Rigid spiroethers targeting the decoding center of the bacterial ribosome

Accepted Manuscript

Ioannis Mavridis, Georgia Kythreoti, Konstantina Koltsida, Dionisios

Vourloumis

PII:

S0968-0896(14)00005-4

DOI:

http://dx.doi.org/10.1016/j.bmc.2013.12.068

BMC 11330

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

19 November 2013

30 December 2013

Please cite this article as: Mavridis, I., Kythreoti, G., Koltsida, K., Vourloumis, D., Rigid spiroethers targeting the

decoding center of the bacterial ribosome, Bioorganic & Medicinal Chemistry (2014), doi:http://dx.doi.org/10.1016/

j.bmc.2013.12.068

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract

Rigid spiroethers targeting the decoding

center of the bacterial ribosome

Leave this area blank for abstract info.

I. Mavridis, G. Kythreoti, K. Koltsida, D. Vourloumis*

Chemical Biology Laboratories, National Center for Scientific Research “Demokritos”, Agia Paraskevi Attikis,

GR-15310, Greece

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.c om

Rigid spiroethers targeting the decoding center of the bacterial ribosome

Ioannis Mavridis^a, Georgia Kythreoti^a, Konstantina Koltsida^aand Dionisios Vourloumis^a,

^aChemical Biology Laboratories, National Center for Scientific Research “Demokritos”, Agia Paraskevi Attikis, GR-15310, Greece

ARTICLE INFO

ABSTRACT

Article history:

Received

Revised

Accepted

Available online

Continuing our efforts towards understanding the principles governing ribosomal recognition

and function, we have synthesized and evaluated a series of diversely functionalized 5,6-, 6,6-

and 7,6-spiroethers. These compounds successfully mimic natural aminoglycosides regarding

their binding to the decoding center of the bacterial ribosome. Their potential to inhibit

prokaryotic protein production in vitro along with their antibacterial potencies have also been

examined.

Keywords:

aminoglycosides

ribosomal A-site

spiroethers

antibacterials

RNA recognition

1. Introduction

binding orientations, directed us to concentrate on the

southeastern (IV, Figures 1A and 1B) as well as the northwestern

Natural aminoglycosides (for example neomycin B and

neamine in Figure 1A) represent exceptional molecular tools for

exploring the biological effects of RNA targets within living

organisms.¹In addition to their profound antibacterial properties,²

associated with their ability to bind with high affinity to the

decoding center (A-site, Figure 1A) of the respective ribosomal

RNA, additional antiviral applications³as well as their potential

use as therapeutics for genetic disorders⁴are fast emerging in the

scientific arena. Unfortunately, the current bias of small-molecule

libraries, employed in high-throughput screening endeavors for

protein targets, obstructs these efforts from identifying chemical

scaffolds privileged for binding RNA.⁵Consequently, our

understanding of the fine structural requirements and their

precise orientations for efficient binding with RNA components

is currently directed from crystallographic information of the

specific macromolecules, co-crystallization experiments with

certain, mainly natural or semisynthetic representatives, as well

as the biological evaluation of novel synthetic analogues.⁶

regions (III, Figures 1A and 1B) of these scaffolds. The former

demonstrated significant improvement of the binding affinities

for the A-site in previously synthesized 7,6-spiroethers,^7bwhile

the latter produced exceptional affinities for the same target when

occupied by substituted triazole moieties.^7d

Recent scientific efforts from our group identified a new series

of spiro-bicyclic scaffolds of various ring sizes (I and II, Figure

1A), which successfully mimicked the natural products’ affinity

for the A-site through the exploitation of spatial, electrostatic and

aromatic interactions.⁷Their conformational rigidity is believed

to play an important role in site specificity by capturing the

required “bioactive” conformation. Inspired by the obtained

promising results of the parent compounds^7a,cwe anticipated that

further functionalization of the spiro-bicyclic skeleton could lead

to improved biological profiles. Examination of the predicted

———

 Corresponding author. Tel.: +30-210-6503624; fax: +30-210-6511766; e-mail: vourloumis@chem.demokritos.gr

their orientation within the A-site,^7dalbeit the highest affinity

conformations were found to overlap with the position of

neamine in the co-crystal structure. Specifically, their

aminocyclitol component was located either proximal to 2-DOS

of neamine, or inverted superimposing with its sugar residue. The

targeted substitution pattern was identified to exhibit promising

potential for aromatic, electrostatic or H-bonding interactions

within the green sphere (Figure 1B) regions of the RNA decoding

site. Consequently, a wider variety of functionalities was selected

to complement conclusions regarding structure-activity

relationships. Moreover, our current study was performed on all,

5,6-, 6,6- and the bulkier 7,6-bicyclic entities, delivering a more

comprehensive picture regarding the chemical space that can be

accessed towards the development of more efficient antibiotics.

2. Results and discussion

2.1 Synthesis

Figure 1. A) Secondary structure of the bacterial decoding-site, depicting

the internal loop (A-site) in 16S rRNA. The four base-changes of the

eukaryotic sequence are indicated by arrows. The recognition site for the 2-

DOS moiety of aminoglycosides is boxed; structures of neomycin B, neamine

(neomycin A), 2-deoxy-streptamine (2-DOS), previously synthesized

conformationally restricted analogues I^7a,cand II^7dand analogues III and IV^7b

designed and synthesized in this study. AA: aminoacid residue; B) Three-

dimensional representation of the spiro-bicyclic scaffold within the A-site.^6f,7

The available targeted space for further functionalization of III and IV is

represented by the green spheres.

Synthesis of the 5,6-spirocyclic analogues III commenced

from known azide 1 (Scheme 1).^7dReduction of the azide

functionality under Staudinger conditions furnished the

corresponding amine, which was further coupled with a series of

appropriately protected aminoacids (list in Scheme 1) producing

the desired conjugates in 41-81% yields. Hydrogenolysis of the

Cbz-groups followed by acidic treatment (TFA) furnished final

products 2-7, as shown in scheme 1, compiling a wide selection

of functionalities and thus a variety of interactions within the

RNA target.

Scheme 1. Reagents and conditions: a) Me₃P (1.0M in THF, 1.5 eq), NaOH

(1.0M, 1.3 eq), THF, 14 h, 0→25 °C, 82%; b) EDCl (1.2 eq), protected

aminoacid (1.0 eq), DIPEA (2.5 eq), HOBt (1.2 eq), THF (0.1M), 12 h, 25

°C, 41-86%; c) 1,4-cyclohexadiene (xs.), Pd/C (10% wt., 0.1 eq), CH₃COOH

(0.01M), 14 h, 25 °C, 68-80%; d) TFA (0.02M), 1 h, 25 °C, 80-96%.

Protected Aminoacids: Boc-Arg(Z)₂-OH, Boc-His-OH, Boc-Lys(Z)-OH, Z-

Pro-OH, Boc-Trp-OH, Boc-Tyr(Bzl)-OH; THF = tetrahydrofuran; EDCl = N-

Scheme 2. Reagents and conditions: a) allyl-^tBu-carbonate (3.5 eq),

Pd(PPh₃)₄(0.1 eq), THF (0.036M), 4 h, 45 °C, 70% for 10, 88% for 11; b)

Grubbs 2^ndgeneration (0.1 eq), CH₂Cl₂(0.01M), 12 h, 25 °C, 90% for 12,

84% for 13; c) OsO₄(0.09 eq), NMO (3.1 eq), acetone /H₂O 4:1 v/v (0.02M),

3-15 h at 25 °C or 8 h, 25 °C, >5:1 ratio for 14 : 16, 71% for 14, 57% for 15

and 37% for 17; d) OsO₄(1.2 eq), TMEDA (1.2 eq), CH₂Cl₂, ‒78→25 °C, 3

h, 75% for 14 and 72% for 15; e) THF / AcOH / H₂O, 3:3:1 v/v/v (0.04M), 50

°C, 8 h, 90% for 18; f) conc. HCl (24.0 eq), MeOH (0.06M), 25 °C, 3 h, 74%

(3-Dimethylaminopropyl)-N′-ethylcarbo-diimide hydrochloride; DIPEA

=

N,N-Diisopropylethylamine; HOBt = 1-Hydroxybenzotriazole hydrate; TFA

= Trifluoroacetic acid; Z = Cbz = carbobenzyloxy; Bzl = benzoyl.

As before, the calculated conformations of the designed

analogues exhibit a high degree of invariability with respect to

for 19 and 74% for 21; g) NaH (4.0 eq), DMF (0.019M), 25 °C, 4 h; h) aq.

0.5N LiOH (5.0 equiv), dioxane (0.027M), 25 °C, 12 h, 60% for 26 and 58%

for 27 for two steps i) aq. 0.5N LiOH / dioxane 1:1 v/v (0.027M), 60 °C, 2 h,

61% for 26, 95% for 27 and 61% for 29. NMO = 4-Methylmorpholine N-

oxide; TMEDA = N,N,N′,N′-Tetramethylethylenediamine; DMF = N,N-

Dimethylformamide.

22-25 was performed under basic conditions (NaH, DMF). Basic

hydrolysis of the cyclic carbamates revealed the free amine at

position 1, distinguished from position 3 for further orthogonal

functionalization. It was later identified that 18-21 could be

directly transformed into 26-29 in one step after basic treatment

with LiOH and heating to 60 °C for 2 hours.

Synthesis of spiro analogues IV was initiated from known

intermediades 8 and 9, which as reported before represent a

diastereoisomeric mixture at C4 (Scheme 2) of 4:1 for 8 and 9:1

for 9.^7b,dAllylation of the tertiary hydroxyl was accomplished

through the use of allyl-^tBu-carbonate and catalytic Pd(PPh₃)₄

furnishing 10 and 11.⁸Careful chromatographic purification at

this stage successfully separated the isomeric mixtures. Only the

major isomers were selected to be independently transformed

further. Specifically, 10 and 11 were cyclized in the presence of

Grubbs second generation catalyst⁹producing 12 and 13 in 90%

and 84% yields respectively. Catalytic dihydroxylation of 12 and

13 furnished a separable mixture of diols (from 12→14+16 and

from 13→15+17), albeit with low diastereoselectivity, especially

in the case of the 7-membered heterocycles (Scheme 2). Its

stoichiometric counterpart¹⁰in the presence of TMEDA produced

only the two major isomers 14 and 15 in 75% and 72%

respectively. Removal of the ketal protecting moiety was

performed under acidic conditions yielding tetraols 18-21.

With key intermediates 26-29 at hand we embarked to the

synthesis of analogues IV, where the additional functionality

would be attached through an amide bond, sacrificing the basicity

of the amino group at this position. We believed that the α-amino

or α-hydroxyl moieties of the aminoacids used would

successfully compensate for that change. Thus, amines 26-29

reacted with the corresponding activated esters, presented in

scheme 3, producing amides 30-36 in good yields.

Hydrogenolysis of Cbz-carbamates was accomplished under

standard conditions with Pd/C furnishing final analogues 37-43.

Scheme 4. a) BH₃(16.6 eq), ‒10→25 °C, 6 h, 93%; b) (COCl)₂(3.7 eq), Et₃N

(3.7 eq), DMSO (3.7 eq), CH₂Cl₂(0.2M), ‒78 °C, 50 min, 78%; c) BH₃(11.5

eq), ‒10→25 °C, 3 h, 80%; d) TFA / CH₂Cl₂1:3 v/v (0.006M), 25 °C, 1 h,

85%; e) CbzCl (3.0 eq), NaHCO₃(5.0 eq), H₂O / dioxane 2:1 v/v (0.1M), 25

°C, 17 h, 73%; f) (COCl)₂(3.0 eq), Et₃N (3.7 eq), DMSO (3.7 eq), CH₂Cl₂

(0.2M), -63 °C, 50 min, 73%; g) TfN₃(2.0 eq), CuSO₄•5H₂O (0.1 eq), K₂CO₃

(2.0 eq), MeOH / H₂O 1:1 v/v (0.059M), 25 °C, 76%; h) BnBr (4.0 eq), NaH

(4.0 eq), TBAI (0.1 eq), DMF (0.11M), 25 °C, 1 h, 80%; i) DIBAL (2.0 eq),

toluene (0.17M), ‒78 °C, 2 h, 90%; j) 26-29 (1.0 eq), aldehydes 45 or 47 or

49 (3.0 eq), AcOH (0.1 eq), MeOH (0.015M), 25 °C, 1 h; k) NaBH₃(CN)

Scheme 3. Reagents and conditions: a) 26-29 (1.0 eq), activated ester (1.35

eq, 0.1M in THF), NaHCO₃(xs), H₂O / dioxane 3:1 v/v (0.028M), 25 °C, 15

h, 33-88%; b) 10% wt. Pd/C (cat), MeOH (0.004M), 25 °C, 3 h, 32-78%.

In the interest of differentiating the two amino groups in the

molecules, cyclization to the corresponding trans-oxazolidinones

(10.0 eq), MeOH (0.015M), 25 °C, 15 h, 24-74% for two steps; l) 10% wt.

Pd/C (0.05 eq), MeOH (0.04M), 25 °C, 2 h, 79-98%. DMSO = Dimethyl

sulfoxide; CbzCl = Benzyl chloroformate; Tf = trifluoromethanesulfonyl; Bn

observation can be made for the 7,6-spirocyclic analogues (38 vs.

40 and 57 vs. 58).

=

Benzyl; TBAI

=

Tetrabutylammonium iodide; DIBAL

=

The basicity of the amino group at position 1 appears to be

very important for binding as indicated by comparison of the

corresponding amides (entries 11-13) with the respective amines

(entries 20-21 and 25), with much higher affinities observed for

the amino-analogues in both, 6,6- and 7,6-series. Compound 57

yields the best binding affinity for the bacterial A-site from all

compounds tested in this study.

Diisobutylaluminum hydride.

For the synthesis of the analogous amines, the construction of

aldehydes 45, 47 and 49 was performed as presented in scheme

4. Thus, di-Cbz-lysine 44 was initially reduced to the

corresponding alcohol and then oxidized, utilizing Swern’s

protocol, to aldehyde 45 in 78% yield. Similarly, tyrosine

derivative 46 was transformed to aldehyde 47 after the exchange

of the Boc protecting group to a Cbz towards a global

deprotection strategy at the end of the synthetic sequence. For

aldehyde 49, (S)-2-hydroxy-4-amino butyric acid (48) was first

transformed to the corresponding azide after its reaction with

freshly prepared TfN₃and catalytic amount of CuSO₄•5H₂O.^7c

Protection of the hydroxyl as a benzyl ether followed by DIBAL-

H reduction furnished aldehyde 49 in 86% yield.

Table 1. Binding affinities and biological activities of

synthetic analogues

Amines 26-29 reacted with aldehydes 45, 47 and 49 under

reductive amination conditions, producing amines 50-55. Global

deprotection was accomplished as before via catalytic

hydrogenation, furnishing final products 56-61.

