 trifluoromagnesate complexes (MgF3
–)e which are both anionic and have tbp geometry. Indeed, 104 some of the AlF3 complexes have been shown in reality to be MgF3
– complexes in solution.[9] 105 The growth in use of these four types of MFx complexes is illustrated in Figure 1. In addition, 106 there are many significant structures of phosphoryl transfer enzyme complexes that include 107 vanadium(V) or tungsten(VI) complexes either as tetrahedral phosphate mimics or as tbp mimics 108 of transition states. The relative growth in use of these six species is presented in Figure 1. The 109
a * The description of the trigonal planar monoanionic termolecular PO3 species as phosphoryl group is in line with long-established usage in biochemistry, biomolecular chemistry, and molecular biology. We note that the IUPAC recommendation is that the species ‘phosphoryl group’ relates to the neutral, diatomic trivalent species ≡PO. b BeF3
–; PDB ligand name, Beryllium trifluoride ion; PDB code, BEF; IUPAC name, Trifluoridoberyllate c AlF4
–; PD ligand code: ALF; IUPAC name Tetrafluoridoaluminate d AlF3; PDB and IUPAC ,name Aluminum fluoride; PDB ligand code: AF3 e MgF3
–; PDB ligand code, MGF; IUPAC name, Trifluoridomagnesate
5
double change from four coordinate tetrahedral PO4 to five coordinate tbp O-MF3-O and six 110 coordinate, octahedral O-MF4-O complexes adds a new dimension to the problem of the atomic 111 description of these complexes. The need to solve this general problem provided the principal 112 motivation for this development of these standardized naming conventions. 113
114
Figure 1. Cumulative numbers of protein structures published in the PDB in successive triennia 115 that contain ligands which are analogues of phosphoryl groups or their transition states (data for 116 mid-2016 extrapolated to 2017). 117
As the development of our protocols progressed, it became apparent to us that a rational, 118 logical set of labels for the 5- and 6-coordinate systems described above could only be 119 established on the basis of a clear definition of the systematic labelling of phosphorus atoms in 120 standard multiple phosphate molecules, that already extends to eight in the case of 121 hexaphosphoinositol bisphosphates.[10] It needed to be followed by a comprehensive system for 122 oxygen atom labelling to include both bridge and non-bridge atoms in linear chains of 123 phosphates, as for the 13 oxygen atoms of 5'-adenosyl 5'''-guanosyl P1,P4-tetraphosphate [11] and 124 the 3 non-isotopically identifiable oxygen atoms of the PO3
– group of terminal phosphates. With 125 those objectives accomplished, our recommendations could then be developed to incorporate the 126 fluorine ligands of MFx systems and also the oxygen atoms of vanadate and tungstate analogues 127 of phosphates and their TSAs. 128
The basic strategy of the recommendations is built on the recognition that a phosphate 129 monoester comprises an alkoxy group and a phosphoryl group (ROH + PO3
–), a monoalkyl 130 diphosphate comprises a phosphate monoester and a second phosphoryl group (ROPO3
– + PO3–), 131
6
a monoalkyl triphosphate comprises a monoalkyl diphosphate and a third phosphoryl group, and 132 so on. For simplicity, we have ignored anionic charges on phosphoryl oxygen atoms and we have 133 treated P=O “double bonds” as P-O single bonds because there is no π-bonding in the phosphoryl 134 group. While we do not seek to claim that our coverage has been exhaustive, we believe that the 135 principles for naming atoms set out here will prove generally applicable to all cognate molecular 136 species which share a geometrical relationship to phosphates, e.g. sulfates, perchlorates, etc. 137
Lastly, we provide an Appendix as a simple guide to the application of Cahn-Ingold-Prelog 138 Rules to label prochiral, non-bridge oxygen atoms in molecules under inspection. 139
2. EXISTING RECOMMENDATIONS 140
Phosphorus Nomenclature and Related IUPAC Recommendations 141
a) The nomenclature of phosphorus-containing compounds of biochemical importance, 142 Recommendations 1976, was published in 1977.[12] It was concerned with the naming of 143 compounds but did not consider the identification of the individual atoms of the phosphate or 144 polyphosphate groups other than to label the phosphates of a nucleoside triphosphate α, β, 145 and γ. It did cover naming of polyphosphates where a bridging oxygen is replaced by a 146 methylene or imino group. A variation on this was proposed in 1980 and revised in 1992.[13] 147
b) A document on the abbreviations and symbols for the description of conformation of 148 polynucleotide chains, Recommendations 1982, was published in 1983.[14] In a related 149 paper, it was proposed that the pro-S oxygen should be OP1 and pro-R should be OP2.[15] 150 This is the reverse of the system proposed here and it is also contrary to CIP nomenclature 151 where Rule 5 gives priority to R over S. We have chosen to adhere to CIP priority Rule 5. 152
c) IUPAC Recommendations for preferred names of derivatives of phosphoric acid are 153 pertinent.[16] They included the application of the CIP rules to chiral phosphates as well as 154 CIP rules for a trigonal bipyramidal and octahedral systems. These are also described in 155 IUPAC inorganic chemistry nomenclature systems for bipyramidal and octahedral 156 structures.[17] 157
158
7
3. RECOMMENDATIONS FOR LABELLING PHOSPHORUS ATOMS IN 159 PHOSPHATES 160
A. Labelling Phosphorus Atoms in Polyphosphate Species 161
A1. Species with One Single Polyphosphate Chain 162 This requires a one-symbol code to describe the position of each phosphorus in a single chain 163 of phosphates. Phosphorus descriptions use a capital letter that serves to discriminate 164 sequential phosphorus atoms in the same chain (PDB usage). 165
a) Phosphorus atoms are named in progression from the RO- end as PA, PB, PG, PD etc.f Hence 166 adenosine 5'-tetraphosphate (PDB ligand: AQP) has phosphorus atoms labelled as PA, PB, 167 PG, PD starting from the ribose 5'-oxygen 168 [Fig. A1a]. 169 Figure A1a 170
b) For P1-(5'-adenosyl)-P5-(5'''-guanosyl) pentaphosphate (PDB Ligand: G5P), phosphorus 171 atoms should be named PA, PB, PG, PD, and PE starting at the 5'-oxygen of the adenosine. 172
c) For the RO- group at the end of a phosphate chain, a nucleoside takes priority over a non-173 nucleoside. Thus in uridine diphosphate glucose (PDB ligand: UPG), PA is bonded to 174 uridine-O5' and PB is bonded to O1'' of glucose [Fig. A1b].g 175
Figure A1b 176
d) A nucleic acid base takes priority over non-nucleic acid base (i.e. adenosine > nicotinamide 177 riboside). Thus in NAD+ (PDB ligand: NAD), PA is bonded to O5' of adenosine with PB 178 bonded to O5''' of the nicotinamide riboside [Fig. A1c]. 179
Figure A1c 180
f PDB usage currently always replaces Pγ with PG as it does not use a Greek/Symbol font. g Here, and throughout, negative charges on phosphates and P=O double bonds are omitted for simplicity.
O
HO OH
AdeO
PO
PO
PO
PO
O O O OO O OO
PD PG PB PA
5'
O
HO OH
UraO
PO
PO
O OO O
PB PA
5'
OHOHO
OH
HO1"
O
HO OH
NicotinamideO
PO
PO
O OO O
PA
5'"5'OAde
HO OH PB
8
e) Nucleosides take priority in their alphabetical order (A > C > G > dT > U). Thus in P1-(5'-181 adenosyl) P4-(5'"-deoxythymidyl) tetraphosphate (Ap4dT) (PDB ligand: 4TA), the 182 phosphorus atoms should be named PA, PB, PG, PD starting at the 5'-oxygen of the 183 adenosine [Fig. A1d]. 184
185
Figure A1d 186
f) Pentoses use CIP priorities: D-ribose > L-ribose > 2-deoxy-D-ribose > 2-deoxy-L-ribose.h 187 Thus a transition state for dAMP kinase should label the four phosphorus atoms PA, PB, PG, 188 PD starting from the adenosine 5'-oxygen [Fig. A1e]. 189
190
Figure A1e 191
g) In phosphonate and phosphoramidate analogues of polyphosphates, phosphorus atoms will 192 be labelled in the same manner as for the parent polyphosphate molecule. Hence for β,γ-193 methylene-GTP (PDB ligand: GCP), phosphorus atoms should be named PA, PB and PG 194 from the 5'-oxygen [Fig. A1f1]. 195
Figure A1f1 196
Likewise, for 2'-deoxyuridine 5'-α,β-imidotriphosphate (PDB ligand: DUP) phosphorus 197 atoms should be named PA, PB and PG from the 5'-oxygen [Fig. A1f2]. 198
199
Figure A1f2 200
201
h This pentose order approximates to CIP Rule 5 priority (R) > (S). This rule will apply particularly to transition states for deoxynucleotide kinases, e.g. where ATP phosphorylates dAMP.
