O-nucleoside, S-nucleoside, and N-nucleoside probes of lumazine synthase and riboflavin synthase

Article

pubs.acs.org/joc

O-Nucleoside, S-Nucleoside, and N-Nucleoside Probes of Lumazine

Synthase and Riboﬂavin Synthase

Arindam Talukdar,^†Yujie Zhao,^†Wei Lv,^†Adelbert Bacher,^‡Boris Illarionov,^‡Markus Fischer,^‡

and Mark Cushman*^,†

^†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and The Purdue Center for Cancer

Research, Purdue University, West Lafayette, Indiana 47907, United States

^‡Institute of Biochemistry and Food Chemistry, Food Chemistry Division, University of Hamburg, D-20146 Hamburg, Germany

S

* Supporting Information

ABSTRACT: Lumazine synthase catalyzes the penultimate step in the

biosynthesis of riboﬂavin, while riboﬂavin synthase catalyzes the last step. O-

Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical

lumazine biosynthetic intermediates have been synthesized in order to

obtain structure and mechanism probes of these two enzymes, as well as

inhibitors of potential value as antibiotics. Methods were devised for the

selective cleavage of benzyl protecting groups in the presence of other easily

reduced functionality by controlled hydrogenolysis over Lindlar catalyst.

The deprotection reaction was performed in the presence of other reactive

functionality including nitro groups, alkenes, and halogens. The target

compounds were tested as inhibitors of lumazine synthase and riboﬂavin synthase obtained from a variety of microorganisms. In

general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and

riboﬂavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed

by free energy calculations using the Poisson−Boltzmann/surface area (MM-PBSA) method were carried out in order to

rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding

as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction.

pentose phosphate pool by the loss of C-4. The riboﬂavin

synthase-catalyzed dismutation of two molecules of 6,7-

dimethyl-8-^D-ribityllumazine (3) results in the formation of

one molecule of riboﬂavin (4) and one molecule of the

substituted pyrimidinedione 1, which can be recycled by

lumazine synthase.²¹⁻²⁹The pyrimidinedione derivative 1 is

therefore both a substrate of lumazine synthase and a product of

riboﬂavin synthase, and analogues of 1 could therefore

conceivably bind to and inhibit both lumazine synthase and

riboﬂavin synthase.

The mechanism of the reaction catalyzed by lumazine synthase

has not been completely elucidated. The currently proposed

mechanism outlined in Scheme 2 involves initial Schiﬀ base

formation between the four-carbon carbohydrate 2 and the

pyrimidinedione derivative 1, resulting in the imine 5, which

eliminates phosphate to form the enol 6 (Scheme 2).

Tautomerization of the enol 6 to the ketone 7, followed by the

addition of the secondary amino group to the ketone, leads to the

formation of the covalently hydrated lumazine 10. Elimination of

water yields the 6,7-dimethyl-8-^D-ribityllumazine (3). A redox

disproportionation reaction of two molecules of 3, followed by a

4 + 2 cycloaddition reaction and two elimination reactions, has

recently been advanced in order to explain the conversion of two

INTRODUCTION

■

Riboﬂavin, also known as vitamin B₂, is the central component of

the cofactors FAD and FMN and is therefore required for a wide

variety of cellular processes. It plays a key role in energy

production and is required for the metabolism of fats,

carbohydrates, and proteins. Animals, including humans, obtain

it from their diet, while a variety of pathogenic Gram-negative

bacteria, including Escherichia coli and Salmonella typhimurium,

lack an eﬃcient riboﬂavin uptake system and are therefore

absolutely dependent on endogenous synthesis of this

vitamin.¹⁻⁴The riboﬂavin biosynthesis gene ribB has recently

been shown to play an essential role in diﬀerent Salmonella

disease models.^5,6Since animals lack the riboﬂavin biosynthetic

pathway, inhibitors of the pathway should be selectively toxic to

the pathogen and not the host. A detailed understanding of the

structure and mechanism of the enzymes involved in the

biosynthesis of riboﬂavin is essential for the rational design of

enzyme inhibitors with antibacterial activity.

Lumazine synthase and riboﬂavin synthase catalyze the last

two steps in the biosynthesis of riboﬂavin (Scheme 1). The initial

steps in the biosynthesis of riboﬂavin lead from GTP to the

pyrimidinedione 1.⁷⁻¹⁵Lumazine synthase catalyzes the

condensation of 3,4-dihydroxy-2-butanone 4-phosphate

(2)¹⁶⁻¹⁸with 5-amino-6-D-ribitylamino-2,4(1H,3H)-

pyrimidinedione (1) yielding 6,7-dimethyl-8-D-ribityllumazine

(3).^19,20The four-carbon phosphate moiety 2 originates from the

Received: May 23, 2012

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molecules of the lumazine 3 to one molecule of riboﬂavin (4) and

one molecule of the pyrimidinedione 1 (Scheme 3).³⁰

Scheme 1

X-ray crystallography of complexes of the purinetrione

inhibitor 15 (PDB code: 1W19) and the phosphonate analogue

16 (PDB code: 1NQW) with lumazine synthase has established

that the phosphate of the hypothetical Schiﬀ base initially binds

far away from the ribityl side chain as depicted roughly in

chemical structure 5 (Chart 1).³¹Although the mechanism

outlined in Scheme 2 is certainly very reasonable, the details of

the pathway, such as the timing of phosphate elimination relative

to the conformational reorganization of the side chain to allow

cyclization, remain unknown.³²For example, it has been

proposed that the whole phosphate side chain rotates toward a

cyclic conformation 8 with assistance from the enzyme before

phosphate elimination and dehydration occur to form the cis

Schiﬀ base 7 directly (Scheme 2).³¹The possible initial

formation of a thermodynamically less stable cis Schiﬀ base, or

the isomerization of the trans Schiﬀ base 17 to the cis Schiﬀ base

7, has not been established. By a semiempirical approach, the

energy barrier for the 17 to 7 isomerization was calculated to be

19.6−21 kcal/mol.³³More recently, as a result of the

temperature-dependent pre-steady-state kinetic experiments of

lumazine synthase from Aquifex aeolicus, it was proposed that one

of the subsequent steps occurring after phosphate elimination

and tautomerization is the rate-determining step.^34,35

Ligand Design. In order to gain insight into the structural

change of the Schiﬀ base side chain occurring after formation of

intermediate 5, metabolically stable substrate analogues are

Scheme 2

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Scheme 3

Chart 1

Figure 1. Hydrogen bonds and distances of the substrate analogue 18

bound in the active site of B. subtilis lumazine synthase. The distances are

in Å.

nucleophilic attack by the ribityl amino group to form the cyclic

lumazine derivative 3. They might therefore be expected to be

lumazine synthase inhibitors, and they might also provide

information about the conformations of reaction intermediates if

they could be crystallized with the enzyme.

Most potent lumazine synthase inhibitors contain a C3−C5

phosphate side chain and are analogues of intermediate 5

(Scheme 2).^37,38Hypothetically, mechanism probes without a

phosphate side chain would be expected to be moderate

lumazine synthase inhibitors compared to the more potent

ones with a phosphate side chain because the phosphate binds to

Arg, Thr, Ala, Gln, and Ser residues in the active sites of lumazine

synthases isolated from a variety microorganisms.^{31,32,39−43}

Accordingly, intermediate 5 is likely to have a higher aﬃnity

for the enzyme than intermediates 6 or 7. Enol 6 is an

intermediate from the hypothetical reaction mechanism after

phosphate cleavage. In the present study, metabolically stable

analogues of lumazine synthase substrate 1 and reaction

intermediate 6 were designed.

necessary that would allow Schiﬀ base formation but would not

allow the cyclization to proceed. The crystal structure of

analogue 18 (PDB dode: 1KYY) bound to Bacillus subtilis

lumazine synthase³⁶has revealed that the ribitylamino nitrogen

has no obvious direct role in binding the ligand to the enzyme

(Figure 1), suggesting that the replacement of the nitrogen with

another atom might not aﬀect binding to the enzyme.

Replacement of the nitrogen atom of the ribitylamine side

chain with oxygen, sulfur, or carbon would not be expected to

produce gross changes in the orientation of ribityl chain of 18

(Figure 1), and the resulting compounds could possibly bind to

both lumazine synthase and riboﬂavin synthase. They obviously

could not complete the catalytic cycle as they cannot undergo the

The proposed intermediate 6 is a Schiﬀ base. Schiﬀ bases are

not stable under acidic and reductive conditions. Bioisosteres 19

and 20 were therefore designed as metabolically stable analogues

of the Schiﬀ base 6 (Chart 2). These amides have partial double

C

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There are indeed many common features of riboﬂavin synthase

and lumazine synthase, and the dual inhibitor concept has, in

principle, a strong theoretical basis. In fact, the sequences and

structures of riboﬂavin synthases of Archaea closely resemble

those of 6,7-dimethyl-8-ribityllumazine synthase, as evidenced by

the crystal structure of a pentameric Methanocaldococcus

jannaschii riboﬂavin synthase in complex with a substrate

analogue.⁴⁴

Chart 2

Molecular Modeling of O- and S-Nucleosides. A 2.4 Å

resolution X-ray structure is available of the substrate analogue

18 (PDB code: 1KYY)³⁶complexed with B. subtilis lumazine

synthase (Figure 1). The structure allows the construction of

hypothetical models of the binding of 21−24 to lumazine

synthase (Figure 2), which were produced by docking these

compounds into the active site of Mycobacterium tuberculosis

lumazine synthase. Docking was performed with GOLD (BST,

version 3.0, 2005; for details, see the Experimental Section). The

energies of the complexes were minimized using the MMFF94s

force ﬁeld while allowing the ligand and the protein structure

contained within a 6 Å diameter sphere surrounding the ligand to

remain ﬂexible with the remainder of the protein structure

frozen. The calculated structures of 21−24 bound to lumazine

synthase suggest that these inhibitors bind in the active site in an

almost identical fashion to 18 (Figure 2).

RESULTS AND DISCUSSION

■

Synthesis of O-Nucleosides. Since both the ribityl hydroxyl

groups and the pyrimidinedione ring have the potential to be

alkylated, the attachment of a side chain on the amino group at C-

5 of the pyrimidinedione moiety required that the ribityl

substituent and the pyrimidinedione ring be generated in

protected forms. The synthesis of the protected ribitol 26

started from ^D-ribose as previously described.⁴⁵Treatment of

chloropyrimidine 25 with the ribitol derivative 26 was carried out

in the presence of the hindered base LiHMDS. The reaction was

attempted in the presence of NaH and BuLi, but LiHMDS gave

the best result. The reaction proceeded in 71% yield to aﬀord the

desired product 27.

bond character between the amide nitrogen and the carbonyl

carbon. Compound 19 has a methacroyl side chain attached to 5-

amino-6-ribitylamino-1H-pyrimidine-2,4-dione, which resem-

bles the intermediate 6.

Compounds 21−24 mimic the intermediate 1, which is a

substrate of the lumazine synthase-catalyzed reaction and a

product of the riboﬂavin synthase-catalyzed reaction. These

compounds therefore have the potential to inhibit both enzymes.

The removal of the isopropylidine groups (Scheme 4) with 0.5

N HCl in THF yielded compound 28. Hydrogenolysis of 28 with

Figure 2. Calculated structures of the O- and S-nucleoside derivatives (21−24) complexed with B. subtilis lumazine synthase. The amino acid residues

involved in the active site are labeled and shown as sticks. The black dashed lines represent the hydrogen bonds between one of the ligands and B. subtilis

lumazine synthase. The color code for compound 21 is green, 22 is red, 23 is magenta, and 24 is blue. The maximum distance between the heavy atoms

participating in the hydrogen bonds was set at 3.8 Å. This ﬁgure was generated by PyMol [DeLano, W. L. (2002), the PyMOL Molecular Graphics

System, DeLano Scientiﬁc, San Carlos, CA, USA]. The ﬁgure is programmed for walleyed viewing.

D

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Pd/C in MeOH containing 2 drops of HCl under atmospheric

pressure aﬀorded the desired O-nucleoside 29.

isolate. This route was eventually abandoned in favor of the

synthesis detailed in Scheme 4.

Compounds 36, 40, and 43 were chosen for synthesis because

they are the oxygen analogues of the corresponding

ribitylaminopyrimidines that were previously reported.⁴⁸In

order to provide a handle for the attachment of side chains at C-5

of the pyrimidine, the reduction of the nitro group in compound

27 was carried out in the presence of sodium hydrosulﬁte

(Scheme 6) in reﬂuxing 1,4-dioxane and methanol to provide

amine 32. The amine 32 was treated with ethyl chlorooxoacetate

in the presence of Et₃N to aﬀord ester 33. Treatment of

compound 33 with 0.5 N HCl in MeOH at room temperature

proceeded to compounds 34 and 35 as a 1:1 mixture of methyl

and ethyl esters. Compounds 34 and 35 were easily separated by

ﬂash column chromatography. Hydrogenolysis of compounds 34

in EtOH and 35 in MeOH in the presence of Pd/C provided the

required products 36 and 37 in good yield. Compounds 40 and

43 were prepared by following a similar protocol. However,

synthesis of 46 presented additional challenges because of the

presence of the alkene in the side chain. Compound 46 is a

bioisostere of an intermediate 6/17 involved in lumazine 3

biosynthesis. The amine 32 was treated with methacrolyl

chloride to aﬀord 44. The double bond present on the side

chain prevented application of the general hydrogenolysis

protocol using Pd/C. Use of Lindlar catalyst (Scheme 4) was

attempted, but unfortunately with compound 45 this led to both

deprotection of the benzyl groups as well as reduction of double

bond in the side chain to provide compound 40 instead of 46. A

modiﬁed protocol was devised in order to selectively remove the

benzyl groups under controlled hydrogenolysis in which 1,4-

cyclohexadiene was used as the source of hydrogen. Addition of

1,4-cyclohexadiene to a reaction mixture containing compound

45 and Lindlar catalyst gave rise to the desired pyrimidinedione

46.

Scheme 4

Synthesis of S-Nucleosides. The syntheses of S-nucleoside

23 and the nitro derivative 24 were described earlier.⁴⁹The

syntheses of additional metabolically stable sulﬁdes that can

structurally mimic the substrates, hypothetical intermediates, and

products involved in 6,7-dimethyl-8-ribityllumazine (3) and

riboﬂavin (4) biosynthesis were undertaken in order to explore

the eﬀects of sulfur incorporation. Following the standard

protocol (Scheme 7), derivatization of the amine 47 with acid

chlorides provided the expected amide products in very low yield.

Reaction of 47 with various acids using peptide coupling

synthesis methods was therefore explored. The reaction

proceeded in only 10% yield in THF, but changing the solvent

to pyridine increased the eﬃciency of the reaction so that

moderate yields of 48 and 50 could be obtained. Removal of the

acetonide protecting groups under acidic conditions aﬀorded the

desired sulﬁde derivatives 49 and 51.

The next goal was to selectively remove the benzyl groups

without reducing the nitro group. Lewis-acid-mediated cleavage

of the benzyl groups proved to be futile. However, a

hydrogenolysis reaction employing Lindlar catalyst provided

solely compound 22 without even a trace of the amine 21

(Scheme 4).

Initially, dimethoxypyrimidine 31 was synthesized through a

nucleophilic substitution reaction of 26 with 4-iodo-5-nitro-2,6-

dimethoxypyrimidine (30) (Scheme 5).^46,47The intended

Scheme 5

Synthesis of N-Nucleosides. The N-nucleosides 56, 60, 63,

66, and 69 were synthesized as metabolically stable analogues of

the hypothetical intermediates 6, 7, 9, and 10. The synthesis of

56 started with a nucleophilic aromatic substitution reaction of

compound 25 with ribitylamine to yield the expected product 52

(Scheme 8). TBDMS protection of the hydroxyl groups of the

ribityl side chain was employed to avoid their reaction during

acylation of the primary amino group attached to the

pyrimidinedione ring in intermediate 54. Compound 53⁵⁰was

then reduced with sodium hydrosulﬁte to amine 54. The amine

54 is very unstable and therefore it was not isolated. Acylation of

the amine 54 with methacrolyl chloride aﬀorded the amide 55.

