A Scalable Synthesis of α-L-Threose Nucleic Acid Monomers

Article abstract of DOI:10.1021/acs.joc.5b02768

Recent advances in polymerase engineering have made it possible to copy information back and forth between DNA and artificial genetic polymers composed of TNA (α-l-threofuranosyl-(3′,2′) nucleic acid). This property, coupled with enhanced nuclease stability relative to natural DNA and RNA, warrants further investigation into the structural and functional properties of TNA as an artificial genetic polymer for synthetic biology. Here, we report a highly optimized chemical synthesis protocol for constructing multigram quantities of TNA nucleosides that can be readily converted to nucleoside 2′-phosphoramidites or 3′-triphosphates for solid-phase and polymerase-mediated synthesis, respectively. The synthetic protocol involves 10 chemical transformations with three crystallization steps and a single chromatographic purification, which results in an overall yield of 16-23% depending on the identity of the nucleoside (A, C, G, T).

Full text of DOI:10.1021/acs.joc.5b02768

Subscriber access provided by NEW YORK UNIV
Article
A Scalable Synthesis of alpha-L-Threose Nucleic Acid Monomers
Sujay P. Sau, Nour Eddine Fahmi, Jen-yu Liao, Saikat Bala, and John C. Chaput
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b02768 • Publication Date (Web): 19 Feb 2016
Downloaded from http://pubs.acs.org on February 24, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
α
A Scalable Synthesis of -L-Threose
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nucleic Acid Monomers
Sujay P. Sau1, Nour Eddine Fahmi2, Jen-Yu. Liao1, Saikat Bala1
and John C. Chaput1*
1Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697
2The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5301
*To whom correspondence should be addressed: John Chaput
Tel. 480-727-0392; E-mail: jchaput@uci.edu
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 28
1
2
3
4
5
6
7
8
9
Abstract. Recent advances in polymerase engineering have made it possible to copy
information back and forth between DNA and artificial genetic polymers composed of
TNA (α-L-threofuranosyl-(3’, 2’) nucleic acid). This property, coupled with enhanced
nuclease stability relative to natural DNA and RNA warrants further investigation into the
structural and functional properties of TNA as an artificial genetic polymer for synthetic
biology. Here, we report a highly optimized chemical synthesis protocol for constructing
multi-gram quantities of TNA nucleosides that can be readily converted to nucleoside 2’-
phosphoramidites or 3’-triphosphates for solid-phase and polymerase-mediated
synthesis, respectively. The synthetic protocol involves 10 chemical transformations
with three crystallization and a single chromatographic purifications and results in an
overall yield of 16-23% depending on the identity of the nucleoside (A, C, G, T).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
TOC Figure:
HO
10 steps
(16-23 %)
H
O
O
BPG
OH
O
HO
OH
HO
DMTO
L-ascorbic acid
B = A,C,T,G
2
ACS Paragon Plus Environment

Page 3 of 28
The Journal of Organic Chemistry
1
2
3
4
Introduction
5
6
7
TNA (α-L-threofuranosyl-(3’,2’) nucleic acid) is an artificial genetic polymer in which
8
9
the natural ribose sugar found in RNA has been replaced by the tetrose sugar of α-L-
threose.1 In contrast to natural DNA and RNA, which have a six-atom backbone repeat
unit connected by 3’,5’-phosphodiester linkages, TNA has a backbone periodicity of five
atoms (or bonds) with phosphodiester linkages occurring at the 2’ and 3’ vicinal
positions of the threose sugar. Despite this major structural difference, TNA is capable
of forming stable antiparallel Watson-Crick duplexes and shows efficient cross-pairing
with complementary strands of DNA and RNA.1-3 The ability to transfer genetic
information between TNA and RNA, coupled with the chemical simplicity of threose
relative to ribose, has fueled interest in TNA as a possible progenitor of RNA during a
hypothetical period in life’s history known as the RNA world.4-6 TNA is also being
explored as a source of nuclease resistant affinity reagents (aptamers) and catalysts for
synthetic biology and molecular medicine, as the constitutional structure of TNA is
stable under biological conditions. Ongoing efforts in both of these areas have inspired
researchers to engineer polymerases that can ‘transcribe’ DNA into TNA and other
polymerases that can ‘reverse transcribe’ TNA back into DNA.7-12 By including a
selective amplification step in the replication cycle, methods are now being developed to
isolate functional TNA molecules by in vitro selection.13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Motivated by a desire to explore the structural and functional properties of TNA by
in vitro selection and atomic-level structure determination studies by solution NMR and
X-ray crystallography, we have systematically evaluated the chemical synthesis of TNA
monomers with the goal of devising a synthetic pathway that is amenable to large-scale
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 28
1
2
3
4
5
6
7
8
9
(multi-gram) synthesis. A new chemical synthesis strategy was necessary because the
original approach (Scheme 1) first described by Eschenmoser and colleagues suffered
from a number of shortcomings that included low overall yield (2-6%), numerous silica
gel purification steps, and poor regioselectivity during nucleoside tritylation.1 While the
two DMT regioisomers (3’ and 2’) 5a and 5b can be chromatographically resolved, only
trace amounts of the 2’-isomer is obtained in the case of adenosine and cytosine, which
necessitates an additional series of protection-deprotection steps to synthesize the
nucleotide 3’-triphosphates of adenosine and cytosine.14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Original strategy developed to synthesize α-L threose nucleosides.15
HO
H
O
O
O
O
O
OAc
OBz
3 steps
Ref 19
3 steps
HO
OH
OH
HO
HO
BzO
1
2
3
BPG
BPG
BPG
O
O
O
2 steps
1 step
OH
ODMT
DMTO
HO
OH
HO
4
5a
5b
Here, we describe a highly optimized chemical synthesis strategy for generating
TNA nucleoside precursors that can be readily converted to 2-phosphoramidites for
solid-phase synthesis or 3’-triphosphates for polymerase-mediated primer-extension
reactions.16,17 Our approach involves a total of 10 chemical transformations with three
crystallization and a single chromatographic purification steps and results in an overall
4
ACS Paragon Plus Environment