O
HO
AdeO
POPO
PO
PO
O OO
OOO
OO
PA PG
5'"5'OAde
HO OHPB PD
O
O
HO
AdeO
POPO
PO
PO
O OO
OOO
OO
PA PG
5'"5'OAde
HO OHPB PD
O
O
HO OH
GuaO
PO
PCH2
PO
O O OO O O
PG PB PA
5'
O
HO
UraO
PNH
PO OO O
PB PA
OP
O
O O
PG
9
A2. Species with Multiple Single Phosphate Chains 202 This requires a one-symbol code to describe the relationship of each phosphate chain to the 203 parent molecule. 204
a) Inositol polyphosphates require a phosphorus label derived from the identity of the oxygen 205 to which each single phosphate is attached. Thus for myo-inositol 1,3,4,5,6-pentakis-206 phosphate (InsP5) (PDB ligand: 5MY) the phosphorus atoms should be labelled P1, P3, P4, 207 P5, and P6 [Fig. A2a1.i For fructose 1,6-bisphosphate (PDB label: FBP) the phosphorus 208 atoms should be labelled P1 and P6 [Fig. A2a2]. 209
Figure A2a1 Figure A2a2 210
i cf. R. F. Irvine & M. J. Schell, Nature Rev. Molec. Cell Biol. 2, 327-338 (2001).
A3. Species with Multiple Single Phosphate and/or Polyphosphate Chains 211 This requires a two-symbol code to describe (i) the position of each phosphorus in a single 212 chain of phosphates, and (ii) the relationship of that phosphate chain to the parent molecule. 213
a) Species with polyphosphates located on multiple oxygen atoms require a two-symbol code 214 to designate their phosphorus atoms, a numerical code for the oxygen bridging to the parent 215 molecule and an alphabetic code for the position of the phosphorus in the phosphate chain. 216 Thus in pppGpp (PDB ligand: 0O2), the 5'-phosphorus atoms should be named PA5, PB5 217 and PG5, and the 3'-phosphorus atoms 218 named PA3 and PB3 [Fig. A3a]. 219
Figure A3a 220
OPO3
OPO3
OPO3
OPO3O3PO
HO
P5
P1
P3
P4
P61
3 O
OH
OHHO
OPO
OO
POOP1
P6
61O
O
O
OH
GuaO
PO
PO
PO
O O OO O O
PB5
5'
PG5 PA5
O PO P
O
OOO OPB3 PA3
3'
10
b) Inositol polyphosphates having polyphosphate moieties require a two-symbol code to 221 designate their phosphorus atoms. A numerical symbol designates the oxygen to which 222 each single phosphate is attached and an alphabetic code designates the position of the 223 phosphorus in the phosphate chain. In the case of 224 monophosphates, the labels P1, P2, etc. should 225 apply to single phosphorus entities while PAn, PBn 226 will apply to diphosphates, as in PP-InsP5 (PDB 227 ligand: I7P) [Fig. A3b].d 228
Figure A3b 229
4. RECOMMENDATIONS FOR LABELLING OXYGEN ATOMS IN PHOSPHATES 230
B1. Non-terminal Phosphates in Molecules with One Single Phosphate Chain 231 This requires a two-symbol code to describe (i) the identity of the oxygen relative to its 232 congeners and (ii) the identity of the parent phosphorus atom. Oxygen codes use a number 233 first to discriminate oxygen atoms bonded to the same phosphorus, followed by a letter to 234 indicate the parent phosphorus. 235
a) The oxygen linking PA to the carbon moiety of the molecule will retain its regular label. 236 Thus in ATP, O5' bonds PA to the ribose [Fig. B1a1]. In Ap4G, O5' bonds PA to adenosine 237 while O5''' bonds PD to guanosine [Fig. B1a2].[11] 238
Figure B1a1 Figure B1a2 239
b) In each non-terminal phosphoryl group (PO3), the bridging oxygen bonding PX to P(X+1) in 240 the chain should be numbered O3X. Hence in ATP, O3A joins PA to PB, and O3B joins PB to 241 PG [Fig. B1b]. 242
Figure B1b 243
244
OPO3
OPO3
OPO3
OO3PO
O3PO
PO
PO
O OOO
PA5 PB5
PA1
PA2
PA3
PA4
PA6
O
HO OH
AdeO
PO
PO
PO
O O OO O O
O5'
5'
O
HO
GuaO
PO
PO
PO
PO
O O O OO O OO5'"5'
OAde
HO OHO5' O5'"
OH
O
HO OH
AdeO
PO
PO
PO
O O OO O O5'
O3A
O1A O2A
O3B
11
c) In each non-terminal phosphoryl group, the two non-bridging oxygen atoms will be 245 labelled 1 and 2 according to their CIP pro-R- and pro-S-chiralities respectively.j Hence, in 246 ATP, PA will have non-bridging oxygen atoms O1A and O2A for the pro-R and pro-S oxygen 247 atoms respectively [Fig. B1b]. 248
d) Bridging atoms in polyphosphate chains should be given priorities On’ < O3A < O3B < O3G 249 < O3D < etc. (Section B3). This provides a relatively direct CIP route to the assignment of 250 paired non-bridging oxygens on non-terminal 251 PB, PG, etc. that can be illustrated here for O1B 252 and O2B [Fig. B1d]. 253
Figure B1d 254
e) In each non-terminal phosphoryl group (PO3), the bridging oxygen bonding PX to P(X+1) in 255 the chain should be numbered O3X. Hence in ATP, O3A joins PA to PB, and O3B joins PB to 256 PG [Fig. B1b]. 257
f) In chains containing a sulfur atom in a non-bridging, non-terminal position, the sulfur will 258 take the name S1A (for substituent on PA), S1B (for substituent on PB), etc. The non-bridging 259 oxygen then is named O2A, O2B, etc., and the bridging oxygen is O3A, O3B, etc., as above. 260 This is shown for guanosine 5'-(Rp)-α-thio-triphosphate (PDB ligand: GAV) [Fig. B1d]. 261
262
263
264
Figure B1f1 265
In modified polyphosphate chains having two-atom bridges replacing an O3N (where N = A, 266 B, etc.) the bridging atoms X and Y will be labelled X3A and Y4A progressively. Thus in 267 β,γ-oxymethylene-ATP (AdoPOPOCH2P), the PB,PG-bridging atoms are O3B and C4B 268 respectively [Fig. B1e]. 269
Figure B1f2 270
j This nomenclature is widely used in the PDB for oxygen atoms on PA and PG in nucleoside triphosphates but is rather variably used for oxygen atoms on PB.