Simultaneous removal of both types of protecting groups

deprotection of the pyrimidinedione system at a relatively late

stage in the synthesis under acidic or basic conditions was

complicated by the fact that the ether linkage between the ribityl

group and the pyrimidine ring was very labile. In most cases, the

reaction gave rise to a mixture of products that were diﬃcult to

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Scheme 6

(TBDMS and methyl) was accomplished by heating inter-

mediate 55 with HBr in aqueous methanol at 55−60 °C to aﬀord

the target compound 56.

according to the reported procedure.⁴⁸The amine 57 was

reacted with 2-chloropropanoyl chloride, 2-chloroacetyl chlor-

ide, or 3,3,3-triﬂuoropropanoyl chloride to aﬀord the corre-

sponding amides 58, 61, and 64 (Scheme 9), which were

deprotected with TBAF to yield 59, 62, and 65. Removal of the

benzyl protecting groups by catalytic hydrogenolysis using

Lindlar or Pearlman’s catalyst and 1,4-cyclohexadiene as a

controlled source of hydrogen provided the desired products 60,

63, and 66. The Lindlar catalyst worked ﬁne for the conversion to

compound 60. Conversion of 62 to 63 and 65 to 66 using Lindlar

catalyst was slow, but the hydrogenolysis reaction could be

expedited by changing the catalyst to Pearlman’s catalyst. For the

synthesis of the compound 69 with a conformationally restricted

side chain resembling the hypothetical carbinolamine inter-

mediate 10, 2-oxopropanoyl chloride was reacted with amine 57

in the presence of Et₃N at room temperature to aﬀord

intermediate 67. The reagent 2-oxopropanoyl chloride was

synthesized by reacting pyruvic acid with α,α-dichloromethyl

methyl ether. Treatment of compound 67 with tetrabutylammo-

The synthesis outlined in Scheme 8 proceeded well except for

the last step. Several alternate methods were tried to cleave the

methyl groups, but none of them aﬀorded the desired product.

For example, heating at reﬂux in HCl/MeOH, reaction with

Amberlite-IR-120 (H⁺)/MeOH, and stirring at room temper-

ature with BBr₃/THF or TMSI/THF were all tried. The best

method to cleave methyl groups in this case proved to be heating

at reﬂux in HBr/MeOH solution. However, puriﬁcation of the

ﬁnal product by ﬂash chromatography (SiO₂) was diﬃcult.

Eluting with EtOAc/EtOH/H₂O/AcOH (67:23:9:1) was

eﬀective but leached silica gel from the column. Fortunately,

the silica could be removed by passing compound 56 twice

through Sephadex LH 20 columns.

In order to avoid the problems faced during removal of the

methyl groups, in subsequent syntheses, the protecting group

was switched to benzyl. The unstable amine 57 was prepared

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hydrogenolysis deprotection protocol using Pd/C. By applying

our Lindlar catalyst/1,4-cyclohexadiene methodology, the

benzylic groups were selectively removed under controlled

hydrogenolysis conditions.

Scheme 7

Lindlar Catalyst and Pearlman’s Catalyst in Chemo-

selective Hydrogenolysis. The benzyl group is one of the

most commonly used groups for the protection of oxygen and

nitrogen functional groups in synthetic organic chemistry, as it is

stable toward many reaction conditions and can be easily

installed. Catalytic hydrogenolysis often oﬀers the mildest

method for removal of the benzyl ether protecting group.

However, hydrogenolysis is often not compatible with the

presence of other reducible functional groups, such as alkenes,

nitro groups, and halogens. Several examples are provided here of

the use of Lindlar catalyst, either with H₂(compounds 22 and

69) or with 1,4-cyclohexadiene (compounds 46, 60, and 78), as

the source of hydrogen. Pearlman’s catalyst was successful with

1,4-cyclohexadiene as the source of hydrogen in the cases of

compounds 63 and 66.

Enzyme Inhibition Studies. The lumazine synthase

substrate analogues and hypothetical intermediate analogues

were tested as inhibitors of the recombinant lumazine synthases

of Bacillus subtilis, Mycobacterium tuberculosis, and Schizosacchar-

omyces pombe, as well as the recombinant riboﬂavin synthase of

Escherichia coli and Mycobacterium tuberculosis. The inhibition

constants and inhibition mechanisms of these inhibitors are listed

in Table 1 along with the previously reported compounds 70−

77, which are also included in Table 1 for comparison. All of the

synthesized metabolically stable oxalamic acid derivatives with S,

O, and NH linkages between the ribityl side chain and

pyrimidinedione ring are analogues of the phosphate elimination

intermediate 6 of the lumazine synthase-catalyzed reaction.

Except for compound 72,⁴⁸these probes without a phosphate

side chain were in general found to be moderately active or

inactive lumazine synthase inhibitors. However, some of the new

compounds did display submicromolar K_ivalues versus some of

the enzyme. These include 23 versus S. pombe lumazine synthase,

24 versus M. tuberculosis riboﬂavin synthase, and 69 versus M.

tuberculosis riboﬂavin synthase. Compound 72 was found to be

an outlier with unusually high inhibitory potency versus E. coli

riboﬂavin synthase (K_i0.0013 μM).

Scheme 8

The relative eﬃcacies of the N-nucleoside, O-nucleoside, S-

nucleoside, and C-nucleoside substrate analogue inhibitors can

be directly accessed by comparison of the nitro compounds 18

(Figure 1) (K_i13 μM⁴⁹), 22 (K_i60 μM, Table 1), 24 (K_i11

μM⁴⁹), and 73 (K_i220 μM⁴⁹) versus M. tuberculosis lumazine

synthase. The S-nucleosides were generally more potent than the

corresponding O-nucleosides, as exempliﬁed by the relative

activities of the S-nucleosides 49 (K_i128 μM) and 51 (K_i198

μM) versus the O-nucleosides 43 (K_i>1000 μM) and 46 (K_i

>1000 μM) against M. tuberculosis lumazine synthase. The S-

nucleoside 23⁴⁹having an amino group at C-5 of the

pyrimidinedione was found to be a relatively potent inhibitor

of the lumazine synthases of S. pombe (K_i0.16 μM), B. subtilis (K_i

2.6 μM), and M. tuberculosis (K_i31 μM) and of the riboﬂavin

synthases of M. tuberculosis (K_i2.5 μM) and E. coli (K_i47 μM),

whereas the C-nucleoside analogue 74⁵¹of the substrate 1 was

shown previously to be completely inactive against B. subtilis

lumazine synthase.⁵¹

nium ﬂuoride removed the TBDMS groups, and during the

process, the intermediate underwent cyclization to yield 68.

Hydrogenolysis of 68 with Lindlar catalyst successfully provided

the desired compound 69.

The structures of some previously reported lumazine synthase

and riboﬂavin synthase inhibitors are listed in Chart 3 for the

purpose of comparison with the presently reported com-

pounds.^{41,42,48,51,52}The crystal structure of 75 bound to M.

tuberculosis lumazine synthase (PDB code: 2C97)⁴¹encouraged

the synthesis of compound 78, which is described in Scheme 10.

Intermediate 80 was obtained by the reaction of 79⁵²with

methacrolyl chloride. The chloride and double bond present in

the intermediate 80 prevented the application of general

Compound 75⁴¹displayed a K_dof 0.72 μM versus M.

tuberculosis lumazine synthase. Compounds 76,⁵²77,⁵²and 78

had very weak or no enzyme inhibitory activity. Although

compound 75 lacks the ribityl side chain, it has a phosphate

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Scheme 9

moiety that contributes positively to binding. The inhibitory

activity is probably due in large part to the presence of the

phosphate group that binds in the phosphate binding pocket of

the enzyme.⁴¹It also has a π−π stacking interaction of the

pyrimidinedione ring with Trp27.

less potent inhibitor (K_i0.61 μM) of E. coli riboﬂavin synthase.⁵⁵

It is possible that the dioxolumazine 81 is a more potent

riboﬂavin synthase inhibitor than 84 because it resembles the

hypothetical intermediate 11 involved in the reaction catalyzed

by riboﬂavin synthase.⁵⁶The related compound 69 is also a

relatively potent inhibitor of M. tuberculosis riboﬂavin synthase

(K_I0.74 μM), although it is not nearly as potent as the dioxo

compound 81. Compound 69, which resembles intermediate 10

in the lumazine synthase-catalyzed reaction, inhibits M. tuber-

culosis lumazine synthase with a K_ivalue of 8.6 μM.

The O-nucleosides 36, 40, and 43 were synthesized as the

oxygen analogues of the N-nucleoside inhibitors 72 (K_i4 μM),

71 (K_i42 μM), and 70 (K_i110 μM) of M. tuberculosis lumazine

synthase. In contrast to the N-nucleoside inhibitors, the O-

nucleosides 36, 40, and 43 were totally inactive versus lumazine

synthase. The N-nucleoside 70 had a K_iof 110 μM versus M.

tuberculosis lumazine synthase, while the corresponding S-

nucleoside 49 had a K_isof 128 μM versus M. tuberculosis

lumazine synthase. Crystallographic studies of 70 bound to M.

tuberculosis lumazine synthase have revealed a water bridge

between the ribitylamino NH and the side chain amino group of

Lys138 (PDB code: 2VI5).⁴⁸The greater activity of the N-

nucleosides may reﬂect the ability of the ribitylamino NH to

The N-nucleoside ethyl oxalate derivative 72⁴⁸proved to be an

unusually potent inhibitor of E. coli riboﬂavin synthase, with a K_i

of 0.0013 μM. It also had a K_iof 4.0 μM against M. tuberculosis

lumazine synthase, whereas the O-nucleoside ethyl oxalate

derivative 36 is completely inactive against all lumazine synthases

and riboﬂavin synthases tested. In contrast, the methyl oxalate

derivative 37 had weak activity (K_iof 868 μM) against M.

tuberculosis riboﬂavin synthase. The potency and the structure of

the compound 72 are reminiscent of the potent 6,7-dihydro-6,7-

dioxo-8-ribityllumazine system 81 (Chart 4), which was

previously shown to be a potent inhibitor of baker’s yeast

riboﬂavin synthase (K_i0.025 μM) and Ashbya gossypii riboﬂavin

synthase (K_i0.009 μM).^53,54It should be noted, however, that 72

(K_i0.0013 μM) is more potent versus E. coli riboﬂavin synthase

than 81 (K_i0.0062 μM) by a factor of 4.8. In this particular case,

the greater ﬂexibility of 72 versus 81 may allow it to better ﬁt the

binding pocket of the enzyme. The previously synthesized,

conformationally restricted compound 84 was also found to be a

H

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MD Simulation and Binding Energy Calculations.

Although the mechanism probes 36 and 72 diﬀer only in the

heteroatom linkage between the ribityl side chain and the

pyrimidinedione ring, they have a very large diﬀerence in their

inhibitory activities against M. tuberculosis lumazine synthase and

E. coli riboﬂavin synthase. In compounds 69 and 81, the C-5 side

chains of 82 and 72 have been conformationally restricted

through cyclization and they have shown good inhibitory

activities against M. tuberculosis lumazine synthase and M.

tuberculosis riboﬂavin synthase (Chart 4). In the case of lumazine

synthase, conformational restriction of the side chain appears to

increase the inhibitory activity. To understand these results, a

series of molecular dynamics simulations followed by free energy

calculations using the Poisson−Boltzmann/surface area (MM-

PBSA) method were carried out.⁵⁷All simulations were

performed using AMBER 9.0. For each ligand, the force ﬁeld

parameters were taken from the general Amber force ﬁeld

(GAFF), whereas the atomic partial charges were derived by

geometry optimization using the Gaussian 03 package.⁵⁸These

analyses have suggested a molecular basis for the interaction of

these compounds with lumazine synthase, and they have

provided a practical method to predict ligand binding aﬃnities

in future applications.

Chart 3

MD Simulations of the M. tuberculosis Lumazine

Synthase Complex with Ligand 70. The X-ray crystal

structure of ligand 70 and M. tuberculosis lumazine synthase

complex is available from the Protein Data Bank (PDB code:

2VI5).⁴⁸The C-5 sp²carbon of the ligand in this structure

appears distorted and deviates from its standard geometry, and

therefore the starting structure was thoroughly energy minimized

to ensure the right ligand conformation before MD simulation.

The whole simulation time was 1000 ps, during which the ligand

remained stable in the binding site and the ligand rmsd remain at

about 0.7 Å (in Figure 3, black). Hydrogen bond analysis

revealed that most of the hydrogen bonds and water bridges that

are apparent in the crystal structure were retained during the

simulation.

Scheme 10

MD Simulations of the M. tuberculosis Lumazine

Synthase Complex with Ligand 81. Ligand 81 binds tightly

to the active site of M. tuberculosis lumazine synthase during the

entire MD simulation process. As shown in Figure 3 (red), the

ligand rmsd remained quite small, around 0.2−0.3 Å, during the

whole simulation time. Figure 4 shows the minimized average

structure of the binding site of lumazine synthase complex with

ligand 81. Compared to ligand 70, the additional ring system in

ligand 81 did not result in much change in the overall binding

mode, and most of the hydrogen bonds to the binding site

residues (including Ala59, Ile60, Glu61, Val81, and Asn114)

were retained with ligand 81, as well as the two water bridges.

The most remarkable diﬀerence was the formation of two

hydrogen bonds with Lys138 and Gln141, which were not

observed with ligand 70. As shown in Figure 5 (top), the

hydrogen bond between the diketone carbonyl oxygen (O-7)

and NH₂of Gln141 formed at 400 ps in the MD simulation when

the side chain of Gln141 rotated 90° to approach the carbonyl

group (O-7) of ligand 81. The distance between the (O-7)

carbonyl oxygen on ligand 81 and the NH₂side chain hydrogen

of Gln141 ﬂuctuates around 1.95 Å during the next 600 ps of the

MD simulation, indicating a tight hydrogen bond between them.

The MD simulation also revealed a stable hydrogen bond

between the NH₂of Lys138 and the (O-6) carbonyl oxygen of

ligand 81. The three hydrogens on the protonated amino group

of Lys138 are constantly rotating and form a hydrogen bond with

function as a hydrogen bond donor toward the bridging water

molecule, in contrast to the C-, O-, and S-nucleosides, which

cannot act as hydrogen bond donors. It seems less likely that the

NH of the ribitylamino group could act as a hydrogen bond

acceptor because it is part of a vinylogous amide system. A related

set of compounds that allows comparison is the S-nucleoside 49

(K_i128 μM), the N-nucleoside 70 (K_i110 μM), and the O-

nucleoside 43 (inactive). The one N-nucleoside that does not ﬁt

the pattern at all is 56, which proved to be inactive versus M.

tuberculosis lumazine synthase.

With regard to the new monocyclic lumazine synthase

intermediate analogues, whose syntheses are outlined in Scheme

8 and Scheme 9, the activity order versus M. tuberculosis lumazine

synthase is 63 (K_i57 μM) > 66 (K_i100 μM) > 60 (K_i279 μM) >

56 (K_i> 1000 μM). In the case of E. coli riboﬂavin synthase, the

order is reversed: 56 (K_i52 μM) > 60 (K_i314 μM) > 66 (K_i>

1000 μM) = 63 (K_i> 1000 μM). The bicyclic compound 69 is

exceptional because it is the most active of the new compounds

versus both M. tuberculosis lumazine synthase (K_i8.6 μM) and E.

coli riboﬂavin synthase (K_i0.74 μM). However, none of the

compounds in this series can match the potency versus M.

tuberculosis lumazine synthase displayed by compounds with

phosphate side chains (e.g., 85, K_i0.0041 μM).³⁷

I

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Table 1. Inhibition Constants versus S. pombe Lumazine Synthase, M. tuberculosis Lumazine Synthase, B. subtilis Lumazine

Synthase, M. tuberculosis Riboﬂavin Synthase, and E. coli Riboﬂavin Synthase

J

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Table 1. continued

K

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Table 1. continued

L

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Table 1. continued

a

b

c

Recombinant β₆₀capsid from B. subtilis. Recombinant riboﬂavin synthase from E. coli. Recombinant homopentameric lumazine synthase from S.

e

d

pombe. Recombinant riboﬂavin synthase from M. tuberculosis. Recombinant homopentameric lumazine synthase from M. tuberculosis. The assays

with lumazine synthase were performed with substrate 2 held constant, while the concentration of the pyrimidinedione substrate 1 was varied. K_sis

the substrate dissociation constant for the equilibrium E + S ⇌ ES. K_catis the rate constant for the process ES → E + P. K_iis the inhibitor

dissociation constant for the process E + I ⇌ EI. K_isis the inhibitor dissociation constant for the process ES + I ⇌ ESI. The reaction mixtures

i

f

g

h

contained 50 mM Tris·HCl, pH 7.0.

the (O-6) carbonyl oxygen of ligand 81 alternately during the

entire simulation time, as shown in Figure 5 (bottom).

