Current Protocols in Nucleic Acid Chemistry || Non-Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling

UNIT 4.55Non-Nucleoside Phosphoramidites ofXanthene Dyes (FAM, JOE, and TAMRA)for Oligonucleotide Labeling

Maksim V. Kvach,1 Dmitry A. Tsybulsky,1 Vadim V. Shmanai,1

Igor A. Prokhorenko,2 Irina A. Stepanova,2 and Vladimir A. Korshun2

1Institute of Physical Organic Chemistry, Minsk, Belarus2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

ABSTRACT

This unit describes the preparation of 5- and 6-carboxy derivatives of the xanthenefluorescent dyes fluorescein (FAM), 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein (JOE),and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion tonon-nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. Theuse of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hy-droxyls facilitates the chromatographic separation of isomers of carboxy-FAM andcarboxy-JOE as pentafluorophenyl esters. Acylation of 3-dimethylaminophenol with1,2,4-benzenetricarboxylic anhydride gave a mixture of 4-dimethylamino-2-hydroxy-2′,4′(5′)-dicarboxybenzophenones, easily separable into individual compounds uponfractional crystallization. Individual isomeric benzophenones are precursors of 5- or6-carboxytetramethylrhodamines. The dyes were converted into 6-aminohexanol- (JOE),4-trans-aminocyclohexanol- (FAM and JOE), and hydroxyprolinol-based (TAMRA)phosphoramidite reagents. Curr. Protoc. Nucleic Acid Chem. 52:4.55.1-4.55.33. C© 2013by John Wiley & Sons, Inc.

Keywords: xanthene fluorophores � fluorescein � JOE � TAMRA � protectinggroups � isomer separation � non-nucleoside phosphoramidites � linkers �

fluorescence quenching � energy transfer

INTRODUCTION

Xanthene fluorescent dyes, fluoresceins and rhodamines, are widely used for labelingof oligonucleotide primers and fluorogenic probes for real-time PCR. Very convenientcompounds for bioconjugation are 5- and 6-carboxy derivatives of these dyes. Unpro-tected TAMRA carboxamides and 3′,6′-O-diacyl derivatives of FAM, as well as JOEcarboxamides, are suitable for preparation of phosphoramidite reagents for direct use inautomated oligonucleotide synthesizers.

The unit contains procedures (suitable for multigram scale-up) for preparation of pentaflu-orophenyl esters of FAM and JOE carboxy-derivatives as individual 5- and 6-isomers(Basic Protocol 1), procedures for 5- and 6-isomers of carboxy-TAMRA (Basic Pro-tocol 2), procedures for syntheses of non-nucleoside FAM, JOE, and TAMRA phos-phoramidites (Basic Protocol 3), and procedures for modified oligonucleotides (BasicProtocol 4). These protocols are based on our studies of FAM (Kvach et al., 2007),JOE (Tsybulsky et al., 2012), and TAMRA (Kvach et al., 2009) synthesis. The SupportProtocol describes synthesis of cyclohexanecarboxylic anhydride, a reagent for phenolichydroxyl protection in FAM and JOE dyes.

CAUTION: Wear laboratory coat, gloves, and protective glasses and carry out all opera-tions involving organic solvents and reagents in a well-ventilated fume cupboard.

Current Protocols in Nucleic Acid Chemistry 4.55.1-4.55.33, March 2013Published online March 2013 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142700.nc0455s52Copyright C© 2013 John Wiley & Sons, Inc.

Synthesis ofModifiedOligonucleotidesand Conjugates

4.55.1

Supplement 52

Non-NucleosidePhosphoramiditesof Xanthene Dyes(FAM, JOE, and

TAMRA) forOligonucleotide

Labeling

4.55.2

Supplement 52 Current Protocols in Nucleic Acid Chemistry

NOTE: Strictly speaking, the numbering (5- and 6-carboxy) in compound names at-tributed to nonfluorescent spirolactone tautomeric/isomeric form [in phosphoramiditereagents; see, e.g., Kvach et al. (2009) and Figure 4.55.12 in Basic Protocol 3 below]should be changed (5- → 4′- and 6- → 5′-) upon conversion in an open, fluorescentform (in oligonucleotides). However, in the vast majority of sources, modified oligonu-cleotides are usually designated using reagent numbering, like “5′-(6-FAM)-NNNN. . .”,which is more suitable for comprehension, versus “5′-(5′-FAM)-NNNN. . . ”. Therefore,the reagent numbering (5- and 6-) will be used here for labeled oligonucleotides.

BASICPROTOCOL 1

SYNTHESIS OF PENTAFLUOROPHENYL ESTERS OF 5- AND 6-CARBOXYDERIVATIVES OF 3′,6′-O-DIACYL-FLUORESCEIN (FAM) AND3′,6′-O-DIACYL-4′,5′-DICHLORO-2′,7′-DIMETHOXY-FLUORESCEIN (JOE)

This protocol contains procedures for pentafluorophenyl esters of 5- and 6-carboxyfluorescein-3′,6′-O-dipivalate. The 5- and 6-isomers can be easily separated inmultigram quantities by column chromatography. The use of the cyclohexylcarbonyl(Chc) protecting group instead of pivaloyl (Piv) facilitates the separation of isomers.Moreover, Chc appears to be the protecting group of choice for preparation of JOE(4′,5′-dichloro-2′,7′-dimethoxy-5(6)-carboxyfluorescein) derivatives. These (5- and 6-isomers, pentafluorophenyl esters) are also easily chromatographically separable. Theresulting compounds are convenient reagents for derivatization of amines with FAM andJOE dyes. See Figs. 4.55.1 and 4.55.2.

Materials

N,N-dimethylformamide (DMF)5(6)-Carboxyfluorescein (4), mixture of isomers (prepare as described by Schreder,

1878; Drechsler and Smagin, 1965; Haralambidis et al., 1990; Ueno et al., 2004;Lee and Grissom, 2009; Xu et al., 2011); available also from Acros and ABCR(http://www.abcr.de)

N,N-Diisopropylethylamine (DIEA)Trimethylacetic anhydride (pivalic anhydride; Sigma-Aldrich, cat. no. 143502)Dichloromethane (DCM)Ethyl acetate (EtOAc)1 M sodium phosphate buffer, pH 7.0 (APPENDIX 2A)Magnesium sulfate (MgSO4) anhydrousPentafluorophenol (Sigma-Aldrich, cat. no. 103799)N,N′-dicyclohexylcarbodiimide (DCC; Sigma-Aldrich, cat. no. D80002)Chloroform (stabilized with amylene; Sigma-Aldrich, cat. no. C2432; Acros, cat.

no. 38376; or equivalent can be replaced with DCM stabilized with amylene;Sigma-Aldrich, cat. no. 270997; Acros, cat. no. 11346; or equivalent)

Silica gelToluene, dryCyclohexanecarboxylic anhydride (3; see Support Protocol for synthesis)Hexane2-Chloro-3-hydroxy-4-methoxybenzaldehyde (11; prepare as described by

Faulkner and Woodcock, 1962 and Lyttle et al., 2001)Selenium dioxide (SeO2)30% hydrogen peroxide, aqueous10% (w/v) NaHSO3 solutionSodium sulfate, anhydrousMethanol (MeOH)Acetyl chlorideDiethyl etherTrimellitic anhydride (Sigma-Aldrich cot. no. B4600)


4.55.3

Current Protocols in Nucleic Acid Chemistry Supplement 52

OHO OH

HO2C

O

O

OO O

HO2C

O

O

O O

OO O

O

O

O O

OO O

O

O

O O

OC6F5O

C6F5O

O4

Piv2O, DIEA, DMF, 72 hr

C6F5OH, DCC,EtOAc, 16 hr

OO O

HO2C

O

O

O O

OO O

O

O

O O

OO O

O

O

O O

OC6F5O

C6F5O

O

56 7


Chc2O, DIEA, DMF, 72 hr

+

+

(41%)

(42%)

(25%)

(28%)

8 9 10

Figure 4.55.1 Synthesis of pentafluorophenyl esters of 3′,6′-O-bis(pivaloyl)-fluorescein-5-carboxylic acid (6), 3′,6′-O-bis(pivaloyl)-fluorescein-6-carboxylic acid (7), 3′,6′-O-bis(cyclohexylcarbonyl)-fluorescein-5-carboxylic acid (9), and 3′,6′-O-bis(cyclohexylcarbonyl)-fluorescein-6-carboxylic acid (10).

25% ammonium hydroxide (aqueous)Methanesulfonic acidTin (IV) chloride (SnCl4)Neutral alumina (Fisher A/2374 or Brockman I from Sigma-Aldrich)2-PropanolTriethylamine (Et3N)Concentrated HClPyridine (>99.5 purity)Cyclohexanecarboxylic anhydride (Support Protocol)0.5 M HClBrine (saturated NaCl)

100-mL, 250-mL, 500-mL and 1-L round-bottom flasksDropping funnelCalcium chloride drying tubesRotary evaporator equipped with a vacuum pump1-L separatory funnel1-L Erlenmeyer flaskMagnetic stirrer and Teflon-coated magnetic stirring barsVacuum filtration system: 350- and 200-mL sintered-glass funnels (25- to 50-μm

porosity) and vacuum adaptersMembrane vacuum pump equipped with a vacuum controller (Vacuubrand

MV10NT VARIO, http://www.vacuubrand.com, or similar)Silica-coated aluminum-backed TLC plates with fluorescent indicatorGlass chromatography columns, 8 × 60–cm, 4 × 60–cmHeat gunOverhead stirrerOil bathReflux condensers1-L beaker

Additional reagents and equipment for TLC (APPENDIX 3D) and columnchromatography (APPENDIX 3E)



Labeling

4.55.4


OHO OH

HO2C

O

O

11

OO O

HO2C

O

O

O O

OO O

O

O

O O

OO O

O

O

O O

OC6F5O

C6F5O

O15

16 17


Chc2O, Py, Δ, 1hr

+

(34%) (22%)

CHOCl

OHOMe

OCHOCl

OHOMe

MeO

Cl Cl

OMe

OHCl

OHOMe

12 13 14

SeO2, H2O2,DCM, H2O HCl, MeOH

84% 61%

O

O

OHO2C

SnCl4, MeSO3H

Cl

MeO OMe

Cl Cl

MeO OMe

Cl

MeO

Cl Cl

OMe

Figure 4.55.2 Synthesis of pentafluorophenyl esters of 3′,6′-O-bis(cyclohexylcarbonyl)-JOE-5-carboxylic acid (16) and3′,6′-O-bis(cyclohexylcarbonyl)-JOE-6-carboxylic acid (17).

Prepare 3′,6′-O-bis(pivaloyl)-fluorescein-5(6)-carboxylic acid, pentafluorophenylester (6 and 7)1. In a 500-mL round-bottom flask place Teflon-coated magnetic stirring bar, DMF

(100 mL), and 5(6)-carboxyfluorescein (4; 20.0 g, 53.2 mmol). Stir until solid dis-solves.

2. Connect a dropping funnel and place the flask in an ice bath and cool. Add N,N-diisopropylethylamine (36.3 mL, 213 mmol) dropwise over a period of 15 min andthen trimethylacetic anhydride (23.7 mL, 117 mmol) over a period of 30 min, whilemaintaining the reaction temperature at 0◦ to 5◦C.

3. Replace the dropping funnel with a calcium chloride drying tube and remove the icebath. Protect the flask from light and stir for 72 hr at room temperature.

4. Evaporate the solution using rotary evaporator. Add DCM (170 mL) and EtOAc(350 mL) and dissolve the residue.

5. Transfer the solution in a 1-L separatory funnel and wash three times, each time with300 mL of 1 M sodium phosphate buffer, pH 7.0. Then, transfer the solution in a 1-LErlenmeyer flask and dry the solution over MgSO4 with magnetic stirring (2 hr).

6. Remove the drying agent by vacuum filtration (using membrane vacuum pump withvacuum controller) through 200-mL sintered-glass funnel (25- to 50-μm porosity)in a 1-L round-bottom flask. Evaporate the solution using rotary evaporator.

7. Dry the residue under vacuum (1 mm Hg) for 12 hr.

8. Add Teflon-coated magnetic stirring bar and EtOAc (400 mL), stir, and dissolve thesolid.

9. Dissolve pentafluorophenol (11.75 g, 63.9 mmol) in EtOAc (50 mL) and DCC(13.15 g, 63.8 mmol) in EtOAc (150 mL).

10. Install a dropping funnel and an ice bath to cool the mixture. Add the pentafluorophe-nol solution dropwise over 20 min followed by the DCC solution dropwise over 1 hr.After an additional 2 hr, remove the cooling bath and stir the mixture overnight atroom temperature.


4.55.5


11. Remove the dicyclohexylurea precipitated by vacuum filtration through 200-mLsintered-glass funnel (25 to 50 μm porosity) in a 1-L round-bottom flask. Wash thesolid with dry EtOAc (50 mL). Evaporate the solution using rotary evaporator.

12. Apply the residue to a short silica gel column (8 × 30 cm) and elute with chloroformor DCM. Collect fractions containing compounds with Rf 0.05 to 0.15 (using tolueneas the TLC solvent), combine, and evaporate to dryness.

Yield, 25.3 g of a mixture of 6 and 7.

CAUTION: Chloroform is hazardous, affecting liver and kidneys. Perform chromatog-raphy in a well-ventilated fume cupboard. DCM, a much less hazardous solvent, can beused instead of chloroform.

Use chloroform or DCM stabilized with amylene, not with ethanol (the latter changesthe eluting ability of the solvent thus making it not suitable for purification of lipophiliccompounds 6 and 7). When using volatile DCM, collect fractions from the column usinginert tubing placed in the collecting Erlenmeyer flask or glass test tube; this preventscooling from DCM evaporation and moisture condensation from air.

13. Dissolve the residue in dry toluene (70 mL) with heating using a heat gun and applythe solution to a silica gel column (8 × 60-cm). Elute with toluene, then with 0.1%EtOAc in toluene, and then with 1% EtOAc in toluene. Collect fractions containingcompounds 6 and then 7, and evaporate to dryness.

Yield: 6 (15.6 g, 41%); 7 (9.5 g, 25%).

14. Recrystallize portions of each compound from toluene-hexane and characterize thecompounds by TLC, melting point, high-resolution mass spectrometry (HRMS), and1H, 19F, and 13C NMR.

3′,6′-Bis(2,2-dimethyl-1-oxopropyloxy)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanth-ene]-5-carboxylic acid, pentafluorophenyl ester (6). Rf 0.12 (toluene); mp 233–234 ◦C(toluene–hexane). 1H NMR (CDCl3) δ 8.88 (br. s, 1H, H-4), 8.47 (dd, 1H, J6,7 = 8.0 Hz,4J4,6 = 1.5 Hz, H-6), 7.37 (d, 1H, J6,7 = 8.0 Hz, H-7), 7.10 (d, 2H, J3

′,4

′= J5′,6

′= 1.8Hz, H-4′,5′), 6.87–6.79 (m, 4H, H-1′,2′,7′,8′), 1.37 (s, 18H, CH3). 13C NMR (CDCl3): δ

176.5 (2C, COBut), 167.6 (C3), 161.2 (CO2C6F5), 158.4 (C7a), 153.1 (2C, C4a′,10a′),151.6 (2C, C3′,6′), 141.3 (m, 2C, 1JCF = 253 Hz, 2JCF = 12 Hz, C2′′,6′′), ∼140 (m, 1JCF

∼240 Hz, C4′′), ∼138 (m, 2C, 1JCF ∼240 Hz, C3′′,5′′), 137.1 (C6), 129.4 (C5), 128.7(2C, C1′,8′), 128.2 (C4), 127.2 (C3a), ∼125 (m, C1′′), 125.1 (C7), 118.1 (2C, C2′,7′),115.2 (2C, C8a′,9a′), 110.7 (2C, C4′,5′), 82.2 (C1), 39.3 (2C, CCH3), 27.2 (6C, CH3).19F NMR (CDCl3) δ –152.11 (d, 2F, J2

′′,3

′′ = J5′′

,6′′ = 18.3 Hz, F-2′′,6′′), –156.71 (t,

1F, J3′′

,4′′ = J4

′′,5

′′ = 21.4 Hz, F-4′′), –161.49 (m, 2F, F-3′′,5′′). HRMS (MALDI+): m/z[M+H]+ calcd for C37H28F5O9

+: 711.1648; found: 711.1642.

3′,6′-Bis(2,2-dimethyl-1-oxopropyloxy)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanth-ene]-6-carboxylic acid, pentafluorophenyl ester (7). Rf 0.05 (toluene); mp 202–203 ◦C(toluene–hexane). 1H NMR (CDCl3): δ 8.45 (dd, 1H, J4,5 = 8.0 Hz, 4J5,7 = 1.4 Hz, H-5),8.21 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.96 (br. s, 1H, H-7), 7.10 (br. s, 2H, H-4′,5′), 6.84 (m,4H, H-1′,2′,7′,8′), 1.36 (s, 18H, CH3). 13C NMR (CDCl3) δ 176.5 (2C, COBut), 167.7(C3), 161.2 (CO2C6F5), 153.6 (C7a), 153.0 (2C, C4a′,10a′), 151.6 (2C, C3′,6′), 141.2(m, 2C, 1JCF = 257 Hz, C2′′,6′′), 140.0 (m, 1JCF = 240 Hz, C4′′), 138.0 (m, 2C, 1JCF =241 Hz, C3′′,5′′), 133.4 (C6), 132.3 (C5), 131.0 (C3a), 128.8 (2C, C1′,8′), 126.4 (C7),125.9 (C4), 124.8 (m, C1′′), 118.1 (2C, C2′,7′), 115.2 (2C, C8a′,9a′), 110.7 (2C, C4′,5′),82.3 (C1), 39.3 (2C, CCH3), 27.1 (6C, CH3). 19F NMR (CDCl3) δ –151.75 (d, 2F, J2

′′,3

′′= J5

′′,6

′′ = 18.3 Hz, F-2′′,6′′), –156.69 (t, 1F, J3′′

,4′′ = J4

′′,5

′′ = 21.9 Hz, F-4′′), –161.60(m, 2F, F-3′′,5′′). HRMS (MALDI+): m/z [M+H]+ calcd for C37H28F5O9

+: 711.1648;found: 711.1655.

IMPORTANT NOTE: See Figure 4.55.3 for atom numbering in fluorescein system andpentafluorophenyl residue.