2.2 Biological activity

Examination of the specificity of our analogues for the

ribosomal decoding site was performed by a previously described

RNA fluorescence assay, based on enhancement of the

fluorescence emission of 2-aminopurine (2AP) attached to the

flexible adenine A1492 of the model oligonucleotide (Table 1,

RNA fluorescence assay).¹¹The activity of the compounds as

functional bacterial-translation inhibitors was evaluated in a

coupled in vitro transcription-translation assay (B_IVT; Table 1,

Bacterial IVT).¹²Table 1 incorporates our results in comparison

to neamine and the values obtained previously for the parent 6,6-

and 6,7-bicyclic analogues^7a,cas well as the isomers of 38 and 40

(entries 12 and 15).^7b

In general, most of the synthetic analogues exhibited very

good binding affinities for the bacterial A-site, validating our

design efforts through docking calculations, as previously

described.^7a,bAlbeit, correlation of the acquired fluorescent

measurements with the ability of the compounds to interfere with

the protein production process in bacteria (B_IVT) yielded several

discrepancies. We speculate that the obtained conformational

changes of the biological target, although measurable by our

affinity screen, are not sufficient to induce the analogous

biological effect. Specific spatial and electrostatic requirements

along with the precise orientation of the synthetic analogues

within the decoding center will be accurately elucidated upon

successful completion of co-crystallization experiments, which

are currently underway.

Overall, 5,6-spirocycles 2-6 appear highly promising,

producing very good binding affinities and reasonable abilities to

interfere with protein production. With the exception of tyrosine

analogue 7, a variety of basic aminoacid substitutions can be

tolerated as long as H-bonding potential and positive charges are

maintained.

6,6-spiro analogues (Table 1, entries: 2, 11, 14 and 20) present

good binding affinities, excellent in the case of 56, but higher

inconsistencies regarding their B_IVTs, demonstrating a more

profound dependence on the fine conformational changes

required for biological activity. Longer chain-, basic-substitutions

(Lys) are preferred over shorter or aromatic (37, 56 vs. 39, 41)

for both, binding affinities and biological activity. The opposite

[a] All final products were quantitated by NMR spectroscopy,

utilizing an internal standard (DiMethylFuran, DMFu).¹³[b]

Concentrations are reported in M. [c] RNA-ligand EC₅₀values

were determined by decoding-site RNA fluorescence assay (average

of 3 replicate experiments per compound, ±10%). [d] Bacterial in

vitro transcription/translation (B_IVT) IC₅₀values were determined by

coupled transcription-translation assay using E. coli extract (average

of 3 replicate experiments per compound, ±15%). [e] Fluorescent

signal from the compound interfering with assay’s measurement;

exact number cannot be obtained.

Table 2. Minimum Inhibitory Concentrations (MICs) against selected bacterial strains

Escherichia coli

Staphylococcus Pseudomonas

entry

compound

aureus^a

aeruginosa^a

ATCC 25922^a

JW 5503-1^a,b

JW 3596-1^a,bJW 0451-2^a,b

1

2

3

4

5

6

7

Paromomycin

4

5

113

73

4

113

300

113

150

113

150

Neamine

14

4

7.2

8.4

3.8

N/A^c

14.7

>112

65

>112

98

>112

131

112

>112

32.5

118

6

>131

>118

>129

>118

57

59

61

>118

N/A^c

>118

236

N/A^c

236

118

>129

>118

129

118

[a] Concentrations are reported in μg/ml. [b] Kanamycin resistant strains. [c] N/A : not active.

The stereochemistry of the di-hydroxyl functionality on the

7-membered heterocycle, although not easily explainable by

our docking calculations (unless we hypothesize different

binding orientations within the biological target), appears to

be of great importance for binding and for biological activity.

Specifically, entries 12 vs. 13 and 15 vs. 16 reveal that

placement of the two hydroxyls on the top phase increases

binding to the bacterial decoding center by almost 10 and 20

fold respectively albeit the exact opposite is observed

regarding their B_IVTIC₅₀values.

natural aminoglycosides to the bacterial ribosome A-site

construct utilized in our fluorescent studies, exemplified by

the evident binding affinities presented. The nature of the

substitution, the absolute stereochemistry as well as the

basicity of the amino-group at position 1 (Figure 1, 2-DOS

numbering) have been studied. Additionally, their potential to

inhibit protein production in vitro for prokaryotes has been

evaluated, illustrating that the complexity of the specific

mechanism cannot always be explained solely based on

binding affinities. Their antibiotic activity in a series of gram

positive and gram negative microorganisms has also been

examined, revealing several examples of promising analogues

for further studies. Realizing the serious socio-economic

challenge of emerging antibiotic resistance and the associated

urgent need for novel potent antibiotics,¹⁹we consider the

identified scaffolds and their proposed RNA target as

important tools for studying the biological processes involved

and the development of effective pharmaceutical agents.

Additional examples, co-crystallisation experiments and

further biological evaluations in prokaryotic as well as

eukaryotic systems²⁰are currently underway.

The synthetic analogues were also evaluated regarding

their antibacterial activity against Gram positive

Staphylococcus aureus (ATCC 29213) as well as Gram

negative Pseudomonas aeruginosa (ATCC 27853) and

Escherichia coli (ATCC 25922) strains. Furthermore, E. coli

K12 kanamycin resistant mutated strains^14,15JW 5503-1

(ΔtolC732::kan),¹⁶JW 0451-2 (ΔacrB747::kan)¹⁷and JW

3596-1 (ΔrfaC733::kan)¹⁸were also selected to identify

potential problems with membrane permeability along with

microbial resistance mechanisms for all tested compounds.

Minimal inhibitory concentrations (MICs) were determined

by the geometric micro-dilution method (see Supplementary

material) and the results for the most active analogues are

presented in table 2. It is interesting to note that none of the

parent spirocycles (entries 2, 3 and 4 in Table 1) exhibited any

antibiotic activity, regardless their good B_IVTvalues.

Compounds 4, 6, 57 and 61 demonstrated very good

antibacterial activities against the S. aureus strain, comparable

to the ones of the natural products. Analogue 6 in particular

was the only one with broad spectrum activity in both, gram

positive and gram negative microorganisms. Specifically, it

demonstrates comparable values to paromomycin’s for S.

aureus, but it also presents superior activities in P. aeruginosa

as well as in the kanamycin resistant E. coli mutant JW 0451-

2.

4. Experimental

4.1 General methods

Unless otherwise noted, solvents and reagents for organic

synthesis were obtained from commercial suppliers and were

used without further purification. Tetrahydrofuran (THF),

toluene and ethyl ether (ether) were distilled from sodium

benzophenone, and methylene chloride (CH₂Cl₂) from

calcium hydride. All reactions were carried out under a dry

argon atmosphere with anhydrous, freshly distilled solvents

under anhydrous conditions unless otherwise noted. All

reactions were magnetically stirred with Teflon stir bars, and

temperatures were measured externally. Reactions requiring

anhydrous conditions were carried out in oven dried (120 C,

24 h) or flame dried (vacuum < 0.5 Torr) glassware. Yields

refer to chromatographically and spectroscopically (¹H NMR)

homogeneous materials. All reactions were monitored by thin

layer chromatography (TLC) carried out on 0.25-mm E.

Merck silica gel plates (60F-254) using UV light as

visualizing agent and 7% ethanolic phosphomolybdic acid or

3. Conclusions

In summary, we have successfully synthesized and

evaluated representative members from three different sub-

families of 5,6-, 6,6- and 7,6-spirocyclic analogues. These

compounds successfully mimic the bonding interactions of

p-anisaldehyde solution and heat as developing agents.

E.Merck silica gel (60, particle size 0.040-0.063 mm) was

used for flash column chromatography. Nuclear magnetic

resonance (NMR) spectra were recorded on a Bruker AM-250

1H), 3.77-3.61 (m, 1H), 3.58-3.45 (m, 2H), 3.44-3.09 (m, 5H),

2.35-2.13 (m, 2H), 2.11-1.78 (m, 4H), 1.78-1.59 (m, 1H),

1.52-1.16 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 167.9, 134.2,

125.8, 118.1, 85.1, 80.0, 74.9, 70.3, 52.1, 51.6, 49.9, 42.8,

30.5, 28.5, 27.9, 25.9; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₁₆H₂₉N₆O₄369.2250, found 369.2238.

or

a Bruker Avance DRX-500 instrument as noted

individually. Unless stated otherwise, NMR experiments were

performed at 25 °C. Chemical shifts are measured in parts per

million () relative to the deuterated solvent used in the

experiment. Multiplicities are designated as singlet (s),

doublet (d), triplet (t), or multiplet (m). Broad peaks are

indicated as “b”.

4.2.3. (S)-2,6-diamino-N-(((2S,5S,6R,7S,8R,10S)-8,10-dia

mino-6,7-dihydroxy-1-oxaspiro[4.5]decan-2-

yl)methyl)hexa namide 4: 12.3 mg, 94% yield. R_f= 0.45 in

1

40% NH₄OH/MeOH. H NMR (250 MHz, D₂O) δ 4.26-4.10

(m, 1H), 3.94 (dd, J = 6.83, 6.71 Hz, 1H), 3.61 (dd, J = 10.06,

10.03 Hz, 1H), 3.51-3.21 (m, 4H), 3.21-3.05 (m, 1H), 2.90

(dd, J = 7.63, 7.49 Hz, 2H), 2.28-1.71 (m, 8H), 1.70-1.52 (m,

2H), 1.46-1.25 (m, 2H). ¹³C NMR (63 MHz, D₂O) δ 170.4,

85.7, 80.6, 75.3, 70.9, 53.3, 52.0, 50.4, 43.1, 39.1, 30.9, 30.4,

29.1, 28.4, 26.3, 21.4; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₁₆H₃₄N₅O₄360.2611, found 360.2611.

4.2. Synthesis of products 2-7: Azide 1 (141 mg, 0.268

mmoles) was dissolved in anhydrous THF and the resulting

solution was cooled to 0 ^°C. Me₃P (408 μl, 0.408 mmoles, 1.5

eq) was added dropwise and stirring was continued for 2 h

while the solution was warming-up to ambient temperature. A

1M solution of NaOH (350 μl, 0.35 mmoles, 1.3 eq) was then

added and the reaction was allowed to stir for an additional 12

h. Removal of the solvents under vacuum followed by column

chromatography (MeOH→10% NH₄OH/MeOH) furnished

110 mg of the desired amine as a white foam in 82% yield

(for spectroscopic data see supporting info).

4.2.4.

(S)-N-(((2S,5S,6R,7S,8R,10S)-8,10-diamino-6,7-di

hydroxy-1-oxaspiro[4.5]decan-2-yl)methyl)pyrrolidine-2-

carboxamide 5: 18.6 mg, 80% yield. R_f= 0.6 in 20%

1

NH₄OH/MeOH. H NMR (500 MHz, D₂O) δ 4.35-4.23 (m,

1H), 4.07-3.98 (m, 1H), 3.51-3.41 (m, 1H), 3.41-3.22 (m, 3H),

3.21-3.07 (m, 2H), 2.93-2.84 (m, 1H), 2.84-2.74 (m, 1H),

2.34-2.22 (m, 1H), 2.22-1.97 (m, 3H), 1.97-1.82 (m, 4H),

1.81-1.67 (m, 1H), 1.57-1.40 (m, 1H). ¹³C NMR (125 MHz,

D₂O) δ 174.3, 89.1, 81.1, 76.5, 74.8, 60.7, 52.8, 51.2, 47.0,

44.0, 34.9, 31.3, 30.6, 29.0, 25.1; ΗRMS-ESI (m/z) [M+H]⁺

calcd for C₁₅H₂₉N₄O₄329.2189, found 329.2179.

The corresponding amine (0.1 mmoles) was dissolved in

anhydrous THF (0.1M) under argon. To this solution EDCl

(1.2 equiv, 0.12 mmoles), protected aminoacid (1.0 equiv, 0.1

mmoles), DIPEA (2.5 equiv, 0.25 mmoles) and HOBt (1.2

equiv, 0.12 mmoles) were consecutively added and stirring

was continued for 12 h. The solvent was removed under

vacuum and the product was re-dissolved with CH₂Cl₂and

extracted with sat. NH₄Cl (2x2 mL), sat. NaHCO₃(2x2 mL)

and brine (2x2 mL). The combined organic extracts were

dried with MgSO₄, filtered, concentrated under vacuum and

purified by flash column chromatography (CH₂Cl₂→10%

MeOH/CH₂Cl₂) to yield the desired conjugate as a white foam

(for spectroscopic data of all intermediates see supporting

info).

4.2.5.

(S)-2-amino-N-(((2S,5S,6R,7S,8R,10S)-8,10-diam

ino-6,7-dihydroxy-1-oxaspiro[4.5]decan-2-yl)methyl)-3-

(1H -indol-3-yl)propanamide 6: 15.8 mg, 96% yield. R_f=

1

0.65 in 20% NH₄OH/MeOH. H NMR (500 MHz, D₂O) δ

7.57 (bs, 1H), 7.47 (bs, 1H), 7.27 (s, 1H), 7.24-6.93 (m, 2H),

4.22-4.04 (m, 1H), 4.04-3.89 (m, 1H), 3.69-3.05 (m, 8H),

2.32-2.09 (m, 1H), 2.07-1.68 (m, 3H), 1.56-1.44 (m, 1H),

1.43-1.11 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 170.2, 136.8,

127.2, 125.7, 122.7, 120.0, 118.8, 112.6, 107.2, 85.8, 80.3,

75.7, 71.4, 54.8, 52.4, 50.6, 43.5, 31.2, 29.2, 28.1, 27.4;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₂₁H₃₂N₅O₄418.2454,

found 418.2452.

The corresponding protected amines (1.0 equiv) were

dissolved in AcOH (0.01M) and Pd/C (10% wt., 0.1 equiv)

was added along with excess of 1,4-cyclohexadiene and the

mixture was stirred at ambient temperature for 14 h. The

whole was filtered through celite and washed with MeOH,

after which the solvents were removed under vacuum. The

products were isolated after purification by flash

chromatography (MeOH→20%→30% NH₄OH/MeOH). Boc-

protected amines were dissolved in TFA (0.02M) and stirred

for 1 h at ambient temperature. Final amines 2-7 were isolated

after removal of the solvent under high vacuum.

4.2.6.

(S)-2-amino-N-(((2S,5S,6R,7S,8R,10S)-8,10-diami

no-6,7-dihydroxy-1-oxaspiro[4.5]decan-2-yl)methyl)-3-(4-

hydroxyphenyl)propanamide 7: 14.4 mg, 97% yield. R_f=

1

0.65 in 20% NH₄OH/MeOH. H NMR (250 MHz, D₂O) δ

7.07-6.88 (m, 2H), 6.78-6.61 (m, 2H), 4.09-3.96 (m, 1H),

3.96-3.85 (m, 1H), 3.52-3.39 (m, 1H), 3.39-3.29 (m, 1H),

3.28-3.02 (m, 3H), 3.03-2.92 (m, 1H), 2.92-2.79 (m, 1H),

2.20-2.08 (m, 1H), 2.01-1.92 (m, 1H), 1.92-1.70 (m, 2H),

1.68-1.54 (m, 1H), 1.38-1.20 (m, 1H); ¹³C NMR (63 MHz,

D₂O) δ 170.2, 154.9, 130.7, 130.7, 126.2, 115.8, 115.8, 85.8,

79.8, 75.3, 71.1, 55.2, 51.8, 50.2, 42.9, 36.5, 30.7, 29.5, 27.9;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₉H₃₁N₄O₅395.2294,

found 395.2285.