According to Table 2, the estimated binding free energy (using

than ligand 70 (−30.98 kJ/mol), which is consistent with the

results of the experimental enzyme inhibition assay. The

enhanced aﬃnity of ligand 81 could be attributed to the

following three factors. First, there is the addition of two

MM-PBSA method) for ligand 81 is −42.93 kJ/mol, much lower

M

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Chart 4

Figure 3. Time-dependent rmsd proﬁle of the ligands in molecular dynamics simulations.

hydrogen bonds with Lys138 and Gln141. Evidence comes from

the analysis of the electrostatic (ΔE_elec) term contribution to the

binding free energy in Table 2. The electrostatic term in ligand

81 (−78.45 kJ/mol) is much lower than that in 70 (−63.10 kJ/

mol), which dominates their diﬀerence in binding free energy.

Second, because of the introduction of the additional

dioxolumazine ring system, the π−π interaction with Trp27 is

expected to be strengthened in ligand 81. In Table 2, the van der

Waals term (ΔE_vdw) of ligand 81 (−38.09 kJ/mol) is quite close

to that of ligand 70 (−39.67 kJ/mol). In ligand 70, the amide side

chain protrudes into a hydrophobic pocket around Phe90 and

Val93, and these hydrophobic interactions are largely compen-

sated by the enhanced π−π interaction with Trp27 in ligand 81.

Third, the enhanced binding of ligand 81 in the active site of

lumazine synthase can be attributed to the change in entropy.

The 1,2-diketone chain is conformationally restricted in ligand

81, which would decrease the entropy loss during binding to the

lumazine synthase binding site. Although the entropy loss is not

explicitly estimated in this paper because of the tremendous

calculation needed, it is an important factor.

MD Simulations of the M. tuberculosis Lumazine

Synthase Complex with Ligand 69. Ligand 69 is also

calculated to bind tightly in the binding site. The ligand rmsd

stays at 0.5 Å (Figure 3, green). Ligand 69 can also form a

hydrogen bond between the carbonyl oxygen (O-6) with NH₂of

Lys138, but not with Gln141. The estimated binding free energy

for ligand 69 is −35.87 kJ/mol, which is a bit higher than that of

ligand 81. The diﬀerence mostly originates from the high

desolvation energy (ΔG_solv) for ligand 69 (88.74 kJ/mol

compared to 73.61 kJ/mol for ligand 81). Since a hydroxyl

group is more polar than a carbonyl group and can act as a

hydrogen bond donor as well as a hydrogen bond acceptor, the

N

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Figure 4. Molecular interactions between ligand 81 and M. tuberculosis lumazine synthase. Residues of the enzyme are labeled and shown as sticks.

Hydrogen bonding interactions are indicated by black dashed lines with key distance. The ﬁgure is programmed for walleyed viewing.

Figure 5. Time dependence of the key distances for the complex of ligand 81 with lumazine synthase during molecular dynamics simulations.

Table 2. Energy Contributions to Free Energy of Binding

entire MD simulation. The ligand rmsd ﬂuctuates mainly

between 0.7 and 1.7 Å, with the highest rmsd up to 2 Å (in

Figure 3, orange). The instability comes from the steric clash

between the long 1,2-dicarbonyl side chain and the lumazine

synthase binding pocket. As a result, the estimated binding free

energy for ligand 36 is −21.41 kJ/mol, which is much higher than

that of ligands 69, 72, and 81. The estimated binding free energy

for ligand 36 is consistent with poor binding.

Ligands 36 and 72 diﬀer only in the heteroatom linkage.

Consequently, the dynamic behavior of ligand 72 is nearly the

same as ligand 36 in the MD simulations. The ligand 72 also

experiences large ﬂuctuations during simulation. The ligand

rmsd ﬂuctuates between 0.5 and 1.0 Å (in Figure 3, blue). The

binding free energy calculated for ligand 72 is only −25.84 kJ/

mol, which is not consistent with its inhibitory activity relative to

the other ligands in Table 2.

Calculated for Each Compound with the M. tuberculosis

a

Lumazine Synthase Complex

compd

ΔE_elec

ΔE_vdw

ΔG_solv

ΔG_bind

K_i(μM)

36

69

70

72

81

−59.18

−86.93

−63.10

−65.25

−78.45

−42.80

−37.69

−39.67

−44.24

−38.09

80.57

88.74

71.78

83.64

73.61

−21.41

−35.87

−30.98

−25.84

−42.93

>1000

8.6 0.7

110 14

4.0 1.7

1.4 0.1

a

All energies are expressed as kJ/mol.

polar C-7 hydroxyl group makes ligand 69 more hydrophilic than

ligand 81 and elevates its desolvation energy.

MD Simulations of the M. tuberculosis Lumazine

Synthase Complex with Ligands 36 and 72. Ligand 36 is

inactive in the experimental inhibitory activity assay. Consistent

with this result, ligand 36 appears to be very unstable during the

O

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temperature over 5 h. The reaction was quenched using ammonium

chloride, THF was removed under vacuum, the residue then dissolved in

CHCl₃(15 mL), and the organic layer was washed with water and brine,

dried, and concentrated. The residue was puriﬁed by silica gel ﬂash

column chromatography, eluting with 15% ethyl acetate in hexane, to

CONCLUSION

■

In conclusion, this article describes a comprehensive study of O-

nucleoside, S-nucleoside, and N-nucleoside probes that has

provided insights into the mechanistic details of lumazine

synthase and riboﬂavin synthase catalysis. The following is a list

of the main results: (1) All of the synthesized metabolically stable

oxalamic acid derivative probes with S, O, and NH linkages

between the ribityl side chain and pyrimidinedione ring that are

analogues of the phosphate elimination products of the lumazine

synthase reaction intermediate 6 are moderate or inactive

lumazine synthase inhibitors. However, compounds containing a

phosphate side chain have very strong aﬃnity for lumazine

synthase. This suggests that lumazine synthase predominantly

catalyzes Schiﬀ base formation and the phosphate elimination

reaction, and the subsequent intermediates have low enzyme

aﬃnity (Scheme 2). (2) Compounds 76, 77, and 78, which do

not contain either the phosphate side chain or the ribityl side

chain, showed very weak or complete absence of any inhibitory

activity. Whereas compound 75⁴¹lacks the ribityl side chain, it

has a phosphate group that binds to the phosphate binding site,

resulting in strong aﬃnity for lumazine synthase (K_dof 0.72 μM

vs M. tuberculosis lumazine synthase). This suggests that the

pyrimidinedione ring alone has low binding aﬃnity, and the

phosphate moiety contributes more positively to the binding of

compound compared to the ribityl side chain. Previously

reported work has shown that the ribityl side chain can be

replaced by a styryl moiety with retention of aﬃnity for the

enzyme, indicating that the ribityl chain is not essential for

binding.⁵⁹(3) Among all of the compounds synthesized, S-

nucleoside 23 showed greater lumazine synthase inhibitory

potency (Table 1) as compared to all the O-nucleoside, N-

nucleoside, and C-nucleoside derivatives. The greater inhibitory

potency of the S-nucleoside 23 and its nitro precursor 24

compared with the O-nucleoside 29 and C-nucleoside 74 could

possibly be due to electronics. The nonbonded electrons on

sulfur would cause 23 to more closely resemble the natural

substrate relative to the C-nucleoside 74. (4) All of the bicyclic

compounds (69, 81, and 84) were found to exhibit stronger

aﬃnity for riboﬂavin synthase than lumazine synthase. (5) The

molecular dynamics simulations provide a method for predicting

ligand aﬃnities to the active site of lumazine synthase. They also

provide insight into the dynamic aspects of ligand binding. (6) A

useful controlled hydrogenolysis methodology has been

developed and applied to the selective cleavage of benzyl

protecting groups in the presence of easily reducible functional

groups. The information revealed in this study will be valuable for

future structure-based drug design of lumazine synthase and

riboﬂavin synthase inhibitors.

1

yield compound 27 (108 mg, 71%) as a colorless oil: H NMR (300

MHz, CDCl₃) δ 7.33−7.24 (m, 10 H), 5.48 (s, 2 H), 5.40 (s, 2 H), 4.79

(dd, J = 3.9 Hz, 11.5 Hz, 1 H), 4.61 (dd, J = 6.2 Hz, 11.5 Hz, 1 H), 4.52−

4.47 (m, 1 H), 4.16−4.01 (m, 3 H), 3.93−3.85 (m, 1 H), 1.39 (s, 3 H),

1.34 (s, 3 H), 1.32 (s, 3 H), 1.23 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ

163.8, 162.0, 135.4, 135.0, 128.6, 128.3, 128.2, 127.6, 109.8, 109.4, 77.7,

75.0, 73.3, 70.3, 69.8, 68.0, 66.8, 27.6, 26.7, 25.3, 25.2. Anal. Calcd for

C₂₉H₃₃N₃O₉: C, 61.37; H, 5.86; N, 7.40. Found: C, 61.44; H, 5.88; N,

7.36.

(2R,3R,4S)-5-(2,6-Bis(benzyloxy)-5-nitropyrimidin-4-yloxy)-

pentane-1,2,3,4-tetrol (28). HCl (0.5 N, 2 mL) was added to a

solution of compound 27 (50 mg, 0.09 mmol) in THF (2 mL), and the

reaction mixture was stirred for 6 h at room temperature. THF was

removed under vacuum, the residue then dissolved in ethyl acetate (10

mL), and the organic layer was washed with water and brine, dried, and

concentrated. The residue was puriﬁed by silica gel ﬂash column

chromatography, eluting with 60% ethyl acetate in hexane, to aﬀord

1

compound 28 (31 mg, 72%) as a colorless oil: H NMR (300 MHz,

CDCl₃) δ 7.35−7.27 (m, 10 H), 5.46 (s, 2 H), 5.38 (s, 2 H), 4.68 (dd, J =

2.9 Hz, 11.3 Hz, 1 H) 4.55 (dd, J = 6.2 Hz, 11.3 Hz, 1 H), 4.06−4.00 (m,

1 H), 3.76−3.63 (m, 4 H); ¹³C NMR (75 MHz, CDCl₃) δ 164.0, 163.5,

161.8, 135.0, 134.7, 128.2, 128.0, 127.7, 127.2, 72.4, 71.3, 70.6, 70.1,

70.0, 69.6, 62.9; ESIMS m/z (rel. intensity) 997 (2MNa⁺, 100), 863

(30), 510 (MNa⁺, 42). Anal. Calcd for C₂₃H₂₅N₃O₉: C, 56.67; H, 5.17;

N, 8.62. Found: C, 56.81; H, 5.21; N, 8.49.

5-Amino-6-((2R,3R,4S)-2,3,4,5-tetrahydroxypentyloxy)-

pyrimidine-2,4(1H,3H)-dione (29). Pd/C (10%, 5 mg) was added to

a solution of compound 28 (45 mg, 0.08 mmol) in MeOH/EtOAc (3 +

1 mL). A hydrogen balloon was attached, and the mixture was stirred at

room temperature for 12 h. The reaction mixture was ﬁltered through

Celite into a solution of MeOH containing HCl. The solution was

concentrated to provide compound 29 (24 mg, 83%) as yellow

amorphous solid: mp 200−204 °C (dec); ¹H NMR (300 MHz, MeOH-

d₄) δ 4.14−4.08 (m, 1 H), 3.97 (dd, J = 4.8 Hz, 9.5 Hz, 1 H), 3.71−3.68

(m, 4 H), 3.52 (dd, J = 4.8 Hz, 12.0 Hz, 1 H); ¹³C NMR (75 MHz,

MeOH-d₄) δ 168.3, 166.0, 158.9, 108.8, 84.1, 73.8, 73.1, 72.5, 63.2;

ESIMS m/z (rel. intensity) 278 (MH⁺, 35); negative ion ESIMS m/z

(rel. intensity) 276 [(M − H⁺)⁻, 62], 171 (100). Anal. Calcd for

C₉H₁₆ClN₃O₇: C, 34.46; H, 5.14; N, 13.40. Found: C, 34.12; H, 5.18; N,

13.52.

5-Nitro-6-((2S,3R,4R)-2,3,4,5-tetrahydroxypentyloxy)-

pyrimidine-2,4(1H,3H)-dione (22). Lindlar catalyst (5 mg) was

added to a solution of compound 28 (45 mg, 0.08 mmol) in MeOH/

EtOAc (3 + 1 mL). A hydrogen balloon was attached, and the mixture

was stirred at room temperature for 12 h. The reaction mixture was

ﬁltered through Celite, which was then washed with 50% aq MeOH (5

mL). The solution was concentrated, and the residue was puriﬁed via

silica gel ﬂash column chromatography, eluting with 10% methanol in

ethyl acetate, to furnish compound 22 as a colorless oil. Compound 22

was washed several times with CH₂Cl₂and THF. Finally, compound 22

was precipitated out by dissolving in MeOH and adding excess diethyl

ether to aﬀord pure compound 22 (22 mg, 79%) as an orange

amorphous solid: mp 165−168 °C (dec); ¹H NMR (300 MHz, MeOH-

d₄) δ 4.15−4.106 (m, 1 H), 4.03−3.94 (m, 3 H), 3.78−3.68 (m, 2 H),

3.68 (dd, J = 9.5 Hz, 14.0 Hz, 1 H); ¹³C NMR (75 MHz, MeOH-d₄) δ

167.9, 164.2, 157.6, 123.0, 79.8, 79.6, 74.5, 68.4, 62.2; negative ion

ESIMS m/z (rel. intensity) 306 [(M − H⁺)⁻, 100], 172 (65). Anal.

Calcd for C₉H₁₃N₃O₉: C, 35.19; H, 4.27; N, 13.68. Found: C, 34.97; H,

4.43; N, 13.90.

EXPERIMENTAL SECTION

■

General. Melting points were determined using capillary tubes and

are uncorrected. NMR spectra were determined at 300 or 500 MHz

(¹H) and 75 or 125 MHz (¹³C) as speciﬁed. High-resolution mass

spectra were recorded on a double-focusing sector mass spectrometer

with magnetic and electrostatic mass analyzers. Silica gel ﬂash

chromatography was performed using 230−400 mesh silica gel.

2,4-Bis(benzyloxy)-5-nitro-6-(((4R,4′R,5S)-2,2,2′,2′-tetra-

methyl-4,4′-bi(1,3-dioxolan)-5-yl)methoxy)pyrimidine (27). A

solution of ^D-ribitol derivative 26 (62 mg, 0.27 mmol) in dry THF (5

mL) was cooled to −40 °C, and LiHMDS (0.3 mL, 1 M solution in

THF, 0.32 mmol) was added. The solution was stirred for 30 min at

room temperature and cooled to −40 °C. Chloropyrimidine derivative

25 (100 mg, 0.27 mmol) dissolved in dry THF (5 mL) was introduced

into the reaction mixture, which was slowly brought to room

2,4-Dimethoxy-5-nitro-6-(((4R,4′R,5S)-2,2,2′,2′-tetramethyl-

4,4′-bi(1,3-dioxolan)-5-yl)methoxy)pyrimidine (31). A solution of

D-ribitol derivative 26 (100 mg, 0.43 mmol) in dry THF (8 mL) was

cooled to −40 °C and n-BuLi (0.3 mL, 2.5 M, 0.86 mmol) was added.

