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Validamycin and Its Derivatives
Validamycin
and Its Derivatives
Discovery, Chemical Synthesis,
and Biological Activity
Xiaolong Chen, Yuele Lu,

Yongxian Fan and Yinchu Shen
Institute of Fermentation Engineering,
Zhejiang University of Technology, Hangzhou, PR China
Elsevier
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Chapter 1
An Introduction to Validamycins
and Their Derivatives
1.1 IMPORTANCE OF ANTIBIOTICS
The term “antibiotics” was first coined by the American microbiologist
Selman Waksman and his colleagues to describe chemical substances pro-
duced by microorganisms and having antagonistic effects on the growth of
other microorganisms. It excluded synthetic antimicrobials (sulfur drugs) and
biological products of nonmicrobial origin having antagonistic effects on bac-
teria. Though antibiotics were introduced into clinical practice only in the
middle of the 20th century, the use of microorganisms for the management of
microbial infections in ancient Egypt, Greece, China, and some other places
in the world is well documented. The modern era of antibiotics started with
the serendipitous discovery of penicillin from the culture filtrate of a fungus,
Penicillium notatum, by Alexander Fleming in 1928 (Fleming, 1929).
In the present scenario, antibiotics available in the market are either
produced by microbial fermentation or are derived via semisynthetic route
using the existing antibiotic backbone structure. They are classified into
different chemically defined groups. Antibiotics target bacterial physiology
and biochemistry, causing microbial cell death or the cessation of growth.
A significant number of these antibiotics affect cell walls or membranes
(e.g., β-lactam and glycopeptides), while several others exert their anti-
bacterial activity by targeting protein synthetic machinery via interaction
with ribosomal subunits, and these include antibiotics such as macrolides,
chloramphenicol, tetracycline, linezolid, and aminoglycosides. Other “mech-
anistic” groups include molecules that interfere with the nucleic acid
synthesis [e.g., fluoroquinolones (FQ) and rifampin], while some others exert
their effects by interfering with the metabolic pathways (e.g., sulfonamides
and folic acid analog) or by disruption of the bacterial membrane structure
(e.g., polymyxins, daptomycin, and others).
The successful use of antibiotics against bacterial diseases of human
beings has led to a large-scale screening of antibiotics’ effect for plant
disease control in the world.
Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00001-0

© 2017 Elsevier Ltd. All rights reserved. 1
2 Validamycin and Its Derivatives
1.2 AGRICULTURAL ANTIBIOTICS FOR PLANT PATHOGENS

Many antibiotics developed for medical purposes were investigated for activity
against plant pathogens. Furthermore, screening of soil organisms for production
of antibiotic substances was started with the prime purpose of plant disease con-
trol. However, the results obtained with antibiotics and antibiotic-containing cul-
ture broth did not fulfill the high expectations. Many of them were too
unstable under field conditions or showed toxic side effects on plants. Most anti-
biotics were rather expensive, even when used as a crude product. In the 1950s,
soon after the introduction of antibiotics in human medicine, the potential of
these "miracle drugs" to work wonders in controlling plant diseases was
explored (Mcmanus and Stockwell, 2000). Nearly 40 antibiotics were screened
for plant disease control. To be a viable candidate for disease control, the antibi-
otic needed to (1) be active on or inside of the plant; (2) tolerate oxidation, UV
irradiation, rainfall, and high temperatures; (3) be safe to plants; and (4) select
for resistant pathogens only at a low or nondetectable rate. Of the screened com-
pounds, fewer than 10 were used commercially and only streptomycin, tetracy-
cline, cycloheximide, and griseofulvin saw significant use worldwide (Kumar
et al., 2005). Streptomycin, the first antibiotic introduced in agriculture, was
used in the United States for the control of pear fire blight. This antibiotic and a
mixture of streptomycin and tetracycline have been used for the control of bac-
terial plant diseases, while cycloheximide and griseofulvin have been used for
the control of fungal plant diseases. Cycloheximide is a very powerful fungicide,
but unfortunately, is highly toxic to plants, which restricts its use against plant
diseases. Griseofulvin is a much less phytotoxic systemic fungicide, but its use
is also restricted, because the relation of its manufacturing cost to its perfor-
mance under field conditions is not quite satisfactory.
Later, in Japan (Misato, 1976), blasticidin S and kasugamycin have been
in practical use for rice blast control instead of mercuric fungicides, and
polyoxins and validamycin have been used to control the sheath blight of the
rice plant instead of arsenic fungicides. Since then, more and more antibio-
tics were found to be applied in plant diseases.
1.3 VALIDAMYCINS: MAGIC AGRICULTURAL ANTIBIOTICS

Validamycin (Asano et al., 1990; Horii et al., 1972; Iwasa et al., 1970; Kameda
et al., 1986), also called as jinggangmycin in China (Agricultural Antibiotic
Group, 1975a, 1975b), is a magic agricultural antibiotic produced by fermenta-
tion with Streptomyces hygroscopicus var. limoneus or S. hygroscopicus var.
jinggangensis, and has been widely used in Asia as the rice and wheat protectant
against Rhizoctonia solani since the 1970s. Sheath blight caused by R. solani is
a major disease of rice and wheat that greatly reduces yield and grain quality.
And validamycin is a nonsystemic antibiotic with fungistatic action and exhibits
remarkable therapeutic effects on the disease by inhibiting the extension of
R. solani without its growth inhibition (Shibata et al., 1982, 1989; Trinci, 1984).
An Introduction to Validamycins and Their Derivatives Chapter | 1 3
Validamycin can also be used for the control of R. solani in potatoes, vegetables,
strawberries, tobacco, ginger, and other crops, and damping-off diseases of
cotton, rice and sugar beet, etc. Besides its excellent control effect, low price,
low drug-resistance, and low toxicity are the other outstanding merits of valida-
mycin, and it is now one of the most important agricultural antibiotics with the
biggest production in China (Shen, 1996).
Validamycin is a mixture of aminoglycoside compounds. There have
been eight members termed A to H (Fig. 1.1) (Asano et al., 1990; Horii
R5OH2C
R6O
OH HO
HO
HN
R3
OH
R1
R2OH2C
R4O
Compound R1 R2 R3 R4 R5 R6
Validamycin A H H H β-D-Glc H H
Validamycin B H H OH β-D-Glc H H
α-D-
Validamycin C H H H β-D-Glc H
Glc
Validamycin D H α-D-Glca H H H H
α-D-Glc(1-4)- β-
Validamycin E H H H H H
D-Glc
Validamycin F H H H β-D-Glc H α-D-Glc
Validamycin G OH H H β-D-Glc H H
β-D-Glc(1-6)- β-
Validamycin H H H H H H
D-Glc
aGlc=Glucopyranosyl.
FIGURE 1.1 Chemical structures of validamycins.

CH2OH CH2OH
CH2 OH
HO
OH OH OH NH2
NH2 OH NH2 OH
OH
OH OH OH
Valienamine Validamine Hydroxyvalidamine
CH 2OH CH2 OH
OH
OH OH
OH OH N
NH2 OH
OH H
OH OH OH
Valiolamine Voglibose
FIGURE 1.2 Chemical structures of valienamine and its related compounds.
et al., 1972; Iwasa et al., 1970; Kameda et al., 1986) isolated from the broth
of S. hygroscopicus var. limoneus, with validamycin A [1L-(1,3,4/2,6)-2,3-
dihydroxy-6-hydroxymethyl-4-[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-hydroxymethyl-
cyclohex-2-enylamino] cyclohexyl β-D-glucopyranoside] as the main member
and the most active. So the content of validamycin A is the most important
quality parameter of the commercial products of validamycin.
Many researchers also focused on the chemical synthesis, biosynthesis
genes, and derivatives of validamycins. Among them, the most interesting
work is that validamycins were lysed to produce valienamine or validamine
(Fig. 1.2) (Asano et al., 1984; Chen et al., 2005a, 2005b; Kameda et al.,
1980, 1981), which could be applied to synthesize valiolamine (Horii et al.,
1985; Kameda et al., 1984), and then voglibose (Babu et al., 2005; Chen
et al., 2006; Floss et al., 1999; Fukase, 1997; Kameda and Horii, 1986; Lu
et al., 2005; Yuan et al., 2005). Voglibose (code number: AO-128, trade
name: Basen), is an N-substituted derivative of valiolamine, which is a
branched-chain aminocyclitol, or pseudoamino sugar. Voglibose has attracted
considerable interest due to its wide range of therapeutic and pharmacologi-
cal properties, which include its excellent inhibitory activity against
α-glucosidases and its action against hyperglycemia and various disorders
caused by hyperglycemia. It has shown strong antiobesity and antidiabetic
activities, as it is a new, potent glucosidase inhibitor and is a drug used for
NIDDM (noninsulin-dependent diabetes mellitus) in Japan, China, and
Korea. Therefore, the industry of validamycin has converted from the agri-
cultural antibiotic to pharmaceutical.
Valienamine could also be used to synthesize N-octyl-4-epi-β-valienamine
(NOEV) (Iwasaki et al., 2006; Ogawa et al., 2004; Suzuki, 2006, 2008,
2013; Suzuki et al., 2007) and N-octyl-β-valienamine (NOV) (Ogawa et al.,
1996, 1998; Suzuki, 2013) (Fig. 1.3), two highly potent drug candidates
OH OH
HO
H HO
H
HO N HO N
(CH2 )7 CH3 (CH2 )7 CH3
HO HO
NEOV NOV
FIGURE 1.3 Chemical structures of NEOV and NOV.
for chemical chaperone therapy. NOEV and NOV are promising thera-
peutic agents for human β-galactosidase deficiency disorders (GM1-
gangliosidosis and Morquio B disease) and β-glucosidase deficiency
disorders (phenotypic variations of Gaucher disease), respectively (Suzuki,
2013). Originally NOEV and NOV had been discovered as competitive
inhibitors, and then their paradoxical bioactivities as chaperones were
confirmed in cultured fibroblasts from patients with these disorders.
Subsequently, GM1-gangliosidosis model mice have been used for con-
firmation of clinical effectiveness, adverse effects, and pharmacokinetic
studies. Orally administered NOEV entered the brain through the blood
brain barrier enhanced β-galactosidase activity, reduced substrate storage,
and improved neurological deterioration clinically. Computational analysis
revealed pH-dependent enzyme chaperone interactions. The recent study
(Suzuki, 2013) indicated chaperone activity of a new 1-deoxygalactonojirimycin
derivative, MTD118, for β-galactosidase complementary to NOEV. NOV also
showed the chaperone effect toward several β-glucosidase gene mutants in
Gaucher disease. Furthermore a commercial expectorant drug, ambroxol, was
found to be a chaperone for β-glucosidase. A few Gaucher patients
responded to this drug with remarkable improvement of oculomotor dys-
function and myoclonus. It is hoped that chaperone therapy will become
available for some patients with Fabry disease, GM1-gangliosidosis,
Gaucher disease, and other lysosomal storage diseases particularly with
central nervous system involvement.
1.4 AIMS OF THE WORK

Validamycins have nice characteristics, such as high and long efficiency,
low toxicity and safe to the environment, and low production cost. Most
important of all, there is no report about discovery of validamycin-
resistant strains of R. solani. Thus, since the production of validamycins in
large scale, the yield was more than 30,000 40,000 ton per year (in 5%
preparations) for 1 1.3 3 107 hm2 rice field against R. solani and the loss
of rice yield was decreased 5000, 000 ton per year at least in China
(Shen, 1996). In recent years, the production of validamycins in other