4.2.1.

(S)-2-amino-N-(((2S,5S,6R,7S,8R,10S)-8,10-diam

ino-6,7-dihydroxy-1-oxaspiro[4.5]decan-2-yl)methyl)-5-

gua nidinopentanamide 2: 21.4 mg, 96% yield. R_f= 0.2 in

1

50% NH₄OH/MeOH. H NMR (500 MHz, D₂O) δ 4.34-4.24

(m, 1H), 4.04 (dd, J = 6.46, 6.38 Hz, 1H), 3.66 (dd, J = 10.2,

10.0 Hz, 1H), 3.58-3.38 (m, 4H), 3.30-3.25 (m, 3H), 2.34-2.23

(m, 2H), 2.16-1.85 (m, 6H), 1.70-1.59 (m, 2H). ¹³C NMR

(125 MHz, D₂O) δ 170.2, 157.4, 85.9, 80.9, 75.8, 71.4, 53.8,

52.5, 50.8, 43.6, 41.0, 31.5, 29.4, 28.8, 28.5, 24.3; ΗRMS-ESI

(m/z) [M+H]⁺calcd for C₁₆H₃₄N₇O₄388.2672, found

388.2664.

4.3. Allylation of tertiary alcohols 8 and 9.

4.3.1. Synthesis of dibenzyl((3aR,4S,5S,7R,7aS)-4-allyl-4-

(allyloxy)hexahydrospiro[benzo[d][1,3]dioxole-2,1'-cyclo-

hexane]-5,7-diyl)dicar bamate 10: To a solution of alcohol 8

(50 mg, 9.1 x 10^-2mmoles, 1.0 eq) in THF (2.5 mL),

Pd(PPh₃)₄(12.5 mg, 1.1 x 10^-2mmoles, 0.1 eq) and allyl-t-

butylcarbonate (50 mg, 3.2 x 10^-1mmoles, 3.5 eq) were

consecutively added and the whole was stirred for 4 h at 45

°C. The resulting brown reaction mixture was concentrated to

dryness under vacuum and was purified by flash

4.2.2.

(S)-2-amino-N-(((2S,5S,6R,7S,8R,10S)-8,10-diam

ino-6,7-dihydroxy-1-oxaspiro[4.5]decan-2-yl)methyl)-3-

(1H -imidazol-5-yl)propanamide 3: 6.1 mg, 94% yield. R_f=

1

0.45 in 20% NH₄OH/MeOH. H NMR (500 MHz, D₂O) δ

8.68 (s, 1H), 7.41 (s, 1H), 4.41-4.28 (m, 1H), 4.27-4.11 (m,

t

chromatography (5→25% EtOAc / Hexanes) to yield 40 mg

of diene 10 as an amorphous white solid in 70% yield. R_f=

0.44 in 30% EtOAc / Hexanes. ¹H NMR (500 MHz, CDCl₃) δ

7.30-7.20 (m, 10H), 5.91-5.73 (m, 2H), 5.29-4.83 (m, 9H),

4.50 (dd, J = 12.8, 4.6 Hz, 1H), 4.19-4.08 (m, 1H), 3.85-3.56

(m, 3H), 3.42 (d, J = 8.9 Hz, 1H), 2.79 (dd, J = 12.8, 8.2 Hz,

1H), 2.30 (dd, J = 12.8, 6.5 Hz, 1H), 2.20 (bs, 1H), 1.77-1.29

(m, 11H); ΗRMS-ESI (m/z) [M+H]⁺calcd for C₃₄H₄₃N₂O₇

591.3070, found 591.3072.

mmoles, 3.1 eq), followed by ΟsO₄(2.5 wt % in BuOH, 0.6

mL, 4.8 x 10^-2mmoles, 0.09 eq) and allowed to stir, at

ambient temperature, for 3 h. The solvent was removed under

reduced pressure and the crude residue was purified by flash

chromatography on silica gel (1.5% MeOH / CH₂Cl₂) in order

to furnish 225 mg of diol 14 as a white amorphous solid

(71%). The minor product 16 was not isolated; R_f= 0.61 σε

1

70% ΕtOAc / CH₂Cl₂. H NMR (500 MHz, Acetone-d6) δ

7.44-7.24 (m, 10H), 6.54 (bd, J = 6.56 Hz, 1H), 5.87 (d, J =

9.0 Hz, 1H), 5.09 (d, J = 12.6 Hz, 1H), 5.06 (s, 2H), 5.04 (d, J

= 12.6 Hz, 1H), 4.35-4.26 (m, 2H), 3.97 (dd, J = 9.8, 9.8 Hz,

1H), 3.88-3.75 (m, 2H), 3.73-3.64 (m, 1H), 3.64-3.53 (m, 3H),

2.19-2.07 (m, 2H), 1.74-1.26 (m, 12H); ¹³C NMR (63 MHz,

Acetone-d6) δ 155.99, 155.64, 137.41, 128.34, 128.30,

127.82, 127.72, 110.59, 85.15, 75.51, 75.27, 68.34, 67.75,

65.82, 65.63, 65.50, 53.84, 49.82, 36.13, 35.99, 34.61, 32.93,

24.83, 23.66, 23.65; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₃₂H₄₁N₂O₉597.2812, found 597.2807.

4.3.2. Synthesis of dibenzyl ((3aR,4S,5S,7R,7aS)-4-

(allyloxy)-4-(but-3-en-1-yl)hexahydrospiro[benzo[d][1,3]di

oxole-2,1'-cyclohexane]-5,7-diyl)dicarbamate

11:

According to the procedure described for alcohol 8, 9

produced 47 mg of diene 10 as an amorphous white solid in

88% yield. R_f= 0.57 in 25% EtOAc / Hexanes. ¹H NMR (250

MHz, CDCl₃) δ 7.39-7.30 (m, 10H), 5.94-5.69 (m, 2H), 5.26-

4.78 (m, 9H), 4.45 (dd, J = 13.0, 4.1 Hz, 1H), 4.14 (dd, J =

12.7, 4.1 Hz, 1H), 3.91-3.67 (m, 3H), 3.44 (d, J = 8.7 Hz,

1H), 2.41-1.91 (m, 3H), 1.82-0.76 (m, 13H); ¹³C NMR (125

MHz, CDCl₃) δ 155.56, 155.55, 137.94, 136.38, 135.41,

128.56, 128.47, 128.16, 128.07, 115.18, 114.98, 111.56,

80.61, 75.57, 66.86, 66.76, 66.75, 64.93, 51.22, 50.18, 36.35,

36.01, 34.92, 29.67, 29.20, 28.27, 24.95, 23.63; ΗRMS-ESI

(m/z) [M+H]⁺calcd for C₃₅H₄₅N₂O₇605.3227, found

605.3222.

4.5.2. Synthesis of diols 15 and 17: According to the

procedure described for the synthesis of 14, olefin 13 (100

mg, 1.7 x 10-1 mmoles, 1.0 eq) produced 39 mg of 15 (37%)

and 60 mg of 17 (57%) as white amorphous solids.

1

15: 57% yield; R_f= 0.43 in 5% MeOH / CH₂Cl₂; H NMR

(500 MHz, Acetone-d6) δ 7.43-7.25 (m, 10H), 6.54 (bd, J =

5.6 Hz, 1H), 6.48 (d, J = 9.7 Hz, 1H), 5.10-5.03 (m, 4H), 4.17

(d, J = 13.3 Hz, 1H), 3.96 (dd, J = 9.8, 9.8 Hz, 1H), 3.90 (dd,

J = 13.4, 5.3 Hz, 1H), 3.85-3.71 (m, 2H), 3.67-3.62 (m, 1H),

3.58-3.53 (m, 1H), 3.47 (d, J = 9.3 Hz, 1H), 3.36 (d, J = 6.4

Hz, 1H), 2.15-2.00 (m, 2H), 1.99-1.92 (m, 1H), 1.79-1.71 (m,

1H), 1.71-1.50 (m, 10H), 1.45-1.33 (m, 2H); ¹³C NMR (63

MHz, Acetone-d6) δ 156.90, 156.59, 138.33, 138.26, 129.18,

129.14, 128.63, 128.54, 128.52, 128.46, 111.30, 84.38, 78.61,

76.16, 75.06, 73.44, 66.69, 66.57, 65.71, 57.44, 50.63, 37.08,

36.93, 36.38, 29.32, 27.91, 27.82, 25.75, 24.44; ΗRMS-ESI

(m/z) [M+H]⁺calcd for C₃₃H₄₃N₂O₉611.2969, found

611.2964. 17: 37% yield; R_f= 0.40 in 5% MeOH / CH₂Cl₂; ¹H

NMR (500 MHz, Acetone-d6) δ 7.42-7.24 (m, 10H), 6.55 (bs,

1H), 6.16 (bd, J = 9.7 Hz, 1H), 5.13-5.00 (m, 4H), 4.22 (dd, J

= 12.4, 8.5 Hz, 1H), 3.96 (dd, J = 9.8, 9.8 Hz, 1H), 3.84-3.66

(m, 3H), 3.65-3.54 (m, 3H), 3.51 (d, J = 9.2 Hz, 1H), 3.37 (d,

J = 4.1 Hz, 1H), 2.44 (dd, J = 14.6, 11.3 Hz, 1H), 2.02-1.82

(m, 3H), 1.77-1.52 (m, 10H), 1.45-1.32 (m, 2H); ¹³C NMR

(250 MHz, Acetone-d6) δ 156.85, 156.53, 138.39, 129.20,

129.18, 128.65, 128.59, 111.34, 84.49, 78.44, 76.24, 74.01,

71.93, 66.74, 66.50, 65.55, 57.09, 50.45, 37.18, 36.90, 36.36,

27.35, 27.22, 25.77, 24.47, 24.41; ΗRMS-ESI (m/z) [M+H]⁺

calcd for C₃₃H₄₃N₂O₉611.2969, found 611.2963.

4.4. Synthesis of spirocyclic intermediates 12 and 13

through Ring Closing Metathesis.

4.4.1. Synthesis of 12: To a stirred solution of diene 10 (50

mg, 8.4 x 10-2 mmoles, 1.0 eq) in CH₂Cl₂(16 mL) at ambient

temperature, was added Grubbs’ second generation catalyst (7

mg, 8.4 x 10-3 mmoles, 0.1 eq) and the solution was allowed

to stir for 12 h (color change from green to dark brown). The

solvent was removed in vacuo and the crude product was

purified by flash chromatography on silica gel (20→30%

EtOAc / Hexanes) to furnish spiro ether 12 (43 mg, 90%

yield) as a white amorphous solid. R_f= 0.26 in 30% EtOAc /

1

Hexanes. H NMR (500 MHz, CDCl₃) δ 7.40-7.28 (m, 10H),

5.85-5.78 (m, 1H), 5.70 (d, J = 10.5 Hz, 1H), 5.15-5.03 (m,

5H), 4.57 (d, J = 15.0 Hz, 1H), 4.07 (d, J = 16.0 Hz, 1H),

3.87 (dd, J = 9.44, 9.44 Hz, 1H), 3.81-3.72 (m, 1H), 3.66-3.56

(m, 1H), 3.36 (d, J = 9.0 Hz, 1H), 2.46 (d, J = 17.7 Hz, 1H),

2.34 (dd, J = 6.48, 6.48 Hz, 1H), 2.06-1.95 (m, 1H), 1.70-1.29

(m, 11H); ΗRMS-ESI (m/z) [M+H]⁺calcd for C₃₂H₃₉N₂O₇

563.2757, found 563.2752.

4.4.2. Synthesis of 13: According to the procedure described

for 12, 64 mg of olefin 13 was isolated as an amorphous white

1

4.6. Removal of the ketal protection from 14-17.

solid in 84% yield. R_f= 0.35 in 33% EtOAc / Hexanes. H

NMR (500 MHz, CDCl₃) δ 7.39-7.28 (m, 10H), 5.68-5.61 (m,

1H), 5.58-5.51 (m, 1H), 5.25 (d, J = 9.5 Hz, 1H), 5.13-5.01

(m, 5H), 4.92 (bs, 1H), 4.04 (dd, J = 16.3, 4.6 Hz, 1H), 3.84

(dd, J = 9.5, 9.5 Hz, 1H), 3.79-3.70 (m, 2H), 3.35 (d, J = 9.0

Hz, 1H), 2.70-2.51 (m, 2H), 2.25-2.18 (m, 2H), 1.88-1.81 (m,

1H), 1.73-1.51 (m, 8H), 1.44 (dd, J = 12.0, 12.0 Hz, 2H), 1.31

(bs, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 155.76, 155.67,

136.58, 136.50, 131.09, 128.63, 128.60, 128.27, 128.22,

111.59, 83.77, 77.27, 75.37, 66.95, 66.89, 64.17, 56.14, 50.06,

36.44, 36.24, 36.17, 33.54, 25.92, 25.12, 23.83, 23.81;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₃₃H₄₁N₂O₇577.2914,

found 577.2917.

4.6.1 Dibenzyl((3R,4S,6S,7S,9R,10S,11R)-3,4,10,11-tetrahy

droxy-1-oxaspiro[5.5]undecane-7,9-diyl)dicarbamate 18:

Ketal 14 (225 mg, 3.8 x 10^-1mmoles, 1 eq) was dissolved in

MeOH (10 mL) and 0.1 mL of concentrated HCl (3.2 eq)

were added. After stirring for 3 h, the reaction was quenched

with Et₃N to pH 8 and the mixture was concentrated to

dryness. Flash column chromatography (5-10% MeOH /

CH₂Cl₂) furnished 173 mg of 18 as a white amorphous solid

1

(90%). R_f= 0.59 in 10% MeOH / CH₂Cl₂. H NMR (500

MHz, Acetone-d6) δ 7.45-7.23 (m, 10H), 6.30 (bs, 1H), 5.94

(d, J = 8.6 Hz, 1H), 5.15-4.97 (m, 4H), 4.45 (bs, 1H), 4.35 (d,

J = 11.6 Hz, 1H), 4.28 (bs, 1H), 4.22 (bs, 1H), 3.78 (d, J =

12.2 Hz, 1H), 3.71-3.60 (m, 3H), 3.59-3.37 (m, 4H), 1.79-1.63

(m, 2H), 0.89-0.77 (m, 2H); ¹³C NMR (500MHz, Acetone-d6)

δ 157.33, 156.89, 138.47, 138.44, 129.20, 128.66, 128.57,

82.97, 77.88, 73.95, 69.23, 68.66, 66.86, 66.59, 66.58, 54.29,

4.5. Upjohn dihydroxylation of olefins 12 and 13.

4.5.1. Synthesis of diol 14: To a solution of olefin 12 (300

mg, 5.3 x 10^-1mmoles, 1.0 eq) in acetone/H₂O (4:1 v/v, 20

mL), was added N-methylmorpholine N-oxide (192 mg, 1.6

53.18, 34.47, 34.35; ΗRMS-ESI (m/z) [M+Na]⁺calcd for

C₂₆H₃₂N₂NaO₉539.2006, found 553.1998.