The solution was stirred for 30 min at room temperature and cooled to

−40 °C. Iodopyrimidine derivative 30 (122 mg, 0.39 mmol) dissolved in

P

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dry THF (8 mL) was introduced into the reaction mixture, which was

slowly brought to room temperature by overnight stirring. The reaction

was quenched using ammonium chloride, THF was removed under

vacuum, the residue then dissolved in CHCl₃(15 mL), and the organic

layer was washed with water and brine, dried, and concentrated. The

residue was column chromatographed with ﬂash silica gel eluting with

20% ethyl acetate in hexane to yield compound 31 (75 mg, 45%) as a

colorless oil along with both the staring materials 30 and 26: ¹H NMR

(300 MHz, CDCl₃) δ 4.81 (dd, J = 4.1 Hz, 1 H), 4.64 (dd, J = 6.0 Hz, 1

H), 4.50−4.47 (m, 1 H), 4.16−4.06 (m, 3 H), 4.05 (s, 3 H), 4.00 (s, 3

H), 3.92−3.86 (m, 1 H), 1.37 (s, 3 H), 1.33 (s, 3 H), 1.31 (s, 3 H), 1.23

(s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 164.4, 163.8, 162.7, 109.7,

109.4, 77.7, 75.1, 73.3, 68.0, 66.7, 55.6, 55.5, 27.5, 26.7, 25.2; EIMS m/z

was puriﬁed by silica gel ﬂash column chromatography, eluting with 70%

ethyl acetate in hexane, to give compounds 34 (16 mg, 53%) and 35 (11

mg, 37%) as a colorless oils. Compound 34: H NMR (300 MHz,

1

acetone-d₆) δ 9.36 (s, 1 H), 7.50−7.30 (m, 10 H), 5.46 (s, 2 H), 5.40 (s,

2 H), 4.74−4.69 (m, 1 H), 4.53−4.48 (m, 1 H), 4.42 (brs, 1 H), 4.31 (q, J

= 7.12 Hz, 2 H), 4.14−4.03 (m, 3 H), 3.77−3.67 (m, 4 H), 1.34 (t, J =

7.12 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 165.6, 165.2, 159.9, 154.8,

136.0, 135.5, 128.6, 128.3, 128.2, 128.1, 95.6, 73.2, 71.5, 69.8, 69.3, 63.8,

63.3, 13.9; ESIMS m/z (rel. intensity) 1137 (2 M + Na⁺, 100), 580

(MNa⁺, 69), 559 (M2H⁺, 12), 558 (MH⁺, 47). Anal. Calcd for

C₂₇H₃₁N₃O₁₀: C, 58.16; H, 5.60; N, 7.54. Found: C, 57.82; H, 5.88; N,

7.21.

1

Compound 35: H NMR (300 MHz, acetone-d₆) δ 9.42 (s, 1 H),

+

(rel. intensity) 400 (M⁺− CH₃, 11), 256 (C₉H₁₀N₃O₆, 20), 101

7.47−7.32 (m, 10 H), 5.46 (s, 2 H), 5.40 (s, 2 H), 4.75−4.68 (m, 1 H),

4.52−4.45 (m, 2 H), 4.19 (brs, 1 H), 4.10−4.08 (m, 2 H), 3.85 (s, 3 H),

3.75−3.70 (m, 3 H), 3.68−3.64 (m, 2 H), 3.58 (m, 1 H), 3.42 (m, 1 H);

¹³C NMR (75 MHz, acetone-d₆) δ 167.4, 167.1, 161.5, 156.2, 137.7,

+

(C₅H₅O₂, 100); CIMS m/z (rel. intensity) 416 (MH⁺, 14), 215 (M⁺−

C₁₁H₁₉O₄, 100). Anal. Calcd for C₁₇H₂₅N₃O₉: C, 49.15; H, 6.07; N,

10.12. Found: C, 49.44; H, 5.88; N, 10.34.

2,4-Bis(benzyloxy)-6-(((4R,4′R,5S)-2,2,2′,2′-tetramethyl-4,4′-

bi(1,3-dioxolan)-5-yl)methoxy)pyrimidin-5-amine (32). Sodium

bicarbonate−sodium carbonate buﬀer (3 mL) (pH = 9.5, prepared by

adjusting the pH value of saturated sodium carbonate aqueous solution

to 9.5 with sodium carbonate and 1 M NaOH) and Na₂S₂O₄(260 mg,

1.48 mmol) were added to a solution of compound 27 (120 mg, 0.211

mmol) in 1,4-dioxane (3 mL). Argon was bubbled for 20 min to degas

the reaction mixture, which was stirred at room temperature for 6 h and

subsequently at 100 °C for 3 h. The reaction mixture was cooled to room

temperature and poured into ice water (5 mL) and extracted with

diethyl ether (3 × 8 mL). The organic layer was combined, washed with

brine, dried, and solvent was distilled oﬀ. The residue was separated by

ﬂash chromatography eluting with 20% ethyl acetate in hexane, to yield

137.6, 129.2, 129.0, 128.8, 128.7, 128.5, 96.9, 73.7, 73.4, 71.8, 70.2, 70.0,

69.3, 64.5, 53.6; ESIMS m/z (rel. intensity) 566 (MNa⁺, 100), 504 (8),

303 (8); negative ion ESIMS m/z (rel. intensity) 542 [(M − H⁺)⁻, 100],

408 (59). Anal. Calcd for C₂₆H₂₉N₃O₁₀: C, 57.45; H, 5.38; N, 7.73.

Found: C, 57.69; H, 5.18; N, 7.66.

Ethyl 2-(2,4-Dioxo-6-((2S,3R,4R)-2,3,4,5-tetrahydroxypenty-

loxy)-1,2,3,4-tetrahydropyrimidin-5-ylamino)-2-oxoacetate

(36). Pd/C (10%, 5 mg) was added to a solution of compound 34 (30

mg, 0.054 mmol) in EtOH (4 mL). A hydrogen balloon was attached,

and the mixture was stirred at room temperature for 12 h. The reaction

mixture was ﬁltered through Celite, which was then washed with EtOH

(15 mL) and the ﬁltrate concentrated to provide compound 36 (14 mg,

70%) as white solid: mp 62−64 °C; ¹H NMR (300 MHz, MeOH-d₄) δ

4.52−4.47 (m, 1 H), 4.46−4.40 (m, 1 H), 4.34 (q, J = 7.10 Hz, 2 H),

4.02−3.98 (m, 1 H), 3.77−3.70 (m, 2 H), 3.67−3.61 (m, 2 H), 1.34 (t, J

= 7.10 Hz, 3 H); ¹³C NMR (75 MHz, MeOH-d₄) δ 164.7, 162.4, 161.1,

159.2, 153.1, 93.2, 74.1, 74.0, 73.1, 72.5, 64.5, 64.1, 14.3; ESIMS m/z

(rel. intensity) 777 (2 M + Na⁺, 100), 400 (MNa⁺, 32), 378 (MH⁺, 16);

negative ion ESIMS m/z (rel. intensity) 752 [(2 M − H⁺)⁻, 42], 376

[(M − H⁺)⁻, 100], 241 (60). Anal. Calcd for C₁₃H₁₉N₃O₁₀·H₂O: C,

39.50; H, 5.35; N, 10.63. Found: C, 39.67; H, 5.17; N, 10.40.

1

compound 32 (82 mg, 72%) as light yellow oil: H NMR (300 MHz,

CDCl₃) δ 7.42−7.18 (m, 10 H), 5.33 (s, 2 H), 5.25 (s, 2 H), 4.59 (dd, J =

10.41 Hz, 3.58 Hz, 1 H), 4.48−4.38 (m, 2 H), 4.13−3.95 (m, 4 H),

3.91−3.82 (m, 1 H), 3.21 (brs, 2 H), 1.46 (s, 3 H), 1.37 (s, 3 H), 1.28 (s,

3 H), 1.22 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 158.5, 158.3, 155.0,

137.2, 136.7, 128.4, 128.3, 128.1, 128.0, 127.8, 109.7, 109.1, 107.7, 77.9,

75.6, 73.2, 68.7, 68.4, 68.0, 65.2, 27.9, 26.8, 25.5, 25.3; ESIMS m/z (rel.

intensity) 560 (MNa⁺, 100), 538 (MH⁺, 50), 480 (9). Anal. Calcd for

C₂₉H₃₅N₃O₇: C, 64.79; H, 6.56; N, 7.82. Found: C, 65.03; H, 6.41; N,

7.64.

Methyl 2-(2,4-Dioxo-6-((2S,3R,4R)-2,3,4,5-tetrahydroxypen-

tyloxy)-1,2,3,4-tetrahydropyrimidin-5-ylamino)-2-oxoacetate

(37). Pd/C (10%, 3 mg) was added to a solution of compound 35 (20

mg, 0.037 mmol) in MeOH (3 mL). A hydrogen balloon was attached,

and the mixture was stirred at room temperature for 12 h. The reaction

mixture was ﬁltered through Celite, which was then washed with MeOH

(15 mL) and concentrated to provide compound 37 (10 mg, 75%) as a

Ethyl 2-(2,4-Bis(benzyloxy)-6-(((4R,4′R,5S)-2,2,2′,2′-tetra-

methyl-4,4′-bi(1,3-dioxolan)-5-yl)methoxy)pyrimidin-5-ylami-

no)-2-oxoacetate (33). Triethylamine (0.3 mL, 2.2 mmol) was added

to a solution of compound 32 (30 mg, 0.055 mmol) in THF (5 mL).

The solution was cooled to 0 °C, and ethyl chlorooxacetate (18 μL, 0.16

mmol) was added dropwise. The reaction mixture was stirred at 0 °C for

2 h and 30 min at room temperature. The solvent was distilled oﬀ under

reduced pressure, and the residue was dissolved in dichloromethane (20

mL) and washed with water (2 × 15 mL). The organic layer was dried

and solvent was distilled oﬀ. The residue was separated by silica gel ﬂash

column chromatography, eluting with 30% ethyl acetate in hexane, to

1

colorless oil: H NMR (300 MHz, MeOH-d₄) δ 4.49−4.42 (m, 2 H),

3.95−3.90 (m, 1 H), 3.88 (s, 3 H), 3.76−3.71 (m, 2 H), 3.64−3.58 (m, 2

H); ¹³C NMR (75 MHz, MeOH-d₄) δ 168.2, 165.3, 160.7, 152.0, 91.3,

74.2, 74.1, 73.4, 72.8, 69.1, 64.5; ESIMS m/z (rel. intensity) 386 (MNa⁺,

94), 256 (93), 179 (100); negative ion ESIMS m/z (rel. intensity) 362

[(M − H⁺)⁻, 100], 348 (8), 231 (7). Anal. Calcd for

C₁₂H₁₇N₃O₁₀·2H₂O: C, 36.10; H, 5.30; N, 10.52. Found: C, 36.33; H,

5.43; N, 10.49.

1

aﬀord compound 33 (36 mg, 96%) as a yellowish oil: H NMR (300

MHz, CDCl₃) δ 8.11 (s, 1 H), 7.41−7.19 (m, 10 H), 5.37 (s, 2 H), 5.32

(s, 2 H), 4.67−4.59 (m, 1 H), 4.47−4.42 (m, 2 H), 4.32 (q, J = 7.16 Hz, 2

H), 4.06−3.96 (m, 3 H), 3.89−3.83 (m, 1 H), 1.34 (s, 3 H), 1.33 (t, J =

7.1 Hz, 3 H), 1.30 (s, 3 H), 1.25 (s, 3 H), 1.19 (s, 3 H); ¹³C NMR (75

MHz, CDCl₃) δ 165.9, 161.6, 160.2, 154.7, 136.2, 136.1, 128.4, 128.2,

128.0, 127.7, 109.8, 109.2, 95.6, 77.7, 75.3, 73.2, 69.5, 69.0, 67.9, 66.0,

63.3, 27.7, 26.8, 25.3, 25.2, 14.0; ESIMS m/z (rel. intensity) 660 (MNa⁺,

100), 446 (2). Anal. Calcd for C₃₃H₃₉N₃O₁₀: C, 62.16; H, 6.16; N, 6.59.

Found: C, 62.34; H, 6.30; N, 6.88.

Ethyl 2-(2,4-Bis(benzyloxy)-6-((2S,3R,4R)-2,3,4,5-

tetrahydroxypentyloxy)pyrimidin-5-ylamino)-2-oxoacetate

(34) and Methyl 2-(2,4-Bis(benzyloxy)-6-((2S,3R,4R)-2,3,4,5-

tetrahydroxypentyloxy)pyrimidin-5-ylamino)-2-oxoacetate

(35). HCl (0.5 N, 1 mL) was added to a solution of compound 33 (35

mg, 0.055 mmol) in MeOH (1 mL), and the reaction mixture was stirred

for 12 h at room temperature. MeOH was removed under vacuum, the

residue then dissolved in ethyl acetate (10 mL), and the organic layer

was washed with water and brine, dried, and concentrated. The residue

N-(2,4-Bis(benzyloxy)-6-(((4R,4′R,5S)-2,2,2′,2′-tetramethyl-

4,4′-bi(1,3-dioxolan)-5-yl)methoxy)pyrimidin-5-yl)-

isobutyramide (38). Triethylamine (0.3 mL, 2.2 mmol) was added to

a solution of compound 32 (115 mg, 0.21 mmol) in THF (5 mL). The

solution was cooled to 0 °C, and isobutyryl chloride (25 μL, 0.24 mmol)

was added dropwise. The reaction mixture was stirred at 0 °C for 6 h.

The solvent was distilled oﬀ under reduced pressure, and the residue was

dissolved in dichloromethane (20 mL) and washed with water (2 × 15

mL). The organic layer was dried and solvent was distilled oﬀ. The

residue was ﬂash column chromatographed with ﬂash silica gel, eluting

with 35% ethyl acetate in hexane, to aﬀord compound 38 (110 mg, 85%)

1

as a yellowish-white solid: mp 150−153 °C; H NMR (300 MHz,

CDCl₃) δ 7.43−7.24 (m, 10 H), 6.53 (s, 1 H), 5.34 (s, 4 H), 4.68 (dd, J =

2.1, 10.5 Hz, 1 H), 4.47−4.39 (m, 2 H), 4.08−4.0 (m, 3 H), 3.92−3.89

(m, 1 H), 2.47 (m, 1 H), 1.39 (s, 3 H), 1.35 (s, 3 H), 1.30 (s, 3 H), 1.26

(s, 3 H), 1.17 (m, 6 H); ¹³C NMR (75 MHz, CDCl₃) δ 175.6, 166.4,

166.1, 161.0, 136.3, 136.2, 128.2, 128.1, 127.9, 127.7, 127.4, 109.6, 109.0,

Q

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The Journal of Organic Chemistry

Article

97.0, 77.5, 75.4, 73.1, 69.2, 68.6, 67.7, 65.7, 35.3, 27.6, 26.7, 25.2, 19.5,

19.3; ESIMS m/z (rel. intensity) 608 (MH⁺, 100), 550 (14); HRMS m/

z calcd for C₃₃H₄₂N₃O₈(MH⁺) 608.2972, found 608.2981. Anal. Calcd

for C₃₃H₄₁N₃O₈: C, 65.22; H, 6.80; N, 6.91. Found: C, 65.40; H, 6.53; N,

7.12.

solid: mp 110−113 °C; ¹H NMR (300 MHz, MeOH-d₄) δ 7.43−7.31

(m, 10 H), 5.38 (s, 4 H), 4.62−4.58 (m, 1 H), 4.46 (dd, J = 2.7, 10.8 Hz,

1 H), 4.08−4.02 (m, 1 H), 3.79−3.76 (m, 2 H), 3.69−3.61 (m, 2 H),

2.36 (q, J = 7.4 Hz, 2 H), 1.17 (t, J = 7.2 Hz, 3 H); ¹³C NMR (75 MHz,

MeOH-d₄) δ 177.4, 168.8, 168.4, 163.3, 138.6, 138.4, 130.0, 129.5,

129.2, 98.7, 74.6, 74.0, 72.5, 71.0, 70.4, 65.0, 30.5, 11.0; ESIMS m/z (rel.

intensity) 536 (MNa⁺, 100), 514 (MH⁺, 10); negative ion ESIMS m/z

(rel. intensity) 512 [M − H⁺)⁻, 4], 462 (100); HRMS m/z calcd for

C₂₆H₃₁N₃O₈Na (MNa⁺) 536.2009, found 536.2011. Anal. Calcd for

C₂₆H₃₁N₃O₈: C, 60.81; H, 6.08; N, 8.18. Found: C, 60.95; H, 6.31; N,

8.02.