Page 5 of 28
The Journal of Organic Chemistry
1
2
3
4
yield of 16-23% depending on the identity of the nucleoside (A, C, G, T). We
5
6
7
8
demonstrate that this new strategy can be used to produce multi-gram quantities of TNA
monomers required to explore the structural and functional properties of TNA polymers.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Results and Discussion
The synthesis of L-threonolactone (2) from L-ascorbic acid (vitamin C) has been
reported previously (Scheme 1).1 Accordingly, L-ascorbic acid undergoes oxidative
cleavage in the presence of hydrogen peroxide and CaCO3 to produce the calcium salt
of L-threonate (6). Compound 6 is then converted to the desired L-threonolactone (2)
using a Dowex cation exchange resin to promote acidification and intramolecular
lactonization. However, due to the limited solubility of 6, both steps require large
volumes of water that must be removed prior to cyclization and crystallization.
Scheme 2. Synthesis of universal glycosyl donor for TNA nucleosides.18
Ca2+
HO
H
O
O
O
O
O
HO
HO
O
a
b
c
HO
OH
OH
HO
OH
HO
1
6
2
O
O
O
OAc
OBz
d-f
OBz
HO
TBDPSO
7
8
aReagents and conditions: (a) i) CaCO3, 30% aq. H2O2, H2O,18 h, 0 oC to rt; ii) active charcoal,
70 oC, 2 h, 85%; (b) oxalic acid, para-toluenesulfonic acid (cat), CH3CN, 2 h, reflux, 93%; (c)
benzoyl chloride, 1:10 pyridine-CH2Cl2, 0.5 h, 0 oC, 64%; (d) tert-butyldiphenylchlorosilane,
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 28
1
2
imidazole, DMAP (cat) CH2Cl2, 18 h, 0 oC to rt; (e) 1M DIBAL-H in toluene, dry 1,2-
dimethoxyethane, 0.5 h, -78 oC; (f) Ac2O-DMAP (5 equiv, 1.5 equiv) in CH2Cl2, 2 h, -78 oC to rt,
95% from 7.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We began by examining the crystallization of 6 from the resulting aqueous reaction
mixture. Literature methods require reducing the volume of aqueous solution prior to
crystallization with methanol;15,19 however, this is a tedious process, especially when the
reaction is performed on a large scale. In an attempt to avoid this step, we examined
possible conditions for obtaining pure 6 without volumetric reduction under reduced
pressure. Through minimal optimization, we found that the addition of 2 volumetric
equivalents of methanol directly to the reaction mixture allowed 6 to precipitate as a
white crystalline material in 85% yield. This subtle change to the protocol resulted in a
yield that compared favorably against previous reports (65-79%),15,17,19 and allowed
large-scale reactions to be performed with substantially higher throughput.
Next, we probed the conversion of 6 to 2 (Scheme 1). While previous literature
methods invoke the use of a Dowex cation exchange resin to generate threonic acid
from 6, such methodology is cost prohibitive for routine large-scale (≥100-gram)
synthesis.15,19 We therefore sought an alternative method for removing calcium from the
reaction mixture and promoting the subsequent in situ cyclization to the threonolactone.
For this purpose, we examined several calcium-precipitating reagents such as
hydrofluoric acid, sulfuric acid, and phosphoric acid. Of these, dilute sulfuric acid
produced the desired compound in crystalline form with yields that are comparable to
what has been achieved previously using Dowex.15,19 However, because of the
6
ACS Paragon Plus Environment

Page 7 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
hygroscopic nature of L-threonolactone (2), crystallization failed whenever the
compound was not properly dried.
7
8
9
As an alternative to dilute H2SO4, we tested oxalic acid as an in situ calcium
exchange reagent. We found that refluxing a heterogeneous mixture of 6 in acetonitrile
with 1 equivalent of oxalic acid and a catalytic amount of para-toluenesulfonic acid
produced the desired L-threonolactone (2) and calcium oxalate as an insoluble
precipitate that could be easily separated by filtration. Using this approach, pure 2 is
obtained as a white solid in 93% yield after co-evaporation with ethyl acetate. In addition
to eliminating the need for an expensive cation exchange resin, oxalic acid provided a
streamlined method for obtaining large quantities of L-threonolactone (2) without the
strict need for rigorous drying of a dilute aqueous solution to an anhydrous state.
A major limitation of the original synthetic route developed by Eschenmoser and
colleagues was the low regioselectivity observed when α-L-threofuranosyl nucleosides 4
are tritylated with DMT-Cl (Scheme 1). In most cases, tritylation produced a mixture of
2’ and 3’ regioisomers that could be separated by careful silica gel chromatography to
generate pure 5a and 5b. In principle, this synthetic strategy would have been ideal if 5a
and 5b were obtained in equal amounts; however, in practice, the tritylation of
adenosine and cytosine α-L-threofuranosyl nucleosides yielded only trace amounts of
the 2’-DMT isomer (5b). Thus, in order to obtain the precursor compounds required to
synthesize TNA 3’-nucleoside triphosphates, a cumbersome strategy of protection and
deprotection was developed to convert 5a into 5b.14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In an effort to generate antiviral compounds based on the structural framework of
TNA nucleosides, Herdewijn and colleagues developed a regioselective strategy that
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 28
1
2
3
4
5
6
7
8
9
defined the 2’ and 3’ hydroxyl positions early in the synthetic pathway.18,20 Accordingly,
the authors reported that 2 could be selectively converted to the 2-O-benzoyl L-
threonolactone derivative (7) by the addition of benzoyl chloride to a solution of 2 and
imidazole in acetonitrile after 8 hours of stirring from -5°C to room temperature (Scheme
2). Although this method proved unsatisfactory in our hands, selective benzoylation of 2
was achieved with the addition of 1 equivalent of benzoyl chloride to 2 in 1:10 pyridine-
dichloromethane solution after stirring for 30 minutes at 0 °C. Following the reaction, we
further discovered that 7 could be precipitated as a pure white powder in 64% yield after
stirring overnight at 4°C from a mixture of dichloromethane and hexanes.
In analogy to previous work on threofuranosyl nucleosides,18 compound 7 was
converted to the universal glycosyl donor 8 following three successive chemical
transformations. Accordingly, 7 was silylated at the 3-hydroxy position with tert-
butyldiphenylchlorosilane followed by DIBAL-H reduction of the lactone to the lactol and
finally acetylation at the anomeric position to give 8 in 96% yield from 7. Filtration of 8
through a short-pad of silica gel proved highly facile and efficient (see 1H-NMR in
supporting information) due to the strong polarity differences between 8 and the non-
washable reagents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
ACS Paragon Plus Environment