Labeling

4.55.6


a

OO O

O

O

O O

O

O

OO O

O

O

O OMeO

Cl Cl

OMe1'

2'

3'4' 5'

4a' 10a'6'

7'8'

8a'1

3

3a

4

5

6

7

7a

1''

2''3''

4''

5''6''

1'''2'''

3'''

4'''

5'''

6'''

9a'

F

F

F

FF

O

O

1'2'

3'4' 5'

4a' 10a'6'

7'8'

8a'1

3

3a

4

5

6

7

7a

1''

2''3''

4''

5''6''

1'''2'''

3'''

4'''5'''

6'''

9a'

F

F

F

FF

aae

a

aa

e

ee

e

Axial (a) and equatorial (e)protons in cyclohexane ring

9, 10 16, 17

Figure 4.55.3 Atom numbering for NMR assignments in pentafluorophenyl esters of 3′,6′-O-bis(cyclohexylcarbonyl)fluorescein and JOE.

Prepare 3′,6′-O-bis(cyclohexylcarbonyl)-fluorescein-5(6)-carboxylic acid,pentafluorophenyl ester (9 and 10)15. In a 1-L round-bottom flask place Teflon-coated magnetic stirring bar, DMF

(100 mL), and 5(6)-carboxyfluorescein (4; 20.0 g, 53.2 mmol). Stir until solid dis-solves.

16. Connect a dropping funnel, place the flask in an ice bath, and cool the mixture. AddN,N-diisopropylethylamine (36.3 mL, 213 mmol) dropwise over a period of 15 minand then cyclohexylcarboxylic anhydride (28.0 g, 117 mmol) over a period of 30 minwhile maintaining the reaction temperature at 0◦ to 5◦C.

17. Repeat steps 3 to 11 from above, exactly as described for the synthesis of 6 and7. Apply the residue on a short silica gel column (8 × 30 cm) and elute withchloroform or DCM. Collect fractions containing compounds with Rf 0.05 to 0.15(toluene), combine, and evaporate to dryness.

Yield, 28.7 g of a mixture of 9 and 10.

See annotations to step 12, above, for further necessary information on this step.

18. Dissolve the residue in dry toluene (60 mL) with heating using a heat gun and applythe solution to a silica gel column (8 × 60–cm). Elute with toluene, then with 0.1%EtOAc in toluene, and then with 1% EtOAc in toluene. Collect fractions containingcompounds 9 and then 10, and evaporate to dryness.

Yield: 9 (17.1 g, 42%); 10 (11.4 g, 28%).

19. Recrystallize portions of each compound from toluene-hexane and characterize thecompounds by TLC, melting point, HRMS, and 1H, 19F, and 13C NMR.

3′,6′-Bis(cyclohexylcarbonyloxy)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-5-carboxylic acid, pentafluorophenyl ester (9). Rf 0.14 (toluene); mp 147–148 ◦C(toluene–hexane). 1H NMR (CDCl3) δ 8.88 (m, 1H, H-4), 8.47 (dd, 1H, J6,7 = 8.0 Hz,4J4,6 = 1.4 Hz, H-6), 7.37 (d, 1H, J6,7= 8.0 Hz, H-7), 7.11 (m, 2H, H-4′,5′), 6.83 (m,4H, H-1′,2′,7′,8′), 2.57 (tt, 2H, Ja,a = 11.2 Hz, Ja,e = 3.6 Hz, H-1a′′′), 2.05 (m, 4H,H-2e′′′,6e′′′), 1.82 (m, 4H, H-3e′′′,5e′′′), 1.70 (m, 2H, H-4e′′′), 1.59 (m, 4H, H-2a′′′,6a′′′),1.33 (m, 6H, H-3a′′′,4a′′′,5a′′′). 13C NMR (CDCl3) δ 174.0 (2C, COCH), 167.6 (C3),161.2 (CO2C6F5), 158.4 (C7a), 152.8 (2C, C4a′,10a′), 151.6 (2C, C3′,6′), ∼141.5 (m,2C, 1JCF ∼247 Hz, C2′′,6′′), ∼140 (m, 1JCF ∼240 Hz, C4′′), ∼138 (m, 2C, 1JCF ∼240Hz, C3′′,5′′), 137.1 (C6), 129.4 (C5), 128.8 (2C, C1′,8′), 128.2 (C4), 127.2 (C3a), 125.4(m, C1′′), 125.1 (C7), 118.2 (2C, C2′,7′), 115.2 (2C, C8a′,9a′), 110.8 (2C, C4′,5′), 82.2


4.55.7


(C1), 43.3 (2C, C1′′′), 29.0 (4C, C2′′′,6′′′), 25.7 (2C, C4′′′), 25.4 (4C, C3′′′,5′′′). 19F NMR(CDCl3) δ –152.12 (d, 2F, J2

′′,3

′′ = J5′′

,6′′ = 18.3 Hz, F-2′′,6′′), –156.72 (t, 1F, J3

′′,4

′′ =J4

′′,5

′′ = 21.4 Hz, F-4′′), –161.50 (m, 2F, F-3′′,5′′). HRMS (MALDI+): m/z [M+Na]+calcd for C41H31F5NaO9

+: 785.1780; found: 785.1799.

3′,6′-Bis(cyclohexylcarbonyloxy)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-6-carboxylic acid, pentafluorophenyl ester (10). Rf 0.05 (toluene); mp 124–126 ◦C(toluene–hexane). 1H NMR (CDCl3) δ 8.45 (dd, 1H, J4,5 = 8.0 Hz, 4J5,7 = 1.2 Hz, H-5),8.20 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.96 (br. s, 1H, H-7), 7.11 (br. s, 2H, H-4′,5′), 6.84(m, 4H, H-1′,2′,7′,8′), 2.57 (tt, 2H, Ja,a = 11.2 Hz, Ja,e = 3.6 Hz, H-1a′′′), 2.05 (m, 4H,H-2e′′′,6e′′′), 1.82 (m, 4H, H-3e′′′,5e′′′), 1.69 (m, 2H, H-4e′′′), 1.59 (m, 4H, H-2a′′′,6a′′′),1.33 (m, 6H, H-3a′′′, 4a′′′, 5a′′′). 13C NMR (CDCl3) δ 174.0 (2C, COCH), 167.7 (C3),161.2 (CO2C6F5), 153.6 (C7a), 152.8 (2C, C4a′,10a′), 151.6 (2C, C3′,6′), ∼141.5 (m,2C, 1JCF ∼249 Hz, C2′′,6′′), ∼140 (m, 1JCF = 240 Hz, C4′′), ∼138 (m, 2C, 1JCF =241 Hz, C3′′,5′′), 133.4 (C6), 132.3 (C5), 131.1 (C3a), 128.8 (2C, C1′,8′), 126.5 (C7),126.0 (C4), 125.4 (m, C1′′), 118.2 (2C, C2′,7′), 115.2 (2C, C8a′,9a′), 110.7 (2C, C4′,5′),82.4 (C1), 43.3 (2C, C1′′′), 29.0 (4C, C2′′′,6′′′), 25.7 (2C, C4′′′), 25.4 (4C, C3′′′,5′′′).19F NMR (CDCl3) δ –151.76 (d, 2F, J2

′′,3

′′ = J5′′

,6′′ = 18.3 Hz, F-2′′,6′′), –156.70 (t,

1F, J3′′

,4′′ = J4

′′,5

′′ = 22.0 Hz, F-4′′), –161.38 (m, 2F, F-3′′,5′′). HRMS (MALDI+): m/z[M+H]+ calcd for C41H32F5O9

+: 763.1961; found: 763.1979.

Prepare 2-chloro-4-methoxyresorcinol (13), a precursor for JOE dye20. In a 1-L round-bottom flask equipped with an overhead stirrer, place DCM (650 mL),

2-chloro-3-hydroxy-4-methoxybenzaldehyde (11; 37.32 g, 0.2 mol), SeO2 (1.78 g,0.016 mol), and 30% aqueous H2O2 (61 mL, 0.60 mol). Stir vigorously for 48 hr atroom temperature.

CAUTION: Selenium dioxide is toxic.

21. Transfer the mixture in a 1-L separatory funnel, separate, and discard aqueous layer.Wash the organic layer with 10% NaHSO3 (200 mL). Then transfer the solution in a1-L Erlenmeyer flask and dry the solution over Na2SO4 under magnetic stirring (2 hr).

22. Remove the drying agent by vacuum filtration through a 200-mL sintered-glassfunnel (25 to 50 μm porosity) in a 1-L round-bottom flask. Evaporate the solutionusing a rotary evaporator to yield 12 a solid with a slight lilac color.

23. In a 1-L round-bottom flask equipped with a magnetic stirrer, ice bath, and droppingfunnel, place Teflon-coated magnetic stirring bar, MeOH (400 mL), and add acetylchloride (20 mL) dropwise over a 15-min) period at room temperature.

24. Add this solution to the crude 12 from step 22, and stir for 30 min at room temperature.

25. Evaporate the solution using rotary evaporator.

26. Add a Teflon-coated magnetic stirring bar and crystallize the residue from diethylether–hexanes. Collect the desired 13 by vacuum filtration through 200-mL sintered-glass funnel (25 to 50 μm porosity).

Yield: 29.3 g (84%).

27. Characterize the compound by TLC, melting point, and 1H and 13C NMR.

2-Chloro-4-methoxyresorcinol (13). Rf 0.36 (MeOH–CHCl3 1:19 v/v); mp 76–77◦C. 1HNMR (DMSO-d6) δ 9.44 (s, 1H, OH), 9.19 (s, 1H, OH), 6.72 (d, J5,6 = 8.0 Hz, 1H, H-5),6.35 (d, J5,6= 8.0 Hz, 1H, H-6), 3.71 (s, 6H, OCH3). 13C NMR (DMSO-d6) δ 148.0 (C1),143.9 (C3), 141.2 (C4), 111.0 (C5), 108.3 (C6), 104.8 (C2), 56.8 (OCH3).

Prepare 3′,6′-O-bis(cyclohexylcarbonyl)-JOE-5(6)-carboxylic acid,pentafluorophenyl ester (16 and 17)28. In a 100-mL round-bottom flask equipped with an oil bath and a magnetic stir-

rer, place a Teflon-coated magnetic stirring bar, 2-chloro-4-methoxyresorcinol (13)



Labeling

4.55.8


(17.45 g, 0.10 mol), trimellitic anhydride (9.60 g, 0.05 mol), and methanesulfonicacid (50 mL). Stir for 15 min to dissolve solids.

29. Add SnCl4 (5.85 mL, 0.05 mol) in one portion. Connect a reflux condenser equippedwith a calcium chloride drying tube and stir at 40◦C for 6 hr.

30. Pour the mixture into vigorously magnetically stirred water (300 mL) in a 1-L beaker.

31. Collect the solid by vacuum filtration through 200-mL sintered-glass funnel (25 to50 μm porosity).

32. Apply the solid on a neutral alumina column (8 × 60 cm) and elute using a stepgradient from 30% (v/v) component 1 (25% v/v aqueous ammonia) to 50% (v/v)component 1 in component 2 (2-propanol) in steps of 30%, then 35%, 40%, 45%,and 50% component 1. Collect fractions containing both isomers of 14 as the secondand the third colored bands on TLC (1:5:14 (v/v/v) Et3N–MeOH–CHCl3). Combinefractions and evaporate to dryness.

33. Dissolve the residue in water (250 mL), and adjust to pH 2 with concentrated HCl.

34. Collect the solid by vacuum filtration through 200-mL sintered-glass funnel (25-to 50-μm porosity). Dry the solid in a desiccator to obtain 14 as a red-brick solid(15.41 g, 61%).

35. In a 100-mL round-bottom flask, place a Teflon-coated magnetic stirring bar, 5(6)-carboxy-JOE (14; 5.05 g, 10.0 mmol), and pyridine (25 mL), and stir until soliddissolves. Next, add cyclohexanecarboxylic anhydride (7.14 g, 30.0 mmol). Connectreflux condenser equipped with a calcium chloride drying tube, stir, and heat at 70◦Cfor 1 hr.

36. Evaporate the solution using a rotary evaporator.

37. Dissolve in EtOAc (150 mL) and place in a 500-mL separatory funnel. Wash with0.5 M HCl (150 mL) and then brine (saturated NaCl; 150 mL). Then, transfer thesolution to a 500-mL Erlenmeyer flask and dry the solution over MgSO4 undermagnetic stirring (2 hr).

38. Remove the drying agent by vacuum filtration through 200-mL sintered-glass funnel(25- to 50-μm porosity) in a 500-mL round-bottom flask. Evaporate the solutionusing a rotary evaporator.

39. Dry the residue under vacuum (1 mm Hg) for 12 hr.

40. Add a Teflon-coated magnetic stirring bar and EtOAc (30 mL), stir, and dissolve thesolid.

41. Dissolve pentafluorophenol (2.02 g, 11.0 mmol) in EtOAc (7 mL) and DCC (2.47 g,12.0 mmol) in EtOAc (7 mL).

42. Connect a dropping funnel and place the flask in an ice bath to cool the mixture.Add pentafluorophenol solution dropwise over 20 min followed by the DCC solution(dropwise over 1 hr). After an additional 2 hr remove the cooling bath and stir themixture overnight at room temperature.

43. Remove the dicyclohexylurea precipitate by vacuum filtration through 50-mLsintered-glass funnel (25- to 50-μm porosity) in a 250-mL round-bottom flask. Washthe solid with dry EtOAc (15 mL).

44. Evaporate the solution using rotary evaporator.


4.55.9


45. Dissolve the residue in dry toluene (10 mL) with heating using a heat gun andapply the solution to a silica gel column (4 × 40 cm). Elute using a step gradientwith the eluant portions prepared manually: 0%, 0.5%, and 1% (v/v) EtOAc intoluene. Collect fractions containing compounds 16 and then 17, and evaporate todryness.

Yield: 16 (3.0 g, 34%); 17 (2.0 g, 22%).

46. Characterize the compounds by TLC, HRMS, and 1H, 19F, and 13C NMR.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-3-oxo-spiro[isobenzofu-ran-1(3H),9′-[9H]xanthene]-5-carboxylic acid, pentafluorophenyl ester (16). Rf 0.50(EtOAc–toluene 1:19 v/v); mp 240–242 ◦C (toluene–hexane). 1H NMR (CDCl3) δ 8.86(m, 1H, H-4), 8.48 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6 = 1.0 Hz, H-6), 7.41 (d, 1H, J6,7 = 8.0Hz, H-7), 6.24 (s, 2H, H-1′,8′), 3.63 (s, 6H, OCH3), 2.69 (tt, 2H, Ja,a = 11.0 Hz, Ja,e =3.7 Hz, H-1a′′′), 2.12 (m, 4H, H-2e′′′,6e′′′), 1.83 (m, 4H, H-3e′′′,5e′′′), 1.72–1.61 (m,6H, H-4e′′′,2a′′′,6a′′′), 1.43–1.26 (m, 6H, H-3a′′′,4a′′′,5a′′′). 13C NMR (CDCl3) δ 172.4(2C, COCH), 167.5 (C3), 160.9 (CO2C6F5), 158.1 (C7a), 149.1 (2C, C2′,7′), 141.3(2C, C4a′,10a′), ∼141 (m, 2C, 1JCF ∼250 Hz, C2′′,6′′), ∼140 (m, 1JCF ∼250 Hz, C4′′),139.7 (2C, C3′,6′), ∼138 (m, 2C, 1JCF ∼250 Hz, C3′′,5′′), 137.4 (C6), 129.5 (C5), 128.3(C4), 125.6 (C3a), 124.9 (m, 2C, C7,1′′), 118.2 (2C, C4′,5′), 115.2 (2C, C8a′,9a′), 106.5(2C, C1′,8′), 82.3 (C1), 56.6 (2C, OCH3), 42.8 (2C, C1′′′), 28.9 (4C, C2′′′,6′′′), 25.6(2C, C4′′′), 25.2 (4C, C3′′′,5′′′). 19F NMR (CDCl3), δ –152.20 (d, 2F, J2

′′,3

′′ = J5′′

,6′′

= 19.8 Hz, F-2′′,6′′), –156.53 (t, 1F, J3′′

,4′′ = J4

′′,5

′′ = 21.5 Hz, F-4′′), –161.40 (m, 2F,F-3′′,5′′). HRMS (ESI+): m/z [M+H]+ calcd for C43H34

35Cl2F5O11+: 891.1393; found:

891.1355.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-3-oxo-spiro[isobenzofu-ran-1(3H),9′-[9H]xanthene]-6-carboxylic acid, pentafluorophenyl ester (17). Rf 0.31(EtOAc–toluene 1:19 v/v). 1H NMR (CDCl3) δ 8.45 (dd, 1H, J4,5 = 8.0 Hz, 4J5,7 =1.1 Hz, H-5), 8.21 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.97 (br. s, 1H, H-7), 6.23 (br. s, 2H,H-1′,8′), 3.63 (s, 6H, OCH3), 2.69 (tt, 2H, Ja,a = 11.0 Hz, Ja,e = 3.7 Hz, H-1a′′′), 2.11(m, 4H, H-2e′′′,6e′′′), 1.83 (m, 4H, H-3e′′′,5e′′′), 1.71–1.60 (m, 6H, H-4e′′′,2a′′′,6a′′′),1.42–1.27 (m, 6H, H-3a′′′,4a′′′,5a′′′). 13C NMR (CDCl3) δ 172.5 (2C, COCH), 167.7(C3), 160.8 (CO2C6F5), 153.6 (C7a), 149.0 (2C, C2′,7′), 141.4 (2C, C4a′,10a′), ∼141(m, 2C, 1JCF ∼250 Hz, C2′′,6′′), ∼140 (m, 1JCF ∼250 Hz, C4′′), 139.7 (2C, C3′,6′),∼138 (m, 2C, 1JCF ∼250 Hz, C3′′,5′′), 132.5 (C5), 126.1 (2C, C4,7), 133.7 (C6), 129.4(C3a), 124.6 (m, C1′′), 118.2 (2C, C4′,5′), 115.2 (2C, C8a′,9a′), 106.6 (2C, C1′,8′), 82.4(C1), 56.6 (2C, OCH3), 42.8 (2C, C1′′′), 28.9 (4C, C2′′′,6′′′), 25.7 (2C, C4′′′), 25.2 (4C,C3′′′,5′′′). 19F NMR (CDCl3) δ –151.57 (m, 2F, F-2′′,6′′), –156.59 (t, 1F, J3

′′,4

′′ = J4′′

,5′′

= 21.1 Hz, F-4′′), –161.56 (m, 2F, F-3′′,5′′). HRMS (ESI+): m/z [M+H]+ calcd forC43H34

35Cl2F5O11+: 891.1393; found: 891.1381.

IMPORTANT NOTE: See Figure 4.55.3 for atom numbering.

SUPPORTPROTOCOL

SYNTHESIS OF CYCLOHEXANECARBOXYLIC ANHYDRIDE

Cyclohexanecarboxylic anhydride (Chc2O) is used for acyl protection of phenolichydroxyls in fluorescein derivatives. The synthesis is shown in Figure 4.55.4.