Southeast Asian countries, such as India, Vietnam, Laos, has been boom-
ing. In addition, three drugs, voglibose, NEOV, and NOV, were derived
from validamycins. These drugs will give a second rise to validamycin
production and make products from the agricultural industry to the medical
industry.
The aims of the work are to review the magic agricultural antibiotics,
validamycins, including their discovery, biosynthesis, production, biological
activities, chemical synthesis, important derivatives, etc., and to provide
references in the research of antibiotics.
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Agricultural Antibiotic Group SIOAPS, 1975a. Classification and identification of jinggangmy-
cin producing strains. Weishengwu Xuebao 15 (2), 110 113.
Agricultural Antibiotic Group SIOAPS, 1975b. Isolation and identification of jinggangmycins.
Weishengwu Xuebao 15 (3), 223 226.
Asano, N., Kameda, Y., Matsui, K., Horii, S., Fukase, H., 1990. Validamycin H, a new pseudo-
tetrasaccharide antibiotic. J. Antibiot. 43 (8), 1039 1041.
Asano, N., Takeuchi, M., Ninomiya, K., Kameda, Y., Matsui, K., 1984. Microbial degradation
of validamycin A by Flavobacterium saccharophilum. Enzymatic cleavage of C N linkage
in validoxylamine A. J. Antibiot. (Tokyo) 37 (8), 859 867.
Babu J.S., Chavan G.J., Khanduri C.H., Kumar Y., Ray P.C., (Ranbaxy Laboratories Limited,
India). assignee. 2005 20050324. Processes for the purification of voglibose and the
preparation and purification of its intermediates. Application: WO patent 2005-IB777,
2005092834.
Chen, X., Zheng, Y., Shen, Y., 2005a. A new method for production of valienamine with micro-
bial degradation of acarbose. Biotechnol. Prog. 21 (3), 1002 1003.
Chen, X., Zheng, Y., Shen, Y., 2005b. Quantitative analysis of valienamine in the microbial deg-
radation of validamycin A after derivatization with p-nitrofluorobenzene by reversed-phase
high-performance liquid chromatography. J. Chromatogr. B Anal. Technol. Biomed. Life
Sci. 824 (1 2), 341 347.
Chen, X., Zheng, Y., Shen, Y., 2006. Voglibose (Basen, AO-128), one of the most important
alpha-glucosidase inhibitors. Curr. Med. Chem. 13 (1), 109 116.
Fleming, A., 1929. Classics in infectious diseases: on the antibacterial action of cultures of a
penicillium, with special reference to their use in the isolation of B. influenzae. Br. J. Exp.
Pathol. 10, 226 236.
Floss H.G., Lee S., Tornus I., (Bayer A.-G., Germany). assignee. 1999 19990329. Preparation of
valiolone as synthon for acarbose and voglibose. Application: WO patent 1999-EP2141,
9950217.
Fukase, H., 1997. Development of voglibose (Basen), an antidiabetic agent. Yuki Gosei Kagaku
Kyokaishi 55 (10), 920 925.
Horii, S., Fukase, H., Kameda, Y., 1985. Stereoselective conversion of valienamine and valida-
mine into valiolamine. Carbohydr. Res. 140 (2), 185 200.
Horii, S., Kameda, Y., Kawahara, K., 1972. Validamycins, new antibiotics. VIII. Isolation and
characterization of validamycins C, D, E, and F. J. Antibiot 25 (1), 48 53.
Iwasa, T., Yamamoto, H., Shibata, M., 1970. Validamycins, new antibiotics. I. Streptomyces
hygroscopicus var. limoneus nov. var., validamycin-producing organism. J. Antibiot.
(Tokyo) 23 (12), 595 602.
Iwasaki, H., Watanabe, H., Iida, M., Ogawa, S., Tabe, M., Higaki, K., et al., 2006. Fibroblast
screening for chaperone therapy in beta-galactosidosis. Brain Dev. 28 (8), 482 486.
Kameda, Y., Asano, N., Teranishi, M., Matsui, K., 1980. New cyclitols, degradation of valida-
mycin A by Flavobacterium saccharophilum. J. Antibiot. 33 (12), 1573 1574.
Kameda, Y., Asano, N., Teranishi, M., Yoshikawa, M., Matsui, K., 1981. New intermediates,
degradation of validamycin A by Flavobacterium saccharophilum. J. Antibiot. 34 (9),
1237 1240.
Kameda, Y., Asano, N., Yamaguchi, T., Matsui, K., Horii, S., Fukase, H., 1986. Validamycin G
and validoxylamine G, new members of the validamycins. J. Antibiot. 39 (10), 1491 1494.
Kameda, Y., Asano, N., Yoshikawa, M., Takeuchi, M., Yamaguchi, T., Matsui, K., et al., 1984.
Valiolamine, a new alpha-glucosidase inhibiting aminocyclitol produced by Streptomyces
hygroscopicus. J. Antibiot. (Tokyo) 37 (11), 1301 1307.
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Valiolamine derivatives. Application: EP patent 1986-303048, 199591.
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impact on the terrestrial environment. Adv. Agron. 87 (05), 1 54.
Lu, C., Ye, W., Hu, S., Pan, Y., Yuan, J., 2005. Preparation of voglibose from valiolamine.
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Mcmanus, P., Stockwell, V., 2000. Antibiotics for plant diseases control: silver bullets or rusty
sabers. Core Discussion Papers Rp (May), 27 51.
Misato, T., 1976. The development of agricultural antibiotics. Environ. Qual. Safety 5 (5),
48 55.
Ogawa, S., Ashiura, M., Uchida, C., Watanabe, S., Yamazaki, C., Yamagishi, K., et al., 1996.
Synthesis of potent β-D-glucocerebrosidase inhibitors: N-alkyl-β-valienamines. Bioorg. Med.
Chem. Lett 6 (8), 929 932.
Ogawa, S., Kobayashi, Y., Kabayama, K., Jimbo, M., Inokuchi, J.-I., 1998. Chemical modifica-
tion of β-glucocerebrosidase inhibitor N-octyl-β-valienamine: synthesis and biological evalu-
ation of N-alkanoyl and N-alkyl derivatives. Bioorg. Med. Chem. 6 (10), 1955 1962.
Ogawa, S., Sakata, Y., Ito, N., Watanabe, M., Kabayama, K., Itoh, M., et al., 2004. Convenient
synthesis and evaluation of glycosidase inhibitory activity of α- and β-galactose-type valie-
namines, and some N-alkyl derivatives. Bioorg. Med. Chem. 12 (5), 995 1002.
Shen, Y.C., 1996. Research and development on jinggangmycins for 25 years. Zhiwu Baohu
(Plant Protect.) 22 (4), 44 45.
Shibata, M., Kido, Y., Honda, Y., Shimizu, K., 1989. Hyphal extension inhibitors I and II with
similar inhibitory spectra to validamycin, isolated from hyphae of Rhizoctonia solani. Agric.
Biol. Chem. 53 (3), 869 873.
Shibata, M., Mori, K., Hamashima, M., 1982. Inhibition of hyphal extension factor formation by
validamycin in Rhizoctonia solani. J. Antibiot. 35 (10), 1422 1423.
Suzuki, Y., 2006. β-Galactosidase deficiency: an approach to chaperone therapy. J. Inherit.
Metab. Dis. 29 (2/3), 471 476.
Suzuki, Y., 2008. Chemical chaperone therapy for GM1-gangliosidosis. Cell. Mol. Life Sci.
65 (3), 351 353.
Suzuki, Y., 2013. Chaperone therapy update: Fabry disease, GM1-gangliosidosis and Gaucher
disease. Brain Dev. 35 (6), 515 523.
Suzuki, Y., Ichinomiya, S., Kurosawa, M., Ohkubo, M., Watanabe, H., Iwasaki, H., et al., 2007.
Chemical chaperone therapy: clinical effect in murine G(M1)-gangliosidosis. Ann. Neurol.
62 (6), 671 675.
Trinci, A.P.J., 1984. Antifungal agents which affect hyphal extension and hyphal branching.
Symp. Br. Mycol. Soc. 9 (Mode Action Antifungal Agents), 113 134.
Yuan J., Shao C., Chen D., Ye W.; (Shanghai Laiyi Biopharmaceutical Research and
Development Center Co., Ltd., Peop. Rep. China; Xinchang Pharmaceutical Factory,
Zhejiang Medicine Co., Ltd.). assignee. 2005 20050303. Preparation of valiolamine as
intermediate of voglibose. Application: CN patent 2005-10024194, 1683320.
Chapter 2
Production of Validamycins
2.1 DISCOVERY OF VALIDAMYCINS
In the course of screening for new antibiotics effective in the control of
sheath blight, a destructive disease of rice plants caused by Pellicularia sasa-
kii (Shirai) S. Ito, validamycins were first discovered in the broth of
Streptomyces hygroscopicus var. limoneus T-7545, which was isolated from
a soil sample collected in Akashi City, Hyogo Prefecture, Japan in 1970
(Iwasa et al., 1970). Five years later, they were also discovered in the broth
of S. hygroscopicus var. jinggangensis Yen. TH82, which was isolated from
a soil sample of Jinggang Mountain in Jiangxi, China (Agricultural
Antibiotic Group, 1975). Since then, they have been widely used in Asia as
the rice and wheat protectant against P. sasakii. Now, eight validamycins
have been purified and identified, including A, B, C, D, E, F, G, and H.
2.2 MICROBES FOR PRODUCING VALIDAMYCINS

2.2.1 General Characteristics for Microbes
Microbes for producing validamycins belong to S. hygroscopicus, including
S. hygroscopicus var. limoneus (Iwasa et al., 1970) and var. jinggangensis
(Agricultural Antibiotic Group, 1975). The strains were similar except in
some respects, such as the morphology and cultural characteristics. The mor-
phological characteristics of the strains are shown in Fig. 2.1. The aerial
mycelium of strain T-7545 was simply branched and terminated in coils of
three to five volutions. The spores were oval or cylindrical and measure
1.01.3 3 1.01.5 μm. Their surfaces were smooth. On the other hand, the
aerial mycelium of strain TH82 was simply branched and terminated in coils.
The spores were oval and not of a uniform size. Their surfaces were smooth.
The cultural characteristics of S. hygroscopicus var. limoneus T-7545 and
var. jinggangensis Yen. TH82 are listed in Table 2.1. The colors of aerial
mycelium are grey to grey and yellow, and black moist areas form in the
aerial mycelium on glucose asparagine agar and starch agar. The colors of
the vegetative mycelium on most of the media are bright yellow to ochre
with, in some cases, a slight greenish tinge. A light yellow to faint yellowish
brown diffusible pigment was noted in various media, but because dark
Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00002-2