Page 9 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of TNA phosphoramidite precursorsa
O
O
NH
NH
a,e
f,g
8
O
O
N
N
O
O
OBz
OH
HO
DMTO
9a
10a
NHBz
N
NHBz
N
b,e
c,e
d,e
f,g
O
O
N
N
O
O
OBz
OH
HO
DMTO
9b
10b
NHBz
NHBz
N
N
N
N
N
N
N
f,g
N
O
O
OBz
OH
HO
DMTO
9c
10c
ODPC
N
ODPC
N
N
N
N
N
h,g
N
NHAc
N
NHAc
O
O
OBz
OH
10d
HO
DMTO
9d
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 28
1
2
aReagents and conditions: (a) thymine, N,O-bis(trimethylsilyl)acetamide (BSA),
trimethylsilyltriflate (TMSOTf), CH3CN, 2 h, 60 oC; (b) N4-benzoylcytosine, BSA, TMSOTf,
CH3CN, 2 h, 60 oC; (c) N6-benzoyladenine, BSA, TMSOTf, toluene, 2.5 h, 95 oC; (d) i) N2-acetyl-
O6-diphenylcarbamoyl-guanine, BSA, 1,2-dichloroethane, 0.5 h 70 oC ii) TMSOTf, CH3CN,
toluene, 1.5 h, 70 oC; (e) 1M TBAF in THF, 1 h, 0 oC, 64% for 9a, 70% for 9b, 43.5% for 9c,
69% for 9d; (f) DMT-Cl, DMAP, pyridine, 18 h, 70 oC; (g) 1M NaOH, THF-MeOH, 20 m, 0 oC,
71% for 10a, 65% for 10b, 63% for 10c; (h) DMT-Cl, AgOTf, 2,4,6-trimethylpyridine, dry CH2Cl2
18 h, 70 oC, 52% for 10d.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
From 8, we constructed a complete set of TNA nucleosides for thymidine (T, 9c),
cytidine (C, 9b), adenosine (A, 9a), and guanosine (G, 9d) using a Vorbrüggen
glycosylation (scheme 3) that involved heating the glycosyl donor and desired
nucleobase (in protected form) in the presence of trimethylsilyl trifluoromethane-
sulfonate (TMSOTf). After work-up, the 3’- tert-butyldiphenylsilyl protecting group was
removed following treatment with tetrabutylammonium fluoride for 1 hour at 0°C.
Glycosylation and desilylation proceeded smoothly for thymine, N4-benzoyl cytosine,
and N2-acetyl-O6-diphenylcarbamoyl guanine to give 9a, 9b and 9d, respectively, in 64-
70% yield after crystallization. However, glycosylation with N6-benzoyl adenine resulted
in a 3:2 mixture of N9 and N7-regioisomers. Surprisingly, similar isomeric mixtures were
obtained when SnCl4 was substituted for TMSOTf, which gave exclusively the N9-
regioisomer for glycosylation with 1,2,3-tribenzoyl threose.15 Although the N9 and N7-
regioisomers are separable by silica gel chromatography, we found that the crude
isomeric mixture could be converted to the thermodynamically favored N9-isomer by
heating in dry toluene in presence of 1 eq. of TMSOTf for 1 hour at 80°C. Further
optimization showed that glycosilation in toluene at 95°C provided the desired N9-
regioisomer as the major product. Crystallization after desilylation gave pure N9-
adenosine nucleoside (9c) in 43.5% yield.
10
ACS Paragon Plus Environment

Page 11 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Compounds 9a-d are key intermediates in the divergent synthesis of TNA
nucleoside-3’-triphosphates and 2’-phosphoramidites. For L-threofuranosyl nucleoside
3’-triphosphates, compounds 9a-d can be phosphorylated using the standard Ludwig
and Eckstein method followed by treatment with concentrated NH4OH to remove the
sugar and nucleobase protecting groups.14 For L-threofuranosyl nucleoside 2’-
phosphoramidites, compounds 9a-d are tritylated with DMT-Cl and treated with 1M
NaOH for 20 minutes at 0°C to remove the 2’-benzoyl group to obtain compounds 10a-d
(scheme 3).17 While the tritylation of 9a-c proceeded efficiently under standard
conditions, tritylation of 9d required rigorous azeotropic removal of water and the use of
AgOTf as the catalyst. Removal of the O6-diphenylcarbamoyl (DPC) group with 90%
TFA or glacial acetic acid allowed tritylation to occur under standard conditions,
suggesting that the bulky DPC group limits addition of the DMT by steric hindrance.21,22
In our laboratory, we routinely synthesize TNA nucleosides 9a-d using the above-
mentioned methodology. In a typical synthesis run, 125 g of 1 is converted into 80 g of 7
with an overall yield of 51%. In contrast to previously described methods, modifications
to the purification protocol of 6 and 2 makes it possible to generate large quantities of
highly pure 7 in just 5 days. The conversion of 7 to 8 is generally performed with 16.5 g
of 7 to obtain 35 g of 8 with an overall yield of 96% in 24 hours. The N-glycosylation of 8
followed by desilylation affords nucleosides 9a-d in 64-70% yield, with the noted
exception of 9c, which is obtained in slightly lower yield (43.5%). We have found that
the synthesis of 9a-d from 7 can be performed on scales as large as 50 grams, which is
a dramatic improvement that will help accelerate the synthesis and characterization of
TNA polymers.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 28
1
2
3
4
5
6
7
8
9
In summary, we provide a scalable and highly optimized synthesis protocol for
constructing L-threofuranosyl nucleosides that are immediate precursors of TNA
triphosphates and phosphoramidite monomers. Several key challenges have been
resolved and purification steps were minimized to increase the yield and throughput of
key intermediates. The conversion of vitamin C to L-threonolactone was optimized to
minimize the cost and labor required for generating a key intermediate in the synthesis
pathway. The universal glycosyl donor 8 was prepared from 7 in three chemical steps
followed by filtration through a short pad of silica gel. The complete set of TNA
nucleosides (9a-d) were synthesized in two reactions from 8 and purified by
crystallization. Final tritylation followed by debenzoylation provided the desired
compounds (10a-d) after silica gel column chromatography. Together, these changes
make it possible to synthesize gram-scale quantities of TNA monomers that can be
used to synthesize TNA polymers.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
12
ACS Paragon Plus Environment