O
HO OH
AdeO
PO
PO
PO
O OO O O5'
O3AO3B
O
PB
O1B O2B
PAPG
O
HO OH
GuaO
PO
PO
PO
O O SO O O5'
O3A
S1A O2A
O
HO OH
AdeO
PO
PCH2O
O OO OP
O
O O
O3BC4B
12
O
HO OH
NicotinamideO
PO
PO
O OO O
PA
5'"5'OAde
HO OHPB
O1AO2A O1B O2B
O3A
In polyphosphate chains with a bridging oxygen replaced by carbon or nitrogen, the 271 prochiralityk designations may change in consequence. Thus in α,β-methylene adenosine 272 5'-triphosphate (PDB ligand: APC) [Fig.B1f], oxygen atoms O1A and O2A are reversed 273 relative to their designation in ATP [Fig. B1b] because of the changed priority of C3A 274 relative to O5'. 275
276
Figure B1f3 277
B2. Non-terminal Phosphates in Molecules with 278
Multiple Phosphate Chains 279 This requires a three-symbol code to describe (i) the identity of the oxygen relative to its 280 congeners and (ii) two symbols for the identity of the parent phosphorus atom (v.s.) 281
a) In each non-terminal phosphoryl group, the two non-bridging oxygen atoms will be 282 labelled 1 and 2 according to their CIP pro-R and pro-S chiralities respectively. Hence in 283 ppGpp (PDB ligand: G4P), PA5 will have non-bridging 284 oxygen atoms O1A5 and O2A5 for the pro-R and pro-S 285 oxygen atoms respectively, and PA3 will have non-286 bridging oxygen atoms O1A3 and O2A3 for the pro-R and 287 pro-S oxygen atoms respectivelyl [Fig. B2a]. 288
b) 289 290
Figure B2a 291
c) In NAD+, the oxygen atoms on PA will be labelled O1A and O2A for the pro-R and pro-S 292 oxygen atoms respectively, and the oxygen atoms on PB will be labelled O1B and O2B for 293 the pro-R and pro-S oxygen atoms respectively [Fig. B2b]. (NB Ade takes priority over 294 Nicotinamide. Fig. A1d). 295
296
Figure B2b 297
k An Appendix has been added on a simple introduction to the use of CIP Rules on prochirality and the assignment of pro-R and pro-S descriptions. l For simplicity, the designation omits the prime symbol from e.g. O2A3'.
O
HO OH
AdeO
PCH2
PO
PO
O O OO O O5'
O1AO2A
C3APA
O
OH
GuaO
PO
PO
O OO O5'
OP
O PO
OOO O
3'
O1A5 O2A5
O1A3O2A3
PA5
PA3
13
d) In ppIns5p, the oxygen atoms on PA5 will be labelled O1A5 and O2A5 for the pro-R and 298 pro-S oxygen atoms respectively [Fig. B2c]. 299
300 301 302 303
Figure B2c 304
305 306
e) In each non-terminal phosphoryl group (PO3), the bridging oxygen bonding PN to P(N+1) 307 (where N = A, B, etc.) in the chain should be numbered O3Nx, where x designates the parent 308 oxygen of the polyphosphate chain. Hence in ppGpp 309 (PDB ligand: P4G), PA5 is joined to PB5 by O3A5, and 310 PA3 is joined to PB3 by O3A3 [Fig. B2d]. 311
312
313
314
Figure B2d 315 316
B3. Non-terminal Phosphates in Molecules with a Doubly-Capped, Single Phosphate 317 Chain 318 This requires a two-symbol code to describe (i) the identity of the oxygen relative to its 319 congeners and (ii) one symbol for the identity of the parent phosphorus atom (v.s.). 320
There are several examples of natural and non-natural dinucleosidyl polyphosphates with 321 phosphate chain lengths from 2 to 6 phosphates. P1-(5'-adenosyl) P4-(5'"-deoxythymidyl) 322 tetraphosphate (Ap4dT) (PDB ligand: 4TA) [Fig. A1d] and P1-(5'-adenosyl)-P4-(5'"-323 guanosyl) tetraphosphate (Ap4G) [Fig. B1a2] have been discussed above. While the 324 accurate, and sometimes tricky, application of CIP rules can deliver an appropriate 325 descriptor for paired non-bridging oxygens, for symmetrical molecules including Ap3A 326 and Ap5A, the oxygens on the central phosphate are stereochemically identical (being 327 related by C2 symmetry). They become a diastereotopic pair only in a chiral environment, 328 as when bound to a protein, for which an additional Rule is needed. The problem can easily 329 be resolved by the application of an additional priority Rule for sequential phosphates in 330 polyphosphate chains. 331
“Bridging atoms in polyphosphate chains are given priorities that increase with increasing 332 separation from the priority nucleoside oxygen: On' < O3A < O3B < O3G < O3D < etc.” 333
O3A5
O3A3
O
OH
GuaO
PO
PO
O OO O5'
O PO P
O
OO
OO
3'
PA5
PA3
PB5
PB3
OPO3
OPO3
OPO3
OO3PO
O3PO
PO P
O
O
OO
O
PA5
O2A5O1A5
1
3 5
O3A5
O3A3
O
OH
GuaO
PO
PO
O OO O5'
O PO P
O
OO
OO
3'
PA5
PA3
PB5
PB3
14
The application of this rule is illustrated for P1-(5'-adenosyl)-P3-(5'"-adenosyl) 334 triphosphate, Ap3A [Fig. B3a]. By extension, this rule also provides a relatively direct 335 assignment for the non-terminal phosphates of ATP, p4A, etc. It is further illustrated in 336 the Appendix (Section 9.5). 337
338
339
340
Figure B3a 341
B4. Terminal Phosphates in Molecules with Multiple Phosphate Chains 342 This requires a two-symbol code to describe (i) the identity of the oxygen relative to its 343 congeners and (ii) the identity of the parent phosphorus atom (v.s.). The three oxygen 344 atoms of a terminal phosphoryl group (PO3) are pro-pro-chiral. They can thus be labelled 345 according to CIP rules in those (rare) cases where they are identified by isotopes 16O, 17O, 346 and 18O. 347
a) In cases of a terminal phosphoryl oxygen being replaced by e.g. sulfur, fluorine, or 348 nitrogen, the remaining two terminal oxygen 349 atoms are prochiral and can be appropriately 350 identified by CIP chirality rules. Thus, in 351 GTPγS (PDB ligand: GSP), the sulfur has 352 priority to be labelled S1G and the oxygen 353 atoms are labelled O2G (pro-R) and O3G (pro-S) 354 respectively [Fig. B4a]. 355
Figure B4a 356 357
b) Prochirality identification can be applied if one of the three oxygen atoms is promoted 358 relative to the other two. In the context of enzyme-bound nucleotides, such promotion can 359 often be identified by co-ordination of the terminal phosphate to a protein-bound metal ion, 360 typically magnesium. Thus for ATP bound in many kinases, the γ-phosphate is coordinated 361 from one of its three oxygen atoms to magnesium. This oxygen is thus designated O1G. The 362 remaining oxygen atoms are now prochiral and can be identified in the priority series: 363 O3B > O1G > O2G > O3G. CIP rules then 364 designate O2G as the pro-R oxygen and O3G 365 as the pro-S oxygen, as illustrated for ATP 366 bound in phosphoglycerate kinase 367 [Fig. B4b; PDB entry: 1VJC]. 368 369
Figure B4b 370
O
HO
GuaO
PO
PO
PS
O O OO O O
OH
O1GO2G
PGPAPB
O
HO OH
AdeO
PO
PO
P
O
O O O
O
O O
PG PB PA
5'
MgO1G
O2G
O3GO3B
PO
PO
PO
O O OO OO5'
OAde
HO OHO5'
O5'"
O Ade
OHHOO5'"O3A O3B
O1A O1B O2G
PA PGPB
O2A O2B O1G
15
c) In the absence of metal ion coordination to the terminal phosphate, H-bond donation from 371 amino acids in the protein provides a means of priority identification for O1N. Hydrogen 372 bonds are considered only if they have a length ≤ 3.0 Å,m priority will be given according 373 to donor atom XH priority with CIP rules (S > O > N). Hydrogen bonding to the amino acid 374 of lowest primary sequence number will identify O1G in ATP, etc. If there is still ambiguity 375 in the assignment, then backbone NH takes priority over sidechain NH.n This selection 376 makes O2G and O3G prochiral. Hence they can be assigned by application of CIP rules.o 377 Thus in human bisphosphoglycerate mutase (PDB entry: 2A9J), the 3-phosphoglycerate 378 has phosphoryl oxygen coordination from Arg100 and Arg116 to O1A, from Arg117 and Asn190 379 to O2A, and from Arg117 to O3A [Fig. B4c1]. After O1A is promoted by amino acid linkage 380 priority, O2A and O3A are assigned by prochirality rules (O3 > O1A > O2A > O3A). 381
In the case of human protein tyrosine phosphatase ptpn5 (C472S mutant), the tyrosine 382 phosphate moiety is coordinated to residues in the loop Ala474-Arg478 (PDB entry: 2CJZ). 383 Consideration of hydrogen bonds ≤ 3.0 Å shows oxygen O1P coordinated to Gly476 and 384 Ile477; oxygen O2P coordinated to Ala474 and Arg478; and oxygen O3P coordinated to Arg478. 385 Thus we can now designate O1A as being coordinated to the lowest numbered amino acid, 386 Ala474 (it is labelled as O2P in 2CJZ).j The oxygen atom priority is O4' > O1A > O2A > O3A, 387 in which O2A and O3A are designated by CIP rules for prochiralityj as shown (O2A being 388 pro-R and O3A is pro-S) [Fig. B4c2]. (NB There are hydrogen H-bonds from Ser472(OH) to 389 O2P and O3P but both are longer than 3.0 Å and thus are ignored). 390
Figure B4c1 Figure B4c2 391
m The Ångstrom as the unit of distance in protein structures is standard throughout biochemistry, bioorganic chemistry, and molecular biology. The IUPAC use of nm as the IS unit of sub-micrometer distance is readily accommodated by the conversion factor 1 nm = 10 Å. n In determining priorities, coordination to an isolated water is ignored, because the presence or absence of a particular isolated water in a crystal structure can be a function of the structural resolution achieved, which makes water a variable object. However, waters coordinated to metal ions can be used. o For CIP Rules see the IUPAC Blue Book p92. For the use of pro-R and pro-S see “Basic Terminology of Stereochemistry (IUPAC Recommendations 1996)” Pure Appl. Chem. 68, 2193-2222 (1996).