According to the procedure described for 26, 140 mg of

tetraol 21 furnished 63 mg of tetraol amine 29 as a white

amorphous solid (61%). R_f= 0.57 in 10% ΝΗ₄ΟΗ / ΜeOH.

¹H NMR (500MHz, CD₃OD) δ 7.49-7.39 (m, 5H), 5.19 (d, J

= 12.6 Hz, 1H), 5.12 (d, J = 12.6 Hz, 1H), 4.35 (d, J = 13.7

Hz, 1H), 3.95 (dd, J = 13.4 Hz, J = 4.6 Hz, 1H), 3.90-3.77

(m, 3H), 3.73 (dd, J = 11.7 Hz, J = 3.8 Hz, 1H), 3.41 (d, J =

9.6 Hz, 1H), 3.23-3.16 (m, 1H), 2.32-2.24 (m, 1H), 2.18-1.98

(m, 3H), 1.90-1.71 (m, 2H); ¹³C NMR (500 MHz, CD₃OD) δ

159.19, 138.12, 130.35, 130.19, 129.91, 129.18, 80.79, 79.06,

75.73, 73.63, 72.31, 68.65, 66.02, 56.44, 52.67, 31.61, 29.22,

27.64; ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₉H₂₉N₂O₇

397.1975, found 397.1971.

4.6.2.

Dibenzyl((1S,3R,4S,5R,6S,9R,10S)-4,5,9,10-tetra

hydroxy-7-oxaspiro[5.6]dodecane-1,3-diyl)dicarbamate

19: According to the procedure described for tetraol 18, 111

mg of ketal 15 furnished 95 mg of tetraol 19 as a white

1

amorphous solid (74%). H NMR (500 MHz, Acetone-d6) δ

7.42-7.18 (m, 10H), 6.63 (d, J = 9.2 Hz, 1H), 6.27 (bs, 1H),

5.10-5.02 (m, 4H), 4.48 (d, J = 13.5 Hz, 1H), 3.75 (dd, J =

13.6 Hz, J = 4.5Hz, 1H), 3.68-3.58 (m, 4H), 3.54-3.45 (m,

2H), 3.30-3.25 (m, 1H), 2.44 (dd, J = 14.0, 14.0 Hz, 1H), 2.21

(dd, J = 24.8, 12.7 Hz, 1H), 1.89-1.67 (m, 3H), 1.67-1.58 (m,

1H); ΗRMS-ESI (m/z) [M+Na]⁺calcd for C₂₇H₃₄N₂NaO₉

553.2162, found 553.2155.

4.8. Synthesis of intermediates 30-36.

4.6.3. Dibenzyl ((1S,3R,4S,5R,6S,9S,10R)-4,5,9,10-tetra

hydroxy-7-oxaspiro[5.6]dodecane-1,3-diyl)dicarbamate

21: According to the procedure described for tetraol 18, 111

mg of ketal 17 furnished 95 mg of tetraol 21 as a white

amorphous solid (74%). R_f= 0.26 in 10% MeOH / CH₂Cl₂. ¹H

NMR (250 MHz, Acetone-d6) δ 7.43-7.22 (m, 10H), 6.41 (bd,

J = 6.5 Hz, 1H), 6.27 (d, J = 9.8 Hz, 1H), 5.13-5.00 (m, 4H),

4.63 (bs, 1H), 4.35 (dd, J = 12.2, 8.3 Hz, 2H), 4.21 (bs, 1H),

3.74-3.46 (m, 6H), 3.38 (bd, J = 8.2 Hz, 1H), 2.98 (bs, 1H),

2.48 (dd, J = 14.8, 10.5 Hz, 1H), 2.26-2.09 (m, 1H), 1.95-1.61

(m, 4H); ¹³C NMR (250 MHz, Acetone-d6) δ 157.34, 157.02,

138.26, 138.24, 129.14, 128.60, 128.50, 80.36, 80.03, 74.27,

74.00, 73.02, 66.77, 66.59, 65.47, 56.31, 52.81, 34.64, 27.97,

27.84; ΗRMS-ESI (m/z) [M+Na]⁺calcd for C₂₇H₃₄N₂NaO₉

553.2162, found 553.2149.

4.8.1. (S)-2,6-bis(carbobenzyloxyamino)-N-((3R,4S,6S,7R,

8S,9R,11S)-3,4,7,8-tetrahydroxy-11-(carbobenzyloxyami-

no)-1-oxaspiro[5.5]undecan-9-yl)hexanamide 30: Amine 26

(14 mg, 3.7 x 10^-2mmoles, 1.0 eq) was dissolved in

dioxane/H₂O (1.33 mL, 3:1), followed by the addition of

saturated NaHCO₃(50 μL) and Z-Lys(Z)-OSu (0.5 mL of

0.1Μ solution in ΤΗF, 5.0 x 10^-2mmoles, 1.35 eq) and the

mixture was stirred at ambient temperature for 15 h. After that

time, the reaction was diluted with EtOAc (5.0 mL) and

extracted with saturated NaCl (3 x 5.0 mL). The organic

extract was concentrated to dryness and purified by flash

chromatography (5→10% ΜeOH / 1% NH₄OH / CH₂Cl₂) to

furnish 22 mg of amide 30 as a white amorphous solid (88%).

1

R_f= 0.25 in 10% ΜeOH / 1% NH₄OH / CH₂Cl₂. H NMR

(500 MHz, Acetone-d6) δ 7.47-7.18 (m, 16H), 6.62-6.46 (m,

1H), 6.42-6.26 (m, 1H), 6.00 (bd, J = 8.3 Hz, 1H), 5.17-4.95

(m, 6H), 4.47 (bs, 1H), 4.39-4.25 (m, 1H), 4.20-4.10 (m, 1H),

3.86-3.59 (m, 5H), 3.54-3.39 (m, 2H), 3.17-3.06 (m, 1H), 2.93

(bs, 1H), 2.01-1.33 (m, 10H); ¹³C NMR (125 MHz, Acetone-

d6): δ 173.31, 157.31, 157.16, 156.91, 138.49, 138.31,

138.10, 129.22, 129.20, 129.17, 128.63, 128.60, 128.56,

128.53, 82.75, 77.85, 74.02, 69.15, 68.58, 66.86, 66.84, 66.65,

66.40, 56.07, 54.25, 51.40, 41.20, 34.31, 33.67, 33.02, 30.29,

23.48; ΗRMS-ESI (m/z) [M+Na]⁺calcd for C₄₀H₅₀N₄NaO₁₂

801.3323, found 801.3312.

4.7. Selective cleavage oft he Cbz-group at position 1.

4.7.1. Benzyl ((3R,4S,6S,7S,9R,10S,11R)-9-amino-3,4,10,11

-tetrahydroxy-1-oxaspiro[5.5]undecan-7-yl)carba-mate 26:

Tetraol 18 (100 mg, 1.9 x 10^-1mmoles, 1.0 eq) was dissolved

in dioxane (9.5 mL) and LiOH (0.5M, 9.5 mL, 5.0 mmoles,

25.7 eq) was added with stirring and the reaction mixture was

heated to 70°C for 4 h. After the completion of the reaction

(TLC monitoring), the solvents were removed under vacuum

and the product was purified by column chromatography (2.5-

5% ΝΗ₄OH / MeOH) to furnish 45 mg of 26 as a white

amorphous solid (61%). R_f= 0.66 in 10% ΝΗ₄OH / MeOH.

¹H NMR (500 MHz, D₂O) δ 7.56-7.34 (m, 5H), 5.24-5.07 (m,

2H), 4.51 (bs, 1H), 4.27 (d, J = 8.1Hz, 1H), 3.95-3.72 (m,

3H), 3.61-3.41 (m, 2H), 3.27-3.13 (m, 1H), 2.16-1.83 (m, 3H),

1.77-1.64 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 158.4, 136.9,

129.2, 128.7, 127.9, 80.1, 78.1, 70.7, 68.3, 67.5, 67.2, 66.0,

52.8, 51.5, 31.7, 29.9; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₁₈H₂₇N₂O₇383.1818, found 383.1814.

4.8.2.

(S)-2,6-bis(carbobenzyloxyamino)-N-((1R,2S,3R,

5S,6S,9S,10R)-1,2,9,10-tetrahydroxy-5-(carbobenzyloxy-

amino)-7-oxaspiro[5.6]dodecan-3-yl)hexanamide

31:

According to the procedure described for 30, 16 mg of amine

29 furnished 21 mg of amide 31 as a white amorphous solid

1

(79%). R_f= 0.28 in 10% ΜeOH / 1% NH₄OH / CH₂Cl₂. H

NMR (250MHz, CD₃OD) δ 7.99 (bd, J = 7.6 Hz, 1H), 7.43-

7.19 (m, 15H), 7.17-6.92 (m, 3H), 5.12 (d, J = 12.5 Hz, 1H),

5.07 (s. 2H), 5.06 (d, J = 12.5 Hz, 1H), 5.05 (s, 2H), 4.23 (dd,

J = 13.0, 7.8 Hz, 1H), 4.12-3.97 (m, 1H), 3.77-3.43 (m, 6H),

3.29-3.18 (m, 1H), 3.09 (dd, J = 12.3, 6.2 Hz, 2H), 2.38-2.07

(m, 2H), 1.86-1.24 (m, 10H); ¹³C NMR (63 MHz, CD₃OD): δ

174.72, 158.94, 158.41, 158.37, 138.44, 138.13, 129.47,

129.45, 129.04, 128.94, 128.88, 128.78, 80.85, 80.83, 74.74,

74.11, 74.01, 67.71, 67.60, 67.34, 65.75, 56.55, 56.45, 51.76,

41.40, 33.84, 33.15, 30.49, 28.60, 28.08, 23.95; ΗRMS-ESI

(m/z) [M+Na]⁺calcd for C₄₁H₅₂N₄NaO₁₂815.3479, found

815.3468.

4.7.2. Benzyl((1S,3R,4S,5R,6S,9R,10S)-3-amino-4,5,9,10 -

tetrahydroxy-7-oxaspiro[5.6] dodecan-1-yl)carbamate 27:

According to the procedure described for 26, 50 mg of tetraol

19 furnished 35 mg of tetraol amine 27 as a white amorphous

solid (95%). R_f= 0.57 in 10% ΝΗ₄ΟΗ / ΜeOH. ¹H NMR (500

MHz, CD₃OD) δ 7.40-7.26 (m, 6H), 5.11 (d, J = 12.5 Hz,

1H), 5.08 (d, J = 12.5 Hz, 1H), 4.57 (d, J = 13.5 Hz, 1H),

3.92-3.81 (m, 1H), 3.79-3.48 (m, 4H), 3.16 (d, J = 9.5 Hz,

1H), 3.08-2.99 (m, 1H), 2.41-2.31 (m, 1H), 2.19 (dd, J =

12.5Hz, J = 12.5Hz, 1H), 2.06-1.90 (m, 1H), 1.87-1.74 (m,

1H), 1.73-1.54 (m, 2H); ¹³C NMR (500MHz, CD₃OD) δ

158.3, 138.3, 129.5, 129.0, 128.8, 80.6, 79.3, 76.9, 73.9, 73.5,

67.6, 66.2, 57.2, 52.9, 33.0, 29.7, 28.3; ΗRMS-ESI (m/z)

[M+H]⁺calcd for C₁₉H₂₉N₂O₇397.1975, found 397.1973.

4.8.3. (S)-2-hydroxy-4-(carbobenzyloxyamino)-N-((3R,4S,

6S,7R,8S,9R,11S)-3,4,7,8-tetrahydroxy-11-(carbobenzyl-

oxyamino)-1-oxaspiro[5.5]undecan-9-yl)butanamide 32:

According to the procedure described for 30, 20 mg of amine

26 furnished 15 mg of amide 32 as a white amorphous solid

1

(47%). R_f= 0.32 in 10% ΜeOH / CH₂Cl₂. H NMR (250

4.7.3. Benzyl((1S,3R,4S,5R,6S,9S,10R)-3-amino-4,5,9,10 -

tetrahydroxy-7-oxaspiro[5.6] dodecan-1-yl)carbamate 29:

MHz, CD₃OD) δ 7.43-7.22 (m, 10H), 6.86 (bd, J = 9.2 Hz,

1H), 5.17-4.97 (m, 4H), 4.58-4.33 (m, 2H), 4.06 (dd, J = 7.6,

3.8 Hz, 1H), 3.84-3.58 (m, 4H), 3.47 (dd, J = 10.7, 5.3 Hz,

1H), 3.39-3.19 (m, 4H), 2.14-1.72 (m, 5H), 1.65 (dd, J = 12.9,

5.7 Hz, 1H); ¹³C NMR (63 MHz, CD₃OD) δ 176.80, 158.95,

158.50, 138.41, 138.26, 129.46, 128.96, 128.94, 128.87,

128.80, 82.92, 78.89, 73.84, 70.86, 69.26, 69.07, 67.59,

67.43,67.40, 54.65, 51.41, 38.12, 35.62, 33.93, 33.84;

ΗRMS-ESI (m/z) [M+Na]⁺calcd for C₃₀H₃₉N₃NaO₁₁

640.2482, found 640.2475.

bobenzyloxyamino)-7-oxaspiro[5.6]dodecan-3-yl)propan-

amide 36: According to the procedure described for 30, 20

mg of amine 29 furnished 14 mg of amide 36 as a white

amorphous solid (34%). R_f= 0.30 in 10% ΜeOH / CH₂Cl₂. ¹H

NMR (250 MHz, CD₃OD) δ 7.42-7.18 (m, 10H), 7.03 (d, J =

8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 5.20-4.94 (m, 4H),

4.30-4.16 (m, 2H), 3.68-3.42 (m, 6H), 3.23 (d, J = 9.1 Hz,

1H), 2.96 (dd, J = 13.6, 6.8Hz, 1H), 2.77 (dd, J = 13.6, 8.1

Hz, 1H), 2.37-2.08 (m, 2H), 1.84-1.45 (m, 4H); ¹³C NMR (63

MHz, CD₃OD) δ 173.95, 158.41, 158.17, 157.27, 138.49,

138.18, 131.47, 129.47, 129.05, 128.95, 128.93, 128.68,

116.21, 80.84, 80.79, 74.75, 74.15, 73.99, 67.61, 67.55, 65.74,

58.31, 56.47, 51.67, 38.81, 33.72, 28.59, 28.09; ΗRMS-ESI

(m/z) [M+Na]⁺calcd for C₃₆H₄₃N₃NaO₁₁716.2795, found

716.2783.