© 2017 Elsevier Ltd. All rights reserved. 9
(A)
(×950) (×10,000×1/1.5)
(B)
(×7500) (×7500)
FIGURE 2.1 Morphology of S. hygroscopicus var. limoneus T-7545 (A) and S. hygroscopicus
var. jinggangensis Yen. TH82 B. (A) Reprinted with permission courtesy of Japan Antibiotics
Research Association (JARA).
brown soluble pigment on proteinaceous media was not observed, T-7545

and TH82 are considered to be nonchromogenic.
Physiological characteristics of T-7545 and TH82 are shown in
Table 2.2. As show in the table, a wide temperature range and rather high
optimum temperature for growth are characteristic for these two organisms.
Starch hydrolysis and milk peptonization tests are positive, whereas tyrosi-
nase, nitrate reduction, cellulose decomposition, and serum liquefaction are
negative for T-7545. On the other hand, for TH82, starch hydrolysis and
milk peptonization tests are positive, whereas cellulose decomposition and
serum liquefaction are negative. Furthermore, nitrate reduction for TH82 is
positive in Czapek’s solution and negative in peptone solution. Gelatin is
slowly liquefied for two strains. Products of T-7545 are validamycins A.
However, products of TH82 are validamycins and other antibiotics.
For T-7545 and TH82, a variety of carbon sources, such as D-xylose,
L-arabinose, L-galactose, D-glucose, D-fructose, L-rhamnose, melibiose, malt-
ose, sucrose, lactose, raffinose, inositol, D-mannitol, mannose, and glycerol,
Production of Validamycins Chapter | 2 11
TABLE 2.1 Cultural Characteristics of S. hygroscopicus var. limoneus

T-7545 and var. jinggangensis Yen. TH82
Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

limoneus T-7545 jinggangensis Yen. TH82
Medium
Czapek’s agar Growth (G): Moderate, R: Yellowish Brown to
colorless, folded Maroon
Reverse (R): Raw Sienna AM: Pink White to Jasmine
(Rda.a, III 17-i) to Sudan Buff to Mouse Grey with
Brown (Rdg., III 15-k) black moist areas
Aerial mycelium (AM): SP: Faint Brownish tinge
Tilleul-Buff (Rdg., XL
17’’’-f) to Light Buff
(Rdg., XV 17’-f), partially
Mouse Grey (Rdg.,
LI 15’’’’) along the
periphery of the colony
Soluble pigment (SP):
Yellow with a faint
Brownish tinge
Glucose Czapek’s agar G: Moderate, colorless to G: Banana-Buff to Maroon
Sulphin Yellow (Rdg., IV
21-i), folded
R: Raw Sienna AM: Faint Yellow to Dust
Grey
AM: Tilleul-Buff to SP: Faint Brownish tinge
Massicot Yellow (Rdg.,
XVI 21’-f), partially Light
Olive-Grey (Rdg., LI
23’’’’’-d) along the
periphery of the colony
SP: Yellow with a faint
Brownish tinge
Glycerol Czapek’s agar G: Moderate, colorless to G: Sulphin Yellow to
Orange-Citrine (Rdg., IV Brown
19-k), folded
R: Raw Sienna AM: Faint Grey to Almond
Yellow to Mouse Grey
AM: Tilleul-Buff to SP: Faint Brownish tinge
Massicot Yellow,
partially Light Olive-grey
SP: Yellow with a faint
brownish tinge
(Continued )
TABLE 2.1 (Continued)

Medium
Glucose asparagine agar G: Colorless G: Grey Green to Brown
R: Old Gold (Rdg., XVI AM: Mouse Grey with
19’-i) to Antimony yellow areas to black
Yellow (Rdg., XV 17’-b) moist areas
to Cinnamon-Brown
(Rdg., XV15’-k)
AM: Light Olive-Grey to SP: Faint Yellow
Mouse Grey, with yellow
patches and black moist
areas
SP: Light brown
Calcium malate agar G: Primuline Yellow G: Colorless
(Rdg., XVI 19’)
R: Primuline Yellow. AM: Faint Yellow
AM: Sparse at first, but
later Tilleul-Buff to Light
Olive-Grey
SP: Pale Yellow SP: Faint Yellow
b
Starch agar G: Colorless to Barium G: Pheasant Brown to Tea
Yellow (Rdg., XVI 23’-d) Brown
R: Deep Colonial Buff AM: Grey White to Mouse
(Rdg., XXX 21’’-b) to Grey with black moist
Snuff Brown (Rdg., XXIX areas
15’’-k)
AM: Cartridge Buff (Rdg., SP: Faint Yellow
XXX 19’’-f) to Mouse
Grey, with black moist
areas
SP: Light Brown.
Hydrolysis of starch was
observed
Gelatin (25 C) G: Very poor
AM: None
SP: None. Liquefaction,
slow
Nutrient gelatin (25 C) Same results as with
gelatin
(Continued )

Medium
Whole egg (37 C) G: Colorless
AM: None
SP: None
Tyrosine agar G: Colorless to Strontium R: Bamboo Shoot Brown
Yellow (Rdg., XVI 23’) to Maroon
AM: Ic43’ to Id64’
R: Pale Ochraceous-Buff SP: Faint Brown
(Rdg., XV 15’-f) to Light
Ochraceous-Buff (Rdg.,
15’-d)
AM: None
SP: None
Yeast extract agar G: Colorless, folded R: Colorless
R: Cream Color (Rdg., AM: Grey White
XVI 19’-f)
AM: White SP: Colorless
SP: Light brown

Nutrient agar (37 C) G: Colorless R: Colorless
R: Colorless AM: None
AM: None SP: Colorless
SP: None
Glucose nutrient agar G: Colorless wrinkled R: Pheasant Brown
(37 C)
R: Cartridge Buff to Pale AM: Ic 11’ to Id 42’ with
Ochraceous-Buff wrinkles
AM: None SP: Pale brown
SP: None
Peptone glucose agar G: Chartreuse Yellow R: Yellowish Brown to
Brown
R: Honey Yellow (Rdg., AM: Grey White to
XXX 19’’) Jasmine Yellow
AM: Thin, Cream Buff SP: Pale brown
SP: Yellow with a
Brownish tinge
(Continued )

Medium
Nutrient broth (37 C) G: Surface growth G: Surface growth
colorless and colorless colorless and colorless
flocculent growth at flocculent growth at
bottom of tubes bottom of tubes
AM: None AM: None
SP: None SP: None
Glucose nutrient broth G: Surface growth
Cartridge Buff, and
colorless flocculent
growth at bottom of
tubes
AM: None
SP: None
Potato plug G: Colorless to Pale G: Colorless to White
Ochraceous-Buff
AM: Tilleul-Buff to AM: Grey White to Green
Mouse Grey. Color of Grey to Pale Brown
the plug turned to Sayal
SP: Faint Yellow
Brown (Rdg., XXIX 15’’-i)
Carrot plug G: Colorless R: Colorless
AM: White to Mouse AM: Grey White to Mouse
Grey. Color of the plug Grey to Faint Brown
turned to Cinnamon-
SP: Pale Yellow
Rufous (Rdg., XIV 11’-i)
to Cinnamon-Brown
Cellulose G: Poor growth, R: Colorless
Chartreuse Yellow (Rdg.,
AM: Mouse Grey and
XXXI 25’’-d) to Reed
grown poorly
Yellow (Rdg., XXX
23’’-d)
AM: Mouse Grey SP: Pale Yellow

Litmus milk (37 C) G: Surface growth Cream
Color to Seashell Pink
(Rdg., XIV 11’-f)
AM: None
SP: Army Brown (Rdg.,
XL 13’’’-i). Peptonization
(Continued )

Medium
with or without weak
coagulation. Reaction of
the medium, weakly
acidic
Löeffler’s medium (37 C) G: Naples Yellow (Rdg.,
XVI 19’-d) becoming
Light Buff
AM: None
SP: None. No
liquefaction
a
Rdg.: Ridgway.
b
Soluble starch 1%, potassium monohydrogen phosphate 0.3%, calcium carbonate 0.3%,
magnesium sulfate 0.1%, ammonium sulfate 0.2%, sodium chloride 0.05%, agar 2%.
TABLE 2.2 Physiological Properties of Strains T-7545 and TH82
Properties
T-7545 TH82

Temperature and Growth occurs at 1545 C, Growth occurs at 1545.
pH rangesa better growth at 3745 C, no Growth occurs at pH 510,
growth at 10 C and 50 C. better growth at pH 67
Growth occurs at pH 510,
no or poor growth at pH 4,
optimum range pH 67
Gelatin Slow liquefaction Slow liquefaction
Starch Hydrolysis. Diameter of Hydrolysis
hydrolyzed area/diameter of
colony 5 33 mm/8 mm
Tyrosinase Negative Nb
reaction
Litmus milk Peptonization. Coagulation, Peptonization. Coagulation,
doubtful. Reaction, weakly doubtful. Reaction, weakly
acidic acidic
Reduction of Negative (in peptone solution Positive in Czapek’s
nitrate to nitrite and Czapek’s solution) solution. Negative in
peptone solution
(Continued )
Properties
T-7545 TH82
Cellulose Negative Negative
decomposition
Chromogenicity Negative Negative
Liquefaction of Negative N
serum
Products Validamycins Validamycins and other
antibiotics
a
On glucose asparagine agar.
b
Not tested.
are well utilized for growth. In view of the relatively high optimum tempera-
ture for growth of T-7545 and TH82, the cultural characteristics at 45 C
were observed and compared with those at 28 C. The characteristics at the
higher temperature were found to be almost the same as those at 28 C.
A few properties, such as sparse formation of white aerial mycelium on
Czapek’s agar, Czapek’s glucose agar, Czapek’s glycerol agar, and filter
paper, and a slightly deeper color of the vegetative mycelium on calcium
malate agar, were noted (Table 2.3).
The characteristics of T-7545 and TH82 are summarized as follows: good
growth and abundant aerial mycelia and coiled chains of spores form at
2545 C; on a variety of media, grey to grey and yellow aerial mycelium,
bright yellow to ocher vegetative mycelia and faint, brownish yellow diffus-
ible pigment form; on certain media, black moist spots form in the aerial
mycelium. Dark brown diffusible pigment is not produced on proteinaceous
media, i.e., the strain is nonchromogenic.
2.2.2 Complete Genomes for the Two Microbes

Recently, complete genomes of the two strains, S. hygroscopicus var. jing-
gangensis 5008 (Wu et al., 2012) and var. limoneus KCTC 1717 (Leea et al.,
2016), were sequenced.
2.2.2.1 General Features of the S. hygroscopicus

var. jinggangensis 5008 Genome
Except for a linear chromosome, the strain 5008 also harbors a linear plas-
mid pSHJG1 and a 73,282-bp large circular plasmid. With a total length of
10,383,684 bp, the genome of strain 5008 is larger than most published
TABLE 2.3 Utilization of Carbon Sources by T-7545 and TH82
Carbon Source T-7545 TH82 Carbon Source T-7545 TH82

Erythritol 6 6 Inositol 11 11
Adonitol 6 N a
D-Mannitol 11 11
Sorbitol 1 6 Dulcitol 6 6
D-Xylose 11 11 Trehalose 11 N
L-Arabinose 11 11 Salicin 6 N
L-Sorbose 6 1 Esculin 6 6
D-Galactose 11 11 Inulin 11 N
Glucose 11 11 Dextran 1 1
D-Fructose 11 11 Mannose 11 11
L-Rhamnose 11 11 Glycerol 11 11
Melibiose 11 11 Na-Acetate 1 6
Maltose 11 11 Na-Succinate 1 11
Sucrose 11 11 Na-Citrate 1 N
Lactose 11 11 Ca-2-ketogluconate 6 N
Raffinose 11 11 Carbon-free control 6 N
Not tested. 1 1 : Good growth. 1 : Fair growth. 6: No or very poor growth.