Page 13 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
Experimental Section
General Methods
7
8
9
Except as otherwise noted, all non-aqueous reactions were carried out in oven-dried
glassware under a balloon pressure of argon or nitrogen. Reagents were commercially
available and used as received; anhydrous solvents were purchased as the highest
grade. Reactions were monitored by thin layer chromatography using 0.25 mm Silicycle
or EM silica gel 60 F254 plates. Column chromatography was performed using Silicycle
40-60 mesh silica gel. Yields are reported as isolated yields of spectroscopically pure
compounds. 1H and 13C NMR spectra were obtained using 400 and 500 MHz NMR
spectrometers. Chemical shifts are reported in parts per million (ppm, δ) referenced to
the 1H resonance of TMS. 13C spectra are referenced to the residual 13C resonance of
the solvent (DMSO-d6, 39.52 ppm, CD3OD, 49.00 ppm). Splitting patterns are
designated as follows: s, singlet; br, broad; d, doublet; dd, doublet of doublets; t, triplet;
q, quartet; m, multiplet.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Calcium-L-threonate (6)15
To a cold (0-5ºC) solution containing 125 g (0.71 mol) of L-ascorbic acid (1) in 1 L of
H2O, 125 g (1.25 mol) of CaCO3 was slowly added with a spatula over 30 min (evolution
of CO2 was observed). To the resulting heterogeneous slurry was added 250 mL of
30% aq. H2O2 dropwise over a period of ca. 1 h with stirring at 0-5°C. The reaction
mixture was allowed to warm to r.t and stirred overnight. The heterogeneous slurry was
filtered and the filter cake was washed with two-100 mL portions of H2O. The filtrate was
treated with 25 g of activated carbon Darco G-60 and then heated to 50°C, until
13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 28
1
2
3
4
5
6
7
8
9
peroxide was no longer detected using Merckoquant 1001-1 peroxide test strips. The
hot suspension was filtered and the solid material was washed with two-50 mL portions
of H2O. The washings and the filtrate were combined and crystallized by the addition of
2 volume equivalents of methanol while stirring for 16 h at 4°C. The solid material was
filtered, washed with two 50-mL portions of MeOH and dried under high vacuum at
40°C. Calcium L-threonate monohydrate (6) was obtained as a white solid: yield 99.4 g
(85%); 1H NMR (400 MHz, D2O) δ 3.60 (dd, 1H, J = 11.6, 7.6 Hz), 3.66 (dd, 1H, J =
11.6, 5.2 Hz), 3.95 (m, 1H) and 4.03 (m, 1H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
L-Threonolactone (2)19
To a suspension of calcium L-threonate (6) (99.4 g, 0.30 mol) in dry acetonitrile (500
mL) was added anhydrous oxalic acid (28.8 g, 0.32 mol) and para-toluenesulfonic acid
monohydrate (1.0 g). The heterogenous mixture was stirred at reflux for 3 h. The hot
mixture was allowed to cool to room temperature and filtered. The filter cake was
washed with 50 mL of acetonitrile and the combined filtrate was evaporated under
reduced pressure to produce a colorless syrup. The residue was dissolved in 100 mL of
EtOAc and evaporated to dryness to give L-threonolactone (2) as a white solid: yield
1
66.5 g (93%); silica gel TLC Rf 0.36 (EtOAc), H NMR (400 MHz, CD3OD) δ 3.93 (dd,
1H, J = 8.8, 7.2 Hz), 4.20 (d, 1H, J = 7.2 Hz), 4.29 (dd, 1H, J = 14.0, 6.8 Hz) and 4.41
(dd, 1H, J = 9.8, 6.8 Hz), 4.82 (s, 2H).
14
ACS Paragon Plus Environment

Page 15 of 28
The Journal of Organic Chemistry
1
2
3
4
2-O-Benzoyl-L-threonolactone (7)18
5
6
7
8
To a cold (0-5ºC) solution containing 66.5 g (0.56 mol) of L-threonolactone (2) in 1.2 L
of CH2Cl2 and 135 mL of anhydrous pyridine under argon was added benzoyl chloride
(72.0 mL, 0.62 mol) dropwise. The mixture was stirred under argon for 30 min at 4°C.
The crude reaction mixture was then sequentially washed with 1 N HCl (3 x 400 mL)
and brine (200 mL). The combined aqueous layer was back extracted with two-100 mL
of CH2Cl2. To the combined organic layers was added two-volume equivalents of
hexane over 1 h and the solution was left stirring for 16 h at 4°C. The product was
collected by filtration and dried under vacuum. 2-O-Benzoy-L-threonolactone (7) was
obtained as a white solid: yield 80.1 g (64%); silica gel TLC Rf 0.30 (1:1 hexanes–
EtOAc); 1H NMR (400 MHz, CDCl3) δ 3.81 (brs, 1H), 4.16 (dd, 1H, J = 9.2, 7.6 Hz), 4.64
(dd, 1H, J = 9.2, 8.0 Hz), 4.73 (q, 1H, J = 7.6 Hz), 5.41 (d, 1H, J = 6.8 Hz), 7.48 (t, 2H, J
= 8.0 Hz), 7.64 (t, 1H, J = 8.0 Hz) and 8.10 (d, 2H, J = 7.6 Hz).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-O-Acetyl-2-O-benzoyl-3-O-tert-butyldiphenylsilyl-L-threofuranose (8)18
To a cold (0-5ºC) solution containing 16.5 g (74.3 mmol) of 2-O-benzoyl-L-
threonolactone (7), 60 mg of DMAP and 10.2 g (150 mmol) of imidazole in 160 mL of
CH2Cl2 was added dropwise tert-butyldiphenylchlorosilane (20 mL, 75.0 mmol). The
reaction mixture was warmed to r.t. and stirred under argon for 16 h. The solvent was
evaporated under reduced pressure and the residue was dissolved in 300 mL of
hexane. The organic phase was sequentially washed with 1 N HCl (100 mL), H2O (100
mL) and brine (100 mL). The organic layer was dried over MgSO4 and evaporated to
give 2-O-benzoyl-3-O-tert-butyldiphenylsilyl-L-threonolactone as a colorless syrup (35
15
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 28
1
2
3
4
5
6
g). The crude material was used directly without further purification; silica gel TLC (Rf
0.50 in 3:1 hexanes–EtOAc);
7
8
9
To a cold (-78°C, acetone/dry ice) solution containing 35.0 g of crude 2-O-
benzoyl-3-O-tert-butyldiphenylsilyl-L-threonolactone in 150 mL of 1,2-dimethoxyethane
was added a 1 M solution of DIBAL-H in toluene (100 mL, 100 mmol) dropwise over a
10 min period. The mixture was stirred under argon for an additional 20 min at -78°C.
TLC showed the reaction to be complete (Rf 0.35 in 3:1 hexanes–EtOAc). A pre-made
solution containing Ac2O (367 mmol) and DMAP (115 mmol) in CH2Cl2
(Ac2O/DMAP/CH2Cl2 = 35 mL/ 14.0 g/ 40 mL) was then added dropwise at -78 ºC. After
10 min the reaction mixture was removed from the dry-ice bath and allowed to stir for
1.5 h while warming to r.t. The mixture was then diluted with 200 mL hexanes and
poured into a cold stirring 1N aq. HCl solution (200 mL). The organic layer was
separated and washed with H2O (100 mL), satd. aq. NaHCO3 (100 mL) and brine (100
mL). The organic layer was dried over MgSO4 and evaporated to give 42 g of crude
product as a yellow syrup. The product was suspended in 25 mL of 10 % EtOAc in
hexanes, passed through a short-pad of silica (50 g) and washed out with 10 % EtOAc
in hexanes (200 mL). Anomeric mixture of 1-O-Acetyl-2-O-benzoyl-3-O-tert-
butyldiphenylsilyl-L-threofuranose (8) was obtained as a colorless syrup: yield 36.0 g
(96%); silica gel TLC Rf 0.50 (3:1 hexanes–EtOAc).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
α
1-(2’-O-Benzoyl- -L-threofuranosyl) thymine (9a)
To a solution containing 34.0 g (67.4 mmol) of 1-O-acetyl-2-O-benzoyl-3-O-tert-
butyldiphenylsilyl-L-threofuranose (8) and 8.9 g (70.5 mmol) of thymine in 140 mL of
16
ACS Paragon Plus Environment