4.8.4.

2S,3R,5S,6S,9S,10R)-1,2,9,10-tetrahydroxy-5-(carbobenzyl

oxyamino)-7-oxaspiro[5.6]dodecan-3-yl)butanamide 33:

(S)-2-hydroxy-4-(carbobenzyloxyamino)-N-((1R,

According to the procedure described for 30, 16 mg of amine

29 furnished 9 mg of amide 33 as a white amorphous solid

(45%). R_f= 0.35 in 10% ΜeOH / CH₂Cl₂.¹H NMR (250 MHz,

CD₃OD) δ 7.41-7.23 (m, 10H), 7.13 (bd, J = 9.6 Hz, 1H),

5.13 (d, J = 12.5 Hz, 1H), 5.06 (s, 2H), 5.04 (d, J = 12.5 Hz,

1H), 4.22 (dd, J = 13.0 Hz, J = 7.8Hz, 1H), 4.05 (dd, J = 7.7,

4.0 Hz, 1H), 3.74-3.49 (m, 6H), 3.30-3.19 (m, 3H), 2.33-2.13

(m, 2H), 2.02-1.61 (m, 6H); ¹³C NMR (63 MHz, CD₃OD): δ

176.80, 158.95, 158.41, 138.47, 138.41, 129.46, 129.04,

128.95, 128.93, 128.82, 80.95, 80.93, 74.73, 74.11, 70.78,

67.58, 67.39, 65.74, 65.73, 56.47, 51.31, 38.04, 35.60, 33.87,

28.75, 28.10; ΗRMS-ESI (m/z) [M+Na]⁺calcd for

C₃₁H₄₁N₃NaO₁₁654.2639, found 654.2637.

4.9. Synthesis of final products 37-43.

4.9.1.

(S)-2,6-diamino-N-((3R,4S,6S,7R,8S,9R,11S)-11-

amino-3,4,7,8-tetrahydroxy-1-oxaspiro[5.5]undecan-9-yl)-

hexanamide 37: Amide 30 (8 mg, 2 x 10^-2mmoles, 1.0 eq)

was dissolved in MeOH (5 mL) and Pd/C (10%wt., 15 mg)

was added. The reaction mixture was degassed (x3), charged

with H₂and stirred for 3 h at ambient temperature. After

filtration through celite and removal of the solvent under

reduced pressure, the product was purified by column

chromatography on silica gel (20% NH₄OH / MeOH)

resulting in the isolation of 7 mg of 37 as a white amorphous

solid (72%). R_f= 0.19 in 20% NH₄OH / MeOH. ¹H NMR

(250 MHz, D₂O) δ 4.49 (ddd, J = 9.9, 5.8, 2.7 Hz, 1H), 4.21

(dd, J = 13.2, 2.5 Hz, 1H), 3.97 (dd, J = 6.7, 6.7 Hz, 1H),

3.92-3.66 (m, 4H), 3.46 (d, J = 9.5 Hz, 1H), 3.39 (dd, J =

12.3, 4.8 Hz, 1H), 2.99 (dd, J = 7.7, 7.7 Hz, 2H), 2.11-1.80

(m, 6H), 1.79-1.61 (m, 2H), 1.45 (dd, J = 15.8, 8.4 Hz, 2H);

¹³C NMR (63MHz, D₂O) δ 170.60, 80.43, 76.55, 72.20, 68.80,

67.44, 66.34, 54.24, 53.78, 50.59, 40.02, 32.84, 31.36, 30.70,

27.42, 22.41; ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₆H₃₃N₄O₆

377.2400, found 377.2403.

4.8.5. (S)-3-(4-hydroxyphenyl)-2-(carbobenzyloxyamino)-

N-((3R,4S,6S,7R,8S,9R,11S)-3,4,7,8-tetrahydroxy-11-(car-

bobenzyloxyamino)-1-oxaspiro[5.5]undecan-9-yl)propane-

amide 34: According to the procedure described for 30, 14

mg of amine 26 furnished 8 mg of amide 34 as a white

amorphous solid (33%). R_f= 0.37 in 10% ΜeOH / CH₂Cl₂. ¹H

NMR (250 MHz, CD₃OD) δ 7.45-7.21 (m, 10H), 7.03 (d, J =

8.4 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 5.18-4.97 (m, 4H),

4.56-4.33 (m, 2H), 4.24 (dd, J = 7.4, 7.4 Hz, 1H), 3.79-3.63

(m, 3H), 3.56 (dd, J = 9.7, 9.7 Hz, 1H), 3.49-3.35 (m, 2H),

2.94 (dd, J = 13.5, 6.9 Hz, 1H), 2.79 (dd, J = 13.5, 7.8 Hz,

1H), 2.04 (dd, J = 13.5, 13.5 Hz, 1H), 1.74-1.49 (m, 3H); ¹³

C

4.9.2.

(S)-2,6-diamino-N-((1R,2S,3R,5S,6S,9S,10R)-5-

NMR (63 MHz, CD₃OD): δ 174.00, 158.52, 158.18, 157.35,

138.21, 131.45, 129.48, 128.96, 128.70, 116.22, 82.81, 78.78,

73.88, 69.22, 69.06, 67.64, 67.57, 67.42, 58.38, 54.59, 51.72,

38.81, 33.87, 33.62; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₃₅H₄₂N₃NaO₁₁716.2795, found 716.2784; ΗRMS-ESI (m/z)

[M+Na]⁺calcd for C₃₅H₄₁N₃NaO₁₁702.2639, found 702.2635.

amino-1,2,9,10-tetra-hydroxy-7-oxaspiro[5.6]dodecan-3-

yl) hexanamide 38: According to the procedure described for

37, 21 mg of amide 31 furnished 8.5 mg of 38 as a white

amorphous solid (64%). R_f= 0.2 in 20% NH₄OH / MeOH. ¹H

NMR (250 MHz, D₂O) δ 4.38 (dd, J = 12.8, 9.1 Hz, 1H),

3.98-3.88 (m, 2H), 3.87-3.74 (m, 1H), 3.73-3.62 (m, 2H), 3.55

(bd, J = 13.0 Hz, 1H), 3.41-3.26 (m, 2H), 2.96 (dd, J = 7.8,

7.5 Hz, 2H), 2.57 (dd, J = 15.8, 11.5 Hz, 1H), 2.16 (ddd, J =

15.2, 7.8, 7.8 Hz, 1H), 1.96-1.81 (m, 4H), 1.78-1.58 (m, 4H),

1.41 (dd, J = 14.9, 7.6 Hz, 2H); ¹³C NMR (63 MHz, D₂O) δ

170.87, 78.94, 78.70, 73.89, 73.09, 71.79, 65.31, 56.60, 54.52,

50.69, 40.31, 31.64, 31.12, 27.69, 27.67, 26.31, 22.71;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₇H₃₅N₄O₆391.2557,

found 391.2552.

4.8.6. (S)-3-(4-hydroxyphenyl)-2-(carbobenzyloxyamino)-

N-((1R,2S,3R,5S,6S,9R,10S)-1,2,9,10-tetrahydroxy-5-car-

bobenzyloxyamino)-7-oxaspiro[5.6]dodecan-3-yl)propan-

amide 35: According to the procedure described for 30, 15

mg of amine 27 furnished 11 mg of amide 35 as a white

amorphous solid (54%). R_f= 0.32 in 10% ΜeOH / CH₂Cl₂. ¹H

NMR (500 MHz, CD₃OD) δ 7.43-7.17 (m, 10H), 7.03 (d, J =

8.1 Hz, 2H), 6.68 (d, J = 8.3 Hz, 2H), 5.09 (s, 2H), 5.06 (d, J

= 13.1 Hz, 1H), 5.00 (d, J = 12.4 Hz, 1H), 4.56 (d, J = 13.5

Hz, 1H), 4.25 (dd, J = 7.2, 7.1 Hz, 1H), 3.73-3.63 (m, 3H),

3.63-3.53 (m, 3H), 3.15 (d, J = 9.5 Hz, 1H), 2.95 (dd, J =

13.6, 6.7 Hz, 1H), 2.78 (dd, J = 13.3, 8.2 Hz, 1H), 2.37 (dd, J

= 13.7, 13.7 Hz, 1H), 2.18 (dd, J = 24.6, 12.4Hz, 1H), 1.75-

1.45 (m, 5H); ¹³C NMR (63 MHz, CD₃OD): δ 173.98, 158.31,

158.18, 157.30, 138.40, 138.18, 131.45, 129.63, 129.47,

129.20, 128.95, 128.79, 128.67, 116.21, 80.47, 79.86, 76.98,

74.01, 73.91, 67.58, 67.55, 66.08, 58.35, 57.48, 51.95, 38.81,

34.15, 29.74, 28.32; ΗRMS-ESI (m/z) [M+Na]⁺calcd for

C₃₆H₄₃N₃NaO₁₁716.2795, found 716.2784.

4.9.3. (S)-4-amino-N-((3R,4S,6S,7R,8S,9R,11S)-11-amino-

3,4,7,8-tetrahydroxy-1-oxaspiro[5.5]undecan-9-yl)-2-hydro

xybutanamide 39: According to the procedure described for

37, 17 mg of amide 32 furnished 7.5 mg of 39 as a white

amorphous solid (78%). R_f= 0.19 in 20% NH₄OH / MeOH.

¹H NMR (250 MHz, D₂O) δ 4.53-4.41 (m, 1H), 4.28 (dd, J =

7.8, 4.1 Hz, 1H), 4.19 (dd, J = 13.0, 2.3 Hz, 1H), 3.90-3.69

(m, 4H), 3.48-3.29 (m, 2H), 3.12 (dd, J = 7.2, 7.2 Hz, 1H),

2.22-1.73 (m, 6H); ¹³C NMR (63 MHz, D₂O) δ 176.56, 80.65,

76.65, 72.11, 70.70, 68.85, 67.53, 66.44, 53.99, 50.26, 37.65,

32.99, 31.93, 30.75; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₁₄H₂₈N₃O₇350.1927, found 350.1923.

4.8.7. (S)-3-(4-hydroxyphenyl)-2-(carbobenzyloxyam- ino)-

N-((1R,2S,3R,5S,6S,9S,10R)-1,2,9,10-tetrahydroxy-5-(car-

4.9.4. (S)-4-amino-N-((1R,2S,3R,5S,6S,9S,10R)-5-amino-

1,2,9,10-tetrahydroxy-7-oxaspiro[5.6]dodecan-3-yl)-2-

hydroxybutanamide 40: According to the procedure

described for 37, 9 mg of amide 33 furnished 2 mg of 40 as a

white amorphous solid (32%). R_f= 0.2 in 20% NH₄OH /

mixture is cooled to -10°C and stirred for 6 h while slowly

warming up to ambient temperature. After consumption of the

starting acid, the reducing reagent is quenched with 0.5 mL of

methanol and concentrated to dryness under reduced pressure.

Purification by column chromatography on silica gel (5%

ΜeOH / CH₂Cl₂) furnished 89 mg of the corresponding

alcohol as a white amorphous solid (93%). R_f=0.38 in 10%

ΜeOH / CH₂Cl₂. ¹H NMR (500 MHz, DMSO-d6) δ 7.36-7.23

(m, 10H), 7.20-7.16 (m, 1H), 6.87-6.82 (m, 1H), 5.00-4.92

(m, 4H), 3.80-3.73 (s, 1H), 2.96-2.85 (m, 2H), 2.50-2.41 (d,

1H), 1.65 (s, 1H), 1.57-1.43 (m, 1H), 1.40-1.28 (m, 2H), 1.26-

1.17 (m, 2H); ¹³C NMR (500 MHz, DMSO-d6) δ 156.05,

155.61, 137.28, 137.22, 128.29, 127.68, 127.33, 65.13, 65.07,

55.04, 40.30, 31.84, 29.29, 22.63.

1

MeOH. H NMR (250 MHz, D₂O) δ 4.39 (dd, J = 12.7, 9.1

Hz, 1H), 4.28 (dd, J = 7.8, 4.2 Hz, 1H), 3.99-3.88 (m, 1H),

3.83-3.64 (m, 3H), 3.56 (bd, J = 12.6 Hz 1H), 3.39 (d, J = 9.1

Hz, 1H), 3.32 (dd, J = 11.3 Hz, J = 5.3 Hz, 1H), 3.11 (dd, J =

7.2, 7.2 Hz, 2H), 2.70-2.50 (m, 1H), 2.27-1.83 (m, 5H), 1.79-

1.56 (m, 2H); ¹³C NMR (63 MHz, D₂O) δ 176.79, 79.04,

78.76, 73.91, 72.93, 71.84, 70.92, 65.27, 56.69, 50.28, 37.84,

32.13, 31.07, 27.65, 26.38; ΗRMS-ESI (m/z) [M+H]⁺calcd

for C₁₅H₃₀N₃O₇364.2084, found 364.2082.

4.9.5. (S)-2-amino-N-((3R,4S,6S,7R,8S,9R,11S)-11-amino-

3,4,7,8-tetrahydroxy-1-oxaspiro[5.5]undecan-9-yl)-3-(4-

hydroxyphenyl)propanamide 41: According to the

procedure described for 37, 9 mg of amide 34 furnished 2 mg

of 41 as a white amorphous solid (35%). R_f= 0.63 in 20%

NH₄OH / MeOH. ¹H NMR (500 MHz, D₂O) δ 7.17 (d, J =

8.2 Hz, 2H), 6.91 (d, J = 8.1 Hz, 2H), 4.49 (m, 1H), 4.18 (d, J

= 11.0 Hz, 1H), 4.10 (dd, J = 8.7, 6.4 Hz, 1H), 3.89-3.82 (m,

2H), 3.80-3.71 (m, 1H), 3.58 (dd, J = 10.0, 10.0 Hz, 1H), 3.42

(d, J = 9.7 Hz, 1H), 3.33 (dd, J = 8.9, 8.9 Hz, 1H), 3.21 (dd, J

= 13.6, 6.1 Hz, 1H), 3.04 (dd, J = 13.2, 9.5 Hz, 1H), 1.96 (dd,

J = 13.6, 5.3 Hz, 1H), 1.83 (dd, J = 13.3, 10.7 Hz 1H), 1.75-

1.68 (m, 1H), 1.51 (dd, J = 24.8, 12.3 Hz, 1H); ¹³C NMR (63

MHz, D₂O) δ 170.18, 156.32, 132.19, 127.05, 117.16, 80.59,

76.71, 72.42, 68.95, 67.69, 66.61, 56.13, 53.95, 50.39, 37.36,

33.06, 30.72; ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₉H₃₀N₃O₇

412.2084, found 412.2081.