1 2 3

CO2H O

O O

SOCl2, 4 hrCl

O

91% 91%

1, Py, benzene, 2.5 hr

Figure 4.55.4 Synthesis of cyclohexanecarboxylic anhydride.



Labeling

4.55.10


Materials

Cyclohexanecarboxylic acid (Sigma-Aldrich, cat. no. 101834)Thionyl chloride (Sigma-Aldrich, cat. no. 320536)Toluene, dryPyridine (>99.5% purity), dry

100-mL, 250-mL, 500-mL and 1-L round-bottom flasksMagnetic stirrer and Teflon-coated magnetic stirring bars250-mL graduated cylinderReflux condensersCalcium chloride drying tubesOil bathGas outlet adapterMembrane vacuum pump equipped with a vacuum controllerVigreux columnClaisen adapterThermometerLiebig condenser4-way vacuum adapter1-L Erlenmeyer flaskVacuum filtration system (350-mL sintered-glass funnel, 25- to 50-μm porosity,

and vacuum adapter)Rotary evaporator equipped with a vacuum pumpHigh-vacuum membrane or an oil vacuum pump

Prepare cyclohexanecarboxylic anhydride (3)1. In a 1-L round-bottom flask place Teflon-coated magnetic stirring bar, cyclohex-

anecarboxylic acid (128.2 g, 1.0 mol) and add thionyl chloride (110 mL, 1.5 mol,measure using graduated cylinder). Add reflux condenser equipped with calciumchloride drying tube and stir at ambient temperature for 1 hr.

2. Add an oil bath and stir the mixture at 60◦C for 3 hr.

3. Reduce the bath temperature to 30◦C. Stop water flow in the reflux condenser.Replace the drying tube with a gas outlet adapter and remove excess thionyl chlorideusing a chemically resistant membrane pump (10 mm Hg vacuum, apply for 30 min).

4. Replace the reflux condenser with a vacuum distilling system (Vigreux column,Claisen adapter with thermometer, Liebig condenser, and 4-way vacuum adapter,with two 100-mL and one 250-mL round-bottom flasks for distillate collection) andperform the distillation using membrane pump vacuum, magnetic stirring, and theoil bath heating. Collect cyclohexanecarbonyl chloride as the major fraction boilingat 71◦ to 73◦C/10 mm Hg. colorless.

Cyclohexanecarbonyl chloride is a liquid; yield 133 g (91%).

5. In a 1-L Erlenmeyer flask, equipped with an oil bath, place a Teflon-coated magneticstirring bar, cyclohexanecarbonyl chloride (from step 4; 50.7 g, 0.346 mol), and drytoluene (350 mL). Stir for 3 min using a magnetic stirrer.

6. Add dry pyridine (56 mL, 0.69 mol) and then cyclohexanecarboxylic acid (44.9 g,0.35 mmol). Connect the reflux condenser equipped with a calcium chloride dryingtube. Stir the mixture for 2.5 hr at 60◦C oil bath temperature, then cool to ambienttemperature.

7. Remove the crystalline solid precipitate by vacuum filtration through 350-mLsintered-glass funnel (25 to 50 μm porosity) in a 1-L round-bottom flask. Washthe solid with dry toluene (50 mL).


4.55.11


8. Evaporate the solution using a rotary evaporator. Transfer the residue in a 250-mLround-bottom flask.

9. Perform the distillation using a vacuum distilling system (Vigreux column, Claisenadapter with thermometer, Liebig condenser, 4-way vacuum adapter, with two 50-mLand one 100-mL round-bottom flasks for distillate collection) and a high-vacuummembrane or an oil pump. Collect cyclohexanecarboxylic anhydride as fractionboiling at 125◦ to 135◦C/1 mm Hg.

Cyclohexanecarboxylic anhydride is a colorless liquid, yield 68.8 g (91%).

10. Characterize the compound by 1H and 13C NMR.

Cyclohexanecarboxylic anhydride (3). 1H NMR (CDCl3) δ 2.38 (tt, 2H, Ja,a = 11.2 Hz,Ja,e = 3.7 Hz, H-1a), 1.94 (m, 4H, H-2e,6e), 1.76 (m, 4H, H-3e,5e), 1.63 (m, 2H, H-4e),1.46 (m, 4H, H-2a,6a), 1.25 (m, 6H, H-3a,4a,5a). 13C NMR (CDCl3) δ 171.9 (2C, CO),44.0 (2C, C1), 28.4 (4C, C2,6), 25.6 (2C, C4), 25.2 (4C, C3,5).

See Figure 4.55.3 for axial (a) and equatorial (e) protons in cyclohexane ring.

BASICPROTOCOL 2

SYNTHESIS OF 5- AND 6-CARBOXYTETRAMETHYLRHODAMINE(TAMRA)

This protocol contains procedures for synthesis of benzophenones 18 and 19 and theirseparation by crystallization (Fig. 4.55.5). These compounds are easily converted toisomerically pure carboxy-TAMRA rhodamine dye (20 and 21).

Materials

3-Dimethylaminophenol (Sigma-Aldrich, cat. no. D144002)Trimellitic anhydride (Sigma-Aldrich, cat. no. B4600)TolueneMethanol (MeOH)Acetic acid, glacialChloroform (stabilized with amylene, Sigma-Aldrich, cat. no. C2432; Acros, cat.

no. 38376; or equivalent)Phosphorus pentoxideHexamethyldisiloxane (Sigma-Aldrich, cat. no. 205389)N,N-dimethylformamide (DMF)5% (w/v) NaOHConcentrated (12 M) hydrochloric acid (HCl)Triethylamine (Et3N)

250- and 500-mL, and 1-L round-bottom flasksMagnetic stirrer and Teflon-coated magnetic stirring bars500-mL graduated cylinderOil bathCalcium chloride drying tubesReflux condensersVacuum filtration system (20-mL sintered-glass funnel, 25 to 50 μm porosity, and

vacuum adapter)Membrane vacuum pump equipped with a vacuum controller (Vacuubrand

MV10NT VARIO, http://www.vacuubrand.com, or similar)Rotary evaporator equipped with a vacuum pumpSilica-coated aluminum-backed TLC plates with fluorescent indicator




Labeling

4.55.12


18

OMe2N NMe2

O

O

OHO2C

OOH

Me2N

CO2H

CO2H

HO NMe2

CO2

CO2H

OOH

Me2N

CO2H

CO2H

OMe2N NMe2

CO2

HO2C

19

20b

21b

HO NMe2

TMSPP, DMF, Δ

99%

HO NMe2

TMSPP, DMF, Δ

99%

16%

10%

OMe2N NMe2

O

O

OMe2N NMe2

O

O

CO2H

HO2C21a

20a

Figure 4.55.5 Synthesis of 5-carboxy-TAMRA (20a), 4′-carboxy-TAMRA (20b), 6-carboxy-TAMRA (21a), and 5′-carboxy-TAMRA (21b).

Prepare 4-dimethylamino-2-hydroxy-2′,4′-dicarboxy-benzophenone (18) and4-dimethylamino-2-hydroxy-2′,5′-dicarboxy-benzophenone (19), precursors forTAMRA dyeThe quality of 3-dimethylaminophenol is important; it must be a loose crystalline solid.The compound in the form of a gummed mass is not suitable. It must be distilledunder vacuum from a 250-mL round bottom flask using a vacuum distilling system[Vigreux column, Claisen adapter with thermometer, wide air condenser (important!),4-way vacuum adapter with two 50-mL and one 100-mL round-bottom flasks for col-lecting distillate], and a high-vacuum membrane pump (1 mm Hg).

1. In a 500-mL round-bottom flask equipped with magnetic stirrer and an oil bath,place a Teflon-coated magnetic stirring bar, toluene (300 mL), and freshly distilled3-dimethylaminophenol (13.70 g, 0.10 mol). Stir until the solid dissolves. Heat an oilbath to 60◦C and add powdered trimellitic anhydride (23.04 g, 0.12 mol). Add refluxcondenser equipped with a calcium chloride drying tube and reflux the mixture for24 hr, then cool to room temperature.

2. Collect the solid precipitated by vacuum filtration through 200-mL sintered-glassfunnel (25 to 50 μm porosity). Wash the solid three times, each time with 50 mLtoluene.

3. Transfer the solid to a 1-L round bottom flask, add MeOH (300 mL), add refluxcondenser, and reflux 10 min. Then add glacial acetic acid (100 mL) and evaporateto dryness on rotary evaporator.

4. To the residue add MeOH (200 mL), reflux for 2 hr, then cool and keep at 4◦Covernight.

5. Collect the solid precipitated by vacuum filtration (using membrane vacuum pumpwith vacuum controller) through 200-mL sintered-glass funnel (25- to 50-μm poros-ity). Wash the solid with MeOH (50 mL).

Compound 18, yield 5.16 g (16%).

6. Evaporate the mother liquid using rotary evaporator, add glacial acetic acid (150 mL),reflux 1 hr, and stir at room temperature overnight.

7. Collect the solid precipitated by same vacuum filtration system. Recrystallize it twicefrom acetic acid to furnish compound 19 [yield 3.35 g (10%)].


4.55.13


8. Characterize the compounds 18 and 19 by TLC, HRMS, melting temperature, and1H and 13C NMR.

4-Dimethylamino-2-hydroxy-2′,4′-dicarboxy-benzophenone (18). Rf 0.34 (Et3N–MeOH–CHCl3 5:20:75 v/v/v); dec.>250◦C (MeOH). 1H NMR (DMSO-d6) δ 13.44 (br. s, 2H,CO2H), 12.38 (s, 1H, OH), 8.49 (d, 1H, 4J3

′,5

′ = 1.7 Hz, H-3′), 8.20 (dd, 1H, J5′,6′ = 7.9Hz, 4J3′,5′ = 1.7 Hz, H-5′), 7.52 (d, 1H, J5′,6′ = 7.9 Hz, H-6′), 6.80 (d, 1H, J5,6 = 9.2 Hz,H-6), 6.20 (dd, 1H, J5,6 = 9.2 Hz, 4J3,5 = 2.5 Hz, H-5), 6.11 (d, 1H, 4J3,5 = 2.5 Hz, H-3),3.00 (s, 6H, CH3). 13C NMR (DMSO-d6) δ 197.7 (CO), 166.1 (2C, CO2H), 164.2 (C2),155.8 (C4), 143.8 (C1′), 133.8 (C6), 132.7 (C5′), 131.7 (C2′ or C4′), 130.7 (C3′), 130.0(C2′ or C4′), 128.3 (C6′), 109.5 (C1), 104.5 (C5), 97.0 (C3), 39.6 (2C, CH3). HRMS(MALDI+): m/z [M+H]+ calcd for C17H16NO6

+ 330.0972; found 330.0975.

4-Dimethylamino-2-hydroxy-2′,5′-dicarboxy-benzophenone (19). Rf 0.52 (Et3N–MeOH–CHCl3 5:20:75 v/v/v); mp 244–245 ◦C (AcOH). 1H NMR (DMSO-d6) δ 13.46 (br. s, 2H,CO2H), 12.41 (s, 1H, OH), 8.14 (dd, 1H, J3′,4′ = 8.3 Hz, 4J4′,6′ = 1.7 Hz, H-4′), 8.06 (d,1H, J3′4′ = 8.3 Hz, H-3′), 7.83 (d, 1H, 4J4′,6′ = 1.7 Hz, H-6′), 6.86 (d, 1H, J5,6 = 9.3Hz, H-6), 6.22 (dd, 1H, J5,6 = 9.3 Hz, 4J3,5 = 2.5 Hz, H-5), 6.11 (d, 1H, 4J3,5 = 2.5 Hz,H-3), 3.01 (s, 6H, CH3). 13C NMR (DMSO-d6) δ 197.4 (CO), 166.5 (2′-CO2H), 166.1(5′-CO2H), 164.4 (C2), 155.9 (C4), 140.1 (C1′), 133.9 (C6), 133.7 (C2′), 133.6 (C5′),130.4 (C3′), 130.2 (C4′), 128.2 (C6′), 109.4 (C1), 104.6 (C5), 97.1 (C3), 39.6 (2C, CH3).HRMS (MALDI+): m/z [M+H]+ calcd for C17H16NO6

+ 330.0972; found 330.0970.

Prepare 5(4′)-carboxy-TAMRA (20) and 6(5′)-carboxy-TAMRA (21)9. In a 250-mL round-bottom flask equipped with an oil bath, add chloroform (100 mL),

phosphorus pentoxide (20.0 g, 70 mmol), and hexamethyldisiloxane (50.0 mL,247 mmol). Add a reflux condenser equipped with a calcium chloride drying tubeand reflux the mixture for 30 min, then cool to room temperature.

CAUTION: Chloroform is hazardous, affecting liver and kidneys.

IMPORTANT NOTE: Use chloroform stabilized with amylene, not with ethanol (ethanolreacts with phosphorus pentoxide). The above procedure for preparing trimethylsi-lylpolyphosphate was adopted from Yokoyama et al. (1982).

10. In a 250-mL round-bottom flask equipped with a magnetic stirrer and an oil bath,place a Teflon-coated magnetic stirring bar, DMF (80 mL), benzophenone [18 (3.29 g,10.00 mmol), from step 5], 3-dimethylaminophenol (1.78 g, 13.00 mmol), andtrimethylsilylpolyphosphate solution (from step 9; 20 mL). Add a reflux condenserequipped with calcium chloride drying tube. Stir and reflux the mixture for 3 hr, thencool to room temperature.

11. Evaporate the reaction mixture using a rotary evaporator. Add 5% NaOH (70 mL)and stir at room temperature overnight.

12. Add water (150 mL) and then add concentrated hydrochloric acid dropwise to pH 2.

13. Collect the solid precipitated by vacuum filtration through 100-mL sintered-glassfunnel (25- to 50-μm porosity). Wash the solid with water (50 mL).

14. Dry the residue under vacuum (1 mm Hg) at 150◦C for 5 hr.

Yield of dye 20, 4.25 g (99%).

15. In a 250-mL round-bottom equipped with magnetic stirrer and an oil bath, placea Teflon-coated magnetic stirring bar, DMF (80 mL), benzophenone (19; 3.29 g,10.00 mmol), 3-dimethylaminophenol (1.78 g, 13.00 mmol), and trimethylsi-lylpolyphosphate solution in (step 9; 20 mL). Add a reflux condenser equippedwith calcium chloride drying tube. Stir and reflux the mixture for 3 hr, then cool toroom temperature.

16. Evaporate the reaction mixture using rotary evaporator. Add 5% NaOH (70 mL) andstir at room temperature overnight.



Labeling

4.55.14


17. Add water (150 mL) and then add concentrated hydrochloric acid dropwise to pH 2.

18. Collect the solid precipitated by vacuum filtration through 100-mL sintered-glassfunnel (25- to 50-μm porosity). Wash the solid with water (50 mL).

19. Dry the residue under vacuum (1 mm Hg) at 150◦C for 5 hr.

Yield of dye 21, 4.27 g (99%).

20. Purify analytical samples of carboxy-rhodamines 20 and 21 using preparative silicagel TLC in 5:15:80 (v/v/v) Et3N–MeOH–CHCl3 as a mobile phase and characterizeas triethylammonium salts by mass spectrometry and 1H and 13C NMR.

3,6-Bis(dimethylamino)-9-[2,4-dicarboxylatephenyl]xanthylium, triethylammonium salt(4′-carboxytetramethylrhodamine, triethylammonium salt) (20·Et3N). Rf 0.14 (Et3N–MeOH–CHCl3 5:20:75 v/v/v). 1H NMR (CD3OD), open chain numbering, δ 8.73 (br.s, 1H, H-3′), 8.18 (d, 1H, J5′,6′ = 7.5 Hz, H-5′), 7.28 (d, 1H, J5′,6′ = 7.5 Hz, H-6′), 7.25(d, 2H, J1,2 = J7,8 = 9.3 Hz, H-1,8), 6.98 (dd, 2H, J1,2 = J7,8 = 9.3 Hz, 4J2,4 = 4J5,7 =1.7 Hz, H-2,7), 6.83 (d, 2H, 4J2,4 = 4J5,7 = 1.7 Hz, H-4,5), 3.23 (br. s, 12H, NCH3), 3.13(q, 6H, J = 7.4 Hz, NCH2), 1.25 (t, 9H, J = 7.4 Hz, CH2CH3). 13C NMR (CD3OD) δ

173.42 (4′-CO2−), 173.06 (2′-CO2

−), 160.64 (C9), 158.75 (2C, C4a,10a), 158.40 (2C,C3,6), 140.82 (C2′ or 4′), 140.47 (C2′ or 4′), 136.30 (C1′), 132.66 (2C, C1,8), 131.81(C3′), 131.38 (C5′), 129.96 (C6′), 114.73 (2C, C2,7), 114.65 (2C, C8a,9a), 97.35 (2C,C4,5), 47.33 (3C, CH2CH3), 40.81 (4C, NCH3), 9.07 (3C, CH2CH3). HRMS (MALDI+):m/z [M+H]+ calcd for C25H23N2O5

+ 431.1601; found 431.1597.

3,6-Bis(dimethylamino)-9-[2,5-dicarboxylatephenyl]xanthylium, triethylammonium salt(5-carboxytetramethylrhodamine, triethylammonium salt) (21·Et3N). Rf 0.25 (Et3N–MeOH–CHCl3 5:20:75 v/v/v). 1H NMR (CD3OD), open chain numbering, δ 8.23 (d,1H, J3′,4′ = 8.0 Hz, H-4′), 8.11 (d, 1H, J3′,4′ = 8.0 Hz, H-3′), 7.97 (br. s, 1H, H-6′),7.27 (d, 2H, J1,2 = J7,8 = 9.3 Hz, H-1,8), 6.96 (dd, 2H, J1,2 = J7,8 = 9.3 Hz, 4J2,4 =4J5,7 = 2.4 Hz, H-2,7), 6.84 (d, 2H, 4J2,4 = 4J5,7 = 2.4 Hz, H-4,5), 3.22 (br. s, 12H,NCH3), 3.09 (q, 6H, J = 7.4 Hz, NCH2), 1.21 (t, 9H, J = 7.4 Hz, CH2CH3). 13C NMR(CD3OD) δ 173.02 (2C, 2′-CO2

−, 4′-CO2−), 162.71 (C9), 158.85 (2C, C4a,10a), 158.49

(2C, C3,6), 142.85 (C2′), 140.21 (C5′), 133.49 (C1′), 132.84 (2C, C1,8), 131.51 (C4′),131.38 (C6′), 130.57 (C3′), 114.95 (2C, C8a,9a), 114.78 (2C, C2,7), 97.38 (2C, C4,5),47.39 (3C, CH2CH3), 40.82 (4C, NCH3), 9.09 (3C, CH2CH3). HRMS (MALDI+): m/z[M+H]+ calcd for C25H23N2O5

+ 431.1601; found 431.1595.