Page 17 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
anhydrous acetonitrile was added 45 mL (145 mmol) of N,O-bis(trimethylsilyl)acetamide
and the mixture was stirred for 30 min at 60°C. TMSOTf (20.0 mL, 108 mmol) was
added dropwise and stirring was continued for another 2 h at 60°C, after which time
TLC analysis (1:1 hexanes–EtOAc) showed the reaction to be complete. The mixture
was cooled to r.t, diluted with 300 mL of EtOAc, and poured into 200 mL of cold sat. aq.
NaHCO3 solution with stirring. The organic layer was separated and washed with H2O
(100 mL) and brine (100 mL), dried over MgSO4 and concentrated under reduced
pressure. The crude nucleoside was obtained as a white foam (41 g) and was used
directly without further purification; silica gel TLC Rf 0.37 (1:1 hexanes–EtOAc).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To a cold (0-5ºC) solution containing 41 g of crude 1-(2’-O-benzoyl-3’-O-tert-
butyldiphenylsilyl-α-L-threofuranosyl) thymine in THF (250 mL) was added dropwise
tetrabutylammonium fluoride (1 M solution in THF, 70.0 mL, 70.0 mmol) and the mixture
was stirred for 1 h at 0–5°C. The solvent was evaporated under reduced pressure and
the residue was dissolved in 300 mL EtOAc. The organic layer was separated and
washed twice with H2O (100 mL) and brine (100 mL), dried over MgSO4 and
concentrated under reduced pressure to give 20 g of the crude nucleoside as a yellow
syrup. The syrup was dissolved in 50 mL CH2Cl2 and crystallized by the addition of 50
mL of hexanes. 1-(2’-O-Benzoyl-α-L-threofuranosyl)thymine (9a) was obtained as a
1
white solid: yield 14.44 g (64 %); silica gel TLC Rf 0.35 (1:2 hexanes–EtOAc); H NMR
(400 MHz, CDCl3) δ 1.90 (s, 3H), 4.05 (brs, 1H), 4.20 (dd, 1H J = 4.4, 10.0 Hz), 4.29 (d,
1H, J = 10.0 Hz), 4.51 (brs, 1H), 5.43 (s, 1H), 5.87 (d, 1H, J = 2.0 Hz), 7.36 (s, 1H), 7.47
(t, 2H, J = 7.6 Hz), 7.61 (t, 1H, J = 7.2 Hz), 8.02 (d, 2H, J = 8.2 Hz) and 8.90 (brs, 1H);
13C NMR (125 MHz, CD3OD): δ 12.5, 74.7, 76.5, 83.9, 91.4, 111.2, 129.7, 130.4, 130.8,
17
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 28
1
2
3
4
5
6
134.8, 138.7, 152.3, 166.4, 166.6; HRMS (ESI-TOF): [M+Na]+ calcd for C16H16N2O6Na,
355.0906; found, 355.0910.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
α
N -Benzoyl-1-(2’-O-benzoyl- -L-threofuranosyl) cytosine (9b)
To a solution containing 33.5 g (66.3 mmol) of 1-O-acetyl-2-O-benzoyl-3-O-tert-
butyldiphenylsilyl-L-threofuranose (8) and 15.0 g (70.0 mmol) of N4-benzoylcytosine in
150 mL of anhydrous acetonitrile was added 40.0 mL (154 mmol) of N,O-
bis(trimethylsilyl)acetamide and the mixture was stirred for 30 min. at 60°C. TMSOTf
(38.0 mL, 199 mmol) was added dropwise and stirring was continued at 60°C for
another 2 h, after which time TLC (1:1 hexanes–EtOAc) showed the reaction to be
complete. The mixture was cooled to room temperature, diluted with 200 mL of EtOAc,
and poured into 200 mL of sat aq. NaHCO3 solution with stirring. The white suspension
was filtered over celite, the organic layer was separated and washed with H2O (150 mL)
and brine (150 mL), dried over MgSO4 and concentrated under reduced pressure. The
crude nucleoside was obtained as white foam (36.0 g) and was used directly without
further purification; silica gel TLC Rf 0.75 (EtOAc).
To a cold (0-5ºC) solution containing 36.0 g of crude N4-benzoyl-1-(2’-O-benzoyl-
3’-O-tert-butyldiphenylsilyl-α-L-threofuranosyl) cytosine in THF (200 mL) was added
dropwise tetrabutylammonium fluoride (1 M solution in THF, 70.0 mL, 70.0 mmol) and
the mixture was stirred at 0–5°C for 1 h. The solvent was evaporated under diminished
pressure and the residue was dissolved in 500 mL EtOAc. The organic layer was
washed with 1N aq. HCl (150 mL) and H2O (150 mL), and then was stirred with brine
(150 mL) at r.t. to precipitate the product. N4-Benzoyl-1-(2’-O-benzoyl-α-L-
18
ACS Paragon Plus Environment