N-(2,4-Dioxo-6-((2S,3R,4R)-2,3,4,5-tetrahydroxypentyloxy)-

1,2,3,4-tetrahydropyrimidin-5-yl)propionamide (43). Pd/C

(10%, 5 mg) was added to a solution of compound 42 (55 mg, 0.08

mmol) in MeOH/EtOAc (3 + 1 mL). A hydrogen balloon was attached,

and the mixture was stirred at room temperature for 12 h. The reaction

mixture was ﬁltered through Celite into a solution of MeOH containing

HCl. The solution was concentrated to provide compound 43 (25 mg,

70%) as yellow amorphous solid: mp 128−131 °C; ¹H NMR (300 MHz,

MeOH-d₄) δ 4.62−4.58 (m, 1 H), 4.49−4.43 (m, 1 H), 4.06−4.02 (m, 1

H), 3.80−3.75 (m, 2 H), 3.67−3.61 (m, 2 H), 2.38 (q, J = 7.4 Hz, 2 H),

1.16 (t, J = 7.3 Hz, 3 H); ¹³C NMR (75 MHz, MeOH-d₄) δ 178.5, 163.7,

162.8, 156.0, 85.0, 72.4, 71.2, 70.7, 64.5, 30.1, 29.4, 10.5. Anal. Calcd for

C₁₂H₁₉N₃O₈: C, 43.24; H, 5.75; N, 12.61. Found: C, 43.10; H, 5.72; N,

12.89.

N - ( 2 , 4 - B i s ( b e n z y l o x y ) - 6 - ( ( 2 S , 3 R , 4 R ) - 2 , 3 , 4 , 5 -

tetrahydroxypentyloxy)pyrimidin-5-ylamino)isobutyramide

(39). HCl (0.5 N, 1 mL) was added to a solution of compound 38 (60

mg, 0.1 mmol) in THF (2 mL), and the reaction mixture was stirred for

12 h at room temperature. THF was removed under vacuum, the residue

then dissolved in ethyl acetate (10 mL), and the organic layer was

washed with water and brine, dried, and concentrated. The residue was

column chromatographed with ﬂash silica gel, eluting with 80% ethyl

acetate in hexane, to produce compound 39 (43 mg, 81%) as white

semisolid: ¹H NMR (300 MHz, MeOH-d₄) δ 7.42−7.29 (m, 10 H), 5.38

(s, 4 H), 4.61 (m, 1 H), 4.58 (dd, J = 4.5, 8.1 Hz, 1 H), 4.46 (dd, J = 6.6,

11.4 Hz, 1 H), 3.78−3.65 (m, 2 H), 3.68−3.62 (m, 2 H), 2.68−2.50 (m,

1 H), 1.17 (d, J = 1.2 Hz, 3 H), 1.15 (d, J = 1.2 Hz, 1 H); ¹³C NMR (75

MHz, MeOH-d₄) δ 180.1, 168.4, 168.0, 162.9, 138.2, 137.9, 129.7, 129.5,

129.4, 129.1, 128.8, 128.6, 98.2, 84.1, 74.2, 73.5, 72.1, 70.5, 70.0, 64.6,

36.2, 19.9; ESIMS m/z (rel. intensity) 550 (MNa⁺, 100), 528 (MH⁺,

68), 394 (36); negative ion ESIMS m/z (rel. intensity) 526 [(M − H⁺)⁻,

28], 392 (100). Anal. Calcd for C₂₇H₃₃N₃O₈: C, 61.47; H, 6.30; N, 7.96.

Found: C, 61.65; H, 6.31; N, 7.80.

N-(2,4-Dioxo)-6-((2S,3R,4R)-2,3,4,5-tetrahydroxypentyloxy)-

1,2,3,4-tetrahydropyrimidin-5-yl)isobutyramide (40). Pd/C

(10%, 5 mg) was added to a solution of compound 39 (55 mg, 0.08

mmol) in MeOH/EtOAc (3 + 1 mL). A hydrogen balloon was attached,

and the mixture was stirred at room temperature for 12 h. The reaction

mixture was ﬁltered through Celite into a solution of MeOH containing

HCl. The solution was concentrated to provide compound 40 (26 mg,

72%) as yellow amorphous solid: mp 116−119 °C; ¹H NMR (300 MHz,

MeOH-d₄) δ 4.47−4.41 (m, 2 H), 3.97−3.90 (m, 1 H), 3.77−3.71 (m, 2

H), 3.64−3.60 (m, 2 H), 2.66−2.59 (m, 1 H), 1.19 (s, 3 H), 1.17 (s, 3

H); ¹³C NMR (75 MHz, MeOH-d₄) δ 180.9, 165.8, 165.26, 156.0, 93.5,

74.2, 73.2, 72.7, 72.0, 64.5, 36.2, 20.0; ESIMS m/z (rel. intensity) 370

(MNa⁺, 100), 300 (11), 219 (16); negative ion ESIMS m/z (rel.

intensity) 346 [(M − H⁺)⁻, 100], 212 (6). Anal. Calcd for C₁₃H₂₁N₃O₈:

C, 44.96; H, 6.09; N, 12.10. Found: C, 45.11; H, 5.93; N, 11.86.

N-(2,4-Bis(benzyloxy)-6-(((4R,4′R,5S)-2,2,2′,2′-tetramethyl-

4,4′-bi(1,3-dioxolan)-5-yl)methoxy)pyrimidin-5-yl)-

propionamide (41). Triethylamine (0.3 mL, 2.2 mmol) was added to a

solution of compound 32 (80 mg, 0.15 mmol) in THF (5 mL). The

solution was cooled to 0 °C and propionyl chloride (16 μL, 0.18 mmol)

was added dropwise. The reaction mixture was stirred at 0 °C for 6 h.

The solvent was distilled oﬀ under reduced pressure, and the residue was

dissolved in dichloromethane (20 mL) and washed with water (2 × 15

mL). The organic layer was dried, and solvent was distilled oﬀ. The

residue was ﬂash column chromatographed with ﬂash silica gel, eluting

with 35% ethyl acetate in hexane, to aﬀord compound 41 (77 mg, 87%)

N-(2,4-Bis(benzyloxy)-6-(((4R,4′R,5S)-2,2,2′,2′-tetramethyl-

4,4′-bi(1,3-dioxolan)-5-yl)methoxy)pyrimidin-5-yl)-

methacrylamide (44). Triethylamine (0.3 mL, 2.2 mmol) was added

to a solution of compound 32 (115 mg, 0.21 mmol) in THF (5 mL).

The solution was cooled to 0 °C and methacrolyl chloride (25 μL, 0.24

mmol) was added dropwise. The reaction mixture was stirred at 0 °C for

6 h. The solvent was distilled oﬀ under reduced pressure, and the residue

was dissolved in dichloromethane (20 mL) and washed with water (2 ×

15 mL). The organic layer was dried, and solvent was distilled oﬀ. The

residue was ﬂash column chromatographed with silica gel, eluting with

35% ethyl acetate in hexane, to aﬀord compound 44 (85 mg, 65%) as

white amorphous solid: mp 118−120 °C; ¹H NMR (300 MHz, CDCl₃)

δ 7.42−7.24 (m, 10 H), 6.81 (s, 1 H), 5.72 (s, 1 H), 5.37 (s, 2 H), 5.34 (s,

1 H), 5.33 (s, 2 H), 4.67 (dd, J = 1.8, 8.1 Hz, 1 H), 4.49−4.42 (m, 2 H),

4.15−4.00 (m, 3 H), 3.89 (dd, J = 4.8, 8.1 Hz 1 H), 1.97 (s, 3 H), 1.38 (s,

3 H), 1.34 (s, 3 H), 1.29 (s, 3 H), 1.24 (s, 3 H); ¹³C NMR (75 MHz,

CDCl₃) δ 167.2, 166.3, 166.2, 161.1, 140.1, 136.3, 128.3, 128.1, 128.0,

127.8, 127.4, 119.8, 109.6, 109.0, 97.0, 77.6, 75.4, 73.2, 69.3, 68.6, 67.8,

65.8, 27.5, 26.7, 25.2, 18.7; ESIMS m/z (rel. intensity) 606 (MH⁺, 100),

550 (59), 538 (21); HRMS m/z calcd for C₃₃H₄₀N₃O₈(MH⁺)

606.2851, found 606.2812. Anal. Calcd for C₃₃H₃₉N₃O₈: C, 65.44; H,

6.49; N, 6.94. Found: C, 65.51; H, 6.62; N, 7.09.

N - ( 2 , 4 - B i s ( b e n z y l o x y ) - 6 - ( ( 2 S , 3 R , 4 R ) - 2 , 3 , 4 , 5 -

tetrahydroxypentyloxy)pyrimidin-5-ylamino)methacrylamide

(45). HCl (0.5 N, 1 mL) was added to a solution of compound 44 (60

mg, 0.1 mmol) in MeOH (1 mL), and the reaction mixture was stirred

for 12 h at room temperature. MeOH was removed under vacuum, the

residue was then dissolved in ethyl acetate (10 mL), and the organic

layer was washed with water and brine, dried, and concentrated. The

residue was ﬂash column chromatographed with silica gel, eluting with

80% ethyl acetate in hexane, to provide compound 45 (36 mg, 69%) as a

colorless oil: ¹H NMR (300 MHz, MeOH-d₄) δ 7.40−7.27 (m, 10 H),

5.84 (s, 1 H), 5.48 (s, 1 H), 5.40 (s, 2 H), 5.38 (s, 2 H), 4.62 (dd, J = 3.0,

11.4 Hz, 1 H), 4.47 (dd, J = 6.6, 11.4 Hz, 1 H) 4.06−4.05 (m, 1 H),

3.79−3.77 (m, 2 H), 3.75−3.73 (m, 2 H), 1.99 (s, 3 H); ¹³C NMR (75

MHz, MeOH-d₄) δ 170.9, 168.5, 168.1, 163.0, 141.1, 138.1, 138.0, 129.5,

129.4, 129.1, 129.0, 128.6, 128.4, 121.7, 98.3, 74.1, 73.5, 72.0, 70.5, 70.0,

69.9, 64.6, 18.9; ESIMS m/z (rel. intensity) 526 (MH⁺, 100), 494 (24),

392 (42); negative ion ESIMS m/z (rel. intensity) 524 [(M − H⁺)⁻, 24],

391 (21), 390 (100). Anal. Calcd for C₂₇H₃₁N₃O₈: C, 61.70; H, 5.95; N,

8.00. Found: C, 61.91; H, 5.90; N, 7.88.

1

as a yellowish-white solid: mp 121−123 °C; H NMR (300 MHz,

CDCl₃) δ 7.38−7.25 (m, 10 H), 6.42 (s, 1 H), 5.31−5.28 (m, 4 H), 4.62

(dd, J = 2.4, 10.5 Hz, 1 H), 4.45−4.41 (m, 2 H), 4.04−3.98 (m, 3 H),

3.86−3.85 (m, 1 H), 2.25 (q, J = 7.4 Hz, 2 H), 1.34 (s, 3 H), 1.30 (s, 3 H),

1.25 (s, 3 H), 1.21 (s, 3 H), 1.14 (t, J = 7.2 Hz, 3 H); ¹³C NMR (125

MHz, CDCl₃) δ 172.6, 166.5, 166.2, 161.1, 136.3, 136.2, 128.7, 128.5,

128.4, 128.1, 127.9, 127.6, 127.5, 109.8, 109.2, 97.1, 77.7, 75.7, 73.4,

69.3, 68.7, 67.8, 65.9, 29.7, 27.7, 26.9, 25.2; ESIMS m/z (rel. intensity)

594 (MH⁺, 100), 554 (27), 245 (35); HRMS m/z calcd for C₃₂H₄₀N₃O₈

(MH⁺) 594.2815, found 594.2825. Anal. Calcd for C₃₂H₃₉N₃O₈: C,

64.74; H, 6.62; N, 7.08. Found: C, 64.88; H, 6.85; N, 6.81.

N - ( 2 , 4 - B i s ( b e n z y l o x y ) - 6 - ( ( 2 S , 3 R , 4 R ) - 2 , 3 , 4 , 5 -

tetrahydroxypentyloxy)pyrimidin-5-ylamino)propionamide

(42). HCl (0.5 N, 1 mL) was added to a solution of compound 41 (60

mg, 0.1 mmol) in MeOH (1 mL), and the reaction mixture was stirred

for 12 h at room temperature. MeOH was removed under vacuum, the

residue then dissolved in ethyl acetate (10 mL), and the organic layer

was washed with water and brine, dried, and concentrated. The residue

was ﬂash column chromatographed with silica gel, eluting with 80%

ethyl acetate in hexane, to aﬀord compound 42 (39 mg, 75%) as white

N-(2,4-Dioxo)-6-((2S,3R,4R)-2,3,4,5-tetrahydroxypentyloxy)-

1,2,3,4-tetrahydropyrimidin-5-yl)methacrylamide (46). Lindlar

catalyst (5 mg) was added to a solution of compound 45 (50 mg, 0.095

mmol) in anhydrous ethanol (4 mL). 1,4-Cyclohexadiene (52 μL, 0.95

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dx.doi.org/10.1021/jo3010364 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

mmol) was added, and argon was bubbled through the reaction mixture

for 10 min. The mixture was stirred at room temperature for 12 h. The

reaction mixture was ﬁltered through Celite, which was then washed

with ethanol (2 × 5 mL). The solution was concentrated, and the residue

was washed several times with CH₂Cl₂and THF. Finally, the product

was precipitated out by dissolving it in MeOH and adding excess diethyl

ether to furnish pure compound 46 (24 mg, 73%) as a white amorphous

solid: mp 170−172 °C (dec); ¹H NMR (300 MHz, MeOH-d₄) δ 5.87 (s,

1 H), 5.46 (s, 1 H), 4.50−4.41 (m, 1 H), 3.96−3.92 (m, 1 H), 3.73−3.71

(m, 3 H), 3.64−3.61 (m, 2 H), 2.01 (s, 3 H); ¹³C NMR (75 MHz,

MeOH-d₄) δ 167.3, 160.8, 149.7, 144.2, 140.0, 119.8, 75.5, 73.2, 69.1,

68.6, 67.9, 65.7, 18.5; ESIMS m/z (rel. intensity) 368 (MNa⁺, 100), 283

(13), 102 (27); negative ion ESIMS m/z (rel. intensity) 344 [M − H⁺)⁻,

100], 325 (3), 210 (5); HRMS m/z calcd for C₁₃H₁₉N₃O₈Na (MNa⁺)

368.1070, found 368.1073. Anal. Calcd for C₁₃H₁₉N₃O₈: C, 45.22; H,

5.55; N, 12.17. Found: C, 45.41; H, 5.27; N, 12.40.

N-(2,4-Dioxo-6-(((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi-

(1,3-dioxolan)-5-yl)methylthio)-1,2,3,4-tetrahydropyrimidin-5-

yl)propionamide (48). Propionic acid (13 μL, 0.18 mmol), 1-ethyl-3-

(3-dimethylaminopropyl) carbodiimide hydrochloride (23 mg, 0.12

mmol), and N-hydroxybenzotriazole monohydrate (16 mg, 0.12 mmol)

were added to a solution of compound 47 (40 mg, 0.12 mmol) dissolved

in pyridine (3.0 mL). The reaction mixture was stirred at rt for 12 h. The

solvent was distilled oﬀ under reduced pressure, and the residue was

dissolved in EtOAc (15 mL) and washed with water (2 × 15 mL). The

organic layer was dried, and solvent was distilled oﬀ. The residue was

puriﬁed by ﬂash column chromatography with silica gel, eluting with 5%

MeOH in ethyl acetate in hexane, to aﬀord compound 48 (30 mg, 65%)

as a colorless oil: ¹H NMR (300 MHz, MeOH-d₄) δ 4.45 (q, J = 6.1 Hz, 1

H), 4.20−4.07 (m, 3 H), 3.89 (dd, J = 4.7, 8.1 Hz, 1 H), 3.37−3.30 (m, 2

H), 2.38 (q, J = 7.6 Hz, 2 H), 1.46 (s, 3 H), 1.38 (s, 3 H), 1.35 (s, 3 H),

1.32 (s, 3 H), 1.19 (t, J = 7.6 Hz, 3 H); ¹³C NMR (75 MHz, MeOH-d₄) δ

177.1, 162.5, 152.6, 152.0, 111.3, 110.8, 79.9, 79.1, 74.5, 68.8, 33.7, 29.8,

27.9, 27.1, 25.6, 25.4, 10.2; ESIMS m/z (rel. intensity) 468 (MK⁺, 100),

452 (MNa⁺, 29), 430 (MH⁺, 5), 268 (13); negative ion ESIMS m/z (rel.

intensity) 428 [(M − H⁺)⁻, 100]; HRMS m/z calcd for

C₁₈H₂₇N₃O₇SNa (MNa⁺) 452.1467, found 452.1461. Anal. Calcd for

C₁₈H₂₇N₃O₇S: C, 50.34; H, 6.34; N, 9.78; S, 7.47. Found: C, 50.46; H,

6.49; N, 9.81; S, 7.61.