To a solution of oxalyl chloride (50 μL, 5.9 x 10^-1mmoles,

3.7 eq) in CH₂Cl₂(1.0 mL) at -63°C there was added dropwise

a solution of 46 μL DMSO in 1.0 mL of CH₂Cl₂followed by a

dropwise addition of a solution of the alcohol (64 mg, 1.6 x

10^-1mmoles, 1.0 eq) in 1.3 mL of CH₂Cl₂and the mixture is

stirred at -78°C for 20 minutes. Et₃N (83 μL, 5.9 x 10^-1

mmoles, 3.7 eq) is then added dropwise and the cooling bath

is removed. The reaction is further diluted with hexanes (5.0

mL) and quenched with 2.0 mL of saturated NaHSO₄solution

with vigorous stirring. The organic phase is separated and the

aqueous is washed with diethyl ether (2x3 mL). The combined

organic extracts are further washed with sat. NaHCO₃(2 x 3

mL), H₂O (3 x 3 mL) and brine (2 x 2 mL), dried with MgSO₄

and concentrated to dryness. The product aldehyde 45 (50 mg,

78% yield) was of sufficient purity for the next steps. R_f=0.73

in 50% ΕtOAc / CH₂Cl₂. ¹H NMR (500MHz, CDCl₃) δ 9.55

(s, 1H), 7.39-7.24 (m, 10H), 5.63-5.53 (m, 1H), 5.16-4.98 (m,

4H), 4.92 (bs, 1H), 4.30-4.18 (m, 1H), 3.24-3.07 (m, 2H),

1.95-1.82 (m, 1H), 1.70-1.28 (m, 5H); ¹³C NMR (500MHz,

CDCl₃) δ 199.51, 156.74, 156.37, 136.65, 136.23, 128.65,

128.61, 128.35, 128.21, 67.23, 66.79, 60.10, 40.37, 29.63,

28.53, 22.09.

4.9.6. (S)-2-amino-N-((1R,2S,3R,5S,6S,9R,10S)-5-amino-

1,2,9,10-tetrahydro-xy-7-oxaspiro[5.6]dodecan-3-yl)-3-(4-

hydroxyphenyl)propanamide 42: According to the

procedure described for 37, 11 mg of amide 35 furnished 3

mg of 42 as a white amorphous solid (35%). R_f= 0.65 in 20%

ΝΗ₄ΟΗ / MeOH. ¹H NMR (250 MHz, D₂O) δ 7.11 (d, J = 8.6

Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 4.42 (d, J = 13.5 Hz, 1H),

4.04 (dd, J = 9.3, 6.2 Hz, 1H), 3.82-3.62 (m, 4H), 3.51 (dd, J

= 10.0, 9.7 Hz, 1H), 3.31-3.20 (m, 2H), 3.15 (dd, J = 13.5,

6.2 Hz, 1H), 2.97 (dd, J = 13.5, 9.4 Hz, 1H), 2.48-2.35 (m,

1H), 1.84-1.63 (m, 3H), 1.60-1.40 (m, 2H); ¹³C NMR (63

MHz, D₂O) δ 170.18, 156.34, 132.20, 127.03, 117.17, 78.80,

78.25, 76.01, 72.95, 72.86, 66.12, 56.93, 56.14, 50.55, 37.36,

31.16, 28.58, 28.01; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₂₀H₃₂N₃O₇426.2240, found 426.2235.

4.11. Synthesis of aldehyde 47: Protected aminoacid 46

(100 mg, 2.6 x 10^-1mmoles, 1.0 eq) was reduced to the

corresponding alcohol (86 mg, 80% yield) according to the

procedure presented for the reduction of 44. R_f= 0.52 in 5%

ΜeOH / CH₂Cl₂. ¹H NMR (250 MHz, CDCl₃) δ 7.48-7.31 (m,

4H), 7.15 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 5.04

(s, 2H), 4.95 (d, J = 6.1 Hz, 1H), 3.83 (bs, 1H), 3.64 (dd, J =

11.0, 3.8 Hz, 1H), 3.53 (dd, J = 11.0, 5.1 Hz, 1H), 3.07 (bs,

1H), 2.79 (d, J = 7.0 Hz, 2H), 1.43 (s, 9H); ¹³C NMR (63

MHz, CDCl₃) δ 157.51, 156.28, 137.10, 130.36, 128.61,

127.98, 127.51, 114.91, 79.69, 70.04, 63.96, 53.79, 36.56,

28.42.

4.9.7. (S)-2-amino-N-((1R,2S,3R,5S,6S,9S,10R)-5-amino-

1,2,9,10-tetrahydroxy-7-oxaspiro[5.6]dodecan-3-yl)-3-(4-

hydroxyphenyl)propanamide 43: According to the

procedure described for 37, 14 mg of amide 36 furnished 3.8

mg of 43 as a white amorphous solid (38%). R_f= 0.57 in 20%

ΝΗ₄ΟΗ / MeOH. ¹H NMR (250 MHz, D₂O) δ 7.10 (d, J = 8.5

Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 4.33 (dd, J = 12.7, 9.2 Hz,

1H), 4.03 (dd, J = 9.3, 6.3 Hz, 1H), 3.96-3.87 (m, 1H), 3.71-

3.59 (m, 2H), 3.56-3.41 (m, 2H), 3.35-3.05 (m, 3H), 2.96 (dd,

J = 13.4, 9.5 Hz, 1H), 2.65-2.42 (m, 1H), 2.24-2.04 (m, 1H),

1.77-1.34 (m, 4H); ¹³C NMR (63 MHz, D₂O) δ 170.16,

156.31, 132.20, 127.06, 117.15, 78.80, 78.55, 73.86, 72.97,

71.74, 65.19, 56.47, 56.10, 50.22, 37.36, 30.88, 27.63, 26.14;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₂₀H₃₂N₃O₇426.2240,

found 426.2233.

To the solution of the resulting protected amine (86 mg,

2.4 x 10^-2mg, 1.0 eq) in CH₂Cl₂(3.0 mL) there was added

TFA (1.0 mL) and the reaction mixture was stirred for 1 h at

ambient temperature. After concentration of the solvents

under reduced pressure the product was purified by column

chromatography on silica gel (2.5% MeOH / CH₂Cl₂) to

furnish 53 mg of the aminoalcohol in 85% yield as a white

1

amorphous solid. R_f= 0.59 in 2.5% MeOH / CH₂Cl₂. H

NMR (250 MHz, CD₃OD) δ 7.43-7.21 (m, 5H), 7.16 (d, J =

8.5 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 5.01 (s, 2H), 4.94 (s,

4H), 3.67 (dd, J = 11.6Hz, J = 3.5Hz, 1H), 3.50 (dd, J =

11.6Hz, J = 6.3Hz, 1H), 3.37 (ddd, J = 13.5Hz, J = 7.0Hz, J

= 3.6Hz, 1H), 3.32-3.27 (m, 1H), 2.86 (d, J = 7.3 Hz, 2H);

¹³C NMR (250MHz, CD₃OD) δ 159.36, 138.58, 131.36,

129.45, 129.22, 128.83, 128.48, 116.31, 70.88, 61.33, 55.87,

35.50.

4.10. Synthesis of aldehyde 45: Di-Cbz-lysine 44 (100

mg, 2.4 x 10^-1mmoles, 1.0 eq) is mixed with a cold solution

of BH₃(1M in THF, 4 mL, 4.0 mmoles, 16.6 eq), the reaction

The resulting amine (100 mg, 3.9 x 10^-1mmoles, 1.0 eq)

was dissolved in H₂O/dioxane (2:1, 4.5 mL) followed by the

addition of solid NaHCO₃(163 mg, 1.95 mmoles, 5.0 eq) and

the mixture was stirred at ambient temperature for 10 minutes.

CbzCl (165 μL, 1.17 mmoles, 3.0 eq) was then added and

stirring was continued for 17 h. The reaction was then diluted

with CH₂Cl₂(10 mL), extracted with HCl (1M, 3x10 mL) and

the organic phase was dried with MgSO4, filtered through

celite, concentrated under reduced pressure and purified by

column chromatography on silica gel (57.510 Acetone /

CH₂Cl₂) to produce 112 mg of the corresponding carbamate in

73% yield as a white amorphous solid. R_f= 0.41 in 10%

Hz, 1H), 4.47 (d, J = 11.3 Hz, 1H), 4.15 (dd, J = 7.9, 4.7 Hz,

1H), 3.51-3.40 (m, 2H), 2.14-1.96 (m, 2H).

The resulting ester (110 mg, 3.4 x 10^-1mmoles, 1.0 eq) is

dissolved in toluene (2.0 mL) and cooled to -78°C followed

by the dropwise addition of DIBAL (1M in toluene, 0.68 mL,

6.8 x 10^-1mmoles, 2.0 eq) under argon. Stirring is continued

for 2 h at the same temperature after which time saturated

KNaC₄H₄O₆·4H₂O solution (Rochelle’s salt) is added

dropwise and the mixture is allowed to stir for 7 h at ambient

temperature. Concentration to dryness under reduced pressure

is followed by dilution with CH₂Cl₂(5.0 mL) and extraction

with Η₂Ο (3 x 5.0 mL). The organic phase is concentrated to

dryness under reduced pressure and purified by column

chromatography on silica gel (20→40% hexanes / CH₂Cl₂) to

produce 64 mg of aldehyde 49 as a clear liquid (90%). R_f=

0.68 in 40% hexanes / CH₂Cl₂. ¹H NMR (500 MHz, CDCl₃) δ

9.72 (s, 1H), 7.49-7.29 (m, 5H), 4.75 (d, J = 11.6 Hz, 1H),

4.61 (d, J = 11.6Hz, 1H), 3.94 (dd, J = 6.4, 5.0 Hz, 1H), 3.53-

3.43 (m, 2H), 2.03-1.89 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃) δ 202.84, 137.08, 128.79, 128.47, 128.29, 80.60,

73.09, 47.09, 29.85.

1

Acetone / CH₂Cl₂. H NMR (500MHz, CDCl₃) δ 7.47-7.27

(m, 6H), 7.11 (d, J = 6.9 Hz, 2H), 6.91 (d, J = 7.9Hz, 2H),

5.09-5.02 (m, 5H), 3.90 (bs, 1H), 3.66 (d, J = 9.1Hz, 1H),

3.61-3.53 (m, 1H), 2.80 (d, J = 5.3Hz, 2H), 2.27 (s, 1H); ¹³C

NMR (500MHz, CDCl₃) δ 157.73, 156.63, 137.17, 136.51,

130.39, 129.97, 128.70, 128.66, 128.27, 128.17, 128.07,

127.59, 115.12, 70.17, 66.94, 64.07, 54.37, 36.57.

Finally, the resulting alcohol (44 mg) is oxidized to the

desired aldehyde 47 (32 mg, 73%) as previously described for

1

45. R_f= 0.70 in 50% ΕtOAc / CH₂Cl₂. H NMR (500 MHz,

4.13. Synthesis of intermediates 50-55.

C₆D₆) δ 9.32 (s, 1H), 7.50-7.21 (m, 10H), 6.99-6.89 (m, 4H),

5.25-5.16 (m, 3H), 4.86 (s, 2H), 4.32 (dd, J = 12.6, 6.2 Hz

1H), 2.99 (dd, J = 13.9, 6.1 Hz, 1H), 2.83 (dd, J = 13.8, 6.5

Hz, 1H).

4.13.1.

(3R,4S,6S,7R,8S,9R,11S)-9-(((S)-2,6-bis(carbobe

nzyloxyamino)hexyl)amino)-11-(carbobenzyloxyamino)-1-

oxaspiro[5.5]undecane-3,4,7,8-tetraol 50: According to the

procedure described for the synthesis of amine 51, reaction of

10 mg of amine 26 with aldehyde 45 furnished 8.5 mg of 50

in 43% yield as a white amorphous solid. R_f= 0.27 σε 10%

4.12. Synthesis of aldehyde 49: Aminoacid 48 (700 mg, 5.9

mmoles, 1.0 eq) is dissolved in a 1:1 mixture of H₂O/MeOH

(10.0 mL) followed by the addition of CuSO₄∙5H₂O (146 mg,

0.58 mmoles, 0.1 eq) and Κ₂CO₃(1.62 gr, 11.7 mmoles, 2.0

eq), and the reaction is stirred at ambient temperature while

ΤfN₃(0.5 Μ solution in CH₂Cl₂, 23.5 mL, 11.8 mmoles, 2.0

eq) is added dropwise. Stirring is continued for 15 h after

which time the reaction is concentrated to a minimum volume

(not to dryness due to the explosive nature of the azides) and

then diluted with CH₂Cl₂(20 mL) and extracted with HCl

(1M, 30 mL). The aqueous phase is back-extracted with

CH₂Cl₂(2x10 mL) and diethyl ether (4x10 mL) and the

combined organic extracts are dried with MgSO₄, filtered

through celite and concentrated to dryness. Column

chromatography on silica gel (82% CH₂Cl₂/ 10% EtOAc / 4%

1

ΜeOH / 1% NH₄OH / CH₂Cl₂. H NMR (500 MHz, CD₃OD)

δ 7.40-7.21 (m, 15H), 5.05 (bs, 6H), 4.47 (bs, 1H), 4.41 (bd, J

= 12.1 Hz, 1H), 3.75 (bd, J = 12.0 Hz, 1H), 3.70-3.65 (m,

2H), 3.54-3.45 (m, 1H), 3.41-3.34 (m, 1H), 3.27 (d, J = 9.4

Hz, 1H), 3.15-3.05 (m, 3H), 2.79-2.63 (m, 1H), 2.37-2.25 (m,

2H), 2.06 (dd, J = 12.2. 12.2 Hz, 1H), 1.93-1.80 (m, 1H),

1.66-1.25 (m, 10H); ¹³C NMR (125 MHz, CD₃OD) δ 158.94,

158.92, 158.65, 138.48, 138.43, 138.27, 129.47, 128.98,

128.93, 128.87, 128.75, 128.71, 82.80, 78.95, 74.55, 69.23,

69.11, 67.60, 67.55, 67.40, 67.31, 59.69, 54.82, 52.50, 52.31,

41.56, 34.07, 32.74, 30.60, 30.52, 24.08; ΗRMS-ESI (m/z)

[M+H]⁺calcd for C₄₀H₅₃N₄O₁₁765.3711, found 765.3702.

MeOH

/ 4% CH₃COOH) delivered 650 mg of the

4.13.2.

(1R,2S,3R,5S,6S,9R,10S)-3-(((S)-2,6-bis(carbobe

corresponding azide (76%) as a yellowish thick oil. R_f= 0.42

in 80% CH₂Cl₂/ 10% EtOAc / 5% MeOH / 5% CH₃COOH.