O
P OO
OHO
O
OH
NH2NArg100
NH2
HNH
N Arg116
NH2
H
H
HNHN
Arg117NHH
H
NHHO
Asn190
O1A
O2A
O3AO3
O
P O
OOTyrosine
O
N
Ala474
HNH
N
NH2
HH
N
NH
H
NHO
O1A
O2A
O3A
Arg478
O
O
Ile476
Gly477
O4'
16
B5. Terminal Phosphates in Molecules with Multiple Phosphate Chains 392 This requires a three-symbol code to describe (i) the identity of the oxygen relative to its 393 congeners and (ii) two symbols for the identity of the parent phosphorus atom (v.s.). 394
a) The rules described above (section B3) for single phosphate chains will apply with the 395 addition of a descriptor symbol designating the point of attachment of that chain to the 396 parent molecule. Thus, for human aldolase reductase (PDB entry: 2J8T) the bound NADP+ 397 (PDB ligand: NAP) has the oxygen atoms of PA2 coordinating no metal and hydrogen 398 bonded to Lys262, Ser263, Val264, Thr265
, and Arg268. Thus the oxygen coordinating Ser263 399 takes priority and is named O1A2. The oxygen atom priorities for PA2 are thus O2 > O1A2 > 400 O2A2 > O3A as shown [Fig. B5a]. 401 402
403
404
405
406
407
Figure B5a 408
B6. Isolated Single Phosphates 409 This requires prioritisation of two oxygen atoms by their coordination features thus 410 allowing the third and fourth oxygen atoms to be assigned their prochirality by CIP rules. 411
Isolated phosphate with no metal ions. In a structure of the small G protein Rab-5c with 412 GDP and Pi ligands in the catalytic site (PDB entry: 1Z0D), the isolated phosphate (PDB 413 ligand: PO4) is not metal coordinated. Thus the relative priorities of its 4 oxygen atoms are 414 determined by H-bonds to amino acid residues. Ignoring H-bonds ≥ 3.0 Å, the PDB file 415 assigns O1 coordinated to Ser30(OH), O2 coordinated to Leu80(NH), and O3 coordinated to 416 Lys34(NH3
+). O4 is only coordinated to ligands at distances ≥ 3.0 Å (oxygen atoms 417 numbered as in 1Z0D) (Fig. B6a). (Hence, the PDB priority order is O1 > O3 > O2 > O4). 418
Figure B6a Figure B6b 419
O
P OO
O
NHHN
Arg268
H2N
NH2
HO2A2
O3A2
O1A2
O2
O
OP
Ade
H
Lys262
N Val264H
O
Thr265
OH
OHO
PO
O O O OO5
PA5PB5
PA2
HO
Ser263
O
PO O
O
OHSer30
NH3Lys34
Leu80
HN
Gly79
O
O2
O1
O4O3
O
PO O
O
OHSer30
NH3Lys34
Leu80
HN
Gly79
O
O2P
O1P
O4P
O3P
17
Assigning the top two oxygen priorities as O1P and O2P respectively (Fig. B6b) converts the 420 two remaining oxygen atoms into a prochiral pair. Promoting the ‘front’ oxygen to 18O 421 gives phosphorus S chirality, thus identifying it as pro-S (O4P). By a similar analysis, the 422 ‘rear’ oxygen (H-bonded to Leu80) is pro-R. Hence, the rear oxygen is designated O3P and 423 the front oxygen is O4P (Fig. B6b) (NB The PDB file assigns PA and PB to the GDP ligand). 424
5. RECOMMENDATIONS FOR LABELLING FLUORINE AND OTHER ATOMS IN 425 PHOSPHATE TRANSITION STATE ANALOGUES 426
C1. Tetrahedral Phosphate Mimics – Trifluoroberyllates 427 These use a two-symbol code that may be expanded to four when there are additional 428 fluorine atoms in the species. 429
a) There are over 100 examples of trifluoroberyllates (BeF3–) in the PDB (PDB ligand: BEF). 430
This phosphate mimic is invariably attached to a carboxylate or terminal phosphate 431 oxyanion. Labelling the three fluorine atoms will follow the same rules as for the three 432 oxygen atoms in a terminal tetrahedral phosphate. Prochiralityj identification can be applied 433 if one of the three fluorine atoms is promoted relative to the other two. In the context of 434 enzyme-bound trifluoroberyllates, such promotion can be generally identified by co-435 ordination of one of the fluorine atoms to a protein-bound metal ion, typically magnesium. 436 For example, in β-phosphoglucose mutase (PDB entry: 2WF8), a BeF3 is coordinated to 437 Asp8, while a catalytic magnesium bridges Asp8 and one fluorine. This fluorine is thus 438 identified as F1Be. The prochiral fluorine atoms F2Be and F3Be are designated by CIP rules, as 439 shown in the example [Fig. C1a]. As there is no other fluorine in this structure, these labels 440 can be abbreviated to F1, F2, and F3 respectively. 441 (Note, that in PDB entry 2WF8, these fluorine 442 atoms were labelled F3, F1, and F2 443 respectively). 444
445
Figure C1a 446 447 448
There is one example of a BeF2 moiety bridging two anionic oxygen atoms. In this case, 449 F1Be and F2Be will correspond to the (pro-R) and (pro-S) stereochemistry assigned by CIP 450 rules. Thus in UMPCMP kinase (PDB entry: 4UKD), BeF2 bonds to ADP O3B, and to UDP 451 O3G [Fig. C1b]. The (pro-R) fluorine is thus F1Be and the (pro-S) fluorine is F2Be.p In this 452
p In the case of an (as yet unidentified) symmetrical species, the priority of the two equivalent fluorine atoms will be based on ligand coordination, as shown in Sections C2b and C3c below.