IMPORTANT NOTE: See Figure 4.55.12 in Basic Protocol 3 below for atom numberingin open form TAMRA derivatives 20a and 21a.

BASICPROTOCOL 3

SYNTHESIS OF NON-NUCLEOSIDE PHOSPHORAMIDITES OF FAM, JOE,AND TAMRA

This protocol contains procedures for 3′,6′-O-diacyl FAM and JOE carboxamides andtheir conversion into phosphoramidites. The preparation of Dmt-protected hydroxypro-linol, its coupling with carboxy-TAMRA, and further conversion to TAMRA phospho-ramidites is also described. See Figures 4.55.6, 4.55.7, 4.55.8, 4.55.9, and 4.55.10.

Materials

3′,6′-O-bis(pivaloyl)-fluorescein-5-carboxylic acid, pentafluorophenyl ester (6;Basic Protocol 1)

3′,6′-O-bis(pivaloyl)-fluorescein-6-carboxylic acid, pentafluorophenyl ester (7;Basic Protocol 1)

3′,6′-O-bis(cyclohexylcarbonyl)-fluorescein-5-carboxylic acid, pentafluorophenylester (9; Basic Protocol 1)

3′,6′-O-bis(cyclohexylcarbonyl)-fluorescein-6-carboxylic acid, pentafluorophenylester (10; Basic Protocol 1)

Dichloromethane (DCM), dry (freshly distilled over CaH2), stabilized withamylene


4.55.15


trans-4-aminocyclohexanol hydrochloride (Sigma-Aldrich, cat. no. 263761)N,N-diisopropylethylamine (DIEA)N,N-diisopropylamino-2-cyanoethoxychlorophosphine (Sigma-Aldrich, cat. no.

302309, also can be prepared as described by Smith et al., 1987)Argon (in steel cylinder)N,N-dimethylformamide (DMF)Sodium sulfate (Na2SO4)Silica gelMethanol (MeOH)TolueneAcetone (Me2CO)Pyridine (>99.5% purity), drySaturated NaHCO3 3′,6′-O-bis(cyclohexylcarbonyl)-JOE-5-carboxylic acid,

pentafluorophenyl ester (16; Basic Protocol 1)3′,6′-O-bis(cyclohexylcarbonyl)-JOE-6-carboxylic acid, pentafluorophenyl ester

(17; Basic Protocol 1)Ethyl acetate (EtOAc)6-Aminohexanol (Sigma-Aldrich, cat. no. A56353)N-Cbz-hydroxy-L-proline (CAS 13504-85-3; Sigma-Aldrich, cat. no. 96310)Concentrated (96%) sulfuric acidTetrahydrofuranSodium borohydride (NaBH4)5% citric acidChloroform (stabilized with amylene; Sigma-Aldrich, cat. no. C2432; Acros, cat.

no. 38376; or equivalent)Dimethoxytrityl chloride (Sigma-Aldrich, cat. no. 38827)10% Pd on carbon (Sigma-Aldrich, cat. no. 520888)Hydrogen (in steel cylinder)Triethylamine (Et3N; >99.5% purity)5-Carboxy-TAMRA (20; Basic Protocol 2)6-Carboxy-TAMRA (21; Basic Protocol 2)Benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP;

Sigma-Aldrich, cat. no. 377848)MgSO4, anhydrideDiisopropylammonium tetrazolide (prepare as described by Caruthers et al., 1987)Bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine (Sigma-Aldrich, cat. no.

305995; also can be prepared as described by Caruthers et al., 1987, or byBannwarth and Trzeciak, 1987)

Calcium chloride drying tubes100-mL, 250-mL, 500-mL, and 1-L round-bottom flasksMagnetic stirrer and Teflon-coated magnetic stirring bars100-mL and 500-mL graduated cylinders500-mL and 1-L separatory funnels250-mL and 500-mL Erlenmeyer flasksVacuum filtration system (various sintered-glass funnels and vacuum adapter)Membrane vacuum pump equipped with a vacuum controller (Vacuubrand

MV10NT VARIO, http://www.vacuubrand.com, or similar)Rotary evaporator equipped with a vacuum pump4 × 20–cm and 3 × 20–cm chromatography columnsSilica-coated aluminum-backed TLC plates with fluorescent indicatorAddition funnels0.2 μm PTFE membrane (Millipore, cat. no. JGWP0470Ø)




Labeling

4.55.16


OO O

O

O

O O

OO O

O

O

O O

OC6F5O

HN

O

6

OO O

O

O

O O

O NH

OH

OO O

O

O

O O

O NH

OCEP

22 23

HO

OO O

O

O

O O

C6F5O

O

OO O

O

O

O O

HN

O

OCEP

724 25

100%

trans-4-aminocyclohexanolDIEA, DCM, DMF, 16 hr

77%

(Pri)2NP(Cl)OCH2CH2CNDIEA, DCM, 1 hr

95%


74%


Figure 4.55.6 Synthesis of phosphoramidite reagents based on 3′,6′-O-bis(pivaloyl)-FAM and trans-4-aminocyclohexanollinker: 5-isomer (23) and 6-isomer (25).

OO O

O

O

O O

OO O

O

O

O O

OC6F5O

HN

O

9

OO O

O

O

O O

O NH

OH

OO O

O

O

O O

O NH

OCEP

26 27

HO

OO O

O

O

O O

C6F5O

O

OO O

O

O

O O

HN

O

OCEP

1028 29

100%


76%


94%


79%


Figure 4.55.7 Synthesis of phosphoramidite reagents based on 3′,6′-O-bis(cyclohexylcarbonyl)-FAM and trans-4-aminocyclohexanol linker: 5-isomer (27) and 6-isomer (29).

Prepare amides of 3′,6′-O-bis(acyl)-fluorescein-5(6)-carboxylic acid (22, 24, 26, and28), precursors for FAM phosphoramidites1. In a 250-mL round-bottom flask equipped with a magnetic stirrer and calcium

chloride drying tube, place a Teflon-coated magnetic stirring bar, activated ester 6or 7 (3.55 g, 5.0 mmol) or 9 or 10 (3.81 g, 5.0 mmol), and dry DCM stabilized withamylene (60 mL). Stir until solid dissolves.

2. Add trans-4-aminocyclohexanol hydrochloride (0.76 g, 5.0 mmol), DIEA (1.74 mL,10.0 mmol), and DMF (60 mL), and stir overnight (16 hr) at room temperature.

3. Transfer the mixture into 1-L separatory funnel, dilute with DCM (200 mL), andwash with water four times, each time with 200 mL. Next, transfer the solution to a500-mL Erlenmeyer flask and dry the solution over Na2SO4 under magnetic stirring(2 hr).


4.55.17


OO O

O

O

O O

OO O

O

O

O O

OC6F5O

HN

O

16

OO O

O

O

O O

O NH

OH

OO O

O

O

O O

O NH

OCEP

30 31

HO

OO O

O

O

O O

C6F5O

O

OO O

O

O

O O

HN

O

OCEP

1732 33

93%


72%


97%


70%


MeO

Cl Cl

OMe MeO

Cl Cl

OMe

Cl

MeO OMe

Cl

Cl

MeO OMe

Cl Cl

MeO OMe

Cl Cl Cl

MeO OMe

Figure 4.55.8 Synthesis of phosphoramidite reagents based on 3′,6′-O-bis(cyclohexylcarbonyl)-JOE and trans-4-aminocyclohexanol: 5-isomer (31) and 6-isomer (33).

OO O

O

O

O O

OO O

O

O

O O

OC6F5O

HN

O

16

OO O

O

O

O O

O NH

OO O

O

O

O O

O NH

34 35

OO O

O

O

O O

C6F5O

O

OO O

O

O

O O

HN

O177363

98%

6-aminohexanolDIEA, DCM, 2 hr

60%


64%


MeO

Cl Cl

OMe MeO

Cl Cl

OMe

Cl

MeO OMe

Cl

Cl

MeO OMe

Cl Cl

MeO OMe

Cl Cl Cl

MeO OMe

96%

6-aminohexanolDIEA, DCM, 2 hr

OHO

CEP

HOO

CEP

Figure 4.55.9 Synthesis of phosphoramidite reagents based on 3′,6′-O-bis(cyclohexylcarbonyl)-JOE and 6-aminohexanol linker: 5-isomer (35) and 6-isomer (37).


5. Apply the residue on a silica gel column (4 × 20–cm) and elute with a manualstep gradient from 0→5% MeOH in toluene. Collect fractions containing majorcompound, combine, and evaporate to dryness.

6. Dry the residue under vacuum (1 mm Hg) for 8 hr. Obtain compounds 22 (3.17 g,100%), 24 (3.06 g, 95%), 26 (3.42 g, 100%), or 28 (3.26 g, 94%) as white amorphoussolids.

7. Characterize the compounds 22, 24, 26, and 28 by TLC, HRMS, and 1H and 13CNMR.



Labeling

4.55.18


60%

46%

O

NMe2

NMe2

O

O

38

NHO2C

Cbz

OH

NCbz

OH

HO NCbz

OH

DmtO

HN

OH

DmtO

N

OH

DmtO

N

OH

DmtO

OMe2N NMe2

O

O

O

O

N

O

DmtO

OMe2N NMe2

O

O

O

O

NMe2

NMe2

O

ON

O

DmtO

O

CEP

CEP

NCbz

OH

MeO

O

39 40 41

42

43

45

44

46

PyBOP, DIEA, DMF, 2 hr

20

21

PyBOP, DIEA,DMF, 2 hr

73%

47%

(Pri2N)2POCH2CH2CN,diisopropylammonium tetrazolide,DCM, 2 hr

(Pri2N)2POCH2CH2CN,diisopropylammonium tetrazolide,DCM, 2 hr

93% 92% 83% 99%

MeOH, H+ NaBH4, MeOH, THF H2, 10% Pd/C, MeOHDmtCl, Py, 16 hr

Figure 4.55.10 Synthesis of phosphoramidite reagents based on TAMRA and trans-4-hydroxyprolinol linker: 5-isomer(44) and 6-isomer (46).

3′,6′-Bis(2,2-dimethyl-1-oxopropyloxy)-5-(4-hydroxycyclohexylaminocarbonyl)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (22). Rf 0.30 (MeOH–toluene 15:85 v/v).1H NMR (DMSO-d6): δ 8.57 (d, 1H, J4

′′,NH = 7.9 Hz, NH), 8.55 (br. s, 1H, H-4), 8.28 (d,

1H, J6,7 = 8.2 Hz, H-6), 7.48 (d, 1H, J6,7 = 8.2 Hz, H-7), 7.27 (s, 2H, H-4′,5′), 6.93 (s,4H, H-1′,2′,7′,8′), 4.56 (d, 1H, J = 4.0 Hz, OH), 3.78 (m, 1H, H-4′′), 3.42 (m, 1H, H-1′′),1.92–1.80 (m, 4H, H-2e′′, 3e′′, 5e′′, 6e′′), 1.48–1.20 (m, 22H, H-2a′′, 3a′′, 5a′′, 6a′′,CH3). 13C NMR (DMSO-d6): δ 175.8 (2C, COBut), 167.8 (C3), 163.5 (CONH), 154.0(C7a), 152.3 (2C, C4a′,10a′), 150.7 (2C, C3′,6′), 136.7 (C6), 135.0 (C5), 129.1 (2C,C1′,8′), 125.6 (C3a), 124.1 (C4), 123.6 (C7), 118.4 (2C, C2′,7′), 115.6 (2C, C8a′,9a′),110.2 (2C, C4′,5′), 81.0 (C1), 68.2 (C1′′), 48.1 (C4′′), 38.5 (2C, CCH3), 34.0 (2C,C2′′,6′′), 30.0 (2C, C3′′,5′′), 26.5 (6C, CH3). HRMS (MALDI+): m/z [M+H]+ calcd forC37H40NNaO9

+: 642.2698; found: 642.2709.

3′,6′-Bis(2,2-dimethyl-1-oxopropyloxy)-6-(4-hydroxycyclohexylaminocarbonyl)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (24). Rf 0.24 (MeOH–toluene 15:85 v/v).1H NMR (DMSO-d6): δ 8.37 (d, 1H, J4

′′,NH = 7.8 Hz, NH), 8.22 (d, 1H, J4,5 = 8.2 Hz,

H-5), 8.14 (d, 1H, J4,5 = 8.2 Hz, H-4), 7.79 (s, 1H, H-7), 7.28 (s, 2H, H-4′,5′), 6.94 (s, 4H,H-1′,2′,7′,8′), 4.51 (br. s, 1H, OH), 3.67 (m, 1H, H-4′′), 3.32 (m, 1H, H-1′′), 1.83–1.72(m, 4H, H-2e′′,3e′′,5e′′,6e′′), 1.31 (s, 18H, CH3), 1.30–1.14 (m, 4H, H-2a′′,3a′′,5a′′,6a′′).13C NMR (DMSO-d6): δ 175.8 (2C, COBut), 167.6 (C3), 163.4 (NHCO), 152.3 (2C,C4a′,10a′), 152.1 (C7a), 150.7 (2C, C3′,6′), 141.2 (C6), 130.0 (C5), 129.3 (2C, C1′,8′),127.3 (C3a), 125.1 (C4), 122.0 (C7), 118.4 (2C, C2′,7′), 115.6 (2C, C8a′,9a′), 110.2 (2C,C4′,5′), 81.1 (C1), 68.1 (C1′′), 48.1 (C4′′), 38.5 (2C, CCH3), 34.0 (2C, C2′′,6′′), 29.9 (2C,C3′′,5′′), 26.5 (6C, CH3). HRMS (MALDI+): m/z [M+Na]+ calcd for C37H39NNaO9

+:664.2517; found: 664.2498.

IMPORTANT NOTE: See Figure 4.55.11 below for atom numbering in fluorescein systemand aminocyclohexanol residue.

3′,6′-Bis(cyclohexylcarbonyloxy)-5-(4-hydroxycyclohexylaminocarbonyl)-3-oxo-spiro[i-sobenzofuran-1(3H),9′-[9H]xanthene] (26). Rf 0.34 (MeOH–toluene 1:9 v/v). 1H NMR(DMSO-d6) δ 8.57 (d, 1H, J4

′′,NH = 7.8 Hz, NH), 8.55 (br. s, 1H, H-4), 8.28 (dd, 1H, J6,7

= 8.0 Hz, 4J4,6 = 1.4 Hz,H-6), 7.49 (d, 1H, J6,7 = 8.0 Hz, H-7), 7.26 (br. s, 2H, H-4′,5′),


4.55.19


a

OO O

O

O

O O

HN

O

OO O

O

O

O OMeO

Cl Cl

OMe1'

2'

3'4' 5'

4a' 10a'6'

7'8'

8a'1

3

3a

4

5

6

7

7a

1''

2''3''4''

5''6''

1'''2'''

3'''

4'''

5'''

6'''

9a'1'

2'

3'4' 5'

4a' 10a'6'

7'8'

8a'1

3

3a

4

5

6

7

7a

1'''2'''

3'''

4'''5'''

6'''

9a'aae

a

aa

(e)

ee

e

26, 27, 28, 29

Axial (a) and equatorial (e)protons in cyclohexane ring of Chcand trans-aminocyclohexanol

RO

HN

O1''

2''

3''

4''

5''

6''

HN

ORO 1''

2''3''4''

5''6''

RO

30, 31, 32, 33

34, 35, 36, 37

4

5

6

7

R = H or CEP

Figure 4.55.11 Atom numbering for NMR assignments in amides of 3′,6′-O-bis(cyclohexylcarbonyl) fluorescein and JOE.

6.92 (s, 4H, H-1′,2′,7′,8′), 4.56 (d, 1H, J= 4.3 Hz, OH), 3.79 (m, 1H, H-4′′), 3.42 (m, 1H,H-1′′), 2.61 (tt, 2H, Ja,a = 11.0 Hz, Ja,e = 3.6 Hz, H-1a′′′), 1.99 (m, 4H, H-2e′′′,6e′′′),1.87 (m, 4H, H-2e′′′,3e′′′,5e′′,6e′′), 1.72 (m, 4H, H-3e′′′,5e′′′), 1.61 (m, 2H, H-4e′′′),1.55–1.18 (m, 14H, H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,5a′′′,6a′′′). 13C NMR (DMSO-d6)δ 173.2 (2C, COCH), 167.8 (C3), 163.5 (CONH), 153.9 (C7a), 152.1 (2C, C4a′,10a′),150.7 (2C, C3′,6′), 136.7 (C6), 135.0 (C5), 129.1 (2C, C1′,8′), 125.7 (C3a), 124.1 (C4),123.6 (C7), 118.4 (2C, C2′,7′), 115.5 (2C, C8a′,9a′), 110.2 (2C, C4′,5′), 81.1 (C1), 68.2(C1′′), 48.1 (C4′′), 41.9 (2C, C1′′′), 34.0 (2C, C2′′,6′′), 30.0 (2C, C3′′,5′′), 28.2 (4C,C2′′′,6′′′), 25.1 (2C, C4′′′), 24.5 (4C, C3′′′,5′′′). HRMS (MALDI+): m/z [M+Na]+ calcdfor C41H43NNaO9

+: 716.2830; found: 716.2808.

3′,6′-Bis(cyclohexylcarbonyloxy)-6-(4-hydroxycyclohexylaminocarbonyl)-3-oxo-spiro[i-sobenzofuran-1(3H),9′-[9H]xanthene] (28). Rf 0.20 (MeOH–toluene 1:9 v/v). 1H NMR(DMSO-d6) δ 8.38 (d, 1H, J4

′′,NH = 7.6 Hz, NH), 8.22 (dd, 1H, J4,5 = 8.1 Hz, 4J5,7

= 1.0 Hz, H-5), 8.14 (d, 1H, J4,5 = 8.1 Hz, H-4), 7.81 (s, 1H, H-7), 7.28 (s, 2H,H-4′,5′), 6.93 (s, 4H, H-1′,2′,7′,8′), 4.51 (d, 1H, J = 4.4 Hz, OH), 3.67 (m, 1H, H-4′′),3.29 (m, 1H, H-1′′), 2.61 (tt, 2H, Ja,a = 11.0 Hz, Ja,e = 3.6 Hz, H-1a′′′), 1.97 (m, 4H,H-2e′′′,6e′′′), 1.84–1.65 (m, 8H, H-2e′′,3e′′,5e′′,6e′′,3e′′′,5e′′′), 1.61 (m, 2H, H-4e′′′),1.54–1.43 (m, 4H), 1.38–1.14 (m, 10H) (H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,5a′′′,6a′′′).13C NMR (DMSO-d6) δ 173.2 (2C, COCH), 167.6 (C3), 163.4 (NHCO), 152.1 (2C,C4a′,10a′), 152.0 (C7a), 150.7 (2C, C3′,6′), 141.1 (C6), 130.0 (C5), 129.2 (2C, C1′,8′),127.4 (C3a), 125.1 (C4), 122.0 (C7), 118.5 (2C, C2′,7′), 115.6 (2C, C8a′,9a′), 110.2 (2C,C4′,5′), 81.1 (C1), 68.1 (C1′′), 48.1 (C4′′), 41.9 (2C, C1′′′), 34.0 (2C, C2′′,6′′), 29.9 (2C,C3′′,5′′), 28.2 (4C, C2′′′,6′′′), 25.1 (2C, C4′′′), 24.5 (4C, C3′′′,5′′′). HRMS (MALDI+):m/z [M+Na]+ calcd for C41H43NNaO9

+: 716.2830; found: 716.2817.