¹H NMR (500 MHz, D₂O) δ 4.30 (dd, J = 7.3 Hz, J = 3.8 Hz,

1H), 3.53-3.44 (s, 2H), 2.10-2.06 (m, 1H), 1.90 (dt, J = 13.6,

6.6 Hz, 1H).

nzyloxyamino)hexyl)amino)-5-(carbobenzyloxyamino)-7-

oxaspiro[5.6]dodecane-1,2,9,10-tetraol 51: Amine 27 (12

mg, 3 x 10^-2mmoles, 1.0 eq) is dissolved in MeOH (2 mL)

followed by the addition of aldehyde 45 (35 mg, 8.8 x 10^-2

mmoles, 2.9 eq), acetic acid (1 μL, 3 x 10^-3mmoles, 0.1 eq)

and ΝaBH₃CN (19 mg, 3 x 10^-1mmoles, 10.0 eq) and the

reaction is allowed to stir for 15 h at ambient temperature. The

mixture is then diluted with CH₂Cl₂(5 mL) and quenched

with HCl (1M, 3 mL ). The organic phase is separated and the

aqueous is washed with CH₂Cl₂(3 x 5.0 mL). The combined

organic extracts are dried with MgSO₄, filtered through celite,

concentrated to dryness under reduced pressure and purified

by column chromatography (10% MeOH / CH₂Cl₂) to furnish

10 mg of amine 51 as a white amorphous solid (44%). R_f=

The corresponding α-hydroxy acid (66 mg, 4.5 x 10^-1

mmoles, 1.0 eq) is dissolved in DMF (4.0 mL) and the

solution is cooled down to 0°C. NaH (99%, 44 mg, 1.8

mmoles, 4.0 eq) is then added and stirring is continued for 10

min at 50°C. BnBr (0.22 mL, 1.8 mmoles, 4.0 eq) and ΤΒΑΙ

(14 mg, 4.5 x 10^-2mmoles, 0.1 eq) are consecutively added

and the reaction is stirred for 1 h at the same temperature.

After complete consumption of the starting material (TLC

monitoring) the reaction is brought to ambient temperature,

diluted with CH₂Cl₂(10 mL) and extracted with HCl (1M,

3x10 mL). The organic phase is dried with Mg₂SO₄, filtered

through celite, concentrated to dryness under reduced pressure

and purified by column chromatography to furnish 82 mg of

the dibenzylated adduct as a yellow oil (80%). R_f= 0.52 in

40% hexanes / CH₂Cl₂. ¹H NMR (500 MHz, CDCl₃) δ 7.47-

7.31 (m, 10H), 5.26 (q, J = 12.2 Hz, 2H), 4.78 (d, J = 11.3

1

0.25 in 10% ΜeOH / 1% NH₄OH / CH₂Cl₂. H NMR (250

MHz, CD₃OD) δ 7.41-7.20 (m, 15H), 5.13-5.00 (m, 6H), 4.56

(d, J = 13.1 Hz, 1H), 3.76-3.62 (m, 3H), 3.61-3.45 (m, 3H),

3.16-3.01 (m, 3H), 2.82 (dd, J = 11.9, 3.8 Hz, 1H), 2.56-2.08

(m, 4H), 1.79-1.23 (m, 10H); ¹³C NMR (63 MHz, CD₃OD): δ

159.01, 158.93, 158.40, 138.47, 138.39, 138.37, 129.48,

128.96, 128.78, 128.76, 80.60, 79.80, 76.96, 74.40, 73.97,

67.58, 67.48, 67.33, 66.06, 59.73, 57.60, 52.21, 52.09, 41.53,

33.94, 32.95, 30.58, 29.88, 28.35, 24.04; ΗRMS-ESI (m/z)

mg of 55 in 35% yield as a white amorphous solid. R_f= 0.32

in 10% ΜeOH / 1% NH₄OH / CH₂Cl₂. H NMR (500MHz,

1

[M+H]⁺calcd for C₄₁H₅₅N₄O₁₁779.8956, found 779.8950.

CD₃OD) δ 7.38-7.24 (m, 15H), 5.16-5.00 (m, 6H), 4.21 (dd, J

= 12.0, 7.5 Hz, 1H), 3.69 (bs, 1H), 3.60-3.51 (m, 3H), 3.51-

3.41 (m, 2H), 3.19 (d, J = 8.9 Hz, 1H), 3.13-3.02 (m, 2H),

2.85-2.76 (m, 1H), 2.55-2.40 (m, 2H), 2.27-2.16 (m, 2H),

1.80-1.22 (m, 12H); ¹³C NMR (126 MHz, CD₃OD) δ 159.01,

158.94, 158.51, 138.50, 138.50, 138.42 129.47, 129.06,

128.93, 128.75, 80.95, 80.81, 74.85, 74.68, 74.14, 67.61,

67.49, 67.32, 65.74, 59.36, 56.57, 52.13, 52.05, 41.51, 33.92,

32.37, 30.57, 28.85, 28.13, 24.01; ΗRMS-ESI (m/z) [M+H]⁺

calcd for C₄₁H₅₅N₄O₁₁779.3867, found 779.3861.

4.13.3. benzyl ((1S,3R,4S,5R,6S,9R,10S)-3-(((S)-4-azido-2-

(benzyloxy)butyl)amino)-4,5,9,10-tetrahydroxy-7-oxaspiro

[5.6]dodecan-1-yl) carbamate 52: According to the

procedure described for the synthesis of amine 51, reaction of

15 mg of amine 27 with aldehyde 49 furnished 5.5 mg of 52

in 24% yield as a white amorphous solid. R_f= 0.32 in 10%

1

ΜeOH / CH₂Cl₂. H NMR (500MHz, CD₃OD) δ 7.44-7.16

(m, 10H), 5.14 (d, J = 12.5, 1H), 5.10 (d, J = 12.5, 1H), 4.64-

4.54 (m, 2H), 3.77-3.63 (m, 3H), 3.56-3.48 (m, 3H), 3.42 (dd,

J = 6.8, 6.8 Hz, 2H), 3.14 (d, J = 9.6 Hz, 1H), 2.85 (dd, J =

12.0, 3.6 Hz, 1H), 2.58 (dd, J = 11.9, 8.2 Hz, 2H), 2.46-2.32

(m, 2H), 2.21 (dd, J = 24.9, 12.8 Hz, 2H), 1.94-1.89 (m, 2H),

1.78-1.55 (m, 4H); ¹³C NMR (125 MHz, CD₃OD) δ 158.44,

139.73, 138.44, 129.46, 129.25, 129.15, 128.94, 128.78,

80.67, 79.93, 77.17, 76.97, 74.77, 74.00, 72.61, 67.56, 66.06,

59.80, 57.70, 51.51, 33.29, 32.62, 29.93, 28.39; ΗRMS-ESI

(m/z) [M+H]⁺calcd for C₃₀H₄₂N₅O₈600.3033, found

600.3030.

4.14. Synthesis of final products 56-61.

4.14.1.

(3R,4S,6S,7R,8S,9R,11S)-11-amino-9-(((S)-2,6-

diaminohexyl)amino)-1-oxaspiro[5.5]undecane-3,4,7,8-

tetraol 56: According to the procedure described for 37, 8.5

mg of amine 50 furnished 4.5 mg of 56 as a white amorphous

solid (87%). R_f= 0.26 in 20% NH₄OH / MeOH. ¹H NMR (500

MHz, D₂O) δ 4.57-4.48 (m, 1H), 4.25 (d, J = 11.1 Hz, 1H),

4.00-3.86 (m, 3H), 3.77-3.69 (m, 1H), 3.58-3.41 (m, 4H),

3.39-3.31 (m, 1H), 3.05 (dd, J = 7.4, 7.4 Hz, 2H), 2.44-2.32

(m, 1H), 2.14 (dd, J = 24.9, 12.4Hz, 1H), 2.00 (dd, J = 13.7,

5.3 Hz, 1H), 1.94-1.68 (m, 5H), 1.63-1.47 (m, 2H); ¹³C NMR

(125 MHz, D₂O) δ 79.14, 75.53, 69.76, 68.22, 66.72, 65.58,

58.09, 52.88, 49.24, 47.28, 39.41, 31.93, 30.54, 26.77, 26.31,

21.81; ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₆H₃₅N₄O₅

363.4729, found 363.4725.

4.13.4.

(1R,2S,3R,5S,6S,9R,10S)-3-(((S)-3-(4-benzyloxy

phenyl)-2-(carbobenzyloxyamino)propyl)amino)-5-(carbo-

benzyloxyamino)-7-oxaspiro[5.6]dodecane-1,2,9,10-tetraol

53: According to the procedure described for the synthesis of

amine 51, reaction of 6.5 mg of amine 27 with aldehyde 47

furnished 3.0 mg of 53 in 74% yield as a white amorphous

solid. R_f= 0.56 in 20% NH₄OH / MeOH. ¹H NMR (500 MHz,

CD₃OD) δ 7.61-7.34 (m, 15H), 7.29 (d, J = 7.9 Hz, 2H), 6.98

(d, J = 7.9 Hz, 2H), 5.31-5.03 (m, 6H), 4.54 (d, J = 13.7 Hz,

1H), 3.91-3.83 (m, 1H), 3.69 (d, J = 13.6 Hz, 1H), 3.64 (bs,

1H), 3.53-3.42 (m, 3H), 3.10 (d, J = 9.5 Hz, 1H), 2.77-2.63

(m, 3H), 2.53-2.38 (m, 2H), 2.32 (dd, J = 13.7, 13.7 Hz, 1H),

2.17 (dd, J = 24.6, 12.2 Hz, 1H), 1.67-1.52 (m, 4H); ¹³C NMR

(125 MHz, CD₃OD): δ 158.92, 158.69, 158.43, 138.89,

138.43, 138.43, 131.78, 131.41, 129.49, 128.95, 128.84,

128.63, 128.55, 115.92, 80.60, 79.85, 76.96, 74.54, 73.99,

71.03, 67.58, 67.28, 66.05, 59.85, 57.62, 54.22, 51.27, 39.63,

33.09, 29.89, 28.36; ΗRMS-ESI (m/z) [M+H]⁺calcd for

C₄₃H₅₂N₃O₁₀770.3653, found 770.3647.

4.14.2.

(1R,2S,3R,5S,6S,9R,10S)-5-amino-3-(((S)-2,6-

diaminohexyl)amino)-7-oxaspiro[5.6]dodecane-1,2,9,10-

tetraol 57: According to the procedure described for 37, 15

mg of amine 51 furnished 8 mg of 57 as a white amorphous

solid (86%). R_f= 0.24 in 20% NH₄OH / MeOH. ¹H NMR (500

MHz, D₂O) δ 4.62 (d, J = 13.2 Hz, 1H), 4.07 (dd, J = 9.8, 9.8

Hz, 1H), 4.04-3.82 (m, 4H), 3.70-3.43 (m, 5H), 3.15 (dd, J =

7.2, 7.2 Hz, 2H), 2.64 (dd, J = 13.5 Hz, 1H), 2.46-2.37 (m,

1H), 2.32 (dd, J = 24.1, 12.0 Hz, 1H), 2.07-1.79 (m, 8H),

1.74-1.57 (m, 2H); ¹³C NMR (125 MHz, D₂O) δ 77.49, 76.69,

74.75, 71.82, 69.76, 65.19, 58.05, 55.48, 48.85, 47.00, 39.19,

30.31, 27.22, 26.82, 26.52, 26.34, 21.55; ΗRMS-ESI (m/z)

[M+H]⁺calcd for C₁₇H₃₇N₄O₅377.2764, found 377.2758.

4.13.5.

(1R,2S,3R,5S,6S,9S,10R)-3-(((S)-3-(4-benzyloxy

phenyl)-2-(carbobenzyloxyamino)propyl)amino)-5-(carbo

benzyloxyamino)-7-oxaspiro[5.6]dodecane-1,2,9,10-tetraol

54: According to the procedure described for the synthesis of

amine 51, reaction of 10 mg of amine 29 with aldehyde 47

furnished 8.5 mg of 54 in 36% yield as a white amorphous

solid. R_f= 0.34 in 10% ΜeOH / CH₂Cl₂. ¹H NMR (500 MHz,

CD₃OD) δ 7.45-7.19 (m, 15H), 7.11 (d, J = 7.1 Hz, 2H), 6.88

(d, J = 7.6 Hz, 2H), 5.13 (d, J = 12.4 Hz, 1H), 5.08-5.00 (m,

2H), 5.03 (s, 2H), 4.97 (d, J = 12.5 Hz), 4.19 (dd, J = 12.2,

7.3 Hz, 1H), 3.90 (bs, 1H), 3.68-3.53 (m, 3H), 3.51-3.40 (m,

2H), 3.20 (d, J = 9.0 Hz, 1H), 2.88-2.64 (m, 3H), 2.56-2.46

(m, 1H), 2.46-2.34 (m, 1H), 2.31-2.15 (m, 2H), 1.84-1.54 (m,

4H); ¹³C NMR (250 MHz, CD₃OD) δ 158.91, 158.65, 158.51,

138.89, 138.48, 138.41, 131.81, 131.38, 131.32, 129.47,

129.43, 129.03, 128.89, 128.81, 128.62, 128.52, 115.93,

80.96, 80.89, 74.95, 74.70, 74.16, 71.06, 67.62, 67.27, 65.73,

59.50, 56.63, 54.26, 51.25, 39.62, 32.71, 28.90, 28.14;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₄₃H₅₂N₃O₁₀770.3653,

found 770.3642.

4.14.3. (1R,2S,3R,5S,6S,9R,10S)-5-amino-3-(((S)-4-amino-

2-hydroxybutyl)amino)-7-oxaspiro[5.6]dodecane-1,2,9,10-

tetraol 58: According to the procedure described for 37, 5.5

mg of amine 52 furnished 3.5 mg of 58 as a white amorphous

1

solid (90%). R_f= 0.60 in 20% NH₄OH/MeOH. H NMR (500

MHz, D₂O) δ 4.43 (d, J = 13.2 Hz, 1H), 4.05-3.98 (m, 1H),

3.90-3.65 (m, 5H), 3.40-3.25 (m, 4H), 3.15-3.06 (m, 2H), 2.45

(dd, J = 12.9, 12.9 Hz, 1H), 2.18-2.11 (m, 2H), 1.93-1.68 (m,

5H); ¹³C NMR (125 MHz, D₂O) δ 77.61, 76.80, 74.74, 71.81,

69.30, 65.19, 65.19, 57.08, 55.51, 49.55, 36.53, 31.66, 27.28,

26.80, 26.46; ΗRMS-ESI (m/z) [M+H]⁺calcd for C₁₅H₃₂N₃O₆

350.2291, found 350.2288.

4.14.4.