OBeF
FF
Asp8O
Mg
OOH
OHO
HO
HO
H F1Be
F2BeF3Be
18
unique and rather complicated example, CIP rules give priority to O5' over O5''' since 453 adenine (A) takes priority over uridine (U) (Section A1d). 454
455
456
457
Figure C1b 458
b) There may be less common species where there is no metal ion coordinating the 459 trifluoroberyllate. In these, the hydrogen bonding priorities set out in B3c can be applied. 460
c) In an example of multiple metal coordination, and where the distances of separation from 461 both metals to fluorine are less than the sum of the two van der Waals radii, the 462 coordinating metal with higher atomic number will take priority. 463
464
C2. Trigonal Bipyramidal Phosphate Transition State Analogues – Trifluoromagnesates 465 and Aluminum Trifluorides 466
This requires a two-symbol code to describe (i) the identity of the fluorine relative to its 467 congeners and (ii) the identity of the core metal ion. 468
a) For AlF3 (PDB code: AF3), MgF3 (PDB code: MGF), and ScF3 tbp transition state 469 analogues (TSA), the three fluorine atoms are invariably equatorial with two axial oxygen 470 ligands to the 5-coordinate metal. Priority identification can be applied when one of the 471 three fluorine atoms is promoted relative to the other two and directional priority for the 472 two axial ligands is established. In the context of enzyme-bound trifluoromagnesates and 473 aluminates, such promotion is readily identified by closest proximity of one fluorine to a 474 protein-bound metal ion, typically a catalytic magnesium. The direction of viewing is 475 determined by CIP priority of one of the apical (oxygen) atoms over the second and 476 viewing down the priority O-metal bond. Thus in the small G protein, Ras (PDB entry: 477 1OW3), MgF3 is axially coordinated to GDP via O3B and to a water, and CIP priority gives 478 O3B > OH2. Thus the fluorine coordinated to the catalytic magnesium is designated F1Mg. 479 F2Mg and F3Mg are then identified in a clockwise progression from F1Mg when viewed from 480 O3B to Mg [Fig. C2a].q 481
q NB These fluorine atoms are labelled F2, F1, and F3 respectively in PDB entry: 1OW3 (Viewing indicated by magenta arrow).
OMgOP
OP
O
O OF
O FO
PA
5'OAde
HO OH
PB
FH
Mg
OH2O
F2Mg
F3Mg
F1Mg
O3B
Ser21'
Asn171
NH2Mg
OO
Asp184
OH2OH2Mg1Mg2
19
482
483
Figure C2a 484
b) In case of multiple metal ion coordination, and where both distances of separation are less 485 than the sum of the two van der Waals radii, the coordinating metal with highest atomic 486 number will take priority. In cases where two fluorine atoms are coordinated to two 487 equivalent metals, as for cAPK (PDB entry: 1L3R) in which the tbp complex of 488 ADP•MgF3 is liganded to two catalytic magnesium atoms, F1Mg is prioritised as the 489 fluorine coordinated to the magnesium of higher priority. Metal priority shall be determined 490 by its amino acid coordination (see Section B3c). Viewing priority is determined by: 491 O3B > O-Ser21'. In cAPK, one catalytic magnesium is coordinated to Asn171, to Asp184, and 492 to a water; the second magnesium is coordinated to Asp184 and to two waters. Hence, the 493 magnesium linked to Asn171 has priority and is thus coordinated to F1Mg; F2Mg and F3Mg 494 follow in clockwise progression [Fig. C2b]. 495
496
497
498
Figure C2b 499
c) In the absence of fluorine coordination to a metal, hydrogen bonding to amino acids can be 500 used to determine fluorine priority (see section B4c).r 501
d) A significant number of structures in the PDB (>24) have a trigonal bipyramidal complex 502 assigned as tetrafluoromagnesate(2–) (PDB ligand: MF4). The best resolved of these (PDB 503 entry: 1WPG, 2.30 Å resolution) has electron density and bond lengths that can be equally 504 well assigned as a regular Asp351-CO2
–.MgF3–.OH2 complex. This can be labelled as for 505
C2a (above) using coordination to a catalytic Mg to give priority to F1.s 506
r No example of a tbp complex of AF3 or MGF (PDB ligand identities for AlF3 and MgF3
– respt.) having a coordinating divalent metal at good resolution has been lodged in the PDB prior to December 2015). s No analytical work has been yet presented to identify the number of fluorides, e.g. by 19F NMR.
HOMgO
PO
PO
O OF
O FO
PA
5'OGua
HO OH
PB
FH
MgF2Mg
F3Mg
F1Mg
O3B
OMgOP
OP
O
O OF
O FO
PA
5'OAde
HO OH
PB
FH
Mg
OH2O
F2Mg
F3Mg
F1Mg
O3B
Ser21'
Asn171
NH2Mg
OO
Asp184
OH2OH2Mg1Mg2
20
C3. Octahedral phosphate transition state mimics 507 This requires a two-symbol code to describe (i) the identity of the fluorine relative to its 508 congeners and (ii) the identity of the core metal ion. 509
a) For tetrafluoroaluminate, AlF4– octahedral TSA analogues (PDB ligand: ALF), the four 510
fluorine atoms are invariably equatorial with two trans-oxygen ligands to the 6-coordinate 511 aluminium. Priority identification can be applied by promoting one of the four fluorine 512 atoms relative to the other three. In the context of enzyme-bound tetrafluoroaluminates, 513 such promotion is invariably identified by closest proximity of one fluorine to a protein-514 bound metal ion, usually magnesium. The direction of viewing is determined by CIP 515 priority of one of the apical oxygen atoms over the second and viewing down the priority 516 O-metal bond. Thus in the structure of βPGM (PDB entry: 4C4R) the fluorine coordinated 517 to the catalytic magnesium is identified as F1Al while F2Al, F3Al, and F4Al follow in 518 clockwise progression viewed from Asp8-Od1 which has priority over glucose-O6. (NB The 519 corresponding PDB designations are F2, 520 F1, F3, and F4 respectively)t
[Fig. C3a]. 521
522
Figure C3a 523
b) There are at least 3 examples of octahedral 524 trifluoroaluminate complexes (in PDB to 2015) having three fluorines in equatorial 525 positions with the fourth equatorial ligand identified as oxygen. An example of this is the 526 transition state analogue for enzymatic hydrolysis of dUTP (PDB entry: 4DL8). Axial 527 priority for viewing is established by the CIP precedence of O3A > OWat401 [Fig. C3b]. One 528 fluorine is coordinated to two catalytic magnesium atoms and so is designated F1B. A 529 progression viewed in the priority direction then 530 identifies the bridging oxygen as the second 531 priority ligand, O1B, with F2B and F3B completing 532 the clockwise equatorial sequence. 533
Figure C3b 534
c) In case of multiple metal coordination, the 535
t p In cases where there are no other fluorines in the system, the Al designation may be omitted.
Al
F F
FF
OO Asp8
O
OHO
HO
HO
OH
Mg
6F2Al
F3Al
F4AlF1Al
Al
F F
OF
OH2O
PO
dUrd O O
PO
OOMg
Mg
O3AWat401
F1B
F2BF3B
O1B
PA
PG
21
coordinating metal with highest atomic number will take priority, where the distance of 536 separation is less than the sum of the two van der Waals radii (as for C2b above). 537
d) In the absence of fluorine coordination to a metal, hydrogen bonding to amino acids will be 538 used to determine fluorine priority. Thus in the fructose 2,6-bisphosphatase reaction of the 539 enzyme PFKFB3, an AlF4
– complex with His253Ne2 has been described (PDB entry: 540 3QPW. Fig. C3c). This octahedral complex is completed by water coordination trans to the 541 histidine nitrogen. The four fluorine atoms are coordinated F1 to water, F2 to Arg252 and 542 Gln388, F3 to His387 and water, and F4 to Arg252 and Asn259 (fluorines numbered as in PDB 543 3QPW). As F2 is coordinated to Ne of Arg252 and F4 is coordinated to Arg252-NH1, F2 takes 544 priority as its H-bonding is to the nitrogen nearer to Cα of the lowest numbered 545 coordinating amino acid. Hence F1Al is coordinated to Arg252 and Gln388 and the 546 progression to F2Al, F3Al, and F4Al proceeds 547 clockwise as viewed from the water apex of 548 the octahedral complex (CIP priority is O > 549 N, magenta arrow) [Fig. C3c].u 550
551
Figure C3c 552
553
u Coordination to an oxygen of an isolated water is ignored. This is because the presence or absence of water in a PDB structure may be a function of the resolution of the structure, and therefore may vary from one structure to another of the same protein-ligand complex. Also note the use of PDB style numbering for atoms in amino acids (which avoids the use of Greek symbols).