Prepare FAM phosphoramidites (23, 25, 27, and 29)8. In a 100-mL round-bottom flask equipped with magnetic stirrer place a Teflon-

coated magnetic stirring bar, amide 22 or 24 (2.57 g, 4.0 mmol) or 26 or 28 (2.78 g,4.0 mmol), and dry DCM (50 mL). Stir until solid dissolves.

9. Evaporate the solvent using rotary evaporator. Add dry DCM (50 mL) and evaporateagain.

IMPORTANT NOTE: Use DCM stabilized with amylene, freshly distilled over CaH2.

10. Add DCM (50 mL), DIEA (0.765 mL, 4.4 mmol), and N,N-diisopropylamino-2-cyanoethoxychlorophosphine (1.04 g, 4.4 mmol), then stir under argon for 1 hr.



Labeling

4.55.20


11. Monitor conversion of the starting compound by TLC [3 parts (v/v) acetone/7 parts(v/v)/toluene containing 1% (v/v) pyridine].

12. Transfer the mixture into 250-mL separatory funnel and wash with saturatedNaHCO3 (50 mL), then brine (60 mL). Transfer the solution to a 250-mL Erlen-meyer flask and dry the solution over Na2SO4 with magnetic stirring (2 hr).


14. Apply the residue on a silica gel column (4 × 20 cm) and elute withtoluene/acetone/pyridine 94:5:1 (v/v/v). Collect fractions containing the major com-pound, combine, and evaporate to dryness. Obtain compounds 23 (2.60 g, 77%), 25(2.51 g, 74%), 27 (2.73 g, 76%), or 29 (2.84 g, 79%) as white amorphous solids.

15. Characterize the compounds 23, 25, 27, and 29, by TLC, HRMS, and 1H, 13C, and31P NMR.

3′,6′-Bis(2,2-dimethyl-1-oxopropyloxy)-5-[4-(N,N-diisopropylamino-2-cyanoethoxypho-sphinyloxy)-cyclohexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanth-ene] (23). Rf 0.68 (Me2CO–toluene 3:7 + 1% pyridine v/v/v). 1H NMR (DMSO-d6): δ

8.58 (d, 1H, J4′′

,NH = 7.6 Hz, NH), 8.55 (br. s, 1H, H-4), 8.28 (dd, 1H, J6,7 = 7.9 Hz,4J4,6 = 1.2 Hz, H-6), 7.49 (d, 1H, J6,7= 7.9 Hz, H-7), 7.27 (s, 2H, H-4′,5′), 6.93 (s, 4H,H-1′,2′,7′,8′), 3.87–3.65 (m, 4H, H-1′′,4′′, POCH2), 3.63–3.52 (m, 2H, PNCH), 2.77 (t,2H, J = 6.0 Hz, CH2CN), 2.04 (m, 1H), 1.96 (m, 1H), 1.90 (m, 2H) (H-2e′′,3e′′,5e′′,6e′′),1.47 (m, 4H, H-2a′′,3a′′,5a′′,6a′′), 1.31 (m, 18H, CCH3), 1.15 (d, 12H, J= 6.7 Hz,CHCH3). 13C NMR (DMSO-d6): δ 175.8 (2C, COBut), 167.8 (C3), 163.6 (CONH), 154.0(C7a), 152.3 (2C, C4a′,10a′), 150.7 (2C, C3′,6′), 136.7 (C6), 135.0 (C5), 129.1 (2C,C1′,8′), 125.6 (C3a), 124.1 (C4), 123.6 (C7), 118.9 (CN), 118.4 (2C, C2′,7′), 115.6 (2C,C8a′,9a′), 110.2 (2C, C4′,5′), 81.0 (C1), 71.7 (d, 2JP,C = 19.2 Hz, C1′′), 57.8 (d, 2JP,C =19.2 Hz, POCH2), 47.6 (C4′′), 42.4 (d, 2JP,C = 13.0 Hz, PNCH), 38.5 (2C, CCH3), 32.6,32.5 (C2′′,6′′), 29.7 (2C, C3′′,5′′), 26.5 (6C, CCH3), 24.2 (d, 2C, 3JP,C = 8.0 Hz), 24.1 (d,2C, 3JP,C = 8.0 Hz) (CHCH3), 19.7 (d, 3JP,C = 7.4 Hz, CH2CN). 31P NMR (DMSO-d6): δ

144.96. HRMS (ESI+): m/z [M+Na]+ calcd for C46H56N3NaO10P+: 864.3595; found:864.3556.

3′,6′-Bis(2,2-dimethyl-1-oxopropyloxy)-6-[4-(N,N-diisopropylamino-2-cyanoethoxypho-sphinyloxy)-cyclohexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanth-ene] (25). Rf 0.60 (Me2CO–toluene 3:7 + 1% pyridine v/v/v). 1H NMR (DMSO-d6):δ 8.39 (d, 1H, J4

′′,NH = 7.8 Hz, NH), 8.22 (d, 1H, J4,5 = 8.0 Hz, H-5), 8.14 (d, 1H,

J4,5 = 8.0 Hz, H-4), 7.79 (s, 1H, H-7), 7.28 (s, 2H, H-4′,5′), 6.94 (s, 4H, H-1′,2′,7′,8′),3.76–3.60 (m, 4H, H-1′′,4′′, POCH2), 3.58–3.50 (m, 2H, PNCH), 2.74 (t, 2H, J = 6.0 Hz,CH2CN), 1.95 (m, 1H), 1.87 (m, 1H), 1.81 (m, 2H) (H-2e′′,3e′′,5e′′,6e′′), 1.42–1.29 (m,22H, H-2a′′,3a′′,5a′′,6a′′, CCH3), 1.12 (m, 12H, CHCH3). 13C NMR (DMSO-d6): δ 175.8(2C, COBut), 167.6 (C3), 163.4 (NHCO), 152.3 (2C, C4a′,10a′), 152.1 (C7a), 150.7 (2C,C3′,6′), 141.1 (C6), 129.9 (C5), 129.2 (2C, C1′,8′), 127.3 (C3a), 125.1 (C4), 122.0 (C7),118.8 (CN), 118.4 (2C, C2′,7′), 115.6 (2C, C8a′,9a′), 110.2 (2C, C4′,5′), 81.1 (C1), 71.85(d, 2JP,C = 17.8 Hz, C1′′), 57.8 (d, 2JP,C = 17.8 Hz, POCH2), 47.6 (C4′′), 42.3 (d, 2JP,C

= 12.6 Hz, PNCH), 38.5 (2C, CCH3), 32.6 (d, 3JP,C = 3.4 Hz), 32.5 (d, 3JP,C = 3.4 Hz)(C2′′,6′′), 29.7, 29.6 (C3′′,5′′), 26.71 (6C, CCH3), 24.2 (d, 2C, 3JP,C = 7.4 Hz), 24.1 (d,2C, 3JP,C = 6.9 Hz) (CHCH3), 19.7 (d, 3JP,C = 6.9 Hz, CH2CN). 31P NMR (DMSO-d6): δ


3′,6′-Bis(cyclohexylcarbonyloxy)-5-[4-(N,N-diisopropylamino-2-cyanoethoxyphosphin-yloxy)-cyclohexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1 (3H) ,9′-[9H] xanthene](27). Rf 0.69 (Me2CO–toluene 3:7 + 1% pyridine v/v/v). 1H NMR (DMSO-d6) δ 8.58(d, 1H, J4

′′,NH = 7.8 Hz, NH), 8.55 (br. s, 1H, H-4), 8.28 (dd, 1H, J6,7 = 8.1 Hz, 4J4,6

= 1.2 Hz,H-6), 7.49 (d, 1H, J6,7= 8.1 Hz, H-7), 7.26 (br. s, 2H, H-4′,5′), 6.92 (s, 4H,H-1′,2′,7′,8′), 3.88–3.67 (m, 4H, H-1′′,4′′, POCH2), 3.63–3.54 (m, 2H, CHCH3), 2.77 (t,


4.55.21


2H, J = 6.0 Hz, CH2CN), 2.61 (tt, 2H, Ja,a = 11.0 Hz, Ja,e = 3.6 Hz, H-1a′′′), 2.07–1.88(m, 8H, H-2e′′,3e′′,5e′′,6e′′,2e′′′,6e′′′), 1.72 (m, 4H, H-3e′′′,5e′′′), 1.60 (m, 2H, H-4e′′′),1.54–1.21 (m, 14H, H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,5a′′′,6a′′′), 1.15 (d, 12H, J = 6.7Hz, CH3). 13C NMR (DMSO-d6) δ 173.2 (2C, COCH), 167.7 (C3), 163.6 (CONH), 153.9(C7a), 152.1 (2C, C4a′,10a′), 150.7 (2C, C3′,6′), 136.7 (C6), 135.0 (C5), 129.1 (2C,C1′,8′), 125.7 (C3a), 124.1 (C4), 123.6 (C7), 118.9 (CN), 118.4 (2C, C2′,7′), 115.5 (2C,C8a′,9a′), 110.2 (2C, C4′,5′), 81.0 (C1), 71.7 (d, 2JP,C = 17.8 Hz, C1′′), 57.8 (d, 2JP,C

= 17.8 Hz, POCH2), 47.6 (C4′′), 42.4 (d, 2JP,C = 12.6 Hz, PNCH), 41.9 (2C, C1′′′),32.6, 32.5 (C2′′,6′′), 29.7 (2C, C3′′,5′′), 28.2 (4C, C2′′′,6′′′), 25.1 (2C, C4′′′), 24.5 (4C,C3′′′,5′′′), 24.2 (d, 2C, 3JP,C = 7.4 Hz), 24.1 (d, 2C, 3JP,C = 7.4 Hz) (CHCH3), 19.7 (d,3JP,C = 6.9 Hz, CH2CN). 31P NMR (DMSO-d6) δ 144.97. HRMS (ESI+): m/z [M+Na]+calcd for C50H60N3NaO10P+: 916.3908; found: 916.3892.

3′,6′-Bis(cyclohexylcarbonyloxy)-6-[4-(N,N-diisopropylamino-2-cyanoethoxyphosphin-yloxy)-cyclohexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1 (3H), 9′-[9H] xanthene](29). Rf 0.62 (Me2CO–toluene 3:7 + 1% pyridine v/v/v). 1H NMR (DMSO-d6) δ 8.39(d, 1H, J4

′′,NH = 7.6 Hz, NH), 8.22 (d, 1H, J4,5 = 8.0 Hz, H-5), 8.14 (d, 1H, J4,5

= 8.0 Hz, H-4), 7.80 (s, 1H, H-7), 7.28 (s, 2H, H-4′,5′), 6.93 (s, 4H, H-1′,2′,7′,8′),3.75–3.60 (m, 4H, H-1′′,4′′, POCH2), 3.59–3.50 (m, 2H, CHCH3), 2.74 (t, 2H, J =5.8 Hz, CH2CN), 2.62 (tt, 2H, Ja,a = 11.0 Hz, Ja,e = 3.6 Hz, H-1a′′′), 2.02–1.68 (m,12H, H-2e′′,3e′′,5e′′,6e′′,2e′′′,3e′′′,5e′′′,6e′′′), 1.61 (m, 2H, H-4e′′′), 1.52–1.19 (m, 14H,H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′, 4a′′′,5a′′′,6a′′′), 1.12 (d, 6H, J = 6.4 Hz), 1.11 (d, 6H, J =6.4 Hz) (CH3). 13C NMR (DMSO-d6) δ 173.2 (2C, COCH), 167.6 (C3), 163.4 (NHCO),152.1 (2C, C4a′,10a′), 152.0 (C7a), 150.7 (2C, C3′,6′), 141.1 (C6), 130.0 (C5), 129.2(2C, C1′,8′), 127.4 (C3a), 125.1 (C4), 122.0 (C7), 118.8 (CN), 118.5 (2C, C2′,7′), 115.6(2C, C8a′,9a′), 110.2 (2C, C4′,5′), 81.1 (C1), 71.7 (d, 2JP,C = 17.8 Hz, C1′′), 57.8 (d,2JP,C = 17.8 Hz, POCH2), 47.6 (C4′′), 42.3 (d, 2JP,C = 12.6 Hz, PNCH), 41.9 (2C, C1′′′),32.6 (d, 2JP,C = 4.0 Hz), 32.5 (d, 2JP,C = 4.0 Hz) (C2′′,6′′), 29.7, 29.6 (C3′′,5′′), 28.2 (4C,C2′′′,6′′′), 25.1 (2C, C4′′′), 24.5 (4C, C3′′′,5′′′), 24.2 (d, 2C, 3JP,C = 7.4 Hz), 24.1 (d, 2C,3JP,C = 6.9 Hz) (CHCH3), 19.7 (d, 3JP,C = 6.9 Hz, CH2CN). 31P NMR (DMSO-d6) δ


IMPORTANT NOTE: See Figure 4.55.11, above, for atom numbering.

Prepare amides of 3′,6′-O-bis(cyclohexylcarbonyl)-JOE-5(6)-carboxylic acid andtrans-4-aminocyclohexanol (30 and 32), precursors for JOE phosphoramidites withrigid linker16. In a 100-mL round-bottom flask equipped with a magnetic stirrer and a calcium

chloride drying tube, place a Teflon-coated magnetic stirring bar, activated ester 16or 17 (892 mg, 1.0 mmol), and dry DCM (5 mL). Stir until solid dissolves.

17. Add trans-4-aminocyclohexanol hydrochloride (150 mg, 1.0 mmol), DIEA (0.34 mL,2.0 mmol), and DMF (10 mL), and stir for 2 hr at room temperature.

18. Transfer the mixture into a 500-mL separatory funnel, dilute with EtOAc (100 mL),and wash successively with water (100 mL), saturated NaHCO3 (100 mL), and brine(100 mL). Transfer the solution to a 250-mL Erlenmeyer flask and dry over MgSO4

with magnetic stirring (2 hr).

19. Remove the drying agent by vacuum filtration through 100-mL sintered-glass funnel(25- to 50-μm porosity) into a 250-mL round-bottom flask. Evaporate the solutionusing a rotary evaporator.

20. Apply the residue onto a silica gel column (3 × 20–cm) and elute with 0→5% MeOHin toluene. Collect fractions containing major compound, combine, and evaporate todryness. Obtain compounds 30 (0.76 g, 92%) or 32 (0.80 g, 97%) as white amorphoussolids.

21. Characterize the compounds 30 and 32 by TLC, HRMS, and 1H and 13C NMR.



Labeling

4.55.22


3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-5-(4-hydroxy-trans-cyc-lohexylaminocarbonyl)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (0). Rf 0.21(acetone–toluene 3:7 v/v). 1H NMR (DMSO-d6) 8.70 (m, 1H, NH), 8.55 (m, 1H, H-4),8.28 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6 = 1.3 Hz, H-6), 7.60 (d, 1H, J6,7 = 8.0 Hz, H-7),6.42 (s, 2H, H-1′,8′), 4.56 (d, 1H, J = 4.3 Hz, OH), 3.67 (m, 1H, H-4′′), 3.56 (s,6H, OCH3), 3.32 (m, 1H, H-1′′), 2.74 (tt, 2H, Ja,e = 3.6 Hz, Ja,a = 10.8 Hz, H-1a′′′),1.99 (m, 4H, H-2e′′′,6e′′′), 1.73 (m, 8H, H-2e′′,3e′′,5e′′,6e′′,3e′′′,5e′′′), 1.62–1.50 (m,6H), 1.40–1.09 (m, 10H) (H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,4e′′′,5a′′′,6a′′′). 13C NMR(DMSO-d6) δ 172.0 (2C, COCH), 167.7 (C3), 164.2 (CONH), 153.2 (C7a), 148.6 (2C,C2′,7′), 140.6 (2C, C4a′,10a′), 138.7 (2C, C3′,6′), 136.7 (C5), 134.9 (C6), 125.7 (C3a),124.2 (2C, C4,7), 116.5 (2C, C4′,5′ or C8a′,9a′), 116.2 (2C, C4′,5′ or C8a′,9a′), 108.3,108.2 (C1′,8′), 81.2 (C1), 68.1 (C1′′), 56.7 (2C, OCH3), 48.1 (C4′′), 41.7 (2C, C1′′′),34.0 (2C, C2′′,6′′), 30.0 (2C, C3′′,5′′), 28.5 (4C, C2′′′,6′′′), 25.2 (2C, C4′′′), 24.5 (4C,C3′′′,5′′′). HRMS (ESI+): m/z [M+Na]+ calcd for C43H45

35Cl2NNaO11+: 844.2262;

found: 844.2250.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-6-(4-hydroxy-trans-cyc-lohexylaminocarbonyl)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (32). Rf

0.17 (acetone–toluene 3:7 v/v). 1H NMR (DMSO-d6) δ 8.37 (d, 1H, J = 5.5 Hz, NH), 8.22(dd, 1H, J4,5 = 8.0 Hz, 4J5,7 = 0.8 Hz, H-5), 8.15 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.85 (m,1H, H-7), 6.42 (s, 2H, H-1′,8′), 4.56 (d, 1H, J = 4.3 Hz, OH), 3.67 (m, 1H, H-4′′), 3.57(s, 6H, OCH3), 3.32 (m, 1H, H-1′′), 2.74 (tt, 2H, Ja,e = 3.6 Hz, Ja,a = 10.8 Hz, H-1a′′′),2.00 (m, 4H, H-2e′′′,6e′′′), 1.75 (m, 8H, H-2e′′,3e′′,5e′′,6e′′,3e′′′,5e′′′), 1.65–1.52 (m,6H), 1.43–1.12 (m, 10H) (H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,4e′′′,5a′′′,6a′′′). 13C NMR(DMSO-d6) δ 172.0 (2C, COCH), 167.6 (C3), 163.6 (CONH), 151.6 (C7a), 148.7 (2C,C2′,7′), 141.1 (C6), 140.4 (2C, C4a′,10a′), 138.7 (2C, C3′,6′), 130.3 (C5), 127.2 (C3a),125.6 (C4), 122.1 (C7), 116.3 (2C, C4′,5′ or C8a′,9a′), 116.2 (2C, C4′,5′ or C8a′,9a′),108.4 (2C, C1′,8′), 81.2 (C1), 68.3 (C1′′), 56.8 (2C, OCH3), 48.3 (C4′′), 41.7 (2C, C1′′′),34.2 (2C, C2′′,6′′), 30.2 (2C, C3′′,5′′), 28.5 (4C, C2′′′,6′′′), 25.2 (2C, C4′′′), 24.5 (4C,C3′′′,5′′′). HRMS (ESI+): m/z [M+Na]+ calcd for C43H45

35Cl2NNaO11+: 844.2262;

found: 844.2272.