5.48 (s, 1 H), 4.45 (q, J = 6.1 Hz, 1 H), 4.18−4.07 (m, 3 H), 3.89 (dd, J =

4.7, 8.1 Hz, 1 H), 3.38−3.34 (m, 2 H), 2.15 (s, 3 H), 1.46 (s, 3 H), 1.37

(s, 3 H), 1.35 (s, 3 H), 1.32 (s, 3 H); ¹³C NMR (75 MHz, MeOH-d₄) δ

170.9, 162.5, 153.1, 152.1, 140.8, 129.2, 121.9, 111.3, 110.8, 79.9, 79.0,

74.5, 68.8, 33.7, 27.9, 27.1, 25.6, 25.4, 18.8; ESIMS m/z (rel. intensity)

464 (MNa⁺, 64), 442 (MH⁺, 100), 166 (26); negative ion ESIMS m/z

(rel. intensity) 440 [(M − H⁺)⁻, 100]; HRMS m/z calcd for

C₁₉H₂₈N₃O₇S (MH⁺) 442.1648, found 442.1655. Anal. Calcd for

C₁₉H₂₇N₃O₇S: C, 51.69; H, 6.16; N, 9.52; S, 7.26. Found: C, 51.92; H,

5.91; N, 9.60; S, 7.02.

N-(2,4-Dioxo-6-((2R,3R,4R)-2,3,4,5-tetrahydroxypentylthio)-

1,2,3,4-tetrahydropyrimidin-5-yl)methacrylamide (51). HCl (1

N, 2 mL) was added to compound 50 (30 mg, 0.068 mmol) dissolved in

MeOH (2 mL). The reaction mixture was stirred for 8 h at room

temperature. The solution was concentrated, and the residue was

washed several times with CH₂Cl₂(3 × 10 mL) and THF (3 × 10 mL).

Finally, compound 51 was precipitated out by dissolving in MeOH and

adding excess diethyl ether to furnish pure compound 51 (19 mg, 78%)

1

as a white amorphous solid: mp 140−143 °C (dec); H NMR (300

MHz, MeOH-d₄) δ 5.88 (s, 1 H), 5.49 (s, 1 H), 3.99 (q, J = 6.1 Hz, 1 H),

3.78−368 (m, 2 H), 3.65−3.59 (m, 2 H), 3.30−3.27 (m, 2 H), 2.0 (s, 3

H); ¹³C NMR (125 MHz, MeOH-d₄) δ 171.2, 163.8, 154.1, 141.0,

129.9, 121.7, 108.6, 74.7, 74.4, 74.0, 64.7, 35.9, 19.0; ESIMS m/z (rel.

intensity) 745 (2MNa⁺, 100), 384 (MNa⁺, 48), 361 (MH⁺, 14); negative

ion ESIMS m/z (rel. intensity) 360 [(M − H⁺)⁻, 100]; HRMS m/z calcd

for C₁₃H₂₀N₃O₇S (MH⁺) 361.1022, found 361.1025. Anal. Calcd for

C₁₃H₁₉N₃O₇S: C, 43.21; H, 5.30; N, 11.63; S, 8.87. Found: C, 43.01; H,

5.44; N, 11.49; S, 9.03.

2,6-Dimethoxy-5-nitro-N-((2S,3S,4R)-2,3,4,5-tetrakis(tert-bu-

tyldimethylsilyloxy)-pentyl)pyrimidine-4-amine (53).⁵⁰Com-

pound 52 (5.2854 g, 15.8 mmol), tert-butyldimethylsilyl chloride

(16.55 g, 109.8 mmol), and imidazole (7.49 g, 109.8 mmol) were

dissolved in dry DMF (115 mL), and the mixture was stirred at room

temperature for 2 days. DMF was then removed under reduced pressure,

and the residue was stirred in ethyl acetate (90 mL) for 2 h. The

precipitated salt was ﬁltered, and the ﬁltrate was concentrated to form a

yellow oil, which was separated by silica gel ﬂash column

chromatography, eluting with hexane/EtOAc (95:5), to aﬀord pure

53 (6.95 g, 57%) as a light yellow oil: ¹H NMR (300 MHz, CDCl₃) δ

9.00 (s, 1 H), 4.10−4.09 (m, 1 H), 4.06 (s, 3 H), 3.96 (s, 3 H), 3.93−3.88

(m, 2 H), 3.83−3.80 (dd, J = 2.57 Hz, J = 5.99 Hz, 1 H), 3.72−3.67 (dd, J

= 5.16 Hz, J = 10.32 Hz, 1 H), 3.56−3.51 (m, 2 H), 0.87 (s, 9 H), 0.86 (s,

9 H), 0.85 (s, 9 H), 0.84 (s, 9 H), 0.11 (s, 3 H), 0.08 (s, 3 H), 0.06 (s, 6

H), 0.04 (s, 3 H), 0.03 (s, 6 H), −0.04 (s, 3 H); ESIMS (MH⁺) m/z 791.

Anal. Calcd for C₃₅H₇₄N₄O₈Si₄: C, 53.12; H, 9.43; N, 7.08; Si, 14.20.

Found: C, 52.89, H, 9.21; N, 7.34; Si, 13.93.

N-(2,4-Dioxo-6-((2R,3R,4R)-2,3,4,5-tetrahydroxypentylthio)-

1,2,3,4-tetrahydropyrimidin-5-yl)propionamide (49). HCl (1 N,

2 mL) was added to compound 48 (30 mg, 0.068 mmol) dissolved in

MeOH (2 mL). The reaction mixture was stirred for 8 h at room

temperature. The solution was concentrated, and the residue was

washed several times with CH₂Cl₂(3 × 10 mL) and THF (3 × 10 mL).

Finally, compound 49 was precipitated out by dissolving it in MeOH

and adding excess diethyl ether to furnish pure compound 49 (17 mg,

2,6-Dimethoxy-N⁴-((2S,3S,4R)-2,3,4,5-tetrakis(tert-butyldi-

methylsilyloxy)-pentyl)pyrimidine-4,5-diamine (54). Compound

53 (6.9 g, 8.73 mmol) was added to a methanol (175 mL) and water (10

mL) mixture. Na₂S₂O₄(11.4 g, 65.4 mmol) was added to the reaction

mixture. The mixture was heated at reﬂux and stirred for 6 h. The

reaction mixture was then cooled to room temperature. The solution

was ﬁltered through Celite, and the ﬁltrate was dried to aﬀord a residue.

The residue was separated by silica gel ﬂash column chromatography

with hexane/ethyl acetate 20:1 as the mobile phase to aﬀord the product

1

70%) as a white amorphous solid: mp 137−140 °C (dec); H NMR

(300 MHz, MeOH-d₄) δ 3.97−3.88 (m, 1 H), 3.78−3.72 (m, 3 H),

3.67−3.56 (m, 3 H), 2.35 (q, J = 7.5 Hz, 2 H), 1.19 (t, J = 7.5 Hz, 3 H);

¹³C NMR (125 MHz, CDCl₃) δ 177.1, 169.5, 162.6, 158.2, 104.8, 73.3,

63.1, 33.3, 29.4, 9.2; ESIMS m/z (rel. intensity) 721 (2MNa⁺, 94), 372

(MNa⁺, 100), 350 (MH⁺, 5); negative ion ESIMS m/z (rel. intensity)

348 [(M − H⁺)⁻, 100]; HRMS m/z calcd for C₁₂H₁₉N₃O₇SNa (MNa⁺)

372.0841, found 372.0843. Anal. Calcd for C₁₂H₁₉N₃O₇S: C, 41.25; H,

5.48; N, 12.03; S, 9.18. Found: C, 41.31; H, 5.48; N, 12.21; S, 9.31.

N-(2,4-Dioxo-6-(((4S,4′R,5R)-2,2,2′,2′-tetramethyl-4,4′-bi-

(1,3-dioxolan)-5-yl)methylthio)-1,2,3,4-tetrahydropyrimidin-5-

yl)methacrylamide (50). Methacrylic acid (13 μL, 0.15 mmol), 1-

ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (20 mg,

0.10 mmol), and N-hydroxybenzotriazole monohydrate (14 mg, 0.10

mmol) were added to a solution of compound 47 (35 mg, 0.10 mmol)

dissolved in pyridine (3.0 mL). The reaction mixture was stirred at room

temperature for 12 h. The solvent was distilled oﬀ under reduced

pressure, and the residue was dissolved in EtOAc (15 mL) and washed

with water (2 × 15 mL). The organic layer was dried, and solvent was

distilled oﬀ. The residue was puriﬁed by ﬂash column chromatography

with silica gel, eluting with 5% MeOH in ethyl acetate in hexane, to

aﬀord compound 50 (28 mg, 68%) as a white amorphous solid: mp 95−

98 °C; ¹H NMR (300 MHz, MeOH-d₄) δ 5.89 (s, 1 H), 5.52 (s, 1 H),

1

54 as unstable yellow oil (6.24 g, 94%) that soon turned purple: H

NMR (300 MHz, CDCl₃) δ 4.03−3.98 (m, 1 H), 3.93−3.89 (m, 1 H),

3.90 (s, 3 H), 3.85 (s, 3 H), 3.84−3.80 (m, 1 H), 3.76−3.64 (m, 2 H),

3.57−3.45 (m, 2 H), 0.88−0.86 (m, 36 H), 0.10−0.00 (m, 24 H); ¹³C

NMR (75 MHz, CDCl₃) δ 161.2, 159.6, 159.1, 100.8, 75.0, 71.7, 64.7,

54.1, 53.6, 43.0, 31.6, 29.6, 26.1, 25.9, 25.3, 22.6, 18.4, 18.3, 18.2, 18.0,

14.1, −4.1, −4.3, −4.6, −4.7, −4.9, −5.3, −5.4; ESIMS (MH⁺) m/z 761.

Anal. Calcd for C₃₅H₇₆N₄O₆Si₄: C, 55.21; H, 10.06; N, 7.36; Si, 14.76.

Found: C, 55.58; H, 10.20; N, 7.14; Si, 14.40.

N-(2,4-Dimethoxy-6-((2S,3S,4R)-2,3,4,5-tetrakis(tert-butyldi-

methylsilyloxy)-pentylamino)pyrimidine-5-yl)methacrylamide

(55). Compound 54 (3.28 g, 4.32 mmol) was mixed with THF (30 mL).

Triethylamine (6.0 mL, 43.3 mmol) was added to the THF solution.

The mixture was cooled to 0 °C, and methacryloyl chloride (678 mg,

6.48 mmol) was added dropwise. The reaction mixture was stirred at 0

S

dx.doi.org/10.1021/jo3010364 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

°C for 0.5 h. The reaction mixture was warmed to room temperature and

stirred for 20 min. The mixture was dried in vacuo to get a residue, and

that was puriﬁed by silica gel ﬂash column chromatography with

hexane/ethyl acetate 20:1 as the mobile phase to aﬀord the product 55

as a colorless oil (2.53 g, 71%): ¹H NMR (300 MHz, CDCl₃) δ 5.84 (s, 1

H), 5.58 (brs, 1 H), 5.45 (s, 1 H), 3.95 −3.91 (m, 2 H), 3.89 (s, 3 H),

3.88 (s, 3 H), 3.78−3.68 (m, 3 H), 3.57−3.51 (m, 1 H), 3.44−3.41 (m, 1

H), 2.06 (s, 3 H), 0.87−0.83 (m, 36 H), 0.08- −0.05 (m, 24 H); ¹³C

NMR (75 MHz, CDCl₃) δ 166.8, 164.3, 162.3, 139.4, 120.9, 94.5, 74.6,

71.6, 64.6, 54.4, 54.0, 43.3, 31.9, 31.6, 29.7, 26.1, 25.9, 22.6, 18.7, 18.4,

18.3, 18.2, 18.0, 14.1, −4.0, −4.3, −4.4, −4.6, −5.2, −5.3, −5.4; ESIMS

(MH⁺) m/z 829. Anal. Calcd for C₃₉H₈₀N₄O₇Si₄: C, 56.48; H, 9.72; N,

6.75; Si, 13.54. Found: C, 56.83; H, 9.40; N, 6.38; Si, 13.21.

N-[2,4-Dioxo-6-((2S,3S,4R)-2,3,4,5-tetrahydroxypentylami-

no]-1,2,3,4-tetrahydropyrimidin-5-yl)methacrylamide (56).

Compound 55 (0.7 g, 0.85 mmol) was dissolved in a solution made

from 48% HBr/H₂O (2:1, 30 mL) and MeOH (30 mL), and the mixture

was stirred at 55−60 °C for 3 h. The solvent was removed in vacuo, the

residue was dissolved in MeOH (10 mL), and ethyl ether (60 mL) was

added. After 24 h in the refrigerator, the precipitate was ﬁltered out as a

light red solid, which was dissolved in water (60 mL), decolorized with

active charcoal, and ﬁltered. The solvent was removed from the ﬁltrate,

and the residue was puriﬁed by silica gel ﬂash column chromatography

(EtOAc/EtOH/H₂O/AcOH 67:23:9:1) twice and Sephadex LH 20

column twice with MeOH to remove silica gel and other inorganic

impurities and provide the product 56 (100 mg, 34%) as a white

semisolid powder: ¹H NMR (300 MHz, D₂O) δ 5.88 (s, 1 H), 5.55 (s, 1

H), 3.87−3.47 (m, 7 H), 1.95 (s, 3 H); ¹³C NMR (125 MHz, D₂O) δ

172.4, 162.6, 153.6, 151.3, 138.1, 123.1, 86.3, 72.2, 72.1, 70.3, 62.3, 44.1,

17.7; ESIMS m/z (rel. intensity) 389 [(M2Na⁺− H⁺)⁺, 100], 367

(MNa⁺, 76); HRMS calcd for C₁₃H₂₀N₄O₇Na (MNa⁺) 367.1230, found

367.1226. Anal. Calcd for C₁₃H₂₀N₄O₇·1.5 H₂O·1 MeOH: C, 41.68; H,

6.75; N, 13.89. Found: C, 41.27; H, 6.35; N, 14.19.

N-(2,4-Bis(benzyloxy)-6-((2S,3S,4R)-2,3,4,5-tetrakis(tert-

butyldimethylsilyloxy)pentylamino)pyrimidin-5-yl)-2-chloro-

propanamide (58). Compound 57 (210 mg, 0.23 mmol) was mixed

with THF (5 mL). TEA (0.3 mL, 2.1 mmol) was added to the THF

solution. The mixture was cooled to 0 °C, and 2-chloropropanoyl

chloride (34 μL, 44 mg, 0.34 mmol) was added dropwise. The reaction

mixture was stirred at 0 °C for 0.5 h. The mixture was dried in vacuo to

yield a residue, and that was puriﬁed by silica gel ﬂash column

chromatography to provide the product 58 as a yellow oil (200 mg,

87%): ¹H NMR (300 MHz, CDCl₃) δ 7.48−7.26 (m, 10 H), 5.42−5.35

(m, 4 H), 4.08−3.65 (m, 7 H), 1.77−1.75 (d, J = 6.7 Hz, 3 H), 0.93−0.90

(m, 36 H), 0.15−0.01 (m, 24 H); ¹³C NMR (75 MHz, CDCl₃) δ 168.9,

164.1, 162.1, 162.1, 160.3 160.2, 137.0, 136.5, 128.3, 128.2, 128.0, 127.8,

127.7, 127.6, 93.5, 93.4, 76.6, 74.5, 71.3, 68.8, 68.3, 64.5, 60.3, 55.7, 43.2,

26.0, 25.9, 22.7, 22.6, 18.4, 18.2, 18.1, 17.9, 14.1; ESIMS m/z (rel.

intensity) 1005.32 [M(Cl³⁷)H⁺, 64.10], 1003.34 [M(Cl³⁵)H⁺, 100];

HRMS calcd for C₅₀H₈₈ClN₄O₇Si₄(MH⁺) 1003.5419, found

1003.5408. Anal. Calcd for C₅₀H₈₇ClN₄O₇Si₄: C, 59.81; H, 8.73; N,

5.58; Si, 11.19. Found: C, 60.03; H, 8.97; N, 5.61; Si, 11.25.