Al
FF
FF OH2
N
HN
N
N
Arg252
NH2
H H
H
H NH
OAsn259
N NH
H OH
His387
NO H
Gln388
ε
1
F1Al
F2Al
F3Al
F4AlHis253
22
6. RECOMMENDATIONS FOR LABELLING VANADATE AND TUNGSTATE 554 ANALOGUES OF PHOSPHATES 555
556
C4. Vanadates 557
Orthovanadate, VO43–
, is encountered as an analogue of phosphate in a variety of forms. 558 They are invariably trigonal bipyramidal and thus mimic a five-coordinate phosphoryl 559 transfer process. 560
a) Monosubstituted vanadate (V). In isolation, vanadate (PDB ligand: VO4) can mimic the 561 transition state for phosphoryl group transfer as a trigonal bipyramidal complex substituted 562 by either one or two axial oxygen ligands that represent nucleophile and leaving group. A 563 typical example is the Xac nucleotide pyrophosphatase/phosphodiesterase structure (PDB 564 entry: 2GSO) where the vanadate is axially coordinated to Thr90. The three equatorial 565 oxygen atoms are numbered O1V, O2V, and O3V with the axial oxygen O4V being trans to the 566 hydroxylic oxygen of Thr90 [Fig. C4a]. The equatorial oxygen coordinated to two zinc ions 567 takes priority and is O1V. The direction of viewing is determined by the priority Thr90 oxygen 568 > O4V (magenta arrow). Thus a clockwise progression 569 identifies O2V at the front and O3V at the rear of the 570 trigonal planar array. 571
Figure C4a 572
573
b) Disubstituted vanadate. A transition state analogue complex for phosphorylation of glucose 574 1-phosphate on O6 by α-phosphoglucomutase has vanadate linearly coordinated by oxygen-3 575 of Ser116 and by oxygen-6 of glucose 1-phosphate (PDB entry: 1C4G). CIP priority analysis 576 gives O6G > O3S. The three equatorial oxygen atoms take priority from O1V by its 577 coordination to cobalt, that substitutes for a native catalytic magnesium. Assignment of O2V 578 and O3V follows a clockwise progression 579 when viewed from O6G (magenta arrow) 580 [Fig. C4b]. 581
582
Figure C4b 583
584
OVO
OO
O
O2V
Thr90Me
Zn ZnO1V
O3V
O4V
OV
O OO
O
O2V
O1V
O3V
O
Ser116
NH
OHHO
OH
OPO3
CoO6G
O3S
23
For the nucleoside-diphosphate kinase from B. burgdorferi, a vanadate transition state 585 complex links ADP and His134 as axial ligands (PDB entry 4DZ6). There is no catalytic 586 metal to coordinate the three equatorial oxygen atoms. Thus, oxygen H-bonded to Lys13 587 takes priority as O1V over oxygen O2V H-bonded to Arg94, while O3V is not H-bonded to any 588 amino acid. These assignments are in accord with those in the PDB entry. 589
c) Trisubstituted vanadate. Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme 590 that catalyzes the hydrolysis of a phosphodiester bond linking a tyrosine residue to a DNA 591 3'-phosphate. Orthovanadate is central in a transition state analogue structure in which 592 vanadium is linked to the tyrosine oxygen, to the 3'-oxygen of the scissile nucleotide, and to 593 His262 of the enzyme (PDB entry: 1RFF). Axial ligand priority is Tyr-O > HisNε2. Equatorial 594 ligand priority is assigned to Thd-O3'. Hence O2V 595 and O3V follow in a clockwise progression when 596 viewed from the Tyr-oxygen [Fig. C4c]. 597
598
Figure C4c 599
c) Cyclic Trisubstituted Vanadate. Trisubstituted vanadate provides a transition state 600 analogue structure for hairpin ribozyme cleavage of a phosphodiester (PDB entry: 1M5O). 601 The axial O2' has CIP priority over the axial O5'. Priority in the three equatorial oxygen atoms 602 is taken by the ribose-O3' leading to 603 assignment of O1V followed by O2V in a 604 clockwise progression [Fig. C4d]. 605
606
607
Figure C4d 608 609
610
OV
N OO
O
O Thy
TyrN
His263O2V
O1V
O3V
VO
OOO2VO1V
O
O
O
Ade
OGua
OHHO
O2'
O5'
O3'
24
C5. Tungstates 611
Tungstate(VI) ion, WO4= (PDB ligand code: WO4) is a mimic of tetrahedral phosphate in a 612
small but significant range of structures in the PDB. In such systems, two oxygen atoms 613 need to be assigned priority to enable the remaining two to be assigned by prochirality rules. 614
a) Isolated tungstate(VI) with two metal ions. In a structure of purple acid phosphatase 615 (PDB entry: 3KBP), an isolated tungstate(VI) ion mimics phosphate. It is coordinated both 616 to zinc and to iron. Zinc, with atomic number 30, takes CIP priority over iron (atomic 617 number 26) and so the two tungstate oxygen atoms coordinated to these metal ions are 618 labelled O1W and O2W respectively (Fig. C5a). The remaining two tungstate oxygen atoms 619 are now prochiral and can be labelled O3W and O4W 620 by CIP rules described above. 621
622
Figure C5a 623
b) Isolated tungstate(VI) with one metal ion. In a structure of a tungstate complex of 624 CheYN59D/E89R, the isolated tungstate(VI) ion is coordinated to manganese and several 625 amino acids (PDB entry: 3RVS). Thus O1W is identified by its coordination to tungsten. 626 Coordination to oxygen gives precedence over coordination to nitrogen. Coordination to 627 oxygen is only considered if the distance of the heavy atoms ≤ 3.0 Å (see Section B3c). 628 Hence O2W is coordinated to Asp59 and takes precedence over the third oxygen that is 629 coordinated to Thr87. The remaining two tungstate oxygen atoms are now prochiral and 630 can be labelled O3W and O4W by CIP rules described above (Fig. C5b). 631
632
Figure C5b 633
634
OFe O
WZn
O
O
Asp164
OO1W
O2W O4W
O3WO
OMn
O
WO
O
OAsp57 O
O1W
O2W
O4W
O3W
3.16Å
2.87Å
OO
Asp59
OThr87
3.05Å 2.70Å
2.52Å
HN Ala88
25
c) Isolated tungstate(VI) with no metal ion. For an isolated WO4– species, a similar 635
procedure of prioritisation by amino acid coordination can be used to identify O1W and 636 O2W. Then O3W and O4W can be assigned by the prochirality procedure. Thus in a 637 structure of Yersinia enterocolitica PTPase complexed with tungstate (PDB entry 3F9A) 638 an isolated tungstate is encircled by a loop of 639 amino acids 404-409 with three of its oxygen 640 atoms coordinated to NHs. As isolated water 641 coordination is ignored,v priority is given to 642 coordination from Arg404 to O1W followed by 643 coordination from Val407 to O2W. Hence, O3W 644 and O4W are now prochiral and can be assigned 645 using CIP rules (Fig. C5c). 646
Figure C5c 647