a
Streptomyces genomes (Table 2.4). The linear chromosome (10,145,833 bp)

of strain 5008, with an average G 1 C% mol content of 71.9%, comprises
8849 predicted protein-coding sequences (locus tagged as SHJG), 6 rRNA
operons (16S23S5S), and 68 tRNA genes (Table 2.4). The replication
origin oriC contains at least 18 DnaA box-like sequences (Jakimowicz et al.,
1998) and is shifted 875 kb away from the center to the right (Fig. 2.2A).
Intriguingly, it only has 14-bp terminal inverted repeats (TIRs), which is
one of the shortest TIRs hitherto found in actinomycetes. Based on a
BLASTCLUST analysis, 4607 (41.6%) of predicted protein coding
sequences (CDSs) are clustered into 924 families. The linear plasmid
pSHJG1 (164,566 bp) (Fig. 2.1B) contains 184 CDSs possibly involved in
replication, partitioning, transfer, and other biological functions. It lacks a
conserved telomere-associated protein (TAP) and TIRs. However, the right-
most 1.2-kb region of pSHJG1 demonstrates a strong homology to the right
arm of the chromosome, implying an evolutionary recombination event
occurred between the linear plasmid and the chromosome. Moreover, the
left terminus of pSHJG1 is equipped with atypical nucleotide sequences
TABLE 2.4 General Features of Seven Completely Sequenced Streptomyces Chromosomesa
Species Length TIR GC Content CDS (No.) Average CDS Coding (%) rRNA Operons tRNA
(bp) (bp) (%) size (bp) (No.) (No.)
S. hygroscopicus 5008 10,145,833 14 71.9 8849 952 83.2 6 68
S. coelicolor A3(2) 8,667,507 21,653 72.1 7825 991 88.9 6 63
S. avermitilis MA-4680 9,025,608 49 70.7 7582 1027 86.3 6 68
S. griseus IFO13350 8,545,929 132,910 72.2 7138 1055 88.1 6 66
S. scabies 87.22 10,148,695 18,488 71.5 8746 1005 86.2 6 75
S. bingchenggensis BCW-1 11,936,683 40,000 70.8 10,023 1031 86.6 6 ND
S. clavuligerus ATCC 27064 6,760,392 ND 72.0 5710 1031 87.1 6 66
a
Data were obtained from GenBank: S. coelicolor A3(2), NC_003888; S. avermitilis MA-4680, NC_003155; S. griseus IFO13350, NC_010572; S. scabies 87.22, NC_013929;
S. bingchenggensis BCW-1, CP002047; S. clavuligerus ATCC 27064, CM000913. ND, not determined.
FIGURE 2.2 Schematic representation of the S. hygroscopicus 5008 chromosome and two
plasmids. (A) The chromosome atlas. The outer scale is numbered in megabases from the left to
the right ends and indicates the core (red) and noncore (blue) chromosomal regions; Circles 1
and 2 (forward and reverse strands), predicted protein coding sequences colored according to
Clusters of Orthologous Groups (COG) function categories; Circles 3 and 4 (forward and reverse
strands), distribution of conserved (red) or strain-specific genes (blue) in 5008 compared with
other Streptomyces chromosomes; Circle 5, distribution of secondary metabolic gene clusters
(red); Circle 6, distribution of tRNA (red) and rRNA operon (blue); Circle 7, GC content; Circle
8, GC bias. Ori, origin of replication. val, validamycin biosynthetic gene cluster. (B) Atlas of lin-
ear plasmid pSHJG1. Circles 1 and 2, predicted coding sequences on the plus and minus strands,
respectively, colored according to COG functional categories; Circle 3, GC content; Circle 4,
GC bias. (C) Atlas of circular plasmid pSHJG2. Circles 1 and 2, predicted coding sequences on
the plus and minus strands, respectively, colored according to COG functional categories; Circle
3, GC content; Circle 4, GC bias.
consisting of several packed palindromes with nonconserved loop

sequences, thereby forming a different secondary structure from its right
end and both chromosome ends. Interestingly, a complete bacterial immune
system CRISPR-Cas (Horvath and Barrangou, 2010) was identified in
pSHJG1, suggesting a resistance to phages and other invading genetic ele-
ments by strain 5008.
Genome-wide comparison among completely sequenced Streptomyces chro-
mosomes revealed highly conserved core regions ranging from 5.50 to 7.25 Mb
[SCLAV0503-SCLAV5245 (5.50 Mb), SCO1209SCO6774 (6.25 Mb),
SGR0954SGR6311 (6.36 Mb), SAV1638SAV7128 (6.48 Mb), SBI25785
SBI889 (7.12 Mb), SCAB12831SCAB78641 (7.25 Mb)], substantially in
proportion to the corresponding chromosomal length. However, the genome of

strain 5008 was predicted to have a relatively small core region (5.56 Mb), with
a left arm of 3.16 Mb and a right arm of 1.43 Mb (Fig. 2.2A). Syntenic analyses
showed that, except for S. scabies, large continuous or separate inversions cen-
tered at oriC were detected in the chromosome of strain 5008, when compared
with other Streptomyces species.
To further identify commonly conserved or species-specific proteins in
strain 5008, orthologs shared among the seven Streptomyces strains were
analyzed by Microbial Genome Database (MBGD) (Uchiyama, 2007). The
results showed that 2954 SHJG proteins (33.3% of the total CDSs), 2899
SCO proteins (37.3%), 2901 SAV proteins (38.3%), 2879 SGR proteins
(40.3%), 2989 SCAB proteins (33.4%), 2989 SBI proteins (29.8%), and
2806 SCLAV proteins (49.1%) could be classified into 2754 clusters. The
major conserved proteins are assigned with functions for transcription, trans-
lation, energy production, and amino acid and carbohydrate metabolisms.
Notably, 1640 strain-specific orthologous clusters including 1749 proteins
for strain 5008 could be detected. Surprisingly, the amfABST cluster
(Capstick et al., 2007) and the ramR-activated gene (rag) cluster for aerial-
mycelium formation and sporulation (San Paolo et al., 2006) were not found
in strain 5008.
In total, 29 gene clusters were determined in the chromosome of strain
5008. Twenty are located in subtelomeric regions with 14 in the left arm
and 6 in the right arm. The validamycin A gene cluster (val) is located at
a region 350 kb away from the left end of the chromosome (Fig. 2.2A).
Among an additional 27 gene clusters putatively for secondary metabo-
lites, 6 were assigned for the biosynthesis of polyketide synthases (PKSs),
8 for non-ribosomal peptide synthases (NRPSs), 5 for hybrid PKS-
NRPSs, 4 for terpenoids, 1 for lantibiotics, and the other 3 for melanin,
norcardamine siderophore, and ochronotic pigment, respectively.
Similar to most Streptomyces, the central carbon metabolism of strain
5008 includes complete glycolysis, the pentose phosphate pathway (PPP),
the tricarboxylic acid (TCA) cycle, and gluconeogenesis pathway with
multiple copies of genes encoding key enzymes for these pathways
(Fig. 2.3). Validamycin A synthesis requires sedoheptulose 7-phosphate
and UDP-glucose derived from carbohydrate metabolism as precursors.
The UDP-glucose synthesis is possibly catalyzed by UDP-glucose-1-
phosphate uridylyltransferases (UGP), SHJG4652, and SHJG7333 (sharing
77% identity), preceded by the isomerization of glucose-6-phosphate to
glucose-1-phosphate catalyzed by phosphoglucomutase (SHJG1995).
Unlike strain 5008, only one copy of the ugp gene is present in other
sequenced Streptomyces genomes, implicating stronger carbon fluxes
from glucose to UDP-glucose for the validamycin A synthesis in strain
5008 (Fig. 2.3).
FIGURE 2.3 Schematic diagram of central carbon and nitrogen metabolisms for validamycin
A production in strain 5008. Red arrows and characters represent metabolic pathways and pre-
cursors directly related to validamycin A biosynthesis, respectively. Numbers in parentheses
indicated copy numbers of annotated proteins or complexes in the metabolic pathways.
Abbreviations: ALD, alanine dehydrogenase; DHAP, dihydroxyacetone phosphate; FBP,
fructose-1, 6-bisphosphate; GAP, glyceraldehyde-3 phosphate; GDH, glutamate dehydrogenase;
Glc, glucose; GlcNAc, N-acetylglucosamine; GOGAT, glutamate synthase; GS, glutamine synthe-
tase; Mal, maltose; PEP, phosphoenolpyruvate.
2.2.2.2 General Features of the S. hygroscopicus subsp.

limoneus KCTC 1717 Genome
The complete genome of S. hygroscopicus subsp. limoneus KCTC 1717
consists of two linear chromosomes of 10,537,932 bp with 71.96% G 1 C
content (Table 2.5). The coding regions that cover 84.62% of the genome
(8,916,963 bp) encode 8983 proteins. The genome also encodes 85 pseu-
dogenes, 18 rRNAs (6 operons), 67 tRNAs, 1 tmRNA, and 1 ncRNA, all
of which make up 1.14% of the genome (Table 2.4). Signal peptides and
transmembrane helices were detected in 576 (6.41%) and 1924 (21.42%)
protein coding genes, respectively. The genome encodes a 27-gene cluster
(val) located from 337,192 to 374,655 nucleotide positions on chromo-
some II (locus tags: SHL15 7990 to SHL15 8016), which is responsible
TABLE 2.5 Genomic Features of S. hygroscopicus subsp. limoneus

KCTC 1717
Features Chromosome I Chromosome II

Genome size (bp) 8,648,026 1,889,906
DNA coding (bp) 7,424,859 1,492,104
G 1 C content (%) 72.1 71.4
Total genes 7586 1569
Protein coding genes 7447 1536
rRNA (operons) 18(6) 0(0)
tRNA 67 0
tmRNA 1 0
ncRNA 1 0
for producing validamycin. The detection and localization of complete val

gene cluster is expected to confer useful genetic information regarding
biosynthesis of aminocyclitols (e.g., validamycin and valienamine).
Moreover, 111 putative gene clusters responsible for production of diverse
secondary metabolites were also detected. Of these, the existence of genes
encoding NRPSs, types IIII PKSs, and a type II PKS/NRPS hybrid
synthase reveals the potential of KCTC 1717 to produce industrially
important natural products.
2.3 PRODUCTION AND ISOLATION OF VALIDAMYCINS

AND RELATED NATURAL COMPOUNDS
2.3.1 Fermentation of Validamycins
Validamycins are weakly basic, water soluble antibiotics produced by
S. hygroscopicus var. limoneus. Cultural conditions for the production of
validamycins with S. hygroscopicus var. limoneus No. 7545 were studied by
Iwasa et al. (1971b).
2.3.1.1 Temperature of Submerged Culture

As the optimum temperature for the growth of T-7545 was found to be fairly
high, it was cultivated at 37 C. Since the fermented broth thus obtained was
turbid and filtration was very difficult, fermentation at 27 C was compared
with that at 37 C. As shown in Table 2.6, the validamycin titer of the filtered
broth obtained in 37 C incubation was a little higher than that at 27 C, but
TABLE 2.6 Effect of Temperature on Validamycin Production
Incubation 27 C 37 C