2-Chloro-N-(2,4-dioxo-6-((2S,3S,4R)-2,3,4,5-tetrahydroxy-

pentylamino)-1,2,3,4-tetrahydropyrimidin-5-yl)propanamide

(60). Lindlar catalyst (5 mg) was added to a solution of compound 59

(50 mg, 0.13 mmol) in anhydrous ethanol (4 mL). 1,4-Cyclohexadiene

(0.1 mL, 1.3 mmol) was added, and argon was bubbled through the

reaction mixture for 10 min. The mixture was stirred at room

temperature for 12 h. The reaction mixture was ﬁltered through Celite,

which was then washed with ethanol (2 × 5 mL). The solution was

concentrated, and the residue was washed several times with CH₂Cl₂

and THF. Finally, compound 60 was precipitated out by dissolving it in

MeOH and adding excess diethyl ether to furnish pure compound 60

(18 mg, 66%) as a white amorphous solid: mp 208−211 °C (dec); ¹H

NMR (300 MHz, MeOH-d₄) δ 4.51 (q, J = 7.0, 13.7 Hz, 1 H), 3.75−3.72

(m, 1 H), 3.67−3.61 (m, 2 H), 3.54−3.46 (m, 2 H), 3.23−3.19 (m, 2 H),

1.18 (t, J = 7.6 Hz, 3 H); ¹³C NMR (125 MHz, MeOH-d₄) δ 172.1,

161.8, 153.2, 150.7, 86.2, 72.9, 72.2, 71.5, 63.0, 54.0, 44.6, 20.6; ESIMS

m/z (rel. intensity) 369 [M(Cl³⁷)H⁺, 32], 366 [M(Cl³⁵)H⁺, 100];

HRMS m/z calcd for C₁₂H₂₀ClN₄O₇(MH⁺) 367.1021, found 367.1023.

N-(2,4-Bis(benzyloxy)-6-((2S,3S,4R)-2,3,4,5-tetrakis(tert-

butyldimethylsilyloxy)pentylamino)pyrimidin-5-yl)-2-chloroa-

cetamide (61). Compound 57 (0.7 g, 0.77 mmol) was mixed with THF

(20 mL). TEA (1 mL, 6.9 mmol) was added to the THF solution. The

mixture was cooled to 0 °C, and 2-chloroacetyl chloride (88 μL, 121 mg,

1.1 mmol) was added dropwise. The reaction mixture was stirred at 0 °C

for 0.5 h. The mixture was dried in vacuo to aﬀord a residue that was

puriﬁed by silica gel ﬂash chromatography to provide the product 61 as a

yellow oil (0.7 mg, 92%): ¹H NMR (300 MHz, CDCl₃) δ 7.57 (s, 1 H),

7.45−7.26 (m, 10 H), 5.44−5.33 (m, 4 H), 4.14 (s, 2 H), 3.99−3.47 (m,

7 H), 0.92−0.88 (m, 36 H), 0.14−0.00 (m, 24 H); ¹³C NMR (75 MHz,

CDCl₃) δ 165.0, 164.0, 162.2, 160.1, 137.0, 136.6, 128.4, 128.3, 128.1,

127.9, 127.8, 127.5, 93.2, 74.5, 71.4, 68.9, 68.2, 64.4, 43.1, 42.5, 26.1,

26.1, 25.9, 18.4, 18.3, 18.2, 18.0, −4.0, −4.4, −4.4, −4.6, −5.2, −5.3,

−5.4; ESIMS m/z (rel. intensity) 991 [M(Cl³⁷)H⁺, 63], 989

[M(Cl³⁵)H⁺, 100]; HRMS m/z calcd for C₄₉H₈₆ClN₄O₇Si₄(MH⁺)

989.5262, found 989.5269. Anal. Calcd for C₄₉H₈₅ClN₄O₇Si₄: C, 59.45;

H, 8.65; N, 5.66; Si, 11.35. Found: C, 59.69; H, 8.73; N, 5.47; Si, 11.07.

N - ( 2 , 4 - B i s ( b e n z y l o x y ) - 6 - ( ( 2 S , 3 S , 4 R ) - 2 , 3 , 4 , 5 -

tetrahydroxypentylamino)pyrimidin-5-yl)-2-chloroacetamide

(62). Compound 61 (40 mg, 0.04 mmol) was mixed with THF (2 mL).

TBAF (1.0 M tetra-n-butylammonium ﬂuoride in THF, 0.2 mmol, 0.2

mL) was added to the reaction mixture. The mixture was stirred at room

temperature for 4 h and then dried in vacuo to produce a residue. The

residue was puriﬁed by silica gel preparative TLC (solvent system:

EtOAc/MeOH 20:1) to yield a white solid 62 (17 mg, 79%): mp 130−

131 °C; ¹H NMR (300 MHz, MeOH-d₄) δ 7.29−7.15 (m, 10 H), 5.25

(d, J = 15.0 Hz, 2 H), 5.18 (d, J = 15.2 Hz, 2 H), 4.09 (s, 2 H), 3.77−3.67

(m, 1 H), 3.66−3.40 (m, 6 H); ¹³C NMR (75 MHz, MeOH-d₄) δ 169.9,

166.3, 163.8, 163.1, 138.5, 138.4, 129.4, 129.0, 128.8, 128.5, 94.0, 74.4,

74.2, 72.9, 70.0, 69.2, 64.6, 44.8, 43.3; ESIMS m/z (rel. intensity) 535

[M(Cl³⁷)H⁺, 36], 533 [M(Cl³⁵)H⁺, 100]; HRMS m/z calcd for

C₂₅H₃₀ClN₄O₇(MH⁺) 533.1798, found 533.1803. Anal. Calcd for

C₂₅H₂₉ClN₄O₇: C, 56.34; H, 5.48; N, 10.51. Found: C, 56.41; H, 5.61;

N, 10.79.

N - ( 2 , 4 - B i s ( b e n z y l o x y ) - 6 - ( ( 2 S , 3 S , 4 R ) - 2 , 3 , 4 , 5 -

tetrahydroxypentylamino)pyrimidin-5-yl)-2-chloropropana-

mide (59). Compound 58 (144 mg, 0.14 mmol) was mixed with THF

(7 mL). TBAF (1.0 M tetra-n-butylammonium ﬂuoride in THF, 0.65

mmol, 0.65 mL) was added to the reaction mixture. The mixture was

stirred at room temperature for 4 h and then dried in vacuo to aﬀord a

residue. The residue was puriﬁed by silica gel preparative TLC (solvent

system: EtOAc/MeOH 20:1) to give a white solid 59 (65 mg, 83%): mp

132−134 °C; ¹H NMR (300 MHz, MeOH-d₄) δ 7.33−7.17 (m, 10 H),

5.24−5.21 (d, J = 8.2 Hz, 4 H), 4.53−4.46 (q, J = 6.8, 13.7 Hz, 1 H),

3.80−3.42 (m, 7 H), 1.55−1.53 (d, J = 6.9 Hz, 3 H); ¹³C NMR (75 MHz,

MeOH-d₄) δ 172.9, 166.3, 163.8, 163.0, 138.5, 138.3, 129.4, 129.4,

129.0, 128.95, 128.9, 128.6, 94.1, 74.4, 74.2, 72.8, 70.0, 69.3, 64.6, 55.2,

44.8, 22.2; ESIMS m/z (rel. intensity) 549 [M(Cl³⁷)H⁺, 34], 547

[M(Cl³⁵)H⁺, 100]; HRMS calcd for C₂₆H₃₂ClN₄O₇(MH⁺) 547.1960,

found 547.1959. Anal. Calcd for C₂₆H₃₁ClN₄O₇: C, 57.09; H, 5.71; N,

10.24. Found: C, 56.85; H, 5.49; N, 10.45.

2-Chloro-N-(2,4-dioxo-6-((2S,3S,4R)-2,3,4,5-tetrahydroxy-

pentylamino)-1,2,3,4-tetrahydropyrimidin-5-yl)acetamide

(63). Compound 62 (40 mg, 0.08 mmol) was mixed with anhydrous

ethanol (5 mL). 1,4-Cyclohexadiene (130 mg, 1.6 mmol) and

Pearlman’s catalyst (40 mg, 20% Pd(OH)₂on carbon) were added to

the reaction mixture. The mixture was stirred at room temperature

under argon for 18 h and then ﬁltered. The ﬁltrate was then dried in

vacuo to yield a residue. The residue was puriﬁed by prep TLC (solvent

system: EtOAc/EtOH/H₂O 15:5:2) to provide a white solid 63 (17 mg,

1

64%): mp 173−174 °C; H NMR (500 MHz, D₂O) δ 4.25 (s, 2 H),

3.85−3.81 (m, 1 H), 3.75−3.69 (m, 2 H), 3.65−3.44 (m, 4 H); ¹³C

NMR (125 MHz, D₂O) δ 171.3, 162.5, 155.8, 153.2, 85.8, 72.19, 72.1,

70.5, 62.3, 43.8, 42.2; ESIMS m/z (rel. intensity) 353 [(M(Cl³⁷) −

H⁺)⁻, 24,], 351 [(M(Cl³⁵) − H⁺)⁻, 100,]; HRMS calcd for

C₁₁H₁₆ClN₄O₇(M − H⁺)⁻351.0708, found 351.0707. Anal. Calcd

for C₁₁H₁₇ClN₄O₇: C, 37.46; H, 4.86; N, 15.88. Found: C, 37.35; H,

4.61; N, 16.04.

T

dx.doi.org/10.1021/jo3010364 | J. Org. Chem. XXXX, XXX, XXX−XXX

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Article

N-(2,4-Bis(benzyloxy)-6-((2S,3S,4R)-2,3,4,5-tetrakis(tert-bu-

tyldimethylsilyloxy)-pentylamino)pyrimidin-5-yl)-3,3,3-tri-

ﬂuoropropanamide (64). Compound 57 (0.58 mg, 0.64 mmol) was

mixed with THF (15 mL). TEA (0.9 mL, 6.9 mmol) was added to the

THF solution. The mixture was cooled to 0 °C, and 3,3,3-

triﬂuoropropanoyl chloride (65 μL, 0.64 mmol) was added dropwise.

The reaction mixture was stirred at 0 °C for 0.5 h. The mixture was dried

in vacuo to provide a residue that was puriﬁed by silica gel ﬂash

chromatography to yield the product 64 as a yellow oil (0.49 mg, 75%):

¹H NMR (300 MHz, DMSO-d₆) δ 7.45−7.29 (m, 10 H), 5.78 (s, 1 H),

5.44−5.28 (m, 4 H), 4.03−4.01 (m, 1 H), 3.87−3.89 (m, 1 H), 3.76−

3.69 (m, 2 H), 3.58−3.50 (m, 3 H), 3.40−3.37 (m, 2 H), 0.90−0.83 (m,

36 H), 0.13−0.02 (m, 24 H); ¹³C NMR (75 MHz, CDCl₃) δ 164.11,

162.1, 161.7, 160.2, 136.9, 136.3, 128.6, 128.4, 128.2, 128.0, 127.8, 127.7,

129.3−118.3 (q, J = 275.6 Hz), 93.4, 74.7, 71.2, 69.2, 68.9, 68.4, 64.5,

43.3, 42.2−41.1 (q, J = 29.3 Hz), 26.0, 25.8, 18.3, 18.2, 18.1, 17.9, −4.1,

−4.3, −4.4, −4.5, −4.7, −4.7, −5.3, −5.4, −5.5; ¹⁹F NMR (300 MHz,

CDCl₃) δ 16.9 (t, J = 11.7 Hz, 3 F); HRMS calcd for C₅₀H₈₆F₃N₄O₇Si₄

(MH⁺) 1023.5526, found 1023.5519. Anal. Calcd for C₅₀H₈₅F₃N₄O₇Si₄:

C, 58.67; H, 8.37; N, 5.47; Si, 10.98. Found: C, 58.83; H, 8.08; N, 5.40;

Si, 11.15.

N - ( 2 , 4 - B i s ( b e n z y l o x y ) - 6 - ( ( 2 S , 3 S , 4 R ) - 2 , 3 , 4 , 5 -

tetrahydroxypentylamino)pyrimidin-5-yl)-3,3,3-triﬂuoropro-

panamide (65). Compound 64 (131 mg, 0.13 mmol) was mixed with

THF (6 mL). TBAF (1.0 M tetra-n-butylammonium ﬂuoride in THF,

0.58 mmol, 0.58 mL) was added to the reaction mixture. The mixture

was stirred at room temperature for 4 h and then dried in vacuo to aﬀord

a residue. The residue was puriﬁed by silica gel preparative TLC (solvent

system: EtOAc/MeOH 20:1) to produce a white solid 65 (61 mg, 84%):

mp 137−138 °C; ¹H NMR (300 MHz, MeOH-d₄) δ 7.32−7.17 (m, 10

H), 5.25−5.23 (m, 4 H), 3.88−3.86 (m, 1 H), 3.78- 3.53 (m, 6 H), 3.37

−3.30 (m, 2 H); ¹³C NMR (75 MHz, MeOH-d₄) δ 166.3, 166.2, 163.8,

163.0, 138.5, 138.3, 129.4, 129.4, 129.0, 128.8, 128.6, 131.3−120.3 (q, J

= 274.4 Hz), 94.0, 74.4, 74.2, 72.9, 70.0, 69.3, 64.6, 54.7, 44.8, 41.7−40.6

(q, J = 29.4 Hz); ¹⁹F NMR (300 MHz, CDCl₃) δ 11.3 (t, J = 11.5 Hz, 3

F); HRMS calcd for C₂₆H₂₉F₃N₄O₇Na (MNa⁺) 589.1886, found

589.1889. Anal. Calcd for C₂₆H₂₉F₃N₄O₇: C, 55.12; H, 5.16; N, 9.89.

Found: C, 54.93; H, 5.23; N, 10.07.

N-(2,4-Dioxo-6-((2S,3S,4R)-2,3,4,5-tetrahydroxypentylami-

no)-1,2,3,4-tetrahydropyrimidin-5-yl)-3,3,3-triﬂuoropropana-

mide (66). Compound 65 (30 mg, 0.05 mmol) was mixed with

anhydrous ethanol (3 mL). 1,4-Cyclohexadiene (80 mg, 1.0 mmol) and

Pearlman’s catalyst (30 mg, 20% Pd(OH)₂on carbon) were added to

the reaction mixture. The mixture was stirred at room temperature

under argon for 24 h and then ﬁltered. The ﬁltrate was then dried in

vacuo to yield a residue. The residue was puriﬁed by prep TLC (solvent

system: EtOAc/EtOH/H₂O 15:5:2) to aﬀord a white solid 66 (15 mg,

72%): mp 189−190 °C; ¹H NMR (500 MHz, D₂O) δ 3.84−3.83 (m, 1

H), 3.77−3.69 (m, 2 H), 3.61−3.54 (m, 4 H) 3.50−3.42 (m, 2 H); ¹³C

NMR (125 MHz, D₂O) δ 167.9, 162.4, 154.9, 152.5, 123.9 (q, J = 274.1

Hz), 85.9, 72.1, 70.3, 62.3, 43.8, 39.6 (q, J = 29.1 Hz); ¹⁹F NMR (300

MHz, DMSO-d₆) δ 12.10 (t, J = 11.7 Hz, 3 F); HRMS (ESI) Calcd for

C₁₂H₁₆F₃N₄O₇(M − H⁺)⁻385.0971, found 385.0976. Anal. Calcd for

C₁₂H₁₇F₃N₄O₇: C, 37.31; H, 4.44; N, 14.50. Found: C, 37.33; H, 4.67;

N, 14.39.

127.7, 93.4, 74.5, 71.4, 68.9, 68.3, 64.5, 43.4, 26.14, 26.08, 25.9, 24.3,

18.4, 18.3, 18.2, 18.0, −4.0, −4.4, −4.5; ESIMS m/z (rel. intensity) 983

(MH⁺, 100), 965 (11), 719 (16); negative ion ESIMS m/z (rel.

intensity) 981 [(M − H⁺)⁻, 100], 911 (65), 847 (35); HRMS m/z calcd

for C₅₀H₈₆N₄O₈Si₄(MH⁺) 983.5601, found 983.5609.