d) Tungstate(VI) coordinated to a substrate ligand. A compound example of tungstate 648 as a dual analogue of phosphate is found in the structure of a protein of the histidine triad 649 family in which adenosyl 5’-ditungstate (PDB ligand: ADW), an analogue of ADP, is 650 coordinated to His112 (PDB entry: 1KPE). This situation calls for labelling of both 651 tungstens and of seven oxygen atoms, since the first tungstate is a trigonal bipyramidal 652 TSA of PA and the second tungstate is a tetrahedral analogue of PB of ADP. Tungsten WA 653 is equatorially linked to the adenosyl 5'-oxygen and axially linked to His112-Nε2. As in the 654 case of polyphosphates (Section B1c), the bridging oxygen to WB is designated O3A. That 655 enables assignment of the two prochiral equatorial oxygen atoms as O1A and O2A (when 656 viewed in the axial direction O3A to Ne2). For WB, oxygen O3A has highest CIP priority 657 because it is coordinated to WA. The oxygen coordinated to Gly105 takes precedence over 658 the oxygen coordinated to Ser107 and 659 is therefore identified as O1B. This 660 enables the prochiral pair of oxygen 661 atoms to be assigned as O2B and O3B 662 as shown (Fig. C5d). 663
Figure C5d 664
665
v Note that once coordination has reduced the number of non-prioritised oxygen atoms to two, this pair is assigned by application of CIP rules on prochirality
O WO
OO
O1WO2W
O4WO3W
NHHN
NH
O
NHOO
NHO
Arg404Ala405
Gly406
Val407
NH
O
Arg409Gly408
O
O
W
O O
NN
His112
OW
O
O Oadenosyl
NH
HNO
Gly105
HNNH
HO
OSer107
WAWB
O5'5'
O2A
O1B
O2B O3B
O3A
O1A
Ne2
26
7. SUMMARY 666
The recommendations presented here have been developed to describe molecules derived 667 from orthophosphoric acid and its derivatives, analogues, and transition state 668 analogues. We have built on existing usage of nomenclature wherever possible, especially 669 on Cahn-Ingold-Prelog Rules. We have introduced only one Rule to override CIP analysis, 670 and that for the purpose of simplicity of use by non-experts in stereochemistry. In our 671 experience, the recommendations have worked well for the most demanding species we 672 have examined, e.g. C3c and C5d. However, we recognise that the recommendations may 673 be equally relevant to other species with tetrahedral geometry, such as sulfates and 674 sulfonamides, or with tbp or octahedral geometries. We also recognise that there may be 675 existing, or as yet non-existent, structures that could require an extension of these 676 recommendations, and we remain receptive for advice on such problems. 677
678
679
680
681
8. REFERENCES AND NOTES 682
[1] J. R. Knowles. Annu. Rev. Biochem. 49, 877-919 (1980). 683
[2] G. Lowe. Acc. Chem. Res. 16, 244-251 (1983). 684
[3] R. S. Cahn, C. Ingold, V. Prelog. Angew. Chem. 78, 413-447 (1966); Angew. Chem. Int. Ed. 685 Engl. 5, 385-414, and 515 (1966). 686
[4] G. A. Orr, J. Simon, S. R. Jones, G. J. Chin, J. R. Knowles. Proc. Natl. Acad. Sci. USA 75, 687 2230-2233 (1978); K.-F. Sheu, P. A. Frey. J. Biol. Chem. 253, 378-380 (1978); R. L. Jarvest, 688 G. Lowe. J. Chem. Soc. Chem. Commun. 364-366 (1979). 689
[5] S. R. Jones, L. A. Kindman, J. R. Knowles. Nature 275(5680), 564-565 (1978). 690
[6] M. W. Bowler, M. J. Cliff, J. P. Waltho, G. M. Blackburn. New J. Chem. 34, 784-794 691 (2010). 692
[7] S. C. Kamerlin, P. K. Sharma, R. B. Prasad, A. Warshel. Quart. Revs. Biophys. 46, 1-132 693 (2013). 694
[8] G. M. Blackburn, Y. Jin, N. G. Richards, J.P. Waltho, Angewandte Chemie Int. Ed. (2016). 695 DOI to be added. 696
[9] N. J. Baxter, L. F. Olguin, M. Goličnik, G. Feng, A. M. Hounslow, W. Bermel, G. M. 697 Blackburn, F. Hollfelder, J. P. Waltho, N. H. Williams. Proc. Natl. Acad. Sci. USA 103, 698 14732-14737 (2006); N. J. Baxter, G. M. Blackburn, J. P. Marston, A. M. Hounslow, M. J. 699 Cliff, W. Bermel, N. H. Williams, F. Hollfelder, D. E. Wemmer, J. P. Waltho. J. Am. Chem. 700 Soc. 130, 3952-3958 (2008); Y. Jin, M. J. Cliff, N. J. Baxter, H. R. W. Dannatt, A. M. 701
27
Hounslow, M. W. Bowler, G. M. Blackburn, J. P. Waltho. Angew. Chem. Int. Ed. 51, 12242-702 12245 (2012). 703
[10] J. Mishra, U. S. Bhalla. Biophys. J. 83, 1298-1316 (2002). 704
[11] M. A. G. Sillero, A. de Diego, E. Silles, F. Pérez-Zúñiga, A. Sillero. FEBS Lett. 580, 5723-705 5727 (2006). 706
[12] Nomenclature of Phosphorus-Containing Compounds of Biochemical Importance 707 (Recommendations 1976) IUPAC-IUB Commission on Biochemical Nomenclature, Proc. 708 Natl. Acad. Sci. USA 74, 2222-2230 (1977). 709
[13] International Union of Biochemistry and Molecular Biology, Biochemical Nomenclature 710 and related documents, 2nd edition, Portland Press 1992, 109-114, 256-264, and 335 [ISBN 711 1-85578-005-4]. (see also IUPAC-IUB Joint Commission on Biochemical Nomenclature, 712 Pure Appl. Chem. 55, 1273-1280 (1983)). 713
[14] Abbreviations and symbols for the description of conformations of polynucleotide chains. 714 Recommendations 1982, Eur. J. Biochem. 131, 9-15 (1983). 715
[15] Newsletter of the Nomenclature Committees of the International Union of Biochemistry 716 and Molecular Biology, Eur. J. Biochem. 122, 437-438 (1982). [also ref 12, p. 265] 717
[16] IUPAC Nomenclature of Organic Chemistry, IUPAC Recommendations and Preferred 718 Names 2013 (H. A. Favre, W. H. Powell, Eds.), Royal Society of Chemistry, Cambridge, 719 UK, 2014 (a) P-93.2.4 p. 1215-1216, (b) P-93.3.3.5 p. 1222-1223, (c) P-93.3.3.7 p. 1225-720 1226, H. A. Favre, W. H. Powell Royal Society of Chemistry (2013). Corrections 721 http://www.chem.qmul.ac.uk/iupac/bibliog/BBerrors.html; p. 432ff P-42.3-4 Phosphorus 722 acids etc.; p. 915ff P-67 Phosphorus acids etc.; p. 992ff P-68.3 Phosphorus compounds; p. 723 1215ff P-93.2.4 Stereochemistry of phosphates etc.; p. 1420ff P-105 Nucleosides; p. 1425ff 724 P-106 Nucleotides. 725
[17] Nomenclature of Inorganic Chemistry: IUPAC Recommendations 2005, Royal Society of 726 Chemistry (2005), Corrections http://www.chem.qmul.ac.uk/iupac/bibliog/RBcorrect.html 727 which has links to later corrections. PDF:http://www.iupac.org/nc/home/publications/iupac-728 books/books-db/book-details.html?tx_wfqbe_pi1[bookid]=5; p.180ff, IR 9.3.3.4, octahedral 729 coordination; p184ff, IR 9.3.3.6, bipyramidal coordination; p. 189ff, IR 9.3.3.8, chiral 730 octahedral; p. 190ff, IR 9.3.3.10, chiral bipyramid. 731
732
28
9. APPENDIX – A GUIDE FOR THE USE OF CAHN-INGOLD-PRELOG RULES 733 FOR PROCHIRALITY 734 Short procedure for procedure for identification of paired non-bridging oxygen atoms (or 735 paired fluorine atoms) using Cahn-Ingold-Prelog Rules for prochirality (enantiotopicity) 736
1) Two non-bridging oxygen atoms bonded to the same phosphorus are enantiotopic if 737 promoting one of them from isotope-16 to isotope-18 generates the opposite enantiomer 738 compared to promotion of the other.w This is illustrated for methyl phenylphosphonate 739 (Fig. X1). Promoting the ‘front’ oxygen (Step a) gives molecule (A) where the 740 phosphorus is a stereogenic centre and is labelled R in Cahn-Ingold-Prelog nomenclature. 741 Promoting the ‘rear’ oxygen (Step b) gives molecule (B) where the stereogenic 742 phosphorus is labelled S. This analysis is based on the CIP priority rule O(Me) > 18O > 743 16O > C; on viewing the face of the P-centered tetrahedron with the lowest priority ligand 744 (C) at the rear (magenta arrow), a clockwise progression from high to low priority is 745 designated R (as shown) and an anticlockwise progression is S. Because A and B are 746 enantiomers, the two non-bridge oxygen atoms are enantiotopic. In extension, the paired, 747 non-bridge oxygen atoms can be labelled (pro-R) for the front one (clockwise 748 progression) and (pro-S) for the rear one (C, right). 749