Period
Validamycin Brothb Validamycin Brothb
(days)
Titera (unit/mL) Titera (unit/mL)
4 20,000 Clear, easy 35,000 Turbid,
filtration difficult
filtration
6 20,000 do 35,000 do
a
Assayed by dendroid-test method.
b
The medium used in this experiment consists of 5.0% glucose, 3.6% soybean flour, 0.5% peptone,
and 0.6% CaCO3, pH 7.0. 60 mL of the medium in each Erlenmeyer flask of 200 mL capacity was
inoculated and cultivated on the rotary shaker (5.0 cm radius) at 220 rpm.
TABLE 2.7 Effect of Medium Volume on Validamycin Production
Incubation Volume of the Mediumb

Period (h)
20 mL 60 mL
pH Validamycin pH Validamycin
Titera (unit/mL) Titera (unit/mL)
66 8.0 10,000 7.4 15,000
90 8.0 15,000 7.8 20,000
114 8.2 20,000 8.0 50,000
138 8.6 20,000 8.2 50,000
a
Assayed by dendroid-test method.
b
Composition of the medium used in this experiment was the same as in Table 2.4.
filtration was more difficult and essentially equivalent results were obtained in
a greenhouse test. So the lower temperature was used to prepare the antibiotic.
2.3.1.2 Effect of Medium Volume on Validamycin Production

Erlenmeyer flasks of 200 mL capacity containing 20 mL or 60 mL of the
fermentation medium were compared. As shown in Table 2.7, 60 mL of the
medium gave superior production.
2.3.1.3 Effect of Medium Composition

on Validamycin Production
(1) Selection of carbon and nitrogen sources. A variety of media containing
glucose, glycerol, or starch as a carbon source and various nitrogen sources
such as peptone, beef extract, corn steep liquor (CSL), soybean flour (SBF),
and corn gluten meal (CGM) were compared by the reversed layer method.
The results are shown in Table 2.8. Good growth occurred in the media
containing glucose or glycerol as the carbon source regardless of nitrogen
sources.
As for the yield of validamycin, glucose was notably superior to other
carbon sources, and when it was used, no remarkable differences were found
among the nitrogen sources. As a result of repeated examinations, CSL and
CGM were judged relatively better than SBF and beef extract.
When starch was adopted as the carbon source, considerable validamycin
production was obtained, although slowly, in the medium containing CSL
and CGM as the nitrogen sources.
(2) Effects of inorganic salts. Effects of FeSO4, MnSO4, ZnSO4, NaCl,
KCl, and NH4Cl on the production of validamycin were investigated in the
following two kinds of media: (a) 5.0% glucose, 3.0% SBF, 1.0% CSL,
0.5% CaCO3; (b) 4.0% glucose, 2.0% corn starch, 2.0% CSL, 3.0% CGM,
1.5 CaCO3. Little effect of these inorganic salts was found in the first
medium, but in the latter medium, remarkable effects of NaCl and NH4Cl
were found as shown in Table 2.9.
(3) Selection of medium for high yield of validamycins. From these and
other results, a medium composed of glucose 2.0%, corn starch 4.0%, CSL
2.0%, CGM 4.0%, NH4Cl 0.5%, NaCl 1.5%, and CaCO3 1.5% was selected,
yielding about 620 μg/mL of validamycins.
2.3.2 Isolation of Validamycins From the Broth

Validamycins A and B were first isolated as follows (Iwasa et al., 1971c).
The culture broth of S. hygroscopicus var. limoneus was acidified with oxalic
acid to pH 34 and the calcium ions in the broth were removed with the
mycelium by filtration. To remove cationic and anionic impurities, the broth
filtrate was first treated with Amberlite IR-120 (H1) and IR-45 (OH2) and
then applied to a column of Dowex-50 X-2. The antibiotics were adsorbed
on the resin and then eluted with aqueous ammonia. The elute concentrate
was chromatographed on Dowex-50 X-2 with pyridineacetic acid buffer at
pH 6.5. Validamycin A was adsorbed on the column and validamycin B
passed through. Validamycin A adsorbed on the resin was eluted with
pyridineacetic acid, pH 7.5, and the elute was concentrated to give pure
crystalline validamycin A. The concentrate of the column effluent was dis-
solved in water, applied to a column of Dowex-1 X-2 and developed with
water to give pure validamycin B. The purity of each antibiotic was confirmed
by thin-layer chromatography (TLC) (Fig. 2.4) and its crystalline derivative.
Later, the crude validamycins were chromatographed on Dowex 1 3 2
column (OH2 form, 100200 mesh), and the column was developed
with water to give the eight components: validoxylamines A and B, and
TABLE 2.8 Production of Validamycins by T-7545 and Its Growth on Various Media
Media
1 2 3 4 5 6 7 8 9 10 11 12
Component Glucose 5 5 5 5
(%)
Glycerol 5 5 5 5
Starch 5 5 5 5
Peptone 1 1 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Beef 0.5 0.5 0.5
extract
CSL 3 3 3 1 1 1 1 1 1
SBF 0.5 0.5 0.5 1 1 1 3 3 3
CGM 3 3 3
NaCl 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3
CaCO3 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Growth a
111 111 1 B1 1 111 111 1 1 B1 1 1 111 111 111 111 111 1 B1 1
pH 7.5 7.25 8.0 8.2 7.3 8.3 7.75 7.15 8.0 7.55 8.1 8.6
b
Validamycin titer 64 12.5 12.5 140 12.5 74 103 12.5 38 64 28 12.5
1 1 1 : Very good growth; 1 1 : Good growth; 1 : Fair growth.

a
b
Five-day culture was assayed by reversed layer method.
TABLE 2.9 Effects of NH4Cl and NaCl on the Production of Validamycin
Salt Added (%) Growth pH Validamycin Titera (μg/mL)

NH4Cl 0.5 111 7.8 345
NaCl 1.5 111 7.45 300
NH4Cl 0.5 1 NaCl 1.5 111 7.1 380
No addition 111 8.4 75
a
Seven-day culture was assayed by reversed layer method.
Rf
1.0
0.8
0.6
0.4
0.2
0
1 2 3
FIGURE 2.4 TLC chromatograph of validamycins A and B. Stationary phase: Silica gel G;
Solvent system: n-PrOH/AcOH/H2O 5 4/1/1; Detection: Naphthoresorcin-H2SO4 reagent.
(1) Crude powder of validamycins; (2) Validamycin A; (3) Validamycin B. Reprinted with
permission courtesy of Japan Antibiotics Research Association (JARA).
validamycins D, A, C, B, F, and E, in order of elution from the column

(Horii et al., 1972). The peak of validamycin C appeared just before valida-
mycin B, and these two components were slightly overlapped. On the other
hand, validamycin D was eluted just before validamycin A. Validamycin
C-rich fraction and fractions mainly containing validamycin D were rechroma-
tographed on a silica gel column (elution with n-PrOH/AcOH/H2O 5 4/1/1)
and Dowex 50W 3 2 column (elution with pyridineacetic acid buffer, pH
6.0). Validamycins E and F were strongly retarded on Dowex 1 3 2 column
owing to their great affinity for the resin, which showed a rather diffuse elution
pattern on a gravity flow. The complete resolution of validamycins E and F
was not accomplished on Dowex 1 3 2 column. Validamycin E-rich
fractions and validamycin F-rich fractions obtained by Dowex 1 3 2
chromatography were further chromatographed on Dowex 50W 3 2 column

(elution with pyridineacetic acid buffer, pH 6.0). In this chromatography,
early fractions contained validamycin F. Then, validamycin E was eluted.
These chromatographic purifications were repeated once more, if necessary.
Finally, chromatographically homogeneous validamycins C, D, E, and F were
obtained by rechromatography on Dowex 1 3 2 (OH2 form, developed
with water).
Validoxylamine A was eluted just before validamycin D on Dowex 1 3 2
column, and crystallization from waterethanol gave the pure validoxyla-
mine A. Although validoxylamine B and validamycin D were almost over-
lapped on Dowex 1 3 2 chromatography, these components were easily
separated on Dowex 50W 3 2 column (eluted with pyridineacetic acid
buffer, pH 6.0). These compounds were identical with validoxylamines A
and B obtained by the hydrolysis of validamycins A and B, respectively. The
chromatograms on Dowex 1 3 2 columns of these components are shown in
Fig. 2.5 (validamycins A, B, C, D, E, F, and validoxylamines A) and
Fig. 2.6 (validoxylamines A and B).
VA-A
D
A
Refractive index
C
F
B E
0 1 2 3 4 5 6 7 8 9 hours
FIGURE 2.5 Chromatogram of authentic mixture of validamycins A, B, C, D, E, F and vali-

doxylamines A. Column: Dowex 1 3 2 (OH form, 100200 mesh, 27 cm 3 15 mm I.D.).
Carrier: Water (gravity flow rate; 1.1 mL/min). Reprinted with permission courtesy of Japan
Antibiotics Research Association (JARA).
VA-A
Refractive index
VA-B
0 10 20 30 40 50 60 min.
FIGURE 2.6 Chromatogram of authentic mixture of validoxylamines A and B. Column:

Dowex 1 3 2 (OH2 form, 100200 mesh, 27 cm 3 15 mm I.D.). Carrier: Water (gravity flow
rate, 1.1 mL/min). Reprinted with permission courtesy of Japan Antibiotics Research Association
(JARA).
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both to Winder and to the citizens; he attended the President in his
expeditions, used wisely such power as he had, and indulged in few
words.[257] At the President’s request he went to Bladensburg to
support and assist Winder; at the President’s order he retired after
the battle began. He returned, after Winder, to the Capitol, hoping to
convert that strong building into a fortress,—a measure not
unreasonable, if the regulars and sailors were rallied to make a
stand there. When Winder decided to retire to Georgetown, the
secretary acquiesced without a word. Then, in pursuance of a
Cabinet decision made a few hours before, he set out in company
with Secretary Campbell for Frederick, and arrived there in the
course of the next day.
Armstrong and Campbell were at Frederick when the British army
began its retreat Thursday night; the President was in Virginia,
sixteen miles up the river; Winder and Monroe, with the remaining
troops, were at Montgomery Court House sixteen miles from
Washington. There they learned, Friday morning, that the British had
marched toward Bladensburg, probably going to Baltimore; and at
about ten o’clock in the morning Winder marched from Montgomery
Court House toward Brookville and Baltimore with all his force.
Passing through Brookville, they camped, Friday night, “about half
way between Montgomery Court House and Ellicott’s upper
mills,”[258] and Winder there left them, starting late that evening
alone for Baltimore, and leaving Monroe and Stansbury in command,
with directions to follow.
Meanwhile the President had crossed the Potomac that morning,
expecting to find Winder and Monroe at Montgomery Court House;
but on arriving there at six o’clock in the evening, and learning that
the army had marched toward Baltimore, he followed as far as
Brookville, about ten miles, where he passed the night.[259] Attorney-
General Rush was with him. Saturday morning, August 27, the
President sent notes to all his Cabinet requesting them to unite in
Washington.[260] The same afternoon he returned with Monroe and
Rush to the city, which they reached at about six o’clock, after three
days’ absence.
Armstrong and Campbell, ignorant of the change in plan, waited
at Frederick for the President’s arrival, while the President and
Monroe, Sunday, August 28, began the task of restoring the
functions of government. The task was difficult, not so much on
account of the British ravages, which had been confined to public
property, as on account of the general irritation and the continued
panic. Hardly had Ross’s army disappeared when a squadron of
British war-vessels, under Captain Gordon of the frigate “Seahorse,”
worked its way up the river, approaching Fort Washington or
Warburton August 27. The commander of that post,
misunderstanding his orders, abandoned it and crossed the river
with his men. Gordon’s squadron reached Alexandria the next day,
and the town capitulated, since it could not resist. Until August 31 the
frigates remained at Alexandria, loading such vessels as were there
with the tobacco and other produce which the warehouses
contained.[261]
The citizens of Washington and Georgetown, expecting to be
visited in their turn, and conscious of their inability to resist, talked of
a capitulation. Public feeling ran strong against the President.
Armstrong was absent. Winder was at Baltimore. Monroe alone was
in a position to act, and upon Monroe the President was obliged to
depend.
“Under these circumstances,” said Monroe in the only authentic
account of the event which remains,[262] “the President requested Mr.
Monroe to take charge of the Department of War and command of the
District ad interim, with which he immediately complied. On the 28th,
in the morning, the President with Mr. Monroe and the Attorney-
General visited the navy-yard, the arsenal at Greenleaf’s Point, and
passing along the shore of the Potomac up toward Georgetown. Mr.
Monroe, as Secretary of War and military commander, adopted
measures under sanction of the President for the defence of the city
and of Georgetown.”
Colonel W[adsworth?] who was placing some guns on the
opposite shore refused to obey an order of Monroe to change their
position. Monroe rode across the bridge and gave the order in
person. The colonel replied that he did not know Mr. Monroe as
Secretary of War or commanding general. Monroe ordered him to
obey or leave the field, and the colonel left the field.
Monroe’s act, whether such was his intention or not, was a coup
d’état. The citizens, unable to punish the President, were rabid
against Armstrong. No one could deny that they had reason for their
anger, although the blame for their misfortunes was so evenly
distributed between every officer and every branch of government
that a single victim could not justly be selected for punishment.
Monroe, instead of giving to Armstrong in his absence such support
as might have sustained him, took a position and exercised an
authority that led necessarily to his overthrow. The influence of such
acts on the citizens was obvious. That evening the first brigade of
militia held a meeting, and passed a formal and unanimous
resolution that they would no longer serve under the orders or
military administration of General Armstrong, whom they denounced
as the willing cause of the fate of Washington.[263] This mutinous
resolution, adopted in the immediate presence of the enemy, was
taken to the President by two officers of the brigade, one of whom at
least was a strong friend of Monroe.[264]
The resolution of the first brigade was communicated to the
President the next morning, Monday, August 29. All the President’s
recorded acts and conversation for months after the capture of
Washington implied that he was greatly shaken by that disaster. He
showed his prostration by helplessness. He allowed Monroe for the
first time to control him; but he did not dismiss Armstrong. At one
o’clock on the afternoon of the same day the Secretary of War
arrived in Washington.[265] The President that evening rode to his
lodgings. Madison preserved a memorandum of their conversation,
and Armstrong also immediately afterward recorded what passed.
[266] The President described to the secretary the violent prejudices
which existed in the city against the Administration, and especially
against himself and the Secretary of War. “Before his arrival there
was less difficulty, as Mr. Monroe, who was very acceptable to them,
had, as on preceding occasions of his absence, though very
reluctantly on this, been the medium for the functions of Secretary of
War;” but since Armstrong had returned, something must be done.
Armstrong replied that he was aware of the excitement, and knew
its sources; that evidently he could not remain if his functions were
exercised by any one else; that he would resign, or retire from
Washington at once, as the President preferred.
Madison deprecated resignation, and recommended “a temporary
retirement, as he suggested;” and after some further conversation, in
which the President complained of the secretary’s mistakes, they
parted with the understanding that Armstrong should leave
Washington the next morning. Armstrong behaved with dignity and
with his usual pride; but he understood, if Madison did not, the
necessary consequences of his retirement, and on reaching
Baltimore sent his resignation to the President. At the same time he
announced it to the public in a letter, dated September 3, containing
comments on the weakness of Madison’s conduct calculated to
close their relations.
Between conscious intrigue and unconscious instinct no clear line
of division was ever drawn. Monroe, by the one method or the other,
gained his point and drove Armstrong from the Cabinet; but the
suspicion that he had intrigued for that object troubled his mind to
the day of his death.[267] Even after Armstrong’s departure, the
dangers and disadvantages of appointing Monroe his successor
were so great that for three weeks the post remained unfilled, until,
after many doubts and hesitations, Monroe wrote to Madison a letter
claiming the appointment of Secretary of War.
“I have thought much of the state of the Departments at this time,”
he informed the President, September 25,[268] “and of the persons
whom it may be proper to place in them, and have concluded that
whatever may be the arrangement with respect to other Departments,
the Department of War ought to be immediately filled. I think also that I
ought to take charge of it.... By taking charge of the Department twice,
and withdrawing from it a second time, it may be inferred that I shrink
from the responsibility from a fear of injuring my reputation; and this
may countenance the idea that the removal of the others was an affair
of intrigue in which I partook, especially in the latter instance, from
selfish and improper motives, and did not proceed from his
incompetency or misconduct. It seems due, therefore, to my own
reputation to go through with the undertaking by accepting
permanently a trust which I have not sought, never wished, and is
attended with great responsibility and hazard. By taking the place, all
clamor will be silenced. It is known, here at least, that I was put into it
when the other could no longer hold it. Those who wished it in the first
instance will be satisfied, and I shall go on with your support and a
favorable expectation of the public that I shall discharge to advantage
its duties.”
While Monroe in private communications with Madison thus
treated Armstrong’s retirement as a “removal,” due to his
“incompetency or misconduct,” and Madison apparently acquiesced
in that view, in public Madison seemed inclined to convey the idea
that Armstrong was not removed or meant to be removed from
office, but rather deserted it. Whichever view was correct, Madison
certainly dreaded the political effect of appearing to remove
Armstrong; and while he gave to Monroe the appointment of
Secretary of War, he wrote, September 29, to Governor Tompkins of
New York, offering him the State Department.
Governor Tompkins declined the offer. Apart from the great need
of his services as governor, the experience of Northern men in
Virginia Cabinets was not calculated to encourage any aspirant to
the Presidency in seeking the position. Monroe remained Secretary
of State as well as Secretary of War. As Secretary of State he had
little or nothing to do, which was partly the cause of his activity in
military matters; but as Secretary of War he was obliged to
undertake a task beyond the powers of any man.
During an entire month after the appearance of the British in the
Patuxent, the United States government performed few or none of its
functions. The war on the frontiers was conducted without orders
from Washington. Every energy of the government was concentrated
on its own immediate dangers, as though Washington were a
beleaguered fortress. Slowly the tide of war ebbed from the Potomac
and Chesapeake, and not until it had wholly subsided could men
cease to dread its possible return.
Captain Gordon’s squadron began its descent of the river
September 1, greatly annoyed[269] by batteries erected on the banks
by Commodore Rodgers, Perry, and Porter, who were sent from
Baltimore, by order of Secretary Jones, for the purpose. Not until
September 6 did Captain Gordon escape from his perilous position
and rejoin the fleet. Meanwhile the shores of Chesapeake Bay
continued to be ravaged with all the severity threatened by
Cochrane. Frederick Chamier, afterward the author of several
popular sea-stories, was then a lieutenant on the “Menelaus” in
Cochrane’s squadron, and his recollections in “The Life of a Sailor”
gave a lively picture of the marauding in which he took part.[270] Like
Napier, Chamier was too tender-hearted for his work. “I am willing to
make oath,” he wrote, in reply to Captain Scott’s contradictions, “that
on the day that the ‘Menelaus’ entered the Potomac, three houses
were burning at the same time on the left-hand bank of the river. We
burnt more than five ourselves.” War was commonly accompanied
by destruction, but the war in the Chesapeake was remarkable for
the personal share taken by the highest officers, especially by
Cockburn and Ross, in directing the actual operation of setting fire to
private and public property.[271]
At last the practice caused a disaster that cost the British navy a
life more valuable than all the property it could destroy or carry away.
The “Menelaus,” commanded by Sir Peter Parker, was sent up
Chesapeake Bay to divert attention from the general movement of
troops and ships on the Potomac.[272] The “Menelaus” took position
off the Sassafras River, which Cockburn had cleared of vessels the
year before. Nothing in the river could injure the navy, but the
“Menelaus” was ordered to make a diversion; and Sir Peter Parker
learned from negroes that two hundred militia, encamped behind a
wood half a mile from the beach, intended to cross the bay for the
protection of Baltimore.[273] One hundred and twenty-four men were
landed at eleven o’clock on the night of August 30, and went in
search of the militia. Instead of half a mile, they were led by their
guides three or four miles, and at last found the militia drawn up,
ready to receive them. Sir Peter Parker ordered an attack, and while
cheering it on in the moonlight was struck by a buckshot, which
severed the femoral artery. The sailors carried him back to the ship,
but he died long before reaching it.[274] His party escaped with a loss
of thirteen killed and twenty-seven wounded besides their captain.
The Americans regretted only that the punishment had fallen on
the wrong person, for Cochrane and Cockburn rather than Parker
were the true marauders; but the lesson was effectual, and the
British became more cautious after Parker’s death. Indeed, their
activity in the Chesapeake centred thenceforward in an effort to
capture Baltimore, which required all their strength.
Baltimore should have been first attacked, but Cockburn’s
influence by diverting Ross to Washington gave the larger city time
to prepare its defence. The citizens themselves, headed by the
mayor, took charge of the preparations; and their first act, contrary to
the course pursued by Armstrong and Winder at Washington, was to
construct intrenchments round the city, and to erect semi-circular
batteries at a number of points, mounted with cannon and connected
by a line of works. After the capture of Washington the citizens toiled
still more earnestly at their task, until a formidable line of redoubts
protected the town, and, though not wholly finished, gave cover to
the militia. The batteries were manned by sailors, commanded by
officers of the navy. The harbor was protected by Fort McHenry,
small but capable of defence, and occupied by a strong force of
regular troops, sailors, and volunteer artillerists numbering about one
thousand, under the command of Lieutenant-Colonel Armistead of
the Artillery.[275]
These precautions made the capture of Baltimore impossible by
such a force as had taken Washington, even though aided by the
fleet. The precise number of troops present in the city, according to
the official return for September 10, was twelve thousand nine
hundred and ninety-one men present for duty, with eight hundred
and ninety-seven officers. The aggregate of present and absent was
sixteen thousand eight hundred and five men.[276] The force was
ample to man the works, but the fortifications chiefly decided the
result. No army on either side during the war succeeded in storming
works in face, except by surprise; and to turn the works of Baltimore
a larger army was required than Ross had at his command.
The militia major-general commanding at Baltimore was no other
than Samuel Smith, senator of the United States. He had lately
passed his sixty-second year. The brigadier-general in the United
States service who commanded the military district was W. H.
Winder, whose defence of Washington ended abruptly August 24,
and who left that neighborhood on the evening of August 26 to take
command of the defences of Baltimore. Winder was a Baltimore
lawyer, only three years Smith’s junior, when Eustis and Madison
gave him a regiment in March, 1812. Smith was not disposed to
accept the idea of subordination to a man of inferior rank, military
and civil, who knew no more of war than Smith knew, and whose
career had been twice marked by unusual ridicule. Winder on
arriving in Baltimore notified Smith that he should take command,
and was astonished when the senator declined to surrender his
authority. Winder appealed to the President and to the governor of
Maryland, his cousin Levin Winder; but nothing could be done to
assist him, and in the end he submitted. Samuel Smith remained in
command.
The British leaders having succeeded in turning their
demonstration up the Patuxent into an attack on Washington, next
decided to make “a demonstration upon the city of Baltimore, which
might be converted into a real attack should circumstances appear to
justify it,”[277] and sailed from the Potomac September 6 for the
Patapsco River. They anchored September 11 off its mouth. From
that point Ross’s army when landed had only fourteen miles to
march, and no water in their way, while Cochrane’s fleet had but
twelve miles to sail. Compared with the approaches to Washington,
the approach to Baltimore was easy.
MILITARY TOPOGRAPHY
OF
BALTIMORE
AND ITS VICINITY
AND OF
PATAPSCO NECK
TO
NORTH POINT
BY ORDER OF
BRIG. GEN. W. H. WINDER
1814.
Ross’s troops were all landed at daylight on the northern point,
and were in motion by eight o’clock September 12, without firing a
shot. Their numbers were differently given by British authorities,—
one reporting them at three thousand two hundred and seventy rank-
and-file;[278] the other reckoning them at upward of five thousand.
[279] Ross made on the Patapsco no such leisurely movements as
on the Patuxent, but began his march at once, and proceeded about
five miles without meeting resistance. The light brigade with the
Eighty-fifth regiment was in advance; the second brigade, under
Colonel Brooke of the Forty-fourth, followed; and the third brigade,
under Colonel Patterson of the Twenty-first, formed the rear. At the
same time the fleet moved up the channel toward Fort Henry.
The city was naturally excited at the news that the British had
arrived. General Smith, on receiving the intelligence September 11,
detached a brigade of Baltimore militia, under General Stricker, to
check the enemy if possible, and Stricker advanced that evening
about seven miles toward North Point. His force numbered about
three thousand two hundred men;[280] and with that body of raw
militia, a part of whom had been routed at Bladensburg only a
fortnight before, General Stricker attempted to fight a battle with five
thousand old soldiers. On the morning of September 12 he formed
his troops in three lines three hundred yards apart, apparently in
close order, without cover or protection of any kind, standing in fields
more or less open, and with an exposed flank.[281] Of all his
arrangements, the only one which showed ordinary caution was to
send a detachment of cavalry and riflemen a mile or two in his front.
As the British advance approached, the American outposts fell back,
and General Stricker sent forward some four hundred men, partly
rifles, as skirmishers. The British advanced guard coming up, the
skirmishing party fired, but was soon driven back. Ross and
Cockburn were walking together with the advance, and after the
firing ceased, Ross turned back alone to order up the light