Page 19 of 28
The Journal of Organic Chemistry
1
2
3
4
threofuranosyl)-cytosine (9b) was obtained as a white solid: yield 19.4 g (70%); silica
5
6
7
1
gel TLC Rf 0.40 (1:2 hexanes–EtOAc); H NMR (400 MHz, DMSO-d6) δ 4.3 (m, 3H),
8
9
5.39 (s, 1H), 5.85 (d, 1H J = 2.8 Hz), 5.99 (s, 1H), 7.41 (d, 1H, J = 7.2 Hz), 7.50-7.64
(m, 5H), 7.72 (t, 1H, J = 7.2 Hz), 8.03 (t, 4H, J = 7.6 Hz), 8.22 (d, 1H, J = 7.6 Hz), and
11.27 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 72.4, 76.6, 81.5, 90.9, 95.7, 128.5,
128.9, 129.5, 132.8, 133.2, 133.9, 145.7, 154.5, 163.4, 164.5, 167.4; HRMS (ESI-TOF):
[M+Na]+ calcd for C22H19N3O6Na, 444.1172; found, 444.1180.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
18
α
N -Benzoyl-9-(2’-O-benzoyl- -L-threofuranosyl) adenine (9c)
A mixture of 40.5 g (80.0 mmol) of 1-O-acetyl-2-O-benzoyl-3-O-tert-butyldiphenylsilyl-L-
threofuranose (8) and 19.1 g (80.0 mmol) of N6-benzoyladenine was co-evaporated with
50 mL of anhydrous toluene and then suspended in 160 mL of anhydrous toluene. To
the suspension was added 42.0 mL (163.8 mmol) of N,O-bis(trimethylsilyl)acetamide
and the mixture was stirred for 30 min. at 95°C. Once all the suspension was dissolved
TMSOTf (22.2 mL, 118.8 mmol) was added dropwise and stirring was continued at
95°C for another 2.5 h, after which TLC (1:1 hexanes–EtOAc) showed complete
consumption of the starting material and a more polar major products corresponding to
the N9 glycosides. The mixture was cooled to room temperature, then poured into
stirring mixture of 150 g ice and 150 mL of sat aq. NaHCO3 solution and stirred for 30
m. The organic layer was separated and washed with H2O (50 mL) and brine (50 mL),
dried over MgSO4 and concentrated under reduced pressure. The crude nucleoside
was obtained as a yellow foam: yield 54.3 g.
19
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 28
1
2
3
4
5
6
7
8
9
To a cold (0-5ºC) solution (ice-bath) containing 54.3 g of N6-benzoyl-9-(2’-O-
benzoyl-3’-O-tert-butyldiphenylsilyl-α-L-threofuranosyl) adenine in THF (160 mL) was
added dropwise tetrabutylammonium fluoride (1 M solution in THF, 80.0 mL, 80.0 mmol)
and the mixture was stirred at 0-5 °C for 30 m. The solvent was evaporated under
diminished pressure and the residue was dissolved in 200 mL of EtOAc. The organic
layer was washed with H2O (50 mL) and brine (50 mL). The combined aqueous layer
was back-extracted with EtOAc (2x50 mL). Combined organic layer was dried over
MgSO4 and concentrated under reduced pressure. The resulting crude material was
dissolved in 150 mL of 1% methanol in CH2Cl2 and crystallized by slow addition of 120
mL of hexanes. N6-Benzoyl-9-(2’-O-benzoyl-α-L-threofuranosyl) adenine (9c) was
obtained as a white solid: yield 15.5 g (43.5%); silica gel TLC Rf 0.35 (EtOAc); 1H NMR
(400 MHz, CDCl3) δ 4.30 (dd, 1H J = 4.0, 10.0 Hz), 4.39 (d, 1H, J = 10.0 Hz), 4.67 (d,
1H, J = 3.2 Hz), 5.63 (s, 1H), 6.07 (d, 1H, J = 1.6 Hz), 7.56 (m, 6H), 8.04 (t, 4H, J = 8.1
Hz), 8.27 (s, 1H), and 8.81 (s, 1H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
6
α
N -Acetyl-O -diphenylcarbamoyl-9-(2’-O-benzoyl- -L-threofuranosyl) guanine (9d)
To a solution containing 8.45 g (26.1 mmol) of N2-acetyl-O6-diphenylcarbamoyl
guanine23 in 200 mL of a mixture of anhydrous dichloroethane and toluene (1:2 v/v) was
added 15.8 mL (64.6 mmol) of N,O-bis(trimethylsilyl)acetamide and the mixture was
stirred for 30 min. at 70°C. The solvent was removed under reduced pressure and the
residue was dissolved in 55 mL of anhydrous toluene. 1-O-Acetyl-2-O-benzoyl-3-O-tert-
butyldiphenylsilyl-L-threofuranose (8) (11 g, 21.8 mmol) in 65 mL anhydrous toluene
was added dropwise using a canula and the mixture was heated to 70°C. TMSOTf (8.5
20
ACS Paragon Plus Environment

Page 21 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
mL, 45.9 mmol) was then added dropwise and the mixture was stirred at 70°C for
another 1.5 h, after which TLC (1:1 hexanes–EtOAc) showed complete consumption of
(8). The mixture was cooled to room temperature, diluted with 300 mL of EtOAc, and
poured into 150 mL of sat aq. NaHCO3 solution with stirring resulting in a purple
suspension. The suspension was filtered and the organic layer was separated and
washed with H2O (150 mL) and brine (150 mL), dried over MgSO4 and concentrated
under reduced pressure. The crude nucleoside was obtained as purple foam (16.12 g)
and was used directly without further purification; silica gel TLC Rf 0.7 (1:2 hexanes–
EtOAc).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To a cold (0-5ºC) solution containing 16.12 g of crude N2-acetyl-O6-
diphenylcarbamoyl-9-(2’-O-benzoyl-3’-O-tert-butyldiphenylsilyl-α-L-threofuranosyl)
guanine in THF (50 mL) was added dropwise tetrabutylammonium fluoride (1 M solution
in THF, 19.0 mL, 19.0 mmol) and the mixture was stirred at 0–5°C. After 15 min TLC
was checked and another 9 mL of TBAF was added and the mixture stirred for 45 min.
The solvent was evaporated under diminished pressure and the residue was dissolved
in 250 mL EtOAc. The organic layer washed with 1N aq. HCl (50 mL), H2O (50 mL) and
brine (50 mL), dried over MgSO4 and concentrated under reduced pressure. The
resulting crude product was dissolved in CH2Cl2 (30 mL) and crystallized by addition of
an equal amount of hexanes. N2-Acetyl-O6-diphenylcarbamoyl-9- (2’-O-benzoyl-α-L-
threofuranosyl) guanine (9d) was obtained as a white solid: yield 8.9 g (69 %); silica gel
1
TLC Rf 0.25 (1:2 hexanes–EtOAc); H NMR (400 MHz, DMSO-d6) δ 2.20 (s, 3H), 4.32
(dq, 2H, J = 3.6, 7.6 Hz), 4.60 (m, 1H), 5.78 (t, 1H, J = 2.0 Hz), 5.96 (d, 1H, J = 4.0 Hz),
6.31 (d, 1H, J = 1.6 Hz), 7.33 (t, 2H, J = 6.0 Hz), 7.43-7.60 (m, 10H), 7.72 (t, 1H, J = 6.0
21
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 28
1
2
3
4
5
6
7
8
9
Hz), 8.03 (dd, 1H, J = 0.8, 6.4 Hz), 8.60 (s, 1H), and 10.81 (s, 1H); 13C NMR (125 MHz,
DMSO-d6): δ 24.6, 73.2, 74.8, 82.3, 87.9, 120.1, 128.7, 128.9, 129.5, 129.6, 134.0,
141.6, 144.3, 150.1, 152.3, 154.2, 155.3, 165.0, 169.2; HRMS (ESI-TOF): [M+Na]+
calcd for C31H26N6O7Na, 617.1761; found, 617.1750.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
α
1-(3’-O-Dimethoxytrityl- -L-threofuranosyl) thymine (10a)
A mixture containing 3.2 g (9.6 mmol) of 1-(2’-O-benzoyl-α-L-threofuranosyl) thymine
(9a), 4.88 g (14.4 mmol, 1.5 eq.) of DMT-Cl and 100 mg of DMAP (0.82 mmol) was
coevaporated twice with 20.0 mL of anhydrous pyridine. The mixture was dissolved in
40 mL of anhydrous pyridine and stirred under argon at 80ºC for 16 h. The solvent was
evaporated under diminished pressure and the residue partitioned between EtOAc (50
mL) and H2O (50 mL). The organic layer was washed with brine (50 mL), dried (MgSO4)
and evaporated.
The residue was dissolved in a mixture of 38 mL of THF and 30.5 mL of MeOH, and
then cooled to 0°C. To the cold solution was added 15.0 mL of ice-cold 1N aq. NaOH
while stirring. After 30 min the reaction was quenched by addition of 100 mL of water.
The volatile solvent was removed under diminished pressure and the residual aqueous
solution was extracted with EtOAc (3 x 50 mL). The combined organic layer was dried
(MgSO4) and evaporated. The residue was purified on a silica gel column (50 g silica
bed), eluting with 50 to 100% EtOAc in hexanes containing 2% Et3N in steps of 5%
increase in EtOAc for every 200 mL. 1-(3’-O-Dimethoxytrityl-α-L-threofuranosyl) thymine
(10a) was obtained as a white foam: yield 3.6 g (71%); 1H-NMR (400 MHz, CDCl3 ): δ
1.82 (s, 3H), 3.30 (d, 1H, J = 9.6 Hz), 3.67 (s, 3H), 3.71 (s, 3H), 3.91 (d, 1H, J = 13.6
22
ACS Paragon Plus Environment