Figure X1 750
2) Two non-bridging oxygen atoms bonded to the same phosphorus are diastereotopic if 751 promoting one of them from isotope-16 to isotope-18 generates a different 752 diastereoisomer compared to promotion of the other.x In the case of adenosine 753 5'-diphosphate (ADP), the two non-bridging paired oxygen atoms on PA are 754 diastereotopic. Promoting the ‘front’ oxygen to 18O generates a new stereogenic centre at 755 PA (D; CIP label S) while promoting the ‘rear’ oxygen to 18O generates a stereogenic 756 centre with the opposite sense at PA (E; CIP label R) [Fig. X2]. NB the D and E 757 stereoisomers are not mirror images because the stereochemistry of the D-ribose is 758 unchanged. Since they are not enantiomers, they are termed diastereoisomers.y 759
Figure X2 760 w These oxygen atoms are spectroscopically and chemically non-equivalent in a chiral environment. x These oxygen atoms are spectroscopically and chemically non-equivalent in any environment. y The term diastereoisomer simply embraces all stereoisomers that are NOT enantiomers
AdoOP
OO3P
16O 16O
PB PA
5'
O5'O3Ad eAdoO
PO
O3P
18O 16O
PB PA
5'
O5'O3AAdoO
PO
O3P
16O 18O
PB PA
5'
O5'O3A
D E
S R
PO
Me
16O16O
PO
Me
18O 16O
PO
Me
16O 18O
a bA B
PO
Me
O O
C
pro-R pro-S
R S
29
3) We can now apply the priority rules described in Section X1 to label the non-bridging 761 oxygen atoms on PA in ADP. This analysis begins with the CIP priority rule that ranks 762 di-coordinate oxygen above mono-coordinate oxygen. Thus O5' and O3A rank above the 763 two non-bridging oxygen atoms. For these bridging oxygen atoms, relative priority is 764 determined by the next atom in the chain: priority is given to the atom with the higher 765 atomic number. In the case of ADP, the sub-adjacent atoms along the chain are PB and C5'. 766 Hence, O3A has priority over O5' as P has a higher atomic number than C. The CIP priority 767 ranking is thus O3A > O5' > 18O > 16O (Fig. X3).z 768
769
770 771 772 Figure X3 773
Viewing the P-centered tetrahedron in stereoisomer (D) from the face with 16O at the rear 774 gives an anticlockwise progression from high to low priority ligands (Fig. X3 left) and so 775 PA in D has S-configuration. Hence, the two paired-oxygen atoms in ADP can be labelled 776 pro-S for the front one (as its promotion to 18O makes PA an S chiral centre) and pro-R 777 for the rear one (as its promotion to 18O makes PA an R chiral centre) (Fig. X3 center). 778 We can now apply CIP Rule 5 that gives R priority over S. Thus the pro-R oxygen is 779 labelled O1A and the pro-S oxygen is labelled O2A (Fig. X3 right). 780
4) The rigorous application of the CIP rules almost inevitably means that there are 781 unexpected outcomes. For example, the stereochemistry of the non-bridging oxygen 782 atoms at PB in guanosine 5'-triphosphate (GTP) and in γ-thioGTP (GSP) have opposite 783 assignments. For GTP, the rules for the in-chain atoms flanking PB identify O5' bonded to 784 C5' thereby taking priority over all oxygen atoms bonded to PG (O1G, O2G, O3G). Hence, 785 the priority sequence for the four GTP oxygen ligands at PB is O3A > O3B and thus the 786 front oxygen is pro-R and the rear oxygen is pro-S (Fig. X4a). Hence the pro-R oxygen is 787 labelled O1B and the pro-S oxygen oxygen is O2B (Fig. X4a). 788
By contrast, for GTPγS, the sulfur atom on PG takes CIP priority over O5' with the 789 consequence that O3A takes priority over O3B (Fig. X4b). The result is that in GTPγS (as 790 presented) the rear oxygen is pro-R (and thus O1B) and the front oxygen is pro-S (and thus 791 O2B) (Fig. 4Xb). This is the opposite 3D spatial outcome compared to GTP. We note that 792 a like situation holds for GTPγF but not for γ-amino-GTP. 793
z NB Labelling a non-bridge oxygen with 18O only gives it priority over the 16O oxygen. It does not change its priority relative to the non-bridging oxygen atoms.
AdoOP
OO3P
18O 16O
PBPA
5'
O5'O3A
D
AdoOP
OO3P
O O
PB PA
5'
O5'O3A
pro-Rpro-S
AdoOP
OO3P
O O
PB PA
5'
O5'O3A
O1AO2A
S
30
Figure X4a Figure X4b 794
5) The above problem is readily resolved by a user-friendly solution. 795 The practical use of these interlinked analogues of nucleoside triphosphates, 796
especially for ATP and GTP, calls for a simplified nomenclature system, designed to 797 deliver comparable labels for diastereotopic oxygens on non-terminal phosphates across 798 the series of phosphoramidate, phosphonate, phosphofluoridate, and phosphorothioate 799 analogs of NTPs. That can be achieved by the use of a new nomenclature rule that gives 800 priority to bridging oxygens in an extended polyphosphate chain based simply on their 801 position in the chain, rather than on their, often convoluted, substituent atom priorities: 802
“Bridging atoms in polyphosphate chains should be given priorities that increase with 803 separation from the priority nucleoside oxygen: On' < O3A < O3B < O3G < O3D < etc.” 804
This rule, already described and used in Section 4B1d, is applied here to resolve the 805 nomenclature discrepancy for O1B and O2B encountered in structures X4a,b (above). In the 806 case of GTP analogs, having O3G replaced by another heavy atom, the rule assigns 807 priorities as: O3B > O3A > O1B > O2B (Fig. X5). This now assigns the same descriptors to 808 the prochiral oxygens at PB as that in Figure X4b (and the contrary to that for Figure 809 X4a). 810
F811 i812 g813 u814 r815 e816 817 Figure X5a Figure X5b 818
The simplicity of use of this rule is also illustrated in naming the diastereotopic pairs of 819 oxygens on PA, PB and PG for adenosyl-5' tetraphosphate, p4A (Fig. X5b). The priority 820 order for the bridging oxygens, On' < O3A < O3B < O3G, results in a consistent naming for 821 the non-bridging oxygens relative to their spatial location as O1A, O1B, and O1G (all 822 frontal) and O2A, O2B, and O2G (all rearwards) (Fig. X5b). 823
O
HO OH
GuaO
PO
PO
PX
O18O
OOO
O
PGPB
PA
5'
O5'O3AO3B
GTP analog (X = BH3, F, NR2, OR, S)
pro-S pro-RO2B O1B
O
OH
AdeO
PO
PO
PO
PO
OOOO OOO O5'
O5'HOPA PG
O3A O3B O3G
O1A O1B O1G
PDPB
O2A O2B O2G
O
HO OH
GuaO
PO
PO
PO
O18O OO O O
PGPB
PA
5'
O5'O3AO3B
O
HO OH
GuaO
PO
PO
PS
O18O OO O O
PG PBPA
5'
O5'O3B
GTP GTPγS
pro-Spro-RO2BO1B O1BO2B
pro-S pro-R