companies in anticipation of more serious resistance. On his way he
was shot through the breast from the wood, and fell in the road,
where he lay till he was found by the light companies hurrying
forward to the scene of the firing. He barely spoke afterward.[282]
The loss of their commanding general was the second heavy
penalty paid by the British for their contempt of militia. Colonel
Brooke immediately took command, and the advance was not
checked; but the loss was not the less serious. When Brooke saw
Stricker’s line stretching across the field, he did not dash at them at
once with the light brigade as Thornton had attacked the larger force
and stronger position at Bladensburg, but deployed the whole army
and formed a regular order of battle. Although his force easily
overlapped and outflanked the American, the engagement that
followed was sharp, and the Americans were not routed without
considerable loss to the British, who reported forty-six killed and two
hundred and seventy-three wounded,—or more than they reported at
Bladensburg. The Americans, though routed, suffered less, losing
only twenty-four killed, one hundred and thirty-nine wounded, and
fifty prisoners, with two field-pieces.
This spirited little battle detained the British so long that they
bivouacked on the field, and passed the night in a drenching rain,
resuming their march the next morning, September 13, when they
found the roads obstructed, and were obliged to move so slowly that
evening arrived before they came in sight of Baltimore.[283] When at
last they saw on the distant heights the long line of intrenchments
that surrounded Baltimore on the side of their approach, they
stopped short. Colonel Brooke had gone forward with the advance,
and was engaged all day, at about a mile and a half distance, in
studying the American lines. He made arrangements for a night
attack, hoping to avoid the effects of the American artillery,[284] and
then waited for the fleet to support him.
The fleet all day bombarded the forts and batteries that covered
the entrance to the harbor, and continued the bombardment till past
midnight. Unlike most naval engagements during the war, this battle
was harmless to either party. The heavier British ships feared to
approach within range, owing to a barrier of sunken vessels, covered
by the guns on shore and by gunboats. Without the heavy ships, the
lighter vessels could not maintain a position. The fort sustained no
great injury, and only four men were killed and twenty-four wounded.
[285] The fleet as far as was reported sustained no injury whatever.
The firing ceased toward midnight, and Admiral Cochrane sent word
to Colonel Brooke that he could do no more.[286]
“Under these circumstances,” reported Colonel Brooke, “and
keeping in view your Lordship’s instructions, it was agreed between
the Vice-admiral and myself that the capture of the town would not
have been a sufficient equivalent to the loss which might probably be
sustained in storming the heights.”
Sir George Prevost at Plattsburg only two days before, with three
times the number of troops and a much smaller number of
opponents, came to the same conclusion. That both officers were
probably wise was shown by the experience of Lieutenant-General
Drummond, a month earlier, in attempting to storm the lines of Fort
Erie. Brooke and Prevost followed the same course in another
respect, for Brooke withdrew his army so rapidly that at noon of
September 14 it had already passed the battle-field of two days
before, and in another day the whole force was re-embarked.[287]
As soon as the wind allowed, the fleet returned to the lower
Chesapeake; and September 19 Admiral Cochrane sailed for Halifax
to prepare for a new expedition. The troops remained till October 14
in their transports in the bay, and then set sail for Jamaica, leaving
Virginia and Maryland to a long repose, which the vexed shores
sorely needed.
CHAPTER VII.
After balancing gains and losses, the result of the campaign
favored Great Britain by the amount of plunder which the navy
obtained in Alexandria, and by the posts which Governor Sherbrooke
occupied between the Penobscot and the Passamaquoddy in Maine.
Considering the effort made and the waste of money, the result was
a total disappointment to the British people; but even these
advantages on land could not be regarded as secure until the British
navy and mercantile marine had summed up their profits and losses
on the ocean.
At the beginning of the year 1814 the American navy had almost
disappeared. Porter in the “Essex” still annoyed British interests in
the Pacific; but of the five large frigates only the “President” was at
sea. January 1 the “Constitution,” Captain Charles Stewart, left
Boston and cruised southward, making a few prizes and destroying a
British fourteen-gun schooner, but fighting no battle and effecting no
object equivalent to her cost. In returning to Boston, April 3, she
narrowly escaped capture by the two British frigates blockading the
port, and with difficulty got into Marblehead harbor. The
“Constitution” did not again go to sea until December 17. During her
cruise of three months, from January 1 to April 3, she made four
prizes.
The “President” regained New York February 18, and was
blockaded during the rest of the year. The “United States” and
“Macedonian” remained blockaded at New London. The
“Constellation” remained blockaded at Norfolk. The corvette
“Adams,” twenty-eight guns, ran the blockade of Chesapeake Bay
January 18, and cruised until August 17, making nine prizes and
several narrow escapes before striking on the Isle of Haut and taking
refuge in the Penobscot as the British forces occupied Castine. The
story of her destruction has been told. Her fate was the same she
would have met had she remained in Washington, where a week
earlier the new forty-four-gun frigate “Columbia” and the new twenty-
two-gun sloop-of-war “Argus” were burned to prevent them from
falling prize to the British army.
This short abstract accounted for all the frigates except the
“Essex,” whose fortune was no happier than that of the larger ships.
October 27, 1812, the “Essex,” Captain David Porter, left the
Delaware, intending to meet Bainbridge and form part of a squadron
under his command. Failing to meet Bainbridge, though constantly
near him, Porter at last decided to sail southward; and when
Bainbridge in the “Constitution” reached Boston February 27, 1813,
the “Essex” had already passed Cape Horn, and was running up the
western coast of South America to Valparaiso.
At Valparaiso Porter arrived March 14, 1813, to the consternation
of commerce. Chili had recently asserted independence of Spain,
and as yet no English war-vessels were stationed in the Pacific. The
chief British interest was the whale fishery which centred in the
Galapagos Islands,—a group lying under the equator, about a
thousand miles from Panama. Although the influence of England
was supreme, on account of her naval power, her commerce, and
her political alliance with the Spanish people, and although Porter
had neither a harbor of his own, nor the support of a diplomatic
officer on the Pacific, he had nothing to fear. He was well received at
Valparaiso, where since 1811 J. R. Poinsett had held the post of
United States Consul-General for Buenos Ayres, Chili, and Peru; but
the “Essex” tarried only for supplies, and soon sailed for the
Galapagos Islands. There she arrived in April, 1813, and in the
course of the summer captured all the British whalers known to be in
those seas. These were twelve in number, and after sending some of
them away, Porter still had a fleet of five armed ships besides his
own, and nothing more to do.
The “Essex” had then been a year at sea, and needed repairs.
Porter determined to take his entire fleet of six vessels about three
thousand miles to the Marquesas Islands,—as though to make a
voyage of discovery, or to emulate the mutineers of the “Bounty.” The
squadron sailed three weeks over the southern seas, until, October
23, the Marquesas Islands were sighted. There Porter remained
seven weeks, amusing himself and his crew by intervention in native
Marquesan politics, ending in his conquest of the principal tribes,
and taking possession of the chief island in the name of his
Government. That he should have brought away his whole crew after
such relaxation, without desertion, was surprising. The men were for
a time in a state of mutiny on being ordered to sea; but they did not
desert, and the squadron sailed, Dec. 12, 1813, for Valparaiso.
Porter would have done better to sail for the China seas or the
Indian Ocean. He knew that British war-vessels were searching for
him, and that Valparaiso was the spot where he would be directly in
their way. He arrived February 3, and five days afterward two British
vessels of war sailed into the harbor, making directly for the “Essex”
with the appearance of intending to attack and board her. The crew
of the “Essex” stood at quarters ready to fire as the larger ship ran
close alongside, until her yards crossed those of the “Essex,” and
Porter probably regretted to the end of his life that he did not seize
the opportunity his enemy gave him; but the British captain, from his
quarter-deck only a few feet away, protested that the closeness of
his approach was an accident, and that he intended no attack. The
moment quickly passed, and then Porter found himself overmatched.
The British frigate “Phœbe,” thirty-six guns, had sailed from
England in March, 1813, under secret orders to break up the United
States fur-establishment on the Columbia River.[288] At Rio Janeiro
the “Phœbe” was joined by the “Cherub,” a sloop-of-war rated at
eighteen guns, and both sailed in search of the “Essex.” The
“Phœbe” was one hundred and forty-three and three quarters feet in
length, by thirty-eight and a quarter in breadth; the “Essex” was one
hundred and thirty-eight and a half feet in length, and thirty-seven
and a quarter in breadth. The “Phœbe” carried a crew of three
hundred men and boys; the “Essex” carried two hundred and fifty-
five. The “Essex” was the better sailer, and the result of an action
depended on her ability to use this advantage. The broadside of the
“Essex” consisted of seventeen thirty-two-pound carronades and six
long twelve-pounders; the “Phœbe” showed only eight carronades,
but had thirteen long eighteen-pounders, one long twelve-pounder,
and one long nine-pounder. At close range, Porter’s battery would
overpower the “Phœbe’s” long guns, but the “Phœbe’s” thirteen long-
range eighteen-pounders could destroy her enemy without receiving
a shot in return. Porter knew all this, and knew also that he could not
depend on Chilian protection. No British captain in such a situation
could afford to be delicate in regard to the neutrality of Chili, which
was not even a recognized nation. At most Porter could hope for
immunity only in the port of Valparaiso.
Captain Hillyar of the “Phœbe” made no mistakes. During an
entire month he blockaded the “Essex” with his two vessels, acting
with extreme caution. At last Porter determined to run out, trusting to
a chase to separate the blockading cruisers; and March 28, 1814,
with a strong southerly wind, he got under way. As he rounded the
outermost point a violent squall carried away his maintopmast. The
loss threw on Porter a sudden emergency and a difficult,
instantaneous decision. He decided to return to harbor. A young
midshipman, David Farragut, who made his first cruise in the
“Essex,” gave his high authority in after years to the opinion that
Porter’s decision was wrong. “Being greatly superior in sailing
powers,” said Farragut, “we should have borne up, and run before
the wind.” The chance of outsailing the “Phœbe,” or separating her
from her consort, was better than that of regaining the anchorage.
The wind did not allow of a return to port, and the “Essex” was run
into a small bay three miles from Valparaiso, and anchored within
pistol-shot of the shore. There Hillyar had her wholly at his mercy. At
first he attacked somewhat timidly. Although Porter could bring only
three long twelve-pounders to bear, he damaged the “Phœbe’s”
rigging until Hillyar, in half an hour, hauled off to repair the injury,—or,
according to Hillyar’s account, the “Phœbe” was prevented by the
freshness of the wind from holding a position.[289] Finally the
“Phœbe” anchored, and began firing her broadsides of long
eighteen-pounders into the “Essex’s” quarter. The “Cherub” kept
under way, using only her bow guns. Reply was impossible. The
crew of the “Essex” fired what guns would bear, and got the ship
under way; but the “Phœbe” kept her distance, throwing thirteen
eighteen-pound shot into the “Essex” every five or ten minutes, until
the “Essex” was cut to pieces and her decks were shambles.
The last attack continued, according to Captain Hillyar, from 5.35
till 6.20 p. m., when the “Essex” struck. The entire battle lasted from
four o’clock until the surrender. The carnage was frightful and
useless. Porter declared that fifty-eight of his crew were killed. Hillyar
claimed one hundred and nineteen unwounded prisoners, while
Porter declared the number of unwounded prisoners to be seventy-
five. The British ships, with five hundred men, lost only fifteen killed
and wounded.
The loss of the “Essex,” like the loss of the “Chesapeake” and
“Argus,” was unnecessary. Porter need not have gone to Valparaiso,
or might have tried to run out at night, or might have fought, even
after the loss of his maintopmast, under less disadvantage. The
disaster completed the unfortunate record of the frigates for the year.
They made some sixteen prizes and busied many British cruisers,
but won no victories and suffered one bloody defeat.
The sloops told a different story. Early in 1814 three of the new
sloops-of-war were ready for sea,—the “Frolic,” the “Peacock,” and
the “Wasp.” They were heavy vessels of their class, about one
hundred and twenty feet long on the main-deck, and thirty-two feet in
extreme breadth; carrying crews of about one hundred and sixty
men, with an armament of twenty thirty-two-pound carronades and
two long eighteen-pounders. Although only one third the tonnage of
the forty-four-gun frigates, and carrying only one third the crew, the
new sloops-of-war threw nearly half the weight of metal,—for the
broadside of the “Constitution” commonly exceeded but little the
weight of seven hundred pounds, while the sloops threw three
hundred and thirty-eight. The difference was due not to the weight,
but to the range. The frigates carried thirty long twenty-four-
pounders; the sloops carried only two long eighteen-pounders. The
sloops were rigged as ships, and built with the usual solidity of war-
vessels, costing about seventy-five thousand dollars each.
The first to sail was the “Frolic,” from Boston, in February. She
captured only two prizes before she was herself taken, April 20, off
Matanzas, after a chase by the thirty-six-gun British frigate
“Orpheus,” assisted by a twelve-gun schooner.
The second sloop-of-war, the “Peacock,” commanded by Lewis
Warrington, sailed from New York in March. Warrington was a
Virginian, thirty-two years old and fourteen years in the service, with
the rank of master-commandant in 1813. Cruising down the coast,
the “Peacock” first ran in to St. Mary’s on the Florida frontier; and
then continuing southward, on the morning of April 29, off the Indian
River Inlet, she discovered a small convoy on its way from Havana to
Bermuda, under charge of the British eighteen-gun brig “Epervier.”
The British brig was no match for the American ship. She was
smaller, and carried only sixteen thirty-two and two eighteen-pound
carronades, with a crew of one hundred and three men and fifteen
boys.[290] The inferiority was something like four to three; but
Captain Wales of the “Epervier” gallantly brought his vessel into
action at the usual close-range of these murderous combats.
Captain Wales told the result in an official report, dated May 8, to
Vice-Admiral Cochrane.[291] The report was not published, the
British Admiralty having become sensitive to the popular outcry
against their naval management.
“At eight a. m.,” reported Captain Wales, “the wind being about
east-south-east, I saw a strange sail in the southwest apparently in
chase of us; at nine, perceiving her to near very fast and to be a
square-rigged vessel-of-war, I shortened sail and hauled to the wind
on the larboard tack to be between her and the convoy, being rather
ahead of them. The wind at this time veering round to the southward
enabled the stranger to lay up for us.... At 9.50 a. m. we weathered
her and exchanged broadsides; having passed her beam, we tacked,
shortened sail, and continued in close action until eleven a. m., when
—five of our larboard guns being disabled by the breeching-bolts
giving way, and three others by shot, and unable to manœuvre so as
to get the starboard guns to bear in consequence of the rigging and
sails being cut to pieces in the early part of the action by star-shot, the
main boom shot away, the foremast wounded in several places, and
several shot between wind and water, with four-and-a-half feet of
water in the hold, and the enemy seemingly in a state to continue the
action—I deemed it prudent to surrender.”
The giving way of the breeching-bolts did not wholly disable the
guns, for Captain Wales specially commended “Mr. Lawrence
Kennedy the Purser, who rendered much service in his exertions at
the after-guns by getting them in a fighting state again when
unshipped by the fighting-bolts coming out of their places.”
At the close of the battle the “Peacock’s” hull had not been
touched; aloft, her foreyard was disabled and a few upper stays were
cut away; of her crew, two men were slightly wounded,—but this was
all the injury sustained in running for three quarters of an hour under
the close fire of nine heavy guns.[292] The “Epervier” was reported by
Captain Warrington as showing forty-five shot-holes in her hull;
masts and rigging much cut up, and twenty-three men killed or
wounded in a crew of one hundred and twenty-eight. The difference
between the force of the two vessels amply accounted for the
capture; but the Admiralty might well show unwillingness to admit the
bad condition of the vessels-of-war to which it intrusted the duty of
convoying British mercantile shipping.[293] So complete was the
“Epervier’s” disaster that no excuse was offered for it, except the
plea that she was in almost every respect inferior to the standard
that British vessels of her class were supposed to maintain.
Captain Warrington saved the “Epervier” and brought her into
Savannah in spite of two British frigates encountered on the way. He
sailed again early in June, and passed the months of July and
August in British waters or in the track of British commerce from the
Faroe Islands to the Canaries. He burned or sunk twelve prizes,
besides making cartels of two more, and brought his ship through
the blockade into New York harbor, October 30, without injury, with
only one man lost and the crew in fine health.[294]
The third new sloop was named the “Wasp” after the famous
victor over the “Hornet.” The new “Wasp” sailed from Portsmouth,
New Hampshire, May 1, under command of Johnston Blakeley. Born
in Ireland in 1781, Blakeley was from infancy a North Carolinian. He
became in 1800 an officer in the navy. Blakeley and the “Wasp” of
1814, like Jones and the “Wasp” of 1813, ran a career in which
tragedy gave a deeper tinge than usual to the bloody colors they
won; but their success was on the whole greater than that of any
other national cruiser from the beginning to the end of the war.
Merely as a story of adventure Blakeley’s career was exciting, but