Page 23 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
Hz), 4.19 (s, 1H), 4.23 (s, 1H), 5.72 (s, 1H), 6.79 (m, 4H), 7.12-7.38 (m, 9H), 7.44 (s,
1H) and 10.92 (s, 1H).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
15
α
N -Benzoyl-1-(3’-O-dimethoxytrityl- -L-threofuranosyl) cytosine (10b)
A mixture of 3.18 g (7.7 mmol) of N4-benzoyl-1-(2’-O-benzoyl-α-L-threofuranosyl)
cytosine (9b), 3.84 g (11.3 mmol, 1.5 eq.) of DMT-Cl and 120 mg DMAP (0.99 mmol,
0.1 eq) was coevaporated twice with 15.0 mL of anhydrous pyridine. The residue was
suspended in 50 mL of anhydrous pyridine and stirred under argon for 16 h at 80ºC.
The solvent was evaporated under diminished pressure and the residue partitioned
between EtOAc (50 mL) and H2O (50 mL). The organic layer was washed with brine (50
mL), dried (MgSO4) and evaporated.
The residue was dissolved in a mixture of 38 mL of THF and 30.5 mL of MeOH, and
then cooled to 0°C. To the cold solution was added 7.7 mL of ice-cold 1N aq. NaOH
while stirring. After 10 min another 7.7 mL of ice-cold 1N aq. NaOH was added. Ten min
after the second addition, the reaction was quenched by addition of 40 mL of 10%
NH4Cl in water. The volatile solvent was removed under diminished pressure and the
residue was diluted by addition of 50 mL EtOAc. The organic layer was separated,
washed with H2O (50 mL) and brine (50 mL), dried (MgSO4) and evaporated. The
residue was purified on a silica gel column (50 g silica bed), eluting with 70-100%
EtOAc in hexane containing 1% Et3N in steps of 5% increase in EtOAc for every 200
mL. N4-Benzoyl-1-(3’-O-dimethoxytrityl-α-L-threofuranosyl) cytosine (10b) was obtained
as a white foam: yield 3.12 g (65%); 1H-NMR (400 MHz, CDCl3): δ 3.32 (d, 1H, J = 10.0
Hz), 3.71 (d, 1H, J = 4.0 Hz), 3.74 (s, 6H), 4.25 (s, 1H), 4.33 (s, 1H), 5.70 (s, 1H), 6.79
23
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 28
1
2
3
4
(m, 4H), 6.75-7.35 (m, 9H), 7.47-7.67 (m, 4H), 7.94 (d, 2H, J = 8.0 Hz), 8.05 (d, 1H, J =
5
6
8.0 Hz), and 9.10 (brs, 1H).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
15
α
N -Benzoyl-9-(3’-O-dimethoxytrityl- -L-threofuranosyl) adenine (10c)
A mixture of 3.39 g (7.6 mmol) of N6-benzoyl-9-(2’-O-benzoyl-α-L-threofuranosyl)
adenine (9c), 3.86 g (11.4 mmol, 1.5 eq.) of DMT-Cl and 93 mg DMAP (0.76 mmol, 0.1
eq) was co-evaporated twice with 7.0 mL of anhydrous pyridine. The residue was
dissolved in 35 mL of anhydrous pyridine and the mixture was stirred at 75°C under
argon for 16 h. The solvent was evaporated under diminished pressure and the residue
partitioned between EtOAc (50 mL) and H2O (50 mL). The organic layer was washed
with brine (50 mL), dried (MgSO4) and evaporated.
The residue was dissolved in a mixture of 38 mL of THF and 30.5 mL of MeOH, and
then cooled to 0 °C. To the cold stirred solution was added 7.6 mL of ice-cold 1 N aq.
NaOH solution. After 10 min another 7.6 mL of ice-cold 1 N aq. NaOH solution was
added. Ten min after the second addition, the reaction was quenched with 40 mL of
10% aq. NH4Cl. The volatile solvent was removed under diminished pressure and the
aqueous residue was diluted with 50 mL of EtOAc. The organic layer was separated,
washed with H2O (50 mL) and brine (50 mL), dried (MgSO4) and evaporated. The
residue was purified on a silica gel column (70 g silica bed), eluting with 30-80% EtOAc
in hexanes containing 1% Et3N. Stepwise elution was performed with 10 % increases in
EtOAc for each 200 mL volume. N6-Benzoyl-9-(3’-O-dimethoxytrityl-α-L-threofuranosyl)
1
adenine (10c) was obtained as a white foam: yield 3.6 g (63%); H-NMR (400 MHz,
DMSO-d6) 3.45 (dd, 1H, J= 5.2, 9.6 Hz), 3.59 (dd, 1H, J=3.2, 9.6 Hz), 3.73 (s, 6H), 4.17
24
ACS Paragon Plus Environment