2,4-Bis(benzyloxy)-7-hydroxy-7-methyl-8-[(2S,3S,4R)-

2,3,4,5-tetrahydroxypentyl]-7,8-dihydropteridin-6(5H)-one

(68). Compound 67 (60 mg, 0.06 mmol) was dissolved in THF (3 mL),

and TBAF (0.07 mL, 0.36 mmol, 1 M solution in THF) was added. The

reaction mixture was stirred at room temperature for 8 h. THF was

removed under vacuum. The residue was extracted with ethyl acetate (3

× 10 mL), washed with water (2 × 10 mL), dried with anhydrous

Na₂SO₄, and concentrated under reduced pressure. The residue was

column chromatographed with ﬂash silica gel, eluting with 5% MeOH in

ethyl acetate, to aﬀord compound 68 (22 mg, 69%) as an oil: ¹H NMR

(300 MHz, MeOH-d₄) δ 8.21 (brs, 1 H), 7.43−7.28 (m, 10 H), 5.43

(ABq, J = 8.4, 15.6 Hz, 2 H), 5.36 (s, 2 H), 4.18−4.10 (m, 1 H), 3.92−

3.80 (m, 2 H), 3.81−3.51 (m, 4 H), 1.39 (s, 3 H); ¹³C NMR (75 MHz,

MeOH-d₄) δ 164.8, 170.0, 157.5, 150.6, 138.4, 137.9, 129.50, 129.46,

129.2, 128.94, 128.88, 126.1, 99.4, 90.8, 79.0, 74.2, 71.5, 70.1, 69.6, 64.7,

45.9, 24.5; ESIMS m/z (rel. intensity) 509 (MH⁺− H₂O, 56), 202

(100); negative ion ESIMS m/z (rel. intensity) 525 [(M − H⁺)⁻, 71],

507 [(M − H⁺− H₂O)⁻, 100]; HRMS m/z calcd for C₂₆H₃₀N₄O₈(M −

H⁺)⁻525.1985, found 525.1984.

7-Hydroxy-7-methyl-8-[(2S,3S,4R)-2,3,4,5-tetrahydroxypen-

tyl)-7,8-dihydropteridin-2,4,6(1H,3H,5H)-trione (69). Lindlar cat-

alyst (5 mg) was added to a solution of compound 68 (20 mg, 0.04

mmol) in MeOH/EtOAc (3 + 1 mL). A hydrogen balloon was attached,

and the mixture was stirred at room temperature for 12 h. The reaction

mixture was ﬁltered through Celite, which was then washed with 50% aq

MeOH (5 mL). The solution was concentrated, and the residue was

washed several times with CH₂Cl₂(3 × 10 mL) and THF (3 × 10 mL).

Finally, compound 69 was precipitated out by dissolving in MeOH and

adding excess diethyl ether to furnish pure compound 69 (10 mg, 77%)

1

as a white amorphous solid: mp 180−183 °C (dec); H NMR (300

MHz, MeOH-d₄) δ 4.20−4.17 (m, 1 H), 4.07−4.01 (m, 1 H), 3.87−3.84

(m, 1 H), 3.77−3.74 (m, 1 H), 3.66−3.54 (m, 3 H), 1.40 (s, 3 H); ¹³C

NMR (125 MHz, MeOH-d₄) δ 164.2, 159.0, 152.5, 141.1, 95.4, 91.9,

79.8, 74.3, 71.2, 64.8, 48.2, 23.8; negative ion ESIMS m/z (rel. intensity)

345 [(M − H⁺)⁻, 10], 327 [(M − H⁺− H₂O)⁻, 100]; negative ion

HRMS m/z calcd for C₁₂H₁₇N₄O₈(M − H⁺)⁻345.1046, found

345.1051. Anal. Calcd for C₁₂H₁₈N₄O₈·1.75H₂O: C, 38.15; H, 5.74; N,

14.83. Found: C, 37.82; H, 5.79; N, 14.42.

N-(6-Chloro-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-

methacrylamide (78). Lindlar catalyst (5 mg) was added to a solution

of compound 80 (40 mg, 0.097 mmol) in anhydrous ethanol (4 mL).

1,4-Cyclohexadiene (53 μL, 0.97 mmol) was added, and argon was

bubbled through the reaction mixture for 10 min. The mixture was

stirred at room temperature for 12 h. The reaction mixture was ﬁltered

through Celite, which was then washed with ethanol (2 × 5 mL). The

solution was concentrated. The residue was washed several times with

CH₂Cl₂and THF. Finally, compound 78 was precipitated out by

dissolving it in MeOH and adding excess diethyl ether to furnish pure

compound 78 (14 mg, 63%) as a white amorphous solid: mp 234−236

°C; ¹H NMR (300 MHz, MeOH-d₄) δ 5.89 (s, 1 H), 5.53 (s, 1 H), 1.99

(s, 3 H); ¹³C NMR (75 MHz, MeOH-d₄) δ 170.9, 162.8, 151.3, 146.0,

140.6, 122.2, 110.4, 18.7; ESIMS m/z (rel. intensity) 252 (MNa⁺, 100),

230 (MH⁺, 4); negative ion ESIMS m/z (rel. intensity) 228 [(M − H⁺)⁻,

100], 192 (32); HRMS m/z calcd for C₈H₈ClN₃O₃Na (MNa⁺)

252.0152, found 252.0153. Anal. Calcd for C₈H₈ClN₃O₃.0.5 MeOH:

C, 41.56; H, 4.10; N, 17.11. Found: C, 41.13; H, 4.01; N, 16.80.

N-[2,4-Bis(dibenzyloxy)-6-chloropyrimidin-5-yl)-

methacrylamide (80). Triethylamine (0.3 mL, 2.2 mmol) was added

to a solution of compound 79 (80 mg, 0.23 mmol) in THF (5 mL). The

solution was cooled to 0 °C, and methacrolyl chloride (28 μL, 0.28

mmol) was added dropwise. The reaction mixture was stirred at 0 °C for

12 h. The solvent was distilled oﬀ under reduced pressure, and the

residue was dissolved in dichloromethane (20 mL) and washed with

water (2 × 15 mL). The organic layer was dried and solvent was distilled

oﬀ. The residue was ﬂash column chromatographed with silica gel,

N-(2,4-Bis(benzyloxy)-6-[(2S, 3S, 4R)-2,3,4,5-tetrakis(tert-bu-

tyldimethy-silanyloxy)pentylamino]-pyrimidin-5-yl)-2-oxopro-

panamide (67). Triethylamine (0.3 mL) was added to a solution of

compound 57 (180 mg, 0.2 mmol) in THF (5 mL). The solution was

cooled to 0 °C, and 2-oxopropanoyl chloride (24 μL, 0.24 mmol) was

added dropwise. The reaction mixture was stirred at 0 °C for 12 h. The

solvent was distilled oﬀ under reduced pressure, and the residue was

dissolved in dichloromethane (20 mL) and washed with water (2 × 15

mL). The organic layer was dried, and solvent was distilled oﬀ. The

residue was column chromatographed with ﬂash silica gel, eluting with

35% ethyl acetate in hexane, to aﬀord compound 67 (135 mg, 70%) as

yellowish oil: ¹H NMR (300 MHz, CDCl₃) δ 8.21 (brs, 1 H), 7.43−7.24

(m, 10 H), 5.75 (m, 1 H), 5.42−5.27 (m, 4 H), 4.0 (m, 2 H), 3.88 (m, 2

H), 3.79 (m, 1 H), 3.59 (m, 1 H), 3.41 (m, 1 H), 2.47 (s, 3 H), 0.94 −

0.86 (m, 36 H), 0.20 −0.05 (m, 24 H); ¹³C NMR (75 MHz, CDCl₃) δ

196.0, 163.7, 162.0, 159.2, 158.1, 137.0, 136.5, 128.5, 128.3, 128.1, 128.0,

U

dx.doi.org/10.1021/jo3010364 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

eluting with 35% ethyl acetate in hexane, to aﬀord compound 80 (70 mg,

73%) as white semisolid: ¹H NMR (300 MHz, CDCl₃) δ 7.27−7.15 (m,

10 H), 5.63 (s, 1 H), 5.24 (s, 1 H), 5.21 (m, 4 H), 1.81 (s, 1 H); ¹³C

NMR (75 MHz, CDCl₃) δ 167.0, 166.8, 161.2, 158.6, 139.3, 135.5,

135.3, 128.3, 128.1, 127.5, 120.8, 110.4, 69.9, 69.4, 18.5; ESIMS m/z

(rel. intensity) 432 (MNa⁺, 100), 410 (MNa⁺, 34) 268 (13); HRMS m/z

calcd for C₂₂H₂₁ClN₃O₃Na (MH⁺) 432.1091, found 432.1090. Anal.

Calcd for C₂₂H₂₀ClN₃O₃: C, 64.47; H, 4.92; N, 10.25. Found: C, 64.22;

H, 5.03; N, 9.94.

Computational Detail. The crystal structure of M. tuberculosis

lumazine synthase and ligand 70 complex was obtained from the Protein

Data Bank (PDB code: 2VI5).⁴⁸To minimize the size of our simulation

system, only one catalysis unit of lumazine synthase was kept, including

subunit A, subunit B, inorganic phosphate, ligand 70, and all

surrounding crystal water. The complex structure of M. tuberculosis

lumazine synthase with other ligands (36, 69, 73, and 81) was built using

2VI5 as the template, followed by an energy minimization to optimize

their geometry.

MD Simulations. All simulations were performed using AMBER

9.0. MD simulations were carried out using the parm99 force ﬁeld at 300

K. An explicit solvent model TIP3P water was used, and the complexes

were solvated with a truncated octahedron periodic box, extended to 8 Å

from the solute atoms. Sodium ions were added as counterions to

neutralize the system. For each ligand, the force ﬁeld parameters were

taken from the general Amber force ﬁeld (GAFF), whereas the atomic

partial charges were derived by geometry optimization using Gaussian

03 package⁵⁸at Hartree−Fock (HF)/6-31G* level and subsequent

single-point calculation of the electrostatic potential, to which the

charges were ﬁtted using the RESP procedure.

Prior to MD simulations, two steps of minimization were carried out;

in the ﬁrst stage, both protein and ligand were kept ﬁxed with a

constraint of 500 kcal/mol, and the positions of the water molecules

were minimized; then in the second stage, the entire system without any

constraint was minimized. The two minimization stages consisted of

3000 steps in which the ﬁrst 1000 were steepest descent (SD) and the

last 2000 conjugate gradient (CG). Molecular dynamics trajectories

were run using the minimized structure as a starting input. The time step

of the simulations was 2.0 fs with a cutoﬀ of 10 Å for the nonbonded

interaction, and SHAKE was employed to keep all bonds involving

hydrogen atoms rigid. Constant volume was carried out for 100 ps,

during which the temperature was raised from 0 to 300 K (using the

Langevin dynamics method); then 1000 ps of constant-pressure MD

were carried out at 300 K. The ﬁnal structure of the complexes was

obtained as the average of the last 400 ps of MD minimized with the CG

method until a convergence of 0.05 kcal/mol·Å.

surface area (SASA) from the MSMS program, where γ = 0.00542 kcal/

(mol·Å²) and β = 0.92 kcal/mol with a solvent probe radius of 1.4 Å:

ΔG_np= ΔSASA + β

(4)

TΔS_soluterepresents the entropic contribution to binding aﬃnity at

temperature T. The ﬁve ligands used in our calculation are structurally

very similar analogues. For a series of compounds with similar structures

and binding modes, the entropy contribution can be omitted if one is

only interested in relative binding aﬃnities. Since this calculation

converges slowly and can have large uncertainties, we omitted the

entropic contribution in this study.

Kinetic Assay of Lumazine Synthase Inhibitors. Assay mixtures

contained 100 mM Tris hydrochloride, pH 7.0, 100 mM NaCl, 2% (v/v)

DMSO, 5 mM dithiothreitol, 100 μM 2, 0.3−2.0 μM lumazine synthase,

variable concentrations of 1 (3−100 μM), and inhibitor (0−200 μM) in

a volume of 0.2 mL. Assay mixtures were prepared as follows. A solution

(176 μL) containing 100 mM NaCl, 5.0 mM dithiothreitol, 112 μM 2,

0.34−2.3 μM lumazine synthase in 100 mM Tris hydrochloride, pH 7.0,

was added to 4 μL of inhibitor in 100% (v/v) DMSO in a well of a 96-

well microtiter plate. The reaction was started by adding 20 μL of a

solution containing 100 mM NaCl, 5.0 mM dithiothreitol, and substrate

1 (30−1000 μM) in 100 mM Tris hydrochloride, pH 7.0. The formation

of 6,7-dimethyl-8-^D-ribityllumazine (3) was measured online for a

period of 30 min at 27 °C with a computer-controlled plate reader at 408

nm (ε_Lumazine= 10 200 M⁻¹cm⁻¹).

Kinetic Assay of Riboﬂavin Synthase Inhibitors. Assay mixtures

contained 100 mM Tris hydrochloride, pH 7.0, 100 mM NaCl, 2% (v/v)

DMSO, 5 mM dithiothreitol, 0.2−1 μM riboﬂavin synthase, variable

concentrations of 3 (3−60 μM), and inhibitor (0−200 μM) in a volume

of 0.2 mL. Assay mixtures were prepared as follows. A solution (176 μL)

containing 100 mM NaCl, 5.0 mM dithiothreitol, 0.23−1.13 μM

riboﬂavin synthase in 100 mM Tris hydrochloride, pH 7.0, was added to

4 μL of inhibitor in 100% (v/v) DMSO in a well of a 96-well microtiter

plate. The reaction was started by adding 20 μL of a solution containing

100 mM NaCl, 5.0 mM dithiothreitol, and substrate 3 (30−600 μM) in

100 mM Tris hydrochloride, pH 7.0. The formation of riboﬂavin (4) was

measured online for a period of 30 min at 27 °C with a computer-

controlled plate reader at 470 nm (ε_Riboﬂavin= 9600 M⁻¹cm⁻¹).

Evaluation of Kinetic Data. The velocity−substrate data were

ﬁtted for all inhibitor concentrations with a nonlinear regression method

using the program DynaFit.⁶⁰Diﬀerent inhibition models were

considered for the calculation. K_iand K_isvalues standard deviations

were obtained from the ﬁt under consideration of the most likely

inhibition model.

Energy Evaluation. To calculate the binding free energy, we

extracted 100 snapshots (at time intervals of 4 ps) for each species

(complex, receptor, and ligand) from the last 400 ps of MD of the

ligand−lumazine synthase complexes. The binding free energies were

calculated using the MM-PBSA method.⁵⁷The binding aﬃnity for a

protein/ligand complex corresponds to the free energy of association in

solution as shown in

ASSOCIATED CONTENT

■

S

* Supporting Information

All experimental procedures and data, H NMR and ¹³C NMR

1

spectra, and computational methods. This material is available

free of charge via the Internet at http://pubs.acs.org.

ΔG_bind= G_complex− (G_{unbound protein}+ G_{free ligand}

In MM-PBSA, the binding aﬃnity in eq 1 is typically calculated using

ΔG_bind= ΔE_MM− (ΔG_solv+ TΔS_solute

)

(1)

AUTHOR INFORMATION

■

Corresponding Author

*Tel: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@

purdue.edu.

)

(2)

where ΔE_MMrepresents the change in molecular mechanics potential

energy upon formation of the complex, calculated using all bonded and

nonbonded interactions. Solvation free energy, ΔG_solv, is composed of

the electrostatic component (G_PB) and a nonpolar component (G_np):

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

ΔG_solv= ΔG_PB+ ΔG_np

(3)

This research was made possible by NIH Grant Nos. GM51469

and R21 NS053634, the Fonds der Chemischen Industrie, the

Hans Fischer Gesellschaft, support from Research Facilities

Improvement Program Grant No. C06-14499 from the National

Center for Research Resources of the National Institutes of

Health.

G_PBwas calculated using the DelPhi program with PARSE radii. The

cubic lattice had a grid spacing of 0.5 Å. Dielectric constants of 1 and 80

were used for the interior and exterior, respectively, and 1000 linear

iterations were performed. The hydrophobic contribution to the

solvation free energy, G_np, was calculated using the solvent-accessible

V

dx.doi.org/10.1021/jo3010364 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Article

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(43) Yu, T. Y.; O’Connor, R. D.; Sivertsen, A. C.; Chiauzzi, C.; Poliks,

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Biochemistry 2008, 47, 13942.

(44) Ramsperger, A.; Augustin, M.; Schott, A. K.; Gerhardt, S.; Krojer,

T.; Eisenreich, W.; Illarionov, B.; Cushman, M.; Bacher, A.; Huber, R.;

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dx.doi.org/10.1021/jo3010364 | J. Org. Chem. XXXX, XXX, XXX−XXX

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O-nucleoside, S-nucleoside, and N-nucleoside probes of lumazine synthase and riboflavin synthase

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