IMPORTANT NOTE: See Figure 4.55.11 above for atom numbering.

Prepare amides of 3′,6′-O-bis(cyclohexylcarbonyl)-JOE-5(6)-carboxylic acid and6-aminohexanol (34 and 36), precursors for JOE phosphoramidites with flexiblelinker22. In a 100-mL round-bottom flask equipped with a magnetic stirrer and a calcium

chloride drying tube, place a Teflon-coated magnetic stirring bar, activated ester 16or 17 (892 mg, 1.0 mmol), and DCM (10 mL). Stir until solid dissolves.

23. Add 6-aminohexanol (117 mg, 1.0 mmol) and DIEA (0.18 mL, 1.0 mmol) and stirfor 2 hr at room temperature.

24. Transfer the mixture into 250-mL separatory funnel, dilute with DCM (50 mL), thenwash with water (50 mL), saturated NaHCO3 (50 mL), and brine (50 mL). Next,transfer the solution to a 250-mL Erlenmeyer flask and dry the solution over MgSO4

with magnetic stirring (2 hr).


26. Apply the residue to a silica gel column (3 × 20–cm) and elute with 0→20% acetonein toluene. Collect fractions containing major compound, combine and evaporate todryness.

27. Dry the residue in vacuum (1 mm Hg) for 8 hr. Obtain compound 34 (0.81 g, 98%)or 36 (0.79 g, 96%) as white amorphous solid.

28. Characterize the compounds 34 and 36 by TLC, HRMS, and 1H and 13C NMR.


4.55.23


3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-5-(6-hydroxyhexylamin-ocarbonyl)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (34). Rf 0.22 (acetone–toluene 3:7 v/v). 1H NMR (DMSO-d6) δ 8.85 (t, 1H, J = 5.5 Hz, NH), 8.55 (m, 1H, H-4),8.28 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6 = 1.3 Hz, H-6), 7.60 (d, 1H, J6,7 = 8.0 Hz, H-7),6.42 (s, 2H, H-1′,8′), 4.35 (t, 1H, J = 5.3 Hz, OH), 3.56 (s, 6H, OCH3), 3.39 (m, 2H,H-6′′), 3.30 (m, 2H, H-1′′), 2.73 (tt, 2H, Ja,e = 3.7 Hz, Ja,a = 10.7 Hz, H-1a′′′), 1.99 (m,4H, H-2e′′′,6e′′′), 1.72 (m, 4H, H-3e′′′,5e′′′), 1.63–1.49 (m, 8H, H-2′′,2a′′′,4e′′′,6a′′′),1.45–1.20 (m, 12H, H-3′′,4′′,5′′,3a′′′,4a′′′,5a′′′). 13C NMR (DMSO-d6) δ 172.0 (2C,COCH), 167.7 (C3), 164.2 (CONH), 153.2 (C7a), 148.6 (2C, C2′,7′), 140.6 (2C,C4a′,10a′), 138.7 (2C, C3′,6′), 136.7 (C5), 134.9 (C6), 125.7 (C3a), 124.2 (2C, C4,7),116.5 (2C, C4′,5′ or C8a′,9a′), 116.2 (2C, C4′,5′ or C8a′,9a′), 108.3, 108.2 (C1′,8′), 81.2(C1), 60.7 (C6′′), 56.8 (2C, OCH3), 41.7 (2C, C1′′′), ∼40 (C1′′), 32.5 (C5′′), 29.1 (C2′′),28.5 (4C, C2′′′,6′′′), 26.5 (C3′′), 25.3 (C4′′), 25.2 (2C, C4′′′), 24.5 (4C, C3′′′,5′′′). HRMS(ESI+): m/z [M+Na]+ calcd for C43H47

35Cl2NNaO11+: 846.2418; found: 846.2436.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-6-(6-hydroxyhexylamin-ocarbonyl)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (36). Rf 0.17 (acetone–toluene 3:7 v/v). 1H NMR (DMSO-d6) δ 8.65 (t, 1H, J = 5.4 Hz, NH), 8.21 (dd, 1H,J4,5 = 8.0 Hz, 4J5,7 = 0.8 Hz, H-5), 8.15 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.83 (br. s, 1H,H-7), 6.44 (s, 2H, H-1′,8′), 4.32 (m, 1H, OH), 3.57 (s, 6H, OCH3), ∼3.30 (m, 2H, H-6′′),3.19 (m, 2H, H-1′′), 2.73 (tt, 2H, Ja,e = 3.7 Hz, Ja,a = 10.8 Hz, H-1a′′′), 1.99 (m, 4H,H-2e′′′,6e′′′), 1.72 (m, 4H, H-3e′′′,5e′′′), 1.63–1.58 (m, 2H, H-4e′′′), 1.57–1.50 (m, 4H,H-2a′′′,6a′′′), 1.46 (m, 2H, H-2′′), 1.41–1.33 (m, 6H, H-3a′′′,4a′′′,5a′′′), 1.30–1.20 (m,6H, H-3′′,4′′,5′′). 13C NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.6 (C3), 164.2 (CONH),151.6 (C7a), 148.7 (2C, C2′,7′), 141.2 (C6), 140.4 (2C, C4a′,10a′), 138.7 (2C, C3′,6′),130.1 (C5), 127.1 (C3a), 125.7 (C4), 122.1 (C7), 116.4 (2C), 116.2 (2C) (C4′,5′,8a′,9a′),108.4 (2C, C1′,8′), 81.1 (C1), 60.6 (C6′′), 56.8 (2C, OCH3), 41.7 (2C, C1′′′), ∼40 (C1′′),32.4 (C5′′), 29.0 (C2′′), 28.5 (4C, C2′′′,6′′′), 26.5 (C3′′), 25.2 (3C, C4′′,4′′′), 24.5 (4C,C3′′′,5′′′). HRMS (ESI+): m/z [M+Na]+ calcd for C43H47

35Cl2NNaO11+: 846.2418;

found: 846.2429.


Prepare JOE phosphoramidites (31, 33, 35, and 37)29. In a 100-mL round-bottom flask equipped with magnetic stirrer, place a Teflon-

coated magnetic stirring bar, amide 30, 32, 34, or 36 (0.41 g, 0.5 mmol) and dryDCM (20 mL). Stir until solid dissolves.

30. Evaporate the solvent using a rotary evaporator. Add DCM (20 mL) and evaporateagain.


31. Add DCM (20 mL), DIEA (0.104 mL, 0.6 mmol), and N,N-diisopropylamino-2-cyanoethoxychlorophosphine (0.142 g, 0.6 mmol). Stir under argon for 1 hr.

32. Monitor conversion of the starting compound by TLC [3 parts (v/v) acetone/7 parts(v/v)/ toluene containing 1% (v/v) pyridine].

33. Transfer the mixture into 250-mL separatory funnel and wash with saturatedNaHCO3 (50 mL) and brine (60 mL). Transfer the solution to a 250-mL Erlen-meyer flask and dry the solution over Na2SO4 under magnetic stirring (2 hr).


35. Apply the residue to a silica gel column (4 × 20–cm) and elute with 5% acetone intoluene plus 1% pyridine. Collect fractions containing major compound, combine,and evaporate to dryness. Obtain compounds 31 (0.37 g, 72%), 33 (0.36 g, 70%), 35(0.31 g, 60%), or 37 (0.33 g, 64%) as amorphous solids.



Labeling

4.55.24


36. Characterize compounds 31, 33, 35, and 37, by TLC, mass spectrometry, and 1H,13C, and 31P NMR.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-5-[4-(N,N-diisopropyl-amino-2-cyanoethoxyphosphinyloxy)-trans-cyclohexylaminocarbonyl]-3-oxo-spiro[iso-benzofuran-1(3H),9′-[9H]xanthene] (31). Rf 0.80 (Et3N–acetone–toluene 1:4:15 v/v/v).1H NMR (DMSO-d6) δ 8.62 (d, 1H, J = 7.8 Hz, NH), 8.58 (br. s, 1H, H-4), 8.29 (dd, 1H,J4,5 = 8.1 Hz, 4J5,7 = 1.2 Hz, H-6), 7.60 (d, 1H, J4,5 = 8.1 Hz, H-7), 6.40 (m, 2H, H-1′,8′),3.84 (m, 1H), 3.77–3.65 (m, 3H) (H-1′′,4′′, POCH2), 3.60 (m, 2H, CHCH3), 3.56 (s, 6H,OCH3), 2.77 (t, 2H, J = 6.0 Hz, CH2CN), 2.73 (tt, 2H, Ja,e = 3.6 Hz, Ja,a = 10.8 Hz,H-1a′′′), 2.05–1.86 (m, 8H, H-2e′′,3e′′,5e′′,6e′′,2e′′′,6e′′′), 1.72 (m, 4H, H-3e′′′,5e′′′),1.63–1.21 (m, 14H, H-2a′′,3a′′,5a′′,6a′′, H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,5a′′′,6a′′′),1.15 (d, 12H, J = 7.2 Hz, CCH3). 13C NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.7(C3), 163.6 (CONH), 153.2 (C7a), 148.6 (2C, C2′,7′), 140.6 (2C, C4a′,10a′), 138.7 (2C,C3′,6′), 136.7 (C5), 135.1 (C6), 125.6 (C3a), 124.2 (2C, C4,7), 119.1 (CN), 116.4 (2C),116.2 (2C) (C4′,5′,8a′,9a′), 108.3 (2C, C1′,8′), 81.3 (C1), 71.8 (d, 2JP,C = 18 Hz, C1′′),58.0 (d, 2JP,C = 18 Hz, POCH2), 56.8 (2C, OCH3), 47.8 (C4′′), 42.4 (d, 2JP,C = 13.5 Hz,PNCH), 41.7 (2C, C1′′′), 32.8, 32.7 (C2′′,6′′), 29.9 (2C, C3′′,5′′), 28.5 (4C, C2′′′,6′′′),25.2 (2C, C4′′′), 24.5 (4C, C3′′′,5′′′), 24.4 (d, 2C, 3JP,C = 7.5 Hz), 24.3 (d, 2C, 3JP,C =7.5 Hz) (CHCH3), 19.8 (d, 3JP,C = 7.5 Hz, CH2CN). 31P NMR (DMSO-d6) δ 144.85.HRMS (ESI+): m/z [M+Na]+ calcd for C52H62

35Cl2N3NaO12P+: 1044.3340; found:1044.3349.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-6-[4-(N,N-diisopropyl-amino-2-cyanoethoxyphosphinyloxy)-trans-cyclohexylaminocarbonyl]-3-oxo-spiro[iso-benzofuran-1(3H),9′-[9H]xanthene] (33). Rf 0.65 (Et3N–acetone–toluene 1:4:15 v/v/v).1H NMR (DMSO-d6) δ 8.39 (d, 1H, J = 7.8 Hz, NH), 8.22 (m, 1H, H-5), 8.15 (d, 1H, J4,5

= 8.1 Hz, H-4), 7.85 (br. s, 1H, H-7), 6.42 (s, 2H, H-1′,8′), 3.75–3.62 (m, 4H, H-1′′,4′′,POCH2), 3.57 (s, 6H, OCH3), 3.56–3.50 (m, 2H, CHCH3), 2.74 (m, 2H, H-1a′′′), 2.02–1.94 (m, 4H, H-2e′′′,6e′′′), 1.90–1.70 (m, 8H, H-2e′′,3e′′,5e′′,6e′′,3e′′′,5e′′′), 1.63–1.50(m, 6H), 1.41–1.15 (m, 10H) (H-2a′′,3a′′,5a′′,6a′′,2a′′′,3a′′′,4a′′′,4e′′′,5a′′′,6a′′′), 1.12(m, 12H, CHCH3). 13C NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.6 (C3), 163.6(CONH), 151.6 (C7a), 148.7 (2C, C2′,7′), 141.1 (C6), 140.4 (2C, C4a′,10a′), 138.7(2C, C3′,6′), 128.9 (C5), 127.2 (C3a), 125.6 (C4), 122.1 (C7), 119.1 (CN), 116.4 (2C),116.2 (2C) (C4′,5′,8a′,9a′), 108.4 (2C, C1′,8′), 81.2 (C1), 71.8 (m, C1′′), 57.9 (d, 2JP,C

= 19.5 Hz, POCH2), 56.8 (2C, OCH3), 47.9 (C4′′), 42.4 (d, 2JP,C = 19.5 Hz, PNCH),41.7 (2C, C1′′′), 32.7 (m, 2C, C2′′,6′′), 29.9 (m, 2C, C3′′,5′′), 28.5 (4C, C2′′′,6′′′), 25.2(2C, C4′′′), 24.5 (4C, C3′′′,5′′′), 24.4 (d, 2C, 3JP,C = 10.5 Hz), 24.3 (d, 2C, 3JP,C =10.5 Hz) (CHCH3), 19.8 (d, 3JP,C = 10.5 Hz, CH2CN). 31P NMR (DMSO-d6) δ 144.79.HRMS (ESI+): m/z [M+Na]+ calcd for C52H62

35Cl2N3NaO12P+: 1044.3340; found:1044.3320.

3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-5-[6-(N,N-diisopropyl-amino-2-cyanoethoxyphosphinyloxy)hexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (35). Rf 0.82 (Et3N–acetone–toluene 1:4:15 v/v/v). 1H NMR(DMSO-d6) δ 8.85 (m, 1H, NH), 8.55 (m, 1H, H-4), 8.28 (dd, 1H, J6,7 = 8.0 Hz, 4J4,6

= 1.2 Hz, H-6), 7.60 (d, 1H, J6,7 = 8.0 Hz, H-7), 6.42 (s, 2H, H-1′,8′), 3.77–3.50 (m,6H, H-6′′, POCH2, CHCH3), 3.56 (s, 6H, OCH3), 3.30 (m, 2H, H-1′′), 2.77–2.68 (m,4H, H-1a′′′, CH2CN), 2.00 (m, 4H, H-2e′′′,6e′′′), 1.72 (m, 4H, H-3e′′′,5e′′′), 1.65–1.47(m, 8H, H-2′′,2a′′′,4e′′′,6a′′′), 1.43–1.20 (m, 12H, H-3′′,4′′,5′′,3a′′′,4a′′′,5a′′′), 1.12 (d,6H, J = 6.8 Hz), 1.11 (d, 6H, J = 6.8 Hz) (CHCH3). 13C NMR (DMSO-d6) δ 172.0(2C, COCH), 167.7 (C3), 164.2 (CONH), 153.2 (C7a), 148.6 (2C, C2′,7′), 140.6 (2C,C4a′,10a′), 138.7 (2C, C3′,6′), 136.7 (C5), 134.9 (C6), 125.7 (C3a), 124.2 (2C, C4,7),119.1 (CN), 116.5 (2C), 116.2 (2C) (C4′,5′,8a′,9a′), 108.3 (2C, C1′,8′), 81.2 (C1), 62.9(d, 2JP,C = 16.0 Hz, C6′′), 58.1 (d, 2JP,C = 19.0 Hz, POCH2), 56.8 (2C, OCH3), 42.4(d, 2C, 2JP,C = 12.0 Hz, PNCH), 41.7 (2C, C1′′′), ∼40 (C1′′), 30.7 (d, 3JP,C = 7.0 Hz,C5′′), 29.0 (C2′′), 28.5 (4C, C2′′′,6′′′), 26.2 (C3′′), 25.3 (C4′′), 25.2 (2C, C4′′′), 24.5 (4C,C3′′′,5′′′), 24.4 (m, 4C, CHCH3), 19.8 (d, 3JP,C = 7.0 Hz, CH2CN). 31P NMR (DMSO-d6)δ 146.23. HRMS (ESI+): m/z [M+Na]+ calcd for C52H64

35Cl2N3NaO12P+: 1046.3497;found: 1046.3505.


4.55.25


3′,6′-Bis(cyclohexylcarbonyloxy)-4′,5′-dichloro-2′,7′-dimethoxy-6-[6-(N,N-diisopropyl-amino-2-cyanoethoxyphosphinyloxy)hexylaminocarbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene] (37). Rf 0.66 (Et3N–acetone–toluene 1:4:15 v/v/v). 1H NMR(DMSO-d6) δ 8.66 (m, 1H, NH), 8.21 (m, 1H, H-5), 8.15 (m, 1H, H-4), 7.83 (br.s, 1H, H-7), 6.44 (s, 2H, H-1′,8′), 3.77–3.50 (m, 6H, H-6′′, POCH2, CHCH3), 3.56(s, 6H, OCH3), 3.19 (m, 2H, H-1′′), 2.77–2.68 (m, 4H, H-1a′′′, CH2CN), 2.00 (m,4H, H-2e′′′,6e′′′), 1.72 (m, 4H, H-3e′′′,5e′′′), 1.65–1.47 (m, 8H, H-2′′,2a′′′,4e′′′,6a′′′),1.43–1.20 (m, 12H, H-3′′,4′′,5′′,3a′′′,4a′′′,5a′′′), 1.11 (d, 6H, J = 6.8 Hz), 1.08 (d, 6H,J = 6.8 Hz) (CHCH3). 13C NMR (DMSO-d6) δ 172.0 (2C, COCH), 167.7 (C3), 164.2(CONH), 151.7 (C7a), 148.7 (2C, C2′,7′), 141.2 (C6), 140.4 (2C, C4a′,10a′), 138.8(2C, C3′,6′), 130.2 (C5), 127.1 (C3a), 125.7 (C4), 122.1 (C7), 119.1 (CN), 116.4 (2C),116.2 (2C) (C4′,5′,8a′,9a′), 108.4 (2C, C1′,8′), 81.2 (C1), 62.9 (d, 2JP,C = 20.0 Hz,C6′′), 58.1 (d, 2JP,C = 22.8 Hz, POCH2), 56.8 (2C, OCH3), 42.4 (d, 2C, 2JP,C = 15.0Hz, PNCH), 41.8 (2C, C1′′′), ∼40 (C1′′), 30.6 (m, 3JP,C = 6.8 Hz, C5′′), 29.0 (C2′′),28.6 (4C, C2′′′,6′′′), 26.2 (C3′′), 25.3 (C4′′), 25.2 (2C, C4′′′), 24.5 (4C, C3′′′,5′′′), 24.4(m, 4C, CHCH3), 19.8 (d, 3JP,C = 10.0 Hz, CH2CN). 31P NMR (DMSO-d6) δ 146.22.HRMS (ESI+): m/z [M+Na]+ calcd for C52H64

35Cl2N3NaO12P+: 1046.3497; found:1046.3496.