Page 25 of 28
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(m, 1H), 4.50 (m, 1H), 5.91 (d, 1H, J= 2.8 Hz), 5.93 (d, 1H, J=4.8 Hz), 6.86 (m, 4H),
7.13-7.33 (m, 9H), 7.56 (t, 2H, J=7.6 Hz), 7.65 (t, 1H, J=7.2 Hz), 8.06 (d, 2H, J=7.2 Hz),
8.56 (s, 1H), 8.75 (s, 1H), 11.22 (brs, 1H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
6
α
N -Acetyl-O -diphenylcarbamoyl-9-(3’-O-dimethoxytrityl- -L-threofuranosyl)
guanine (10d)15
5.2
g
(8.74 mmol) of N2-acetyl-O6-diphenylcarbamoyl-9- (2’-O-benzoyl-α-L-
threofuranosyl) guanine (9d) was co-eveporated with 20 ml of anhydrous pyridine under
reduced pressure and the solid residue was dried under vacuum for 18 h. To the
residue was added 5.92 g (17.48 mmol, 2 eq) of DMT-Cl, anhydrous dichloromethane
(60 mL), 2.3 mL of 2,4,6- trimethylpyridine (17.48 mmol, 2 eq) and 675 mg (2.62 mmol,
0.3 eq) silver triflate. The mixture was stirred under nitrogen for 18 h. TLC (3:1 hexanes-
EtOAc) showed the reaction to be complete. The solvent was removed under
diminished pressure and the residue dissolved in 150 ml of dichloromethane. The
organic layer was washed with 0.2(N) HCl (300 mL), brine (60 mL), dried (MgSO4) and
evaporated. The residue was purified on a silica gel column (4x15 cm), eluting with 100
ml 99:1 DCM- Et3N to 300 ml 97:2:1 DCM-MeOH-Et3N.. The product was obtained as a
pale yellow powder: yield 5.8 g (74.3 %).
The compound (3.0 g, 3.34 mmol) was dissolved in 45 mL of THF-MeOH-water
(5:4:1) and cooled to 0-5o C in a water-ice bath. Ice-cold 1 N aq. NaOH (2.1 mL) was
added dropwise to the mixture and stirred for 10-15 min TLC (hexane-EtOAc 2:1)
showed that the reaction was not complete. Another 2.1 mL of cold 1 N aq. NaOH was
added dropwise and stirring continued for another 15-20 min. The reaction was
25
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 28
1
2
3
4
5
6
7
8
9
quenched with 15 mL of 10% aq. NH4Cl and the crude product was extracted with
EtOAc (2 x 50 mL). The combined organic layer was dried over MgSO4 and evaporated
under reduced pressure. The residue was purified on a silica gel column (4 x 15 cm)
using a gradient elution with 100 ml of (85:14:1) Hexane-EtOAc-Et3N followed by 100 to
300 ml of (75:24:1) Hexane-EtOAc-Et3N and (65:34:1) Hexane-EtOAc-Et3N. N2-Acetyl-
O6-diphenylcarbamoyl-9-(3’-O-dimethoxytrityl-α-L-threofuranosyl) guanine (10d) was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
obtained as a pale yellow powder: yield 1.9 g (71 %); H-NMR (400 MHz, CDCl3 ): δ
2.17 (s, 3H), 2.73 (s, 1H), 3.42 (s, 2H), 3.71 (s, 6H), 4.33 (m, 1H), 4.45 (s, 1H), 5.83 (d,
1H, J = 2.4 Hz), 6.78 (m, 4H), 7.12-7.45 (m, 19H), 8.19 (s, 1H), 8.81 (s, 1H).
Acknowledgments:
We wish to thank members of the Chaput lab and J. Heemstra for helpful comments
and suggestions. This work was supported by the DARPA Folded Non-Natural
Polymers with Biological Function Fold F(x) Program under award number N66001-14-
2-4054 and a grant from the National Science Foundation grants (1304583).
Supporting Information
NMR data for compounds 2, 6, 7, 8, 9a-d and 10a-d. This material is available free of
charge via the internet at http://pubs.acs.org
26
ACS Paragon Plus Environment

Page 27 of 28
The Journal of Organic Chemistry
1
2
3
4
References
5
6
7
(1) Schoning, K. U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A.
Science 2000, 290, 1347.
8
9
(2) Yang, Y.-W.; Zhang, S.; McCullum, E. O.; Chaput, J. C. J. Mol. Evol. 2007, 65, 289.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) Ebert, M.-O.; Mang, C.; Krishnamurthy, R.; Eschenmoser, A.; Jaun, B. J. Am. Chem.
Soc. 2008, 130, 15105.
(4) Orgel, L. E. Science 2000, 290, 1306.
(5) Joyce, G. F. Nature 2002, 418, 214.
(6) Chaput, J. C.; Yu, H.; Zhang, S. Chem. Biol. 2012, 19, 1360.
(7) Chaput, J. C.; Szostak, J. W. J. Am. Chem. Soc. 2003, 125, 9274.
(8) Chaput, J. C.; Ichida, J. K.; Szostak, J. W. J. Am. Chem. Soc. 2003, 125, 856.
(9) Kempeneers, V.; Vastmans, K.; Rozenski, J.; Herdewijn, P. Nucleic Acids Res. 2003,
31, 6221.
(10) Horhota, A.; Zou, K.; Ichida, J. K.; Yu, B.; McLaughlin, L. W.; Szostak, J. W.; Chaput, J.
C. J. Am. Chem. Soc. 2005, 127, 7427.
(11) Yu, H.; Zhang, S.; Dunn, M.; Chaput, J. C. J. Am. Chem. Soc. 2013, 135, 3583.
(12) Dunn, M. R.; Larsen, A. C.; Fahmi, N.; Zahurancik, W. J.; Meyers, M.; Suo, Z.; Chaput,
J. C. J. Am. Chem. Soc. 2015, 137, 4014.
(13) Yu, H.; Zhang, S.; Chaput, J. C. Nat. Chem. 2012, 4, 183.
(14) Zou, K.; Horhota, A.; Yu, B.; Szostak, J. W.; McLaughlin, L. W. Org. Lett. 2005, 7,
1485.
(15) Schoning, K.-U.; Scholz, P.; X., W.; Guntha, S.; Delgado, G.; Krishnamurthy, R.;
Eschenmoser, A. Helv. Chim. Acta 2002, 85, 4111.
(16) Zhang, S.; Yu, H.; Chaput, J. C. Curr. Protoc. Nucleic Acid Chem. 2013, 52, 4.54.
(17) Zhang, S.; Chaput, J. C. Curr. Protoc. Nucleic Acid Chem. 2012, 4, 4.51.
(18) Dumbre, S. G.; Jang, M.-Y.; Herdewijn, P. J. Org. Chem. 2013, 78, 7137.
(19) Wei, C. C.; Bernardo, S. D.; Tengi, J. P.; Borgese, J.; Weigele, M. J. Org. Chem. 1985,
50, 3462.
(20) Wu, T.; Froeyen, M.; Kempeneers, V.; Pannecouque, C.; Wang, J.; Busson, R.; De
Clercq, E.; Herdewijn, P. J. Am. Chem. Soc. 2005, 127, 5056.
27
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 28
1
2
3
4
5
(21) Milecki, J.; Foldesi, A.; Fischer, A.; Adamiak, R. W.; Chattopadhyaya, J. J. Label.
Compd. Radiopharm. 2001, 44, 763.
6
7
8
9
(22) Jang, M.-Y.; Froeyen, M.; Herdewijn, P. Tetrahedron Lett. 2013, 54, 6084.
(23) Zou, R.; Robins, M. J. Can. J. Chem. 1987, 65, 1436.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28
ACS Paragon Plus Environment