(1R,2S,3R,5S,6S,9R,10S)-5-amino-3-(((S)-2-ami-

no-3-(4-hydroxyphenyl)propyl)amino)-7-oxaspiro[5.6]do-

decane-1,2,9,10-tetraol 59: According to the procedure

described for 37, 10 mg of amine 53 furnished 5 mg of 59 as a

white amorphous solid (98%). R_f= 0.58 in 20% ΝΗ₄ΟΗ /

MeOH. ¹H NMR (500 MHz, D₂O) δ 7.17 (d, J = 7.5 Hz, 2H),

6.87 (d, J = 7.9 Hz, 2H), 4.42 (d, J = 13.6 Hz, 1H), 3.89-3.74

(m, 4H), 3.73-3.66 (m, 1H), 3.49-3.43 (m, 2H), 3.34 (d, J =

10.4 Hz, 1H), 3.30-3.24 (m, 2H), 3.07 (dd, J = 14.5, 6.1 Hz,

1H), 2.90 (dd, J = 14.1, 8.3 Hz, 1H), 2.49-2.40 (m, 1H), 2.18-

2.11 (m, 1H), 2.05 (dd, J = 24.1, 12.1 Hz, 1H), 1.80-1.68 (m,

4.13.6.

(1R,2S,3R,5S,6S,9S,10R)-3-(((S)-2,6-bis(carbo-

benzyloxyamino)hexyl)amino)-5-(carbobenzyloxyamino)-

7-oxaspiro[5.6]dodecane-1,2,9,10-tetraol 55: According to

the procedure described for the synthesis of amine 51,

reaction of 12 mg of amine 29 with aldehyde 45 furnished 8.0

3H); ¹³C NMR (125 MHz, D₂O) δ 155.47, 131.03, 125.60,

116.31, 77.46, 76.67, 74.73, 71.78, 69.70, 65.16, 58.06, 55.43,

50.31, 46.60, 35.90, 27.20, 26.78, 26.22; ΗRMS-ESI (m/z)

[M+H]⁺calcd for C₂₀H₃₄N₃O₆412.2448, found 412.2444.

The coupled in vitro transcription-translation assay was

carried out in a 96-well format as previously described.¹²

Specifically, the tested compounds were incubated at different

concentrations ranging from 1mM to 500nM or 5nM with

pBESTluc (Promega) plasmid DNA encoding luciferase at a

concentration of 200 ng/µl, and a mixture of bacterial extract,

premix and amino acids, in a total reaction volume of 10 µl.

Reactions were incubated at 37 °C for 30 min. After cooling

on ice for 5 min, luciferin substrate (Promega) was added and

luminescence measured immediately with an Infinite M200

(Tecan) luminescence counter. IC₅₀values were determined

from Relative Luminescence Units (RLU) versus log(c) plots

by fitting to a variable slope dose-response equation (Figure

S2). Three replicate experiments were run, in duplicate, per

compound. Counter screens were also performed in the

presence of each compound (for figures and titration curves

see supporting information).

4.14.5. benzyl((S)-1-(((1R,2S,3R,5S,6S,9S,10R)-5-amino-

1,2,9,10-tetrahydroxy-7-oxaspiro[5.6]dodecan-3-yl) amino)

-3-(4-hydroxyphenyl)propan-2-yl)

carbamate

60:

According to the procedure described for 37, 10 mg of amine

54 furnished 5 mg of 60 as a white amorphous solid (98%). R_f

1

= 0.62 in 20% ΝΗ₄ΟΗ / MeOH. H NMR (500 MHz, D₂O) δ

7.18 (bs, 2H), 6.88 (bs, 2H), 4.37 (bs, 1H), 3.93 (s, 1H), 3.89-

3.76 (m, 2H), 3.68 (bs, 1H), 3.54 (d, J = 12.1 Hz, 1H), 3.47

(bs, 2H), 3.38-3.22 (m, 2H), 3.11-3.03 (m, 1H), 2.95-2.86 (m,

1H), 2.61-2.52 (m, 1H), 2.19-2.08 (m, 2H), 2.06-1.95 (m, 1H),

1.74-1.55 (m, 2H); ¹³C NMR (125 MHz, CD₃OD) δ 155.70,

131.30, 125.86, 116.57, 77.42, 72.90, 70.75, 70.12, 64.46,

58.05, 55.31, 50.54, 46.92, 36.16, 26.65, 26.38, 26.21, 25.02;

ΗRMS-ESI (m/z) [M+H]⁺calcd for C₂₀H₃₄N₃O₆412.2448,

found 412.2443.

4.15.3. Minimum Inhibitory Concentration

Minimal inhibitory concentrations (MICs) were

determined by geometric microdilution method according to

published protocols. Briefly, 96-well plates were incubated at

37 °C, and bacterial growth was monitored at 600 nm after 18

h of growth. Experiments were run in triplicate using

ampicillin (16 μg/ml) as a positive control and no compound

as a negative control.

4.14.6.

(1R,2S,3R,5S,6S,9S,10R)-5-amino-3-(((S)-2,6-di-

aminohexyl)amino)-7-oxaspiro[5.6]dodecane-1,2,9,10-tetra

ol 61: According to the procedure described for 37, 7 mg of

amine 55 furnished 3 mg of 61 as a white amorphous solid

1

(79%). R_f= 0.21 in 20% NH₄OH / MeOH. H NMR (500

MHz, D₂O) δ 4.37 (dd, J = 12.1, 9.5 Hz, 1H), 3.91 (bs, 1H),

3.85 (dd, J = 9.7, 9.7 Hz, 1H), 3.88-3.82 (m, 2H), 3.54 (d, J =

12.8 Hz, 1H), 3.49-3.40 (m, 2H), 3.38-3.26 (m, 3H), 2.99-2.91

(m, 2H), 2.60-2.51 (m, 1H), 2.23-1.99 (m, 3H), 1.86-1.55 (m,

6H), 1.53-1.39 (m, 2H); ¹³C (125 MHz, D₂O) δ 77.18, 77.17,

72.67, 70.52, 69.92, 64.23, 57.75, 55.10, 48.84, 47.02, 39.17,

30.31, 26.51, 26.41, 26.07, 24.79, 21.55; ΗRMS-ESI (m/z)

[M+H]⁺calcd for C₁₇H₃₇N₄O₅377.2764, found 377.2760.

Acknowledgments

This research was supported by Marie Curie Actions,

Excellence Teams (EXT) Grants, Contract number MEXT-

CT-2006-039149. I.M. thanks the ‘Leonidas Zervas’

Foundation for financial support (2011 Organic Synthesis

Award). G.K. acknowledges support by the Action

‘Supporting Postdoctoral Researchers’ grant LS9_1057 of the

Operational Program ‘Education and Lifelong Learning’.

4.15. Biological Methods

4.15.1. RNA Specific

Gel-purified and desalted RNA oligonucleotides, carrying

the bacterial decoding-site sequence, were purchased from

Dharmacon Inc. Double-stranded constructs were obtained by

annealing complementary strands in sodium cacodylate buffer

(30mM, pH 6.8) at 65 °C for 3 min and snap cooling on ice.

References and notes

1. Thomas, J. R.; Hengenrother, P. J. Chem. Rev. 2008, 108, 1171.

2. (a) Auerbach, T.; Bashan, A.; Harms, J.; Schluenzen, F.; Zarivach, R.;

Bartels, H.; Agmon, I.; Kessler, M.; Pioletti, M.; Franceschi, F.;

Yonath, A.; Curr. Drug Targets Infect. Disord. 2002, 2, 169; (b)

Moazed, D.; Noller, H. F.; Nature 1987, 327, 389; (c) Vicens, Q.;

Westhof, E. ChemBioChem 2003, 4, 1018; (d) Hermann, T. Angew.

Chem. Int. Ed. 2000, 39, 1890; (e) Purohit, P.; Stern, S. Nature 1994,

370, 659; (f) Ramakrishnan, V. Cell 2002, 108, 557; (g) Ogle, J. M.;

Brodersen, D. E.; Clemons, W. M.; Tarry, M. J.; Carter, A. P.;

Ramakrishnan, V. Science 2001, 292, 897.

Titrations of tested compounds to the 2AP-labelled RNA

duplex (Figure S1A) were performed, with concentrations

ranging from 1mM to 1nM. Fluorescence was measured on a

Cary Eclipse (Varian) fluorescence spectrophotometer at 25

°C. Emission spectra were recorded at 1µM RNA

concentration in 1-cm path-length quartz cells. The excitation

wavelength used was at 310 nm, while emission was

monitored between 320 and 450 nm and normalized at 370

nm. Half-maximal response concentration (EC₅₀) values were

calculated by fitting a dose response curve to the fluorescence

intensity plotted against the ligands’ log(c) (Figure S1B).

Three to six replicate experiments were run per compound. As

a control, tested compounds were also added to 2AP-labeled

single-stranded oligonucleotide which showed no significant

increase in fluorescence. Additionally, spermidine, a known

non-specific RNA binder, was titrated in parallel, as a

negative dose-response control. The difference in

fluorescence between the single stranded and double stranded

2AP-labelled RNAs was also used to monitor structure

formation in the decoding-site loop hybrid (for figures and

titration curves see supporting information).

3. Houghton, J. L; Green, K. D.; Chen, W.; Garneau-Tsodikova, S.

ChemBioChem 2010, 11, 880.

4. (a) Hainrichson, M.; Nudelman, I.; Baasov, T. Org. Biomol. Chem.

2008, 6, 227; (b) Kandasamy, J.; Atia-Glikin, D.; Belakhov, V.;

Baasov, T. Med. Chem. Commun. 2011, 2, 165; (c) Brendel, C.;

Klahold, E.; Gartner, J.; Huppke, P. Pediatric Research 2009, 65, 520;

(d) Kondo, J.; Hainrichson, M.; Nudelman, I.; Shallom-Shezifi, D.;

Barbieri, C.M.; Pilch, D.S.; Westhof, E.; Baasov, T. ChemBioChem

2007, 8, 1700.

5. Guan, L.; Disney, M.D. ACS Chem. Biol. 2012, 7, 73.

6. (a) Schroeder, R.; Waldsich, C.; Wank, H. EMBO J. 2000, 19, 1; (b)

Carter, A. P.; Clemons, W. M.; Brodersen, D. E.; Morgan-Warren, R.

J.; Wimberly, B. T.; Ramakrishnan, V. Nature 2000, 407, 340; (c)

Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science 1996,

274, 1367; (d) Tor, Y.; Hermann, T.; Westhof, E. Chem. Biol. 1998, 5,

R277; (e) Blount, K. F.; Zhao, F.; Hermann, T.; Tor, Y. J. Am. Chem.

Soc. 2005, 127, 9818; f) Francois, B.; Russell, R. J. M.; Murray, J. B.;

Aboul-ela, F.; Maquida, B.; Vicens, Q.; Westhof, E. Nucleic Acids

Research 2005, 33, 5677.

7. a) Katsoulis, I. A.; Pyrkotis, C.; Papakyriakou, A.; Kythreoti, G.;

Zografos, A. L.; Mavridis, I.; Nahmias, V. R.; Anastasopoulou, P.;

Vourloumis, D. ChemBioChem 2009, 10, 1969; b) Stathakis, C. I.;

4.15.2. Bacterial IVT

Mavridis, I.; Kythreoti, G.; Papakyriakou, A.; Katsoulis, I. A.; Cottin,

T.; Anastasopoulou, P.; Vourloumis, D. Biorg. Med. Chem. Lett. 2010,

20, 7488; c) Cottin, T.; Pyrkotis, C.; Stathakis, C. I.; Mavridis, I.;

Katsoulis, I. A.; Anastasopoulou, P.; Kythreoti, G.; Zografos, A. L.;

Nahmias, V. R.; Papakyriakou, A.; Vourloumis, D. ChemBioChem

2011, 12, 71; d) Katsoulis, I. A.; Kythreoti, G.; Papakyriakou, A.;

Koltsida, K.; Anastasopoulou, P.; Stathakis, C. I; Mavridis, I.; Cottin,

T.; Saridakis, E.; Vourloumis, D. ChemBioChem 2011, 12, 1188.

8. Haight, A. R.; Stoner, E. J.; Peterson, M. J.; Grover, V. K. J. Org.

Chem. 2003, 68, 8092.

T.; Mori, H.; Perna, N. T.; Plunkett, G.; Rudd, K. E.; Serres, M. H.;

Thomas, G. H.; Thomson, N. R.; Wishart, D.; Wanner, B. L.; Nucleic

Acids Res. 2006, 34, 1.

15. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.;

Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H.; Molecular

Systems Biology 2006 doi:10.1038/msb4100050.

16. Morona, R.; Reeves, P.; Bacteriology J. 1982, 150, 1016.

17. Okusu, H.; Ma, D.; Nikaido, H.; Bacteriology J. 1996, 178, 306.

18. Chen, L.; Coleman, W. G.; Bacteriology J. 1993, 175, 2534.

19. Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K. M.; Wertheim, H.

F. L.; Sumpradit, N.; Vlieghe, E.; Hara, G. L.; Gould, I. M.; Goossens,

H.; Greko, C.; So, A. D.; Bigdeli, M.; Tomson, G.; Woodhouse, W.;

Ombaka, E.; Peralta, A. Q.; Qamar, F. N.; Mir, F.; Kariuki, S.; Bhutta,

Z. A.; Coates, A.; Bergstrom, R.; Wright, G. D.; Brown, E. D.; Cars, O.

Lancet Infect. Dis. 2013, 13, 1057.

9. Trnka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001, 34, 18.

10. Donohoe, T. J.; Moore, P. R.; Waring, M. J. Tetrahedron Lett. 1997,

38, 5027.

11. Shandrick, S.; Zhao, Q.; Han, Q.; Ayida, B. K.; Takahashi, M.;

Winters, G. C.; Simonsen, K. B.; Vourloumis, D.; Hermann, T. Angew.

Chem. 2004, 116, 3239; Angew. Chem. Int. Ed. 2004, 43, 3177.

12. a) Simonsen, K. B.; Ayida, B. K.; Vourloumis, D.; Takahashi, M.;

Winters, G. C.; Barluenga, S.; Qamar, S.; Shandrick, S.; Zhao, Q.;

Hermann, T. ChemBioChem 2002, 3, 1223; b) Vourloumis, D.;

Takahashi, M.; Winters, G. C.; Simonsen, K. B.; Ayida, B.; Barluenga,

S.; Qamar, S.; Shandrick, S.; Zhao, Q.; Hermann, T. Bioorg. Med.

Chem. Lett. 2002, 12, 3367.

20. Kythreoti, G.; Vourloumis, D. Anal. Biochem. 2011, 412, 102.

Supplementary Material

Supplementary material with NMR spectra of all

intermediates and final products along with titration curves is

included.

13. Gerritz, S. W.; Sefler, A. M. J. Comb. Chem. 2000, 2, 39.

14. Riley, M.; Abe, T.; Arnaud, M. B.; Berlyn, M. K.; Blattner, F. R.;

Chaudhuri, R. R.; Glasner, J. D.; Horiuchi, T.; Keseler, I. M.; Kosuge,

Article Doi

Rigid spiroethers targeting the decoding center of the bacterial ribosome

DOI: 10.1016/j.bmc.2013.12.068

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2013.12.068

Full text of DOI:10.1016/j.bmc.2013.12.068

Products guided by the article

R&D Labs maybe for 1569042-73-4

Relevant to this article

Hot Product