Prepare Dmt-protected hydroxyprolinol (41), a pseudosugar unit for TAMRAphosphoramidites37. In a 250-mL round-bottom flask equipped with addition funnel, magnetic stirrer,

and ice bath place a Teflon-coated magnetic stirring bar, N-Cbz-hydroxy-L-proline(13.3 g, 50 mmol), and MeOH (60 mL). Add concentrated H2SO4 (1.92 mL,34.5 mmol) dropwise and stir for 2 hr. Remove the ice bath and stir at ambienttemperature for 48 hr.

38. Evaporate the solution using rotary evaporator to ∼20 to 30 mL.

39. Add EtOAc (250 mL), transfer the solution into 500-mL separatory funnel, washwith water (three times with 50 mL) and saturated NaHCO3 (twice with 200 mL).Then, transfer the solution to a 500-mL Erlenmeyer flask and dry the solution overNa2SO4 under magnetic stirring (2 hr).


41. Dry the residue in vacuum (1 mm Hg) overnight. Obtain ester 39 as colorless oil(13.0 g, 93%).

42. Characterize compound 39 by TLC and 1H NMR.

N-Cbz-hydroxy-L-proline, methyl ester (39). Rf 0.50 (CHCl3–Et3N 9:1 v/v). 1H NMR(DMSO-d6) δ 7.40–7.25 (m, 5H, ArH), 5.13–5.05 (m, 2H, CH2Ph), 4.98 (br. s, 0.5H),4.95 (br. s, 0.5H) (OH), 4.38–4.26 (m, 2H, COCH, CHOH), 3.64 (s, 1.5H), 3.54 (s,1.5H) (OCH3), 3.53–3.43 (m, 1H), 3.42–3.36 (m, 1H) (NCH2), 2.16 (v, 1H), 1.94 (m, 1H)(CHCH2CHOH).

43. In a 250-mL round-bottom flask equipped with addition funnel and magnetic stirrer,place a Teflon-coated magnetic stirring bar, ester 39 (3.63 g, 13 mmol), and THF(90 mL). Stir until the compound dissolves. Add NaBH4 (1.18 g, 31 mmol) in oneportion and then MeOH (4.0 mL) dropwise. Stir for 16 hr.

44. Add additional MeOH (20 mL) dropwise, then stir for 1 hr until hydrogen evolutionceases.

45. Add water (200 mL), transfer the solution into 1-L separatory funnel, and extract withEtOAc (200 mL). Wash the organic layer with 5% citric acid (twice with 100 mL)



Labeling

4.55.26


and water (100 mL). Then transfer the solution in a 250-mL Erlenmeyer flask anddry the solution over Na2SO4 with magnetic stirring (2 hr).


47. Apply the residue to a silica gel column (4 × 20–cm) and elute with 0→20% MeOHin CHCl3. Collect fractions containing major compound, combine and evaporate todryness.

48. Dry the residue in vacuum (1 mm Hg) overnight. Obtain compound 40 (3.00 g, 92%)as colorless oil.

49. Characterize compound 40 by TLC and 1H and 13C NMR.

(3R,5S)-3-Hydroxy-5-hydroxymethyl-1-benzyloxycarbonylpyrrolidine (40). Rf 0.30(CHCl3–Et3N 9:1 v/v). 1H NMR (DMSO-d6) δ 7.40–7.29 (m, 5H, ArH), 5.12–5.00 (m, 2H,CH2Ph), 4.87 (m, 1H, CHOH), 4.67 (m, 1H, CH2OH), 4.24 (m, 1H, CHOH), 3.86 (m, 1H,CHCH2OH), 3.53–3.42 (m, 2H, CH2OH), 3.37–3.25 (m, 2H, NCH2), 1.98 (m, 1H), 1.84(m, 1H) (CHCH2CHOH). 13C NMR (DMSO-d6): 154.4 (CO), 137.1 (C1), 128.3 (2C, C3),127.7 (C4), 127.4 (2C, C2) (Ph), (68.2, 67.7) (1C, CHOH), (65.8, 65.6) (1C, CH2Ph),(62.2, 61.4) (1C, CH2OH), (58.0, 57.3) (1C, NCH2), (55.2, 54.8) (1C, CHCH2OH), (37.1,36.3) (1C, CHCH2CHOH).

50. In a 250-mL round-bottom flask place diol 40 (2.77 g, 11 mmol) and dry pyridine(160 mL), and evaporate away half of the volume on a rotary evaporator.

51. Add ice bath and magnetic stirrer, place inside the flask Teflon-coated magneticstirring bar, then add dimethoxytrityl chloride (4.1 g, 12 mmol) in one portion. Stirfor 2 hr, remove the cooling, stir for next 16 hr.

52. Add MeOH (1.0 mL) and stir for 1 hr.

53. Add EtOAc (300 mL), transfer the solution into a 1-L separatory funnel, and washwith water (200 mL), EtOAc (200 mL), saturated NaHCO3 (twice with 200 mL),5% citric acid (twice with 100 mL), saturated NaHCO3 (100 mL), and water (100mL). Then transfer the solution in a 500-mL Erlenmeyer flask and dry the solutionover Na2SO4 under magnetic stirring (2 hr).

54. Remove the drying agent by vacuum filtration through 200-mL sintered-glass funnel(25- to 50-μm porosity) in a 500-mL round-bottom flask. Evaporate the solutionusing rotary evaporator.

55. Apply the residue to a silica gel column (4 × 30–cm) and elute with 0→20% acetonein CHCl3. Collect fractions containing major compound, combine, and evaporate todryness.

56. Dry the residue under vacuum (1 mm Hg) overnight. Obtain compound 41 (5.06 g,83%) as white foam.

57. Characterize compound 41 by TLC, HRMS, and 1H and 13C NMR.

(3R,5S)-3-Hydroxy-5-(4,4′-dimethoxytrityloxymethyl)-1-benzyloxycarbonylpyrrolidine(41). Rf 0.31 (CHCl3–EtOAc 1:1 v/v). 1H NMR (DMSO-d6) δ 7.38–7.15 (m, 13H, ArH(Dmt, Ph)), 7.08 (m, 1H, ArH (Dmt)), 6.85 (d, 4H, J 8.2, ArH (Dmt)), 5.07 (s, 1H, OH),4.92 (m, 2H, CH2Ph), 4.31 (m, 1H, CHOH), 4.01 (m, 1H, CHCH2ODmt), 3.72 (s, 6H,OCH3), 3.42 (m, 2H, CH2ODmt), 3.21 (m 1H), 3.06 (m, 1H) (NCH2), 2.09–1.87 (m, 2H,CHCH2CHOH). 13C NMR (DMSO-d6): 158.0 (2C, C4′ (COCH3), Dmt), 154.2 (CO),145.0 (C1, Dmt), (137.1, 136.6) (1C, C1, Ph), (135.8, 135.7) (1C), 135.6 (C1′, Dmt),129.5 (4C, C2′,6′, Dmt), 128.3, 128.2 (C3,5, Ph), 127.7 (2C, C3,5, Dmt), 127.7 (C4, Ph),127.5 (2C, C2,6, Dmt), (127.5, 127.2) (C2,6, Ph), 126.6 (C4, Dmt), 113.1 (4C) (C3′,5′,


4.55.27


Dmt), 85.2 (Ar3C (Dmt)), (68.4, 67.8) (1C, CHOH), (65.9, 65.6) (1C, CH2Ph), (64.4,63.4) (1C, CCH2ODmt), (56.0, 55.2) (1C, NCH2), 55.0 (2C, OCH3, Dmt), (55.4, 54.9)(1C, CH2ODmt), (37.7, 36.6) (1C, CHCH2CHOH). HRMS (MALDI+): m/z [M]+: calcdfor C34H35NO6+: 553.6498; found: 553.6501.

58. In 250-mL round-bottom flask equipped with magnetic stirrer place Teflon-coatedmagnetic stirring bar, compound 41 (5.0 g, 9.0 mmol), and MeOH (150 mL). Stiruntil oil dissolves.

59. Add a gas inlet and displace air with argon. Add 10% Pd on carbon (300 mg) in oneportion. Displace argon with hydrogen; keep excess hydrogen pressure at ∼0.1 bar.Stir the mixture for 2 hr.

CAUTION: Hydrogen gas can be explosive when mixed with air. Avoid flame and heaters.

60. Remove the catalyst by vacuum filtration through 0.2 μm PTFE membrane in a250-mL round-bottom flask. Evaporate the solution using rotary evaporator.

61. Dry the residue under vacuum (1 mm Hg) overnight. Obtain compound 42 (3.77 g,99%) as white foam.

62. Characterize compound 42 by TLC and 1H NMR.

(3R,5S)-3-Hydroxy-5-(4,4′-dimethoxytrityloxymethyl)-pyrrolidine (42). Rf 0.30 (CHCl3–MeOH 9:1 v/v). 1H NMR (DMSO-d6) δ 7.42–6.80 (m, 13H, ArH), 4.35 (m, 1H, OCH),3.62 (m, 1H, NCH), 3.13–2.88 (m, 4H, OCH2, NCH2), 1.87 (m, 1H, CHCH2CH), 1.65(m, 1H, CHCH2CH).

Prepare amides of Dmt-hydroxyprolinol and 5(6)-carboxy-TAMRA (43 and 45),precursors for TAMRA phosphoramidites63. In a 100-mL round-bottom flask equipped with a magnetic stirrer and a calcium

chloride drying tube, place a Teflon-coated magnetic stirring bar, carboxytetram-ethylrhodamine [5-carboxy-TAMRA (20) or 6-carboxy-TAMRA (21); 1.72 g, 4.0mmol], and DMF (22 mL). Stir until solid dissolves.

64. Add PyBOP (2.08 g, 4.0 mmol) and DIEA (1.39 mL, 8.0 mmol), and stir for 5 min.

65. Add compound 42 (1.68 g, 4.00 mmol) in DMF (8 mL) in one portion and stir for 2 hr.

66. Transfer the mixture into a 1-L separatory funnel, dilute with EtOAc (350 mL),and wash with water (50 mL), saturated NaHCO3 (350 mL), and brine (200 mL).Next, transfer the solution to a 500-mL Erlenmeyer flask and dry over MgSO4 undermagnetic stirring (2 hr).

67. Remove the drying agent by vacuum filtration through 200-mL sintered-glass funnel(25- to 50-μm porosity) in a 500-mL round-bottom flask. Evaporate the solutionusing rotary evaporator.

68. Apply the residue on a silica gel column (3 × 20–cm) and elute with acetonecontaining 1% Et3N. Collect fractions containing major compound, combine, andevaporate to dryness.

69. Dry the residue in vacuum (1 mm Hg) overnight. Obtain compounds 43 (1.98 g,60%) or 45 (1.52 g, 46%) as dark red amorphous foam.

70. Characterize compounds 43 and 45 by TLC, mass spectrometry, and 1H and 13CNMR. See Figure 4.55.12 for atom numbering.

1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-5-yl-c-arbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (43). Rf

0.25 (Et3N–MeOH–acetone 2:20:78 v/v/v). 1H NMR (CDCl3), spirolactone form num-bering, δ 8.14 (br. s, 1H, H-4′), 7.79 (d, 1H, J6

′,7

′ = 7.8 Hz, H-6′), 7.43 (d, 2H, J2′ ′ ′ ′,3′ ′ ′ ′



Labeling

4.55.28


OMe2N NMe2

O

O

1''

2''

3''4'' 5''

4a'' 10a''6''

7''8''

8a''1'

3'

3a'

4'

5'

6'

7'7a'

1''''

2''''3''''

4''''

5'''' 6''''

1'''2'''

3'''4'''

5'''

6'''

9a'

43, 44, 45, 46

R = H or CEP

N

OR

O

O

20b.Et3N, 21b.Et3N

OMe

OMe

2

34

5

OMe2N NMe2

CO2

1

2

34 5

4a 10a

6

78

8a9

2'

3'4'

5'

6'1'

9a

O2CEt3NH

Figure 4.55.12 Atom numbering for NMR assignments in open chain and lactone forms ofTAMRA derivatives.

= J5′ ′ ′ ′,6′ ′ ′ ′ = 7.4 Hz, H-2′′′′,6′′′′), 7.31 (m, 4H, H-2′′′,6′′′), 7.27 (m, 2H, H-3′′′′,5′′′′), 7.18(m, 2H, H-7′,4′′′′), 6.81 (m, 4H, H-3′′′,5′′′), 6.75–6.66 (m, 2H, H-1′′,8′′), 6.49 (m, 2H,H-4′′,5′′), 6.44 (m, 2H, H-2′′,7′′), 4.72 (m, 1H, H-3), 4.49 (m, 1H, H-5), 3.82–3.54 (m,9H, OCH3, OCH2, H-2a), 3.30 (m, 1H, H-2b), 2.98 (m, 12H, NCH3), 2.34–2.26 (m, 1H,H-4a), 2.15–2.08 (m, 1H, H-4b). 13C NMR (CDCl3) δ 169.2 (OCO), 169.1 (NCO), 158.5(2C, C4′′′), 153.8 (2C, C4a′′,10a′′), 153.0 (2C, C3′′,6′′), 151.0 (C7a′), 145.1 (C1′′′′),138.3 (C3a′ or C5′), 136.2 (2C, C1′′′), 133.2 (C6′), 130.1 (5C, C2′′′,6′′′, C5′ or C3a′),129.4 (2C, C1′′,8′′), 128.2 (2C, C3′′′′,5′′′′), 127.9 (2C, C2′′′′,6′′′′), 126.9 (C4′′′′), 125.3(C7′), 124.9 (C4′), 113.2 (4C, C3′′′,5′′′), 109.7 (2C, C2′′,7′′), 107.6 (2C, C8a′′,9a′′),98.2 (C4′′ or C5′′), 98.1 (C4′′ or C5′′), 85.9 (Ar3C (Dmt)), 70.6 (C5), 63.5 (C2), 59.1(OCH2), 56.2 (C3), 55.3 (2C, OCH3), 40.4 (4C, NCH3), 36.8 (C4). HRMS (MALDI+):m/z [M+H]+ calcd for C51H50N3O8

+ 832.3592; found 832.3595.

1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-6-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-hydroxypyrrolidine (45). Rf

0.16 (Et3N–MeOH–acetone 2:20:78 v/v/v). 1H NMR, spirolactone form numbering,(CDCl3) δ 8.01 (d, 1H, J4′,5′ = 8.0 Hz, H-4′), 7.76 (d, 1H, J4′,5′ = 8.0 Hz, H-5′), 7.34 (m,2H, H-2′′′′,6′′′′), 7.27 (br. s, 1H, H-7′), 7.26–7.19 (m, 4H, H-2′′′,6′′′), 7.15–7.05 (m, 3H,H-3′′′′,4′′′′,5′′′′), 6.80–6.70 (m, 6H, H-1′′,8′′,3′′′,5′′′), 6.51–6.41 (m, 4H, H-2′′,4′′,5′′,7′′),4.60 (m, 1H, H-3), 4.39 (m, 1H, H-5), 3.78–3.40 (m, 9H, OCH3, OCH2, H-2a), 3.21 (m,1H, H-2b), 2.96 (m, 12H, NCH3), 2.16 (m, 1H, H-4a), 2.04 (m, 1H, H-4b). 13C NMR(CDCl3) δ 169.3 (OCO), 169.1 (NCO), 158.4 (2C, C4′′′), 154.3 (C4a′′ or C10a′′), 154.2(C4a′′ or C10a′′), 153.4 (2C, C3′′,6′′), 147.4 (C7a′), 145.0 (C1′′′′), 141.6 (C6′), 136.4(C1′′′), 135.9 (C1′′′), 130.2 (4C, C2′′′,6′′′), 130.1 (C3a′), 129.9 (2C, C1′′,8′′), 129.0(C5′), 128.0 (2C, C3′′′′,5′′′′), 127.8 (2C, C2′′′′,6′′′′), 126.8 (C4′′′′), 126.7 (C4′), 124.6(C7′), 113.1 (4C, C3′′′,5′′′), 110.3 (C2′′ or C7′′), 110.2 (C2′′ or C7′′), 108.4 (C8a′′ orC9a′′), 108.3 (C8a′′ or C9a′′), 97.9 (2C, C4′′,5′′), 85.8 (Ar3C (Dmt)), 70.4 (C5), 63.6(C2), 58.9 (OCH2), 56.2 (C3), 55.2 (2C, OCH3), 40.4 (4C, NCH3), 36.5 (C4). HRMS(MALDI+): m/z [M+H]+ calcd for C51H50N3O8

+ 832.3592; found 832.3594.

Prepare hydroxyprolinol-based TAMRA phosphoramidites (44 and 46)71. In a 250-mL round-bottom flask equipped with a magnetic stirrer and a calcium

chloride drying tube, place a Teflon-coated magnetic stirring bar, amide 43 or 45(0.83 g, 1.0 mmol), and dry DCM (100 mL). Stir until solid dissolves.

72. Evaporate the solvent using rotary evaporator. Add DCM (100 mL) and evaporateagain.


73. Add DCM (100 mL), diisopropylammonium tetrazolide (0.26 g, 1.5 mmol), andbis(N,N-diisopropylamino)-2-cyanoethoxyphosphine (476 μL, 1.5 mmol). Stir underargon for 1 hr.


4.55.29


74. Monitor conversion of the starting compound by TLC [using 98 parts (v/v) acetone/2parts (v/v) Et3N).

75. Transfer the mixture into 500-mL separatory funnel, dilute with DCM (100 mL),wash with saturated NaHCO3 (100 mL), and wash with brine (100 mL). Then,transfer the solution in a 500-mL Erlenmeyer flask and dry the solution over Na2SO4

under magnetic stirring (2 hr).

76. Remove the drying agent by vacuum filtration through 100-mL sintered-glass funnel(25- 50-μm porosity) in a 500-mL round-bottom flask. Evaporate the solution usingrotary evaporator.

77. Apply the residue to a silica gel column (3 × 20–cm) and elute with 1% (w/v) Et3Nin acetone. Collect fractions containing major compound, combine, and evaporate todryness. Obtain compounds 44 (0.75 g, 73%) or 46 (0.48 g, 47%) as red amorphoussolids.