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Validamycin and its Derivatives.

Discovery, Chemical Synthesis, and

Carbohydrates in Drug Discovery and Development:

N-Sulfonated-N-Heterocycles: Synthesis, Chemistry, and

Environmental Technology and Sustainability: Physical,

Wearable Physical, Chemical and Biological Sensors:

Green Sustainable Process for Chemical and

Systems Immunology and Infection Microbiology 1st

Logarithm And Its Applications 1st Edition - eBook PDF

Imidazole-Based Drug Discovery (Heterocyclic Drug

Xiaolong Chen, Yuele Lu,

Copyright r 2017 Elsevier Ltd. All rights reserved.

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Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00001-0

1.2 AGRICULTURAL ANTIBIOTICS FOR PLANT PATHOGENS

1.3 VALIDAMYCINS: MAGIC AGRICULTURAL ANTIBIOTICS

Validamycin F H H H β-D-Glc H α-D-Glc

FIGURE 1.1 Chemical structures of validamycins.

1.4 AIMS OF THE WORK

(Shen, 1996). In recent years, the production of validamycins in other

2.2 MICROBES FOR PRODUCING VALIDAMYCINS

Validamycin and Its Derivatives. DOI: http://dx.doi.org/10.1016/B978-0-08-100999-4.00002-2

brown soluble pigment on proteinaceous media was not observed, T-7545

TABLE 2.1 Cultural Characteristics of S. hygroscopicus var. limoneus

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

TABLE 2.1 (Continued)

Cultural Characteristics S. hygroscopicus var. S. hygroscopicus var.

TABLE 2.2 Physiological Properties of Strains T-7545 and TH82

TABLE 2.2 (Continued)

2.2.2 Complete Genomes for the Two Microbes

2.2.2.1 General Features of the S. hygroscopicus

TABLE 2.3 Utilization of Carbon Sources by T-7545 and TH82

Carbon Source T-7545 TH82 Carbon Source T-7545 TH82

Not tested. 1 1 : Good growth. 1 : Fair growth. 6: No or very poor growth.

Streptomyces genomes (Table 2.4). The linear chromosome (10,145,833 bp)

consisting of several packed palindromes with nonconserved loop

proportion to the corresponding chromosomal length. However, the genome of

2.2.2.2 General Features of the S. hygroscopicus subsp.

TABLE 2.5 Genomic Features of S. hygroscopicus subsp. limoneus

Features Chromosome I Chromosome II

for producing validamycin. The detection and localization of complete val

2.3 PRODUCTION AND ISOLATION OF VALIDAMYCINS

2.3.1.1 Temperature of Submerged Culture

TABLE 2.6 Effect of Temperature on Validamycin Production

Incubation 27 C 37 C

TABLE 2.7 Effect of Medium Volume on Validamycin Production

Incubation Volume of the Mediumb

2.3.1.2 Effect of Medium Volume on Validamycin Production

2.3.1.3 Effect of Medium Composition