78. Characterize the compounds 44, and 46 by TLC, HRMS, and 1H, 13C, and 31P NMR.

1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-5-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-(N,N-diisopropylamino-2-cya-noethoxyphosphinyloxy)pyrrolidine (44). Rf 0.60 (1% Et3N in acetone). 1H NMR(MeCN-d3), spirolactone form numbering, δ 8.05 (br. s, 1H, H-4′), 7.82 (m, 1H, H-6′),7.46 (m, 2H, H-2′′′′,6′′′′), 7.36–7.18 (m, 8H, H-7′,2′′′,6′′′,3′′′′,4′′′′,5′′′′), 6.84 (m, 4H,H-3′′′,5′′′), 6.75–6.62 (m, 2H, H-1′′,8′′), 6.51 (m, 4H, H-2′′,4′′,5′′,7′′), 4.55 (m, 2H,H-3,5), 4.15–3.45 (m, 13H, OCH3, POCH2, DmtOCH2, NCH, H-2a), 3.21 (m, 1H, H-2b),2.98 (m, 12H, NCH3), 2.75 (t, 1H, J = 5.9 Hz, CH2CN), 2.63 (m, 1H, CH2CN), 2.37–2.20(m, 2H, H-4), 1.17–1.04 (m, 12H, CHCH3). 13C NMR (MeCN-d3) δ 169.6 (OCO), 169.5(NCO), 159.6 (2C, C4′′′), 154.1 (2C, C4a′′,10a′′), 153.8 (2C, C3′′,6′′), 153.1 (C7a′),146.3 (C1′′′′), 139.5 (0.5C, C5′ or C3a′), 139.4 (0.5C, C5′ or C3a′), 137.1 (2C, C1′′′),134.4 (m, C6′), 130.9 (m, 4C, C2′′′,6′′′), 129.9 (m, 3C, C3a′ or C5′, C1′′,8′′), 128.9 (2C,C2′′′′,6′′′′), 128.8 (2C, C3′′′′,5′′′′), 127.7 (C4′′′′), 125.6 (C7′), 125.1 (m, C4′), 119.4 (m,CN), 114.0 (4C, C3′′′,5′′′), 110.3 (2C, C2′′,7′′), 107.8 (2C, C8a′′,9a′′), 98.8 (2C, C4′′,5′′),86.6 (Ar3C (Dmt)), 73.6 (m, C5), 64.1 (m, C2), 59.5–58.6 (m, 2C, CH2ODmt, POCH2),56.9 (m, C3), 55.8 (2C, OCH3), 44.0 (m, 2C, CHCH3), 40.5 (4C, NCH3), 36.4 (C4),23.1 (m, 4C, CHCH3), 20.9 (m, 2C, CH2CN). 31P NMR (MeCN-d3) δ 147.35, 147.22(diastereomers). HRMS (ESI+): m/z [M+Na]+ calcd for C60H66N5NaO9P+ 1054.4490;found 1054.4492.

1-{3′,6′-Bis(dimethylamino)-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-6-yl-carbonyl}-(3R,5S)-5-[(4,4′-dimethoxytrityl)oxymethyl]-3-(N,N-diisopropylamino-2-cya-noethoxyphosphinyloxy)pyrrolidine (46). Rf 0.35 (1% Et3N in acetone). 1H NMR(MeCN-d3), spirolactone form numbering, δ 8.01 (m, 1H, H-4′), 7.74 (m, 1H, H-5′), 7.40–7.04 (m, 10H, H-7′,2′′′,6′′′,2′′′′,3′′′′,4′′′′,5′′′′,6′′′′), 6.86–6.62 (m, 6H, H-1′′,8′′,3′′′,5′′′),6.51–6.41 (m, 4H, H-2′′,4′′,5′′,7′′), 4.54–4.39 (m, 2H, H-3,5), 3.79–3.39 (m, 14H, OCH3,POCH2, DmtOCH2, NCH, H-2), 3.03–2.86 (m, 13H, NCH3, CH2CN), 2.57 (t, 1H, J= 5.9 Hz, CH2CN), 2.36–2.19 (m, 2H, H-4), 1.21–0.86 (m, 12H, CHCH3). 13C NMR(MeCN-d3) δ 169.6 (OCO), 169.2 (NCO), 159.5 (C4′′′), 159.4 (C4′′′), 154.0–153.0 (m,4C, C3′′,6′′,4a′′,10a′′), 146.0 (C1′′′′), 144.3 (C6′), 137.3 (C1′′′), 136.7 (C1′′′), 131.0(2C), 130.7 (2C) (C2′′′′,6′′′′), 129.9 (C5′), 129.5 (m, 3C, C3a′,1′′,8′′), 128.7 (m, 4C,C2′′′′,3′′′′,5′′′′,6′′′′), 127.6 (C4′′′′), 125.9 (m, C4′), 123.7 (0.5C), 123.6 (0.5C) (C7′), 113.8(4C, C3′′′,5′′′), 109.9 (m, 2C, C2′′,7′′), 107.0 (2C, C8a′′,9a′′), 99.0 (m, 2C, C4′′,5′′), 86.4(Ar3C (Dmt)), 73.5–73.0 (m, C5), 63.5 (m, C2), 59.2 (m, POCH2), 58.4 (m, CH2ODmt), 56.8 (m, C3), 55.8 (2C, OCH3), 43.8 (m, 2C, CHCH3), 40.4 (4C, NCH3), 35.9 (C4),24.8 (m, 4C, CHCH3), 20.9 (m, 2C, CH2CN). 31P NMR (MeCN-d3) δ 147.39, 146.55(diastereomers). HRMS (ESI+): m/z [M+Na]+ calcd for C60H66N5NaO9P+ 1054.4490;found 1054.4494.




Labeling

4.55.30


BASICPROTOCOL 4

AUTOMATED SOLID-PHASE SYNTHESIS, PURIFICATION, ANDCHARACTERIZATION OF OLIGONUCLEOTIDES LABELED WITH FAM,JOE, AND TAMRA

The protocol describes synthesis of FAM-, JOE-, and TAMRA-modified oligonu-cleotides. These can be prepared using any automated DNA synthesizer containingbottles for modifying reagents (we used ASM 800 DNA Synthesizer from Biosset).

Materials

Dried phosphoramidites 23, 25, 27, 29, 31, 33, 35, 37, 44, and 46 prepared asdescribed in Basic Protocol 3

Dry acetonitrileDichloromethane (DCM)CPG supports dA, dG, dC, dT, BHQ-1 (Glen Research)Conventional phosphoramidite monomers of dA, dG, dC, and dT (Glen Research)Reagents for oligonucleotide synthesis (also see APPENDIX 3C):

0.45 M 1H-tetrazole in MeCN (Glen Research)Cap A solution: acetic anhydride in tetrahydrofuran/pyridine (Glen Research)Cap B solution: 1-methylimidazole in tetrahydrofuran (Glen Research)Deblocking solution: 3% trichloroacetic acid in DCM (Glen Research)

25% (v/v) ammoniat-Butylamine (tBuNH2; Sigma-Aldrich, cat. no. 391433)0.1 M ammonium acetate (APPENDIX 2A)Acetonitrile (MeCN)

Automated DNA synthesizer (ASM 800 from Biosset, http://www.biosset.com;also see APPENDIX 3C)

2-mL cryovialsSpeedvac concentrator (Eppendorf)5-μm Discovery C18 HPLC column (25 cm × 4.6 mm; Supelco)1-mL quartz cuvettes with Teflon capsUV/Vis spectrophotometer

Additional reagents and equipment for automated DNA synthesis (APPENDIX 3C),denaturing polyacrylamide gel electrophoresis (PAGE; UNIT 10.4), ethanolprecipitation of DNA (Moore and Dowhan, 2002), column chromatography(APPENDIX 3E), HPLC purification (UNIT 10.5). and MALDI-TOF massspectrometry (UNIT 10.1)

Synthesize modified oligonucleotides1. Prepare 0.1 M solution of modifying phosphoramidite in dry acetonitrile [1:1 (v/v)

acetonitrile–DCM v/v for phosphoramidite 31] and place in DNA synthesizer.

2. Place the appropriate nucleoside or BHQ-1 column (for 0.2-μmol-scale solid-phasesynthesis) into the DNA synthesizer. Input the desired nucleotide sequence, increasecoupling time for modifying phosphoramidites to 7.5 min, and start the synthesizeraccording to the manufacturer’s protocol.

3. Perform the nucleotide chain assembly and introduction of modifying reagent(s).

4. Cleave an oligomer from solid support using 25% ammonia (1 mL, 1 hr) and collectthe solution in a 2-mL cryovial.

Use “TAMRA cocktail,” 1:1:2 tBuNH2/MeOH/H2O, for TAMRA-containing oligonu-cleotides.

5. Close tightly and keep for deprotection at 60◦C for 6 hr, then cool to roomtemperature.


4.55.31


6. Evaporate to dryness using Speedvac concentrator.

Purify modified oligonucleotides by PAGE and HPLC7. Purify the crude product by denaturing PAGE (UNIT 10.4) and desalt the oligonu-

cleotide by ethanol precipitation (Moore and Dowhan, 2002), size-exclusion filtration(APPENDIX 3E), or affinity cartridge protocols (UNIT 10.7).

8. Purify each oligonucleotide on a 5-μm Discovery C18 HPLC column (UNIT 10.5).Starting from 0.1 M ammonium acetate, pH 7.0, use a linear gradient of 3.3%MeCN/min at a flow rate of 1 mL/min for 30 min.

9. Collect the chromatographic peaks corresponding to each product in a 2-mLpolypropylene microcentrifuge tube and evaporate using Speedvac concentratorwithout heat. Desalt (UNIT 10.7) and evaporate again.

10. Dissolve modified oligonucleotide in Milli-Q water and quantify by measuring UVabsorbance at 260 nm. Register UV/Vis spectrum in a quartz cuvette to confirm thepresence of a dye (or a dye and the BHQ-1 quencher).

11. For further characterization of modified oligonucleotides use MALDI-TOF massspectrometry (UNIT 10.1) and fluorescence spectrometry.

COMMENTARY

Background InformationModified phosphoramidites containing flu-

orescent dyes are reagents of choice to produce5′-labeled oligonucleotides during automatedsynthesis (UNITS 4.2 & 4.10). Several reagentsare commercially available. However, they arerather expensive, and their synthesis is de-scribed solely in patents.

Spectral and photophysical studies showthat 6-isomers have slightly narrower emis-sion band and fluorescence quantum yieldsthan 5-isomers (Table 4.55.1). The differ-ence in emission spectra between 5- and 6-isomers decreases in the sequence FAM > JOE>> TAMRA (see references cited in Table4.55.1). The use of a 4-trans-aminocyclohex-anol rigid linker versus a flexible linker basedon 6-aminohexanol prevents JOE fluorescencequenching with dG nucleosides present inclose proximity to a 5′-dye residue (Tsybulskyet al., 2012). FAM is a common energy donorfor TAMRA. In energy transfer primers con-taining on their 5′-termini a (6-FAM)dTn(6-TAMRA) dye system (n = 0, 2, 4, 6, 8, 10,12, 14), the linker with n = 2 was foundpreferable in terms of energy transfer effi-ciency (Kvach et al., 2009). 5′-Dye,3′-BHQ-1-modified oligonucleotides are suitable as real-time PCR probes; moreover, JOE dye is a goodquencher for FAM (Ryazantsev et al., 2012).

Critical Parameters andTroubleshooting

The synthesis of xanthene fluorescent dyesand their phosphoramidites requires experi-

ence with routine chemical laboratory tech-niques such as vacuum distillation, solventevaporation, extraction, filtration, TLC, andcolumn chromatography. Anhydrous condi-tions must be observed during phospho-ramidite preparation. The synthesis and iso-lation of modified oligonucleotides requiresexperience with DNA synthesis, gel elec-trophoresis, and HPLC. Characterization ofcompounds and oligonucleotide conjugatesrequires knowledge of NMR (1H, 13C, 19F,31P), UV, fluorescence, and mass spectroscopy(MALDI-TOF and ESI-TOF). Do not forget touse the TAMRA cocktail for removal of pro-tecting groups from TAMRA-containing con-jugates. There are many hazardous compoundsand solvents used in this protocol; thus, labo-ratory safety is of primary concern when re-peating the procedures.

Anticipated ResultsModerate yields for steps of dye molecule

assembly and separation of isomers are verygood in terms of expense and as comparedto literature precedents (e.g., the yield of 11→ 14 conversion is 40% versus 4% as re-ported by Lyttle et al., 2001). Yields of amidesfrom pentafluorophenyl esters are excellent(93% to 100%). Condensation of TAMRAacids with hindered secondary amine Dmt-hydroxyprolinol gives modest yields of amides(60% and 46%). Yields on phosphitylationsteps are typically good (70% to 79%), ex-cept for 35 (60%), 37 (64%), and 46 (47%).Yields for the condensation step of modified



Labeling

4.55.32


Table 4.55.1 Spectral and Photophysical Properties of Xanthene Dyes as 5′-Labels ofOligonucleotides

DyeAbsorbance

maximum (nm)Emission

maximum (nm)Fluorescencequantum yield

Reference

5-FAM 494 521 0.48–0.58 Kvach et al. (2007)

6-FAM 494 517–518 0.48–0.58

5-JOE 522–526 555–560 0.60–0.80 Tsybulsky et al. (2012)

6-JOE 523–529 551–557 0.60–0.75

5-TAMRA 561–562 586–589 0.30–0.65 Kvach et al. (2009)

6-TAMRA 560–562 585–588 0.30–0.70

phosphoramidites are >90%. Although dyephosphoramidites are stable for many monthsat –20◦C in dark, we recommend bulk prepa-ration and storage of amide precursors. Por-tions of these should be used for preparationof phosphoramidites in amounts that will beconsumed in the next 2 to 4 months. Then,further portions of amide should be convertedinto portions of phosphoramidite for use overthe next 2 to 4 months, and so forth.

Time ConsiderationsThe complete synthesis of each modify-

ing phosphoramidite can be accomplished in1 to 3 weeks, depending on scale, the natureof the linker (hydroxyprolinol linker requiresseveral days to synthesize versus commer-cially available 6-aminohexanol and trans-4-aminocyclohexanol), and the nature of the dye(JOE requires time-consuming preparation of2-chloro-4-methoxyresorcinol, 13). The timefor oligonucleotide synthesis is practically thesame as for nonmodified oligonucleotides (theonly change is an additional 7 min for the con-densation step). PAGE and RP-HPLC purifi-cation of modified oligonucleotides and real-time PCR probes can be performed in 1 to2 days.

AcknowledgmentsThis work was supported by the Molecular

and Cellular Biology Program of the RussianAcademy of Sciences, Russian Foundation forBasic Research (projects 08-03-90900, 10-04-00998, 13-04-01317), and Belarusian Founda-tion for Basic Research (project B06R-004).

Literature CitedBannwarth, W. and Trzeciak, A. 1987. A simple and

effective chemical phosphorylation procedurefor biomolecules. Helv. Chim. Acta 70:175–186.

Caruthers, M.H., Barone, A.D., Beaucage, S.L.,Dodds, D.R., Fisher, E.F., McBride, L.J.,

Matteucci, M., Stabinsky, Z., and Tang, J.-Y. 1987. Chemical synthesis of deoxyoligonu-cleotides by the phosphoramidite method. Meth.Enzymol. 154:287-313.

Drechsler, G. and Smagin, S. 1965. Zur Darstel-lung von Trimelliteinen und 5′-Carboxy-fluoresceinen. J. Prakt. Chem. 28:315-324.

Faulkner, J.K. and Woodcock, D. 1962. Somederivatives of isovanillin. J. Chem. Soc. 1962:4737-4738.

Haralambidis, J., Angus, K., Pownall, S., Dun-can, L., Chai, M., and Tregear, G.W. 1990.The preparation of polyamide-oligonucleotideprobes containing multiple non-radioactive la-bels. Nucl. Acids Res. 18:501-505.

Kvach, M.V., Tsybulsky, D.A., Ustinov, A.V.,Stepanova, I.A., Bondarev, S.L., Gontarev, S.V.,Korshun, V.A., and Shmanai, V.V. 2007. 5(6)-Carboxyfluorescein revisited: New protectinggroup, separation of isomers, and their spec-tral properties on oligonucleotides. Bioconjug.Chem. 18:1691-1696.

Kvach, M.V., Stepanova, I.A., Prokhorenko, I.A.,Stupak, A.P., Bolibrukh, D.A., Korshun, V.A.,and Shmanai, V.V. 2009. Practical synthesis ofisomerically pure 5- and 6-carboxytetramethy-lrhodamines, useful dyes for DNA probes. Bio-conjug. Chem. 20:1673-1682.

Lee, M. and Grissom, C.B. 2009. Design, synthesis,and characterization of fluorescent cobalaminanalogues with high quantum efficiencies. Org.Lett. 11:2499-2502.

Lyttle, M.H., Carter, T.G., and Cook, R.M. 2001.Improved synthetic procedures for 4,7,2′,7′-tetrachloro- and 4′,5′-dichloro-2′,7′-dimethoxy-5(and 6)-carboxyfluoresceins. Org. Proc. Res.Dev. 5:45-49.

Moore, D. and Dowhan, D. 2002. Purification andconcentration of DNA from aqueous solutions.Curr. Protoc. Mol. Biol. 59:2.1.1–2.1.10.

Ryazantsev, D.Y., Tsybulsky, D.A., Prokhorenko,I.A., Kvach, M.V., Martynenko, Y.V.,Philipchenko, P.M., Shmanai, V.V., Korshun,V.A., and Zavriev, S.K. 2012. Two-dye andone- or two-quencher DNA probes for real-timePCR assay: Synthesis and comparison with aTaqManTM probe. Anal. Bioanal. Chem. 404:59-68.


4.55.33


Schreder, J. 1878. Uber eine Fluorescein-Carbonsaure. Ber. Dtsch. Chem. Ges. 11:1340-1344.

Smith, L.M., Kaiser, R.J., Sanders, J.Z., and Hood,L.E. 1987. The synthesis and use of fluores-cent oligonucleotides in DNA sequence analy-sis. Meth. Enzymol. 155:260-301.

Tsybulsky, D.A., Kvach, M.V., Stepanova,I.A., Korshun, V.A., and Shmanai, V.V.2012. 4′,5′-Dichloro-2′,7′-dimethoxy-5(6)-carboxyfluorescein (JOE): Synthesis andspectral properties of oligonucleotide conju-gates. J. Org. Chem. 77:977-984.

Ueno, Y., Jiao, G.-S., and Burgess, K. 2004. Prepa-ration of 5- and 6-carboxyfluorescein. Synthesis2004:2591-2593.

Xu, K., Liu, F., Ma, J., and Tang, B. 2011. A newspecific fullerene-based fluorescent probe fortrypsin. Analyst 136:1199-1203.

Yokoyama, M., Yoshida, S., and Imamoto, T. 1982.Organic reactions using trimethylsilyl polyphos-phate (PPSE): A convenient synthesis of nitrilesfrom carboxamides. Synthesis 1982:591-592.

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Current Protocols in Nucleic Acid Chemistry || Non-Nucleoside Phosphoramidites of Xanthene Dyes (FAM, JOE, and TAMRA) for Oligonucleotide Labeling